vol.4.no.1.indd journal of tropical crop science vol. 4 no. 1, february 2017 www.j-tropical-crops.com 32 mohammad wahyu sautomo, muhamad syukur evaluation of progress selection of f2 f6 population, a cross between two lowland tomato genotypes mohammad wahyu sautomo, muhamad syukur* department of agronomy and horticulture, bogor agricultural university, jalan meranti, ipb darmaga campus, west java, indonesia 16680 *coresponding author, email:mhsyukur@gmail.com abstract one of the parameters that can be measured from the activities of selection is progress selection. the population of a plant which has been selected is expected to be good against a derivative of a crop which was selected. the aim of this research is to study the results of progress selection varieties superior of tomato plants down the generations in the population f2 005001 until f6 005001-4-1-12-3 with a standard tomato cultivar, “ratna”. the result showed that the selected genotypes have shown improvements over the standard cutivar in terms of weight per fruit, fruit weight per plant, the number of fruits per plant, fruit thickness and the age of harvested plants in the population f2-005001 until f6 005001-4-1-12-3. the mean against the character being observed in the population f2 005001 until f6 005001-4-1-12-3 indicated that the result is better if compared with both parents, p1 (ssh-5 ) and p2 (intan) and the standard cultivar “ratna”. the value of heritability a wider sense indicate its value being on each character of being selected, while the value of heritability in a more narrow sense showed a low value on every character. the value of progress against character selection of weights per fruit, the weight of the fruit per plant, the number of fruit per plant, thick fl esh fruit and the age of harvest showed increased slow progress in a genotype f2 005001 followed by a period of rapid progress in a genotype f4 005001-4-1 and very slow in a genotype f6 005001-4-1-12-3. keywords: progress selection, population, genotype, mean, heritability introduction production of tomatoes, solanum lycopersicum, in 2010-2011 increased by 7% from 891,616 tons (2010) to 954,046 tons (2011) with productivity average of 16.65 tons per ha (dirjenhorti, 2012). national demand for tomatoes consistently increases every year, so many superior varieties are developed which are able to produce high production. high productivity is one of the major breeding objectives (hidayat et al., 1997). however, with the limitations of high environmental factors conducive to tomato growing, high yielding varieties of tomatoes were developed that are able to adapt in lowland with high productivity. one of the stages in the assembling of superior varieties of tomatoes is by selection, a process of separation of plants from a mixed population based on the appearance of certain characters (arif, 2008). the purpose of selection is to select particular desired phenotypes to obtain a better genotype (wahdina, 2004). there are two forms of selection for character enhancement, namely the selection between the existing population to enhance the desired character, and selection in the population to create new varieties, resulting in the off spring of the crosses usually consisting of segregation (syukur et al., 2012). evaluation of the selection between the existing populations is conducted to determine the response of a population selection to the expected characters. one of the measurable parameters of the selection activity is the progress of selection. the estimates of selection progress will depend largely on the value of heritability, the standard intersection of selected populations, and the intensity of selection (mejaya et al., 2010). selected crop populations are expected to provide better results for derivatives of selected plants. heritability value is the ratio of genotype with the total amount of phenotypes. generally, narrow sense of heritability gets much attention because the additive eff ect of each allele is inherited from the elder to its derivatives (memen et al., 2007). in this study, evaluation of selected characters is expected to get good selection progress. this study aims to study the progress of selection of superior varieties of tomato plants between generations in the population f2 f6 with the “ratna” variety as the comparing variety. journal of tropical crop science vol. 4 no. 1, february 2017 www.j-tropical-crops.com 33evaluation of progress selection of f2 f6 population, a cross .......... materials and methods the research was conducted at the leuwikopo experimental station and plant breeding education laboratory of agronomy and horticulture department, bogor agricultural university, indonesia, from december 2013 until april 2014. the materials used in this research were tomato parents ssh-5 (40 plants), tomato parents intan (40 plants), and fi ve generations of selfi ng derivatives (f2 005001) (100 plants), the third derivative (f3 005001-4) (100 plants), the fourth derivative (f4 005001-4-1) (100 plants), the fi fth derivative (f5 005001-4-1 -12) (100 plants), and the sixth derivative (f6 005001-4-1-12-3) (100 plants). two seeds of tomato were planted per hole on seedling trays containing media soil mixture and manure (1:1 v/v). gandasil d at the rate of 2 g.l-1 was applied in a liquid form. watering was conducted twice a day, morning and afternoon. land clearing was done before it was prepared and leveled after which the land was then divided into seven plots for each varieties tested. the size of each bed was 1 m x 5 m with 80 cm as the distance between beds. fertilizers were applied after planting with half dose 50 kg n per ha (5 g urea per plant), 100 kg p2o5 per ha (16 g tsp per plant) and 120 kg k2o per ha (20 g kcl per plant) (harjadi and sunaryono, 1989). plant pest and disease control were conducted twice a week using mancozeb 80% or propinep 70% (2 g.l-1), profenofos (2 ml.l-1) and dicofol (2 ml.l-1). harvesting was conducted when the fruit skin were yellow reddish in colour. results and discussion harvest age heritability values are referred to as low if less than 20%, medium if between 20-50% and high if the value is more than 50% (sujiprihati et al., 2003). the width sense of heritability value character of tomato harvest age was in the medium range of 40.66 to 54.03%, while the narrow sense of heritability was in the low range of 6.24 to 15.13% (table 5). based on the additive ratio contained in table 1, it is known that the value of the additive variety of harvest age characters is low. the low variety of additives aff ecting the age of harvest suggests that selection done in the early generations did not provide much genotype progress. a study by arif et al. (2012) showed thatthe width sense of heritability value in the character of harvest age is in the medium-high range, while heritability in the narrow sense is in the low range. our fi ndings are in line with this study, where the width sense of heritability was in the medium range and narrow sense of heritability was in the low range. the mean value of the harvest age character decreases from the genotype f2 005001 f6 005001-4-1-12-3 which was in the range of 67 to 65 dap. the decline in the mean values on the character of harvest age indicated that there was a great selection of progress from the parents to the progenies. idris et al. (2011) reported that there was a positive correlation between harvest age of maize and seed weight per ear. our study had shown a similar result where increasing harvest age in each genotype aff ects the weight increase per fruit in each genotype. the addition of harvested lifespan had an enormous impact on the opportunities for plants to accumulate more organic matter and photosynthates in the fruit ripening process. weight of fruit per plant heritability value of the broad mean of fruit weight character per plant was in the medium range of 18.75 to 61.24%. a high heritability value is caused by a relatively homogenous environment (arifi n, 2008), the low heritability value in the f3 005001-4 genotype (18.75%) was infl uenced by environmental factors table 1. component variety (?) and heritability of tomato harvest age population harvest age range (days) mean (days) diversity h2bs (%) h 2 ns (%) a ratio p1 (ssh-5) 65 – 76 70.25 13.69 p2 (intan) 55 – 64 57.93 5.55 f2 005001 58 – 75 65.43 16.48 41.64 6.24 0.15 f3 005001-4 58 – 75 66.81 19.50 50.68 8.96 0.18 f4 005001-4-1 56 – 75 63.14 16.21 40.66 16.94 0.42 f5 005001-4-1-12 56 – 73 67.48 16.70 42.42 16.11 0.38 f6 005001-4-1-12-3 56 – 79 65.29 20.93 54.03 15.13* 0.28 “ratna” 57 – 73 62.60 11.78 note : * = based on extrapolation of the additive ratio journal of tropical crop science vol. 4 no. 1, february 2017 www.j-tropical-crops.com 34 mohammad wahyu sautomo, muhamad syukur (table 2), including pest and diseases. the severe pest and disease attack occurredd in the population of f3 005001-4 had directly aff ected the fruit yield per plant. table 2. various components and heritability of fruit weight per tomato plant population fruit weight per plant range (g) mean (g) diversity h2bs (%) h 2 ns (%) a ratio p1 (ssh-5) 30.05 63.25 49.23 151.55 p2 (intan) 54.42 805.53 309.28 53996.48 f2 005001 89.28 1023.23 231.53 35557.66 23.86 11.09 0.46 f3 005001-4 83.17 768.35 235.22 33322.38 18.75 17.96 0.96 f4 005001-4-1 94.7 1276.53 389.80 69841.74 61.24 29.54 0.48 f5 005001-4-1-12 69.86 975.02 430.46 56990.66 52.29 24.24 0.46 f6 005001-4-1-12-3 384.76 1193.03 806.15 40090.63 32.47 14.29* 0.44 “ratna” 23.27 1257.83 434.25 108786.46 note : * = based on extrapolation of additive ratio the narrow sense of heritability value of character of fruit weight per plant was in the low range of 11.09 to 29.54%. according to masruroh et al. (2009), the value of heritability in the narrow sense indicates the magnitude of additive infl uence on phenotype appearance. a narrow sense of heritability value indicated a low proportion of additive variance (table 2). this was less favorable in the selection process because of the low possibility to improve fruit weight per plant. the number of fruits per plant the width sense of heritability value of the character of the number of fruit was in the medium range of 44.32 to 51.95%, except in the genotype f3 0050014 which is in the low range of 10.16% (table 3). the narrow sense of heritability value of the number of fruit was in the low range of 7.4 to 26.33%. the low value of heritablitas in genotype f3 005001-4 was caused by pest and disease attack resulting in a decrease in population hence infl uenced the value of genotype varieties. based on the results on the number of fruits per plant it can be seen that the highest variety of values was in the genotype f2 005001 (51.95%) (table 3) whereas the value of the highest narrow sense of heritability is in the genotype f4 005001-4-1 (26.33%). according to gaswanto et al. (2009) the narrow sense of heritability becomes more important in an inheritance to its derivatives due to the diversity caused by the role of the additive gene as part of the total genetic diversity. the higher narrow sense of heritability value of the genotype f4 005001-4-1 indicated the eff ect of higher additive varieties so that the nature of the number of fruits will be inherited from the off spring of the crosses occurring in the f4 005001-4-1 generation. thickness of fruit flesh the width sense of heritability value of the thickness of the fruit fl esh was in the medium range of 24.64 table 3. various components and heritability of the number of fruits per plant population the number of fruit per plant range (fuits) mean (fuits) diversity h 2 bs (%) h 2 ns (%) a ratio p1 (ssh-5) 2 – 12 3.75 9.94 p2 (intan) 3 – 31 11.78 52.54 f2 005001 2 – 43 11.25 65.01 51.95 13.55 0.26 f3 005001-4 3 – 26 11.38 34.78 10.16 7.40 0.73 f4 005001-4-1 6 – 36 16.20 59.07 47.11 26.33 0.56 f5 005001-4-1-12 8 – 40 19.00 56.11 44.32 19.64 0.44 f6 005001-4-1-12-3 10 – 41 26.29 58.15 46.27 14.81* 0.32 “ratna” 2 – 45 18.24 135.13 note : * = based on extrapolation of additive ratio journal of tropical crop science vol. 4 no. 1, february 2017 www.j-tropical-crops.com 35evaluation of progress selection of f2 f6 population, a cross .......... to 55.88%, while the narrow sense of heritability value was in the low range of 8.18 to 14.96% (table 4). based on the thickness and heritability of tomato fl esh (table 4), the highest mean value of heritability was in f4 005001-4-1 genotype (55.88%) and the lowest value was in f6 006001-4-1-12-3 genotype (24.64%). the highest narrow sense of heritability value is in the genotype f3 005001-4 (14.96%). this indicated that eff ective selection was performed on the f4 005001-4-1 generation. however, in the fi nal population of the f6 005001-4-1-12-3 genotype the selection was not eff ective. weight per fruit the width sense of heritability value of the character of weight per fruit was in the medium range of 29.28 to 39.60%, in the genotype f2 005001f5 0050014-1-12. however heritability decreased to as low as 19.18% in the genotype f6 005001-4-1-12-3 (table 5) while the narrow sense of heritability value of weight character per fruit was in the low range of 11.72 to 26.63%. a similar study on chilly by syukur (2012) indicated a high weight per fruit. however, the results of this study are somewhat diff erent and it is thought to be due to diff erences in population and the methods employed. the value of heritability shows the eff ectiveness of selection of a character. according to arifi n (2008) heritability estimates are used as a fi rst step in the selection work of segregated populations. the low diversity value of the genotype f6 005001-4-1-123 shows that selection of weight per fruit in the f6 generation will result in a low selection progress. according to arif et al. (2012) the narrow sense of heritability in approaching the width sense of heritability shows that the proportion of the additive diversity is greater than the dominant diversity. this is in accordance with the additive ratio (table 1), which shows a high diversity of additives compared to the dominant. progress selection progress selection is a value that becomes the parameter of success on the selection of a population. in simple terms the progress of selection is the table 4. the thickness of tomato fl esh and its heritability population thickness of fruit fl esh range (mm) mean (mm) diversity h2bs (%) h 2 ns (%) a ratio p1 (ssh-5) 0.96 4.03 2.17 0.72 p2 (intan) 0.90 4.03 2.32 0.36 f2 005001 1.50 4.83 3.04 0.76 28.86 8.92 0.31 f3 005001-4 2.04 5.45 3.08 0.84 35.95 14.96 0.42 f4 005001-4-1 2.11 5.95 3.81 1.22 55.88 8.69 0.16 f5 005001-4-1-12 2.57 6.15 4.50 0.80 32.89 8.18 0.25 f6 005001-4-1-12-3 3.19 6.98 5.37 0.72 24.64 8.62* 0.35 “ratna” 1.51 7.02 4.59 1.55 note : * = based on extrapolation of additive ratio table 5. various components and heritability weights per tomato fruit population weight per fruit range (g) mean (g) variation h2bs (%) h 2 ns (%) a ratio p1 (ssh-5) 15.03 59.09 25.09 9.95 p2 (intan) 18.13 50.52 32.66 81.90 f2 005001 6.98 40.41 20.15 76.03 39.60 11.72 0.30 f3 005001-4 10.40 43.62 20.49 70.01 34.40 19.13 0.56 f4 005001-4-1 20.35 48.37 31.95 69.69 34.11 26.4 0.77 f5 005001-4-1-12 24.24 64.18 35.72 64.94 29.28 21.63 0.74 f6 005001-4-1-12-3 36.44 66.62 48.87 56.82 19.18 13.43* 0.70 “ratna” 12.21 55.73 29.18 100.12 note : * = based on extrapolation of additive ratio journal of tropical crop science vol. 4 no. 1, february 2017 www.j-tropical-crops.com 36 mohammad wahyu sautomo, muhamad syukur diff erence between the mean of the descendants of the selection with the median selection of the population (gratitude et al., 2012). the value of selection of weight per fruit, weight of fruit per plant, number of fruits per plant, fruit fl esh thickness and plant harvest age showed a slow increase in genotype f2 005001 followed by a period of rapid advancement in genotype f4 005001-4-1, and progressively slowed down in genotype f6 005001-4-1-12-3. this is in line with zulfarosda et al. (2012) where the weight of fruit per grain, fruit weight per plot, fl owering age, and harvest age of ten tomato genotypes tested had slow selection progress. susiana (2006) demonstrated that most chili characters had high genetic expectation values except for fl owering age, harvest age, marketable fruit weight and thick bark of fruit having low genetic progress value (0%). the age of tomato harvest showed the decline of the days of each generation with values from 0.45 to 1.48 (table 6). this means that each generation experiences an acceleration of harvest time between 0.45 days to 1.48 days after planting. graph of the mean age of harvest (figure 1) shows the average yield of harvest age of each generation was between the second harvest age of p1 (ssh-5) which has a long harvesting age character with p2 (intan) table 6. progress of selection of tomato yield characters population harvest age fruit weight per plant fruit number per plant s gm gt s gm gt s gm gt f2 005001 -4.56 -0.28 -0.45 231.80 25.70 36.80 13.75 1.86 1.92 f3 005001-4 -4.81 -0.43 -0.70 156.80 28.16 57.69 7.62 0.56 0.77 f4 005001-4-1 -4.94 -0.84 -1.20 381.11 112.58 137.40 12.70 3.34 3.56 f5 005001-4-1-12 -6.03 -0.97 -1.16 312.70 75.79 101.83 13.00 2.55 2.59 f6 005001-4-1-12-3 -5.89 -1.08 -1.48 260.31 37.19 50.34 11.41 1.69 1.99 note : gm = progress selection based on diff erential selection; gt = progress selection based on theory ( i =1.76) which had the character of harvest age. in the study of barmawi et al. (2013) the value of the genetic progress of the harvest age is low suggesting that there was a slow progress in the selection in every generation of selection. estimated progress of selection will be highly dependent on the heritability value, standard intersection of selected population and the selection intensity (mejaya et al., 2010). the comparison of selection progress using the diff erence between the selected middle value and the middle of the initial population (diff erential selection) using the selection intensity has a small diff erence, where the value of selection progress using diff erential selection always has lower value than the value of selection progress by using intensity of selection (table 6 and table 7). this was because the diff erential selection used had not been standardized for the deviation, so the diff erence of the units in comparing the selection power of two or more characters could not be used diff erential magnitude of selection. quantitative characters are infl uenced by many genes whose infl uence is cumulative (murti et al., 2015). the value of selection of weight per fruit, the weight of fruit per plant and the number of fruit figure 1. the mean values of harvest age of tomato from population f2-f6 and both parents. journal of tropical crop science vol. 4 no. 1, february 2017 www.j-tropical-crops.com 37evaluation of progress selection of f2 f6 population, a cross .......... per plant had the highest selection progress on the genotype f4 005001-4-1 and decreased in the next generation. in contrast to the progress of selection on the thickness of fruit fl esh and fruit harvest age, the highest thickness of the fl esh was observed on the genotype f3 005001-4 with a value of 0.22 indicating a progressively diff erential selection and 0.24 was progressively selection with the intensity of selection (table 7). while the highest selection of fruit harvest age on genotype f6 005001-4-1-12-3 with a value of 1.08 in progress diff erential selection and 1.48 in progress selection with the intensity of selection (table 7). fruit weight per plant with cycles of 36.80 to 137.40 (table 6). this means that each generation contains additional fruit per plant with an added value of 36.80 grams per plant up to 137.40 grams per plant. graph of the median fruit-per-plant value (figure 2) shows an improvement in the larger f6 005001-4-1-12-3 genotype compared to both parents p1 (ssh-5) and p2 (intan), and the genotype of each generation. in the study of idris et al. (2011) fruit weight per plant is positively correlated with the weight per fruit, so the increase in fruit weight per fruit could potentially increase yield. there was an increased progress of high selection of fruit weight per plant on genotype f4 005001-4-1 and genotype f5 005001-4-1-12 (table 6). this is due to the mean value and narrow sense of heritability value that infl uenced the selection of a population. on the other hand, the additive ratio level in the f3 005001-4 genotype of 0.96 (table 2) gives a greater impact on subsequent generations, so the selection of the f3 005001-4 genotype provides higher upgrades in the next generation. the number of fruits per plant shows the progress of selection of each cycle with values from 0.77 to 3.56 (table 6). this means that each generation has an additional number of fruits per plant with an additional value of 0.77 fruits up to 3.56 fruits per plant. graph of the mean value of the number of fruits per plant (figure 3) shows an increase in the number of fruits per generation with the genotype f6 005001-4-1-12-3 showing the highest number of fruits when compared with both parents ie p1 (ssh-5) fruit per plant is very low and p2 (intan), as well as the genotype of each generation. the parental crossover of p1 (ssh-5) had a low number of fruits per plant compared to the p2 (intan) parent which yielded a high number of fruits per plant producing saplings that inherit the number of fruits per plant with average yields between the two ages in genotype f2 005001 (figure 3). this shows that the infl uence of p2 (intan) is stronger than p1 (ssh5). therefore it is possible that the gene p2 (intan) is dominant for the character of the number of fruits table 7. progress of selection of advanced tomato yield characters population thickness of fruit fl esh weight per fruit s gm gt s gm gt f2 005001 1.20 0.11 0.14 13.17 1.54 1.80 f3 005001-4 1.49 0.22 0.24 12.10 2.32 2.82 f4 005001-4-1 1.58 0.14 0.17 11.63 3.07 3.88 f5 005001-4-1-12 1.32 0.11 0.13 12.18 2.64 3.07 f6 005001-4-1-12-3 1.06 0.09 0.13 8.81 1.18 1.78 note : gm = progress selection based on diff erential selection; gt = progress selection based on theory (i =1.76) figure 2. the mean values of fruit weight per plant of population f2-f6 and both parents journal of tropical crop science vol. 4 no. 1, february 2017 www.j-tropical-crops.com 38 mohammad wahyu sautomo, muhamad syukur per plant. the results of research by masruroh et al. (2009) showed that the number of fruit on the tomato crossing lv6123 x lv5152 fully infl uenced the action of additive gene. the existence of additive gene action provides an increase in the number of fruit per plant that occurs after the best population selection. the thickness of the tomato fl esh shows a progress in the selection of each cycle with values from 0.13 to 0.24 (table 7). this means that every generation had increased thickness in fruit fl esh with an additional value of 0.13 to 0.24 mm per generation of selection. graph of mean value of thickness of fruits fl esh (figure 4) shows an increase in the thickness of fruit fl esh of each generation with genotype f6 005001-41-12-3 having the largest fruit thickness as compared to the two parents namely p1 (ssh-5) which has thin fl esh and p2 (intan) with character of medium fl esh thickness. the weight per fruit shows the progress of selection of each cycle with the values of 1.78 to 3.88 (table 7). each generation had an average weight per fruit ranging from 1.78 grams to as high as 3.88 grams per fruit in the genotype f4 005001-4-1. the weight character per fruit is one of the most important selection criteria that correlated with other characters and showed the potential outcomes (zulfarosda et al., 2012). graph of weight mean value per fruit (figure 1) shows a selection progress on genotype f6 0050014-1-12-3 which has the biggest weight per fruit when compared to both parents p1 (ssh-5) and p2 (intan), as well as the genotype of each generation. increased progress of low weight selection per fruit was shown in genotype f2 005001 and f3 005001-4 when compared to both parents (figure 5). this is consistent with the data in table 1 where there is a modest advancement of mean values on the genotype f2 005001 and f3 005001-4. the low advancement of mean values in this initial genotype was due to the high value of variance indicating high levels of heterozygosity compared with the fi nal genotype. the high genetic diversity of early generations in a population indicated that selection can be performed on the desired variables according to the purpose of plant breeding activities performed (susiana, 2006). the result of the t-test was conducted on population f6 005001-4-1-12-3 as genotype of selection result figure 3. the mean values of the number of fruits per plant of population f2-f6 and the two parents figure 4. the mean values of the tomato fl esh thickness of population f2-f6 and both parents figure 5. the mean values of weight per fruit of the f2-f6 population and the two parents journal of tropical crop science vol. 4 no. 1, february 2017 www.j-tropical-crops.com 39evaluation of progress selection of f2 f6 population, a cross .......... with “ratna” as varieties of comparison on weight per fruit, fruit weight per plant and number of fruit table 8. test the t-student tomato yield characters of population f6 005001-4-1-12-3 agains t the standard cultivar “ratna” populasi average fruit weight (g) fruit weight per plant (g) number of fruits per plant fruit fl esh thicnkess (mm) harvest age (day) f6 005001-4-1-12-3 48.87 ± 7.54 806.15 ± 200.23 26.29 ± 7.63 5.37 ± 0.85 65 ± 5 “ratna” 29.18 ± 10.01 434.25 ± 329.83 18.24 ± 11.62 4.59 ± 1.24 63 ± 3 t-test 10.09* 6.09* 3.79* 3.45ns 3.65ns prob > |t| 0.000 0.000 0.000 0.001 0.000 note : * = signifi cantly diff erent to “ratna”; ns = not signifi cantly diff erent from “ratna” per plant there is a real diff erence (table 8). this result demonstrated that the weight character per fruit, the weight of the fruit per plant and the number of fruits per plant f6 005001-4-1-12-3 has a better value compared to the standard cultivar “ratna”. however t-test results was conducted on population f6 005001-4-1-12-3 with “ratna” on the thickness of fruit fl esh and harvest age showed no signifi cant diff erence. this is due to the low selection progress in harvest age and fl esh thickness as well as the good character of the “ratna” variety that has a thick fruit fl esh that is large and the age of the shorter harvest. the selection improvement of thick character of fruit fl esh and harvest age did not show tangible results when compared with “ratna”. the characters that have low selection progress, generally indicated that the character was a quantitative that was controlled by many genes (komariah and amalia 2012). conclusion the result of this research demonstrated that there were progress of selection on character of weight per fruit, fruit weight per plant, number of fruit per plant, fruit fl esh thickness and plant harvest age in population f2-005001 to f6 005001-4-1-12-3. the mean values of the characters observed in the population f2 005001 f6 005001-4-1-12-3 showed better results when compared to both p1 (ssh-5) and p2 (intan) parents, and to the standard cultivar “ratna”. the width sense of heritability value indicates the current value of each selected characterwhile the narrow sense of heritability value indicated a low value on each character. the value of selection of weight per fruit, weight of fruit per plant, number of fruits per plant, thickness of fruit fl esh and harvest age showed a slow increasd in the genotype f2 005001 and followed by a period of rapid advancement in genotype f4 005001-4-1 and progressively slowed down in the genotype f6 005001-4-1-12-3. references arif, a.b. 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(2006). pendugaan nilai heritabilitas, variabilitas dan evaluasi kemajuan genetik beberapa karakter agronomi genotipe cabai (capsicum annuum l.) f4. thesis. institut pertanian bogor, bogor. syukur, m., sujiprihati, s., and yunianti, r. (2012). “teknik pemuliaan tanaman”. penebar swadaya, jakarta. wahdina. (2004). evaluasi kemajuan seleksi generasi f3 dan f4 persilangan kedelai varietas slamet x gh-09. thesis. institut pertanian bogor, bogor. zulfarosda, r., kendirini, n., and respatijarti. (2012). potensi hasil sepuluh genotip tomat (lycopersicon esculentum l.) di karangploso malang. jurnal produksi tanaman 1, 450-455. invigoration increased quality and storability of true seed of shallot .......... journal of tropical crop science vol. 9 no. 2, june 2022 www.j-tropical-crops.com 145 invigoration improved quality and storability of true seed of shallot (allium ascalonicum l.) qudus sabha adhinugrahaa, eny widajatib, endah retno palupib a seed science and technology program, graduate school, faculty of agriculture, ipb university, bogor 16680, indonesia b department of agronomy and horticulture, graduate school, faculty of agriculture, ipb university, bogor 16680, indonesia *corresponding author; email: endah_retno@apps.ipb.ac.id abstract seed invigoration is a pre-planting seed treatment by balancing the seed water potential and stimulating their metabolic activities so that the seeds germinate simultaneously. in this study, invigoration treatments were applied to improve the quality of the true seeds of shallots (tss) that had started to deteriorate. invigoration increases the moisture content of the seeds so the seeds need to be re-dried to extend their storability. the aims of the study were to determine the best invigoration treatment and the drying method to maintain the benefit of invigoration, and to prolong tss storability after invigoration and drying. this research was conducted at the laboratory of seed quality testing, ipb university, indonesia, and consisted of three experiments, i.e., invigoration of deteriorating tss to improve their viability and vigor, drying after invigoration to extend seed storability, and seed storability after invigoration and drying treatments. the first experiment was arranged in completely randomized design with onefactor (invigoration solutions), i.e. control, medium containing 3% kno3, 50 ppm ga3, 0.5 % znso4 or -10 bar peg6000. the second experiment was conducted using a two-factor completely randomized design, drying temperature (20˚c, 30˚c, and 40˚c), and drying duration (8 hours, 16 hours, 32 hours, and 48 hours). the third experiment was arranged in a nested design with two factors. the first factor was a seed storage condition, i.e., ambient temperature, air-conditioned room, and refrigerator. the second factor was seed packages, i.e., aluminum foil and polypropylene plastic. non-invigorated seeds were used as control. the studies were conducted on two shallot varieties separately, “trisula” and “lokananta”, except in the third experiment which only used “lokananta”. the results showed that invigoration in 3 % kno3 and 50 ppm ga3 effectively improved the vigor of deteriorating tss. drying the seeds at 40˚c for 8 hours was the most effective method to reduce seed moisture content without reducing the benefit of invigoration. storing the invigorated tss in an air-conditioned room with aluminum foil packaging, or in a refrigerator with aluminum foil or plastic packaging until 14 weeks maintained the benefit of invigoration. the quality of the invigorated tss was maintained until 14 weeks in an air-conditioned room with aluminum foil packaging, or in a refrigerator with aluminum. keywords: seed drying, seed storage, viability, vigor introduction the true seed of shallot (tss) is an alternative planting material in shallot cultivation other than bulb seeds. botanical seeds have advantages over the bulb seeds, including pathogen-free, less expensive, and allow longer-term storage compared to bulb seeds (askari-khorasgani and pessarakli, 2019). only 3 kg of botanical seeds is required per ha, in contrast to 2 tons of bulb seeds. tss have no dormancy, and can be stored for more than one year (sopha et al., 2015, rosliani et al., 2018). at 6% moisture content, tss can be stored for up to 12 months at 4-15˚c with relative humidity (rh) of 40-60% (selvy and saraswathy, 2017). physiological and biochemical changes occur during storage, such as decreased germination and vigor index, decreased activity of dehydrogenase and amylase enzymes, and increased cell membrane leakage (umesh et al., 2014). at a later stage of deterioration, seed quality decreases, indicated by reduced seed viability and vigor (brar et al., 2019a). seed invigoration is generally used as a pre-planting treatment to improve germination and seedling development by balancing the water potential of the seeds and stimulating the metabolic activities so that the seeds germinate simultaneously (ilyas, journal of tropical crop science vol. 9 no. 2, june 2022 www.j-tropical-crops.com 146 qudus sabha adhinugraha, eny widajati, endah retno palupi, 2012). invigoration on tss has been studied using various solutions such as kno3 (muruli et al., 2016), ga3 (agung and diara, 2017), znso4 (saranya et al., 2017; kamanga et al., 2021), polyethylene glycol (peg) (arin et al., 2011). invigoration by soaking tss in 0.5 % znso4 for 10 hours increased germination from 53% to 85.5% (kamanga et al., 2021), while immersion in 3% kno3 for 24 hours increased germination from 75% to 81%, increased vigor index and mean germination time (muruli et al., 2016). brar et al. (2019b) reported that invigorating one-yearold tss using 50 ppm ga3 for 16 hours improved viability (78%), vigor index-i (germination length x seed germination of 1302.8), and field emergence (65.93%). invigoration treatment increases seed moisture content, therefore the seeds need to be redried to extend their storability. re-drying chili seeds at 35°c and 75% relative humidity for 48 hours after hydropriming (soaking the seeds in distilled water for a certain period of time) maintained the positive effect of hydropriming without affecting the quality of the seeds. however, re-drying seeds at a low temperature (15°c) occurs slowly and causes a decrease or a delay germination (demir et al., 2005). sedghi et al. (2012) reported that a temperature of 20˚c 30˚c is the ideal for drying following seed invigoration of calendula officinalis l. the role of invigoration treatment in extending seed storability is still debated. butler et al. (2009) and wood and hay (2010) reported that invigoration extends the storability of digitalis purpurea and rhododendron griersonianum seeds, while hussein et al. (2015) and malek et al. (2019) reported the contrary, invigoration harms seed storability. butler et al. (2009) reported that digitalis purpurea seeds invigorated with peg6000 at osmotic pressure of -1 mpa and dried properly had an extend storability. on the other hand, hussein et al. (2015) reported that invigorated rice seeds then stored at a temperature of 25˚c harmed seed storability compared to non-invigorated seed. invigorated rice seeds (priming) only had 20-30% germination after being stored for 210 days at 25°c, as opposed to non-invigorated rice seeds, which maintained the germination up to 80% after being stored for 210 days at 25°c. the results of the previous studies indicated that the effect of invigoration on the seed storability varied with species. invigoration followed by appropriate drying does not only improve seed viability and vigor but also extends seed storability. this technique would be very useful in managing seed production, especially those seeds which only be produced once a year such as tss. this study aims to obtain the optimal invigoration treatment and drying methods for tss, and to determine their storability post-invigoration. material and methods location our study was conducted from april 2021 to march 2022 at the seed quality testing laboratory, leuwikopo experimental station (06˚56’s dan 106˚73’ e), department of agronomy and horticulture, faculty of agriculture, ipb university. materials the materials used were two varieties of tss, “trisula” and “lokananta”. seed of var. “trisula” was produced by the brastagi vegetable crops research station, harvested on august 24, 2018. the tss was 32 months after harvest (mah) when was used for this research, during which time the seed was stored in a refrigerator (4±2°c) with polypropylene plastic packaging. seed of var. “lokananta” was produced by pt east west seed indonesia with an expired date in april 2021, and previously was stored in an air-conditioned room (18±2°c) with aluminum foil packaging. invigoration was applied using kno3, ga3, znso4, and polyethylene glycol (peg6000). aerated invigoration bottles were used for invigoration at 20± 2˚c. following invigoration, aluminum foil and polypropylene (pp) plastic packaging were used to store seeds. germination was carried out in germination boxes with 3-ply of filter paper and put in germination room with temperature of 20± 2˚c. treatment experiment 1. invigoration to improve viability and vigor this experiment was arranged using a one-factor completely randomized design consisting of five treatments, namely no treatment (control), kno3 at 3% (muruli et al., 2016), ga3 at 50 ppm (brar et al. 2019b), znso4 at 0.5% (kamanga et al. 2021), and -10 bar peg 6000 (arin et al., 2011). peg 6000 at -10 bar was prepared by mixing 273 g of peg in 1-l of distilled water at 20˚c (michel, 1983). a total of 400 grains of tss each from the “trisula” and “lokananta” varieties were soaked in 200 ml of four types of aerated invigoration solutions in plastic bottles until the seeds were completely immersed; all treatments were repeated four times. each treatment was incubated at 20˚c for 24 hours except for invigoration in znso4, which was only incubated for 10 hours (saranya et al., 2017). the seeds were air-dried at room conditions (26-32˚c, 61-79% rh) for 5 hours (kamanga et al., 2021) germinated in a germinator at 20±2˚c. invigoration increased quality and storability of true seed of shallot .......... journal of tropical crop science vol. 9 no. 2, june 2022 www.j-tropical-crops.com 147 experiment 2. drying of tss after invigoration to extend storability the experiment was arranged using a two-factor completely randomized design. the first factor was the drying temperatures: 20˚c, 30˚c, and 40˚c. the second factor was the duration of drying: 8 hours, 16 hours, 32 hours, and 48 hours. the treatment was applied to two varieties, “trisula” and “lokananta”, separately. the treatment was repeated four times so that there were 48 experimental units for each variety. four-hundred tss that have been invigorated using 200ml of kno3 at 3% were air-dried at ambient conditions (26-32˚c, rh 61-79%) for 5 hours. the initial moisture content was determined at a temperature of 103˚c for 17 hours. after the initial seed moisture content had been determined, the seeds were dried at 20˚c, 30˚c, or 40˚c with drying duration according to the treatment, then the seeds were germinated at 20± 2˚c. experiment 3. the storability of tss after invigoration and drying the experiment was arranged using a two-factor nested design. the first factor was the storage condition: 28±2˚c, rh 73±7% (ambient condition); 18±2˚c, rh 61±7% (air-conditioned room); and temperature 8±2˚c, rh 24±8% (refrigerator). the second factor was the type of packaging, aluminum foil and polypropylene (pp) plastic packaging. the experiment was repeated four times so that there were 28 experimental units per observation. only tss var. “lokananta” was used in experiment 3, due to the limited amount of var. “trisula” seeds for the study. true shallot seeds (4.6 g) that had been invigorated with 1.6l kno3 at 3% for 24 hours were air-dried at ambient condition (26-32˚c, 61-79% rh) for 5 hours, followed by an oven drying at 40˚c for 16 hours. after the seeds reaching the moisture content safe for storage, then were packaged according to the treatments. seed quality testing was carried out every two weeks for 14 weeks. seed germination and seedling growth measurements measurement of seed viability and vigor was based on germination percentage (gp), vigor index (vi), germination speed (gs), radicle emergence (re), t50, electrical conductivity (ec), mean emergence time (met), and field emergence (fe). germination percentage (gp) was conducted using 50 seeds on 3-ply filter paper and repeated four times in a standard germinator at a temperature of 20± 2˚c. observations and measurements were made on day 6 and day 12. the criteria for normal seedlings are those having long and straight roots, having long and green cotyledons, and forming an angle in the middle (ista, 2018). gp is calculated using the formula: emergence (fe). germination percentage (gp) was conducted using 50 seeds on 3-ply filter paper and repeated four times in a standard germinator at a temperature of 20± 2c. observations and measurements were made on day 6 and day 12. the criteria for normal seedlings are those having long and straight roots, having long and green cotyledons, and forming an angle in the middle (ista, 2018). gp is calculated using the formula: gp �%� � number of ns i � number of ns iitotal seeds germinated x 100% where: ns i: normal seedlings on the first count (day 6) ns ii: normal seedlings on the second count (day 12) vigor index (vi) was obtained based on the percentage of normal germination on the first count (day 6) of the germination test. vi is calculated using the formula below: vi �%� � number of normal seedlings on the first counttotal seeds germinated x 100% germination speed (gs) was measured every day for 12 days by counting the number of normal seedlings and the difference in hours of each observation (%ns/etmal). sp is calculated using the formula: gs (%ns/etmal) = ∑ � %��������� � where: t : observation time %ns : percentage of normal germination at each observation time etmal : observation time every 24 hours radicle emergence (re) was measured using 50 seeds per experimental unit on filter paper and repeated four times. radicle emergence was measured 72 hours after germination (kamanga et al., 2021), when a radicle has emerged at least 2 mm (ista, 2018). t50 value is measured based on the time required to reach the percentage of normal germination of 50% of the total gp, and is expressed in days. measurements were carried out every day for 12 days. t50 is calculated by the formula of coolbear et al. (1984): t50 (days) = �� � ����%����������� ��� � ��� where: n 50% is the number of germinated seeds (50% of the total germinate), nj and ni are the cumulative number of seed germinated by adjacent counts at times tj (day) and ti (day), respectively, when ni < n50% < nj. electrical conductivity (ec) was measured by immersing 50 tss grains into 50 ml of distilled water for 24 hours according to method of dias et al. (2006) and covered with aluminum foil. each experimental unit was replicated four times. ec is calculated using the formula: ec (µs.cm−1.g−1) = ������������ �� ������ � ����� ��µ�.�� ��.� ��� ���� ��������� mean emergence time (met) is the number of days required for the seedlings to emerge in the field. met was carried out every day until 14 dap. met was calculated based on the number of seedlings that appeared with a hypocotyl length of at least 2 cm per day which was recorded every day until 14 dap. mean emergence time = ʃ ����������� �� ������ ��������� where: n : number of seeds that appear at –t t : days after planting (dat) field emergence (fe) is the percentage of seeds that grow into normal seedlings in the field. observations were made on 14th days after planting (dat). seedlings are considered to be normal if the hypocotyl that appears above the soil surface has reached a minimum of 2 cm. data analysis analysis of variance was carried out using statistical analysis system (sas) version 9.1.2 at α=0.05; tukey test was used for further analysis. correlation between simple linear regression was performed on the measured variables. where: ns i: normal seedlings on the first count (day 6) ns ii: normal seedlings on the second count (day 12) vigor index (vi) was obtained based on the percentage of normal germination on the first count (day 6) of the germination test. vi is calculated using the formula below: emergence (fe). germination percentage (gp) was conducted using 50 seeds on 3-ply filter paper and repeated four times in a standard germinator at a temperature of 20± 2c. observations and measurements were made on day 6 and day 12. the criteria for normal seedlings are those having long and straight roots, having long and green cotyledons, and forming an angle in the middle (ista, 2018). gp is calculated using the formula: gp �%� � number of ns i � number of ns iitotal seeds germinated x 100% where: ns i: normal seedlings on the first count (day 6) ns ii: normal seedlings on the second count (day 12) vigor index (vi) was obtained based on the percentage of normal germination on the first count (day 6) of the germination test. vi is calculated using the formula below: vi �%� � number of normal seedlings on the first counttotal seeds germinated x 100% germination speed (gs) was measured every day for 12 days by counting the number of normal seedlings and the difference in hours of each observation (%ns/etmal). sp is calculated using the formula: gs (%ns/etmal) = ∑ � %��������� � where: t : observation time %ns : percentage of normal germination at each observation time etmal : observation time every 24 hours radicle emergence (re) was measured using 50 seeds per experimental unit on filter paper and repeated four times. radicle emergence was measured 72 hours after germination (kamanga et al., 2021), when a radicle has emerged at least 2 mm (ista, 2018). t50 value is measured based on the time required to reach the percentage of normal germination of 50% of the total gp, and is expressed in days. measurements were carried out every day for 12 days. t50 is calculated by the formula of coolbear et al. (1984): t50 (days) = �� � ����%����������� ��� � ��� where: n 50% is the number of germinated seeds (50% of the total germinate), nj and ni are the cumulative number of seed germinated by adjacent counts at times tj (day) and ti (day), respectively, when ni < n50% < nj. electrical conductivity (ec) was measured by immersing 50 tss grains into 50 ml of distilled water for 24 hours according to method of dias et al. (2006) and covered with aluminum foil. each experimental unit was replicated four times. ec is calculated using the formula: ec (µs.cm−1.g−1) = ������������ �� ������ � ����� ��µ�.�� ��.� ��� ���� ��������� mean emergence time (met) is the number of days required for the seedlings to emerge in the field. met was carried out every day until 14 dap. met was calculated based on the number of seedlings that appeared with a hypocotyl length of at least 2 cm per day which was recorded every day until 14 dap. mean emergence time = ʃ ����������� �� ������ ��������� where: n : number of seeds that appear at –t t : days after planting (dat) field emergence (fe) is the percentage of seeds that grow into normal seedlings in the field. observations were made on 14th days after planting (dat). seedlings are considered to be normal if the hypocotyl that appears above the soil surface has reached a minimum of 2 cm. data analysis analysis of variance was carried out using statistical analysis system (sas) version 9.1.2 at α=0.05; tukey test was used for further analysis. correlation between simple linear regression was performed on the measured variables. germination speed (gs) was measured every day for 12 days by counting the number of normal seedlings and the difference in hours of each observation (%ns/ etmal). sp is calculated using the formula: gs (%ns/etmal) = emergence (fe). germination percentage (gp) was conducted using 50 seeds on 3-ply filter paper and repeated four times in a standard germinator at a temperature of 20± 2c. observations and measurements were made on day 6 and day 12. the criteria for normal seedlings are those having long and straight roots, having long and green cotyledons, and forming an angle in the middle (ista, 2018). gp is calculated using the formula: gp �%� � number of ns i � number of ns iitotal seeds germinated x 100% where: ns i: normal seedlings on the first count (day 6) ns ii: normal seedlings on the second count (day 12) vigor index (vi) was obtained based on the percentage of normal germination on the first count (day 6) of the germination test. vi is calculated using the formula below: vi �%� � number of normal seedlings on the first counttotal seeds germinated x 100% germination speed (gs) was measured every day for 12 days by counting the number of normal seedlings and the difference in hours of each observation (%ns/etmal). sp is calculated using the formula: gs (%ns/etmal) = ∑ � %��������� � where: t : observation time %ns : percentage of normal germination at each observation time etmal : observation time every 24 hours radicle emergence (re) was measured using 50 seeds per experimental unit on filter paper and repeated four times. radicle emergence was measured 72 hours after germination (kamanga et al., 2021), when a radicle has emerged at least 2 mm (ista, 2018). t50 value is measured based on the time required to reach the percentage of normal germination of 50% of the total gp, and is expressed in days. measurements were carried out every day for 12 days. t50 is calculated by the formula of coolbear et al. (1984): t50 (days) = �� � ����%����������� ��� � ��� where: n 50% is the number of germinated seeds (50% of the total germinate), nj and ni are the cumulative number of seed germinated by adjacent counts at times tj (day) and ti (day), respectively, when ni < n50% < nj. electrical conductivity (ec) was measured by immersing 50 tss grains into 50 ml of distilled water for 24 hours according to method of dias et al. (2006) and covered with aluminum foil. each experimental unit was replicated four times. ec is calculated using the formula: ec (µs.cm−1.g−1) = ������������ �� ������ � ����� ��µ�.�� ��.� ��� ���� ��������� mean emergence time (met) is the number of days required for the seedlings to emerge in the field. met was carried out every day until 14 dap. met was calculated based on the number of seedlings that appeared with a hypocotyl length of at least 2 cm per day which was recorded every day until 14 dap. mean emergence time = ʃ ����������� �� ������ ��������� where: n : number of seeds that appear at –t t : days after planting (dat) field emergence (fe) is the percentage of seeds that grow into normal seedlings in the field. observations were made on 14th days after planting (dat). seedlings are considered to be normal if the hypocotyl that appears above the soil surface has reached a minimum of 2 cm. data analysis analysis of variance was carried out using statistical analysis system (sas) version 9.1.2 at α=0.05; tukey test was used for further analysis. correlation between simple linear regression was performed on the measured variables. where: t : observation time %ns : percentage of normal germination at each observation time etmal : observation time every 24 hours radicle emergence (re) was measured using 50 seeds per experimental unit on filter paper and repeated four times. radicle emergence was measured 72 hours after germination (kamanga et al., 2021), when a radicle has emerged at least 2 mm (ista, 2018). t50 value is measured based on the time required to reach the percentage of normal germination of 50% of the total gp, and is expressed in days. measurements were carried out every day for 12 days. t50 is calculated by the formula of coolbear et al. (1984): t50 (days) = emergence (fe). germination percentage (gp) was conducted using 50 seeds on 3-ply filter paper and repeated four times in a standard germinator at a temperature of 20± 2c. observations and measurements were made on day 6 and day 12. the criteria for normal seedlings are those having long and straight roots, having long and green cotyledons, and forming an angle in the middle (ista, 2018). gp is calculated using the formula: gp �%� � number of ns i � number of ns iitotal seeds germinated x 100% where: ns i: normal seedlings on the first count (day 6) ns ii: normal seedlings on the second count (day 12) vigor index (vi) was obtained based on the percentage of normal germination on the first count (day 6) of the germination test. vi is calculated using the formula below: vi �%� � number of normal seedlings on the first counttotal seeds germinated x 100% germination speed (gs) was measured every day for 12 days by counting the number of normal seedlings and the difference in hours of each observation (%ns/etmal). sp is calculated using the formula: gs (%ns/etmal) = ∑ � %��������� � where: t : observation time %ns : percentage of normal germination at each observation time etmal : observation time every 24 hours radicle emergence (re) was measured using 50 seeds per experimental unit on filter paper and repeated four times. radicle emergence was measured 72 hours after germination (kamanga et al., 2021), when a radicle has emerged at least 2 mm (ista, 2018). t50 value is measured based on the time required to reach the percentage of normal germination of 50% of the total gp, and is expressed in days. measurements were carried out every day for 12 days. t50 is calculated by the formula of coolbear et al. (1984): t50 (days) = �� � ����%����������� ��� � ��� where: n 50% is the number of germinated seeds (50% of the total germinate), nj and ni are the cumulative number of seed germinated by adjacent counts at times tj (day) and ti (day), respectively, when ni < n50% < nj. electrical conductivity (ec) was measured by immersing 50 tss grains into 50 ml of distilled water for 24 hours according to method of dias et al. (2006) and covered with aluminum foil. each experimental unit was replicated four times. ec is calculated using the formula: ec (µs.cm−1.g−1) = ������������ �� ������ � ����� ��µ�.�� ��.� ��� ���� ��������� mean emergence time (met) is the number of days required for the seedlings to emerge in the field. met was carried out every day until 14 dap. met was calculated based on the number of seedlings that appeared with a hypocotyl length of at least 2 cm per day which was recorded every day until 14 dap. mean emergence time = ʃ ����������� �� ������ ��������� where: n : number of seeds that appear at –t t : days after planting (dat) field emergence (fe) is the percentage of seeds that grow into normal seedlings in the field. observations were made on 14th days after planting (dat). seedlings are considered to be normal if the hypocotyl that appears above the soil surface has reached a minimum of 2 cm. data analysis analysis of variance was carried out using statistical analysis system (sas) version 9.1.2 at α=0.05; tukey test was used for further analysis. correlation between simple linear regression was performed on the measured variables. where: n 50% is the number of germinated seeds (50% of the total germinate), nj and ni are the cumulative number of seed germinated by adjacent counts at times tj (day) and ti (day), respectively, when ni < n50% < nj. electrical conductivity (ec) was measured by immersing 50 tss grains into 50 ml of distilled water journal of tropical crop science vol. 9 no. 2, june 2022 www.j-tropical-crops.com 148 qudus sabha adhinugraha, eny widajati, endah retno palupi, for 24 hours according to method of dias et al. (2006) and covered with aluminum foil. each experimental unit was replicated four times. ec is calculated using the formula: ec (µs.cm−1.g−1) = emergence (fe). germination percentage (gp) was conducted using 50 seeds on 3-ply filter paper and repeated four times in a standard germinator at a temperature of 20± 2c. observations and measurements were made on day 6 and day 12. the criteria for normal seedlings are those having long and straight roots, having long and green cotyledons, and forming an angle in the middle (ista, 2018). gp is calculated using the formula: gp �%� � number of ns i � number of ns iitotal seeds germinated x 100% where: ns i: normal seedlings on the first count (day 6) ns ii: normal seedlings on the second count (day 12) vigor index (vi) was obtained based on the percentage of normal germination on the first count (day 6) of the germination test. vi is calculated using the formula below: vi �%� � number of normal seedlings on the first counttotal seeds germinated x 100% germination speed (gs) was measured every day for 12 days by counting the number of normal seedlings and the difference in hours of each observation (%ns/etmal). sp is calculated using the formula: gs (%ns/etmal) = ∑ � %��������� � where: t : observation time %ns : percentage of normal germination at each observation time etmal : observation time every 24 hours radicle emergence (re) was measured using 50 seeds per experimental unit on filter paper and repeated four times. radicle emergence was measured 72 hours after germination (kamanga et al., 2021), when a radicle has emerged at least 2 mm (ista, 2018). t50 value is measured based on the time required to reach the percentage of normal germination of 50% of the total gp, and is expressed in days. measurements were carried out every day for 12 days. t50 is calculated by the formula of coolbear et al. (1984): t50 (days) = �� � ����%����������� ��� � ��� where: n 50% is the number of germinated seeds (50% of the total germinate), nj and ni are the cumulative number of seed germinated by adjacent counts at times tj (day) and ti (day), respectively, when ni < n50% < nj. electrical conductivity (ec) was measured by immersing 50 tss grains into 50 ml of distilled water for 24 hours according to method of dias et al. (2006) and covered with aluminum foil. each experimental unit was replicated four times. ec is calculated using the formula: ec (µs.cm−1.g−1) = ������������ �� ������ � ����� ��µ�.�� ��.� ��� ���� ��������� mean emergence time (met) is the number of days required for the seedlings to emerge in the field. met was carried out every day until 14 dap. met was calculated based on the number of seedlings that appeared with a hypocotyl length of at least 2 cm per day which was recorded every day until 14 dap. mean emergence time = ʃ ����������� �� ������ ��������� where: n : number of seeds that appear at –t t : days after planting (dat) field emergence (fe) is the percentage of seeds that grow into normal seedlings in the field. observations were made on 14th days after planting (dat). seedlings are considered to be normal if the hypocotyl that appears above the soil surface has reached a minimum of 2 cm. data analysis analysis of variance was carried out using statistical analysis system (sas) version 9.1.2 at α=0.05; tukey test was used for further analysis. correlation between simple linear regression was performed on the measured variables. mean emergence time (met) is the number of days required for the seedlings to emerge in the field. met was carried out every day until 14 dap. met was calculated based on the number of seedlings that appeared with a hypocotyl length of at least 2 cm per day which was recorded every day until 14 dap. mean emergence time = emergence (fe). germination percentage (gp) was conducted using 50 seeds on 3-ply filter paper and repeated four times in a standard germinator at a temperature of 20± 2c. observations and measurements were made on day 6 and day 12. the criteria for normal seedlings are those having long and straight roots, having long and green cotyledons, and forming an angle in the middle (ista, 2018). gp is calculated using the formula: gp �%� � number of ns i � number of ns iitotal seeds germinated x 100% where: ns i: normal seedlings on the first count (day 6) ns ii: normal seedlings on the second count (day 12) vigor index (vi) was obtained based on the percentage of normal germination on the first count (day 6) of the germination test. vi is calculated using the formula below: vi �%� � number of normal seedlings on the first counttotal seeds germinated x 100% germination speed (gs) was measured every day for 12 days by counting the number of normal seedlings and the difference in hours of each observation (%ns/etmal). sp is calculated using the formula: gs (%ns/etmal) = ∑ � %��������� � where: t : observation time %ns : percentage of normal germination at each observation time etmal : observation time every 24 hours radicle emergence (re) was measured using 50 seeds per experimental unit on filter paper and repeated four times. radicle emergence was measured 72 hours after germination (kamanga et al., 2021), when a radicle has emerged at least 2 mm (ista, 2018). t50 value is measured based on the time required to reach the percentage of normal germination of 50% of the total gp, and is expressed in days. measurements were carried out every day for 12 days. t50 is calculated by the formula of coolbear et al. (1984): t50 (days) = �� � ����%����������� ��� � ��� where: n 50% is the number of germinated seeds (50% of the total germinate), nj and ni are the cumulative number of seed germinated by adjacent counts at times tj (day) and ti (day), respectively, when ni < n50% < nj. electrical conductivity (ec) was measured by immersing 50 tss grains into 50 ml of distilled water for 24 hours according to method of dias et al. (2006) and covered with aluminum foil. each experimental unit was replicated four times. ec is calculated using the formula: ec (µs.cm−1.g−1) = ������������ �� ������ � ����� ��µ�.�� ��.� ��� ���� ��������� mean emergence time (met) is the number of days required for the seedlings to emerge in the field. met was carried out every day until 14 dap. met was calculated based on the number of seedlings that appeared with a hypocotyl length of at least 2 cm per day which was recorded every day until 14 dap. mean emergence time = ʃ ����������� �� ������ ��������� where: n : number of seeds that appear at –t t : days after planting (dat) field emergence (fe) is the percentage of seeds that grow into normal seedlings in the field. observations were made on 14th days after planting (dat). seedlings are considered to be normal if the hypocotyl that appears above the soil surface has reached a minimum of 2 cm. data analysis analysis of variance was carried out using statistical analysis system (sas) version 9.1.2 at α=0.05; tukey test was used for further analysis. correlation between simple linear regression was performed on the measured variables. where: n : number of seeds that appear at –t t : days after planting (dat) field emergence (fe) is the percentage of seeds that grow into normal seedlings in the field. observations were made on 14th days after planting (dat). seedlings are considered to be normal if the hypocotyl that appears above the soil surface has reached a minimum of 2 cm. data analysis analysis of variance was carried out using statistical analysis system (sas) version 9.1.2 at α=0.05; tukey test was used for further analysis. correlation between simple linear regression was performed on the measured variables. result and discussion invigoration to improve viability and vigor invigoration treatments increased the vigor of tss var. “trisula”, and increased the viability and vigor of var. “lokananta” (table 1). invigoration in 50 ppm ga3 or 3% kno3 increased the vigor of var. “trisula”, as indicated by the increase in vigor index of 71.5% and 64.9%, and germination speed of 15.0 % ns/etmal and 15.1 % ns/etmal, respectively. however, the invigoration did not increase seed viability based on the germination percentage (table 1). similar results were reported by muruli et al. (2016) in that ga3 at 50 ppm increased the highest vigor index-i (seed germination x germination length) (from 911 to 1342) compared to other invigoration media, while kno3 at 3% accelerated germination time from 5.6 days to 4.6 days. selvarani and umarani (2011) also reported that invigoration of tss with kno3 at 3 % increased germination speed from 20.5 % ns/etmal to 25.9 % ns/etmal. invigoration using peg6000 at -10 bar in var. “lokananta” resulted in the highest germination percentage (96.50 %) which was not significantly different from other invigoration treatments. invigoration using peg6000 at -10 bar increased the activity of α-amylase enzymes and antioxidant enzymes such as superoxide dismutase, catalase, and peroxidase (yuan et al., 2010, sheteiwy et al., 2016). the increase activities of the α-amylase enzyme in seeds accelerated food mobility and increased seed viability (yuan et al., 2010). on the other hand, kno3 at 3% increased the tss vigor of var. “lokananta” better than other invigoration treatments, as shown by the highest germination speed (20.28% ns/etmal) and the lowest t50 of 4.07 days. it is suspected that the nitrate content within kno3 enhances the synthesis of the nitrate reductase and antioxidant enzymes such as superoxide dismutase, catalase, and ascorbate peroxidase, which play important roles in increasing the speed of germination (lara et al., 2014). in addition to increasing viability and vigor, invigoration reduced membrane leakage as indicated by the lower electric conductivity (matthews et al., 2018). in tss “trisula”, invigoration using ga3 at 50 ppm resulted in the lowest electric conductivity of 9.54 µs.cm−1. g−1, which was not significantly different from the other invigoration treatments, while in “lokananta”, treatment with 0.5% znso4 resulted in the lowest electric conductivity (9.54 µs .cm−1.g−1) which was not significantly different from peg6000 at -10 bar (table 1). the results of this study was in line with the results obtained by brar et al. (2019) in which invigoration of tss with ga3 at 50 ppm, kno3 at 0.5%, thiram at 2 g.kg−1, and kh2po4 at 0.5% significantly reduced cell membrane leakage, as indicated by their electric conductivity values. amooaghaie et al. (2010) reported that hydropriming and osmo-priming treatments could increase the stability of tomato seed cell membranes by up to 23%. according to siri et al. (2013) increased cell membrane stability was associated with an increase in antioxidant enzymes during invigoration. the increase in total antioxidant activity, dehydro-ascorbate, and catalase in invigorated seeds, promoted the defense mechanism in protecting cell membranes. according to orhan et al. (2011), antioxidants can protect cell membranes from the damaging effects of ros, such as single oxygen, superoxide, peroxyl, and hydroxyl. “trisula” and “lokananta” demonstrated different responses to invigoration treatments. “trisula” seeds used in this study were 32 month after harvest, which could have reached the critical period, the third period in seed life time (sadjad, 1993), during which time the seed vigor declines more rapidly than the viability. the “lokananta” seeds had just expired but invigoration increased quality and storability of true seed of shallot .......... journal of tropical crop science vol. 9 no. 2, june 2022 www.j-tropical-crops.com 149 is possible to be re-labelled if germination percentage is at least 70% (table 1). based on vigor index and electric conductivity, the increase in “trisula” vigor was higher than “lokananta”, while the germination speed showed the opposite. these data indicated that invigoration treatment was more effective when applied to seeds with low vigor than those with high vigor. the difference in the effectiveness of invigoration is in line with the research results of muruli et al. (2016) and brar et al. (2019b) that the seed quality improvement in response to invigoration was higher in seeds with low vigor than high vigor. invigoration did not increase mean emergence time and field emergence; in “trisula” the mean emergence time was 8-10 days and it was 8-9 days in “lokananta” (table 2). field emergence of the “trisula” and “lokananta” was 21-35% and 24-52%, respectively, except for the “trisula” treated with peg6000 at -10 bar which only had 7% field emergence due to high fungal attacks. correlation analysis between variables in “trisula” showed that vi, gs, and re had a negative and significant correlation with met, but the correlation table 1. quality of tss in response to invigoration treatments treatments gp vi gs t50 re ec (%) (%) (%ns/etmal) (days) (%) (µs.cm−1.g−1) “trisula” control 80.50 a 35.00 b 11.90 b 6.45 a 51.50 a 178.85a peg6000 -10bar 78.50 a 46.50 ab 12.70 ab 6.31 a 56.00 a 110.94b 3% kno3 76.50 a 64.00 a 15.10 a 5.03 a 73.50 a 96.03b 50 ppm ga3 77.50 a 71.50 a 15.00 a 5.03 a 78.50 a 91.02b 0.5% znso4 76.00 a 49.00 ab 12.58 ab 5.72 a 76.00 a 91.87b f-test ns ** ** * ns ** cv (%) 6.67 16.14 13.28 15.57 14.74 14.97 “lokananta” control 84.00 b 57.50 b 14.05 c 5.73 a 92.50 a 113.46 a peg6000 -10bar 96.50 a 79.00 a 19.13 ab 4.59 bc 89.00 a 67.44 bc 3% kno3 88.50 ab 86.50 a 20.28 a 4.07 c 92.50 a 79.39 b 50 ppm ga3 94.50 ab 79.00 a 17.48 ab 5.08 b 94.50 a 75.28 b 0.5% znso4 93.00 ab 77.50 ab 17.18 b 5.03 b 90.50 a 57.56 c f-test * ** ** ** ns ** cv (%) 7.15 17.25 13.96 12.52 5.65 12.58 note: values within the same column followed by the same letter are not significantly different based on the tukey test at α=0.05. * = significant at p < 0.05, ** = significant at p < 0.01, ns = not significantly different. gp = germination percentage, vi = vigor index, gs = germination speed, re = radicle emergence, ec = electric conductivity. table 2. mean emergence time and field emergence of tss in response to invigoration treatments treatments “trisula” “lokananta” met (days) fe (%) met (days) fe (%) control 9.71 ab 34.50 a 8.75 ab 34.00 a peg6000 -10bar 10.00 a 7.00 b 8.72 ab 30.00 a 3% kno3 8.90 b 35.00 a 8.14 b 51.50 a 50 ppm ga3 8.70 b 21.00 a 8.93 ab 31.50 a 0.5% znso4 9.09 ab 29.00 a 9.11 a 24.00 a f-test * ** ns ns cv (%) 7.29 30.80 5.76 24.17 note: values within the same column followed by the same letter are not significantly different based on tukey’s test at α=0.05, * = significant at p < 0.05, ** = significant at p < 0.01, ns = not significantly different. met = mean emergence time up to 14 dap, fe = field emergence at 14 dap. journal of tropical crop science vol. 9 no. 2, june 2022 www.j-tropical-crops.com 150 qudus sabha adhinugraha, eny widajati, endah retno palupi, was not significant on fe (table 3). this indicated that the higher the vi, gs, and re values in “trisula”, the faster the time for seedlings to emerge in the field, although the correlation between the two was not very strong (-0.47 to -0.53). meanwhile, t50 was significantly and positively correlated with met, which indicated that the higher the t50 value, the longer the time to seed emergence in the field. tss drying to extend tss storability post-invigoration seed drying is a critical step in maintaining the positive effect of invigoration. inappropriate post-invigoration drying of seeds can cause seed decline or even death due to the deterioration process (farooq et al., 2018). seed drying needs to be conducted until a safe moisture content of 8% had been reached (ministry of agriculture, 2017). drying the seeds at a temperature of 20°c and 30°c for up to 48 hours could not reach the required moisture content whereas at 40°c for 8 hours could reach an mc <8% (table 4). in general, the drying temperature did not affect the viability and vigor of “trisula” (table 5). although the drying temperature had a significant effect on vi, it did not show a clear pattern of influence, while drying time affected viability and vigor shown by gp and vi. drying time of 8 to 32 hours did not affect gp and vi, but drying for 48 hours decreased gp and vi (table 5). these data indicate that drying at 40˚c is relatively safe as long as the drying does not exceed 32 hours. according to farooq et al. (2010), the decrease in the seed quality due to drying for too long occurred because of the decrease in α-amylase and dehydrogenase enzymes activities, leading to a decrease in metabolic activities of the seeds. there were differences in the initial seed quality of “trisula” and “lokananta”. the age of “trisula” seeds was 32 mah, so the seeds might have entered a critical period of seed storage, whereas “lokananta” seeds had expired. the drying temperature did not affect viability and vigor as shown by gp and gs (table 6). however, the length of drying duration as a table 3. linear regression between seed quality testing in the laboratory and seedling emergence time in the field of “trisula” and “lokananta” varieties parameters “trisula” “lokananta” r r2 equation r r2 equation mean emergence time re -0.470* 0.208 y = -0.018x + 10.50 0.128tn 0.016 y = 0.012x + 7.560 vi -0.531* 0.279 y = -0.022x + 10.46 -0.191tn 0.036 y = -0.007x + 9.307 gp 0.039tn 0.001 y = 0.005x + 8.866 0.267tn 0.071 y = 0.021x + 6.809 gs -0.509* 0.215 y = -0.175x + 11.64 -0.275tn 0.075 y = -0.057x + 9.753 t50 0.568 * 0.276 y = 0.401x + 6.987 0.382tn 0.146 y = 0.321x + 7.158 field emergence re 0.131tn 0.017 y = 0.098x + 18.7 -0.252tn 0.063 y = -0.757x + 103.7 vi 0.045tn 0.002 y = 0.036x + 23.38 0.179tn 0.032 y = 0.213x + 17.99 gp 0.115tn 0.013 y = 0.284x + 3.201 -0.327tn 0.107 y = -0.781x + 105.5 gs 0.169tn 0.027 y = 1.180x + 9.403 0.151tn 0.022 y = 0.958x + 17.31 t50 -0.170 tn 0.028 y = -2.437x + 39.21 -0.295tn 0.087 y = -7.508x + 70.97 note: * = significantly different according to the pearson correlation test. re = radicle emergence, vi = vigor index, gp = germination percentage, gs = germination speed. table 4. decreases in the moisture content (%) of tss “trisula” and “lokananta” after invigoration and drying at different temperatures and duration drying duration (hours) “trisula” drying temperature “lokananta” drying temperature 20oc 30oc 40oc 20oc 30oc 40oc 8 10.7 12.9 7.2 9.8 22.3 6.4 16 10.9 11.2 7.2 5.3 9.3 6.6 32 9.2 11.7 6.4 4.2 11.0 6.2 48 9.2 10.7 6.3 3.6 10.3 5.2 invigoration increased quality and storability of true seed of shallot .......... journal of tropical crop science vol. 9 no. 2, june 2022 www.j-tropical-crops.com 151 single factor or its interaction with drying temperature, affected the viability and vigor as shown by gp, gs, vi and t50 (tables 6 and 7). in general, drying up to 32 hours did not reduce the seed viability and vigor of post-invigoration. at low temperatures (20˚c) prolonged drying (48 hours) reduced seed quality. it is suspected that a deterioration process occurs because of the high moisture content. these data indicate that the tss quality post-invigoration can be maintained by drying for 8-32 hours. however, longer drying duration will risk reducing the seed quality, so drying for 8 hours at 40˚c, which can reduce the mc by 8%, is the best drying method. table 5. germination percentage, vigor index, germination speed, and t50 of tss of “trisula” variety as a response to temperature and drying time post-invigoration. drying temperature (˚c) gp vi gs t50 (%) (%) (%ns/etmal) (day) 20 66.00 a 35.50 b 11.39 a 6.22 a 30 68.88 a 46.63 a 11.77 a 6.22 a 40 68.13 a 35.25 b 10.97 a 6.34 a f-test ns ** ns ns cv (%) 10.23 20.82 9.64 5.45 drying duration (h) 8 68.83 a 42.50 a 11.08 a 6.26 a 16 69.00 a 41.50 a 11.62 a 6.36 a 32 74.17 a 42.83 a 11.56 a 6.16 a 48 58.67 b 28.33 b 11.24 a 6.27 a f-test ** ** ns ns cv (%) 10.23 20.82 9.64 5.45 note: the values within the same column followed by the same letter are not significantly different based on tukey’s test at α =0.05, ** = significant at p < 0.01, ns = not significantly different. gp = germination percentage, vi = vigor index, gs = germination speed. table 6. germination percentage and speed of germination of tss “lokananta” in respond to drying temperature and duration of post-invigoration seeds treatments gp (%) gs (%ns/etmal) drying temperature (˚c) 20 83.38 a 13.84 a 30 83.50 a 13.36 a 40 87.75 a 13.58 a f-test * ns cv (%) 6.41 9.19 drying duration (h) 8 89.67 a 14.87 a 16 89.00 ab 14.08 a 32 83.33 bc 13.53 a 48 77.50 c 11.89 b f-test ** ** cv (%) 6.41 9.19 note: the values within the same column followed by the same letter are not significantly different based on tukey’s test at α =0.05, * = significant at p < 0.05, ** = significant at p < 0.01, ns = not significantly different. gp = germination percentage, gs = germination speed. journal of tropical crop science vol. 9 no. 2, june 2022 www.j-tropical-crops.com 152 qudus sabha adhinugraha, eny widajati, endah retno palupi, the tss storability after invigoration and drying the quality of the dried tss post-invigoration can be maintained for 14 weeks after storage (was), as indicated by vigor parameters such as re, vi, and gs (figure 1). this result is in line with the research of srinivasan and saxena (2001) which showed that the improvement of radish seed quality after invigoration using peg6000 -0.75 mpa could be maintained for 10 months of storage. basra et al. (2003) showed that canola seeds invigorated using peg10000 for 4 hours and then stored in plastic packaging (pp) at 8˚c for 6 months resulted in better gp than control. during storage, the viability of invigorated “lokananta” was similar to that of non-invigorated tss (figure 1b). this is in line with the results of an experiment i which figure 1. quality of true shallot seeds “lokananta” after invigoration for 14 weeks after storage at different storage condition and packaging material. gp = germination percentage, re = radicle emergence, vi = vigor index, gs = germination speed. kf = room conditions and aluminum foil; kp = room conditions and pp plastic; af = ac and aluminum foil chamber; ap = ac room and pp plastic; lf = refrigerator and aluminum foil, lp = refrigerator and pp plastic; up = without invigoration. table 7. vigor index and t50 of “lokananta” variety in respond to the interaction between temperature and drying duration post-invigoration drying duration (h) vi (%) t50 (days) 20˚c 30˚c 40˚c 20˚c 30˚c 40˚c 8 67.00 a 74.00 a 71.50 a 5.55 a 5.67 a 5.65 a 16 60.00 ab 73.50 a 66.50 a 5.89 ab 6.09 ab 5.83 ab 32 60.50 ab 41.00 bcd 53.50 abc 6.02 ab 6.67 bc 6.26 abc 48 41.50 bcd 19.00 d 34.50 cd 7.67 d 7.11 cd 6.96 cd f-test ** * cv (%) 18.24 5.53 note: the values within the same column followed by the same letter are not significantly different based on tukey’s test at α =0.05, * = significant at p < 0.05, ** = significant at p < 0.01. vi = vigor index. invigoration increased quality and storability of true seed of shallot .......... journal of tropical crop science vol. 9 no. 2, june 2022 www.j-tropical-crops.com 153 showed that invigoration of the “lokananta” with 3% kno3 for 24 hours was more effective in increasing seeds vigor than viability. the positive effect of invigoration during storage can be maintained if the seeds are stored in proper conditions. invigorated tss stored in a refrigerator or air-conditioned room using aluminum foil or plastic packaging can maintain the positive effect of invigoration better than invigorated tss stored in room conditions (figure 1). the positive effect of invigoration during storage will gradually disappear if stored under room conditions. invigorated tss stored at room conditions had decreased vigor quality since the eighth week. the quality of invigorated tss, stored at room conditions using plastic packaging declined faster than those packaged using aluminum foil. the decreased vigor of invigorated tss stored at room temperature could be caused by high temperature and high relative humidity (rh) (28±2⁰c, 73±7%),which lead to the accumulation of reactive oxygen species (ros), loss of membrane integrity, and depleted seed reserve, and resulted in seed damage (liu et al. 2016). seed storage at high rh could also increase lipid peroxidation and seed deterioration (wang et al., 2018). butler et al. (2009) and wood and hay (2010) had reported that invigoration can extend the storability of seeds. however, hussein et al. (2015) and malek et al. (2019) reported the contrary, invigoration can negatively affect seed storability. the response of seed storability to invigoration treatment is influenced by the initial vigor of the seed (powell et al., 2000). invigoration can adversely affect seeds with high initial vigor, while invigoration treatment can extend the storability of seeds on seeds with low initial vigor. in this study, invigoration could improve tss quality as indicated by improvement in seed viability and vigor (tables 1 and 2). after storage in the various environment for 14 weeks, the invigorated tss demonstrated better seed quality than the noninvigorated tss (figure 1). referring to powel et al. (2000) the positive effect of invigoration can be maintained during storage for tss with low initial vigor (35-57.5%). thus, invigoration could be used as seed pretreatment to enhance field emergence and to prolong the storage, even though long storage post-invigoration is not recommended. our study had demonstrated that invigoration treatment with kno3 at 3%, drying at 40°c for 8 hours followed by storage in a refrigerator with aluminum foil packaging can improve the quality and storability of tss. this results would be useful to manage the availability of quality tss which can only be produced once a year. conclusion seed invigoration in medium containing 3 % kno3 and 50 ppm ga3 increased seed vigor, did not affect seed viability of the true shallot seed “trisula”, but increased seed vigor and viability of “lokananta”. true shallot seeds with lower vigor is more responsive to the invigoration treatment than those with high vigor. drying at 40˚c for 8 hours is effective to reduce seed moisture post invigoration and to maintain seed quality after invigoration. the quality of tss post-invigoration can be maintained for 14 weeks after storage in an air-conditioned room (18±2°c, rh 61±7%) using aluminum foil packaging, or in refrigerator (8±2°c, rh 24±8%) using aluminum foil or plastic packaging. references agung, i.g.a.m.s., and diara, i.w. (2017). pre-sowing treatment enhanced germination and vigour of true shallot (allium cepa var. aggregatum) seeds. international journal of environment, agriculture and biotechnology 2, 3262-3267. doi: dx.doi.org/10.22161/ijeab/2.6.62 alboresi, a., gestin, c., leydecker, m.t., bedu, m., meyer, c., and truong, h.n. (2005). nitrate, a signal relieving seed dormancy in arabidopsis. plant cell and environment 28, 500-512. doi: 10.1111/j.1365-3040.2005.01292.x amooaghaie, r., nikzad, k., and shareghi, b. (2010). the effect of priming on emergence and biochemical changes on tomato seeds under sub optimal temperatures. seed science and technology 38, 508-512. doi: 10.15258/ sst.2010.38.2.22 arin, l., polat, s., deveci, m., and salk, a. (2011). effects of different osmotic solutions on onion seeds emergence. african journal of agricultural research 6, 986-991. doi: 10.5897/ajar10.1046 basra, s.m.a., ullah, e., warraich, e.a., cheemat, m.a., and afzal, i. 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(2021). development of seed vigour testing method using single count of radicle emergence for true seed of shallot (allium ascalonicum b.). international journal of food science and agriculture 5, 152-162. doi:10.26855/ ijfsa.2021.03.019 [ministry of agriculture] decree of the minister of agriculture no. 10 of 2017 concerning technical guidelines for shallot seed certification. lara, t., lira, j.m., rodrigues, a.c., rakocevic, m., and alvarenga, a.a. (2014). potassium nitrate priming affects the activity of nitrate reductase and antioxidant enzymes in tomato germination. journal of agricultural science 6, 72-80. doi: dx.doi.org/10.5539/jas.v6n2p72 liu, j., gui, j., gao, w., ma, j., and wang, q. (2016). review of the physiological and biochemical reactions and molecular mechanisms of seed aging. acta ecologica sinica 36, 4997-5006. doi: 10.5846/stxb201502040289 matthews, s., wagner, m., kerr, l., and powell, a.a. (2018). potential for early counts of radicle emergence and leakage of electrolytes as quick test to predict the percentage of normal seedlings. seed science and technology 46, 1-18. doi: doi.org/10.15258/sst.2018.46.1.0 malek, m., ghaderi-far, f., torabi, b., sadeghipour, h.r., and hay, f.r. (2019). the influence of seed priming on storability of rapeseed (brassica napus) seeds. seed science and technology 47, 87-92. doi: doi.org/10.15258/ sst.2019.47.1.09 mendes, c.r., de moraes, d.m., de souza lima, m.g., and lopes, m.f. (2009). respiratory activity for the differentiation of vigor on soybean seed lots. revista brasileira de sementes 31, 171-176. doi: doi.org/10.1590/ s0101-31222009000200020 michel, b.e. (1983). evaluation of the water potentials of solutions of polyethylene glicol 8000 both in the absence and presence of other solutes. plant physiology 72, 66-70. doi: 00320889/83/72/0066/05/$00.50/0 invigoration increased quality and storability of true seed of shallot .......... journal of tropical crop science vol. 9 no. 2, june 2022 www.j-tropical-crops.com 155 muruli, c.n., bhanuprakash, k., and channakeshava, b.c. (2016). impact of seed priming on vigour in onion (allium cepa l.). journal of applied horticulture 18, 68-70. doi: 10.37855/ jah.2016.v18i01.15 orhan, i., kartal, m., abu-asaker, m., senol, f.s., yilmaz, g., and sener, b. (2009). free radical scavenging properties and phenolic characterization of some edible plants. food chemistry 114, 276-281. doi: 10.1016/j. foodchem.2008.09.005 powell, a.a., yule, l.j., hai-chun j., groot, s.p.c., bino, r.j., and pritchard, h.w. (2000). the influence of aerated hydration seed treatment on seed longevity as assessed by the viability equations. journal of experimental botany 51, 2031-2043. rosliani, r., hilman, y., sulastrini, i., yufdy. m.p., sinaga, r., and hidayat, i.m. (2018). evaluation of the packages tss seed production technology of bima brebes varieties in the highland. jounal of horticulture 28, 67-76. doi: 10.21082/jhort.v28n1.2018.p67-76 saranya, n., renugadevi, j., raja, k., rajashree, v., and hemalatha, g. (2017). seed priming studies for vigour enhancement in co onion. journal of pharmacognosy and phytochemistry 6, 77-82. sadjad, s. (1993). “dari benih kepada benih”. grasindo. sedghi, m., amanpour-balaneji, b., and gholi-toluie, s. (2012). dessication tolerance in hdro-primed calendula officinalis l. seeds as influenced by slow and rapid drying back conditions. annals of biological research 3, 3563-3569. selvarani, k., and umarani, r. (2011). evaluation of seed priming metdhods to improve seed vigour of onion (allium cepa cv. agregatum) and carrot (daucus carota). international journal of agricultural technology 7, 857-867. selvy, d.t., and saraswathy, s. (2017). seed viability, seed deterioration and seed quality improvement in stored onion seeds: a review. journal of horticultutal science and biotechnology. doi: doi.org/10.1080/1462031 6.2017.1343103 sheteiwy, m.s., fu, y., hu, q., nawaz, a., guan, y., li, z., huang, y., and hu, j. (2016). seed priming with polyethylene glicol induces antioxidative defense and metabolic regulation of rice under nano-zno stress. environmental science and pollution research. doi: 10.1007/s11356-0167170-7 siri, b., vichitphan, k., kaewnaree, p., vichitphan, s., and klanrit, p. (2013). improvement of quality, membrane integrity and antioxidant systems in sweet pepper (capsicum annuum l.) seeds affected by osmopriming. australian journal of crop science 7, 2068-2073. sopha, g.a., sumarni, n., setiawati, w., and suwandi. 2015. sowing technique of true shallot seed to produce seedling and set of shallot. journal of horticulture 25, 318-330. doi: 10.21082/jhort. v25n4.2015.p318-330 srinivasan, k., saxena, s. (2001). priming seeds for improved viability and storability in raphanus sativus cv. chinese pink. indian journal of plant physiology 6, 271-274. umesh, h., vasudevan, s.n., bhanuprakash, k., and shakuntala, n.m. (2014). physiological and biochemical changes during seed ageing in onion (allium cepa l.). green farming 5, 653656. wang, w., he, a., peng, s., huang, j., cui, k., and nie, l. (2018). the effect of storage condition and duration on the deterioration of primed rice seeds. frontiers in plant science 9, 1-17. doi: 10.3389/fpls.2018.00172 wood, l.p., hay, f.r. (2010). priming increases the storability and changes the water sorption properties of rhodondendron griersonianum seeds. seed science and technology 38, 682691. doi: 10.15258/sst.2010.38.3.16 yuan, s.y., jian, s.y., tian, w.m., yi, l.x., xiang, g., rong, h., and jun, m. (2010). effects of seed priming on germination seedling growth under water stress in rice. acta agronomica sinica 36, 1931-1. doi: 10.1016/s18752780(09)60085-7 1. lianah_vol2_2014.cdr abstract this article presents an overview of the current knowledge of (blume) planch, atetrastigma glabratum host of raf�esiaceae, by providing information on botany and ecology of , and conservationt. glabratum status by propagating the plants vegetatively and monitoring its growth and . the study wasin-situ ex-situ conducted in the protected rainforests at mount prau, candiroto, north kedu, central java between 20092014. shoot growth and expansion in stem diameter were faster than whereas the number ofin-situ ex-situ leaves was similar in both locations. ex-situ conservation and cultivation is very important to protect these rare, unique, and endangered species from extinction. introduction tetrastigma planch (vitaceae) is the sole host species of the raf�esia (raf�esiaceae), a genus of parasitic plants that have the largest �ower of all �owering plants, with �owers reaching up to 150 cm inr. arnoldii diameter (nais 2001). raf�esia plants live inside the roots and stems of as the raf�esiaceaetetrastigma entirely lack leaves, stems, roots and therefore are completely dependent on their host plants for nutrients and water. the centres of species diversity in raf�esia are located in borneo and sumatra, also malaysia, thailand and the philippines (nais, 2001). eight out of the 18 identi�ed species were found in borneo and sumatra, two from java, indonesia. one species was reported from thailand, three from the peninsular malaysia, and two from the philippines. tetrastigma is a widespread genus of approximately 100 species occurring from asia to oceania (ren & wen, 2007). the name for the genus comes from the greek words 'tetras' meaning four and 'stigma' which is the tip of the female pistil in a �ower, in reference to the four-lobed stigma. the species are found in subtropical and tropical regions of asia and australia and suited to grow in hillsides and valleys of shady and moist primary rainforests. , also known and recorded ast. glabratum cissus glabrata, is not recorded from australia although �ve other species have been identi�ed in queensland and new south wales (north of sydney) (anonim 2013; 2014). habitat loss has severely affected the distribution of the endemic and native plant species in the tropics. ex-situ conservation and cultivation has become increasingly important to protect these rare, unique, and endangered species from extinction. this article presents an overview of the current knowledge of (blume) planch, atetrastigma glabratum host of raf�esia (zuhud, 1998), by providing information on botany and ecology of , its traditionalt. glabratum uses, and and conservation status byin-situ ex-situ propagating the plants vegetatively and report some observation on growth and development of t. glabratum. the study was conducted in the protected rainforests at mount prau, candiroto, north kedu (7.3713900°s, 109.9866700° e) and at village blumah, sub-district plantungan located at the foot slopes of mount prau between 2009 to 2014. blumah village is bound to the north by tlogo payung village; to the west by subdistrict bulang and lampin river; to the east by village kediten; and to the south by district wonosobo (anon 2009). the village is located about 95km north east of semarang city, central java. the location is classi�ed as humid with relative humidity of 60-80%, soil ph of 6.9, slopes of up to 60°, temperatures of 20-25°c day and 16-20°c night (lianah, 2013). botanical description nd ecologya t. glabratum are climbing perennial vines with tendrils (figure 1 ) and the ability to grow to a height of up to 20a m, while their leaves consist of �ve lea�ets, serrated and arranged palmately compound (figure 1a). the stems are reddish (figure 1 ); their small �ower clustersa appear in the axils of the leaves, and are whitish in colour (fig 1 ).b description and ecology of indonesian species tetrastigma glabratum (blume) planch, a host of raf�esiaceae lianah* iain walisongo, jl walisongo 4-5, semarang, indonesia 50185. *corresponding author; email: lianahkuswanto@yahoo.co.id 1 short communication journal of tropical crop science vol. 1 no. , 20142 october www.j-tropical-crops.com figure 1. t. glabratum lea�ets, stem and tendril (a), �ower cluster (b). the species appears to be endemic to the mount prau area as it has not been found in other areas (lianah, 2013). it is growing in a mountainous area with an elevation >1300 m above sea level (lianah 2013). the, local names of include walikadep (lianah,t. glabratum 2013), gang putih, akar darik-darik, bantengan, oyod epek and oyon waliran (hyne, 1987). int. glabratum mount prau were found climbing on meranak trees ( (bl.) a. dc. meranak iscastanopsis acuminatissima the predominant tree growing at higher elevation on mount prau (lianah, 2013). they are large trees of up to 40m height with a large canopy: the diameter of a mature tree can be more than 100cm. traditional uses the local people at blumah village have been using the liquid exudates from the stems of fort. glabratum generations. the exudate is believed to have a refreshing and stimulating effects, increase children's appetites and has been used as an important medicinal product in that area (personal communication: supangat, 2011). the research in indonesia on the medicinal properties of is still at the earlyt. glabratum stages. this is in contrast to another tetrastigma species, , a chinese endemic speciest. hemsleyanum (ren and wen, 2007) that has been studied for many years in china and has been demonstrated to be effective against in�ammatory disorders, as an analgesic and antipyretic (liu et al , 2002).. table 1. and vegetative growth of from may to october 2010.in-situ ex-situ t. glabratum vegetative growth in-situ 1) ex-situ 2) survival (%) 100 75 leaf number 20-31 20-31 increase in shoot growth (mm) 23-28 13-29 increase in stem diameter (mm) 3.7-8.9 1-3 biomass (g) 2500 800 1) protection forests at mount prau, candiroto, north kedu. blumah village, candiroto, north kedu 2) . observation n t. glabratum growth ndo a development this description is based on our research and observations of this plant between june – october 2010 (�ve months) as the plant grew in its natural habitat at mount prau and at blumah village greenhouse. our research demonstrated that this species is hard-toroot (lianah, 2013). propagation from stem cuttings resulted in only seven percent of the planted cuttings rooted and survived (lianah, 2013) despite optimizing the vegetative propagation environment for hard-to-root species. the majority of the stem cuttings experienced browning and �nally died. preliminary examination at the biology laboratory at the university of gadjah mada, indonesia, indicated that the leaves and stem of t. glabratum contains high concentration of phenols and �avonoids (lianah, 2013). a number of naturally occurring compounds and enzymes, including phenols, have been reported to affect rooting (krisantini, 2005). plant growth at the protected rainforests at mountin-situ prau and at blumah village is described in tableex-situ 1. plant growth was measured every month for �ve months. generally shoot growth and expansion in stem diameter were faster than (table 1). thein-situ ex-situ number of leaves was similar in both locations as the plants in the greenhouse at blumah village received plenty of sunlight as opposed to the shaded environment in-situ. tetrastigma glabatrum is a slow-growing plant, and it needs about �ve years' growth from cuttings until the diameter of the plant stem reaches 2-3 cm and can be harvested (lianah, 2013). therefore, this plant is increasingly rare because people take those �ve-yearold plants, and they are not available to reproduce. further study is currently under way to optimise the propagation of this hard-to-root species and to measure 2 lianah journal of tropical crop science vol. 1 no. , 20142 october www.j-tropical-crops.com a b propose to educate the community to preserve this endangered species. acknowledgment the author thanks isdn and iain walisongo indonesia for their funding support for this research. the information on local knowledge of growth,tetrastigma ecology and traditional uses from mr supangat and the late mr solikhin of blumah village is highly appreciated. the author thanks dr krisantini for her advice on the contents of this manuscript, mr. ken hayes for his technical advice on vegetative propagation, and dr malcolm wegener of the university of queensland, australia for his valuable comments and manuscript editing. anon. (2009). pro�l kph kedu utara. bkph candiroto, perum perhutani kph kedu utara. anon. (2010). “an alphabetical list of plant species cultivated in the bogor botanical garden, indonesia”. institute for species centre for plant c o n s e r v a t i o n , b o g o r b o t a n i c a l g a r d e n , indonesia. anon. (2013). census of the queensland flora 2013: va s c u l a r p l a n t s . h t t p s : / / d a t a . q l d . g o v. a u / dataset/census-of-the-queensland-�ora-2013. accessed 3 of october 2014. rd anon. (2014). plant databases royal botanic garden and domain trust, sydney, new south wales, australia. http://www.rbgsyd.nsw.gov.au/plant_info/plant_ databases accessed 3 of october 2014.. rd hyne, k. (1987). ”tumbuhan berguna indonesia” vol, iii. balitbang kehutanan. jakarta, indonesia. krisantini. (2005). “adventitious root formation in grevillea (proteaceae), an australian native species”. unpublished phd thesis, the university of queensland, australia li c.l. and wu z. y. (1995). a taxonomical study on tetrastigma (miq.) planch. in china. chinese journal of applied environmental biology 1:307–333. lianah (2013). “implikasi lingkungan pemanfaatan tumbuhan walikadep ( )tetrastigma glabratum untuk bahan obat tradisional”. unpublished phd thesis, university of diponegoro, indonesia. liu d., ju j. h, lin g., xu x. d., yang j. s. and tu g. z. ( 2 0 0 2 ) . n e w c g l y c o s y l � a v o n e s f r o m tetrastigma hemsleyanum acta(vitaceae). botanica sinica : 227–229.44 nair, j. (2001). “raf�esia of the world”. natural history publications. 259 p. borneo, malaysia. ren h. and wen j. (2007). tetrastigma. : flora ofin “ china”. beijing (z.y. wu, raven p.h. eds). st. louis missouri botanical garden press :12 195–208. z u h u d e . a . m . ( 1 9 9 8 ) . “ r a f � e s i a i n d o n e s i a : keanekaragaman, ekologi dan .pelestariannya” the indonesian wildlife fund, indonesia. 3d ...................escription and ecology of indonesian species journal of tropical crop science vol. 1 no. , 20142 october www.j-tropical-crops.com 6. tuan_vol2_2014.cdr abstract brunonia australis rhodanthe(goodeniaceae) and floribunda (asteraceae) are two potential australian native �ower species desired by �oricultural markets. however, the species are dif�cult to propagate. this study examined internal factors that constraint seed germination, seed viability and physiological dormancy. the study was conducted during april to may 2009 at the gatton nursery, the university of queensland gatton campus to identify the underlying reasons for poor germination and to provide recommendations for improving propagation effectiveness. seeds of b. australis collected in 2003 germinated readily irrespective of germination media, whereas seeds collected in 2007 and 2008 with high proportion of viable seeds could only germinate with the presence of ga3 (100 mg.l ) in germination media though with relatively -1 low rates (8.75% and 10.00% respectively) of seed germination. seeds of collected in 2003r. floribunda showed a signi�cant improvement in germination in agar media supplemented with 100 mg.l ga (67.5%) -1 3 compared to control treatment (10.0%). the results suggest that physiological dormancy occurs in both species. the use of ga is recommended for improving3 germination rates of the two species. further studies on the effects of different ga concentrations to improve3 seed germination should be conducted. keywords: dormancy, germination, gibberellic acid, tz, viability. introduction brunonia australis rhodanthe(goodeniaceae) and floribunda (asteraceae) are recognized as economically important wild�ower species of australia (barker et al., 2002; bunker, 1994; joyce, 2005; peacock and smithwhite, 1978; stewart, 1996). nonetheless, like other native �owers the two species have a poor ability to germinate and this is considered as a barrier for commercial development (bunker, 1994; johnston et al., 1999). seed germination is controlled by both internal and external factors (benech-arnold and sanchez, 2004; khan, 1982), in which the former comprises physiological dormancy (genetic control of dormancy) and physical dormancy (a coat-induced dormancy) . in addition, seed(bewley and black, 1994) viability, i.e. the ability of seed to germinate, is also an internal factor that affects germination of seeds (mayer and poljakoff-mayber, 1989). gibberellic acid (ga), particularly ga has recently been3 proven to play an important role in regulating seed germination. gas can replace the requirement for environmental factors that are required for germination of dormant seeds, including light and temperature (da silva et al, 2005). according to benech-arnold and sanchez an increase in the seed ga level can(2004) stimulate metabolism processes in the embryo as well as in the endosperm, leading to germination. research carried out at the university of queensland centre for native floriculture (cnf) has shown that some samples of the two aforementioned species failed to germinate regardless of favorable external factors (temperature, humidity, light and nutrient) (cave, 2009; johnston, 2009; robert, 2003). consequently, the internal factors including seed dormancy and/or viability are assumed as the limitations for seed germination which need further studies. for this reason, the research direction was focused on i n v e s t i g a t i n g t h e m e c h a n i s m s l e a d i n g t o l o w germination rates and to formulate suitable technical methods to improve propagation ef�ciency. this is also in line with the recent strategies of the queensland government and the cnf, a joint initiative of the government and the university of queensland (uq). the strategies focus on the discovery and development of potential �oricultural species to foster germplasms available to the industry via exploitation, selection, breeding and overcoming technical obstacles such as germination constraints, diseases problems and poor postharvest quality .(johnston, 2003; joyce, 2005) the overall objective of this research was to formulate reliable seed germination strategies for brunonia australis rhodanthe floribundaand based on understanding their physiological characters and germination responses. seed viability (tz) test and germination experiments with the use of ga at 1003 mg.l were carried out to determine whether or not seed -1 the physiological dormancy and germination responses of brunonia and to gibberellic acid treatmentaustralis rhodanthe floribunda tuan m. ha thai nguyen university of agriculture and forestry faculty of agronomy, thai nguyen city, vietnam email: haminhtuan@tuaf.edu.vn journal of tropical crop science vol. 1 no. , 20142 october www.j-tropical-crops.com 30 viability and/or seed dormancy are the limiting factors of seed germination in the two species. materials and methods selecting and processing seeds seeds of tested in this study consistbrunonia australis of three groups: (i) seeds collected from the wild (gps coordinate: 27°42'748''s; 145°47'790”e) on 20 november 2003 (ba1); (ii) ba2 (19 november 2007) and (iii) ba3 (4 april 2008) collected at the uq gatton campus. seeds of (rf) wasrhodanthe floribunda collected on 16 february 2003 from site 3 (gps coordinate: 27°57'748”s; 148°00'834”e) on the roadside of the moonie highway, queensland. seeds of each group were collected from at least 50 individual plants. only mature seeds close to the point of natural dispersal were collected. following collection, seeds were stored in paper envelopes at 15°c and 15 – 20% relative humidity in a cold room of the cnf laboratory until required. experimental flow chart as discussed above, this study was carried out to test the two possibilities, dormancy and viability, as the internal factors that in�uence germination of the two species (figure 1). viability and germination tests physical damages of the seeds were checked using a stereomicroscope. only intact seeds were then selected for germination and viability tests. to a v o i d c o n t a m i n a t i o n p r o b l e m s d u r i n g t h e germination tests reported by previous studies on native �oricultural species (johnston et al., 2004; mullins et al., 2002), seeds were disinfected in chlorine (2000 mg.l ) -1 plus one drop of tween 20 for 10 minutes then rinsed ® with sterile distilled water (dw) three times before being placed into sterile germination media. in this test, two treatments were used for each seed sample with four replicates of 20 seeds each sown into 9cm diameter plastic petri dishes containing (i) 1% agar water (control) and (ii) 1% agar water and ga (100 mg.l ). petri dishes3 -1 were sealed with plastic bands to avoid agar figure 1. experimental �ow chart to examine poor germination of (ba) and and seeds.brunonia australis rhodanthe floribunda the photo of tz staining patterns of seeds was obtained from international seed testing association (ista) handbook on flower seed testing (ripka, 2008). dotted arrows represent conclusions based on the test results. journal of tropical crop science vol. 1 no. , 20142 october www.j-tropical-crops.com 31the physiological dormancy and germination responses of brunonia.......... desiccation. seeds of all treatments were placed in dark and air-conditioned room with temperature of 25°c. germination, de�ned as radical emergence by at least 1 mm, was scored every week. seeds that did not germinate by the end of the germination experiment was subjected to germination testing using tz method. the seeds were imbibed for 24 hours in distilled water (dw) at ambient temperatures. imbibed seeds were placed into 1.0% 2,3,5-triphenyl tetrazolium chloride (tz) solution and held at 40°c in dark for 24 hours. after rinsing with dw, seeds were cut longitudinally to expose the embryo. deep red to deep pink embryos were recorded as viable, whereas white to pale pink embryos were recorded as non-viable (copeland and mcdonald, 1995; hampton and tekrony, 1995). experimental design and analysis the study was set up in a completely randomized factorial design with four replicates each for ba1, ba2, ba3 and rf seed group. each replicate consists of 20 seeds, totaling 80 seeds for each seed group. the data of germination rates was analyzed by using balanced anova of the irristat statistical package (irri release 5, manila, the philippines, 2005) with least signi�cant differences (lsd) calculated at 5% level of signi�cance. graphpad prism (release 6, graphpad software inc., la jolla, ca 92037 usa) was used to illustrate germination trend over time and compare germination rates amongst selected treatments. results and discussion brunonia australis there were noticeable differences in germination rates of the three groups of (ba) seedsbrunonia australis (table 1). ba1 seeds (seeds collected in 2003 from the wild) germinated readily on the �rst week, i.e. 70% germination on 1% agar medium plus ga (100mg.l )3 -1 and 78.8% on 1% agar without ga . the germination3 increased gradually until the �fth week, i.e. 87.5% and 80.0% for ba1 with and without ga respectively (table3, 1). table 1. percentage of seeds germinated over �ve weeks.brunonia australis week 1 week 2 week 3 week 4 week 5 ba1 with ga3 70.00 78.75 85.00 85.00 87.50 ba1without ga3 78.75 78.75 78.75 78.75 80.00 ba2 with ga3 0.00 0.00 2.50 6.25 8.75 ba2 without ga3 0.00 0.00 0.00 0.00 0.00 ba3 with ga3 0.00 2.50 5.00 6.25 10.00 ba3 without ga3 0.00 0.00 0.00 0.00 0.00 treatments percentage of seeds germinated over time (weeks) (%) * p-value n.s * note: ba1, ba2 and ba3 are seeds of collected in 2003, 2007 and 2008,brunonia australis respectively; ga concentration: 100 mgl ; date sown: 9 april 2009; n.s. not signi�cant; * p < 0.05.3 -1 however, there was no signi�cant difference in the germination rates between ba1 with and without ga ,3 suggesting that ga at 100 mg.l did not have3 -1 stimulating effect on germination of the ba1 seeds. ba1 seeds which were collected in 2003 might have experienced a suf�cient dormancy period which has enabled the seeds to germinate under the favorable temperature and humidity condition regardless of the ga treatment.3 in contrast, ba2 seeds (collected in 2007) and ba3 seeds (collected in 2008) sown on agar with 100 mg.l -1 ga did not germinate until week 2 and week 3,3 respectively. after that the germination rate rose slightly to 10 % and 8.8%, respectively, by the �fth week (table 1). ba2 and ba3 seeds without ga did not germinate3 after �ve weeks (table 1). ga treatment signi�cantly3 increased both ba2 and ba3 germination (p <0.05). non-germinated ba2 and ba3 seeds were subjected to a viability test using the tz staining technique. the results indicated high proportions of viable seeds, i.e. 82 % in ba2 and 81% in ba3. therefore, it was concluded that the seeds of collected in 2007 (ba2) andb. australis 2008 (ba3) have a dormancy character, and that ga at3 100 mg.l had a stimulating effect on germination of -1 these seeds. these results demonstrated that the more recent harvested seeds (ba2 and ba3) experienced a certain dormancy period before being able to germinate under favorable environments. to break dormancy, addition of stimulants such as gas is necessary. journal of tropical crop science vol. 1 no. , 20142 october www.j-tropical-crops.com 32 tuan m. ha as described above, the seeds of collectedb. australis in 2003 (ba1) germinated readily without a need for addition of ga , whereas oneand two-year-old seeds3 (ba2, ba3) required ga for germination even though3 only a small percentage of seeds germinated (figure 2 the same as table 1). further studies to test the effects of different ga concentrations on germination of the3 newly harvested seeds are recommended. rhodanthe floribunda rhodanthe seeds treated with and without ga started3 germinating at the �rst week with the percentages of 60 % and 5 %, respectively. the germination rates increased gradually until the �fth week, reaching 67.5% for ga -treated seeds and 10 % for control seeds.3 therefore ga -treated seeds had a higher germination3 percentage compared to the control seeds (p <0.05) (figure 3). figure 2. germination rates over time of (ba) seeds. vertical bars representb. australis standard errors of means (se). it can be be concluded that has dormancyr. floribunda character, and the use of ga at 100 mg.l can3 -1 overcome dormancy and stimulate seed germination. conclusion the study has established that both brunonia australis and seeds have dormancyrhodanthe floribunda character. the addition of ga at 100 mg.l to the3 -1 germination media can stimulate germination of r. floribunda collected in 2003 and oneand two-year-old b. australis seeds. for this reason, adding ga to3 germination media is recommended to increase propagation ef�ciency of the two species. different concentrations of ga should be tested to determine the3 o p t i m u m g a c o n c e n t r a t i o n t o i m p r o v e s e e d3 germination. acknowledgement the author would like to express sincere thanks to the staff of gatton seed technology laboratory (the university of queensland, qld, australia) for conducting the viability test, mr. john swift for laboratory figure 3 germination rates of (rf) seeds over time. seeds were collected on 16 february. rhodanthe floribunda 2003. the seeds were sown on 9 april 2009. vertical bars represent standard errors of means (se). journal of tropical crop science vol. 1 no. , 20142 october www.j-tropical-crops.com 33the physiological dormancy and germination responses of brunonia.......... safety procedure, mr. allan lisle for statistical advice, and ms. vishu wickramasinghe for her assistance in germination media preparation. the author specially thank dr margaret johnston for her valuable information on the research methods and constructive comments and advice for this manuscript. references barker, j., greig, j., peate, n., courtney, b., salkin, e., schaumann, m., armstrong, j., and thomlinson, g. (2002). "everlasting daisies of australia: identi�cation, propagation, cultivation". 196 p. c.h. jerams and associates. australia. benech-arnold, r. l. and sanchez, r. a. eds. (2004). "handbook of seed physiology: application to agriculture" 479 p. crc press.. bewley, j. d., and black, m., eds. (1994). "seeds: physiology of development and germination". 446 p. springer. bunker, k. v. (1994). overcoming poor germination in australian daisies (asteraceae) by combination of gibberellin, scari�cation, light and dark. scientia horticulturae , 243-252.59 cave, r. (2009). pers.com. (m. t. ha, ed.), centre for native flowers, the university of queensland, australia. copeland, l. o., and mcdonald, m. b. (1995). "principles of seed science and technology“. 409 p. chapman and hall, inc.third edition. da silva, e.a., toorop, p.e., nijsse, j., bewley, j.d. and hilhorst, h.w.m. (2005). exogenous gibberellins inhibit coffee ( cv. rubi) seedcoffea arabica germination and cause cell death in the embryo. journal of experimental botany , 1029 – 1038.56 hampton, j. g., and tekrony, d. m., eds. (1995). "handbook of vigour test methods". 117 p. the international seed testing association, zurich, switzerland. johnston, m. (2009). pers.com. (m. t. ha, ed.), centre f o r n a t i v e f l o w e r s , t h e u n i v e r s i t y o f queensland, australia. johnston, m., kibbler, h., fletcher, t. and webber, j. 2000. the introduction to commercial �oriculture of recalcitrant australian native plants. acta hort (ishs) , 31-36.541 johnston, m. e. (2003). the centre for native f l o r i c u l t u r e “ p r o c e e d i n g s o f t h e 5i n t h international symposium on new floricultural c r o p s ” ( a . f. c . to m b o l a t o a n d g . m . diastagliacozzo, eds.), pp. 165-169, parana, brazil. international society for horticultural science. johnston, m. e., bauer, l. m., o'brien, s. d., and kochanek, j. (2004). dormancy issues for australian �oricultural species. "proceedingsin of the �fth australian workshop on native seed biology" (s. w. adkins, ed.), pp. 89-96. australian centre for minerals extension and research brisbane, queensland, australia. joyce, d. c. (2005). queensland's center for native floriculture. "proceedings of the internationalin symposium on harnessing the potential of horticulture in the asian-paci�c region“ pp. 4752. khan, a. a., ed. (1982). "the physiology and biochemistry of seed development, dormancy and germination", 547 p. elsevier biomedical press. mayer, a. m., and poljakoff-mayber, a., eds. (1989). "the germination of seeds" 208 p. edition.. third pergamon press, jerusalem, israel. mullins, r. g., koch, j. m., and ward, s. c. (2002). practical method of germination for a key jarrah forest species: snottygobble (persoonia l o n g i f o l i a e c o l o g i c a l m a n a g e m e n t &) . restoration , 97-103.3 peacock, w. j., and smith-white, s. (1978). cytogeography of . , 31-brunonia australis brunonia 1 44. ripka, z., ed. (2008). "ista handbook on flower seed testing." ista, bassersdorf, switzerland. robert, j. (2003). pers.com. (m. t. ha, ed.), centre for native flowers, the university of queensland, australia. stewart, a. (1996). gardening on the wild side, the new australian bush garden. ,australian plants 18 287-296. journal of tropical crop science vol. 1 no. , 20142 october www.j-tropical-crops.com 34 tuan m. ha leaf and flower characterization of abiu (pouteria caimito radlk.) at two locations .......... journal of tropical crop science vol. 10 no. 1, february 2023 www.j-tropical-crops.com 1 leaf and flower characterization of abiu (pouteria caimito radlk.) at two locations in bogor region, west java, indonesia. muhamad ramdana, slamet susanto*a, deden derajat matraa, dhika prita hapsaria a department of agronomy and horticulture, faculty of agriculture, ipb university jalan meranti, kampus ipb dramaga, bogor 16680 *corresponding author; e-mail: ssanto@apps.ipb.ac.id research article abstract abiu is a species introduced into indonesia from the tropics of the amazon and has been developing for several years. information on the characteristics of the abiu plant are still limited compared to other introduced fruit species. this research aims to identify the characteristics of the abiu plants in two locations in the bogor region, west java, indonesia. the research was conducted at balumbang jaya village, the sub-district of dramaga, the village of mekarsari, the sub district of cileungsi, bogor, west java. further observation was conducted at postharvest laboratory and micro technic laboratory, department of agronomy and horticulture ipb. the field study was conducted in january to july of 2018. this research was descriptive and no treatment was applied on the plant samples. sampling was done randomly with as many as 15 plants at each location. observations and measurements were conducted on the number of branches, leaves and flowers on 10 samples per plants. the results showed that there is morphological diversity of flowers and leaves at the two locations. the length, width, and weight of leaves and flowers in balumbang jaya were larger than those in mekarsari. different environments may contribute to the diversity of abiu leaves and flowers in the two locations. keyword: agroclimate, identification, morphology, sapotaceae introduction plant resources are of fundamental importance for human survival and development. various species of tropical fruits need to be continuously explored and domesticated, one way is by introducing native and unknown being through the introduction of the native and unknown species. new fruit species as germplasms from around the world have been introduced to many regions of indonesia. the agroclimate similarity between the area of the origin of the introduced fruits and agro-climates in the territory of indonesia is an important consideration in germplasm introduction. abiu plant (pouteria caimito radlk.) originates from the area of the tropical amazon (clement, 1989). abiu has been introduced to indonesia in the last several decades; however, information on the development of variations, cultural practices, and the potential of abiu in indonesia is still lacking compared to the other introduced commercial fruits in the markets. abiu is a tropical species that is found to grow at an altitude of 500-2500 m above sea level and is generally propagated by seeds. abiu is from the sapotaceae family which has berry fruits (campbell and ledesma, 2003). abiu species includes evergreen trees which have a short flowering and fruiting include age compared to other sapotaceae plants. abiu grows optimally in soil with good drainage, sandy to heavy clay texture, and neutral ph (lim and ramsay, 1992). the first flowers appear when the plant is three-years-old, and the fruit is ready for harvest about three months later (campbell and ledesma, 2003). abiu fruits have elliptical, clear white flesh and contain 1-4 seeds. a soft texture and yellow peel color at the fully ripe stage are exhibited at harvest time (campbell et al., 2010). abiu fruit has beneficial elements for human health such as antioxidants (franca et al., 2016; montero et al., 2018), antimicrobial (abreu et al., 2019, fernadez et al., 2020), and phenolic compounds (filho et al., 2018). the antioxidants in the abiu leaves function journal of tropical crop science vol. 10 no. 1, february 2023 www.j-tropical-crops.com 2 muhamad ramdan, slamet susanto, deden derajat matra, dhika prita hapsari by refusing free radicals and preventing degenerative disease (sousa et al., 2019). every 100 g of abiu fruit contain protein 0.81 g, fat 0.49 g (aguiar and souza, 2014), carbohidrate 14.5-36.3 g, thiamine 0.02-0.04 mg, ascorbic acid 1149 mg, and vitamin b 0.2 mg (love da paull, 2011). observations and measurements of the morphological characters can be used to measure and describe the variabilities in plants. development and improvement of abiu cultivation can be started by a description of the plant’s morphological characteristics. tthis study aims to identify the leaf and flower characteristics of the abiu (pouteria caimito radlk.) plants grown at two locations in bogor region, west java, indonesia. materials and methods the field experiment was conducted at balumbang jaya village, the sub-district of dramaga mekarsari village, the sub-district of cileungsi, and the bogor district, west java. the research was conducted from january to june 2018. plants in balumbang jaya were three years old, with a planting distance of 3 x 4 m. plants in mekarsari were 8 years old with a spacing of 4 x 5 m. compound npk fertilizer (16:16:16) at 500 g per plant was applied two times during the experiment, i.e. in january and may in each location. irrigation and pest control were applied as needed. this study was descriptive research, there were no treatments applied to the plants. a total of 15 plants in each location were selected for this experiment. in each plant 10 leaves and 10 flowers were sampled to be observed. all the samples were labeled properly. the characterization of the leaf and flower of the abiu plant was based on several observed variables. the variables were taken from non-destruction samples and destruction samples. the observation and measurement from nondestructive samples were conducted on (1) flower organs: flower length and fruit set. the fruit set is calculated as the number of flowers that were successfully developed into young fruits (± 2 cm in size) on each brunch; (2) stem organ: the angle of branches to the main stem; (3) leaf organ: leaf length, leaf width, leaf shape, leaf tip shape and leaf base shape. shape variables refer to plant botany described in descriptor for avocado (ipgri, 1995) in a modified version. destructive samples were used to describe the flower morphology by cutting the flower using a knife, pincette and observation using a stereo microscope (carton scw-e). the measurement of flower parts was done using a trinocular compound microscope (olympus bx51) and dp2-bsw program. data were classified into qualitative and quantitative. qualitative data were presented in the form of percentage values from the total samples. quantitative data and the percentage of the qualitative data were statistically analyzed using minitab program. the t-test was run at a level (α = 5%) to compare the average values between the two locations. result and discussion study site environment experimental fields were located in two sites, balumbang jaya village at balumbang jaya sub-district , and mekarsari village at cileungsi sub-district, bogor district, west java, indonesia. balumbang jaya village is located at latitude 6o30” − 6 o45” s, and longitude 106 o30” − 106 o45” e and with an altitude ± 300 m asl. mekarsari village is located at latitude 6°.43” − 6°.41”s and longitude 106° 05” – 106°.98” e with an altitude ± 70 m asl. during the experiment, balumbang jaya village had an average air temperature of 25.1°c, rainfall 241 mm per month, and air humidity of 86%, while mekarsari had an average air temperature of 25.9°c, rainfall 232 mm per month, and air humidity 83% (bmkg, 2018). both locations are classified as having humid tropical climates. the statistical analysis results of the statistical analysis indicated that the mean of almost all growth variables is greater at balumbang jaya than those at mekarsari, although some variables are not significantly different. vegetative variables such as leaf length, leaf width, and the angle of brunches at balumbang jaya are significantly different than those at mekarsari. generative variables such as stamen diameter, pistil diameter, and fruit set also have significantly different values between locations. the difference in variables between the two location was probably due to differences in altitude, where balumbang jaya has a higher altitude than mekarsari (table 1). hovenden and schoor (2006) reported that the leaf morphology of nothofagus cunninghamii is related to altitude, and related to temperature. genotype had a significant effect on leaf morphology of this species, but in the field, there was also an effect from altitude. in terms of generative variables, fabbro and korner (2003) reported that alpine grown in higher altitude allocated three times more of their above-ground biomass to floral structures than alpine grown in lowland area. leaf morphology leaf morphology was observed based on the shapes of leaf, leaf base, and leaf tip. the abiu leaves in both locations had a lanceolate shape, however, the leaf and flower characterization of abiu (pouteria caimito radlk.) at two locations .......... journal of tropical crop science vol. 10 no. 1, february 2023 www.j-tropical-crops.com 3 table 1. morphology of abiu in balumbang jaya and mekarsari, bogor, west java, indonesia character balumbang jaya mekarsari new growth flush colour pale green, yellowish pale green, yellowish lamina shape lanceolate lanceolate leaf tip acute 54.7%, acuminate 45.3% acute 34.7%, acuminate 65.3% leaf bases acute 64%, cuneate 36% acute 80.7%, cuneate 19.3% leaf size (length, width) (cm) 16.87; 5.6 14.57; 4.51 angle of brunch on stem (o) 68.3 53.1 flower length (mm) 6.42 5.89 number of sepal 4 4 number of petal 8 8 number of stamen 4 4 stamen length (mm) 3.76 3.08 anther size (length, diameter) (mm) 0.89; 0.51 0.76; 0.46 pistil size (length, diameter) (mm) 4.86; 0.44 4.45; 0.36 ovary size (length, diameter) 0.96; 1.23 0.89; 1.28 number of flowers per plant 64.9 76.35 number of fruits 2.82 1.7 fruit set (%) 4.34 2.23 harvest time (weeks after anthesis) 10.02 10.08 figure 1. morphology of abiu: a) the whole plant, b) leaves and branches, and c) fruit journal of tropical crop science vol. 10 no. 1, february 2023 www.j-tropical-crops.com 4 muhamad ramdan, slamet susanto, deden derajat matra, dhika prita hapsari shapes of the leaf base and the leaf tip varied. the shape of the leaf base of the abiu plant is classified as acute and cuneate, while the shape of the leaf tip of the abiu plant is classified as acute and acuminate. the shape of the base of the abiu leaves at the two locations was dominated by acute as much as 64.00% at balumbang jaya and as 80.70% at mekarsari. the shape of the base of the abiu leaves at balumbang jaya was mostly (54.70%) acute, whereas at mekarsari are mostly (65.30%) acuminate (table 2). in mekarsari. however, plant age may also be a factor affecting leaf size. the abiu plants at mekarsari are eight years old whereas at balumbang jaya they are three years old. performance of the abiu leaves are presented in figure 1a. morphology of flower the flower size at balumbang jaya was larger than that of mekarsari. in both locations the flower has table 2. percentages of the shape of leaf, of leaf base, and of leaf tip at the two locations location leaf shape leaf base shape leaf tip shape lanceolate (%) acute (%) cuneate (%) acute (%) acuminate (%) balumbang jaya 100.00 64.00 36.00 54.70 45.30 mekarsari 100.00 80.70 19.30 34.70 65.30 cv (%) 0.00 31.66 22.02 16.78 13.55 significance ns * * * * leaf size and angle of the branches on the main stem the leaf size of abiu plants and the angle of branches on the stem were significantly different between locations. abiu leaves (their length and width) were longer at balumbang jaya than at mekarsari (table 2). leaf size differences may be caused by the environmental conditions of the two locations. the leaf is the plant organ most sensitive to environmental condition such as temperature, rainfall, nutrient deficiency and pest and disease attacks. environmental conditions in balumbang jaya of slightly lower temperatures and higher rainfall possibly contributed to better plant growth than those figure 2. leaf morphology of abiu: a) leaf bases acute, b) leaf bases cuneate, c) leaf tip acute, d) leaf tip acuminate four sepals and eight petals. the sepals overlap to form calyx to protect the other young organs. the four sepals are placed alternately. sepals in the outer place were smaller than those in the inner ones. the flower petals of the abiu plants are arranged close together in the form of corolla tubes at the side near the receptacle and then they split into eight sheets (four sheets bigger and four sheets smaller) at their edges. moreover, four stamens stick out from the inner part of the petal sheet, with the size at balumbang jaya being larger than that of at mekarsari. (table 3). the size of the abiu flower organ at balumbang jaya was larger than that at mekarsari, except for the diameter of the ovary (table 4). different environmental conditions may also affect the growth of the flowers. leaf and flower characterization of abiu (pouteria caimito radlk.) at two locations .......... journal of tropical crop science vol. 10 no. 1, february 2023 www.j-tropical-crops.com 5 these different conditions may be attibuted to the different leaf sizes at the different locations which indicated that the abiu plant at balumbang jaya grew better than at mekarsari. the performance of abiu flower is presented at figure 1b and 1c. flower number and fruit set the inflorescence of the abiu is arranged in single or group. abiu flowers appear on the leaf base and on the print sites of the fall down leaves along the branches, so that it is called ramiflorous. beside those, abiu flowers may appear on the main stem so that is called cauliflorous. the percentage of cauliflorous flowers are very small compared with that of ramiflorous flowers. the number of flowers and the harvest time of the abiu fruits were not different between two locations. however, the fruit set and the number of fruits at those locations table 3. leaf size and the angles of brunches on stem at balumbang jaya and mekarsari location leaf size stem length (cm) width (cm) angle of branches (º) balumbang jaya 16.87 5.60 68.30 mekarsari 14.57 4.51 53.10 cv (%) 14.59 14.73 26.22 significance * * * note: ns = not significant; * = significant according to t-test at p <0.05 table 4. morphology of abiu sepal, petal, and stamen at two locations in bogor, west java location flower length number of sepal number of petal stamen big small total number length (mm) mm) balumbang jaya 6.42 4 4 4 8 4 3.76 mekarsari 5.89 4 4 4 8 4 3.08 cv (%) 12.69 0 0 0 0 0 20.75 significance * ns ns ns ns ns * note: ns = not significant; * = significant according to t-test at p <0.05 table 5. morphology of abiu flower (anther, pistil, and ovary) at two locations location anther pistil ovary length diameter number length diameter length diameter (mm) (mm) (mm) (mm) (mm) (mm) balumbang jaya 0.89 0.51 1 4.86 0.44 0.96 1.23 mekarsari 0.76 0.46 1 4.45 0.36 0.89 1.28 cv (%) 18.14 23.01 0 10.89 18.88 15.06 15.97 significance * * ns * * * ns note: ns = not significant; * = significant according to t-test at p <0.05 figure 3. flower morphology of abiu: a) inflorescences; b) flower parts; c) a close-up of flower parts. journal of tropical crop science vol. 10 no. 1, february 2023 www.j-tropical-crops.com 6 muhamad ramdan, slamet susanto, deden derajat matra, dhika prita hapsari were differed significantly. the average of fruit set and fruit number at balumbang jaya each was 4.34% and 2,82 fruit higher than those at mekarsari namely 2.23% and 1.70 fruit (table 5). heinz (1983) stated that environmental conditions influence the growth of abiu. this indicates that the environmental conditions at balumbang jaya, such as lower temperature and higher rainfall were more favorable to support the process of fruit formation than at mekarsari. lora et al. (2009) stated that higher temperature might increase the speed of stigma desiccation on the second day of the flower cycle, so that pollination process and fruit set fruitset may have been less effective compared to those before. on the other hand, lopez and dejong (2007) reported that fruit development rates of peaches were high under higher temperatures; however the trees may not have been capable of supplying resources rapidly enough to support the potential fruit growth rates that accompanied the higher temperatures. abiu takes about 11 weeks to develop from ovule to harvest (figure 4). generally, the shape of abiu fruit is round and oval and has white flesh with a soft texture. the taste of the fruit is sweet when it is ripe (75 – 100% yellow). abiu fruit harvested in fully-ripe conditions have soluble solid content (ssc) value 10.04°brix, while at 12 days after storage period the ssc had increased to 12.33°brix (arif et al. 2022). abiu fruit diameter is 6 – 7 cm and the weight varies between 150 – 250 grams. one abiu fruit has 1 – 2 seeds. the seeds of abiu are blackish brown (figure 5) and firm like sapodilla seeds. conclusion our study revealed that the abiu plants grown in the two geographical location have morphological variations. the morphological diversity of flowers and leaves occurred at the individual level. statistical tests showed that the length, width, and weight of leaves and flowers from the plants in balumbang jaya were longer and larger than those grown in mekarsari. the plants at balumbang jaya also have more fruit set and fruit numbers per plant. environmental conditions may cause diversity in the character of leaf and flower table 6. abiu flower formation at two locations in bogor, west java location number of flowers per plant number of fruits fruit set harvest time (%) (waa) balumbang jaya 64.90 2.82 4.34 10.02 mekarsari 76.35 1.70 2.23 10.08 cv (%) 62.01 64.67 68.49 10.08 significance ns * * ns note: waa = week after anthesis. ns = not significant; * = significant according to t-test at p <0.05 figure 4. abiu fruit growth, development and maturation. figure 5. an abiu fruit and abiu seeds. leaf and flower characterization of abiu (pouteria caimito radlk.) at two locations .......... journal of tropical crop science vol. 10 no. 1, february 2023 www.j-tropical-crops.com 7 at both locations. however, genetic and phenological studies are required to identify the diversity of abiu for further development and commercialization of this species. references abreu, m. m., nobrega, p.d.a., sales, p.f., oliviera, f.r.d., and nascimento, a. a. (2019). antimicrobial and antidiarheal activities of methanolic fruit peel extract of pouteria caimito. pharmacognosy journal 11, 944-950. aguiar, j.p.l. and souza, f.c.a. (2014). soluble and insoluble fibrin some amazonian fruit with low energy density. food and nutrition sciences 5, 1415-1419. arif, a.b., susanto, s., widayanti, s. m., and matra, d. d. (2022). effect of ripening stage on postharvest quality of abiu (pouteria caimito) fruit during storage. agricultural and natural resources 56, 441-454. [bmkg] badan meterologi klimatologi dan geofisika. (2018). “data cuaca desember 2017 – mei 2018”. stasiun klimatologi bogor. campbell, r.j. and ledesma, n. (2003). the sapodilla and green sapote’s potential in tropical america. journal of tropical horticulture 46, 55-56. campbell, r.j., ledesma, n., zill, g., and herrera, j.c. (2010). collection pouterias (pouteria spp.), sapodilla (manilkara zapota) and caimito (chrysophyllum caimito) for the creation of new market. journal of the american pomological society 6, 24-27. clement, c.r. (1989). a center of crop genetic diversity in western amazonia. bio science 39, 623-631. fernandez, i.m., chagas, e.a., maldonado, s.a.a., takahashi, j.a., aleman, r.s., filho, a.a.m., santos, r.c.d., ribeiro, p.r.e., fantes, j.a.m., chagas, p.c., and melo, a.c.g.r. (2020). antimicrobial activity and acetilcolinesterase inhibition of oils and amazon fruit extracts. journal medicinal plant research 14, 8897. franca, c.v., perfeito, jps., resck, i.s., gomes, s.m., fagg, c.w., castro, c.f.s., simeoni, l. a., and silveira, d. (2016). potential radical scavenging activity of pouteria caimito leaves extracts. journal of applied pharmaceutical science 6, 184-188. filho, a.a.m., costa, a.m.d.c., fernandez, i.m., santos, r.c.d., chagas, e.a., chagas, p. c., takahashi, j.a., and perraz, v.p. (2018). fatty acids, physical-chemical properties, minerals, total phenols and antiacetylcholinesterase of abiu seed oil. chemical engineering transactions 64, 1-7. [ipgri] international plant genetic resources institute. (1995). “descriptors for avocado (persea spp.)”. international plant genetic resources institute. heinz, h. (1983). some field observation on pouteria caimito, p. campechiana, diospyros digyna and solanum topiro under humid tropical condition (turrialba, costa rica). journal of the american society for horticultural science 27, 111-116. hovenden, m.j., and schoor, j.k.v. (2006). the response of leaf morphology to irradiance depends on altitude of origin in nothofagus cunninghamii. new phytologist 169, 291-297. lim, t.k., and ramsay, g. (1992). abiu – a new fruit with potential for the northern territory. acta horticulturae 321, 99-105. lora, j., testillano, p.s., risueño, m.c., hormaza, j.i., and herrero, m. (2009). pollen development in annona cherimola mill. (annonaceae). implications for the evolution of aggregated pollen. bmc plant biology 9, 129-139. love, k. and paull, r.e. (2011). “abiu”. college of tropical agriculture and human resources, university of hawai. montero, i.f., chagas, e.a., filho, a.a.d.m., saravia, s.a.m., santos, r.c., chagas, p.c., ednalva, and duarte, d.r.d.s. (2018). evaluation of total phenolic compounds and antioxidant activity in amazon fruit. chemical engineering transactions 64, 649-654. sousa, l.c.r.d., junior, a.r.d.c., carvalho, m.g.d., silva, t.m.s.d., and ferreira, r.o. (2019). uplc-qtof-ms analysis of extracts from the leaves of pouteria caimito (sapotaceae) and their antioxidant activity. journal of bioscience and medicine 7, 91-101. 1. sandra a aziz.cdr abstract ta a is acc ( (linn.) o. kuntze)tacca leontopetaloides tropical plant that widely used for starch in someis its countries as well as medicinal plant. tacca has advantages as it can grow on sand in tropical seashore areas and rain forests. farmers in madura island indonesia cultivated tacca by using mother tubers as propagules and harvest the daughter tubers, but no studies have been conducted on the growth of mini-tubers and their cultivation. the purpose of this study was to �nd the best combination of planting media miniand the size of tuber for growing tacca. the experiment was set in a completely randomized block design with factortwo s and three replications e :. th planting media sandare rice-hull charcoal : cow manure (1:1:1 v/v), soil : sand : cow manure (1:1:1 v/v), soil : rice-hull charcoal : cow manure (1:1:1 v/v), and soil : cow manure (1:1 v/v) mini-tuber weight 1-5 and 5.1-20 g. the; are result showed polynesian arrowroot grow best on sand : rice hull charcoal : cow manure (1:1:1 v/v) and rice hull charcoal : cow manure (1:1 v/v) media. both propagules sizes can be used, but plants from large mini tuber had better growth and produced larger mother and daughter tubers. key words: mini tuber tuber,, parent polynesian arrowroot, ,organic media planting secondary tuber introduction polynesian arrowroot or tacca leontopetaloides (linn.) o. kuntze heyneisfrom taccaceae family ( , 1987; meena and yadav, 2010), is an annual tuber ( s p e n n e m a n , 1 9 9 4 ; w i l k i n e t a l . , 2 0 0 5 ) , hermaphrodite dioecious (wilkin et al., 2005) and an underutilized source of starch in indonesia.this plant is herbaceous (abdullah et al., 2014; booth et al., 2004) plant other than grass or sedges, leaves compound or lobed, stem with longitudinal ridges and hollow (booth et al., 2004). abdullah et al. (2014) stated that polynesian arrowroot possess the most stunning in�orescence with whisker-like �liform bracteoles and the colour of the two conspicuous inner involucral bracts range from white, green, purple, brown to near black colour. the true �owers of are dark purple, brown, or neart. leontopetaloides black in colour and they are actinomorphic and hermaphroditic with six stamens. ast. integrifolia potted plants and exterior landscaping need 30% shade level for the longest �ower stalk, whereas shade level of 70% is most suitable to produce a good quality potted plant with intense �ower colour and suitable �ower stalk length in malaysia. full sunlight will delay and slow down the growth of plants and cause death of plants. in india tubers are usuallyt. leontopetaloides cooked, boiled with leaves of guava or tamarind to avoid the irritating property of the tubers, or roasted as vegetables (misra and misra, 2014). in hawaii the tubers are used as famine food (bevacqua, 1994), and it is a staple food in mozambique (bruschi et al. 2014). ndouyang et al. (2014) stated that the tacca genus has toxic alkaloid taccalonolid e and a. furthermore ukpabi et al. (2009) stated that the bitterness in comes from taccalint. leontopetaloides (3,5,7,4'-tetrahydroxy-�avylium-3-xyloside), a phenolic compound, that should be processed before eaten by livestock (ubwa et al , 2011).. some medicinal uses of this plant has been documented, such as to reduce chest pain and to cure rashes on newborn baby (nandwani et al., 2008), to relieve pain of the body and stomach, and it is considered as antidote of food poisoning, and possesses analgesic, antipyretic and antiin�ammatory activities (jiang et al., 2014). sandra ari�n aziz* , rendy susanto a a department of agronomy and horticulture, faculty of agriculture, bogor agricultural university jl. meranti, kampus ipb darmaga, bogor 16680, indonesia *corresponding author; email: sandraaziz@yahoo.com journal of tropical crop science vol. no. , 2012 1 february 5 www.j-tropical-crops.com 1 research article are (linn.) o. kuntze mini tuber growthtacca leontopetaloides affected by media composition and tuber size? phytochemical screening of t. leontopetaloides showed that their leaves contain alkaloids, saponins and tannins whereas the tuber only contains alkaloids (borokini and ayodele, 2012).the tuber also contains �avonoid (ukpabi et al., 2009), and this �nding is differed from habila et al. (2011) that reported no �avonoids in . habilat. leontopetaloides e t a l . ( 2 0 11 ) , h o w e v e r, r e p o r t e d t h a t t. leontopetaloides has antioxidant activities of 86% at 125 μg.ml , reducing potential and total phenolic -1 content express in terms of gallic acid equivalent (gae) 0.217nm, 6.90 mg, compared with gallic acid standard (1.268nm). jiang et al. (2014) stated that 120 constituents have been isolated from t. leontopetaloides, including steroidals, phenolics, �avonoids, sesquiterpenoids, triterpenoids and starch. steroidals and diarylheptanoids showed potent bioactivities, such as cytotoxic, microtubules t a b i l i z i n g , n f k b a c t i v a t i o n a n d p pa r transcriptional and insecticidal activities. the starch from has high amylase contentt. leontopetaloides and showed potential uses in food and drug system. huang et al. (2002) stated that leaf extract from t. leontopetaloides can be used against slugs and snails. evaluation of the physicochemical properties of fresh and stored (4 months) tacca tubers showed that the bitter brown skinned tuber had 28.2529.00% dry matter, 25.00-27.25% starch 40-43 mg/100 g ascorbic acid, 3.15-3.58% crude �avonoid extract,and a density of 1.67 g.ml density (ukpabi -1 et al., 2009). the proximate composition of the tuber �esh was 1.10-1.50% protein, 2.70-2.73% ash, 0.280.68% �ber, 0.08-0.10% fat and 95.02-95.42% total carbohydrate on dry matter basis (ukpabi et al., 2009), and marcel et al. (2012) reported that the tubers have 35% starch content. people still use starch from tacca tubers as staple food in madura island, east java, indonesia. tacca has many bene�ts, one of them is that it grows on sand in tropical seashore areas, rain forest and subtropical rain forest ( , meena andheyne 1987; yadav, 2010). the vast idle tropical seashore areas in indonesia can be made more productive by producing starch from .t. leontopetaloides spennemann (1994) stated that t. leontopetaloides was found to grow on very �ne to medium soil with low to medium humus content. t. leontopetaloides height of leaves and �owercan reach 0.2-1.2 m and 0.8-1.8 m, respectively, when grown understory of coconut tree, orartocarpus altilis pandanus tectorius. pate et al. (2014) study in nigeria reported that intra-row spacing of 20 and 25 cm, application of npk and poultry manure produced higher tuber production of polynesian arrowroot than using cow manures. media used in this research should resemble the media where polynesian arrowroot was found in nature, media combination that have high porosity i.e. sand and rice hull charcoal for porosity, soil and cow manure as clay source, binding agent, as well as nutrient source. mualim (2009) reported that cow manure application increased soil ph, p o , k o, decreased c-organic,2 5 2 and change the soil texture from clay to dusty clay in talinum triangulare cultivation. farmers in madura island cultivated tacca by using mother tubers as propagules and harvest the daughter tubers, but there has no report on using mini tuber produced from seedlings and their cultivation.this cultivation tradition decreases the harvest because the use of big tuber as propagule. mini tubers can befound on land cultivated with t. leontopetaloides, but they are usually lay idle, or disturb the planting distance needed for producing big tuber, and were treated as weeds. spennemann (1994) stated that in marshall islands traditional and modern cultivation of polynesian arrowroot only harvest the big tubers, while the small tubers are left behind in the holes. a new tuber formed close to the one of the previous year. although the tubers are generally described as small, there are reports of larger tubers, weighing over 500 g. the number of tubers of each mature plant can range from one to well over thirty, with numbers between ten and twenty being the most common. the plants tend to show preponderance for small tubers, with a few big ones added. in onion cultivation in nigeria, large onion bulb ( ) can be used for growth, highallium cepa yield and better quality of onion seeds (ashagrie et al., 2014). hussain et al. (2001) reported that medium size bulbs had greater leaf production than small and large bulbs. this research aims to �nd media composition and tuber sizes that can be used to cultivate polynesian arrowroot. materials and methods treatments the experiment was conducted in bogor, indonesia from october 2013 to may 2014 and laid out in c o m p l e t e l y r a n d o m i z e d b l o c k d e s i g n w i t h two sfactor , compositionof planting media and mini t u b e r w e i g h t , w i t h t h r e e r e p l i c a t i o n s e. t h composition of :planting media sand ricetested are hull charcoal : cow manure (1:1:1 v/v), soil : sand : cow manure (1:1:1 v/v), soil : rice hull charcoal : cow manure (1 1:1 v/v), and soil : cow manure (1:1 v/v): . 2 sandra ari�n aziz, rendy susanto journal of tropical crop science vol. no. , 2012 1 february 5 www.j-tropical-crops.com the tuber propagules are grouped into 1-5 (small tubers) (large tubers)and 5.1-20 g . the propagules came from sumenep, madura island. the shade percentage was 35%. duncan multiple range test was used on variables that were signi�cantly different (p<0.05). results and discussion composition of planting media and polynesian arrowroot growth plants grown on soil : cow manure (1:1 v/v) media had the lowest values on all growth variables observed (figure 1 and 2). plants on sand : rice-hull charcoal : cow manure (1:1:1 v/v) media were 36.9047.98% taller those on soil : cow manure (1:1 v/v), and those grown in soil : rice hull charcoal : cow manure (1:1:1 v/v) were than31.13-37.34% taller those grown in soil : cow manure (1:1 v/v) (data not shown). leaf emerged on 6 wat (weeks after planting) in soil : rice-hull charcoal : cow manure media (1:1:1 v/v). leaf number wasaffected by media combination at 16-18 wap. leaf number of the plants grown on sand : rice hull charcoal : cow manure (1:1:1 v/v) was 58.8 % greater than that in soil : sand : cow-62.5 manure (1:1:1 v/v). the plants did not form new leaves between 19-22 wap, but leaf number on sand : rice hull charcoal : cow manure media (1:1:1 v/v) was than that in soil : cow31.5-44.5% greater manure (1:1 v/v). in addition, individual leaf area of the plants grown in sand: rice hull charcoal : cow manure media (1:1:1 v/v) was larger and the plants grew faster. shoot diameter was affected by media composition on 1522 wat. shoot diameter of the plants grown in sand: rice hull charcoal : cow manure (1:1:1 v/v) was 51.38-71.17% greater than that inthose in soil : cow manure (1:1 v/v), likely due to a larger leaf area. relative growth rate was not affected by media composition. tacca grown on sand : rice hull charcoal : cow manure (1:1:1 v/v) had higher22.98% rgr than those grown on soil : cow manure (1:1 v/v), likely due to greater leaves, petiole, tuber, and root dry weight. net assimilation rate was affected by how much the leaves shaded each other. tacca grown on sand : rice hull charcoal : cow manure (1:1:1 v/v) had 104.7 higher nar than those grown on soil : cow% manure (1:1 v/v), likely due to greater leaf area. 3 journal of tropical crop science vol. no. , 2012 1 february 5 www.j-tropical-crops.com are (linn.) o. kuntze mini tuber..........tacca leontopetaloides 4 figure 1. growth variables of at different media combination (a-d): (a) plant height (cm) att. leontopetaloides 4 and 22 wap; (b) number of leaves at 6, 18, 22 wap; (c) leaf area (cm ) at 22 wap; (d) relative 2 growth rate (g per day) at 18-24 wap; and (e) net assimilation rate (g.cm per day) at 18-24 wap -2 where a = sand : rice hull charcoal : cow manure; b = soil : sand : cow manure; c = soil : rice hull charcoal : cow manure; d = soil : cow manure. journal of tropical crop science vol. no. , 2012 1 february 5 www.j-tropical-crops.com sandra ari�n aziz, rendy susanto weight difference between total tuber produced to initial mini tuber was affected signi�cantly by media combination on 1-24 wap (data not shown).the weight of daughter tubers on sand : rice-hull charcoal : cow manure (1:1:1 v/v) was 216.54% than that on soil : cow manure (1:1 v/v), whereas on sand : rice hull charcoal : cow manure (1:1:1 v/v) was 401.80% greater than that on soil : cow manure (1:1 v/v) (figure 2). figure 2. tuber production at different media combination (a-d) at 24 wap: (a) mothert. leontopetaloides tuber weight (g); (b) mother tuber diameter (cm); (c) mother tuber thickness (cm); (d) daughter tuber weight (g); (e) daughter tuber diameter (cm); (f) daughter tuber thickness (cm); (g) weight differences between parent and secondary tubers (g); (h) weight differences between total tuber produced and parent tubers (g), where a = sand : rice hull charcoal : cow manure; b = soil : sand : cow manure; c = soil : rice hull charcoal : cow manure; d = soil : cow manure. 5 journal of tropical crop science vol. no. , 2012 1 february 5 www.j-tropical-crops.com are (linn.) o. kuntze mini tuber..........tacca leontopetaloides tuber sizes and polynesian arrowroot growth plant height was affected by tuber size at 13-22 wat (data not shown). the plants from larger tubers (5.120 g) were 65.09-81.73 % taller than those from the smaller tubers (1-5 g). leaf number was affected by tuber size at 19-20 wap. plants from small tubers (15 g) had 20.4-5.0% more leaves than those from large tubers (5.1-20 g). however, the leaf area was 47.13% smaller. shoot diameter of the plants from 5.1-20 g tubers was signi�cantly larger than the plants from 1 5 gtubers. no signi�cant effect of tuber size on rgr and nar (figure 3). figure 3. growth variables of small (1-5 g) and large (>5-20 g) tubers: (a) plant height at 4t. leontopetaloides and 22 wap; (b) number of leaves at 6 and 22 wap; (c) leaf area (cm ) at 13 and 24 wap; (d) 2 relative growth rate (g per day) at 18-24 wap; (e) net assimilation rate (g.cm per day) at 18-24 -2 wap. 6 journal of tropical crop science vol. no. , 2012 1 february 5 www.j-tropical-crops.com sandra ari�n aziz, rendy susanto the tuber size signi�cantly affected the sizes of the daughter tuber harvested, mother tuber diameter, mother and daughter tuber thickness (figure 4, data not shown). large tubers (5.1-20 g) had signi�cantly greater mother and daughter tuber weight, mother and daughter tuber thickness than small tubers (1-5 g), i.e. 166.52, , and ,54.61 29.86, 59.93% respectively. large propagules (5.1-20 g) produced 166.52 and 54.61 % larger mother and daughter tubers, respectively, than small propagules (1-5 g), and the mother and daughter tubers produced were 29.86 and 59.93% thicker than the initial mini-tuber propagules. figure 4. small (1-5 g) and large (>5-20 g) tuber production at 24 wap: (a) mother tubert. leontopetaloides weight (g); (b) mother tuber diameter (cm); (c) mother tuber thickness (cm); (d) daughter tuber weight (g); (e) daughter tuber diameter (cm); (f) daughter tuber thickness (cm); (g) parent and secondary tubers weight differences (g); (h) weight differences between total tuber produced and parent tuber weight differences (g). 7 journal of tropical crop science vol. no. , 2012 1 february 5 www.j-tropical-crops.com are (linn.) o. kuntze mini tuber..........tacca leontopetaloides media composition in�uenced tacca growth and tuber production. in madura island, polynesian arrowroot mostly found grow on sand in the seacoast that has good drainage. this condition is similar to rice hull charcoal media in this experiment. rice hull charcoal (saleh, 2010) and cow manure (mualim, 2009) has higher c-organic, n, and k 0 than sand,2 which resulted in better plant growth and greater tuber weight. t. leontopetaloides grown on soil : cow manure media (1:1 v/v) had the lowest value on all growth growthvariables due to slow leaf emergence, more leaves of smaller sizes, low rgr and nar, that was the opposite of on sand : rice hullt. leontopetaloides charcoal : cow manure (1:1:1 v/v) media. media composition with high porosity supported better t. leontopetaloides growth. large tubers associated with high carbohydrate content (source) promoted better growth (sink). soemonoet et al.(1986) found on that plants fromamorphopallus campanulatus large tuber propagules grow faster, had an increased lai and signi�cantly produced larger tuber weight per plant and per hectare. f a r m e r s i n m a d u r a i s l a n d c u l t i v a t e d t. leontopetaloides by using mother tubers as propagules and harvest the big daughter tubers. this cultivation tradition decreases the harvest because the large tubers are used as propagule.t. leontopetaloides are harvested mostly from the wild, and limited information available on culture and types of propagules used for cultivation.the results of this study showed that mother tuber size became larger when using mini tuber, and produced daughter tubers. therefore mini tubers can be used as propagules to produce tacca tubers for consumption in the future. conclusion polynesian arrowroot grow best on media combination of sand : rice hull charcoal : cow manure (1:1:1 v/v) and rice hull charcoal : cow manure (1:1 v/v). both mini tuber sizes can be used as propagules, but larger mini tuber size produced better growth and larger mother and daughter tubers. references abdullah, t.l., misrol, m.z., stanslas, j., abd aziz, m., and goh, l.p. (2014). effect of shade and morphological characterization of janggut adam ( sp.) as a promising nativetacca ornamental and medicinal plant.transanction of the malaysian society of plant physiology 22, 13-15. ashagrie, t., belew, d., alamerew, s., and getachew, y. (2014). effects of planting time and mother bulb size on onion ( l.)allium cepa seed yield and quality at kobo woreda, nothern ethiopia. international journal of agricultural research 9, 231-241. bevacqua, r.f. (1994). origin of horticulture in s o u t h e a s t a s i a a n d t h e d i s p e r s a l o f domesticated plants to the paci�c islands by polynesian voyagers: the hawaiian islands case study. , 1226-1229.hortscience 29 booth, r., harwood, r. k., and mangion, c. p. (2004). "field key for the monsoon rainforest flora of the darwin region". 95 pp. northern university press. b o r o k i n i , t. i . , a n d a y o d e l e , a . e . 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(2005). a plastid gene phylogeny of the yam genus, dioscorea: roots, fruits and madagascar. systematic botany 30, 736-749. journal of tropical crop science vol. no. , 2012 1 february 5 www.j-tropical-crops.com 5. m. tuan.cdr abstract to m a t o ( m i l l . )l y c o p e r s i c o n e s c u l e n t u m (solanaceae) is one of the most economically important vegetable crops in the red river delta ( ) of northern vietnam. the recent marketrrd demands for tomatoes in both domestic and overseas markets have led to an increasing cultivated area for the crop in the . to supportrrd growers in producing healthy and quality produce, this paper presents in detail the agronomic requirements of tomatoes regarding temperatures, light, water, suitable soil type and nutrients. cultivation techniques are discussed to provide a g e n e r a l g u i d e f o r d e v e l o p i n g a p p r o p r i a t e management strategies, depending on speci�c soil types, cropping seasons, cultivars and availability of resources. in addition, six major pests have been i d e n t i � e d , p r o v i d i n g u s e f u l i n f o r m a t i o n f o r developing an integrated pest management ( )ipm program for tomato production in the region. keywords: agronomic requirements, cultivation techniques, management strategies, market d e m a n d s , p e s t i d e n t i � c a t i o n , s u s t a i n a b l e production. introduction vegetable production provides major livelihoods of many people in rural and peri-urban areas of vietnam (ha and nguyen, 2013; ha et al., 2015c). to m a t o ( m i l l . )l y c o p e r s i c o n e s c u l e n t u m (solanaceae) is among the most commercially important vegetable crops for both domestic and export markets in vietnam (johnson et al., 2008; tran, 2005; van wijk, 2008). compared to rice, tomato production could generate double economic ef�ciency (tran et al., 2008) which helps to create more job opportunities for farmers (tran, 2005). the red river delta ( ), a subtropical plainrrd region in northern vietnam, is the main vegetable production area where tomato cultivated area accounts for a signi�cant proportion (pham et al., 2002; van wijk, 2008). the recent increasing market demands have led to high percentage of growers shifting from rice to tomato production (van wijk, 2008). this paper introduces agronomic requirements, cultivation methods and major pests on tomatoes in the . these would be used as a general guide torrd support production and de�ne appropriate pest management strategies. tomato agronomic requirements tomatoes can adapt to various climatic conditions, however the optimum temperatures for their growth and development lie between 21 and 27 c (hanson, o 2001; shankara et al., 2005). in addition, tomatoes are day-length neutral plants (nuruddin, 2001). light intensity of 400-500 μmol.m s is optimal for growth -2 -1 . and development. high light intensity may cause fruit cracking, sunscald and green shoulders (jones, 1999). though tomatoes have high water requirement (peet, 2008) water surplus may cause fruit rot (jones, 1999) and bacterial wilt (nuruddin, 2001). according to jones (1999) tomato is most sensitive to water de�cit at the �owering stage. bud and �ower drops might occur under a prolonged dry period. various soil types can be used for tomato production, preferably well-drained sandy loam soil (hanson, 2001; jones, 1999; nuruddin, 2001; peet, 2008). suitable soil ph is between 6.0-7.0. if the ph is less than 5.5, plant disorders such as blossom-end-rot may occur (hanson, 2001). tuan m. ha* thai nguyen university of agriculture and forestry, thai nguyen city, vietnam corresponding author; email haminhtuan@tuaf.edu.vn* : 33 short communication agronomic requirements and production methods of tomatoes in the red river delta of vietnam j 2 1 februaryournal of tropical crop science vol. no. , 2015 www.j-tropical-crops.com due to their rapid growth in a long production period, tomatoes have high requirements of nutrients. for instance, to produce 1 ton of fruits the crop requires 1.36 3.63 kg n; 0.23 1.36 kg p o ; 2.27 5.45 kg2 5 k o (peet, 2008)2 production methods cropping calendar three traditional growing seasons in the wererrd reported by tran (2005) (table 1): table 1. winter – spring cropping seasons for tomatoes in the red river delta, vietnam seasons early season main season late season sowing seeds july/ august mid-september end of october november planting august/september november december harvesting end of october or december (depending on cultivars) february or march march or april due to the recent high market demands for tomatoes, growers have started two additional seasons, spring to summer (sowing from early january to february), and summer to autumn (sowing from july to august) seasons with the harvesting times of may/june and october/ november, respectively. special attention however needs to be paid to pest management in these seasons since hot temperatures and high humidity are favorable for development of pests and diseases (tran, 2005). cultivar selection there is a range of varieties used for the winterspring season crop such as local varieties, indian cultivars, 3 and some exotic f1 hybrids such assb s902, delta, 2000, 5 and s901. some cultivarsvl hp are suitable for hot and rainy seasons such as 4,kbt so sb12, 2, s901 (nguyen, 2004), and virusresistant such as vl vl dv3500, 642, 2926, kim cuong, savior, trang nong-05 (le, 2013). six new cultivars produced by the fruits and vegetables research institute are also recommended, depending on the market demands including lai 09 and 10 for main and late seasons, 2, 20,hpt vr fm and 29 for early and late seasons, and 5 forfm xh early season ( , 2009).vista soil preparation and planting density seed-bed preparation: prepare raised seedbeds (15 cm high) with a 3-4 cm rice straw layer to improve drainage and to avoid soil-borne disease problems. sow seeds in rows (750-900 seeds.m or a 6 cm -2 distance between seeds). cover seedbeds with a thin compost or a rice straw mulch layer to conserve moisture (hanson et al., 2001). field preparation: prepare raised beds (1 m wide) with furrows (50 cm wide) in between. bed height may range from 20 cm (in dry seasons) to 35 cm (in wet seasons). mulching materials such as rice straw can be used to prevent weed development, keep moisture and reduce leaching of fertilizers (hanson et al., 2001). planting density: depending on plant growth habit, the asian vegetable research and development center ( ) recommended the followingavrdc spacing: plant habit spacing plant density (number of plants per ha) table 2. tomato plant spacing according to plant habits (hanson et al., 2001). 33,333 16,666 2 rows per bed (1 m wide); 40 cm between plants one row per bed (1 m wide); 40 cm between plants indeterminate (pruned) determinate (not pruned) j 2 1 februaryournal of tropical crop science vol. no. , 2015 www.j-tropical-crops.com 34 tuan m. ha fertilization and irrigation depending on soil conditions, the amount of fertilizers applied per hectare are n, p o and k o of2 5 2 60 – 120 kg; 60 -140 kg and 60 – 120 kg, respectively, as recommended by the whereavrdc half of each element is applied at planting as the basal fertilizer. the second half is applied after the �rst fruit-set. further, combination of organic matter (farmyard manure) and chemical fertilizers is recommended (hanson et al., 2001). tran (2005) recommends the following fertilizer formula for tomatoes in the :rrd irrigation should be provided on regular basis to maintain �eld moisture at around 60% (tran, 2005). in dry seasons, weekly watering is needed for the �rst month after transplanting and then every 10 days until the end of the crop season. water-logging should be avoided in rainy seasons (hanson et al., 2001). staking & pruning bamboo poles or wooden stakes (1.2 m) can be used for staking tomatoes to keep foliage off the ground (hanson et al., 2001; tran, 2005). staking is provided when the plants reach 30 cm tall. plants should be rebound every 5-7 days. moreover, old and damaged leaves should be removed to maximize light interception (peet and welles, 2005), air circulation (hochmuth, 2008; koske et al., 2008) and to decrease disease incidence (navarrete, 2000). shoot topping (pruning) should also be carried out 5-8 weeks prior to harvest (peet and welles, 2005). harvesting timing of harvesting is important as it affects fruit nutritional composition and quality. fruit maturity can be categorized into four stages (table 4), and harvesting at stages 3 and 4 is recommended to obtain quality fruits (shankara et al., 2005). however, for long distance markets, fruits can be harvested at earlier stages where less than 10% of fruit surface is tannish-yellow, pink or red (hanson et al., 2001). identi�cation of major pests on tomatoes in the red river delta, vietnam pest management is an integral component in agricultural production to reduce crop losses due to pest damages. pests are de�ned as “organisms such as weeds, insects, bacteria, fungi, viruses and animals which unfavorably in�uence human lifestyles” (ha, 2014e). the insects, diseases and weeds that have been reported as the major pests that caused serious problems in tomato production in the ofrrd vietnam are presented in table 5. j 2 1 februaryournal of tropical crop science vol. no. , 2015 www.j-tropical-crops.com table 3. fertilization method for tomatoes in the red river delta, vietnam method of fertilizer application amount of fertilizer applied per hectare remarks mix the fertilizers then apply into holes, cover with a soil layer before transplanting seedlings into the holes. additional fertilizer is applied when roots have developed, at �owering, and at fruit setting stage. 15-20 tons of decomposed manure; 400-500 kg of ; 195-200 kg ofssp ;potassium 70 kg of urea. 10 tons of decomposed manure; 130-200 kg urea. basal (applied once) top-dressing (4 5 times) table 4. maturity stages of tomato fruits (shankara et al., 2005) stage description seeds are white in color (immature) and can be cut when fruits are sliced. there is no juice inside the fruits. seeds have tanned color (mature) and some juice present. seeds are pushed aside when cut. the color inside is still green. juice color becomes red. 1 2 3 4 35a ..........gronomic requirements and production methods of recently, eco-friendly production practices have been embraced as one of the appropriate strategies in agriculture (bosch et al., 2015; ha, 2007, 2014b; ha et al., 2015a, 2015b) particularly in vietnam vegetable production (ha, 2011, 2014a, 2014c, 2014d) in which is an essential part in cropipm protection towards sustainable production systems (ha, 2014e). therefore, developing an programipm for tomatoes in the is of crucial importance torrd ensure high product yields and quality in addressing the market demands, particularly in terms of food safety, and other bene�ts such as improved environment, human health, more secure income and livelihoods for smallholder farmers in the region. details of the program for tomatoes will beipm described in the forthcoming paper. conclusion this paper has presented an overview of the agronomic requirements of tomatoes, which include temperatures, light intensity, water, soil types and nutrients. special attention should be paid to its high water requirement at �owering time, and balanced amount of fertilisers to warrant plants' healthy growth, high yield and quality. suitable cultivars should be selected in hot seasons to reduce fruit sunscald and cracking. in addition, a general guide on cultivation techniques for tomatoes to support tomato producers having appropriate management strategies, depending on their soil types, cropping seasons, cultivars and availability of resources. this paper has described six major pests of tomato crops in the region. speci�c advice on productionrrd guidelines of new cultivars, and forecast of weather condition and emergence of potential pests should be updated from local departments of plant protection and extension stations. references bosch, o.j.h., nguyen, n.c, ha, t.m., and banson, k.e. 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(2008). “the market for vegetables in north vietnam”. 47 p. applied plant research, wageningen university, wageningen. j 2 1 februaryournal of tropical crop science vol. no. , 2015 www.j-tropical-crops.com 38 tuan m. ha 4. sudradjat_vol2_2014.cdr abstract the oil palm ( )s are highlyelaeis guineensis jacq. ef�cient . it produces �ve to seven timesoil producers more vegetable oil per hectare than soybean, sun�ower, and rapeseed. cthe obje tives of this research were to study the role of organic and n, p, and k fertilizers on growth of immatureone-year-old oil palm on marginal soil in jonggol. the research was conducted at ipb cargill teaching farm of oil palm, jonggol,(ictf) bogor, west java from march 2013 to march 2014. the experiment was ed factorial experimentarrang in a following a randomized block design with three replications. the �rst factor was organic fertilizer (manure) consist of 0 , 15 and 30 kg pering (o) (o ) (o )1 2 tree per year. the second factor was inorganic fertilizer consist of 0ing (s ); 0.25 kg n + 0.25 kg p o + 0.39 kg0 2 5 k o (s ) 0.50 k n + 0.50 kg p o + 0.78 kg k o (s )2 2 5 21 2and g per tree demonstrated that a. the results pplication of manure up to 30 kg did not affect theper tree per year growth of one year-old oil palm 0.50 k n. application of g + 0.50 kg p o + 0.78 kg k o highest2 5 2 per tree gave the result in increasing frond production, stem girth, and leaf nutrient concentration. the results of leaf analysis and plant growth responses suggested that rate ofthe s manure in this studyand inorganic fertilizers applied are not suf�cient to improve young oil palm growth in marginal soil in jonggol. keywords: cow ,manure critical nutrient level, inorganic fertilizer, soil fertility, vegetative growth introduction the oil palm ( ) belongs toelaeis guineensis jacq. which the family arecaceae ef�cient .is a highly oil producer the oil palm produces �ve to seven times more vegetable oil per hectare than soybean, sun�ower, and rapeseed (basiron and weng, 2004; obi and udoh, 2012). indonesia is the major oil palm produc in theer world oil palm plantation in indonesia has rapidly. the increased from four decades ago. the area of oil palm plantation in indonesia has expanded expon ntiallye from about 80 000 ha in 1965 to 9.8 ha in 2012, million and produce 23.5 t of rude alm ilmillion onnes c p o (directorate general of estate crops, 2013). the demand for edible vegetable and palm oil has increased the increase of per capita consumptiondue to and population growth. the demand of palm oil is expected to double from present consumption of around 120 to 240 by 2050 (corley, 2009). if oilmillion tonnes palm production does not expand to contribute theto increas of demand, it will result in a shortage of supplye of palm oil. production can be increased by area expansion and yield intensi�cation in existing plantations. yield improvement can be achieved by improv crop management. increase in yield canement of reduce the amount of land expansion for palm oil production. the main issue that will occur in the future in expansion area is shifting the use of agricultural lands of fertile land to marginal lands s. most of oil palms were planted in poor fertility status (ng et al., 2011;soil with obi and udoh, 2012). therefore application of organic and inorganic (synthetic) fertilizers are important to supply nutrient n this marginals for the crops grown o soil. m soundaximizing oil productivity of the oil palm through agricultural s providingpractice and balanced nutrition raised oil palm yields (ng, 2001). application of inorganic fertilizer provide plant nut ients that can notr be suppl by soil.ied the nitrogen fertilizer is one of the most expensive nutrient inputs in oil palm production (amir et al. 2001). therefore, it is very important to manage fertilizer application and use the nitrogen ef�ciently. organic fertilizer can improve the physical, chemicalsoil and biological properties such as improving water capacity, aeration, porosity, soil agregation, cation exchange capacity and microorganism activity in the soil (yang et al., 2004; and , ).leszczynska malina 2011 studies investigating the role of organic residues on young oil palm growth were conducted by khalid (1999; 2000) in malaysia. fertilizerorganic application was in�uence of manure, nitrogen, phosphorus and potassium fertilizer application on growth of one-year-old oil palms on marginal soil in jonggol, bogor, indonesia. sudradjat , yan sukmawan , sugiyanta a b a* a bogor agricultural university, department of agronomy and horticulture, bogor, indonesia 16680 b post graduate student, bogor agricultural university department of agronomy and horticulture, bogor, indonesia 16680 corresponding author; email: * sudradjat_ipb@yahoo.com journal of tropical crop science vol. 1 no. , 20142 october www.j-tropical-crops.com 18 effective to increase growth of oil palm seedlings (uwumarongie-ilori et al., 2012 )., ovie et al. 2014 the obje tive of this research was to study the effect ofc manure and single element of n, p, and k fertilizer application youngon growth of one-year-old oil palms on marginal .soil in jonggol material nd methodsa experimental ites the research was conducted ipb-cargill teachingat farm located in jonggol district, west java, indonesia ( )6.453144°s, 107.039198°e from march 2013 to march 2014. soil type in the research area is ultisols with an altitude 113 m above sea level. the rainy seasonof ± the usually occurs between november and february and t each yearhe dry season between june to september . average rainfall in the research area was 302.6 mm per month total rainfall from march 2013 to marchwith a 2014 3 631 mm. average air temperature was 28.8 cof , o and average relative humidity was 77.3%. materials the oil palm tenera var. damimas.study used oneyear-old plants iwere planted n december 2012 in 9.2 m x 9.2 m x 9.2 m triangular pattern with a population of 136 . kg organic fertilizertrees per hectare sixty of in the form of decomposed cow manures, 500 g rock phosphate and 500 g dolomite were applied to each planting hole. experimental esign and reatmentsd t the experiment arrang in awas ed factorial experiment following a randomized block design with three replications. the �rst factor was organic fertilizer (cow manure) consist of 0, 15, and 30 kg per tree per year,ing applied once in march 2013. the second factor was single element s0fertilizers consist of 0ing ( ), 0.25 kg n + 0.25 kg p o + 0.39 kg k o ( ) 0.50 k n + 0.50 kg2 5 2 s1 and g p o + 0.78 kg k o ( ) the2 5 2 s2 per tree. n, p, and k fertilizer treatment applied three times,were i.e. in march, june and december 2013. the soil analysis was conducted on the levels of total nitrogen, available p, exchangable k bulk density, and organic matter content. scoring was conducted monthly from march 2013 to march 2014 on frond production, stem girth and leaf area hlorophyll and leaf nutrient concentration. leaf c were measured at 6 and 12 month after treatment (mat). the youngest fully expanded leaf the top ofon the leaf crown was assigned as number 1, the next down as number 2, and so on (legros et al., 2009). leaf area was measured based on a measurement method developed by .hardon et al. (1969) the effect of treatments were analyzed by anova and further analysed using duncan multiple range test (dmrt) and considered signi�cant at <0.05.p results nd discussiona the chemical and physical properties are presentedsoil in table 1. based on the soil criteria for oil palms the soil, parameter value criteria* ph: h2o 5.00 low kcl 4.30 low organic c (%) 1.83 low total n (%) 0.17 low c/n 10.76 intermediate avaible p (ppm) 7.60 very low ca (me.100 g -1 ) 5.70 intermediate mg (me.100 g -1 ) 4.74 intermediate k (me.100 g -1 ) 0.20 very low na (me.100 g -1 ) 0.23 low cation exchange capacity (me. 100 g -1 ) 21.59 intermediate base saturation (%) 50.35 intermediate exchangeable al (me.100 g -1 ) 3.45 exchangeable h (me.100 g -1 ) 1.14 texture: sand (%) 24.65 silt (%) 49.43 clay (%) 25.92 loam *criteria by indonesian oil palm research institute. table 1. hysical and chemical of jonggol soilp properties journal of tropical crop science vol. 1 no. , 20142 october www.j-tropical-crops.com 19in�uence of manure, nitrogen, phosphorus and potassium fertilizer.......... in jonggol ic soilwas acid and low in organic matter. the total n, available p, and exchangable k are low or verylow, soil bulk density is 1.20 g cm thus indicating poor -3 soil fertility. manure application had no signi�cant effect on the vegetative growth of oil palm from the beginning to the end of the experiment. a ofpplication n, p, and k fertilizer signi�cantly increased onwardsthe growth of oil palm matat �ve . response of lowoil palm to fertilization for severalwas months after transplanting, likely due to transplanting shock. it took time for the seedlings to build an effective root system . application of n,(goh and hardter, 2003) p, and k fertilizer gave the resultsat level highests2 to improve one year-oldthe growth of oil palm onyoung marginal soil in jonggol. the effect of manure and fertilizer application on palm oil vegetative growth and frond production manure application did not signi�cantly affect frond production, n, p, and k fertilizer signi�cantlywhereas increased frond on 5 and 10 mat (table 2).production the highest frond production was achieved by application of n, p, and k fertilizer and . n,s at s level1 s2 p, and k fertilizer and s2 increasing fronds at levels1 production 26.3 and 21.0 % respectively-32.1% -42.7 , , compared control effect of n, p, and kto . fertilizersthe were only at 5 and 10 ; this may besigni�cant mat associated with of fertilizer application. pplicationtime a of single component n, p, and k fertilizer weres conducted three times i e in march 2013 (0 ), june, . . mat 2013 (3 ) and december 2013 (9 ) somat mat , frond production shows a response to n, p, and ksigni�cant fertilizer at one to two months after application.s f prond roduction is also affected by climatic factors such as rainfall (figure 1) water is important for plants. tabl thee 2. fects of and n, p, k fertilizer (s) on frond productionef manure application 1 3 5 7 9 10 11 12 0 kg 0.80 1.60 1.82 0.36 2.18 1.44 2.27 2.69 15 kg 0.87 1.73 1.96 0.56 2.41 1.73 2.51 2.93 30 kg 0.76 1.76 2.02 0.36 2.34 1.73 2.40 2.98 s0 0.78 1.62 1.67b 0.33 2.11 1.31b 2.35 2.89 s1 0.73 1.73 2.11a 0.40 2.32 1.73a 2.24 2.80 s2 0.91 1.73 2.02a 0.53 2.50 1.87a 2.58 2.91 treatment time (mat) frond production (frond.month -1 ) rates of manure (kg.tree -1 ) n, p, k fertilizer (s) note: -mat = month after treatment; = without ap lication of n, p, k fertilizer-s0 p = ap lication of 0.25 kg n + 0.25 kg p o + 0.39 kg k o-s1 p 2 5 2 = ap lication of 0.50 k n + 0.50 kg p o + 0.78 kg k o-s2 p g 2 5 2 dmrt-values followed by different letters within a column are signi�cantly different at 95% figure 1. effect of n, p, k fertilizer rainfall on frond productionapplication and correlation with (mm) . journal of tropical crop science vol. 1 no. , 20142 october www.j-tropical-crops.com 20 sudradjat, yan sukmawan, sugiyanta as raw material for photosynthesis, part of the plant cell, and solvent frond production in afor the nutrients. month is in�uenced by rainfall one to two months earlier. f prond roduction ranged from 1.5 to 3 per months under suf�cient rainfall (>100 mm per month) and can dramatically 0.5 per months or lessdecrease underto lower rainfall (<100 mm per month). however, based on observations in the �eld the plants still formed a few spears under low rainfall. when rainfall is suf�cient the spear open sooner so that production increasess frond to 2.5 per (9 ). the fewermonth mat frond, table 2 production during dry season is probably related to the poor nutrients absorption by the plants. water plays an important role in dissolving nutrients that can be absorbed by plant roots in the soil so that water shortages will nutrient uptake result indisrupt , which will a stunted plant growth. an increasing rate of manure application up to 30 kg per plant per year tends to result in increasegreatest of stem girth s2 treatment resulted in t highest(table 3). he growth girth, i.e. 57.09 cm, but not signi�cantlyof stem different with 53.20 cm. the increases1 treatment, i.e. in werestem girth due to s1 and s2 treatment 11.9 % and 20.1% respectively compared control at 12, , to dat. palm trunks serve as support structures of leaves, �owers, and fruit; vascular system transportthat s nutrients, water and ; and as an organassimilate accumulation of nutrients (corley and tinker 2003)., oil palm trunks represent approximately 50% of the total above-ground biomass when the plants reach the age of 10 years (corley and tinker 2003). the large girth, stem will support high fruit production . thein the future increase girth is in�uenced by the availability ofin stem n, p, and k collectively (corley and tinker, 2003). n is a major constituent amino acids, amides,itrogen of proteins, and nucleotides role in, and plays a signi�cant the formation of chlorophyll and increase vegetative growth (ra hman et al., 2008). phosphorus plays rolec as molecular component of the energy transferor i.e. atp adp nad and nadph which energy-rich, , are compound that controls various reactions in plants such as photosynthesis, respiration, protein synthesis and amino acids, and nutrient transport (booromand and grough 2012) potassium plays role as an enzyme, . activator, maintaining osmotic potential and water uptake, photosynthesis, and translocationassimilate from to sinkleaf (taiz and zeiger, 2010). application of manure and fertilizer did notn, p, k signi�cantly increase leaf area (table ). leaf area is3 one of the important variables for the growth of palm oil. l deaf area etermines sunlight interception that affect the rate of photosynthesis (hardon et al. 1969). the, wider the leaf area, the higher the rate of photosynthesis is due to an increasing of the of leavesportion that capture sunlight. even though the effects were not signi�cant, application of manure increased bulk density and soil c, n, p and k levels . similar results were reported by(table 4) uwumarongie-ilori et al. (2012). application of organic fertilizer is bene�cial for long-term production thedue to slow release nature of the nutrients (ermadani and muzar, 2011). the effect of manure and fertilizer application on leaf stomatal density, and leaf n, p and k content manure and n, p, and k fertilizer application had no effect on leaf stomatal density (table ). leaf stomatal5 density ranges from 198-211 mm . for comparison, the -2 table 3. effects of and n, p, k fertilizer (s) on stem girth and leaf areamanure application 0 4 8 12 0 4 8 12 15 kg 23.79 30.02 39.61 53.13 0.32 0.86 0.71 1.29 30 kg 23.43 29.62 41.81 55.98 0.32 0.81 0.67 1.21 s0 23.86 28.67 36.20b 47.53b 0.32 0.74 0.70 1.09 s1 23.71 29.63 40.52ab 53.20ab 0.34 0.83 0.63 1.21 s2 23.21 30.51 41.93a 57.09a 0.33 0.87 0.66 1.29 time (mat) stem girth (cm) leaf area (m 2 ) rates of manure application (kg.tree -1 ) n, p, k fertilizer (s) treatment note: mat = month after treatment;= without aplication of n, p, k fertilizer-s0 = ap lication of 0.25 kg n + 0.25 kg p o + 0.39 kg k o-s1 p 2 5 2 = ap lication of 0.50 k n + 0.50 kg p o + 0.78 kg k o-s2 p g 2 5 2 dmrt-values followed by different letters within a column are signi�cantly different at 95% journal of tropical crop science vol. 1 no. , 20142 october www.j-tropical-crops.com 21in�uence of manure, nitrogen, phosphorus and potassium fertilizer.......... average leaf stomatal density was 146 mm in nigeria -2 and 175 mm in malaysia (corley and tinker 2003) -2 , . a mverage leaf stomatal density in this study was 201 m -2 at 6 and 208 m at 12 .mat m mat -2 manure and n, p, and k fertilizer did not signi�cantly affect leaf chlorophyll content (table ). chlorophyll5 plays an important role in photosynthesis. chlorophyll tabel 4. effects of onmanure application soil bulk density, soil organic c, soil total n, p and k rate of manure application (kg.tree -1 ) bulk density (g cm -3 ) organic c (%) total n (%) total p (ppm) total k (ppm) 0 1.05 1.12 0.10 114.52 54.00 15 1.02 1.15 0.10 125.97 60.60 30 0.98 1.28 0.11 145.98 75.00 table 5. effects of and n, p, k fertilizer (s) on stomata density and chlorophyll contentmanure application leaf l 6 12 6 12 0 199.08 208.90 0.032 0.042 15 206.07 207.20 0.036 0.042 30 198.52 208.62 0.035 0.042 s0 203.61 206.92 0.033 0.042 s1 198.14 206.92 0.034 0.041 s2 201.91 210.88 0.037 0.043 n, p, k single fertilizer (s) treatment time (mat) stomatal density (mm -2 ) chlorophyll content (mg.cm -2 ) manure manure (kg.tree -1 ) note: mat = month after treatment;= without ap lication of n, p, k fertilizer-s0 p = ap lication of 0.25 kg n + 0.25 kg p o + 0.39 kg k o-s1 p 2 5 2 = ap lication of 0.50 k n + 0.50 kg p o + 0.78 kg k o-s2 p g 2 5 2 note: mat = month after treatment;= without ap lication of n, p, k fertilizer-s0 p = ap lication of 0.25 kg n + 0.25 kg p o + 0.39 kg k o-s1 p 2 5 2 = ap lication of 0.50 k n + 0.50 kg p o + 0.78 kg k o-s2 p g 2 5 2 dmrt-values followed by different letters within a column are signi�cantly different at 95% table 6. effects of and n, p, k fertilizer (s) on leaf nutrient concentrationmanure application journal of tropical crop science vol. 1 no. , 20142 october www.j-tropical-crops.com treatment manure (kg.tree ) -1 0 15 30 n, p, k single fertilizer (s) s0 s1 s2 n (%) 2.64 2.56 2.67 2.35 2.73 2.79 p (%) 0.20 0.20 0.20 0.19 0.20 0.20 k (%) 1.11 1.17 1.31 1.09b 1.20ab 1.31a n (%) 2.14 2.12 2.26 2.03b 2.15b 2.33a p (%) 0.19 0.19 0.20 0.19 0.20 0.20 k (%) 0.84 0.88 0.89 0.80b 0.86b 0.95a time (mat) 126 22 sudradjat, yan sukmawan, sugiyanta absorbs light and pass it to the reaction center of photosystem (karacan 2006). chlorophyll content in, this study ranged from 0 032 0 043 mg.cm .. to . -2 leaf analysis showed that manure application had no signi�cant effect on n, p and k n,leaf content (table 6). p, and k fertilizer only affected the n at 12 , andleaf mat leaf k at 6 and 12 but did not affect leaf p contentsmat, (table 6). pplication n, p, and k fertilizera of at levels2 resulted in the the increased of leafhighest n and k contents oil. critical nutrient levels in the leaves of young palm 2.75%s according to ochs and olivin (1977) are for n, 0 16% for p and 1 25% for k. reatment. . s2 t resulted in contentsleaf n and k above the critical nutrients levels n 6 . leaf p in this studyo mat content was not signi�cantly affected by manure and inorganic fertilizers treatment and the level is classi�ed as suf�cient according to ochs and olivin (1977). the results of leaf analysis and plant growth responses suggested that rate of organic and inorganic fertilizers applied are notin this study suf�cient to improve young oil palm growth in marginal soil. high rate of fertilizer application is usually recommended for marginal soils and soil with low fertility ( , ,vanlauwe et al. 2001; ng et al. 2011). conclusions application of up to 30 kg didmanure per tree per year not affect the growth of one year-old oil palm. application of g0.50 k n + 0.50 kg p o + 0.78 kg k o2 5 2 per tree highestgave the increase in frond production, stem girth, application ofand leaf nutrient concentration. manure, n, p, and k fertilizer in this study did not affect leaf stomatal density, leaf chlorophyll and leaf n, p, and k content. references amir, h.g., shamsuddin, z.h., halimi, m.s., ramlan, m.f., and marziah, m. (2001). effects of azospirillum inoculation on n �xation and growth2 of oil palm plantlets at nursery stage. journal of oil palm research , 42-49.13 basiron y , weng . 2004 . the oil palm and its, . , c. k ( ) sustainability. 1-journal of oil palm research 16, 10. boroomand, n., grouh, m. s. h. (2012). macro elements nutrition (npk) of medicinal plants. j med plant resournal of icinal earch 6, 22492255. corley r h v , tinker . 2003 . the oil palm, . . . , p. b ( ) “ ”. 592 p. londonblackwell science ltd., 4 edition. . th corley r h v. 2009 . how much oil palm do we need?, . . 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(2006). monitoring of changing chlorophyll content of l. andbuxus sempervirens eunymus japonica l. leaves affected with air pollutants in ankara. world j agricournal of ultural science , 1-6.2 khalid, h., zin, z. z., anderson, j. m.. (1999). mineralization of soil organic carbon and nitrogen in relation to residue management following replanting of an oil palm plantation. j oilournal of palm research 11, 72-88. khalid, h., zin, z. z., anderson, j. m.. (2000). soil nutrient dynamics and palm growth performance in relation to residue management practices following replanting of oil palm plantations. j oil palm researchournal of 12, 25-45. legros s , mialet-sera i , caliman j p , siregar f a ,, . , . , . . , . . clement-vidal a , and fabre d , dingkuhn., . , . ( )2009 . phenology, growth and physiological adjustments of oil palm ( ) toelaeis guineensis sink limitation induced by fruit pruning. annals botany 104, 1183-1194. leszczynska malina 2011, d. , j. k. ( ). effect ofand organic matter from various so rces on yield andu quality of plant on soils contaminated with heavy metals. ,ecol chem eng a.ogy of ical ineering 18 501-507. ng, s. k. (2001). nutrition and nutrient management of the oil palm, new thrust for the future perspective. “ipi prii k in nutrientin: management for sustainable crop production in india”, pp 415-429. international potash institute. ng p h c , gan , and goh 2011 . soil, . . . , h. h. , k. j. ( ) nutrient changes in ultisols under oil palm in johor, malaysia. journal of oil palm environment 2, 93-104. journal of tropical crop science vol. 1 no. , 20142 october www.j-tropical-crops.com 23in�uence of manure, nitrogen, phosphorus and potassium fertilizer.......... obi j c , udoh . 2012 . nutrient budget for, . . , b. t ( ) optimal oil palm ( jacq.) yield onelaeis guineensis coastal plain sands soils of akwa ibom state nigeria. 289-open journal of soil sciences 2, 298. ochs, r., olivin, j. (1977). le diagnostic foliaire pour le controle de la nutrition des plantations de palmiers à huile: prélèvement des échantillions foliares. , 211-216.oléagineux 32 ovie, s ekabafe, m.o nkechika, a udegbunam, o.., ., ., n. (2014). in�uence of composted oil palm bunch waste on soil ph, nitrogen, organic matter status and growth of oil palm seedlings under water stress condition. continental journal of agronomy , 1-15.8 rachman, i. a., djuniwati, idris . 2008 . pengaruhs., , k ( ) bahan organik dan pupuk npk terhadap serapan hara produksi jagung di inceptisol ternate. jurnal tanah dan lingkungan 7-13 .10, (in indonesia )n taiz, l., zeiger, e. (2010). plant physiology .“ ” 690 pp. sunderland: sinauer associates, 5 edition, th sunderland. uwumarongie-ilori e g , sulaiman-ilobu ,, . . , b. b. ederion , imogie , imoisi , garuba ,, o. , a. , b. o. , n. u g b a h . 2 0 1 2 . ve g e t a t i v e g r o w t h, m ( ) performance of oil palm ( )elaeis guineensis seedlings in response to inorganic and organic fertilizer. greener journal of agri sciencecultural 2, 26-30. vanlauwe b , diels , sanginga , merckx ., . , j. , n. , r ( )2001 . “integrated plant nutrition management in sub-saharan africa”, 384 p. cabi publishing, wallingford. yang, s. m., li, f. m., malhi, s. s., wang, p., suo, d. r., wang, j. g. (2004). long-term fertilization effects on crop yield and nitrate nitrogen accumulation in soil in northwestern china. ,agron jomy ournal 96 1039-1049. journal of tropical crop science vol. 1 no. , 20142 october www.j-tropical-crops.com 24 sudradjat, yan sukmawan, sugiyanta 5. aminah_vol2_2014.cdr abstract a study on identi�cation of �owering weeds as predatory insect alternative foods was conducted at bantimurungbulusaraung national park, south sulawesi, indonesia in february 2013 among the plants that naturally grow. in the national park area andcassia siamea clerodendron paniculatum, which were source of nectar for insects, particularly butter�ies. visits of predatory insect weedsto �owering and species of both insects and �owering weeds were recorded. preferredthe �owering weeds predatory insect arevisited by the s c. striata a.conyzoides(papilionaceae), (asteraceae) and l. camara s. nodiflora(verbenaceae) followed by (asteraceae), (malvaceae) ands. rhombifolia l. crustacea (scrophulariaceae). number of adulta large s and pupae sp. n theof was found icoccinella bantimurung-bulusaraung national park. keywords : nectar, pollen, alternative host, weeds, indonesia introduction modern agricultural system has reduced the population and activities of predatory insects. the development of strategies to conserve and maximise the abundance of insects is crucial to enhance biological control of agricultural crop pests. flowering weeds in a naturally diverse habitat thecan potentially bene�t predatory insects by providing alternative source of pollens and nectar particularly during off-season planting (hassan, 1998; nentwig, 1998; fox and landis, 2002). even during the growing seasons vegetable crops are normally ; t grown untilharvested before they �ower hose �owering do not �ower so sources of foodall year round (nectar, pollen) for insects were not always available. pollen and nectar from �owering weeds play an important role increas longevity, fecundity, andto e survival for predatory insects (jervis and kidds, 1996). flowering weeds also provide a suitable microclimate for the predatory insects (landis , 2000).et. al. the predator insects can be seen either with the nakedy eye or magnifying to the nectar orglasses feed on pollen euphorbiae and umbelliferaes of species. buckwheat ( ,fagopyrum esculentum polygonaceae) nectar is normally abundant in the morning and attracted different species of nectar feeders (lee and heimpel, 2002). the larvae/ and adult stages of predatorynymph insects especially holometabolous invary considerably their nutritional requirement and food ecology (wackers et al., 2007). one of the predatory insects that commonly found in the crop production area is coccinelids. adults were �rst attracted tococcinella sp �oral color and odor before they feed on the pollen. pollen can improve the longevity and fecundity of the other predatory insect such as mirids macrolophus pygmaeus to control white�ies (vandekerkhove and de clerqc, 2010). increased habitat diversity in crops can increase population densities of locally available predators to enhance biological control of pests conservation which. involves protection and maintenance of natural enemy population has proved crucial for maintaining native natural enemies in ecosystems .(sharma et al, 2013) the use of persistent pesticide can kill insects particularly predatory insect that live around the crop p r o d u c t i o n a r e a s . h a b i t a t m a n a g e m e n t f o r enhancement of predatory insects is an important component to reduce pesticide (pickettapplication and bugg p application methods can be, 1998). esticide modi�ed, e.g. pesticides can only be applied when the pest population exceeds speci�ed levels (sharma et al, 2013). conservation of natural enemies can also be achieved by changing the active ingredient, rates, formulations, timing, and location of pesticide applications or by maintaining refuges .(hulls and beers, 1985) predatory insects such as coccinellids are normally found to live on the cultivated crops and on the weeds that produces nectar and pollens. reduced pesticide application could increase the roles of insect natural enemies to control crop pests (nentwig, 1998). bantimurungbulusaurung national park in south sulawesi which spreads from maros to pangkep districts ( ) is found to be the4.9000° s, 119.7500° e the potential of flowering weeds as refugia for predatory insects at bantimurung-bulusaraung national park, south sulawesi sri nur aminah ngatimin nurariaty agus annie p. saranga*, , department of plant pest and disease, faculty of agriculture, hasanuddin university makassar 90245, indonesia * : sri�rnas@gmail.comcorresponding author; email journal of tropical crop science vol. 1 no. , 20142 october www.j-tropical-crops.com 25 natural habitat for predatory insects from ncoleoptera , hemiptera , hymenoptera dipteran n and n orders (aminah ngatimin and syatrawati, 2010; aminah ngatimin, saranga and junaedi, 2014). flowering weeds can provide shelters or refugia to support the lives of predatory insects particularly during the extreme environmental condition as well as during non-growing seasons (landis et al, 2000). the objectives of research towere identify the species of predatory insect and s that servethe �owering weed as alternative source of foods thefor predatory insects at marosbantimurung-bulusaraung national park, district south sulawesi, indonesia., material and methods a survey was conducted in bantimurung-bulusaraung national park, maros district south sulawesi between february 2013.to april the elevation of the park is about 500 m above the sea level with average precipitation of 1 500-2 100 mm year, maximum and minimum air, , per temperature ranging from 23-32°c, relative humidity ranging 65-88% and soil ph 5 47.of . the national park covers the area of 43,750 ha and contains large and steep karst area, a small pond and has more than 250 caves. the area of the national park covered in the study was approximately 700 . teakm 2 ( ), bit i ( ) and johartectona grandis vitex cofassust ( )cassia siamea were planted on some area of the national park. samples were taken systematically according to the methods developed by landis et al (2000). the survey was conducted at ten sampling points, i.e. two points near the lake, two points on the grass �elds, two points among teak, bitti and other forestry trees, two on the wild bushes, and the rest in the butter�y cultivation area. predatory insects collection all predatory insects that use �owering weeds as food sources and refugia were recorded. observationvisual on the patter of ' to ingn predatory insects visit �ower weeds was conducted 8 3every day from 0 00-1 00 hours in �ve consecutive days per month between february to april 2013. insects from the ten sampling points were identi�ed on site using naked eye or magnifying glass. small insects such as ants and coccinellids were collected using tweezers and kept in glass bottles containing 90% alcohol. pupae were collected along with the leaves or other plant parts and kept in petri dishes using �lter paper as bases. all collected insects were brought to bantimurung-bulusaurung laboratory for further identi�cation using insect identi�cation reference by kalshoven (1981), kristensen et al. (1991) and hill (1994). all pupae of coccinelids were returned back to the natural habitat after identi�cation. the diversity of predatory insects was calculated using the shannon index diversity (langmack , 2001).et al. flowering weeds collection the �owering weeds visited by predatorythat were insects collected and identi�ed us everaarstwere ing (1981), soerjani et al. (1987) and van steenis (1988) as references. results and discussion flowering weeds as predatory insectsbio-resource for plant species that naturally grow in the national park i n c l u d e ( f a b a c e a e ) ,c a e s a l p i n i a p u l c h e r r i m a clerodendr paniculatumum (verbenaceae) and hibiscus rosa-chinensis (malvaceae). they grow mainly around the limestone hills of the park. our study demonstrated that the predatory insects visited �owering weeds, particularly those with broad leaves. different insects s eha different preferenc to �owering weed species, whereas different weed species were visited by insects for different functions, i.e. as refugia, source of nectar and pollen plant. canopies and root surfaces also function as refugia for pupae of coccinellids. , anda. conyzoides c. striata l. camara were visited by the predatory insects for their nectar (table 1). using magnifying glass we observed that coccinellids visited asteraceae, papilionaceae and verbenaceae �owers and fed on their nectar, clearly seen by the feeding movement of their mouth. using binocular microscope at the laboratory, pollens were found on the mandibles of the coccinellids. the elongated proboscis of the butter�ies from pieriade and papilionidae was observed when they visited the weed �owers. predatory insects chose the ,a. conyzoides c. striata and as nectar sourcel. camara their preferred s (table 1) whereas was the main source of pollenm. udicap for pollinator insects (honey bees). these �ndings con�rmed that the f are natural resourcelowering weeds of nectar and pollen . their function as non-for insects prey food takasu and lewis,increased insect survival ( 1995; hassan 1998; nentwig, 1998, lewis et al , 1998 .). previous studies conducted by soejono (2006) and bàrberi et al 2010 �owers( ) reported that a. onyzoidesc were source of athe nectar for ,coccinella arquata potential predator t control mirid bugshat s (cytorhynus lividipennis) o .n horticultural crops similar �nding on the functions of weeds as refugia, source of nectar and pollen for insects were reported by journal of tropical crop science vol. 1 no. , 20142 october www.j-tropical-crops.com 26 sri nur aminah ngatimin urariaty agus, nnie p. saranga, n a nentwig (1998). we observed the movement of the insect's mouthparts while feeding on the �owers of asteraceae, papilionaceae and verbenaceae species. nentwig (1998) reported that predatory insects fed on b o t h n e c t a r a n d p o l l e n s f r o m a s t e r a c e a e , papilionaceae verbenaceaeand �owers, and pollens from �owersmimosaceae . nectar �owering weeds carbohydrateof the vary in their composition.the viscosity and concentration of sugar solution the predatory insectsaffect the ease of to feed them sugar provides and(karise et. al., 2006). energy extends the span of (takasulife microplitis croceipes and lewis, 1995). hover ies-� syrphidae have a strong respond to sucrose and glucose other insectsover and concentration of sugar the response rate ofaffect predatory insects (lundgren, 2009). the nectar concentration season and plant healthvary with status (lundgren, 2009). ectarn concentration is usually higher during the dry season (lundgren, 2009). nectar is not only sugar but nitrogenoussource of also substances improvethat longevity, fecundity and foraging of predatory insects (wackers and fadamiro, 2005; jervis and kidds, 1996). the weeds performance and insect visitors our study revealed that the f were notlowering weeds only visited by the predatory insects but also by insect pollinators such as honey bees and butter�ies our. survey coccinellidae,found predatory insects from syrphidae and formicidae offamily. adults coccinella sp. 0 122were mostly found on ( . ),c. triata l. amaras c ( . ) ( . ,0 048 and 0 028 table 2).a. onyzoidesc studies by nentwig (1998) and peggie and amir (2009) reported that l. camara attracted honey bees and b u t t e r � i e s a n d f l o r a l.p. d e m o l i o n c . c y l l as characteristic abundanceand nectar within the �owers are the main reason for insect visiting a �ower. floral and extra �oral nectar are food source forimportant s many coccinellids. , which normallystethorus puctillum die within 4-5 days on water alone can survive for 43 2, . days on a diet from borage �owers borago officinalis (nentwig, 1998). of sp.adults was found tococcinella be the ( . ), followed bydominant insect in 0 122c. triatas l. amara a. onyzoidesc c( . ) ( . ,0 048 and 0 028 table 2). we found pupae on the stems0.184 individual c. triatas and barks (table 2). has a yellow long corollc. striata a and aphids colony on the buds.was found living �oral flower anatomy, colour and odor (fox and landis, 2002) presence of preys and honeydew (bàrberi et, the al., 2010) predatoryare amongst the factors that attract insects. honeydew is an important indicator of aphid presence on the plants. honeydew is nutritious and can serve as an alternative food for the predatory insect. in hover-�y (syrphidae) for example, predatory insect with short proboscis unable to the nectar ofwere feed on �owers with long corolla (jervis and kidd, 1996; wackers et al , 2007) and coccinellids prefer a �at. , �ower base (nentwig, 1998). loral morphology andf �ower base size also affects insect visits (lundgren, 2009) reduce nectar evaporation,. long corolla tends to making it more attractive to insect visitors especially predator (lundgren, 2009).y insects we observed aphid colony on whereas manyc. striata coccinellids pupae onwere found , al. amarac �owering weed that is mostly found growing nearby or around were found toc. striata. coccinella sp. pupae live s, and they fed on aphidson stem and near the root . a study by reported thatmacleod (2004) theet al. densities of the predators carabids and staphylinids (agrostis stolonifera dactylis glomerata holcus, , lanatus lolium perenneand ) on a grassy bank habitat during winter (i.e. extreme environment) were greater in the bank than in the boundary of the �eld in which the bank was situated. nthe ba k was protected them from the rain and �oods. insect such asother dolichoderus sp chose the as their refuges near theirl. crustaceae nets macleod et al 2004). the weeds species visited( ,. table 1. flowering weed visited by predatory insects in bantimurung-bulusaraung national parkspecies , south sulawesi journal of tropical crop science vol. 1 no. , 20142 october www.j-tropical-crops.com 27 nectar pollen refugia asteraceae ageratum conyzoides l. synedrella nodiflora (l.) gaertn capparidaceae cleome rutidosperma dc malvaceae sida rhombifolia l. mimosaceae mimosa pudica l. papilionaceae crotalaria striata dc scrophulariaceae lindernia crustacea (l.) f.v.m verbenaceae lantana camara l. plant family species function t ..........he potential of flowering weeds as refugia for predatory insects by predatory insects depend on the nectar availability and of refug (schellhorn and silberbauer,the vicinity ia 2002). conclusion c. striata a.conyzoides(papilionaceae), (asteraceae) and (verbenaceae) �owers were preferred byl. camara the predatory insects, followed by s. nodiflora (asteraceae), (malvaceae) ands. rhombifolia l. crustacea coccinella(scrophulariaceae) sp. ha a. d high number of adult and pupae on the �owering weeds in bantimurung-bulusaraung national park. this study demonstrated that could be used as�owering weeds one of the maintainingmanagement tools for predatory insect .population references aminah ngatimin, s. n. and syatrawati. 2010 . the( ) diversity of soil arthropods in cabbage farm in malino, south sulawesi. “proceeding of thein: n a t i o n a l s e m i n a r o n b e l o w g r o u n d biodiversity”, university of lampung, bandar lampung, indonesia. aminah ngatimin, s. n., saranga, a.p. and junaedi. the impact of leaf extract of extractlantana camara on soybean's predatory insects. “proceedingin: of the asean conference on science and technology”, 18-19 august 2014, bogor, indonesia ( )in-press bàrberi p , burgio g , dinelli g , moonen a c , otto s ,. . . . . . vazzana c zanin g (2010). functional io. and . b d i v e r s i t y i n t h e g r i c u l t u r a l a n d s c a p e :a l r b welationships etween eeds and arthropods fauna. 388-401.weed research 50, everaarst, a p 1981 . weeds of vegetables in the. . 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( ) “ ” 495p. pradnya paramitha, jakarta ., indonesia journal of tropical crop science vol. 1 no. , 20142 october www.j-tropical-crops.com 29t ..........he potential of flowering weeds as refugia for predatory insects journal of tropical crop science vol. 8 no. 3, october 2021 www.j-tropical-crops.com 154 tran loc thuy, tran ngoc thach, tran thi thanh xa, chau thanh nha, vo thi tra my, nguyen thi thao nguyen and nguyen thuy kieu tien heat stress affects seed set and grain quality of vietnamese rice cultivars during heading and grain filling period tran loc thuy*, tran ngoc thach, tran thi thanh xa, chau thanh nha, vo thi tra my, nguyen thi thao nguyen and nguyen thuy kieu tien cuu long delta rice research institute/ tan thanh ward, thoi lai district, can tho city, vietnam. *corresponding author; email: tranlocthuy@gmail.com abstract environmental stress trigger a variety of rice plant response, ranging from alters seed set, grain yield and grain quality during flowering and grain filling stage. efforts are required to improve our understanding of the impact of heat stress on rice production, which are essential strategies in rice cultivation. this article investigated the seed set, yield components and grain yield of vietnamese rice cultivars (indica germplasm) under high temperature environment during the flowering and grain filling stage. six rice cultivars, including popular cultivars and new cultivars of cuu long delta rice research institute, and one popular extraneous cultivar with differences in maturing time, were grown in pots at high temperature (ht) and natural temperature condition as control (ct). all rice cultivars were subjected to the high temperature starting from the heading stage to the harvest maturity, applied by greenhouse effect. the greenhouse has about 25 cm window opening on 3 sides for air ventilation. the seed set rate of the heat-sensitive rice genotypes decreased significantly under ht, leading to a significant reduction in grain yield. the lowest seed set was rrecorded in “om4900” (44.3%) and "om18" (39.9%) under high temperature environment. the lower yield in all rice cultivars at an elevated temperature resulted in a dramatic decrease of filled grains and contributed to a loss of 1000-grain weight. "om892" is a potential rice cultivar for heat tolerant breeding program due to the seed set percentage was above 80% in both ht and ct conditions. high temperature during the grain filling stage resulted in a decreased amylose and increased chalkiness for all om cultivars. keywords: grain quality, high temperature, om rice, seed set introduction rice is one of the most important crops in the world, especially in asia, where approximately 90% of the rice production is consumed (fao, 2008). rice consumption has been increasing in africa and latin america as well (jagadish et al., 2007). unfortunately, climate change is predicted to have negative impacts on food production, food quality and food security (ceccarelli et al., 2010). high temperature caused by climate change is exposing most of the world’s crops, including rice, at least at some stages of their life cycle. global warming might increase agricultural production in some temperate countries, but possibly reduce it in most asian countries (rosenzweig et al., 1993). in the report of shah et al. (2014), the increase in the daily mean temperature of 2oc would make a dramatic reduction in rice grain yield. wheat and rice are more sensitive to high temperature during reproductive stages compared to vegetative stages (djanaguiraman et al., 2020). many studies have discussed the impact of the rising temperature on rice cultivation. the critical temperature differs according to variety, growth stage, duration of critical temperature and diurnal change (sridevi and chellamuthu, 2015). plant reproductive processes are complex and sensitive to environmental changes, including high temperatures, which ultimately affect fertilization and post-fertilization processes leading to decreased yields. high temperature affects anther dehiscence, pollination and pollen germination which then leads to spikelet sterility and yield loss (yoshida et al., 1981). flowering is one of the most susceptible stages in the life cycle of rice, and rice spikelet at anthesis exposed to above 35 °c for 4–5 days induces sterility, with no seed produced (thuy et al., 2020) high temperature during the grain filling period limits grain yield and quality in rice. high night temperature heat stress affects seed set and grain quality of vietnamese rice cultivars during .......... journal of tropical crop science vol. 8 no. 3, october 2021 www.j-tropical-crops.com 155 and day high temperature adversely affect rice productivity (haixian et al., 2011). rice quality is mainly evaluated by several traits, including milling, appearance, cooking, eating, nutritional qualities and other indicators. in addition to genetic factors, these traits are also affected by the grain positions on the rachis and environmental factors such as water status and high temperature. previous studies showed that grain quality was significantly affected by the different positions of grains, which is particularly relevant to heavy-panicle rice varieties (cheng et al., 2000). in rice production and breeding practices, heading date and yield traits are generally considered as key indicators for the response to temperature variation; for instance, early heading and yield loss have been commonly observed at high temperatures. the identified heat-tolerant varieties based on high seedset rate and the phenotype protocols could be used in future genetic studies and breeding programs focusing on heat tolerance. throughout this study, we determined the heat tolerant cultivars for future warmer circumstances by phenotype protocols. in addition, our investigation presented yield and grain quality parameters response to the temperature of cuu long delta rice research institute cultivars which could provide a reference for building crop index based weather. materials and methods crop cultivation six cultivars of cuu long delta rice research institute (om cultivars) and one popular extraneous cultivar of clrri in mekong delta were used in this experiment (table 1). the selected cultivars include three popular cultivars, three new cultivars and one heat tolerant cultivar. depending on the growth duration, the sowing date of each cultivar was adjusted to make the synchronization of anthesis in all cultivars. three hills (3 plants/hill) of each cultivar were sowed to pot (25 cm height x 25 cm diameter x 25 bottom diameter) filled with soil. rice plants were given additional fertilizer at the ratio n-p2o5k2o=106-4 (g/m2). twenty pots for each cultivar were placed in the net house until heading, i.e., when the first panicle was extruded out of its flag leaf sheat. high temperature treatment the experimental design was a split-plots with ten replications. the main plots and the subplots were two temperature regimes (high temperature and control) and 6 cultivars (table 1), respectively. this study was conducted in the net house; control (ct) and in the greenhouse; high temperature treatment (ht). in ht condition, heat stress was applied by using greenhouse effect to increase the temperature inside which opened about 25 cm on 3 sides for air ventilation. in ct condition, the temperature inside the net house was similar to the natural condition. the temperature treatment started at heading stage, half pots of each cultivar were transferred to ht while half of it remained in ct. the temperature treatments were applied from heading to harvest maturity. measurement of air temperature the air-temperature in net house and green house was measured with thermo recorders ‘ondotori’ (tr55i-pt, t and d, japan). the temperature sensors were installed in a force-ventilated radiation shield and placed at 2 m height above the ground. the diurnal air-temperature was recorded every 10-minutes from the heading stage to harvest maturity. table 1. details on the growth duration, sowing date and the heading date of seven cultivars in the net house of cuu long delta rice research institute, can tho, vietnam. no. rice cultivar growth duration (days) sowing date heading date note 1 “om5451” 99 20-nov-20 25-jan-21 popular cultivar of clrri in mekong delta 2 “om8923” 99 20-nov-20 25-jan-21 heat tolerant cultivar of clrri (thuy and shaitoh, 2017) 3 “om8” 99 20-nov-20 25-jan-21 new cultivar of clrri 4 “om426” 99 20-nov-20 25-jan-21 new cultivar of clrri 5 “om18” 99 20-nov-20 25-jan-21 new cultivar of clrri 6 “om4900” 103 15-nov-20 25-jan-21 popular long growth duration cultivar of clrri 7 “jasmine85” 103 15-nov-20 25-jan-21 popular extraneous cultivar of clrri in mekong delta journal of tropical crop science vol. 8 no. 3, october 2021 www.j-tropical-crops.com 156 tran loc thuy, tran ngoc thach, tran thi thanh xa, chau thanh nha, vo thi tra my, nguyen thi thao nguyen and nguyen thuy kieu tien yield and yield components at physiological maturity, 30 hills were sampled diagonally from each treatment to determine grain yield/ hill and yield components; i.e., the number of panicle per hill , 1000-grain weight (g), number of spikelet per panicle, and percentage of filled grain (%). the grain yield was adjusted to the standard moisture (14%). heat tolerance analysis the heat tolerant cultivars were classified base on the spikelet fertility (seed set) at the maturity stage according to irri (2002). heat tolerance scoring system in rice was defined base on the percentage of seed set (fertility) with the following scale; high tolerant (>80%), tolerant (61-80%), moderately tolerant (41-60%), susceptible (11-40%), highly susceptible (<11%). chalky percentage three replications (20-30g each) of milled grains of each treatment were used for the determination of chalky grains. grains covering white parts covering more than 10% of their total surface area, for example, a white belly, white center, or white back, were recorded chalky grains. amylose content three replications (0.1g) of rice starch of each treatment were prepared from rice seeds by diluted alkali method and gelatinized by treatment with 1 mol.l-1 naoh. amylose content was measured by a colorimetric method using an iodine-potassium iodide solution (rolando et al., 2004). statistical analysis data of yield, yield components and grain quality were analyzed as two-way completely randomized design. tukey’s hsd at a probability level of 0.5 and 1.0 % was used to compare the difference between treatment and genotype. sas version 9.2 (spss inc.) was used for data analysis. results all rice cultivars were subjected to high temperature environment after heading. the maximum temperature in heading time in ht and ct was higher and less than 35oc, respectively. the physiological effects of high temperature on sterility and grain yield when the temperature over 35oc had been documented (matsui et al., 2001). during the day, the temperature in green house (ht) was always around 4.0-8.5oc higher than net house (ct) (figure 1). the temperature of both in ht and ct were changed by the natural air temperature. the max-temperature was 35.8-40.0oc and 29.5-34.9oc in ht and ct, respectively. the minimum temperature was not much different between ht and ct. the seed set rate decreased in all tested cultivars under high temperature (table 2). the seed set rate of om cultivars ranged from 73.4% to 89.4% in the max-temperature in day around 30.5-31.4oc during the anthesis. when the max-temperature in day increased to 37.8-38.6oc the seed set of these cultivars ranged from 55.7-80.8%. most om cultivars were tolerant and moderate tolerant to high temperature. “jasmine85” was also the same as om cultivars in heat tolerant score. “om8923” kept the figure 1. the maximum and minimum temperature at high temperature environment and control condition after the rice heading stage.   figure 1. the maximum and minimum temperature in the high temperature and control environment after the rice heading stage. heat stress affects seed set and grain quality of vietnamese rice cultivars during .......... journal of tropical crop science vol. 8 no. 3, october 2021 www.j-tropical-crops.com 157 percentage of seed set in the range of high tolerant score under both conditions. the magnitude of genotypic effects on yield traits varied greatly, showing an association of stronger effects with increased daily-mean temperature (table 3). the grain yield of all cultivars decreased significantly in the range 13.5-46.3% under the high temperature condition. the highest reduction was in "om5451" with 46.3% yield loss by heat stress. the grain yield in “om8923” was the least affected by high temperature. the number of panicle per hill and the number of spikelet per panicle were not affected by the temperature regime. the significant decrease in filled grains and 1000-grain weight under high temperature condition lead to lower yield of all cultivars under high temperature condition. the percentage of grain chalkiness in most cultivars increased significantly under high temperature (figure 2a). the difference in the percentage of grain chalkiness between ct and ht varied widely among six cultivars, and ranged from 0.2-12.2% and 3.440.6% in ct and ht, respectively. the chalkiness in “om5451” and “om426” were strongly affected by high temperature during the grain filling stage. the chalky ratio of “jasmine85” on ht and ct was similar in 7.6 and 6.6%, respectively. high temperature during grain filling had a considerable influence on amylose content in hulled rice endosperm (figure 2b). there is a significant reduction of amylose content in ht compared to ct. amylose content at maturity of om cultivar was 19.5 and 20.4 % at ht and ct condition, respectively. “om8” was the most affected in amylose by heat stress with 10.9% lower in ht compared to ct. discussions the optimum temperature range for rice development fluctuates between 27-32oc (xinyou et al., 1996). the temperatures of >35oc negatively affect to flowering pattern and spikelet fertility (matsui et al., 2001). in this study, the maximum temperature after the heading in ht was remained higher than 35oc and lower than 35oc in ht and ct, respectively. the temperature in ht reached over 35oc from 09.00 to 16.30 that is the anthesis time of indica rice group. the temperature in ht was always higher than the critical temperature for rice development during flowering and grain filling stage. in ct, air temperature was in the optimum range to rice development. the flowering stage in most rice genotypes occur over a 5 days period, and the anthesis duration varies in different rice genotype (prasad et al., 2006). during the flowering stage, temperatures higher than the optimal temperature induce spikelet sterility and thus decrease the rice yield (nakagawa et al., 2003). the anthesis time in day is important due to the fact that spikelet sterility is induced by high temperature during or soon after anthesis (1~3 h in rice) (satake and yoshida, 1978). in this study, the fertility of om rice cultivars decreased when the temperature reached over 35oc. these results were consistent with the studies of yoshida et al. (1981) in that the temperature above 35°c at the flowering stage induces floret sterility. spikelet sterility was induced by high temperature particularly on the flowering day. during the flowering day, high temperature during anthesis time was the most detrimental to spikelet fertility, followed by high temperature right before anthesis. high temperature after anthesis had little influence on spikelet fertility as reported by satake and yoshida (1978) that spikelets exposed to high temperature one hour after figure 2. the quality of rice with different cultivars in high temperature and control condition. * and ** show significant difference based on anova at α=0.05 or α=0.01, respectively.   figure 2. the quality of different cultivars of rice in the high temperature and control condition. * and ** show significant differences based on anova at α=0.05 or α=0.01, respectively. journal of tropical crop science vol. 8 no. 3, october 2021 www.j-tropical-crops.com 158 tran loc thuy, tran ngoc thach, tran thi thanh xa, chau thanh nha, vo thi tra my, nguyen thi thao nguyen and nguyen thuy kieu tien table 2. the seed set rate and scale of heat tolerance of several rice cultivars under high temperature and control condition rice cultivar temperature condition seed set rate (%) scale of heat tolerance “om5451” ct 89.3 high tolerant ht 56.8 moderately tolerant “om8923” ct 89.4 high tolerant ht 80.2 high tolerant “om8” ct 84.8 high tolerant ht 61.5 tolerant “om426” ct 84.1 high tolerant ht 70.5 tolerant “om18” ct 73.4 tolerant ht 60.1 tolerant “om4900” ct 75 tolerant ht 55.7 moderately tolerant “jasmine85” ct 90.1 high tolerant ht 69.9 tolerant note: the maximum temperature during anthesis was 37.8-38.6oc in the high temperature environment and 30.5-31.4oc in the control condition. table 3. yield and yield components of seven rice cultivars in the high temperature and control condition. rice cultivar number of panicle/ hill number of spikelets/ panicle 1000-grain weight (g) filled grains (%) yield/hill (g) “om5451” ct 13 118 25.1 89.3 34.3 ht 12 109 24.8 56.8 18.4 ““om8”923” ct 12 138 25.5 89.4 37.8 ht 12 134 25.0 80.2 32.2 “om8” ct 16 89 26.5 84.8 32.0 ht 15 86 26.2 61.5 20.7 “om426” ct 13 89 24.9 84.1 24.3 ht 12 86 24.7 70.5 18.0 “om18” ct 11 165 26.4 73.4 35.2 ht 12 162 26.1 60.1 30.4 “om4900” ct 12 135 25.0 75 30.3 ht 12 127 25.0 55.7 21.2 “jasmine85” ct 11 106 25.4 90.1 26.7 ht 12 99 25.2 69.9 20.9 cultivar ** ns ** ** ** temperature ns ns ** ** ** cultivar*temperature ns ns ** ** ns note: * and ** show significant difference based on anova at α=0.05 or α=0.01, respectively; ns = not significant. heat stress affects seed set and grain quality of vietnamese rice cultivars during .......... journal of tropical crop science vol. 8 no. 3, october 2021 www.j-tropical-crops.com 159 anthesis had normal fertility. the major causes of high temperature-induced sterility were attributed to the disturbed pollen shedding and the decreased viability of pollen grains, resulting in decreased number of germinated pollen grains on a stigma (thuy et al., 2020). in this study, yield losses under high temperature were attributed to spikelet sterility and reduction of grain weight (table 3.) which was the same finding of prasad et al. (2006). flowering is the growth stage that is the most susceptible to high temperature in rice, and poor germination of pollen grains on stigma is partially responsible for the spikelet sterility (satake and yoshida, 1978). the decrease of grain weight is related to the formation of imperfect grains and the reduction in dry mass accumulation and translocation (tashiro and wardlaw, 1991). chalky, an opaque in the rice grain, is an important quality characteristic in rice and occurs most commonly when rice is exposed to high temperature during development (areum et al., 2009). the primary cause for chalky grain is an imbalance between sink and source abilities of carbohydrate metabolism, as a result of high temperature at the ripening stage (wang et al., 2006). endosperm chalkiness is both qualitative and quantitative characteristics of improper grain filling in determining grain appearance quality (peng et al., 2014). copper at el. (2008) showed that high ambient temperature from the anthesis (r4) to single grain maturity stage (r8) in reproductive development induced increases in chalky kernels. in this study, the mean daily temperature from r4 to r8 in ht was higher than that in ct with the increases of 2.1-4.8°c. thus, the percentage of chalky grains observed the same trend as the previous report. in the report of tabassum et al. (2021), the number of days with max-temperature above 34oc was more causative to chalky ratio than days with lower temperature. in this study, the max-temperature was always above 35oc corresponding to the increase of chalkiness percentage in all om cultivars (figure 2a). amylose content in all om cultivars was reduced under high temperature conditions (figure 2b.). our findings are in agreement with the previous findings that high temperature during grain filling affects the total starch content and reduced amylose content in endosperm starch (cheng et al., 2000). the previous study suggested that the change in amylose/amylopectin ratio under high temperature attributed to reducing in activity of granule-bound starch synthase and the role enzyme responsible for amylose biosynthesis (ahmed et al., 2015). conclusions this study suggested that the reduction of grain yield in om cultivars was due to the negative effects of high temperatures on filled grain (seed set) rate and grain weight. high temperatures at the anthesis time have a negative impact on the seed set ability of om cultivars. all om cultivars were in moderately to high tolerant range to high temperature. the most sterile-tolerant cultivar to heat stress was “om892”. consistent with the previous reports, the grain quality of om cultivars parameter during grain filling stage changed at high temperature. “om8923” is sterile-tolerant when subjected to high temperatures during the anthesis stage, therefore this cultivar could be used as a breeding material to develop heat tolerant cultivars for the future warmer circumstances. references ahmed, n., tetlow, i. j., nawaz, s., iqbal, a., mubin, m., rehman, m. s. m. u., butt, a., lightfoot d. a. and meakawa, m. (2015). effect of high temperature on grain filling period, yield, amylose content and activity of starch biosynthesis enzymes in endosperm of basmati rice. journal of the science of food and agriculture 95, 2237-2243. areum, c., song, j., kee, j. k. and ho j. l. (2009). quality of head and chalky rice and deterioration of eating quality by chalky grain. journal of crop science and biotechnology 12, 239-244. ceccarelli, s., grando, s., maatougui, m., michael, m., slash, m., haghparast, r., rahmanian, m., taheri, a., al-yassin, a., benbelkacem, a., labdi, m. and mimoun, n. m. (2010). plant breeding and climate change. journal of agricultural science 148, 627-637. cheng, f. m., ding, y. s. and zhu, b. y. (2000). the formation of amylose content in rice grain and its relation with field temperature. acta ecologica sinica 20, 646-652. copper, p. w., siebenmorgen, t. j. and counce, p. a. (2008). effects of night time temperature during kernel development on rice physicochemical properties. cereal chemistry 85, 276-282. djanaguiraman, m., narayanan, s., erdayani, e. and prasad, p. v. v. (2020). effect of high temperature stress during anthesis and grain filling periods on photosynthesis, lipids and journal of tropical crop science vol. 8 no. 3, october 2021 www.j-tropical-crops.com 160 tran loc thuy, tran ngoc thach, tran thi thanh xa, chau thanh nha, vo thi tra my, nguyen thi thao nguyen and nguyen thuy kieu tien grain weight in wheat. bmc plant biology 20, 268. fao. (2008). "food outlook global market analysis". http://www.fao.org/3/ai466e/ai466e05.htm. italy (june 1, 2021). haixia, l., zhen, x., meixia, h., zhenmei, w., hua, h., changxi, y. and hanlai, z. (2011). different effects of night versus day high temperature on rice quality and accumulation pfrofiling pf rice grain proteins during grain filling. plant cell report 30, 1641-1659. irri. (2002). “standard evaluation system for rice”. 54 pp. irri, philiphine. jagadish, s. v. k., craufund, p. q. and wheeler, t. r.( 2007). high temperature stress and spikelet fertility in rice (oryza sativa l.). journal of experimental botany 58, 1627-1635. mastui, t., omasa, k. and horie, t. (2001). comparison between anthers of two rice (oryza sativa l.) cultivars with tolerance to high temperature at flowering or susceptibility. plant production science 4, 36-40. nagakawa, h., horie, t. and matsui, t. (2003). “effects of climate change on rice production and adaptive technologies”. irri, manila, philippine. peng, b., wang, l., fan, c., jiang, g., luo, l., li, y. and he, y. (2014). comparative mapping of chalkiness components in rice using five populations across two environments. bmc genetics 15, 49. prasad, p. v. v., boote, k. j., aleen, j. l. h., sheehy, j. e. and thomas, j. m. g. (2006). species, ecotype and cultivar differences in spikelet fertility and harvest index of rice in response to high temperature stress. field crop research 95, 398-411. rolando, j. g., allberto, l. and brigitte, p. (2004). physico-chemical and cooking characteristics of some rice varieties. brazilian archives of biology and technology 47, 71-76. rosenzweig, c., party, m. l., fischer, g. and frohberg, k. (1993). "climate change and world food supply" research report no.3 environmental change unit. university of oxford, england. shah, f., nie, l., cui, k., shah, t., wu, w., chen, c., zhu, l., ali, f., fahad, s. and huang, j. (2014). rice grain yield and component response to near 2 oc of warming. field crop research 157, 98-110. sridevi, v. and chellamuthu, v. (2015). impact of the weather on rice. international journal of applied research 9, 825-831. tabassum, r., dosaka, t., morita, r., ichida, h., ding, y., abe, t. and tanaka, t. k. (2021). the conditional chalky grain mutant ‘flo 11-2’ of rice (oryza sativa l.) is sensitive to high temperature and useful for studies on chalkiness. plant production science 24, 230-243. tashiro, t. and wardlaw, i. f. (1991). the effect of high temperature on kernel dimensions and the type and occurrence of kernel damage in rice. australian journal of agricultural research 42, 485-496. thuy, t. l., lee, c. k., jeong, j. h., lee, h. s., yang, s. y., im, y. h. and hwang, w. h. (2020). impact of heat stress on pollen fertility rate at flowering stage in korean rice. korean journal of crop science 65, 22-29. thuy, t. l. and shaitoh, k. (2017). response of fourteen vietnamese rice (oryza sativa l.) cultivars to high temperature during grain filling period under the field condition. agronomy 7, 57. satake, t. and yoshida, s. 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"high temperature stress on rice, a review". research paper series 67. irri. 18 suwarto journal of tropical crop science vol. 5 no. 1, february 2018 www.j-tropical-crops.com reduction of urea fertilizer uses through application of livestock manures in an integrated farming of maize and cattle abstract crops and livestock have historically been grown together in an integrated farming; this system facilitates sharing of nutrient and land resources, and benefiting both operations. the current study examined integrated farming of maize and cattle in waleran village, east java, indonesia. in maize cultivation, the farmers apply 10 tonnes of the manure, 300 kg urea (46% n) and 300 kg npk phoska (n:p2o5:k2o = 15%:15%:15%) per hectare. however, more nitrogen had actually been applied when manure is added, increasing the amount of nitrogen by 180 kg n.ha-1 which consequently a waste. therefore, more efficient application of nitrogen in maize production needs to be studied. a field experiment was conducted from september 2017 to february 2018 using maize “pioneer 32”. the study was organised in a completely randomized block design consists of 10 m x 10 m plots and four replications. the crops were fertilized with nitrogen at 46, 91, 137 and 183 kg.ha-1. two seeds of maize were planted per hole with planting distance of 70 cm between rows and 40 cm within row. plant growth were measured weekly at four to eight weeks after planting; yield variables were measured at harvest. the increasing nitrogen levels did not significantly influence the maize growth or yield. the manure contributed about 114 kg.ha-1 nitrogen supply. the lowest total nitrogen fertilizer applied (159 kg.n ha-1) resulted in the optimum leaf area index (4.65) and leaf nutrient content of n (2.70%) and p (0.26%) which is above to the critical leaf nutrient content with maize grain yield of 6.19 ton ha-1. the manure in integrated farming could reduce the use of urea due nitrogen contribution from the livestock manures. keywords: maize growth, nitrogen levels, leaf area index, leaf nutrient content introduction maize is the second important of food crop in indonesia after rice; the main uses are for food and feed. the highest maize production area in indonesia in 2015 was east java (bps, 2015), with the largest area of maize production is on dry land in the tuban regency of the east java province. almost all farmers in tuban regency, especially in grabagan village integrated relay planting of maize/chilli/groundnut with cattle. chilli was planted at 53 days after maize; one row chilli at every three rows of maize. at 73 days after planting, the maize leaves under cob was pruned to feed the cattle, then groundnut was planted among the maize and chilli. at 97 days afer planting the maize stems section above the cob was cut to feed the cattle, either fresh or dried. maize planting season was six month duration started at november to april each year. based on interviews with 23 farmers conducted by the author, every fam has two cattle animals, and at least 10 tonnes of farm manure can be produced from two cattle a year (unpublished report). cattle manure is an important source of nitrogen (sharifi et al., 2011). manure from these two cattle is sufficient to supply one hectare of crops during the planting seasons throughout the year. the amount of nitrogen applied to the crops is an important factor that affects maize yield (okumura et al., 2011). the farmers in grabagan, tuban, use livestock manure as organic fertilizers for maize production, whereas the maize wastes are used for livestock feed. manure differs from most commercial fertilizers in that it consists of a mix of organic nitrogen compounds that require conversion to inorganic nitrogen by microorganisms before they are available to crops. one of the challenges of manure management is to estimate the rate of nitrogen release from manure organic material and the fraction of organic nitrogen that is available to crops. the average dosage of the manure applied in the study location was 10 ton.ha-1. hartatik and suwarto* department of agronomy and horticulture, faculty of agriculture, bogor agricultural university, jl. meranti, kampus ipb darmaga, bogor 16680, indonesia. *corresponding author: wrtskm@yahoo.com 19reduction of urea fertilizer uses though application of livestock manures in .......... journal of tropical crop science vol. 5 no. 1, february 2018 www.j-tropical-crops.com widowati (2010) estimated that the nitrogen available for use by maize in manure varied by 0.4% 2.04%. based on these figures (hartatik and widowati, 2010) 10 ton.ha-1 manure is estimated to contain 40 to 204 kg nitrogen. in order to reach the best maize growth and yield, farmers fertilized the maize with urea and npk phonska. the farmers have a perception that ‘more fertilizers will give more growth and yield of maize’. due to this perception, the average dosage of urea and npk phonska applied by the farmers is relatively high, i.e. 300 kg of urea and 300 kg of npk phonska per hectare, which is equal to 183 kg n, 45 kg p2o5, and 45 kg k2o per hectare. low soil nitrogen is often the limiting factor in increasing maize production in indonesia. however, farmers in tuban apply more urea than a crop needs, resulting in inefficient use of nitrogen. okumura et al (2011) stated that nitrogen fertilization is required for maize, but then stated that n is to be applied when there is a major requirement for n because nitrogen is readily available compared to other nutrients such as p and k. however, application of excessive amount of nitrogen is not economical, could have negative consequences to plant metabolism, and the excess nitrogen can potentially cause problems to the environment. therefore, it is important to understand the optimum time of nitrogen application, and level at which to apply nitrogen for maximum availability to the crop. nitrogen is highly volatile and inappropriate application to field crops not only contributes towards pollution but also increases the cost of production (akmal et al., 2010). the benefits of using manure as a fertilizer requires proper assessment of the available nutrients in manure, calculating the appropriate rate to provide the needed nutrients to the crop and applying the manure uniformly in the field. this study evaluated the practices of maize production in a central production of maize in grabagan, east java, and determined the efficient dosage of fertilizers, especially from urea, for maize production. the contribution of manure as a source of nitrogen was examined to develop a production system with low external input for a sustainable agriculture, which is the main concept of an integrated farming system. material and methods an experiment was conducted on the maize production area of village of waleran, district of grabagan, tuban regency, east java, indonesia, located at 112o00’ east longitude and 7o00 south latitude during september 2017 to february 2018. the soil has a relatively neutral ph of 6.07. the experiment used a completely randomized block design with a single factor of nitrogen (n) fertilizer dosages. four levels of n fertilizer dosages were tested, i.e. 45, 91, 137, and 183 kg n.ha-1. each treatment was repeated four times. the source of n fertilizers were a combination of urea (45%n) and npk phonska (15%n 15%p2o5 15% k2o). urea was applied at 0, 100, 200, and 300 kg.ha-1 , and npk phonska at 300 kg.ha-1. the other source of n was the manure applied during land preparation at a rate of 10 ton.ha-1. at the end chilli harvest, or when the dry seasons started, the land was prepared by cleaning from weeds followed by a minimum tillage, digging hole, and manuring for next maize planting seasons. every planting hole was applied with an average of 280 g of manure, or 10 tonnes per hectare. one-third of npk phonska was applied at 15 days after planting, then at 25 and 40 days after planting, respectively 1/3 of npk phonska was mixed with ½ dosage of urea were applied. the fertilizer was inserted into a hole at about 7 to10 cm distance from the maize plant and covered with soil. n, p2o5, k2o levels in the manure and in the soil were analysed at plant and soil analysis laboratory of department agonomy and horticulture, bogor agricultural university. the n, p2o5, k2o of the soil in the study location was also analyzed before planting. the size of the experimental plots was 10 m x 10 m. seeds of the maize hybrid “pioneer 32” were planted spaced at 70 cm between rows and 40 cm within rows; two seeds were planted in one planting hole. the npk phoska fertilizer was applied at 15, 25, and 40 days after planting, whereas urea was applied at 25 and 40 days after planting. measured growth variables included plant height, stem diameter, and leaf number which were observed weekly at four to eight weeks after planting (wap). at the tasseling phase of maize (8 wap) measurements of the ninth leaf length (l) and width (w) were taken to determine leaf area and leaf area index. the area of one maize leaf (cm2) was calculated by the formula = l * w * 0.98 (sitompul and guritno, 1995), then multiplied with the number of leaves per plant to get a total leaf area per plant. leaf area index was calculated as the projected area of leaves over a unit of land (m2) (waring and runnin, 2007). the nutrient content of n, p, and k of the ninth leaf was also taken evaluate the nutrient status in the plant. yield variables measured at harvest time included weight of maize cob, weight of seeds per plant and weight of 100 seeds at 22% moisture content. the weight of seeds per plant was then converted to the moisture contents of 13% following the standard of 20 suwarto journal of tropical crop science vol. 5 no. 1, february 2018 www.j-tropical-crops.com moisture content in maize grain trading. the converted value was then used to estimate maize yield per hectare by multifliying with the plant population and correction factor of 0.7. the correction factor was applied because the effective land was estimated to be about 70% due to the rocky condition of the soil in the study site. data were analysed using anova and significant differences between treatments were further analysed with least significant differences (lsd) to determine the effect of the different n fertilizer dosages on the growth variables. results maize growth plant height (table 1), stem diameter (table 2), and number of leaf (table 3) of maize increased during 4 to 8 wap. the dosage of nitrogen levels of 45, 91, 137, and 183 kg n.ha-1 did not significantly affected the growth variables. leaf area index at 8 wap was also not significantly influenced by the n fertilizer dosage (table 4). the weekly increment of plant height (table 1) was normal for all levels of nitrogen applied; the plant height range at 8 wap was 230 to 243 cm. yulisma (2011) reported that at 4, 6 and 8 wap plant height of “pioneer 21” at nangroe aceh darussalam was 88, 179 and 230 cm respectively. surbakti et al. (2013) stated that the maize plant height of “pioneer 12” at 6 wap at deli serdang, north sumatra was in the range between 188 to 190 cm. stem diameter of maize in all the nitrogen fertilizer dosages were in the range of 17.3 to 18.4 mm (table 2) were also insignificant. bora and murdolelono (2006) reported that stem diameter of maize cultivated on dryland at belu, nusa tenggara timur, was in the range of 14.3 to 16.0 mm. the maize stem diameter was relatively stable starting at 6 wap. the number of leaf per plant is a genetic characteristic of the maize variety; this might be one of the reasons that the nitrogen fertilizers levels did not affect the number of leaf per plant. the number of leaf per plant in this study was 15.2 (table 3). depending on the maize variety, the number of leaves can range from 8 to 18; for example efendi and suwardi (2010) reported the number of leaves on maize cultivar “bisi 2” and “pioneer 21” were 13.5 and 12.8, respectively. all dosages of n fertilizer resulted in a similar leaf area index (lai) which was in the optimal range averaging 4.65 (table 4). the lai value was calculated from the average of leaf area per plant. the average leaf area within nitrogen dosage treatments of 45, 91, 137 and 183 kg.ha-1 were 6,970, 7,640, 7,242 and 8,174 cm2, respectively. the leaf area values of the maize table 1. maize plant height at different dosages of n fertilizers n dosage (kg.ha-1) source of n plant height (cm) urea (kg.ha-1) npk phonska (kg.ha-1) 4 wap 5 wap 6 wap 7 wap 8 wap 45 0 300 85.0 129.9 161.2 199.1 230.2 91 100 300 86.9 133.7 164.7 204.9 240.7 137 200 300 87.2 131.7 162.1 200.7 234,1 183 300 300 87.5 135.6 167.3 208.7 243.2 average 86.7 132.7 163.8 203.4 237.1 table 2. maize stem diameter at different dosages of of n fertilizers n dosage (kg.ha-1) source of n stem diameter (mm) urea (kg.ha-1) npk phonska (kg.ha-1) 4 wap 5 wap 6 wap 7 wap 8 wap 45 0 300 15.8 18.0 18.7 18.3 18.4 91 100 300 15.9 18.0 18.7 18.1 18.0 137 200 300 15.7 17.9 19.0 18.2 18.4 183 300 300 15.5 17.1 18.1 17.8 17.3 average 15.7 17.8 18.6 18.1 18.0 21reduction of urea fertilizer uses though application of livestock manures in .......... journal of tropical crop science vol. 5 no. 1, february 2018 www.j-tropical-crops.com crops in this study were higher than those reported by yulisma (2011) in reuleut aceh utara of 5,996 cm2. in another study (efendi and suwardi, 2010) the maximum maize lai was with the nitrogen dosage treatment of 150 kg.ha-1 which was 4.2 for cultivar “pioneer 22” and 4.0 for cultivar “bisma 2”. maize yield yield components of maize showed no significant responses to the nitrogen fertilizer treatments. the number of seeds per cob, seed weight per plant, and estimated grain yield slightly increased with the increase of nitrogen level of 137 kg.ha-1 and then decreased (table 5). the trend of maize yield with increasing dosages of nitrogen was similar to those of stem biomass under the cob (table 4). the maize grain yield in the range of 6.20 to 6.89 ton.ha-1 were categorized as normal according to the indonesian ministry of agriculture (2018). the maize yield in this study was higher than those reported by yusalima (2011) at nangroe aceh darussalam (5.25 ton.ha-1). the potential yield of hybrid maize according to the indonesian ministry of agriculture (2018) ranged 6 to 8 ton.ha-1, which is higher than a traditional maize variety with the range of 3 to 4 ton.ha-1. therefore the maize grain yield of “pioneer 32” at the all levels nirogen fertilizer in this study was normal. discussion at the optimal lai (4.32 – 5.07), photosyntesis should be maximal and result in sufficient energy for the plant to have normal growth of stems and leaves. in this study an optimal lai has been achieved, therefore, all maize “pioneer 32” growth variables including plant height, stem diameter, and number of leaf were in the normal range. this means that all n dosages of urea applied was sufficient to support to normal maize growth. the critical content of nitrogen and phosporus of the maize leaf at tasseling phase are 1.40% and 0.16%, respectively. the levels of nitrogen and phosphorus in the leaf of all n dosages (table 6) were higher than the critical. however, the potassium content of leaf (1.45 to 1.58%) was still lower than the critical content (2.00%). it means that soil nitrogen and phosphorus were likely to be sufficient for corn growth, whereas the soil potassium was not. potassium is an essential nutrient and is also the most abundant cation in plants (gul et al., 2015); it plays essential roles in enzyme activation, protein synthesis, photosynthesis, osmoregulation, stomatal movement, energy transfer, phloem transport, cation-anion balance, and stress resistance. the leaf potassium content lower than critical point in this study was probably related to water availability. sometime, at the planting seasons maize leaves showed water stress symptoms. wang et al. (2013) reported during drought stress, root growth and the rates of k+ diffusion in the soil towards the roots table 3. maize leaf number per plant at different dosages of n fertilizers n dosage (kg.ha-1) source of n number of leaf per plant urea (kg.ha-1) npk phonska (kg.ha-1) 4 wap 5 wap 6 wap 7 wap 8 wap 45 0 300 8.3 9.7 11.3 13.1 15.2 91 100 300 8.5 9.9 11.4 13.1 15.2 137 200 300 8.1 9.9 11.4 13.0 15.0 183 300 300 8.1 9.6 11.2 13.0 15.2 average 8.3 9.8 11.3 13.1 15.2 table 4. maize leaf area index and total biomass at different dosages of n fertilizers n dosage (kg.ha-1) source of n leaf area index (8 wap) stem biomass under cob at harvest (g per plant)urea (kg.ha-1) npk phonska (kg.ha-1) 45 0 300 4.32 64.18 91 100 300 4.74 67.48 137 200 300 4.49 67.80 183 300 300 5.07 63.98 average 4.66 65.86 22 suwarto journal of tropical crop science vol. 5 no. 1, february 2018 www.j-tropical-crops.com were both restricted, thus limiting k acquisition. the resulting lower k concentration can further reduce the plant resistance to drought stress, as well as k absorption. the leaf nitrogen content was not significantly different with any of the n treatments. in this case, the manure applied at 10 ton.ha-1 was important in contributing nutrient supply for the crops. laboratory analysis demonstrated that the cattle manure contains 1.14% of nitrogen, 2.30% of phosphorus, and 1.18% of potassium. based on this analysis results, the maize crop had been supplied with 114 kg nitrogen, 230 kg phosphorus, and 118 kg potassium per hectare from the 10 tonnes of manure applied. total amount of the nitrogen levels applied to the maize land that originated from the urea and the manure were 159, 204, 249 and 294 kg.ha-1. the total levels of phosphorus and potassium were 275 and 163 kg.ha-1, respectively. based on the leaf nutrient content (table 5) and leaf area index (table 4), the amount of nitrogen applied at the lowest level was already sufficient for supporting maize growth. this was indicated by all of the growth variables observed and the fact they were not significantly different among the treatments. the main stem under cob after cob harvested and the yield of maize at harvest slightly increased at the nitrogen fertilizer dosage of 135 kg.ha-1. it is probably correlated with the potassium nutrient content of the leaf (table 6). potassium (k) is an essential nutrient that affects most of the biochemical and physiological processes that influence plant growth and metabolism (wang et al., 2013). out of all the mineral nutrients, potassium (k) plays a particularly critical role in plant growth and metabolism. many studies suggest that k has no effect on stomatal control and photosynthetic rates under well-watered conditions, but k starvation could favor stomatal opening and promote transpiration (benlloch-gonzalez et al., 2008). okumura et al. (2011) stated that increase in nitrogen level applied normally promotes higher maize yield. ifia (2002) reported that nitrogen amount used in maize production in china is 130 kg.ha-1; in usa 150 kg ha-1. in brazil, increasing nitrogen level of 0, 40, 80, 120, 160, and 200 kg.ha-1 presented quadratic response during vegetative and reproductive stages, and grain protein showed an increase until n level of 160 kg ha-1 (okumura et al., 2011). nitrogen in excessive amounts can have negative consequences in plant metabolism and produce problems in agronomical, economical and environmental scales (okumura et al, 2011). in a study by donner and kucharik (2003), when the application rate of nitrogen fertilizer was increased by 30%, the corn yield increased 4%, but the amount of nitrate lost through leaching increased by 53%. furthermore, the yield decreased by 10% when the application rate of nitrogen fertilizer was reduced table 5. yield components of maize at different dosages of n fertilizers n dosage (kg.ha-1) source of n number of seeds per cob seed weight at harvest at 22% moisture content (g per plant) seed weight at 13% moisture content (g per plant) estimated grain yield (ton per ha) urea (kg.ha-1) npk phonska (kg.ha-1) 45 0 300 563 141.1 126.5 6.20 91 100 300 587 148.0 132.7 6.50 137 200 300 615 156.8 140.6 6.89 183 300 300 614 145.9 130.8 6.41 average 147.9 132.6 6.50 table 6. nitrogen, phosphorus and potassium nutrient content of the nineth leaf at maize tasseling phase n dosage (kg.ha-1) source of n nitrogen (%) phosphorus (%) potassium (%)urea (kg.ha-1) npk phonska (kg.ha-1) 45 0 300 2.70 0.26 1.45 91 100 300 2.82 0.29 1.50 137 200 300 2.92 0.30 1.58 183 300 300 2.95 0.31 1.45 average 2.85 0.29 1.45 23reduction of urea fertilizer uses though application of livestock manures in .......... journal of tropical crop science vol. 5 no. 1, february 2018 www.j-tropical-crops.com by 30%, the leaching loss was 37% less. shariffi et al. (2011) reported that applying manure at at an equivalent to 150 kg n.ha−1per year increased the yield of phleum pratense l. champ; however, the excess nitrogen that accumulates in the soil if a double the amount of manure was applied might result in a decrease in yield. research by akmal et al. (2010) demonstrated that maize grain yield increases in quadratic model with increased n application to a plateau level. considering soil fertility status and cropping systems, the 150 kg.ha-1. n application to maize cultivar “jalal” in peshawar was required for maximum grain production (akmal et al., 2010). referring to the study of okumura et al. (2011), donner and kucharik (2003), shariffi et al. (2011) and akmal et al. (2010), the nitrogen level of 159 kg.ha-1 would be optimum for maize production at grabagan. therefore, the combination of manure at 10 ton.ha-1 and npk phonska 300 kg.ha-1 without urea, which had already supplied nitrogen of 159 kg.ha-1 to the maize, could be recommended. the use of urea as external fertilizer input could be reduced by application of livestock waste in this integrated farming system. conclusion the nitrogen fertilizer level of 45, 91, 137, and 183 kg n ha-1 combined with the manure at 10 ton.ha-1 are equivalent to supplying 159, 205, 251 and 297 kg.ha-1 of nitrogen to maize crops. the nitrogen levels did not significantly influence the maize growth and yield. application of manure of livestock waste in an integrated farming of maize and cattle can reduce the use of urea fertilizers as an external input of nitrogen in grabagan village. with regular application of the manure with nutrient content as analysed in this study at every planting season applying npk phonska at 300 kg.ha-1 should be sufficient to support maize growth to achieve maize grain yield of 6.20 ton per hectare. acknowledgement author thanked adm ltd. and yayasan gizi yasmin who funded the research through the program of increasing productivity, value add and increasing revenue of the corn farmers through an integration system with cattle farms in grabagan, tuban, east java, indonesia. references akmal m., rehman, h.u., farhatullah, asim, m., and akbar, h. (2010). response of maize varieties to nitrogen application for leaf area profile, crop growth, yield and yield components. pakistan journal of botany 42, 1941-1947 benlloch-gonzalez m., arquero o., fournier j.m., barranco d., and benlloch m. (2008). k+ starvation inhibits water-stress-induced stomatal closure. journal of plant physiology 165, 623–630. bora c.y. and murdolelono, b. (2006). influence of fertilizing to ahuklean corn cultivation in besikama, belu, nusa tenggara timur. berita biologi 8, 53 -59. bps [biro pusat statistik]. (2015). produksi jagung menurut provinsi (ton), 1993-2015. https://bps. go.id/linktabledinamis/view/id/868. [january 12, 2018] donner s.d. and kucharik j.a. (2003). evaluating the impacts of land management and climate variability on crop production and nitrate export across the upper mississippi basin. global biogeochemical cycle 17. doi: 10.1029/ 2001gb001808. efendi r. and suwardi. (2010). respon tanaman jagung hibrida terhadap tingkat takaran pemberian nitrogen dan kepadatan populasi in “prosiding pekan serealia nasional 2010” pp 230-240. http://balitsereal.litbang. pertanian.go.id/ wp-content/uploads/2016/12/ pros2010new400.pdf [january 12, 2018] gul s., m. khan, h., khanday, b. a., and nabi, s. (2015). effect of sowing methods and npk levels on growth and yield of rainfed maize (zea mays l.). scientifica doi: 10.1155/2015/198575 hartatik, w. and widowati l.r. (2010). “pupuk organik dan pupuk hayati”. balai besar litbang sumberdaya pertanian. bogor. international fertilizer industry association (ifia). 2002. “fertilizer use by crop”. http://www. fertilizer.org [january 12, 2018]. [kementerian pertanian]. (2018). mengenal varietas jagung p32 singa si tongkol tangguh. http:// cybex.pertanian.go.id/materipenyuluhan/ detail/ 11110/ mengenal-varietas-jagung-p32singa-si-tongkol-tangguh. [january 12, 2018]. 24 suwarto journal of tropical crop science vol. 5 no. 1, february 2018 www.j-tropical-crops.com okumura r.s, takahashi, h. w., costa dos santos, d. g., da silva lobato a. k., de cinque mariano, d., marques, o. j., da silva, m.h., de oliveira neto, l. c. f., and de lima junior, j.a. (2011). influence of different nitrogen levels on growth and production parameters in maize plants. journal of food, agriculture and environment 9, 510-514. sharifi m., zebarth b.j., burton d.l., rodd v., and grant c.a. (2011). long-term effects of semi solid beef manure application to forage grass on soil mineralizable nitrogen. soil science society of america journal 75, 649–658. doi: 10.2136/sssaj2010.0089. sitompul s.m. and guritno, b. (1995). “analisis pertumbuhan tanaman”. universitas gadjah mada press. yogyakarta. surbakti m.f., ginting, s., ginting, j. (2013). pertumbuhan dan produksi jagung (zea mays l.) varietas pioneer-12 dengan pemangkasan daun dan pemberian pupuk npkmg. jurnal online agroekoteknologi 1, 523 – 534. wang m., zheng, q., shen, s. and guo, s. (2013). the critical role of potassium in plant stress response. international journal of molecular sciences 14, 7370 – 7390. waring r. and running, s. (2007). water cycle in “forest ecosytem” (r. waring r and running,s., eds), pp 19 -57. 3rd edition. academic press. https://doi.org/10.1016/b978012370605-8.50007-4. [january 12, 2018]. yulisma ( 2011). pertumbuhan dan hasil beberapa varietas jagung pada berbagai jarak tanam. penelitian pertanian tanaman pangan 30, 196 86 juang gema kartika, shinta wulandari karyana journal of tropical crop science vol. 4 no. 3, october 2017 www.j-tropical-crops.com the use of trellis and mulch increased fruit production of spaghetti squash (cucurbita pepo l.) abstract spaghetti squash is squash of american origin that has spaghetti-like characteristic of fruit flesh, unlike any other squash varieties. this study aims to investigate the use of trellis and mulch to increase the spaghetti squash production. the experiment was conducted at the experimental station of centre of tropical horticulture, bogor from april to november 2016. the experiment used a completely randomized block design with four replicates. two factors were tested, i.e. the use of trellis and mulch as the main plots, and accessions of the spaghetti squash as a sub-plot. the treatments were the use of trellis and mulch, mulch without the trellis, the trellis without mulch, and without the trellis and mulch. the spaghetti squash accessions used in this experiment were accessions from taiwan (f1) and from america. each experimental unit consists of five plants with a total a total of 160 plants. the results of this experiment showed that the growing squash using mulch and trellis had the greatest number of fruits per plant, more marketable fruits and had the largest fruit weight as compared with the other treatments. taiwan accession had more fruits per plant and total marketable fruits, the largest fruit weight, the longest and widest fruits with large fruit diameter. taiwan accession produced fruits at lower nodes of the plants, and fruits can be harvested earlier than the american accession. correlation analysis indicated that the more fruits per plant, the more number of fruits that are marketable, the greater fruit weight and more fruits can be harvested from the higher nodes of the plants. from agronomical point of view, taiwan spaghetti squash would be preferable to obtain the high fruit production provided the crops were mulched and grown with trellis. keywords: cucurbitacea, trellis, mulch, plant culture introduction spaghetti squash, cucurbita pepo l, belongs to the cucurbitaceae family and has been widely cultivated in various parts of the world such as america, japan and taiwan with a variety of cultivation techniques, from traditional to hydroponics (npgs, 2006). cucurbitacea consists of around 800 species including cucumbers, melons, watermelons, and pumpkins. twenty genera are used for culinary purposes, which usually includes consumption of the mature fruit flesh, whole immature fruits, and/or seeds. three genera, cucumis (cucumbers, melons), cucurbita (pumpkins, squash), and citrullus (watermelons), rank among the top ten in economic importance among the vegetable crops of the world (schaffer and paris, 2003). cucurbitaceae are herbaceous, tendrilbearing vines that are adapted to warm climates; the leaves are large palmate shape, roots are fibrous with large prominent fruits. under favorable conditions, the plants grow and spread quickly, as stems can elongate 30 cm or more within 24 h (schaffer and paris, 2003). spaghetti squash has a unique characteristic which differentiates from the other cucurbits; the fruit flesh splits apart like spaghetti when boiled, steamed, or roasted. this happens due to the breakdowns of pectic compounds that are present in the fruit flesh when cooked. as the pectic chains break up the pumpkin flesh started to look like spaghetti (ishii, 2015). pectic or pectin is one of the polysaccharide inside plant cell walls that function in complex physiological processes such as growth and differentiation of cells, as well as the development and stiffness of plant tissues (voragen et al., 2009). pumpkins and squash rank high overall in nutritional value, most familiarly in the carotenoid content of the fruit flesh (schaffer and paris, 2003). the spaghetti squash has essential vitamins and minerals required for human body metabolism such as vitamins a, c, and e as well as minerals sodium and potassium. the pulps of the squash can potentially substitute juang gema kartika*, shinta wulandari karyana department of agronomy and horticulture, faculty of agriculture, bogor agricultural university. jl. meranti, darmaga campus, bogor 16680, indonesia. *corresponding author; email: ika_juang@yahoo.com 87the use of trellis and mulch increased fruit production of spaghetti .......... journal of tropical crop science vol. 4 no. 3, october 2017 www.j-tropical-crops.com spaghetti made from wheat, in addition it contains low fat. one serving of spaghetti squash contains 42 calories while one serving of wheat pasta contains 221 calories (self nutrition data, 2014). the uniqueness and benefits of spaghetti squash can add variety of highly nutritious horticultural produces in indonesia. spaghetti squash is not a native to indonesia and not widely known by indonesian growers. very limited information is available on squash culture. therefore in order to introduce squash as a new horticultural crop a standard of production guidelines should be prepared to the crop can grow and produce fruits in the tropics.the current study was conducted to examine whether or not the use of trellis and mulch can increase fruit production of two accessions of spaghetti squash. the use of trellis for cucurbitaceae have advantages, including keeping the plant leaves dry and avoid fungal diseases, the fruits are easier to harvest, cleaner and uniform in colour compared to when fruits rested on the ground. in addition mulching keep soil humidity and inhibit weed growth (hinds et al., 2015). the use of trellis improved the yield and quality of cucumber; improved photosynthetic efficiency has also been correlated to the increased yield (solangi et al., 2009; hanna. et al., 1987). the use of organic mulch provides valuable nutrients as the mulch breaks down, and improves soil physical properties (hinds et al., 2015). materials and methods this experiment was conducted at the tropical horticulture experimental research center, tajur, bogor, from june to november 2016. this experiment used split plot design with trellis and mulch as the main plot, and squash accessions as sub plot. the main plots consist of the use trellis and mulch, trellis without mulch, without trellis with mulch, and without trellis and without mulch. plastic mulch was used, and two accessions, from taiwan (f1) and from american, were tested in the study. each experimental unit consists of five plants with a total of 160 plants. squash seeds were sown until the seedlings had grown at least two true leaves prior to planting prior to transplanting into raised beds with plant spacing of 1.2 m between rows and 1.4 m within rows. the type of trelliss used was para-para or bamboo stakes (7 x 1.2 x 1.7 m). bamboo trellis was used to support the growth of the climbing plants, by tying the branches to the trellis using ropes to direct the plant growth upwards. npk fertilizer at 10 g.l-1 was applied weekly. fruits were harvested when ripened and skin has hardened to avoid cut and bruishes during handling. ripened fruits are uniformly yellowish in colour. according to squash harvesting guideline by the usa department of agriculture, forestry and fisheries (2011) squash fruits can be harvested at ±100 days after planting (dap), or 30-40 days after the pollination of the female flowers. scoring was conducted on the total number of fruits per plant, marketable and non-marketable fruits per plant, the smallest and largest fruit weight (g), fruit length and diameter, skin thickness and fruit thickness (cm) using a digital caliper, lowest and highest harvesting nodes, total soluble solids (tss) using hand refractometer, and time to harvest (weeks after planting, wap). results and discussion fruits had to be harvested early at 6 weeks after planting (wap) as most of the plants were infected by fruit rot disease erwinia. the infection had caused the plants to set flower early, i.e. at 3 wap. squash usually flower at 8 wap, or 30 to 40 days after pollination, and fruits can be harvested at 13 wap or about 100 days (department of agriculture, forestry and fisheries, 2011). the erwinia disease might have spread from the cabbage crops grown adjacent to the squash block. according to maynard (2007), cucurbita fruits take about two to three weeks to reach its maximum size and take more than three weeks to reach maturity before the fruits are ready for harvest. earlier harvest made the fruits had not physiologically matured and not as sweet as the mature fruits, although the fruits have reached their maximum size. in addition, the fruit pulps do not completely break when cooked. fruit skin thickness, fruit tss content and fruit weight were significantly affected by squash accessions and growing methods (table 1). total fruit production and marketable fruits table 2 shows that the number of fruit per plant and marketable fruits were significantly different between accessions. taiwan accession produced about 30% more fruits per plant than the american accession (table 2). differences in the number of fruits harvested might have been caused by the types of seed used in this study. the seeds of taiwan accession are f1 produced by known you seed, whereas the seeds of the american accession come from independent seed producer in america. there was a significant difference in the fruit production and marketable fruits per plant with the use of mulch and strellis (table 2). corn and peanut grown using mulch as ground cover reduced nutrient 88 juang gema kartika, shinta wulandari karyana journal of tropical crop science vol. 4 no. 3, october 2017 www.j-tropical-crops.com erosion by up to 75% compared with those grown without mulch (wahjunie and listyarini, 1998). shetty et al. (1998) showed that cucumber plants (cucumis sativus) grown using trellis resulted in better quality and quantity of fruits than those grown without trelliss. in this study, crops that were grown without trellis produced fewer numbers of marketable fruits compared with plants that were grown with trelliss. agu (2004) reported that growing squash (cucurbita maxima) using trellis resulted in more fruits than that without trellis. this is in line with our results showing a higher number of fruits when plants were grown with both mulch and trellis. the plants grown with mulch and trellis had more total and marketable fruits compared to those grown with mulch only. the use of mulch can prevent soil and nutrient erosion, whereas the use of trellis potentially increases the rate of photosynthesis as the trained plants had more exposure to sunlight. chukwudi and agbo (2014) reported in their studies that pumpkin bells with 90 cm trellis height had the best vegetative growth, flowering and crop productivity compared to other treatments in the study. the use of mulch, however, can not avoid some fruits to rot even though the fruits had not been in contact directly with the soil. the fruits got rotten when they grow on the ground and exposed to the water puddles retained by the mulch. fruits that were off the ground can also rot during wet and heavy rain, which had occurred during this research. fruit weight per plant the american spaghetti squash produced smaller fruit size than the taiwanese accession. beany et al. (2002) reported that the weight of spaghetti squash of american accession “hasta la pasta and vegetable” ranged from 1.1-1.4 kg per fruit. the weight of spaghetti squash fruit from the america in this study ranged from 621.9-1021.3 g per fruit, and the smallest spaghetti squashfruit was only a half of the weight of the spaghetti squash reported in beany et al. (2002). the largest squash fruit weight of the taiwan accessions was 360 grams greater than the weight of the spaghetti squash reported in beany et al. (2002) study, but smaller than the standard squash weight of 2.5 kg per fruit (known you seed, 2016). table 3 shows the crop growing methods significantly influenced all variables of fruit weight. the use of mulch and trellis resulted in the highest fruit weight compared to other growing methods, and this might be related to less nutrient erosion due to mulch treatment (wahjunie and listyarini, 1998). the use of trellis without mulch resulted in smaller fruit weight compared to without trellis and without mulch, possibly because without trellis and without mulch the plants grow horizontally on the ground and each node can produce roots and allowing the plants to absorb more nutrients than the crops that grow vertically with trellis. table 1. recapitulation of the quantitative characteristics of the spaghetti squash fruits variable pr>f cv (%) accession use of trellis and mulch interaction number of fruits 0.00** 0.04* 0.97ns 26.45a) number of rotten fruit 0.19 ns 0.71ns 0.93ns 25.46b) σ marketable fruits 0.02* 0.04* 0.99ns 23.42b) smallest fruit weight 0.00** 0.47ns 0.00** 15.46 largest fruit weight 0.00** 0.01* 0.62ns 09.09 fruit length 0.00** 0.11ns 0.71ns 08.88 fruit diameter 0.00** 0.23ns 0.66ns 05.83 fruit flesh thickness 0.33 ns 0.73ns 0.58ns 12.96 skin thickness 0.09 ns 0.61ns 0.05* 11.26 low node harvest 0.00** 0.76ns 0.08ns 14.55 high node harvest 0.81ns 0.12ns 0.89ns 21.28 total soluble solids 0.43 ns 0.72ns 0.04* 11.02 earliest time to harvest 0.00** 0.47ns 0.53ns 04.22 final harvest time 0.01* 0.88ns 0.12ns 05.18 note : cv: coefficient of variation; pr = probability; ns = not significantly different (α=5%). * = significantly different (α=5%). and ** = very significantly different (α=1%). transformation: a) √x. b) √x+0.5. 89the use of trellis and mulch increased fruit production of spaghetti .......... journal of tropical crop science vol. 4 no. 3, october 2017 www.j-tropical-crops.com fruit length, fruit diameter, fruit flesh and epicarp thickness the squash accession and the use of trellis and mulch resulted in different fruit length and diameter (table 4). the taiwan accession had a larger fruit length of 21.6 cm compared to the american spaghetti squash of 18.0 cm. the fruit length of taiwan accession in this study is similar to the description by the seed manufacturer, which is about 23 cm (known you seed, 2016). the fruit diameter of the american accession was smaller than that of taiwan accession, as no fruit thinning was conducted, resulting in nonuniform size (length and diameter) of the fruits. limiting the number of fruits per plant can affect the length and diameter of the fruit; the fewer the amount of fruit left on the plant, the greater the size of the fruits (anna, 2009). table 4 shows that crops with mulch and trellis produced fruits of similar size compared to other cultural techniques. this suggests that the squash crop could produce fruits of a similar size although they were grown with a different cultivation system. spaghetti squash with trellis and without mulch were able to produce fruit sizes similar to the fruits produced with mulch and without trellis. these results demonstrated that spaghetti squash could be cultivated at a lower cost, without the use of trellis or mulch, to produce acceptable fruit sizes. fruit formation and stem node number fruit formation was evaluated by recording the node number along the main stem at which the fruit developed. the taiwan squash were harvested from lower nodes than than those in american accession, i.e. in the node 10th and 13th nodes whereas fruits from the american accession were harvested from the 17th and 19th nodes. crops of taiwan accession had better vegetative growth than american accession. good plant growth results in the fruit to set in the lower nodes of the plants. both accessions had similar fruits formed on the higher nodes. the use of trellis and mulch (table 5) had a significant effect on the number of fruits formed on the high nodes. crops grown with trellis and mulch had fruits table 2. effects of trellis and mulch on fruit production of two accessions of spaghetti squash treatment number of fruits per plant number of rotten fruits per plant number of marketable fruits per plant accession taiwan 3.2a 0.5 2.7a america 2.0b 0.8 1.6b use of trellis and mulch mulch and trellis 3.7a 0.6 3.1a mulch without trellis 3.4a 0.6 2.8a trellis without mulch 1.9b 0.9 1.2b without trellis and mulch 1.6b 0.5 1.6ab note : values followed by the same letters within the same column are not significantly different according to dmrt at α = 5%. table 3. effects of trellis and mulch on fresh weight of spaghetti squash fruits treatment smallest fruit weight (g) largest fruit weight (g) accession taiwan 1,024.6a 1,760.2a america 621.9b 1,021.3b use of trellis and mulch mulch and trellis 845.8b 1,620.1a mulch without trellis 764,5b 1,555.1a trellis without mulch 794.4b 1,124.5c without trellis and mulch 1,005.7a 1,361.3b note : values followed by the same letters within the same column are not significantly different according to dmrt at α = 5%. 90 juang gema kartika, shinta wulandari karyana journal of tropical crop science vol. 4 no. 3, october 2017 www.j-tropical-crops.com harvested from the 24th node whereas those cultivated without trellis and without mulch had fruits harvested from 17th node. the number of fruits harvested from the lower nodes of the crops grown with trellis and with mulch was similar. tss (total soluble solid) content and fruit harvest time squash fruits of taiwan accession had a higher tss than fruits of american accession (table 6). the tss of both accessions was between 4.1 to 4.6 °brix (table 6). the use of trellis and mulch resulted in a significant difference in the fruit tss. according to harril (1994) tss of 6 is considered “poor”, whereas the good tss value is around 14°brix. the fruit tss of both accessions grown with trellis and mulch were all <6, indicating that the harvested spaghetti squash fruits in this study were not sweet. the use of trellis and mulch affected time to harvest in which the taiwan accession had an earlier harvest than the american accession. taiwan fruits were harvested earlier at 6 wap whereas america accession fruits were harvested at 9 wap. spaghetti squash is usually harvested at 13 wap (100 days) or 30-40 days after the pollination of female flowers (department of agriculture, forestry and fisheries, 2011) but in this study the fruits had to be harvested earlier at 42 days to avoid loss due to rotting fruit. the female and male flowers appeared at 3 wap, whereas based on the department of agriculture, forestry and fisheries (2011) flowers appear after one to two months after planting. according to maynard (2007), cucurbita fruits take about two to three weeks to reach its maximum size and takes about three weeks or more to reach maturity until final harvest. the recommended optimum temperature of the taiwan accession squash based on information from known you seed is 15°c – 25°c. based on this information early harvest will likely result in fruits with reduced sweetness. correlation analysis table 8 shows the linear correlation analysis between the quantitative characters recorded in this study. the number of fruits per plant was positively correlated table 4. effects of trellis and mulch on fruit characteristics of two spaghetti squash accessions treatment length of fruit (cm) diameter of fruit (cm) thickness of flesh (mm) thickness of epicarp (mm) accession taiwan 21.6a 12.6a 22 2 america 18.0b 11.0b 19 2 using of trellis and mulch mulch and trellis 20.5 12.2 21 2 mulch without trellis 20.1 11.8 21 2 trellis without mulch 20.6 12.2 21 2 without trellis and mulch 19.4 11.7 21 2 note : values followed by the same letters within the same column are not significantly different according to dmrt at α = 5%. table 5. effects of trellis and mulch on the number of squash fruits harvested from different nodes treatment the lowest node the highest node accession taiwan 10.8a 20.6 america 17.6b 21.9 use of trellis and mulch mulch and trellis 14.6 24.0a mulch without trellis 13.7 22.4ab trellis without mulch 14.7 18.5ab without trellis and mulch 13.8 17.6b note : values followed by the same letters within the same column are not significantly different according to dmrt at α = 5%. 91the use of trellis and mulch increased fruit production of spaghetti .......... journal of tropical crop science vol. 4 no. 3, october 2017 www.j-tropical-crops.com with the number of marketable fruits, the highest harvest node and the largest fruit weight. this means that the more fruits harvested, the higher the number of marketable fruits, the many fruits are harvested in high node and the heavily harvested fruit weights. according to sultana et al. (2015) fruit weight correlated positively with the number of fruit per plant and can be used as one of the useful selection criteria for determining fruit yield per plant of cucurbita moschata l. the number of rotten fruits (table 7) positively correlated to the lowest harvesting node and the final harvest time. this indicates that many rotten fruits were harvested from low nodes and harvested in the final week of harvest. figure 6 shows the rotting fruit that contacts the soil surface as it appears on the low node. the number of rotten fruits was also not correlated to the number of marketable fruits and to the smallest fruit weight. this means, the more rotten fruits harvested, the less the marketable yields which resulted from smaller fruit weights that were harvested. the number of marketable fruits was positively correlated with the highest harvest node and the largest fruit weight. this means that more and more harvestable fruits are harvested, so too many fruit weights are of great weight. lee (2004) reported that on the hamigua melon fruits harvested table 6. effects of trellis and mulch on total soluble solids (tss) and time to harvest of two accessions of spaghetti squash treatment tss (°brix) early harvest time (wap) final harvest time (wap) accession taiwan 4.6a 6.9b 8.3a america 4.1b 9.4a 9.6b use of trellis and mulch mulch and trellis 4.5a 7.5a 9.2a mulch without trellis 4.5a 7.8a 8.8a trellis without mulch 3.7b 7.8a 8.8a without trellis and mulch 4.6a 7.9a 8.5a note : values followed by the same letters within the same column are not significantly different according to dmrt at α = 5%. table 8. the linear correlation between the quantitative characters of the spaghetti squash ʃfpp ʃrf ʃmf lhn hhn sfw lfw fl fd tss ft st fht eht ʃrf -0.11 ʃffs i0.93** -0.46** lhn -0.44** i0.36* -0.52** hhn i0.74** i0.07 i0.63** 0.11 sfw -0.03 -0.52** i0.16 0.58** -0.29 bfw i0.65** -0.33 i0.70** -0.75** 0.33 0.61** l i0.20 0.05 i0.17 -0.59** -0.05 0.58** 0.82** d i0.23 0.07 i0.19 -0.56** -0.02 0.55** 0.78** 0.91** tss i0.22 -0.26 i0.30 -0.38* 0.09 0.21 0.54** 0.41* 0.37 ft i0.08 -0.15 i0.13 -0.46* -0.14 0.37 0.53** 0.73** 0.79** 0.35 st -0.13 -0.06 -0.11 0.14 -0.19 -0.14 -0.17 0.05 0.12 0.03 0.33 fht -0.50** i0.38* -0.58** 0.85** -0.08 -0.62** -0.79** -0.61** -0.58** -0.44* -0.39* 0.26 eht -0.52** i0.32 -0.58** 0.81** -0.16 -0.52** -0.76** -0.59** -0.58** -0.47* 0.34 0.31 0.96** fht i0.41* -0.37* -0.37* 0.73** 0.17 -0.54** -0.52** -0.36 -0.29 -0.36 -0.20 0.12 0.78** 0.73** note : **very significantly different (α=1%); * significantly different (α=5%); ʃfpp = number of fruits per plant; ʃrf = the number of rotten fruit; ʃmf = the number of marketable fruits; lhn = the lowest harvest node; hhn = the highest harvest node; sfw = the smallest fruit weight; lfw = the largest fruit weight; fl = fruit length; fd = fruit diameter; tss = total soluble solid; ft = flesh thickness; st = skin thickness; fht = fruit harvest time; eht = early harvest time; fht = final harvest time table 7. the linear correlation between the quantitative characters of the spaghetti squash note : **very significantly different (α=1%); * significantly different (α=5%); ʃfpp = number of fruits per plant; ʃrf = the number of rotten fruit; ʃmf = the number of marketable fruits; lhn = the lowest harvest node; hhn = the highest harvest node; sfw = the smallest fruit weight; lfw = the largest fruit weight; fl = fruit length; fd = fruit diameter; tss = total soluble solid; ft = flesh thickness; st = skin thickness; fht = fruit harvest time; eht = early harvest time; fht = final harvest time 92 juang gema kartika, shinta wulandari karyana journal of tropical crop science vol. 4 no. 3, october 2017 www.j-tropical-crops.com on the branches of the 8th node on the main stem produced 2.8 kg per fruit, resulting in a higher productivity of 43.4 ton.ha-1 compared to those with fruits harvested from lower nodes (nodes 5 th, 6 th, and 7 th) the marketable fruits were negatively correlated with harvest nodes and early harvest. this means more harvestable fruits are harvested, the fewer fruits harvested from the lowest crop node and few being harvested in the early weeks of the plant harvest. the fruits harvested from the low nodes of the plants were smaller in length and diameter. the largest fruit weight was positively correlated with fruit length, fruit diameter, and thickness of flesh; this means the heavier squash fruit, the longer and wider the size of the fruit and the thickness of the flesh. the greatest fruit weight was negatively correlated with the time to harvest; this shows that longer growing period resulted in larger and heavier fruits. fruit diameter was positively correlated with thickness of flesh; meaning the greater the diameter of the fruit the thicker the flesh of squash. conclusion growing spaghetti squash with trellis and mulch produced more total and marketable fruits per plant and heavier fruits compared with the other cultural methods. the taiwanese accession had more fruits per plant, more marketable fruits, heavier and larger fruit weight than the american accession, and the fruits can be were harvested earlier from the low nodes of the plant. the correlation analysis showed that the more the number of fruits per plant, the more the number of marketable fruits, the greater fruit weight, and the fruits can be harvested from the higher nodes of the plant. fruits from taiwanese accession had the largest fruits with mulch, with or without trellis. references agu, c. m. (2004). growth and yield responses of pumpkin (cucurbita maxima) to poultry manure applications and staking techniques southeastern nigeria. journal of sustainable agriculture 24, 5-10. anna, y. n. s (2009). “pengaruh jumlah buah per tanaman dan pangkas pucuk (toping) terhadap kualitas buah pada budidaya melon (cucumis melo l.) dengan sistem hidroponik”. thesis. bogor agricultural university. beany, a.h., stoffella, p. j., roe n., and picha, d. h. (2002). “production, fruit quality, and nutritional value of spaghetti squash”. ashs press. usa. department of agriculture, forestry and fisheries. (2011). squash (cucurbita moschata) production. agriculture, forestry and fisheries republic of south africa. hanna, h.y., adams, a.j., and stony, r.n. (1987). increased yield in slicing cucumbers with vertical trainings of plants and reduced plants spacing. hort science 22, 32-34. harrill, r. (1994). using a refractometer to test the quality of fruits and vegetables. pineknoll publishing. keedsyville. usa. hinds, j., wang, k.h., and hooks, c.r.r. (2015). growth and yield of zucchini squash (cucurbita pepo l.) as influenced by a sunn hemp living mulch. biological agriculture and horticulture 32, 21-33. https://doi.org/10.1080/01448765.2 015.1017736 [march 1, 2017]. known-you seed co. (2016). pumpkin (winter squash). http://www.knownyou.com/en_index. jsp?bodyinclude=productdetail&pid=9c8 b16ec5695090b8ab8e3428c1aebdbp405. [june 11, 2016]. maynard, l. (2007). cucurbit crop growth and development in “proceedings of indiana cca conference”. indiana. national plant germplasm system (npgs). (2006). taxon: cucurbita pepo l. grin global. https:// npgsweb.ars-grin.gov/gringlobal/taxonomy. [may 20, 2016]. self nutrition data. (2014). squash winter spaghetti, cooked, boiled, drained, or baked without salt. http://nutritiondata.self.com/ facts/vegetablesand-vegetable-products /2655/2. [june 11, 2016]. schaffer, a.a. and paris, h.s (2003). melons, squashes and gourds in “encyclopedia of food sciences and nutrition” (b. caballero, p. finglas, f. toldra, eds.) pp 3817-3827. academic press. shetty, n. v. and wehner, t. c. (1998). evaluation of oriental trellis cucumbers for production in north carolina. hortscience 33, 891-896. solangi, a.h., baloch, j.a. and iqbal, z. (2009). effect of vertical trailing on vegetative, reproductive 93the use of trellis and mulch increased fruit production of spaghetti .......... journal of tropical crop science vol. 4 no. 3, october 2017 www.j-tropical-crops.com and yield of luffa as intercrop in coconut field. pakistan journal of botany 41, 2537-2541. voragen, a. g. j., coenen, g. j., verhoef, g. j., and schols, h. a. (2009). pectin, a versatile polysaccharide present in plant cell walls. journal of structure chemistry 20, 263-275. wahjunie, d. e. and listyorini, e. (1998). peranan mulsa dan guludan dalam erosi hara. journal habitat 9, 7-11. 94 sugiyanta, galih angga kusuma journal of tropical crop science vol. 4 no. 3, october 2017 www.j-tropical-crops.com june_2014.cdr journal of tropical crop science (issn 2356-0169; e-issn 2356-0177) is published four-monthly by department of agronomy and horticulture, faculty of agriculture, bogor agricultural university, indonesia. publication details, including instructions for authors and subscription information: www.j-tropical-crops.com any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by the publisher. the accuracy of the content should be independently veri�ed with primary sources of information. the publisher shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the content. permission to make digital or hard copies of part or all of a work published in journal of tropical crop science is granted for personal or educational/classroom use provided that copies are not made or distributed for pro�t or commercial advantage. ©department of agronomy and horticulture, faculty of agriculture, bogor agricultural university, indonesia. all rights reserved. journal of tropical crop science volume number 1 20141 june on the cover the cover image shows sun�owers by darda effendi editorial board krisantini sintho wahyuning ardie sandra a. aziz robert j. hampson satriyas ilyas tri koesoemaningtyas rohana p mahaliyanaarachchi awang maharijaya maya melati roedhy poerwanto bambang sapto purwoko sudarsono muhamad syukur hugo volkaert malcolm wegener managing editor krisantini graphic design syaiful anwar features editor damayanti buchori dadang sisir mitra agus purwito ernan rustiadi short communication tropical and subtropical fruits in india sisir mitra heliconia cultivar registration dave skinner, jan hintze, bryan brunner research articles estimation of genetic parameter for quantitative characters of pepper ( l.)capsicum annuum muhamad syukur, syaidatul rosidah irrigation volume based on pan evaporation and their effects on water use ef�ciency and yield of hydroponically grown chilli eko sulistyono, abe eiko juliana evaluation of commercial sun�ower (helianthus annuus ) cultivars in bogor, indonesia, forl. ornamental and nursery production syarifah iis aisyah, khotimah, krisantini different growth partitioning and shoot production of talinum triangulare treated with organic and inorganic fertilizer sandra ari�n aziz, leo mualim, sitta azmi farchany cloning and characterization of p5cs1 and p5cs2 genes from l. under droughtsaccharum officinarum stress hayati minarsih iskandar, dwiyantari widyaningrum, sony suhandono journal of tropical crop science (issn 2356-0169; issn 2356-0177) is published four-monthly (one volume per year) byedepartment of agronomy and horticulture, faculty of agriculture, bogor agricultural university, ipb darmaga campus, bogor, indonesia 16680. send all inquiries regarding printed copies and display advertising to or to secretary, department ofinfo@j‐tropical‐crops.com agronomy and horticulture; telephone/fax 62-251-8629353. permission to reprint: permission to make digital or hard copies of part or all of a work published in isjournal of tropical crop science granted for personal or educational/classroom use provided that copies are not made or distributed for pro�t or commercial advantage and that copies bear the full citation and the following notice on the �rst page: “copyright department of agronomy and horticulture, faculty of agriculture, bogor agricultural university”. for all other kinds of copying, request permission in writing from head of school, department of agronomy and horticulture of�ce, ipb darmaga campus, bogor, indonesia 16680. © department of agronomy and horticulture, faculty of agriculture, bogor agricultural university. all rights reserved. printed in the republic of indonesia.in s t t t u i b o g o r perta n ia n abstract talinum triangulare or waterleaf is an underutilized tropical plant, mostly found as weeds, and has been used more as medicinal plant than as vegetable in indonesia. the study of cultivationtalinum triangulare has been explored to increase the shoot production as functional vegetables. the effects of organic fertilizer applications at 0.50, 0.75, 1.00, 1.25 and 1.50 of the standard rate on waterleaf growth were tested in a leuwikopo research station, bogor, west java, indonesia. standard rate of organic fertilizer consisted of 12.3 t..ha of cow manure, 226.8 kg. .ha of guano, and-1 -1 5.5 t.ha of rice hull ash that is equal to 100 kg urea, 60 kg-1 sp-36 and 100 kg kcl.ha . net assimilation rate (nar)-1 of the organic fertilizer-treated plants was lower than the inorganic fertilizer-treated with plant at two to four weeks after planting (wap). however, the plants treated with 0.75-1.25 organic fertilizer had a higher nar than those treated with inorganic fertilizer at four to six wap. plants treated with 0.75 rate of organic fertilizer had similar relative growth rate (rgr) to plants treated with the inorganic fertilizer at two to four wap, whereas plants treated with 1.00, 1.25 and 1.50 standard rate had higher rgr than plants treated with inorganic fertilizer at 4-6 wap. plants treated with 1.50 organic fertilizer rate had 34.55% more marketable shoots compared to those treated with inorganic fertilizer whereas those treated with 0.50 rate of organic fertilizer had 179.54% at 6 wap. the percentage of marketable shoots to total fresh weight of the organic fertilizer-treated plants was lower than the inorganic fertilizer-treated plants. keywords: , leafy vegetables, organictalinum triangulare introduction talinum triangulare or waterleaf is an underutilized tropical plant from portulacacea family found mostly as weeds, and has been used more as medicinal plant than as vegetable in indonesia. the name of waterleaf came from the high water content of the plant, which is 95-98 % (susanti et al., 2008; mualim et al., 2009). waterleaf is classi�ed into functional vegetables. the edible part of the plants are the leaves which contain primary metabolites such as protein and vitamin c, and secondary metabolites such as phenolic compounds that have high anti-oxidant activities (yang et al., 2006; andarwulan et al., 2010). phytochemical study by aja et al. (2010a) reported that each 100g dry weight of waterleaf contain �avonoid (0.070%), alkaloid (0.056%), saponin (0.001%), tannin (0.001%) and a total phenol of 0.489 mg eq galic acid (gae).g fresh weight with lipid-1 peroxidation inhibition of 97.1%. dpph, abts and frap tests showed trolox equivalen (te) values of 7.4, 1.03 and 28.3 te/ g. ha fresh weight, respectively-1 (andarwulan et al. 2010). waterleaf methanol extract, showed teac test value of 79 μmol te/g (yang et al., 2006). the leaves of waterleaf contains carbohydrate (10.87 mg.g ), steroid (106.61 mg.g ), protein (35.20-1 -1 mg.g ), fat (35.20 mg.g -karoten (114.15 mg.g ), and-1 -1 -1), β crude �ber (120 mg.g ) (aja et al., 2010 b).-1 to date there is still no available information on the effects of organic fertilizer application on waterleaf growth partitioning and shoot production. organic farming is believed to produce better quality vegetables over conventional farming that uses inorganic fertilizers. organically produced vegetables had a higher concentration of polyphenol antioxidants (carbonaro et al., 2002; young et al., 2005; benbrook et al., 2008; abuzahra et al., 2007), vitamin c, fe, mg, and p than those produced by conventional systems (worthington, 2001). in addition, organic farming products had high sugar content (stert, 2005; hallmann and rembialkowska, 2007). vegetable quality is indicated by the content of vitamin, antioxidant, minerals and functional components including pigments and polyphenols (ali et al., 2009). studies on the effect of manure (ibeawuchi et al., 2006; susanti et al., 2008), and npk (mualim et al., 2009; mualim and aziz, 2011) on waterleaf growth has been reported. application of 5 ton chicken manure.ha-1 resulted in high phytochemical waterleaf quality (susanti et al., 2009). potassium was reported to be the limiting factor in waterleaf anthocyanin production; the highest anthocyanin (39.60 mol.plant ) was obtained from-1 application of 100 kg sp-36.ha dan 100 kg kcl.ha-1 -1 (mualim et al., 2009). the aims of this study were (1) to determine the relative growth rate (rgr) and net assimilation rate (nar); (2) to determine if application of organic fertilizer can increase marketable shoots; (3) to investigate the effects of organic and inorganic fertilizer application on the proportion of marketable shoots to other part of the waterleaf. materials and methods treatments the experiment was set in randomized block design with research article different growth partitioning and shoot production of talinum triangulare treated with organic and inorganic fertilizer s a. aziz , l mualim , s a farchanyandra eo itta zmi ab a a department of agronomy and horticulture, bogor agricultural university, darmaga campus, bogor, indonesia 16680a corresponding author; e-mail addressb sandraaziz@yahoo.com 55 17 journal of tropical crop science vol. 1 no. 1, 2014june www.j-tropical-crops.com �ve rates of organic fertilizer treatment, i.e. 0.50, 0.75, 1.00 (the standard rate), 1.25 and 1.50 of the standard rate, which is 12.3 t.ha of cow manures + 226.8 kg.ha-1 -1 of guano + 5.5 t.ha of hulls. one rate of organic fertilizer-1 treatment equals to 100 kg urea, 60 kg sp-36 and 100 kg kcl.ha and was set as control. plant spacing was 100-1 cm x 50 cm. rice hull charcoal at 2 t.ha was applied at-1 the same time with the organic fertilizer treatment application (mualim et al., 2009). net assimilation rate (nar) and relative growth rate /rgr (south, 1995) were measured at two, four and six wap, and shoot harvesting at six wap. marketable shoots are shoots of a minimum of 15-cm length with leaf buds and were harvested at six wap. data obtained from organic and inorganic fertilizer treatments were compared using t-dunnet test. results t h e e x p e r i m e n t w a s c o n d u c t e d a t l e u w i k o p o experimental farm of bogor agricultural university, indonesia (gps). the soil type is latosol with �at topography, light intensity of 302 cal/cm /minute, average2 temperature of 25.8 c, and rainfall intensity of 6.5 mm pero week. soil ph (h2o) and (kcl) was 4.6 (acidic) and 4.1 (very acidic), respectively, c/n ratio of 15 (low), cec of 8.97 cmol(+)/kg, soil base saturation of 57%, and soil texture consisting of sand, loam, and clay of 19%, 13% and 68%, respectively. talinum plants �owered at three to six wap. the corganic and n levels of the organic vs inorganic fertilizertreated leaves at six wap were 49.3 vs 50.3% and 1.81 vs 2.42%, the p and k levels were(p<0.01), respectively; 0.27 vs 0.21 and k 5.56 vs 4.92% (p<0.05), respectively. organic fertilizer-treated plants had a lower plant biomass (figure 1a) and leaf photosynthetic activity (figure 2a) than inorganic fertilizer-treated plants up to four wap, and the plants treated with a half rate of organice fertilizer had the lowest of both. however, the biomass of the organic fertilizer-treated plants showed an increase at four to six wap (figure 1b). at six wap the plants treated with a standard, 1.25, and 1.5 standard rates of organic fertilizer had a greater biomass than those treated with inorganic fertilizer (figure 1b). research article figure 1. relative growth rate (g.day ) of organic fertilizer-treated vs to inorganic fertilizer-treated plants-1 at (a) two to four wap (0.09g.day ); (b) four to six wap (0.02g.day ).-1 -1 a b 55 s. a. aziz, l. mualim, s. a. farchany18 rela�ve growth rate (g.day ‐1 ) 0,02 0,02 rates of organic ma� er applica�on 0,01 0,50 0,75 1,00 1,25 1,50 0,50 0,75 1,00 1,25 1,50 rates of organic ma� er applica�on ‐0,01 ‐0,02 ‐0,02 ‐0,04 rela�ve growth rate (g.day ‐1 ) plants treated with 1.5 standard rate of organic fertilizer had the greatest fresh weight of marketable shoots at six wap (figure 3a). however, the dry weight was lower than those treated with the inorganic fertilizer (figure 3b). this indicated that the plants treated with 1.5 standard rate of organic fertilizer had the highest water content. the organic fertilizer-treated plants had a higher percentage of marketable shoots to the total biomass journal of tropical crop science vol. 1 no. 1, 2014june www.j-tropical-crops.com than the inorganic fertilizer-treated at four wap; and plants treated with the standard rate had the highest (figure 4b), i.e. 20.42% higher than inorganic fertilizertreated. at two and six wap the proportion was dominated by other parts of the plant. 55 19different growth partitioning and shoot production of talinum.......... figure 2. net assimilation rate (g.cm .day ) of organic fertilizer-treated compared to inorganic fertilizer-treated-2 -1 plants at (a) two to four wap (1.22 g.cm .day ) and (b) at four to six wap (0.5 g.cm .day )-2 -1 -2 -1 a b a b figure 3. (a) fresh weight of marketable shoots (g.plant ) of the organic fertilizer-treated plants compared to-1 those treated with inorganic fertilizer (146.22 g.plant ) at six wap; (b) dry weight of marketable shoot-1 weight of plants treated with organic fertilizer compared to those treated with inorganic fertilizer (9.06 g.plant ) at six wap.-1 journal of tropical crop science vol. 1 no. 1, 2014june www.j-tropical-crops.com 55 s. a. aziz, l. mualim, s. a. farchany20 a b c table1. growth and shoot production of treated with organic and inorganic fertilizertalinum triangulare note: values followed by different letters in the same row indicate signi�cant differences by duncan's multiple range test, p<0.05; value followed by (+) indicate signi�cant difference by t-dunnet test wap plant height (cm) shoot diameter (cm) marketable shoot weight (g) stem weight (g) leaf fresh weight (g) shoot fresh weight (g) root dry weight (g) stem dry weight (g) leaf dry weight (g) shoot dry weight (g) cultivar 2 3 4 5 6 2 3 6 6 2 4 6 2 4 6 2 4 6 2 4 6 2 4 6 2 4 6 24.79 31.41 40.49 47.41 49.52 30.15 36.89 70.38 26.75 38.42 218.05 124.91 155.41 460.30 296.20 0.85 1.66 1.63 2.56 6.33 3.87 5.55 13.91 11.00 8.67 22.51 32.22 b c b c b b b 27.81 35.07 43.09 48.93 53.12 30.69 40.67 94.10 26.57 34.00 229.67 140.42 95.73 520.80 343.70 0.60 3.00 1.94 2.33 4.37 4.41 4.21 12.65 13.00 6.28 27.93 34.79 ab bc b abc ab b b 24.89 33.67 43.14 49.25 53.17 34.12 43.57 108.34 25.52 48.98 252.19 166.26 109.00 547.90 430.40 0.51 1.88 2.77 1.84 3.98 4.55 4.96 14.53 14.65 7.52 29.33 44.13 ab bc b abc a b b 28.98 35.50 44.60 49.81 56.57 31.87 42.65 130.64 46.51 78.70 209.19 208.89 131.18 464.90 498.10 0.65 2.05 2.30 2.23 4.13 5.14 4.89 11.12 12.51 8.06 19.20 38.82 ab b b ab a b a rate of organic fertilizer 0.5 0.75 1.00 1.25 1.50 26.33 37.71 47.89 54.85 58.73 34.14 44.03 196.74 45.84 37.11 266.56 296.99 211.59 596.50 702.20 0.88 2.34 2.60 2.13 5.51 4.30 6.18 14.52 15.18 9.46 31.90 42.72 a a+ a+ a a a a inorganic fertilizer 24.90 34.53 41.43 46.47 50.67 32.59 41.29 146.22 31.68 40.12 230.42 205.61 94.31 515.10 402.00 0.63 2.14 2.11 2.86 4.04 4.26 4.78 14.94 18.05 7.52 31.23 42.56 figure 4. percentage of marketable shoots to total biomass (fresh weight) of the organic fertilizer-treated vs inorganic-fertilizer treated plants at (a) two wap; (b) four wap; and (c) six wap. journal of tropical crop science vol. 1 no. 1, 2014june www.j-tropical-crops.com plants treated with 1.50 standard rate of organic fertilizer had better performance than plants treated with inorganic fertilizers or with other rates of organic fertilizer for all other variables. this treatment also produced 34.55% more marketable shoots than the inorganic fertilizer treatment, and 179.5% of the 0.50 organic fertilizer treatment (table 1). the total marketable shoots of the organic fertilizer-treated plants were similar to those treated with inorganic fertilizer. discussions the low rgr and nar at two to four wap might be related to the slow availability of the nutrients from the decomposing organic fertilizer, and to the fact that the plants had reached the generative phase. this condition showed that waterleaf had a good access to nutrients from the inorganic fertilizer at two to four wap. the leaf corganic and nitrogen content of the inorganic fertilizertreated plants were initially lower than the organic fertilizer-treated, i.e. 49.3 vs 50.3%, and 1.81 vs 2.42% respectively (p<0.01). however, at four to six wap the nutrient accessability from organic fertilizer increased. p and k levels of the organic fertilizer-treated leaves were signi�cantly higher (p<0.05) than the inorganic fertilizertreated leaves at six wap. conclusions out of all rates of organic fertilizers tested plants treated with 0.75 rates had similar rgr to the inorganic fertilizertreated plants at two to four wap. plants treated with 1.00, 1.25 and 1.50 fertilizer rates had higher rgr than inorganic fertilizer at four to six wap. marketable shoots from 1.50 organic fertilizer-treated plants was 25.67% greater than those treated with inorganic fertilizer, and it was 179.54% of those treated with 0.50 rate of organic fertilizer at six wap. all plants treated with organic fertilizer produced more marketable shoots than those treated with inorganic fertilizer. references abu-zahra, t.r., al-ismail, k. and shatat, f. 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(2010). flavonoid content and antioxidant activity of vegetables from indonesia. food chemistry 1231-1235.121, anwar, m., patra, d.d., chand, s., alpesh, k., naqvi, a.a. and khanuja, s.p.s. (2007). effect of organic manures and inorganic fertilizer on growth, herb and oil yield, nutrient accumulation, and oil quality of french basil. communications in soil science and plant analysis , 1737-1746.36 benbrook, c., zhao, x., yanez, j., davies, n. and andrews, p. (2008). new evidence con�rms the nutritional superiority of plant-based organic foods. state of science review. the organic center: foster, ri. carbonaro, m., mattera, m., nicoli, s., bergamo, p. and cappelloni, m. (2002). modulation of antioxidant compounds in organic vs conventional food (peach, l., and pear,prunus persica pyru scommunis journal of agricultural and foodl.). chemistry 9-11.50, h a l l m a n n , e . a n d r e m b i a l k o w s k a , e . 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(2010). respon pertumbuhan kolesom terhadap pemupukan p (waterleaf growth respond to p fertilizer). fakultas pertanian, institut pertanian bogor, bogor, indonesia. shipley, b. and keddy, p.a. (1988). the relationship between relative growth rate and sensitivity to nutrients stress in twenty-eight species of emergent macrophytes. ,journal of ecology 76 1101-1110. shipley, b. (2006). net assimilation rate, speci�c leaf area and leaf mass ratio: which is most closely correlated with relative growth rate? a metaanalysis. , 565-574.functional ecology 20 south, d.b. (1995). relative growth rates: a critique. south african forestry journal , 43-48.173 susanti, h., aziz, s.a. and melati, m. (2008). produksi biomassa dan bahan bioaktif kolesom (talinum triangulare (jacq.) willd.) dari berbagai asal bibit dan dosis pupuk kandang ayam. (biomass and bioactive compound production of waterleaf ( (jacq.) willd) from differenttalinum triangulare propagule and chicken manure fertilizer rate). indonesian journal of agronomy 48-5536, susanti, h. (2012). produksi protein dan antosianin pucuk kolesom ( (jacq.) willd.)talinum triangulare dengan pemupukan nitrogen+kalium dan interval panen (protein and anthocyanin productions of waterleaf shoot ( (jacq.) wild.)talinum triangulare at different levels of nitrogen+potassium and harvest intervals). institut pertanian bogor, bogor, indonesia. susanti, h., aziz, s.a., melati, m. and susanto, s. (2011). protein and anthocyanin productions of waterleaf shoot ( (jacq.) willd.) attalinum triangulare different levels of nitrogen+ potassium and harvest intervals. indonesian journal of agronomy 38, 119123. worthington, v. (2001). nutritional quality of organic versus conventional fruits, vegetables, and grains. the journal of alternative & complementary medicine 7, 161-173. yang, r.y., tsou, s., lee, t.c., wu, w.j., hanson, p.m., kuo, g., engle, l.m. and lai, p.y. (2006). distribution of 127 edible plant species for antioxidant activities by two assays. journal of the science of food and agriculture , 2395-2403.86 55 s. a. aziz, l. mualim, s. a. farchany22 journal of tropical crop science vol. 1 no. 1, 2014june www.j-tropical-crops.com the 29 international horticultural congress th sustaining lives, livelihoods and landscapes (http://www.ihc2014.org) brisbane, australia, 17-22 august 2014 morphology and starch production potential of sago palm (metroxylon spp) .......... journal of tropical crop science vol. 9 no. 1, february 2022 www.j-tropical-crops.com 31 morphology and starch production potential of sago palm found in village haripau, east mimika subdistrict, mimika, papua province, indonesia muhammad iqbal nurulhaqa, muhammad hasjim bintorob, supijatnoc acollege of vocational studies, ipb university bdepartment of agronomy and horticulture, ipb university cdepartment of agronomy and horticulture, ipb university *corresponding author; email: muhammadiqbalnurulhaq@apps.ipb.ac.id abstract the largest sago palm (metroxylon sagu rottb.) growing area in the world (85%) is in indonesia, and 95% of sago area in indonesia is in papua and west papua. field observation of accessions of sago palm was conducted at the sago growing area at hiripau village. the aim of the study was to determine the diversity of sago palm accessions from hiripau village, and compare their morphology and starch production. based on interview with the local farmers, the four accessions of sago palm dominated the hiripau area are nakowai, mapartaro, tuhai, and korearipi. these accessions vary in the spine characteristics, features of the trunk, leaf, pith, and starch production. sago mapartaro leaves are the largest (23.56 m2) and the leaflet areas are almost twice the size of the other accessions. tuhai has the highest starch yield but it has high ash content resulting in low starch quality. keywords: sago variability, leaf spine, starch yield introduction sago palm (metroxylon spp) belongs to the arecaceae family; sago grows to form clumps with many suckers. the main part of the sago palm is the trunk that has accumulation of carbohydrates. saitoh et al., (2008) reported, the production dry starch in one trunk reaches 835 kg. the leaves on sago palms are similar to those of other types of palms. the leaves are made up of leaflets that attached along the rachis. in some types of sago, there are spines attached to the midrib, leaflets, trunk whereas some types of sago are spineless. indonesia is the center of diversity for sago palm in the world (abbas et al., 2010; flach 1983; bintoro et al., 2018). in indonesia, sago can be found not only in papua or maluku island, but also in sumatra, borneo, celebes (sulawesi), and java island. the sago palm in indonesia comprise 85% of the sago worldwide, and 95% (5.2 million ha) of sago area in indonesia is in papua and west papua (bintoro et al., 2018). the morphological and genetic diversity in these regions is inferred to be high (dewi et al., 2016) due to the crosses that occur naturally (schuilling, 2009). according to abbas (2018) papua exhibited the largest genotype diversity in their population based on wx gene markers, and papua it is considered to be the center of sago palm genetic diversity in indonesia. the local people in papua and west papua have a simple method to classify sago. they differences based on shape (trunk shape, spine or spineless) and choose the best sago with the highest pith content based on local experience. because sago grows as a natural stand, it is possible to have very high sago diversity due to cross-pollination (pratama, 2018). sago trees growing in a certain area could be from one or more species. samples from each putative species (henceforth called ‘accessions’) are obtained and they represent the diversity of sago in the area. dewi et al. (2016) reported the diversity in morphology of sago palm based on morphological characteristics such as shoots color, crown shape, trunk height, number of leaflets, existence of spine, starch content, pith and starch color. from these morphological characteristics, 12 accessions from the wild stand of sago palm in south sorong district have been identified (dewi et al., 2016). in addition, limbongan (2007) reported sago diversity based on the presence of spines, plant height, stem circumference, and starch color. mimika district in papua province has an extensive growth of wild sago trees, i.e. 382,189 ha, and it has journal of tropical crop science vol. 9 no. 1, february 2022 www.j-tropical-crops.com 32 muhammad iqbal nurulhaq, muhammad hasjim bintoro, supijatno natural of sago stand. currently there are limited studies on sago diversity in indonesia and in the world. the objective of this study was to determine the diversity and production potential of sago (metroxylon spp) in hiripau village, east mimika subdistrict, mimika district, papua province, indonesia. materials and methods field observations were conducted in the natural sago stand area of hiripau village from july to september 2016. data were collected through field observations and interviews with sago farmers. each sago accession observed had entered the ready-tocut (harvest) or ripe phase. the location of accessions were marked using garmin gps 64s series. not all people in the hiripau know about sago plants in the sago region. in general, people only recognize sago at their maturity phase (harvest time). there is a huge gap in the knowledge on sago between the older and younger generation of hiripau, therefore only the older people (local term: porapoka) in the area were interviewed and particularly, the community leaders of the hiripau people. the accessions of sago were observed using the following parameters: 1) trunk (height, diameter of trunk at base (bottom), middle, and top, bark thickness); 2) leaf (number of leaves, number of leaflets, leaf area, width of leaflets, length of rachis, length and width of the petiole); 3) spine or spineless, spine spacing, spine pattern, length of spine); 4) leaf color, bark, and pith of each accession were also observed and determined using the royal horticulture society color chart 2015; 5) sampling of the pith of each accession was carried out using a sample ring on three parts of the trunk (bottom, middle, and top). the carbohydrate content of sago starch and analysis of chemical composition of sago starch was conducted at the food technology laboratory ipb bogor using the aoac method (2006). all data obtained were expressed in average. standard deviation and coefficient of diversity were calculated using minitab 16. results and discussion general environment natural sago stand area in hiripau village is located at an altitude of 10-26 meter above sea level. the area is semi-permanently flooded. the sago area is adjacent to the big river (wania river) that in the rainy season, the water overflows and floods the sago growing area. the area has a gley type mineral soil with a top layer rich in organic matter from the decomposition of dead leaves and plants. the surface of the soil is also covered with mud deposits from the wania river overflow and in steady conditions ranging from 0-15 cm. in general, the sago trees in hiripau village are naturally found in wetlands; this ecology seems to be optimal for sago. according to muhidin et al. (2016a) sago grows better in wetlands than in dry land. sago area in hiripau village is located in lowland areas. sago grown in the lowlands more quickly produce flowers compared to those grown in the highlands. sago grown on the plateau has a longer vegetative phase (muhidin et al., 2016b), so sago that grow in the lowlands, such the village of hiripau, can be harvested earlier compared to sago growing on the highlands. the vegetation in hiripau village is heterogeneous, consisting of various types of timber trees in various stages of development (mature trees and seedlings), bamboo, and palm trees, but forest areas are dominated by sago stands that form clusters. the humidity in the sago area is between 72% -81% with the air temperature of 27.6 to 31.60c. local community interview based on field observations and interviews, four local accessions of sago are recognized by the local community. the accessions of sago were given local names as follows: nakowai, mapartaro, tuhai, and korearipi. of the four accessions, one of them is unbounded sago (korearipi). during the field observation, flowering sago accessions were rarely observed. this is because the harvesting of sago had been conducted before the sago plants started flowering. morphology of sago accessions the trunk condition of each accession varies. generally, the accessions (except for tuhai) have dead stump that attaches to the surface of the chief plant trunk (figure 1). the size of the trunk (height, diameter) also varies. nakowai has a high trunk, whereas mapartaro has large and short trunks (table 1). nakowai and mapartaro are the most accessible accessions in the sago forest in hiripau. both are spine accessions with almost identical features. the striking difference of both accessions is the size of the morphology and starch production potential of sago palm (metroxylon spp) .......... journal of tropical crop science vol. 9 no. 1, february 2022 www.j-tropical-crops.com 33 trunk. hiripau people recognize nakowai with its tall trunk, while mapartaro is characterized with a large but short trunk. the hiripau people recognize the accessions of sago based on whether they have spine or are spineless. the spine accessions are differentiated by size and trunk shape. the visible features of the trunk is the basis for the people of hiripau village to distinguish sago accession (figure 2). nakowai is 12.28 m tall while mapartaro is only 7.9 m. however, the diameter and circumference of the mapartaro rod is greater than that of nakowai, tuhai and korearipi. mapartaro also has along the trunk. tuhai has a unique trunk shape with the seedling growing in the middle of the trunk so that the rods are seen in a continuous manner. table 1. characteristic of trunks of the four sago accessions in hiripau village. accession height (m) diameter (cm) bark thickness (cm) trunk circumference (cm) nakowai 12.28 45.60 1.54 140.00 mapartaro 7.90 54.30 1.54 55.00 tuhai 11.80 45.60 1.65 125.00 korearipi 13.20 40.30 1.90 120.00 average 11.30 46.45 1.66 135.00 standard deviation 2.43 5.80 0.17 15.81 diversity coefficient (%) 20.69 12.48 10.15 11.71 the sago accessions from hiripau village do not vary in leaf shapes; korearipi has a white band in the middle of the midrib (leaf bone) in the back. the number of leaves at the harvest stage among the four accessions is at 14-22 (table 2). the number of leaves of the four accessions ranged from 14-18 midribs, which is consistent with schuiling (2009) that sago in the adult vegetative phase has the average number of leaf of 14 to 22 per tree. sago tree begins to initiate flowers at the harvest stage. the new leaves that grow will be smaller in size with short rachis and the number of leaflets is also fewer than the previous phase. the number of leaflets do not vary among the four accessions. mapartaro has the longest rachis but shorter petiole size compared to the other accessions. the number of leaflets on the left and right sides of the leaves of each accession varies. in nakowai, mapartaro, tuhai and korearipi there are more leaflets on the left side than the right side of the leaves. mapartaro has the largest lea and leaflet size compared to the other accessions (table 3). the mapartaro leaves are the largest (23.56 m2) compared to the other accessions. the leaf and the leaflet areas of mapartaro are almost twice the size of the other accessions. the area of the leaves is influenced by the length of the rachis. the longer rachis will contain more leaflets in the midrib making the leaves more capacious. although there are more leaflets on the left than the right side (table 2) the width of the leaflets on the right side is broader than the leaflets on the left side of the rachis (table 3). the difference in the number of leaflets on the left and right sides of the rachis is due to the sinistrorse phyllotaxis in sago plants (nakamura and goto 2015). sinistrorse phyllotaxis can be seen by looking at the direction of the leaf arrangement. if the direction of the leaf composition is counter-clockwise, it is called sinistrorse, and if it is clockwise it is called dextrorse. another difference in the morphology of the leaves is the presence of a white band on the back of korearipi leaves (figure 3). the white band extends from the table 1. characteristic of trunks of the four sago accessions in hiripau village. accession height (m) diameter (cm) bark thickness (cm) trunk circumference (cm) nakowai 12.28 45.60 1.54 140.00 mapartaro 7.90 54.30 1.54 55.00 tuhai 11.80 45.60 1.65 125.00 korearipi 13.20 40.30 1.90 120.00 average 11.30 46.45 1.66 135.00 standard deviation 2.43 5.80 0.17 15.81 coefficient diversity (%) 20.69 12.48 10.15 11.71 the hiripau people recognize the accessions of sago based on whether they have spine or are spineless. the spine accessions are differentiated by size and trunk shape. the visible features of the trunk is the basis for the people of hiripau villageto distinguish sago accession (figure 2). nakowai is 12.28 m tall while mapartaro is only 7.9 m. however, the diameter and circumference of the mapartaro rod is greater than that of nakowai, tuhai and korearipi. mapartaro also has a remnant of petiole spine that sticks along the trunk. tuhai has a unique trunk shape with the seedling growing in the middle of the trunk so that the rods are seen in a continuous manner. figure 1. trunk variations: nakowai (a), mapartaro (b), tuhai (c), and korearipi (d) figure 2. hiripau method to distinguish sago accessions a b c d figure 1. trunk variations: nakowai (a), mapartaro (b), tuhai (c), and korearipi (d) journal of tropical crop science vol. 9 no. 1, february 2022 www.j-tropical-crops.com 34 muhammad iqbal nurulhaq, muhammad hasjim bintoro, supijatno table 1. characteristic of trunks of the four sago accessions in hiripau village. accession height (m) diameter (cm) bark thickness (cm) trunk circumference (cm) nakowai 12.28 45.60 1.54 140.00 mapartaro 7.90 54.30 1.54 55.00 tuhai 11.80 45.60 1.65 125.00 korearipi 13.20 40.30 1.90 120.00 average 11.30 46.45 1.66 135.00 standard deviation 2.43 5.80 0.17 15.81 coefficient diversity (%) 20.69 12.48 10.15 11.71 the hiripau people recognize the accessions of sago based on whether they have spine or are spineless. the spine accessions are differentiated by size and trunk shape. the visible features of the trunk is the basis for the people of hiripau villageto distinguish sago accession (figure 2). nakowai is 12.28 m tall while mapartaro is only 7.9 m. however, the diameter and circumference of the mapartaro rod is greater than that of nakowai, tuhai and korearipi. mapartaro also has a remnant of petiole spine that sticks along the trunk. tuhai has a unique trunk shape with the seedling growing in the middle of the trunk so that the rods are seen in a continuous manner. figure 1. trunk variations: nakowai (a), mapartaro (b), tuhai (c), and korearipi (d) figure 2. hiripau method to distinguish sago accessions a b c d figure 2. hiripau method to distinguish sago accessions table 2. leaf characteristics of sago accessions in hiripau village accession leaf number leaflet number leaf length (cm) rachis length (m) petiole length (cm) adaxial-left side adaxial-right side nakowai 14.00 74.00 79.00 6.90 5.07 183.00 mapartaro 14.00 85.00 88.00 8.80 8.01 79.00 korearipi 17.00 85.00 88.00 6.72 5.67 105.00 tuhai 18.00 72.00 75.00 6.22 4.99 123.00 average 15.75 79.00 82.50 7.16 5.94 122.50 standard deviation 2.06 6.98 6.56 1.13 1.42 44.19 diversity coefficient (%) 13.06 8.83 7.95 15.79 23.86 36.08 table 3. leaf area and leaflet area of sago accessions at hiripau village. accession leaf area (m2) leaflet area (cm2) adaxial-left side adaxial-right side nakowai 13.00 11.15 23.15 mapartaro 23.56 16.35 36.00 tuhai 12.13 10.32 10.60 korearipi 13.23 9.71 10.77 average 15.48 11.88 20.13 standard deviation 5.41 3.04 12.11 diversity coefficient (%) 34.95 25.54 60.13 petiole to the rachis on the back of the midrib. the band on the back of the midrib is found only in the spineless accessions. this feature has also been found in other spineless sago accessions in the eastern indonesian archipelago (north sulawesi and maluku), one accession with a slightly black band (roe) and three accessions with brown band (tewasen / rumbia, beka bawes, roku mamo) (ehara et al., 2000). based on research conducted by ehara (2009), sago diversity in eastern indonesia is genetically correlated to geographical distribution. accessions found in the same location are most likely to be similar in morphology. figure 3. white band (black arrow) on the abaxial of korearipi leaf midrib. morphology and starch production potential of sago palm (metroxylon spp) .......... journal of tropical crop science vol. 9 no. 1, february 2022 www.j-tropical-crops.com 35 spines are found only in three accessions: nakowai, mapartaro, and tuhai. spines were present on every single leaf midrib except during the harvest phase. the spines can be seen as traces or have been broken off in the leaf midrib during harvest phase (figure 4). spines in the midrib (petiole and rachis) of seedlings have different densities (table 4). spines are also present on the side of the mother plant and leaflets from the sucker. the color of the mother plant leaves and leaflet can be described as green and yellow-green based on the rhs color chart 2015 (table 5). the color of the sucker leaflet is more varied and can be described as green, yellow-green and grayed-purple. the color of the bark and pith are as follows: brown and greyedpurple for the bark, and white and orange-white for the pith. figure 4. variations of spine traces at adult phase of sago leaves: nakowai (a), mapartaro (b), tuhai table 4. variation in the spines on mother plants and sucker of different accessions of sago from hiripau village. accession spine spacing of the mother plant (cm) spine spacing of the sucker (cm) length of the spine (cm) nakowai 4.54 12.11 4.81 mapartaro 7.10 9.08 4.08 tuhai 3.89 6.15 3.86 average 5.18 9.11 4.25 standard deviation 1.72 2.98 0.49 diversity coefficient (%) 32.78 32.70 11.70 the spines traces on the leaves at the nakowai, and mapartaro troughout the petiole to the mid of rachis, while the tuhai spines traces is only at the base of petiole. the spine traces pattern of each accession varies both on the mother plant petiole and on the sucker. the spine on the sucker forms a scattered or random pattern (spotted) and surround the petioles (rimmed). the traces on the mother plant form regular pattern (straight) and random. spines are present on both sides of each leaflet for all accessions, except korearipi. in mapartaro the spines of the dead leaves are still attached to the trunk (figure 1b). spines are also present on both sides of the leaflet and midrib of the leaves in the mother plant leaves and the sucker, except on korearipi. the density of spines on the petiole and rachis in the sucker varies from 6-12 cm (table 4). according to novero et al., (2012), the appearance of spines in sago is epigenetic, which means that the spines in sago plants is controlled by genes, expression of which is caused by other factors, in addition to changes in basic dna sequences. environmental factors could influence the morphology of sago plants. the green color of the leaves of the parent plant is similar to those of the leaflets and midrib leaves, as well as the bark and pith, whereas the color of the sucker leaflet svaries. in mapartaro the leaves on the sucker is red purple while on the other accessions, the leaves look green or greenish yellow. the color of leaflets on new leaves is one of the sago characters that is easy to distinguish and can be used as a distinguishing characteristic because it is not influenced by the environment (dewi et al., 2016). the leaflet color is one way for people in the south sorong to differentiate the various accessions of sago palm. sago pith color on sago spine accessions (nakowai, mapartaro and tuhai) tends to be almost similar (table 5). the pith of these accessions is white. based on the royal horticulture society (2015) the pith color of nakowai is pale yellow green, while the accessions of mapartaro and tuhai are pure white. the pith color of the korearipi is orange-white or yellowish pink. the color differences indicate the activity levels of polyphenol-oxidase on the pith of each accession. konuma et al., (2012) reported that the brownish red color of starch and sago pith journal of tropical crop science vol. 9 no. 1, february 2022 www.j-tropical-crops.com 36 muhammad iqbal nurulhaq, muhammad hasjim bintoro, supijatno table 5. observation of leaf and leaflet color* of the sago plant accessions. accessions mother plant leaflet sucker leaflet bark mother plant leaves pith nakowai nn137b 152a 200b 143b 155c mapartaro 143a 183a 185a 143a n155d tuhai 137a 137a 200a 144a n155c korearipi 144a 143a 200b 144a 159a *color code according to rhs2015, where 125-143 = green group; 159 = orange-white group; 144-154 = yellow-green group; 183-n187 = greyed –purple group; 155-nn155 = white group; 200-n200 = brown group indicates high levels of polyphenol-oxidase activity. differences in the color of the pith and starch indicate the differences in the type of sago growing in the ecosystem. starch production the average production of dry starch of sago accessions from hiripau village was 174.83 kg per plant, with the lowest dry starch content coming from nakowai and the highest from tuhai (table 6). the lowest yield of starch comes from nakowai while the highest comes from korearipi. the average water content of starch was 16.2%. according to dewi et al. (2016) the high production of sago starch is influenced by genetics and environment. each accession has a different production of starch seen from the difference in the yield of each accession. the difference in yield is also influenced by the water content in the sago pith causing low yield of sago starch in the pith. the fat, protein, and carbohydrate contents of the four accessions of sago from hiripau village did not vary significantly. with regard to ash content in dry starch produced, tuhai has the highest at 2.1%, indicating the presence of inorganic materials. among the four accessions, only nakowai has an ash content that is within the indonesian national standard (sni = 0.5%). high ash levels determine the quality of food. the proportion of ash content in foods is influenced by several factors, such as plant species, soil nutrient state, plant maturity, climate, growing area, and age of sago cutting (adisti, 2016). based on research conducted by konuma et al. (2012) sago that grows in waterlogged land, in acidic conditions, and areas with high concentration of sulfur, will produce starch with high ash content. table 6. potential starch production of the four sago accessions from hiripau village. accession rendemen (%) water content (%) starch production (kg per plant) nakowai 9.00 15.60 104.38 mapartaro 12.00 15.70 116.47 tuhai 20.00 17.70 275.04 korearipi 23.00 15.60 203.45 average 16.00 16.20 174.83 standard deviation 6.58 1.03 80.10 diversity coefficient (%) 41.14 6.41 45.79 table 7. chemical composition of sago starch among different accessions from hiripau village accession ash content (%) fat (%) protein (%) carbohydrate (%) nakowai 0.23 0.16 0.52 83.10 mapartaro 0.63 0.29 0.53 82.70 tuhai 2.10 0.29 1.53 78.10 korearipi 0.58 0.18 0.53 83.08 average 0.89 0.23 0.78 81.75 standard deviation 0.82 0.07 0.50 2.44 diversity coefficient (%) 93.71 30.33 64.53 2.98 morphology and starch production potential of sago palm (metroxylon spp) .......... journal of tropical crop science vol. 9 no. 1, february 2022 www.j-tropical-crops.com 37 conclusion four sago palm accessions in hiripau have been identified by the local farmers, i.e. nakowai, mapartaro, tuhai and korearipi. the four sago accessions vary in the leaf, spine, and trunk morphology. the starch production can reach > 200 kg dry starch per trunk. tuhai has the highest potential yield but had high ash content, indicating low starch quality. acknowledgement the authors would like to thank the government of mimika district, papua province for funding this research and the head of hiripau clan, mr. benedictus mapeko, for his invaluable information and permission to study the sago accessions in the hiripau area. references abbas, b., renwarin, y., bintoro, m.h., surahman, m., and ehara, h. (2010). genetic relationship of sago palm in indonesia based on chloroplast dna (cpdna) markers. biodiversitas 10, 168174. abbas, b. (2018). sago palm genetic resource diversity in indonesia. in “sago palm: multiple contribution to food security and sustainable livelihoods” (h. ehara, y. toyoda, and d.v. johnson, eds.), pp. 61-71. springer nature. adisti, f.w. (2016). “characteristic of sago starch (metroxylon spp.) from sorong and south of sorong district, west papua”. 53 pp. bogor agricultural institute, bogor, indonesia. [aoac] the association official analytical chemist. (2006). “official methods of analysis”. association of official analytical chemists. washington. bintoro, m. h., nurulhaq, m. i., pratama, a. j., ahmad, f. and ayulia, l. (2018) growing area of sago palm and its environment. in “sago palm: multiple contributions to food security and sustainable livelihoods” (h. ehara, y. toyoda and d.v. johnson, eds.), pp. 17–29. springer nature. dewi, r.k., bintoro, m.h., and sudrajat. (2016). morphology and potential production of various sago accessions (metroxylon spp) in south of sorong, west papua. jurnal agronomi indonesia 44, 91-97. ehara, h. (2009). potentials of sago palm as carbohydrate resource for strengthening food security program. jurnal agronomi indonesia 37, 209-219. ehara, h., susanto, s., mizota, c., hirose, s., and matsuno, t. (2000). sago palm (metroxylon sagu, arecaceae) production in the eastern archipelago of indonesia: variation in morphological characteristics and pith drymatter yield. economy botany 54, 197-206. flach, m. (1983). “the sago palm". fao plant production and protection paper 47. 85 pp. fao, rome, indonesia. konuma, h., rolle, r., and boromthanarat, s. (2012). color characteristics of sago starch as they relate to the growth environment of the sago palm (metroxylon sagu rottb.). journal of agricultural technology 8, 273-287. limbongan, j. (2007). morphology of several potential types of sago in papua. jurnal litbang pertanian 26,16-24. muhidin., leomo, s., jaya, m., sumarlin., and arli, w.o. (2016a). the effect of elevation gradient on the phenological aspect of growth and production of sago palm (metroxylon sagu rottb). aensi journals 10, 28-34. muhidin., leomo, s., alam, s., and wijayanto, t. (2016b). comparative studies on different agroecosystem base on soil physicochemical properties to development of sago palm on dryland. international journal of chemtech research 9, 511-518. nakamura, s., and goto, y. (2015). leaf. in “the sago palm: the food and enviromental challenges of 21st century”. (the society of sago palm studies, eds.), pp 66-75. kyoto university press. novero, a.u., mabras, m.b., and esteban, h.j. (2012). epigenetic inheritance of spine formation in sago palm (metroxylon sagu rottb.). plant omics journal 5, 559-566. pratama, a.j., bintoro, m.h., and trikoesoemaningtyas. (2018). variability and relationship analysis of sago accessions from natural population of papua based on morphological characters. sabrao journal of breeding and genetics 50, 461-474. journal of tropical crop science vol. 9 no. 1, february 2022 www.j-tropical-crops.com 38 muhammad iqbal nurulhaq, muhammad hasjim bintoro, supijatno saitoh, k., bintoro, m.h., oh-e, i., jong, f.s., louw, j., and sugiyama, n. (2008). starch productivity of sago palm in indonesia. sago palm 16, 102 108. schuiling, d.l. (2009). “growth and development of true sago palm (metroxylon sago rottb.) with special reference to accumulation of starch in the trunk” 259 pp. wageningen university. journal of tropical crop science vol. 6 no. 3, october 2019 www.j-tropical-crops.com 164 defiyanto djami adi, iskandar lubis, suwarto and sugiyanta determination of the optimum rates for n, p, and k fertilizer for upland rice variety “ipb 9g” defiyanto djami adia, iskandar lubisb, suwartob and sugiyantab a graduate school, department of agronomy, ipb university, darmaga campus, bogor 16680, indonesia b department of agronomy and horticulture, faculty of agriculture, ipb university, bogor 16680, indonesia corresponding author; email: iskandarlbs@yahoo.com abstract a study was conducted to determine the optimum rates of n, p, k fertilizers on “ipb 9g” upland rice variety from march to july 2018 at the sawah baru experimental field of ipb university, west java, indonesia. three paralel experiments were carried out in a randomized complete block design to test five fertilizing levels, i.e. 0, 50, 100, 150 and 200% of the reference rate. the reference rates of fertilizer application are 100% n = 200 kg urea ha-1, 100% p = 100 kg sp36 ha-1, and 100% k = 100 kg kcl ha-1. the applied fertilizers were urea (46% n), sp36 (36% p2o5) and kcl (60% k2o). fertilizers were applied three times, 40% at planting, 30% at 4 wap and the rest 30% at 8 wap; 100% p and 100% k were applied at planting. increasing n fertilizer from 0 to 200% of the reference rate increased plant height, dry weight biomass at heading and at harvest, number of productive tillers, harvest index, and grain weight per clump. p increased dry weight of the above ground biomass, whereas k fertilizer increased the leaf area, canopy dry weight, number of grain per panicle, panicle length, harvest index and grain weight per clump. the optimum rate of n and p were 107.09 kg urea ha-1 and 63.3 kg p2o5 kg.ha -1, respectively, whereas the optimal rate for k fertilizer for “ipb 9g” could not be determined with this study. keywords: oryza sativa, grain weight, growth phase, harvest index, plant growth rate introduction rice (oryza sativa l.) plays an important role in the food supply in asia, including indonesia. approximately 95% of the indonesian population consumes rice as a staple food (syakhril et al., 2014). norsalis (2011) reported that consumption of rice can cover 63% of the total energy adequacy and 37% of protein. efforts to increase rice production in indonesia is still focused on intensification of culture as massive conversion of agricultural land into non-agriculture has significantly decreased the rice production area (ministry of agriculture, 2004). around 5.1 million ha of dry land spread across various provinces in indonesia has potential to be used to grow upland rice (toha, 2007; hidayat, 2011). the current data from bps in 2014, however, showed that the potential area has drop into 3,292,578 ha. the common problems of developing dry land area is caused by low levels of soil fertility. the use of new rice varieties which are resistant to pests and diseases, and management of efficient application of fertilizer can potentially increase rice productivity in the dry land area. ipb university has released new superior varieties of upland rice, named “ipb 8g” and “ipb 9g”. “ipb 9g” variety has a potential yield of 9.09 tons.ha-1 with an average yield of 6.09 ton.ha-1. both varieties have resistance to several races of blast disease infection (food crop research and development, 2017). superior varieties in general require balanced nutrients, especially n, p and k, to reach their potential high yields. the availability of n, p and k on soil is the most limiting factor to rice growth and yield (munawar, 2011). putra (2012) reported that fertilizing with 200 kg npk, 50 kg urea, and 201 kg per ha could increase the yield of upland rice “situ patenggang” to 3.4 t.ha-1, or 58% higher compared to the application of 200 kg urea single fertilizer, 100 kg of sp 36 and 50 kg.ha-1 of kcl per ha. according to ramadhan (2014) application of npk fertilizer can significantly improve rice growth. in addition, krismawati and firmansyah (2014) also stated that the use of fertilizers n, p, k could increase plant height, number of tillers, panicle, grain weight, and yield of “situ patenggang” variety. most rice growers tend to use the excessive amount of fertilizer to increase yield. the objective of this study is to determine the optimum doses of n, p, and k fertilizers for upland rice “ipb 9g”. andika determination of the optimum rates for n, p, and k fertilizer for upland rice .......... journal of tropical crop science vol. 6 no. 3, october 2019 www.j-tropical-crops.com 165 material and methods time and place this study was carried out at the sawah baru experimental station of department of agronomy and horticulture, ipb university from march to july 2018. “ipb 9g” seeds were used for this study. experimental design the experiment consisted of three parallel experiments, each to determine the optimum fertilization of n, p, and k (table 1). each trial tested five levels of fertilizer doses, i.e., 0, 50, 100, 150 and 200% of the reference rate, which are 100% n = 200 kg urea; 100% p = 100 kg sp36; and 100% k = 100 kg kcl per ha (doberman and fairhust, 2000). the three experiments consisted of a total of 45 plots organized in a completely randomized block design; each treatment was replicated three times. fertilizer application the fertilizers used in this study were urea (46% n), sp36 (36% p2o5), kcl (60% k2o). the n fertilizer was applied at three stages, 40% at planting, 30% at 4 week after planting (wap), and the rest of 30% at 8 week after planting. both p and k fertilizers were applied 100% at planting. measurement and observation the physical and chemical properties of the soil were analysed prior to and after the experiment. growth parameters measured are height (cm), number of tillers, leaf area (cm2), canopy dry weight (g), yield and yield components, namely the number of productive tillers, number of grains per panicle, harvest index and grain weight per clump (g). determination of optimum fertilizer dose the optimum n, p, and k fertilizer doses were determined by calculating the relative yield, which was the value of a variable compared to the maximum value of the variable obtained from each dose, using the following formula of suminar (2016) : yi/ymax x 100% where yi = the values of the treatment variables n, p, and kith ymax = maximum values of the nutrients n, p, k variables the determination of the recommended options was conducted at the end of the study using a multinutrient response approach based on the grain weight per clump variable (jannah, 2012; rohmawati, 2013). this approach was used to determine fertilizer recommendations using the quadratic model of several fertilization experiments. the relative results were converted into quadratic equations, then used to determine the selection of recommendations. the maximum fertilizer dose was determined using the quadratic regression model y = ax2 + bx + c with y = relative plant values, x = fertilizer dosage, whereas a, b, c are constant values. the determination of the table 1. fertilizer doses of the experiment with n, p, and k fertilizer fertilizer % reference doses 0 50 100 150 200 n fertilizer experiment (kg.ha-1) n 0 46 92 138 184 p2o5 36 36 36 36 36 k2o 60 60 60 60 60 p2o5 fertilizer experiment (kg.ha -1) n 92 92 92 92 92 p2o5 0 18 36 54 72 k2o 60 60 60 60 60 k2o fertilizer experiment (kg.ha -1) n 92 92 92 92 92 p2o5 36 36 36 36 36 k2o 0 30 60 90 120 journal of tropical crop science vol. 6 no. 3, october 2019 www.j-tropical-crops.com 166 defiyanto djami adi, iskandar lubis, suwarto and sugiyanta maximum fertilizer dosage was calculated by using the derivative formula of the regression equation: dy/ dx = 2a + xb = -b/2a (suminar, 2016). results and discussion general condition based on information from the meteorology, climatology and geophysics agency (bmkg) dramaga climatology station, bogor (2018) sawah baru experimental site is located on 207 m above the sea level. the highest weekly rainfall during the experiment was 150 mm, air temperature was 25.5027°c and humidity was 76-90%. irrigation was applied according to weather condition (figure 1). soil in the experimental site is classified as latosol, containing 9.7% sand, 38.5% dust, 51.8% clay, and categorized as acidic with h2o ph of 5.0 (table 2). total of n-content, p, k, c-organic and ca were classified as moderate, very high, low, low and medium, with medium cation and mg exchange capacity (table 2). in general, the level of soil fertility in the experimental land is classified as moderate, therefore additional fertilizer was expected to increase the growth and yield of upland rice “ipb 9g”. the results of soil analysis at the end of this study showed that soil n, p and k increased compared to pre experiment (table 3). this result showed that fertilizing with n, p, k increased nutrient availability in the soil even though n and p tended to decrease due to the uptake by the crops, or washed by rainwater. in this experiment, availability of soil n and p was classified from low to high, whereas the availability of k was classified as moderate to very high. a. rainfall b. temperature c. humidity figure 1 rainfall (a), average temperature (b), and humidity (c). source: bmkg dramaga climatology station, bogor   0 20 40 60 80 100 120 140 160 1 3 5 7 9 11 13 15 a m ou nt o f r ai nf al l ( m m ) wap (weeks after planting) 70 75 80 85 90 95 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 h un id ili ty (% ) wap (weeks after planting) 24,5 25,0 25,5 26,0 26,5 27,0 27,5 1 3 5 7 9 11 13 15 te m pe ra tu re (° c ) wap (weeks after planting) figure 1. rainfall (a), average temperature (b), and humidity (c). source: bmkg dramaga climatology station, bogor determination of the optimum rates for n, p, and k fertilizer for upland rice .......... journal of tropical crop science vol. 6 no. 3, october 2019 www.j-tropical-crops.com 167 plant height, number of tillers and leaf area increasing of n doses from 0 to 200% from the reference rate linearly increased plant height, but it was not significantly affect the number of tillers and leaf area (table 4). since the soil nutrient status was categorized as moderate (0.27%), n supply is still required. nitrogen is the basic component of chlorophyll and can increase the plants’ photosynthetic capacity, hence plant growth (anhar et al., 2016). n is also involved in the formation of protoplasm, as a constituent of plant cell structure and cell division (hernita et al., 2012). increasing the dose of p from 0 to 200% of the reference rate had positive correlation with development of leaf area. according to karamanos et al. (2010) leaf area affects light interception during photosynthesis, so greater leaf area will increase photosynthesis, resulting in better plant growth. increasing the k dose from 0 to 200% of the reference rate increased the leaf area, but did not affect plant height and number of tillers (table 4). k also plays an important role in water absorption and can improve plant resistance to drought (karamanos et al., 2010). water shortages that last for a long time can hamper cell enlargement due to a decrease in the rate of photosynthesis, table 2. sawah baru soil analysis prior to the experiment* soil characteristic value status ph 5.0 acid c-organic (%) 1.77 low n-total (%) 0.27 moderate c/n 7 low p2o5 bray (ppm) 17.0 very high ktk 19.21 moderate ca (me/100g) 6.94 moderate mg (me/100g) 1.62 moderate k (me/100g) 0.16 low na (me/100g) 2.30 very high base saturation (%) 57.40 moderate * data was obtained from seameo biotrop soil laboratory table 3. sawah baru soil analysis after the experiment* treatment (%) nutrients status n (%) p (ppm) k (ppm) n (%) p (ppm) k (ppm) n0 0.20 8.17 79.21 low moderate very high n50 0.20 9.04 99.01 low moderate very high n100 0.24 11.13 59.41 moderate high very high n150 0.23 11.30 84.16 moderate high very high n200 0.20 10.78 71.78 low moderate very high p0 0.24 6.96 47.03 moderate low high p50 0.17 5.22 39.60 low low high p100 0.27 4.87 56.93 moderate low high p150 0.29 5.74 37.13 moderate low high p200 0.36 6.43 49.50 moderate low high k0 0.24 4.35 34.65 moderate low moderate k50 0.26 5.39 44.55 moderate low high k100 0.18 9.22 42.08 low moderate high k150 0.24 4.87 51.98 moderate low high k200 0.20 5.04 44.55 low low high *data was obtained from laboratory of the department of soil sciences and land resources of ipb journal of tropical crop science vol. 6 no. 3, october 2019 www.j-tropical-crops.com 168 defiyanto djami adi, iskandar lubis, suwarto and sugiyanta protein synthesis, and resulted in smaller leaf area (toha et al., 2002). k is also important for enzyme activation during photosynthesis, and regulating stomata opening and closing (song and banyo, 2011). canopy biomass dry weight increasing the n dose from 0 to 200% of the reference rate tended to increase dry weight of canopy biomass at the vegetative phase, especially at heading and harvesting time. bustami et al. (2012) demonstrated that optimum application of fertilization had a good impact on plant growth and dry weight of canopy biomass. furthermore, increasing of n dose also related to n content in the soil. in this study, soil analysis was categorized as moderate (0.23-0.27%) before and after n application. sarif et al. (2015) reported that n deficiency decreases plant growth, whereas excessive n causes pollution to the environment. treatments with p and k fertilizer doses of 0-200% of the reference increased canopy dry weight at the vegetative, primordia, heading and harvesting phases. soil nutrients availability will support absorption by the plants resulting in a better canopy growth. anisyah et al. (2014) stated that availability of p in the soil is associated with the application of p fertilizer. k availability supports photosynthesis through photophosphorylation which produces atp and nadph (novizan, 2002), resulting in greater dry weight of plant biomass (gardner et al., 2017). yield and yield components increasing n fertilizer doses from 0 to 200% from the reference rate increased the number of productive tillers, harvest index and grain weight per clump, whereas the number of grains per panicle was not significantly affected. number of tillers in the treatment of 200 kg.n-1 showed the highest value compared to the other n treatments. however, increasing of n dose reduced harvest index; application of n at 200% table 4. the effects of increasing n, p, k doses from 0 to 200% of the reference rate on plant height at 10 wap, number of tillers at 8 wap and leaf area at heading phase treatment (%) plant height (cm) number of tillers (stem) leaf area (cm2) 10 wap 8 wap heading n0 108.71 12.20 1799.38 n50 114.05 13.33 4786.35 n100 118.46 12.93 3198.66 n150 120.53 15.20 4501.70 n200 124.73 15.13 5585.15 pr>f 0.0112 0.0853 0.0707 response patternt l* ns ns p0 127.51 11.30 1724.87 p50 124.72 11.93 2559.52 p100 124.97 11.53 2404.40 p150 124.35 13.00 2212.93 p200 122.15 12.20 2846.69 pr>f 0.3545 0.4713 0.0064 response patternt ns ns l** k0 121.89 10.45 1786.23 k50 124.19 11.67 2748.58 k100 125.72 10.67 3224.81 k150 129.15 11.73 2920.28 k200 127.98 10.93 3766.72 pr>f 0.2399 0.2566 0.0139 response patternt ns ns l* note: t: orthogonal polynomial test for fertilizer dosage; l:linear; ns: not significantly different; *: significantly different at α = 0.05; **: significantly different at α=0.01 determination of the optimum rates for n, p, and k fertilizer for upland rice .......... journal of tropical crop science vol. 6 no. 3, october 2019 www.j-tropical-crops.com 169 table 5. the rice dry weight of canopy biomass at the vegetative, primordia, heading and harvesting phase treatment (%) the dry weight of the canopy biomass (g) vegetative primordia heading harvest n0 5.34 21.61 35.90 20.35 n50 8.42 24.94 49.82 24.87 n100 10.52 27.97 59.27 28.17 n150 10.28 32.61 66.61 27.59 n200 10.16 27.73 81.84 28.68 pr>f 0.0717 0.2058 <.0001 0.0395 response patternt l+ ns l** l* p0 9.92 24.17 49.59 18.05 p50 11.64 27.24 62.44 21.27 p100 13.04 28.69 64.48 20.56 p150 14.77 35.51 68.61 25.03 p200 16.19 36.65 79.16 25.25 pr>f 0.0070 0.0357 0.0022 0.0045 response patternt l** l* l** l** k0 8.93 17.38 50.41 22.96 k50 10.18 34.54 59.02 27.16 k100 11.61 31.05 59.68 25.17 k150 11.85 36.99 67.16 28.71 k200 13.50 37.45 86.63 28.25 pr>f 0.0066 0.0202 <.0001 0.0133 response patternt l** l* l** l* t: orthogonal polynomial test for fertilizer dosage; l: linear ; ns: not significantly different; *: significantly different at α = 0.05; **: significantly different at α=0.01 of the reference rate produced a low harvest index compared to 100% of the reference. proportionally, grain weight was lower than canopy dry weight, whereas the harvest index value tended to decrease in line with increasing of total dry weight of plants. n is the main constituent of protein ingredients, and has important roles in chlorophyll formation and photosynthesis processes, resulting in the promotion of plant height, number of tillers, increasing size and grain, improving plant and grain quality, increasing protein content, as well as the amount and the percentage of grain contents (misbahudi et al., 2017). manurung and ismunadji (1988) reported that the range of rice crop index to obtain high yield was ± 0.5. p fertilizer 0 to 200% of the reference rate generally did not affect the number of productive tillers, the number of grain per panicle, harvest index, and grain weight per clump. however, the excessive nutrient levels over plant needs only caused an increasing of p level in the soil and no longer effect on plant growth. similar with n, optimum level of p correlates positively with p availability in the soil (fitriatin et al., 2008). furthermore, setiawati et al. (2016) reported that phosphate levels in the soil can be increased by fungi; fungi possess phosphatase enzymes which can hydrolize inositol hexaphosphate. increasing of k doses from 0 to 200% from the reference rate increased grain number per panicle and grain weight per clump and harvest index, but did not affect the number of grain per panicle. k plays an essential role in translocating assimilates from leaves to all plant organs whereas can accelerate photosynthesis (muslim, 2017). the best timing of potassium absorption on rice plant is tillering which can increase the number of panicles and grain, and in the primordial phase which helping the increment of both weight and yield of grain (salbiah et al., 2012). hardjowigeno (2003) stated that potassium is very important in plant physiology processes. lubis (2018) demonstrated that application of higher concentration of k increased the circumference of belitung taro (xanthosoma sagittifolium (l.) stem. journal of tropical crop science vol. 6 no. 3, october 2019 www.j-tropical-crops.com 170 defiyanto djami adi, iskandar lubis, suwarto and sugiyanta recommendations for n, p and k for rice “ipb 9g” fertilizer recommendations were made based on grain weight per upland rice clump of the “ipb 9g”. the yields were converted into relative results so that the three trials could be compared. fertilization recommendations cannot be determined by a multinutrient response approach as the observation characters with quadratic curves are only found in the n and p experiments on the grain weight per clump (figure 2). therefore the fertilizer recommendations were determined using the optimum dose range of the growth variables. based on the quadratic equation of relative results (figures 2a and 2b) the optimum dose of n was 107.9 kg n.ha-1, and the optimum dose of p was 63.3 kg p2o5.ha -1. the optimum dose of k could not be determined because a negative value of -11.27 kg k2o ha -1 was obtained. the relative yield of “9g ipb” upland rice, which consists of relative value of n, p and k, are described in figure 2a, 2b and 2c, respectively. the relative yield showed that grain weight per clump was significantly affected by n and p fertilization treatments with quadratic response pattern, whereas the grain weight per clump was affected by k fertilization treatment with linear response pattern. conclusion the optimum dose of k fertilizer can not be determined from this experiment. the optimum doses of n and p fertilization for upland rice “ipb 9g” in sawah baru were 107.09 kg n.ha-1 and 63.3 kg p2o5. ha -1, respectively. table 5. yield and yield components treatment (%) number of productive tillers number of grain per panicle harvest index grain weight per clump (g) n0 9.56 210.03 0.40 49.73 n50 10.44 210.38 0.37 100.72 n100 11.44 215.59 0.41 89.38 n150 11.11 211.51 0.35 83.16 n200 13.89 226.69 0.26 105.06 pr>f 0.0033 0.5447 0.0062 0.0027 response patternt l** ns l** l** p0 9.89 202.69 0.41 65.48 p50 12.44 224.69 0.43 93.41 p100 11.44 220.29 0.37 93.13 p150 11.78 245.20 0.42 98.62 p200 12.11 231.69 0.41 88.20 pr>f 0.1799 0.1625 0.2246 0.1404 response patternt nsd nsd nsd nsd k0 11.22 216.54 0.45 93.48 k50 13.00 229.28 0.35 87.69 k100 12.44 232.10 0.43 97.25 k150 12.22 237.35 0.41 104.42 k200 13.22 260.46 0.47 116.86 pr>f 0.3675 0.0254 0.0186 0.0368 response patternt ns l* q* l* t: orthogonal polynomial test for fertilizer dosage; l: linear ; ns: not significantly different; *: significantly different at α = 0.05; **: significantly different at α=0.01 determination of the optimum rates for n, p, and k fertilizer for upland rice .......... journal of tropical crop science vol. 6 no. 3, october 2019 www.j-tropical-crops.com 171 a. b. c figure 2. the relative value of grain weight per experimental group; n (a), p (b), and k (c) based on the quadratic equation of relative results (figures 2a and 2b) the optimum dose of n was 107.9 kg n.ha-1, and the optimum dose of p was 63.3 kg p2o5.ha-1. the optimum dose of k could not be determined because a negative value of -11.27 kg k2o ha-1 was obtained. the relative yield of “9g ipb” upland rice, which consists of relative value of n, p and k, are described in figure 2a, 2b and 2c, respectively. the relative yield showed that grain weight per clump was significantly affected by n and p fertilization treatments with 44.47 91.22 75.69 94.70 58.50 y = -0.0038x2 + 0.8139x + 47.838 r² = 0.7262 0 20 40 60 80 100 120 0 50 100 150 200 r el at iv e va lu e (% ) 100% = 92 kg n per ha 82.28 83.75 87.37 78.00 77.89 y = -0.0005x2 + 0.0633x + 82.454 r² = 0.6137 0 20 40 60 80 100 120 0 50 100 150 200 r el at iv e va lu e (% ) 100 % = 36 kg p2o5 per ha 74.19 80.88 86.36 96.63 116.81 y = 0.0009x2 + 0.0203x + 75.32 r² = 0.988 0 20 40 60 80 100 120 0 50 100 150 200 r el at iv e va lu e (% ) 100% = 60 kg k2o per ha figure 2. the relative value of grain weight per experimental group; n (a), p (b), and k (c) references anhar, r., hayati, e., and efendi, e. 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(2015). pertumbuhan dan hasil tanaman sawi (brassica juncea l.) akibat pemberian berbagai dosis pupuk urea. jurnal agrotekbis 3, 585-591. setiawati, m.r., sofyan, e.t., and mutaqin, z. (2016). pengaruh pupuk hayati padat terhadap serapan n dan p tanaman, komponen hasil dan hasil padi sawah. jurnal agroteknologi 8, 120-130. song, a.n., and banyo, y. (2011). konsentrasi klorofil daun sebagai indikator kekurangan air pada tanaman. jurnal ilmiah sains 11, 166-173. suminar, r. (2016). penentuan dosis optimum pemupukan n, p, dan k pada sorgum (sorghum bicolor l. moench) dengan metode multinutrient response [thesis]. institut pertanian bogor. toha, h.m., permadi, k., and munarso, s.j. (2002). pengaruh pemberian pupuk kalium dan nitrogen terhadap hasil padi dan mutu beras varietas ir64. penelitian pertanian tanaman pangan 21, 20-29. toha, h.m. (2007). peningkatan produktivitas padi gogo melalui penerapan pengelolaan tanaman terpadu dengan introduksi varietas unggul. jurnal penelitian pertanian tanaman pangan 26, 180-186. krisantini sticky note 271 pp. (tambahkan sebelum pt mandar maju.) � krisantini cross-out krisantini sticky note 39-54. krisantini sticky note 240 pp. institut pertanian bogor determination of the optimum rates for n, p, and k fertilizer for upland rice .......... journal of tropical crop science vol. 6 no. 3, october 2019 www.j-tropical-crops.com 173 syakhril, r. and arsyad, h. (2014). pengaruh pupuk nitrogen terhadap penampilan dan produktivitas padi inpari sidenuk. agrifor 13, 85-92. journal of tropical crop science vol. 9 no. 1, february 2022 www.j-tropical-crops.com 8 r.a.d. widyastuti, rahmat budiarto, kus hendarto, hayane adeline warganegara, indah listiana, yoyon haryanto, and helvi yanfika fruit quality of guava (psidium guajava ‘kristal’) under different fruit bagging treatments and altitudes of growing location r.a.d. widyastutia, rahmat budiartob, kus hendartoa, hayane adeline warganegaraa, indah listianaa, yoyon haryantoc, and helvi yanfikaa a faculty of agriculture, lampung university, jl. sumantri brojonegoro no. 1, bandar lampung, lampung, indonesia. b department of agronomy, faculty of agriculture, universitas padjadjaran, sumedang, 45363, indonesia c agricultural development polytechnic, jl. aria surialaga no. 1, pasirjaya, bogor, west java, indonesia *corresponding author; email: radianawidyatuti@yahoo.co.id abstract fruit quality is an important aspect that requires attention and more study when it comes to commercializing tropical fruits, including guava ‘kristal’. this study aimed to evaluate the effect of bagging treatment and altitude of growing orchard on fruit quality of guava (psidium guajava var. ‘kristal’). this study was conducted at two local orchards managed by small scale farmers, at gunung batu (1000 meter above sea level, m asl) and brajaselebah (25 m asl), lampung province, indonesia. nested design was applied to test 5 types of bagging treatments and 2 levels of land altitude. fruit quality was assessed by measuring both physical and chemical characteristics. fruit size indicated by fruit weight and diameter was significantly larger in lowland compared to highland. the total soluble solid (tss) was significantly higher in lowland compared to highland, while titratable acid (ta) showed an opposite result. vitamin c of the fruits varied from140 to 146 mg per 100 g. different types of fruit bagging and land altitudes did not affect the physical and chemical characteristics of “kristal” guava fruits. fruit scar intensity was higher in lowland, especially in fruit without bagging. fruit bagging is highly recommended for farmer due to the success of this treatment to reduce fruit scar intensity, thus resulting in good fruit quality, irrespective of the growing locations. keywords: land altitude, fruit quality, total soluble solid, titratable acid, vitamin c introduction guava (psidium guajava) is one of the popular tropical fruits worldwide due to its delicious taste and nutritious content. earlier studies reported that guava is rich in vitamin c, comparable to citrus and pomelo (kalsum et al., 2015; romalasari et al., 2017). one of the popular guava varieties in indonesia is ‘kristal’; it was introduced from vietnam and recently widely cultivated due to the suitability to agroclimatic factors in indonesia. several studies have been conducted to improve guava tree growth and to increase the production by applying biofertilizer (widyastuti et al., 2021a), organic fertilizer (widyastuti et al., 2021b), artificial shading (budiarto et al., 2019), and canopy manipulation (widyastuti et al., 2019a; budiarto et al., 2018). more efforts should be made to improve fruit quality in guavas, in addition to increasing fruit production (musyarofah et al., 2020). fruit quality is an important issue, especially for products that are consumed in raw and fresh, such as guavas. guava fruit quality derived from conventional farming may be subjected to pesticide residue and inappropriate pesticide dosage. in order to achieve profitable agribusiness and minimize the negative impact on the environment, sustainable agriculture practices are recommended (listiana et al., 2021). in a sustainable agriculture, there is a need to replace chemical pesticide with more environmental-friendly substances such as biopesticide. in addition to biopesticide, physical treatments to minimize the deterioration of fruit quality, such as fruit bagging, are needed. fruit bagging is suitable in guava organic systems (neto et al., 2020). fruit bagging can improve fruit coloration (kim et al., 2008) and reduce the damage from pest, disease, sunburn, mechanical injuries and agrochemical residues (jing et al., 2008; sharma et al., 2014; xu et al., 2018; campbell et al., 2021). fruit bagging has been widely used in numerous fruit fruit quality of guava (psidium guajava ‘kristal’) under different fruit bagging .......... journal of tropical crop science vol. 9 no. 1, february 2022 www.j-tropical-crops.com 9 commodity, such as apple (frank 2018; teixeira et al., 2011), pomegranate (bagle, 2011), mango (sarker et al., 2009), litchi (debnath and mitra 2008), loquat (ko et al., 2010), pear (wang et al., 2011) and guava (sharma et al., 2020; parameswara and susanto, 2019; morera-montoya et al., 2010). more studies should be conducted to improve the success of fruit bagging in maintaining fruit quality (ali et al., 2021). to achieve the optimum fruit bagging treatment, it is necessary to evaluate this technique on both low and high altitude areas, where guavas are grown. the difference of altitude of the growing location may cause variation of agroclimatic, yield, growth and plant metabolite content (efendi et al., 2021; widyastuti et al., 2019b). earlier study has implied that metabolite content is an important variable related to ground cherry fruit quality (mubarok et al., 2021). unfortunately, there is still limited study reporting the effect of guava fruit bagging in different growing location. therefore, this study aimed to evaluate the fruit quality of ‘kristal’ guava in response to different fruit bagging treatment and altitude of growing location in indonesia. material and methods study site this study was conducted between march to june 2021 at two local guava orchards managed by small scale farmers, at gunung batu (1000 meter above sea level, m asl) and brajaselebah (25 m asl), lampung province, indonesia. the orchards grow three-year-old guava trees (psidium guajava var. kristal) that were being managed under intensive cultures, as indicated by triannual pruning, monthly weed control, precisely determined fertilizer doses, i.e., 40 kg of organic fertilizer and 250 g npk per tree. this study was arranged in a nested design, with two factors, i.e., bagging treatment and altitude of growing location. measured fruits were collected from 5 bagging treatments: (1) white plastic bag, (2) black plastic bag, (3) blue plastic bag, (4) transparent plastic bag and (5) no plastic bag/control. the guava trees in this study were grown at low altitude (100 m asl) or high altitude orchard (800 m asl). fruits with no fruit scar were bagged when the fruit diameter reached 2 cm. the bagging treatment was maintained until the day of harvesting. the bagging was done on the same day, during a sunny morning. there were 10 combination treatments and were replicated 10 times, so that there were a total of 100 fruits from 20 individual trees prepared for observation. at harvest, fruits were manually picked and stored in a close container during the transfer to the laboratory. the fruits were measured for diameter, weight, softness, scar intensity, vitamin c content, titratable acid content, total soluble solid, and ratio of total soluble solid to titratable acid. fruit diameter (cm) was measured using digital caliper on the widest part of the fruit, i.e., the middle. fruit weight (g) was measured using digital analytical balance and expressed in wet basis. fruit softness was measured using penetrometer and expressed in mm per 50 g per second. this measurement was conducted in three spots within a fruit, i.e., upper, middle and lower parts. fruit scar intensity (%) was measured based on the ratio of the scar area to the total peel area. the entire fruit peel was detached from the fruit and then scanned using imagej software for area calculation. vitamin c (mg per 100 g-1) content was measured in guava juice by following standard iodine titration method (rahman et al., 2015). titratable acid (%) was measured by using 0.1 n naoh titration method and indicator of phenolphthalein following sadler and murphy (2010). total soluble solid (0brix) was measured by subjecting the guava juice on refractometer lens (pal-1 atago). data analysis all measurements were subjected to analysis of variance (anova) and duncan post hoc test at α = 5%, if any significant differences were found. statistical analysis was done using the program star version 2.0.1. result and discussion the altitude of growing location significantly affected all fruit quality variables, except fruit softness and vitamin c. additionally, fruit bagging significantly affected both fruit scar intensity and total soluble solids of kristal’ guava. guava fruit quality with different bagging treatments is similar, both at low and high altitudes (table 1). the difference of bagging treatment showed slight variation in fruit diameter, i.e., ranging from 7.887.85 cm (figure 1a). in contrast, the fruit diameter was significantly higher in fruit harvested from lowland (8.03 cm) than fruit from highland (7.65 cm) (figure 1a). the greater diameter observed in lowland-originated ‘kristal’ guava fruit was supported by greater fruit weight obtained. the fruit weight of lowland fruit was 259 g and it was significantly higher than the highland ones, i.e., 217 g (figure 1b). there was approximately 19% weight improvement among lowland compared to highland fruits. this finding was supported by results from previous studies (e.g. journal of tropical crop science vol. 9 no. 1, february 2022 www.j-tropical-crops.com 10 r.a.d. widyastuti, rahmat budiarto, kus hendarto, hayane adeline warganegara, indah listiana, yoyon haryanto, and helvi yanfika musyarofah et al., 2020; widyastuti et al., 2019a). fruit weight is an important attribute of ‘kristal’ guava. most consumers prefer the ‘kristal’ guava fruit with a weight of more than 250 g than the small/light one (unpublished data). thus, the production of heavier fruit (>250 g) could potentially improve the income and profit of fruit farmers. the fruit softness of ‘kristal’ guava varied from 0.420.46 mm.g-1.s-1 (figure 1c). the fruit softness is related to the composition of the cell wall, such as cellulose, hemicellulose, lignin, and pectin; where the decline of these variables lead to the softer fruit texture (jain et al., 2003). in contrast to fruit softness, the fruit scar intensity was significantly affected either by land altitude and fruit bagging treatment (table 1). the intensity of fruit scar was significantly higher in the control fruits compared to bagged fruit, irrespective of color variation of the bags. the control fruit had fruit scar intensity of about 7.7%, while transparent bagged fruit had 0.88%, followed by black, white and blue bagging treatment of 1.75%, 1.85%, and 2.5%, table 1. the result of analysis of variance of ‘kristal’ guava fruit quality in response to different fruit bagging treatments and altitudes treatment fw fd fs fsi tss ta tss/ta vc fruit bagging (f) ns ns ns ** ** ns ns ns land altitude (l) ** ** ns ** ** ** ** ns interaction f x l ns ns ns ns ns ns ns ns note: fwfruit weight, fdfruit diameter, fsfruit softness, fsifruit scar intensity, tsstotal soluble solid, tatitratable acid, tss/taratio of tss to ta, vcvitamin c, ns – not significantly different, * * significantly different. figure 1. physical quality of ‘kristal’ guava fruit in response to two factors, i.e., fruit bagging treatment and land altitude. note: 1a – fruit diameter, 1b – fruit weight, 1cfruit softness, 1d fruit scar incidence. the different alphabet above the rectangular bar is significantly different among the same factor based on the duncan’s multiple range test at α= 0.0-5; the error bar represents the standard deviation. figure 1. physical quality of ‘kristal’ guava fruit in response to two factors, i.e., fruit bagging treatment and land altitude. note: 1a – fruit diameter, 1b – fruit weight, 1cfruit softness, 1dfruit scar incidence. the different alphabet above the rectangular bar is significantly different among the same factor based on the duncan’s multiple range test at α= 0.0-5; the error bar represents the standard deviation. fruit quality of guava (psidium guajava ‘kristal’) under different fruit bagging .......... journal of tropical crop science vol. 9 no. 1, february 2022 www.j-tropical-crops.com 11 respectively (figure 1d). the higher scar intensity in control fruits could be attributed to natural scarring pest infections in the orchards. the bagging practice was proven to reduce the damage of scarring pest on fruit appearance quality. this finding was in agreement with previous studies in taiwanese guava (moreramontoya et al., 2010), apple (frank, 2018; teixeira et al., 2011), pomegranate (bagle 2011), mango (sarker et al., 2009), litchi (debnath and mitra, 2008), loquat (ko et al., 2010) and pear (wang et al., 2011). besides physical quality, there was also the assessment of chemical fruit quality, as indicated by total soluble solid (tss), titratable acid (ta) and vitamin c content. the highest tss of 8.86 0brix was recorded in ‘kristal’ guava fruits that were covered by white plastic, whereas the lowest tss was from the control fruit, i.e., 8.55 0brix (figure 2a). however, the variation of tss among four bag colors was not significant and ranged from 8.74-8.86%. in response to different growing altitude, the fruit from lowland had significantly higher tss (8.90 0brix) compared to those from the highland (8.57 0brix). this finding was similar to the result of previous studies (musyarofah et al., 2020). in contrast, high-altitude fruits had a significantly higher ta (figure 2b). this finding was in agreement with an earlier study (solarte et al., 2014), however it was in contrast with another study (parracoronado et al., 2018). present findings implied that kristal guava fruit had more acid and taste more sour compared to the fruit from lowland. fruit maturation stage highly influenced the level of tss and ta in fruit, where the ta decreased and tss increased as fruit ripening occurred (dolkar et al., 2017). chemical quality of ‘kristal’ guava fruit in response to fruit bagging treatments and land altitude. note: 1atotal soluble solid, 1btitratable acid, 1cratio of total soluble solid to titratable acid, 1dvitamin c. the different alphabets above the rectangular bar show significantly different among the same factor based on the duncan’s multiple range test at α =0.05; the error bar represents the standard deviation. figure 2. chemical quality of ‘kristal’ guava fruit in response to fruit bagging treatments and land altitude. note: 1atotal soluble solid, 1btitratable acid, 1cratio of total soluble solid to titratable acid, 1dvitamin c. the different alphabets above the rectangular bar show significantly different among the same factor based on the duncan’s multiple range test at α =0.05; the error bar represents the standard deviation. figure 2. chemical quality of ‘kristal’ guava fruit in response to fruit bagging treatments and land altitude. note: 1atotal soluble solid, 1btitratable acid, 1cratio of total soluble solid to titratable acid, 1dvitamin c. the different alphabets above the rectangular bar show significantly different among the same factor based on the duncan’s multiple range test at α =0.05; the error bar represents the standard deviation. journal of tropical crop science vol. 9 no. 1, february 2022 www.j-tropical-crops.com 12 r.a.d. widyastuti, rahmat budiarto, kus hendarto, hayane adeline warganegara, indah listiana, yoyon haryanto, and helvi yanfika the fruit taste evaluated by consumers did not solely depend upon the ta or tss of the fruit. both tss and ta simultaneously influenced the taste of ‘kristal’ guava fruit (susanto et al., 2019). this finding revealed that only the altitude of growing location that significantly influenced the ratio of tss and ta (table 1). the ratio of tss/ta in fruit from lowland was 17.46 while fruit from lowland was 14.89 (figure 2c). the fruits from lowland have sweeter taste compared those from highland. further studies are required to confirm the range of tss/ta ratio that suit consumer preference. the content of vitamin c with different fruit bagging systems and altitude was similar. the vitamin c content of guava fruit from different bagging treatment varied from 140 to 146 mg per 100 g, and from 141 to 145 mg per 100 g in different land altitude (figure 2d). in contrast to the present finding, earlier studies reported the significant variation of vitamin c content in guava in during ripening and at different geographical sites (gull et al., 2012) and cultural practice (musyarofah et al., 2020). the high content of vitamin c in guava fruit is important for human health (lee and kader, 2000) and its role as a strong antioxidant (macan et al., 2019). conclusion our study reported the fruit quality of “kristal” guava that were grown on lowland and highland with different bagging treatments. fruit weight and diameter was significantly higher when guava trees were grown on lowland compared to those grown on highland. the total soluble solid was significantly higher in lowland than highland, whereas titratable acid was lower. vitamin c content was not significantly affected by fruit bagging treatments and altitude. fruit scar intensity in both lowland and highland was reduced with fruit bagging. fruit scar intensity was higher in lowland than in highland, especially without fruit bagging. acknowledgement this work was financially supported by the institute for research and community service, university of lampung, through applied research scheme, fiscal year 2021, contract number 1620/un26.21/pn/2021. references ali, m.m., anwar, r., yousef, a.f., li, b., luvisi, a., bellis, l.d., aprile, a., and chen, f. 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(2018). the effects of fruit bagging on residue behavior and dietary risk for four pesticides in apple. scientific reports 8, 14348. doi: 10.1038/ s41598-018-32358-6 3. endah r. palupi_vol2_2014.cdr abstract large scale exploitation of for biofueljatropha curcas production is restrained by low productivity hence economically not pro�table. one of the main constraints is due to limited number of female �owers in an in�orescence which eventually develop into fruits that bear seed. investigation on �oral biology and reproductive potential of the species had been carried out, but seems to vary among sites the research was. conducted to study reproductive success and the compatibility among indonesian accessions.jatropha the research was set up in two experiments. the �rst experiment was conducted at the seedjatropha orchard, pakuwon, sukabumi, west java from april to july 2007 using four-yearold trees of accessions from lampung, banten, west java, and central java. the second experiment was conducted at leuwikopo experimental station of bogor agricultural university from april to july 2008 using one-year-old trees of accessions from lampung, bengkulu, palembang, and kediri. flowers were obtained from controlled self and cross-pollinated as well as left for open-pollination. the result showed that reproductive success varied among accessions; the greatest success was 0.73 obtained from a west java accession and the least was 0.53 from banten accession. the accessions werejatropha categorized as partially self-incompatible as indicated by index of self-incompatibility (isi) that ranged 0.930.99. banten accession produced highly viable and vigorous seeds regardless of pollination methods; accessions of central java produced higher viability seeds when cross-pollinated within accession, whereas those from west java and lampung produced higher viability seeds when self-pollinated. keywords: accessions, , self-jatropha curcas pollination, self-incompatibility, seed viability introduction the need of renewable energy has encouraged many parties to �nd sources of biofuel. isjatropha curcas considered as one of the potential species not only for biofuel production but also biogas and compost (openshaw, 2000, tiwari et al., 2007, achten et al., 2008). d speciesrought tolerant can survive in marginal land thus avoid competition with food crops which can only grow in fertile land. however, large scale exploitation is restraint to low productivity due to the low number of female �owers, i.e. averaged 10 female �owers per in�orescence (adikarsih and hartono, 2007). moreover a long-term systematic breeding program has not been set up which make the species is still considered as wild and exhibits variability in productivity among individuals (achten et al., 2008). the biodiesel development program in indonesia has been set up to grow plantations consisting of 1.5 million ha of oil palm, 1.5 million ha of , 0.75 million hajatropha of sugar cane and 1.5 million ha of cassava. the development of 1.5 million ha of plantationsjatropha during 2007-2010 required about 900 ton of seeds (hasnam, 2007) or about 0.6 kg.ha . seed procurement -1 is also facing several limitations that contributed to low seed viability such as time of harvest in relation to fruit maturity, seed processing and drying (sudjindro, 2008). the project was started with the exploration of jatropha germplasm in nine provinces which consisted of 54 districts and 200 000 stem cuttings and seeds had been collected from these areas. the collected germplasm were then grouped into 12 accessions, i.e. west sumatera, lampung, banten, west java, central java, east java, west nusa tenggara, east nusa tenggara, south sulawesi, gorontalo and maluku. variations in plant morphology including leaf thickness, leaf shape and size, leaf color, pedicel length, fruit shape and size, seed weight and size, number of seed per capsule were found among the accessions (hasnam, 2007). reproductive success and compatibility among accessions of jatropha curcas in indonesia. endah r. palupi *, ade s. ahmad , ro�q affandi , abdul qadir , and eni randriani a a a a b departmen agronomy and horticulture, bogor agricultural university, bogor, indonesia 16680 a indonesian centre for estate crops research and development, sukabumi, indonesia b *corresponding author; email: erpalupi@yahoo.co.id research article 11 journal of tropical crop science vol. 1 no. , 20142 october www.j-tropical-crops.com the germplasm were then planted in three locations: pakuwon, sukabumi, west java representing wet areas, asembagus, situbondo, and east java representing dry areas, and muktiharjo, pati central java representing moderately dry areas. after a mass selection the �rst generation of improved-population was obtained in 2006, i.e. ip-1p (pakuwon), ip-1a (asembagus), and ip-1m (muktiharjo) with productivity of about four to �ve t.ha .year at the fourth year after -1 -1 planting. the second generation of the improvedpopulation (ip-2) had a higher productivity of about six to seven t.ha .year at the fourth year after planting. the -1 -1 third generation (ip-3) was expected to have productivity of about eight to ten t.ha .year after the -1 -1 fourth year. after the third generation of improvedpopulation a long-term breeding plan will be required to increase productivity further, using either conventional or molecular techniques in order to become ajatropha more economically attractive (heliyanto et al., 2008). conventional technique for plant improvement through controlled pollination or hybridization requires information on �owering phenology and pollination system as well as compatibility among accessions. hartati (2008) studied the �owering phenology of jatropha in pakuwon, sukabumi during april-october and reported that pollination was best conducted in either late morning (when the stigma has completely splayed), or afternoon on the �rst day of anthesis, although the pistillate (female) �ower lasts for several days. this research was aimed at studying �owering biology, reproductive success of several accessions and the compatibility among accessions of injatropha indonesia. materials and methods the research was set up in two experiments. the �rst experiment was conducted at the seedjatropha orchard, pakuwon, sukabumi, west java, about 450 m above sea level, with latosol soil and type b climate (oldeman) from april to july 2007. four-year-old plants from four accessions were used i.e. accessions from lampung, banten, west java, and central java with 20 plants from each accession. the second experiment was conducted at leuwikopo experimental station, bogor agricultural university, about 250 m above sea level, from april to july 2008. one-year-old plants of accessions from lampung, bengkulu, palembang, and kediri (from stem cuttings) taken from the seed orchard in pakuwon were used with 20 plants from each accession. type of in�orescence, ratio of male to female �owers, as well as time and duration of blooming were observed. reproductive success, which was de�ned as the number of ovules that develop into viable seeds (wiens et al., 1987) was observed in open-pollinated population during the �rst and second experiment which was started with the number of in�orescence per plant and number of female or hermaphroditic �ower per in�orescence (fl/in�). fruit set (fr/fl) was calculated as the ratio of female/hermaphroditic �owers that developed into mature fruits in an in�orescence, whereas number of ovule per �ower (o/fl) was counted as averaged of 20 �owers per accession, which resulted in three ovules per �ower. therefore seed set (sd/o) was calculated as the proportion of the three ovules that developed into viable seeds in a fruit. reproductive success was calculated from fr/fl ratio and sd/o ratio. the �owers were tagged with color threads when once opened. the number and time of �ower or fruits abortion were observed every alternate day. the experiment was arranged in a completely randomized design and analyzed using duncan's multiple range tests (dmrt) t o c o m p a r e t h e r e p r o d u c t i v e s u c c e s s a m o n g accessions. the compatibility among accessions in the �rst experiment (four-year-old plants) was recorded from four types of hand controlled-pollinations, i.e.: 1. intra-self-pollination (sp-intra): pistillate �owers were pollinated with pollen from staminate �owers of the same in�orescence. 2. inter-self-pollination (sp-inter): pistillate �owers were pollinated with pollen from staminate �owers of different in�orescence of the same plant. 3. intra-cross-pollination (cp-intra): pistillate �owers were pollinated with pollen from staminate �owers of other plants within the same accession. 4. inter-cross-pollination (cp-inter): pistillate �owers were pollinated with pollen from staminate �owers of other accession. accession from south sulawesi was used as pollen donor for the four selected accessions because it had high staminate �ower production. all pistillate �owers were bagged at 06.00-07.00hrs in the morning and pollinated during 10.00-12.00hrs. based on the results from the �rst experiment selfand cross-pollination of the second experiment (on oneyear-old plants) were arranged as follows: 1. self-pollination: pistillate �owers were pollinated with pollen from staminate �owers of the same or different in�orescence of the same plant. 2. cross-pollination: pistillate �owers were pollinated with pollen from staminate �owers of other accession. ip-1p was used as pollen donor for the four selected accessions. the observation on each accession included fruit set from selfand cross-pollination, index of self12 endah r. palupi, ade s. ahmad, ro�q affandi, abdul qadir, and eni randriani journal of tropical crop science vol. 1 no. , 20142 october www.j-tropical-crops.com incompatibility. seed quality was determined based on germination percentage, and germination speed was analyzed according to ista (2007). the data was analyzed using split-plot design, with accession as the main plot and pollination type as the sub-plots. index of self-incompatibility (isi) was de�ned as the ratio of fruit set from self-pollination to cross-pollination. the isi values were then classi�ed into: isi 1: self-≥ compatible; 0.2<isi<1: partially self-incompatible; isi<0.2: mostly self-incompatible; isi=0: completely self-incompatible (zapata and arroyo, 1978). results and discussion the �rst in�orescence of the one-year-old plants was commonly unisexual. staminate in�orescences were observed in lampung and kediri accessions whereas pistillate in�orescences in bengkulu and palembang accessions. the subsequent in�orescences in all plants were bisexual. in most cases staminate and pistillate �owers were born on the same in�orescence (monoecious) and rarely staminate and hermaphroditic (andromonoecious) (figure 1). most of the plants from central java produced staminate and hermaphroditic �owers. the �ower sex was indicated by the size of the �ower bud; the hermaphroditic �ower buds was the largest followed by pistillate and staminate �ower buds. the staminate �owers of the four-year-old plants usually bloomed around six days earlier than the pistillate �owers and �owering continued for several days. the pistillate �owers bloomed only for two to �ve days and the �owering period coincided with that of staminate �owers. on the other hand, the pistillate �owers of the one-year-old plants bloomed 2 days earlier than the staminate �owers and last for 2-6 days, followed by the staminate �owers for several days. in this case only late blooming pistillate �owers coincided with the blooming staminate �owers. raju and ezradanam (2003) reported that is a cross pollinating species,jatropha therefore different blooming time of male and female �owers within an in�orescence is bene�cial to prevent sel�ng. battacharya et al. (2005) added that spp.apis were the most frequent visitor of �owers at thejatropha same time they deposited pollen grains on the stigmatic surface, thus generating cross-pollination. figure 1. in�orescence: (a) male (red arrow)jatropha and female (yellow arrow) �owers (b) male (red arrow) and hermaphroditic (yellow arrow) �owers the number of in�orescence of the four-year-old plants varied among accessions and was fewer compared to that of the one-year-old plants. however, the a b note: numbers of the same set of experiment and the same column followed by different letters were signi�cantly different based on dmrt at = 0.05 1 number of blooming in�orescence per plant 2 percentage of staminate in�orescence: in�orescence that produces only staminate �ower 3 number of female or hermaphroditic �owers per in�orescence 4 number of maturing fruits per in�orescence 5 fruit to �ower ratio (female or hermaphroditic): the proportion of female or hermaphroditic �ower that develop into mature fruit 6 number of �lled seeds per fruit 7 seed to ovule ratio: the proportion of ovule that develop into viable seed (based on three ovules per �ower) 8 reproductive success 13reproductive success and compatibility among accessions of jatropha.......... table 1. reproductive characters and reproductive success of each accession (from open pollination) accassion lampung banten west java central java average lampung bengkulu palembang kediri average in�/pl 1 3.90b 5.15a 2.65c 3.40bc 5.57 6.02 6.29 5.02 5.73 st. in�.(%) 2 14.1 21.4 15.1 17.1 16.9 1.7 0 0 0.9 0.7 fl/in� 3 1.93 5.33 3.50 4.63 4.60 8.88a 7.35ab 9.10a 6.34b fr/ln� 4 3.80 3.69 2.62 3.98 3.52 5.63 5.82 6.46 4.75 5.67 fr/fl 5 0.77ab 0.69bc 0.75bc 0.86ab 0.62 0.80 0.73 0.73 0.72 sd/fr 6 0.36 2.27 2.28 2.57 2.37 2.74 2.76 2.80 2.66 2.74 sd/o 7 0.78 0.76 0.76 0.85 0.79 0.88 0.94 0.92 0.86 0.90 rs 8 0.61b 0.53b 0.59b 0.73a 0.56 0.74 0.68 0.66 0.66 four-year-old plants one-year-old plants journal of tropical crop science vol. 1 no. , 20142 october www.j-tropical-crops.com percentage of staminate in�orescence was higher in the older (averaged 16.9%) than the younger plants (averaged 0.7%) (table 1). the four-year-old plants were planted in the �eld with 2 x 2 m spacing; while the one-year-old plants were planted in polybags. it is possible that the higher light intensity in the �eld led to the formation of more male �owers. investigation on environmental factors affecting sex ratio will be valuable to increase production of female �owers which could lead to an increase in fruit yield. accession from banten produced the greatest number of in�orescence whereas that of west java had the fewest. the number of pistillate or hermaphroditic �ower per in�orescence as well as the number of fruits per in�orescence was not signi�cantly different among accessions, ranged 3.5-5.33 in the four year-old and ranged 6.3-9.1 in the one-year-old plants (table 1). again, the data showed that the performance of the oneyear-old was better than the four-year-old plants. however, the fruit to �ower ratio (fruit set) from both plants was similar (table 1), ranged 0.62-0.86 indicating that the number of pistillate or hermaphroditic �ower per in�orescence was a major yield component and at the same time the limiting factor of kernel production. the data implied that increasing the number of pistillate �ower per in�orescence would be the key to increase the productivity. not all pistillate or hermaphroditic �owers can develop into mature fruits. bhattacharya et al (2005) reported that fruit set from pistillate �owers was only 50%, due to fruit abortion during development. in this research, abortion at �owering stage, i.e. �owers that aborted within 4 days after anthesis, was around 5% in the four-year-old and 1% in the one-year-old plants. the abortion during fruit and seed development ranged 9-33%. the average number of seeds per fruit in both experiments ranged 2.27-2.57 and 2.66-2.80 although on average three ovules were found in one ovary. hermaphroditic �owers in this experiment can produce up to four seeds per fruit. the seed to ovule ratio (seed set) was 0.76-0.85 in the four-year-old plants and 0.860.92 in the one-year-old plants, and did not vary among accessions. the data indicated that seed set was reasonably high although not all ovules developed into viable seeds. insuf�cient pollination could be the cause of the less than maximum seed set and could be overcome by increasing the population of the insect pollinators. social bees, i.e. andapis cerana apis dorsata were reported as effective pollinators for jatropha in indonesia (rianti et al., 2010), hence could be employed to increase seed production. in nigeria, however, where the most effective insectjatropha pollinators were not social bees, i.e. chrysomya chloropyga eristalis tenax(diptera-calliphoriade) and (diptera-syrphidae) (alamu et al., 2013), introduction and maintenance of the insect population could be more dif�cult. data on reproductive success showed similar range from the four-year-old and the one-year-old plants, between 0.53-0.74, meaning that from 100 ovules produced, only 53-74 developed into viable seeds. this data indicated that seed production was lower than the yield potential; therefore cultural techniques need to be explored to increase fruit and seed set. abortion at the �owering stage following controlled pollination was low, i.e. < 3% in cross-pollination and self-pollination during both experiments. however, abortion at the fruit development stage was as high as 32% in the four-year-old and 20% in the one-year-old plants. fruit abortion in self-pollination was similar to that in cross-pollination. the data indicated that the abortion was not due to incompatibility as fruit set from self-pollination was not signi�cantly different from that of fruit set (%) seed set (%) 1 fruit set (%) seed set (%) lampung 74.3 54.3 65.7 51.4 1.13 banten 61.4 42.9 65.7 40.5 0.93 west java 70.0 59.5 64.3 51.4 1.09 central java 64.3 44.8 78.6 57.1 0.82 lampung 81.1 88.3 85.1 86.2 0.95 bengkulu 80.2 89.1 80.2 91.0 1.00 palembang 72.0 90.2 78.1 86.1 0.92 kediri 75.2 87.1 89.3 86.0 0.84 one-year-old plants accession self-pollination cross-pollination isi 2 four-year-old plants table 2. fruit set and seed set from self and cross-pollination note: seed set was calculated from the number of sound seed divided by the number of total ovule assuming that 1 there were three ovules per �ower isi (index of self-incompatibility): ratio of fruit set from self-pollination to cross-pollination 2 14 endah r. palupi, ade s. ahmad, ro�q affandi, abdul qadir, and eni randriani journal of tropical crop science vol. 1 no. , 20142 october www.j-tropical-crops.com cross-pollination (table 2). fruit abortions occurred at different stages of development, but mostly were during the early stages when the size was less than half of the mature fruit. observation on the causes of the abortion would be useful to �nd measures to increase kernel production. fruit set and seed set of the four pollination types in the four-year-old plants (sp-intra, sp-inter, cp-intra and cp-inter) were not signi�cantly different, so the data was presented as selfand cross-pollination (table 2). fruit set did not vary among accessions either in the �rst or second experiments. it was surprising that cross pollination did not result in higher fruit and seed set than self-pollination, which indicated that was notjatropha an obligate cross pollinated species. these results were in contrast to that reported by raju and ezradanam (2002) in india that fruit set from hand cross-pollination was higher (96%) than that of hand self-pollination (77%). kaur et al (2011) also reported that in the central region of punjab the fruit set from cross-pollination was higher (93.2%) than that of self-pollination (72.2%). the average index of self-incompatibility in this research was 0.99 and 0.93 in the four-year-old and one-year-old plants, respectively, so is categorized as ajatropha partially self-incompatible species, supporting the research result of wang and ding (2012) that j. curcas has both self-pollination and cross-pollination systems. this is contradictory to heller (1996) who stated that jatropha was a cross-pollinating species. given the data it is possible that the seeds produced in any jatropha plants in indonesia were the results of selfpollination with ants as the effective pollinator ( etluo al. 2012). the germination percentage of the self-pollination seeds was not signi�cantly different from the crosspollination seeds, supporting the idea that wasjatropha not an obligate cross pollinating species. the four-yearold accession from banten produced highly viable seeds from both selfand cross-pollination. lampung and central java accessions produced highly viable seeds (> 80%) from self-pollination, whereas cross pollinated seeds from west java accession had higher viability than self-pollinated (table 3). slightly different results were obtained from the oneyear-old plants in which cross-pollination produced better quality seeds than self-pollination as indicated by germination rate of > 80% and germination speed of ≥ 9.46 %.day (table 4). in general banten, bengkulu and -1 kediri accessions produced highly viable seeds regardless of pollination systems. it would be interesting to learn the reproductive success of the selfand crosspollinated progenies. a study in malaysia showed positive heterosis from crosses of particular superior parents (islam et al, 2011) which could potentially lead to the development of hybrid varieties of jatropha curcas. 15reproductive success and compatibility among accessions of jatropha.......... table 3. seed germination (%) and germination speed (%.day ) based on pollination systems -1 and accessions from four-year-old plants note: numbers followed by the same letter of the same column and row were not signi�cantly different based on dmrt at = 0.05 accession lampung banten west java central java lampung banten west java central java sp-intra 96.67a 90.48ab 72.22de 95.83a 8.53ab 8.00abc 6.60cd 8.34ab sp-inter 82.22abcde 92.31abcde 73.33cde 88.89abcd 7.47bcd 8.18a 6.39d 7.99abc cp-intra 81.82abcde 90.91abcde 89.58abcde 85.97abcde 7.42bcd 8.13abcde 8.33ab 8.24ab cp-inter 69.84e 97.44a 77.08bcde 40.48f 6.55cd 9.30a 7.20bcd 3.53e germination (%) germination speed (%.day ) -1 journal of tropical crop science vol. 1 no. , 20142 october www.j-tropical-crops.com accession germ 1 . (%) gr 2 (%.day -1 ) germ 1 . (%) gr 2 (%.day -1 ) germ 1 . (%) gr 2 (%.day -1 ) lampung 80.54 9.67 73.71 8.73 86.41 10.34 bengkulu 85.02 10.35 84.90 8.54 85.28 10.32 palembang 80.98 9.82 78.61 9.53 81.20 9.46 kediri 78.49 9.84 85.42 9.72 87.51 10.78 cross-pollinationopen-pollination self-pollination table 4. germination (%) and germination speed (%.day ) based on pollination system and accessions from -1 one-year-old plants note: germination percentage indicating seed viability 1 germination speed indicating seed vigor 2 16 endah r. palupi, ade s. ahmad, ro�q affandi, abdul qadir, and eni randriani the germination speed, which is an indication of seed vigor, showed similar trends to germination percentage in which selfand cross-pollination resulted in nonsigni�cantly different rates. the one-year-old plants tend to demonstrate higher germination speed than the four-year-old plants. banten accession germinated at a faster rate (table 4) regardless of pollination types. selfpollinated seeds from west java were slower to germinate compared to cross-pollinated ones. these data were in line with data on germination percentage that cross pollination between accessions could give negative effect on seed viability. in general, although banten accession had the lowest reproductive success, hence seed production, this accession produced high quality seeds, which is an important character for a conventional breeding program. conclusion reproductive success varied among accessions, the highest was from west java of 0.73 and the lowest from banten of 0.53. jatropha sccessions (lampung, banten, west java, central java, bengkulu, palembang and kediri) are partly self-incompatible with self-incompatibility index range of 0.93-0.99. pollen source did not affect fruit set and seed set, but affected seed germination and speed of germination. acknowledgment we thank the indonesian centre for estate crops research and development for the permission to use seeds from seed orchard in pakuwon, westjatropha java for this research. references achten w.m.j., vercot l., franken y.j., mathijs e., singh v.p., aerts a., and muys b. (2008). jatropha bio-diesel production and use. biomass and bioenergy , 1063-1084.32 alamu o.t., amao, a., oke, o.a., and suleiman, r.a. (2013). foraging behavior of three insect pollinators of in samaru, zaria,jatropha curcas nigeria. international journal of advance agricultural research 1, 87-91. bhattacharya a., datta k., and datta s.k. (2005). floral biology, �oral resources constraints and pollination limitation in l.jatropha curcas pakistan journal of biological sciences , 456-8 460. hartati rr s. (2008). “persilangan jarak pagar (jatropha curcas l)”. lokakarya nasional iv akselerasi i n o v a s i te k n o l o g i j a r a k p a g a r m e n u j u kemandirian energi. malang, 6 nopember 2008, indonesia. hasnam (2007). “status perbaikan dan penyediaan bahan tanaman jarak pagar ( l.)”.jatropha curcas prosiding lokakarya ii. status teknologi tanaman jarak pagar ( l.).jatropha curcas puslitbang perkebunan, bogor, indonesia heller j. 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(euphorbiaceae). hayati journal of bioscience , 38-42.17 sudjindro (2008). “permasalahan benih jarak pagar (jatropha curcas l)”. lokakarya nasional iv akselerasi inovasi teknologi jarak pagar menuju kemandirian energi. malang, 6 nopember 2008, indonesia. tiwari a.k., kumar a, and raheman h. (2007). biodiesel production from jatropha (jatropha curcas) with high free fatty acids: an optimized process. biomass and bioenergy , 569–575.31 wang x.r. and ding g.j. (2012). reproductive biology c h a r a c t e r i s t i c o f j a t r o p h a c u r c a s (euphorbiaceae). revista de biologica tropical 60, 1525-33 wiens d., calvin c.l., wilson c.a., davern c.i., frank d., and scavey s.r. (1987). reproductive success, spontaneous embryo abortion, and genetic load in �owering plants. ,oecologia 71 501-509. berlin. z a p a t a t. r . a n d a r r o y o m . t. k . ( 1 9 7 8 ) . p l a n t reproductive ecology of a secondary deciduous tropical forest in venezuela. , 221-biotropica 10 30. journal of tropical crop science vol. 1 no. , 20142 october www.j-tropical-crops.com water requirement estimation of one-whorl rubber (hevea brasiliensis müll. arg.) .......... journal of tropical crop science vol. 8 no. 3, october 2021 www.j-tropical-crops.com 161 water requirement estimation of one-whorl rubber (hevea brasiliensis müll. arg.) planting materials andi nur cahyo* indonesian rubber research institute, jl. palembang-pangkalan balai km. 29, sembawa, banyuasin 30953, south sumatera, indonesia *corresponding author; email: nurcahyo.andi@yahoo.co.uk abstract fulfilling water requirement is one of the important factors for a successful production of rubber tree planting materials. research on the irrigation requirement for young rubber trees is crucial to determine the amount of water required for an optimum plant growth. the aim of this study was to determine the amount of water needed by one whorl rubber planting material to compensate the amount of evapotranspiration, as well as to estimate the crop coefficient value (kc). the research was conducted at the indonesian rubber research institute on july 2021. daily evapotranspiration (etc) of rubber planting materials of clone “pb 260”, “rric 100”, and “irr 112” planted in polybag size 13 cm x 35 cm were measured by weighing the planting materials daily. evapotranspiration for the reference crop was collected from the indonesian rubber research institute climatological station. our study showed that the amount of water required by each rubber planting material was 92.21 ml per day per polybag when the mean of daily reference evapotranspiration (eto) was 3.67 mm per day. therefore, the crop coefficient (kc) of one whorl rubber planting material arranged sparsely was ± 0.32. this kc value can be used as a base to calculate water requirement of one whorl rubber planting material based on the daily reference evapotranspiration (eto). keywords: crop coefficient, evapotranspiration, plant water requirement, rubber planting material, water balance introduction the largest area of rubber plantation in indonesia is located in south sumatera. most of the rubber farmer in south sumatera also produce rubber planting materials for their own plantation or to sell to others farmer. to produce rubber planting materials, sufficient amount of water should be available every day for several months to fulfill their water requirement for producing young plants with vigorous growth. this condition requires the rubber planting material producer to prepare adequate irrigation system especially during dry season. in south sumatera (located at 1o 4o s and 102o 106o e), rainfall is not evenly distributed throughout the year (cahyo et al., 2011; cahyo et al., 2016). in this area, three to four months with rainfall less than 100 mm per month usually occur during a normal year. moreover, during el-nino event, dry months could occur for more than four months (saputra et al., 2016). fluctuation of monthly rainfall of the last 20 years collected from the indonesian rubber research institute climatological station is presented in figure 1. occurrence of three to four dry months during a year in south sumatera is a constraint for rubber tree growth. without irrigation, three to four dry months causes moderate drought stress because the amount of water from rainfall is not enough to fulfill rubber water requirement. therefore, irrigation during dry months is critical for the optimal plant growth, especially for the production of rubber planting materials. rubber planting materials that are grown without irrigation during dry season show stagnant growth in stem girth. water is an essential component for photosynthesis (cahyo et al., 2020; carr, 2012; taiz and zeiger, 2002), therefore insufficient water supply results in low growth rate and low quality rubber planting materials. due to the occurrence of long dry months in south sumatera, the determination of nursery location, particularly with regard to the availability of water supply for the rubber planting materials is critical (wijaya et al., 2011; wijaya, 2008). ideally, water source for irrigation is from a river or channel that never dries during dry season and therefore irrigation is still efficient during dry season. if ponds or reservoirs are used as source of water for irrigation, accurate calculation of irrigation requirement is needed. this is to assure that water from the reservoir is channeled journal of tropical crop science vol. 8 no. 3, october 2021 www.j-tropical-crops.com 162 andi nur cahyo to the rubber planting materials efficiently and sufficiently in order to fulfill plant water requirement during dry season. available water is determined as the level of soil water content in between field capacity and permanent wilting point (allen et al., 1998; doorenbos and pruitt, 1977; savva and frenken, 2002; waller and yitayew, 2016). at the above field capacity, water will be moved to other places by percolation or seepage processes. on the contrary, if the soil water content decreases below the permanent wilting point, plant will be unable to absorb water from the soil (kirkham, 2014). therefore, the amount of irrigation should be provided to the plants accurately to achieve effective and efficient usage of water. the optimum amount of water needed by plants can be estimated by measurement of plant (crop) evapotranspiration. in the absence of rainfall, in order to maintain soil water content at optimum amount for plant to grow, the amount of water that should be added to the soil (plant water requirement) should be equal to the amount of water lost by evapotranspiration (etc). in the presence of rainfall, the plant water requirement should be calculated by using water balance method. in addition, the value of crop evapotranspiration (etc) is depended on the value of reference evapotranspiration (eto) and crop coefficient (kc) (allen et al., 1998; mangmeechai, 2020; savva and frenken, 2002). by determining etc and eto, the kc for one whorl rubber planting material planted in a polybag can also be determined. hence, in the future plant water requirement can be easily calculated using determined kc and eto data collected from climatological station. the aim of this research was to determine the amount of water required by rubber planting material with one whorl of leaves to compensate the amount of evapotranspiration as well as to estimate the rubber planting material coefficient value (kc). by determining kc value, the plant water requirement can be calculated accurately, hence water use efficiency can be optimized. material and methods experimental site and plant materials the experiment was conducted on july 2021 at the indonesian rubber research institute nursery located at sembawa, south sumatera, indonesia. a total of 15 plant materials representing three rubber clones (“pb 260”, “rric 100”, and “irr 112”) with five replicates per clone were used for the study. the plant materials were 3-month-old with one whorl of fully expanded leaves. the one-whorl rubber plant materials were planted in the 13 cm × 35 cm size polybags filled with ultisol soil as the planting medium. the moisture content of the planting medium in each pot was maintained at 18.5 and 33.7% by adding 450 ml of water into the polybags once every two days, except when there was rainfall. climate data the climate data around the nursery were collected from the climatological station located at 02o55’40” s and 104o32’16” e with the altitude of 10 m above sea level. the climate data used for this research included rainfall (collected from ombrometer observatorium) and the reference crop evapotranspiration generated from class a evaporation pan. calculation of plant water requirement (evapotranspiration, etc) to calculate evapotranspiration (etc), the water balance method was used due to the occurrence of rainfall during measurement of etc. in the water balance calculation, the change of storage (ds/dt) was equal to input minus output of water. the input parameter for this calculation was rainfall (p) and the output parameters were run off (r), evapotranspiration (etc), and percolation (pe). the calculation of water balance is illustrated in equation 1 (sutanto, 2011).   (1)  (2) in this research, the rainfall recorded was very low (≤ 1 mm.day-1), hence the run off (r) and percolation (pe) was neglected. therefore, the water balance calculation was simplified into equation 2.    (1)  (2) where: i = input o = output = change of the storage (soil water content) p = rainfall r = run off etc = crop evapotranspiration pe = percolation the change of water storage was measured daily at 07.30 am by weighing the polybags containing water requirement estimation of one-whorl rubber (hevea brasiliensis müll. arg.) .......... journal of tropical crop science vol. 8 no. 3, october 2021 www.j-tropical-crops.com 163 rubber plants. the daily change of the weight of the polybags indicated the daily change of soil water content due to the daily evapotranspiration. the daily evapotranspiration in kg was converted to mm by dividing the volume of water loss due to transpiration by the evapotranspiration area of both plant (leaves) and the planting medium (soil top surface). calculation of crop coefficient (kc) to calculate crop coefficient, evaporation data from class a evaporation pan (epan) of the indonesian rubber research institute climatological station was used to determine the reference crop evapotranspiration (eto). the eto was calculated using the formula presented in equation 3 (allen et al., 1998; ayutthaya, 2010; doorenbos and pruitt, 1977; mesike and esekhade, 2014; savva and frenken, 2002).  (3) (4) where: eto = reference crop evapotranspiration kp = pan coefficient with the value as 0.85 for class a evaporation pan epan = class a evaporation pan the crop coefficient (kc) of one-whorl rubber plants in polybag was calculated using equation 4 (allen et al., 1998; doorenbos and pruitt, 1977; fisher, 2012; mangmeechai, 2020; savva and frenken, 2002).   (3) (4) where: etc = crop evapotranspiration eto = reference crop evapotranspiration kc = crop coefficient statistical analysis a completely randomized design was used to analyze the effect of the clone types on the water requirement and crop coefficient of one whorl rubber plant materials. if the rubber clone types significantly affected the observed variables, the duncan multiple range test (dmrt) was employed with significance level of 95% to differentiate the means of each variable. statistical analysis was conducted using sas version 9 program software (sas institute inc., 2002). results and discussion  during the study period, the observed reference evapotranspiration (eto) fluctuated between 1.46 to 5.08 mm per day. within the same time frame, rainfall only occurred on 13 july 2021 as high as 1 mm per day. the fluctuation of eto and rainfall is presented in figure 2. figure 2 shows that rainfall only occurred in the evening of the first day of observation with as high as 1 mm. this amount of rainfall was lower than the eto, hence we assumed that run off and percolation did not occur during the research duration. therefore, in the water balance calculation we only used the rainfall (p), evapotranspiration (etc), and change of storage ( ). in addition, based on initial soil water content data at the first day of the research and daily weight of the polybags, the weight soil and water inside the polybags was calculated to determine daily soil water content. daily soil water content data was required to maintain soil water content at available level between permanent wilting point and field capacity, hence the amount of daily etc was not reduced by the low water content of the soil medium. for clay loam soil used in this research, the permanent wilting point was about (4) where: etc = crop evapotranspiration eto = reference crop evapotranspiration kc = crop coefficient statistical analysis a completely randomized design was used to analyze the effect of the clone types on the water requirement and crop coefficient of one whorl rubber plant materials. if the rubber clone types significantly affected the observed variables, the duncan multiple range test (dmrt) was employed with significance level of 95% to differentiate the means of each variable. statistical analysis was conducted using sas version 9 program software (sas institute inc., 2002). results and discussion during the study period, the observed reference evapotranspiration (eto) fluctuated between 1.46 to 5.08 mm per day. within the same time frame, rainfall only occurred on 13 july 2021 as high as 1 mm per day. the fluctuation of eto and rainfall is presented in figure 2. figure 2 shows that rainfall only occurred in the evening of the first day of observation with as high as 1 mm. this amount of rainfall was lower than the eto, hence we assumed that run off and percolation did not occur during the research duration. therefore, in the water balance calculation we only used the rainfall (p), evapotranspiration (etc), and change of storage ( ). figure 1. fluctuation of monthly rainfall in indonesian rubber research institute, sembawa, south sumatera. 0 50 100 150 200 250 300 350 jan feb mar apr may jun jul aug sep oct nov dec r ai nf al l ( m m p er m on th ) figure 1. fluctuation of monthly rainfall in indonesian rubber research institute, sembawa, south sumatera. journal of tropical crop science vol. 8 no. 3, october 2021 www.j-tropical-crops.com 164 andi nur cahyo 18.5% and the field capacity was about 33.7% (ardika and cahyo, 2020; cahyo et al., 2020; stevanus et al., 2017). hence, the soil moisture content in this research was maintained in between 18.5 and 33.7%. the fluctuation of daily soil water content of the soil inside the polybags is presented in figure 3. figure 3 shows that soil water content was maintained above permanent wilting point (18.5%), hence the etc was maintained at normal rate. if the soil was too dry, the etc would have dropped due to the decrease in evaporation rate. evaporation reduces as the soil dries out (weiss et al., 2021). in this study, the plant water requirement was equal to the amount of etc added by the rainfall. daily etc was equal to the difference of the polybag plant material weight at day n and day n+1. the weight of the polybag was observed when soil water content was in available level for the plant. the weight loss was the amount of water loss due to etc or equal to plant water requirement. the results of the plant water requirement calculation are presented in table 1. table 1 shows that the average of plant water requirement for a one-whorl rubber polybag planting material was 92.21 ml per day per polybag. the plant water requirements for all three clones were not significantly different. it indicates that in the nursery, the irrigation can be adjusted at uniform rate for all three rubber clones. the amount of plant water requirement (92.21 ml per day per polybag) was only applied to a one-whorl rubber plant. in this research the average leaf area for a one-whorl rubber planting material was 834.16 cm2. for 6-month-old rubber planting materials with two-leaf whorls, the leaf area is estimated to be twice than the above value. therefore, the plant water requirement for two-whorl rubber plant is also estimated to be double (around figure 2. reference of evapotranspiration (eto) and rainfall (mm per day) in july 2021 in addition, based on initial soil water content data at the first day of the research and daily weight of the polybags, the weight soil and water inside the polybags was calculated to determine daily soil water content. daily soil water content data was required to maintain soil water content at available level between permanent wilting point and field capacity, hence the amount of daily etc was not reduced by the low water content of the soil medium. for clay loam soil used in this research, the permanent wilting point was about 18.5% and the field capacity was about 33.7% (ardika and cahyo, 2020; cahyo et al., 2020; stevanus et al., 2017). hence, the soil moisture content in this research was maintained in between 18.5 and 33.7%. the fluctuation of daily soil water content of the soil inside the polybags is presented in figure 3. figure 3. fluctuation of soil water content of soil planting medium in the polybags figure 3 shows that soil water content was maintained above permanent wilting point (18.5%), hence the etc was maintained at normal rate. if the soil was too dry, the etc would have dropped due to the decrease in evaporation rate. evaporation reduces as the soil dries out (weiss et al., 2021). in this study, the plant water requirement was equal to the amount of etc added by the rainfall. daily etc was equal to the difference of the polybag plant material weight at day n and day n+1. the weight of the polybag was observed when soil water content was in available level for the plant. the weight loss was the amount of water loss due to etc or equal to plant water requirement. the results of the plant water requirement calculation are presented in table 1. table 1 shows that the average of plant water requirement for a one-whorl rubber polybag planting 0,0 1,0 2,0 3,0 4,0 5,0 6,0 m m p er d ay rainfall eto 10 15 20 25 30 35 0,0 0,2 0,4 0,6 0,8 1,0 1,2 1,4 1,6 1,8 s oi l w at er c on te nt ( % ) w ei gh t ( kg ) water weight (kg) soil weight (kg) soil water content (%) figure 2. reference of evapotranspiration (eto) and rainfall (mm per day) in july 2021 figure 2. reference of evapotranspiration (eto) and rainfall (mm per day) in july 2021 in addition, based on initial soil water content data at the first day of the research and daily weight of the polybags, the weight soil and water inside the polybags was calculated to determine daily soil water content. daily soil water content data was required to maintain soil water content at available level between permanent wilting point and field capacity, hence the amount of daily etc was not reduced by the low water content of the soil medium. for clay loam soil used in this research, the permanent wilting point was about 18.5% and the field capacity was about 33.7% (ardika and cahyo, 2020; cahyo et al., 2020; stevanus et al., 2017). hence, the soil moisture content in this research was maintained in between 18.5 and 33.7%. the fluctuation of daily soil water content of the soil inside the polybags is presented in figure 3. figure 3. fluctuation of soil water content of soil planting medium in the polybags figure 3 shows that soil water content was maintained above permanent wilting point (18.5%), hence the etc was maintained at normal rate. if the soil was too dry, the etc would have dropped due to the decrease in evaporation rate. evaporation reduces as the soil dries out (weiss et al., 2021). in this study, the plant water requirement was equal to the amount of etc added by the rainfall. daily etc was equal to the difference of the polybag plant material weight at day n and day n+1. the weight of the polybag was observed when soil water content was in available level for the plant. the weight loss was the amount of water loss due to etc or equal to plant water requirement. the results of the plant water requirement calculation are presented in table 1. table 1 shows that the average of plant water requirement for a one-whorl rubber polybag planting 0,0 1,0 2,0 3,0 4,0 5,0 6,0 m m p er d ay rainfall eto 10 15 20 25 30 35 0,0 0,2 0,4 0,6 0,8 1,0 1,2 1,4 1,6 1,8 s oi l w at er c on te nt ( % ) w ei gh t ( kg ) water weight (kg) soil weight (kg) soil water content (%) figure 3. fluctuation of soil water content of soil planting medium in the polybags water requirement estimation of one-whorl rubber (hevea brasiliensis müll. arg.) .......... journal of tropical crop science vol. 8 no. 3, october 2021 www.j-tropical-crops.com 165 180 ml per day per polybag) compared to that of onewhorl rubber plant. the results of the calculation for one-whorl rubber plant material water requirement in this study can also be used to determine crop coefficient of the 3-month-old one whorl rubber young tree by dividing etc by eto. eto data was generated from class a evaporation pan data (epan) available at the indonesian rubber research institute. considering the position of the evaporation pan and the average wind speed, as well as the relative humidity, the pan coefficient (kp) used in this study was 0.85 (savva and frenken, 2002). the unit of eto generated from epan data was mm per day, hence the etc data in this research should be converted from ml per day per polybag to mm per day. etc is the water loss by soil surface (evaporation) and by plants (transpiration) (allen et al., 1998; weiss et al., 2021), hence to convert etc unit from ml per day per polybag to mm per day, the volume of water loss should be divided by the surface area of soil and plants leaves. the average of total evapotranspiration area (surface area of soil and plants leaves) obtained in this study was 892.73 cm2. the results of calculation of etc in mm per day are presented in table 2. table 2 shows that although the volume of plant water requirements of all clones was not significantly different, the etc in mm per day of clone “pb 260” was significantly lower than clone “irr 112” and “rric 100”. the differences in the volume of water requirement can be attributed to the leaf area of “pb 260” which was relatively wider than “irr 112” and “rric 100”. the relatively similar etc (expressed in ml per day per polybag) resulted in lower etc in mm per day for clone “pb 260”. based on daily etc and eto data, kc can be determined. the results of kc calculation are presented in table 3. table 3 shows that the average kc of the three rubber clones were 0.32. furthermore, the kc values among three rubber clones were not significantly different. this value was far lower than the kc values stated by savva and frenken (2002), namely 0.95 for initial stage and 1.00 for middle and end stage of rubber tree lifespan. the low kc value in this study could be attributed to the sparsely arrangement of 3-monthold rubber plants in the nursery. on the contrary, the high kc stated by (savva and frenken, 2002) were generated from rubber tree planted on the ground. we planted one-whorl rubber planting materials in polybags to give reference to the producers of rubber plant materials on how much they should provide water for their nursery based on etc calculation using determined kc for rubber planting materials and eto data from climatological station. the unit of the result of this calculation is mm per day, hence to give sufficient water into the polybag, the etc should be converted from mm per day to ml per day per polybag by multiplying the etc to the sum of evaporation and transpiration area (approximation of leaves and soil surface area). for larger plants with denser canopy, the etc and kc would need to be increased; mature table 1. plant water requirement (etc) of three different rubber clones (ml per day per polybag) clone etc (ml per day per polybag) average rep. 1 rep. 2 rep. 3 rep. 4 rep. 5 “pb 260” 80.81 89.94 108.17 93.31 88.44 92.13ns “irr 112” 90.03 99.99 92.71 97.58 87.12 93.49ns “rric 100” 87.62 107.35 83.12 96.44 80.58 91.02ns average 86.15 99.09 94.67 95.78 85.38 92.21 note: values followed by ns were not significantly different according to dmrt at α = 0.05. rep. = replicate table 2. plant water requirement (etc) of three different rubber clones (mm per day) clone etc (mm per day) average rep. 1 rep. 2 rep. 3 rep. 4 rep. 5 “pb 260” 0.89 0.57 0.97 1.07 0.82 0.86b “irr 112” 0.97 1.28 1.14 1.02 1.17 1.12a “rric 100” 1.04 1.40 1.03 1.06 1.15 1.14a average 0.97 1.08 1.05 1.05 1.05 1.04 note: values followed by different letter were significantly different according to dmrt at α = 0.05. rep. = replicate journal of tropical crop science vol. 8 no. 3, october 2021 www.j-tropical-crops.com 166 andi nur cahyo rubber trees usually have a kc of 1.00. conclusion water requirement should be determined accurately to avoid drought stress, as well as to avoid excess water given to the plant to achieve optimum water use efficiency. to determine plant water requirement, etc can be estimated using eto from climatological station and determined kc for rubber planting material. our study has determined that the plant water requirement for 3-month-old one whorl young rubber tree is 92.21 ml per day per polybag this amount of water requirement can be changed based on the change of eto value and the size of the plant. in addition, our study has determined that the kc value for one whorl young rubber tree in polybag size of 13 cm x 35 cm was 0.32. references allen, ri. g., pereira, l. s., raes, d., and smith, m. (1998). “fao irrigation and drainage paper no. 56 on crop evapotranspiration; a guideline for computing crop water requirements”. fao food and agriculture organization of the united nations. ardika, r., and cahyo, a. n. (2020). soil water content below 33.7% progressively reduces the latex yield of rubber pb 60, a study in sembawa, south sumatra, indonesia. journal of tropical crop science 7, 97–103. https://doi. org/10.29244/jtcs.7.03.97-103 ayutthaya, s. i. n. (2010). “change of whole-tree transpiration of mature hevea brasiliensis under soil and atmospheric droughts; analyzed in intermittent and seasonal droughts under the framework of the hydraulic limitation hypothesis”. [thesis]. universite blaise pascal. cahyo, a. n., ardika, r., and wijaya, t. (2011). water consumption and rubber production on various planting space arrangement system and their relationship with soil water content. indonesian journal of natural rubber research 29, 110– 117. https://doi.org/10.22302/jpk.v29i2.243 cahyo, a. n., babel, m. s., datta, a., prasad, k. c., and clemente, r. (2016). evaluation of land and water management options to enhance productivity of rubber plantation using wanulcas model. agrivita journal of agricultural science 38, 93–103. https://doi. org/10.17503/agrivita.v38i1.583 cahyo, a. n., murti, r. h., and putra, e. t. s. (2020). dampak kekeringan terhadap proses fisiologis, pertumbuhan, dan hasil tanaman karet (hevea brasiliensis müll. arg.). warta perkaretan 39, 57–72. cahyo, a. n., stevanus, c. t., and syafaah, a. (2020). perhitungan kebutuhan irigasi pembibitan batang bawah karet berdasarkan neraca air di sembawa, sumatera selatan. jurnal penelitian karet, 38, 37–48. https://doi.org/10.22302/ppk. jpk.v38i1.689 carr, m. k. v. (2012). the water relations of rubber (hevea brasiliensis ): a review. experimental agriculture, 48, 176–193. https://doi. org/10.1017/s0014479711000901 doorenbos, j., and pruitt, w. o. (1977). “crop water requirements”. food and agriculture organization of the united nations. fisher, d. k. (2012). simple weighing lysimeters for measuring evapotranspiration and developing crop coefficients. international journal of agricultural and biological engineering 5, 35– 43. kirkham, m. b. (2014). field capacity, wilting point, available water, and the nonlimiting water range. in “principles of soil and plant water relations” (s. r. coombs, ed.). 2nd ed., pp. table 3. crop coefficient (kc) of three different rubber clones clone kc average rep. 1 rep. 2 rep. 3 rep. 4 rep. 5 “pb 260” 0.29 0.18 0.30 0.35 0.27 0.28ns “irr 112” 0.29 0.39 0.34 0.31 0.35 0.34ns “rric 100” 0.33 0.43 0.30 0.33 0.35 0.35ns average 0.31 0.33 0.31 0.33 0.32 0.32 note: values followed by ns were not significantly different according to dmrt at α = 0.05. rep. = replicate water requirement estimation of one-whorl rubber (hevea brasiliensis müll. arg.) .......... journal of tropical crop science vol. 8 no. 3, october 2021 www.j-tropical-crops.com 167 153–170. elsevier. https://doi.org/10.1016/ b978-0-12-420022-7.00010-0 mangmeechai, a. (2020). effects of rubber plantation policy on water resources and land use change in the northeastern region of thailand. geography, environment and sustainability 13, 73–83. https://doi.org/10.24057/20719388-2019-145 mesike, c. s., and esekhade, t. u. (2014). rainfall variability and rubber production in nigeria. african journal of environmental science and technology 8, 54–57. https://doi.org/10.5897/ ajest2013.1593 saputra, j., stevanus, c. t., and cahyo, a. n. (2016). the effect of el-nino 2015 on the rubber plant (hevea brasiliensis) growth in the experimental field sembawa research centre. widyariset 2, 37–46. https://doi.org/10.14203/ widyariset.2.1.2016.37-46 sas institute inc. (2002). “the sas system for windows” version 9. sas institute inc. savva, a. p., and frenken, k. (2002). “crop water requirements and irrigation schedulingirrigation manual module 4”. fao. stevanus, c. t., ardika, r., and saputra, j. (2017). the effect of tillage and cover crop on soil physical properties and young rubber plant growth. jurnal penelitian karet 35, 139–148. https://doi.org/10.22302/ppk.jpk.v35i2.357 sutanto, s. j. (2011). evapotranspiration calculation and determination of evaporation effect in the soil using stable isotopes (18o and 2h) and hydrometric measurements. jurnal teknik hidraulik 2, 98–192. taiz, l., and zeiger, e. (2002). “plant physiology”. 3rd ed. sinauer associates. waller, p., and yitayew, m. (2016). “irrigation and drainage engineering”. springer international publishing. weiss, o., minixhofer, p., scharf, b., and pitha, u. (2021). equation for calculating evapotranspiration of technical soils for urban planting. land 10, 622. https://doi.org/10.3390/ land10060622 wijaya, t. (2008). kesesuaian tanah dan iklim untuk karet. warta perkaretan 27, 34–44. wijaya, t., cahyo, a. n., and ardika, r. (2011). “anticipation and efforts to cope with la nina climate anomaly in rubber plantation”. the role of science and technology on climate change anticipation on the perspective sustainable agriculture seminar. department of agriculture, sriwijaya university. journal of tropical crop science vol. 8 no. 2, june 2021 www.j-tropical-crops.com 80 betty betty, sandra arifin aziz, ketty suketi the effects of different rates of chicken manure and harvest intervals on the bioactive compounds of bitter leaf (vernonia amygdalina del.) betty bettya,b, sandra arifin aziz*c, ketty suketic a study program of agronomy and horticulture, graduate school of ipb university, bogor 16680, indonesia b department of biology education, sanata dharma university, jogjakarta 55281, indonesia c department of agronomy and horticulture, faculty of agriculture, ipb university, bogor 16680, indonesia *corresponding author; email: sandraaziz@apps.ipb.ac.id abstract bitter leaf (vernonia amygdalina del.) is a valuable functional vegetable and traditional medicinal plant in many tropical countries including indonesia. bitter leaf is commonly used to remedy hypertension and diabetes among indonesian people. the leaf extract comprises bioactive compounds such as sesquiterpene lactones, steroid glycosides, and flavonoids. despite its growing market demand, there is insufficient data on agronomic practice in order to obtain optimum yield with high bioactive compounds. the experimental design used was a randomized complete block design with two factors namely chicken manure rates (0, 2.5, 5, and 7.5 kg per plant) and harvest intervals (2 and 3 months). there was a significant difference in fresh and dry weights of leaves, chlorophyll a and b, carotenoids, nitrogen concentration, anthocyanins, and flavonoids among different manure treatment. the highest concentration and production of bioactive compounds on the bitter leaf was found on the highest treatment of 7.5 kg chicken manure per plant. the harvest interval of three months significantly increased fresh and dry weights of leaves, concentration of anthocyanins, phosphorus, and potassium, uptake of phosphorus and potassium, and production of anthocyanins and flavonoids. cultivation using chicken manure with frequent period of pruning can enhance the production of biomass as well as secondary metabolites in bitter leaf. keywords: flavonoids,functional vegetable, medicinal plant, nutrients uptake, secondary metabolites introduction vernonia amygdalina, commonly known as bitter leaf, belongs to the family asteraceae. it is an evergreen tree that can grow up to 10 m but are commonly seen as shrub as a result of frequent pruning. vernonia amygdalina is well known as a traditional medicinal plant across tropical countries (grubben and denton, 2004). each part of the plant can be used for treating various diseases (oyeyemi et al., 2017). the fresh leaf of the plant is usually consumed to treat diabetes, malaria, fever, and high blood pressure (asante et al., 2016). studies show that leaf extracts of v. amygdalina contain sesquiterpene lactones (kupchan et al., 1969) saponins, flavonoids, steroids, and coumarins (tona et al., 2004). limited data are available on effective cultivation of bitter leaf, and in order to comply to the good agriculture practice for medicinal crops (ministry of agriculture act number 54, 2012), we aimed to determine the effects of organic fertilizer on bitter leaf quality and production. application of organic fertilizers could potentially enhance both production of harvested plant parts and manufacture of secondary metabolites (dumas et al., 2003; abd-alla et al., 1999). antioxidant concentration in tomatoes was higher when treated with organic fertilizer than with inorganic fertilizer (dumas et al., 2003). organic fertilizer can change the chemical and physical properties of soil resulting in increased efficiency of nutrient absorption in faba bean, soybean, and lupin (abd-alla et al., 1999). additionally, organic fertilizers, such as manure, is proven to improve physical, chemical, and biological soil properties, and to ensure the sustainability of soil health (khaliq et al., 2006). application of organic fertilizer was found to promote vegetative growth, yield potential, and phytochemical content of corchorus olitorious (ghoneim and elaraby, 2003). the use of chicken manure provided the effects of different treatment rates of chicken manure and harvest .......... journal of tropical crop science vol. 8 no. 2, june 2021 www.j-tropical-crops.com 81 the best plant growth and yield results because it has higher macroand micro-nutrients contents than compost and cow manure. chicken manure was also found to increase secondary metabolite compounds. according to ibrahim et al. (2013), total phenolics, flavonoids, ascorbic acid, saponins, and glutathione, and increase antioxidant activity in labisia pumila increased after treatment with chicken manure. karimuna et al. (2015) also showed that the application of chicken manure raised the fresh and dry weight of murraya paniculata, however, it correlated negatively with the total flavonoid content. additionally, in murraya paniculata, the combination of chicken manure and rice-hull charcoal treatment resulted in a higher leaf production and total flavonoid content than without organic fertilizer application (taufika et al., 2017). besides fertilizer application, harvest interval was also reported to influence the content of secondary metabolites on cultivated plants. taufika et al. (2017) reported that murraya paniculata contained the highest concentration of anthocyanins and flavonoids at 4-month harvest intervals compared to 2and 3-month harvest intervals. generally, the best harvest interval in talinum triangulare that generates the highest anthocyanin concentration is at 10 days after planting (susanti et al., 2011). however, the optimum harvest interval for bitter leaf to produce a high concentration of secondary metabolites is still unknown. in this study, we determined treatment rate and harvest interval that results in greater growth and yield in v. amygdalina. the results of this study will be useful in cultivating these plants efficiently in order to meet increasing demands of bitter leaves. materials and methods study location the experiment was conducted at cikarawang organic experimental field station, bogor agricultural university, bogor, indonesia from december 2017 to june 2018. the average temperature during the conduct of experiment was between 25.4 to 26.6°c, with the average monthly rain fall of 181.2 to 358.9 mm. the soil and chicken manure chemistry test are presented in table 1. the analyses of nitrogen, phosphorus, and potassium of the leaves were carried out in the testing laboratory, department of agronomy and horticulture at ipb university. the analyses of chlorophyll, carotenoids, anthocyanins, and flavonoids were performed in the postharvest laboratory, department of agronomy and horticulture at ipb university. sampling and treatments a total of 120 v. amygdalina plants (figure 1) for this study originated from stem cuttings with length of approximately 30 cm. the plants were grouped into 40 experimental units composed of 3 plants each and planted with a 1 m × 1 m planting distance. figure 1. vernonia amygdalina one month after pruning a randomized block design with two factors, i.e. chicken manure treatment rates (0 or control, 2.5, 5, and 7.5 kg per plant) and harvest intervals (2 and 3 months) was used in this study. each treatment was repeated five times. the chicken manure treatment was applied a week before planting. data collection harvesting was executed by cutting the main stem or branches from 50 cm above the ground. leaves were collected, air-dried for 3 days at room temperature, then dried in the oven at 60 ℃ for 24 hours. the dried leaves were pulverized for nutrients and phytochemical analysis. nitrogen concentration of the leaf was analyzed using the kjeldahl method (eviati and sulaeman, 2009). one milligram of leaf powder was mixed with 3 ml sulfuric acid at 350°c for 3–4 hours. the clear extract was diluted with ion-free water until 50 ml. ten ml of the solution was mixed with 10 ml of 40% sodium hydroxide. freed nh3 was captured using 1% boric acid until the solution turned green from red using journal of tropical crop science vol. 8 no. 2, june 2021 www.j-tropical-crops.com 82 betty betty, sandra arifin aziz, ketty suketi conway indicator. the green solution was then titrated with 0.05 n sulfuric acid until it turned light pink. the wet ashing method (eviati and sulaeman, 2009) was used to analyze leaf phosphorus and potassium concentrations. leaf powder of 0.5 mg was mixed with 5 ml hno3 and 0.5 ml hclo4 overnight. the mixture was heated at 100°c for 1 hour, raised to 150°c for 2 hours, and to 200°c until white sediment or clear solution was formed. the solution was added with ion-free water and left overnight. phosphorus concentration was further measured using shimadzu uv-1800 spectrophotometer, while potassium concentration was further measured using atomic absorption spectrophotometry agilent 240 fs aa. chlorophyll, carotenoid, and anthocyanin analysis followed the method described in sims and gamon’s (2002). a total of 20 ± 2 mg fresh leaf observed as 1st to 3rd from apex (tjhia et al., 2018) were collected, weighed and crushed with 2 ml of 85:15% acetone tris hcl, and centrifuged at 12000 rpm for 5 minutes. three milliliters of acetone tris was added to 1 ml supernatant. the absorbance of the solution was measured using shimadzu uv-1201 uv-vis spectrophotometer at 457, 537, 647, and 663 nm. the formula to quantify pigment concentrations according to sims and gamon (2002) were used: chlorophyll a (mg.g-1) = [((0.01373×a663)– (0.000897×a537)–(0.003046×a647)× df/fresh weight sample)×893.5]/1000 chlorophyll b (mg.g-1) = [((0.02405×a647)– (0.004305×a537)–(0.005507×a663) ×df/fresh weight sample)×907.5]/1000 anthocyanin (mg.100g-1) = [((0.08173×a537)– (0.00697×a647)–(0.002228 × a663) ×df/fresh weight sample)×0.20708×100]/100 carotenoids (mg.g-1) = [((a470–(17.1×(chlorophyll a + chlorophyll b) – 9.479× anthocyanin))/119.26×df/ fresh weight sample)×550]/1000 where a= absorbance; df = dilution factor flavonoids were analyzed following the protocol of chang et al. (2002). extraction was made by mixing 100 mg of leaf powder with 1 ml of ethanol 100% and put into the a 60°c oven and left for 60 minutes. the mixture was centrifuged for 5 minutes at 12000 rpm. one milliliter of the supernatant was added to 1.9 ml ethanol, 0.1 ml 10% aluminum chloride, 0.1 1 m ml potassium acetate, and 2.8 ml distilled water. the mixture was incubated at room temperature for 30 minutes. the absorbance of the mixture was measured at wavelength 415 nm. a standard curve was constructed by 0–400 mg/l quercetin. statistical analysis data were analyzed using the fisher test, followed by the duncan multi-range test at a significance level of 5% using star (statistical tool for agricultural research) nebula (irri, 2018). results data obtained from soil chemistry test using two months as harvest interval with four different rates of chicken manure treatment are shown in table 1. adding 7.5 kg of chicken manure to v. amygdalina plants results in highest retention of nitrogen, c-organic, k2o and p2o5. these soil components decreased consistently as treatment of chicken manure was reduced (table 1). these trends were also similar to those from the three-months harvest interval (table 2). the effect of chicken manure rates as the rates of chicken manure were increased, the fresh and dry weight of the leaf, nitrogen concentration and uptake, and concentration of chlorophyll a, chlorophyll b, and carotenoids in the bitter leaf also increased (table 3). moreover, analysis of leaf anthocyanins showed that increasing the rates of chicken manure could increase the production of leaf anthocyanins. meanwhile, concentrations of phosphorus, potassium, anthocyanins, and flavonoids, uptake of phosphorus and potassium were not affected by the chicken manure application. the effect of harvest interval the effect of harvest interval on v. amygdalina plants are shown in table 4. the fresh and dry weight of leaves, uptake of nitrogen, phosphorus, and potassium, concentration of anthocyanins, and production of flavonoids rose with longer harvest interval of 3-months. on the other hand, concentrations of phosphorus and potassium of bitter leaf were slightly higher at 2-month harvest interval. concentrations of nitrogen, chlorophyll a, chlorophyll b, carotenoids, and flavonoids were not affected by the harvest interval treatment (table 4). the effects of different treatment rates of chicken manure and harvest .......... journal of tropical crop science vol. 8 no. 2, june 2021 www.j-tropical-crops.com 83 discussion administering higher rate of chicken manure promoted higher leaf production in v. amygdalina as demonstrated in the increasing fresh and dry weight of leaves. according to nwite and alu (2017), the ability of chicken manure in improving the physical, chemical, and biological properties of soil can support the ideal growth of corn. the result of the soil chemistry test (table 1 and 2) in this study proved that as the chicken manure rate increased, the soil acidity is reduced. the plant samples in this study grow better in the neutral than acidic soil. with the application of chicken manure, the soil acidity was reduced and the concentration of macronutrients was increase (duruigbo et al., 2007) . the degree of soil acidity will affect the solubility and availability of ions for plants. if the ph of the soil becomes more acidic, aluminum, iron, manganese, copper, and zinc ions will dissolve even more (duruigbo et al., 2007). the increasing availability of macro nutrients in soil by application of higher chicken manure rates are shown in tables 1 and 2. the nitrogen, phosphorus, and potassium concentration in the leaves (table 3) were also increasing as the concentration of soil nutrients increased. finally, the uptakes of three major nutrients, nitrogen, phosphorus, and potassium, also increased. ewulo et al. (2008) study demonstrated that application of chicken manure reduces soil density, increases humidity, and increases nutrient concentration of the soil, which causes the uptake of n, p, and k nutrients to increase. a similar finding to ewulo et al. (2008) was reported in tomato, i.e. table 1. soil chemical properties in set-up with harvest interval of two-months tested at the end of the experiment chemical indicator chicken manure soil chemistry before treatments 2 months harvest interval 0 2.5 5 7.5 ph2o 7.97 5.92 slightly acidic 6.39 slightly acidic 6.64 neutral 6.9 neutral 6.9 neutral pkcl 5.34neutral 5.61 neutral 6.26 slightly alkaline 6.63 alkaline 6.6 alkaline n (%) 1.64 0.25 moderate 0.25 moderate 0.35 moderate 0.41 moderate 0.42 moderate c-organic (%) 19.12 2.17 moderate 2.05 moderate 2.68 moderate 2.92 moderate 3.35 high k2o (mg k2o/100g) 1.60% 31.4moderate 12.43 low 94.93 very high 133.13 very high 144.08 very high p (mg p2o5 /100g) 5.14% 82.1 very high 74.12 very high 406.74 very high 744.44 very high 816.98 very high table 2. soil chemical properties in set-up with harvest interval of three-months tested at the end of the experiment chemical indicator 3 months harvest interval 0 2.5 5 7.5 ph2o 6.4 6.38 6.71 6.76 slightly acidic slightly acidic neutral neutral pkcl 5.58 5.83 6.46 6.54 neutral neutral slightly alkaline alkaline n 0.24 0.29 0.32 0.38 (%) moderate moderate moderate moderate c-organic 2.17 2.38 2.68 3.02 (%) moderate moderate moderate high k2o 2.28 50.27 137.83 141.55 (mg k2o /100g) very low high very high very high p 89.82 129.58 322.4 461.06 (mg p2o5/100g) very high very high very high very high journal of tropical crop science vol. 8 no. 2, june 2021 www.j-tropical-crops.com 84 betty betty, sandra arifin aziz, ketty suketi increasing soil density would reduce the absorption of n, p, k, ca, and mg (adekiya and ojeniyi, 2002). in addition to root morphology, soil structure and moisture are important factors in nutrient absorption from the soil (baldwin, 1975). the increasing nitrogen concentration and uptake is correlated with the increase in leaf production of bitter leaf. nitrogen is a determinant element of plant growth. according to evans (1989), nitrogen increases plant growth which in turn increases photosynthetic activity. leaf photosynthetic capacity is associated with nitrogen concentrations as the proteins in the calvin and thylakoid cycles represent most of the nitrogen in the leaves. therefore, high nitrogen can increase photosynthesis which then promotes vegetative and generative growth, which ultimately increases crop production. the increase of mineral concentration and uptake was accompanied by an increase in the concentration of chlorophyll a, chlorophyll b, and carotenoids. according to tatjana et al. (2007), increase in pigment concentration is influenced mainly by an increase in the higher nitrogen concentration, along with the increase in the fertilizer rate found in wheat. marschner (2012) also stated nutrients that are directly related to chlorophyll are nitrogen, because nitrogen is one of the main components of chlorophyll. this was supported also by findings of suarez (2007) that the chlorophyll concentration in corn increases along with the increase use of nitrogen from chicken manure. higher chlorophyll concentrations indicate a higher rate of photosynthesis (butterly and buzzell, 1977), and high photosynthesis rates increases plant biomass (arntz et al., 1998). in our study, the higher the rate of fertilizer treatment, the greater the concentration of pigments, thereby increasing the rate of photosynthesis. as a result, vegetative growth also increases. the anthocyanin and flavonoids concentration were not significantly different among rates of treatment (table 3). however, the production of anthocyanins and flavonoids was increasing as the rates increased because the production resulted from the multiplication table 3. the effects of chicken manure rates on leaf and dry weights, and metabolite contents of vernonia amygdalina plants variables chicken manure rates (kg per plant) 0 2.5 5 7.5 leaf fresh weight (g/plant) 397.11±63.41 a 661.53±265.42 b 828.32±295.92 c 850.53±247.46 c leaf dry weight (g/plant) 64.10±9.37 a 107.29±47.74 b 117.57±43.03 bc 136.37±54.10 c nitrogen concentration (%) 4.07±0.22 a 3.96±0.20 ab 4.18±0.27 ab 4.24±0.16 ab phosphorous concentration (%) 0.36±0.06 a 0.35±0.06 a 0.35±0.05 a 0.36±0.04 a potassium concentration (%) 3.21±0.75 a 3.59±0.56 a 3.50±0.82 a 3.93±0.37 a nitrogen uptake (g/plant) 3.22±1.98 a 3.75±2.60 ab 4.27±1.79 bc 4.88±2.20 c phosphorous uptake (g/plant) 0.28±0.13 a 0.34±0.23 a 0.38±0.19 a 0.45±0.24 a potassium uptake (g/plant) 2.40±1.35 a 3.17±1.74 a 3.77±2.44 a 4.56±2.27 a chlorophyll a (mg.g-1) 1.53±0.19 b 1.60±0.12 ab 1.72±0.10 a 1.76±0.04 a chlorophyll b (mg.g-1) 0.52±0.06 a 0.54±0.04 ab 0.58±0.04 bc 0.60±0.01 c carotenoids (mg.g-1) 0.39±0.05 a 0.42±0.03 ab 0.46±0.02 bc 0.47±0.01 c anthocyanins (mg.100g-1) 0.08±0.01 a 0.09±0.02 a 0.09±0.01 a 0.09±0.02 a flavonoids (mg.g-1) 22.66±0.31 a 23.33±1.50 a 22.24±0.39 a 21.68±1.51 a flavonoids production (mg/plant) 1706.7±399.8 a 2357.1±1085.5 b 2360.1±693.8 b 2726.8±1073.6 b anthocyanins production (mg/ plant) 0.51±0.24 a 0.72±0.44 b 0.81±0.43 b 0.88±0.39 b note: values followed by different letters within the same row showed significant differences in duncan multiple range test (α= 5%) the effects of different treatment rates of chicken manure and harvest .......... journal of tropical crop science vol. 8 no. 2, june 2021 www.j-tropical-crops.com 85 of the concentration and the leaf production. highest production of both anthocyanins and flavonoids and the highest leaf production were associated with the highest rate of chicken manure. ibrahim et al. (2013) noted that the use of chicken manure could increase the production of phenolic compounds, especially flavonoids. this, according to ibrahim and jaafar (2011) and weibel et al. (2000) is due to the fact that organic fertilizers, including chicken manure, generally contains several important micronutrients for plant metabolism. moreover, according to bímová and pokluda (2009), some chemical reactions in plant metabolism involve micronutrients both directly and indirectly. plants that are cultivated organically tend to produce high secondary metabolites. in this study, we found higher leaf production in plants that had longer harvest interval i.e. 3 months. longer harvest interval provided sufficient time for the plant to recover after pruning. similar results have been reported in the continuously and regularly pruned murraya paniculata (taufika et al., 2017) and talinum triangulare (susanti et al., 2011). susanti et al. (2011) also reported that the response of talinum triangulare to harvest interval is related to process recovery, rejuvenation, and source-sink. longer harvest intervals will allow the plants to regrow longer before the next harvesting. higher leaf production caused higher nutrient uptake because nutrient uptake was obtained from multiplication between dry weight and nutrient concentration per plant (table 4). the longer harvest interval causes an increase in nutrient uptake, but decrease in the nutrient concentration. it was also confirmed by soil chemistry test that soil with harvest interval of 3-months had lower nutrient concentration (table 2) than soil with harvest interval of 2-months (table 1). crop’s dry weight and nutrient uptake is determined by harvest intervals, and dry weight and nutrient uptake for n, p, and k have a linear relationship with harvest interval (allhands and overman, 1995). while longer harvest intervals can increase dry weight, it can also cause a decline in the quality of leaves, reflected by a decreased nutrient concentration, protein, or fibers in the leaves (allhands and overman, 1995). harvest interval of three-month generated higher leaf anthocyanin concentration than the two-month harvest interval, whereas flavonoids concentrations in both harvest intervals were similar. however, both anthocyanins and flavonoids production tend to be higher in the longer harvest interval. according to aziz (2015), bioactive compound production is the result of the bioactive compound concentration multiplied by the biomass of the harvested organ (e.g. leaves). the high production of bioactive compounds does not only depend on the high concentration of the bioactive compounds but also on the fresh or dry weight of the harvested organ. in this case, the longer table 4. the effects of harvest intervals on leaf fresh and dry weights and metabolite contents of vernonia amygdalina plants variables harvest intervals 2 months 3 months leaf fresh weight (g/plant) 494.4±114.9 b 874.3±284.9 a leaf dry weight (g/plant) 72.5±12.8 b 140.1±46.0 a nitrogen concentration (%) 4.1±0.2 a 4.0±0.1 a phosphorous concentration (%) 0.4±0.0 b 0.3±0.0 a potassium concentration (%) 3.8±0.7 b 3.2±0.7 a nitrogen uptake (g/plant) 2.3±0.7 b 5.7±1.5 a phosphorous uptake (g/plant) 0.2±0.0 b 0.5±0.2 a potassium uptake (g/plant) 2.1±0.6 b 4.8±2.0 a chlorophyll a (mg.g-1) 1.6±0.1 a 1.6±0.1 a chlorophyll b (mg.g-1) 0.5±0.0 a 0.5±0.0 a carotenoids (mg.g-1) 0.4±0.0 a 0.4±0.0 a anthocyanins (mg.100 g-1) 0.08±0.0 b 0.1±0.0 a flavonoids (mg.g-1) 22.5±1.2 a 22.4±1.2 a flavonoids production (mg/plant) 1595.0±218.6 b 2980.3±662.1 a anthocyanins production (mg/plant) 0.3±0.1 b 1.0±0.2 a note: values followed by different letters within the same row showed significant differences according to duncan multiple range test (α= 5%) journal of tropical crop science vol. 8 no. 2, june 2021 www.j-tropical-crops.com 86 betty betty, sandra arifin aziz, ketty suketi growing duration resulted in more biomass, therefore, its bioactive compound production were also higher than those produced with the two-months harvest interval. conclusion our study demonstrated that the application of organic manures was essential in improving the growth and secondary metabolites production of v. amygdalina plants. the chicken manure rate that resulted in optimum growth and high production of secondary metabolites was 7.5 kg per plant. the harvest interval of 3 months produced a higher concentration of secondary metabolites than the harvest interval of 2 months. acknowledgment we would like to thank the lpdp (indonesia endowment fund for education) under the indonesian ministry of finance for funding this research as the part of master scholarship program. references abd-alla, m.h., yan, f., and schubert, s. 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(2018). correlations between leaf nitrogen, phosphorus and potassium and leaf chlorophyll, anthocyanins and carotenoids content at vegetative and generative stage of bitter leaf (vernonia amygdalina del.). journal journal of tropical crop science vol. 8 no. 2, june 2021 www.j-tropical-crops.com 88 betty betty, sandra arifin aziz, ketty suketi of tropical crop science 5, 1-4. doi: 10.29244/ jtcs.5.1.25-33 tranaviciene, t., siksnianiene, j.b., urbonaviciute, a., vaguseviciene, i., samuoliene, g., duchovskis, p., and sliesaravicius, a. (2007). effect of nitrogen fertilizer on wheat photosynthetic pigment and carbohydrate contents. biologija 53, 80-84. taufika, r., aziz, s.a., and melati, m. (2017). flavonoid production of orange jessamine (murraya paniculata l.) at different rates of organic fertilizer and harvest interval. buletin penelitian tanaman rempah dan obat 27, 2736. (in indonesian). tona, l., cimanga, r.k., mesia, k., musuamba, c.t., de bruyne, t., apers, s., hernans, n., van miett, s., pieters, l., totte, j., and vlietinck, a.j. (2004). in-vitro anti plasmodial activity of extracts and fraction from seven medicinal plants used in the democratic republic of congo. journal of ethnopharmacology 93, 2732. weibel, f.p., bickel, r., leuthold, s., and alfoldi, t. (2000). are organically grown apples tastier and healthier? a comparative field study using conventional and alternative methods to measure fruit quality. acta horticulturae 517, 417-42. journal of tropical crop science vol. 10 no. 1, february 2023 www.j-tropical-crops.com 8 yuni nurfiana, m. rahmad suhartanto, endah retno palupi, y. aris purwanto ultrafine bubbles water priming to improve viability and vigor of bean (phaseolus vulgaris) seeds yuni nurfianaa, m. rahmad suhartantob, endah retno palupib, y. aris purwantob a seed science and technology program, graduate school, faculty of agriculture, ipb university, bogor 16680, indonesia b department of agronomy and horticulture, graduate school, faculty of agriculture, ipb university, bogor 16680, indonesia c department of mechanical and biosystem engineering, graduate school, faculty of agricultural engineering and technology, ipb university, bogor 16680, indonesia *corresponding author; email: tantosuhartanto63@gmail.com abstract priming is a physiological technique of hydration of seed to improve metabolic processes before germination to accelerate germination and seedling growth under normal and stressful conditions. this research is aimed to study the pretreatment using ultra-fine bubble (ufb) water to increase the viability and vigor of seeds before planting (pre-planting) and during storage (pre-storage). this research was conducted at the laboratory of seed quality testing, ipb university, indonesia. the research consisted of two experiments, i.e., ufb water priming to increase the viability and vigor of bean seeds and their storability after priming. the first experiment was arranged using a completely randomized design with a combination of seed lot treatments (l1: initial germination percentage (gp) about 80%, l2: initial gp around 70%, and l3: initial gp around 60%) with priming (p0: no priming/control, p1: soaked in distilled water for 60 minutes, p2: distilled water for 120 minutes, p3: 8 ppm ufb water for 60 minutes, p4: 8 ppm ufb water for 120 minutes, p5: 20 ppm ufb water for 60 minutes, p6: ufb water 20 ppm for 120 minutes). the second experiment was arranged using a nested design with the main factor being the storage room condition and priming as the second factor nested in the main factor. the storage room condition factors consisted of km: room conditions (temperature 28±50c, rh 73±7%) and ac: airconditioned room (18±20c, rh 61±7%). the priming factor consisted of two levels, namely p0: without priming and p1: priming with 20 ppm ufb water soaked for 120 minutes. the results showed that priming treatment with ufb water 20 ppm for 120 minutes as a pre-planting treatment could increase the viability of bean seeds, particularly for seeds with low initial viability, and primed bean seeds with an initial viability of approximately 80% were able to retain their viability for 16 weeks of storage in an airconditioned room. keywords: germination, pre-planting, pre-storage, seed storability, seed quality introduction quality seed is one of the keys to the success of agricultural production. bean seed (phaseolus vulgaris l.) is one of the essential commercial horticultural crops with high market demand, so good quality seed for planting materials is required. bean seeds undergo a period of storage before reaching the farmers for planting. good handling during storage will maintain the storability of the seeds, but the longer the seeds are stored, the seeds will deteriorate or decline so that their viability of the seeds will decrease. the process of seed deterioration can be classified into chronological decline caused by time factors and physiological decline caused by storage environmental factors. invigoration is one way to increase seed viability and vigor. ilyas (2012) stated that invigoration could be in the form of osmoconditioning priming and matriconditioning. osmoconditioning priming is a preplanting treatment developed to increase germination. mohajeri et al. (2016) showed that the invigoration of bean seeds is carried out by immersing the seeds in osmotic solutions such as cacl2, kcl, nacl, and peg. the results have shown that beans seeds soaked for 6 hours in cacl2 had the highest percentage of germination at 95% while the control was 84%. the research of yuanasari et al. (2015) exhibited that black soybean seeds that were stored for 22 months in a storage room at a temperature of 12±2ºc with a humidity of ±60% had a germination rate of 76.67% ultrafine bubbles water priming to improve viability and vigor of bean .......... journal of tropical crop science vol. 10 no. 1, february 2023 www.j-tropical-crops.com 9 after osmoconditioning treatment using peg-6000 for 12 hours germination rate increased rate to 86.00%. invigoration is used not only as a pre-planting treatment but also as a pre-storage treatment to maintain seed vigor during storage. invigoration is used for marketable seeds to increase germination uniformity in the field. waqas et al. (2019) stated that priming is a physiological technique of hydration and seed drying to improve metabolic processes before germination to accelerate germination, seedling growth, and plant yields under normal and stressful conditions. nawaz et al. (2013) explained that the increase in seed germination after priming treatment was caused by the cell cycle activation process and reduced the endosperm covering so that the seeds remained in the second phase of imbibition and were ready to germinate. research by utami et al. (2013) on long beans seeds invigorated by immersion for 2 hours in water (hydropriming), cacl2, kno3, and ascorbic acid can increase seed vigor index and can be maintained until the end of short-term storage up to 15 weeks both in air conditioning room and room temperature. likewise, the results of yan (2017) on chinese cabbage seeds before priming had a germination rate of 69.4±2.4%, after hydropriming and stored for six months at 40c germination rate increased to 78.2±2.5, while after storage at 200c the germination rate was still 76.2±3.4% and at a storage temperature of 30 ̊c germination rate was 74.8±2.2%. hydropriming by giving constant water bubbles can also increase the viability and vigor of rice seeds. high viability after hydropriming can be maintained for up to 60 days of storage in aluminum foil packaging at a low temperature of -4 ̊c (wang et al., 2018). new technology with great potential for increasing seed viability and vigor is ultra-fine bubbles (ufb) water in the form of micro and nano-sized fine bubbles water (liu et al., 2016). ufb water has been widely studied for improving seed viability and vigor in several commodities, including soybean (purwanto et al., 2019), gmelina arborea (siregar et al., 2020), and white jabon (fata et al., 2020) seeds. numerous researches on priming was conducted by using ufb water to treat pre-planting seeds instead of pre-stored. ufb water increased seed viability and vigor, including invigoration with the hydropriming technique. this study aims to use ufb water to improve seed quality (viability and vigor) and evaluate its effect after the beans are stored. material and methods location the research was carried out from april 2021 to june 2022 at the seed quality testing laboratory, leuwikopo experimental field, department of agronomy and horticulture, faculty of agriculture, and biosystem engineering laboratory, department of mechanical and biosystem engineering, faculty of agricultural engineering and technology, ipb university, bogor, indonesia. materials and methods the material used was “rofi” variety beans with three lots of different quality seeds produced by pt east west seed indonesia, which consisted of lots with germination capacity of about 80% (l1), 70% (l2), and 60% (l3). aluminum foil packaging is used to store seeds after priming. the equipment required is seed germination equipment (eco germinator type ipb 72-l, plastic, and stencil paper as germination media), equipment for determining seed moisture content (oven 1300c, analytical balance, porcelain cup), and equipment for making ufb water (ufb water generator 9fzin-10, ides and pure oxygen with concentrations of 8 ppm and 20 ppm). experiment 1. priming to increase viability and vigor of bean seeds the experiment was arranged in a completely randomized design with one factor with a combination of seed lot treatments (l1: initial gp about 80%, l2: initial gp around 70%, and l3: initial gp around 60%) and priming type (p0: no priming/control, p1: soaked in distilled water for 60 minutes, p2: distilled water for 120 minutes, p3: ufb water 8 ppm for 60 minutes, p4: ufb water 8 ppm for 120 minutes, p5: ufb water 20 ppm for 60 minutes, p6: ufb water 20 ppm for 120 minutes). the combinations obtained were 21 and repeated four times so that there were 84 experimental units. two hundred bean seeds from each seed lot were soaked in 250 ml of distilled water, ufb water 8 ppm, and 20 ppm according to the priming treatment that had determined for 60 minutes and 120 minutes. ufb water 8 ppm used oxygen from the room room, while ufb water 20 ppm used pure oxygen injection. seeds that have been treated are dried to reach the initial moisture content for 7×24 hours at a temperature of 18±2˚c and humidity of 61±7%. seeds were germinated using the between paper method at seed germinator type ipb72-l at 25±5˚c and rh 70±7% in the laboratory. the growth and time of emergence of seeds from treated seeds were also tested in the field by planting one seed in journal of tropical crop science vol. 10 no. 1, february 2023 www.j-tropical-crops.com 10 yuni nurfiana, m. rahmad suhartanto, endah retno palupi, y. aris purwanto each planting hole. experiment 2. the storability of bean seeds after priming the experiment was arranged using a nested design, with the main factor being the storage room condition and priming as the second factor nested in the main factor. the storage room condition factors consist of km: room temperature (temperature 28±50c, rh 73±7%) and ac: air-conditioned room condition (18± 20c, rh 61±7%). the priming factor consisted of two levels, namely p0: without priming and p1: priming with ufb water 20 ppm soaked for 120 minutes. the treatment was repeated four times so that there were 16 experimental units for each observation. the treatment was applied to seed lots with an initial gp of around 80%. the storage period was for 16 weeks, and the seeds’ viability and vigor were tested every two weeks. statistical analysis variance analysis was carried out using statistical analysis system (sas) enterprise guide 7.1 at α=0.05; the duncan test was used for further analysis. result and discussion priming to increase viability and vigor of bean seeds the results of the analysis of variance showed that the combination of treatments between seed lots and priming had a very significant effect on moisture content (mc), germination percentage (gp), vigor index (vi), maximum growth potential (mgp), seedling growth rate (sgr), growth speed (gs), t50, normal seedling dry weight (nsdw), electrical conductivity (ec), mean emergence time (met) and field emergence (fe) (table 1). after priming, the seeds were dried to minimize seed moisture content. the drying process lasted 24 hours and seven times 24 hours. drying for 24 hours after priming for 60 minutes resulted in a seed moisture content of approximately 22-29%, whereas priming for 120 minutes resulted in a moisture content of approximately 30-35%. the still-high moisture content needs drying to attain the initial moisture content of seeds before priming, which is 11-12%. at all initial viability levels, 7 x 24 hours of drying reduced seed moisture content to 10-12%, comparable to seed moisture content without priming. according to the germination percentage parameter, seeds with high initial viability or lot 1 (initial germination percentage 82.5%) were insensitive to priming treatment; even priming with distilled water and ufb water 20 ppm for 60 minutes lowered germination percentage (69.50% and 71.00%, respectively). priming seeds with medium initial viability or lot 2 (initial germination percentage 77.5%) did not significantly affect seed viability, and lowered germination percentage after priming with distilled water for 120 minutes (64.5%). priming with ufb water 20 ppm for 120 minutes significantly improved the germination percentage of seed lots with low initial viability or lot 3 (from 69.5% to 90.50%). (table 2). this outcome is consistent with siregar’s (2020) research findings. invigoration or priming of albizia chinensis with high initial viability did not improve table 1. recapitulation of variance of priming effect on the quality of three bean seed lots that have different initial viability no. variable lot×priming cv 1 moisture content (mc) ** 0.97 2 germination percentage (gp) ** 8.80 3 maximum growth potential(mgp) ** 4.83 4 seedling growth rate (sgr) ns 14.77 5 t50 ** 3.62 6 vigor index (vi) ** 14.98 7 germination speed (gs) ** 14.69 8 normal seedling dry weight (nsdw) ** 11.75 9 electrical conductivity (ec) ** 10.42 10 mean emergence time (met) ns 6.29 11 field emergence (fe) ** 10.97 note: * = significant at p < 0.05. ** = significant at p < 0.01; ns = not significantly different according to the f test with 95% confidence level. ultrafine bubbles water priming to improve viability and vigor of bean .......... journal of tropical crop science vol. 10 no. 1, february 2023 www.j-tropical-crops.com 11 germination percentage, whereas priming with ufb water increased germination percentage in seeds with poor initial viability. all priming treatments failed to raise the maximum growth potential in all lots of bean seeds, and in the case of lot 2, priming with distilled water for 120 minutes decreased maximum growth potential values (table 2). the maximum growth potential number was derived from normal and abnormal germination computation. therefore, even though it was high it did not influence the germination percentage or seed vigor. in contrast, priming with ufb water 20 ppm for 120 minutes raised normal seedling dry weight in lots 1 and 3, but had no significant effect on lot 2. priming can boost seed vigor. the improvement in seed vigor was demonstrated by the vigor index, t50, growth speed, and electrical conductivity (table 3). priming with ufb water 8 ppm and ufb water 20 ppm for 120 minutes raised the vigor index in all seed lots examined, however priming with ufb water 20 ppm for 60 minutes had no effect on seed lots with high initial viability. all seed lots exhibited an increase in t50 following priming with ufb water 8 ppm for 120 minutes, ufb water 20 ppm for 60 minutes, and 120 minutes. priming with distilled water for 120 minutes, ufb water 8 ppm for 60 minutes, and aqua dest for 60 minutes enhanced t50 in lots 2 and 3, whereas priming with aquadest for 60 minutes increased t50 in the seeds with the lowest quality. after priming with ufb water 20 ppm for 120 minutes, growth speed was increased in lots 1 and 2. white jabon seeds were able to improve growth speed, consistent with the findings of fata’s research (2020) on ufb. significantly different from the control, seed priming with ufb water 20 ppm reduced the electrical conductivity value in lot 1. the lower the electrical conductivity score implies that the viability and vigor of the seeds are still high. the priming treatment in lots 2 and 3 had a favorable effect on the electrical table 2. germination percentage, maximum growth potential, normal seedling dry weigh, and seedling growth rate in response to various priming treatments. seeds lot priming gp mgp nsdw sgr (%) (%) (g) (g/kn) l1 p0 82.50 abcd 93.00 abc 2.63 bcd 0.064 p1 69.50 efgh 90.00 abcd 2.12 fg 0.061 p2 82.50 abcd 96.00 a 2.82 ab 0.069 p3 80.00 bcde 94.00 abc 2.59 bcde 0.065 p4 80.00 bcde 95.00 ab 2.70 bc 0.068 p5 71.00 efgh 88.00 bcd 1.97 fg 0.056 p6 92.00 a 95.50 ab 3.22 a 0.069 l2 p0 77.50 cdef 91.00 abcd 2.49 bcdef 0.064 p1 69.50 efgh 84.50 de 2.17 efg 0.062 p2 64.50 gh 79.50 e 2.12 fg 0.066 p3 72.00 defgh 87.00 cd 2.41 bcdefg 0.068 p4 76.00 def 88.00 bcd 2.48 bcdef 0.066 p5 70.50 efgh 91.00 abcd 2.08 fg 0.059 p6 87.00 abc 94.50 abc 2.82 ab 0.065 l3 p0 69.50 efgh 96.00 a 2.05 fg 0.059 p1 77.50 cdef 92.50 abc 2.33 cdefg 0.060 p2 61.00 h 89.00 abcd 1.48 h 0.056 p3 70.50 efgh 89.50 abcd 2.19 defg 0.063 p4 67.00 fgh 90.00 abcd 2.02 fg 0.061 p5 73.00 defg 93.50 abc 2.12 fg 0.058 p6 90.50 ab 93.50 abc 2.66 bc 0.059 note: values within the same column followed by the same letter are not significantly different based on the dmrt. gp = germination percentage. mgp = maximum growth potential. nsdw = normal seedling dry weight. sgr = seedling growth rate journal of tropical crop science vol. 10 no. 1, february 2023 www.j-tropical-crops.com 12 yuni nurfiana, m. rahmad suhartanto, endah retno palupi, y. aris purwanto figure 1. moisture content of bean seeds without priming and priming as a response to the conditions of the seed storage space during 16 weeks of storage: (a) air-conditioned room, (b) room temperature figure 2. germination of bean seeds without priming and priming in response to the condition of the seed storage space during 16 weeks of storage: (a) air-conditioned room, (b) room temperature conductivity parameter. except for priming with ufb water 8 ppm for 60 minutes, all priming procedures in lot 2 and lot 3 reduced the electrical conductivity value compared to the control. the field emergence of seeds revealed that none of the priming treatments increased field emergence in any of the seed lots that were examined. priming with ufb water 20 ppm for 120 minutes was the treatment that improved seed quality the most at the three initial viability levels examined (vigor index, t50, and electrical conductivity; see table 3). high seed vigor refers to the capacity of seeds to survive in suboptimal environments. very fine ufb water can reach the seeds through the seed coat, enhancing seed respiration. according to liu et al. (2016), as more oxygen is transported into the seed, the seed’s respiration will increase, allowing it to spend more energy for germination. in addition, gomes and garcia (2013) demonstrated that ufb water generates hydroxyl radicals that can induce the development of reactive oxygen species (ros) in seeds, which act as physiological regulators for germination signaling and can enhance gibberellin levels in seeds. shelf life of bean seeds after priming after priming and prior to storage, the initial moisture content of seeds is approximately 13% (figure 1). according to government regulations, this moisture content are not safe for storing bean seeds which is 11% (ministry of agriculture, 2019). this increased moisture content led to decreased seed viability and vigor during storage of 16 was (weeks after storage) relative to the control (figures 2, 3, and 4). the high initial moisture content enables the seeds to have a high respiration process. the germination percentage of seeds without priming could be maintained up to 16 was both in the room and in the air-conditioned room (>80%), however, seeds with ufb water 20 ppm for 120 minutes decreased the germination percentage. however, the germination percentage treated with priming and stored in a room with air conditioning increased over time. during the storage of 16 was, the germination percentage of seeds treated with priming and stored at room temperature continued to decline (figure 2). this may arise because the seed lot utilized ultrafine bubbles water priming to improve viability and vigor of bean .......... journal of tropical crop science vol. 10 no. 1, february 2023 www.j-tropical-crops.com 13 possesses high initial viability and vigor (around 80% initial germination percentage). according to research by powell et al. (2000), priming with water immersion for 28 hours on cauliflower seeds with high initial vigor has a negative effect on seed shelf life, however, priming on seeds with low vigor can extend seed shelf life. based on t50 (50% normal germination time), the vigor of priming and control seeds stored in both the air-conditioned and room temperature did not differ substantially (figure 3). intriguingl the vigor index of priming seeds increased throughout storage in both storage conditions (figure 4), whereas the vigor index of non-primed seeds decreased during storage in room temperature circumstances. after being stored in an air-conditioned room, priming and non-priming seeds had the same vigor index. seed quality can be maintained during storage if the seeds are stored under optimal conditions. the storage of priming seeds at high temperatures and rh accelerates seed respiration, utilizing food reserves. the seed quality of beans stored in the room temperature was lower than beans that are not primed. the decline in viability and vigor the room temperature-stored bean stored bean seeds could be induced by high temperature and relative humidity (28±2⁰c, 73±7%). according to wang et al. (2018), situations of high rh can result in seed degeneration due to an increased lipid peroxidation. after priming, seed storage in an air-conditioned room is preferable to storage at room temperature. according to adhinugraha et al. (2022), the true seed of shallot (allium ascalonicum l.) stored in an airconditioned environment following revitalization can be maintained for 14 weeks. this study revealed that priming and storing bean seeds in an air-conditioned room boosted their quality despite their high moisture content. table 3. vigor index, t50, growth speed, electrical conductivity, mean emergence time, and field emergence in response to various priming treatments lot benih priming vi t50 gs ec met fe (%) (day) (%kn/etmal) (µs cm−1 g−1) (day) (%) l1 p0 46.95 cde 4.08 b 19.80 bcde 24.01 bc 5.38 a 59.00 abcde p1 44.90 cdef 4.09 b 19.70 bcde 22.31 bcde 5.05 a 63.00 abcd p2 67.33 a 3.99 bc 19.00 bcde 20.66 cdef 4.95 a 51.50 efg p3 53.50 bc 3.91 bcd 21.28 abcd 21.02 cdef 5.18 a 64.00 abc p4 68.00 a 3.67 efgh 21.93 abc 21.55 cde 5.05 a 63.00 abcd p5 54.78 bc 3.48 hi 22.15 abc 21.81 cde 5.25 a 67.50 a p6 71.03 a 3.01 l 25.48 a 17.52 f 5.00 a 65.00 ab l2 p0 38.50 def 4.10 b 17.48 cdef 28.70 a 5.30 a 53.00 defg p1 50.23 bcd 4.08 b 13.63 f 22.48 bcde 5.18 a 53.00 defg p2 42.38 cdef 3.83 cdef 16.48 def 20.89 cdef 5.00 a 37.00 h p3 46.75 cde 3.87 cde 17.55 cdef 20.89 cdef 5.08 a 55.50 bcdefg p4 67.56 a 3.59 gh 18.80 bcde 21.97 bcde 4.98 a 54.50 cdefg p5 52.30 bc 3.22 jk 19.70 bcde 20.63 cdef 4.83 a 58.50 abcdef p6 62.43 ab 3.10 kl 22.53 ab 19.94 def 5.05 a 56.50 bcdefg l3 p0 32.82 f 4.33 a 17.48 cdef 28.77 a 5.28 a 51.50 efg p1 50.23 bcd 3.77 defg 16.28 ef 23.69 bcd 5.03 a 48.00 g p2 35.92 ef 3.58 gh 17.33 cdef 23.96 bc 5.18 a 48.50 fg4 p3 46.50 cde 3.63 fgh 19.35 bcde 25.78 ab 5.00 a 53.00 defg p4 45.45 cde 3.68 efgh 17.95 bcdef 23.21 bcd 4.90 a 49.00 efg p5 60.83 ab 3.34 ij 19.63 bcde 22.11 bcde 5.25 a 55.00 bcdefg p6 61.55 ab 3.04 kl 21.43 abc 18.72 ef 4.93 a 54.00 cdefg note: values within the same column followed by the same letter are not significantly different based on the dmrt. vi = vigor index, t50 = time required to 50% germination, gs = growth speed, ec = electrical conductivity, met = mean emergence time, fe = field emergence. journal of tropical crop science vol. 10 no. 1, february 2023 www.j-tropical-crops.com 14 yuni nurfiana, m. rahmad suhartanto, endah retno palupi, y. aris purwanto figure 3. t50 of bean seeds without priming and priming as a response to the condition of the seed storage space during 16 weeks of storage: (a) air-conditioned room, (b) room temperature figure 4. vigor index of bean seeds without priming and priming as a response to the condition of the seed storage space during 16 weeks of storage: (a) air-conditioned room, (b) room temperature ultra-fine bubble water provides oxygen to the seeds, hence boosting seed respiration. when seeds are stored, respiration will continue, resultdeclining seed viability. the reduced viability and vigor of seeds in priming treated with ufb water 20 ppm for 120 minutes may also be owing to the influence of extremely high ros, which destroys the seeds’ macromolecules. ros can induce oxidative damage to macromolecules, dna damage, and dna splitting/ shortening, according to tomizawa et al. (2005). ishibashi et al. (2009) discovered that excessive concentrations of reactive oxygen species (ros) might act as damaging substances; nevertheless, under optimal conditions, ros will play an essential role in relaxing cell walls and signaling crucial plant growth activities. a study in rice, lettuce and radish reported that priming shortened the seed shelf life, (hill et al. 2008, hussein et al. 2015, malek et al. 2019). while other researchers found that priming boosted and prolonged the shelf life of seeds of digitalis purpurea, rhododendron griersonianum, long beans, chicory, rice, and shallots (butler et al. 2009; wood and hay 2010; utami 2013; yan 2017; wang et al. 2018; adhinugraha et al. 2022). this results demonstrate that priming with ufb water 20 ppm for 120 minutes enhanced the viability of bean seeds stored in an airconditioned environment until week 16. meanwhile, priming bean seeds stored at room temperature was insufficient to preserve the seeds’ shelf life until 16 was. the failure to lower the moisture content until they are safe for storage is believed to be the leading cause of the deterioration in seed quality following priming. if seeds treated with priming are not followed by a decrease in safe storage moisture content, seed deterioration will be accelerated. in order to preserve the viability and vigor of the primed seeds during storage, the seeds should preferably be dried and stored at low humidity soon after priming. conclusion priming ufb water 20 ppm for 120 minutes as a preplanting treatment can increase the viability of bean seeds, particularly for seeds with low initial viability (germination percentage about 60%), however, the ultrafine bubbles water priming to improve viability and vigor of bean .......... journal of tropical crop science vol. 10 no. 1, february 2023 www.j-tropical-crops.com 15 effect is insignificant for seeds with greater initial viability. the priming treatment boosted seed vigor in all evaluated seed lots. references [ministry of agriculture] decree of the ministry of agriculture republic of indonesia. adhinugraha, q.s., widajati, e., and palupi, e.r., (2022). invigoration improved quality and storability of the true seed of shallot (allium ascalonicum). journal of tropical crop science 9, 145-155. butler, l.h., hay, f.r., ellis, r.h., smith, r.d., and murray, t.b. (2009). priming and re-drying improve the survival of mature seeds of digitalis purpurea during storage. annals of botany 103, 1261-1270. doi: 10.1093/aob/mcp059 fata, n.a.n., supriyanto, n., rustam, e., and sudrajat, d.j. (2020). invigoration treatment of white jabon (neolamarckia cadamba (roxb.) bosser) seeds using polyethylene glycol and ultrafine bubbles. jurnal perbenihan tanam hutan 8, 11–24. doi:10.20886/bptpth.2020.8.1.11-24 gomes, m.p., and garcia, q.s. (2013). reactive oxygen species and seed germination. biologia 68, 351-57. doi:10.2478/s11756-013-0161-y hill, h.j., bradford, k.j., cunningham j.d., and taylor, a.g. (2008). primed lettuce seeds exhibit increased sensitivity to moisture during aging. acta horticulturae 782, 135-141. doi:10.17660/actahortic.2008.782.14 hussein, s., zheng, m., khan, f., khaliq, a., farhad, s., peng, s., huang, j., cui, k., and nie, l. (2015). the benefit of rice seed priming is offset permanently by prolonged storage and storage conditions. scientific reports 5, 1-12. doi: 10.1038/srep08101 ilyas, s. (2012). “ilmu dan teknologi benih: teori dan hasil-hasil penelitian”. ipb press. ishibashi, y., tawaratsumida, t., zheng, s.h., yuasa, t., and iwaya, i.m. (2009). nadph oxidases act as a key enzyme in barley germination and seedling growth (hordeum vulgare l.). plant production science 13, 45–52. doi: 0.1626/ pps.13.45 liu, s., oshita, s., makino, y., wang, q., kawagoe, y., and uchida, t. (2016). oxidative capacity of nanobubbles and its effect on seed germination. acs sustainable chemistry and engineering 4, 1347–1353. doi:10.1021/ acssuschemeng.5b01368 malek, m., ghaderi-far, f., torabi, b., sadeghipour, h.r., and hay, f.r. 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(2015). peningkatan viabilitas benih kedelai hitam (glycine max l. merr) melalui invigorasi osmoconditioning. jurnal produksi tanaman 3, 518-527. 2. willy bayuardi_vol2_2014.cdr abstract the phenomenon of heterosis provides a great opportunity for plant breeders to gain greater performance and yield in the hybrids compared to their inbred line parents. in most cross-pollinated crops like maize, heterosis plays an important role as the performance of the hybrids is of a great value. heterosis gain much interest until recently and current studies still attempt to elucidateone of these is utilizing molecular markers to estimate genetic distances among inbred lines followed by developing putative groups. in a wellde�ned heterotic group, between-groups mating are expected to produce better hybrids than within-groups mating. some studies of marker-aided heterotic group development resulted in functional heterotic groups; while some other reported that the groups failed to provide evidence of heterosis in the hybrids. considering the prediction of hybrids' performance will depend on genetic background of inbred lines, and markers technology are continuously improved, there is still a good opportunity to obtain useful heterotic groups for a particular breeding population. keywords: maize breeding, genetic distance, heterosis, molecular markers. hybrid maize breeding excellent maize hybrid varieties are obtained through development and use of inbred lines with superior combining ability for grain yield and other agronomic traits. hybrid maize breeding includes development of stable, vigorous, high-yielding inbred lines with the extensive evaluation of combining ability during the process of developing the lines, followed by use of selected inbred lines in development of improved hybrids (singh, 1987). characterization of maize lines for their combining ability is routinely conducted for numerous traits, including adaptation to drought and low nitrogen stress conditions (betran et al., 2003; medici et al., 2004) (welcker et al., 2005), soil acidity , a�atoxin accumulation , resistance to(williams et al., 2008) pathogens causing lodging (moreno-gonzalez et al., 2004) (bynum et al., 2004), mite , striga hermonthica and , maizestriga asiatica (gethi and smith, 2004) weevil , and many more.(dhliwayo et al., 2005) combining ability of maize lines is also important for value adding traits, including nutritional characteristics, such as grain's iron and zinc density ,(long et al., 2004) pro vitamins a, protein, oil and starch content, and of course, grain yield. the increase in size, vigor, or productivity of a hybrid plant over its parents is known as hybrid vigor or heterosis (allard, 1960; poehlman, 1995). genetic diversity is important in maize breeding because crosses among two similar inbred lines are expected to result in small levels of heterosis. inbred lines are therefore typically developed from two or more genetically different populations to obtain high levels of heterosis in their hybrids (singh, 1987). instead of naturally exists in the germplasm, strong evidences revealed that heterotic pattern were developed by the plant breeders (tracy and chandler, 2006). the use of molecular plant breeding tools has become routine in many variety development programs. these methods can enhance breeding ef�ciency by replacing phenotypic with genotypic selection during some stages of the breeding process, thereby reducing overall phenotyping costs, biases (e.g. caused by environment factors and genotype by environment interactions), and measurement errors (moose and mum, 2008). in hybrid maize breeding programs, development of heterotic groups and assignment of inbred lines into the established heterotic groups are very important. development of heterotic groups and assignment of experimental inbred lines to heterotic groups before making hybrid crosses is time and cost ef�cient, because the number of crosses to be made and evaluated will decrease substantially. the use of molecular marker data and genetic distances (gds) to predict heterosis, however, has given inconsistent results (collard and mackill, 2008). molecular marker approaches for heterotic groups development molecular marker methods have been used extensively to estimate genetic distance between maize populations and lines and to assign maize inbreds to heterotic groups. the molecular marker technologies have been the usefulness of molecular markers approach for developing heterotic groups in maize willy b. suwarno* department of agronomy and horticulture, faculty of agriculture, bogor agricultural university jl. meranti kampus ipb dramaga, bogor 16680, indonesia. *corresponding author; email: bayuardi@gmail.com 4 journal of tropical crop science vol. 1 no. , 20142 october www.j-tropical-crops.com evolving rapidly, thus the preferred molecular marker types for these and other applications have been changing every few years. beginning around 1991, researchers commonly used restriction fragment length polymorphism (rflp) markers for genetic relationship studies in maize. the use of rflps was followed by a preference for use of ampli�ed fragment length polymorphism (aflp) markers, and after that simple sequence repeat (ssr) or microsatellites have been the most commonly used markers for diversity work in maize. most recently, single-nucleotide polymorphism (snp) markers are becoming the markers of choice for many applications in maize breeding. rflp markers were proven as a convenient method to distinguish maize populations based on geographic origins . to(gauthier et al., 2002; rebourg et al., 1999) some extent, researchers also found that rflps were also suitable for predicting heterotic groups and measuring genetic diversity between lines. several studies found that rflp is useful for assigning inbreds into heterotic groups, indicated by consistency of the gds grouping with pedigree information (boppenmaier et al., 1993; dubreuil and charcosset, 1999; li et al., 2000; livini et al., 1992; melchinger et al., 1991) and scas estimation . however, different(pinto et al., 2003) studies reported that rflp is only effective for predicting f performance for crosses between lines1 from the same heterotic group (boppenmaier et al., 1993; melchinger et al., 1992). on the other hand, warburton et al. (2005) had dif�culty to �gure out potential heterotic groups using rflps only as they could not differentiate the testers from opposite heterotic groups. genetic distance based on rflp markers could have positive correlation with f values, hybrid performance, sca, and heterosis . however,(betran et al., 2003) correlation between gds and heterosis were low for pairs from different heterotic groups (benchimol et al., 2000). correlation between gd and performancebased relationship was high (rebourg et al., 2001; williams and hallauer, 2000), especially when the parents were distantly related .(dudley et al., 1991) genetic similarity (gs) and f values were highly correlated for related lines (ajmonemarsan et al., 1992; messmer et al., 1993), and gs mean for line combinations from different population was smaller than unrelated line combinations from the same population (livini et al., 1992; marsan et al., 1998; melchinger et al., 1991). studies that demonstrated successful heterotic grouping using aflp marker were reported by chitto, et al. (2000), li et al. (2004) and marsan et al. (1998). on the contrary, a study by we and dai (2000) reported that aflp's based gds have low reliability to predict hybrid yield and heterosis because there is either no correlation between them for unrelated lines (wu and dai, 2000) (marsan et al.,or the correlation is too small 1998). in a study with 51 european maize lines representing �int and dent heterotic groups, gs estimates from rflps and aflps were correlated for both related and unrelated pairs (lubberstedt et al., 2000). low correlation between gd and grain yield could be caused by small range of gd due to previous selection for combing ability . kumar (1999)(barbosa et al., 2003) suggests that in order to obtain signi�cant relationship between gds and hybrid performance several conditions must be met, i.e. adequate phenotypic evaluation and choice of markers used for analysis, and suf�cient number of parental inbred lines (stuber et al. in kumar, 1999). gd revealed by ssrs tends to show greater degree of polymorphism compared to aflps (heckenberger et al., 2006). finding an appropriate set of ssr loci is also an important consideration because it could provide similar accuracy with the use of a greater number of loci .(enoki et al., 2005) more recent studies on genetic distances and heterotic grouping have been using ssr markers. several studies found that ssr markers perform high polymorphism and could be used for predicting heterotic groups, i.e. for cimmyt and iita lines (dhliwayo et al., 2009), tropical maize lines (aguiar et al., 2008), and reid yellow dent and lancaster sure crop lines (choukan et al., 2006; jambrovic et al., 2008). in contrast, some studies revealed that ssr markers could not predict heterotic groups precisely (adetimirin et al., 2008; barata and carena, 2006; xia et al., 2004; xia et al., 2005), where one of the possible reasons is because the lines studied have incorporated mixed composition from quite diverse populations (xia et al., 2004; xia et al., 2005). ssr-based genetic distances were correlated well with speci�c combining ability in temperate inbreds (zheng et al., 2008) (phumichai et al.,and tropical hybrids 2008). however, evidence of correlation in interand intrapopulational crosses were vary. amorim et al. (2006) reported that interpopulational crosses tend to have more signi�cant correlation between gds and grain yield, whereas barbosa et al. (2006) reported low correlation at interpopulation level. meanwhile, ssrmarker gds are not signi�cantly correlated with heterosis in the study of ciimmyt and iita inbred lines (dhliwayo et al., 2009) (xu etand chinese inbred lines al., 2004) comparison studies between markers revealed that aflps and ssrs found that there were signi�cant correlation between marker-based and morphologicalbased gds for both marker platforms (beyene et al., 2005), as well as the correlation between those two 5 journal of tropical crop science vol. 1 no. , 20142 october www.j-tropical-crops.com the usefulness of molecular markers approach for developing.......... platforms' gds (beyene et al., 2005; heckenberger et al., 2003). both marker platforms can successfully differentiate earlyfrom late-maturing -resistantstriga inbred lines . another successful(menkir et al., 2005) result was obtained from a study with 18 tropical maize inbred lines, which reported that aflps, ssrs, and rflps' gd were correlated with hybrid performance and heterosis in intra population crosses (garcia et al., 2004), however, in some cases, aflp and ssr gds were poorly correlated and could(laborda et al., 2005) not perform satisfying heterotic groups for both tropical maize .(laborda et al., 2005; menkir et al., 2004) bernardo et al. (2000) suggested that ssrs are more reliable than rflps to study the genetic relationships in maize. low correlation between gd and grain yield could be caused by small range of gd due to previous selection for combing ability . kumar (1999)(barbosa et al., 2003) suggests that in order to obtain signi�cant relationship between gds and hybrid performance several conditions must be met, i.e. adequate phenotypic evaluation and choice of markers used for analysis, and suf�cient number of parental inbred lines (stuber et al. in kumar, 1999). gd revealed by ssrs tends to show greater degree of polymorphism compared to aflps (heckenberger et al., 2006). finding an appropriate set of ssr loci is also an important consideration because it could provide similar accuracy with the use of a greater number of loci .(enoki et al., 2005) recently, snps are known as new marker technology provides automation and more precise results. clustering of genotypes based on ssr markers has successfully differentiated the tropical from temperate maize germplasm, and provided consistency with pedigree and information and combining ability (lu et al, 2009). another study reported that snp loci should be available in large number to replace highly polymorphic ssrs (hamblin et al., 2007). moreover, large parental genetic diversity makes precise measurement of the association among lines more dif�cult (hamblin et al., 2007). prospects on developing heterotic groups for a novel breeding population heterotic groups for tropical maize are not yet as establish as those for temperate maize, and molecular markers can be a good advance to help in setting up such groups. an applicable heterotic pattern can bene�t a maize breeding program for development of goodperforming hybrids. well-developed heterotic groups will allow plant breeders to acquire maximum bene�t from heterosis, yet the question is whether the use of molecular markers approach for building such groups is ef�cient. as mentioned above, there are several studies con�rming the usefulness of molecular markers approach for developing heterotic groups, despite of some unsuccessful ones. it is important to note that the heterotic pattern can vary from population to population, since such pattern is developed by breeders rather than naturally exists (tracy and chandler, 2006). the predictability of f performance using a marker-aided1 heterotic pattern depends on the genetic background of inbred lines that one working on (boppenmaier et al, 1993). other important consideration is selection of marker platform to be used. in some of the previous studies, rflp, aflp, and ssr markers had been reported helpful for developing heterotic groups. as markers technology advances rapidly, exploitation of the most current marker technology such as snp for various genetic studies is interesting to many researchers. a recent methods, genotyping by sequencing (gbs) (elshire, 2011), allows utilization of thousands of snp markers for heterotic pattern studies, which may increase the probability to develop a useful one. one interesting exercise would be to estimate how many markers are needed to perform such analysis (k. pixley, cimmyt, pers. comm.), followed by de�ning a core set of snps that suitable for estimation of genetic distances among lines having certain adaptation (e.g. lowland tropical, temperate, or highland). with the availability of recent bioinformatics tools with capability to handle large data sets, genetic distance between inbred lines can be calculated in a timely manner. a putative heterotic group developed using markerbased gds can be further re�ned using estimates of speci�c combining ability (sca) and discriminant analysis of principal components (dapc). a hierarchical clustering using the complete linkage method can be used as dapc's prior information. as expected, results from the analysis based on sca (actual) estimates were more satisfactory than that based on gd (predictive) data in terms of f yield1 comparison among betweenversus within groups mating (suwarno., et al, 2014). previous studies suggested that marker-based gds are correlated with heterosis and f yield only up to a certain1 distance point, and after that there are no associations between those (melchinger, 1999; suwarno, 2014). this implies that heterosis is not expected to arise from crosses among two genetically similar lines, yet it is challenging to predict heterosis and f yield for crosses1 among two genetically dissimilar lines using molecular marker information. considering the usefulness of h e t e r o s i s i n h y b r i d m a i z e p r o d u c t i o n f u r t h e r investigations using more recent approaches should be conducted to better understand this phenomenon. acknowledgements the author would like to thank dr. kevin pixley (genetic 6 journal of tropical crop science vol. 1 no. , 20142 october www.j-tropical-crops.com willy b. suwarno resources program, cimmyt) for his valuable suggestions to improve this manuscript. references adetimirin, v.o., vroh-bi, i., menkir, a., mitchell. s.e., and kresovich, s. 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(2008). m o l e c u l a r g e n e t i c d i s t a n c e a n d h y b r i d performance between chinese and american maize ( l.). inbreds.zea mays australian journal of agricultural research , 1010-1020.59 10 journal of tropical crop science vol. 1 no. , 20142 october www.j-tropical-crops.com willy b. suwarno growth and yield of kalimantan landrace rice and “ipb 8g” as affected by time .......... journal of tropical crop science vol. 6 no. 2, june 2019 www.j-tropical-crops.com 129 growth and yield of kalimantan landrace rice and "ipb 8g" as affected by dose and time of nitrogen fertilizer application faisala, iskandar lubisb, ahmad junaedib and sugiyantab a north sulawesi assessment institute for agricultural technology, manado 95013, indonesia b department of agronomy and horticulture, ipb university, bogor 16680, indonesia. *corresponding authors: iskandarlbs@yahoo.com abstract the study aimed to determine the effect of nitrogen fertilizer dose and the frequency of application on the growth and production of landrace rice kalimantan and a new rice cultivar developed by bogor agricultural university, “ipb 8g”. the study was conducted in december 2017 until june 2018 at sawah baru experimental field, bogor agricultural university, west java, indonesia. the experimental design was a split-split plot consisting of three treatment factors, i.e. rice cultivars, fertilization time and fertilizing doses. the three rice cultivars used in this trial were landrace cultivars “mayas” and “samarinda”, and “ipb 8g”. the rates of nitrogen were 75 and 150 kg n.ha-1 applied at different phases of rice growth i.e. 50% at planting, 25% at the vegetative phase, and 25% at the primordial phase, and without n fertilizer as control. another time of application was 40% at planting, 20% during the vegetative phase, 20% at the primordial phase, and 20% at heading (four times). each treatment consisted of three replications totalling 54 experimental units. the n fertilizer application gave a significant effect on all growth parameters except for the harvest index. time of fertilizer application significantly affected rice panicle number per hill and harvest index. there were significant differences in the growth of the three rice cultivars, except for the number of tillers. rice crops fertilized three times at 75 kg n.ha-1 had the highest number of tillers at five week after planting, but it was not significantly different from n at 150 kg.ha-1. the highest grain yield of 2.9 t.ha-1 was obtained from “ipb 8g” cultivar fertilized with 75 kg.ha-1 of nitrogen. keywords : local rice, “mayas”, “samarinda”, fertilization introduction rice is the main staple food in indonesia with a population of 255.46 million and rice consumption rate of 124.89 kg per capita per year (pusdatin kementan, 2016). the rice consumption increases along with the increase in the population. according to the bps projection (2014), the indonesian population will reach up to 305.65 million by 2035 which would propose a big challenge to meet the national demand for rice. one of the ways of fulfilling this demand is by increasing rice production. efforts to increase national rice production could be achieved by optimizing the use of dry lands, both those that have been and have not been used for agricultural land (abdurachman et al., 2008). the current production of upland rice in indonesia is 3,631,345 tons of dry milled grains which accounts for 4.8% of national rice production or 33.39 quintal per ha (bps, 2016). the productivity of upland rice is still low due to the many limiting factors in its production management. new technological innovation in upland rice farming such as the use of superior cultivar and the correct dose of fertilizer should be investigated. according to nasirah and damanik (2015) superior upland rice cultivars have high yields, resistance to major diseases, earlier production, and have acceptable taste. one of the new superior upland rice cultivar with high productivity is “ipb 8g”. the development of new type superior rice cultivar does not necessarily affect the demand for local rice varieties. local rice varieties have important traits, including resistance to diseases (wijayanto, 2013; ichsan et al., 2015), so they are popular and still produced in many regions in indonesia. the availability of local rice cultivar assists breeders in developing new rice cultivar. it is very important to preserve the rice local varieties as assets of national genetic resources, so they can be utilized in breeding journal of tropical crop science vol. 6 no. 2, june 2019 www.j-tropical-crops.com 130 faisal, iskandar lubis, ahmad junaedi and sugiyanta programs (sitaresmi et al., 2013; kurniawan and widodo, 2009). “mayas” and “samarinda” are kalimantan landrace rice which local communities continue to grow for their good taste, fragrant aroma and high economic value, high production and the lowest percentage of empty grain compared to other kalimantan cultivars, abung and timur (arinta and lubis, 2016). however, kalimantan landrace rice production in general is still low. investigation on the suitable cultivation technology is needed to for the landrace rice, including determination of the optimal rates of fertilizers, and the frequency of fertilizer application. nitrogen (n) fertilizer plays an important role in increasing rice production; sufficient availability of n during rice generative phase is very important in slowing down the leaf aging process, thus maintaining an optimum photosynthesis during the grain filling phase. n deficiency affects plant height, reduced number of tillers, and number of grains per panicle (siregar and marzuki, 2011). leaf color is an indicator of n status in plants as a plant deficient in n will exhibit chlorosis (yellowing) in the older leaves (siregar and marzuki, 2011; syahril et al., 2017). upland rice genotypes might have different response to high dose of n fertilizer, and low n did not affect their yield components (fageria et al., 2010). hirzel et al. (2011) reported that rice fertilized with n at 120 or 140 kg.ha-1 had the highest grain production. the highest productivity was obtained when n was applied in stages, i.e. 33% at planting, 33% at tillering stage, and 34% at panicle initiation. another option is 50% n at planting, and 50% at panicle initiation. baretto et al. (2012) showed that n fertilizer at 150 kg.ha-1 applied 50% at planting and 50% at tillering increased production of two upland rice cultivars, “brs mg curinga” and “brs primavera”. time of fertilizer application determines the production and rice harvest index. gebremariam and baraki (2016) demonstrated that the application of n fertilizer 33% at planting, 34% at the time of tiller formation, and 33% at the initiation of panicles, gave the best yield and harvest index. moreover, jemberu et al. (2015) in their study on upland rice reported that application of 50% nitrogen fertilizer at planting and 50% at tillering phase resulted in a higher production compared to 50% n applied at planting and at the primordial stage, 25% each at tillering and flowering stage. therefore, it is important to study the correct time of application and the optimal dose of n fertilizer on kalimantan landrace rice. this study investigated the application time and rates of n on rice growth and yields of landrace rice kalimantan as compared to a new rice cultivar “ipb 8g”. materials and methods the research was conducted at the sawah baru experimental station, dramaga, department of agronomy and horticulture, ipb from december 2017 until june 2018. fertilizers used were urea, sp36, and kcl. the equipment used in this experiment was li-3000c portable leaf area meter, cameras, stationeries, and moisture meter. the experiment was organized in a split-split plot design consisting of three treatments, i.e. rice cultivar, fertilization time, and the different doses of fertilizer. three rice cultivars were tested, local “mayas”, local “samarinda”, and “ipb 8g”. the nitrogen fertilizers were applied at 0, 75 and 150 kg n.ha-1 at different proportion and at different stages of the crop growth. the first method of application is 50% n at planting, 25% n at vegetative stage, and 25% at primordia stage. with the second method of application, n was applied at four stages of the crop growth, i.e. 40% n at planting, 20% n during the vegetative phase, 20% at the primordia stage and 20% at heading (50% flowering). each treatment consisted of three replications, so altogether there were 54 experimental units consisting of 10 m2 plots for each treatment. the growth parameters measured were plant height (cm), performed weekly by measuring plant height from the base of the stem at the surface of the ground to the longest leaf in one hill. number of tillers, the number of tillers per hill was measured on five rice hill samples per plot. destructive samplings were collected from two hills per plot at vegetative, panicle initiation, heading and maturity. leaf area (cm2) was measured by li3000c portable leaf area meter. dry weight (g) was from two sample plants per plot, and then dried in an oven at 80° c for 48 hours. 1000-grain weight (g), number of grain per panicle, and the number of grains per panicle were calculated from the average of three sample panicles per hill from three sample clusters. the number of panicles per hill is calculated on all panicles in one cluster from three sample plants. the panicle length (cm) was measured from the base of the panicle to the tip of the panicle before the rice grains were separated after the crops were harvested. three panicles per hill of three sample clusters were measured. grain weight per hill and estimated yield per hectare were obtained by measuring the yields from 1 m x 1.5 m plots, then multiplied the yield to one hectare area. soil analysis was conducted at biotrop seameo soil laboratory, bogor, indonesia, to determine the levels of organic c, n, c/n ratio, p2o5, k, cec and growth and yield of kalimantan landrace rice and “ipb 8g” as affected by time .......... journal of tropical crop science vol. 6 no. 2, june 2019 www.j-tropical-crops.com 131 base saturation using the soil analysis method by sulaeman et al. (2005). results and discussion soil properties the soil type in the research site is latosol with a ph of is 5.4, which is classified as low. the soil has low organic c level (1.33%), moderate level of n (0.23%), low c / n ratio (6), high p2o5 (122.2 ppm), very low level of k (0.19 cmol.kg-1), moderate level of cec (18.26 cmol.kg-1), and high saturation base (66.81%). plant height different rice cultivars showed different responses to nitrogen fertilizer rates (table 1). “ipb 8g” has a shorter life cycle (10 weeks) than “mayas” and “samarinda” cultivars (table 1). “samarinda” was the tallest cultivar with the height of 110.6 cm at week 9 and 128.7 cm at week 13 (table 1). this is in line with the results of arinta and lubis’s (2018) that “samarinda” is classified as tall, whereas “mayas” and “ipb 8g” are classified as medium height. differences in plant height among cultivars or varieties are likely due to the differences in genetic traits (nazirah and damanik, 2015), which can affect plant adaptation to the environment (alavan et al, 2015). n application increased the rice height as shown in figure 1. there was a significant effect of the different rates of n on plant height of the three rice cultivars starting at 3 wap. these results are in line with alim (2012) which reported that application of nitrogen increases plant height, and mahajan et al. (2010) reported that nitrogen enhances vegetative growth through increasing cell division and enlargement in the meristem tissue, resulted in expansion of the width or diameter of plant organs (gardner et al. 1991) number of tillers time and dose of nitrogen application interacted in affecting the number of tillers (table 2), as also reported by amrutha et al. (2016). tiller growth is strongly influenced by nitrogen; urea application at planting can supply nitrogen requirement for the optimal growth (abu et al., 2017). jemberu et al. (2015) showed that the application of 50% nitrogen fertilizer at planting and 50% in the early stages of tillering to upland rice gave the greatest number of tillers, and there was no interaction between the rice variety and time of application of nitrogen fertilizer. leaf area leaf area varies with rice cultivars and rates of n fertilizer (table 3). at the vegetative and the primordial stage, there was no significant difference in the leaf area amongst the three rice cultivars as affected by the different rates of n fertilizer. however, n fertilization at 150 kg.ha-1 significantly increased leaf area during the heading stage (2952.5 cm2) (table 3). “mayas” and “samarinda” had a relatively large leaf area compared to “ipb 8g”’s at vegetative and primordia stage, but there were no significant differences amongst the cultivars at the heading stage. the leaf area of all cultivar showed similar growth pattern, with an increase in leaf area in the early stages, and decreased after entering the heading phase. maya and “samarinda” have greater leaf area than “ipb 8g” as these two local rice cultivars have a longer leaves. similarly, wahyuti et al. (2013) reported that local superior cultivar “rojolele” and “pandan wangi” have longer leaves compared to table 1. height of different rice cultivars at different rates of n fertilizer treatment plant height (cm) weeks after planting 3 5 7 9 11 13 cultivar “mayas” 32.8b 58.8b 80.5c 95.0b 107.6b 107.7b “samarinda” 37.2a 66.2a 93.2a 110.6a 124.3a 128.7a “ipb 8g” 36.1a 63.2a 88.4b 97.0b n doses (kg.ha-1) 0 33.3c 56.4c 80.2b 93.6b 102.3c 106.2b 75 35.6b 63.9b 88.4a 102.4a 119.4a 121.0a 150 37.3a 68.0a 93.5a 106.5a 126.1a 127.3a note: the values followed by the same letters within the same column are not significantly different according to tukey test at 5% journal of tropical crop science vol. 6 no. 2, june 2019 www.j-tropical-crops.com 132 faisal, iskandar lubis, ahmad junaedi and sugiyanta “ir64” and “fatmawati”. longer rice leaves will result in a drooping leaf canopy, which causes leaves to be less efficient in utilizing solar radiation. the application of n at 150 kg.ha-1 resulted in the greatest leaf area which was significantly different from the control (without fertilizer), but it was not significantly different from those fertilized with n at 75 kg.ha-1 at the vegetative and the primordial phase. at the heading stage there were significant differences between the application of 0 kg.ha-1, 75 kg.ha-1 and 150 kg.ha-1. the leaf area in one hill of rice is influenced by the number of tillers as the greater the number of tillers the greater the leaf area. these results confer with rahman et al. (2014) that an increase in leaf area is caused by an increase in the number of tillers and leaf sizes in accordance with increasing doses. shoot and grain dry weight “samarinda” fertilized with 150 kg.ha-1 n produced the greatest shoot dry weight in even though it was not significantly different from that of “ipb 8g” (table 5). according to bustami et al. (2012) the shoot dry weight is related to plant height and number of tillers. nitrogen application significantly affects dry matter production during the plant growth cycle (haque and haque, 2016). according to dordas et al. (2008) nitrogen is the most important nutrient for crop production because it greatly affects the production of shoot dry weight, leaf area development and photosynthetic efficiency. in the primordial stage “ipb 8g” fertilized with 150 kg n.ha-1 had the highest dry weight, and the shoot dry weight was higher than those at the vegetative phase. dry matter production has a higher correlation with plant height and number of tillers at seed filling, flowering, and physiological maturation stages than the initial stages of growth. increase in dry weight is an effect of an increase in nitrogen doses (fageria, 2007; 2011). rice yield components the rice yield components, i.e. 1000-grain-weight, the number of grains per panicle, length of panicles, number of panicles per hill, percentage of empty grain, grain yield and the harvest index are described in table 5. the different rice cultivars showed differences in the weight of 1000-grains and panicle length, but table 2. the effect of dose and time of fertilization on the average number of tillers treatment time of observation (wap) 3 4 5 6 n dose (kg.ha-1) w1 w2 w1 w2 w1 w2 w1 w2 0 6.0c 6.0c 7.3b 7.6b 7.9c 8.8c 8.1a 8.2a 75 6.2bc 7.6ab 8.1b 10.3a 9.6bc 11.7ab 9.8a 11.3a 150 7.7a 7.2abc 11.2a 10.6a 13.1a 11.9ab 11.7a 11.3a note: the values followed by the same letters within the same column are not significantly different according to the tukey test level at 5%. wap: weeks after planting. w1: n fertilizer were applied three times, 50% dose at planting, 25% at vegetative phase, and 25% at primordial phase. w2: n fertilizers were applied four times, 40% at planting, 20% during the vegetative phase, 20% at the primordial phase, and 20% at heading. table 3. leaf area of different rice cultivars fertilized with different doses of nitrogen at the vegetative, primodial and 50% blooming stage. treatment leaf area (cm2) at different stage of growth vegetative primordia heading cultivar “mayas” 933.7a 3457.6a 2335.9a samarida 909.9a 2791.3ab 2489.9a “ipb 8g” 271.7b 2256.5b 2256.4a n dose (kg.ha-1) 0 376.7c 1963.7b 1785.5c 75 753.3a 3017.1a 2344.0b 150 985.2a 3524.6a 2952.5a note: the values followed by the same letters within the same column are not significantly different according to tukey test at 5% growth and yield of kalimantan landrace rice and “ipb 8g” as affected by time .......... journal of tropical crop science vol. 6 no. 2, june 2019 www.j-tropical-crops.com 133 did not show significant differences in the number of grains. “ipb 8g” had the highest 1000-grain-weight compared to “mayas” and “samarinda”. the local rice cultivars have relatively small seed sizes so the 1000-grain-weight is lighter than that of superior cultivar (rusdianyah et al, 2015). the panicle length of “ipb 8g” (figure 1c) are longer than those of “mayas” (figure 1a) and “samarinda” (figure 1 b). according to jahan et al. (2014) the panicle length variation was due to the different genetic backgrounds of each variety. similarly, getachew and birhan (2015) reported different number of panicles in rice cultivars “ediget”, “gumara” and “nerica-4”. n application at 150 kg.ha-1 resulted in a significantly higher number of panicles per hill relative to the control, but it was not significantly different from application of n at 75 kg.ha-1. n fertilization can increase the number of panicles and the number of grains per panicle (tayefe et al., 2014; firouzi, 2015). rice cultivars, the time of fertilization, and the dose of fertilizer significantly affected the percentage of empty grain. “mayas” had a lower percentage of empty grain compared to “samarinda”, but it was not significantly different from “ipb 8g”. the difference percentage of empty grain of each cultivar is likely due to the genetic makeup of the different cultivars. according to asaad and warda (2016), the differences in yield components amongst rice cultivar, including percentage of empty grains, is due to differences in the physical characteristics of each variety and the environment in which they grow. a split n application where 40% n is applied at planting, 20% n during the vegetative stage, 20% at the primordial stage, and 20% at heading (w2), showed that the application of nitrogen fertilizer at heading stage had improved fruit filling and reduced percentage of empty grain. according to bah et al. (2009) n fertilization at 55 days after planting increased the amount of filled grain. the table 4. effect of interactions between cultivars and nitrogen fertilization dose on rice shoot and grain dry weight at vegetative and primordial stage rice cultivar shoot and grain dry weight (g) vegetative stage primordial stage n (kg.ha-1) 0 75 150 0 75 150 “mayas” 2.6c 7.0b 8.3ab 18.2cd 28.7bc 28.0bc “samarinda” 3.7c 7.2b 10.a 21.4cd 38.2ab 42.9a “ipb 8g” 1.6c 2.5c 2.6c 10.2d 26.6bc 37.0ab note: the values followed by the same letter within the same column are not significantly different according to tukey test at 5% figur 2. length of rice panicles “samarinda” (a), “mayas” (b) and “ipb 8g” (c) journal of tropical crop science vol. 6 no. 2, june 2019 www.j-tropical-crops.com 134 faisal, iskandar lubis, ahmad junaedi and sugiyanta application of 150 kg.ha-1 nitrogen fertilizer resulted in the lowest percentage of empty grain compared to without nitrogen, whereas khalifa (2012) showed that treatment without nitrogen produced the lowest seed filling percentage. each rice variety showed different responses to the increase in nitrogen doses and time of nutrient application; this difference in responses could be influenced by differences in morphological and physiological characters between cultivars. according to soplanit and nukuhaly (2012), supplying sufficient n during the generative phase is very important in slowing down the aging process of leaves, so photosynthesis process can be maintained during the grain filling phase. the rice yields varied with rice cultivar and nitrogen dose. “ipb 8g” had the highest yield of 2.9 t.ha-1 which was significantly different from “samarinda”, but it was not significantly higher than “mayas” (table 5). “ipb 8g” had high yield due to the heavier 1000-seeds weight, longer panicles, and the greater weight per hill compared to the local cultivars. the results of a study by arinta and lubis (2018) showed that the higher yield of “ipb 8g” compared to local cultivars was due to more number of grains per panicle and greater 1000-grain weight. “ipb 8g” is a new plant type (npt) that has high yield potential. according to abdullah et al. (2008), npt with high yield potential generally have medium number of tillers but all productive (12-18), number of grain per panicle of 150–250, percentage of rice grain of 85– 95%, weight of 1,000-grain of 25−26 g, have sturdy and short stems (80−90 cm), early maturing (110– 120 days), erect leaves, green to dark green, have dense and deep roots, good quality rice grains, and resistant against major pests and diseases. nitrogen treatment had a significant effect on the yield; rice fertilized with n at 75 kg.ha-1 had the highest grain yield and was significantly higher compared to control, but it was not significantly different from those fertilized with n at 150 kg.ha-1. the result shows that increasing n rate up to 75 kg.ha-1 increased the yield, but the effect diminished as the n rate was increased to 150 kg.ha-1. this results confirms the study by firouzi (2015) that rice yield increases with increasing nitrogen fertilizer doses from 0 to 75 kg.ha-1, but the yield did not increase with higher n doses (150 kg.ha-1). moreover, hirzel et al. (2011) also showed that an optimal dose of nitrogen had a positive effect on grain yield compared to without nitrogen. application of n at several stages of rice growth is one of the important fertilizer recommendations. however, time of application, especially at critical stages, varies depending on the type of rice cultivars. the application of n during the critical stage can optimize the distribution of n in the leaves, thus maintaining photosynthesis, particularly during the seed filling stage (bah et al., 2009) “mayas” had the highest harvest index, but it was not significantly different from “ipb 8g”. according to fageria (2007) the harvest index varies among cultivars and is greatly influenced by environmental table 5. rice 1000-grain-weight, number of grains per panicle, panicle length, number of panicles per hill, empty grain, and grain yield of different rice cultivars at different rates and time of fertilizer application treatment 1000-grain weight (g) number of grains per panicle length of panicles (cm) number of panicles per hill empty of grain (%) grain yield (t. ha-1) harvest index cultivar “mayas” 18.2c 242.7a 21.1b 9.9a 13b 2.5ab 48.8a “samarinda” 20.6b 232.7a 21.9b 7.1b 16a 2.4b 33.4a “ipb 8g” 26.8a 257.3a 28.3a 7.7b 14ab 2.9a 47.0a frequency of fertilizer application 3 21.9a 240.3a 23.8a 7.8a 16a 2.7a 46.1a 4 21.8a 244.8a 23.7a 8.6a 13b 2.5a 40b n dose (kg.ha-1) 0 21.5a 257.6a 23.9a 7.1b 17a 2.1b 4.0a 75 22.2a 236.5a 23.7a 8.5a 14b 2.9a 4.0a 150 21.7a 233.6a 23.7a 9.0a 13b 2.8a 4.0a note: the values followed the same letters within the same column are not significantly different according to tukey test at 5%. growth and yield of kalimantan landrace rice and “ipb 8g” as affected by time .......... journal of tropical crop science vol. 6 no. 2, june 2019 www.j-tropical-crops.com 135 and nutritional factors. frequency of nitrogen application showed significant effects on harvest index; n fertilization applied three times resulted in a higher harvest index compared to when n was applied four times. this showed that nitrogen application time at each growth stage has influences on the harvest index. according to fageria (2010) time of nitrogen application affects the rice harvest index, the harvest index varies from 45 when all n was applied at planting to 53 when two third of n was applied at planting, and 1/3 is applied at panicle initiation. conclusion n fertilizer significantly affected rice height, number of tillers, leaf area, dry weight and yields. the kalimantan rice cultivar mayas, samarinda, and “ipb 8g” have similar number of tillers, but vary significantly in the other growth traits. the new rice cultivar “ipb 8g” had the highest grain yield of 2.8 ton per ha. nitrogen fertilizer at 75 kg.ha-1 applied three times resulted in the highest grain yields, therefore nitrogen fertilization at this level to “ipb 8g” and kalimantan landrace cultivars is already sufficient. acknowledgement the author would like to thank the agriculture research and development agency of the ministry of agriculture for providing scholarships and funding this research. references abdurachman, a., dariah, a. and mulyani, a. 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(2013). prospek penerapan bioteknologi dalam pemanfaatan dan pengembangan biodiversitas padi lokal sulawesi tenggara. jurnal agroteknos 3, 41-47. june_2014.cdr journal of tropical crop science (issn 2356-0169; e-issn 2356-0177) is published four-monthly by department of agronomy and horticulture, faculty of agriculture, bogor agricultural university, indonesia. publication details, including instructions for authors and subscription information: www.j-tropical-crops.com any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by the publisher. the accuracy of the content should be independently veri�ed with primary sources of information. the publisher shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the content. 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registration dave skinner, jan hintze, bryan brunner research articles estimation of genetic parameter for quantitative characters of pepper ( l.)capsicum annuum muhamad syukur, syaidatul rosidah irrigation volume based on pan evaporation and their effects on water use ef�ciency and yield of hydroponically grown chilli eko sulistyono, abe eiko juliana evaluation of commercial sun�ower (helianthus annuus ) cultivars in bogor, indonesia, forl. ornamental and nursery production syarifah iis aisyah, khotimah, krisantini different growth partitioning and shoot production of talinum triangulare treated with organic and inorganic fertilizer sandra ari�n aziz, leo mualim, sitta azmi farchany cloning and characterization of p5cs1 and p5cs2 genes from l. under droughtsaccharum officinarum stress hayati minarsih iskandar, dwiyantari widyaningrum, sony suhandono journal of tropical crop science (issn 2356-0169; issn 2356-0177) is published four-monthly (one volume per year) byedepartment of agronomy and horticulture, faculty of agriculture, bogor agricultural university, ipb darmaga campus, bogor, indonesia 16680. send all inquiries regarding printed copies and display advertising to or to secretary, department ofinfo@j‐tropical‐crops.com agronomy and horticulture; telephone/fax 62-251-8629353. permission to reprint: permission to make digital or hard copies of part or all of a work published in isjournal of tropical crop science granted for personal or educational/classroom use provided that copies are not made or distributed for pro�t or commercial advantage and that copies bear the full citation and the following notice on the �rst page: “copyright department of agronomy and horticulture, faculty of agriculture, bogor agricultural university”. for all other kinds of copying, request permission in writing from head of school, department of agronomy and horticulture of�ce, ipb darmaga campus, bogor, indonesia 16680. © department of agronomy and horticulture, faculty of agriculture, bogor agricultural university. all rights reserved. printed in the republic of indonesia.in s t t t u i b o g o r perta n ia n abstract increasing world sugar demand might be ful�lled with extensi�cati n which include the use ofland o dry area. development of drought tolerance and high productivity sugarcane could be byvariety achieved plant genetic engineering. under drought condition, proline will be accumulated and functioned as an osmoregulator in plant. δ -5-1-pyrroline carboxylate synthase (p5cs) is one of the important enzyme ins proline biosynthesis. this enzyme is encoded by p5cs gene family. we cloned two homologous genesp5cs from sugarcane, (accession number :sop5cs1 kf178299) and (accession number :sop5cs2 kf178300), which encode 729 and 716 amino acid polypeptide . the between these genes wass identity two 74% based on nucleotide sequence . thes sop5cs1 gene had 98% with (accessionidentity sbp5cs1 number : gq377719.1) and had 99%sop5cs2 identity with (accession number : ef113257.1). in thissap5cs experiment, sugarcane were exposed toplantlets medium containing peg 6000 (40%) for 12, 24, 48, and 72 hours. roline concentrationp was measured after treatment genes were zedand expression analy by real time qpcr. the results showed that the prolineconcentration was increased 12 folds (9 8 umol g ) after. . -1 48-hours stress treatment. ighest expression ofthe h s o p 5 c s 1 o c c u r e d a t 2 4 h o u r s t r e a t m e n t w i t h approximately 16 times from plant without peg (control plant) and decreased gradually at 48 and 72 hours treatment. ighest expression of occuredthe h sop5cs2 at 24-hours drought stress with approximately 3 6 folds. compared to control. in drought treatment, the expression level was higher than and hassop5cs1 sop5cs2 increased signi�cantly at 12-hours treatment. ti is suggested that the gene contributes moresop5cs1 signi�cantly to the production of proline during drought stress than . , could potentialsop5cs2 sop5cs1hence y be used as a marker to screen sugarcane variety for drought tolerance for the development of transgenicand plant tolerant to drought. keywords: cloning, drought, expression, p5cs, sugarcane introduction sugarcane is mainly used as raw material for sugar and bioethanol production. this crop productivity and growth has been in�uenced by enviromental factors such as climate and soil condition. drought and salinity could decrease sugarcane productivity and growth (silva et al., 2011). in contrast, the use of marginal and barren land for crop plantation is needed to ful�ll high world sugar demand. research to improve plant stress tolerance is crucial to boost the sugarcane productivity and extend its range. plant genetic engineering is one of the potential methods for improving plant quality. proline accumulation is an adaptive response when plants are exposed to abiotic stresses. proline acts as osmotic adjustment and osmoprotectant which function to maintain homeosta condition, stabilize cellsis structure, and prevent protein degradation (verbruggen and hermans, 2006; taiz and zeiger, 2006). glutamate (glu) and ornithine (orn) are the proline precursors (verbruggen and hermans, 2006; roosens et al., 1998). glutamate pathway is the main route for proline biosynthesis during abiotic stresses. in glutamate pathway, proline is synthesized from glutamate via -ɣ glutamic semialdehyde (gsa) and δ -pyrroline-5-1 carboxylate. this process is catalyzed by two enzymes, δ -pyrroline-5-carboxylate synthethase (p5c ) and δ -1 1s pyrroline-5-carboxylate reductase (p5cr) (verbruggen and hermans, 2008; hare and cress, 1997; kishor et al., 2005). p5cs, a rate-limiting enzyme, plays an important role in proline biosynthesis. p5cs is a bifunctional enzyme that exhibits -glutamyl kinase and glutamic-semialdehydeɣ ɣ dehydrogenase activities. this enzyme is encoded by two homologous genes (verbruggen andp5cs hermans, 2008; hare and cress, 1997; kishor et al., 2005). the genes had been cloned andp5cs characterized from several plants such as arabidopsis thaliana , solanum licopersicum(savoüre et al., 1995) (fujita et al., 1998) ( et al., ;, oryza sativa igarashi 1997 hur 2004 zhu 1998et al., ; et al., ) and, sorghum bicolor ( et al ). in some species, these genes showedsu ., 2011 different expression patterns. both of genes hadp5cs different functions in plant development stage and stress response ( and ).verbruggen hermans, 2008 in , the expression of was induced bya thaliana atp5cs1. drought and salt stress while was ubiquitouslyatp5cs2 expressed in dividing and differentiating cells ( etsavoure al., ). had two closely related1995 s licopersicum p5cs. research article cloning and characterization of and genes fromp5cs1 p5cs2 saccharum officinarum l. under drought stress hayati minarsih iskandar dwiyantari widyaningrum sony suhandono ac b , , b abiotechnology research for estate crop, jalan taman kencana no. 1, bogor, indonesiainstitute genetics and molecular biology division, school of life science and technology, institut teknologi bandung,b jalan ganesha no. 10, bandung, indonesia c corresponding author; email: hmiskan@yahoo.com 5555 23 journal of tropical crop science vol. 1 no. 1, 2014june www.j-tropical-crops.com genes, and . the expression oftompro1 tompro2 tompro1 was induced by salt stress the, whilst tompro2 transcript was undetec able (fujita et al.,t 1998). in , gene was a housekeepingo sativa osp5cs1. gene while osp5cs2 was induced by salt, drought, and, cold stress ( et al., ; et al., ; etigarashi 1997 hur 2004 zhu al., ). in , the transcript was1998 s bicolor sbp5cs1. signi�cantly up-regulated under abiotic stress while the sbp5cs2 was a housekeeping gene which function in proline biosynthesis under normal condition (su et al., 2011). previous study showed that overexpression of vap5csfrom vigna aconitifolia increased stress tolerance and proline content on transgenic tobacco ( et al., ).kishor 1995 this study aims to characterize twoclone and homologous p5cs genes and �nd the more potential canditate gene that might be used to improve drought tolerance in sugarcane. in this research, two homologous p5cs genes had been cloned from sugarcane followed by its characterization using bioinformatic analysis. we studied the expression pattern of genes duringsop5cs drought stress and demonstrated its relation with proline accumulation to �nd the more potential candidate among those genes for sugarcane genetic engineering. material and method plants materials and drought stress treatment for cloning full-length isolat on of andand i sop5cs1 sop5cs2 genes, month old�ve after acclimatization of sugarcanes were not wateredplants of psjt 941 variety in seven days in order to simulate drought conditions. the sugarcanes were planted in polybags containing a mixture of , sand and manure the control plants weresoil . watered until sampled t were used fhree-month old sugarcane planlets or expression study of and genessop5cs1 sop5cs2 under drought stress and proline measurement. the planlets were incubated in [ms+peg] medium in 12, 24, 48, and 72 hours in order to simulate drought stress. we used peg 6000 with 40% (w/v) concentration. for control, the planlets were incubated in ms medium in 12, 24, 48, and 72 hours. the leaf samples were collected after incubation three biological replicates for eachwith treatment. isolation of total rna and cdna synthesis total rna was extracted from leaf samples using total r n a ( p l a n t ) i s o l a t i o n k i t g e n e a i d f o l l o w i n g manufacturer's intruction. cdna was synthesized using revert-aid cdna synthesis kit fermentas and super( ) script iii cdna synthesis kit invitrogen according to the manufacturer's instruction. cloning of sop5cs1 and sop5cs2 genes we used two different primer pairs (p5cs1-f: 5'g a g c c a c t ta g c g a g g a a g 3 ' ; p 5 c s 1 r : 5 ' g g t c c t t c t g c t t g t ta c g a 3 ' ; p 5 c s 2 f : 5 ' agaggggagaccgaa gaccaggag-3'; p5cs2-r: 5'-tacagaatgaaccaccagaatgat-3') in order to clone the sop5cs1 and sop5cs2 genes from sugarcane. primers were designed using primer3 (www.frodo.wit.mit.edu). primer p5cs1-f and p5cs1-r were designed based on est database number t20007 : 9719 from kegg (kyoto encyclopedia of genes and genomes) and nucleotide sequence of genesbp5cs1 (genbank accession number : gq377719.1). primer p5cs2-f and p5cs2-r were designed based on sop5cs (genbank accession number : ef155655.1). the 50 ul pcr mixture contained 2 ul (250 ng) cdna, 0 2 mm dntp, 0 5 um primers, 5 ul 10x. . kapa2g gc buffer ( 1.5 mgcl kapa2gcontained mm ), and 1 unit2 robust hotstart dna polymerase. we used touchdown pcr program that was set as followed: initial denaturation at 95 c for 5 minutes, 10 cycles of 95 c foro o 30 s, 60 c for 30 s, 72 c for 3 minutes, 10 cycles ofo o touchdown program (95 c in 30 s, annealing temperatureo between 60 c to 50 c in 30 s, 72 in 3 minutes), 10 cycleso o for 95 c for 30 s, 50 c for 30 s, 72 c for 3 minutes,o o o folllowed by �nal extension of 72 c for 7 minutes. pcro were done using veriti thermal cycler applied biosystem. the fragment of gene was clonedsop5cs1 to pgem-t easy vector (promega) while fragment of sop5cs2 gene was cloned to pjet 1.2/blunt vector (fermentas) then transformed to dh5 competente.coli cells. the positive recombinant plasmids were subjected for sequencing. bioinformatics we used several bioinformatic analysis methods in order to analyze the sequences and predict the p5cs proteins. homology analysis of nucleotide sequence wassop5cs done using blastn. multiple sequence alignment was made by clustalw. phylogenetic trees were made using clustalx and mega5 using neighboroining method.j amino acid sequence of protein weres sop5cs predicted using bioedit. in order to predict sop5cs proteins celular localization, we used targetp and protcomp from softberry (www.softberry.com). real time q pcr (rtpcr) analysisuantitative q rtpcr was used for expression analysis. the 20 ulq rtpcr mixture contained 10 ul 2x power sybr greenq (applied biosystem), 0 1 um primers, and 2 ul cdna. template. we used different pairs of primer for each gene. f o r g a p d h g e n e , w e u s e d g a p d h f ( 5 ' tgcacccatgtt cgttgt-3') and gapdh-r : 5'ccatcaacagtcttctgggt-3'. -spesi�csop5cs primers were used as followed : (p5csa-f: 5'gagccacttagcgaggaag-3' ; p5csa-r: 5'ttctgcccagtg acaacag-3') for andsop5cs1 (p5csb-f: 5'-ggagaccgaagaccagga-3' and p5csb-r: 5'-tcacaatgatcacctcgtacc-3') for sop5cs2. we used relative quantitative real time pcr 5555 h , d sayati minarsih iskandar wiyantari widyaningrum, ony suhandono24 journal of tropical crop science vol. 1 no. 1, 2014june www.j-tropical-crops.com program that was set as followed : initial denaturation at 95 c in 10 minutes, 40 cycle of rtpcr (95 c in 15s ando oq 57 c in 15s), followed by melt curve program witho different melting temperature (δtm = 0 3 c) at every. o cycle from 60 c to 90 c. real time qpcr were doneo o using applied biosystem step one plus real time pcr. in rt-qpcr, we did three technical replicates for each genes. the relative expression ratio of andsop5cs1 sop5cs2 gene were calculated with pfaf� method (pfaf�, 2001). the pfaf� method was used because of the ef�ciency of the gapdh primers as gene reference, was different with the ef�ciency of p5cs1 and p5cs2 primers. the ef�ciency of gapdh, p5cs1, and p5cs2 primers were 1.8, 2.11, and 2.0. proline determination free proline was extracted and calculated using bates (1973) method 0 5 gram fresh lea was homogenized: . ves in 10 ml of 3% sulfosalicylic acid. the homogenate was sentrifuged to separate supernatant (�ltrate) and solid molecul. 2 ml of �ltate was reacted with 2 ml acidninhidrin and 2 m glacial acetic acid. the mixtures werel boiled in 100 for 1 hour, kept on ice, and then wereoc extracted using 4 ml toluene. proline absorbance was measured using life science spectrophotometer at λ 520 nm. proline concentration was calculated using equation (i) : y = o ; proline concentration m). the equationd520 (μ was based on proline standard curve with r = 0.9987.2 we continued to measure proline concentration using equation (ii) based on bates (1973) : results sequence analysis and phylogenetic tree two sugarcane genes were isolated from 5' utr top5cs 3' utr region by rt-pcr and registered in ncbi (genebank accession number : kf178299 and genebank accession number : kf178300). the sop5cs1 sop5cs2and encoded 716 and 729 amino acid polypeptides which was predicted would be located in cytoplasm. sequence analysis showed that sop5cs1 shared 75 6% in nucleotide sequence and. identity s shared 75 7% similarity protein sequence with. s sop5cs2 sop5cs1. blastn results showed that shared 96% similarity with (genebanksbp5cs1 accession number : gq377719.1; protein id : acu65226) while had 98% withsop5cs2 identity sop5cs (genebank accession number : ef155655), 98% with (genebank accessionidentity sap5cs number : eu113257), and 93% withidentity sbp5cs2 (genebank accession number : gq377720). the two protein sequences were aligned withsop5cs atp5cs, osp5cs, and sbp5cs. multiple sequence alignment result (figure 1) showed that both of sop5cs protein sequences had conserved regions such as atp binding site, glutamate-5-kinase (g5k) domain, nad(p)h binding site, putative leucine zipper domain, and glutamate semialdehyde dehydrogenase (gsa-dh) domain. onserved phe (f) residue also found inthe c sop5cs1 sop5cs2(position 141) and (position 128) protein sequence. the residue was functioned in proline feedback inhibition regulation ( et al ).hong ., 2000 phylogenetic tree (figure 2) was made based on the p5cs protein sequences in order to study the evolutionary relationship. it was constructed by clustalx and mega5 using nj method with 1000 bootstraps. it shows that there were two clusters separating monocots and dicots p5cs protein. two proteinsof aresop5cs located within the monocot cluster owever these. h , proteins were in different sub-cluster which ds divide monocot p5cs1 and p5cs2. the sharedsop5cs1 homology with sbp5cs1, zmp5cs, and osp5cs2 while sop5cs2 similar to sop5cs, sap5cs, and sbp5cs2.is expression analysis real time quantitative pcr (rt-qpcr) was performed in order to analyze the expression of andsop5cs1 sop5cs2 genes during drought stress. the expression of genes on drought treatment plants weresop5cs compared to control plant. both genes were induced by drought stress. the expression of weresop5cs1 increased signi�cantly up to 11 folds at 12-hours drought treatment then continously increased to achive the highest ratio, 16 folds, at 24-hours treatment. however, sop5cs1 transcript gradually decreased to 13 folds and 10 folds at 48-hours and 72 hours drought treatment (figure 3a.). the expression of not signi�cantly increasesop5cs2 did during drought stress. transcript was inducedsop5cs2 to achieve the highest ratio, 3.3 folds, at 24-hours drought stress. the transcript was down-expression of sop5cs2 regulated to 1.4 folds at 48-hours drought stress. the gene expression was increased again up to 2.8 folds at 72-hours treatment (figure 3b.). the expression of sop5cs1 sop5cs2was compared to treat plantof ed during drought condition (figure 4). hehence, t expression of was signi�cantly higher thansop5cs1 sop5cs2 sop5cs. the expression pattern of genes were similar to genes.sbp5cs 55 25cloning and characterization of and genes fromp5cs1 p5cs2 .......... journal of tropical crop science vol. 1 no. 1, 2014june www.j-tropical-crops.com figure 1. multiple sequence alignment between two sop5cs protein sequence with sop5cs (protein id : abm30223), sbp5cs1 (protein id : acu65226), sbp5cs2 (protein id : acu65227), osp5cs1 (protein id : baa19916), osp5cs2 (protein id : bag94966), atp5cs1 (protein id : np_001198715.1), atp5cs2 (protein id : np_191120.2). boxes ([]) show predicted conserved region. 5555 h , d sayati minarsih iskandar wiyantari widyaningrum, ony suhandono26 journal of tropical crop science vol. 1 no. 1, 2014june www.j-tropical-crops.com figure 2. phylogenetic relationship of sop5cs1 and sop5cs2 (boxes) with sap5cs (s. arundinaceum p5cs), sop5cs ( p5cs), sbp5cs2 ( p5cs1), osp5cs1 ( p5cs1), tap5css. officinarum s. bicolor o. sativa ( p5cs), osp5cs2 ( p5cs2), zmp5cs ( p5cs), sbp5cs ( p5cs1),t. aestivum o. sativa z. mays s. bicolor vap5cs ( p5cs), gmp5cs ( p5cs), jcp5cs ( p5cs), rcp5cs (v. aconitifolia g. max j. curcas r. communis p5cs), atp5cs1 ( p5cs1), and atp5cs2 ( p5cs2). the phylogenetic tree had twoa. thaliana a. thaliana cluster separating monocots (i) and dicots p5cs (ii). a b figure 3. the expression of ( ) and ( ) in treatment and control plantsop5cs1 sop5cs2a b figure 4. comparison of two genes expression during drought treatmentsop5cs 55 27cloning and characterization of and genes fromp5cs1 p5cs2 .......... journal of tropical crop science vol. 1 no. 1, 2014june www.j-tropical-crops.com proline concentration the proline concentration was measured using the same samples for expression analysis in order to determine the corelation between proline accumulation with sop5cs genes transcript level. the proline content of treatment plant was higher than control plant since 12-hours drought treatment (figure 5). the proline concentration was signi�cantly increased at 48-hours treatment then achieved the highest peak (9 8 umol gram ) at 72-hours. . -1 drought stress. figure 5. comparison of proline content between treatment and control plant discussion the p5cs enzyme from sugarcane was encoded by two homologous genes as observed in other plantp5cs species initial. , we cloned the genes that weresop5cs2 related with and genes databasesop5cs sbp5cs2 on in ncbi genebank. we found another cdna fromp5cs sugarcane using kgenes search by kegg (kyoto encyclopedia of genes and genome). this data shared high homology with gene from sweet sorghum.sbp5cs1 both of the isolated genes had complete codingsop5cs region which encode two closely related sop5css protein .s the sop5cs proteins shared high similarity with sbp5cs2 as shown in multiple alignment result. the sop5cs1 sbp5cs1 sop5cs2similar to whil had high, st homology with .these proteins had conservedsbp5cs2 motif atp and nad(p)h binding site, glu-5-kinase and gamma-glutamyl phosphatase reductase domain, and leucine zipper region that were located at the same position (su et al., 2011). the result indicated that the sop5cs proteins would have same expressionthe pro�le with proteins.sbp5cs in phylogenetic tree, the p5cs protein from many plant species were clustered into two groups p5csdividing on monocots and dicots. the andsop5cs1 sop5cs2 protein were clustered in different sub-groups within monocots p5cs. this result indicated that sugarcane p5cs protein was encoded by p5cs gene family. turchetto-zolet ( ) studied the evolutionary2009 relationship of p5cs protein. in their phylogenetic tree, monocots and dicots p5cs were clustered separately. further study showed that there were two sub-clusters in monocots p5cs separating p5cs1 and p5cs2 protein. this event could result in one or more copies of a gene in genome which had new function or paralogous gene (grassi et al., 2008). there were some previous studies about genep5cs from , , and reporteda thaliana o sativa s bicolor. . . that about s ousexpression pro�le of two homolog p5cs genes from the plant species under abiotic stresses such as drought, salt, and cold. e focus on genehere, w p5cs expression under drought stress. in ,a thaliana atp5cs1. had a role in abiotic stress response and prolin accumulation. this gene expression increased during drought stress. atp5cs2 was especially expressed in dividing cell. the gene was ubiquitously expressed both in normal and stress condition. however the expression of atp5cs2 was signi�cantly lower than during abioticatp5cs1 stress such as drought. in , the waso sativa osp5cs1. highly expressed in reproductive and vegetative organs especially during normal condition. in contrast, the osp5cs2 transcript was highly induced by drought stress. the genes had similar expression pattern withsbp5cs atp5cs osp5cs sbp5cs1and genes. the gene was highly expressed in mature plant during abiotic stress including drought condition. the transcript wassbp5cs2 highly detected in dividing cell. the was alsosbp5cs ubiquitously expressed both in normal and drought condition however the expression of wassbp5cs1 extremely higher than . su ., (2011)sbp5cs2 et al suggested that had an important role in prolinesbp5cs1 5555 h , d sayati minarsih iskandar wiyantari widyaningrum, ony suhandono28 journal of tropical crop science vol. 1 no. 1, 2014june www.j-tropical-crops.com 55 29cloning and characterization of and genes fromp5cs1 p5cs2 .......... accumulation during abiotic stress while sbp5cs2 played a role in proline biosynthesis duringregular normal condition. transcriptionalor unstressed regulation is a major key in gene expression. study about sbp5cs sbp5cspromoters showed that both of genes had cis-elements such as mycconsensusat, wrky, and mybcore. these elements functioned as binding site for transcription factors which nvolved in cold,i wound, and drought stress ( et al., ). two closelysu 2011 related p5cs genes had different function ins bicolor. plant development, growth, and stress response that might be caused the different regulation of -elementby cis in sbp5cs promoter ( et al , ).alberts . 2008 current study reported one type gene fromp5cs sugarcane (iskandar et al 201 ; patade et al., 2012).., 1 iskandar et al (201 ) reported the expression of in. p5cs1 young and mature culm internodes of sugarcane after 15days water de�cit treatment. the expression of p5cs was higher than control, and generally not signi�cantly different between the young and mature culm internodes. expression analysis showed that both of andsop5cs1 sop5cs2 were induced by drought stress. the sop5cs1 transcript was signi�cantly increased, when compared to control, during peg incubat . however,ion the expression of was not signi�cantlysop5cs2 increased during drought condition. our study showed that transcript and proline accumulation hadsop5cs1 different pattern. the expression of had beensop5cs1 signi�cantly increased at 12-hours drought treatment then achieved the highest peak at 24-hours drought treatment. transcript level was graduallysop5cs1 decreased after 24-hours treatment. in contrast, proline was signi�cantly accumulated at 48-hours drought treatment.the highest proline concentration was achieved at 72-hours drought treatment. the expression and activity of p5cs protein might be inhibited by feedback inhibition regulation of high proline concentration. the proline was continously produced after 48-hours, when transcript repressed, might be causedsop5cs1 by proline biosynthesis by ornithine pathway. study in a. thaliana showed that ornithine pathways played a role in proline biosynthesis during drought and salt stress ( et al. 1998).roosens , based on the similarity of sop5cs with sbp5cs genes and expression analysis data, we suggest that the sop5cs1 gene contributes more signi�cantly to the production of proline during drought stress than sop5cs2 sop5cs1. the could potential be used as aly marker to screen sugarcane variety for drought tolerance and may also potentially be useful for the development of tran genic plants tolerant to drought. further study abouts sop5cs promoters is needed in order to determine the sop5cs1 sop5cs2and gene regulation. acknowledgement this research was supported by research colaboration between indonesian biotechnology research institute for estate crops ( )bpbpi , pt. riset perkebunan nusantara (rpn), indonesian center for estateand crops research and development (puslitbangbun) with funding from dipa apbn 2012. the authors are also grateful to . asmini budiani, mr. soekarno mismanadr putra, mrs. niyyah fitrant , and mrs. marini for theiri assistance and support during research. references alberts, b., johnson, a., lewis, j., raff, m., roberts, k. and walter, p. 2008 . molecular biology of the( ) cell, fifth edition. g .arland science, new york bates, l.s., waldren, r.p. and teare, i.d. 1973 . rapid( ) determination of free proline for water-stress studies. , 205-207.plant and soil 39 fujita t , maggio a , garcia-rios m, . , . , ., bressan, r.a. and csonka, l.n. 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email: ylurwanu.cpp@buk.edu.ng research article introduction rice is considered as one of the major staple cereal foods and forms a signifi cant component of the energy resource of the human race. rice is an economically and scientifi cally important crop and mostly produced and consumed in asia and african countries (islam et al. 2000; dede et al. 2019). currently, 35–40 % more yield productivity is required to meet global demands (fao 2017). the available rice stock in these countries is stored to adjust the demands and supply for both seed and food (islam et al., 2000). to date, the seed of other cereals crops like wheat, maize, and barely have received adequate attention for storage and post-harvest, however, the research for rice is still not completely yet (misra et al., 1995). storage facilities in asia and africa are still far from satisfactory. the farmers keep their stock in various ways, some of the means of storage are out-ofdate; therefore, weak and practically unserviceable. increasing rice production and subsequent reduction of its international importation and endemic plant pathogens continue to be a challenge in safeguarding plant health in nigeria. therefore, early and accurate diagnosis and pathogen surveillance is the most important approach to tackle this matter. the assessment of seed health standard in rice is very important for farmers and food security as it is a fi rst line approach in managing seed-borne diseases of plants. furthermore, the quality of planted seeds has a critical infl uence on the ability of crops to become established and to realize their full yield potential and value (mcgee 1995). the importance of seed quality in understanding the full potential of a variety is well andika journal of tropical crop science vol. 6 no. 3, october 2019 www.j-tropical-crops.com 146 bolanle tolani edun, yahuza lurwanu, mustapha sunusi, ali a. sulaiman known. the three signifi cant aspects of seed quality are genetic and physical purity, high germination percentage and vigor, and free from seed-borne diseases and insects (seshu and dadlani 1989). seed vigor is recognized as an essential seed quality parameter distinct from germination (seshu et al., 1988). seed-borne diseases may not only introduce new pathogens to aff ect the quantity or quality of the crop yield but also contaminate the soil permanently (anselme, 1981). fungi are commonly identifi ed as causal agents of many seed-borne diseases (nsemwa and wolff hechel 1999). studies from biruma et al. (2003) and kanobe et al. (2004) showed a wide range of seed-borne fungi pathogens on the farmer’s stored rice seeds, of which many farmers mainly rely on to propagate the next planting session. most seedborne diseases caused by the fungi pathogens are disastrous as they may decrease seed germination, causing seed discoloration and produce toxins that may be detrimental to man and domestic animals. distribution of some seedborne fungi associated with rice seeds has been reported in many countries, including nigeria (suleiman and omafe 2013); pakistan (butt et al. 2011); egypt (madbouly et al. 2012); bangladesh (ora et al. 2011); cameroon (nguefack et al. 2007) and chad republic (serferbe et al. 2016). the main objectives of this study were i) to identify seed-borne fungi associated with rice and evaluate its role for seedling abnormalities ii) to investigate the possibility of combining germination and seed health tests to assess the infl uence of seed treatments on germination and seedling vigor. materials and method experimental site the experiment was carried out at the research and teaching laboratory and screen house of the department of crop protection, faculty of agriculture, bayero university, kano, (11° 58” n, 8° 25” e and 47m above sea level) northwestern , nigeria. source of experimental materials the study was conducted using seven rice varieties commonly grown in the local area. these are “faro 52” (wita), “faro 44” (sipi), “faro 60” (improved varieties), “kwandala” , “jamila” , ex-china, and “jif” (local varieties). out of the seven cultivars, four rice seed samples collected from green spore agric. limited (“faro 44” , “faro 52” , “faro 60” and “jamila” ) while the remaining three varieties (“jif” , “kwandala” , and ex-china) collected from rice farmers in kura and bichi local government areas, respectively. the three seed dressing chemicals used were apron star 42 ws, dress force 42ws, and zeb-care 80%wp. detection of seed-borne fungi using blotter method to perceive the seed-borne fungal pathogens associated with the seeds using blotter method was used following the international rules for seed testing as used by ahmed et al. (2013). modifi ed from ista to perceive the seed-borne fungal pathogens associated with the seeds. in this method, three layers of blotting papers (whatmantm fi lter paper no.1) were soaked in sterile distilled water and placed at the bottom of the 90 mm diameter plastic petri dishes. four hundred seeds from each sample were taken randomly and then put on the moist fi lter paper at the rate of 25 seeds per plate. incubation was conducted at 28 ± 2°c under 12/12 alternating cycles of light and darkness in the incubation room for seven days. during the incubation period, petri dishes were checked and wetting of fi lter paper done as the need arise. after incubation, seeds were examined under the stereomicroscope for the presence of seed-borne fungi. number of infected seeds expressed in percentage. two selected rice varieties within the highest disease incidence were tested on agar plate method and seed treatment. identifi cation of fungi each seed was observed under stereo-microscope at ×16 and × 25 magnifi cations to identify the seedborne fungi. the most associated fungi were detected by observing their growth characters on the incubated seeds and identifi ed following the identifi cation keys from (ahmed et al., 2013). temporary slides were also prepared and observed under a compound microscope for proper identifi cation. the fungi were identifi ed up to species level, following the identifi cation keys of (fakir et al., 2002). the results presented as percent incidence for the individual fungal pathogen indentifi ed. agar plate method in this technique, one hundred seeds tested for each variety and replicated ten times. surface sterilized seeds as above were plated (10 seeds/petri dishe) on the potato dextrose agar (pda) medium. the plated seeds were incubated for seven days at 28 ± 2 °c in darkness. at the end of the incubation period, fungi developed from the seeds on the agar medium, and then were sub-cultured into fresh medium for seed health, quality test, and control of seed-borne fungi of some .......... journal of tropical crop science vol. 6 no. 3, october 2019 www.j-tropical-crops.com 147 sporulation. the fungi were examined and identifi ed based on colony characteristics, and morphology of fruiting bodies under a compound microscope after seven days incubation. examination of incubated materials the slide of various fruiting structures of the fungi was prepared and observed under a compound microscope (× 100) for identifi cation following (barnett and hunter, 1998). dry inspection of seed samples four hundred seeds from each sample were visually inspected and graded into nine categories. the categories were: (1) good seeds, (2) spotted seeds, (3) discolored seeds, (4) deformed seeds, (5) varietal mixture (6) broken seeds, (7) insect damaged seeds, (8) chaff y seeds and (9) inert matter. the result of diff erent seed categories was expressed in percentage. fungicide seed treatment seeds were rinsed with sterile distilled water in a clean plastic bowl to remove impurities before chemical seed treatment. fungicides were applied to the seeds following the manufacturer‘s recommendation. the three fungicides used are apron star 42 ws (20% w/w thiamethoxam + 20% w/w metalaxyl-m + 2% w/w difenoconazole), dress force 42ws (20% imidacloprid + 20% metalaxyl-m + 2% tebuconazole) and zeb-care 80%wp (mancozeb 80%wp). 0.025g of each chemical used in treating 10g of rice seeds of “jamila” and “faro 44” varieties with a high percentage of disease incidence that had been selected for this experiment. the control consisted of untreated rice seeds, that were allowed to dry after treatment. germination of treated-seeds in plastic pots planting media used in the screen house was sterilized sandy soil so as to meet ista regulations for grain size, ph, and conductivity (only recommended for seeds >5mm). plastic pot (30 cm diameter) 10 l in volume and about 16.5 cm depth was used. the treated seeds have been supplied with suffi cient water to prevent from drought condition. vigor test a vigor test was done using the sand pot method (ista 2001). shoot and root length were measured using a ruler (30 cm length) after 14 days of sowing. fifteen seedlings (30 seedlings per treatment) were randomly selected for the measurement of shoot and root length. the seedling vigor was determined using the formula of (abdul-baki and anderson, 1972): vigor index = (mean of root length + mean of shoot length) × % of seed germination. data analysis all the collected data was analyzed using descriptive statistics and presented as percentage (%). results and discussion dry inspection of rice seed samples the highest number of visually healthy seed (94.16 %) recorded from “jif” variety and lowest (64.77 %) from “jamila” , respectively (table 1). the highest number of deformed seed (1.8 %) recorded from variety “faro 44” , and lowest (0.47 %) recorded for “jif” . the highest number of apparently healthy seed and lowest deformed seeds were observed in the seeds of “jif” variety (table 1), suggesting it as the best among all the varieties. survey and determination of collected seed health status from diff erent regions in bangladesh also have been conducted by previous studies. fakir et al. (2002) recorded that collected seeds in bangladesh consist of 91.20 to 98.89 % pure seed, 3.72 to 37.71 % spotted seed, and 8.4615.50 % deformed seed. following study from fakir et al. (2003) also reported a wide variety of components among the collected seed samples of rice from diff erent regions of bangladesh. another study from uddin (2005) showed that farmer’s seeds (variety upazilla) in noakhali can be categorized into healthy seed (44.33 to 59.42 %), spotted seed (27.84 to 44.77 %), discolored seed (3.93 to 8.94 %), partly fi lled seed (0.43 to 2.35 %), deformed seed (0.91 to 3.98%), unfi lled seed (0.001 to 0.68%), varietal mixture (0.26 to 2.22%), other plant parts (0.001 to 0.36%), inert matter (0.50 to 0.34%) and insect damaged seed (0.05 to 0.75%). to date, seed health survey at bogra district, bangladesh showed that seed from farmer’s storages can be grouped into good seed, spotted seeds, deformed seeds, discolored seeds, chaff y grains and insect damaged seeds recording: 77.84 %, 16.32 %, 3.22 %, 1.03 %, and 0.56 % respectively (akter and hossain, 2016). fungal pathogens identifi ed from rice seed samples the present study has also focused on the survey of seed-borne fungi associated with rice varieties mostly journal of tropical crop science vol. 6 no. 3, october 2019 www.j-tropical-crops.com 148 bolanle tolani edun, yahuza lurwanu, mustapha sunusi, ali a. sulaiman grown in kano state, nigeria. some of the fungal species were isolated and identifi ed with the blotter method while others were obtained by the agar plate method. during our investigation, eight species of fungi identifi ed on the seeds of seven rice varieties (table 2). the identifi ed fungi were fusarium spp., bipolaris oryzae, aspergillus fl avus, curvularia lunata, aspergillus niger, and nigrospora oryzae., rhizoctonia spp., and rhizopus spp. highest seed infection recorded for a. fl avus, a. niger, fusarium spp., followed by rhizoctonia spp and rhizopus spp., while b. oryzae, c. lunata, n. oryzae recorded the lowest percentage of seed-borne infection. the aspergillus species, the one recorded with high frequency, tends to produce toxic substances apart from the deteriorating eff ect of root rotting. this result was in consistance with fi ndings of (javaid et al., 2002; ibiam et al., 2006), that reported the presence of aspergillus species but, not as main pathogen. many of the isolated fungi from our study have been reported to be associated with seeds of other crops (tsopmbeng and fomengia, 2015). some of them are also known to cause seed rot, decrease seed germination and cause pre and post damping off as well as seedling death (al-kassim and monawar, 2000) when fusarium spp and aspergillus fl avus are found in high frequency. this high frequency of the detected seed-borne pathogen from our study has been reported in previous studies on the seeds of rice in india (archana and prakash 2013) and pakistan (khan et al., 1988; javaid et al., 2002). soil borne mycofl ora associated with rice and their infl uence on growth was also recorded in abakaliki, ebonyi state in southeastern nigeria (utobo et al., 2011). although, table 1. dry inspection of rice seed samples diff erent grades of seeds (%) hs ds ss bs ids cs des vm im variety ex-china 66.29 18.46 6.50 0.00 1.92 0.9 1.11 1.04 3.78 faro52 86.35 1.52 9.13 0.55 0.00 0.078 1.33 0.00 0.26 faro44 90.53 1.20 3.18 2.16 0.00 1.13 1.8 0.00 0.00 “jamila” 64.77 2.10 28.4 1.18 0.00 1.7 1.6 0.00 0.25 faro60 82.76 1.10 12.99 0.13 0.00 1.31 1.69 0.00 0.02 “kwandala” 77.72 0.96 19.13 0.33 0.00 0.95 0.55 0.00 0.36 “jif” 94.16 0.99 2.49 0.63 0.057 1.04 0.47 0.00 0.16 note: hs = healthy seed = good looking seed free from spots and abnormalities, ds = discolored seed, ss = spotted seeds, bs= broken seeds, ids = insect damaged seeds, cs = chaff y seeds, des = deformed seeds, vm = varietal mixture and im = inert matter table 2. fungal pathogens identifi ed from rice seed samples s/no. variety fungi identifi ed 1 “kwandala” fusarium spp.,rhizopus spp.,a. fl avus, a. niger 2 “jif” bacterial growth, fusarium spp., a. fl avus, aspergillus niger. 3 “faro 60” rhizopus spp., a.niger, a. fl avus, rhizoctonia spp. 4 “faro 44” a. fl avus, a. niger, fusarium spp, bipolaris oryzae, curvularia lunata, rhizoctonia spp., rhizopus spp. 5 “faro 52” rhizoctonia spp., fusarium spp., a. fl avus, a. niger, rhizopus spp., curvularia lunata. 6 “jamila” bacterial growth, rhizopus spp., n. oryzae, a. fl avus, a. niger, fusarium spp., c. lunata. 7 ex-china bacterial growth seed health, quality test, and control of seed-borne fungi of some .......... journal of tropical crop science vol. 6 no. 3, october 2019 www.j-tropical-crops.com 149 no such kind of soil borne pathogens related to rice crops were recorded in our study area. quality test: purity analysis of rice seed samples a seed purity analysis was conducted and concluded: 100 % pure seed in “faro 44” rice variety, and the lowest pure seed (95.18%) was recorded in variety ex-china (table 3). the lower purity of cultivar exchina compared to other cultivars was caused by lousy storage practices by the farmers while high purity “faro 44” was obtained from applied seed technology in commercial company. another study from chowdhury’s (2012) about seed quality status reported that hyv has the highest percentage of purity. haque et al. (2007) also found 99.01 % pure seed from the trained farmers and minimum (96.19 %) in untrained farmers. percentage of seed purity was determined by uddin (2005) in upazilla under noakhali district that ranged from 95.59 to 99.39 %. in convex with our fi ndings, fakir et al. (2002) categorized the percentage of pure seeds ranging from 91.20 to 98.89 % collected from trained farmer’s stored rice seed. germination test using blotter method of rice seed samples the result in figure 1 shows the mean percentage of germination of diff erent rice seeds, ranged from 0 % to 90.50%. the highest germination was recorded in variety “jif” (90.50%), while the lowest germination found in variety ex-china (0%). the variety “jamila” has the highest percentage incidence of fungal mycelia growth examined using blotter method (14.25%), and variety ex-china recorded the lowest rate (1.25%). this research clearly showed that “jif” has the highest germination percentage even though it has been sourced from farmer-stored seed within the study area. the zero germination in the variety exchina could be a result of poor storage conditions that lead to the development of seed-borne pathogens, insects, mechanical damage at both handling and storage, or aging which renders the seed to be not viable or dormant. some of these fungal pathogens are known to cause seed rot, decreasing of seed germination, pre and post damping off , and seedling death (al-kassim and monawar, 2000). according to lamrani et al. (2013), alternaria padwickii colonizes variety of seeds thus reducing the percentage germination and causes seed rot. species of the genus curvularia, in particular, c. lunata has been reported to infect the embryo of the seeds, therefore, reducing the percentage germination of rice seeds (imolehin, 1987; bautista and opina, 1991). c. lunata was reported in diff erent rice varieties and identifi ed as one of seed-borne pathogens (butt et al., 2011; utobo et al. , 2011; ashfaq et al., 2015). eff ect of seed treatment on seed vigor index the variety “faro 44” and “jamila” were selected as test sample of seed dressing trial using three diff erent chemicals due to the facts they are most commonly grown variety in kano. “faro 44” is an improved variety while “jamila” is local. another consideration is these both varieties have higher mycelia growth than others and this indicator is suitable to measure the increasing of vigor index based on the chemical treatment. the vigor index of treated seeds using zeb-care was increased up to 62.78% over the untreated control (table 4). the control (untreated variety) from “jamila” has a vigor index score of 928.71, while “faro 44” has the vigor index of 904.11. variety “jamila” treated with zeb-care has 991.49 vigor index and it indicated that there was an increase of vigor index with 62.78% followed by zeb-care treatment on “faro 44” with an increase of 31.78%. the lowest increase in vigor index (5.28%) was found in variety “faro 44” treated with the apron star (table 4). the variety “faro 44” treated with dress force shown a negative value of -6.11 over the untreated which directly implies that there is a decrease in vigor index over control. table 3. seed quality and purity test among the rice seed samples diff erent components under purity analysis (%) variety pure seed other seed inert matter ex-china 95.18 1.04 3.78 “faro 52” 99.74 0.00 0.26 “faro 44” 100.00 0.00 0.00 “faro 60” 99.98 0.00 0.02 “jamila” 99.75 0.00 0.25 “jif” 99.84 0.00 0.16 “kwandala” 99.64 0.00 0.36 journal of tropical crop science vol. 6 no. 3, october 2019 www.j-tropical-crops.com 150 bolanle tolani edun, yahuza lurwanu, mustapha sunusi, ali a. sulaiman following the obtained values, the effi cacy of seed dressing chemicals was hypothesized benefi t the variety “jamila” more. the zeb-care dressing chemical displayed a more positive eff ect (+31.78 and +62.78) on both cultivars (“jamila” and “faro 44”) followed by apron star (+5.28 and +31.17) and lastly dressed force (-6.11 and +22.40) respectively. similar result also has been reported on previous studied by (bhuiyan et al., 2005; hossain and hossain 2012) whereas vigor index increased in vegetable treated seed using bau bio-fungicide. furthermore, shultana et al. (2009) also evaluated wheat seeds treated with bavistin and showed higher vigor index on 2843 followed by bau bio-fungicide treated seed on 2661. conclusion based on the result obtained in this experiment, it can be concluded that farmers are strongly advised to use any of the following varieties “jif” , “jamila” and “faro 44” in kano state as it has been tested and found to be suitable in both seed quality and purity, it will also help to improve rice production and reduce the threats due to seed-borne fungal diseases. the use of zeb-care treated seed, which is often neglected by farmers, is encouraged as the seed dressing fungicide (zeb-care 80wp) signifi cantly increased seed germination and seedling vigor. acknowledgement the authors thank mr. muhammad nura abdulkadir of plant pathology laboratory, crop protection department, bayero university, kano for laboratory assistance. references abdul-baki, a.a. and anderson, j.d. (1972). physiological and biochemical deterioration of seeds. seed biology 2, 283-316. ahmed, m., hossain, m., hassan, k. and dash, c.k. (2013). seed health and quality test of three rice varieties for the detection of fungi associated with seed sample. universal journal of plant science 1, 37-42. akter, m. and hossain, i. (2016). quality of some hybrid seeds of rice and control of seed-borne fungi in bangladesh. journal of the bangladesh agricultural university 13, 161-168. figure 1: the result of germination test using blotter method among the rice seed samples table 4. eff ect of seed treatment on increase vigor index over control on rice seed samples % vigor index increased over control treatment variety seed source zebcare apron star dress force “faro 44” green spore agric. ltd. +31.78 +5.28 -6.11 “jamila” green spore agric. ltd. +62.78 +31.17 +22.40 seed health, quality test, and control of seed-borne fungi of some .......... journal of tropical crop science vol. 6 no. 3, october 2019 www.j-tropical-crops.com 151 al-kassim, m. and monawar, m. (2000). seed-borne fungi of some vegetable seeds in gazan province and their chemical control. saudi. journal of biological science 7, 179-184. anselme, c. (1981). the importance in cultivation of pathogenic organisms transmitted by seeds. seed science and technology (netherlands) 9, 689-695. archana, b. and prakash, h. (2013). survey of seedborne fungi associated with rice seeds in india. international journal of research in pure and applied microbiology 3, 25-29. ashfaq, m., shaukat, m., akhter, m., haider, m., mubashar, u. and hussain, s. (2015). comparison of fungal diversity of local and exotic rice (oryza sativa l.) germplasm for their seed health. journal of animal and plant science, 25, 1349-1357. barnett, h.l. and hunter, b.b. (1998). illustrated genera of imperfect fungi". 218 pp. american phytopathological society press. bautista, e. and opina, o. (1991). isolation, identifi cation and pathogenicity tests of seedborne fungi associated with cowpea seeds. pest management council of the philippines, manila (philippines), 8-11 may 1991. bhuiyan, a., khokon, m. and hossain, i. (2005). 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(2007). study of seed health, germination and seedling vigor of farmers produced rice seeds. international journal of sustainable crop production 2, 34-39. hossain, m. and hossain, i. (2012). eff ect of seed treatment with diff erent botanicals, bavistin and bau bio-fungicide on germination and seedling vigor of groundnut. bangladesh agronomy journal 16, 87-94. ibiam, o., umechuruba, c. and arinze, a. (2006). seed borne fungi associated with seeds of rice (oryza sativa l.) in storage and from the fi eld in ohaozara and onicha local government area of ebony state. world journal of biotechnology 7, 1062-1072. imolehin, e. (1987). the rice seed multiplication centres in relation to seed borne pathogens of rice. a case study of ondo state rice multiplication centres. nigerian journal of plant protection 11, 37-42 islam, m.s., jahan, q., bunnarith, k., viangkum, s. and merca, s. 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(1988). evaluation of seed health testing techniques for the assessment of seedborne mycofl ora of rice. pakistan journal of agricultural research 9, 502-505. lamrani, n., elabdellaoui, f., touhami, a.o., benkirane, r. and douira, a. (2013). etude de la mycofl ore des grains de trois variétés de riz et eff et d’alternaria padwickii (ganguly) mb ellis sur les grains pré-germés. bulletin de l’institut scientifi que, rabat, section sciences de la vie 35, 1-7. madbouly, a.k., ibrahim, m.i., sehab, a.f. and abdel-wahhab, m.a. (2012). co-occurrence of mycofl ora, afl atoxins and fumonisins in maize and rice seeds from markets of diff erent districts in cairo, egypt. food additives and contaminants: part b, 5, 112-120. mcgee, d.c. (1995). epidemiological approach to disease management through seed technology. annual review of phytopathology, 33, 445-466. misra, j., gergon, e. and mew, t. (1995). storage fungi and seed health of rice: a study in the philippines. mycopathologia, 131, 13-24. nguefack, j., nguikwie, s., fotio, d., dongmo, b., zollo, p.a., leth, v. et al. (2007). fungicidal potential of essential oils and fractions from cymbopogon citratus, ocimum gratissimum and thymus vulgaris to control alternaria padwickii and bipolaris oryzae, two seedborne fungi of rice (oryza sativa l.) journal of essential oil research, 19, 581-587. nsemwa, l. and wolff hechel, h. (1999). occurrence of seed-borne fungal pathogens in rice seeds from the southern highlands of tanzania. african crop science journal 7, 217-222. ora, n., faruq, a., islam, m., akhtar, n. and rahman, m. (2011). detection and identifi cation of seed borne pathogens from some cultivated hybrid rice varieties in bangladesh. middle-east journal of scientifi c research 10, 482-488. serferbe, s., tsopmbeng n.g. and kuiate, j. r. (2016). seed-borne fungi associated with rice seeds varieties in bongor, chad republic. international journal of current microbiology and applied sciences, 5, 161-170 seshu, d. and dadlani, m. (1989). role of women in seed management with special reference to rice. seshu, d., krishnasamy, v. and siddique, s. (1988). seed vigor in rice. rice seed health. los banos, philippines: international rice research institute, 315-329. shultana, r., hossain, i., ahmed, s. and mamun, m. (2009). effi cacy of bau-biofungicide in controlling leaf spot of wheat (triticum aestivum). eco-friendly agricultural journal 2, 392-395. suleiman, m. and omafe, o. (2013). activity of three medicinal plants on fungi isolated from stored maize seeds (zea mays l.). global journal of medicinal plant research 1, 77-81. tsopmbeng, n. and fomengia, d. (2015). fungi associated with seeds of huckleberry (solanum scabrum mill.) grown in the western highlands of cameroon. journal of agricultural technology 11, 791-801. uddin, m. (2005). the quality of farmers stored rice seed of begumgonj upazilla. ms thesis, department of plant pathology, bangladesh agricultural university, mymensingh. utobo, e., ogbodo, e. and nwogbaga, a. (2011). seedborne mycofl ora associated with rice and their infl uence on growth at abakaliki, southeast agro-ecology, nigeria. libyan agricultural research center journal international 2, 7984. sensory evaluation of the quality of kaffir lime (citrus hystrix dc.) leaves .......... journal of tropical crop science vol. 8 no. 2, june 2021 www.j-tropical-crops.com 71 sensory evaluation of the quality of kaffir lime (citrus hystrix dc.) leaves exposed to different postharvest treatments rahmat budiartoa,b, roedhy poerwantoc, edi santosac, darda efendic, andria agustad a department of agronomy, faculty of agriculture, universitas padjadjaran, sumedang 45363, indonesia b study program of agronomy and horticulture, graduate school, ipb university, bogor 16680, indonesia c department of agronomy and horticulture, ipb university, bogor 16680, indonesia d research center for chemistry, indonesian institute for science, south tangerang 15314, indonesia *corresponding author; email: rahmat.budiarto@unpad.ac.id abstract this study aimed to evaluate the sensory attributes such as aroma, color and texture of kaffir lime leaves in response to various postharvest treatment. the hedonic test approach was conducted by inviting 70 untrained panelists on seven postharvest treatments, i.e. (d1) post-sortation fresh leaves, (d2) presortation fresh leaves, (d3) cold-storage leaves, (d4) low temperature-storage leaves, (d5) brown dry leaves, (d6) fresh leaf-flour, and (d7) brown dry leafflour. the result showed that most of panelist agreed that aroma was the most important quality attribute that determined the level of preference in kaffir lime leaf products. among seven tested products, the aroma, color, texture and overall impression of d1 was the most favorite one, while d4, d5, and d7 were assessed as low preference products. this work showed the importance of sortation and cold storage to maintain consumer likeliness. the criteria for sortation were green, clean, pest-disease free, scar free, high uniformity and fresh condition of leaves. cold-storage at -20oc maintained the color and texture of kaffir lime leaves better than low temperature storage (5oc). to make leaf flour, the use of fresh green leaves was significantly better than brown dry ones. this finding might become the baseline data for the development of kaffir lime leaf product in the future. keywords: aroma, leaf flour, hedonic test, sortation, untrained panelists. introduction the leaves of kaffir lime (citrus hytrix dc.) tree is considered economically important due to many used bring about by the naturally pleasant fragrance (budiarto et al., 2019a). the leaves display unique morphological characteristics, i.e., bifoliate leaflets with dark green shiny in adaxial and pale green in abaxial (budiarto et al., 2021a; 2021b). the leaves can be used as spice and raw material for essential oil (sato, 1990). in indonesia, the use of kaffir lime leaves as spice is more popular than as raw material for essential oil (budiarto et al., 2019a). spices from kaffir lime leaves can be found in numerous indonesian and other asian dishes such as beef soup of rawon, coconut milk-beef soup of soto, coconut milk-beef dish of rendang, curry, fried tempe, laksa and tom yum (setiyoningrum et al., 2018). in recent years, the high demand for spice is associated with a rapid promotion of exotic and traditional food ingredients by the food industry to support the modern healthy lifestyle (unido and fao 2005). thus, it is important that production and quality of kaffir lime leaves should be well monitored. previous study has reported the success of mild shading to boost production through harvesting more and larger leaves being harvested (budiarto et al., 2019b). proper postharvest handling of kaffir lime leaves in farms is important in order to maintain quality and to reduce losses. improper handling during post-harvest period has been seen in previous practices and usually results in reduced yields (irtwange, 2006). in general, the yield loss in the citrus fruit industry as a result of careless post-harvest handling can reach 25% (asni, 2015). to reduce the yield loss, sortation (yildiz, 1994) and proper cold storage is promising, especially for aromatic horticultural commodity with its perishable characteristic (magray et al., 2017; tananuwong and lertsiri, 2010). currently, the only post-harvest handling being practiced locally involves leaf stripping (mitil) and packing in plastic woven sacks (budiarto et al., 2019b). additional postharvest treatment such as sortation and appropriate storage handling should be formulated to help the quality assurance of products. journal of tropical crop science vol. 8 no. 2, june 2021 www.j-tropical-crops.com 72 rahmat budiarto, roedhy poerwanto, edi santosa, darda efendi, andria agusta in the kaffir lime leaf spice industry, quality is not fully defined and elaborated yet. in the citrus fruit commodities industry, however, the quality is well characterized by attributes such as fruit size, weight, color, shape, texture and contamination level (sutopo, 2011). the indonesian government has issued a quality guideline for mandarin citrus fruit in sni 31652009 (balitjestro, 2021). as preliminary study, the quality of product can be assessed simply based on consumer acceptance and preference. sensory evaluation can be used to determine the quality of product before chemical testing is carried out (putri et al., 2020). sensory evaluation by using hedonic scale test is a technique used to analyze the level of consumer preference for a product (lyon et al., 1999). sensory-hedonic test is commonly reported in citrus fruit preference studies (morales et al., 2020; reis et al., 2017; sawamura et al., 2004). however, there is still limited similar study specifically for kaffir lime leaf spices. the aim of this study was to evaluate the sensory attributes, such as aroma, color and texture of kaffir lime leaves in response to various postharvest by using hedonic test approach. material and methods sampling the work was carried out in march 2019 at postharvest laboratory, agriculture development polytechnic of bogor, indonesia. fully developed leaves of kaffir lime were harvested from pasir kuda experimental farm of ipb university, indonesia (-6.609042, 106.783605 and 263 meters above sea level). seven postharvest treatments were set up namely (d1) post-sortation fresh leaves, (d2) pre-sortation fresh leaves, (d3) cold-storage leaves, (d4) low temperature-storage leaves, (d5) brown dry leaves, (d6) fresh leaf-flour, and (d7) brown dry leaf-flour (figure 1). approximately 500 grams of leaves and or leaf flour was prepared in every treatment. only green, clean, pest disease-free, scar-free, with high uniformity and fresh condition of leaves were selected for d1. the sortation and the hedonic test were done during the harvesting day. pre-sortation leaves for d2 were composed of fresh leaves with low uniformity, including green normal leaves, those with gall, leaves with yellow/brown spots, and pest disease-damaged leaves. pre-sortation leaves was prepared at the same day with hedonic test. to prepare cold-storage leaves for d3, fresh green leaves were harvested from the field at 10 days before the day of hedonic test. the leaves were stored in freezer at -20oc for 10 days. low temperature-storage leaves for d4 were harvested at the same day as the cold-storage leaves. these d4 leaves were stored at 50c for 10 days. brown dry leaves for d5 were picked 10 days prior to test, and then stored in open air condition at 25-310c for 10 days. both fresh leaf-flour and brown dry leafflour for d6 and d7, respectively, were prepared one day before the test, by using milling machine with 1 mm sieve size. data collection the hedonic test method following protocols documented by meilgaard et al., (2007) and setyaningsih et al. (2010) was used to test the post-harvest quality of kaffir lime leaves through 70 randomly chosen untrained panelists composed figure 1. seven post-harvest treatments tested on kaffir lime leaves, i.e. (d1) post-sortation fresh leaves, (d2) pre-sortation fresh leaves, (d3) cold-storage leaves, (d4) low temperature-storage leaves, sensory evaluation of the quality of kaffir lime (citrus hystrix dc.) leaves .......... journal of tropical crop science vol. 8 no. 2, june 2021 www.j-tropical-crops.com 73 of 17-29 years old students. untrained panelists is defined as people who have no formal sensory training experience, but are still able to differentiate and show any preferences to the product being tested (hersleth et al., 2005). the number of untrained panelists invited in certain hedonic experiment could vary from 25 to 100 people (ayustaningwarno, 2014). previous study by mareta (2019) and putri et al. (2020) did hedonic test with 35 and 20 untrained panelists, respectively. in the current study, among the 70 panelists, there were 47 males and 23 females. the use of untrained panelists was favoured due to the lack of sufficient trained panelists. all seven kaffir lime leaf products were labelled prior to testing and were presented at the same time to the panelists. all invited panelists were asked to assess the aroma, color, texture and overall impression of product based on five hedonic scoring scales, similar to previous studies (mareta et al., 2019, basyuni et al., 2019; jariyah et al., 2018; baba et al., 2015), from highly like to highly dislike (1 for highly dislike, 3 for dislike, 5 for neutral, 7 for like and 9 for highly like). after the test, panelists were also asked to answer briefly three questions regarding (i) how often they used kaffir lime leaf product; (ii) which quality attributes are important when assessing kaffir lime leaf products, and (iii) which kaffir lime leaf product was their favorite. drinking water was provided to serve as neutralizing agent every time the panelists finished testing one product. the data from the hedonic test results were tabulated for analysis of variance (anova) using statistical analysis software (sas) version 9.4. when significant difference in values were detected, tukey test using 95% confidence level was conducted. result and discussion profile of panellists among the panelists, only 8% had never known and used kaffir lime leaves, while approximately 92% of them had experienced using kaffir lime leaves. of the 92%, 43% are monthly users, 41% are weekly users and 4% are daily users (figure 2). the variation of gender and experience of the panelists was thought to increase the representation and accuracy of the results. attributes for hedonic test the results of the hedonic test could be considered as the baseline data for product development in the future (ayustaningwarno, 2014). the rate of likes or dislikes given by a panelist for the tested product is a good indicator consumer preference (kotler and armstrong, 2001). in-depth understanding of consumer preferences greatly influences consumer decisions in choosing and buying a product. the results of the consumer preference test could be used to determine the order of the product attributes in determining product quality (simamora, 2003). in this study, four attributes, namely aroma, color, texture and overall impression, were used to assess the quality of kaffir lime leaves postharvest. figure 2. the number of panellists based on their experience to use kaffir lime leaf product aroma is defined as the response of human sensory to volatile compounds from sample that enters the nasal cavity and sensed by the human olfactory system (kemp et al., 2009). volatile compounds could be easily reached the olfactory system at the upper part of the nose and then interacted with one or more olfactory receptors in the nasal organs (tarwendah, 2017). as a species of citrus, kaffir lime is known to be rich in essential oil throughout the plant body, especially in the leaves (budiarto et al., 2019a; othman et al., 2016). in this study, based on aroma, d1 or post-sortation fresh leaves is the most preferred kaffir lime leaf product (figure 3a). the second preferred product was the d6 or fresh leafflour and d2 or pre-sortation fresh leaves. the aroma of the cold-storage leaves (d3) was relatively neutral and it was not significantly different from the d2 product. in contrast, panelists showed dislike to the aroma of kaffir lime leaves stored in low temperatures (d4), brown dry leaves (d5) and brown dry leaf-flour (d7). approximately 52 panelists believed that aroma was the most important attribute among five attributes in kaffir lime leaf product (figure 4a). better understanding of aroma in herb and spice commodity was important since this attribute highly determines consumer acceptability and palatability (tananuwong and lertsiri, 2010). among other citrus species, the aroma of kaffir lime leaf is distinct and strong, so that it can be used either for spices or essential oil. the major portion of l-citronellal is reported to be key factor for production of specific aroma of kaffir lime journal of tropical crop science vol. 8 no. 2, june 2021 www.j-tropical-crops.com 74 rahmat budiarto, roedhy poerwanto, edi santosa, darda efendi, andria agusta leaves that seemed like the combination of citrus and lemongrass aroma (sato, 1990). other important metabolites determining as main composition of kaffir lime leaves essential oil were citronellol (efendi et al., 2021), linalool (gonzalez-mas et al., 2019), and caryophyllene (gonzalez-mas et al., 2019; riyadi, 2012). previous study reported that pre harvest factor, including agroclimatic variables, soil and leaves nutrient status could affected the yield and main composition of kaffir lime leaves essential oil (efendi et al., 2021). the yield of essential oil could be obtained from steam distillation, with a yield ranged from 0.5-0.6% (budiarto et al., 2019). our results show that there is lower preference for products of kaffir lime leaves that have undergone post-harvest treatment, such as storage, drying, and flour milling process. milling process using fresh green leaves was better than brown dry leaves in maintaining desirable aroma as manifested in the reduced preference of panelists by 12.8% and 58.9%, respectively. to support the production of leaf flour that had been initiated in production area (budiarto et al., 2019a), it was seen in this study that there is a higher potential to develop when using fresh green leaves, instead of brown dry leaves. at 10 days after harvesting, better storage performance was noticed at regime -200c than at 50c, as indicated by the higher desirable aroma. however, the preference for aroma from cold storage leaves was still lower than fresh leaves. this is a common result supported by earlier study showing that likeliness and freshness were reported to decline due to storage effects (obenland et al., 2008). the least preferred aroma was observed in brown dry leaves that were obtained from open air drying for about 10 days after treatment. this finding could be attributed to the loss of volatile oil and the change of metabolite profile in remained oil. setiyoningrum et al., (2018) reported the significant reduction of essential oil yield of kaffir lime, for almost 50%, due to drying. as the leaves are exposed to heat and subsequently dry out, there is a breakage on trichomes and other leaf tissues resulting in the loss of essential oil into the air (diaz-maroto et al., 2002; 2003). color was chosen by 19 panelists as the most important attribute for kaffir lime leaves (figure 4a). in terms of color, panelists determined the d1 product as the most preferred product, due to its evenly green look (figure 1), followed by d3, indicating the success of frozen treatment to maintain appearance quality of kaffir lime (figure 3b). these findings agree with earlier study by wingler and roitsch (2008) that show the importance of cold treatment to delay senescence or further deterioration. although the pre-sortation leaves (d2) were green in color, panelists could have been put off by the presence of yellow/brown spots as a result of pest attack on certain leaf parts. the leaves which were stored at low temperature (d4) were not favored by the panelists because they had a brownish yellow color which was thought as indicator of leaf deterioration. green fresh leaf-flour (d6) was figure 3. the result of hedonic test on aroma, color, texture and overall impression of seven postharvest treated kaffir lime leaves, i.e (d1) post-sortation fresh leaves, (d2) pre-sortation fresh leaves, (d3) cold-storage leaves, (d4) low temperature-storage leaves, (d5) brown dry leaves, (d6) fresh leafflour, (d7) brown dry leaf-flour. sensory evaluation of the quality of kaffir lime (citrus hystrix dc.) leaves .......... journal of tropical crop science vol. 8 no. 2, june 2021 www.j-tropical-crops.com 75 more preferable than brown leaf-flour (d7), as proved by the higher color preference. the brown dry leaves (d5) product was determined as the least preferred one in term of color. post-harvest treatment such as milling and drying results in coloration that was least preferred by panelists (figure 3b). in post-milling condition, the leaves were getting softer and less green compared to the fresh ones (setiyoningrum et al., 2018; arslan et al., 2010; buchaillot et al., 2009). the lowering of green color intensity was also noticed in post-drying condition rather than in fresh condition (naidu et al., 2016). moreover, earlier studies also noted the increasing yellow intensity after milling and drying treatments (mcguire, 1992). the decline of green intensity followed by the increase of yellow intensity is attributed to the breakdown of chlorophyll molecules in leaves that are sensitive to the heat (buchaillot et al., 2009). this phenomenon is obviously observed with greater heat exposure duration and regime of heat treatment (schwartz and lorenzo, 1991). it was likely that heat exposure stimulates the transformation of chlorophyll into pheophytin that is identical with brown color (singh and sagar, 2010; rudra et al., 2008). texture was the third preferred attribute of kaffir lime leaf products (figure 4a). statistical analysis showed that the most preferred texture was the d1, however this result was not significantly different than the fresh leaf-flour (d6) and the cold-storage leaf (d3) (figure 3c). this finding proved the success of cold storage to maintain texture of leaves. the pre-sortation leaves (d2) had lower texture preference because of the presence of pest and disease damaged causing the uneven leaf surface. the least preferred texture was from the brown dry leaf product (d5) because of coarseness due to loss of water during the drying process (figure 4a). the least preferred attribute of kaffir lime leaf was overall impression, that is a combination of aroma, color and texture. the order of panelists preference level based on the overall impression was postsortation fresh leaves (d1) ≥ fresh leaf-flour (d6) > cold-storage leaves (d3) > pre-sortation fresh leaves (d2) > brown dry-leaf flour (d7) ≥ low temperaturestorage leaves (d4) > brown dry leaves (d5) (figure 3d). the only products that received a very high preference were the d1 and the d6. there was neutral impression for the d2 and d3. d4 and d7 product were not preferred, while d5 was the least preferred among all products. the fresh leaves from the d1 set-up was the highly preferred kaffir lime leaf product by most of the panelists indicating the success of sortation process during post-harvest period. however, this postharvest treatment is rarely implemented yet by the local farmers (budiarto et al., 2019a). the importance of sortation to eliminate undesired product in horticulture commodity is frequently emphasized in earlier studies (yossi et al., 2017; kader 2013; alipour et al., 2013). undesired products usually show malformation, over ripening, pest-pathogen attacks and rotten condition (magray et al., 2017). none of the panelists preferred kaffir lime stored at low temperature (d4), brown dry leaves (d5), and brown dry leaf-flour (d7) as their favorite products. it was likely that these products had declined in quality due to senescence, so that it was no longer suitable for consumer. leaf senescence is a natural phase of final leaf development that involves a series of biochemical and physiological process, mostly in the form of macro-molecule degradation, organelles breakdown, nutrient reallocation and eventually cell death (guo, 2013; lim and nam, 2007; guo and gan, 2005). both abiotic and biotic stress could trigger the occurrence of leaf senescence (wingler and roitsch, 2008). improper post-harvest treatment allows leaves to exposed to abiotic and biotic stress leading to the occurrence of severe senescence. figure 4. the main attribute (a) among aroma, texture, color and overall impression; and the favourite product (b) among seven postharvest treated kaffir lime leaves, i.e. (d1) post-sortation fresh leaves, (d2) pre-sortation fresh leaves, (d3) cold-storage leaves, (d4) low temperature-storage leaves, (d5) brown dry leaves, (d6) fresh leaf-flour, and (d7) brown dry leaf-flour. journal of tropical crop science vol. 8 no. 2, june 2021 www.j-tropical-crops.com 76 rahmat budiarto, roedhy poerwanto, edi santosa, darda efendi, andria agusta so far, no similar studies have been conducted to assess leaf quality of kaffir lime, especially in response to different post-harvest handling, leading to the lack definition of quality in kaffir lime leaf as leaf spices. previous studies on kaffir lime highlighted the leaf morphological (budiarto et al., 2021a), leaf allometric modelling (budiarto et al., 2021b), leaf production improvement by shading and pruning (budiarto et al., 2019b), leaf essential oil quality (efendi et al., 2021), and current status of production, post-harvest and marketing of leaf in kaffir lime production center area (budiarto et al., 2019a). this finding might fill the research gap and become the baseline data for the development of kaffir lime leaf product in the future. conclusion in the hedonic test conducted to assess the quality of post-harvest kaffir lime leaves, majority of 70 untrained panelists agreed that aroma is the most important attribute that determines their level of preference in kaffir lime leaf products. among the seven kaffir lime leaf products tested, the post-sortation fresh leaves was the most favorite one, while low temperaturestorage leaves, brown dry leaves, and brown dry leafflour were assessed as low preference products. in term of product diversification, kaffir lime leaf powder could be made from green leaves, instead of brown dry leaves. this hedonic assay emphasized the importance of sortation and cold storage to prolong the quality of kaffir limes leaves. the criteria for sortation were a green, clean, pest-disease free, scar free, high uniformity and fresh condition of leaves. coldstorage at -20oc maintained the color and texture of kaffir lime leaves better than low temperature storage (5oc). further study related to the alteration of essential oil content and composition in response to post-harvest treatment was prerequisite to have better understanding in maintaining the quality of kaffir lime leaves. acknowledgement this work was financially supported by the pmdsu research no. 1520/it3.11/pn/2018, fiscal year 2018. authors also express their gratitude to the postharvest laboratory, bogor agricultural development polytechnic in general, to ms. neni musyarofah and students in particular, for their participation in the hedonic test. references alipour, h., hoseinbeyki, a., jahed, m., rahnama, h., and sharifnia, m. 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(2008). metabolic regulation of leaf senescence: interactions of sugar signalling with biotic and abiotic stress sensory evaluation of the quality of kaffir lime (citrus hystrix dc.) leaves .......... journal of tropical crop science vol. 8 no. 2, june 2021 www.j-tropical-crops.com 79 responses. plant biology 10, 50-62. doi: 10.1111/j.1438-8677.2008.00086.x yildiz, f. (1994). initial preparation, handling, and distribution of minimally processed refrigerated fruits and vegetables. in “minimally processed refrigerated fruits and vegetables” (r.c. wiley, ed.). springer, boston, ma. https://doi.org/10.1007/978-14615-2393-2_2 yossy, h., pranata, j., wijaya, t., hermawan, h., and budiharto, w. (2017). mango fruit sortation system using neural network and computer vision. procedia computer science 116, 596603. doi: 10.1016/j.procs.2017.10.013 journal of tropical crop science vol. 8 no. 2, june 2021 www.j-tropical-crops.com 124 md. waliullah, md. mokter hossain and md. habibur rahman influence of sowing dates and sowing methods on growth and seed yield of black cumin (nigella sativa l.) md. waliullah, md. mokter hossain* and md. habibur rahman department of horticulture, faculty of agriculture, bangladesh agricultural university, mymensingh-2202, bangladesh *corresponding author: mokter.agr@bau.edu.bd abstract nigella sativa l. is widely used medicinal plant throughout the world. in bangladesh it is mainly used as spices in preparing various food items. sowing time and sowing methods are the influential factors to produce higher seed yield of black cumin. therefore, this study was undertaken to find out a suitable sowing date and method to produce higher seed yield of black cumin. the two-factor experiment comprised with four sowing dates (1st november, 15th november, 1st december and 15th december) and two sowing methods (line sowing and broadcast seeding). the experiment was carried out following randomized complete block design with three replicates. results showed that plant growth, yield contributing traits and yield of black cumin significantly influenced by sowing dates and methods. it was observed that the line sowing method compared to broadcast seeding and 1st december sowing among the other sowing dates exhibited higher plant growth with greater production of seed. the combine effect of sowing time and sowing methods showed significantly influenced on black cumin seed production. it was observed that the 1st december sowing with line sowing method increased the seed yield as compared to other sowing dates and methods. from the findings of this study it can be concluded that 1st december following line sowing method would maximize plant growth, yield contributing traits and seed yield of black cumin in bangladesh. keywords: growth, nigella sativa, seed yield, yield contributing traits. introduction black cumin (nigella sativa l.) is an annual spicy herb and belongs to the family ranunculaceae. it is also referred to as black seeds in the world (rahman, 2014; koli, 2013). the word nigella originated from the latin word niger which means blackish referring to its seed colour. the word sativa derived from the latin word ‘serere’ which meaning it is sown, planted or cultivated (jansen, 1981). it is believed to have originated in the mediterranean region and subsequently spread to europe, asia and africa (zohary et al., 2012). it is cultivated in many parts of the world including the middle east, north africa and asia where maximum diversity is found (tierra, 2005). the major producing countries are india, sri lanka, bangladesh, afghanistan, pakistan, egypt, iran, iraq, syria, turkey and ethiopia (malhotra, 2004; wallace, 2013). black cumin is commonly known as ‘kalozira’ in bangladesh and some other countries of the world and is cultivated in the winter season. in bangladesh, it is grown well in faridpur, sariatpur, madaripur, pabna, sirajganj, jessore, kusthtia and natore districts (ali et al., 2015). recently cultivation of kalozira is gaining popularity in bangladesh mainly because of its demand as a commodity for export. the area and production of black cumin are 42 thousand acres and 17 thousand tons, respectively (bbs, 2016). as herb, black cumin has a rich nutritional value; it contains monosaccharides. the seed is rich in fatty acids, proteins and carbohydrates. it contains all essential amino acids and rich source of vitamins and minerals (atta, 2003). various factors are responsible for improving the growth and yield of black cumin per unit area. among them, sowing dates and methods are the most important factors for affecting the growth and yield of black cumin. for successful production of any crop, appropriate planting time/sowing time is very important (haq et al., 2015). mahmood et al. (2012) revealed that the early sowings were the best for black cumin seed yield while sadeghi et al. (2009) also reported that early sowing as compared to late resulted in higher seed yield. however, early sowing has been favorable for disease, and leads to early flowering, resulting poor quality of seed (sharangi and roychowdhury, 2014). the optimum temperature for germination is 16.19˚c to 22.14˚c for black cumin in bangladesh reported by (saeidnejad et al., 2012). influence of sowing dates and sowing methods on growth and seed yield of .......... journal of tropical crop science vol. 8 no. 2, june 2021 www.j-tropical-crops.com 125 planting or sowing date is also one of the most important agronomic factors involved in producing high yielding small grain cereal crops, which affects the timing and duration of the vegetative and reproductive stages. fahim et al. (2017) reported that it is generally cultivated as a relay crop or in the fallow land by broadcasting in bangladesh. few research works have been done so far on black cumin production. however, to realize the yield potential of black cumin, agricultural practices will have to be optimized for its production. sowing date is the key factor affecting the yield and yield components of cumin (fahim et al., 2017). mahmood et al. (2012) also reported that the different planting methods improved the seed yield up to 38% than that of direct (broadcast seeding) sowing. from the above aspect, the study is therefore, conducted to find out the influence of different sowing dates and methods on the growth and yield attributes of black cumin. materials and methods experimental site the study was conducted at the horticulture farm, department of horticulture, bangladesh agricultural university, mymensingh during november 2016 to april 2017. the experimental area is located at 24.600 n and 90.500 e latitude. the elevation of the area is approximately 19 m from average sea level. the soil of the experimental area was sandy loam belonged to the old brahmaputra floodplain alluvial tract. the experimental site was medium high elevation and the ph of the soil was 6.7. the study area was situated in the sub–tropical zone, characterized by heavy rainfall during the months of april to september and scanty rainfall during october to march. rabi season (october to march) is characterized by low temperature but plenty of sunshine. planting materials, treatments and experimental design the seed of bari kalozira-1 was collected from the spice research centre (src), bangladesh agricultural research institute (bari), bogra. the two-factor experiment having four sowing dates (d1: 1st november, d2: 15 th november, d3: 1 st december and d4: 15 th december) and two sowing methods (m1: line sowing and m2: broadcast seeding method). the experiment was laid out in randomized complete block design with three replications. each block was divided into eight-unit plots and all treatment combinations were assigned randomly. the size of a unit plot was 1.5 m x 1.0 m. the line to line distance and block to block distances were 20 cm and 50 cm, respectively. experimentation and data collection the land was ploughed to bring a good tilth condition. weeds and stubbles were removed from the field. at the time of first ploughing, cow dung was applied at the rate of 5 t.ha–1. the land was fertilized with 125, 95 and 75 kg.ha–1 of urea, triple super phosphate (tsp) and muriate of potash (mop).ha–1, respectively. before seed sowing the seeds were soaked in tap water for 48 hours. seeds were treated with bavistin @ 2 g.kg–1 of seeds before sowing. the seeds were sown 20 cm apart according to the treatments in both line and broadcast methods. seeds were covered with good pulverized soil just after sowing and gently pressed by hands to help the quick germination of seeds. seed sowing was done as per treatment. after sowing slight watering was provided for quick germination. seed germination completed within 7 days and excess plants were thinned out twice at 15 and 30 days after seed sowing maintaining a spacing of 5 to 8 cm between the plants. weeding was done to keep the plots free from weeds. irrigation was provided immediately after weeding it was continued till the establishment of the plants. mature seed capsules were harvested at different dates according to maturity of capsule as per treatments. harvesting started from 14th march 2017 and continued up to 4th april, 2017. after harvest, the capsules were sun dried and seeds were collected. data collection plant height was measured in centimeter (cm) by a meter scale from the ground level to the tip of the tallest leaves. the data on plant height was recorded at 30, 45, 60, 75, 90 and 105 days after sowing (das). days to 50% flowering, fruit setting and harvesting were counted from the date of sowing. the number of capsules per plant, number and weight of seeds per capsule, per plant and per plot were recorded. statistical analysis the collected data on various parameters were statistically analyzed. analysis of variance was done with the help of mstat–c computer package program. the mean differences among the treatments were evaluated with least significant difference (lsd) test at both 1 and 5% level of significance. journal of tropical crop science vol. 8 no. 2, june 2021 www.j-tropical-crops.com 126 md. waliullah, md. mokter hossain and md. habibur rahman results plant height the plant height of black cumin at different days after sowing showed significant variations among the sowing dates. at 105 das, it was found that 1st december sowing produced the highest plant height (43.20 cm) followed by 15th november (35.53 cm) and the lowest plant height (29.38 cm) recorded from 15th december sowing (figure 1). in case of sowing methods, plant height was always higher in line sowing method as compared to broadcast seeding method (figure 2). the combined effects of sowing dates and methods at different das was statistically significant. at 105 das, the highest plant height (44.47 cm) obtained when line sowing was applied in 1st december (d3m1) the lowest plant height (29.88 cm) recorded from 15th december sowing in broadcast seeding (d4m2) (table 1). days to 50% flowering, fruit setting and harvesting seed sowing time significantly influenced flowering, fruit setting and even harvesting of black cumin. days to 50% flowering showed significant different among figure 1. effect of sowing dates on plant height of black cumin at different days after sowing. vertical bars represent lsd at 5% level of probability. figure 2. effect of sowing methods on plant height of black cumin at different days after sowing. vertical bars represent lsd at 5% level of probability. 0 10 20 30 40 50 60 30 45 60 75 90 105 p la nt h ei gh t ( cm ) days after sowing (das) d1 (1st november) d2 (15th november) d3 (1st december) d4 (15th december) 0 10 20 30 40 50 60 30 45 60 75 90 105 p la nt h ei gh t ( cm ) days after sowing (das) m1 (line sowing) m2 (broadcast seeding) figure 1. effect of sowing dates on plant height of black cumin at different days after sowing. vertical bars represent lsd at 5% level of probability. figure 2. effect of sowing methods on plant height of black cumin at different days after sowing. vertical bars represent lsd at 5% level of probability. figure 1. effect of sowing dates on plant height of black cumin at different days after sowing. vertical bars represent lsd at 5% level of probability. figure 2. effect of sowing methods on plant height of black cumin at different days after sowing. vertical bars represent lsd at 5% level of probability. 0 10 20 30 40 50 60 30 45 60 75 90 105 p la nt h ei gh t ( cm ) days after sowing (das) d1 (1st november) d2 (15th november) d3 (1st december) d4 (15th december) 0 10 20 30 40 50 60 30 45 60 75 90 105 p la nt h ei gh t ( cm ) days after sowing (das) m1 (line sowing) m2 (broadcast seeding) influence of sowing dates and sowing methods on growth and seed yield of .......... journal of tropical crop science vol. 8 no. 2, june 2021 www.j-tropical-crops.com 127 the sowing dates. it was noticed that the earlier sowing d1(1st november) required longer time for days to 50% flowering (75 days), 50% fruit setting (89.50 days) and days to harvesting (133.00 days). the late sowing d4 (15th december) gave earlier flowering (71.50 days), fruit setting (86.67 days) and harvesting (112.50 days) (table 2). sowing methods also significantly impacted flowering, fruit setting and harvesting of black cumin. it was observed that broadcast seeding produced early flowering, fruit setting and harvesting as compared to line sowing method (table 3). the combined effects of sowing dates and methods significantly influenced on flowering time, fruit setting as well as harvesting of black cumin. it was found that the combination of late sowing with broadcast seeding (d4m2) produced early flowering, fruit setting and harvesting as compared to early sowing following line sowing method (d1m1) (table 4). number and weight of capsule the number of capsules per plant and weight of capsule of black cumin significantly affected by the effect of sowing dates and sowing methods. the highest number of capsule per plant(11.40) and weight of individual capsule (0.267 g) produced from the 1st december sowing (d3) plant while those traits were table 1. combined effects of sowing dates and methods on plant height of black cumin at different days after sowing (das) treatment combinations plant height (cm) at different das 30 45 60 75 90 105 d1 (1st november) m1 (line sowing) 3.77 6.26 15.77 27.81 35.12 36.97 m2 (broadcast seeding) 3.74 5.24 14.11 26.18 32.77 34.80 d2 (15th november) m1 (line sowing) 4.18 6.96 16.22 28.42 36.28 37.28 m2 (broadcast seeding) 3.85 6.37 14.37 27.98 34.78 35.78 d3 (1st december) m1 (line sowing) 4.23 7.48 17.50 33.33 43.47 44.47 m2 (broadcast seeding) 4.00 7.36 16.90 32.42 42.93 43.93 d4 (15th december) m1 (line sowing) 2.80 5.57 14.95 27.50 29.57 30.87 m2 (broadcast seeding) 2.71 4.93 11.20 23.88 28.33 29.88 lsd0.01 0.11 0.37 0.61 1.31 0.82 0.85 level of significance ** ** ** ** ** ** note: ** indicates significant at 1% level of probability table 2. effect of sowing dates on days to flowering, fruit setting and harvest of black cumin sowing dates days to 50% flowering days to 50% fruit setting days to harvest (days) d1(1st november) 75.00 89.50 133.00 d2(15th november) 74.00 88.50 128.50 d3(1st december) 73.00 87.50 121.00 d4(15th december) 71.50 86.67 112.50 lsd0.01 0.41 0.39 1.10 level of significance ** ** ** ** indicates significant at 1% level of probability table 3. effect of sowing methods on days to flowering, fruit setting and harvest of black cumin sowing methods days to 50% flowering days to 50% fruit setting days to harvest (days) m1 (line sowing) 74.50 89.42 126.00 m2 (broadcast seeding) 72.25 86.67 121.50 lsd0.01 0.29 0.28 0.78 level of significance ** ** ** note: ** indicates significant at 1% level of probability journal of tropical crop science vol. 8 no. 2, june 2021 www.j-tropical-crops.com 128 md. waliullah, md. mokter hossain and md. habibur rahman the lowest (6.13 per plant and 0.218g from the 15th december sowing (d4) (table 5). similarly, the plants produced from line sowing method (m1) produced significantly the highest number of capsules per plant (9.42) and weight of single capsule (0.255g) than that of broadcast seeding method (m2) (table 6). significant variations were observed on number of capsule per plant and single capsule weight due to the combined effects of sowing dates and methods. the number of capsules per plant and single capsule weight were highest in the combination of line sowing at 1st december (d3m1) while it was lowest with broadcast seeding at 15th december (d4m2) (table 7). the weight of capsule per plant was significantly influenced by the effect of sowing time. it was found that the 1st december sowing plant showed the highest weight of capsules per plant (2.35 g) while 15th december sowing plant recorded the lowest weight of capsules per plant (1.38 g) (figure 3). there was a significant variation for weight of per plant due to the effect of sowing method. it was observed that the line sowing method produced the highest (1.89 g) and broadcast seeding gave the lowest (1.64 g) weight of capsules per plant (figure 4). significant variation was found due to the combined effects of sowing dates and methods of sowing on weight of capsule. the highest weight of capsule (2.48 g per plant) was recorded from the line sowing method at table 4. combined effects of sowing dates and methods on days to flowering, fruit setting and harvest of black cumin treatment combinations days to 50% flowering days to 50% fruit setting days to harvest (days) d1 (1st november) m1 (line sowing) 76.00 91.00 135.00 m2 (broadcast seeding) 74.00 88.00 131.00 d2 (15th november) m1 (line sowing) 75.00 90.00 130.00 m2 (broadcast seeding) 73.00 87.00 127.00 d3 (1st december) m1 (line sowing) 74.00 89.00 124.00 m2 (broadcast seeding) 72.00 86.00 118.00 d4 (15th december) m1 (line sowing) 73.00 87.67 115.00 m2 (broadcast seeding) 70.00 85.67 110.00 lsd0.01 0.58 0.55 1.56 level of significance ** ** ** note ** indicates significant at 1% level of probability table 5. effect of sowing dates on number and weight of capsules of black cumin at harvest sowing dates no. of capsules per plant weight of single capsule (g) d1(1st november) 7.47 0.227 d2(15th november) 9.77 0.253 d3(1st december) 11.40 0.267 d4(15th december) 6.13 0.218 lsd0.01 0.34 0.02 level of significance ** ** note: ** indicates significant at 1% level of probability table 6. effect of sowing methods on number and weight of capsules of black cumin at harvest sowing methods no. of capsules per plant weight of single capsule (g) m1 (line sowing) 9.42 0.255 m2 (broadcast seeding) 7.97 0.228 lsd0.01 0.24 0.012 level of significance ** ** note: ** indicates significant at 1% level of probability influence of sowing dates and sowing methods on growth and seed yield of .......... journal of tropical crop science vol. 8 no. 2, june 2021 www.j-tropical-crops.com 129 1st december sowing (d3m1) followed by broadcast seeding at the same date of sowing (2.21 g per plant) while the broadcast seeding at 15th december (d4m2) gave the lowest weight of capsules (1.37 g per plant) (table 7). yield contributing traits and yield of black cumin significant variations observed in number of seeds per capsule, weight of seeds per plant, per plot as well as per hectare due to the effect of sowing dates, methods and their combinations. among the sowing dates, 1st december sowing produced the highest seeds per capsule (68.60) and the maximum seed weight (1.60 g per plant, 64.31g per plot and 428.73 kg.ha-1, respectively). those parameters were the lowest in case of the 15th december planting (51.97 seeds per capsule, 0.67 g per plant, 40.49 g per plot and 269.94 kg.ha-1, respectively) (table 8 and figure 4). sowing methods significantly impacted the yield contributing traits and seed yield of black cumin. it was noticed that the maximum number of seeds (61.93 per capsule), seed weight (1.17 g per plant, 59.61 g per plot and 397.43 kg.ha-1, respectively) obtained in line sowing method as compared to broadcast seeding method (58.65 per capsule, 1.01 g per plant, 40.49 g per plot and 269.94 kg.ha-1, respectively) (table 9 and figure 4). the combined effects of sowing dates and sowing methods were significantly impacted on yield contributing traits and yield of black cumin seeds. the highest number of seeds (71.73 per capsule) and the maximum seed weight (1.66 g per plant, 78.33 g per plot and 522.22 kg.ha-1, respectively) obtained when black cumin sown in 1st december following line sowing method (d3m1). all the traits performed inferior from the combination of d4m2 (15th december sowing with broadcast seeding) and resulted the minimum seeds (51.47 per capsule, 0.61 g per plant, 35.68 g per plot and 237.84 kg.ha-1, respectively) (table 10). discussion sowing dates and methods are the most important factors for desirable plant height. plant height of black cumin had statistically significant due to sowing date and method as singly or their interactions where 1st december sowing under line sowing method as singly or their interaction were more efficient than other factor(s) affecting in the present study while plant height increased significantly in increasing days after sowing. the result also revealed that the delay sowing at 15th december decreased the plant height. this result revealed that the third sowing date d3 (1st december) had more significant for enhance the plant height in this study which might be due to the proper environment prevailing at this time which ultimately enhance the growth of plant. similar observations were also reported by giridha (2015); jafari (2013) and more other scientists at home and abroad. similarly, black cumin grown under line sowing method received proper space which might increase the photosynthesis activity by using sufficient light from the atmosphere and also received more nutrients from the soil. our results are in agreement with the findings of fahim et al. (2017); mahmood et al. (2012). days to 50% flowering, fruit setting and harvest also influenced significantly due to the individual or their interaction effect of sowing dates and methods while haq et al. (2015); el–mekawy (2012) noticed due to sowing times and fahim et al. (2017); mahmood et al. (2012); claimed due to sowing methods were obtained significant performance for black cumin. a b figure 3. effect of sowing dates (a) and sowing methods (b) on capsule weight of black cumin at harvest. vertical bar represents lsd at 5% level of probability. a b figure 4. effect of sowing dates (a) and sowing methods (b) on seed weight of black cumin at harvest. vertical bar represents lsd at 5% level of probability. figure 3. effect of sowing dates (a) and sowing methods (b) on capsule weight of black cumin at harvest. vertical bar represents lsd at 5% level of probability. journal of tropical crop science vol. 8 no. 2, june 2021 www.j-tropical-crops.com 130 md. waliullah, md. mokter hossain and md. habibur rahman table 7. combined effects of sowing dates and methods on number and capsule weight of black cumin at harvest treatment combinations no. of capsules per plant weight of capsule (g per plant) weight of single capsule (g) d1 (1st november) m1 (line sowing) 7.87 1.74 0.237 m2 (broadcast seeding) 7.07 1.38 0.217 d2 (15th november) m1 (line sowing) 10.47 1.95 0.273 m2 (broadcast seeding) 9.07 1.59 0.233 d3 (1st december) m1 (line sowing) 12.80 2.48 0.283 m2 (broadcast seeding) 10.00 2.21 0.250 d4 (15th december) m1 (line sowing) 6.53 1.39 0.227 m2 (broadcast seeding) 5.73 1.37 0.210 lsd0.01 0.49 0.20 0.024 level of significance ** ** ** note: ** indicates significant at 1% level of probability table 8. effect of sowing dates on seed yield of black cumin sowing dates no. of seeds per capsule seed weight per plot (g) seed yield (kg.ha–1) d1(1st november) 55.03 46.87 312.44 d2(15th november) 65.57 51.15 340.97 d3(1st december) 68.60 64.31 428.73 d4(15th december) 51.97 40.49 269.94 lsd0.01 1.14 3.15 17.12 level of significance ** ** ** note ** indicates significant at 1% level of probability table 9. effect of sowing methods on seed yield of black cumin sowing methods no. of seeds per capsule seed weight per plot (g) seed yield (kg.ha–1) m1 (line sowing) 61.93 59.61 397.43 m2 (broadcast seeding) 58.65 41.79 278.62 lsd0.01 0.81 2.23 12.11 level of significance ** ** ** note: ** indicates significant at 1% level of probability above researcher reported that the early sowing always superior than that of delay sowing while line or bed sowing perform well than other sowing methods. these findings are in agreement with those of haq et al. (2015); el–mekawy (2012); mahmood et al. (2012). similarly, plants in line sowing get more sunlight and other favorable conditions from the atmosphere, proper soil nutrient from the soil which ultimately delayed flowering, fruit setting and harvest time of black cumin. similar observations with the present study were also reported by fahim et al. (2017) and mahmood et al. (2012). number of capsules of black cumin was also statistically significant due to the effect of sowing dates and methods where third sowing at 1st december was more effective to get more number of black cumin capsule. the higher capsule number with december sowing may be due to the favorable environment during the cultivation of black cumin, enabled the plant to acquire higher growth and production of photosynthates in the source and better partitioning to the sink. similar observations were reported by abad et al. (2015); haq et al. (2015); islam and akhtar (2013); meena et al. (2011) and more other scientists at home and abroad. the decrease in capsule number was also recorded in delay sowing at 15th december. a month delay in sowing caused severe reduction in the number of capsules per plant and hence early sowing was reported to be influence of sowing dates and sowing methods on growth and seed yield of .......... journal of tropical crop science vol. 8 no. 2, june 2021 www.j-tropical-crops.com 131 figure 4. effect of sowing dates (a) and sowing methods (b) on seed weight of black cumin at harvest. vertical bar represents lsd at 5% level of probability. a b figure 3. effect of sowing dates (a) and sowing methods (b) on capsule weight of black cumin at harvest. vertical bar represents lsd at 5% level of probability. a b figure 4. effect of sowing dates (a) and sowing methods (b) on seed weight of black cumin at harvest. vertical bar represents lsd at 5% level of probability. critical in nigella reported by giridha (2015); jafari (2013); el-mekawy (2012) which is also supported the present findings. decrease in capsule number due to delay sowing was also reported by haq et al. (2015); vaseghi et al. (2013); islam and akhtar (2013); meena et al. (2012). besides, line sowing had higher capacity to enhance the number and weight of capsules in black cumin plant also reported by fahim et al. (2017) and mahmood et al. (2012) while their findings are strongly supported the present findings. they found that the line or bed sowing get more favorable weather condition and soil nutrient from the soil comparatively than that of broadcast seeding which ultimately resulted the higher production of capsule of black cumin. seed production of black cumin as number and weight were also affected significantly by the effect of sowing dates and methods. this study showed that the third sowing at 1st december had highly efficient to produce more seeds per capsule or per plant, per plot or hectare. the above result revealed that the third sowing at 1st december growing plant might be get proper or favorable weather condition which enhanced the adaptability with the growing region as well as they produced more number and highest weight of seeds. besides, higher adaptable plant can also accumulate the more soil nutrients to the root zone which ultimately enhanced the growth plant growth and confirm the higher number of weight of seeds. results obtained from the study were in compatible with haq et al. (2015); jafari (2013); islam and akhtar (2013); meena et al. (2012). in another observation, line sowing had also more effective to produce more seeds as number and weights as compared to that of broadcast seeding. this results might be due to growing plant in line sowing method get proper space which was helpful for getting the proper air and sunlight, proper humidity, appropriate soil nutrient etc. which improved the growth of plant and finally seed yield is increased. our results are in agreements with many previous findings of fahim et al. (2017); mahmood et al. (2012). besides, tallest plant with numerous branches facilitated more capsules per plant increased seed number and weight per unit area enhanced the final yield of black cumin seed were also reported by mahmood et al. (2012) and meena et al. (2011 and 2012) in black cumin. conclusion plant growth and seed yield of black cumin varied based on the sowing dates and sowing methods. the proper growth and development of black cumin plants are somehow restricted in broadcast seeding method while line sowing method in 1st december sowing provided favorable space for the growth of plants which ultimately enhance higher crop growth and better seed yield of black cumin. this study showed that 1st december sowing following the line sowing method improve plant growth as well as seed yield of black cumin in bangladesh. references abad, f.j.h., nezami, a., kafi, m., and shabahang, j. (2015). effects of sowing time on yield of black seed (nigella sativa l.) ecotypes under mashhad conditions. iranian journal of field crops research 12, 632–640. ali, m.m.k., hasan, m.a., and islam, m.r. (2015). influence of fertilizer levels on the growth and yield of black cumin (nigella sativa l.). the agriculturists 13, 97–104. journal of tropical crop science vol. 8 no. 2, june 2021 www.j-tropical-crops.com 132 md. waliullah, md. mokter hossain and md. habibur rahman table 10. combined effects of sowing dates and methods on seed yield of black cumin treatment combinations no. of seeds per capsule seed weight per plant (g) seed weight per plot (g) seed yield (kg.ha–1) d1 (1st november) m1 (line sowing) 56.00 1.10 56.66 377.71 m2 (broadcast seeding) 54.07 0.87 37.08 247.18 d2 (15th november) m1 (line sowing) 67.53 1.19 58.16 387.73 m2 (broadcast seeding) 63.60 1.02 44.13 294.20 d3 (1st december) m1 (line sowing) 71.73 1.66 78.33 522.22 m2 (broadcast seeding) 65.47 1.55 50.29 335.24 d4 (15th december) m1 (line sowing) 52.47 0.72 45.31 302.04 m2 (broadcast seeding) 51.47 0.61 35.68 237.84 lsd0.01 1.61 0.08 4.46 24.21 level of significance ** ** ** ** ** indicates significant at 1% level of probability atta, m.b. (2003). some characteristics of nigella (nigella sativa l.) seed cultivated in egypt and its lipid profile. food chemistry 83, 63–68. bbs. (2016). “bangladesh bureau of statistics”. 40 pp. statistic division, ministry of planning, government people’s republic of bangladesh. el–mekawy, m.a.m. (2012). growth and yield of nigella sativa l. plant influenced by sowing date and irrigation treatments. american–eurasian journal of agriculture and environmental science 12, 499–505. fahim, a.h.f., naher, s., wadud, m.a., and sarker, m.b. (2017). response of black cumin to different seed rate and sowing method. international journal of agricultural papers 2, 1–6. giridha, k. (2015). “effect of sowing time, plant density, nitrogen and phosphorus levels on growth, yield and quality of black cumin (nigella sativa l.) in vertisols of andhra pradesh”. [thesis]. department of plantation, spices, medicinal and aromatic crops, college of horticulture, hyderabad. haq, m.z., hossain, m.m., haque, m.m., das, m.r., and huda, m.s. (2015). blossoming characteristics in black cumin genotypes in relation seed yield influenced by sowing time. american journal of plant sciences 6, 1167– 1183. islam, n., and akhtar, r. (2013). effect of sowing dates and sowing methods on kalonji (nigella sativa) in “annual report 2011–12”. pp. 69. ayub agricultural research institute (aari), directorate of agricultural information pakistan. jafari, a. (2013). the evaluation effects of plant density and sowing dates on yield and yield components of black cumin (nigella sativa) under ilam climate condition in “proceedings of 2nd national congress on medicinal plants”, pp 456, tehran, iran. jansen, p.c.m. (1981). “spices, condiments and medicinal plants in ethiopia, their taxonomy and agricultural significance”. 327 pp. centre for agricultural publishing and documentation. koli, s.a. (2013). “effect of variety and plant spacing on seed yield and yield attributes of black cumin (nigella sativa l.)”. [thesis]. department of agricultural botany, sher–e– bangla agricultural university. mahmood, t., idress, m., aslam, m., rehman, h.s., akram, h.m., sattar, a., abbas, s., and ferdosi, m.f.h. (2012). growth and yield attributes of black cumin (nigella sativa l.) as affected by sowing dates and methods. mycopath 10, 83– 86. malhotra, s.k. (2004): nigella. in “handbook of herbs and spices” (k.v. peter, eds.), pp. 1–19. woodhead publishing ltd. meena, s.s., anwer, m.m., mehta, r.s., lal, g., kant, k., sharma, y.k., vishal, m.k., jingar, k.l., and meena, s.r. (2011). performance of nigella (nigella sativa l.) as influenced by sowing dates and crop geometry in semi-arid eco-system. international journal of seed spices 1, 8-12. meena, s.s., mehta, r.s., lal, g., kant, k., sharma, y.k., saxena, s.n., and anwere, m.m. (2012). influence of sowing dates and sowing methods on growth and seed yield of .......... journal of tropical crop science vol. 8 no. 2, june 2021 www.j-tropical-crops.com 133 essential oil, fatty oil and seed yield of nigella (nigella sativa l.) as influenced by sowing dates and crop geometry. indian journal of horticulture 69, 591–593. rahman, m.a. (2014). “effect of nitrogen and phosphorous on morphological parameters and yield of black cumin (nigella sativa l.)”. [thesis]. department of agricultural botany, sher–e– bangla agricultural university. sadeghi, s., rahnavard, a., and ashrafi, z.y. (2009). study importance of sowing date and plant density effect on black cumin (cuminum carvi) yield. botany research international 2, 94–98. saeidnejad, a.h., kafi, m., and pessarakli, m. (2012). evaluation of cardinal temperatures and germination responses of four ecotypes of bunium persicum under different thermal conditions. international journal of agriculture and crop sciences 4, 1266–1271. sharangi, a. b., and roychowdhury, a. (2014). phenology and yield of coriander (coriandrum sativum l.) at different sowing dates. journal of plant sciences 9, 32–42. tierra, m. (2005). nigella sativa,. http://www. planetherbs.com/articles/nigella.html [march 9, 2005]. vaseghi, a., ghanbari, a., heydari, m., and davazdahemami, s. (2013). effect of sowing date and growing season on agronomical characters of isfahanian and indian black cumin in “proceedings of 2nd national congress on medicinal plants”, pp 494, tehran– iran. wallace, r.k. (2013). modern research on black cumin. http://www.doshaguru.com [july 17, 2014]. zohary, d., hopf, m. and weiss, e. “domestication of plants in the old world: the origin and spread of domesticated plants in southwest asia, europe, and the mediterranean basin”. 251 pp. oxford university press. pp. 164–165. vol.6, no.3.indd journal of tropical crop science vol. 6 no. 3, october 2019 www.j-tropical-crops.com 182 versi putra jaya hulu, heni purnamawati, anas dinurrohman susila, slamet susanto sources and rates of potassium for drip irrigation of polyethylene-mulched bean production versi putra jaya hulua, heni purnamawati*b, anas dinurrohman susilab, slamet susantob a postgraduate school, ipb university, bogor, 16680, indonesia. b department of agronomy and horticulture, faculty of agriculture, ipb university, bogor 16680, indonesia corresponding author; email: henipurnamawati1@gmail.com abstract common bean is a type of vegetable that can be consumed in the form of young pods. potassium is one of the macro nutrients needed to achieve maximum yield in common bean. therefore this research is aimed at determining a suitable potassium source as well as an appropriate rate for drip irrigation in common bean using polyethylene mulch. the study was conducted from january to may 2018 at university farm, ipb university. this experiment was set up as a nested randomized block design with two factors i.e. potassium sources as the main factor ( kcl, zk, and npk), and potassium rates (0, 37.5, 75, 112.5, 150 kg of k2o per ha). fertilizer rates were nested in the main factor and fertilizers were applied fetilization through a drip irrigation fertigation system with emitters placed close to the roots of plants. fertilizer sources had signifi cant eff ects on the variables of growth and bean yields. npk resulted in a better growth and yield compared with kcl and zk. fertilizer sources that have very signifi cant eff ect were likely related to the shape and solubility of the fertilizers, the ease of application, and also completeness of nutrient content in fertilizer. based on the pod weight per plot, the productivity per hectare of bean pods produced in this study reached 7.52 ton.ha-1. potassium (k2o) rate did not have signifi cant eff ects on the variables of growth and yields of the bean. keywords: fertigation, fertilizer, phaseolus vulgaris, pod weight, yield and yield components introduction common bean (phaseolus vulgaris l.) is an introductory crop in indonesia. common bean originates from western mexico-guatemala (kay, 1979). common bean pods can be consumed in the form of young pods; young bean pods have a sweet taste which make them suitable for vegetable consumption. common beans have a fairly high nutrient content, i.e. every 100 g of beans contains 35 g of calories, 2.4 g of protein, 0.2 g of fat, 7.7 g of carbohydrates, 65 g of calcium, 44 g of phosphorus, 1.1 g of iron, and water 88.9 g (ipgri, 2003). community demands for beans continue to increase along with population growth. the average consumption per capita from 2013 to 2015 had been increasing from 0.782, 0.826, 1.143 kg/capita/year (pusdatin, 2017). on the other hand, the statistical data from direktorat jenderal hortikultura (2018) showed that the production of bean vegetables has been declining from 2013 to 2016, by 327.38, 318.21, 291.31, 275.51 tons, respectively. the expansion of productive land has not keep up with the increasing population growth. it is important to carry out agricultural intensifi cation to increase production using more effi cient water and fertilizer uses. one of the methods to increase effi ciency of watering and fertilizer is by using fertigation system. weeds can reduce fertilizer intake and increase competition with the main crops for sunlight. weeds can also be the host of plant diseases. the use of land cover or mulch could potentially solve these problems. one of the types of mulch that can be used is polyethylene. bosland and votava (2012) stated that the use of polyethylene mulch has several advantages, which are to increase the yield and quality of crop yields, accelerate fruit maturity, maintain moisture and soil structure, increase soil temperature (stable), reduce evaporation or evaporation of soil, suppress weed growth, reduce nutrient loss, reduce soil compaction, and control pests and diseases. the disadvantage is the increase in cost of production at the beginning of planting, and fertigation through drip irrigation can compensate for this initial cost. mulched plant beds do not get rainwater directly, except when heavy rain fl oods the beds. to obtain high-quality beans, the ideal growing conditions and cultivation techniques need to be maintained by supplying nutrients. nutrients available journal of tropical crop science vol. 6 no. 3, october 2019 www.j-tropical-crops.com 183sources and rates of potassium for drip irrigation of polyethylene-mulched bean .......... in the soil are often insuffi cient, so additional fertilizers are necessary. the soil nutrient content varies, therefore the fertilizer requirements of each soil type are also diff erent, indicating more specifi c research was needed to determine the optimum fertilizer rate. potassium (k) is very important to crops as it aff ects metabolic, physiological processes, and k is required in large quantity. in bean potassium can stimulate the growth of new shoots, and maintain effi cient use of water by crops. sumpena and hilman (2000) stated that beans are very sensitive to potassium defi ciency, which can cause slow and dwarf plant growth, decrease yields or number of pods, decrease in the quality of pods, reduced taste and nutritional content, and plant stems can break easily. determining the correct application method and source of fertilizers can increase bean production. a study by sousa et al. (2013) showed that the conventional potassium fertilization rate of 65.80 kg.ha-1 k2o would provide an estimate of maximum productivity of peanuts of 1,092.22 kg.ha-1, whereas fertigation at 69.39 kg ha-1.k2o could increase production to 1,530.68 kg.ha-1. potassium chloride (kcl) and potassium sulfate (zk) provides a single nutrient, while npk supplies macro nutrients n, p and k. two types of kcl fertilizer are available, kcl 80 which has k2o content of 60% in a form of red powder, and kcl 90 which has k2o content of 90% in a form of white powder. zk fertilizers in the market can be found in two types, i.e. zk 90 with k2o content of 50% and zk 96 which k2o content of 53%. zk is in a form of white powder or granules (pt pupuk kujang, 2017). npk has diff erent compositions of nutrients. npk 16-16-16, npk 15-15-15, or npk 12-12-12 indicate the percentage of macro nutrient content. research on potassium (k) fertilization in bean plants through fertigation with drip irrigation in the fi eld has not been widely done. this study investigated three types of k fertilizer to determine the best source of k fertilizer and rates for fertigation through a drip irrigation system in common bean which were grown with polyethylene mulch. materials and methods experimental site this experiment was conducted from january to may 2018 at university farm, ipb university at an altitude of ±230 meters above sea level. the soil analysis before the experiment was carried out at the testing laboratory; pod nutrient content analysis was conducted at post harvest laboratory of department of agronomy and horticulture, ipb university. treatments this experiment used nested randomized block design with two factors. the fi rst factors were potassium source, i.e., kcl, zk, and npk 16:16:16. the second factors were potassium rates that consisted of fi ve levels, i.e. 0, ¼x, ½x, ¾x, dan x. the x rate level is the nutrient content of k2o in each fertilizer used. in this experiment, the fertilizer rate was nested in the main factor (source of fertilizer). the general recommendation for fertilization in beans consisted of urea (46% n) at 300 kg.ha-1, or 138 kg n.ha-1, sp-36 (36% p2o5) at 200 kg ha -1 or 72 kg p2o5.ha -1, and kcl (60% k2o) at 250 kg.ha -1 or 150 kg k2o.ha -1 (puslitbanghorti, 2016). in total there were 15 combination of treatments with three replications i.e. 45 experimental units. each experimental unit consisted of 20 plants so that the total population was 900 plants. the materials used in this study were cultivar of common bean “ladju”, urea fertilizer, sp-36 fertilizer, kcl fertilizer, zk fertilizer, npk 16 fertilizer, manure, insecticide/fungicide, and polyethylene mulch. installation of drip irrigation systems was carried out by assembling components consisting of head units and in-fi eld unit. the components of the head unit are reservoir, engine pump, pressure regulator (prv), disc fi lter, venturi injector, pvc ball valve, pvc fi tting, and pvc pipe fi tting. head unit was connected to the in-fi eld unit component, i.e. ldpe line, dripline 16 mm, neple tee, elbow, check valves, and joiners. the experimental beds were 45 plots of 1.5 m x 5 m (7.5 m2) each bed. the height of beds is 0.3 m and space between beds is 0.6 m. total land area is 400 m2. basic fertilizers were applied preplant (1 week before planting) before the installation of polyethylene mulch, consisting of 100% sp-36, 40% urea, and kcl, zk, and npk according to the treatment (table 1). fertigation was carried out weekly from 1 to 7 wap (weeks after planting), so that the percentage of n and k at each treatment time was 8.6%. the growth variables measured were stem diameter (measured every week from 1 wap to 10 wap), number of days to 50% anthesis i.e. when 50% plant population per plot had fl owering, and stomatal density i.e. when 50% plant population had fl owering was observed on a sample of leaves as high as 1 meter above the ground. the number of stomata was determined using a microscope. journal of tropical crop science vol. 6 no. 3, october 2019 www.j-tropical-crops.com 184 versi putra jaya hulu, heni purnamawati, anas dinurrohman susila, slamet susanto stomatal density was calculated using the formula: sd = number of stomata / (π.r2) where π = constant (3.14) and r2 (radius of the fi eld of view on 400x magnifi cation) the yield variables measured were pod number and pod length, pod weight per plant, and pod weight per plot (7.5 m2) using the following formula: analysis of nutrient content of potassium in pods from the third harvest was measured using atomic absorbtion spectrophotometer (aas) using a method by eviati and sulaeman (2009). measurements were made on two plants taken compositely from control (0 kg k2o.ha -1), medium rate of k2o (75.0 kg.ha -1), and highest rate of k2o (150 kg.ha -1). during the course of the study weeds around the area of the crops were removed manually; insecticides to control caterpillar (plusia signata/plusia chalcites) and grasshoppers was sprayed when necessary. data analysis data was analysed with anova using the statistical analysis system (sas) v 9.4 with a confi dence interval of 95%. separation between means were tested further with the orthogonal polynomial test to determine the pattern of the response. the interaction between treatments was tested using combined analysis using star (statistical tool for agricultural research) nebula program. correlation analysis to determine the relationship between observational parameters used statistical product and service solution (spss) v 23. results general condition the result of soil analysis before the experiment showed a ph h2o of 4.81 which is classifi ed as acidic, and available k of 0.57 cmol kg-1(high) (table 2). based on bmkg data from the darmaga bogor climatology station the agroclimate zone from january to may the average temperature was 25.426.6oc with relative humidity (rh) was classifi ed as high (81-86%) (table 3). rainfall was relatively high during the study. based on oldeman climate classifi cation january and march are humid months, whereas february, april and may are wet months. the highest rainfall in april was 432.1 mm. stem diameter k fertilizer sources signifi cantly increased stem diameter at 2 wap, and the eff ects were highly signifi cant at 4 and 6 wap (table 4). npk produced crops with the largest stem diameter. k rates and their interaction with k sources did not signifi cantly aff ect stem diameter (table 4). table 1. the rates of fertilizer before planting and through fertigation with drip irrigation rate (kg k2o. ha -1) preplant (g) drip irrigation (g) sp-36 urea kcl zk npk 16 urea kcl zk npk 16 0 150.0 90.0 135.0 37.5 150.0 90.0 18.8 22.5 70.3 135.0 28.1 33.8 105.5 75.0 150.0 90.0 37.5 45.0 140.6 135.0 56.3 67.5 210.9 112.5 150.0 90.0 56.3 67.5 210.9 135.0 84.4 101.3 316.4 150.0 150.0 90.0 75.0 90.0 281.3 135.0 112.5 135.0 421.9 table 2. the soil physical and chemical properties before experiment soil parameters extract value criteria ph h2o 4.81 acid organik carbon (%) walkley and black 1.83 low total n (%) kjeldahl 0.21 medium available p (p2o5 ppm) bray i 103.92 very high available k (cmol.kg-1) nh4oac 1m ph 7.00 0.57 high cec (cmol.kg-1) nh4oac 1m ph 7.00 19.00 medium source: analytical laboratory of the department of agronomy and horticulture of ipb journal of tropical crop science vol. 6 no. 3, october 2019 www.j-tropical-crops.com 185sources and rates of potassium for drip irrigation of polyethylene-mulched bean .......... number of days to 50% anthesis days to anthesis was determined from the days required for 50% of the total population of plants to fl ower. table 5 shows that k source and rates did not signifi cantly aff ect the number of days of plants to fl ower. flowering age of beans in this study ranged from 36.75 to 40.44 days after planting (dap). virisya (2014) research also showed that the number of days to 50% anthesis of beans was between 35-53 dap. stomatal density stomatal density is the number of stomata per unit area of view on a microscope. almost all of the stomata at the bottom surface of the leaves was open at 50% anthesis. table 5 shows that the source and rate of k fertilizer did not have a signifi cant eff ect on the stomatal density with values between 336.31 to 397.45 mm-2. yield components table 6 shows that the sources of k fertilizer had very signifi cant eff ects on crop yields, i.e. pod number per plant, pod length per plant, pod weight per plant, and pods weight per plot (table 7), while the rate of k fertilizer and their interaction with k sources did not aff ect all yield variables. npk application resulted in the highest value for all yield variables (table 6). the pod number of the plants treated with npk was 33.73 per plant (table 6) whereas the pod weight was 5,640 g per plot (table 7), which was two times higher than pods from plants treated with kcl and zk. the use of npk increased the pod weight very signifi cantly by 504 g per plant, which was almost fi ve times more than the pods yield from the plants treated with other fertilizers. table 3. the monthly agro-climate data * month average temperature (oc) rh (%) rainfall (mm) rainfall criteria** light intensity (cal cm-2) january 25.7 81 189.2 humid month 311.5 february 25.4 86 358.9 wet month 399.5 march 26.0 83 122.7 humid month 597.4 april 26.3 85 432.1 wet month 504.0 may 26.6 82 284.1 wet month 519.0 note: * bmkg-climatology station darmaga bogor; ** rainfall is based on oldeman climate classifi cation table 4. the average stem diameter at the various k sources and k rates fertilizer treatment stem diameter (mm) at weeks (wap) 2 4 6 8 10 source of kx kcl 1.60 b 3.20 b 4.90 b 7.30 b 10.10 ab zk 1.70 ab 3.40 a 5.40 a 7.70 ab 9.40 b npk 1.90 a 3.70 a 5.80 a 8.60 a 10.90 a f-test * ** ** * * rate of k on the source of k (kg k2o.ha -1) 0 1.70 3.40 5.30 8.10 10.50 37.5 2.00 3.50 5.50 8.20 10.30 75.0 1.70 3.40 5.40 7.80 10.00 112.5 1.60 3.40 5.10 7.30 9.70 150.0 1.50 3.40 5.50 8.30 10.20 responsey ns ns ns ns ns k sources x rates ns ns ns ns ns xthe values followed by the same letter in the same column is not signifi cant diff erent at p<0.05 of dmrt; yresponse regression by orthogonal polynomial test; ns : not signifi cant, *: signifi cant at p<0.05, **: signifi cant at p<0.01. journal of tropical crop science vol. 6 no. 3, october 2019 www.j-tropical-crops.com 186 versi putra jaya hulu, heni purnamawati, anas dinurrohman susila, slamet susanto table 5. the number of days to 50% anthesis and stomatal density at the various k sources and k rates fertilizer treatment the number of days to 50% stomatal density anthesis (dap) (per mm2) source of kx kcl 36.75 378.43 zk 39.20 372.99 npk 39.00 347.86 f-test ns ns rate of k (kg k2o.ha -1) 0 37.63 336.31 37.5 40.44 377.07 75.0 37.33 344.80 112.5 39.00 397.45 150.0 37.63 376.50 responsey ns ns k sources x rates ns ns xthe values followed by the same letter in the same column are not signifi cantly diff erent at p<0.05 of dmrt; yresponse regression by orthogonal polynomial test; ns: not signifi cant, *: signifi cant at p<0.05, **: signifi cant at p<0.01. table 6. eff ect of diff erent k sources and k rates on yield components of beans treatment pod numberper plant pod length per plant (cm) pod weight per plant (g) source of kx kcl 13.16 b 12.18 b 123.02 b zk 16.20 b 12.25 b 185.60 b npk 33.73 a 13.44 a 504.00 a f-test ** ** ** rate of k (kg k2o.ha -1) 0 21.40 12.95 350.80 37.5 26.33 12.54 232.50 75.0 20.78 12.89 272.10 112.5 16.04 12.36 190.60 150.0 21.40 12.36 380.00 responsey ns ns ns k sources x rates ns ns ns xthe values ber followed by the same letter in the same column is not signifi cantly diff erent at p<0.05 of dmrt; yresponse regression by orthogonal polynomial test; ns : not signifi cant, *: signifi cant at p<0.05, **: signifi cant at p<0.01. journal of tropical crop science vol. 6 no. 3, october 2019 www.j-tropical-crops.com 187sources and rates of potassium for drip irrigation of polyethylene-mulched bean .......... table 7. the pods weight per plot (7.5 m2) at diff erent sources and rates of k treatment pods weight per plot (g) source of kx kcl 2,525 b zk 2,411 b npk 5,640 a f-test ** rate of k (kg k2o.ha -1) 0 4,248 37.5 3,234 75.0 3,568 112.5 2,874 150.0 4,339 responsey ns k sources x rates ns xthe values followed by the same letter in the same column is not signifi cant diff erent at p<0.05 of dmrt; yresponse regression by orthogonal polynomial test; ns : not signifi cant, *: signifi cant at p<0.05, **: signifi cant at p<0.01. figure 1. n, p, and k of the bean pods from diff erent sources of fertilizer journal of tropical crop science vol. 6 no. 3, october 2019 www.j-tropical-crops.com 188 versi putra jaya hulu, heni purnamawati, anas dinurrohman susila, slamet susanto nutrient content of pods sampling for testing nutrient contents of pods was carried out from the third harvest from plants treated with three rates of potassium, i.e. control (0 kg k2o. ha-1), medium (75 kg k2o.ha -1), and highest (150 kg k2o.ha -1). due to the limitation of the sample number, the data collection was carried out in a composite. the average value diff erence of nutrient contents in plant pods with diff erent sources of fertilizer (figure 1) showed that an increase in k rate for each source of fertilizer tend to increase bean n and k, but not p. bean n, p and k were 95%, 0.61%, and 2.99%, respectively. correlation analysis of plant growth and yield results of the correlation analysis showed that there was a relationship between plant growth variables and yields. stem diameter at 6 wap indicates the fi nal phase of vegetative growth in plants, characterized by the emergence of fl owers. stem diameter 6 wap had a positive correlation with pod number per plant, pod length per plant, pod weight per plant, and pod weight per plot (table 8). variables that was not signifi cantly correlated with other variables was the number of days to 50% anthesis. farming economic analysis in this study beans were harvested three times. the yield of this study was calculated from the average pod weight per plot in each fertilizer source treatment multiplied by 45 plots (in area of 400 m2). kcl at 37.5 kg k2o.ha -1 resulted in pod weight of 3.92 kg per plot, whereas npk at 150 kg k2o. ha -1 resulted in yield of 6.99 kg per plot (table 7). the results of the analysis showed that the highest r/c ratio was obtained from npk, which was 1.69. the value of r/c ratio is the value of the comparison of revenues and costs incurred, and an agricultural business is considered feasible if the r/c ratio > 1. discussion the diff erent sources of k fertilizer have similar levels of k2o. the total n and p2o5 from kcl and zk treatment was 104 g n and 54 g p2o5 per plot, respectively, which are diff erent from those from npk treatment. however, there was no signifi cant diff erences in the total n and p2o5 of the pods with each source of fertilizer. this could be caused by the high content of n and p2o5 in the soil. table 8. the result of pearson correlation test for growth and yield variables variables sd 6 wap nda pn pl pw nda 0.11 pn 0.41** 0.13 pl 0.38* 0.23 0.61* pw 0.56** 0.10 0.83** 0.65** pw per plot (7.5 m2) 0.53** 0.15 0.70** 0.65** 0.72** note: signifi cant by correlation pearson test; **: signifi cant at p<0.01; *: signifi cant at p<0.05 sd: stem diameter; nda: number of days to 50% anthesis; pn: pod number per plant; pl: pod length per plant; pw: pod weight per plant. tabel 9. farming economic analysis of bean production with drip irrigation source fertilizer pods weight per plot (kg)* price per kg yield cost benefi t r/c ratio (idr) kcl 3.92 12,000 2,116,800 2,190,997 (-) 74,197 0.97 zk 3.48 12,000 1,877,040 2,219,677 (-) 342,627 0.85 npk 6.99 12,000 3,769,800 2,232,717 (+) 1,537,083 1.69 note: *obtained from the pod weight valueper plot multiplied by the number of plots (45 plots or 400 m2). the price of beans is assumed to be normal in the market, which is idr 12,000 kg-1. investment in drip irrigation of idr 328,567 is the result of the calculation of idr 4,928,500 divided by 15. the drip irrigation is assumed to last for 5 years of use, or 15 growing seasons. journal of tropical crop science vol. 6 no. 3, october 2019 www.j-tropical-crops.com 189sources and rates of potassium for drip irrigation of polyethylene-mulched bean .......... the high level of acidity of the soil in this research area might be the cause of the non-signifi cant growth response in beans fertilized with kcl or zk fertilizers. legumes are very sensitive to soil acidity. zk and kcl fertilizers contain 17% s and 2.5% cl. mikkelsen and bruulsema (2005) reported that kcl and zk fertilizers tends to reduce soil ph. similarly, starast et al. (2003) stated that fertilization using fertilizers containing sulfur and ammonium can reduce soil ph. hydrolyzed sulfur and ammonium produce h+ ions which caused soil ph to decrease. npk application resulted in better vegetative growth and yields compared to kcl and zk. research by hamdani et al. (2019) regarding the sources and rates of k fertilizer applied through drip irrigation on chili also showed that npk was a better choice compared to kcl and zk. futhermore, the same research also showed that npk was the best source of nitrogen compared to urea and za (deli et al., 2019). the use of npk resulted in the largest stem diameter. balanced nutrient content of n, p2o5 and k2o played a role in growth and increase in plant stem diameter. gardner et al. (1991) stated that suffi cient availability of macronutrients n, p, k, and water aff ected the growth of stem. common bean that reached the age of 6 wap have begun to fl ower, and at this stage the growth rate of the stem slowed down. this was due to photosynthate allocation to the generative organs,resulting in limited photosynthate distribution for leaves, stems, and roots. in generative stage the role of k elements was greater for fruit formation process (du et al., 2006, amisnaipa et al., 2009). hamdani et al. (2019) showed that in chilli higher rates of k reduced the number of days to 50% anthesis. increasing the rates of k fertilizer can aff ect the acceleration of the growth rate which in turn aff ects the acceleration of the plant to enter the generative phase or fl owering time. the sources and rates of k fertilizer, which did not aff ect number of days 50% anthesis in this study, was possibly due to the high soil k content. karapanos et al. (2008) stated that time to fl ower is determined by many factors, including enviroment, cultivation management, and pests and disease infestation. the sources and rates of k fertilizer did not infl uence leaf stomatal density, which was possibly related to high nutrient content of k in the soil. one of the function of stomata is to reduce water loss due to evaporation. state of the stomata which almost all opened at the time of observation showed that the availability of water and k+ ions for plants were suffi cient (not experincing excessive evaporation). k plays a role in regulating stomata for co2 fi xation in photosynthesis. marschner (2012) states that the accumulation of k in guard cells increases cell osmotic pressure so that it can be absorb water from the surrouding cells. this causes turgor in the guard cell. k absorption can increase the pressure of the guard cell turgor so that it causes opening of the stomata which co2 fi xation during photosyntesis. the research of bhuvaneswari et al. (2013) showed that k fertilization can increase the durability of chili plants against water shortages and diseases and improve the quality of crop yields. the stomatal density in this study which ranged from 336.31 to 397.45 mm-2 was not much diff erent from stomatal density in the lower surface of the leaves of cowpea were 301.81-387.18 mm-2 (purnamawati et al., 2018). futhermore, liana (2019) also obtained stomatal density in cowpea ranging from 257.32 to 341.40 mm-2. in this study the pod harvests was carried out three times. crops treated with npk had the highest yield for all yield components. the result of research by yusdian and mulyadi (2017) showed that application of npk 16 with a rate of 450 kg.ha-1 in common bean produced the highest bean pods weight of 264.90 g per plant. npk application in this study resulted in the pods weight reaching 504 g per plant, or 7.52 tons. ha-1.the productivity of beans in this study is far below the national bean productivity which is 11.75 tons ha-1 (direktorat jenderal hortikultura, 2018). at the time of this research the amount of rainfall during the early fl owering period during april was relatively high (432.1 mm), which might be the cause of the low yield in this study. high rainfall during fl owering caused the fl owers to fall, thus reduced pod formation. high rainfall also caused high humidity, a condition that is favourable for growth of fusarium oxysporum that causes fusarium wilt. fusarium wilt infestation had been recorded since 3 wap, and the percentage of infection reached 75% by the end of the experiment. the mycelium of fusarium is in the form of white threads; it is soil-borne and infected the roots. this fungus attack was later demonstrated by yellowing of plants, wilting, and dwarfi ng (srivastava et al., 2018). according to souza et al. (2012) some bean crops can survive the attack, but will likely produce small number of fruits. it is important to check the soil condition prior to bean planting. in addition, the use of pgpr (plant growth promoting rhizobacter) biofertilizer could be tested as it was proven eff ective for inhibiting fusarium growth, particularly in beans (stanzin et al., 2018). this research also tested the nutrient contents of plant pods, which were collected at the third pod yield. the bean pods had 4.95% n, 0.61% p, and 2.99% k. islam et al. (2016) reported that n pods in the beans without fertilizer was 3.06%, whereas beshir et al. journal of tropical crop science vol. 6 no. 3, october 2019 www.j-tropical-crops.com 190 versi putra jaya hulu, heni purnamawati, anas dinurrohman susila, slamet susanto (2016) reportedthat the bean pods contained 0.48% p and 3.57% k. the increase in fertilizer rates from the three diff erent sources of k fertilizers increased n and k in bean pods. similar results were reported by michalojc and buczkowska (2009) in eggplant. potassium in plant tissues doen not bind in a compound, but are free as k+ ions (marschner, 2012). therefore, if the absorption of k elements was excessive, there can be a condition of luxurious consumption in the plant tissue. jones et al. (1991) stated that red onion plants absorb more amounts of k than needed and the excess k can cause plants to be lacking on mg and ca. correlation test results between plant growth and components of crop beans showed that the variable stem diameter growth of 6 wap was the most signifi cant and positively correlated with crop yield variables. the high level of correlation between variables also showed that the increase in stem diameter was always followed by increase in the pod number, pod length, and pod weight. based on the results of this research fertilizer rates did not signifi cantly aff ect the growth variables, whereas fertilizer sources had signifi cant eff ects. this can be caused by the high content of k nutrients in the soil so that the eff ects of k fertilizer rates was not signifi cant. general rate of fertilizer recommendations cannot be used as a rigid guideline as soil condition vary with locations. determination of the recommended rate of fertilizer should be based on the site-specifi c soil analysis results, therefore it is important to conduct soil analysis, the soil nutrient conditions, the availability of nutrients, and soil ph prior to planting. source of fertilizer that had signifi cant eff ect was likely related to the form and solubility of the type of fertilizer used and nutrient content of each fertilizer. in a fertigation system, before channeled through an irrigation line, fertilizer must be fi rst dissolved. the disadvantage of using kcl fertilizer was that pink crystals require longer time to dissolve and must be fi ltered, otherwise they leave insoluble deposits. zk fertilizer in the form of white powder also requires a considerable amount of time in the dissolution process even though it does not leave deposits, while npk in the form of granules are easily dissolved even though they still need fi ltering. research by subhan et al. (2009) reported that fertilizing with npk resulted in the best tomato growth, n, p, and k uptake, fresh and dry weight, and fruit yield. npk fertilizer has macro-secondary nutrients mgo and cao which are not present in the other fertilizer sources. mgo and cao can correct the acidity of the soil to suit the ph needed by plants by neutralizing organic acids and can increase the eff ectiveness and effi ciency of absorption of nutrients that already exist in the soil both from organic matter and inorganic fertilizer (gardner et al., 1991). in addition, according to hardjowigeno (2007) mg and ca are very important for the formation of chlorophyll which plays a role in the process of photosyntesis, formation of cell walls, and plant cell division. the use of fertigation through drip irrigation in polyethylene-mulched crop provides convenience in the cultivation of beans. fertigation can increase the effi ciency of nutrient use because fertilizer is applied in a small but continuous amount. fertigation through drip irrigation can potentially reduce the use of fertilizer by 25%-50% (kumar et al., 2016). fertilization problems that often occur, i.e. loss of nutrients due to leaching, can be minimized by applying mulch. the use of conventional irrigation in mulched crop could reduce the water received by the crops, and watering through drip irrigation could eliminate this problem. drip irrigation can also prevent excessive watering and nutrient leaching. based on the research of shedeed et al. (2009) the use of fertigation through drip irrigation caused the absorption of n and k nutrients by tomato plants more effi ciently compared to the fertigation method through furrow irrigation. tomato yields were 28% higher in the use of drip irrigation methods compared to furrow irrigation. the use of polyethylene mulch also reduce costs for weeding. the area around the main crop is free of weeds which can reduce compete for light and nutrient with the main crops. berke et al. (2005) stated that the use of polyethylene mulch signifi cantly suppressed weed growth in the chili planting area, therefore reducing labour costs for weeding. the disadvantages in drip irrigation is in initial capital costs for setting up the system, but once set up the system can last for 5 years. farming economic analysis carried out in this study can be used as a reference comparison between the production costs incurred and the production results obtained.the results of the farming economic analysis in this study demonstrated that the use of npk the most feasible option and gave the best yield with the r/c ratio > 1. conclusion application of potassium nutrient from npk fertilizer by fertigation in a drip irrigation system gave signifi cantly better plant growth and yield compared to kcl and zk fertilizer. the diff erent rates of k fertilizer in this study did not signifi canlty aff ect the growth and yield of beans. journal of tropical crop science vol. 6 no. 3, october 2019 www.j-tropical-crops.com 191sources and rates of potassium for drip irrigation of polyethylene-mulched bean .......... references amisnaipa, susila, a.d., situmorang, r., purnomo, d.w. (2009). penentuan kebutuhan pupuk kalium untuk budidaya tomat menggunakan irigasi tetes dan mulsa polyethylene. indonesian journal of agronomy 37, 115-122. berke, t., black, l.l., talekar,ns , wang, j.f., gniff ke, p., green, s.k., wang, t.c., morris, r. (2005). “suggested cultural practices for chili pepper”. avrdc publication 5, 620. beshir, h.m., bueckert, r., tar’an, b. (2016). eff ect of temporary drought at diff erent growth stages on snap bean pod quality and yield. african crop science journal 24, 317-330. bhuvaneswari, g, sivaranjani, r, reeth, s, ramakrishnan, k. (2013). application of nitrogen and potassium effi ciency on the growth and yield of chilli (capsicum annum l.). journal of current microbiology and aplied sciences 2, 329-337. bosland, p.w., votava, e.j. (2012). “peppers: vegetable and spice capsicums”. 2nd ed. 230 pp. cabi pub. deli, s.z.a., susila, a.d., purwono, suketi, k. nitrogen sources and rates for drip irrigated polyethylene mulched chilli pepper. journal of tropical crop science 6, 112-120. direktorat jenderal hortikultura. (2018). “produktivitas sayuran di indonesia”. kementerian pertanian republik indonesia” p 137. http://epublikasi. setjen. pertanian.go.id/ download/fi le/438statistik-pertanian-2018 [june 1, 2019]. du, z, zhou, j., wang, h., du, c., chen, x. (2006). potassium movement and transformation in an acid soil as eff ected by phosphorus. soil science society of american journal 70, 20572064 eviati, and sulaeman. (2009). “petunjuk teknis analisis kimia tanah, tanaman, air, dan pupuk”. 2nd ed. 234 pp. balai penelitian tanah, bogor. gardner, f.p., pearce, r.b., mitchell, r.l. (1991). “fisiologi tanaman budidaya” (s. herawati, trans.). 428 pp. ui press. hamdani, k.k., susila, a.d., purwono, suketi, k. (2019). potassium sources and rates for drip irrigated polyethylene mulched chilli pepper. journal of tropical crop science 6, 89-97. hardjowigeno, s. (2007). “ilmu tanah”. 6th ed. akademika press, jakarta. [ipgri] international plant genetic resources institute. (2003). “descriptors for green bean (phaseolus vulgaris l.)”. rome, italy. islam, m.a., boyce, a.n., rahman, md m., azirun, m.s., ashraf, m.a. (2016). eff ects of organic fertilizers on the growth and yield of bush bean, winged bean, and yard long bean. brazilian archives of biology and technology 59, 2-9. jones, j.b., wolf, b., mills, h.a. (1991). “plant analysis handbook: a practical sampling, preparation, analysis, and interpretation guide”. 213 pp. micro-macro publishing, inc. usa. kay, d.e. (1979). “food legumes”. london (gb): tropical products institute. 435p. liana, d. (2019). “determination of the optimum rate of n fertilizers with the addition of goat manure for the production of cowpea (vigna unguiculata [l.] walp.). thesis. institut pertanian bogor. marschner, p. (2012). “mineral nutrient on higher plants”. 651 pp. academic press inc. mikkelsen, r.l., bruulsema, t.w. (2005). fertilizer use for horticultural crops in the u.s. during the twentieth century. hort technology 15, 24-30. pt pupuk kujang. (2017). “mengenal unsur kalium bagi tanaman”. http://www.pupuk-kujang. co.id/publikasi/petaniku/197-mengenal-unsurkalium-bagi-tanaman [june 9, 2019]. purnamawati, h., kartika, j.g., zandroto, f.v., iska, f.r. (2018). evaluation of source and sink capacity of fi ve cowpea varieties (vigna unguiculata[l.] walp) grown in to diff erent altitudes. in “proceeding of the 2nd international conference on tropical agriculture” pp 171-178. 4 desember 2014. switzerland: springer cam. pusdatin. pusat data dan sistem informasi pertanian. (2017). “statistik konsumsi pangan tahun 2017”. http://epublikasi.setjen.pertanian. go.id/download/file/388-statistik-konsumsijournal of tropical crop science vol. 6 no. 3, october 2019 www.j-tropical-crops.com 192 versi putra jaya hulu, heni purnamawati, anas dinurrohman susila, slamet susanto pangan-2017 [september 5, 2019]. puslitbanghorti. pusat penelitian dan pengembangan hortikultura. (2016). “budidaya tanaman buncis”.http://hortikultura.litbang.pertanian. go.id/teknologi-detail-46.html [october 4, 2017]. shedeed, s.i., zaghloul, s.m., yassen, a.a. (2009). eff ect of method and rate of fertilizer application under drip irrigation on yield and nutrient uptake by tomato. ozean journal of applied sciences 2, 139-147. sousa, g.g.de, azefedo, b.m.de, oliviera, j.r.r.de, mesquita, t.de o., viana, t.v.de a., gomes do ó, l.m. (2013). potassium fertilization applied by fertigation and conventionally in peanut crop. brazilian journal of agriculture and environmental engineering 17, 1055–1060. souza, e.d.t., silveira, p.m., filho, a.c.c., junior, m.l. (2012). fusarium wilt incidence and common bean yield according to the preceding crop and the soil tillage system. pesquisa agropecuária brasileira 47, 1031-1037. srivastava, s., kadooka, c., uchida, j.y. (2018). fusarium species as phatogen on orchids. microbiological research 207, 188-195. subhan, nurtika, n., gunadi, n. (2009). respon tanaman tomat terhadap penggunaan pupuk majemuk npk 15-15-15 pada tanaman latosol pada musim kemarau. jurnal hortikultura 19, 40-48. stanzin, d., shah, t.a., bhat, n.a., sofi , p.a., baba, z.a., catoo, m.a. (2018). evaluation of pgpr isolates for the management of beans wilt caused by fusarium oxysporum f. sp. phaseoli. international journal of current microbiology and applied science 7, 355-365. starast, m., karp, k., moor, u., vool, e., paal, t. (2003). eff ect of fertilization on soil ph and growth lowbush blueberry (vaccinium angustifolium ait.). “14th international symposium of fertilizers, fertilizers in context with resource management in agriculture?”. june 22-25, 2003. debrecen, hungary. proceedings of the conference. debrecen, hungary. virisya, i.r. (2014). “uji daya hasil 12 genotipe buncis (phaseolus vulgaris l.) di tajur bogor”. thesis. institut pertanian bogor. yusdian, y., mulyadi, m. (2017). growth response and results of bean (phaseolus vulgari l.) cultivar lebat-3 because of anorganic fertilizer dose and plant distance. paspalum jurnal ilmiah pertanian 5, 7-14. journal of tropical crop science vol. 8 no. 2, june 2021 www.j-tropical-crops.com 110 manamno workayehu, wassu mohammed and tesfaye abebe estimation of heterosis among potato (solanum tuberosum l.) crosses in ethiopia manamno workayehu*a, wassu mohammedb and tesfaye abebec a adet agricultural research center, department of potato breeding, p.o. box 8, bahir dar, ethiopia. b haramaya university, school of plant sciences, p. o. box 138, dire dawa, ethiopia. c holetta agriculture research center, eiar *corresponding author; email: wmanamno@gmail.com abstract determination of heterosis in tetraploid potato crosses is necessary for identification of superior genotypes for variety development or breeding program. however, producing heterotic potato genotypes through local crossing is not a common practice in ethiopia. hence, this study was conducted to estimate the magnitude of better, and standard check heteroses in potato clones that are produced from local crossing. this will help identify potential of hybrid for future breeding schemes. heterosis of 75 clones were evaluated using a 9 x 9 simple lattice design. results of the analysis of variance showed significant differences for all the growth characters, except medium-sized tubers and specific gravity of tuber. these results indicate the presence of genetic variability among the samples. the clones exhibited as high as 55.62 and 58.31 t.ha-1 marketable and total tuber yield, respectively. these clones also displayed mid, better parent and standard heterosis of 118.8%, 90.5% and 239.1%, respectively, for marketable tuber yield. this result indicates the presence of high magnitude heterosis which could be used to exploit the hybrid vigor. in addition, mid, better parent, and standard check heterosis were estimated at 79.36%, 61.04% and 209.17% for total tuber yield, respectively. our results show the highest chance of getting heterotic offspring than parental and standard check varieties to developing new potato varieties. the information generated from this study would be valuable for researchers who intend to develop highyielding varieties of potato. keywords: clone, heterosis, tuber yield introduction the cultivated potato (solanum tuberosum l.) is a highly heterozygous autotetraploid crop (2n = 4x = 48), with a genome size of 844 mb (muthoni et al., 2012). the complexities of tetrasomic inheritance in cultivated potato arise due to diploid gametes in which diallelic interactions can be transmitted to the next generation, sister chromatids can occur because of double reduction and two alleles be identical by descent (baptiste, 2014). understanding the complexities of tetrasomic inheritance in the potato and their implications in potato breeding will go a long way in enhancing efficiency in a conventional breeding program (bradshaw, 2006). heterosis is a phenomenon whereby there is increased vigor, size, fruitfulness and speed of development, resistance to disease and insect pests in plants. it is also the opposite of inbreeding depression (shull,1952). autotetraploid potato can be severe by inbreeding depression when self-crossing occurs (park et al., 2009). the parents involved in the successful crosses, which are moderately distant parents, can dispose the hybrid to higher heterosis (manosh et al. 2008). hence, heterosis breeding based on the identification of the parents and their cross combinations can produce the highest level of transgressive segregates (falconer, 1960). heterosis can be quantified in terms of the midparent, the high or better-parent and standard check heterosis. exploitation of heterosis in potato can allow the identification of better progenies that will produce high yield and are resistant to abiotic and biotic stress. this happens by reducing inbreeding within mutually heterotic gene pools. heterosis is better for the yield and yield stability in potato and is a key feature in the success of producing hybrid cultivars. it has also been used as an important phenomenon in cultivar improvement because it is a major yield factor in plant breeding and can change in agriculture by improving agronomic traits of crops to meet the world’s food needs (duvick, 1999). in ethiopia generating heterotic clones through local crossing is little effort due to too much dependence estimation of heterosis among potato (solanum tuberosum l.) crosses in ethiopia .......... journal of tropical crop science vol. 8 no. 2, june 2021 www.j-tropical-crops.com 111 on materials from international potato center (cip) (getachew et al., 2016). despite this, heterosis for yield and its component traits have not extensively studied in potato genotypes that are created by local crossing. therefore, producing heterotic potato hybrids can help to supply food for burgeoning populations and improve environmental health of food production system in a country. hence, investigating the magnitude of heterosis that exists among clones generated through hybridization is vital to develop better potato varieties and to guide the choice of desirable parents for developing superior hybrids or future breeding programs (faizaan et al., 2016). this study was conducted to estimate the magnitude of heterosis for different traits in potato clones that are obtained from local crossing. materials and methods study site the experiment was conducted at adet agricultural research station, during the main growing season in 2018. adet agricultural research center is located at longitudes ranging from 37° 28’ 38’’ to 37° 29’ 50’’ e and latitudes ranging from 11° 16’ 19’’ to 11° 17’ 28’’ n in northern highlands of ethiopia, with an average altitude of 2240 meters above sea level (andualem et al., 2013). the mean annual total rainfall during the growing season was 1432 mm with the average minimum and maximum temperatures of 10.81 to 25.55. sampling a total of 81 genotypes of potato were used in the study (figure 1, 2, 3). seventy-five of these genotypes were progenies produced from biparental crossing of ethiopian potato varieties by adet agricultural research center in 2015. in addition, five high yielding parent varieties, belete, “ater ababa” , gera, “shenkola” , “jalene” and “dagim” , were used as the standard check varieties due to their high yielding and improved traits for comparison in this experiment (table 1). the experiment was laid out as a 9 x 9 simple lattice table 1. clones and parents of potato used in the experiment during rainy season in 2018. trt clone trt clone trt clone trt clones 1 j x a.277 22 j x a.42 43 j x a.27 64 b x a.248 2 b x a.153 23 b x a.15 44 ge xsh.186 65 j x a.18 3 j xa.296 24 j x a.49 45 j x a.130 66 j x a.123 4 b x a.174 25 b x a.60 46 b x a.163 67 b x a.207 5 j x a.94 26 j x a.77 47 j x a.67 68 j x a.186 6 b x a.225 27 “gera” (f) 48 “shenkola” (m) 69 b x a.129 7 ge x sh.65 28 j x a.31 49 ge x sh.206 70 j x a.122 8 “belete” (f) 29 ge x sh.101 50 j x a.146 71 j x a.243 9 j x a.140 30 j x a.333 51 b x a.8 72 ge x sh.90 10 b x a.74 31 b x a.228 52 j x a.102 73 ge x sh.317 11 j x a.170 32 j xa.266 53 b x a.213 74 j x a.196 12 b x a.112 33 j x a.143 54 j x a.245 75 j x a.250 13 j x a.21 34 j x a.326 55 j x a.345 76 j x a.119 14 b x a.184 35 “dagim” (s) 56 b x a.201 77 j x a.246 15 b x a.164 36 j x a.188 57 “ater ababa” (m) 78 j x a.165 16 j x a.120 37 j xa.60 58 j x a.135 79 “jalene” (f) 17 j x a.187 38 b x j.16 59 b x a.603 80 b x a.97 18 b x a.44 39 j x a.34 60 j x a.201 81 ge x sh.96 19 j x a.39 40 ge x sh.319 61 b x a.55 20 b x a.198 41 b x a.140 62 j x a.9 21 ge x sh.29 42 j x a.23 63 ge x sh.100 note: trt = treatment number; j x a=“jalene” x “ater ababa”; b x a= “belete” x “ater ababa”; ge x sh = “gera” x “shenkola”; (f) = female parent; (m) = male parent; (s) = standard check variety; numbers followed crosses indicated the codes of clone. journal of tropical crop science vol. 8 no. 2, june 2021 www.j-tropical-crops.com 112 manamno workayehu, wassu mohammed and tesfaye abebe design. each clone was planted in plot size of 4.5 m2 (net) which contained two rows in plot with twenty plants per plot. medium-sized and well-sprouted potato tubers were planted at the spacing of 75 cm between rows and 30 cm between plants. the recommended dose of fertilizer was applied at a rate of 81/69 n/p2o5 per hectare. the whole phosphorus fertilizer was applied during planting, but n source (urea) was applied at planting, 2 weeks after emergence and at flowering at an equal 1/3 rates. earthing up was executed two times throughout the entire growing period, one at 30 days and another one at 60 days after planting. fungicide (ridomil®, a.i. mancozeb and metalaxyl-m) was applied once when symptom occurred on experiment to control potato late blight disease. data collection morphological, phenological, and agronomical data were collected during the growth period of the crop, following huaman et al. (1977). the data of days to emergence(de), days to flowering (df), days to maturity (dma), main stem number (sn), plant height (ph), tuber number per plant (tnp), tuber yield per plant (typ), very small tuber numbers (vsn), medium sized tubers (mdn), large sized tubers (ltn), tuber dry matter content (dm), tuber starch content (sc), tuber specific gravity (sg), average tuber weight (aw), marketable tuber number (mtn), marketable tuber yield (my), unmarketable yield (umy) and total tuber yield (ty) were collected from sixteen plants per plots. data analysis the quantitative data was subjected to analysis of variance (anova) using sas statistical software 9.0 (sas, 2000). means for significant treatments were compared by fisher’s least significant differences (lsd) at 5% (p<0.05). estimation of heterosis heterosis were computed using the excel microsoft program 2019. quantitative traits of potato progenies were used to estimate mid-parent, better parent heterosis and standard heterosis according to the procedures suggested by fehr (1987), bitzer and fu (1972) and by falconer and mackay (1996) respectively. the value of the clones was computed for trait using the following formula:   mid-parent heterosis (mph) (%) = x100 where mp = mid parent value for the crosses f= mean value cross (clones) heterobeltiosis or better parent heterosis (%) = ( ) x100 where bp = mean of better parent (desirable one) of the respective cross. economic heterosis or standard check heterosis (%) = ( x100 where sc = mean value of standard check variety f = is the mean performance of hybrids over replications. mp (t) = , bp (t) = and sc (t) = where f1 = mean of the hybrid for a specific trait bp = mean of better parent in the cross r = number of replications ems = error mean square. the significance of heterosis was carried out by adopting student ‘t’ test according to wynne et al. (1970). heterobeltiosis was tested by ‘t’ test according to sarawgi and shrivastava (1988). the t values obtained were tested against the tabular t-value at error degree of freedom. results and discussion our results demonstrated that the analysis of variance due to clones and its components (parents and standard check) were highly significant for all the traits except medium sized tubers (%) and tuber specific gravity (g.cm3). the significant differences among clones obtained from crossing indicates that there is a chance of obtaining clones that are better performing than their parents and standard check variety for different traits. the clones are expected to be highly heterozygous in which additive and nonadditive gene actions and in most case, both operate (ross, 1986; arndt, 1990). estimation of heterosis the result of heterosis for 75 clones are presented in table 3. 4 and 5. many of the clones showed positive and negative significance of heteroses for phenology, quality, yield and yield-related traits in the current study. heterosis (%) over mid-parent both positive as well as negative heterosis of 75 potato offspring was observed (table 3). mid parent (average) heterosis was significant by some clones for phenological, yield and yield component traits suggesting the presence of directional dominance for the expression of these traits. a total of 5 clones out of 75 showed desirable negative significant heterosis estimation of heterosis among potato (solanum tuberosum l.) crosses in ethiopia .......... journal of tropical crop science vol. 8 no. 2, june 2021 www.j-tropical-crops.com 113 table 2. family mean-values of evaluated clones for quantitative traits clones value dma ph tn/p typ avw mky tty dm sc j x a (41) min 88 33.1 7 0.23 16.61 6.58 10.8 14.13 8.59 max 101 66.0 25 0.97 71.38 43.8 44.1 27.68 20.67 mean 95 49.2 14 0.59 42.27 23.3 27.0 21.18 14.88 “jalene” (f) mean 93 50.2 15 0.60 40.96 23.0 27.4 18.15 12.18 “ater ababa” (m) mean 91 48.1 13 0.49 39.32 17.1 22.4 22.18 15.76 b x a (24) min 88 32.3 6 0.27 26.45 8.73 11.7 15.63 9.93 max 102 73.3 23 1.03 78.10 55.6 58.3 29.00 21.84 mean 95 46.6 12 0.62 53.55 26.9 30.3 21.73 15.36 belete mean 99 52.9 8 0.88 106.80 41.0 42.6 24.3 17.66 ge x sh (10) min 90 41.3 8 0.30 35.11 2.51 16.8 13.78 8.28 max 99 71.6 17 1.03 103.30 33.9 45.7 25.48 18.70 mean 94 53.9 12 0.63 56.49 21.7 29.2 21.57 15.22 gera(f) mean 99 65.8 13 0.89 72.87 37.1 39.3 20.98 14.69 “shenkola” (m) mean 101 66.6 10 0.82 82.11 36.0 37.6 23.75 17.17 “dagim” (c) mean 92 57.4 10 0.43 48.94 16.4 18.9 21.3 14.99 g-mean 95 49.3 13 0.60 48.74 24.5 28.0 21.47 15.14 note: j x a = “jalene” cross with “ater ababa” ; b x a = “belete” cross with “ater ababa”; ge x sh= “gera” cross with “shenkola” ; “dagim” (c) = standard check variety; min= minimum, max = maximum; g mean = grand mean, dma = days to 90% maturity; ph (cm) = plant height, tn/p = tuber number per plant; typ (g) = tuber yield per plant; avw (g) = average tuber weight; mky = marketable yield (t.ha-1); tty = total yield (t.ha-1); dm = tuber dry matter content (%); sc = tuber starch content (%) figure 1. mature and harvested potato berries from local crossing figure 2. potato seedlings produced from local crossing figure 3. potato tubers produced from crossings of white skin female and red skin male potato parents   figure 1. mature and harvested potato berries from local crossing figure 1. mature and harvested potato berries from local crossing figure 2. potato seedlings produced from local crossing figure 3. potato tubers produced from crossings of white skin female and red skin male potato parents   figure 2. potato seedlings produced from local crossing journal of tropical crop science vol. 8 no. 2, june 2021 www.j-tropical-crops.com 114 manamno workayehu, wassu mohammed and tesfaye abebe for days to maturity and ranged from -10 to -9.2% by j x a.266 and ge x sh.100 clones, respectively. in the case of plant height (cm), it is evident that only 10 offspring showed desirable positive significant heterosis ranging from -37.6 to 45.2% for clones ge x sh.186 and b x a.164, respectively. with respect to heterosis for plant height, positive heterosis of hybrids is considered desirable (biswas et al., 2005). potato clones that manifested positively significant heterosis had tallest plant height which can produce heterotic tuber yield due to over dominance or epistatic gene interaction that involve dominance and additive gene effect (shawn, 2012). the heterosis over mid parent for tuber number per plant and average tuber weight ranged between -50 to109% and -64.4 to 77.5%. from a total of 75 clones, 15 exhibited positively significant heterosis for tuber number per plant and eight (8) clones observed positively significance for average tuber weight. most populations showed positively significant heterosis for tuber number per plant than heterosis of average tuber weight due to negative correlation between these two traits (maris,1989). instead of marketable and total tuber yield, mid parent heterosis ranged from -93.1 to 118.8% and -64.1 to 79.4%, respectively. this result is higher than biswas (2010) who found mid parent heterosis to range from -63.5 to 61.7% for tuber yield. among 75 clones only 18% of genetic materials showed above 50% mid parent heterosis by tuber yield in this study, and a total of 19 clones were expressed positively significant mid parent heterosis for this trait. the high correlation between heterozygosity and yield on tetraploid potato due to overdominance was explained by mendoza and haynes (1974). the maximum and minimum mid parent heterosis for dry matter and starch content ranged between -38.4 to 37.2 and -48.0 to 47.9%, respectively. only four clones recorded positively significant mid parent heterosis but none of them showed negative significance by these two traits. most of the clones had not shown any significance for tuber dry matter and starch content that could be attributed to internal cancellation of positive and negative effects and the dominance not being of unidirectional in nature (mather, 1982). heterosis (%) over better-parent the analysis of better parent heterosis in 75 clones are presented in table 4. the better heterosis for days to maturity and plant height ranged from -11.6 (b x a.97) to 8.2% and -39 to 38.7% (b x a.164) accordingly. negatively significant better parent heterosis were observed for seven clones for days to maturity indicating earliness of clones than the best parent. in case of plant height only three clones manifested positively significant better parent heterosis but 24 were exhibited negatively significant heterosis. the better parent heterosis for tuber number per plant and average tuber weight ranged from -56.7 to 73.1% (b x a.44) and -75.7 to 73.2% (j x a.102), respectively. among 75 clones only six (6) were expressing positively significant heterosis for tuber number per plant whereas 16 clones exhibited negatively significant heterosis. the highest and lowest heterosis for marketable and total tuber yield ranged from -93.2 to 90.5% (j x a.119) and -72.6 to 61.0% (j x a.119), respectively, but the minimum and maximum positively significant better heterosis for marketable tuber yield ranged between 35.7 to 90.5% by 10 clones due to over-dominance or intra-allelic interaction at one or multiple loci that can be the base of heterosis (shawn, 2012). this indicated the presence of higher variability for tuber yield in the progeny than parental varieties. this could be because offspring were represented by a segregating population from the cross between highly heterozygous parents. on the other hand, heterosis for dry matter and starch content ranged between -42 to 24.8% and -51.8 to 31.1% accordingly. only two clones namely j x figure 1. mature and harvested potato berries from local crossing figure 2. potato seedlings produced from local crossing figure 3. potato tubers produced from crossings of white skin female and red skin male potato parents   figure 2. potato seedlings produced from local crossing estimation of heterosis among potato (solanum tuberosum l.) crosses in ethiopia .......... journal of tropical crop science vol. 8 no. 2, june 2021 www.j-tropical-crops.com 115 table 3. mid parent heterosis in 75 potato clones clone dma ph tn/p avw mky tty dm sc j x a.277 -1.1 2.8 35.7** 0.0 53.5** 35.3* 18.4 23.7 j x a.296 7.1* 9.8 14.3 22.5 61.6** 40.6** 35.9** 46.2** j x a.94 2.7 -31** -50** 0.0 -45.7* -49** 11.5 14.8 j x a.140 0.0 1.1 -14.3 -12.5 -30.9 -28.4* 0.1 0.0 j x a.170 8.2* 6.4 78.6** -5.0 75.3** 72.1** 24.2* 31.2* j x a.21 6.0 21.7* 50.0** -5.0 50.4** 42.7** 14.9 19.2 j x a.120 3.8 12.5 57.0** -22.5 5.5 16.6 13.2 17.0 j x a.187 1.6 34.3** 7.1 25.0 51.7** 34.6* -12.4 -15.9 j x a.39 1.1 9.8 -21.4 47.5** 28.9 16.5 1.0 1.4 j x a.42 0.5 3.4 14.0 5.0 40.9* 29.1* 15.8 20.3 j x a.49 0.5 15.3 14.3 35.0* 76.4** 53.3** 13.8 17.8 j x a.77 3.3 22.5** 28.6* 12.5 78.9** 47.8** 16.5 21.3 j x a.31 -2.7 -5.1 28.6* -5.0 38.1* 32.8* 21.3* 27.3* j x a.333 7.6* -31.6** 0.0 -58** -67.1** -56.5** -30 -38.5 j x a.266 9.2** 24.6** -21.4 67.5** 57.1** 37.2** 37.2** 47.9** j x a.143 8.7** -4.1 -7.1 -32.5 -53.5** -38.5** -18.6 -23.8 j x a.326 -2.7 18.8* 14.0 2.5 50.7** 32.5* 20.3 26.1 j x a.188 8.7 -23.9* -36** 12.5 -6.7 -21.7 -23.3 -29.9 j x a.60 1.1 -32.7** -28.6* -27.5 -44.2* -45.5** 6.5 8.4 j x a.34 -0.5 4.7 7.0 22.5 37.4* 27.4* 14.0 17.9 j x a.23 -4.9 -8.3 -36** 40.0* -13.2 -13.8 -16.1 -20.7 j x a.27 4.4 -10.6 -21.4 10.0 1.8 -5.0 -11.11 -14.3 j x a.130 -4.9 -22.8** 0.0 0.0 14.8 -1.1 -0.8 -1.0 j x a.67 -0.5 1.6 21.43 -25.0 -14.7 -3.5 19.4 25.0 j x a.146 2.7 -12.0 -50** 2.5 -52.2** -51.4** 0.2 0.2 j x a.102 3.8 21.1* -7.1 77.5** 92.4** 66.7** -15.3 -20.0 j x a.245 1.1 14.2 0.0 72.5** 104.5** 72.2** 14.1 18.1 j x a.345 2.7 7.6 -7.1 0.0 -8.9 -8.7 17.8 22.9 j x a.135 0.5 0.4 -21.4 -22.5 -46.5* -39.8** -17.6 -22.6 j x a.201 -1.1 18.3* 50.0** 7.5 63.1** 64.0** 19.0 24.5 j x a.9 7.6* -9.6 29.0* -10 16.0 16.3 6.3 8.1 j x a.18 1.1 2.4 36.0** -32.5 11.9 -6.8 16.2 20.8 j x a.123 -0.5 -10.7 7.0 -12.5 -7.9 -10.6 11.3 14.6 j x a.186 6.0 -6.8 -28.6* -7.5 -19.4 -30.8* -11.6 -14.9 j x a.122 8.7** -20.3* -21.4 12.5 -9.3 -13.5 -1.6 -2.0 j x a.243 1.6 -17.0 14.0 -12.5 9.3 6.5 11.8 15.3 j x a.196 -2.7 -14.0 14.0 -48** -51.6** -40** -19.5 -25.1 j x a.250 1.6 -11.8 -21.4 2.5 -14.6 -19.4 -7.3 -9.3 j x a.119 1.1 23.1** 7.0 55.0** 118.8** 77.2** 18.0 23.2 j x a.246 2.2 -0.7 -21.4 25.0 11.3 0.3 -18.2 -23.4 j x a.165 5.4 -4.8 -21.4 -5.0 -6.3 -8.4 13.8 17.8 journal of tropical crop science vol. 8 no. 2, june 2021 www.j-tropical-crops.com 116 manamno workayehu, wassu mohammed and tesfaye abebe table 3. mid parent heterosis in 75 potato clones (continued) clone dma ph tn/p avw mky tty dm sc b x a.153 5.8 -22.0* 27.3 -51** -7.3 2.5 11.3 19.5 b x a.174 3.7 -11.9 -27.3 -38.4* -40.6** -39.8* -24.2 -26.2 b x a.225 0.5 -7.9 0.0 -39.7* -46.9** -30** -16.3 -16 b x a.74 -3.7 -2.3 27.3 -34** -5.1 -1.6 -15.1 -14.5 b x a.112 -3.2 -28** -9.1 -43** -45.9** -42** -2.6 1.7 b x a.184 6.8** -18.4 -9.1 -33** -29.0* -29.7* -14.6 -13.8 b x a.164 5.3 45.2** 45.5** -10.96 91.7** 79.4** 5.8 12.5 b x a.44 5.8 4.2 109** -43** 25.8* 26.9* 3.3 9.3 b x a.198 -3.2 7.0 9.1 -5.48 39.6** 32.8* -13.3 -12.1 b x a.15 0.5 -4.3 27.3 -34** 17.8 21.8* -9.1 -6.7 b x a.60 -6.3 12.3 -45.5* 6.9 -23.6 -28.6* -16.9 -16.7 b x a.228 -6.3 6.0 0.0 -6.9 12.6 8.9 -14.2 -13.3 b x j.16 3.7 -19.5* 0.0 -1.4 24.8 17.1 -3.5 0.5 b x a.140 -5.3 -36.1 -45.5* 4.1 -34.1** -37** -35.4 -40.6 b x a.163 4.7 10.4 54.6** -37.0* -0.5 12.3 -18.5 -18.9 b x a.8 0.0 -31** -9.1 -62** -61.9** -57** -27.1 -29.9 b x a.213 -4.2 -12.8 -9.1 4.1 9.8 3.3 -7.0 -4.0 b x a.201 2.1 -21.1* 9.1 -39.7* -29.4* -27.9* -17.7 -17.8 b x a.603 -3.7 -15.7 9.1 -5.5 14.7 7.6 -5.1 -1.5 b x a.55 1.6 25.2** 18.2 -8.2 20.9 19.5 11.3 19.5 b x a.248 7.4** -36** -9.1 -64** -69.9** -64** -33.6 -38.3 b x a.207 -3.2 -20.6* -18.2 -8.2 -11.9 -18 -7.0 -3.9 b x a.129 -2.1 -25.9* 45.4** -57.5* -44.5** -34** -19.2 -19.7 b x a.97 -7.9 -3.8 0.0 -28.8 -19.4 -24.6* 19.9 30.7* ge x sh.65 -1.0 -27.6* -25.0 -29.5 -43.9** -41.6* 3.0 3.7 ge x sh.29 -3.0 -23.6* -25.0 -7.7 -93.1** -28.7* -2.9 -3.6 ge x sh.101 -8.5* -32.0** 0.0 -47** -49.4** -44.7* -6.9 -8.6 ge x sh.319 -9.5* 8.15 33.3* -32.1 -7.4 -6.6 -0.2 -0.2 ge x sh.186 -3.5 -37.6* -33.3* -53** -55.6** -56** -38.4 -48.0 ge x sh.206 -2.5 -6.5 -8.33 32.1* -12.1 18.9* 5.0 6.2 ge x sh.100 -10.0** -19.8 42.0* -46** -28.9** -21.4 13.9 17.4 ge x sh.90 -9.0** -20.7 8.0 -39.7* -23.6* -17.9 10.0 12.1 ge x sh.317 -9.0** -24.1* 0.0 -55** -61.5** -42** -1.2 -1.5 ge x sh.96 -4.0 -10.4 25.0 -32.1 -12.0 -9.1 -0.4 -0.5 “belete” 99.0 52.9 8.0 106.8 40.98 42.63 24.2 17.7 “atera ababa” 91.0 50.2 13.0 39.4 17.05 22.4 22.2 15.8 “jalene” 93.0 50.2 15.0 41.0 23.0 27.4 18.2 12.2 “gera” 99.0 65.8 13.0 72.9 37.1 39.3 21.0 14.7 “shenkola” 101.0 66.6 10.0 82.1 36.0 37.6 23.8 17.2 maximum 9.2 45.2 109.0 77.5 118.8 79.4 37.2 47.9 minimum -10.0 -37.6 -50.0 -64.4 -93.1 -64.1 -38.4 -48.0 note: values with *and ** are significantly different at at p< 0.05 and p<0.01 probability levels, respectively. dma = days to maturity; ph = plant height; tn/p = tuber number per plant; avw= average tuber weight; mky = marketable tuber yield; tty = total tuber yield; dm = tuber dry matter; sc = tuber starch content. estimation of heterosis among potato (solanum tuberosum l.) crosses in ethiopia .......... journal of tropical crop science vol. 8 no. 2, june 2021 www.j-tropical-crops.com 117 table 4. better parent heterosis in 75 potato clones clone dma ph tn/p avw mky tty dm sc j x a.277 -2.2 0.7 27.0 -2.4 33.7 22.9 7.6 9.6 j x a.296 5.9 7.6 7.0 19.5 40.7** 27.7 23.5* 29.6* j x a.94 1.6 -33** -53* -2.4 -52.7* -53.4* 1.3 1.7 j x a.170 7.0* 4.3 67.0** -7.3 52.6** 56.3** 12.9 16.3 j x a.21 4.8 19.2 40.0** -7.3 31.0 29.7* 4.5 5.7 j x a.120 2.7 10.2 43.0** -24.4 -8.1 5.9 2.9 3.7 j x a.187 0.5 31.6* 0.0 22 32.1 22.3 -20.3 -25.4 j x a.39 0.0 7.6 -30.0* 43.9** 12.2 5.8 -8.1 -10.2 j x a.42 -0.5 1.3 3.0 2.4 22.7 17.3 5.3 6.6 j x a.49 -0.5 13.0 7.0 31.7 53.6** 39.3** 3.5 4.4 j x a.77 2.2 20.0 20.0 9.8 55.8** 34.3* 6.0 7.5 j x a.31 -3.8 -7.0 20.0 -7.3 20.2 20.7 10.2 12.9 j x a.333 6.5 -33** -7.0 -59** -71** -61** -36.3 -45.5 j x a.266 8.1* 22.1* -27.0 63.4** 36.9* 24.7 24.8* 31.1* j x a.143 7.5* -6.0 -17.0 -34.2 -60** -44** -26.0 -32.5 j x a.326 -3.8 16.5 3.0 0.0 31.3 20.4 9.3 11.7 j x a.188 7.5* -25** -40* 9.8 -18.7 -28.9 -30.2 -37.9 j x a.60 0.0 -34** -37* -29.3 -51.4* -51** -3.2 -3.9 j x a.34 -1.6 2.6 -3.0 19.5 19.6 15.8 3.6 4.5 j x a.23 -5.9 -10.2 -43.0* 36.6 -24.4 -21.7 -23.7 -29.7 j x a.27 3.2 -12.4 -27.0 7.3 -11.4 -13.7 -19.2 -24 j x a.130 -5.9 -24.4* -7.0 -2.4 0.0 -10.1 -9.8 -12.3 j x a.67 -1.6 -0.4 13.0 -26.8 -25.7 -12.3 8.5 10.8 j x a.146 1.6 -13.7 -57.0* 0.0 -58** -56** -8.9 -11.2 j x a.102 2.7 18.7 -13.0 73.2** 67.6** 51.5** -23.0 -28.8 j x a.245 0.0 11.9 -7.0 68.3** 78.1** 56.5** 3.7 4.7 j x a.345 1.6 5.4 -17.0 -2.4 -20.7 -17.0 7.1 8.9 j x a.135 -0.5 -1.6 -30.0* -24.4 -53** -45** -25.1 -31.4 j x a.201 -2.2 16.0 4.00** 4.9 42.0* 49.0** 8.2 10.3 j x a.9 6.5 -11.4 17.0 -12.2 1.0 5.7 -3.4 -4.2 j x a.18 0.0 0.3 23.0 -34.2 -2.6 -15.3 5.6 7.1 j x a.123 -1.6 -12.5 -3.0 -14.6 -19.8 -18.8 1.2 1.6 j x a.186 4.8 -8.6 -33.0* -9.8 -29.8 -37.1* -19.6 -24.6 j x a.122 7.5* -21.9* -27.0 9.8 -21.0 -21.4 -10.5 -13.1 j x a.243 0.5 -18.7 3.0 -14.6 -4.8 -3.2 1.7 2.2 j x a.196 -3.8 -15.7 3.0 -48.8* -58** -46** -26.9 -33.6 j x a.250 0.5 -13.5 -27.0 0.0 -25.7 -26.8 -15.7 -19.6 j x a.119 0.0 20.6 -3.0 51.2* 90.5** 61.0** 7.3 9.2 j x a.246 1.1 -2.7 -30.0* 22.0 -3.1 -8.9 -25.6 -32.1 j x a.165 4.3 -6.7 -27.0 -7.3 -18.4 -16.8 3.5 4.4 b x a.153 1.5 -25.5* 8.0 -66** -34** -21.8 10.7 13.1 b x a.174 -0.5 -15.9 -38.0* -58** -58** -54** -24.6 -30.2 journal of tropical crop science vol. 8 no. 2, june 2021 www.j-tropical-crops.com 118 manamno workayehu, wassu mohammed and tesfaye abebe table 4. better parent heterosis in 75 potato clones (continued) clone dma ph tn/p avw mky tty dm sc b x a.225 -3.5 -12.0 -15.0 -59** -62** -47** -16.7 -20.5 b x a.74 -7.6 -6.7 8.0 -55** -33** -25.0 -15.5 -19.1 b x a.112 -7.1 -32** -27.0 -61** -62** -56** -3.1 -3.8 b x a.184 2.5 -22.0* -23.0 -54** -50** -46** -15 -18.5 b x a.164 1.0 38.7* 23.0 -39** 35.7** 36.8** 5.3 6.4 b x a.44 1.5 -0.5 73** -61** -10.9 -3.2 2.8 3.4 b x a.198 -7.1 2.2 -8.0 -36** -1.2 1.3 -13.7 -16.8 b x a.15 -3.5 -8.6 8.0 -55** -16.6* -7.1 -9.6 -11.8 b x a.60 -10.0** 7.3 -54.0** -27.1 -45.9* -45.5** -17.3 -21.2 b x a.228 -10.0** 1.3 -15.0 -36.5** -20.3* -16.9 -14.6 -17.9 b x j.16 -0.5 -23.1* -15.0 -32.7** -11.6 -10.7 -4.0 -5.0 b x a.140 -9.1* -39** -54.0** -29.0** -53.4** -52.0** -35.7 -43.8 b x a.163 0.5 5.5 31.0* -57.0** -29.6** -14.3 -18.9 -23.2 b x a.8 -4.0 -34** -27.0 -73.8** -73.0** -67.5** -27.5 -33.7 b x a.213 -8.1 -16.7 -23.0 -29.0** -22.3* -21.3 -7.4 -9.1 b x a.201 -2.0 -24.7* -8.0 -58.9** -50.0* -45.0** -18.1 -22.2 b x a.603 -7.6 -19.5* -8.0 -35.5** -18.8 -18.0 -5.6 -6.8 b x a.55 -2.5 19.6 0.0 -37.4** -14.4 -8.9 10.7 13.1 b x a.248 3.0 -39.0** -23.0 -75.7** -78.7** -72.6** -34.0 -41.7 b x a.207 -7.1 -24.1* -31.0* -37.4** -37.6** -37.4* -7.4 -9.1 b x a.129 -6.1 -29.2* 19.0 -71.0** -60.7** -49.7** -19.6 -24.0 b x a.97 -12.0** -8.2 -15.0 -51.4** -42.9** -42.5** 19.3 23.7 ge x sh.65 -2.0 -28.0** -31.0* -32.9** -44.7** -42.9** -3.1 -3.8 ge x sh.29 -4.0 -24.0** -35.0* -12.2 -93.2** -30.3* -8.5 -10.5 ge x sh.101 -9.0** -32.0** -12.0 -50.0** -50.2** -45.9** -12.3 -15.2 ge x sh.319 -10.0** 7.5 19.0 -35.4** -8.7 -8.6 -6.0 -7.4 ge x sh.186 -4.5 -38.0** -38.0* -54.9** -56.3** -57.3** -42.0 -51.8 ge x sh.206 -3.5 -7.0 -19 25.6 -13.4 16.3 -1.2 -1.5 ge x sh.100 -11.0** -20.0* 27.0 -48.8** -30.0* -23.1 7.3 8.9 ge x sh.90 -9.0** -21.0* -4.0 -42.7** -24.7* -19.7 3.3 4.0 ge x sh.317 -10.0** -25** -8.0 -57.3** -62.0** -43.7** -7.0 -8.6 ge x sh.96 -5.0 -10.9 15.0 -35.4** -13.3 -11.1 -6.2 -7.7 “belete” 99.0 52.9 8.0 106.8 41.0 42.6 24.2 17.7 “ater ababa” 91.0 50.2 13.0 39.4 17.1 22.4 22.2 15.8 “jalene” 93.0 50.2 15.0 41.0 23.0 27.4 18.2 12.2 “gera” 99.0 65.8 13.0 72.9 37.1 39.3 21.0 14.7 “shenkola” 101.0 66.6 10.0 82.1 36.0 37.6 23.8 17.2 maximum 8.1 38.7 73.1 73.2 90.5 61.0 24.8 31.1 minimum -11.6 -39.0 -56.7 -75.7 -93.2 -72.6 -42.0 -51.8 note: values with *and ** showed significant differences at p< 0.05 and p<0.01 probability levels, respectively. values in “belete”, “jalene” and “gera” = mean of female parents, whereas in “ater ababa” and “shenkola” = mean of male parents. dma= days to maturity, ph= plant height; tn/p= tuber number per plant; avw= average tuber weight; mky= marketable tuber yield; tty= total tuber yield; dm= tuber dry matter; sc= tuber starch content. estimation of heterosis among potato (solanum tuberosum l.) crosses in ethiopia .......... journal of tropical crop science vol. 8 no. 2, june 2021 www.j-tropical-crops.com 119 a.266 and j x a.296 manifested positively significant better parent heterosis for quality traits. most of the potato progenies in the three families expressed low or equal amount of tuber dry matter and starch content with parents due to the parents involved in these crosses were very closely related by this trait, or distantly related and the incompatibility of allele’s combinations can result on low heterosis manosh et al. (2008). heterosis % over standard check variety the maximum positively significant and negatively non-significant economic heterosis ranged between 10.9 to -4.9% for days to maturity (table 5). in the case of plant height, the economic heterosis was between -43.8 to 27.7%. only clone b x a.164 manifested positively significant standard check heterosis but 31 clones were negatively significant. the highest and lowest economic heterosis for tuber number per plant and average tuber weight ranged from -40 to 150% and -65.3 to 110.2%, respectively. a total of 23 clones exhibited positively significant heterosis by tuber number per plant. only 14 clones expressed positive significance for average tuber weight. in terms of marketability and total tuber yield, economic heterosis ranged between -84.7 to 239.1% and -42.6 to 209.2% respectively. a total of 35 clones exhibited positively significant heterosis for marketable and total tuber yield in tested clones. the number of clones that recorded above 50 percent of heterosis in the current study for total tuber yield were 10 (mid parent), 7 (better parent) and 35 (standard heterosis). luthra (2005) reported above 50% heterosis with significantly positive heterosis for 18 crosses out of 120 by tuber yield. analysis of economic heterosis for quality traits (dry matter and starch content) were between -35.3 to 36.2% and -44.8 to 45.7%, respectively (table 4). from a total of 75 clones, only five exhibited positively significant heterosis, but no clones showed negatively significant for these quality traits. in this study the most promising clones with high yield were found in biparental crosses of “jalene” with “ater ababa” followed “ater ababa” with “belete” (table 2). but low yielding and inferior genotypes were found in crosses of “gera” with “shenkola” varieties due to the same origin of pedigree (kp90134) for parental varieties or far linked parents (manosh et al., 2008) that attributed non-allelic interaction with the large number of decreasing alleles (mather,1982). clones such as j x a.119, j x a.170, j x a.245, j x a.102 and b x a.164 showed the highest heterosis for tuber yield among tested materials. offspring (clones) derived from crossing “belete” with “ater ababa” and “gera” with “shenkola” did not manifest positively or negatively significant heterosis on mid and better parent for quality traits. this indicates equality or inferiority of progenies than their parents for these traits or because of crossing with a homozygote parent by this trait which might have generated homozygote hybrids. these results agree with reports of kumar (2008) and manivel (2010) who found nonsignificant or low heterosis for tuber dry matter and starch content. accordingly, many authors reported the three heteroses on potato populations. parmar et al. (2015) reported the range of -28.62 to 51.11%, -35.91 to 48.44% and -40.44 to 0.66% for mid parent, better parent heterosis and standard heterosis for plant height, respectively. baptiste (2014) reported the highest mid parent heterosis of 36.05 and highest best parent heterosis of 34.44 for tuber yield among one family progenies. baptiste (2014) also reported mid parent heterosis ranging from -42.03 to 160.08% and better parent heterosis of -47.04 to125.00% among 41 clonal progenies by tuber yield. biswas (2010) noted mid parent heterosis (-63.5 to 61.7%), better parent heterosis (-68.6 to 21.4%) and standard heterosis (-72.4 to 13.9%) for tuber yield from 30 clones. luthra (2006) reported 120 hybrids and 29 parents of potato based on progeny mean and heterosis, with only 4 offsprings exhibiting significant positive heterosis for tuber yield. conclusion heterosis by cross pollination between tetraploid potato varieties would help to develop better hybrids with high yield potential acceptable to the consumers. in this study, most clones that showed positively significant heterosis (mid, better parent and standard check) for tuber number per plant, average tuber weight and tuber yield were found in biparental crosses of “jalene” with “ater ababa”, followed by “ater ababa” with “belete”. low yielding clones were found in progenies of “gera” and “shenkola” due to same origin of pedigree. these findings would help researchers find the critical areas for the development of new potato varieties that some of the investigators were not able to explore. a new theory could be handy for many researchers to develop better hybrids through conventional crossing. finally, the study results indicate that the highest chance of getting heterotic potato offspring were generated from local crossing relatively with high yield than parent and standard check varieties. besides, further investigation can be done to exploit hybrid vigor for effective improvement in yield potential of the traits of the best crosses. journal of tropical crop science vol. 8 no. 2, june 2021 www.j-tropical-crops.com 120 manamno workayehu, wassu mohammed and tesfaye abebe table 5. standard check heterosis for different traits in potato clones. clone dma ph tn/p avw mky tty dm sc j x a.277 -1.1 -12.0 90** -18.4 87.4** 78.6** 12.1 15.2 j x a.296 7.1* -6.0 60** 0.0 97.3** 85.6** 28.6* 36.2* j x a.94 2.7 -41.0** -30 -18.4 -33.8 -32.3 5.5 6.9 j x a.140 0.0 -13.5 15 -28.6 -15.6 -5.5 -5.3 -6.8 j x a.170 8.2* -8.9 150.0** -22.5 113.9** 127.1** 17.6 22.3 j x a.21 6.0 4.2 110.0** -22.5 83.6** 88.4** 8.8 11.1 j x a.120 3.8 -3.7 115.0** -36.7* 28.8 53.9* 7.2 9.0 j x a.187 1.6 15.0 50.0* 2.0 85.2** 77.7** -17.0 -21.6 j x a.39 1.1 -6.0 5.0 20.4 57.3* 53.8* -4.3 -5.5 j x a.42 0.5 -11.5 55.0* -14.3 72.0** 70.5** 9.6 12.1 j x a.49 0.5 -1.3 60.0** 10.2 115.3** 102.4** 7.8 9.7 j x a.77 3.3 4.9 80.0** -8.2 118.4** 95.1** 10.3 13.0 j x a.31 -2.7 -18.7* 80.0** -22.5 68.5** 75.3** 14.8 18.7 j x a.333 7.6* -41.0** 40.0 -65** -59.9* -42.6** -33.7 -42.7 j x a.266 9** 6.7 10 36.7* 91.7** 81.1** 29.9** 37.9* j x a.143 8.7* -17.9 25 -44.9* -43.2 -18.8 -22.9 -29.0 j x a.326 -2.7 1.7 55.0* -16.3 84.0** 74.9** 13.9 17.5 j x a.188 8.7* -34.8* -10.0 -8.2 13.9 3.3 -27.4 -34.7 j x a.60 1.1 -42.0** -5.0 -40.8* -31.9 -28.0 0.8 1.0 j x a.34 -0.5 -10.4 45.0 0.0 67.7** 68.2** 7.9 9.9 j x a.23 -4.9 -21.5* -15 14.3 6.0 13.8 -20.5 -26.1 j x a.27 4.4 -24.0** 10.0 -10.2 24.3 25.4 -15.9 -20.1 j x a.130 -4.9 -34.0** 40.0 -18.4 40.2 30.6 -6.1 -7.8 j x a.67 -0.5 -13.0 70.0** -38.8* 4.2 27.4 13.0 16.4 j x a.146 2.7 -25.0** -35.0 -16.3 -41.6 -35.8 -5.2 -6.6 j x a.102 3.8 3.7 30.0 44.9* 134.9** 120.0** -19.8 -25.2 j x a.245 1.1 -2.2 40.0 40.8* 149.7** 127.4** 8.0 10.0 j x a.345 2.7 -7.9 25.0 -18.4 11.2 20.6 11.5 14.5 j x a.135 0.5 -14.1 5.0 -36.7* -34.6 -20.6 -22.0 -27.9 j x a.201 -1.1 1.3 110.0** -12.2 99.1** 116.5** 12.7 16.0 j x a.9 7.6* -23.0** 75.0 -26.5 41.6 53.5* 0.6 0.7 j x a.18 1.1 -12.4 85.0** -44.9* 36.6 23.1 10.0 12.6 j x a.123 -0.5 -24.0** 45.0 -28.6 12.4 18.0 5.4 6.8 j x a.186 6.0 -20.2 0.0 -24.5 -1.6 -8.6 -16.3 -20.7 j x a.122 8.7* -32.0** 10.0 -8.2 10.7 14.2 -6.8 -8.7 j x a.243 1.6 -29.0** 55.0* -28.6 33.4 40.6 5.9 7.4 j x a.196 -2.7 -26.0** 55.0* -57** -40.9 -21.0 -23.8 -30.2 j x a.250 1.6 -25.0** 10.0 -16.3 4.2 6.4 -12.2 -15.5 j x a.119 1.1 5.4 45.0 26.5 167.1** 134.0** 11.7 14.8 j x a.246 2.2 -15.0 5.0 2.0 35.9 32.4 -22.5 -28.6 j x a.165 5.4 -18.48 10.0 -22.5 14.3 20.9 7.8 7.8 estimation of heterosis among potato (solanum tuberosum l.) crosses in ethiopia .......... journal of tropical crop science vol. 8 no. 2, june 2021 www.j-tropical-crops.com 121 table 5. standard check heterosis for different traits in potato clones (continued) clone dma ph tn/p avw mky tty dm sc b x a.153 9.0** -31.0** 40.0 -26.5 64.0* 76.7* 26.3* 26.3* b x a.174 7.1* -22.5* -20.0 -8.2 5.2 3.8 -14.0 -14.0 b x a.225 3.8 -19.0 10.0 -10.2 -6.0 20.4 -4.9 -4.9 b x a.74 -0.5 -14.1 40.0 -2.0 67.9* 69.6** -3.6 -3.6 b x a.112 0.0 -37.0** -5.0 -14.3 -4.2 0.4 10.6 10.6 b x a.184 10.0** -28.0** 0.0 0.0 25.7 21.2 -3.1 -3.1 b x a.164 8.7* 27.7** 60** 32.7* 239.1** 209.2** 20.1 20.1 b x a.44 9.0** -8.4 125.0** -14.3 122.5** 118.8* 17.3 17.3 b x a.198 0.0 -5.9 20.0 40.8* 146.9** 129.0** -1.5 -1.5 b x a.15 3.8 -15.8 40.0 -2.0 108.5** 110.0** 3.2 3.2 b x a.60 -3.3 -1.2 -40.0 59.2** 35.2 23.1 -5.6 -5.6 b x a.228 -3.3 -6.8 10.0 39.0 99.2** 87.8** -2.6 -2.6 b x j.16 7.1* -29.0** 10.0 46.9* 120.8** 101.9** 9.5 9.5 b x a.140 -2.2 -44.0** -40.0 55.1** 16.5 8.5 -26.6 -26.6 b x a.163 8.2* -2.86 70.0** -6.1 76.0* 93.6** -7.5 -7.5 b x a.8 3.3 -39.0** -5.0 -42.9* -32.6 -26.6 -17.3 -17.3 b x a.213 -1.1 -23.0** 0.0 55.1** 94.2** 78.0** 5.6 5.6 b x a.201 5.4 -30.6* 20.0 -10.2 24.9 24.3 -6.6 -6.6 b x a.603 -0.5 -26.0** 20.0 40.8* 102.8** 85.4** 7.8 7.8 b x a.55 4.9 10.12 30.0 36.7* 113.8** 105.9** 26.29* 26.3* b x a.248 11.0** -44.0** 0.0 -46.9* -46.8 -38.0 -24.7 -24.7 b x a.207 0.0 -30.0** -10.0 36.7* 56.0* 41.4 5.6 5.6 b x a.129 1.1 -35.0** 55.0* -36.7* -1.8 13.7 -8.2 -8.2 b x a.97 -4.9 -15.4 10.0 6.1 42.7 30.0 36.0** 36.0** ge x sh.65 7.6* -16.4 -10.0 12.2 25.0 19.1 8.1 8.1 ge x sh.29 5.4 -11.8 -15.0 46.9* -84.7** 45.3 2.0 2.0 ge x sh.101 -0.5 -21.6* 15.0 -16.3 12.6 12.7 -2.2 -2.2 ge x sh.319 -1.6 24.8** 55.0* 8.2 106.4** 90.5** 4.8 4.8 ge x sh.186 4.9 -28.0** -20.0 -24.5 -1.1 -11.0 -35.3 -35.3 ge x sh.206 6.0 7.9 5.0 110.0** 95.9** 142.4** 10.2 10.2 ge x sh.100 -2.2 -7.5 65.0** -14.3 58.3* 60.3** 19.6 19.6 ge x sh.90 -0.5 -8.5 25.0 -4.1 70.3* 67.4** 15.1 15.1 ge x sh.317 -1.1 -12.4 20.0 -28.6 -14.2 17.3 3.8 3.8 ge x sh.96 4.4 3.4 50.0* 8.2 96.0** 85.3** 4.6 4.6 “dagim” (s) 92.0 57.4 10.0 48.9 16.4 18.9 21.3 15.0 maximum 10.9 27.7 150.0 110.2 239.1 209.2 36.2 45.7 minimum -4.9 -43.8 -40.0 -65.3 -84.7 -42.6 -35.3 -44.8 g-mean 0.5 -5.3 4.1 -9.7 0.7 -1.6 -0.7 0.6 note: “dagim” (s)= standard check variety; g-mean= grand mean; dma= days to maturity, ph= plant height; tn/p= tuber number per plant; avw= average tuber weight; mky= marketable tuber yield; tty= total tuber yield; dm= tuber dry matter; sc= tuber starch content. journal of tropical crop science vol. 8 no. 2, june 2021 www.j-tropical-crops.com 122 manamno workayehu, wassu mohammed and tesfaye abebe acknowledgements i am greatly indebted to adet agricultural research center for the financial support, haramaya university for providing the scholarship, adet potato team for planting and data recording. finally, i would like to express my sincere gratitude to dr wassu mohammed, senior researcher and lecturer at haramaya university and dr. tasfaye ababa, senior researcher at holetta research center for technical assistance to write up this manuscript. references arndt, g.c., and peloquin, s.j. 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(2013). heritability, variance components and genetic advance of some yield and yield related traits in ethiopian collections of finger millet (eleusine coracana (l.) gaertn) genotypes. african journal of biotechnology 12, 5529-5534. june_2014.cdr journal of tropical crop science (issn 2356-0169; e-issn 2356-0177) is published four-monthly by department of agronomy and horticulture, faculty of agriculture, bogor agricultural university, indonesia. publication details, including instructions for authors and subscription information: www.j-tropical-crops.com any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by the publisher. the accuracy of the content should be independently veri�ed with primary sources of information. the publisher shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the content. permission to make digital or hard copies of part or all of a work published in journal of tropical crop science is granted for personal or educational/classroom use provided that copies are not made or distributed for pro�t or commercial advantage. ©department of agronomy and horticulture, faculty of agriculture, bogor agricultural university, indonesia. all rights reserved. journal of tropical crop science volume number 1 20141 june on the cover the cover image shows sun�owers by darda effendi editorial board krisantini sintho wahyuning ardie sandra a. aziz robert j. hampson satriyas ilyas tri koesoemaningtyas rohana p mahaliyanaarachchi awang maharijaya maya melati roedhy poerwanto bambang sapto purwoko sudarsono muhamad syukur hugo volkaert malcolm wegener managing editor krisantini graphic design syaiful anwar features editor damayanti buchori dadang sisir mitra agus purwito ernan rustiadi short communication tropical and subtropical fruits in india sisir mitra heliconia cultivar registration dave skinner, jan hintze, bryan brunner research articles estimation of genetic parameter for quantitative characters of pepper ( l.)capsicum annuum muhamad syukur, syaidatul rosidah irrigation volume based on pan evaporation and their effects on water use ef�ciency and yield of hydroponically grown chilli eko sulistyono, abe eiko juliana evaluation of commercial sun�ower (helianthus annuus ) cultivars in bogor, indonesia, forl. ornamental and nursery production syarifah iis aisyah, khotimah, krisantini different growth partitioning and shoot production of talinum triangulare treated with organic and inorganic fertilizer sandra ari�n aziz, leo mualim, sitta azmi farchany cloning and characterization of p5cs1 and p5cs2 genes from l. under droughtsaccharum officinarum stress hayati minarsih iskandar, dwiyantari widyaningrum, sony suhandono journal of tropical crop science (issn 2356-0169; issn 2356-0177) is published four-monthly (one volume per year) byedepartment of agronomy and horticulture, faculty of agriculture, bogor agricultural university, ipb darmaga campus, bogor, indonesia 16680. send all inquiries regarding printed copies and display advertising to or to secretary, department ofinfo@j‐tropical‐crops.com agronomy and horticulture; telephone/fax 62-251-8629353. permission to reprint: permission to make digital or hard copies of part or all of a work published in isjournal of tropical crop science granted for personal or educational/classroom use provided that copies are not made or distributed for pro�t or commercial advantage and that copies bear the full citation and the following notice on the �rst page: “copyright department of agronomy and horticulture, faculty of agriculture, bogor agricultural university”. for all other kinds of copying, request permission in writing from head of school, department of agronomy and horticulture of�ce, ipb darmaga campus, bogor, indonesia 16680. © department of agronomy and horticulture, faculty of agriculture, bogor agricultural university. all rights reserved. printed in the republic of indonesia.in s t t t u i b o g o r perta n ia n 5 eko sulistyono*, abe eiko juliana department of agronomy and horticulture, faculty of agriculture, bogor agricultural university, indonesia. corresponding author; email: ekosulistyono@ipb.ac.id* irrigation volume based on pan evaporation and their effects on water use ef�ciency and yield of hydroponically grown chilli abstract this study was conducted to determine irrigation volume based on pan evaporation and their effects on growth, yield, irrigation water use ef�cency (iwue) of chilli in sandponic systemgrown in the greenhouse. the amount of water used was based on pan evaporation. irrigation treatments consisted of four coef�cient 0.5, 1, 1.5, 2 epan. plants were watereds, i.e. daily until soil reached during .�eld capacity the �rst week i applied onrrigation treatments were the second week until four months later. total irrigation quantities varied from 9.4 to 37.8 plant . chilli fruit yield varied from 3.98l. -1 to 90.51 plant . the highest total fruit yield and iwueg. -1 was obtained from 2 epan treatment. irrigation treatment had signi�cant (p<0.01) on yield and there wereeffects positive linear relations between the yield and the amount of irrigation volume signi�cantlyirrigation water applied. increased p andlant height, number of lateral branches, number of leaves (p<0.01). key words: plant height, lateral branches, fruit introduction water is a basic requirement for crop growth and production. water requirement for the plant is equal to evapotranspiration. crop evapotranspiration can be determined based on class a pan evaporation. previous studies reported positive correlation between crop water requirements and class a pan evaporation (ertek et al., 2006). therefore the evaporation pan can be used to determine the volume of irrigation (ertek et al., 2006). previous studies have demonstrated that the quantity of irrigation water in�uenced chili yield and water use ef�ciency (liu et al., 2012). reducing irrigation by 22–43% reduced crop evapotranspiration by 11–25% (liu et al., 2012). total dry mass was reduced by 1.17–38.66% in time-space de�cit irrigation treatments compared to optimum irrigation. the root to shoot ratio of the optimally irrigated plants was lower than other treatments and had the highest total fresh fruit yield (19.57 t.ha ). de�cit irrigations increased water use−1 ef�ciency (i.e. yield/evapotranspiration) of hot pepper from a minimum of 1.33% to a maximum of 54.49% ( c h e n g e t a l . , 2 0 1 0 ) . wa t e r s t r e s s r e d u c e d photosynthesis through stomatal closure and impaired mesophyll conductance (pascual et al., 2010). the maximum and minimum values of the yield and yield components were recorded from treatment full irrigation level with paired-row planting method and 0% of etc irrigation level with paired-row planting method, respectively, with the exception of plant height (gadissa and chemeda, 2009). water use ef�ciency (wue) and irrigation water use ef�ciency (iwue) values signi�cantly increased with the application of 11day-interval irrigation (p < 0.05). the highest wue and iwue values obtained from 11-dayinterval irrigation ( et al., 2009).gercek total dry mass of fruit was reduced by 34.7% in drought treatments compared to full watered treatment. at harvest, water-stressed plants had 21% lower root dry weight mass but higher root/shoot ratio other than full watered treatment (kulkarni and phalke, 2009). chili plants had almost double productivity and higher water use ef�ciency under high soil temperature and high soil moisture (yaghi et al., 2013). during severe water stress, photosynthesis decreased due to stomatal closure, and slower maximum carboxylation rate (v )cmax and ribulose 1,5-bisphosphate (rubp) regeneration capacity mediated by maximum electron transport rate (j ) (campos et al., 2013). fruit yield increasedmax gradually as soil water content levels increased from 40–55% to 70–85% �eld capacity, but decreased when soil water content increased above 70–85% �eld c a p a c i t y. t h e w a t e r c o n s u m p t i o n i n c r e a s e d progressively with the increase in soil water levels, but water-use ef�ciency was the highest when soil moisture was 55–70% �eld capacity (zhu et al., 2012). irrigation is also important for controlling plant disease. soil moisture and relative humidity in the atmosphere are critical in determining survival of mycelium and other propagules in the soil and on the plant. plant infections by p. capsici and disease development were favored by rainfall and high soil moisture, and periodic �ooding contributes to a more severe disease (sanogo and ji, 2013). chemical compounds in chili fruits are affected by genetic, harvest time and irrigation control. the capsaicin contents of different species andcapsicum lines were affected by harvest time and drying parameters. capsaicin contents were within the range 0.50–4.20%. the highest capsaicin content was obtained from the solar tunnel drying at the second harvest in the local line acı çiçek 52 (yaldiz et al., 2010).capsicum concentration of antioxidant components and tasterelated properties varied among the pepper cultivars at both the mature and whole colored ripened stages 9 journal of tropical crop science vol. 1 no. 1, 2014june www.j-tropical-crops.com 5 ( et al., 2011).ghasemnezhad irrigation volumes are determined based on pan evaporation. pan coef�cient is de�ned as the ratio of evapotranspiration references (eto) to the pan evaporation. pan coef�cients varied from 0.35-0.85 with an average of 0.7 (fao, 1986). crop evapotranspiration (et) is de�ned as kc x eto, where eto = k pan x e pan. therefore et = kc x k pan x e pan. this study aimed to obtain the value of kc x k pan or the so-called crop and pan coef�cients (k ). k value of this research can becp cp used as a basis for determining the irrigation volume of hydroponically grown chili. this study was also aimed at studying the effects of irrigation volume on chili growth and yield, and to determine the best irrigation methods to improve chili water use ef�ciency. materials and methods the experiment was conducted in greenhouse at darmaga experimental station (6° 24's, 106° 33e; elevation 240 m), bogor agricultural university, bogor, west java, indonesia. chili plants were grown hydroponically using sand media. plants were fertilized with 32n:10p: 10k o and supplied with micronutrients2 during vegetative growth, followed by 10n:55p:10 k o2 plus micronutrients during generative growth. the treatments were four irrigation volumes based on pan evaporation (eoa), i.e. 0.5, 1, 1.5, and 2 eo. pan evaporation values were obtained by measuring the decrease in water height in the evaporation pan. irrigation volume was calculated by multiplying surface area of the pot by 0.5, 1, 1.5, and 2 eo, respectively. the experiment was arranged in a randomized block design and replicated three times, with �ve plants for each experimental unit. seeds were sown in a plastic tray and were grown for six weeks before transplanting into 28-cm pots. percolation holes were made on the pot sides with a height of a third height measured from the bottom of the pot. seedlings were fertilized using foliar fertilizer at 1g.l weekly.-l hydroponics fertilizer was dissolved in irrigation at a concentration of 2g.l and applied twice a week.-1 observations included plant height, number of branches, number of leaves and fruit weight at harvest. the dry matter of root and canopy was weighed on 1, 2 and 3 months after planting, respectively. irrigation water use ef�ciency (iwue) was calculated based on the ratio of fruit weight and irrigation volume. results irrigation volume treatment of 2 eo (twice pan evaporation) resulted in the highest plant height, the greatest leaf number and branches at 8 and 12 weeks after planting (wap) (table 1). irrigation volume of 1.5 eo increased plant dry matter by 99 % compared with 0.5 eo irrigation volume at 4 weeks after planting. on 8 and 12 wap irrigation volume of 2 eo increased plant dry matter as much as 23%, 102% and 374% compared with 1.5, 1.0 and 0.5 eo, respectively (table 2). fruit yield increased gradually as irrigation volume increased from 0.5 eo to 1.0 eo, 1.5 eo and 2.0 eo. the highest fresh fruit yield was obtained from the irrigation volume of 2.0 eo (table 3). water use ef�ciency of 2.0 eo irrigation volume was signi�cantly higher compared with 0.5 eo and 1.0 eo irrigation volume, respectively (table 3). discussion irrigation volume treatment of 2 eo (twice pan evaporation) resulted in the highest plant height, the greatest leaf number and branches at 8 and 12 wap (table 1). 4 wap-old plants 0.5 eo 1.0 eo 1.5 eo 2.0 eo 8 wap-old plants 0.5.eo 1.0 eo 1.5 eo 2.0 eo 12 wap-old plants 0.5 eo 1.0 eo 1.5 eo 2.0 eo i rigar tion volume plant height (cm) leaf number branch number 47.89 57.48 61.49 62.23 63.77 96.95 108.78 117.91 80.73 118.23 123.04 135.02 c b ab a c b ab a c b ab a 22.33 27.60 35.53 32.20 29.83 95.33 178.17 250.33 79.78 257.78 366.22 556.22 b ab a ab d c b a d c b a 2.67 4.93 7.00 8.27 9.42 43.67 84.42 101.00 71.22 223.56 351.00 503.22 a a a a d c b a d c b a table 1 plant height, leaf number, and branch number in each irrigation volume. note: data at the same column followed by the same letter was not signi�cant based on lsd (0.05); wap = weeks after planting 5 eko sulistyono, abe eiko juliana10 journal of tropical crop science vol. 1 no. 1, 2014june www.j-tropical-crops.com 5 these effects were not observed earlier. water requirement at the early growth was normally lower than that in the mid and late growth as young plants have fewer numbers of leaves. gadisa and chemeda (2009) showed that plant height increased signi�cantly with increasing levels of irrigation. shongwe et al. (2010) reported that there were signi�cant (p < 0.05) increases in leaf number, plant height, chlorophyll content, canopy size, fresh and dry mass tops and fruit length at the highest moisture level (1.00 × �eld capacity), whereas at (0.80 × �eld capacity) resulted in smaller increases in each of the parameters. irrigation volume of 1.5 eo increased plant dry matter by 99 % compared with 0.5 eo irrigation volume at 4 weeks after planting. otherwise, on 8 and 12 weeks after planting (wap), irrigation volume of 2 eo increased plant dry matter as much as 23%, 102% and 374% compared with 1.5, 1.0 and 0.5 eo irrigation volume, respectively (table 2). this shows that the crop and pan coef�cient (k ) value for the initial growth phase was 1.5 butcp increased to 2.0 at the middle and late growth. the greatest dry matter allocation was in the leaves in all treatments. at four weeks after planting root and shoot ratio were similar in all treatments. in times of drought, carbon was mostly allocated for root development (ehlers and goss, 2003). kulkarni and phalke (2009) reported that water-stressed plants had 21% lower root dry weight mass but higher root to shoot ratio other than full watertreated plants. fruit yield increased gradually as irrigation volume increased from 0.5 eo to 1.0 eo, 1.5 eo and 2.0 eo. the highest fresh fruit yield was obtained from the irrigation volume of 2.0 eo (table 3). the similar results was reported by zhu et al. (2012) that fruit yield increased gradually as soil water content levels increased from 40–55% to 70–85% �eld capacity, but decreased when soil water content was higher than 70–85% �eld capacity. the water consumption increased progressively with the increase in soil water content levels, but water-use ef�ciency was the highest when soil moisture was at 55–70% �eld capacity. cheng et al. (2010) also reported that the highest total fresh fruit yield (19.57 t.ha ) was−1 obtained in the optimum irrigation treatment. irrigation water use ef�ciency of 2.0 eo irrigation volume was signi�cantly higher compared with 0.5 eo and 1.0 eo irrigation volume, respectively (table 3). .table 2 plant dry matter at each irrigation volume root (g) 0.50 0.69 0.84 1.09 1.38 1.91 2.57 2.59 1.79 3.84 5.38 6.89 b ab ab a a a a a c cb ab a stem (g) 1.25 1.67 2.35 1.95 2.46 5.23 10.27 12.69 3.11 7.87 13.24 21.65 b ab a ab b b a a d c b a leaf (g) 1.49 1.89 3.37 2.68 1.25 4.36 6.27 8.20 1.94 4.72 7.89 10.91 b ab a ab c b ab a d c b a plant (g) 3.25 4.26 6.47 5.73 5.15 12.03 19.75 24.38 7.14 17.16 30.25 41.88 b ab a ab d c b a d c b a root/shoot 0.18 0.20 0.15 0.23 0.37 0.20 0.16 0.12 0.35 0.30 0.25 0.21 ns ns ns ns a b b b a ab ab b 4 wap-old 0.5 eo 1.0 eo 1.5 eo 2.0 eo 8 wap-old 0.5.eo 1.0 eo 1.5 eo 2.0 eo 12 wap-old 0.5 eo 1.0 eo 1.5 eo 2.0 eo i rigar tion volume note: data at the same column followed by the same letter was not signi�cant (ns) based on lsd (0.05); wap = weeks after planting. fruit weight (g plant )-1 3.98 21.84 57.69 90.51 c c b a 0.5 eo 1.0 eo 1.5 eo 2.0 eo i rigar tion volume 0.42 1.15 2.03 2.39 c c ab a irrigation water use ef�ciency (g. l )-1 table 3 fruit weight and irrigation water use ef�ciency in each irrigation volume. note: data at the same column followed by the same letter was not signi�cant based on lsd (0.05). conclusion the highest total fruit yield and iwue was obtained from the 2 epan treatment. irrigation treatment had signi�cant effects (p<0.01) on yield and there were positive linear correlations the amount ofbetween the yield and 5 11i ....................rrigation volume based on pan evaporation journal of tropical crop science vol. 1 no. 1, 2014june www.j-tropical-crops.com irrigation water. height, number of lateral branches,plant number of leaves increased with increasing volumes of irrigation and these effects were signi�cant (p<0.01) since the second weeks after planting. references butcher j.d., crosby, k. m., yoo, k. s., patil, b., jifon, j. l. and rooney, w. l. 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(2013). productivity of pepper crop ( l.) as affected by organiccapsicum annuum fertilizer, soil solarization, and endomycorrhizae. annals of agricultural sciences , 131–13758 zhu j., peng, q., liang, x., wu. w. and hao, w.(2012). leaf gas exchange, chlorophyll �uorescence, and fruit yield in hot pepper ( l.)capsicum annuum grown under different shade and soil moisture during the fruit growth stage. journal of integrative agriculture , 927–93711 55 eko sulistyono, abe eiko juliana12 journal of tropical crop science vol. 1 no. 1, 2014june www.j-tropical-crops.com june_2014.cdr journal of tropical crop science (issn 2356-0169; e-issn 2356-0177) is published four-monthly by department of agronomy and horticulture, faculty of agriculture, bogor agricultural university, indonesia. publication details, including instructions for authors and subscription information: www.j-tropical-crops.com any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by the publisher. the accuracy of the content should be independently veri�ed with primary sources of information. the publisher shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the content. permission to make digital or hard copies of part or all of a work published in journal of tropical crop science is granted for personal or educational/classroom use provided that copies are not made or distributed for pro�t or commercial advantage. ©department of agronomy and horticulture, faculty of agriculture, bogor agricultural university, indonesia. all rights reserved. journal of tropical crop science volume number 1 20141 june on the cover the cover image shows sun�owers by darda effendi editorial board krisantini sintho wahyuning ardie sandra a. aziz robert j. hampson satriyas ilyas tri koesoemaningtyas rohana p mahaliyanaarachchi awang maharijaya maya melati roedhy poerwanto bambang sapto purwoko sudarsono muhamad syukur hugo volkaert malcolm wegener managing editor krisantini graphic design syaiful anwar features editor damayanti buchori dadang sisir mitra agus purwito ernan rustiadi short communication tropical and subtropical fruits in india sisir mitra heliconia cultivar registration dave skinner, jan hintze, bryan brunner research articles estimation of genetic parameter for quantitative characters of pepper ( l.)capsicum annuum muhamad syukur, syaidatul rosidah irrigation volume based on pan evaporation and their effects on water use ef�ciency and yield of hydroponically grown chilli eko sulistyono, abe eiko juliana evaluation of commercial sun�ower (helianthus annuus ) cultivars in bogor, indonesia, forl. ornamental and nursery production syarifah iis aisyah, khotimah, krisantini different growth partitioning and shoot production of talinum triangulare treated with organic and inorganic fertilizer sandra ari�n aziz, leo mualim, sitta azmi farchany cloning and characterization of p5cs1 and p5cs2 genes from l. under droughtsaccharum officinarum stress hayati minarsih iskandar, dwiyantari widyaningrum, sony suhandono journal of tropical crop science (issn 2356-0169; issn 2356-0177) is published four-monthly (one volume per year) byedepartment of agronomy and horticulture, faculty of agriculture, bogor agricultural university, ipb darmaga campus, bogor, indonesia 16680. send all inquiries regarding printed copies and display advertising to or to secretary, department ofinfo@j‐tropical‐crops.com agronomy and horticulture; telephone/fax 62-251-8629353. permission to reprint: permission to make digital or hard copies of part or all of a work published in isjournal of tropical crop science granted for personal or educational/classroom use provided that copies are not made or distributed for pro�t or commercial advantage and that copies bear the full citation and the following notice on the �rst page: “copyright department of agronomy and horticulture, faculty of agriculture, bogor agricultural university”. for all other kinds of copying, request permission in writing from head of school, department of agronomy and horticulture of�ce, ipb darmaga campus, bogor, indonesia 16680. © department of agronomy and horticulture, faculty of agriculture, bogor agricultural university. all rights reserved. printed in the republic of indonesia.in s t t t u i b o g o r perta n ia n the heliconia society international (hsi, website: www.heliconia.org) is a non-pro�t corporation formed in 1985 because of rapidly developing interest around the world in these plants and their close relatives. the purpose of hsi is to increase the enjoyment and understanding of heliconia and related plants (in the f a m i l i e s c a n n a c e a e , c o s t a c e a e , l o w i a c e a e , marantaceae, strelitziaceae, and zingiberaceae) of the order zingiberales through education, research and communication. interest in zingiberales and information on the cultivation and botany of these plants is rapidly increasing. hsi will centralize this information and distribute it to members. the heliconia society international is the of�cial international cultivar registration authority (icra) for the genera heliconia, musaceae, etlingera, costus, cheilocostus, chamaecostus, and paracostus. a cultivar name must comply with the standards as found on the website of the international society for h o r t i c u l t u r a l s c i e n c e ( i s h s ) a t http://www.ishs.org/sci/icra.htm. a cultivar name is "accepted" if it is compliant with the naming standards of the ishs. the name becomes "established" only after it has been published in hard copy with a description in a publication that meets the requirements of the ishs. the following information is required to register a new cultivar: 1. cultivar name 2. parentage of cultivar 3. origin of cultivar 4. description of cultivar with characteristics that distinguish it from other similar cultivars or species 5. photo of the in�orescence and/or other plant parts that distinguish it 6. citation of the earliest dated hard copy publication, if any, of the cultivar the heliconia society international will periodically publish accepted cultivar names of heliconias, costaceae and etlingera and will provide registration services, following the rules of the ishs. contact can be made through the hsi website (www.heliconia.org or cultivar registry@heliconia.org). references www.heliconiasocietypr.ort/cul�var_registra�on.htm www.heliconia.org/registry/costaceaechecklist.pdf dave skinner, jan hintze*, bryan brunner heliconia society international *corresponding author; email: admin@heliconia.org heliconia society cultivar registration 2 3heliconia society cultivar registration journal of tropical crop science vol. 1 no. 1, 2014june www.j-tropical-crops.com xviii heliconia society international conference 2014 25 27 august, 2014 graha cakra hotel, denpasar, bali http://www.heliconia.org/conference.html june_2014.cdr journal of tropical crop science (issn 2356-0169; e-issn 2356-0177) is published four-monthly by department of agronomy and horticulture, faculty of agriculture, bogor agricultural university, indonesia. publication details, including instructions for authors and subscription information: www.j-tropical-crops.com any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by the publisher. the accuracy of the content should be independently veri�ed with primary sources of information. the publisher shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the content. permission to make digital or hard copies of part or all of a work published in journal of tropical crop science is granted for personal or educational/classroom use provided that copies are not made or distributed for pro�t or commercial advantage. ©department of agronomy and horticulture, faculty of agriculture, bogor agricultural university, indonesia. all rights reserved. journal of tropical crop science volume number 1 20141 june on the cover the cover image shows sun�owers by darda effendi editorial board krisantini sintho wahyuning ardie sandra a. aziz robert j. hampson satriyas ilyas tri koesoemaningtyas rohana p mahaliyanaarachchi awang maharijaya maya melati roedhy poerwanto bambang sapto purwoko sudarsono muhamad syukur hugo volkaert malcolm wegener managing editor krisantini graphic design syaiful anwar features editor damayanti buchori dadang sisir mitra agus purwito ernan rustiadi short communication tropical and subtropical fruits in india sisir mitra heliconia cultivar registration dave skinner, jan hintze, bryan brunner research articles estimation of genetic parameter for quantitative characters of pepper ( l.)capsicum annuum muhamad syukur, syaidatul rosidah irrigation volume based on pan evaporation and their effects on water use ef�ciency and yield of hydroponically grown chilli eko sulistyono, abe eiko juliana evaluation of commercial sun�ower (helianthus annuus ) cultivars in bogor, indonesia, forl. ornamental and nursery production syarifah iis aisyah, khotimah, krisantini different growth partitioning and shoot production of talinum triangulare treated with organic and inorganic fertilizer sandra ari�n aziz, leo mualim, sitta azmi farchany cloning and characterization of p5cs1 and p5cs2 genes from l. under droughtsaccharum officinarum stress hayati minarsih iskandar, dwiyantari widyaningrum, sony suhandono journal of tropical crop science (issn 2356-0169; issn 2356-0177) is published four-monthly (one volume per year) byedepartment of agronomy and horticulture, faculty of agriculture, bogor agricultural university, ipb darmaga campus, bogor, indonesia 16680. send all inquiries regarding printed copies and display advertising to or to secretary, department ofinfo@j‐tropical‐crops.com agronomy and horticulture; telephone/fax 62-251-8629353. permission to reprint: permission to make digital or hard copies of part or all of a work published in isjournal of tropical crop science granted for personal or educational/classroom use provided that copies are not made or distributed for pro�t or commercial advantage and that copies bear the full citation and the following notice on the �rst page: “copyright department of agronomy and horticulture, faculty of agriculture, bogor agricultural university”. for all other kinds of copying, request permission in writing from head of school, department of agronomy and horticulture of�ce, ipb darmaga campus, bogor, indonesia 16680. © department of agronomy and horticulture, faculty of agriculture, bogor agricultural university. all rights reserved. printed in the republic of indonesia.in s t t t u i b o g o r perta n ia n introduction india is the second largest producer of fruits in the world with an annual production of 81.2 million tonnes contributing 12.6% of world fruit production next only to china (21.2%). of late, with growing population and realization of commercial and nutritional values, the demand and requirement for fruits has increased manifold globally and so is in india. since then, india has managed a steady growth in area expansion and production of fruits. even in 201213, increase in area20 and production of fruits in india was 4.1 and 6.4 times over 201112 of these, tropical and subtropical fruits20 contribute a major share, and the important fruits are mango, banana, papaya, citrus, guava, pineapple, litchi, sapota and pomegranate. among the minor fruits (less area in cultivation than the major fruits) are jackfruit, bael, aonla, carambola, syzygiums, passion fruit and tamarind are also growing in many states of india. current status globally, india is the leading producer of important tropical fruits such as banana (25.6%), mango and guava (44%), and papaya (42.6%). in terms of production in india, banana occupies the �rst with 32.6% of the total fruit production followed by mango (22.1%), citrus (12.4%) and papaya (6.6%) respectively though maximum area is registered in mango (2500 thousand ha ha) followed by citrus (1042 thousand ) banana (776 thousand ) and guava (236 thousand ) (table 1).ha ha mango mango is the most popular and the choicest fruit of india occupying about 35.8% of the total area under fruits accounting for 22.1% of the total fruit production. india has more than 1200 mango varieties. the important mango varieties grown in india are dashehari, chausa, langra, kesar, himsagar, fazli, alphonso, amrapali etc. banana banana is suitably grown in almost all the states of india and available round the year. cultivation of banana using t i s s u e c u l t u r e d p l a n t l e t s o f g r a n d n a i n e h a s revolutionised the banana industry in india. dwarf cavendish, robusta, poovan, red banana are the common varieties grown all over besides many other indigenous genotypes are also grown in all parts of the country. banana occupies only 11.1% of total cultivated area under fruits but it contributes 32.6% of total fruit production. citrus productivity of citrus is highest in karnataka (22.0 ha ),t. -1 followed by in rajasthan (18.4 ha ) in arunachalt. -1 pradesh and punjab, limes and lemon are produced round the year. pramanali, vikram, shai sabarti, phule sharbati, jai devi and seedless lime are the common lime varieties grown in india while balaji and rasraj are con�ned to only few states. common lemon varieties like eureka, kagzhi kalan and assam lemon are grown all over the country. sweet lime grown in india includes mithachikna and mithotra. in india, mosambi, valencia and blood red dominates in all the sweet orange growing areas. best quality mandarins are produced in the country. nagpur mandarin, coorg mandarin, khasi mandarin, darjeeling mandarin, kinnow mandarin etc have their own unique qualities. guava guava accounts for 3.9 % of the total fruit production from an area of 3.4 % of the total area under fruits in india. sardar, allahabad safeda, shweta, lalit, (pink �esh), allahabad surkha (deep pink �esh), khaja (bengal safeda) are the varieties growing in different states of india. papaya india is the leading producer of papaya in the world accounting for 42.6% of the total world production followed by brazil (12%) and indonesia (7.2%). pink flesh sweet, coorg honey dew, sunrise solo, taiwan, pusa delicious, pusa dwarf, pusa nanha, arka surya, arka prabhat, co1, co3, co4, and co6 are the varieties grown in different parts of the country. good papain yielding varieties like co2, co5 and co7 have also been developed and are successfully grown. pineapple kew, giant kew, queen, mau i ius are the only fewr t varieties successfully grown in different parts of the country. though the current productivity of pineapple in india is only 14.9 ha , karnataka has registered at. -1 journal of tropical crop science vol. 1 no. 1, 2014june www.j-tropical-crops.com short communication sisir mitra* chair, section tropical and subtropical fruits international society for horticultural science *corresponding author; email: sisirm@vsnl.net tropical and subtropical fruits in india 1 productivity of 62.7 hat. -1 litchi india is the second largest producer of litchi after china in the world. in india it is cultivated in about 77000 andha production is about 497000 of which the largest area andt production is from bihar. about 50 varieties of litchi are grown in india of which shahi, china, bombai, rose scented, muzaffarpur etc. are grown commercially. pomegranate ganesh, bhagwa, jyoti, g-137, ruby are the important varieties developed in india. year-round availability of pomegranate in maharashtra and tamil nadu is possible due to its suitable agro-climatic condition and improved production technologies and tamil nadu has the highest productivity of 31.3 hat. -1 table 1: area, production, productivity and major producing states of important tropical and subtropical fruits in india. area (000 )ha production t(000 ) productivity t.h( )-1 major producing statesfruit banana mango citrus papaya guava pineapple pomegranate litchi 776.0 2500.0 1042.0 132.0 236.0 105.0 113.2 77.8 26509.0 18002.0 10090.0 5382.0 3198.0 1571.0 754.0 497.3 34.20 7.20 9.70 40.70 13.60 14.90 6.60 6.4 ta m i l n a d u ( 1 9 . 4 % ) , g u j a r a t ( 1 7 . 1 % ) , maharashtra (13.6%), andhra pradesh (12.2%), karnataka (9.5%). andhra pradesh (24.5%), uttar pradesh (24.4%), karnataka (10%), bihar (7.6%). andhra pradesh (37.7%), madhya pradesh 11.8(%), punjab (10%), maharashtra (8.5%). andhra pradesh (30.7%), gujarat (22.1%), karnataka (8.6%), madhya pradesh (7.7%). madhya pradesh (25.0%), maharashtra (9.5%), uttar pradesh (9.1%), bihar (9.1%). we s t b e n g a l ( 1 9 . 7 % ) , a s s a m ( 1 7 . 1 % ) , karnataka (10.8%), tripura (10.5%). maharashtra (54.8%), karnataka (20.2%), gujarat (10.6%). bihar (45.6%), west bengal (17.1%), jharkhand (7.2%). sisir mitra2 references mitra, s.k., gurung, m.r. and pathak, p.k. (2007). guava production and improvement in india: an overview. acta horticulturae , 59-68.787 mitra, s.k. and dutta ray, s.k. 2005 . advances in the production and research of lychee in india.( ) acta horticulturae 665, 47-52. journal of tropical crop science vol. 1 no. 1, 2014june www.j-tropical-crops.com journal of tropical crop science vol. 7 no. 3, october 2020 www.j-tropical-crops.com 128 molla gebreyohannes hailu, ni made armini wiendi, and diny dinarti increasing ploidy level of garlic (allium sativum l.) “tawangmangu baru” in-vitro using colchicine abstract “tawangmangu baru” garlic variety is known to have low productivity. the variety is still highly demanded due to its strong flavour and aroma; however, its production has not yet been able to fulfill the local needs of central java due to the small size and limited production area. this study aimed to determine the effect of concentration and time duration of colchicine treatment towards increasing the ploidy level of “tawangmangu baru” garlic variety for genetic variability. the experimental design used in this study was a complete randomized design with two factorials and 12 combinations. the first factor was concentration of colchicine, i.e. 0.00, 0.02, 0.04, 0.06, 0.08 and 0.10%, and the second factor was the immersion time, i.e. 24 and 48 hours. the result indicated that, 4.72% callus induction was obtained in bds + 0.4 mg.l-1 2,4-d + 2.0 mg.l-1 kinetin; and 4.0% callus proliferation were obtained in both bds + 1.5 mg.l-1 2,4-d + 1.0 mg.l-1 kinetin and ms +1.5 mg.l-1 2,4-d and 1.0 mg.l-1 kinetin. the untreated plantlets showed higher mortality rate compared to the explants with 48 hours colchicine treatment. higher number of shoots were recorded in 0.1% colchicine at 48 hours and lower shoots in 24 hours, whereas 0.1% colchicine at 24 and 48 hours showed the highest ploidy level of total nuclear dna analyzed by flow cytometry. the genetic diversity of the “tawangmangu baru” garlic was successfully enhanced by colchicine and immersion treatment. mutant lines with tetraploid and mixoploid plants were obtained. the putative lines obtained at 0.1% colchicine treatment were subcultured to produce new mutants before testing the phenotype. the application of colchicine at 24 and 48 hours treatment improved the genetic potential of “tawangmangu baru” garlic variety in vitro. the application of colchicine increased the ploidy level and an increase in ploidy is expected to make the bulb size larger. larger tuber size will increase the tuber weight, and also the overall garlic productivity and production in the future. molla gebreyohannes hailua, ni made armini wiendi*b, and diny dinartib a plant breeding and biotechnology, ipb university, bogor 16680, indonesia b department of agronomy and horticulture, faculty of agriculture, ipb university, bogor, indonesia *corresponding author; email: nmarmini@gmail.com keywords: ploidy induction, 2,4-d, kinetin, colchicine, “tawangmangu baru” introduction the production of garlic has not been able to meet the demand for food consumption in indonesia and causing a considerable gap between consumption and domestic production (agustina, 2019). in indonesia, the production of garlic is limited by lack of farmers’ interest on garlic farming, low prices of imported garlic, bigger clove size of imported garlic, viral diseases ( putra et al., 2015), narrow land area, high production costs, low quality of garlic seeds, high garlic consumption demand (pharmawati and waitiani 2013) and less competency in producing garlic with large bulb size (nur, 2018). these constraints have led to a limited quantity of garlic in indonesia. to fulfill the national garlic demand, the indonesian government has been importing a large amount of garlic from china (pharmawati and waitiani, 2013; nur, 2018). garlic import in indonesia was 94% in 2018 and 95% in 2019 due to the low price of imported garlic and reduction of importing rules of 2005 in line with the asean free trade area (afta) process (kementerian pertanian, 2019b; agustina, 2019). ‘“tawangmangu baru”’ is a javanese local garlic cultivar commonly planted in the highlands of the tawangmangu, central java known by its strong flavor, low yield, good growth, and robust stems (arisuryanti et al., 2018). the variety is grown at 1,000 meter above sea level (m.a.s.l.). it has a purplish-white clove which is suitable for cooking due to its strong aroma. the variety is widely planted in the areas of tegal, wonosobo, banjarnegara, temanggung, magelang, magetan, and karanganyar (harinta et al. 2018). the plant height reaches 60 to 80 cm and its harvesting time is in between 110 to 140 days. the bulb of the “tawangmangu baru” garlic variety is characterized by its oval shape, hard neck, tapered tip with a flat journal of tropical crop science vol. 7 no. 3, october 2020 www.j-tropical-crops.com 129increasing ploidy level of garlic (allium sativum l.) “tawangmangu baru” in-vitro .......... base. the variety has the potential to produce about 8-12 tonnes per hectare (samadi, 2000). the current national yield of “tawangmangu baru” in indonesia is 8.7 tonnes/hectare, while the yield of garlic in china is 27 tonnes/hectare (kementrian pertanian, 2019a). increasing the production of “tawangmangu baru” can be used to fulfill the local demand of the region and other provinces in indonesia (harinta et al., 2018; kementrian pertanian, 2019b). vegetative propagation of garlic offers limited chances for creating genetic variation and gives a low multiplication rate in the field. due to the floral organ abortion and chromosomal abnormalities, pollination it is difficult to happen naturally in garlic, hence the crop is reproduced asexually, and garlic breeding mainly depends on the clonal selection and mutation breeding (su-ping et al., 2014; tchórzewska et al., 2017; cheng et al., 2012; mahajan et al., 2015). colchicine is an alkaloid and secondary metabolite extracted from the plant colchicum autumnale which is the most widely used doubling agent either in garlic or other species (alan et al., 2004; alan et al., 2007) with an average doubling efficiency of 70% (jakše and bohanec, 2003). colchicine is a chemical mutagen senso latu, as its main effect is on ploidy and not on genes (spencer-lopes et al., 2018). colchicine affects chromosome doubling on mitosis cells by binding to the microtublin positive end leading to the disruption of the polymerization of microtublin (roughani et al., 2017; zhang et al., 2016; moghbel et al., 2015). the spindle fibers consisting of microtubules used to pull the sister chromatids to opposite poles of the cell. however, without the action of spindle fibers, the mitotic process is disrupted. this disruption results in dna replication without cell division (roughani et al., 2017). wei et al. (2007) reported that the increased number of chromosomes brings the change in plant morphology and functions. according to many studies, colchicine is the most effective mutagen for producing polyploid plants, but the responses to colchicine differ depending on the plant genotype (heo et al., 2016). colchicine is effective to induce ploidy in plant genome at a concentration of 0.001% 1% in a period of 6–72 hours (amanah et al., 2016). ploidy induction of diploid garlic genome can be induced by treating garlic stem discs with up to 0.75% colchicine with higher duplication at 0.5% and lower duplication rate at 0.75% due to higher toxicity (dixit and chaudhary, 2014). polyploidy has been induced by colchicine in several crops (datta, 1990). polyploid plants retain long stomata (münzbergová, 2017), larger and thicker leaves (jaskani et al., 2005; moghbel et al., 2015; roughani et al., 2017; sajjad et al., 2013), high chloroplast number (jaskani et al., 2005), lower number of stomata compared to control (sajjad et al., 2013), higher secondary metabolites and increased sugar composition (corneillie et al., 2019; dixit and chaudhary, 2014) used in overcoming hybridization barriers, improving stress tolerance, and restoring fertility in wide hybrids (roughani et al., 2017). polyploid seeds are often larger than diploid seeds because of their larger cell size (münzbergová, 2017; tavan et al., 2015; moghbel et al., 2015). moghbel et al. (2015) described polyploidy induced by colchicine in vitro results in an increase in dna content and affects the epidermal surface cells. polyploids have thicker cell size but have low cell number per leaf blade as compared to the diploids (corneillie et al., 2019). by using this technique, through induction of ploidy to obtain polyploidy plants, it is expected that tetraploid plants which have more higher morphology and larger tubers are expected to be obtained. the main objective of this research was to improve the genetic potential of “tawangmangu baru” garlic variety by using colchicine treatment at different concentration and time duration of immersions in vitro for inducing polyploidy. the application of colchicine helps to increase the ploidy level and an increase in ploidy is expected to make the bulb size larger in “tawangmangu baru” garlic variety. larger tuber size will increase the tuber weight, and also the overall garlic productivity and production. materials and methods planting material the research increasing ploidy level of “tawang mangu baru” garlic variety by colchicine in vitro was conducted in the micro technic laboratory, department of agronomy and horticulture from may 2019 to march 2020. the incubation culture room was arranged with temperature 22-24 oc with a 24 hours photoperiod per day from fluorescent lamps with 1000 lux light intensity. bulbs of six-months-old “tawangmangu baru” garlic variety were purchased from maju karanganyar, solo farmers’ group. the explant preparation and sterilization process for planting was conducted through the procedures as described in sinha et al. (2016), khan et al. (2017) and salam et al., (2018). clumps of garlic bulbs were separated into individual clove during the start of the tissue culture process. the outermost layer of the bulbs was carefully peeled, washed with detergent three times followed by rinsing in 2.0 mg.l-1 of dithane (a.i. mancozeb) and agrimycin (a.i. oxytetracyline) for 20 hours. after the treatment by fungicide and bactericide, the surface of the cloves were washed with sterile water three times followed by sterilization with 50% of clorox for the whole bulb, 30% for the half-disc, and 20% of clorox for the explant (0.5 cm) at 30 minutes, 30 journal of tropical crop science vol. 7 no. 3, october 2020 www.j-tropical-crops.com 130 molla gebreyohannes hailu, ni made armini wiendi, and diny dinarti minutes and 20 minutes respectively. the bulbs were taken out and chopped using a sterile surgical blade after treated by 50% and 30% clorox. immediately after clorox treatment, five explants per bottle were then cultured in prepared ms0. the size of the explants was measured about 0.5 cm length with intact meristem. these segments were used as explants for increasing the induction of polyploids. explants were placed with the abaxial side on a 200 ml bottle composed of ms macroand micronutrients, 30 g.l-1 sucrose, 7 mg.l-1 agar with ph 5.8. five-hundred mg.l-1 cefotaxime was used in the ms to control the fungal contamination of the media. after ms culture, the explants were induced at 0, 0.02, 0.04, 0.06, 0.08 and 0.10% concentrations treated at 24 and 48 hours for all treatments. after 24 and 48 hours of colchicine treatment, the mv0 garlic explants were cultured in complete bds media and 0.4 mg.l-1 2,4-d for two months to produce callus. due to zero formation of callus after mv0, the mv0 explants again were sub-cultured after two months interval for callus formation and regeneration in fresh bds media supplemented with 2,4-d (0.4 mg.l-1) and kinetin (2.0 mg.l-1) and stayed for two months. the third sub-culture were performed for all explants in another fresh bds media supplemented with 2,4-d (0.4 mg.l-1), kinetin (2.0 mg. l-1), and asparagine (25 mg.l-1) to ensure the somatic embryogenesis for a month. after the 3rd sub-culture, all the explants were transferred into another fresh bds and ms media with increased 2,4-d (1.5 mg.l-1) and kinetin (1.0 mg.l-1) levels to ensure the proliferation and regeneration of callus for polyploid analysis. for this research, two trials were performed to get a large number of calluses after the “tawangmangu baru” garlic variety failed to form callus in bds + 0.4 mg.l-1 2,4-d in the first trial. the second trial also performed on bds + 0.4 mg.l-1 2,4d and 0.5 mg.l-1 kinetin to get enough callus for shoot proliferation and regeneration for ploidy analysis. the cytological analysis was conducted at the micro-technic laboratory, department of agronomy and horticulture, and flow cytometry analysis at lipi research center. the materials used in this research include basal dunstan short (bds) media, murashige and skoog 1962 (ms) media, sterile water, aqua desitillata sterile (distilled water with more quality), dithane, agrimycin, cefotaxime, rubber, micropipette with different sizes, measuring glass, 70 and 96% alcohol, volumetric flash, 15-mm petri dishes, rectangular buckets, plastics wrap, shelves, growth chamber or a windowless room with environmental control. the tools used in tissue culture include; digital ph meter, micro-organism killing autoclave, pressurized autoclave, laminar airflow cabinet, sensitive balance, dissecting instruments (scalpels with removable blades, and forceps). photos were conducted using flow cytometry (complete set of bd accuri c6 plus, partec, germany), bi-nuclear microscope, and digital camera to display the status of the cells and structures of callus. experimental design and analysis the experimental design used in this study was a factorial completely randomized design with two factors and 12 treatments. the first factor was concentration of colchicine in percentage, i.e. 0.00, 0.02, 0.04, 0.06, 0.08 and 0.10%. the second factor was the time duration (immersion time) at 24 and 48 hours. in this research, there were four replications with 180 explants in each replication. anova for each experiment and means were compared using the fisher test (p< 0.05) through minitab 2018 software. the lc 20%, lc 30%, and lc 50% of the dead plants were calculated by probit analysis in minitab software. callus induction and proliferation efficiency were performed through direct observation; while the nuclear dna status in each putative line was analyzed through flow cytometry analysis. callus induction and proliferation after the basal disc meristem grown in ms0 (ms basal medium without growth hormone) for a week, followed by colchicine treatment at 24 and 48 hours, the explants were cultured in bds media supplemented with 0.4 mg.l-1 2,4-d for two months. after two months, all the explants were transferred into bds media augmented with 0.4 mg.l-1 2,4-d and 2.0 mg.l-1 kinetin to induce callus and organogenesis in two months. after four months from colchicine treatment, all the explants were sub-cultured into bds media supplemented with 0.4 mg.l-1 2,4-d, 2.0 mg.l-1 kinetin, and 25 m. l-1 asparagine for a month. after five months of in vitro culture, all the explants were transferred to another fresh ms and bds mediums supplemented with 1.5 mg.l-1 2,4-d and 1.0 mg.l-1 kinetin. the percentage of callus induction, shoot proliferation, regeneration, and the number of shoots was calculated based on the following formula: the number of surviving and dying plants in each treatment were counted every week after colchicine treatment. data collection was taken every week to know the number of dying and surviving explants at different colchicine concentrations and time intervals. journal of tropical crop science vol. 7 no. 3, october 2020 www.j-tropical-crops.com 131increasing ploidy level of garlic (allium sativum l.) “tawangmangu baru” in-vitro .......... result and discussion callus induction and shoot proliferation with 2,4-d and kinetin treatment after the basal disc meristem grown in ms0 for a week followed by colchicine treatment at 24 and 48 hours, the explants were cultured in bds media supplemented with 0.4 mg.l-1 2,4-d alone for two months (1st in vitro culture). after two months all the explants were transferred to bds media augmented with 0.4 mg.l-1 2,4-d and 2.0 mg.l-1 kinetin supposed to produce callus and organogenesis in two months. after four months from colchicine treatment, all the explants were transferred into bds media supplemented with 0.4 mg.l-1 2,4-d, 2.0 mg.l-1 kinetin, and 25 mg.l-1 asparagine for a month to ensure somatic embryogenesis. all the explants were unable to produce callus at 0.4 mg.l-1 2,4-d alone. there was no response of 2,4-d alone to callus production and organogenesis. 4.72% explants were produced callus in 0.4 mg.l-1 2,4-d and 2.0 mg.l-1 kinetin combination within two weeks as shown in figure 1. the produced calluses were embryogenic callus with white, friable, and transparent characteristics. however, the produced calluses were failed to proliferate into shoot and regenerate under these concentrations of growth hormones. the meristematic basal discs of “tawangmangu baru” garlic variety induced callus in 0.4 mg.l-1 (2,4-d) and 2.0 mg.l-1 kinetin combination after the explants failed to induce callus in 2,4-d alone. auxins and cytokinins in combination are effective in producing callus instead of auxins alone in “tawangmangu baru” garlic. however, at low concentrations of auxins (0.4 mg.l-1) and kinetin (2.0 mg.l-1), the induced callus were unable to proliferate and regenerate. some calluses were produced somatic embryo; however, the produced somatic embryos were died due to the low concentration of 2,4-d after four months. after five months of in vitro culture, all the explants were transferred to another fresh complete ms and bds mediums supplemented with 1.5 mg.l-1 2,4-d and 1.0 mg.l-1 kinetin. after transferring the explants into these growth hormones, 4.0% of the failed callus to proliferate in 0.4 mg.l-1 (2,4-d) and 2.0 mg.l-1 kinetin, started to proliferate rapidly in bds+1.5 mg.l-1 2,4-d + 1.0 mg.l-1 kinetin and ms+1.5 mg.l-1 2,4-d + 1.0 mg.l-1 kinetin after two weeks. the callus proliferation occurred very fast in bds medium supplemented with 1.5 mg.l-1 2,4-d + 1.0 mg.l-1 kinetin compared to ms medium supplemented with 1.5 mg.l-1 2,4-d + 1.0 mg.l-1 kinetin. the development of a somatic embryo into a whole plant is likely affected by the non-optimal growth hormone or media composition during the callus growth. somatic embryogenesis the number of dead and survived explants as a result of colchicine treatment and immersion time were calculated through the method used by (sajjad et al., 2013). the brown and non-growing explants were considered as dead explants, whereas, the live explants were considered as survived plants. to evaluate the number of dying and surviving plants at different time intervals, the data analysis and comparison for dying and surviving plants as a result of colchicine treatment were taken at 2, 4, 5, and 7 months. flow cytometry analysis after the colchicine treated plants had grown for 60 days on bds medium and were induced by colchicine at 24 and 48 hours in vitro, putative polyploids of plants were investigated by flow cytometry. putative polyploid shoots were selected based on morphology, as they appeared to be thicker, darker and showed slow growth. flow cytometric analysis was carried out using a bd accuri c6 plus (partec, germany). the different flow cytometer parameters were adjusted with untreated control to secure well defined and reproducible readings. twenty-four shoots were selected from 12 treatments (0, 0.02, 0.04, 0.06, 0.08, and 0.10%) induced at 24 and 48 hours. from each treatment, 2 plants were detected by flow cytometry to know the total nuclear dna status of the induced plants. the middle dark green part of the leaf of the plants was taken for sample preparation. the plant part taken for sample preparation was chopped manually through a razor blade approximately for 2 minutes to release the intact nuclei of the plant tissue. 0.5 cm2 of leaf was taken from the middle part of the leaf to extract the nuclei of the plant tissue. the leaf sample was poured by 250 µl cystain pi absolute p (nuclei extraction buffer) to extract the intact nuclei representative of the cells within the plant tissues. after the tissues ground well, the solution was transferred to the puppet filtered by 30 µm filter size and added 300 cystain pi absolute p (staining buffer) for colouring. the staining buffer was prepared from 60 µl propidium iodide (a red fluorescent stain that preferentially intercalates the double-stranded dna with 540 nm wavelength excitation and 615 nm emission), 20 µl cystain pi absolute p, and 60 µl ribonuclease (rnase) to degrade the rna molecule from the sample tissue as the target to study in the plant tissue was the nuclear dna status of plant tissue. from the solution that contains propidium, staining buffer, and rnase, 300 µl solution was added to the extracted sample tissue for colouring of the plant tissues. immediately after adding the staining buffer, the homogenized sample tissue was detected by bd accuri c6 plus flow cytometry (partec, germany) to determine the amount of nuclear dna. journal of tropical crop science vol. 7 no. 3, october 2020 www.j-tropical-crops.com 132 molla gebreyohannes hailu, ni made armini wiendi, and diny dinarti can be affected by ethylene concentration, where low concentrations can stimulate, while high concentrations inhibit (trigiano and gray, 2004). the effect of colchicine treatment on mortality and survivability of garlic “tawangmangu baru” based on the total number of plantlets, the colchicine treated plantlets at 24 and 48 hours have no significant difference in mortality and survivability compared to the untreated plantlets as indicated in tables 2 and 3. the untreated plantlets showed a higher mortality rate compared to the treated explants at 48 hours. the explants induced at colchicine 0.08% for 24 hours also resulted in high mortality compared to the other treated explants. the high mortality rate in control plantlets and 0.08% at 24 hours was may be due to the genetic and growth medium condition figure 1. the effect of 2,4-d and kinetin on “tawangmangu baru” somatic embryogenesis; a) bds + 0.4 mg. l-1 2,4-d+ 0.06% colchicine after 3 weeks; b) bds + 0.4 mg.l-1 2,4-d + 2.0 mg.l-1 kinetin + 0.02% colchicine after two weeks; (c) bds + 1.5 mg.l-1 2,4-d + 1.0 mg.l-1 kinetin + +0.02% colchicine; (d) ms + 1.5 mg.l-1 2,4-d+1.0 mg.l-1 kinetin after two weeks regenerate. some calluses were produced somatic embryo; however, the produced somatic embryos were died due to the low concentration of 2,4-d after four months. after five months of in vitro culture, all the explants were transferred to another fresh complete ms and bds mediums supplemented with 1.5 mg.l-1 2,4-d and 1.0 mg.l-1 kinetin. after transferring the explants into these growth hormones, 4.0% of the failed callus to proliferate in 0.4 mg.l-1 (2,4-d) and 2.0 mg.l-1 kinetin, started to proliferate rapidly in bds+1.5 mg.l-1 2,4-d + 1.0 mg.l-1 kinetin and ms+1.5 mg.l-1 2,4-d + 1.0 mg.l-1 kinetin after two weeks. the callus proliferation occurred very fast in bds medium supplemented with 1.5 mg.l-1 2,4-d + 1.0 mg.l-1 kinetin compared to ms medium supplemented with 1.5 mg.l-1 2,4-d + 1.0 mg.l-1 kinetin. the development of a somatic embryo into a whole plant is likely affected by the non-optimal growth hormone or media composition during the callus growth. somatic embryogenesis can be affected by ethylene concentration, where low concentrations can stimulate, while high concentrations inhibit (trigiano and gray, 2004). a b c d figure 1. the effect of 2,4-d and kinetin on “tawangmangu baru” somatic embryogenesis; a) bds+ 0.4 mg.l-1 2,4-d+ 0.06% colchicine after 3 weeks; b) bds + 0.4 mg.l-1 2,4-d + 2.0 mg.l-1 kinetin + 0.02% colchicine after two weeks; (c) bds + 1.5 mg.l-1 2,4-d + 1.0 mg.l-1 kinetin + +0.02% colchicine; (d) ms + 1.5 mg.l-1 2,4-d+1.0 mg.l-1 kinetin after two weeks table 1. callus induction on bds medium supplemented with 2,4-d and kinetin after colchicine treatment of “tawangmangu baru” explant in vitro culture growth medium colchicine (%) callus induction (%) callus proliferation (%) shoot regeneration from callus (%) no. of shoots (%)(direct without callus no. of roots (%) bds + 0.4 mg.l-1 2,4-d 0 0.02 0.04 0.06 0.08 0.10 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 100 100 100 100 100 100 0 0 0 0 0 0 bds+0.4 mg.l-1 2,4-d + 2.0 mg.l-1 kinetin 0.00 0.02 0.04 0.06 0.08 0.10 0.80 1.56 1.56 0.50 0 0.30 0 0 0 0 0 0 0 0 0 0 0 0 100 0 0 0 100 0 0 0 0 0 0 0 bds+1.5 mg.l-1 2,4-d + 1.0 mg.l-1 kinetin 0.00 0.02 0.04 0.06 0.08 0.10 0.50 1.0 1.0 0.30 0 0.30 0 100 100 100 0 100 0 0 0 0 0 0 100 0 0 0 100 0 0 0 0 0 0 0 ms+1.5 mg.l-1 2,4-d + 1.0 mg.l-1 kinetin 0.00 0.02 0.04 0.06 0.08 0.10 0.30 0.50 0.50 0.30 0 0 0 100 100 100 0 0 0 0 0 0 0 0 100 0 0 0 100 0 0 0 0 0 0 0 total (%) 4.72 4.00 0 95.30 0 note: *the total percentage of induced callus, callus proliferation, shoot regeneration from callus, number of shoots, and number of roots that are produced in different growth mediums and growth hormones. journal of tropical crop science vol. 7 no. 3, october 2020 www.j-tropical-crops.com 133increasing ploidy level of garlic (allium sativum l.) “tawangmangu baru” in-vitro .......... (stress), light, temperature, and other cellular and physiological phenotypic traits of the explants. the quick growth of untreated plants at the beginning of in vitro culture may lead to higher death as compared to the treated plants. based on the total number of survivability of the mutagenic plantlets, the colchicine treated plants survived well compared to the untreated plants. the mean of the colchicine treated plantlets was higher in all treatments except the control and the plantlets induced at 0.08% for 24 hours (table 3). this showed that there was lower survivability in control diploid plants and slightly at higher colchicine treatment. the lethal concentration (lc) the lc 20%, lc 30%, and lc 50% were calculated at 2, 4, 5, and 7 months from colchicine treatment because the number of dying and surviving explants were observed after two months from colchicine treatment. the colchicine concentration needed for lc 20%, lc 30%, and lc 50% for the experiment at two and four months were 0.03%, 0.08%, and 0.16% respectively. there was no difference at two and four months for the lethal concentration. whereas, at 7 months, the lc 20%, lc 30%, and lc 50% were 0.01%, 0.08%, and 0.2% respectively. there was a high mortality of plants at the early stages and lower mortality after four months at lc 20% due to the stability of the plantlets. however, there was high mortality of plants as the time increases from 2 months to 7 months in vitro culture at 0.1%. in vitro plants had decreasing survival as the concentration of colchicine and in vitro culture increases with time. colchicine treated explants can survive even at higher colchicine concentrations, but there is also a higher mortality of explants in vitro culture. this is also supported by dixit and chaudhary (2014), amanah et al. (2016) that colchicine is effective in inducing diploid plants at a concentration of 0.001 1% in a period of 6–72 hours depending on the response of each plant species. the untreated plants had a greater number of shoots compared to the treated plants at 24 hours (table 5). however, a lower number of shoots were observed in untreated plants in 48 hours. the higher colchicine concentration resulted in a lower number of shoots table 2. mortality rate of garlic “tawangmangu baru” explants after colchicine treatment months colchicine immersion time (hours) (%) 24 48 2 0 0.02 0.04 0.06 0.08 0.10 1.25 ± 0.96a 0.25 ± 0.50a 0 ± 0a 0.25 ± 0.50a 2.50 ± 4.36a 0.50 ± 0.58a 3.00 ± 4.76a 0.5 ± 0.58a 1.00 ± 1.41a 0.25 ± 0.50a 0 ± 0a 0 ± 0a 4 0 0.02 0.04 0.06 0.08 0.10 1.25 ± 0.96a 0.25 ± 0.50a 0 ± 0a 0.25 ± 0.50a 2.50 ± 4.36a 0.50 ± 0.58a 3 ± 4.76a 0.5 ± 0.58a 1.00 ± 1.41a 0.25 ± 0.50a 0 ± 0a 0 ± 0a 5 0 0.02 0.04 0.06 0.08 0.10 1.75 ± 1.71a 0.50 ± 0.58a 0 ± 0a 0.50 ± 1.00a 2.75 ± 4.27a 0.50 ± 0.58a 3.25 ± 4.57a 1.00 ± 0.82a 1.25 ± 1.50a 0.25 ± 0.50a 0.50 ± 1.00a 0.50 ± 1.00a 7 0 0.02 0.04 0.06 0.08 0.10 2.00 ± 1.41a 0.50 ± 0.58a 0.25 ± 0.50a 0.50 ± 1.00a 3.00 ± 4.24a 0.75 ± 0.50a 3.25 ± 4.57a 1.00 ± 0.82a 1.25 ± 1.50a 0.25 ± 0.50a 0.75 ± 0.96a 0.75 ± 1.50a note: *values followed by the same letters on the same column showed no significant difference based on the fisher test at α=5%. values are means ± sd. journal of tropical crop science vol. 7 no. 3, october 2020 www.j-tropical-crops.com 134 molla gebreyohannes hailu, ni made armini wiendi, and diny dinarti due to the higher toxicity of colchicine at 24 hours. high colchicine concentration leads to higher plant death as compared to the untreated plants. the colchicine treated explants showed greater and thicker shoot size after 5 weeks. there was no root production in all the colchicine treated and untreated explants. shoot and root growth responses to growth hormones and colchicine treatments there was no root production in all the colchicine treated and untreated explants. after colchicine treatment at 24 and 48 hours, the treated and untreated explants were planted in bds media supplemented with 0.4 mg.l-1 2,4-d and 2.0 mg.l-1 of cytokinin; bds + 0.4 mg.l-1 2,4-d and 0.5 mg.l-1 of cytokinin in vitro supposed to produce callus, shoot table 3. survivability rate among the “tawangmangu baru” explants after colchicine treatment months colchicine immersion time (hours) (%) 24 48 survivability rate 2 0 13.75 ± 0.50a 12.0 ± 0.58a 0.02 14.75 ± 0a 14.50 ± 1.41a 0.04 15.00 ± 0.50a 14.00 ± 0.50a 0.06 14.75 ± 4.36a 14.75 ± 0a 0.08 12.50 ± 0.58a 15.00 ± 0a 0.10 14.50 ± 4.76a 15.00 ± 0a 4 0 0.02 0.04 0.06 0.08 0.10 13.75 ± 0.5a 14.75 ± 0a 15.99 ± 0.50a 14.75 ± 4.36a 12.50 ± 0.58a 14.50 ± 4.76a 12.00 ± 0.58a 14.50 ± 1.41a 14.00 ± 0.50a 14.75 ± 0a 15.00 ± 0a 15.00 ± 0a 5 0 13.25 ± 0.58a 12.00 ± 0.82a 0.02 14.50 ± 0a 14.00 ± 1.5a 0.04 15.00 ± 1.00a 13.75 ± 0.96a 0.06 14.50 ± 4.27a 14.25 ± 1.00a 0.08 12.25 ± 0.58a 14.50 ± 1.00a 0.10 14.50 ± 4.76a 14.50 ± 0a 7 0 0.02 0.04 0.06 0.08 0.10 13.00 ± 0.58a 14.50 ± 0.50a 14.75 ± 1.00a 14.50 ± 4.24a 12.00 ± 0.50a 14.25 ± 4.57a 11.75 ± 0.82a 14.00 ± 1.50a 13.75 ± 0.96a 14.25 ± 0.96a 14.25 ± 1.50a 14.25 ± 0a note: *values followed by the same letters on the same column showed no significant difference based on the fisher test at α=5%. values are means ± sd table 4. the lethal concentration* of colchicine treatment at different time intervals after colchicine treatment in vitro lethal concentration (%) time after in vitro culture (months) 2 4 5 7 20 30 50 0.03 0.08 0.16 0.03 0.08 0.16 0.01 0.06 0.15 0.01 0.08 0.20 note: *the percentage of lethal concentrations were calculated by probit analysis in the minitab software. journal of tropical crop science vol. 7 no. 3, october 2020 www.j-tropical-crops.com 135increasing ploidy level of garlic (allium sativum l.) “tawangmangu baru” in-vitro .......... proliferation, and regeneration at different trials. for this research, two trials were performed to get a large number of calluses in different growth hormones concentrations. however, there was no formation of callus at bds media supplemented with 0.4 mg.l-1 2,4-d alone (trial 1) and bds + 0.4 mg.l-1 2,4-d and 0.5 mg.l-1 of cytokinin (trial 2) as indicated in figure 2. this indicated that “tawangmangu baru” garlic cannot form a callus at a low concentration of 2,4d alone and in combination with a low concentration of kinetin. the genotype should be tested in other auxins and cytokinin other than 2,4-d and kinetin. the explants did not produce callus and root within the seven months in vitro culture due to the inappropriate 2,4-d and kinetin combinations and concentrations in the bds medium. the results from the in vitro culture showed that the interaction between the concentration and immersion time of colchicine did not have a significant effect on the root production of the "tawangmangu" baru garlic variety. the failure of root production in this genotype may result from the concentration and combination of the growth hormones, genotype, and other in vitro culture conditions or through endogenous ethylene production. the colchicine treated explants showed greater and thicker shoot size after 5 weeks as shown in figure 2. the control diploid plants showed the fastest growth over the colchicine treated explants. while the induced plants showed higher leaf and shoot size with slow growth over the control of diploid plants after treated by colchicine in vitro. there was also a clear difference in terms of shoot size within the colchicine treated plants as shown in figure 2. colchicine causes an increase in ethylene content. ethylene is a hormone that stimulates cell elongation and cell division through the action of gibberellin. ethylene itself may inhibit the auxin and cytokinin action so that the plantlets did not demonstrate fast growth and development (ayu et al., 2019). the explants treated at 0.06% colchicine showed larger and thicker leaf and shoot size over the other treatments. the explants treated at 0.06% produced a larger shoot size level over the other treatments. a higher concentration of colchicine leads to more negative effect on the treated plants due to the disruption of the genetic content of the plants and cellular inhibition to certain physiological processes in plants (ayu et al., 2019). ploidy verification through flow cytometry flow cytometry measures dna quantity in nuclei from fresh leaves. ploidy level analysis is laborious and time-consuming. although it is a quick and hardly destructive technique, it measures nuclei in phases of high and low ploidy levels (foshi et al., 2013; yenchon, 2014). after the colchicine treated plants had grown for 60 days on bds medium and were induced by colchicine at 24 and 48 hours in vitro, putative polyploid plants were investigated by flow cytometry. after two months, the induced plants were selected based on their larger shoot size to know the ploidy level of the different treatments. 24 plants were selected from 12 treatments (0, 0.02, 0.0, 0.06, 0.08, and 0.10%) induced at 24 and 48 hours. from each treatment, 2 plants were detected by flow cytometry to know the total nuclear dna status of the induced plants. the nuclear dna contents of leaves derived from the cultures were measured with flow cytometry. nuclear suspensions of leaf cells were obtained from 0.5 cm2 pieces of fresh leaves cut with a razor blade in a buffer solution using the cystain uv precise p kit following the manufacturer’s protocol. leaves of “tawangmangu baru” (2n= 2x = 16) were used as an internal standard. each sample was first analyzed alone to identify its fluorescence intensity profile and then analyzed with an internal standard to measure their ratio of mean cell fluorescence intensities. the dark green part of the leaf of the plants was taken for sample preparation. the plant part taken for sample table 5. the average number of shoots per treatment after seven months from colchicine treatment in vitro concentration (%) immersion time (hours) 24 48 no. of st 0.00 7.25 ± 0.975a 4.75 ± 1.893ab 0.02 6.75 ± 1.500ab 3.25 ± 3.770b 0.04 6.00 ± 1.826ab 4.00 ± 3.650ab 0.06 5.75 ± 2.870ab 6.75 ± 0.957ab 0.08 3.00 ± 3.830b 6.75 ± 1.500ab 0.10 4.00 ± 3.270ab 7.00 ± 0.816a note: values followed by the same letters on the same column showed no significant difference based on the fisher test at α=5%. values are means ± sd journal of tropical crop science vol. 7 no. 3, october 2020 www.j-tropical-crops.com 136 molla gebreyohannes hailu, ni made armini wiendi, and diny dinarti preparation was chopped manually through a razor blade approximately for 2 minutes to release the intact nuclei of the plant tissue. 0.5 cm2 of the leaf was taken from the middle part of the leaf to extract the nuclei of the plant tissue. the leaf sample was poured by 250 µl cysstain pi absolute p (nuclei extraction buffer) to extract the intact nuclei representative of the cells within the plant tissues. the extraction was made manually by grinding the plant tissues through a laser blade in the petri dish. after the tissues ground well, the solution was transferred to the puppet filtered by 30 µm filter size and added 300 cysstain pi absolute p for colouring. the staining buffer was prepared from 60 µl propidium iodide, 20 µl cysstain pi absolute p, and 60 µl ribonuclease (rnase) to degrade the rna molecule from the sample tissue as the target to study in the plant tissue was the nuclear dna status of plant tissue. from the solution that contains propidium, staining buffer, and rnase, 300 µl solution was added to the extracted sample a b c d e f figure 2. the shoot and root growth and development of garlic explants treated at 24 and 48 hours in vitro; a. control (bds + 0.4 mg.l-1 2,4-d + 0.5 mg.l-1 kinetin; b: bds + 0.4 mg.l-1 2,4-d + 0.5 mg.l-1 kinetin + 0.02% colchicine; c: bds + 0.4 mg.l-1 2,4-d + 0.5 mg.l-1 kinetin+ 0.04% colchicine; d: bds + 0.4 mg.l-1 2,4-d+ 0.5 mg.l-1 kinetin +0.06 % colchicine; e: bds + 0.4 mg.l-1 2,4-d + 0.5 mg.l-1 kinetin + 0.08% colchicine; f: bds + 0.4 mg.l-1 2,4-d + 0.5 mg.l-1 kinetin + 0.1% colchicine the explants treated at 0.06% colchicine showed larger and thicker leaf and shoot size over the other treatments. the explants treated at 0.06% produced a larger shoot size level over the other treatments. a higher concentration of colchicine leads to more negative effect on the treated plants due to the disruption of the genetic content of the plants and cellular inhibition to certain physiological processes in plants (ayu et al., 2019). ploidy verification through flow cytometry flow cytometry measures dna quantity in nuclei from fresh leaves. ploidy level analysis is laborious and time-consuming. although it is a quick and hardly destructive technique, it measures nuclei in phases of high and low ploidy levels (foshi et al., 2013; yenchon, 2014). after the colchicine treated plants had grown for 60 days on bds medium and were induced figure 2. the shoot and root growth and development of garlic explants treated at 24 and 48 hours in vitro; a. control (bds + 0.4 mg.l-1 2,4-d + 0.5 mg.l-1 kinetin; b: bds + 0.4 mg.l-1 2,4-d + 0.5 mg.l-1 kinetin + 0.02% colchicine; c: bds + 0.4 mg.l-1 2,4-d + 0.5 mg.l-1 kinetin+ 0.04% colchicine; d: bds + 0.4 mg.l-1 2,4-d+ 0.5 mg.l-1 kinetin +0.06 % colchicine; e: bds + 0.4 mg.l-1 2,4-d + 0.5 mg.l-1 kinetin + 0.08% colchicine; f: bds + 0.4 mg.l-1 2,4-d + 0.5 mg.l-1 kinetin + 0.1% colchicine table 6. the ploidy level of “tawangmangu baru” putative lines after flow cytometry analysis colchicine (%) immersion time (hrs) chromosomal counting ploidy level 0.00 24 16 diploid 0.02 24 mixoploid mixoploid 0.04 24 16 diploid 0.06 24 16 diploid 0.08 24 mixoploid mixoploid 0.10 24 32 tetraploid 0.00 48 16 diploid 0.02 48 mixoploid mixoploid 0.04 48 16 diploid 0.06 48 16 diploid 0.08 48 mixoploid mixoploid 0.10 48 32 tetraploid journal of tropical crop science vol. 7 no. 3, october 2020 www.j-tropical-crops.com 137increasing ploidy level of garlic (allium sativum l.) “tawangmangu baru” in-vitro .......... a b c d e f g h i j k l figure 3. flow cytometric analysis of nuclei of “tawangmangu baru” garlic induced by colchicine; a, g: control; b, h: 0.02% colchicine; c, i: 0.04% colchicine; d, j: 0.06% colchicine; e, k: 0.08% colchicine; f, l : 0.1% colchicine at 24 hours (a to f) and 48 hours (g-l). journal of tropical crop science vol. 7 no. 3, october 2020 www.j-tropical-crops.com 138 molla gebreyohannes hailu, ni made armini wiendi, and diny dinarti tissue for colouring of the plant tissues. immediately after adding the staining buffer, the homogenized sample tissue was detected by bd accuri c6 plus flow cytometry (partec, germany) to determine the amount of nuclear dna in each treated line rapidly and efficiently. discussion after colchicine treatment, all the explants were unable to produce callus in bds basal medium with 0.4 mg.l-1 2,4-d alone. after failing the explants to produce callus in bds medium and 0.4 mg.l-1 2,4-d, the explants were sub-cultured into bds medium supplemented with 0.4 mg.l-1 2,4-d and 2.0 mg.l-1 kinetin and the explants were produced an embryogenic callus with very white, friable, and globular structure. however, the produced calluses were failed to proliferate and regenerate under these growth hormones. at bds medium with 2,4 d (0.4 mg.l-1) and kinetin (2.0 mg.l-1), the callus was unable to achieve the somatic embryogenesis and organogenesis. however, the calluses were proliferated in 1.5 mg.l-1 2,4-d and 1.0 mg.l-1 kinetin rapidly after two weeks. the response for callus proliferation was higher in the bds medium supplemented with 1.5 mg.l-1 2,4-d + 1.0 mg.l-1 kinetin compared to ms medium supplemented with 1.5 mg.l-1 2,4-d + 1.0 mg.l-1 kinetin. the developmental processes, such as callusing and proliferation are controlled by plant growth regulators (pgrs). pgr concentration is vital for an optimum response as too low concentration may not trigger the callus induction and shoot proliferation from callus. the combination of kinetin and 2,4-d causes slightly better responses in callus induction than the use of 2,4-d alone in the “tawangmangu baru” garlic. the initiation of embryogenic cells requires an appropriate medium that contains specific plant growth regulators. the meristematic basal discs of “tawangmangu baru” garlic induced callus in the first sub-culturing in 0.4 mg.l-1 (2,4-d) and 2.0 mg.l-1 kinetin combination after the explants failed to induce callus in 2,4-d alone. auxins and cytokinins in combination are effective in producing callus instead of auxins alone in “tawangmangu baru” garlic. however, at low concentrations of auxins (0.4 mg.l-1) and kinetin (2.0 mg.l-1), the induced callus unable to proliferate and regenerate. after five months of in vitro culture, all the explants were transferred to another fresh ms and bds mediums supplemented with 1.5 mg.l-1 2,4-d and 1.0 mg.l-1 kinetin. after transferring the explants into these growth hormones, the failed callus to proliferate in 0.4 mg.l-1 and 2.0 mg.l-1 kinetin started to proliferate rapidly after five weeks. the response for callus proliferation was seen very fast in bds medium supplemented with 1.5 mg.l-1 2,4-d + 1.0 mg.l-1 kinetin compared to ms medium supplemented with 1.5 mg.l-1 2,4-d + 1.0 mg.l-1 kinetin. mortality is a very important indicator when evaluating the efficiency of the induction of polyploidy plants after soaking bulblets in colchicine. explants can show stress symptoms due to colchicine concentration but the symptoms are variable based on the colchicine treatment. based on the total number of plantlets, the most suitable treatment to induce polyploidy was at 0.06% colchicine treatment for 24 and 48 hours because it did not cause the death of the plants compared to the other treatments. as described in table 3, the colchicine treated plantlets survived well in all treatments except the control, and the plantlets induced at 0.08% for 24 hours. untreated plantlets showed a higher mortality rate compared to the treated plantlets at both 24 and 48 hours. moreover, the explants induced at colchicine 0.08% for 24 hours resulted in high mortality as indicated in table 2. the high mortality rate in control plantlets and 0.08% at 24 hours was perhaps due to the genetic and physiological state of the explants. heo et al. (2016) obtained a 50% survival rate at 0.05% concentration of colchicine immersed at 9 hours and he reported that low survival rate and decreased root formation at higher colchicine concentration and longer immersion time were perceived. the meristematic basal disc of tawangmnagu baru garlic explants could survive even at higher colchicine concentration and immersion time if the other growth medium and hormonal compositions were contented. amanah et al. (2016) reported colchicine as an effective mutagen to induce plants at a concentration of 0.001% 1% in a period of 6–72 hours, depending on the response of each plant species. lc50 (lethal concentration 50 %) is used to determine the optimum concentration to be used in the mutagenesis experiment; it causes 50 % lethality in the plant organism used for irradiation in defined time duration. lc50 varies with the plant species, the type and status of the material, and the stage where the lethality is measured (bahadur et al., 2015). according to amanah et al. (2016) colchicine is effective to induce plants at a concentration of 0.001% 0.001 to 1% in a period of 6–72 hours depending on the response of each plant species. dixit and chaudhary (2014) observed the maximum toxicity of garlic at 0.75% (w/v) colchicine and a maximum number of putative tetraploids at 0.5% (w/v) colchicine in 36 hours treatment. dixit and chaudhary (2014) reported all concentrations of colchicine proved lethal to garlic meristematic basal disc of explants when applied for 48 h or 72 hrs after one-week post-treatment incubation. journal of tropical crop science vol. 7 no. 3, october 2020 www.j-tropical-crops.com 139increasing ploidy level of garlic (allium sativum l.) “tawangmangu baru” in-vitro .......... polyploid plants possess large and thicker leaves with a low number of stomata and high chloroplast numbers compared to the diploid plants (ayu et al., 2019; münzbergová, 2017; sajjad et al., 2013; roughani et al., 2017; jaskani et al. 2005). tuwo and indrianto (2016) stated that plantlets at a lower colchicine level grew faster than plantlets which have higher colchicine levels cultured at the same time. according to widoretno (2016) research study, colchicine has also a significant effect on patchouli growth and shoot regeneration cultured on ms medium. as shown in figure 2, the colchicine treated explants possess larger and thicker leaves with curled shoots compared to the control diploid plants. dixit and chaudhary (2014) suggested polyploid plants possess thick green leaves, wider, and greener leaves with larger stomata compared to the control diploids. according to many researcher’s investigation, colchicine is an effective mutagen for producing polyploid plants, but they showed that responses to colchicine differ depending on the plant genotype (heo et al., 2016). the degree of genetic variability obtained with in vitro induction depends on the genotype, type of explant, growth hormone, and the culture conditions used (luciani et al., 2006; mubarrat et al., 2018; hassan et al., 2014). the root meristem helps to have continuous growth as a result of cellular extension in the root system (ayu et al., 2019). limiting the growth and development of roots may affect the plant’s ability to absorb nutrients from the growth media leading to a hindered growth due to cell division failure. cytokinins inhibit rooting and affect treatment efficiency in the growth medium. many economically important plant species cannot induce root easily. the reluctance to root could be due to a high level of cytokinin persisting in stem tissues from previous treatments (trigiano and gray, 2004). however, rooting can be induced by the transfer of the shoots once or twice to medium without pgrs to allow cytokinin levels to drop. the degree of genetic variability obtained from in vitro culture extremely depends on the genotype, type of explant, growth hormone (particular chemical structure and composition), and the culture conditions used (luciani et al., 2006; mubarrat et al., 2018; hassan et al., 2014). the results from the in vitro culture showed that the interaction between the concentration and immersion time of colchicine did not have a significant effect on the root production of the "tawangmangu baru" garlic. the failure of root production in this genotype may result from the concentration and combination of the growth hormones, genotype, and other in vitro culture conditions. for this research, two trials were performed to get a large number of calluses, shoot, and root induction. one trial was on bds with 0.4 mg.l-1 2,4-d alone (1st sub-culture) and the second trial was on bds with 0.4 mg.l-1 2,4-d and 0.5 mg.l-1 kinetin for two months (1st subculture). in the first trial, after two months all the explants were transferred into bds media augmented with 0.4 mg.l-1 2,4-d and 2.0 mg.l-1 kinetin (2nd subculture) supposed to produce callus and organogenesis in two months. in the second sub-culture, 4.72% explants were produced callus within two weeks after 1st sub-culture. after four months, all the explants were transferred into bds media supplemented with 0.4 mg.l-1 2,4-d, 2.0 mg.l-1 kinetin, and 25 mg.l-1 asparagine for a month to ensure somatic embryogenesis. still, all the explants were unable to produce callus, shoot, and root at 0.4 mg.l-1 2,4-d alone and in combination with kinetin. there was no response of 2,4-d alone to callus production and organogenesis. the second trial was sub-cultured again in the same hormonal combination and concentration to ensure somatic embryogenesis, however, there was no somatic embryogenesis at all. occasionally mixtures of auxins usually at lower concentrations than those used singly can promote root initiation whereas single auxins have no activity (trigiano and gray, 2004). plant species or genotype has an adverse effect in shoot and root growth and differentiation, and adventitious root induction in plant tissue culture. in some genotypes, endogenous ethylene causes leaf crinkling, folding, restriction of root growth, and reduces tendril growth after a month in plant tissue culture (trigiano and gray, 2004). the flow cytometry analysis showed a diploid level of chromosome in all treatments except the plants induced in 24 hours with 0.1% colchicine concentration as shown in figure 3. the cytological analysis with flow cytometry revealed that tetraploid “tawangmangu baru” garlic variety (2n=4x=32) stained with propidium iodide showed higher chromosome number compared to the control (2n=2x=16). the tetraploid “tawangmangu baru” garlic plants were characterized with thick dark-green leaves and showed slow growth compared to the other treatments. the counted nuclear dna events with a rate of 50 nuclei per second resulted in a short peak in all the polyploidy plants. the shortest peak in the dna histogram may represent the total nuclei in g0 and g1 phases. mechanical chopping does not produce significant dna hydrolysis or nuclear degradation. the colchicine treated “tawangmangu baru” garlic showed in growth reduction and most of the clones were diploids. dixit and chaudhary (2014) reported that the regenerated garlic plantlets showing reduced growth were all haploids, while those with frizzed leaves were all polyploids. the growth abnormalities as a result of colchicine treatment indicate the success of polyploid induction (dixit and chaudhary, 2014). in treatment 0.02% at 24 hrs, 0.08% at 24 and 48 hrs resulted in mixoploid plants journal of tropical crop science vol. 7 no. 3, october 2020 www.j-tropical-crops.com 140 molla gebreyohannes hailu, ni made armini wiendi, and diny dinarti that produced due to the incidence of chimera. some cells may not be affected by colchicine treatment and remain as diploid (2n). only partial induction of chromosome cells remain doubled by colchicine treatment (amanah et al., 2016). colchicine causes microtubule depolymerization and inhibits cell division by blocking the microtubule development (zhang et al., 2016; roughani et al., 2017; moghbel et al., 2015; amanah et al., 2016). colchicine inhibits the addition of tubulin subunits which merge at the end of the microtubule polymers (amanah et al., 2016). microtubule depolymerization causes the disturbance of the spindle thread formation. the disturbance of spindle fibers results in the separation of chromosomes but the cells fail to produce new cells and cause the doubling of chromosomes (wiendra et al., 2011). results in garlic are promising and research on in vitro mutation breeding technology has been established (broertjes, 2012); however, all cells exposed to mutagen do not incur mutations (amanah et al., 2016). but, those that do incur mutations will give rise to cells reveals the mutation (bahadur et al., 2015; amanah et al., 2016). the research showed that 50% of the putative plants were diploid and 33.3% of the plants were mixoploid in both 24 and 48 hours, and that 16.6% of the putative “tawangmangu baru” plants duplicated their genome at 0.1% colchicine in both 24 and 48 hours. this ploidy value indicates that polyploid garlic candidates have been successfully induced using the meristematic basal discs as an explant. the application of colchicine increased the ploidy level, and an increase in ploidy is expected to make the bulb size larger in the “tawangmangu baru” garlic variety. larger tuber size will increase the tuber weight and overall garlic productivity and production. conclusion the meristematic basal disc of “tawangmangu baru” garlic variety can be propagated by in vitro tissue culture. however, the degree of genetic variability obtained from in vitro culture depends on the plant genotype, type of explant, growth hormone, and the culture conditions. the genotype failed to form callus on bds medium supplemented with 0.4 mg.l-1 2,4-d alone and bds with 0.4 mg.l-1 2,4-d and 0.5 mg.l-1 kinetin, but it formed a transparent, friable, and white callus within two weeks after culturing on 0.4 mg.l-1 2,4-d and 2.0 mg.l-1 kinetin on bds medium. cell and shoot proliferation were better on bds + 2,4-d (1.5 mg.l-1) and 1.0 mg.l-1 kinetin. the lc 20%, lc 30%, and lc 50% for the concentration of colchicine in “tawangmangu baru” garlic at two months were 0.03%, 0.08%, and 0.16% respectively. there was no difference at two and four months for the lethal concentration, whereas, the lc 20%, 30%, and 50% at 7 months was 0.01%, 0.08%, and 0.2% respectively. there was a high mortality of plants at the early stages and lower mortality after four months at lc 20% due to the stability of the plantlets. the “tawangmangu baru” basal disc explants treated by colchicine showed a higher survivability rate in the range of 0.00% 0.1% with immersion time of 24 and 48 hours. the genetic diversity of the “tawangmangu baru” garlic was successfully enhanced by colchicine concentration and immersion treatment. the flow cytometry analysis indicated that 50% of the putative plants were diploid and 33 % of the plants were mixoploid in both 24 and 48 hours. seventeen percent of the putative “tawangmangu baru” duplicated their genome at 0.1% colchicine treatment for 24 or 48 hours, indicating that polyploid garlic have been successfully induced using the meristematic basal discs as an explant. the tetraploids possessed thicker and darker green leaves with slow growth compared to the untreated control plants. the application of colchicine treatment at 24 or 48 hours improved the genetic potential of “tawangmangu baru” garlic variety in vitro. references agustina, t. 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(2016). production of tetraploid plants of trollius chinensis bunge induced by colchicine. czech journal of genetics and plant breeding 52, 34–38. the effects of application of erythrina bruci biomass and inorganic fertilizers .......... journal of tropical crop science vol. 10 no. 2, june 2023 www.j-tropical-crops.com 139 the effects of application of erythrina bruci biomass and inorganic fertilizers on wheat productivity in southwestern ethiopia. zelalem addis musse* south agricultural research institute in bonga agricultural research center, p.o. box 101, bonga, ethiopia *corresponding author; email: zelalemaddis660@gmail.com abstract soil fertility depletion is emerging as a serious challenge causing low crop yields and food insecurity in ethiopia. an experiment was conducted in two cropping seasons 2016 to 2018 to investigate the effects of the application of erythrina biomass and nitrogen fertilizer on soil properties and wheat yields in southwestern ethiopia. treatments were the recommended n and p fertilizers, 50% of the standard rate of erythrina + 50% of the recommended n, 25% erythrina + 75% n, 75% of the standard rate of erythrina + 25% of the standard rate of n, 100% of the standard rate of erythrina, and 50% of the standard rate of erythrina, and without fertilizer as the control. the experiment was arranged in a randomized complete block design with three replications. application of erythrina and nitrogen fertilizer increased soil organic carbon (oc), total nitrogen (tn), and cation exchange capacity (cec) compared to before fertilizer application or the control. plots amended with 25% erythrina + 75% of the recommended n provided the highest above ground biomass (8.98 t.ha-1) and grain yield (3.453 t.ha-1) and it was higher than the 100% chemical fertilizer treatment. conversely, the lowest above ground biomass (5.44 t.ha-1) and grain yields (1.958 t.ha-1) were obtained from the control. our study demonstrated that an integrated nutrient management which combines organic and chemical fertilizer can improve soil properties and increase wheat yield in the highlands of southwestern ethiopia. keywords: organic amendment, soil properties, organic fertilizer, leguminous trees introduction soil fertility depletion is emerging as a serious challenge causing low crop yields and food insecurity in ethiopia. the decline in soil fertility is the main challenge to crop production in the south region. continuous cultivation, inadequate application of organic nutrient sources, and soil erosion account for reduced soil fertility (tamado and mitiku, 2017). organic fertilizers can improve the physico-chemical properties of the soil (abiy, 2018). the use of organic fertilizers such as farmyard manures, compost, green manures, and biomass of leguminous trees is an alternative way to improve soil fertility and soil physical properties (abebe, 2018). wassie (2012) reported that application of erythrina biomass at 10 t.ha-1 + half of the recommended rate of n and p, or 23 kg.ha-1 of n and 23 kg.ha-1 of p increased wheat grain yield by 189%. in addition, the cost of inorganic fertilizer could be reduced by half using erythrina biomass as organic supplement while obtaining superior yield of wheat than either source applied alone. erythrina is one of the locally available trees and can be used as the source of organic matters for the crop. erythrina is grown as live fence and farm boundary plant at chena woreda of kaffa zone. it is a legume species, an n-fixing, easily propagate, nutrient rich leguminous tree and can be easily exploited as source of organic source fertilizer by small scale farmers for crop production and soil fertility amendment. application of inorganic fertilizers has been promoted to overcome low soil fertility and reduced crop yields. the raising costs of fertilizers with lack of financial resources farmers limits the required use of inorganic fertilizers for optimum yield. moreover, the efficiency of the chemical nitrogen fertilizers is very low in ethiopia, and it varies with crops, soil types, and management practices. therefore, it is important to study if the application of organic in combination with inorganic fertilizers can improve soil fertility, increase nutrient use efficiency and crop yield. several studies have shown that green manure and leguminous trees could improve soil fertility and increase crop yields. however, this practice has not widely adopted and used in ethiopia. one of the possible reasons could be the exotic origin of the plants species with farm-level hesitancy for adopting. journal of tropical crop science vol. 10 no. 2, june 2023 www.j-tropical-crops.com 140 zelalem addis musse the other reason is the limited effort made by researchers and extension personnel to identify and deploy them in the right niches (wassie et al., 2009). leaves of erythrina are commonly used as animal feed during dry season and its litter is excellent source of organic matter that can maintain soil fertility (eyasu, 2002). however, the information is scarce on the use of erythrina for improving soil fertility. so, it is essential to study the effect of application erythrina biomass alone or in combination with the inorganic fertilizers on the yield of wheat in the south region to determine the effects of erythrina biomass and inorganic fertilizers for wheat production. material and methods description of study area the experiment was conducted at chena district of kaffa zone, south-western ethiopia, in 2016-2018 main cropping seasons, i.e. from july to november. the site in chena district is located at latitude of 7°05.67´ n, longitude of 35°42.989´ e and altitude of 2135 meters above sea level. the rainfall pattern of this area is characterized by bimodal distribution with small rainy season ‘belg’ (march-june) and main rainy seasons ‘meher’ (july-november). the average annual rainfall data from wushwush meteorological station was 1367 mm. the maximum temperature of cropping season varied from 22.1 to 26.60c; the minimum temperature varied from 10.0 to 13.10c. the soil analysis of the study sites before sowing of the crop showed that the soil texture of the experimental fields was clay loam with medium cec, low organic carbon content, total n, and available p (table 1). experimental design and experimentation the experiment was conducted in 2016-2018 on the farmers‘ field of chena woreda of kaffa zone. wheat variety “daphe” was used as a test crop. a cultural practice was carried out based on the recommendation of the crop (wasihun, 2022). the experiment consists of seven treatments: control (no fertilizer), recommended n and p (46 kg.ha-1 of n, 46 kg.ha-1 of p), 50% of the recommended rate of erythrina + 50% of the recommended rate of n, 25% of the recommended rate of erythrina + 75% of the recommended rate of n, 75% of the recommended rate of erythrina + 25% of the recommended rate of n, 100% of the recommended rate of erythrina and 50% of the recommended rate of erythrina. the experiments was laid out in randomized block design with three replication (plot). each plot was 5m x 5m (25 m2) with the net harvestable area of 4 m x 4 m (16 m2) having 1 m space between plots and 1 m between blocks. rows were being spaced 0.2 m interval. all data was subjected to analysis of variance. sources of n and p was urea and triple super phosphate (tsp). half dose of p and n were applied at planting and the remaining half dose was applied 35 days later. erythrina biomass consisting of young leaves and twigs were chopped into small figure 1. geographical location of the study area at chena district of kaffa zone, south-western ethiopia. the effects of application of erythrina bruci biomass and inorganic fertilizers .......... journal of tropical crop science vol. 10 no. 2, june 2023 www.j-tropical-crops.com 141 pieces to enhance decomposition. the chopped parts were then incorporated into the soil one month before planting of testing crop. a total of 15 composite soil samples from 15 spots, collected from 0-20 cm soil depth, were taken using auger before planting for analysis of texture, ph, cec (cation exchange capacity), oc (organic carbon), total n (total nitrogen), and the available p. erythrina nutrient analysis erythrina leaves and twigs were sampled from six randomly selected healthy trees for nutrient analysis. three branches were selected, the lower, the middle and the upper tree canopy. the samples were mixed and composite samples were analyzed for n, p and k content. samples were air dried and milled to pass through 1 mm diameter mesh size. total n content was analyzed using micro-kjeldahl’s method and p and k contents was determined using dry ashing methods that described by anderson and ingram (1996). the young twigs and leaves of erythrina bruci contain 4.83% n, 0.38% p and 2.24% k. plant growth measurements agronomic parameters including tiller number, grain yield, above-ground biomass, plant height, spike length and thousand seed weight were measured. the data were subjected to analysis of variance using the sas system version 9.4 (sas, 2000) and the significance of means was established using the least significant difference method (lsd). results and discussion soil analysis results the soil properties after the application of treatments was shown in table 2. the soil chemical properties (organic carbon, total nitrogen, available phosphorus, and cec had changed compared to before planting. integration of leguminous tree and inorganic fertilizer increases both cec and organic carbon in the soil. the changes in cec and organic carbon indicated that erythrina biomass was decomposing and contributed nutrients to the soil. effects of erythrina biomass and chemical fertilizer application on wheat yield and yield components the effects of erythrina biomass on wheat yield and yield component in both years are significant (p <0.05, table 3). the maximum grain yield (3.453 t.ha-1), above ground biomass (8.98 t.ha-1) and tiller number (8.22) was obtained by the application of 25% erythrina with 75% n as compared to control that gives the lowest yields (table 3). application of 100% erythrina gives the highest spike length and table 1. initial soil physicochemical characteristics of the experimental sites. soil parameters value rating references textural class clay loam ph 6.03 slightly acidic hazelton and murphy (2007) organic carbon (%) 1.52 low hazelton and murphy (2007) total n (%) 0.11 low tekalign (1991) available p (mg.g-1) 7.10 low tekalign (1991) cec (cmol.kg-1) 18.05 medium landon (1991) table 2. soil properties after erythrina + chemical fertilizer application treatment ph-h2o oc (%) tn (%) cec (me.100g-1) av.p (ppm) control 5.05 3.57 0.31 18.4 4.21 recommended np 5.86 4.81 0.42 25.6 3.67 50% erythrina + 50% n 6.05 5.00 0.42 23.2 5.05 25% erythrina + 75% n 5.76 4.28 0.37 21.8 4.68 75% erythrina + 25% n 5.93 4.95 0.42 24.0 4.87 100% erythrina n 6.02 5.47 0.48 25.8 4.11 50% erythrina n 5.77 3.57 0.31 20.4 3.18 note: oc=organic carbon, tn=total nitrogen, cec=cation exchange capacity, av.p =available phosphorus; 100% erythrina = 4 t.ha-1 fresh biomass. journal of tropical crop science vol. 10 no. 2, june 2023 www.j-tropical-crops.com 142 zelalem addis musse plant height as compared to control. the increase of plant height and spike length in response to the increasing n from both erythrina and nitrogen may be accredited to the increase of availability of n that enhanced wheat vegetative growth. our results agree with moniruzzaman et al. (2009) who recorded maximum plant height of french snap bean from the application of 120 kg n.ha-1 and the minimum height was recorded from the control treatment. another finding conducted by mostafa and zohair (2014) demonstrated that application of 100 kg n.ha-1 and chicken manure to snap bean in sandy soil significantly increased plant height by scoring the highest value (22.8) as compared to control (11.7). on the other hand, one-thousand seed weight (tsw) was significantly affected at (p<0.05) by the application 50% erythrina with nitrogen. maximum value of the one-thousand seed weight (44.37 g) was recorded from the application of 50% erythrina as compared to control treatment (table 3). the result agrees with hossain et al. (2018) who reported that an application of combined organic (5 t.ha-1 bio-slurry) and inorganic fertilizers increasing one-thousand seed weight in treated plots over the control/untreated plots. conclusion our study demonstrated that application of erythrina biomass at 25% of the recommended dose combined with 75% nitrogen (34.5 kg.ha-1 n) resulted in the highest yield and yield components, and it was higher over the 100% chemical fertilizer treatment. these results showed that the requirements for the chemical nitrogen could be reduced by the application of erythrina biomass. therefore, application of 1 t.ha-1 erythrina biomass with 34.5 kg.ha-1 n inorganic nitrogen can be suggested for wheat production in chena district of kaffa zone, south-western ethiopia. future studies should be conducted across similar agro-ecologies to potentially reduce the use of chemical fertilizer without reducing yields. acknowledgments the author thank bonga agricultural research center for their supports and facilitation of the study and publication. references abebe, a. (2018). nitrogen release dynamics of erythrina abyssinica and erythrina brucei litter as influenced by polyphenol, lignin and nitrogen contents. journal of environment and earth science 8, 11-19. abiy, g., getahun, y., and shiferaw, b. (2018). effect of different rates of erythrina biomass transfer combined with different nitrogen and phosphorus rates on yield and yield components of wheat at masha woreda, southwestern ethiopia. international journal of research in agricultural sciences 5, 23483997. anderson, j.m. and ingram, s.j. (1996). “tropical soil biology and fertility: a handbook of methods” 2nd edition. cab international, oxford, uk. table 3. effect of erythrina and chemical fertilizer on yield and yield component on wheat treatments ph (cm) tn sl (cm) tsw (g) above ground biomass (t.ha-1) grain yield (t.ha-1) control 75.62b 4.75c 7.30b 35.32d 5.44c 1.958c recommended np 78.77ab 6.77b 7.85a 42.42ab 7.78ab 2.887b 50% erythrina+ 50% n 80.47a 6.90ab 7.77a 42.03ab 7.96ab 2.963ab 25% erythrina+ 75% n 78.28ab 8.22a 7.52ab 37.80cd 8.98a 3.453a 75% erythrina + 25% n 76.53b 6.73b 7.66ab 41.62ab 7.67ab 3.021ab 100% erythrina 80.68a 6.52b 7.78a 40.40bd 7.68ab 2.993ab 50% erythrina 77.60ab 6.03bc 7.65ab 44.37a 7.06ab 2.766b lsd 1.83 0.72 0.22 1.98 0.641 0.274 cv% 3.68 17.41 4.55 7.69 13.47 15.09 note: means followed by the same letter (s) within the column are not significantly different according to lsd at p ≤ 0.05. ph= plant height, tn= tiller number, sl= spike length, tsw = one-thousand seed weight. the treatment of 100% erythrina equals 4 t.ha-1 erythrina biomass; the recommended np is 46 kg n.ha-1 or 100 kg.ha-1 urea, and 46 kg.ha-1 p2o5 or 100 kg.ha -1 tsp. the effects of application of erythrina bruci biomass and inorganic fertilizers .......... journal of tropical crop science vol. 10 no. 2, june 2023 www.j-tropical-crops.com 143 eyasu, e. (2002). “farmer’s perception of soil fertility change and management”. sos sahel and institute of sustainable development, addis ababa. ethiopia. hazelton, p., and murphy, b. (2007). “interpreting soil test results: what do all the numbers mean?” 2nd ed. 152p. csiro publishing. hossain, m.n, sarker, u.k., uddin, m.r., rehana, s., hoque m.m.i., and islam, m.a. (2018). effects of bio-slurry with chemical fertilizer on the performance of some high yielding varieties of boro rice (oryza sativa l.). journal of archives of agriculture and environmental science 3, 109-115. landon, j.r. (1991). "tropical soil manual: handbook for soil survey and agricultural land evaluation in the tropics and subtropics." longman scientific and technical, longman group. united kingdom. moniruzzaman, m., halim, g.m.a., and firoz, z.a. (2009). performances of french bean as influenced by plant density and nitrogen application. journal of agricultural research 34, 105-111. mostafa, n.f., and zohair, m.m. (2014). influence of organic nitrogen on the snap bean grown in sandy soil. international journal of agriculture and biology 16, 65-72. tamado,t., and mitiku, w. (2017). effect of combined application of organic and mineral nitrogen and phosphorus fertilizer on soil physicochemical properties and grain yield of food barley (hordeum vulgare l.) in kaffa zone, southwestern ethiopia. momona ethiopian journal of science 9, 242-261. tekalign, t. (1991). “soil, plant, water, fertilizer, animal manure and compost analysis”. working document no. 13. international livestock research center for africa. addis ababa. wasihun, y. (2022). review on sesame (sesamum indicum l.) production challenges and opportunities in ethiopia. world journal of agriculture and soil science 8, 2641-6379. wassie, h. (2012). appraisal of erythrina brucei as a source for soil nutrition on nitisols of south ethiopia. international journal of agriculture and biology 14, 371-376. wassie, h., shiferaw, b., and kelsa, k. (2009). integrated soil fertility management options for sustainable crop production: review of research findings from southern regional state of ethiopia. in “improved natural resource management technologies for food security, poverty reduction and sustainable development”. proceedings of the 10th conference ethiopian society of soil science pp. 163–175. eiar, addis ababa, ethiopia. response of lettuce (lactuca sativa l.) to aquaculture wastewater treatment .......... journal of tropical crop science vol. 10 no. 1, february 2023 www.j-tropical-crops.com 79 response of lettuce (lactuca sativa l.) to aquaculture wastewater treatment fia sri mumpunia, nani yulianti*b, yeni maryanib a aquaculture study program, faculty of agriculture, djuanda university, bogor b agrotechnology study program, faculty of agriculture, djuanda university, bogor jl. ciawi 1 toll road, post 35, bogor 16720, indonesia. corresponding author; e-mail:nani.yulianti@unida.ac.id abstract the fish feed waste and litters are organic matter that can be a source of nutrients for plants. this study is aimed to determine the growth and production of lettuce (lactuca sativa l.) treated with wastewater from hard-lipped barb culture of several population densities. the research was carried out from april through june of 2020 at the greenhouse of the agrotechnology study program, faculty of agriculture, djuanda university, bogor, indonesia. the controlled study used a randomized block design with one factor, wastewater from fish population density of 10, 20 and 30 per 21 l of water, and ab mix as a control. plants treated with the ab mix were significantly superior to those plants treated with the hard-lipped barb farming wastewater, demonstrated by taller plants, more and larger leaves, larger stem diameter and leaf area, longer roots, greater fresh and dry weight, as well as a higher leaf nitrate content. the aquaculture wastewater at the tested fish density in this study did not support optimal growth of lettuce, so lettuce production is still very low compared to those grown with the ab mix. keywords: hard-lipped barb, c/n ratio, nutrition introduction vegetables are consumed by 97.29% of the total indonesian rural and urban communities (bps, 2017). vegetables are a source of vitamins, minerals, dietary fibers, and antioxidants that are all important for human health. lettuce (lactuca sativa l.) is one of the leafy vegetables belonging to the asteraceae family with a very high economic value after cabbage, cauliflower, and broccoli (wardhana et al., 2016). according to the central statistics agency (2017), vegetable crop production in indonesia in 2014-2015 had positive developments, especially in the lettuce, with a total production of to 290,993 tons in 2014, an increase of 15.64% to 336,494 tons in 2015. the demand for vegetables is increasing with the growing population, but agricultural land is shrinking due to the conversion of productive agricultural land into residential land and factory buildings, or housing. it is important to develop cultivation techniques, such as hydroponics, that are not completely dependent on ordinary agricultural land. the advantages of hydroponic farming systems include more controlled plant environment, the uses of sterile planting media, efficient uses of water and fertilizers, protection from rain and direct sunlight so that the crops can grow in all seasons (silvina and syafrinal, 2008). substrate hydroponics is a plant culture method that uses soilless media so that the roots grow on a porous substrate supplied with nutrient solution requirements (nelson, 2009). ab mix is a commercial source of the nutrients that are usually used in hydroponic culture. the chemical fertilizers are required for plant growth, but the excess can be detrimental to the environment. on the other hand, the fertilizer prices are increasingly expensive. the wastewater from fish feeds and manures can be a potential source of nutrients for crops, particularly nitrogen, in the form of ammonia, nitrites, and nitrates (darmawan, 2010). the uses of aquaculture wastewater could potentially reduce the discharge of effluents into water environment. hard-lipped barb is a freshwater fish endemic to indonesia which has high nutritional values. high density fish farming increases nitrogen in the wastewater, which comes from the accumulation of left-over feeds and fish feces (darmawan, 2010). our study was conducted to determine the growth and production of hydroponic lettuce treated with wastewater from hard-lipped barb culture at various fish population densities. journal of tropical crop science vol. 10 no. 1, february 2023 www.j-tropical-crops.com 80 fia sri mumpuni, nani yulianti, yeni maryani materials and methods location the research was carried out from april through june of 2020 at the experimental greenhouse of the agrotechnology study program, faculty of agriculture, djuanda university, bogor. the temperature in the greenhouse ranged from 28-33°c with the light intensity ranging from 6,833 to 24,967 lux. materials the study used ”green curly” lettuce seeds, seedling trays, 45-l buckets, 50 x 85 cm and 60 x 100 cm pe plastics, measuring cups (1 l and 50 ml), calipers, analytical balances, hygrometers, lux meters, phmeter, tds-meter, and 32 aquarium measuring 30 cm x 30 cm x 30 cm. the materials used include rockwool, 30 cm x 30 cm polybags, husk charcoal, and wastewater from hard-lipped barb (osteochilus hasseltii cv) culture. experimental design the study was arranged in a randomized block design with a single factor, i.e. the density of bonylip barb culture in its wastewater, i.e. 10, 20, and 30 fishes per 21 l of water, and a control of ab mix (5 ml/liter of water). each aquarium was treated with 250 ml of wastewater, or ab mix solution. the research was started by sowing lettuce seeds in a 3x3 cm rockwool media with a 5 mm sowing depth. aquaculture wastewater was obtained by siphoning an aquarium containing lipped barbs. the ab mix was in the form of crystals which were prepared as a stock solution. lettuce seedlings were transplanted at 15 days after sowing. the planting medium used was husk charcoal, which was put into 30x30 cm polybags, or about 1.4 kg per polybag. plants were watered every morning and evening every day. replanting was conducted on the plants that did not grow and was conducted at 7, 14, and 21 days after planting (dap). weeding was conducted fortnightly by manually pulling weeds that grew in and around the polybags of plants. pest and disease control was carried out manually without the use of pesticides to avoid contamination of plants from harmful chemicals. lettuce was harvested at about 42 dap. the growth measurements were conducted weekly recording the number of leaves that were fully opened, the plant height, the leaf length, leaf width, the stem diameter and also the leaf area from 7 dap to 42 dap. plant height was measured from the base of the stem to the growing point using a ruler. the leaf length was measured from the base of the leaf to the tip of the leaf, whereas leaf width was measured from side to side of the widest leaf following the radius of the leaf segment using a ruler or tape measurement. lettuce stem diameter was measured each week from 21 to 42 dap using a caliper. leaf area was measured on the 5th widest leaf from the shoot using the gravimetric method. leaf nitrate was measured using a nitrate meter (horiba). root lengths, shoot, and root fresh and dry weight were measured using a digital scale after drying. the chlorophyll content analysis was carried out at the laboratory of the department of agronomy and horticulture, faculty of agriculture, bogor agricultural university. data was analyzed by analysis of variance at 5% level using spss; further test was conducted using duncan’s multiple range test (dmrt) at 5% level. results and discussion chemical and physical properties of the wastewater the fish culture wastewater had a ph higher than the ab mix, and a ec lower than the ab mix at the same temperatures (table 1). amongst the wastewater, the ammonia content from the wastewater with a fish population density of 30 was the highest (2.21 ppm), followed by a density 20 at 1.99 ppm and density of 10 (0.95 ppm, table 1). a high ammonia content in the water can inhibit fish growth. a study by pitrianingsih et al. (2014) demonstrated that water ammonia concentration >0.3 mg/l resulted in fish with decreased appetite, and even caused the fish to die. however, for plant culture, the higher the ammonia, the more nitrogen is available for the plants. for example, damayanti et al. (2018) reported that the addition of ammonium (nh4 +) and nitrate (no3 -) increased plant height, the number of leaves, the leaf area, and the fresh weight. therefore, the system developed here has the potentials to create a symbiotic mutualism between the fish and the crops. rockwool can hold 92% of water so it can store nutrients for a long time (agustina, 2019). husk charcoal has good porosity which was a beneficial property for aeration and drainage so that plant roots could absorb nutrients optimally (perez, 2008). the lettuce in this study survived under high temperatures of the nutrient solution and the greenhouse. according to priandoko et al. (2000) the optimal temperature of nutrient solution is 12-22°c, however, in this study it was 27.3°c, whereas the response of lettuce (lactuca sativa l.) to aquaculture wastewater treatment .......... journal of tropical crop science vol. 10 no. 1, february 2023 www.j-tropical-crops.com 81 greenhouse temperature was 28-33°c. the optimum temperature for lettuce growth was 15-25°c (amalia, 2013; aini et al., 2010). according to fariudin et al. (2017) leaf lettuce was relatively able to adapt to high temperatures in the lowland. lettuce height and leaf number at 7-42 dap the lettuce height treated with ab mix was significantly taller those treated with lipped barb farming wastewater (table 2). there was no significant differences in the plant height treated with wastewater of different fish densities (table 2). leaf size lettuce leaf size with the ab mix treatment was larger than the those treated with lipped barb wastewater (table 4). a study by perwitasari et al. (2012) reported that higher nitrogen promoted leaf expansion. leaf length of the plants in different wastewater treatments were not significantly different from 13 to 42 dap (table 4). table 4. lettuce leaf length and leaf width at 14, 28 and 42 days after planting (dap) after treatment with ab mix and fish culture wastewater at several table 1. chemical and physical properties of the fish culture wastewater at several population density parameter ab mix density 10 density 20 density 30 ammonia (ppm) 0.95 1.99 2.21 ph 7.4 8.4 8.6 8.5 ec (ppm) 1888 439 469 485 temperature (oc ) 27.2 27.2 27.2 27.3 table 2. lettuce plant height at 7 to 42 days after planting (dap) after treatment with ab mix and fish culture wastewater at several population density treatment plant height (cm) 7 dap 14 dap 21 dap 28 dap 35 dap 42 dap ab mix (control) 9.48c 11.99b 16.73b 22.93b 35.16b 43.74b density 10 7.16a 9.03a 12.21a 15.90a 21.40a 23.63a density 20 8.17b 9.80a 12.64a 17.19a 22.84a 25.38a density 30 7.86ab 9.49a 12.53a 16.32a 21.77a 24.39a note: the average values in the same column followed by the same letter are not significantly different according to the dmrt at the 5% level. the wastewater treatment did not significantly affect the number of leaves at 7 to 14 dap. it is possible that at the beginning of growth the amount of nutrients available were sufficient for plant growth, but became deficient at later stages (21-42 dap). the ab mix contain macro nutrients and micronutrients, while the nutrients contained in the wastewater of the lipped barb cultivation consisted of only nitrogen. a study by muhadiansyah et al. (2016) reported that lettuce that was grown without micronutrients zn, mo, fe, mn, co and b had fewer leaves than those supplied with micronutrients. at 21 dap, however, the number of leaves on the plants treated with the ab mix were not significantly different from those treated with wastewater with a fish density of 30 (table 2). the nitrogen content in the ab mix with the wastewater at a density of 30 was higher than in wastewater at a density 20 and 10, indicated by a high ammonia content (table 1). population density. in a study by meriyanto et al. (2017) n concentration of 900 ppm is optimum for crop growth, and that n is the most important nutrient for promoting plant stem and leaf growth. the ph value determines the availability of the nutrients. at an alkaline ph, the n availability decreases hence disrupt the roots function to absorb nutrients (fariudin et al., 2017). in addition, the alkaline environment reduced the activity of microorganisms that decompose organic matter (saputra et al., 2016). according to megasari (2017) a neutral ph is optimal for crop growth. ab mix has a total nutrient of 1888 ppm with a ph of 7.4, whereas the wastewater from fish population density of 10, 20 and 30 had 439 ppm, 469 ppm, and 485 ppm, respectively, with a ph of > 8 (table 1). this points to the lower quality of the lettuce production, journal of tropical crop science vol. 10 no. 1, february 2023 www.j-tropical-crops.com 82 fia sri mumpuni, nani yulianti, yeni maryani indicated by smaller leaf and stem diameter compared to those treated with the ab mix (table 4). plant productivity root length, shoot and root fresh and dry weight were significantly reduced when treated with wastewater (table 5). the vegetative growth rate was synergistically related to root length, fresh weight, and dry weight. long dan fibrous roots indicate the amount of nutrients and the amount of water absorbed. in the wastewater treated plants it is suspected that a lot of the nutrients and water were lost due to the evaporation as the temperature in the greenhouse was quite high. the evaporation from the ab mix treatment was presumably less as the large leaves shaded each other. greater fresh shoot weights are related to the optimal root growth that supported the function of absorbing nutrients and water (fariudin et al., 2017). lettuce leaf quality the quality of lettuce in this study was based on the leaf content of nitrate and chlorophyll. lettuce nirate was the highest with ab mix, and there was no significant differences in the nitrate and chlorophyll levels with different wastewater treatments (table 6). the role of chlorophyll is to absorb light for photosynthesis. one of the factors that affects the chlorophyll content of a plant is leaf morphology (setiari and yulita, 2009). the chlorophyll content of the lettuce in this study was not affected by the ab mix or the wastewater treatment, likely due to the thin nature of the lettuce leaves. according to rahmi (2017) chlorophyll compared to kale, green spinach, and mustard green leaves. in lettuce with ab mix treatment, large leaf areas did not have a positive correlation to the chlorophyll content. nitrogen is absorbed by plants in the form of ammonium (nh4 +) and nitrate (no3 -) through several stages of the nitrogen cycle. nitrogen cycle is a process of converting nitrogen into various other chemical forms; starting from n fixation, ammonification, nitrification, and denitrification. the successfully absorbed nitrate was reduced back to nitrogen gas (n2), known as denitrification, and then released into the air to complete the nitrogen cycle, and this cycle continues as long as the plant grows (damayanti et al., 2018). ammonia is converted to ammonium in the table 3. lettuce number of leaf at 7 to 42 days after planting (dap) after treatment with ab mix and fish culture wastewater at several population density treatment number of leaves 7 dap 14 dap 21 dap 28 dap 35 dap 42 dap ab mix (control) 4.44 5.25 6.56c 11.50b 22.94b 31.63b density 10 4.50 5.00 5.75a 8.19a 13.38a 16.63a density 20 4.25 4.94 6.00ab 8.06a 13.31a 16.13a density 30 4.19 5.13 6.31bc 8.81a 14.63a 17.75a note: the average values in the same column followed by the same letter are not significantly different according to the dmrt at the 5% level. table 4. lettuce leaf length and leaf width at 14, 28 and 42 days after planting (dap) after treatment with ab mix and fish culture wastewater at several population density. treatment leaf length (cm) at leaf width (cm) at stem diameter (mm) leaf area (cm2) 14 28 42 14 28 42 42 42 (days after planting) ab mix (control) 11.16b 18.41b 22.84b 7.93b 18.89b 27.01b 16.56 b 122.96b density 10 7.84a 12.18a 14.27a 4.43a 7.04a 8.16a 5.06a 38.17a density 20 8.44a 12.10a 15.03a 4.50a 6.13a 8.47a 4.78a 37.75a density 30 8.36a 12.16a 13.71a 4.64a 6.61a 7.96a 4.56a 39.22a note: the average values in the same column followed by the same letter are not significantly different according to the dmrt at the 5% level. response of lettuce (lactuca sativa l.) to aquaculture wastewater treatment .......... journal of tropical crop science vol. 10 no. 1, february 2023 www.j-tropical-crops.com 83 ammonification process. if conditions are favorable, the nitrification process will occur. nitrification is the conversion of ammonium to nitrate by involving microorganisms. factors that affect the nitrification process include planting media, oxygen availability, temperature, and ph. lettuce in this study was grown on husk charcoals which is a highly porous substrate, thus provided good aeration that supports nitrification. the average temperature of the greenhouse during the research was relatively high, i.e., 28-33°c and the temperature of the nutrients can reach 27.3°c. our study demonstrated that lettuce can still grow at these temperature ranges. high temperatures and alkaline ph can inhibit the activity of microorganisms in the nitrification process. bacteria can still grow and reproduce at a ph of 6.5-7.5 (neutral) with temperatures of 25-35°c (anisa and welly, 2017). the nitrate in the fish culture wastewater was the end product of the ammonia oxidation, assisted by nitrobacter sp, which was then then utilized by the plants for growth. the low nitrate levels in the wastewater indicates that the oxidation of the ammonia was slow, presumably due to the inhibition of the microbial activities and too alkaline water. conclusion plants treated with the ab mix grew significantly better and had a higher leaf nitrogen content than those treated with the aquaculture wastewater. the aquaculture wastewater at the tested fish density in this study did not support optimal growth of lettuce, so lettuce production is still very low compared to those grown with the ab mix. acknowledgement the authors thanked the directorate of resources, directorate general of higher education, ministry of education, culture, research and technology, which has funded this research through the university’s flagship applied research grant for the 2022 implementation year. references agustina, r. (2019). “the effect of hydroponic media and nutrient composition on the growth of green lettuce leaves (lactuca sativa l.)” [thesis]. universitas malikussaleh, aceh. aini, r.q., yaya, s., muhammad, n.h. (2010). application of kpd bionutrients to curly lettuce (lactuca sativa l.). jurnal sains dan teknologi kimia 1, 73-79. table 5. fresh weight and dry weight of lettuce shoots and roots treated with fish culture wastewater at several population density. treatment shoot root total shoot + root root length (cm) fw (g) dw (g) fw (g) dw (g) fw (g) dw (g) ab mix (control) 38.95b 447.53b 21.13b 32.66b 3.23b 240.09b 12.18b density 10 10.88a 14.36a 1.36a 0.59a 0.12a 7.48a 0.74a density 20 14.10a 21.60a 1.40a 0.55a 0.12a 11.08a 0.76a density 30 11.73a 27.00a 1.72a 1.03a 0.18a 14.01a 0.96a note: the average value in the same column followed by the same letter is not significantly different according to the dmrt at the 5% level. table 6. lettuce leaf nitrate and chlorophyll contents treated with fish culture wastewater at several population density. treatment nitrate(ppm) chlorophyll a (mg/g) chlorophyll b (mg/g) ab mix (control) 7791.67b 0.90 0.43 density 10 4175.00a 0.89 0.44 density 20 4383.33a 0.89 0.44 density 30 3950.00a 0.93 0.45 note: the average value in the same column followed by the same letter is not significantly different according to the dmrt at the 5% level. journal of tropical crop science vol. 10 no. 1, february 2023 www.j-tropical-crops.com 84 fia sri mumpuni, nani yulianti, yeni maryani amalia, d.,r.,n. (2013). “efek temperatur terhadap pertumbuhan gracilaria verrucosa”. [thesis]. universitas jember. jember anisa, a., and welly, h. (2017). pengolahan limbah domestik menggunakan moving bed biofilm reactor (mbbr) dengan proses aerobikanoksik utuk menurunkan nitrogen. jurnal teknik its 6, f361-f366. badan pusat statistika. (2017). “konsumsi buah dan sayur susenas maret 2016”. kementerian pertanian, jakarta. damayanti, d.,p.,o., tri, h.,and slameto. (2018). pengaruh ammonium (nh4 +) dan nitrat (no3 -) terhadap pertumbuhan dan kandungan minyak atsiri tanaman kemangi (ocimum basilicum) dengan sistem hidroponik. agritrop 16,163175. darmawan, w.,p.,j. (2010). “pemanfatan air buangan lele dumbo sebagai media budidaya daphnia sp.” [thesis]. bandar lampung: universitas lampung. fariudin, r., endang, s., and sriyanto, w. (2012). “pertumbuhan dan hasil dua kultivar selada (lactuca sativa l.) dalam akuaponik pada kolam gurami dan kolam nila” [thesis]. universitas gadjah mada. yogyakarta fitriasari, n. (2020). “pengaruh komposisi pupuk urea dan kipahit terhadap pertumbuhan, produksi dan kualitas berbagai aksesi kemangi (ocimum basilicum l.)” [thesis]. universitas djuanda, bogor. megasari, r. (2017). “teknologi aquaponik tanaman dan ikan nila pada tiga jenis media tanam dan frekuensi pemupukan” [thesis]. universitas hasanuddin. makassar. meriyanto, busroni, a., and sari, a. (2017). pengaruh pemberian berbagai konsentrasi larutan nutrisi hidroponik terhadap pertumbuhan dan hasil tanaman selada merah (lactuca sativa l.) dengan sistem deep flow technique (dft). jurnal triagro 2, 28-37. muhadiansyah, t.,o., setyono, and sjarif, a.,a. (2016). efektivitas pencampuran pupuk organik cair dalam nutrisi hidroponik pada pertumbuhan dan produksi tanaman selada (lactuca sativa l.). jurnal agronida 2, 37-46. nelson r. (2009). methods of hydroponic production. journal of aquaponics 4, 24-30. perez le. 2008. “hydroponics for the home”. inter-american institute for cooperation on agriculture. san jose. perwitasari, b., mustika, t., catur, w. (2012). pengaruh media tanam dan nutrisi terhadap pertumbuhan dan hasil tanaman pakchoi (brassica juncea l.) dengan sistem hidroponik. jurnal agrovigo 5, 4–25. pitrianingsih,c., suminto, and sarjito. (2014). pengaruh bakteri kandidat probiotik terhadap perubahan kandungan nutrien c, n, p dan k media kultur lele dumbo (clarias gariepinus). journal of aquaculture management and technology 3, 247-256. priandoko, a.,d., parwanayoni, s.,and sundra, i.,k. (2000). kandungan logam berat (pb dan cd) pada sawi hijau (brassica rapa l. subsp. perviridis bailey) dan wortel (paucus carrota l. var. sativa hoffim) yang beredar di kota denpasar. jurnal simbiosis 1, 9-20. purnomo, j., dwi, h., and trijono, d.,s. (2016). budidaya cabai rawit sistem hidroponik substrat dengan variasi media dan nutrisi. journal of sustainable agriculture 31,129-136. rahmi n. (2017). “kandungan klorofil pada beberapa jenis tanaman sayuran sebagai pengembangan praktikum fisiologi tumbuhan” [thesis]. universitas islam negeri ar-raniry. bamda aceh. saputra, a.d., haeruddin, and niniek, w. (2016). efektifitas kombinasi mikroorganisme dan tumbuhan air lemna minor sebagai bioremediator dalam mereduksi senyawa amoniak, nitrit dan nitrat pada limbah pencucian ikan. diponegoro journal of maquares 5, 8090. setiar,i n., yulita, n. (2009). eksplorasi kandungan klorofil pada beberapa sayuran hijau sebagai alternatif bahan dasar food supplement. bioma 11, 6-10. silvina, f., syafrinal. (2008). penggunaan berbagai medium tanam dan konsentrasi pupuk organik cair pada pertumbuhan dan produksi mentimun jepang (cucumis sativus) secara hidroponik. jurnal sage 7, 7-12. response of lettuce (lactuca sativa l.) to aquaculture wastewater treatment .......... journal of tropical crop science vol. 10 no. 1, february 2023 www.j-tropical-crops.com 85 siregar, j., sugeng, t., and diding, s. (2015). pengujian beberapa nutrisi hidroponik pada selada (lactuca sativa l.) dengan teknologi hidroponik sistem terapung (thst) termodifikasi. jurnal teknik pertanian lampung 4, 65-72. wardhana, i., hudaini, h., insan, w. (2016). respon pertumbuhan dan produksi tanaman selada (lactuca sativa l.) pada pemberian dosis pupuk kandang kambing dan interval waktu aplikasi pupuk cair super biotik. agritrop jurnal ilmu pertanian 14, 165-185. yama, d.i., hendro, k. (2020). pertumbuhan dan kandungan klorofil pakcoy (brassica rappa l.) pada beberapa konsentrasi ab mix dengan sistem wick. jurnal teknologi 12, 21-30. journal of tropical crop science vol. 10 no. 1, february 2023 www.j-tropical-crops.com 38 faza yasmin saidah, heni purnamawati, iskandar lubis evaluation of source and sink capacity of new cowpea varieties faza yasmin saidahᴬ, heni purnamawatiᴮ, iskandar lubisᴮ ᴬ agronomy and horticulture, graduate school, faculty of agriculture, ipb university, bogor 16680, indonesia ᴮ department of agronomy and horticulture, faculty of agriculture, ipb university, bogor 16680, indonesia *corresponding author; email: h_purnama@apps.ipb.ac.id abstract cowpea (vigna unguiculata (l.) walp) is a perennial species originating from sub-saharan africa. cowpea has long been cultivated in indonesia and is classified as a species tolerant of drought and acidic soil. cowpea shows its adaptation to acidic soil with a ph of 4.83 by being able to produce 50% to 60% of seed weight under optimum conditions. this enhances the potential of cowpea to be used and developed as one of the food options. cowpea's adaptability research was carried out with the aim to optimise of cowpea productivity by studying the relationship between the source and sink of cowpea. this research was conducted from december 2020 to march 2021 at the cikabayan experimental station, bogor agricultural university. the experiment was set up in a completely randomized block design. four cowpea varieties were evaluated, “albina” ipb, “arghavan” ipb, and “uno” ipb. the measured parameters consisted of photosynthesis rate, stomatal conductance, plant growth rate, the net assimilation rate of the number of pods, pod weight, number of seeds per pod, dry seed weight, the weight of 100-seeds, and productivity. the cowpea varieties did not show significant differences in the rate of photosynthesis, stomatal conductance, plant growth rate, and net assimilation rate. photosynthesis rate in the three cowpea varieties ranged from 29.20 to 31.77 mol. m⁻².s⁻1 at 50% flowering, and ranged from 17.01 to 19.79 mol.m⁻². s⁻1 at the first harvest. the three cowpea varieties in this study showed no differences in their source-sink capacity and productivity. keywords: cowpea varieties, pods, productivity. introduction indonesia’s soybean production in 2015 was 963.18 thousand tons, decreased to 859.65 thousand tons in 2016, and drop further to 538.73 thousand tons in 2017. in 2018 soybean production had increased to 650.00 thousand tons, but drop to 424.19 thousand tons in 2019 (ministry of agriculture, 2021). soybean consumption in indonesia far exceeds the domestic soybean production capacity, so it must supply 2,670,086.4 tons of soybeans through imports (bps, 2019). soybean in indonesia is predominantly consumed as tempeh, a cake made from fermented soybeans. there are other legumes that can be an alternative to making tempeh besides soybeans, such as cowpeas. cowpea (vigna unguiculata (l.) walp) is a perennial species originating from sub-saharan africa (boukar et al., 2015). cowpea has long been cultivated in indonesia and is classified as a species tolerant of drought and acidic soil (trustinah, 2015). cowpea can adapt to various types of land ecology (karsono, 1998; trustinah et al., 2001), including to acidic soil with a ph of 4.83, by being able to produce 50% to 60% of the seed weight under optimum environment (setyowati and sutoro, 2010a). cowpea production in indonesia is relatively low at only 1.5 – 2 ton.ha¹־ (balitkabi, 2005). there is little information about the availability of high yielding cowpea varieties, source-sink activities and capacities, and how to increase cowpea production and productivity. it is known that there are two varieties of cowpea based on their growth patterns, i.e., creeping and shrubs. ndiaga (2000) concluded that cowpea cultivars with different plant morphology require different optimum planting densities to fully express potential seed yield. the optimal planting density of cowpea depends on many factors such as rainfall, humidity and cultivar type, available nutrients, and management (el naim and jabereldar, 2010). information on the relationship between source and sink related to seed filling in cowpea varieties in indonesia is still very limited. crop production is determined by the amount of dry matter accumulation and the partition or distribution of the dry matter into parts to be harvested. the increase in crop yields can be done by increasing the accumulation of dry evaluation of source and sink capacity of new cowpea varieties .......... journal of tropical crop science vol. 10 no. 1, february 2023 www.j-tropical-crops.com 39 matter, or increasing the harvest index. our study was conducted to increase the cowpea productivity by measuring the relationship between the source and sink of cowpea plants. three varieties of cowpea used in this study were released by ipb university breeder, prof. m. syukur, in 2020; all have a bushtype growth pattern. the seeds have fat content 0.901.36%, protein content of 23.98-24.26%, and fiber content of 3.87-4.44% (syukur et al., 2020). material and methods the study was conducted from december 2020 to march 2021 at the cikabayan experimental station, bogor agricultural university, bogor regency, west java, indonesia, located at an altitude of about 250 meters above sea level. the materials used in this study were cowpea variety "arghavan", "uno", and "albina". manures 2 ton.ha¹־, urea 30 kg.ha¹־, sp6 100 kg.ha¹־, kcl 100 kg.ha¹־, and dolomite 500 kg.ha¹־ were supplied to the crops according to production guidelines by fadillah (2019), liana (2019) and siregar, (2020). pesticides were used to control pests and diseases when required. the equipment used included analytical balance, oven, li-cor 6400xt portable photosynthesis system, image-j application, and a spad meter. for the chlorophyll measurement, scissors and a cooler box were used to carry fresh leaf samples prior to analysis. the study tested a one-factor (cowpea variety) organized in a completely randomized block design with four replications, totalling 12 experimental units. after the beds are prepared, manure and dolomite are spread over the planting bed one week prior to planting. cowpea seeds were planted 2 seeds per hole with a plant spacing of 60 cm x 50 cm. crop maintenance included watering, thinning, weeding, and controlling pests and diseases. cowpeas were harvested when the pod colour have turned brown and had dried up. harvesting was carried out starting at 10 until 14 weeks after planting (wap). measured parameters include morphological characters, physiological characters, and production components. physiological characters were measured when 50% of the population flowered, and at the first harvest at 10 weeks after planting, consisting of rate, stomatal conductance, plant growth rate, net assimilation rate, and total carbohydrate content. production components include the number of pods, pod weight, number of seeds per pod, dry seed weight, the weight of 100 seeds, and productivity. crop growth rate (cgr) was carried out when 50% of population have flowered, and at the time of the first harvest. the plant growth rate was calculated using the formula (rajput et al., 2017): maintenance included watering, thinning, weeding, and controlling pests and diseases. cowpeas were harvested when the pod colour have turned brown and had dried up. harvesting was carried out starting at 10 until 14 weeks after planting (wap). measured parameters include morphological characters, physiological characters, and production components. physiological characters were measured when 50% of the population flowered, and at the first harvest at 10 weeks after planting, consisting of rate, stomatal conductance, plant growth rate, net assimilation rate, and total carbohydrate content. production components include the number of pods, pod weight, number of seeds per pod, dry seed weight, the weight of 100 seeds, and productivity. crop growth rate (cgr) was carried out when 50% of population have flowered, and at the time of the first harvest. the plant growth rate was calculated using the formula (rajput et al., 2017): ��� � w2 � w1p t2 � t1 where cgr = crop growth rate (mg.m-2.day) p = plant area (m²) w2 = plant dry weight at t2 (g) w1 = plant dry weight at t1 (g) t1 = time to 50% of flowering (day) t2 = time to harvest (day) net assimilation rate (nar) is the net assimilation result of assimilation per unit leaf area and time. measurements were made when 50% of the plants flowered and at the time of the first harvest. nar calculation using the formula (shon et al., 1997): where nar = net assimilation rate (g cm⁻² hari⁻ ˡ) w1 = the dry weight of the plant when 50% of the plant's flower (g) w2 = the dry weight of the plant at the time of the first harvest (g) a1 = total leaf area when 50% of the plants have flowered (cm²) a2 = total leaf area at first harvest (cm²) t1 = time to 50% of flowering (day) t2 = time to harvest (day) data was analyzed using analysis of variance (anova) a level of 5% with sas design version 9.4. using the f test. if the treatments are significant, further tests are carried out using the duncan multiple range test (dmrt) at the level of significance = 5%. in addition, a correlation test was also carried out between all plant characteristics and pod production. correlation analysis is carried out with a simple correlation according to pearson as follows: ��� � 𝑛𝑛ʃ𝑋𝑋𝑋𝑋𝑛𝑛𝑋𝑋 � �ʃ𝑋𝑋𝑋𝑋��ʃ𝑛𝑛𝑋𝑋�√�𝑛𝑛ʃ𝑋𝑋𝑋𝑋� � �𝑋𝑋𝑋𝑋����𝑛𝑛ʃ𝑛𝑛𝑋𝑋� � �𝑛𝑛𝑋𝑋��� the value of r < 0 indicates that each plant characteristic has a close relationship with the production of pods and seeds but is negative, while the value of r > 0 indicates that each plant characteristic has a close relationship with the production of pods and seeds and is positive. the closer the r value is to 0, the less each plant characteristic has a relationship where cgr = crop growth rate (mg.m-2.day) p = plant area (m²) w2 = plant dry weight at t2 (g) w1 = plant dry weight at t1 (g) t1 = time to 50% of flowering (day) t2 = time to harvest (day) net assimilation rate (nar) is the net assimilation result of assimilation per unit leaf area and time. measurements were made when 50% of the plants flowered and at the time of the first harvest. nar picture 1. cowpea at 7 days (a), 21 days (b) and 55 days (c) after planting. journal of tropical crop science vol. 10 no. 1, february 2023 www.j-tropical-crops.com 40 faza yasmin saidah, heni purnamawati, iskandar lubis calculation using the formula (shon et al., 1997): where nar = net assimilation rate (g.cm-2 per day) w1 = the dry weight of the plant when 50% of the plant’s flower (g) w2 = the dry weight of the plant at the time of the first harvest (g) a1 = total leaf area when 50% of the plants have flowered (cm²) a2 = total leaf area at first harvest (cm²) t1 = time to 50% of flowering (day) t2 = time to harvest (day) data was analyzed using analysis of variance (anova) a level of 5% with sas design version 9.4. using the f test. if the treatments are significant, further tests are carried out using the duncan multiple range test (dmrt) at the level of significance = 5%. in addition, a correlation test was also carried out between all plant characteristics and pod production. correlation analysis is carried out with a simple correlation according to pearson as follows: the value of r < 0 indicates that each plant characteristic has a close relationship with the production of pods and seeds but is negative, while the value of r > 0 indicates that each plant characteristic has a close relationship with the production of pods and seeds and is positive. the closer the r value is to 0, the less each plant characteristic has a relationship with the production of pods and seeds. the value of r = -1 ≤ r ≤ 1. result and discussion cowpea are rich in the phytonutrients and minerals (kirigia et al., 2018), therefore it is a potential crop that still needs to be developed further. according to horn and shimelis (2020) cowpea has high adaptability and tolerance to drought, low soil fertility, and tolerance to acidic soils (setyowati and sutoro, 2010b). in addition, cowpeas can grow better in tropical areas with sandy and dry soils such as saffron areas when compared to soybeans (sheahan, 2012). based on bmkg data from the bogor climatology station in january to april 2021 (table 1), the highest average rainfall occurred in february, which was 626.7 mm per month, and this occurred during midplanting. rainfall increased at harvest time in april. the length of irradiation during the planting period ranged from 19.5 to 116.9 hours per month, being the highest in april (116.9 hours per month) which occurred at harvest. the average temperature during the growing season ranged from 20.5-21.4ºc and the average air humidity during the growing season ranged from 85.1-92.5%. the relative humidity during the study was normal in the tropical study area and is suitable for cowpea growth. according to karsono (1998a) the adaptation area of cowpea is in the tropics with optimum temperatures ranging from 25ºc –30ºc. figure 2. flowers of cowpea flowers: “albina” (a), “arghavan” (b) and “uno” (c). evaluation of source and sink capacity of new cowpea varieties .......... journal of tropical crop science vol. 10 no. 1, february 2023 www.j-tropical-crops.com 41 soil conditions in the study area were classified as acidic (medium n-total, high available p, very low k-dd and low cec). this ph is suitable for cowpea that can grow well in soils with a ph of 5.0-6.5 (karsono, 1998b). the soil physical and chemical properties are in table 2. according to hardjowigono (2015), the total n-level of >0.5% is considered high (table 2). photosynthetic rate and stomatal conductance the rate of co₂ exchange and stomata conductance were measured by a portable photosynthesis system licor li-6400xt on the leaf located on the third node from the topmost leaf. stomata conductance describes the activity of stomata in regulating co₂ during the photosynthesis process and also controls the rate of transpiration in controlling the process of tissue water loss. the rate of photosynthesis is related to the chlorophyll content of the leaves which plays a role in absorbing energy from sunlight which is then transferred to chlorophyll (porra et al., 1993). chlorophyll is a pigment that plays an important role in photosynthesis and is mostly found in leaves. table 2 showed no significant difference between varieties in the rate of photosynthesis and stomatal conductance. in general, the conductance capacity of stomata is related to the rate of photosynthesis. photosynthesis rate in the three cowpea varieties ranged from 29.20 to 31.77 mol m⁻²s⁻1 at 50% flowering, and ranged from 17.01 to 19.79 mol m⁻²s⁻1 at the first harvest. the rate of photosynthesis shows the capacity of plant sources, i.e., the ability of plants to produce assimilates. the rate of photosynthesis of plants is influenced by sunlight received by plants through the leaves (pantilu et al., 2012). the average stomatal conductance is 0.28 μmol.m⁻².s⁻10.74 μmol.m⁻². s⁻1. according to hassan et al. (2009) as chlorophylls play a very important role in photosynthesis, they will impact the biomass production. taiz and zeiger (2002) reported that the more and wider the stomata opening, the higher the co2 gas exchange, as well as stomatal conductance. crop growth rate and net assimilation rate photosynthetic activity is related to source capacity which is characterized by the growth rate of leaf area index, chlorophyll content, and stomata density. leaf area index, leaf-specific weight, and chlorophyll content play a role in determining the ability of plants to absorb solar radiation and the process of photolysis of water. stomata aperture are important for the smooth entry and exit of co2 and water for photosynthesis (purnamawati and manshuri, 2015). crop growth rate shows the production of biomass per unit area in a certain duration (atmaja, 2020). the cowpea growth rate was measured from the difference in plant dry weight at 50% of plants that had flowered and at the time of the first harvest. the growth rates of the three cowpea varieties were not significantly different. the average plant growth rate in the three varieties ranged from 19.50 – 24.77g.m²־ per day (table 3). the increase in the growth rate of cowpea is possible to increase the source capacity (leaf growth) to meet the needs of sinks (pod formation) (liana, 2019). the crop growth rate is an important factor in table 1. agro-climatic data between january to april 2021 month january february march april rainfall (mm) 384.0 626.7 186.8 357.8 irradiation (hours per month) 19.5 34.5 115.2 116.9 relative humidity (%) 88.1 92.5 85.1 85.9 temperature (ºc) 20.5 20.5 21.6 21.4 source: bmkg data january-april 2021 from bogor climatology station table 2. soil chemical properties before cowpea planting soil parameter method value status water content (%) gravimetry 5.58 low* ph h₂o 5.05 acidic** total n (%) kjedahl 0.20 medium** available p (ppm) bray i 34.15 high* cec (cmol.kg-1) nh₄oac 1m, ph 7.00 14.42 low* k-dd (mol k.kg-1) nh₄oac 1m, ph 7.00 0.07 very low* note: soil chemical property criteria according to *soil research institute (2005);** seameo biotrop soil laboratory 2018. journal of tropical crop science vol. 10 no. 1, february 2023 www.j-tropical-crops.com 42 faza yasmin saidah, heni purnamawati, iskandar lubis analyzing plant growth because it shows the amount of accumulated dry matter produced per unit area in a certain period. the higher the dry matter produced, the higher the plant growth rate will be. net assimilation rate is the ability of plants to produce dry matter assimilated per unit leaf area per unit time. the net assimilation rate shows the increase in plant dry weight resulting from the increase in leaf area at a certain time interval. the net assimilation rate was not affected by the three cowpea varieties used and the three varieties were not significantly different. the average net assimilation rate in the three varieties ranged from 3.28 to 3.87 g.cm⁻² per day. the cowpea net assimilation rate in this study was related to plant dry weight and plant leaf area (table 3). cowpea yield and yield component the yield components of cowpea measured in this experiment included pod dry weight, pod length, number of seeds per pod, seed dry weight, and 100seed weight. the yield of pods and seeds per plant in the three varieties did not show significant differences. the highest pod length, pod weight and number of seeds per pod was produced by “arghavan”. the highest dry seed production was produced by “albina”. cowpea seeds in all three varieties produced pithy seeds. the pithy seeds are thought to be caused by the element potassium which plays an important role in translocating the assimilates. seed filling in cowpea pods is also influenced by environmental factors (afitu et al., 2016). the yield of the three cowpea varieties were not significantly different (table 5). the pod weights of the three varieties were not significantly different. the average weight of the pods per plant is 32.80 – 36.47 g. the average length of the pods was 20.09 – 21.21 cm. the average number of seeds per pod in the three varieties ranged from 10.42 to 10.67. the highest production variables including dry seed weight and 100-seed weight were produced by the “albina”. the average dry seed weight was 20.28 – 23.98 g. weight of 100-seeds in the three varieties were significantly different; “albina” produced the highest 100-seed weight compared to the other two cowpea varieties. the average weight of 100-seeds in the three varieties ranged from 13.08 – 14.16 g (table 5). this is in line with the research of rabbani (2021) that the weight of 100 seeds of “albina” is higher than the “uno”. the average 100-seed weight ranged from 13.33 to 14.37 g. the average weight of 100 soybean seeds of the “anjasmoro” and “grobogan” varieties ranges from 14.40 – 18.23 g (atmaja, 2020), so cowpeas can be used as an alternative to soybeans. table 6 shows that variety has no significant effect on cowpea productivity. the productivity of “arghavan” is 0.87-ton.ha¹־, “albina” 0.88-ton.ha¹־, and “uno” 0.79 ton.ha¹־. the low productivity is thought to be due to the low plant population, which in this study was 33,333 per hectare from the spacing of 60 cm x 50 cm. the results of rabani’s research (2021) also showed that the productivity of cowpea “albina” was 0.84 ton.ha¹־ and “uno” was 0.80 ton.ha¹־ with a plant population of 40,000 per hectare. the low productivity of cowpeas in this study could table 3. photosynthesis rate and stomatal conductance of the three cowpea varieties treatment photosynthetic rate (μmol.m⁻².s⁻1) stomatal conductance (μmol.m⁻².s⁻1) 7 wap 10 wap 7 wap 10 wap “arghavan” 31.77 17.01 0.70 0.28 “albina” 29.20 19.79 0.61 0.30 “uno” 30.09 19.27 0.74 0.30 hsd 5% ns ns ns ns note: ns = not significant according to orthogonal polynomial contrast at 5%. wap = weeks after planting. table 4. growth rate and net assimilation rate of the three cowpea varieties cowpea variety crop growth rate(g.cm-2 per day) net assimilation rate (g.cm-2 per day) “arghavan” 19.50 3.28 “albina” 21.84 3.50 “uno” 24.77 3.87 hsd 5% ns ns evaluation of source and sink capacity of new cowpea varieties .......... journal of tropical crop science vol. 10 no. 1, february 2023 www.j-tropical-crops.com 43 be caused by the low intensity of sunlight and too high humidity due to the high and prolonged rainfall intensity during the study period. liana (2019) also reported that the low cowpea production (0.32 ton. ha¹־) was caused by the high rainfall during the trial period. according to gardner et al. (1991), the yield component is strongly influenced by the growing management, technology, genotypes, and environment, and environmental factors strongly affect the ability of plants to achieve their genetic potential. correlation between characters based on the measured characteristics including the rate of photosynthesis, plant growth rate, net assimilation rate, and yield components, the correlation test results are obtained as shown in table 7. the correlation coefficient values are classified into several categories, i.e. very low (0.00 – 0.19), low (0.20 – 0.39), moderate (0.40 – 0.59), strong (0.60 – 0.79), very strong (0.80 – 1.00). based on correlation analysis, the characters measured in this study did not correlate much with each other, neither the rate of photosynthesis, the rate of plant growth, the rate of net assimilation, nor yield components. only dry seed weight was closely correlated with productivity (0.999). this shows that the amount of dry weight of the seeds produced affects the productivity. the measured characters were negatively correlated with productivity, presumably due to the large population of plants, so high shade levels resulted in inhibited pod filling, which shows a high source capacity while the resulting sink is low. table 5. yield components of the three cowpea varieties treatment pod weight per plant (g) pod length (cm) number of seeds per pod seed weight per plant (g) 100-seeds weight (g) “arghavan” 35.83 21.21 10.67 22.49 13.18b “albina” 36.47 20.19 10.50 23.98 14.16a “uno” 32.80 20.09 10.42 20.28 13.08b hsd 5 % ns ns ns ns * note: ns = not significant according to orthogonal polynomial contrast at 5%. * = significantly different at the 5% level. table 6 cowpea productivity treatment productivity (ton.ha¹־) “arghavan” 0.87 “albina” 0.88 “uno” 0.79 hsd 5% ns note: ns = not significant according to orthogonal polynomial contrast at 5%. table 7. correlation analysis between plant characters pr-1 pr-2 cgr pod dry weight seed dry weight 100-seed weight pr-1 pr-2 cgr -0.259 0.341 pod dry weight 0.080 -0.226 -0.286 seed dry weight -0.027 -0.008 -0.473 -0.011 100-seed-weight -0.039 0.242 -0.122 -0.277 0.384 productivity -0.025 -0.012 -0.459 -0.023 0.999 0.401 notes: pr-1 = photosynthetic rate at 7 weeks after planting, pr-2 = photosynthetic rate at 10 weeks after planting, cgr = crop growth rate, nar = net assimilation rate. journal of tropical crop science vol. 10 no. 1, february 2023 www.j-tropical-crops.com 44 faza yasmin saidah, heni purnamawati, iskandar lubis conclusion the three cowpea varieties in this study, “arghavan”, “albina” and “uno”, showed no differences in the source-sink capacity and production per plant. the difference was only in the weight of 100-seeds where the “albina” had the highest 100-seed weight of 20.33 g. references atmaja, i.s.f. (2020). “effect of nitrogen fertilization on physiology and characteristics of seed filling rate of several soybean varieties”. bogor, bogor agricultural institute. [balitkabi] research institute for legumes and tubers. (2005). “description of superior varieties of legumes and tubers”. malang research institute for legumes and tubers. [bps] central bureau of statistics. (2019). “soybean imports by main country of origin”. http://www. bps.go.id. [ november 20, 2020]. boukar o., bhattacharjee r., fatokun c., kumar p, and gueye, b. (2013). cowpea in “genetic and genomic resources of grain legume improvement” (m. singh, h.d. upadhyaya, and i.s. bisht, eds.). 1st ed. p 137–156. london, elsevier. boukar, o., fatokun, c.a., roberts, p.a., abberton, m., huynh, b.l., close, t.j., kyai-boahen, s., higgins, t.j.v, and ehlers, j.d. (2015). cowpea in “grain legumes” (a.m.d. ron, ed.). pp. 219250. springer. new york. doi: 10.1007/978-14939-2797-5_7. el naim, a.m,, and jabereldar, a.a. 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[september 13, 2020] effects of gibberellin on physical and chemical quality of oil palm .......... journal of tropical crop science vol. 10 no. 2, june 2023 www.j-tropical-crops.com 87 effects of gibberellin on physical and chemical quality of oil palm (elaeis guineensis jacq.) fresh fruit bunches imelya ramanisa 0000-0002-3569-6939, sudradjat*b 0000-0001-5824-8795, deden saprudinc 0000-0002-7758-9744 a department of agronomy and horticulture, graduate school, faculty of agriculture, ipb university, bogor 16680, indonesia b department of agronomy and horticulture, graduate school, faculty of agriculture, ipb university, bogor 16680, indonesia c department of biochemistry, graduate school, faculty of mathematic and natural science, ipb university, bogor 16680, indonesia *corresponding author; sudradjat@apps.ipb.ac.id research article abstract the quality of crude palm oil (cpo) is influenced by the quality of fresh fruit bunches, crop culture, and postharvest handling. a delay in fruit processing can cause physical damages to the fresh fruit bunches. gibberellic acid (ga3) can potentially reduce the physical damage due to delayed processing of the fresh fruit bunches. our study aims to determine how ga3 affects the physical and chemical quality of oil palm fresh fruit bunches. the fresh fruit bunch samples were collected from the ipb-cargill palm oil education and research, jonggol, bogor, indonesia. this study used a randomized complete block design that consisted of four concentrations of ga3: 0, 12.5, 25 and 37.5 ppm. ga3 application reduced fruit loss, respiration rate, and maintain fruit moisture and firmness, increased the oil content, and stabilize the free fatty acid content. ga3 concentration of 12.5 ppm is the optimal concentration. based on the correlation analysis, fruit softness has a strong correlation with free fatty acids. keywords: crude palm oil, concentration, fresh fruit bunches, respiration. introduction palm oil (elaeis guineensis jacq.) is a very important crop in indonesia. it is evident from the area of oil palm plantations which continues to increase every year, starting in 2012 with only 9,572,715 ha up to 15,380,981 ha in 2022 (ditjenbun, 2022). the increase in area is in line with the increase in oil palm production. production data for 2012 showed that the total crude palm oil (cpo) production was 26,015,518 tons, while in 2020 production was 48,235,405 tons (ditjenbun, 2022). cpo is crude palm oil that is produced from extraction or from the pressing process of palm fruits and has not been refined. cpo is generally used as a raw material for a variety of products including cosmetics, chemicals, cooking oil, margarine, chocolate, ice cream, biscuits, and animal feed. in addition, cpo is an alternative biodiesel fuel (depperin, 2007). fresh fruit bunches (ffb) of oil palm are susceptible to bruising and other physical damages during harvest and at all stages of postharvest handling. the damage to the oil palm fruit results in an accelerated hydrolysis process so that the levels of free fatty acids (ffa) increase, whereas cpo with high ffa is considered as low-quality (maulana and susanto, 2015). the high content of ffa in cpo will cause various losses due to low refining value resulting in rancidity and odour (fajri and ihsan, 2019). the presence of oil ffa can cause side reactions through alkaline-catalyzed transesterification and can inhibit ester production and glycerol separation (suriaini et al., 2021). according to sni, the standard quality palm oil has an ffa content of <5% (sni 01-29012006). good quality ffb should have an oil content of 22.1% to 22.2% with an ffa content of 1.7% to 2.1% during processing (depperin, 2007). oil palm fruit damages can occur during harvesting, transportation, loading and unloading. several factors can accelerate the formation of ffa after the fruit bunches were harvested e.g., fruits injured by impact or hit by harvesting tools; drops of loose journal of tropical crop science vol. 10 no. 2, june 2023 www.j-tropical-crops.com 88 imelya ramanis, sudradjat, deden saprudin fruits; delays due to transportation, and delayed fruit collection (pahan, 2008). other factors that affects the delay in processing including shortage of manpower, particularly during periods of high production (april to july), and during long holidays. therefore, it is crucial to streamline the postharvest handling to reduce physical damage of the ffb. one method to overcome this problem is to apply growth regulator gibberellic acid (ga3) immediately after ffb are harvested (sudradjat, 2017). gibberellic acid has been widely used to inhibit fruit ripening, and studies on the effect of ga3 on the physical quality of oil palm ffb was reported by sudradjat in 2017. the current study is a methodological improvement from the previous study and was conducted on the actual storage condition of oil palm fruits in the field. sudradjat et al. (2021) showed that the application of ga3 at 25 ppm can keep the physical quality of ffb by reducing fruit respiration rates, fruit weight loss, and by maintaining fruit mesocarp softness. the current study aims to determine the effect of ga3 in maintaining the physical and chemical quality of fresh oil palm fruits, and to determine the correlation between the measured variables. material and methods the study was conducted at the ipb-cargill palm oil education and research station, jonggol, bogor, indonesia, the postharvest laboratory of the department of agronomy and horticulture ipb, and the biochemistry laboratory of the department of biochemistry of ipb from january to march 2022. the study used ffb of the “d×p dami mas” variety from the ipb-cargill oil palm plantation. ga3 hormone powder 10% (sun neo), pp indicator solution and 0.1n naoh were used for field and laboratory works. this study was arranged using a randomized complete block design with four ga3 concentrations, i.e., 0 ppm as control or g0, 12.5 ppm as g1, 25 ppm as g2, and 37.5 ppm as g3) repeated three times. each experimental unit consisted of 6 ffb, totalling 72 ffb. ffb used was fraction 2 with an average weight of 15-20 kg. harvesting was conducted in the morning to reduce the transpiration rates. the harvested ffb was transported to an open field to dry. ga3 powder was dissolved in water to make the above concentrations. ga3 solution was sprayed to ffb that had been harvested at an approximate volume of 500 ml using a sprayer on the surface of the bunches. oil extraction was carried out using a hydraulic bottle jack (haisbuan, 2020) measurement of physical quality consisted of the weight loss of fresh fruit bunches (%), fruit loss (kernel), and fruit mesocarp softness (mm.g-1.sec1) using penetrometer stanhope-seta, followed by physiological observations of fruit which was the fruit respiration rate (ml.co2 -1.kg-1.hour-1) using a combustible gas detector xp-3140, mesocarp water content (%), and free fatty acid levels (%). all variables were measured at 6 daa except for free fatty acid levels which were measured at 1, 3, and 5 daa. collected data were analyzed by analysis of variance (anova) followed by the duncan multiple range test (dmrt) 5% for the significance. correlation analysis was done between variables. the software used was microsoft office excel 2010, r studio ver. 9.1. and sas 9.1 portable. in addition, a correlation test was also carried out between variables at 5 daa, this was because the ffa was analyzed only up to 5 daa. the correlation analysis was conducted with a simple correlation according to pearson (saidah et al., 2023) as follows: manpower, particularly during periods of high production (april to july), and during long holidays. therefore, it is crucial to streamline the postharvest handling to reduce physical damage of the ffb. one method to overcome this problem is to apply growth regulator gibberellic acid (ga3) immediately after ffb are harvested (sudradjat, 2017). gibberellic acid has been widely used to inhibit fruit ripening, and studies on the effect of ga3 on the physical quality of oil palm ffb was reported by sudradjat in 2017. the current study is a methodological improvement from the previous study and was conducted on the actual storage condition of oil palm fruits in the field. sudradjat et al. (2021) showed that the application of ga3 at 25 ppm can keep the physical quality of ffb by reducing fruit respiration rates, fruit weight loss, and by maintaining fruit mesocarp softness. the current study aims to determine the effect of ga3 in maintaining the physical and chemical quality of fresh oil palm fruits, and to determine the correlation between the measured variables. material and methods the study was conducted at the ipb-cargill palm oil education and research station, jonggol, bogor, indonesia, the postharvest laboratory of the department of agronomy and horticulture ipb, and the biochemistry laboratory of the department of biochemistry of ipb from january to march 2022. the study used ffb of the “d×p dami mas” variety from the ipb-cargill oil palm plantation. ga3 hormone powder 10% (sun neo), pp indicator solution and 0.1n naoh were used for field and laboratory works. this study was arranged using a randomized complete block design with four ga3 concentrations, i.e., 0 ppm as control or g0, 12.5 ppm as g1, 25 ppm as g2, and 37.5 ppm as g3) repeated three times. each experimental unit consisted of 6 ffb, totalling 72 ffb. ffb used was fraction 2 with an average weight of 15-20 kg. harvesting was conducted in the morning to reduce the transpiration rates. the harvested ffb was transported to an open field to dry. ga3 powder was dissolved in water to make the above concentrations. ga3 solution was sprayed to ffb that had been harvested at an approximate volume of 500 ml using a sprayer on the surface of the bunches. oil extraction was carried out using a hydraulic bottle jack (haisbuan, 2020) measurement of physical quality consisted of the weight loss of fresh fruit bunches (%), fruit loss (kernel), and fruit mesocarp softness (mm.g-1.sec-1) using penetrometer stanhope-seta, followed by physiological observations of fruit which was the fruit respiration rate (ml.co2-1.kg-1.hour-1) using a combustible gas detector xp-3140, mesocarp water content (%), and free fatty acid levels (%). all variables were measured at 6 daa except for free fatty acid levels which were measured at 1, 3, and 5 daa. collected data were analyzed by analysis of variance (anova) followed by the duncan multiple range test (dmrt) 5% for the significance. correlation analysis was done between variables. the software used was microsoft office excel 2010, r studio ver. 9.1. and sas 9.1 portable. in addition, a correlation test was also carried out between variables at 5 daa, this was because the ffa was analyzed only up to 5 daa. the correlation analysis was conducted with a simple correlation according to pearson (saidah et al., 2023) as follows: ��� � 𝑛𝑛ʃ𝑋𝑋𝑋𝑋𝑛𝑛𝑋𝑋 � �ʃ𝑋𝑋𝑋𝑋��ʃ𝑛𝑛𝑋𝑋�√�𝑛𝑛ʃ𝑋𝑋𝑋𝑋� � �𝑋𝑋𝑋𝑋����𝑛𝑛ʃ𝑛𝑛𝑋𝑋� � �𝑛𝑛𝑋𝑋��� r values of < 0 indicates that each variables have a close negative correlation, while the value of r > 0 indicates that each variables has a close positive correlation. the values of r = -1 ≤ r ≤ 1 (saidah et al., 2023). result and discussion weight loss of fresh fruit bunches ga3 treatment significantly reduced fruit weight loss starting one day after application (daa, table 1). the highest accumulative percentage of fruit weight loss at 6 daa occurred in the control treatment, which was 29.82%. the treatment with the lowest weight loss was ga3 at 25 ppm, which was 23.45%. r values of < 0 indicates that each variables have a close negative correlation, while the value of r > 0 indicates that each variables has a close positive correlation. the values of r = -1 ≤ r ≤ 1 (saidah et al., 2023). result and discussion weight loss of fresh fruit bunches ga3 treatment significantly reduced fruit weight loss starting one day after application (daa, table 1). the highest accumulative percentage of fruit weight loss at 6 daa occurred in the control treatment, which was 29.82%. the treatment with the lowest weight loss was ga3 at 25 ppm, which was 23.45%. weight loss (wl) occurs due to physico-chemical changes in the fruits, including the loss of water during storage until the fruits ripened (sutrisno et al., 2008; aditama, 2014, tarigan et al., 2019). fruit fall fruit falling (ff) is reflected by the number of fall kernels (table 2). fruit ripening is influenced by lipase enzyme that plays important roles in oil synthesis, thus affecting free fatty acid (ffa) levels in the fruits. the fallen fruits contain higher ffa than the intact fruits effects of gibberellin on physical and chemical quality of oil palm .......... journal of tropical crop science vol. 10 no. 2, june 2023 www.j-tropical-crops.com 89 (morcillo et al., 2013). our study demonstrated that ga3 suppressed fruit loss from 2 to 6 daa, whereas the control consistently had the greatest number of fall kernels (table 2). ga3 at 12.5 or 25 ppm were effective to maintain kernels to remain intact until 6 daa. in the final observation, the fallen fruits from ga3 at 12.5 or 25 ppm was 378 and 374 kernels, respectively, whereas without ga3 it was 399 kernels (table 2). according to manurung et al., (2022) application of ga3 at 15 ppm can reduce fruit loss rates. the process of fruit loss (abscission) is related to the ratio of auxin and ethylene content in the abscission zone, with low auxin and high ethylene resulted in senescence or abscission (taiz and zeiger, 2006). ethylene induces the synthesis and secretion of cell wall-degrading hydrolases. hydrolase enzyme may increase due to rna transcription and cause damage to the cell walls of the abscission zone (salisbury and ross, 1996). ga3 roles is to delay the formation of the separating layer in the abscission zone (tuan et al., 2013) and to promote carbohydrate mobilization to fruits, hence reducing fruit loss (bons et al., 2015). fruit respiration rate table 3 showed that ga3 treatment can reduce fruit respiration rate (rr), except at 2-3 daa. the highest respiration rate indicates the climacteric peak. according to aditama (2014), climacteric fruits including oil palm experience a sudden increase in respiration rate before ripening, and the respiration rate decreases gradually after harvesting. ga3 can effectively delay fruit maturity by reducing the respiration rate, so the ffb can be preserved for longer during storage before further processing. almost all ga3 treatments in this study reduced respiration rates compared to the control. fruit respiration rate is influenced by various internal and external factors; included in the internal factors are stages of fruit development, skin layer, cohesiveness of the cells, and fruit’s physical damage table 1. effect of ga3 application on fresh fruit bunch weight loss ga3 (ppm) fruit bunch weight loss with ga3 application at 1 2 3 4 5 6 days after application 0 kg 22.74 22.04 20.26 18.66 18.40 17.39 16.11 (%) 3.14a 11.00a 18.16a 19.27a 23.94a 29.82a 12.5 kg 22.49 22.16 20.60 18.99 18.16 17.10 16.63 (%) 1.51b 8.25a 15.45a 18.92a 23.51a 25.60a 25.0 kg 22.74 22.13 20.51 19.23 18.82 17.81 17.44 (%) 2.71a 9.84a 15.48a 17.31a 21.83a 23.45a 37.5 kg 22.42 21.74 20.66 19.24 18.80 17.36 16.29 (%) 3.10a 7.92a 14.43a 16.19a 22.73a 27.48a dmrt ** ns ns ns ns ns note: means in the same column followed by the same letter are not significantly different based on duncan multiple range test (dmrt); *=significant at α=0.05; **= highly significant at α=0.01; ns= not significant. table 2. the effect of ga3 application on the loss of fresh fruit bunches ga3 (ppm) number of fall kernels at 1 2 3 4 5 6 days after application 0 31a 131a 233a 274a 376a 399a 12.5 27a 113b 193b 267a 359b 378b 25.0 25a 110b 192b 269a 350b 374b 37.5 27a 76c 173c 245b 328c 382ab dmrt 5% ns ** ** * ** * note: means in the same column followed by the same letter are not significantly different based on dmrt at α=0.05. daa=days after ga3 application. journal of tropical crop science vol. 10 no. 2, june 2023 www.j-tropical-crops.com 90 imelya ramanis, sudradjat, deden saprudin (ahmad, 2013). respiration rate is also highly affected by ethylene, that hormone is controlled by its immediate precursor, 1-aminocyclopropane1-carboxylic (maduwanthi and marapana, 2019). external factors that affect fruit respiration rates include ambient temperature, humidity, and air composition (ahmad, 2013). research by marlina et al. (2014) demonstrated that salak fruits placed at different temperatures had different respiration rates; the higher the temperature, the higher the respiration rate. fruit mesocarp water content ga3 significantly affect fruit mesocarp water content (wc) at 3 daa (table 4). ga3 at 12.5 ppm was relatively better for reducing the water content of fruit mesocarp than the other treatments, especially at 4 daa. in contrast, the control consistently had the highest water content. higher ga3 concentrations resulting in higher water content of the fruit mesocarp. hassan et al., (2009) reported that the water content of the fruit is related to the level of maturity of the fruit: ripened fruits have higher the water content. the increase in water content occurs because the respiration rate increases due to higher ethylene production (sutrisno et al., 2008). according to mulyadi et al., (2017), fruit water content is affected by plant genetics, humidity, fruit maturity, and post-harvest treatments. loss of water can cause a decrease in cell turgidity which results in shrinkage and decrease in the quality of fruits (aji, 2016). fruit mesocarp firmness fruit firmness (ffs) during the ripening changes cell wall composition due to changes in cell turgor (winarno and wirakatakusuma, 1979). ga3 treatment can maintain fruit firmness during the storage period. fruit mesocarp firmness in the ga3 at 12.5 ppm was significantly different than those in other treatments (table 5). according to besada et al. (2008), application of ga3 before and after harvesting can maintain fruit firmness for up to several weeks compared to without ga3. the reduction of fruit firmness may be affected by several factors such as increased water content and ethylene level. acuna and mitcham (2008) reported that the decrease in pear hardness is affected by high ethylene levels and temperatures. it is also related to weight loss, water loss, and transpiration processes which cause the mesocarp to wither and wrinkle (wills et al., 2007). according to wang et al., (2018), increased fruit firmness not only relates to the rate of ethylene production but also the hydrolytic enzyme activities, degradation of pectin, cellulose, and hemicellulose. table 3. effect of ga3 application on fruit respiration rate ga3 (ppm) respiration rate (ml.co2 -1.kg-1.hour-1) 1 2 3 4 5 6 days after application 0 52.77a 34.36a 25.07a 33.94a 26.98a 21.80a 12.5 40.21b 33.42a 22.55a 17.94b 22.34b 14.05c 25.0 36.45c 32.26a 24.24a 17.44b 26.31a 18.75b 37.5 52.08a 31.98a 22.40a 19.65b 25.49a 18.27b dmrt 5% ** ns ns ** * ** note: means in the same column followed by the same letter are not significantly different based on dmrt at α=0.05. table 4. effect of ga3 application on fruit mesocarp water content ga3 (ppm) fruit mesocarp water content (%) at 1 2 3 4 5 6 days after application 0 33.19a 30.54a 32.00a 33.18a 36.00a 39.31a 12.5 29.32a 30.73a 31.24a 28.11b 30.24b 33.49b 25.0 30.37a 31.56a 31.29a 32.15a 31.10b 38.37a 37.5 32.29a 31.67a 30.34a 33.26a 32.73ab 36.82a dmrt 5% ns ns ns ** * * note: means in the same column followed by the same letter are not significantly different based on dmrt at α=0.05; daa: days after ga3 application effects of gibberellin on physical and chemical quality of oil palm .......... journal of tropical crop science vol. 10 no. 2, june 2023 www.j-tropical-crops.com 91 free fatty acid level ffa content is a quality indicator used to assess cpo quality. cpo quality decreases as ffa increases. ga3 treatment maintains cpo quality by reducing ffa, as shown in table 6 that the control treatment had the highest ffa levels on each observation day. increasing concentrations of ga3 reduced the ffa, and the differences between ga3 concentrations were significant at 5 daa (table 6). our study demonstrated that g1 (ga3 at 12.5 ppm) is the best treatment and the mesocarp ffa with this treatment met the sni01-2901-2006 standard of < 5%. correlation between variables there are several categories of correlation coefficient value e.g., very low (0.00 0.19), low (0.20 0.39), moderate (0.40 0.59), strong (0.60 0.79), and very strong (0.80 – 1.00). the results in table 7 showed that the measured variables that have strong correlation are fruit mesocarp firmness, fruit respiration rate, mesocarp water content, and ffa levels. the correlation between these variables was positive/ unidirectional, which means that if one variable increases, the other variable will also increase. based on table 7, ffa levels and fruit respiration rate have a moderate unidirectional correlation. the strong, unidirectional relationship between variables consisted of ffs-wc, ffs-rr, and ffawc. water serves as a catalyst in fat hydrolysis increase, so increases in the water content in the fruit mesocarp will promote hydrolysis that results in the increases in ffa levels as a breakdown of triglycerides by the lipase enzymes (aji, 2016). ffa and ffs have a very strong unidirectional relationship: an increase in the fruit softness during the storage period indicates fruit ripening (jiang et al., 2020), and ffa levels increased with fruit maturity (hasibuan, 2020). conclusion ga3 application can maintain the physical and chemical quality of oil palm fresh fruit bunches. ga3 at 12.5 ppm is the recommended concentration to minimize losses due to delays or waiting time in processing the oil palm fruit through reduction of fruit loss, fruit respiration rate, fruit mesocarp water content, and maintaining fruit firmness up to 6 daa and ffa levels up to 5 daa, hence prolonged the ffb shelf life. references acuna, m.v., and mitcham, e. j. (2008). ripening of european pears: the chilling dilemma. postharvest biology and technology 49, 187– 200. table 5. effect of ga3 application on fruit firmness between treatments ga3 (ppm) fruit mesocarp firmness (µm-1.g-1.sec-1) 1 2 3 4 5 6 days after application 0 71a 78a 82a 97a 98a 106a 12.5 48b 58b 60b 75b 74c 81c 25.0 64a 78a 79a 84a 87b 94b 37.5 68a 73a 77a 84a 98a 101ab dmrt 5% ** * ** * ** ** note: means in the same column followed by the same letter are not significantly different based on dmrt at α=0.05. table 7. correlation coefficient of ga3 application between variables at 5 daa variable wl ff rr wc ffs ff 0.241ns rr 0.010 ns 0.229 ns wc 0.276 ns 0.280 ns 0.479 ns ffs 0.091 ns -0.125 ns 0.681* 0.664* ffa 0.124 ns 0.093 ns 0.584* 0.651* 0.879* note: *: significant α = 5%, ns: non-significant, wl: weight loss, ff: fruit fall, rr: respiration rate, wc: water content, ffs: fruit firmness, ffa: free fatty acid. journal of tropical crop science vol. 10 no. 2, june 2023 www.j-tropical-crops.com 92 imelya ramanis, sudradjat, deden saprudin aditama, f.z. 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(2018). fruit softening: revisiting the role of pectin. trends plant science 23, 302-310. wills, r., mcglasson, b., graham, d., and joyce, d. (2007). “postharvest: an introduction to the physiology and handling of fruit, vegetables and ornamentals”. 227 pp. university of new south wales press. winarno, f.g., and wirakartakusuma, m.a. (1979). “fisiologi lepas panen”. 97 pp. sastra hudaya. evaluation of the efficacy of selected insecticides against wheat aphids on .......... journal of tropical crop science vol. 10 no. 2, june 2023 www.j-tropical-crops.com 133 efficacy of selected insecticides against wheat aphids on irrigated wheat in north western amhara, ethiopia mequanint andualem mekonnen* 0000-0002-8294-2354, gebremariam asaye emrie 0000-0001-7832-7749 adet agricultural research center p.o. box 08, bahir dar, ethiopia. *corresponding author; email: mequanint09@gmail.com abstract aphid infestations cause significant losses in wheat production. the experiment was conducted at the koga irrigation site during 2021 to evaluate the efficacy of commercial insecticides for the management of wheat aphids. seven types of insecticides with unsprayed control were laid in a completely randomized block design with three replications. the result showed a reduced aphid infestation with maximum grain yield were from dimethoate, imidacloprid, lambda-cyhalotrin, profenfos + lambdacyhalothrin, imidacloprid + lambda-cyhalothrin, and profenofos-sprayed plots. however, the greater net benefit and marginal rate of return were provided by the applications of imidacloprid and dimethoate insecticides. the economically feasible insecticide application can be considered as an effective aphid management practice in wheat production. keywords: wheat, aphid, insecticide introduction wheat (triticum aestivum l.) is the most widely cultivated cereal crop in the world, with more than 220 million ha planted annually under wide ranges of environmental conditions (faostat, 2022). the global wheat production amounts to an annual average of around 750 million tons (t) (faostat, 2022). thirty-two (32) african countries produce an average of 26.1 million tons of wheat per year on 9.9 million hectares of land, accounting for only 3.5% of the world wheat production system. among african wheat-producing countries, ethiopia is the secondlargest producer, accounting for about 18.98% of total production on the continent. according to recent estimates, 4.96 million tons of wheat were produced on 1.75 million hectares of land (faostat, 2022). wheat is a major imported commodity all over the african continent. based on an average of five years (2016–2020) of faostat data, 48.2 million tons of wheat were imported into africa every year in the form of grain, flour, and macaroni. the average wheat self-sufficiency in africa is about 17%, indicating that the region is significantly dependent on imports. this makes the continent highly vulnerable to global market and supply shocks. for example, the occurrence of the covid-19 pandemic and the russian-ukraine war significantly disrupted the global wheat trade. the fact that ethiopia has become increasingly dependent on external sources of food supply has become a major concern for policy makers and agricultural researchers. therefore, the question of how to make ethiopia self-reliant in food production has received major attention in recent years. wheat is a key crop for ensuring food security. ethiopia’s wheat production is insufficient to meet domestic demand, forcing the country to import more than 1.6 million metric tons annually. ethiopia’s government intends to reduce imports by boosting domestic production. one of the strategies is to produce wheat under irrigation. remarkable achievements are recorded in the past. however, aphids are a serious pest of irrigated wheat in ethiopia, causing production losses ranging from 15% to 93% depending on variety and season (damte, 2015). it can be managed by planting resistant varieties or by employing cultural practices such as alternate host destruction, adjusting planting dates, and producing a healthy, stress-free crop (by maintaining adequate soil moisture and fertilization) (chemeda, 2015; singh and rajaram, 2002). the varieties released so far, either for rainfed or irrigated wheat production, in ethiopia were developed in the absence of wheat aphid or under an insecticideprotected condition in the presence of the pest (damte, 2015). therefore, in the absence of resistance varieties and the buildup of aphid populations against cultural practices, the management of wheat aphids relies primarily on the application of insecticides. currently, insecticide-based management is the most essential measure for controlling aphids in agricultural production since it can effectively suppress aphid journal of tropical crop science vol. 10 no. 2, june 2023 www.j-tropical-crops.com 134 mequanint andualem, gebremariem asaye populations in a short period of time (luo et al., 2022). hence, the objectives of this study were to evaluate the efficacy of different insecticides on aphids to avoid yield loss in irrigated wheat. materials and methods the experiment was conducted in koga irrigation site during 2021. koga irrigation scheme is located within the abay river basin in mecha woreda in west gojjam zone of amhara region. it is located between 11°20’n _ 11°30’n (latitude) and 37°3’e _37°9’e (longitude) in the west of merawi town, 35 km south of bahir dar. it lies at an altitude of 1979 m.a.s.l. the soil of the study area was dominantly sandy clay soil type. the average ph and organic matter content of the soil were 5.5 and 3.4 g.kg-1, respectively. the annual mean rainfall in the area was about 1507 mm. the average maximum and minimum temperatures were 27.5°c and 12°c, respectively. the monthly average relative humidity (rh) was 58.5%. seven commercial insecticides registered in ethiopia (table 1) with untreated control were laid down in completely randomized block design (rcbd) with three replications. each plot was consisting of 10 rows of 3 m length. the space between furrow, rows, plots and replications were 0.4 m, 0.2 m, 1 m and 1.5 m, respectively. a widely cultivated variety ‘kekeba’ was used as a test variety. planting was done at the mid december with the recommended seeding rate of 125 kg.ha-1. recommended fertilizer rate (92 kg of p2o5 per hectare) was applied during planting. furthermore, the recommended n rate of 138 kg.ha-1 was applied in split at tillering and near booting. weeds were managed through hand weeding. the experimental fields were irrigated regularly with furrow irrigation every 14 days. insecticides were used at the rate of factory recommendation per hectare when the aphid population reaches economic threshold level. the insecticides were sprayed using hand sprayer of 2.5 liter capacity. spraying was started at heading stage of the crop and repeated in every two weeks interval for three times. all sprays were applied when the wind velocity was low to avoid any drift effect from the sprayed plot to neighboring ones. the crop was protected from the infections of foliar fungal disease including rust through application of tilt (propiconazole) and natura (tebuconazole) fungicide based on the factory recommendation rate in all experimental fields uniformly to avoid the yield losses due to disease. data collection aphid count data on aphid population were recorded starting from initial occurrence up to maturity of the crop. numbers of aphids per plant were counted on ten (10) randomly selected plants from each plot with only live aphids counted. aphid count was taken one day before each application of insecticide and after 24 and 72 hours of each insecticides spraying from the base of plant up to the top. number of grains per spike it was determined by counting the number of grains per spike expressed as average of ten randomly selected spikes in a harvestable plot. grain yield per ha (tonnes per ha) at maturity, the dried crop were harvested by the help of sickle from harvestable rows of each plot. wheat bundles of each plot were sun dried and then threshed separately. the grain weight of each plot was recorded in kg by the help of sensitive balance. simultaneously, moisture content of each plot were measured by the help of moisture tester. finally the yield obtained from each plot were converted in to t/ ha and adjusted in to 12.5% grain moisture content. table 1. commercial insecticides used as treatments no. trade name active ingredient recommended rate 1 agro plus imidacloprid + lambda-cyhalothrin 400 ml.ha-1 2 nehisa dimethoate 400 g.l-1 750 ml.ha-1 3 agro-lambacin super 315 ec profenfos 30% + lambda-cyhalothrin 1.5% 400 ml.ha-1 4 hunter abamectin + acetamiprid 500 ml.ha-1 5 profit 72% ec profenofos 750 ml.ha-1 6 gain 20 sl imidacloprid 110 ml.ha-1 7 karate 5% ec lambda-cyhalotrin 400 ml.ha-1 evaluation of the efficacy of selected insecticides against wheat aphids on .......... journal of tropical crop science vol. 10 no. 2, june 2023 www.j-tropical-crops.com 135 one-thousand grain weight (g) 1000 seeds were randomly sampled in a harvested grains of each plot and counted by the help of seed counter. subsequently, the weight of counted seed were measured using analytical balance. all the collected agronomic data were subjected to analysis of variance as suggested by gomez & gomez using sas software (gomez and gomez, 1984). significant means were separated by using lsd method at 5% probability. relative yield losses losses were calculated separately for each of the treatments with different levels of aphid populations, based on the following formula (savary and willocquet, 2014). 3  5 profit 72% ec profenofos 750 ml.ha-1 6 gain 20 sl imidacloprid 110 ml.ha-1 7 karate 5% ec lambda-cyhalotrin 400 ml.ha-1 data collection aphid count data on aphid population were recorded starting from initial occurrence up to maturity of the crop. numbers of aphids per plant were counted on ten (10) randomly selected plants from each plot with only live aphids counted. aphid count was taken one day before each application of insecticide and after 24 and 72 hours of each insecticides spraying from the base of plant up to the top. number of grains per spike it was determined by counting the number of grains per spike expressed as average of ten randomly selected spikes in a harvestable plot. grain yield per ha (tonnes per ha) at maturity, the dried crop were harvested by the help of sickle from harvestable rows of each plot. wheat bundles of each plot were sun dried and then threshed separately. the grain weight of each plot was recorded in kg by the help of sensitive balance. simultaneously, moisture content of each plot were measured by the help of moisture tester. finally the yield obtained from each plot were converted in to t/ha and adjusted in to 12.5% grain moisture content. one-thousand grain weight (g) 1000 seeds were randomly sampled in a harvested grains of each plot and counted by the help of seed counter. subsequently, the weight of counted seed were measured using analytical balance. all the collected agronomic data were subjected to analysis of variance as suggested by gomez & gomez using sas software (gomez and gomez, 1984). significant means were separated by using lsd method at 5% probability. relative yield losses losses were calculated separately for each of the treatments with different levels of aphid populations, based on the following formula (savary and willocquet, 2014). relative yield loss yield of protected plot yield of un protected plot yield of protected plot 100 partial budget analysis the cost and benefit of each treatment were analyzed partially, and marginal rate of return were computed by considering the variable cost available in the respective treatment (cimmyt, 1988). to compare the benefits of insecticide treatment, yield and economic data were collected. the grain yield obtained from each treatment was adjusted downwards by 10%. following harvesting, the total gross benefit of the field was computed by multiplying the wheat grain yield with the local market price (birr 35 per kg). variable costs such as insecticide and labour costs for insecticide application were included. the price of profenfos partial budget analysis the cost and benefit of each treatment were analyzed partially, and marginal rate of return were computed by considering the variable cost available in the respective treatment (cimmyt, 1988). to compare the benefits of insecticide treatment, yield and economic data were collected. the grain yield obtained from each treatment was adjusted downwards by 10%. following harvesting, the total gross benefit of the field was computed by multiplying the wheat grain yield with the local market price (birr 35 per kg). variable costs such as insecticide and labour costs for insecticide application were included. the price of profenfos + lambda-cyhalothrin and lambda-cyhalothrin was similar 3300 etb per litter. while the price dimethoate was 2490 etb per litter. the most expensive pesticide used as a treatment was imidacloprid, which cost 4560 etb per litter. the price of abamectin + acetamiprid and imidacloprid + lambda-cyhalothrin per litter was 3900 and 3930 etb, respectively. in addition, the labour cost per man-day was 200 etb. total variable cost (tvc) is the sum of all variable costs associated with insecticides and labour. the net benefit (nb) is calculated by subtracting all variable costs from the gross benefit, as follows: nb = gb -tvc. for dominance analysis, the variable costs that varied for each treatment were ranked in ascending order. the marginal rate of return (mrr) was calculated for two non-dominated treatments (say 1 and 2) using the formula: [change in net benefit (nb2−nb1)/change in tvc (tvc2−tvc1)] × 100. marginal rate of return (mrr) provides the value of benefit obtained per the amount of additional cost incurred percentage. results and discussions insecticides and wheat aphid population aphid infestations on wheat plants first appeared in the first week of march, approximately 65 days after sowing (das), when the crop reached flowering stage (zadoks stage 65), and the population continued to grow until the first week of april on unsprayed control plot (figure 1). damte (2015) reported similar results, with the rwa population rapidly increasing from the second week of march and peaking in the last week of march on unsprayed wheat. the main reason for this is most likely a rise in temperature in march (figure 2). temperature is a key environmental variable that regulates insect population growth through its influence on development, fecundity, and survival (deutsch et al., 2008; wang et al., 2021). the number of aphids per plant counted prior to any insecticide spray was between 5 and 10, except in the abamectin + acetamiprid treated plot (figure 1). however, aphid count data collected after the first spray revealed a reduced trend, with approximately 1 aphid per plant in dimethoate, lambda-cyhalotrin, and imidacloprid + lambda-cyhalothrin sprayed plots (figure 1). aphid numbers increase on unsprayed plots and ineffective insecticide (abamectin + acetamiprid) sprayed plots during the second phase of assessment (2 weeks after the first spray). even though the aphid population increased from counts conducted after first spray, the numbers of aphids were reduced (between 1 and 5) on effective insecticide-sprayed plots after the second spray (figure 1). similar trends were observed in the third phase of assessment (one month later than the first spray). as with all others, aphid numbers per plant are high before spraying and reduced after spraying. the difference is that aphid populations reach a peak (40) in both the unsprayed and abamectin + acetamiprid sprayed plots (figure 1). generally, the aphid count data showed that all the insecticides except abamectin + acetamiprid were superior to untreated control in all date of assessments. however, the mean number of aphids/ tiller differed between effective treatments across all assessments. dimethoate, lambda-cyhalotrin, and imidacloprid + lambda-cyhalothrin were found to be the most effective. profenfos + lambda-cyhalothrin, profenfos, and imidacloprid were the next best. our findings confirm the results of ahmad et al. (2016), who found that dimethoate and imidacloprid have excellent insecticidal activity against wheat aphids. shafique et al. (2016) used lambda-cyhalothrin and imidacloprid insecticides as foliar applications against wheat aphids and found that both insecticides effectively journal of tropical crop science vol. 10 no. 2, june 2023 www.j-tropical-crops.com 136 mequanint andualem, gebremariem asaye controlled aphids. asif et al. (2017) also reported that imidacloprid, lambda-cyhalothrin and profenofos + cypermethrin has outstanding insecticidal activity against sucking insect pest complex of cotton. the effects of aphid on grain yield, one thousand seed weight, and grains per spike insecticide application had a significant (p < 0.001) effect on the grain yield and one-thousand seed weight (table 2). however, grains per spike were not significantly affected by wheat aphid infestation, even though there were numerical differences between treatments. the highest yield (3.8 t.ha-1) of wheat was obtained with the treatment dimethoate (table 2). however, except for abamectin + acetamiprid, the yields obtained from dimethoate were not significantly different from the rest of the insecticides (table 2). the lowest wheat yield (1.8 t.ha-1) was obtained from unsprayed control. statistically, the wheat yield (2.5 t.ha-1) produced from experimental plots treated with abamectin + acetamiprid was not significantly figure 1. the effects of insecticides on aphid population. bfs= before the first spray; 24 hafs= 24 hours after the first spray; 72 hafs= 72 hours after the first spray; bss= before the second spray; 24 hass= 24 hours after the second spray; 72 hass= 72 hours after the second spray; bts= before the third spray; 24 hats= 24 hours after the third spray; 72 hats= 72 hours after the third spray.                          figure 2. minimum (---) and maximum ( ) temperatures of the study location during 2021                           evaluation of the efficacy of selected insecticides against wheat aphids on .......... journal of tropical crop science vol. 10 no. 2, june 2023 www.j-tropical-crops.com 137 different from the unsprayed control (table 2). the result is in agreement with those of ahmad et al. (2016), who reported the highest wheat yield from imidacloprid and dimethoate insecticide applications. in their study, shafique et al. (2016) also reported that imidacloprid and lambda-cyhalothrin applications improved wheat yield compared to an unsprayed control. similar to grain yield, dimethoate (44.3 g) and the unsprayed control (29.6 g) yielded the highest and lowest thousand seed weights, respectively (table 2). one thousand seed weights obtained from imidacloprid (42.9 g), lambda-cyhalotrin (43.5 g), and combinations of imidacloprid plus lambda-cyhalothrin (44.0 g) treated plots did not differ significantly from dimethoate (table 2). generally, the unsprayed control had a lighter seed weight, fewer grains per spike, and a lower grain yield. as a result, the avoidable loss in grain yield was 51.8%. damte (2015) reported a reduction in one thousand seed weight and grains per spike, resulting in a 15 to 93% percentage yield loss due to russian wheat aphid, depending on variety and season. partial budget analysis the partial budget analysis indicates that some insecticide treatments resulted in high net benefits and marginal rates of return (table 4). among the insecticides, imidacloprid and dimethoate provided the greatest net benefit and marginal rate of return. although the net benefit calculated from other insecticide treatments is lower than that of imidacloprid and dimethoate, it is two times greater than the unsprayed control (table 4). table 2. the effects of different insecticides against wheat aphid on yield and yield components of wheat insecticides gps tsw (g) gy(t/ha) ryl abamectin + acetamiprid 41.1 33.3d 2.5bc 33.3 dimethoate 400 g.l-1 45.0 44.3a 3.8a 0.0 imidacloprid + ambdacyhalothrin 37.3 44.0ab 3.3ab 12.3 imidacloprid 38.3 42.9abc 3.5a 7.9 lambda-cyhalotrin 38.1 43.5abc 3.6a 5.3 profenfos 30% + lambda-cyhalothrin 1.5% 42.3 41.6c 3.4a 9.7 profenofos 38.1 41.8bc 3.3ab 12.3 untreated 36.3 29.6e 1.8c 51.8 cv (%) 9.1 3.2 14.6 significance ns ** ** note:values followed by the different letters within one column are highly significant (**) according to lsd at 0.05; ns = notsignificant; gps= grain per spike; tkw= thousand seed weight in gram; gy= grain yield in ton per hectare; ryl= relative yield loss; cv = coefficient of variation. table 3. partial budget analysis of all treatments items adjusted yield (10%) t.ha-1 gross benefit (etb.ha-1) total cost of insecticide (etb.ha-1) total cost of labour total variable cost net benefit mrr% control 1.62 56700 0 0 0 56700 imidacloprid 3.06 107100 501.6 1800 2301.6 104798.4 2089.7 profenfos 30% + lambdacyhalothrin 1.5% 2.97 103950 1320 1800 3120 100830 d lambda-cyhalotrin 2.97 103950 1320 1800 3120 100830 d imidacloprid + lambdacyhalothrin 2.25 78750 1572 1800 3372 75378 d dimethoate 400 g.l-1 3.42 119700 1867.5 1800 3667.5 116032.5 13757.8 abamectin + acetamiprid 3.15 110250 1950 1800 3750 106500 d profenofos 3.24 113400 2250 1800 4050 109350 d note: etb= ethiopian birr; t= tone; ha= hectare; mrr= marginal rate of return; d= dominated journal of tropical crop science vol. 10 no. 2, june 2023 www.j-tropical-crops.com 138 mequanint andualem, gebremariem asaye conclusion results in this study demonstrated that the application of insecticides dimethoate, imidacloprid, lambda-cyhalotrin, profenfos + lambda-cyhalothrin, imidacloprid + lambda-cyhalothrin, and profenofos significantly decreased the aphid population and increased yield by 40 to 52% as compared to the untreated control. the results also showed that aphids in the irrigated wheat caused up to 52% grain yield loss if not controlled. despite the fact that the applications of imidacloprid and dimethoate are more profitable, wheat producers can also use lambda-cyhalotrin, profenfos + lambda-cyhalothrin, imidacloprid + lambda-cyhalothrin, and profenofose if they need to rotate insecticides to prevent the emergence of insecticide resistance. references ahmad, h., mir, i. a., sharma, d., srivastava, k., ganai, s. a., and sharma, s. (2016). seasonal incidence and management of wheat aphid, sitobion avenae (f.). indian journal of entomology 78, 148-152. asif, m. u., muhammad, r., akber, w., and tofique, m. (2017). relative efficacy of some insecticides against the sucking insect pest complex of cotton. the nucleus 53, 140-146. chemeda, t. f. (2015). a review on distribution, biology and management practice of russian wheat aphid diuraphis noxia (mordvilko) (homoptera: aphididae). journal of biology, agriculture and healthcare 5, 70-81. cimmyt.1988). “from agronomic data to farmer recommendations: an economics training manual” no. 27. economics program, international maize, and wheat improvement center. damte, t. (2015). occurrence of, and yield response to, russian wheat aphid, diuraphis noxia (hemiptera: aphididae) in irrigated wheat in ethiopia. international journal of tropical insect science 35, 3-10. deutsch, c. a., tewksbury, j. j., huey, r. b., sheldon, k. s., ghalambor, c. k., haak, d. c., and martin, p. r. (2008). impacts of climate warming on terrestrial ectotherms across latitude. proceedings of the national academy of sciences 105, 6668-6672. food and agriculture organization of the united nations. (2022). “fao statistical database”. https://www.fao.org/faostat/en/?data#home gomez, k.a., and gomez, a.a. (1984). “statistical procedure for agricultural research”. a wiley interscience publications. luo, k., zhao, h., wang, x., and kang, z. (2022). prevalent pest management strategies for grain aphids: opportunities and challenges. frontiers in plant science 12, 790919. savary, s., and willocquet, l. (2014). “simulation modeling in botanical epidemiology and crop loss analysis”. doi: 10.1094/ phi-a-2014-0314-01 shafique, m. a., ahmed, k. s., haider, n., khan, r. r., and majeed, m. z. (2016). field evaluation of different insecticides against wheat aphid (schizaphis graminum rondani) and comparative yield assessment for different wheat cultivars. academic journal of entomology 9, 1-7. singh, r. p., and rajaram, s. (2002). breeding for disease resistance in wheat. in “bread wheat improvement and production”, p.141-156. fao. rome. wang, y., yan, j., sun, j., shi, w., harwood, j. d., monticelli, l. s., tan z., and chen, j. (2021). effects of field simulated warming on feeding behavior of sitobion avenae (fabricius) and host defense systems. entomology and genetics 41, 567-578. june_2014.cdr journal of tropical crop science (issn 2356-0169; e-issn 2356-0177) is published four-monthly by department of agronomy and horticulture, faculty of agriculture, bogor agricultural university, indonesia. publication details, including instructions for authors and subscription information: www.j-tropical-crops.com any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by the publisher. the accuracy of the content should be independently veri�ed with primary sources of information. the publisher shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the content. permission to make digital or hard copies of part or all of a work published in journal of tropical crop science is granted for personal or educational/classroom use provided that copies are not made or distributed for pro�t or commercial advantage. ©department of agronomy and horticulture, faculty of agriculture, bogor agricultural university, indonesia. all rights reserved. journal of tropical crop science volume number 1 20141 june on the cover the cover image shows sun�owers by darda effendi editorial board krisantini sintho wahyuning ardie sandra a. aziz robert j. hampson satriyas ilyas tri koesoemaningtyas rohana p mahaliyanaarachchi awang maharijaya maya melati roedhy poerwanto bambang sapto purwoko sudarsono muhamad syukur hugo volkaert malcolm wegener managing editor krisantini graphic design syaiful anwar features editor damayanti buchori dadang sisir mitra agus purwito ernan rustiadi short communication tropical and subtropical fruits in india sisir mitra heliconia cultivar registration dave skinner, jan hintze, bryan brunner research articles estimation of genetic parameter for quantitative characters of pepper ( l.)capsicum annuum muhamad syukur, syaidatul rosidah irrigation volume based on pan evaporation and their effects on water use ef�ciency and yield of hydroponically grown chilli eko sulistyono, abe eiko juliana evaluation of commercial sun�ower (helianthus annuus ) cultivars in bogor, indonesia, forl. ornamental and nursery production syarifah iis aisyah, khotimah, krisantini different growth partitioning and shoot production of talinum triangulare treated with organic and inorganic fertilizer sandra ari�n aziz, leo mualim, sitta azmi farchany cloning and characterization of p5cs1 and p5cs2 genes from l. under droughtsaccharum officinarum stress hayati minarsih iskandar, dwiyantari widyaningrum, sony suhandono journal of tropical crop science (issn 2356-0169; issn 2356-0177) is published four-monthly (one volume per year) byedepartment of agronomy and horticulture, faculty of agriculture, bogor agricultural university, ipb darmaga campus, bogor, indonesia 16680. send all inquiries regarding printed copies and display advertising to or to secretary, department ofinfo@j‐tropical‐crops.com agronomy and horticulture; telephone/fax 62-251-8629353. permission to reprint: permission to make digital or hard copies of part or all of a work published in isjournal of tropical crop science granted for personal or educational/classroom use provided that copies are not made or distributed for pro�t or commercial advantage and that copies bear the full citation and the following notice on the �rst page: “copyright department of agronomy and horticulture, faculty of agriculture, bogor agricultural university”. for all other kinds of copying, request permission in writing from head of school, department of agronomy and horticulture of�ce, ipb darmaga campus, bogor, indonesia 16680. © department of agronomy and horticulture, faculty of agriculture, bogor agricultural university. all rights reserved. printed in the republic of indonesia.in s t t t u i b o g o r perta n ia n syarifah iis aisyah* khotimah, krisantini, department of agronomy and horticulture, bogor agricultural university, bogor, indonesia 16680, tel: 62-251-629353; fax: 62-251-629353. *corresponding author; email: syarifahiis@yahoo.com evaluation of commercial sun�ower ( ) cultivarshelianthus annuus l. in bogor, indonesia for ornamental and nursery production abstract a wide range of imported ornamental sun�ower seeds have been marketed in indonesia. a �eld evaluation was conducted on seven sun�ower cultivars to investigate their germination, growth and development in humid tropical environment in bogor, west java, indonesia and to determine their potential uses. most of the tested cultivars, except for 'sungold double' and 'velvet queen', demonstrated good germination and growth. 'eclipse' was highly susceptible to stem rot, which resulted in only 30% of the plants survived. 'little leo', 'teddy bear' and 'sungold double' were naturally compact, dwarf, and produced attractive blooms so they might be suitable as pot plants. 'hallo', 'velvet queen', 'sunburst', and 'eclipse's plant height were more than 100 cm, had long stem and large blooms. these cultivars might be suitable as landscape plants. keywords: morphological characteristics, west java, pot plants introduction sun�ower ( linn., family asteraceae)helianthus annuus is an annual plant that is known for its large and bright yellow color in�orescences. sun�ower is native to north america (whipker et al., 1998) and is widely cultivated for their valuable oil, as cut �owers, and ornamental plants. a sun�ower is actually a composite of hundreds of tiny �owers clustered together. the disk �owers form the center and normally have both male and female components. the outer layer around the disk is ray �orets. helianthus has about 67 species (heiser, 1978) which includes the annuals and the perennials (anonim, 2005). sun�ower is also commonly used in the landscape for borders or screening (schoellhor et al., 2004). commercial cultivars of sun�ower are drought tolerant, need full sun, and are capable of growing in a wide range of soil conditions (schoellhorn et al., 2004). introduction of ornamental plants are necessary for diversity in the urban landscape (suleiman et al, 2009). imported sun�ower seeds, both oil and non-oil, have been marketed in indonesia. however, limited information is available on the hardiness characteristics of imported ornamental sun�ower that were introduced to the warm and wet tropical west java province. west java is the most populated province in indonesia and where most of the urban landscape is growing. field trial evaluation to assess the performance of the newly introduced cultivars, which is an important component before imported seeds are distributed and marketed, are lacking. even though sun�owers are known to have wide adaptability to a wide variety of soils and climatic conditions (warrick, undated) many imported seeds has been reported to have low �eld establishment rate and low crop productivity and disadvantaged the farmers and growers. the objectives of this study were to evaluate the seed germination, growth and development of seven imported ornamental sun�ower cultivars and to produce recommendations for gardeners and nurseries in indonesia, particularly in west java. materials and methods the experiment was conducted at the bogor agricultural university farm at leuwikopo, darmaga (6°24's, 106°33e; elevation 250m) in june to october 2006. the sun�ower cultivars 'little leo', 'teddy bear', 'sungold double', 'hallo', 'velvet queen', 'sunburst' (mr fothergill's, england), and 'eclipse' (australia) were used the temperature ranged from 25-32°c. average during the dry season (march to august) to 23-28°c during the rainy season (september – february). seeds thisused in study were obtained from a commercial seed store in bogor. seeds were soaked in water for about 30 minutes prior to sowing on trays with pasteurized sand and cocopeat (1:1 by volume) media. fifteen-day-old seedlings with a two or three sets of true leaves were transplanted into a 0.5l plastic container with pasteurized soil: coco peat: husk (1:1:1 by volume) media and fertilized with 2g of top-dressed 15n-15p-15k fertilizer. plants were placed under plastic house and were fertilized weekly with 2 g.l 20n-6.5p-12.4k-1 (gandasil d®; kalatham co.) until the plants had �rst visible bud. plant spacing was 30 x 30cm. plants were watered three or four times per week or as needed to keep the media moist. data on germination percentage were collected at ten day after seed sowing. colors of the seed-coat and hypocotyl of each cultivar were recorded. scoring was 55 13 journal of tropical crop science vol. 1 no. 1, 2014june www.j-tropical-crops.com conducted on plant height and stem diameter at the �rst visible �oral buds, the number of in�orescence at anthesis, the number of days from the transplanting date to the �rst visible �oral bud and to anthesis. number of in�orescence per plant, in�orescence and disk �oret diameter were measured at anthesis. sun�ower seeds were harvested at 12 weeks after transplanting, and scoring on 100-seed-weight was conducted after the seeds were dried at room temperature (25 c) for 7 days.° a completely randomized design with 15 single-plant replications was used for this experiment. data obtained were subjected to analysis of variance using the general linear model procedure in minitab version 15 followed by duncan multiple range test (dmrt) at 5%. results 'sungold double' and 'velvet queen' seeds had low germination rate, i.e. 36.6 and 40% respectively (table 1). other cultivars had germination rate of >60% (table 1). the main cause of death was stem rot fungal infection caused by at the seedling stage.sclerotium the height of 'velvet queen', 'sunburst'and 'eclipse' reached > 2m at anthesis whereas 'teddy bear' and 'little leo' were < 1m (table 2). 'little leo' had the earliest time (15 days after transplanting) to the �rst visible �oral bud, whereas 'hallo' had the latest, i.e. 22 days after transplanting (table 2). table 1. germination rate, seed-coat and hypocotyl color of seven sun�ower cultivars. tabel 2. plant height, stem diameter and days to visible floral bud of seven sun�ower cultivars *) 'eclipse', 'hallo' 'velvet queen' had the largestand in�orescence whereas 'teddy bear' had the smallest (table 3). all cultivars except for 'teddy bear' and 'sungold double' have both disk and ray �owers. 'velvet queen' and 'hallo' had the greatest number of in�orescence per plant at anthesis (table 3). 'velvet queen' had the greatest 100-seed-weight whereas 'hallo' had the lowest (table 3). all in�orescence except for 'teddy bear' and 'sungold double's, consist of yellow ray and disk �owers whereas 'teddy bear' and 'sungold double' have yellow ray �owers only. 'sunburst' in�orescences were multicolor (table 4). discussion seed germination all cultivars except for 'sungold double' and 'velvet queen' had good germination rate of > 60% (table 1). 'sungold double' and 'velvet queen' germination rate was 36.6 and 40%, respectively (table 1). all seeds had similar storage environment prior to planting and no physical damages on the seeds were noted. the main cause of young plant death was stem rot caused by sclerotinia infection. the symptoms of the stem rot was determined using a reference on sun�ower diseases gulya et al. (1993).by this fungal has been reported to be a major disease in sun�ower worldwide germination rate (%) 60.0 90.0 36.7 70.0 40.0 66.7 70.0 black grey dark grey black brown light brown black reddish green yellowish green yellowish green red red red red ‘little leo’ ‘teddy bear’ ‘sungold double’ ‘hallo’ ‘velvet queen’ ‘sunburst’ ‘eclipse’ cultivar seed-coat color hypocotyl color 55 s kyarifah iis aisyah, hotimah, krisantini14 plant height (cm) at ‘little leo’ ‘teddy bear’ ‘sungold’ ‘hallo’ ‘velvet queen’ ‘sunburst’ ‘eclipse’ cultivar days to visible floral bud visible floral bud anthesis stem diameter at visible floral bud (mm) 30.5 25.7 44.1 80.8 67.4 84.7 79.9 d d c a b a a 95.7 75.8 127.3 141.1 200.0 204.5 216.4 cd d bc b a a a 15.1 19.6 20.7 22.3 16.6 16.7 21.1 a b bc c a a bc 7.3 5.9 6.2 7.2 7.8 7.2 7.4 b a a b b b b *) values followed by different letters within a column are signi�cantly different according to duncan multiple range test at 5%. journal of tropical crop science vol. 1 no. 1, 2014june www.j-tropical-crops.com worldwide ( karov et al, 2011) andgulya et al., 1993; caused rot in seedlings of a wide range of plants in many agricultural production areas in west java. first symptoms were observed in the form of leave wilting followed by stem and root rot. a study conducted by karov et al. (2011) con�rmed the presence of ascomycetes sclerotinia sclerotiorum sclerotinia(lib.) de bary and minor jagger in the infected sun�ower. the color of seeds and hypocotyl vary with cultivars (table 1). the notable cultivar was 'sunburst' that had red hypocotyl and stem. 'hallo' also had red hypocotyl but it turned green as the plants get older. moh (1971) reported the correlation of seed-coat color with hypocotyl color in beans ( l.) and used this correlationphaseolus vulgaris as an early screening in plant breeding. plant growth and development 'velvet queen', 'sunburst'and 'eclipse' can be classi�ed as tall; their height reached > 2m at anthesis. 'teddy bear' and 'little leo' were dwarf with the height of < 1m (table 2). 'teddy bear' was the smallest of the seven cultivars tested with a stem diameter of less than 6 mm (table 2). 'little leo' had the earliest time to the �rst visible �oral bud, i.e. 15 days after transplanting or about 30 days after seed sowing whereas 'hallo' had the latest, i.e. 22 days after transplanting or about 37 days after seed sowing (table 2). inflorescence characteristics number and size of in�orescences vary among cultivars. 'eclipse', 'hallo' dan 'velvet queen' had the largest in�orescence whereas 'teddy bear' had the smallest (table 3). all cultivars except for 'teddy bear' and 'sungold double' have both disk and ray �owers. 'velvet queen' and 'halo' had the greatest number of in�orescence per plant at anthesis (table 3). 'velvet queen' had the greatest 100-seed-weight whereas 'halo' had the lowest (table 3). all cultivars except for 'teddy bear' and 'sungold double' formed seeds. seed weight is one of the important criteria of seed vigor as it indicates the amount of dry matter in the seed (moshatati and gharineh, 2012). high seed weight correlated with better seedling establishment and growth (moshatati and gharineh, 2012). however, these characters were not included in this study. the color and shapes of ray and disk �ower vary among sun�ower cultivars (table 3). all cultivars except for 'teddy bear' and 'sungold double' have typical outer ray and disk �owers whereas 'teddy bear' and 'sungold double' have �uffy and quilted yellow ray-like �owers only. the color of 'sunburst' in�orescences varied from pale to bright yellow, and some were bicolor yellow and red (table 4). these information are important for both growers and landscapers to create �oral arrangements or landscape. 'little leo', 'teddy bear' and 'sungold double' plants were dwarf; their height was < 100cm and they had attractive bright bloom colors. therefore these cultivars would be tabel 3. disk flower and in�orescence diameter, number of in�orescence/plant, and 100-seed weight 1) 1) values followed by different letters within a column are signi�cantly different according to duncan multiple range test at 5%; 2) 'teddy bear'and 'sungold' did not produce seeds. tabel 4. types and colours of sun�ower in�orescence diameter (cm) ‘little leo’ ‘teddy bear’ ‘sungold’ ‘hallo’ ‘velvet queen’ ‘sunburst’ ‘eclipse’ cultivar no of in�orescence/plant at anthesis 100 seed weight (g)in�orescence disk 10.5 8.8 10.6 13.1 13.3 9.8 13.6 b c b a a b a 5.0 4.5 4.9 3.7 5.6 a a a b a 9.2 8.7 12.9 15.2 16.2 8.3 8.2 c c b a a c c 2.3 1.6 2.4 2.0 2.3 a b a ab a in�orescence types single double double single single single single bright yellow dark yellow dark yellow dark yellow dark red shades of yellow and pink bright yellow green black black black or dark brown black ‘little leo’ ‘teddy bear’ ‘sungold double’ ‘hallo’ ‘velvet queen’ ‘sunburst’ ‘eclipse’ cultivar color diskcolor 55 15e l.)..........valuation of commercial sun�ower (helianthus annuus journal of tropical crop science vol. 1 no. 1, 2014june www.j-tropical-crops.com suitable as bedding or potted plants, possibly without the need to apply growth retardant that is frequently used for su n �ower to obtain short and compact plant s (koutroubas et al 2004, krisantini et al. 2006). double �owers look more attractive than single �owers. therefore 'teddy bear'and 'sungold double' might also be potential for cut �owers. 'halo', 'velvet queen', 'sunburst', and 'eclipse' plants were tall (height > 100cm) and had large in�orescence (table 2). however, 30% of 'sunburst' and 'eclipse' lodged and needed supports at anthesis, likely because the plants were tall and grown in pots rather than on the ground. 'sunburst' had large variation of bloom colors that were different from the yellow bloom sun�ower normally have. 'sunburst'and éclipse'are potentials for �eld-grown landscape plants. conclusion seed germination of and'sungold double' 'velvet queen' in this experiment was poor, i.e. 40% or less so this factor has to be carefully taken into account for commercial production. 'eclipse' appeared to be most susceptible to sclerotinia; 70% or the seedlings were infected and died at the seedling stage. 'little leo', 'teddy bear' and 'sungold double' were dwarf and have attractive bloom color, hence have potentials as ornamental pot plants. 'halo', 'velvet queen', 'sunburst', and 'eclipse' have potentials to be used as landscape plants or cut �owers. references anonym (2005). the biology of l.helianthus annuus biology document 2005-01. plant b i o s a f e t y of�ce, plant product directorate, canadian food inspection agency, ontario, canada. gulya, t., rashid, k., maširević, s., 1997. sun�ower d i s e a s e s “ s u n � o w e r te c h n o l o g y a n di n production” (a. a. schneiter, ed.) pp. 21-65. agronomy monogram 35, asa, cssa, and sssa, madison, wi, usa. heiser, c.b., jr. (1978). taxonomy of andhelianthus origin of domesticated sun�ower. : “sun�owerin science and technology” (j.f. carter, ed.) pp 3153. american society of agronomy, crop science society of america, soil science society of america, usa. karov, i., mitrev, s., masirevic, s., and kovacevik, b. 2011. first appearance of white mould on sun�ower caused by in thesclerotinia minor republic of macedonia. , 19-26.helia 34 koutroubas, s., vssiliou, g., fotiadis, s., alexoudis, c. (2004). response of sun�ower to plant growth regulators. fourth international crop science congress, greece. krisantini, rakhmania, n., rani, i., aisyah, s.i., and sukma, d. (2006). peningkatan keragaan bunga matahari ( l.). dengan aplikasihelianthus annuus retardan. laporan hibah penelitian, departemen agronomi dan hortikultura, ipb, indonesia. moh, c.c. (1971). mutation breeding in seed-coat colors of beans ( l.). ,phaseolus vulgaris euphytica 20 119-125. moshatati, a., gharineh, m.h. (2012). effect of grain weight on germination and seed vigor of wheat. international journal of agriculture and crop sciences , 458-460.4 schoellhorn, r., alfarez, e. and frank, m. (2004). warm climate production guidelines for specialty cut flowers: sun�ower. commercial floriculture update. university of florida, florida, usa. suleiman, m.k., zaman, s., al-dossery, r.r. and jacob, s. (2009). germination studies in horwoodia dicksoniae international journal ofturril. environmental studies , 221-225.66 warrick, b.e. (undated). sun�ower production guide. agronomy publication, texas a&m agrilife research and extension center, st angelo, texas, usa. whipker, b., dasoju, s., and mc call, i. (1998). guide to successful pot sun�ower production. horticulture i n f o r m a t i o n l e a � e t 5 6 2 . d e p a r t m e n t o f horticultural science, north carolina state university, north carolina, usa. 55 s kyarifah iis aisyah, hotimah, krisantini16 journal of tropical crop science vol. 1 no. 1, 2014june www.j-tropical-crops.com journal of tropical crop science vol. 10 no. 2, june 2023 www.j-tropical-crops.com 94 habtemariam teshome, kassa sisay and adise degu teff and wheat yield variation with phosphorus application in jamma district, ethiopia. habtemariam teshome* 0000-0001-5801-9116, kassa sisay 0000-0002-6151-6751, adise degu, tesfaye wubu, tadesse hailu sirinka agricultural research center, p.o. box 74, woldia *corresponding author; email: teshomehabte@gmail.com abstract different plant species has different nutrient requirements and utilize nutrients in different ways. biological activity contributes to p solubilization through mineralization, wheathering, and other physicochemical reactions so that the soil plow layer is the major source of soil availabile p for crops. ethiopia ministry of agriculture and natural resource and ata recommended six types of blended fertilizers (npsznb, npksb, npksznb, npszn, npsb and nps) for the amhara region. our study was conducted in jamma districts of eastern amhara, ethiopia, during 2018 cropping season to attest the yield of teff (dega teff) and wheat (danda’a) due to phosphorus fertilizer application. fertilizer treatments tested were the recommended dose of n only, recommended dose of np, 50 kg.ha-1 nps, 100 kg.ha-1 nps, and 150kg. ha-1 nps, set up in a. randomized complete block design with three replication, and conducted on four sites. our study demonstrated that applications of n and p fertilizers significantly improved grain yield of wheat and teff, and that there was significant yield differences (p < 0.05) between different rates of p fertilizer. therefore, even though the soil p levels were shown to be sufficient, application of both n and p fertilizer in the jamma district is essential to increase yield of teff and wheat. key words: fertilizer, nutrient, phosphorous, teff, wheat introduction declining soil fertility status of the soil, inadequate land size, and low crop and livestock productivity are the major challenges of ethiopian agricultural sector (agegnehu and amede, 2017). maintaining soil fertility and increasing crop yield on smallholder farms in ethiopia is a major concern, as fertilizer recommendations were typically either based on extremely generic parameters for each type of crop or, more frequently, on a single general recommendation for all crops (100 kg urea.ha-1 and 100 kg dap.ha1). the same dosages of fertilizers for different crops had resulted in nutritional deficiencies, or excesses with regard to plant needs. different plant species has different nutrient requirements and utilize nutrients in different ways. how, where, and when plants utilize nutrients can greatly affect the overall yield and plant production. understanding a crop’s nutritional needs can be crucial for a farmer looking to increase crop yields and cut input expenses. phosphorus is an essential nutrient in the process of plants converting the sun’s energy into food, fiber and oil. phosphorus plays a key role in photosynthesis, the metabolism of sugars, energy storage and transfer, cell division, cell enlargement and cell enlargement (hagose and aberha, 2016). phosphorus is the most vital nutrient next to n and p is particularly the most yield limiting nutrient in areas where soil acidity or soil alkalinity is a problem due to p fixation in the soil. phosphorus is crucial for a healthy root growth, early shoot growth, speeds ground coverage for erosion protection, enhances the quality of fruit, vegetable and grain crops, and is vital to seed formation (hodges, 2010). phosphorus is often an important limiting factor for crop yields (girma et al., 2017). compared to the other major nutrients, phosphorus requirement is less but is critical particularly in the early developmental stages of growth, and for energy transfer in the crop during the entire growing season (hodges, 2010). the soil reactivity, the level of soil phosphorus deficit, the rate and method of application, the requirements of the particular crops, and other soil variations, may all have an impact on the phosphorus availabilities. adequate phosphorus nutrition enhances many aspects of plant physiology, including the fundamental processes of photosynthesis, root growth particularly development of lateral roots and fibrous rootlets (gebreslassie and hailemariam, 2016). in highly variation in the yield of teff and wheat due to phosphorus application on .......... journal of tropical crop science vol. 10 no. 2, june 2023 www.j-tropical-crops.com 95 p-fertilized soils, the p concentration in soil solution is high, and the depletion zone readily replenished, however, the replenishment is inhibited when the content of p in the solution is low especially for soils with a low buffer capacity, and the quantity of p ions in soil solution at any given time generally represents less than 1% of p annually taken up by crops (kpongor, 2007). most (99%) of the phosphorus taken up by plants is bound to soil constituents before uptake (schneider and morel, 2000). when the soil is low in phosphorus, plants become stunted with a limited root system, stunted, have thin stems, and older leaves may turn purple because of the accumulation of anthocyanins or purple pigments. biological activity contributes to p solubilization through mineralization, weathering and other physicochemical reactions so that the plow layer is the major source of soil availabile p for crops. in regions of developed countries with intensive livestock production, disposal of animal manures on a relatively small land has led to massive acculation of soil available p, soil organic matter, and buildup of organic p (shi et al., 2013). the balance between p addition as fertilizer and p removal by crops is an important consideration when examining depletion, maintenance or build-up of soil p levels and fertility. when the same p rates year after year were applied, residual soil p levels may end up being too high for crops with low p requirements, and conversely it may be insufficient for crops with high p requirements. fertilizer application should be specific for crop species grown in different regions. ethiopia ministry of agriculture and natural resource and ata (2016) recommended six types of blended fertilizers (npsznb, npksb, npksznb, npszn, npsb and nps) for the amhara region including the study district. according to this recommendation, phosphorous fertilizer was 100% recommended for the districts. so, it is very important to re-examine the soil p status and crop response to applied p fertilizer in affecting crop yield in jamma district of eastern amhara. materials and methods description of experimental sites the trials were carried out in jamma district (figure 1) during the primary farming season of 2018. jamma district lies between the geographical coordinates of 10o 23’ to 10o 27’ n latitudes and 39o 07’ to 39o 24’ e longitudes with altitude of 2630 meters above sea level (masl) and the rainfall patterns of the district during growing season of the crop is indicated in figure 2. the principal feature of rainfall in the area is bimodal, with two distinct rainy seasons. the main rainy season is called “kiremt” which represents the long rainy season (meher) extends from mid-july to early september, while the short rainy season is called “spring” from february to mid-may. the dominant soil type of the study district is vertisols. wheat, teff, and faba bean are the three main crops that are produced extensively throughout the study sites.     figure 1. location map of the study sites figure 2. monthly rainfall of jamma district, ethiopia, in 2018. experimental set-up and procedure standard cultivation techniques were used to prepare the test sites before planting: soil on the test fields were plowed using equipment drawn by oxen. after plowing broad bed furrows (bbf) was prepared manually to drain excess water in the experimental sites. the experiment tested five levels of phosphorous (0, 19, 38, 57 and the recommended p2o5 (69 and 46 kg.ha-1); the full recommended nitrogen (115 and 92 kg.ha-1 n) were applied for all treatments for wheat and teff crops, respectively. 0 50 100 150 200 250 300 350 400 jan feb mar apr may jun jul aug sep oct nov dec m on th ly r ai nf al l ( m m ) figure 1. location map of the study sites     figure 1. location map of the study sites figure 2. monthly rainfall of jamma district, ethiopia, in 2018. experimental set-up and procedure standard cultivation techniques were used to prepare the test sites before planting: soil on the test fields were plowed using equipment drawn by oxen. after plowing broad bed furrows (bbf) was prepared manually to drain excess water in the experimental sites. the experiment tested five levels of phosphorous (0, 19, 38, 57 and the recommended p2o5 (69 and 46 kg.ha-1); the full recommended nitrogen (115 and 92 kg.ha-1 n) were applied for all treatments for wheat and teff crops, respectively. 0 50 100 150 200 250 300 350 400 jan feb mar apr may jun jul aug sep oct nov dec m on th ly r ai nf al l ( m m ) figure 2. monthly rainfall of jamma district, ethiopia, in 2018. experimental set-up and procedure standard cultivation techniques were used to prepare the test sites before planting: soil on the test fields were plowed using equipment drawn by oxen. after plowing broad bed furrows (bbf) was prepared manually to drain excess water in the experimental sites. the experiment tested five levels of phosphorous (0, 19, 38, 57 and the recommended p2o5 (69 and 46 kg.ha-1). full recommended nitrogen (115 and 92 kg.ha-1 n) were applied for all treatments for wheat and teff crops. the treatments are crops (wheat and teff) and locations with the specific recommended n, the journal of tropical crop science vol. 10 no. 2, june 2023 www.j-tropical-crops.com 96 habtemariam teshome, kassa sisay and adise degu recommended (crop and location specific) n and p, i.e., 115 kg.ha-1 n and 69 kg.ha-1 p2o5 for wheat and 92 kg.ha-1 n and 46 kg.ha-1 p2o5 for teff (abebe et al., 2020; samuel et al., 2016), 50 kg.ha-1 nps (n was be adjusted to recommended dosage), 100 kg.ha-1 nps (n was be adjusted to the recommended dosage), and 150 kg.ha-1 n, p, s (n was be adjusted to recommended dosage). phosphorus was applied as triple supper phosphate (tsp) for the recommended rate of n and p, and p and nps for the rest rates of phosphorous, whereas nitrogen was from nps and urea. nitrogen rates were equal for all treatments for each crop in each location. a randomized complete block design was used to apply the treatments on plots with a size of 5m × 5m (25 m2) for teff, and 5m x 4.5m (22 m2) for wheat. each treatment had three replicates for each site (farm); four sites per location for teff and five sites per location for wheat. broad bed furrow (80 cm x 40 cm) were prepared for wheat production. spaces between treatments and blocks were 0.5 m and 1 m, respectively, for both crops. spacing between rows were 20 cm for wheat, whereas teff was planted as broadcast. sowing was done second week of july for teff and wheat trials. nitrogen was applied half at planting and the other half at tillering stage for teff and wheat just after weeding with the presence of small rainfall. other standard agronomic practices were applied as per the recommendations for the crop and as farmers practice (abebe et al., 2020; samuel et al., 2016). soil data collection and analysis surface soil samples (0-20 cm depth) were collected randomly in a zig-zag pattern before sowing from the entire experimental field. the soil analysis was carried out in the soil testing laboratory of sirinka agricultural research center for selected chemical and physical soil properties (texture, ph, oc, total n and available p). the texture of the soil was determined by the hydrometer method (bouyoucos, 1962). potentiometric analysis was used to determine the ph of the soils in water suspension at a ratio of 1 : 2.5 (soil to liquid) (van-reewijk and clark, 1992), microkjeldahl digestion method was used to calculate total nitrogen (tn) (jackson,1958). olsen method was used for the determination of available p from the soil sample (olsen et al., 1954). using the wet digestion method, the soil’s organic carbon content was examined (bayu and rethman, 1934). yield data collection harvesting was done from the second week of december for wheat and first week of january for teff. to measure total above-ground biomass and grain yields the central bbf was harvested for wheat and the teff aboveground biomass was harvested using quadrants (4m x 4m). at the maturity stage, the net plot area’s entire plant parts (only excluding boarder row), including leaves and stems, and seeds, were picked, and the biomass was calculated (dry matter basis). grain yield was measured by harvesting the crop from the net (middle) plot area to avoid border effects, after threshing seeds were cleaned and weighed. finally, grain yield was adjusted to standard moisture contents of wheat (12.5%) as described in the following formula: adjusted yield = actual yield x (100 – m) / (100 – d) where m is the measured actual grain moisture content and d is the designated wheat standard moisture content (12.5%). data analysis analysis of variance was carried out for the yield following statistical procedures appropriate for the experimental design using sas version 9.4. whenever treatment effects was significant, the means was separated using the least significant difference (lsd) procedures test at 5% level of significance. result and discussion physico-chemical properties of the soil the results of soil analysis showed that the soil in all experimental sites had moderate total nitrogen content according to tekalign (1991). the soil has organic matter ranges from 0.86%-1.61 % which is categorized low level (berhanu, 1980) (table 1). based on usda textural classification the soil textural class of the experimental site was clay. the soil test result reveals that the available phosphorus content of the soil, as per the olsen’s method and based on the rating of cottenie (1980) was moderate to high. according to wiens (2017) when soil p levels are near sufficient, the application of p fertilizer at rates to account for previous crop removal may be the desirable approach, without expectation of a yield response. teff yield response to applied phosphorous fertilizer the combined analysis indicated that the teff grain yield was significantly (p ≤ 0.05) increased with different p rates (table 2), but the yield increases were significant only in the two out of four sites. the highest teff grain yield (1.720 t.ha-1) were recorded variation in the yield of teff and wheat due to phosphorus application on .......... journal of tropical crop science vol. 10 no. 2, june 2023 www.j-tropical-crops.com 97 with the application of recommended rate of n and p fertilizer (46 kg.ha-1 p2o5 ns 92 kg.ha -1 n), and the yields were not significantly different from the yields with 38 kg.ha-1 and 57 kg ha-1 p2o5 with 46 kg.ha -1 n. application of 46 kg.ha-1 p2o5 and 46 kg.ha -1 n resulted in 9% teff yield increment in comparison to the control (without p) (table 2). according to the combined statistical analysis above ground biomass yield of teff with p treatments was not increased over control, even though the highest biomass yield was obtained from the application of 46 g kg.ha-1 p2o5 (table 4). wheat yield response to applied phosphorous fertilizer application of different rates of p significantly affected (p ≤ 0.05) grain and biomass yield of wheat (table 3). except at farm 3 (table 3) and farm 2 (table 5) application of different rate of p fertilizer increased yield of wheat in comparison with control; therefore, application of p with n fertilizer increased yield of wheat in vertisols of jamma district. the highest grain yield (2.64 t.ha-1 ) and biomass yield (6.08 t.ha1) were obtained from the application of 69 kg.ha-1 p2o5 with 115 kg.ha -1 n and 57 kg.ha-1 p2o5 with 115 kg.ha-1 n respectively. application of 69 kg.ha -1 p2o5 with 115 kg.ha -1 n resulted 22% and 26% grain yield and biomass yield advantage over the control, respectively (table 3 and 5). the results of this study demonstrated that the use of chemical p fertilizer in combination with n consistently enhanced grain yield of wheat and teff compared to inorganic n fertilizer applied alone. these results, however, is not supported by pre-planting soil test results which shows a high available phosphorus in the study sites (table 1). the result obtained falsifies the thought that there is no increase in yield when p fertilizer was applied to soil with sufficient levels of p. this research result were similar to the previous refining trials of wheat in the same district (samuel et al., 2016). the finding of this research disagreed with the results of selassie et al. (2014) who reported that in areas with table 1. soil properties at planting site ph % om %tn available p (ppm) textural class teff 6.2-6.9 0.86-1.04 0.18-0.28 15.7-22.4 clay wheat 6.0-6.1 1.07-1.61 0.15-0.24 11.0-22.3 clay note: om=organic matter; tn= total nitrogen; p=phosphorus table 2. effect of p fertilizer on teff grain yield (t.ha-1) in 2018. fertilizer treatment (kg.ha-1) farm 1 farm 2 farm 3 farm 4 average 0p2o5 1.952 bc 1.142 a 1.356 b 1.848 a 1.575 b 19p2o5 1.822 c 1.223 a 1.462 ab 1.772 a 1.570 b 38p2o5 2.009 b 1.256 a 1.342 b 1.785 a 1.598 ab 46p2o5 2.280 a 1.236 a 1.681 a 1.680 a 1.720 a 57p2o5 2.113 ab 1.257 a 1.485 ab 1.765 a 1.655 ab cv (%) 5.0 12.6 8.1 8.9 9.4 note: cv= coefficient of variation. means within the same column followed by the same letters are not significantly different at 5% level of significance. table 3. effect of p combined with n fertilizer with on wheat grain yield (t.ha-1) in 2018 fertilizer treatment (kg.ha-1) farm 1 farm 2 farm 3 farm 4 farm5 average 92 n only 2.140 b 2.095 b 3.165 a 1.921 b 1.477 b 2.160 b 19p2o5+115n 2.817 a 2.243 ab 3.362 a 2.276 ab 1.616 b 2.463 a 38p2o5+115n 2.774 a 2.379 ab 3.513 a 2.154 ab 1.794 ab 2.523 a 57p2o5+115n 2.305 ab 2.742 a 3.397 a 2.138 ab 1.852 ab 2.487 a p2o5+115n 2.498 ab 2.546 ab 3.597 a 2.370 a 2.218 a 2.646 a cv (%) 11.0 12.6 8.9 10.8 17.2 11.8 note: cv= coefficient of variation. means within one column followed by the same letters are not significantly different at 5% level of significance. journal of tropical crop science vol. 10 no. 2, june 2023 www.j-tropical-crops.com 98 habtemariam teshome, kassa sisay and adise degu high initial soil phosphorus application of p fertilizer is wastage for wheat production. on the other hand, as phosphate fertilizers are applied continuously over time, the amount of phosphorous nutrients in the soil tends to rise, especially in the labile forms that can release phosphorus into the soil solution (brady and weil, 2002). even if the soil data revealed high range of available p (table 1), both crops responded positively to the applied p fertilizers. our results agreed with alemayhu (2014) and fissehaye et al. (2009) findings in that grain yield and yield related parameters were significantly affected by p application and excess p supply could result in low grain yield of teff. liben et al. (2005) also reported that there is a linear increase in growth parameters for wheat by the application of np fertilizers but the response is higher for n than p fertilizer at northwestern ethiopia. application of 23 up to 92 kg p2o5.ha -1 resulted in a grain yield increment of 171203% on vertisols in the central ethiopia (tamene et al., 2017). our findings showed that application of both nitrogen and phosphorus fertilizer increased the yield of both teff and wheat crops. the increase in grain yield with increasing levels of p fertilizer was likely due to better plant growth and health which eventually increased the final grain yield. conclusion application of different rates of p and n significantly increased wheat and teff grain yield over fertilizing with n only. wheat biomass were significantly affected by different rates of p and n fertilizer but biomass yield of teff were not affected. therefore, even though the soil p levels were shown to be sufficient, application of both n and p fertilizer in the jamma district is essential to increase yield of teff and wheat. further studies should be directed to determine the optimal phosphorus and nitrogen fertilizer rates for teff and wheat production in the districts. acknowledgement we would like to express our sincere appreciation to sirinka agricultural research center for funding the research and for supporting most of the research materials and equipment to undertake the research. references agegnehu, g., and amede, t. (2017). integrated soil fertility and plant nutrient management in tropical agro-ecosystems: a review. pedosphere 27, 662–680. table 4. effect of p fertilizer on teff biomass yield (t. ha-1) in 2018 fertilizer treatment (kg.ha-1) farm 1 farm 2 farm 3 farm 4 average 0p2o5 7.200 ab 5.889b 6.903bc 7.768 6.940ab 19p2o5 6.507 b 6.396ab 7.275bc 7.692 6.967ab 38p2o5 7.147 ab 6.322ab 6.728c 7.225 6.856b 46p2o5 7.627 a 6.089b 8.347a 7.440 7.376a 57p2o5 7.520 a 6.750a 7.618ab 7.365 7.313ab cv (%) 5.0 5.5 6.3 8.5 7.9 note: cv= coefficient of variation. means within one columns followed by the same letters are not significantly different at 5% level of significance. table 5. effect of p combined with n fertilizer on wheat biomass yield (t.ha-1) in 2018 fertilizer treatment (kg.ha-1) farm 1 farm 2 farm 3 farm 4 farm5 average 92 n only 4.533d 4.533 7.167c 1.921b 3.767b 4.800c 19p2o5+115n 6.233 ab 4.733 7.900b 2.276ab 3.867ab 5.560b 38p2o5+115n 6.567 a 5.333 9.033a 2.154ab 4.233ab 5.953ab 57p2o5+115n 5.667 bc 5.567 8.700a 2.370a 5.167a 6.087a 69p2o5+115n 5.000 cd 6.033 8.467ab 2.138ab 4.300ab 5.693ab cv (%) 7 19 5 10.9 17 12 note: cv= coefficient of variation, means within one column followed by the same letters are not significantly different at 5% level of significance. variation in the yield of teff and wheat due to phosphorus application on .......... journal of tropical crop science vol. 10 no. 2, june 2023 www.j-tropical-crops.com 99 alemayhu, b. (2014). effect of phosphorus rates and varieties on grain yield, nutrient uptake and phosphorus efficiency of tef [eragrostis tef (zucc.) trotter]. american journal of plant sciences 5, 262. bayu, w., and rethman, n. f. (1934). an examination of the degtjareff method for determining soil organic matter, and a proposed modification of the chromic acid titration method, soil science 37, 29-38. bouyoucos, g.j. (1962). hydrometer method improved for making particle size analyses of soils 1. agronomy journal 54, 464-465. brady, n.c., and weil r. r. (2002). phosphorus and potassium in “the nature and properties of soils”. 352 pp. prentice-hall of india, delhi. cottenie, a. (1980). “soil and plant testing as basis of fertilizer recommendations”. fao soil bulletin no. 38/2. debele, b. (1982). “the physical criteria and their rating proposed for land evaluation in the highland regions of ethiopia”. world soil resources reports. fao. fessehaye, y., bovenhuis, h., rezk, m.a., crooijmans, r., van arendonk, j.a. and komen, h., (2009). effects of relatedness and inbreeding on reproductive success of nile tilapia (oreochromis niloticus). aquaculture 294,180-186. girma, t., beyene, s., and biazin, b. (2017). effect of organic and inorganic fertilizer application on soil phosphorous balance and phosphorous uptake and use efficiency of potato in arbegona district, southern ethiopia. journal of fertilizers and pesticides 8, 1-6. hagose, g., and aberha, h. (2016). a review on effect of phosphorus fertilizer on crop production in ethiopia. journal of biological agriculture healthcare 6, 117-120. haileselassie, b., habte, d., haileselassie, m., and gebremeskel, g. (2014). effects of mineral nitrogen and phosphorus fertilizers on yield and nutrient utilization of bread wheat (triticum aestivum) on the sandy soils of hawzen district, northern ethiopia. agriculture, forestry and fisheries 3, 189-198 hodges, s.c., (2010). “soil fertility basics” pp. 6-9. soil science extension, north carolina state university. jackson, m.l. (1958). “soil chemical analysis” pp. 23-45 prentice-hall. new jersey, u.s.a. van-reewijk, l.p. and clark, f.e, (1992). procedures for soil analysis. international soil reference center (isric) 9, 3. kpongor, d.s. (2007). “ecology and development” series no. 51, pp.51-57. liben, m., assefa, a., tadesse, t., and mariye, a., (2005). response of bread wheat to nitrogen and phosphorous fertilizers at different agro-ecologies of northwestern ethiopia in “proceedings of the 12th regional wheat workshop for eastern, central and southern africa”. tekalign, t. 1991. soil, plant, water, fertilizer, animal manure and compost analysis. working document no. 13. addis ababa: international livestock research center for africa tekalign, t. (1991). “soil, plant, water, fertilizer, animal manure and compost analysis”. working document no. 13. international livestock reaserch center for africa. addis ababa. ministry of agriculture and natural resources and agricultural transformation agency of ethiopia. (2016). “soil fertility status and fertilizer recommendation atlas of amhara national regional state, ethiopia”. olsen, s.r., cole, f.s., and dean, l.a. (1954). “estimation of available phosphorus in soils by extraction with sodium bicarbonate” pp. 132-135.usda circular, accra, ghana. adissie, s., teshome, h., getu, a., and hailu, t. (2016). refining fertilizer recommendations for bread wheat production in south wollo of amhara region in “proceedings of the 9th and 10th annual regional conferences on completed research activities on soil and water management research”. pp 9–20. schneider, a. and morel, c., (2000). relationship between the isotopically exchangeable and resin‐extractable phosphate of deficient to journal of tropical crop science vol. 10 no. 2, june 2023 www.j-tropical-crops.com 100 habtemariam teshome, kassa sisay and adise degu heavily fertilized soil. european journal of soil science 51,709-715 shi, y., lalande, r., hamel, c., ziadi, n., gagnon, b. and hu, z., (2013). seasonal variation of microbial biomass, activity, and community structure in soil under different tillage and phosphorus management practices. biology and fertility of soils 49, 803-818. tamene, l., amede, t, kihara, j., tibebe, d., and schulz, s. (2017). “a review of soil fertility management and crop response to fertilizer application in ethiopia: towards development of siteand context-specific fertilizer recommendation”. international center for tropical agriculture (ciat) publication no 443. 86 p. addis ababa. ethiopia. wiens, j.t. (2017). “agronomic and environmental effects of phosphorus fertilizer application methods”. [thesis]. university of saskatchewan. canada. journal of tropical crop science vol. 10 no. 1, february 2023 www.j-tropical-crops.com 46 mei meihana, benyamin lakitan, m. umar harun, susilawati, erna siaga, laily ilman widuri, kartika kartika proline accumulation and growth of bean leaf (phaseolus vulgaris l.) with biochar application in the shallow water table environment mei meihanaa, benyamin lakitanb,c, m. umar harunb, susilawatib, erna siaga*d, laily ilman widurie, kartika kartikaf a stiper sriwigama, palembang 30137, indonesia b faculty of agriculture, universitas sriwijaya, indralaya 30662, indonesia c research center for sub-optimal lands (pur-plso), sriwijaya university, palembang 30139,indonesia d faculty of plant and animal sciences, universitas bina insan, lubuklinggau 31626, indonesia e faculty of agriculture, universitas jember, jember 68121, indonesia f research center for biology, research organization for life sciences, national research and innovation agency, bogor 16911, indonesia *corresponding author; email: ernasiaga@univbinainsan.ac.id abstract agronomic constraints for vegetable cultivation in riparian wetlands are low soil quality and shallow water table conditions. this study aims to identify the effect of biochar application and shallow water table on proline accumulation and plant growth of bean leaf (phaseolus vulgaris l.) on the generative stage. this study was carried out from april to august 2018 in factorial randomized block design. the first factor was doses of biochar (0, 1, and 2 kg.m-2), and the second was shallow water table condition (20 cm and 10 cm below the soil surface) given in the generative stage. the results showed that biochar application and the shallow water table significantly affected leaf parameters; proline accumulation and increases in the sucrose levels occurred on the leaves. proline content increased started at 3 days after treatment (dat) and decreased at 7 dat.until recovery day (at 7 dat). the biochar application of 2 kg.m-2 provided good aeration in the soil, which enhanced the bean’s ability to survive under excess water. proline accumulation is one of the adaptation mechanisms of beans to abiotic stress in shallow water table conditions. keywords: crop adaptability, ameliorant, submerged land, wetlands. introduction the riparian wetland is classified as suboptimal land but has high potential for rice, food crops, and vegetables. the total riparian wetland area in indonesia is approximately 13.29 million ha and spread across several regions: 2.79 ha in sumatera, 3.58 ha in kalimantan, 6.31 ha in papua, and 0.61 ha in sulawesi (alwi et al., 2017). the commonly cultivated vegetables in the riparian wetland are tomato, chili, eggplant, beans, corn, kale, lettuce, and basil (emile et al., 2012; white, 2013). the main constraints of plant cultivation in riparian wetlands are unpredictable flooding periods during the rainy season and drought during the dry season due to climate change (lakitan et al., 2018c). flooding and drought stress decreased crop yields (baileyserres et al., 2012). the whole riparian wetland area is flooded during the rainy season for several months. during high flooding, plants could be cultivated by utilizing floating cultivation systems (siaga et al., 2018). however, farmers need additional costs to construct the raft. in riparian wetlands, rice cultivation starts when the water level has receded or at the end of the flooding season and is harvested at the beginning of the dry season (lakitan et al., 2018a). at the time, local farmers (1) the crops that were planted after the rice harvest immediately experience water logging in the vegetative stage, and drought stress in the generative stage. this was due to the soil dries gradually; (2) the third crop, planted after second crop harvest, requires additional water supply during for both vegetative and generative stages, but this crop will probably under shallow water table stress in the generative stage if rainy season occurs earlier than expected. therefore, it is crucial to understand vegetable crops that can adapt to this environment. proline accumulation and plant growth analysis of bean leaf (phaseolus vulgaris l.) .......... journal of tropical crop science vol. 10 no. 1, february 2023 www.j-tropical-crops.com 47 plants have several mechanisms to survive under shallow water table conditions, including anatomical and morphological adaptations and metabolic mechanisms (ashraf, 2012). the metabolic mechanism known as osmotic adjustments, such as the enhancement of proline concentration in leaf tissue (singh et al., 2000). proline (c5h9no2) is a photogenic amino acid compound accumulated both in normal conditions (kishor et al., 2015) and abiotic stress due to non-optimal environments such as drought (dehydration), salinity, waterlogging, and extreme temperature (sarker et al., 2005; gupta et al., 2014; fichmann et al., 2015). proline accumulation occurs due to the activation of proline biosynthesis and the inactivation of proline degradation. proline is an osmoprotectant compound that is synthesized in plants via the glutamine and ornithine pathways (delauney et al., 1993). in the primary pathway, glutamine is synthesized into proline through two intermediate compounds, glutamine semialdehyde (g.s.a.) and proline-5-carboxylate (p5c); then p5c is reduced to proline by enzyme p5c reductase (p5cr) (delauney et al., 1993). by accumulating proline in the cell, the osmotic potential of the cell decreases, thereby increasing the cell’s capacity to maintain turgidity (turner, 2018). cell turgidity is essential for photosynthesis, enzyme activity, and cell enlargement (claussen, 2005). previous studies have reported that environmental stress causes proline accumulation in peas (karatas et al., 2014), tobacco (borgo et al., 2015 ), tomatoes (gharsallah et al., 2016), and lentils (ankita et al., 2017). therefore, proline is a crucial indicator for detecting plants’ adaptability to shallow water table stress. after flooding, another crucial issue in the riparian wetland is the low physical, chemical, and biological soil quality. riparian wetlands are sub-optimal land for crop cultivation because of having low chemical attributes such as low ph (alwi et al., 2017), deficiency of macronutrients, and containing excessive amounts of toxic metals (lakitan et al., 2018c). according to noor et al. (2006), most of the riparian wetlands soils are classified as alluvial that have poor drainage. poorly drained soil often has poor aeration, so oxygen availability is limited for the aerobic microorganisms’ activities in the soil. the low availability of oxygen is also caused by the soil which is still wet after the flood recedes. in wet soil, pores are still filled with water so that oxygen becomes limited for plants and for aerobic microbial activities in the soil. this reduces the physical quality as well as the biological quality of the soil. to increase fertility and enhance the physical properties of soil, ameliorant materials such as agricultural lime, manure and biochar could be applied to soil (barrow, 2012; nariratih et al., 2013; goulding, 2016; kartika et al., 2018). therefore, ameliorant materials addition could increase plant growth (lakitan et al., 2018a). this study focused on applying biochar to improve soil quality and the adaptability of beans to the shallow water table. material and methods this study used 72 common bean cultivars. bean seeds were soaked in warm water for 3 hours and sown into 40 cm x 25 cm polybags which contained 14.7 cm-3 of a mixture of soil, chicken manure, and biochar (the height of growing media ±30 cm). soil and chicken manure were mixed in a ratio of 2:1 (v/v). the biochar was applied according to treatment dose. this research used crushed wood charcoal with a size of approximately 1mm. biochar was applied to the soil one week prior to planting time and mixed evenly on the surface of growing media. plants were irrigated twice daily and supplied 6 g n.p.k. fertilizer per plant at transplanting. pest and diseases were controlled using pesticides. experimental site and experimental design the study was carried out in the macan kumbang sub-district (2o58’24.2 “s 104o43’12.5”e), palembang, indonesia, from april to july 2018. this study was arranged in a factorial randomized block design. the first factor was biochar doses consisting of 0, 1, and 2 kg.m-2; the second factor was one control, and two different water table depths were 20 cm below the soil surface and 10 cm below the soil surface for 1 week. each treatment was repeated three times. three samples for proline and sucrose analysis and 12 for all destructive analysis (0, 3, 6, and 7 days after transplanting, dat. in the shallow water table treatment, plants were prepared in a 3.0m x 1.5m x 0.25m pond at their generative stage, i.e., when about 50% of the plants have developed fully bloomed flowers). proline and sucrose analysis leaf proline and sucrose analysis was conducted at the integrated research laboratory of the faculty of agriculture, universitas sriwijaya. the analyses were carried out in several stages: before water table treatments (d0), three days after treatment (d3), six days after treatment (d6), and 7 days of the recovery period (r7). the method of proline analysis followed the method of bates (1973). meanwhile for method of sucrose analysis followed somogyi method (nelson, 1944). journal of tropical crop science vol. 10 no. 1, february 2023 www.j-tropical-crops.com 48 mei meihana, benyamin lakitan, m. umar harun, susilawati, erna siaga, laily ilman widuri, kartika kartika plant growth and yield measurements the relative leaf expansion rate (rler.) was measured based on leaf area data, which was measured continuously for 14 days starting at the day the treatment was initiated until the recovery period. the leaves observed are healthy young leaves that have been in full bloom. leaf area was measured every morning from 6 to 6:30 am. leaf area measured non-destructively following lakitan et al. (2017) formula in which la = 1.5198 x leaf length x leaf width. specific leaf fresh weight (slfw) and specific leaf water content (slwc) were measured based on data, i.e., leaf area, fresh weight, and leaf dry weight. leaf samples were taken simultaneously with leaf sampling to measure proline contents. slfw and slwc were measured by weighing fresh leaves immediately after being picked, then drying in an oven at 80oc until reaching the constant dry weight. slfw is a comparison between the fresh weight of leaves (fw) and leaf area (la), slwc is calculated based on the difference in fresh weight (fw) and dry weight (dw) divided by leaf area (la) (meihana et al., 2017; widuri et al., 2017). the weight and number of pods were measured continuously from the 1st harvest to the 17th harvest. statistical analysis differences in the effect of biochar applications and shallow water table were analyzed of variance (anova) using statistical analysis system (s.a.s.) 9.2. for significant treatments on any measured parameters, differences among mean values were tested using the least significant differences (l.s.d.) test at p ≤ 0.05 result and discussion proline accumulation due to shallow water table and biochar application the results showed that the shallow water table affected the proline content of the leaves. the significant effects started showing three days after treatment (dat). on the water table 20 cm below the soil surface and control, proline accumulation was decreased and gradually increased on the 6 dat (figure 1). contrary to the water table 10 cm below the soil surface (w2), proline accumulation significantly increased on the 3 dat the proline increase was likely due to the dry season when the temperature was in the range of 32-35oc, and plants accumulated the proline as a response to the high temperature. figure 1. proline and sucrose contents are affected by biochar application and shallow water table. proline accumulation and plant growth analysis of bean leaf (phaseolus vulgaris l.) .......... journal of tropical crop science vol. 10 no. 1, february 2023 www.j-tropical-crops.com 49 the shallow water table treatment showed positive impacts until the 3 dat, as indicated by the reduction of proline content compared to the others treatment. however, bean growth decreased after 6 dat, and proline content significantly increased. the mechanism of survival in bean was by producing proline in the leaves, or osmotic adjustment. increasing proline accumulation is a mechanism for beans to survive in excess water stress conditions. in stress conditions, plants accumulate proline rapidly because of proline’s high solubility (bhaskara et al., 2015) and compatibility with the cell environment (kishor et al., 2015), so proline is an indicator to detect plant adaptability to stress. the proline content in bean leaves decreased when the plant entered the recovery period, i.e., when the shallow water table treatment was stopped. generally, proline accumulates on young leaves (claussen, 2005; zegaoui et al., 2017); proline accumulation indicates the physiological adaptation mechanism of the plant to excess water conditions (hypoxia) (ashraf, 2012). by accumulating proline in the cell, the osmotic potential of the cell decreases, thereby increasing the cell’s capacity to maintain turgidity (turner, 2018). cell turgidity is essential for photosynthesis, enzyme activity, and cell enlargement (claussen, 2005). compared to the water table of 20 cm below the soil surface, the water table of 10 cm below the soil surface makes more parts of roots to be in a submerged condition. the closer the water table is to the soil surface, the more parts of the roots are submerged in the water. in excess water condition, gas exchange between soil and air becomes limited (subbaiah and sachs, 2003). as a result, the supply of o2 in the soil becomes low (hypoxia), and in a long time, it will become anoxia (ashraf, 2012). the limited supply of o2 causes metabolic disorders in plants. to survive in this condition, plants can adapt anatomically and morphologically and make osmotic adjustments (ashraf, 2012). osmotic adjustment occurs by increasing the concentration of proline in leaf tissue (singh et al., 2000; abraham et al., 2003; fichman et al., 2015). another finding of this research indicated proline content of beans which growing in the soil without biochar application increased (figure 1); on the contrary proline content of plants growing in the ground mixed with 1 kg.m-2 of biochar and 2 kg.m2of biochar sharply decreased at the 3 dat however, proline content increased at the 6 dat of the shallow water table for 1 kg.m-2 of biochar and 2 kg.m-2 of biochar but decreased for controlled plants. biochar application on 2 kg.m-2 gave a positive effect on plant resistance under shallow water table stress, likely due to the role of biochar in the physical properties of the soil. some benefits of biochar applications on soil physical properties, i.e., facilitating drainage in poorly drained soils (herath et al., 2013), forming macro aggregates in the soil (jien et al., 2013; wong et al., 2016), and increasing soil pore space (sun and lu, 2014; hardie et al., 2014). the 2 kg.m-2 of biochar is better in providing aeration on the part of the soil that is not submerged in water (10 cm near the soil surface) than the soil mixed with 1 kg.m-2 of biochar. therefore, the oxygen supply in well-aerated soil (that is not submerged in water is maintained for root respiration. the change of proline and sucrose contents are physiological mechanisms in the plant when experiencing environmental stress conditions. this research showed that shallow water tables also affected the sucrose contents of the leaves (figure 1). sucrose contents increased at 3 dat and decreased at the 6 dat of stress. despite this, in the control conditions, sucrose content fell from 3 to 6 dat the decrease in sucrose content is likely due to the translocation of sucrose from the source (leaves) to the sink (pods). biochar application at 1 kg.m-2 produced higher proline and sucrose than the 2 kg.m-2 (figure 1). plant growth analysis biochar application and shallow water table treatments significantly interact with plant growth analysis except on the relative water content (rwc) in 6 dat (table 1). leaves and water content variables tend to be more affected by water table conditions rather than biochar application. a shallow water table generally decreased specific leaf fresh weight (slfw) and specific leaf water content (slwc) but increased specific leaf area (sla) and leaf area (la). slfw and slwc showed a downward trend during exposure to shallow water tables until the recovery period; however, the decline was not significant. this condition was a physiological response of plants to the shallow water table. this decrease is not only caused by excess water but also affected by the age of the leaf. after entering the recovery period, the plant is not in the treatment pond. in that condition, the plant cannot compensate for the water loss rate due to high transpiration. after being outside the pond, soil moisture decreased due to evaporation. in recovery time, the bottom root section has not been able to absorb water properly because the bottom roots are damaged during exposure to a shallow water table. in this situation, water absorption rests on the upper root not submerged. because of the upper part of the soil loses a lot of water due to evaporation, the water content in plants, especially journal of tropical crop science vol. 10 no. 1, february 2023 www.j-tropical-crops.com 50 mei meihana, benyamin lakitan, m. umar harun, susilawati, erna siaga, laily ilman widuri, kartika kartika in leaves, decreases. water is needed by plants to maintain leaf turgidity (turner, 2018). in this case, the slwc of the plant becomes low, so the slfw decreases. this phenomenon has been reported on the previous study. lakitan et al. (2018b) found those leaves of beans reached it maximum size in approximately nine days. since proline accumulation is one of the osmotic adjustment mechanisms for plants that experience excess water, slfw and slwc are essential variables that respond to these conditions. however, in this study, the increase in leaf proline content was not followed by the rise of these two variables (figure 1), showing that slfw and slwc are more affected by disruption of water absorption due to root damage that experiences shallow water table conditions. at the beginning of treatment, 1 kg.m-2 of biochar in the control plant showed significantly higher la than the 0 kg.m-2 of biochar-water table treatment. after three days, 1 kg.m-2 of the biochar-water table 20 cm below the soil surface and 2 kg.m-2 of the biochar-control plant than the other treatments. during the recovery period, the interaction of 0 kg.m-2 of the biochar-water table of 10 cm below the soil surface reached higher la compared with 0 kg.m-2 of biochar-control, 1 kg.m-2 of biochar-water table 10 cm below the soil surface, and 2 kg.m-2 of biochar-water table 20 cm below the soil surface. on the day treatment started, higher rwc were obtained in the biochar application compared to controls. at the 3 dat, 2 kg.m-2 of biochar-water table 10-cm below the soil surface got too higher rwc. than the others. it showed that 2 kg.m-2 biochar was better for guaranteeing water availability in the media for plant growth. in this research, we recognized the benefit of biochar for improving soil physic characteristics such as facilitating soil drainage (herath et al., 2013), creating macro aggregate in soil (jien et al., 2013; wong et al., 2016), and increasing soil pore (sun and lu, 2014; hardie et al., 2014). rwc in each treatment did not show a significantly different at the 6 dat, whereas 2 kg.m-2 of biocharwater table 10-cm below the soil surface resulted in the highest of rwc application 2 kg.m-2 of biochar 10cm below the soil surface showed the most elevated of rwc in 3, 6, and 7 dat, respectively. at the beginning of treatment sla in 1 kg.m-2 of biochar increased. at the 3 dat, sla in 1 kg.m-2 of biochar 10 cm below the soil surface significantly increased compared to 0 kg m-2 of biochar-20 cm below the soil surface. it revealed that the water table at 10 cm below the soil surface in three days positively affected leaf area development. the temperature on the day before treatment is relatively high (32oc–35oc), increasing the evapotranspiration rate. common beans obtained sufficient water supply for leaf development at the beginning shallow water table period. however, at the 6 dat, the highest sla was reached on 2 kg.m-2 of biochar-control plant, indicating that biochar could increase water retention of soil media to provide water for the plant. sufficient water for plants increases leaf turgor and leaf area expansion. after the recovery period, 0 kg m-2 of biochar 20 cm below the soil surface got too higher sla than 0 kg.m-2 of biochar-10 cm below the soil surface and control. the effect of the water table 20 cm below the soil surface was not as severe as the water table 10 cm below the soil surface. the water table 10 cm below the soil surface causes the roots to be submerged and damaged. during the recovery period, common beans might be in optimal condition, but plant roots could completely absorb the water supplied during the watering time. while in the 20 cm below the soil surface, fewer roots were damaged, so they recovered more quickly. leaf turgidity also revealed the effect of biochar and water table treatment. low leaf turgidity affects the relative leaf expansion rate. the turgor of actively dividing cells determines leaf turgidity. if the cell turgor decreases, cell elongation becomes obstructed, and rler decreases. rler on treated and non-treated plants (control plants) began to drop dramatically until the 6 dat and finally stopped at the end of the recovery period (figure 2). genetic factors might also cause the termination of the leaf expansion rate. this research revealed that in less than 14 days, the leaves had reached their maximum size. adding biochar increased rler at the control and water table 20 cm below the soil surface. the shallow water table stress condition significantly affected plant dry weight on the 6th day of treatment (figure 3). it showed a rapid dry weight increase on the water table 20 cm below the soil surface due to an increment of leaf, stem, and fruit weight. the dry weight increased continuously until 7 dat the dry weight of the plants increased due to the application of biochar. the higher the dose of biochar, the higher the dry weight obtained (figure 3). proline accumulation and plant growth analysis of bean leaf (phaseolus vulgaris l.) .......... journal of tropical crop science vol. 10 no. 1, february 2023 www.j-tropical-crops.com 51 table 1. leaves and water content-related variables during water table stress condition and recovery day treatment slfw (mg cm-²) slwc (mg cm-²) sla (cm² mg-¹) la (cm²) rwc (%) d0 b0w0 30.02±8.94 a 27.20±7.85 a 0.035±0.010 b 134.77±35.51 b 90.58±2.94 ab b0w1 22.67±5.14 ab 20.61±4.44 ab 0.045±0.010 ab 136.50±26.06 b 85.91±4.26 b b0w2 27.90±1.45 ab 25.01±0.93 ab 0.035±0.001 ab 151.61±17.62 ab 91.93±5.72 ab b1w0 23.67±3.05 ab 20.90±3.22 ab 0.042±0.006 ab 189.65±13.57 a 93.29±1.93 a b1w1 20.71±4.33 b 19.15±3.92 b 0.049±0.009 a 162.13±15.83 ab 91.77±5.61 ab b1w2 22.89±5.26 ab 20.84±4.77 ab 0.045±0.009 ab 142.10±37.90 b 92.24±4.84 ab b2w0 21.36±1.54 b 19.00±1.35 b 0.047±0.003 ab 149.05±36.20 ab 85.64±2.46 b b2w1 21.02±4.35 b 19.26±3.62 b 0.049±0.012 ab 146.37±20.74 ab 94.08±5.45 a b2w2 21.94±3.00 b 19.87±2.65 b 0.046±0.006 ab 169.94±28.98 ab 89.87±3.58 ab d3 b0w0 23.91±4.15 bdc 21.44±3.43 bc 0.042±0.007 abcd 179.40±27.73 abc 88.93±2.55 ab b0w1 30.06±5.32 a 26.72±4.68 a 0.034±0.007 d 178.80±25.58 abc 91.79±3.25 ab b0w2 26.88±1.03 ab 23.87±0.83 ab 0.037±0.001 dc 141.81±3.650 c 90.19±3.15 ab b1w0 20.20±0.92 d 18.13±0.92 c 0.049±0.002 a 187.04±18.13 ab 91.93±4.65 ab b1w1 24.28±3.05 bdc 21.37±2.71 bc 0.042±0.006 abcd 207.87±27.60 a 92.57±2.99 ab b1w2 20.49±2.33 d 18.50±2.00 c 0.049±0.005 a 143.84±23.18 c 88.18±7.07 ab b2w0 22.09±2.30 bdc 19.31±1.83 bc 0.046±0.004 abc 219.57±30.82 a 86.77±9.00 b b2w1 21.09±0.66 dc 18.93±0.62 c 0.047±0.002 ab 149.93±11.97 bc 93.26±2.27 ab b2w2 26.23±4.69 abc 23.64±4.11 ab 0.039±0.006 bdc 152.17±36.34 bc 95.26±1.05 a d6 b0w0 27.88±5.25 a 24.21±4.14 a 0.037±0.007 b 132.12±30.02 d 89.11±1.51 a b0w1 24.70±3.32 ab 21.65±3.02 abc 0.040±0.005 ab 171.00±0.265 bcd 86.25±2.87 a b0w2 21.32±2.89 b 18.84±2.37 bc 0.047±0.006 ab 230.15±57.15 a 87.86±2.45 a b1w0 21.55±4.21 b 18.93±3.47 bc 0.047±0.010 ab 183.16±25.48 abc 85.29±1.02 a b1w1 26.51±2.25 ab 23.67±2.05 ab 0.038±0.003 ab 185.28±5.620 ab 88.52±0.89 a b1w2 23.51±4.71 ab 20.34±4.06 abc 0.044±0.009 ab 133.42±19.90 cd 85.48±0.61 a b2w0 20.45±0.83 b 17.99±0.63 c 0.049±0.002 a 183.34±31.65 abc 86.99±8.56 a b2w1 23.79±3.21 ab 21.07±2.98 abc 0.043±0.005 ab 156.24±27.68 bcd 89.01±4.65 a b2w2 25.90±3.91 ab 21.81±3.36 abc 0.039±0.006 ab 187.46±30.86 ab 91.42±1.65 a d7 b0w0 25.06±4.45 ab 20.74±4.19 ab 0.040±0.008 bc 132.82±11.87 bcd 87.73±3.27 abc b0w1 19.29±1.59 c 15.61±1.01 c 0.052±0.005 a 152.33±37.82 abcd 81.23±5.53 cd b0w2 26.07±0.66 a 22.07±0.50 a 0.038±0.001 c 186.27±46.42 a 88.16±1.45 abc b1w0 21.54±1.69 abc 17.63±1.20 abc 0.046±0.004 abc 154.85±13.34 abcd 76.83±7.62 d b1w1 21.81±1.66 abc 18.28±1.29 abc 0.046±0.004 abc 178.69±9.230 ab 88.83±2.33 abc b1w2 21.89±2.41 abc 18.44±2.17 abc 0.046±0.005 abc 114.03±14.74 cd 87.35±0.37 abc b2w0 20.49±4.88 c 17.29±4.16 c 0.050±0.013 ab 159.36±28.69 abc 84.76±2.51 bcd b2w1 23.75±3.60 abc 19.9±3.94 abc 0.043±0.006 abc 107.78±12.32 d 95.55±14.2 a b2w2 21.68±2.50 abc 17.93±2.41 abc 0.047±0.006 abc 142.62±44.05 abcd 91.92±3.13 ab z means followed by the same letters within columns of each treatment, and the measured trait is not significantly different based on l.s.d. at p < 0.05. b = biochar; b0 = 0 kg m-2; b1 = 1 kg m-2; b3 = 2 kg m-2. w = water table; w0 = control; w1= 20 cm bss, w2= 10 cm bss; bss = below soil surface journal of tropical crop science vol. 10 no. 1, february 2023 www.j-tropical-crops.com 52 mei meihana, benyamin lakitan, m. umar harun, susilawati, erna siaga, laily ilman widuri, kartika kartika figure 2. continuous measurement of relative leaf expansion rate (rler) in beans at 1 to 14 days after treatments. rler began to drop significantly on the sixth day after treatment and finally stopped at the end of the recovery period. two water table depths were 20 cm (a) and 10 cm below the soil surface (b) for one week; bss = below soil surface. proline accumulation and plant growth analysis of bean leaf (phaseolus vulgaris l.) .......... journal of tropical crop science vol. 10 no. 1, february 2023 www.j-tropical-crops.com 53 figure 3. plant biomass at different water table (a) and biochar (b) applications. at day 0 (d0) and three days after treatment (d3) water table had no effects on plant dry weight, but at six (d6) and seven days (d7) after treatment plant dry weight significantly increased with water table 20 cm bss (w1). biochar application increased plant dry weight at d0, d3, d6, and r7. the highest dry weight was obtained by application of 2 kg.m-2 of biochar (b2). journal of tropical crop science vol. 10 no. 1, february 2023 www.j-tropical-crops.com 54 mei meihana, benyamin lakitan, m. umar harun, susilawati, erna siaga, laily ilman widuri, kartika kartika conclusion proline accumulation is an adaptation mechanism of beans to the shallow water table. the biochar application of 2 kg.m-2 provides good aeration in soil, enhancing the bean’s ability to survive under shallow water table conditions. beans can be categorized as tolerant crops to shallow water tables for vegetable cultivation in the riparian wetlands on the rainy season. acknowledgment this work was 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(2017). variation in relative water content, proline accumulation and stress gene expression in two cowpea landraces under drought. journal of plant physiology 218, 26-34. doi: 10.1016/j. jplph.2017.07.009. june_2014.cdr journal of tropical crop science (issn 2356-0169; e-issn 2356-0177) is published four-monthly by department of agronomy and horticulture, faculty of agriculture, bogor agricultural university, indonesia. publication details, including instructions for authors and subscription information: www.j-tropical-crops.com any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by the publisher. the accuracy of the content should be independently veri�ed with primary sources of information. the publisher shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the content. permission to make digital or hard copies of part or all of a work published in journal of tropical crop science is granted for personal or educational/classroom use provided that copies are not made or distributed for pro�t or commercial advantage. ©department of agronomy and horticulture, faculty of agriculture, bogor agricultural university, indonesia. all rights reserved. journal of tropical crop science volume number 1 20141 june on the cover the cover image shows sun�owers by darda effendi editorial board krisantini sintho wahyuning ardie sandra a. aziz robert j. hampson satriyas ilyas tri koesoemaningtyas rohana p mahaliyanaarachchi awang maharijaya maya melati roedhy poerwanto bambang sapto purwoko sudarsono muhamad syukur hugo volkaert malcolm wegener managing editor krisantini graphic design syaiful anwar features editor damayanti buchori dadang sisir mitra agus purwito ernan rustiadi short communication tropical and subtropical fruits in india sisir mitra heliconia cultivar registration dave skinner, jan hintze, bryan brunner research articles estimation of genetic parameter for quantitative characters of pepper ( l.)capsicum annuum muhamad syukur, syaidatul rosidah irrigation volume based on pan evaporation and their effects on water use ef�ciency and yield of hydroponically grown chilli eko sulistyono, abe eiko juliana evaluation of commercial sun�ower (helianthus annuus ) cultivars in bogor, indonesia, forl. ornamental and nursery production syarifah iis aisyah, khotimah, krisantini different growth partitioning and shoot production of talinum triangulare treated with organic and inorganic fertilizer sandra ari�n aziz, leo mualim, sitta azmi farchany cloning and characterization of p5cs1 and p5cs2 genes from l. under droughtsaccharum officinarum stress hayati minarsih iskandar, dwiyantari widyaningrum, sony suhandono journal of tropical crop science (issn 2356-0169; issn 2356-0177) is published four-monthly (one volume per year) byedepartment of agronomy and horticulture, faculty of agriculture, bogor agricultural university, ipb darmaga campus, bogor, indonesia 16680. send all inquiries regarding printed copies and display advertising to or to secretary, department ofinfo@j‐tropical‐crops.com agronomy and horticulture; telephone/fax 62-251-8629353. permission to reprint: permission to make digital or hard copies of part or all of a work published in isjournal of tropical crop science granted for personal or educational/classroom use provided that copies are not made or distributed for pro�t or commercial advantage and that copies bear the full citation and the following notice on the �rst page: “copyright department of agronomy and horticulture, faculty of agriculture, bogor agricultural university”. for all other kinds of copying, request permission in writing from head of school, department of agronomy and horticulture of�ce, ipb darmaga campus, bogor, indonesia 16680. © department of agronomy and horticulture, faculty of agriculture, bogor agricultural university. all rights reserved. printed in the republic of indonesia.in s t t t u i b o g o r perta n ia n abstract the research was conducted at leuwikopo experimental field and plant breeding laboratory of ipb, dramaga, bogor, west java, indonesia from october 2012 to april 2013. crossing population between c15 and c2 genotype were used to study genetic parameters for quantitative characters in pepper. all the characters were not controlled by maternal effect, except fruit length. broad-sense heritability were high for plant height, stem diameter, dichotomous height, day to �ower , day toing harvest ; and medium for fruit length and diameter.ing narrow-sense heritability was high for stem diameter, dichotomous height, day �owering; medium for plantto height, day to harvest ; and low for fruit length anding diameter. the ratio of additive variance was high for all the characters, except for fruit length and fruit diameter. keyword :s heritability, heterosis, heterobeltiosis, additive variance, dominant variance introduction estimation of genetic parameters for quantitative characters is important for plant breeding programs. analysis of genetic parameters is used to obtain information on gene actions, genetic variability, heritability, and other genetic information. the genetic information is of great importance to allow more ef�cient and effective plant selection (syukur et al., 2012). there are potentials to increase pepper production in the tropics through breeding. pepper has wide range of genetic resources which are crucial for plant breeding program (syukur et al., 2012). pepper breeding has been conducted through hybridisation, followed by selection (hilmayanti et al. 2006). the selection process has high, probability to bring genetic improvements if the selected characters have high heritability. previous studies have reported that broad and narrowsense heritability for quantitative characters range from low to high (ben-cham and paran, 2000). broadsense heritability, which captures the proportion of phenotypic variation due to genetic values, was high for dichotomous (arif et al., 2012); day to �ower (lestari et al., 2006), day to harvest (arif et al., 2012), fruit length and diametering (syukur et al., 2010a). narrow-sense heritability was low for day to harvest , medium for dichotomous (arif et al.,ing 2012); and high for fruit length and diameter. the objective of the study was to estimate genetic parameters for quantitative characters in pepper. materials and methods the research was conducted at leuwikopo research station (6° 24' s, 106° 33'e) and plant breeding education laboratory, department of agronomy and horticulture, bogor agricultural university at darmaga, bogor, west java, indonesia, in october 2012 to april 2013. genotypes of ipb c-15 (source: avrdc), ipb c2 (source: department of agronomy and horticulture, ipb), f1 (ipb c15 x 1pb c2), f1 ipb c2 ipb c15of and crossing, bcp1 (fi x 1pb c15), bcp2 (f1 x 1pb c2), and were used in this study twenty plants each of p1,f2 . p2, f1, and f1r; 50 plants each of bcp1 and bcp2; and 126 plants of f2 were planted. pepper seeds were sown on trays with pasteurised media and grown up to 6 weeks after sowing. the growing area was covered with black and silver plastic mulch prior to planting. seedlings were then transplanted to the �eld with planting distance of 50 cm x 50 cm. organic fertiliser at 1 kg per plant, urea at 200 kg ha , sp-36 at 150 kg ha-1 1 -1and kcl at 150 ha were applied one week before planting. pest and diseases were controlled by alternate weekly application of insecticide and fungicide application, or as required, at the recommended doses. plants were fertilized weekly using 10 g.l npk mutiara-1 16n-16p-16k at 250 ml per plant. scoring was conducted on the following quantitative characters: day to �owering (das), day to harvesting (das), plant height (cm), dichotomous height (cm), fruit length (cm) and fruit diameter (mm). broad-sense heritability was estimated using a method developed by allard (1960): h = (( )/2bs v – (v + v + v )/3f2 f1 p1 p2 vf2) x 100%; where h = broad-sense heritability, v = p1 2 bs p1 population variance, v = p2 population variance, v =p2 f1 f1 population variance, and v = f2 population variance.f2 narrow-sense heritability was estimated using a method developed by warner (1952): h = ((2v (v +2ns f2 bcp1 v ))/v ) x100%; where h = narrow-sense heritability,bcp2 f2 ns 2 research article muhamad syukur*, syaidatul rosidah department of agronomy and horticulture, faculty of agriculture, bogor agricultural university, jl. meranti, kampus ipb dramaga 16680, bogor, indonesia corresponding author* ; email: muhsyukur@ipb.ac.id estimation of genetic parameter for quantitative characters of pepper ( l.)capsicum annuum 3 4 journal of tropical crop science vol. 1 no. 1, 2014june www.j-tropical-crops.com v = bcp1 population variance, v = bcp2bcp1 bcp2 population variance, and v = f2 population variance.f2 estimated heritability value was considered low if h <2 20%, medium if 20% < h < 50%, and high if h > 50%2 2 (halloran et al., 1997). maternal effects were tested by comparing means of f1 and f1r using t-test at =5%. additive variance ratio was calculated using formula of a = (h /h ) x 100% a h2 2 2ns bs ns; where is additive variance ratio, is narrow-sense heritability, and is broad-senseh2bs heritability. heterosis was calculated using formula of mid parent ; where is meanheterosis = (x /mp) x 100% xf1 f1 average of and is mean average of both parents.f1 mp heterobeltiosis was measured using the formula = (x /hp) x 100%, x f1 hpf1 f1where is average of and is mean average of the best parents. results normality test was conducted to study the frequency distribution of f2. all f2 variables had a normal frequency distribution. continuous and normal distribution showed that the characters were controlled by polygenic genes. f2 population had the highest variability, followed by bcp1 and bcp2 populations. p1, p2, f1 and f1r populations were more uniform than bcp1, bcp2, and f2 (table 1). the highest variability in f2 population was due to the maximum segregation in the population. p1 a n d p 2 p o p u l a t i o n s w e r e h o m o z y g o u s a n d homogeneous, which means that each individual had the s a m e g e n e t i c c o m p o s i t i o n . f 1 a n d f 1 r w e r e heterozygous and each individual had the same genetic composition (homogeneous). the maternal effect test showed that there was no signi�cant difference between f1 and f1r for all variables (table 2). this suggests that female parent did not affect the inheritance of the character, which indicates that these characters are controlled by genes in the nucleus. broad-sense heritability of all characters ranged from 43.01% to 79.5%, or classi�ed as moderate to high. broad-sense heritability estimates were high for plant height, stem diameter, dichotomous height, day to �owering and day to harvesting, whereas it was were moderate for fruit length and fruit diameter. this was a re�ection of the role of genetic variance. genetic variances were greater than the environmental variance tabl , ,e means variance and standard deviation of quantitative characters in pepper population1. characters plant height (cm) means variance standar deviation steam diameter (mm) means variance standar deviation dichotomous height (cm) means variance standar deviation day to �owering (das) means variance standar deviation day to harvesting (das) means variance standar deviation fruit length (cm) means variance standar deviation fruit diameter (mm) means variance standar deviation 50.87 146.89 12.12 8.30 4.18 2.04 14.98 17.84 4.22 91.08 68.56 8.28 137.24 40.36 6.35 13.74 4.33 2.08 15.29 4.66 2.16 63.65 214.62 14.65 10.87 6.93 2.63 15.72 19.66 4.43 98.95 85.10 9.22 141.46 48.89 6.99 12.70 3.65 1.91 16.65 5.04 2.24 49.08 138.48 11.68 9.44 3.50 1.87 8.32 2.50 1.58 92.60 14.93 3.86 139.90 6.32 2.51 10.10 2.91 1.71 15.85 3.07 1.75 54.32 95.98 9.80 9.18 2.29 1.51 20.70 4.55 2.13 85.20 20.62 4.54 130.30 26.93 5.19 14.20 1.22 1.10 17.27 3.40 1.84 52.18 89.23 9.45 9.09 2.60 1.61 12.39 5.03 2.24 92.00 30.44 5.52 137.89 36.55 6.05 13.17 2.10 1.45 17.25 1.37 1.17 59.82 87.80 9.37 9.60 3.94 1.99 14.76 7.71 2.78 94.90 42.10 6.49 138.70 22.94 4.79 11.23 4.62 2.15 16.23 4.12 2.03 54.78 185.58 13.62 9.76 5.65 2.38 11.17 8.01 2.83 95.42 41.68 6.46 142.58 34.86 5.90 12.72 2.84 1.69 17.64 4.49 2.12 p2 f1p1 population f1r bcp1 bcp2 f2 note: = ipb , = ipb , = ipb x ipb , = ipb x ipb , = x ipb ,p1 c15 p2 c2 f1 c15 c2 f1r c2 c15 bcp1 f1 c15 bcp2 f1 f2 f1 sel�ng= x ipb c2, = 5e e.........stimation of genetic parameter for quantitativ journal of tropical crop science vol. 1 no. 1, 2014june www.j-tropical-crops.com p1 p2 f1 mp heterosis (%) heterobeltiosis (%) 49.08 54.32 52.18 51.70 0.93 -3.94 9.44 9.18 9.09 9.31 -2.36 -3.71 variance component ph (cm) sd (mm) 8.32 20.70 12.39 14.51 -14.61 -40.14 dh (cm) 92.60 85.20 92.00 88.90 3.49 6.80 df (das) 139.90 130.60 137.89 135.25 1.95 7.29 dh (das) 10.10 14.20 13.37 12.15 8.40 -7.25 fl (cm) 15.85 17.27 17.25 16.56 4.17 -0.12 fd (cm) environment variance (σ2e) phenotype variance )(σ2p genetic variance )(σ2g additive variance )(σ2a additive variance ratio broad-sense heritability (h )2bs narrow-sense heritability (h )2ns 107.23 214.62 107.39 96.77 90.11 50.04 45.09 2.79 6.93 4.14 4.03 97.34 59.74 58.15 variance component ph sd 4.03 19.66 15.63 13.47 86.18 79.50 68.51 dh 22.00 85.10 63.10 59.96 95.02 74.15 70.46 df 23.27 48.89 25.62 22.56 88.06 52.40 46.14 dh 2.08 3.65 1.57 0.12 7.64 43.01 3.28 fl 2.61 5.04 2.43 0.93 38.27 48.21 18.45 fd for all characters (table 3). narrow-sense heritability for all character ranged from 3.28% to 70.46%, or classi�ed as low to high. narrowsense heritability estimates were high for stem diameter, dichotomous height, and day to �owering; moderate for plant height and day to harvesting, and low for fruit length and fruit diameter. this was a re�ection of the role of additive variance, which was re�ected in the ratio of additive variance. the ratio of additive variance was high for all the characters, except for fruit length and fruit diameter (table 3). heterosis values were positive for plant height, day to �owering, day to harvesting, fruit length and fruit diameter, while was negative for fruit length. however, heterobeltiosis was positive in day to �owering and day to harvesting (table 4). negative heterobeltiosis means the tabl f1 an f1re standard deviation and mean value t-test of quantitative character in d2. means, note: = ipb x ipb , = ipb x ipb , = , = 0.05f1 c15 c2 f1r c2 c15 ns no signi�cant p <* table and heritability of quantitative characters in pepper3. variance component note: ph = , sd = , dh = , df = ,plant height stem d dichotomous height day to �oweringiamater day to harvesting fruit length fruit diameterdh = , fl = , fd = tabl heterosis an heterobeltiosise d of quantitative characters in pepper4. note: = ipb = ipb = = , ph = , sd = , dh =p1 c15 p2 c2 f1 c15xc2 mp iamater, , , mid parent plant height stem d dichotomous height day to �owering day to harvesting fruit length fruit diameter, df = , dh = , fl = , fd = muhamad syukur, syaidatul rosidah6 plant height (cm) steam diameter (mm) dichotomous height (cm) day to �owering (das) day to harvesting (das) fruit length (cm) fruit diameter (mm) 52.18 9.45+ 9.09 1.61+ 12.39 2.24+ 92.00 5.52+ 137.89 6.05+ 13.17 1.45+ 17.25 1.17+ 59.82 9.37+ 9.60 1.99+ 14.76 2.78+ 94.90 6.49+ 138.70 4.79+ 11.23 2.15+ 16.23 2.03+ -0.60ns ns-1.77 ns-2.03 -1.08ns -0.35ns 2.28* 1.33ns characters f1 f1r t-value value of f1 was lower than that of the best parent. heterosis and heterobeltiosis for day to �ower and day to harvesting were expected to be negative as the selection for this character was directed to obtain earliness character. discussion if a character is in�uenced by the female parent, the offspring of the reciprocal will likely be different (from the female parent), and the offspring will show traits of its female parent. f1 and f1 reciprocal (f1r) population cannot be merged because segregation of the f2 population will be different and does not follow mendelian segregation law. however, if there was no signi�cant difference between f1 and f1r then the two populations can be merged for the purposes of this analysis. a study by arif . (2012) showed that dichotomous height andet al day to harvesting were not affected by maternal characters. in addition, arif et al. (2011) reported there was no maternal effect for �ower position, young fruit journal of tropical crop science vol. 1 no. 1, 2014june www.j-tropical-crops.com color, young stems color and texture of the pepper fruit surface. therefore the offspring and reciprocal can be merged to obtain the next offspring, and the f2 segregation will follow mendel's laws. heritability estimate of a character is important to predict whether the character is heavily in�uenced by genetic or environmental factors. high heritability suggests that genetic factors had greater in�uence over environment on phenotypic appearance. high heritability value, particularly the narrow-sense heritability, plays an important role in increasing the effectiveness of selection (syukur et al., 2012). heritability value is between 0 and 1. heritability value of close to 0 means the variance of phenotypes is primarily caused by environmental factors; values close to 1 means that it was mainly caused by genotype. values closer to 1 indicate higher heritability whereas values closer to 0 indicates lower heritability (syukur et al., 2012). broad-sense heritability was moderate to high for all characters. several studies on pepper showed that broad-sense heritability estimates were high for dichotomous height (syukur et al., 2010b ), stem diameter ( ),syukur et al., 2010b; nsabiera et al., 2013 plant height ( ),syukur et al., 2010b; nsabiera et al., 2013 day to �owering (lestari et al. 2006; syukur et al., 2010c;, syukur et al., 2011; nsabiera et al., 2013), day to harvesting (syukur et al., 2011; arif et al. 2012; nsabiera, et al., 2013 syukur ., 2010a; syukur), fruit length ( et al et al., 2010b; syukur et al., 2011; nsabiera et al., 2013) and fruit diameter (manju and sreelathakumary, 2002; sreelathakumary and rajamony, 2004; lestari et al., 2006; syukur ., 2010a; syukur ., 2010b; syukuret al et al et al., 2010c; syukur et al., 2011). narrow-sense heritability was low to high for all characters. narrow-sense heritability was low for fruit length and fruit diameter, while the other characters were moderate to high. several studies showed that narrowsense heritability estimates in chilli were moderate to high for dichotomous height ( ), plant heightsyukur et al., 2010 (ben-cham and paran, 2000; marame et al., 2008; singh et al. 2014 singh et al. 2014, ,), day to �owering ( ), day to harvesting (ben-cham and paran, 2000; marame et al., 2008 ben-cham and paran, 2000; marame), fruit length ( et al., 2008; syukur et al., 2010a; singh et al. 2014, ) and fruit diameter (ben-cham and paran, 2000; syukur et al., 2010a; singh et al. 2014, ). the appearance of heterosis effects was caused by the accumulation of dominant genes whereas heterobeltiosis was due to the over-dominant gene on the character. genetic distance among two parents is one of the factors that lead to high heterosis value (syukur et al., 2012). daryanto et al. (2010) and sitaresmi et al. (2010) reported correlation between the parental genetic distance and heterosis. parental genetic distance that is far apart produces high heterosis values. the role of dominant effect on the observed character can also be seen from the ratio of additive variance. fruit length and fruit diameter have a low ratio of additive variance. this ratio indicates that dominant variance was more in�uential in controlling the character. it can also be seen from the high heterosis values in both fruit length and fruit diameter. conclusion all the characters except for fruit length were not controlled by maternal effect. broad-sense heritability was high for plant height, stem diameter, dichotomous height, day to �owering, and day to harvesting, and was moderate for fruit length and fruit diameter. narrow-sense heritability was high for stem diameter, dichotomous height, and day to �owering; moderate for plant height and day to harvesting; and low for fruit length and fruit diameter. the ratio of additive variance was high for all the characters, except for fruit length and fruit diameter. acknowledgement we thank the indonesian ministry of agriculture who provided funding for this research through the kkp3n program in 2012. references allard, r.w. (1960). principles of plant reeding. j wileyb & sons, new york, usa. arif, a.b., sujiprihati, s. and syukur, m. (2011). inheritance of several qualitative characters in three group pepper. , 73-bulletin plasma nutfah 17 79. arif, a.b., sujiprihati, s. and syukur, m. (2012). estimation of several genetic parameter on quantitative characters of hybridization between big and curly chilli ( l.).capsicum annuum jurnal agronomi indonesia ,119-12440 . ben-cham, a. and paran j. (2000). genetic analysis of quantitative traits in pepper (capsicum annuum l.). journal of american society of horticultural science , 66-70.125 daryanto a , 2010 ., . sujiprihati, s. and syukur, m. ( ) heterosis and combining ability of chilli genotypes ( l.) for agronomy characters incapsicum annuum half diallel crosses. ,jurnal agronomi indonesia 38 113-121. halloran, g.m., knight, r., mcwhirter, k.s. and sparrow, d.h.b. (1979). plant breeding. australian vice chancellors committee, brisbane, australia. 5 7e e.........stimation of genetic parameter for quantitativ journal of tropical crop science vol. 1 no. 1, 2014june www.j-tropical-crops.com hilmayanti, i., dewi, w., murdaningsih, rahardja, m., rostini, n. and setiamihardja, r. (2006). the inheritance of �owering time and fruit size characters in pepper ( l.).capsicum annuum zuriat , 86-93.17 lestari, a.d., dewi, w., qosim, w.a., rahardja m.,, rostini, n. and setiamihardja, r. (2006). genetic variability and heritability of yield components and yield characters of �fteen red pepper genotypes. zuriat , 94-102.17 manju, p.r., and sreelathakumary, i. (2002). genetic variability, heritability and genetic advance in hot chilli ( jacq.).capsicum chinense journal of tropical agriculture , 4-6.40 marame f , desalegne, l singh h , fininsa c, . ., , . , . and sigvald r. 2008 . genetic components and, ( ) heritability of yield and yield related traits in hot pepper. research journal of agriculture and biological sciences 803-809.4, nsabiera, v., m. ochwo-ssemakula, . seruwagi, .m p ojiewo . gipson (2013). combining abilityc and p., for �eld resistance to disease, fruit �eld and �eld factors among hot pepper ( l.)capsicum annuum genotypes in uganda. international journal of plant breeding 7, 12-21. singh, p., cheema, d.s., dhaliwal, m.s., garg, n.and (2014). heterosis and combining ability for earliness, plant growth, yield and fruit attributes in hot pepper ( l.) involvingcapsicum annuum genetic and cytoplasmic-genetic male sterile lines. scientia horticulturae 168, 175–188. sitaresmi t., sujiprihati, s. and syukur, m. (2010). combining ability of several introduced and local chilli pepper ( l.) genotypescapsicum annuum and heterosis of the offsprings. jurnal agronomi indonesia , 212–217.38 sreelathakumary i rajamony . 2004 . variability,, . and , l ( ) heritability and genetic advance in chilli (capsicum annuum l.). 35-journal of tropical agriculture ,42 37. syukur m , sujiprihati, siregar . 2010, . s. and , a ( b). estimation of genetic parameters of agronomic characters and yield evaluation on f4 pepper using augmented design ,. 9-jurnal agrotropika 15 16. syukur, m., sujiprihati, s. yunianti, r. (2012). teknikand pemuliaan tanaman. penebar swadaya, jakarta. syukur m , sujiprihati, yunianti, kusumah, . s., r. and , d.a ( ) estimation of genetic variance and. 2011 . heritability for yield component characters in chili pepper genotypes ,. 148-156.j agrivigorurnal 10 syukur, m., s., r. and nida, k.sujiprihati, yunianti, (2010c). the estimation of variant component, heritability, and correlation to determine selection criteria in the f5 population of pepper (capsicum annuum jurnal hortikultura indonesial.). , 74-1 80. syukur m , sujiprihati, yunianti, undang., s., r. and. ( a)2010 . diallel analysis using hayman method to study genetic parameters of yield components in pepper ( l.).capsicum annuum h jayati ournal of bioscience 17, 183-188. warner, j.n. 1952. a method of estimating heritability. agronomy journal , 427-430.44 5 muhamad syukur, syaidatul rosidah8 journal of tropical crop science vol. 1 no. 1, 2014june www.j-tropical-crops.com ultrafine bubbles water priming to improve viability and vigor of bean .......... journal of tropical crop science vol. 10 no. 1, february 2023 www.j-tropical-crops.com 27 effects of gibberellic acid (ga3) application on the plant growth and seed production of pinto peanut (arachis pintoi krap & greg) robi rahmat dani, juang gema kartika*, maryati sari department of agronomy and horticulture, faculty of agriculture, ipb university jl. meranti, ipb darmaga campus, bogor 16680, indonesia *corresponding author; email: juangkartika@apps.ipb.ac.id abstract pinto peanut (arachis pintoi krap & greg.) is a legume usually used as a cover crop, bio mulch in fruit and vegetable plantations, ornamental plants, and animal feed. pinto peanut has many benefits; through symbiosis with rhizobacteria, they can fix nitrogen, as ground cover can reduce the risk of landslides, inhibit weed growths, and is a source of nectar for bees. arahcnis pintoi can be propagated vegetatively or generatively, but generative propagation is hard to conduct in the tropics because it takes a long time for the plants to produce seeds. our study was conducted to determine the effect of ga3 application on the seed production of a. pintoi. the experiment was arranged in a single-factor randomized complete block design with ga3 concentrations of 0, 75, 150, 225, and 300 ppm. an orthogonal polynomial test was conducted to determine the effective concentrations for ga3 on seed formation and viability. harvested seeds were stored for one month, then sown on sand medium; the seeds were soaked in 1% kno3 solution to break seed dormancy prior. our study showed that the ga3 effective concentration range from 130.69 ppm to 137.16 ppm, indicated by the increase in the number of flowers at 105, 120, 135, and 150 dat up to 28.59% compared to control. ga3 at 126.80 ppm can also increase the number of harvested pods by 18.16%. the effect of ga3 on vegetative growth was shown by the increase in the growth of new individual plants concentration, i.e. 53.25 new individual plants with ga3 application of 141.88 ppm. keywords: dormancy, gibberellin acid, growth regulators, kno3, legume cover crop. introduction pinto peanut (arachis pintoi) is a low-growing perennial species that spreads by stolons or runners. the leaf of pinto peanut has four leaflets in each leaf, usually round-tipped and light green; the flowers are predominantly yellow. the seed production occurs in pods; at the base of a flower, a peg grows to a length of 5 to 30 cm, and penetrates the soil surface where it will produce a pod (sanchez et al., 2020). pintoi peanut pods usually contain one seed; however, two or three seeds may be observed (sanchez et al., 2020). one of the main characteristics of pintoi peanut pods is that they drop from the pegs after maturity; therefore it requires soil separation from the pods to obtain the seeds. arachnis pintoi grows by forming a strong weave with roots or tendrils that will grow when the stem is in direct contact with the soil. in the tropics and sub-tropics, pinto peanuts can grow in the lowlands and highlands under 70–80% shade. in indonesia, arachnis pintoi is popularly known as an ornamental peanut (balittan, 2004). arachnis pintoi has a fast growth rate during the vegetative phase and a slow growth rate during the generative phase; they bloom throughout the year with 40–65 flowers per m2 per day (sumiahadi, 2014). after pollination, the ovary in the gynophore elongates up to 27 cm and penetrates the soil up to 7 cm, and forms a pod that usually contains a seed (balittan, 2004). the deeper the root area, the smaller the root density, the root zone with a large density occurs at 1–10 cm below the soil surface (pronaningrum, 2016). arachnis pintoi can be propagated using seeds, cuttings, and stolon (balittan, 2004). the germplasm of pinto peanut shows wide variability in adaptation, dry matter yield, nutrient content, and seed production (carvalho and quesenberry, 2012). seed production studies of a. pintoi in indonesia has not been widelycarried out because it takes about 8 months for pinto peanut to produce seeds, with a yield of 1.76 – 2.1 ton.ha-1 (fanindi et. al, 2012). seed production of journal of tropical crop science vol. 10 no. 1, february 2023 www.j-tropical-crops.com 28 robi rahmat dani, juang gema kartika, maryati sari a. pintoi increased with longer forage harvest time. the average forage production harvested at 6, 12, and 18 months was 1.8, 5.2, and 5.9 tonnes.ha-1, respectively (aminah et. al, 1994). seed formation in the highlands can obtain seeds with high germination rates, whereas pinto peanuts grown in the lowlands produce fewer seeds with low germination rates (neef et al., 2004). the seeds of a. pintoi come from the gynophore extension, which forms a seed-filled pod (figure 4). seed formation can be promoted by gibberellic acid (ga3) treatment; elevated levels of gibberellins can promote seed development, flowering, stem elongation, and leaf growth (salisbury and ross, 1995). ga3 application to plants can increase the auxin biosynthesis through proteolytic enzymes that are formed and release tryptophan compounds as auxin precursors. gibberellic acid (ga3) can increase the percentage of flowers in pods (salisbury and ross, 1995). ga3 at a concentration of 80 to 160 ppm applied to peanut crops can prevent flower abscission, reducing flower drops and increasing pod production (yennita, 2014). the formation of seeds in a. pintoi can potentially be increased by applying ga3 exogenously to the plants. according to putra (2012), the application of 100 ppm ga3 can increase bulb production, the number of flowers, and the number of seed bunches of shallot “super phillips”. a study in soybean by irwan et al. (2019) reported that ga3 application at 350 ppm increased the number of seeds. seed production of a. pintoi is important for producing planting materials, particularly for the remote locations. our current study provides information on the extent of ga3 treatment for a. pintoi to produce viable seeds that are needed for propagation. material and methods the study was conducted at the pasir sarongge experimental field, university farm ipb, pacet district, cianjur regency, west java, which has an altitude of about 1200 meters above sea level (m asl). the experiment was conducted from august 2020 may 2021, during which the average rainfall was at 274.9 mm per month (bmkg, 2021). a single-factor randomized complete block design was used for the experiment which included five ga3 concentrations, namely 0, 75, 150, 225, and 300 ppm (parts per million). an orthogonal polynomial follow-up test was conducted to determine the effective concentrations for ga3 on seed formation and seed viability in every concentration with a significant or very significant effect. a plot of land measuring 2 m x 1 m was used with for planting material as many as 12 pols measuring 15 x 30 cm or comparable to a cover of 27% of the beds. the experimental site was cleared, and a plot of beds measuring 2 x 1 m with 30 cm of distance between the plots and 40 cm of distance between replicates were prepared. the plot beds were treated with 2 tons.ha-1 of cow manure,1 ton.ha-1 agricultural lime,100 kg.ha-1 urea fertilizer,150 kg.ha-1 sp-36, and 150 kg.ha-1 kcl. urea fertilizer was applied at the beginning of planting and 4 weeks after planting (wap). weed control was conducted manually throughout the study duration. varying concentrations (75, 150, 225, and 300 ppm) of ga3 were prepared. for instance, 75 ppm of ga3 was prepared by dissolving 75 mg ga3 in 1 l of water. plants were treated/sprayed with ga3 at 30 dap (days after planting) and repeated every 30 day-interval. a dose of 1000 l.ha-1 of ga3 was usually given in the morning. seeds from the plants were harvested after six months by disassembling the beds. after 1 month of storing the seeds, a seed nursery was prepared where seeds are sown on sand media that has been sifted and sterilized by drying in direct sunlight, followed by steaming for 1 hour, and redrying. before sowing, the seeds were immersed in 1% kno3 solution for 2 hours to break seed dormancy. growth and production measurement data included the percentage of soil cover, the addition of new runners, flower production, harvested seeds, percentage of seed formation, seed weight, and seed germination percentage. results and discussion growth and acceleration of soil coverage after application of ga3 the acceleration of soil cover is an important aspect in developing a. pintoi as a bio mulch. the soil cover of 100% was achieved at 60 days after treatment (dat) or 90 days after planting (dap), at which time observations were completed. the acceleration of soil cover in all treatment plots did not vary, which means that the application of ga3 had no significant effect on the acceleration of soil cover in all plots at 0, 15, 30, 45, and 60 dat (table 1). there were rapid growths during 45 dat and 60 dat, which significantly increased the percentage of soil cover. accelerated soil cover can be due to the rainy season, which promotes vegetative growth. planting of a. pintoi in the wet or rainy season can increase plant growth, leading to the highest increase in ultrafine bubbles water priming to improve viability and vigor of bean .......... journal of tropical crop science vol. 10 no. 1, february 2023 www.j-tropical-crops.com 29 forage production, which is highly correlated with water availability (fanindi et al., 2012). during the dry season, the production of a. pintoi tends to be lower (fanindi et al., 2012). the speed of land coverage by a. pintoi in our study is considered fast. a previous study conducted on the same bed area by simbolon (2018) reported that 98% soil cover of a. pintoi occurred at 135 dap, whereas pronaninggrum (2017) reported 100% soil cover of a. pintoi occurred at 105 dap. in this study, 98% of soil cover occurred at 75 dap (60 dat) and >100% at 90 dap (75 dat). the difference in land cover acceleration was thought to be caused by differences in location and location, climate, and soil conditions. both previous studies were conducted in the lowlands. the soil analysis of the pasir sarongge experimental field had a c-organic content of 3.53%, compared to the 2.38% in the pronaninggrum (2017) study. according to a study by balittan (2004), a. pintoi can grow well in soils that have c-organic > 3%. arachnis pintoi growth the growth of new runners or individual plants is an important factor in accelerating land cover. the more a. pintoi individuals were formed, the faster the plant could cover an area. arachis pintoi runners are stolon that has tap roots. the growth of new individuals can be determined by counting the final number of individuals formed at harvest. ga3 resulted in a significant increase in the final number of individuals and the total new growth of individuals after 180 dap (table 2). the orthogonal polynomial test showed that ga3 concentration had a significant quadratic effect on the total addition of new individuals with the equation y = -0.0025x2 + 0.7094x + 58.336, and the minimum, optimum, and maximum concentrations were found, 137.88 ppm, 141.88 ppm, and 145.88 ppm (figure 1). ga3 application can increase the growth of new individual plants by 53.25% than the plot without treatment; application at 141.88 ppm can be done after 90 dap or 60 dat when the growth has reached 100% coverage to promote the vegetative growth before entering the generative phase. gibberellic acid (ga3) application increased the number of a. pintoi individuals so that soil cover would be faster. gibberellins regulate the process of plant growth and development which specifically plays table 1. the percentage of land coverage of arachis pintoi in 0, 15, 30, 45, and 60 dat. concentration of ga3 (ppm) the percentage of land cover 0 dat 15 dat 30 dat 45 dat 60 dat 0 34.51 77.88 92.98 97.33 100 75 36.08 78.24 93.91 98.65 100 150 36.86 73.47 93.87 97.97 100 225 34.65 79.59 93.98 98.75 100 300 34.51 75.85 94.37 98.43 100 f-test ns ns ns ns ns kk % 12.42 7.29 3.00 1.22 0 note: (ns) not significant according to test. dat = days after treatment. table 2. runner growth of a. pintoi at 180 dap concentration of ga3 (ppmp) runner growth initial (0 dap) number of new individual plants (180 dap) 0 50 59.25 75 44 89.75 150 53 126.75 225 50 76.00 300 48 52.50 f-test ns ** cv % 19.11 16.36 notes: ** = very significant ; ns: no significant; dap= days after planting journal of tropical crop science vol. 10 no. 1, february 2023 www.j-tropical-crops.com 30 robi rahmat dani, juang gema kartika, maryati sari an important role in plant stem elongation (tiwari et al., 2011). arachis pintoi, as a legume ground cover, can also be useful as a refugia plant and become a trap for natural enemies such as nepottetix apicalis, nilaparvata lugens, sogatella furcifera, pachydiplosis oryzae, and locusta migratoria. another study reported that planting a. pintoi as ground cover can increase the number of productive tillers in rice cultivation (erdiansyah et al., 2018). effect of ga3 on the number of flowers the bright flower colour of a. pintoi is one of the factors that make it an attractive ornamental ground cover for home and public gardens. arachis pintoi can flower throughout the year. the application of ga3 stimulates to plant growth and development, such as the formation of flowers and fruit (chen et al. 2014; ramaiah et al., 2014). the application of ga3 can increase production and the number of flowers formed in peanut plantations (yennita, 2014). the number of flowers showed a significant difference after the ga3 treatment entered the planting age of 135 dat (table 3). ga3 treatment did not affect the number of flowers per bed at 0, 15, 30, 45, 60, 75, and 90 dat, but had a significant effect on the number of flowers per bed at the later stage of growth, i.e. 105, 120, 135, and 150 dat (table 3). the results of the orthogonal polynomials analysis showed that the ga3 treatment has a quadratic response to the number of flowers at 105 dat (figure 2) with the equation y = -0.0013x2 + 0.3398x + 104.51, and the minimum, optimum, and maximum concentrations were found, at 126.7 ppm, 130.69 ppm and 134.69 ppm, respectively. the quadratic response was also shown at 120 dat (figure 2) with the equation y = -0.0015x2 + 0.4115x + 105.14, and there were minimum, optimum, and maximum concentrations of 133.33 ppm, 137.1667 ppm, and 141.1667 ppm, respectively. the further test of orthogonal polynomials at 135 dat also showed a quadratic response to ga3 application (figure 2 c) with the equation y = -0.0014x2 + 0.3721x + 111.06 and the minimum, optimum, and maximum concentrations were 128.89 ppm, 132.89 ppm, and 136.89 ppm, respectively. the application of ga3 at 150 dat showed a quadratic response to the number of flowers (figure 2 d) with the equation y = -0.0017x2 + 0.4526x + 116.86, and the minimum, optimum, and maximum concentrations were 129.11 ppm, 133.11 ppm, and 137.11 ppm, respectively. ga3 at 150 ppm increased the number of flowers by 23.08 % to 28.59% than the plots without treatment. ga3 application according to the optimum concentration of 130.69 ppm – 137.16 ppm can be conducted at 120 dap or 90 dat to increase flower production (table 3). the increase in flower formation of a. pintoi can be influenced by gibberellic acid, which has an important role at the initiation stage in flowers and at the early flower development stage, which allows ga3 to influence cell differentiation (yasmin et al., 2014). the results showed that a higher concentration of gibberellins increased the number of flowers formed (table 3). in addition to uses as ornamental ground covers, the yellow pinto peanut's attractive flower shape has insect-repellent properties. according to kurniawati figure 1. the orthogonal polynomial graph showing growth of new individual plants of a. pintoi.   figure 1. the orthogonal polynomial graph showing growth of new individual plants of a. pintoi. y = -0.0025x2 + 0.7094x + 58.336 r² = 0.8079 0 20 40 60 80 100 120 140 0 75 150 225 300 n um be r of n ew in di vi du al p la nt s ga3 concentration (ppm) ultrafine bubbles water priming to improve viability and vigor of bean .......... journal of tropical crop science vol. 10 no. 1, february 2023 www.j-tropical-crops.com 31 ta bl e 3. n um be r o f a ra ch ni s pi nt oi fl ow er s pe r b ed a t 0 , 1 5, 3 0, 4 5, 6 0, 7 5, 9 0, 1 05 , 1 20 , 1 35 , a nd 1 50 d ay s af te r g a 3 tre at m en t c on ce nt ra tio n of g a 3 (p pm ) n um be r o f fl ow er s pe r b ed 0 d at t 15 d at t 30 d at 45 d at 60 d at 75 d at 90 d at 10 5 d at 12 0 d at 13 5 d at 15 0 d at 0 14 .5 0 37 .5 0 10 1. 50 14 5. 00 12 8. 75 10 9. 75 11 6. 00 11 0. 25 10 9. 25 11 6. 00 12 3. 25 75 16 .7 5 36 .7 5 89 .7 5 13 0. 00 14 5. 00 13 0. 00 12 1. 25 10 4. 00 11 2. 75 11 4. 00 12 4. 00 15 0 16 .0 0 38 .5 0 77 .0 0 13 7. 25 14 3. 00 12 3. 50 13 4. 50 14 9. 50 15 3. 00 15 6. 50 16 0. 25 22 5 17 .0 0 35 .0 0 70 .0 0 11 5. 25 13 4. 25 10 4. 00 99 .5 0 10 6. 50 11 0. 25 11 1. 75 13 2. 25 30 0 8. 00 36 .0 0 82 .0 0 14 0. 25 13 2. 75 11 0. 50 11 2. 00 95 .0 0 96 .2 5 97 .7 5 98 .5 0 fte st ns ns ns ns ns ns ns ** ** ** ** c v % 18 .2 7 11 .6 5 28 .4 6 22 .0 6 18 .1 6 23 .3 2 17 .7 1 8. 75 7. 62 11 .9 5 12 .5 8 n ot e: n s= no nsi gn ifi ca nt ; ( ** ) h ig hl y si gn ifi ca nt ; d at (d ay s af te r t re at m en t). t d at a w as tr an sf or m ed to l og (x ) f or a na ly si s; th e da ta p re se nt ed is b ef or e tra ns fo rm at io n journal of tropical crop science vol. 10 no. 1, february 2023 www.j-tropical-crops.com 32 robi rahmat dani, juang gema kartika, maryati sari and martono (2015), insects prefer flowers that are small in size, have an open shape, and have a long flowering duration. therefore, arachis pintoi have these features that allow them to be used by insects as refugia plants. refugia plants function as natural habitats, and alternative habitats for insects so they do not infest the main crops (leksono and yanuwiadi, 2013). effect of ga3 application on seed production ga3 application had significant effects on the total number of pods with a quadratic effect on the total number of pods harvested at 6 months (y = -0.001x2 + 0.2536x + 109.51) (table 8, figure 6). the minimum concentration was 122.8 ppm, an optimum concentration of 126.8 ppm, and a maximum concentration of 130.8 ppm (figure 6). the optimum concentration of 126.80 ppm could be recommended when the plants are at 150 dat or 120 dap to increase the total number of pods harvested. ga3 did not affect the number of intact, half-full, and empty pods at all concentration = (table 4). arachnis pintoi pods are formed from gynophores that extend and penetrate the soil. the gynophores are derived from the extension of the flower stalk (figure 4). figure 4. elongated gynophores of a. pintoi that have formed pods harvested pods are classified into intact, half-full, and empty (figure 5). in a. pintoi, intact pods fully contain seeds, half-full pods contain seeds that are slightly wrinkled and do not fill the pods, and empty pods do not contain seeds or small wrinkled seeds (figure 5). the yield of harvested pods of a. pintoi was dominated by empty pods, resulting in reduced seed production. the high number of empty and half-full pods could be due to insufficient photosynthates figure 2. orthogonal polynomial graph of the number of a. pintoi flowers per bed at 105, 120, 135, and 150 days after treatment (dat) 7 ga3 treatment did not affect the number of flowers per bed at 0, 15, 30, 45, 60, 75, and 90 dat, but had a significant effect on the number of flowers per bed at the later stage of growth, i.e. 105, 120, 135, and 150 dat (table 3). the results of the orthogonal polynomials analysis showed that the ga3 treatment has a quadratic response to the number of flowers at 105 dat (figure 2) with the equation y = -0.0013x2 + 0.3398x + 104.51 and the minimum, optimum, and maximum concentrations were found, at 126.7 ppm, 130.69 ppm and 134.69 ppm, respectively. the quadratic response was also shown at 120 dat (figure 2) with the equation y = -0.0015x2 + 0.4115x + 105.14 and there were minimum, optimum, and maximum concentrations of 133.33 ppm, 137.1667 ppm, and 141.1667 ppm, respectively. figure 2. orthogonal polynomial graph of the number of a. pintoi flowers per bed at 105, 120, 135, and 150 days after treatment the further test of orthogonal polynomials at 135 dat also showed a quadratic response to ga3 application (figure 2 c) with the equation y = -0.0014x2 + 0.3721x + 111.06 and the minimum, optimum, and maximum concentrations were 128.89 ppm, 132.89 ppm, and 136.89 ppm, respectively. the application of ga3 at 150 dat showed a quadratic response to the number of flowers (figure 2 d) with the equation y = -0.0017x2 + 0.4526x + 116.86 and the minimum, optimum, and maximum concentrations were 129.11 ppm, 133.11 ppm, and 137.11 ppm, respectively. ga3 at 150 ppm increased the number of flowers by 23.08 % to 28.59% than the plots without treatment. ga3 application according to the optimum concentration of 130.69 ppm – 137.16 ppm can be conducted at 120 dap or 90 dat to increase flower production (table 3). the increase in flower formation of a. pintoi can be influenced by gibberellic acid, which has an y = -0.0013x2 + 0.3398x + 104.51 r² = 0.4356 0 50 100 150 200 0 75 150 225 300 fl ow er n um be r ga3 concentration (ppm) 105 dat y = -0.0014x2 + 0.3721x + 111.06 r² = 0.53190 50 100 150 200 0 75 150 225 300 fl ow er n um be r ga3 concentration (ppm) 135 dat y = -0.0015x2 + 0.4115x + 105.14 r² = 0.5822 0 50 100 150 200 0 75 150 225 300 fl ow er n um be r ga3 concentration (ppm) 120 dat y = -0.0017x2 + 0.4526x + 116.86 r² = 0.7315 0 50 100 150 200 0 75 150 225 300f lo w er n um be r ga3 concentration (ppm) 150 dat ultrafine bubbles water priming to improve viability and vigor of bean .......... journal of tropical crop science vol. 10 no. 1, february 2023 www.j-tropical-crops.com 33 distributed throughout the pods to form seeds. the assimilate is described by plant dry weight, which was similar between treatments. empty pods can be caused by various factors, such as lack of calcium absorption, which plays an important role in seed development, quality, and production (gashti et al., the number of harvested pods was not positively correlated with the number of pithy seeds. the high percentage of wrinkled seeds could be due to the time of harvest being too early so that the seeds have not been filled to the maximum. in-ground peanut (arachis hypogea) plantations where plants figure 5. classification of pods of a. pintoi (a) intact pod, (b) half-full pod, and (c) empty pod table 4. effect of ga3 application on harvested seed yield at 150 days after treatment (dat). concentration of ga3 (ppm) quantity of harvested seed total intact half-fullt emptyt 0 113.75 33.25 36.50 44.00 75 109.75 39.50 20.75 49.50 150 139.00 34.75 39.50 64.75 225 110.25 36.00 29.00 45.25 300 95.75 33.00 26.50 36.25 f-test * ns ns ns cv % 19.31 11.75 12.82 11.57 notes: ns = non-significant ; *= significant; t data was transformed to log(x) for analysis; the data presented is before transformation 2012). empty pods can also be caused by plant pathogenic nematodes, which are carriers of soilborne fungi such as rhizoctonia solani, sclerotium rolfsii, fusarium sp., and aspergillus sp (wicks et al., 2011). ga3 treatment did not affect the yield of a. pintoi pods, as indicated by the results that were not different from the control treatment. the application of ga3 in the range of 150 ppm increased the number of pods harvested by 18.16%. based on the results, the higher the concentration, the lower the yield of the harvested pods. this may be because the high concentration of ga3 used can inhibit plant growth. administration of ga3 by spraying in the study of azizi et al. (2012) showed that the lowest yields of harvested soybeans treated with high concentrations of gibberellins at 200 ppm to 375 ppm. in this study, the application of ga3 did not affect the number and percentage of intact and wrinkled seeds (table 5). are harvested young, the percentage of wrinkled seeds is high (rahmianna et al., 2007). wrinkling and incomplete development of seeds can be caused by the less optimal intake of assimilates. when the rate of absorption of assimilates by the seeds is low, the filling time to develop intact seeds is high (christian et al., 2016). the seed filling time can also be influenced by environmental factors, such as an increase in temperature that can increase the amount of assimilating, so it is necessary to determine the effective harvesting age related to the harvesting of a. pintoi seeds. effect of ga3 application on the weight of harvested pods the weight of the harvested pods was calculated based on the pods that had been through a 30day storage, and the seeds had been dried before storage. drying was conducted to reduce the moisture journal of tropical crop science vol. 10 no. 1, february 2023 www.j-tropical-crops.com 34 robi rahmat dani, juang gema kartika, maryati sari content of the seeds so the seeds could be stored longer without rotting. the dry weight of the seeds is influenced by the remaining water content contained in the seeds (kurniawan and purnamwati, 2017). the weight of harvested seeds and the weight of 100 seeds were not affected by the use of ga3 application after six months of planting (table 6). similar seed weight between treatments might be due to the similar level of moisture content as a result of the drying and storage processes. during seed storage there can be a decrease in carbohydrate, lipid, and protein levels, which can affect seed weight loss (begum et al., 2013). the weight of 100 seeds is also correlated with the shapes of the seed. treatment with ga3 at 150 ppm had a lighter 100-seed, so it can be estimated that this treatment resulted in more seed production than those of the other treatments. arachnis pintoi seed germination the application of ga3 did not have a significant effect on the percentage of germination (table 7). seed germination was carried out after the seeds were kept for 1 month of storage, because seeds from the leguminosae, such as ground peanuts (arachis hypogea) can have physiological dormancy after ripening. seed dormancy is defined as the condition of seeds that do not grow or germinate within a certain period in favourable environmental condition (widajati et al., 2013). the cause of physiological dormancy is that the embryo is not fully developed or immature (hidayat and wardiyati, 2019). breaking seed dormancy to increase germination can be done chemically, such as soaking the seeds in 1% kno3 for 2 hours immersion before the seeds were planted. this treatment can increase the absorption of water in the seeds (candra et al., 2017) and stimulate enzyme activities related to germination hence enhancing seed germination (hartawan, 2016). arachnis pintoi seeds can germinate 10-15 days (balitan, 2004). seeds treated with ga3 at 150 ppm germinated of 76.25%, which is similar to the control treatment of 73.75%. these results agree with neef et al. (2004) that a. pintoi seeds grown for six months in the highlands had a 68-73% germination rate. seeds that do not germinate in our study might still experience seed dormancy, or the seeds have become non-viable. seed dormancy can be caused by genetic factors, adverse environmental figure 6. quadratic orthogonal polynomial of the total harvested pods 9 figure 5. classification of pods of a. pintoi (a) intact pod, (b) half-full pod, and (c) empty pod table 4. effect of ga3 application on harvested seed yield at 150 dat concentration of ga3 (ppm) quantity of harvested seed total intact half-fullt emptyt 0 113.75 33.25 36.50 44.00 75 109.75 39.50 20.75 49.50 150 139.00 34.75 39.50 64.75 225 110.25 36.00 29.00 45.25 300 95.75 33.00 26.50 36.25 f-test * ns ns ns cv % 19.31 11.75 12.82 11.57 notes: ns = non-significant ; *= significant; t data was transformed to log(x) for analysis; the data presented is before transformation figure 6. quadratic orthogonal polynomial of the total harvested pods the yield of harvested pods of a. pintoi was dominated by empty pods, resulting in reduced seed production. the high number of empty and half-full pods could be due to the insufficient flow of photosynthates distributed throughout the pods to form seeds. the assimilate is described by plant dry weight which were similar between treatments. empty pods can be caused by various factors such as lack of calcium absorption, which plays important role in seed development, quality, and production (gashti et al., 2012). empty pods can also be caused by plant pathogenic nematodes which are carriers of soil-borne fungi such as rhizoctonia solani, sclerotium rolfsii, fusarium sp., and aspergillus sp (wicks et al. ,2011). y = -0.001x2 + 0.2536x + 109.51 r² = 0.5777 0 50 100 150 0 75 150 225 300 to ta l h ar ve st ed p od s ga3 concentration (ppm) table 5. quantity and percentage of intact and wrinkled seeds of arachnis pintoi with and without ga3 treatment. concentration of ga3 (ppm) seed quantity seed percentage (%) intactt wrinkledt intact wrinkled 0 33,25 47,50 45,28 54,72 75 39,50 34,75 52,28 47,72 150 34,75 54,75 42,09 57,91 225 36,00 44,25 43,21 56,79 300 33,00 33,75 49,40 50,60 f-test ns ns ns ns cv % 11,75 14,46 20,97 18,19 note: ns = non-significant;. t data was transformed to log(x) for analysis; the data presented is before transformation ultrafine bubbles water priming to improve viability and vigor of bean .......... journal of tropical crop science vol. 10 no. 1, february 2023 www.j-tropical-crops.com 35 conditions, internal hormone imbalance, and physical barrier such as seeds having thick seed coats. seed dormancy can also be related to unfavourable storage conditions and prolonged storage durations (baskin and baskin, 2014). conclusion the use of the growth-regulating hormone ga3 had a significant effect on the generative growth of a. pintoi; application of ga3 at the optimum concentration range of 130.69 – 137.16 ppm increased the number of flowers at 105, 120, 135, and 150 dat by 23.08% 28.59%. ga3 at 126.80 ppm increased the number of harvested pods by 18.16%, whereas ga3 at 141.88 ppm increased the growth of new individual plants by 53.25 %. references [balittan] badan penelitian tanah. (2004). kacang hias (arachis pintoi) pada usaha tani lahan kering. pusat litbang tanah dan agroklimat. bogor. indonesia. [bmkg] badan meterologi klimatologi dan geofisika. 2021. “data iklim wilayah pacet bulan agustus 2020-februari 2021”. stasiun klimatologi dramaga, bogor. aminah, a.g., khairuddin, and kadir, m.y.a. (1994). effect planting material and harvesting time on seed production of arachis pintoi in malaysia. proceeding of forage regional working group of south east asia. arifin, z., yudono, p., and toekidjo. (2014). pengaruh konsentrasi ga3 terhadap pembungaan dan kualitas benih cabai merah keriting (capsicum annum l). vegetalika 4,128-140. doi:10.22146/ veg.1604. azizi, k.h., moradii, j., heidari, s., khalili, a., and felzian, m. (2012). effect of different concentrations of gibberellic acid on seed yield and yield components of soybean genotypes in summer intercropping. international journal of agriculture 4, 291-300 baskin, c.c., and baskin, j.m. (2014). “seeds : ecology, biogeography, and evolution of dormancy and germination”. 2nd ed. academic press. san diego table 6. weight of intact seed, wrinkles seed, and 100-seed weight. concentration of ga3 (ppm) 100-seed weight (g) seed weight (kg.ha-1) intactt wrinkledt 0 10.26 17.30 8.95 75 11.61 22.68 7.41 150 9.61 17.01 9.88 225 10.87 19.60 9.00 300 10.58 17.16 6.80 f-test ns ns ns cv % 7.02 14.00 22.12 note: ns = non-significant; t data was transformed to log(x) for analysis; the data presented is before transformation tabel 7. percentage of seed germination concentration of ga3 (ppm) percentage of seed germination 3 dapt 6 dapt 9 dapt 12 dap 15 dap 0 22.50 27.50 41.25 48.75 73.75 75 23.75 30.00 35.00 43.75 68.75 150 18.75 26.25 37.50 51.25 76.25 225 13.75 15.00 21.25 33.75 57.50 300 17.50 20.00 26.25 35.00 57.50 f-test ns ns ns ns ns cv % 24.02 22.68 14.20 28.25 19.83 note: ns= non-significant. t analysis of variance was carried out on data that has been transformed to log(x) and the data presented is data before the transformation. 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(2014). pengaruh gibberellic acid (ga3) terhadap kacang tanah (arachis hypogea l) pada fase generatif. proceeding biology education conference: biology, science, enviromental, and learning 1, 93-97. morphological character of clove “raja” (syzygium aromaticum l.), .......... journal of tropical crop science vol. 10 no. 1, february 2023 www.j-tropical-crops.com 71 morphological character of clove “raja” (syzygium aromaticum l.), endemic to maluku, indonesia anna yuliana wattimena, asri subkhan mahulette*, marlita herlin makaruku, muhammad fajri aditthia lating agrotechnology study program, faculty of agriculture, pattimura university, poka campus, ambon 97234, indonesia *corresponding author; email: mahulette_07@yahoo.co.id abstract clove “raja” is one of the local maluku clove germplasm, which has characteristics similar to cultivated cloves and wild types. until now, very limited information that are available on the morphological character of clove “raja”. this study aims to provide basic information on the morphological diversity of “raja”. the study was conducted at the location of the distribution of cloves “raja” in mamala village, leihitu sub-district, central maluku district, maluku province, in june-august 2022. the descriptors used referred to tropical fruit descriptors, with some modifications. the clove “raja” that were characterized belonged to the farmers and consisted of 30 accessions of cloves that were over twenty years old. the characterization variables included the characters of trees, stems, branches, leaves, flowers, fruits, and seeds. the results of hierarchical cluster analysis (hca) on 30 plant samples based on 54 morphological characters obtained two accession groups with a dissimilarity coefficient of 41%. the results of the principle component analysis (pca) got a total diversity of 70.5% where the first group consisted of accessions of cloves “raja” group i having identifiable characters in the form of leaf area, leaf length, leaf width, and leaf tip shape; while the second group of clove accessions of “raja” group i in the form of stem circumference, petiole length, flower length, fruit length, fruit width, and fruit weight. characteristics of accession of “raja” cloves group ii had distinctive morphological traits in the form of crown shape, upper surface of leaves, lower surface of leaves, leaf thickness, leaf texture, old leaf colour, shoot colour, leaf tip colour, leaf aroma, leaf spiciness, flower stalk length, and seed colour. keywords: ambon, biplot, cluster analysis, dendro gram, endemic introduction cloves are native species to indonesia, originating from the maluku islands (milind and deepa, 2011; mahulette et al., 2022; hariyadi et al., 2020a). cloves are classified as essential oil-producing plants of the myrtaceae family, which are widely used as raw materials for cigarettes and as spices. as a crucial oil-producing plant, cloves are commonly used in pharmacology as a raw material for medicines, cosmetics and perfume industries, food shields and food preservatives (ulanowska and olas, 2021; kaufman, 2015; nejad et al., 2017; hassan et al., 2020). based on data from ditjenbun (2020), the area and production of cloves in indonesia continue to increase, but the increase in scope and output of cloves has not met the growing demand for cloves. the area of cloves in indonesia in 2018 was recorded at 569, 052 ha, then increased to 570,353 ha in 2020. clove production in indonesia also increased from 131,014 tons in 2018 to 137,758 tons in 2020. the increase in area and production has not met the growing demand for cloves, so clove imports are carried out to meet the shortage of supply. according to data from the ditjenbun (2020), the volume of indonesian clove imports from 2015 to 2018 has increased from 11 tons in 2015 to 13.373 tons in 2018. to overcome these problems, increasing the volume of clove production in indonesia is necessary, including using potential germplasm of local cloves growing in their distribution area. the maluku islands are known as “the centre of origin” for the distribution of cloves in the world (mahulette et al., 2019; mahulette et al., 2022; milind and deepa, 2011; alfian et al., 2019), and store a relatively high genetic diversity of cloves. according to data from the ditjenbun (2020), maluku has been the largest clove-producing province in indonesia since 2015, with an average contribution of 15.37%. according to journal of tropical crop science vol. 10 no. 1, february 2023 www.j-tropical-crops.com 72 anna yuliana wattimena, asri subkhan mahulette, marlita herlin makaruku, muhammad fajri aditthia lating bps maluku (2018), the area of clove plantations in maluku in 2016 was recorded at 43,620.3 ha, then increased to 43,780.1 in 2017, with total production increasing from 20,805.6 tons in 2016 to 21,159.6 in 2017. although the area of development is not as comprehensive as other provinces in indonesia, the clove productivity in maluku is higher, affecting the production. one of the causes of the increased productivity of cloves in maluku is the high diversity of superior clove germplasm, which is supported by the suitability of the agro-climate. cloves in maluku consist of many types with specific characteristics, but the most traded are “tuni”, ““hutan””, and “raja” cloves. cloves “tuni”, “hutan” and “raja” are the local names of cloves in maluku. (mahulette et al., 2019; hariyadi et al., 2020b; alfian et al., 2019). according to mahulette et al. (2022) and hariyadi et al. (2020a), “tuni” are classified as aromatic cloves, which have been released by the indonesian ministry of agriculture as a superior variety since 2013, while “forest” and “raja” are classified as non-aromatic cloves. while the status of “forest” cloves is temporarily in the stage of preparation for release as a variety, the study of clove “raja” is still limited. information on the morphological diversity of “tuni” and forest cloves has been widely published, but is still minimal on “raja” cloves. clove “raja” is a non-aromatic clove with an intermediate character; the morphological shape resembles cultivated type cloves from the aromatic group while the flowers have similarities to wild non-aromatic cloves, or the wild types (alfian et al., 2019; mahulette et al., 2022; pool et al., 1986). according to pool and bermawie (1986) and alfian et al. (2019), the “raja” clove is probably the result of a cross between the cultivated and the wild type cloves so it is likely to have specific morphological characters. based on the description above, a study was conducted to inform on the morphological character of clove “raja” to provide the basic information in the context of the future development of clove “raja” in maluku. material and methods place and time the morphological characterization of “raja” cloves was carried out at the distribution location of the “raja” cloves, namely in mamala village, leihitu subdistrict, central maluku district, maluku province (s: 03033’34.8”, e: 128011’41.7”, altitude 18.3 mdpl) in june-august 2022. materials and methods the material used in the morphological observations consisted of thirty accessions of “raja” clove plants that belonged to farmers. thirty plants of clove “raja” was were selected and measured; the trees more than 20-years-old with the criteria for healthy growth and the similar crown shape. the characterization was carried out in the village of mamala, the location of “raja” clove distribution with a total area of ±10 ha. equipment for morphological observation consisted of a haga meter to measure tree height; a tape measure for measuring the length, width and circumference of the rod; a measuring ruler to measure leaf length, flower length, fruit and seed length; a caliper to measure flower, fruit, and seed diameters; digital scales for weighing flowers, fruit and seeds; easy leaf area software for leaf area measurement; royal horticultural society (rhs) colour chart 2015 for determining the colour scale of leaves, fruit, seeds. research methods the morphological characterization of “raja” cloves used a survey method, and the selected sample was determined randomly. observation of morphological characters refers to tropical fruit descriptors (ipgri 1980), mahulette et al. (2019) dan mahulette et al. (2022), which was modified. research implementation the morphological characters measured included the surfaces of trees, stems, branches, leaves, flowers, fruits, and seeds (table 1). morphological characterization was carried out on thirty samples of “raja” clove plants, where ten pieces of leaves, flowers, fruit, and seeds were taken for measurement from each plant. the criteria for the leaves measured were selected from the fourth leaf, which was counted from the shoots (ruhnayat, 2007; mahulette et al.. 2022; mahulette et al., 2019) and free from pests and diseases, while ripe flowers picked were selected from flower arrangements with criteria that one or two flowers had bloomed (tresniawati dan randrian, 2011). measurement of fruit and seeds was carried out when the fruit had entered physiological maturity, which was marked by a change in the colour of the fruit to purple-black. morphological characters are characterized as nominal or measurement variables. the nominal variable is then scored for hierarchical cluster analysis (hca) and principle component analysis (pca). morphological character of clove “raja” (syzygium aromaticum l.), .......... journal of tropical crop science vol. 10 no. 1, february 2023 www.j-tropical-crops.com 73 data analysis analysis of the data from the morphological characterization used hierarchical cluster analysis (hca) to obtain the percentage of morphological similarity among all samples of the “raja” clove plants. the data from the description was followed by principle component analysis (pca) to obtain the distinctive characteristics of clove “raja” (descriptor of clove “raja”). hierarchical cluster analysis (hca) and principle component analysis (pca) were performed using r stat 3.1.0 software. result and discussion morphological characters of clove “raja” in mamala village, central maluku regency based on hierarchical cluster analysis (hca) morphological characterization of “raja” clove accessions in mamala village, central maluku regency, was conducted against 54 morphological characters. the results of the overall grouping of clove accessions based on hierarchical cluster analysis (hca) are presented in figure 1. according to hartati et al. (2022) and mahulette et al. (2022), hierarchical cluster analysis (hca) can be used to characterize the morphology of clove plants and can be used to select high-yielding parent trees. furthermore, according to ahmadizadeh and felenji (2011) and wang et al. (2014), hca is widely used in breeding studies, especially to identify morphological characters and yield components, especially in the selection of accessions with high production potential. based on hierarchical cluster analysis (hca) results, “raja” cloves in mamala village, central maluku regency, can be divided into two large groups with 41% dissimilarity coefficients or only 59% similarity. the first group consisted of 20 accessions grouped with a coefficient of dissimilarity of 14% (similarity 86%). the second group consisted of 10 accessions with a coefficient of dissimilarity of 6% (similarity of 94%). accessions to the second group on the dendrogram were also divided into two groups, where the first group consisted of 11 accessions with a dissimilarity coefficient of 8% (92% similarity), and the second group consisted of 9 accessions with a dissimilarity coefficient of 10% (90% similarity). according to maji and shaibu (2012) and rosmaina et al. (2021), plant accessions can be grouped into the same group if they have many characteristics in common. furthermore, according to jan et al. (2012) and karuwal et al. (2021), accession groups that have many similarities are accession groups that tend to have a closer kinship. in contrast, accession groups that have many differences are accessions that tend to have somewhat distant kinship relationships. the differences in morphological characters in the “raja” clove population obtained in the study are thought to be caused by several factors, including genetic and environmental factors and the interaction between the two. clove “raja” has cross-pollination properties, so natural pollination in the long term can cause the emergence of new variants in the population. according to mahulette et al. (2019c) and singh et al. (2013), variations in a plant population can provide information on the diversity of genetic traits and the level of variability to assist in selecting superior genotypes. the morphology of the clove “raja” accession group is shown in figure 2-4. in contrast, the results of the measurement of the morphological character of the “raja” clove accession group in mamala village, central maluku regency, are presented in table 2. table 1. variables measuring the morphological character of clove germplasm plant parts observation variable tree height, habitus, canopy shape, canopy width (north to south and east to west). stem stem circumference, main stem shape (single/dividing). branch branching direction, branch angle, lowest branch length. leaf leaf size (index), leaf length and width, leaf shape, old leaf colour and shoot colour, leaf tip and base shape, leaf stalk length and colour, leaf surface texture, leaf edge shape, leaf veins, leaf thickness. flower number of flowers per stalk, flower length and diameter, flower shape, flower stalk length, the colour of flower buds, colour of ripe flowers picked, colour and shape of flower crowns, the weight of mature flowers picked. fruit fruit length and diameter, fruit weight, shape and colour of ripe fruit. seed seed length and diameter, seed weight, seed shape and colour. journal of tropical crop science vol. 10 no. 1, february 2023 www.j-tropical-crops.com 74 anna yuliana wattimena, asri subkhan mahulette, marlita herlin makaruku, muhammad fajri aditthia lating figure 1. dendrogram of grouping of 30 clove “raja” samples (code: r1-r3) in mamala village, central maluku regency, based on 54 morphological characters. figure 2. the morphology of cloves “raja” group i-a, morphological parts: leaf (a), flower arrangement (b), flower bud (c), fruit (d), seed (e). figure 3. morphology of cloves “raja” group i-b, morphological parts: leaf (a), flower arrangement (b), flower (flower bud) (c), fruit (d), seed (e). morphological character of clove “raja” (syzygium aromaticum l.), .......... journal of tropical crop science vol. 10 no. 1, february 2023 www.j-tropical-crops.com 75 group i-a. this group consisted of 11 accessions grouped with 92% similarity (8% dissimilarity coefficient). members of this group have a leaf length of 13.34 cm, a leaf width of 5.28 cm, and a leaf area of 58.39 cm2, and the leaf colour is moderate green/green group (131b). the flower buds of group members have a flower length of 3.13 cm, a diameter of 3.68 mm, a weight of 0.58, and the number of flowers in each arrangement is 13.64. the colour of the ripe flower buds is brilliant yellow-green/yellowgreen group (150b). this group has fruit with a length of 3.81 cm, a diameter of 1.61 mm, a weight of 6.68 g, and a fruit colour of dark red/red-purple group (59a). seeds belonging to this group have a length of 2.70 cm, a diameter of 0.91 mm, a weight of 2.00 g, and seed colour is moderate red/greyed red group (181b). group i-b. this group consisted of 9 accessions grouped with 90% similarity (10% dissimilarity coefficient). members of this group have a leaf length of 11.28 cm, a leaf width of 3.98 cm, and a leaf area of 38.12 cm2, and the leaf colour is moderate green/green group (131b). the flower buds of group members have a flower length of 3.09 cm, a diameter of 3.62 mm, a weight of 0.97 g, and the number of flowers in each arrangement is 12.44. the colour of the ripe flower buds is brilliant yellow-green/yellowgreen group (150b). this group has fruit with a length of 3.86 cm, a diameter of 1.69 mm, a weight of 7.03 g, and a fruit colour of dark red/red-purple group (59a). seeds belonging to this group have a length of 3.06 cm, a diameter of 0.88 mm, a weight of 2.05 g, and seed colour is moderate red/greyed red group (181b). group ii. the second group of the clove “raja” population consisted of 10 accessions with a 97% similarity level (3% dissimilarity coefficient). members of this group have a leaf length of 11.15 cm, a leaf width of 3.87 cm, and a leaf area of 35.54 cm2, and the colour of the leaves is deep yellowish green/green group (141b). the flower buds of group members have a flower length of 2.20 cm, a diameter of 3.51 mm, a weight of 0.42 g, and the number of flowers in each arrangement is 15.03. the colour of the ripe flower buds is brilliant yellow-green/yellow-green group (150b). this group has fruit with a length of 3.21 cm, a diameter of 1.39 mm, a weight of 4.11 g, and a fruit colour deep red/red-purple group (60a). the seeds of this group have a length of 2.80 cm, a diameter of 0.85 mm, and a weight of 1.71 g, the colour of the seeds is a deep purplish red/red-purple group (59b). characterization of clove “raja” in mamala village, central maluku regency, based on principle component analysis (pca) principle component analysis (pca-biplot and variable pca) was conducted on 30 accessions of clove “raja”. the pca variable was performed on 36 characters with diverse morphological data (figure 5a). the results show a diversity of morphological characters with a total variety of 70.5%, where the symbol indicates the characterizing nature with the most extended vector. these characters are shown as the characters that contribute the most to the grouping. pca used can identify and classify groups of plant characters that contribute a lot to the collection. according to rosmaina et al. (2021) and yugandhar et al. (2018), pca can determine the characters that contribute the most to the total variation of characters analyzed. according to mahulette et al. (2022) and sharma et al. (2018), principal component analysis can determine independent character traits from the overall analyzed characters and provide different grouping results. the results of the pca-biplot (figure 5b) show that the characteristics of “raja” cloves group i are divided into two groups of characters : clove “raja” group i-a in the form of leaf area, leaf length, leaf width, leaf tip shape; and clove “raja” group i-b in the form of stem circumference, leaf stalk length, flower length, fruit length, fruit width, fruit weight. the characteristics figure 4. clove “raja” group ii, morphological parts: leaf (a), flower arrangement (b), flower bud (c), fruit (d), seeds (e). journal of tropical crop science vol. 10 no. 1, february 2023 www.j-tropical-crops.com 76 anna yuliana wattimena, asri subkhan mahulette, marlita herlin makaruku, muhammad fajri aditthia lating of clove “raja” group ii consisted of morphological characters : crown shape, upper surface of the leaf, lower surface of the leaf, leaf thickness, leaf texture, old leaf colour, shoot colour, leaf tip colour, leaf aroma, leaf spiciness, flower stalk length, seed colour. the characterizing characters obtained in the morphological characterization of “raja”’s cloves can be used as a reference in describing plant groups. these characters can be used to distinguish the morphological characters of “raja” cloves in the population. according to purnobasuki et al. (2014), characterizing characters obtained from pca results can be used as a reference in “raja” plant descriptors to assist in identifying certain plant groups. the use of pca in previous studies has been widely used in studying morphological variations in plantation crops such as cloves (mahulette et al. 2022, 2019b). these results indicate that although they are still of the same type, there are still differences between accession groups’ characterizing characteristics. this suggests that the clove plant is cross-pollinated, so it is possible for new variants to emerge in the population. pca can help describe the characters that most contribute to the grouping of “raja” cloves in the population. conclusion morphological characterization of clove “raja” accessions based on hierarchical cluster analysis (hca) on 30 plant samples based on 54 morphological characters obtained two accession groups with a 41% dissimilarity coefficient. the first group consisted of two accession groups and the second group consisted of one accession group. the principle component analysis (pca) results obtained morphological diversity in the distribution population of “raja” cloves with a total variety of 70.5%. the first group consisted of accessions of cloves “raja” group i having identifiable characters in the form of leaf area, leaf length, leaf width, and leaf tip shape, while the second group of clove accessions of “raja” group i in the form of stem circumference, petiole length, flower length, fruit length, fruit width, fruit weight. characteristics of accession of cloves “raja” group ii are distinctive morphological traits in the form of crown shape, an upper surface of the leaf, a lower surface of the leaf, leaf thickness, leaf texture, old leaf colour, shoot colour, petiole tip colour, leaf aroma, leaf spiciness, flower stalk length, seed colour. figure 5. principle component analysis: variable pca (5a), pca-biplot (5b) 30 clove accession “raja” in mamala village, central maluku regency, based on 36 morphological characters. accession of clove “raja”, r1-r30; morphological characters, plant height (m1), stem circumference (m2), main stem (m3), crown shape (m4), canopy width u-s (m5), canopy width t-b (m6), lowest branch height (m7), leaf size (m8), leaf length (m9), leaf width (m10), leaf area (m11), petiole length (m12), leaf tip shape (m13), upper leaf surface (m14), lower leaf surface (m15), leaf thickness (m16), leaf texture (m17), dark leaf colour (m18), shoot colour (m19), petiole tip colour (m20), leaf aroma (m21), leaf spiciness (m22), flower stalk length (m23 ), weight of flower stalk (m24), number of flowers/series (m25), flower length (m26), flower tube diameter (m27), the weight of ripe flowers picked (m28), fruit length (m29), fruit width (m30), fruit weight (m31), mature fruit colour (m32), seed length (m33), seed width (m34), seed weight (m35), seed colour (m36). morphological character of clove “raja” (syzygium aromaticum l.), .......... journal of tropical crop science vol. 10 no. 1, february 2023 www.j-tropical-crops.com 77 acknowledgement the authors are grateful to pattimura university for funding this research. this research is a leading applied research based on the rector of the university of pattimura decree 840/un13/sk/2022. references ahmadizadeh, m., and felenji, h. 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(2014). diversity evaluation of morphological traits and allicin content in garlic (allium sativum l.) from china. euphytica 198, 243–254. doi: 10.1007/ s10681-014-1097-1. yugandhar, r., kota, s., and kiran, u. (2018). principal component analysis for agro-morphological and quality characters in germplasm of rice (oryza sativa l.). international journal of advanced and biological research 8, 268–27. journal of tropical crop science vol. 10 no. 2, june 2023 www.j-tropical-crops.com 144 punjung medaraji suwarno, abdul qadir, aldi kamal wijaya, willy bayuardi suwarno agronomic performance of ipb maize hybrids and combining ability of the lines punjung medaraji suwarnoa, abdul qadira b 0000-0003-2989-1911, aldi kamal wijayaa, willy bayuardi suwarno*b 0000-0002-7189-7906 a seed industrial technology study program, vocational school, ipb university. jl. kumbang no. 14, bogor 16128, indonesia b department of agronomy and horticulture, faculty of agriculture, ipb university. jl. meranti kampus ipb darmaga, bogor 16680, indonesia *corresponding author; email: willy@apps.ipb.ac.id abstract maize is a significant food crop consumed in numerous countries worldwide. increasing the yield potential of maize can be achieved through the breeding of high-yielding hybrid varieties. the objectives of this study were to assess the performance of hybrid means and parent combining ability for grain yield and other agronomic traits using a line x tester mating design. the study took place at the gunung gede experimental station of ipb university, bogor, from june to october 2022. the experiment was conducted using a randomized complete block design with a single genotype factor and three replications. twenty-two hybrids were utilized as genetic materials, including 20 hybrid candidates and two check varieties. six maize lines (h1, l26, nei, p2b, and p42) were employed as line parents, while l15 and p10 were used as tester parents in the line x tester analysis. the neighbor-joining tree analysis indicated that the female parent has a significant influence on the agronomic and yield performance of the hybrids. correlation analysis revealed that the yield is more strongly influenced by ear diameter rather than ear length, and it is more affected by the number of kernel rows than the number of kernels per row. three selected hybrids with favorable traits were nei x b1, l15 x l26, and l26 x p10. the parent l26 demonstrated relatively good general combining ability for yield and may be considered for future maize breeding programs. keywords: agronomic traits, correlation, maize breeding, yield introduction maize (zea mays l.) is extensively utilized as a food source, animal feed, and raw material for various industrial products. due to its cost-effectiveness and widespread consumption, maize is an excellent candidate for fortification, especially in regions where vitamin deficiencies pose significant public health concerns (ranum et al., 2014). in indonesia, the national average maize productivity in 2021 was 5.7 t.ha-1, which is lower than the productivity in the usa (11.1 t.ha-1) (bps, 2021; fao, 2023). according to bantacut et al. (2015), the limited availability of superior maize varieties can contribute to low maize productivity. therefore, it is crucial to develop maize varieties with higher productivity. hybrid maize breeding programs offer a means of obtaining highyielding maize varieties. several traits related to yield include ear length, ear diameter, number of kernel rows, and number of kernels per row. a single-cross hybrid variety is derived from crossing two inbred lines; this type of variety usually has superiorities over mid-parents, a phenomenon called heterosis (hallauer et al., 2010). hybrid varieties are more uniform than open-pollinated varieties (acquaah, 2009). schroeder et al. (2013) reported that the use of maize hybrids in smallholder agriculture offers potential in terms of economic development and food security, but only if the framework conditions offer a favorable environment for the adoption of the hybrids. worku et al. (2016) stated that single-cross hybrids account for most corn acreage because of their high yield and uniformity in appearance. soehendi and syahri (2013) stated that some hybrid maize varieties have relatively higher yields than the composite varieties. naturally, maize has a high level of heterozygosity (ganal et al., 2011). heterosis in cross-pollinated crops such as maize is usually higher than the self-pollinated crops such as rice. hybrid maize which has a high yield and is adapted to certain environmental condition can increase in national productivity (haryati and sinaga, 2018). syukur et al. (2015) suggest that prospective hybrid parental lines can be developed using various concepts, such agronomic performance of ipb maize hybrids and combining ability of the lines journal of tropical crop science vol. 10 no. 2, june 2023 www.j-tropical-crops.com 145 as variety, character, or gene. the development of inbred maize lines typically involves the process of self-pollination for a minimum of seven generations, starting from a heterozygous and heterogeneous basic population (salamah et al., 2017). general combining ability indicates average performance of an inbred line, whereas the specific combining ability is the interaction effects of a combination of two inbred lines (hallauer et al., 2010). the combining ability analysis is employed to identify the better combiners which can be hybridized to exploit heterosis and to select better crosses for direct use or further breeding work (murtadha et al., 2018). the line x tester mating design is used to estimate the potential of male and female lines to produce the desired cross combination. this design is used by plant breeders to determine the appropriate crossing method in hybrid variety development programs. tamirat et al. (2014) stated that in the maize breeding program, analysis of general combining ability (gca), specific combining ability (sca), and heterosis would help to identify best inbred lines for hybrid development and hybrid combinations for better specific combining ability. the objectives of this study were to estimate hybrid mean performance and parent combining ability for grain yield and other agronomic traits using line x tester mating design. materials and methods the research was conducted at the gunung gede experimental station of ipb university from june to october 2022. the experiment was arranged in a randomized complete block design with a single factor of genotype and three replications. the evaluated maize genotypes consisting of 20 test hybrids developed in ipb university, namely l26 x p13, p10 x p2b, l15 x h1, nei x p10, l15 x nei, p42 x p10, l15 x p10, l15 x l26, p2b x h1, nei x b1, l22 x p2b, p10 x p13, b1 x l152, p10 x h1, l15 x p2b, l15 x p42, l22 x nei, l26 x p10, l15 x l22, nei x p42, and two commercial hybrid varieties as checks, namely “bisi 18” and “nk perkasa”. the test hybrids were bred for high yield potential and had yellow grain color. all parental lines were developed by ipb university, except nei (nei 9008) which was obtained from the indonesia cereal research institute (iceri). both check varieties are yellow single cross hybrids. “bisi 18” is a widely grown variety in indonesia produced by pt bisi international tbk., whereas “nk perkasa” is produced by pt syngenta. the plot size was a single row of 5 meters, each row contained a maximum of 20 plants, and the plant spacing was 75 cm x 25 cm. the plot size was a single row of 5 meters, and the plant spacing was 75 cm x 25 cm. measurements were conducted on agronomic traits including plant height, ear height, stem diameter, leaf length, and leaf width, and yield-related traits including ear diameter, ear length, number of kernel rows, number of kernels per row, and yield estimates. plant height was collected by measuring from the plants from the soil surface to the base of the male flower at the end of the vegetative phase. stem diameter was measured using a caliper on the first node of the aerial roots, measured at the end of the flowering phase. leaf length was measured on the first leaf above the ear leaf, measured from the node where the leaf was attached to the tip of the leaf, whereas leaf width was measured in the middle of the leaf lamina. ear height is the height from the ground surface to the base of the main ear. ear diameter was measured in the middle of the ear, whereas ear length from the base to the tip of the ear. the yield estimate at 15% moisture content was calculated by multiplying the average ear weight by the maximum number of plants per hectare, assuming 20% moisture content at weighing and a shelling percentage of 80%. the 15% final adjusted moisture content and 80% shelling percentage were used by menkir et al. (2022). all plants in each plot were measured. quantitative data were analyzed with anova (analysis of variance) followed by the t-dunnett test at 0.05 level to compare the test hybrids with the check varieties. repeatability on the entry-mean basis was calculated as a percentage of genotypic variance to phenotypic variance. correlation analysis was performed to elucidate the relationship between agronomic and yield traits. the following software packages were used for data analysis: microsoft excel for data entry and basic calculations; sas ondemand for academics (welcome.oda.sas.com) for anova, t-dunnett, and correlations; agd-r (data. cimmyt.org/dataverse/cimmytswdvn) for combining ability analysis, pbstat-ppb (www.pbstat.com) for repeatability calculation, and pbstat-cl for cluster analysis. results and discussion hybrid performance the genotype had a highly significant effect (p<0.01) on plant height, stem diameter, leaf length, leaf width, ear height, ear diameter, ear length, number of kernel rows, number of kernels per row, and yield, while blocks vary on plant height, stem diameter, leaf length, and ear height (table 1). this showed that at least two genotypes were varied for a particular trait. furthermore, the block effect was significant in plant height, stem diameter, leaf length, and ear journal of tropical crop science vol. 10 no. 2, june 2023 www.j-tropical-crops.com 146 punjung medaraji suwarno, abdul qadir, aldi kamal wijaya, willy bayuardi suwarno height (data not shown). this indicated that there were differences in average trait values based on the planting location. coefficients of variations for most the check varieties, "bisi 18" and "nk perkasa," exhibited relatively high mean values for both agronomic and yield-related traits, as shown in table table 1. means of agronomic traits of the maize hybrids no. hybrids plant height (cm) stem diameter (cm) leaf length (cm) leaf width (cm) ear height (cm) 1. b1 x l152 175.55 2.10 84.93 9.73 98.00 2. l15 x h1 159.18c 2.34b 89.73 9.42 110.93 3. l15 x l22 170.42 2.28 91.08 8.87 114.53 4. l15 x l26 175.42 2.39ab 80.88 9.65 102.93 5. l15 x nei 197.50 2.16 93.42 10.72a 112.27 6. l15 x p10 173.67 2.17 97.80 9.32 105.20 7. l15 x p2b 160.25 2.29 86.75 9.60 99.00 8. l15 x p42 183.78 2.32b 85.50 9.47 117.33 9. l22 x nei 179.47 2.30 84.87 9.43 103.47 10. l22 x p2b 169.32 2.23 83.18 9.42 95.87 11. l26 x p10 162.17 2.21 88.65 9.93 91.73 12. l26 x p13 172.15 2.26 81.10 9.73 103.33 13. nei x b1 191.23 2.12 88.42 10.47 102.60 14. nei x p10 171.92 2.20 88.72 10.12 90.47 15. nei x p42 187.63 1.96 88.65 10.18 112.47 16. p10 x h1 141.38c 2.15 72.34 8.66 94.60 17. p10 x p13 133.15c 2.25 74.92 8.95 79.93cd 18. p10 x p2b 142.97c 2.09 92.28 9.10 83.53a 19. p2b x h1 155.48c 2.25 84.95 9.40 89.60 20. p42 x p10 142.02c 2.22 86.15 9.37 89.80 21. “bisi 18” 197.15 2.05 85.73 9.10 115.87 22. “nk perkasa” 167.85 2.01 84.77 9.28 109.27 f-test ** ** * * ** cv (%) 8.87 5.53 7.54 6.37 10.66 r (%) 76.80 58.66 56.19 52.21 66.37 note: * = significant at 0.05 level, ** = significant at 0.01 level, a = significantly higher than “bisi 18” at 5% level, b = significantly higher than “nk perkasa” at 5% level, c = significantly lower than “bisi 18” at 5% level, d = significantly lower than “nk perkasa” at 5% level, cv: coefficient of variation, r: repeatability. traits are considerably low (less than 10%) for a field experiment, except for ear length (10.66%) and yield (24.80%), indicating that the experiment results are reliable (table 1). hybrid l15 x l26 had the widest stem diameter (2.39 cm), while p10 x p13 has the shortest ear height (79.93 cm). enujeke (2013) stated that plant height among the varieties may be attributed to differences in genetic characteristics of the individual varieties, including rapid growth rates, and tallness or shortness of plants. altitude, types of soil, and climate, affected the performance of the genotypes (azrai et al., 2022). 1 and table 2. nei x b1 had the highest average on ear diameter (5.77 cm) and was significantly higher than both checks while l26 x p10 had the highest mean for the number of kernel rows (18.1). the yield of all test hybrids was not significantly different with both checks, with nei x b1 having the highest average (10.98 t.ha-1) (table 2). aprilianti et al. (2016) reported that the largest ear diameter does not always guarantee that the ear will have the highest number of kernel rows. therefore, three selected hybrids nei x b1, l15 x l26, and l26 x p10. agronomic performance of ipb maize hybrids and combining ability of the lines journal of tropical crop science vol. 10 no. 2, june 2023 www.j-tropical-crops.com 147 a neighbor-joining tree of maize hybrids based on agronomic and yield-related traits is shown in figure 1. several pairs of hybrids having the same female parents are clustered together. for example, l15 x p10 and l15 x l22; p10 x p13 and p10 x h1; l15 x p2b and l15 x h1; l15 x p42 and l15 x l26; nei x p42 and nei x b1. this indicates that the female parent has a substantial effect on the agronomic and yield performance of the hybrids. the check varieties “nk perkasa” and “bisi 18” are also clustered together, indicating that they shared some similarities in agronomic and yield traits. according to mohsin et al. (2014), the increment in the maize grain yield was due to increases in ear length, ear diameter, and 1000-grain weight. yield is a quantitative trait and is influenced by various factors, including genetics and environment. qualitative traits, on the other hand, are mostly less affected by the environment. kernel shape and seed color are among the qualitative traits of maize grain (nugroho et al., 2018) and grain yield is the ultimate end product of the yield-contributing components, physiological and morphological processes taking place in plants during growth and development (tariq et al., 2014). correlation among agronomic and yield traits maize yields had highly significant (p<0.01) correlations with two yield components traits, ear diameter (r=0.618), and the number of kernel rows (r=0.552), and significant (p<0.05) correlations with the other two yield component traits, the number of kernel rows (r=0.457) and ear length (r=0.448) (table table 2. means of yield-related traits of the maize hybrids no. hybrids ear diameter (cm) ear length (cm) number of kernel rows number of kernels per row yield estimate (t.ha-1) 1. b1 x l152 5.63a 19.00 15.60 37.80 10.44 2. l15 x h1 4.77 17.87 13.33cd 34.93 6.05 3. l15 x l22 4.97 19.07 15.07 36.47 9.07 4. l15 x l26 5.37 17.73 16.80 35.80 10.19 5. l15 x nei 5.07 19.47 14.67 40.87 7.78 6. l15 x p10 5.00 18.53 14.07 37.20 7.71 7. l15 x p2b 4.90 17.47 14.67 33.93 6.30 8. l15 x p42 5.43 19.93 16.67 39.60 10.17 9. l22 x nei 5.10 17.80 15.07 33.13 8.45 10. l22 x p2b 5.10 19.20 15.33 34.53 8.14 11. l26 x p10 5.50 18.40 18.13ab 34.80 8.68 12. l26 x p13 5.13 17.67 16.00 33.07 7.99 13. nei x b1 5.77ab 16.87 15.40 35.00 10.98 14. nei x p10 4.80 17.40 14.13 34.27 6.10 15. nei x p42 5.60a 18.47 16.87 36.20 9.78 16. p10 x h1 4.23cd 15.20c 12.80cd 32.80 5.37 17. p10 x p13 4.63 14.33c 13.47cd 32.60 7.92 18. p10 x p2b 5.10 17.00 15.07 33.80 5.79 19. p2b x h1 5.40 16.73 15.07 33.47 5.47 20. p42 x p10 5.53 16.67 16.67 33.27 6.57 21. “bisi 18” 4.90 19.67 15.33 40.00 8.23 22. “nk perkasa” 5.03 17.60 15.87 37.87 8.22 f-test ** ** ** * * cv (%) 5.00 8.09 4.81 8.81 24.80 r (%) 83.88 63.83 89.14 45.81 54.43 note: * = significant at 0.05 level, ** = significant at 0.01 level, a = significantly higher than “bisi 18” at 5% level, b = significantly higher than “nk perkasa” at 5% level, c = significantly lower than “bisi 18” at 5% level, d = significantly lower than “nk perkasa” at 5% level, cv: coefficient of variation, r: repeatability. journal of tropical crop science vol. 10 no. 2, june 2023 www.j-tropical-crops.com 148 punjung medaraji suwarno, abdul qadir, aldi kamal wijaya, willy bayuardi suwarno 3). this indicates that yield is more determined by ear diameter rather than ear length and was affected more by the number of kernel rows than the number of kernels per row. with the agronomic traits, the yield had a highly significant correlation with plant height (r=0.621) and a significant correlation with ear height (r=0.457). this indicated that plant growth had a relationship with yield. however, even though the correlation between plant height and yield is positive and significant, genotypes with too tall plant stature and high ear positions are not desired as they may be more prone to lodging. a multiple-trait selection approach may be employed in breeding programs to identify such genotypes. plant height demonstrated a strong correlation (r=0.61) with the number of kernels per ear row, as reported by alan et al. (2013). additionally, karomah et al. (2018) found a very strong and significant correlation between plant height and ear height (r=0.935). furthermore, ziyomo and bernardo (2013) established a significant correlation between plant height and yield. mohammadi et al. (2003) stated that two yield-related traits, namely the weight of 100-seeds and the number of kernels per ear had a high correlation with the total seed weight. zarei et al. (2012) stated that there was a significant correlation between yield and ear length, 100-seed weight, and number of seeds per row. the research results of salamah et al. (2017) showed that production characters had a significant correlation with plant height, ear height, ear length, and ear diameter. leaves are substantial parts of plants, the larger the leaves, the higher their potential for photosynthesis. in our study, leaf length had a significant positive correlation with ear length, whereas leaf width has a strong positive correlation with ear diameter (table 3). combining ability of inbred lines the estimates of general combining ability (gca) analysis result showed that the parent nei had a table 3. correlation coefficients among agronomic and yield-related traits of the maize hybrids ph sd ll lw eh ed el nr nk sd -0.221 ll 0.405 -0.165 lw 0.605** -0.159 0.397 eh 0.763** -0.071 0.322 0.183 ed 0.388 -0.133 0.324 0.551** 0.105 el 0.758** -0.063 0.557** 0.313 0.717** 0.357 nr 0.306 -0.081 0.140 0.346 0.162 0.788** 0.422 nk 0.719** -0.304 0.426* 0.278 0.727** 0.198 0.764** 0.209 ye 0.621** -0.097 0.049 0.364 0.457* 0.618** 0.448* 0.552** 0.457* note: * = significant at 5% level, ** = significant at 1% level, ph = plant height, sd = stem diameter, ll = leaf length, lw = leaf width, eh = ear height, ed = ear diameter, el = ear length, nr = number of kernel rows, nk = number of kernels per row, ye = yield estimate. table 4. general combining ability effects of the maize inbred lines genotype ph sd ll lw eh ed el nr nk ye line h1 -11.21 0.00 0.00 -0.47 1.14 -0.50* 0.00 -2.14** -0.19 -1.35 l26 4.30 0.00 0.00 0.16 -0.63 0.32 0.00 2.09** -0.01 1.81 nei 17.65 0.00 0.00 0.67* 0.69 -0.12 0.00 -0.86 0.27 -0.30 p2b -10.10 0.00 0.00 -0.21 -2.60 -0.06 0.00 -0.41 -0.19 -1.06 p42 -0.64 0.00 0.00 -0.15 1.40 0.36 0.00 1.32 0.13 0.91 tester l15 11.01 0.06 0.00 0.11 8.86 0.00 0.61 0.00 1.34 0.70 p10 -11.01 -0.06 0.00 -0.11 -8.86 0.00 -0.61 0.00 -1.34 -0.70 note: * = significant at 5% level, ** = significant at 1% level, ph = plant height, sd = stem diamater, ll = leaf length, lw = leaf width, eh = ear height, ed = ear diameter, el = ear length, nr = number of kernel rows, nk = number of kernels per row, ye = yield estimate. agronomic performance of ipb maize hybrids and combining ability of the lines journal of tropical crop science vol. 10 no. 2, june 2023 www.j-tropical-crops.com 149 table 5. specific combining ability effects of the maize inbred lines line tester ph sd ll lw eh ed el nr nk ye h1 l15 -0.61 0.01 1.95 0.00 0.00 0.01 -0.21 0.04 -0.62 -0.01 h1 p10 -0.01 -0.01 -8.34* 0.00 0.00 -0.02 -0.87 -0.09 -0.42 0.00 l26 l15 -0.51 0.02 -3.29 0.00 0.00 0.00 -0.27 -0.14 -0.36 0.00 l26 p10 0.75 0.01 1.31 0.00 0.00 0.01 0.60 0.19 0.29 0.00 nei l15 0.74 -0.03 4.13 0.00 0.00 0.01 0.52 0.06 1.49 0.00 nei p10 0.23 0.00 1.35 0.00 0.00 -0.01 0.14 -0.08 -0.03 0.00 p2b l15 -0.62 0.00 0.18 0.00 0.00 -0.01 -0.39 -0.05 -1.01 -0.01 p2b p10 0.06 -0.02 3.45 0.00 0.00 0.00 -0.05 0.05 -0.03 0.00 p42 l15 1.40 0.01 -0.56 0.00 0.00 0.00 0.74 0.01 1.05 0.02 p42 p10 -1.44 0.01 -0.17 0.00 0.00 0.01 -0.20 0.01 -0.36 -0.01 note: * = significant at 5% level, ** = significant at 1% level, ph = plant height, sd = stem diamater, ll = leaf length, lw = leaf width, eh = ear height, ed = ear diameter, el = ear length, nr = number of kernel rows, nk = number of kernels per row, ye = yield estimate. significant value on leaf width (0.67) while parent l26 had a significant value on the number of kernel rows (2.09) (table 4). this indicated that parent l26 was suitable for increasing the number of kernel rows. positive but insignificant gca for yield was also observed on parent l26. positive gca values on yieldrelated traits observed herein are desired since they are correlated with yield. parent with high gca values was suitable and more effective in the development of synthetic variety as well as hybrid varieties. good general combiners could effectively be used in future breeding programs for the development of highfigure 1. neighbor-joining tree of maize hybrids based on agronomic and yield-related traits. journal of tropical crop science vol. 10 no. 2, june 2023 www.j-tropical-crops.com 150 punjung medaraji suwarno, abdul qadir, aldi kamal wijaya, willy bayuardi suwarno yielding hybrids with desirable traits (amiruzzaman et al., 2013). the estimates of specific combining ability analysis results showed that none of the hybrids had significant values, except h1xp10 which showed a significant negative value on leaf length (-8.34). there results indicated that there was no significant interaction between parents in affecting the growth and yield characters (table 5), therefore in this case, the interaction value is not meaningful for the prediction of hybrid performance. according to murtadha et al. (2018) a negative sca value indicated the unsuitability of both parents as a specific combiner for the trait. furthermore, the higher variability of gca as compared to sca for several traits observed suggested that the hybrid prediction of such traits based on gca may be sufficient. conclusion three selected hybrids with desirable traits were nei x b1, l15 x l26, and l26 x p10. among them, nei x b1 exhibited the largest ear diameter (5.77 cm) and yield estimate (10.98 t.ha-1). l15 x l26 had the largest stem diameter (2.39 cm), while l26 x p10 had the highest number of kernel rows (18.1). the yield of these selected hybrids surpassed that of the check varieties, "bisi 18" and "nk perkasa," which yielded 8.23 and 8.22 t.ha-1, respectively. the analysis using a neighbor-joining tree indicated that the female parent significantly influenced the agronomic and yield performance of the hybrids. correlation analysis revealed that yield was more strongly determined by ear diameter rather than ear length, and it was more affected by the number of kernel rows rather than the number of kernels per row. the gca analysis results suggested that the parent l26 demonstrated relatively good general combining ability for maize yield. acknowledgment the authors would like to express their sincere gratitude to ipb university for the financial support provided for this study. special thanks are extended to the staff members at gunung gede experimental station of ipb university for their invaluable assistance in conducting the experiment. references acquaah, g. 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(2016). grain yield performance and flowering synchrony of cimmyt’s tropical maize (zea mays l.) parental inbred lines and single crosses. euphytica 211, 395-409. zarei, b., kahrizi, d., aboughadareh, a.p., and sadeghi, f. (2012). correlation and path coefficient analysis for determining interrelationships among grain yield and related characters in corn hybrids (zea mays l.). international journal of agriculture and crop sciences 4, 1519-1522. ziyomo, c., and bernardo, r. (2013). drought tolerance in maize: indirect selection through secondary traits versus genome wide selection. crop science 53, 1269-1275. effects of citrus aurantifolia linn and xylopia aethopica (dunal) a. rich extracts .......... journal of tropical crop science vol. 10 no. 1, february 2023 www.j-tropical-crops.com 17 effects of citrus aurantifolia linn and xylopia aethopica (dunal) a. rich extracts on leaf blight disease of taro (colocasia esculenta l. schott) ugwuja, f. n.*a, offor, v.n.a, akanwa, f.e.a, achi, n.kb. and onifade, a. k.c a department of plant science and biotechnology, michael okpara university of agriculture, umudike, nigeria b department of biochemistry, michael okpara university of agriculture, umudike, nigeria c department of microbiology, federal university of technology, akure, nigeria *corresponding author; email: ugwuja.faustina@mouau.edu.ng abstract phytophthora colocasiae raciborski, an oomycete phytopathogen, has been known for several decades as the causal agent of the most infectious and devastating disease of colocasia esculenta (l.) schott, known as taro leaf blight (tlb). investigations were conducted in a screenhouse to determine the effects of fruit extracts of citrus aurantifolia and xylopia aethiopica on the incidence and severity of tlb. the experiment was set up in a completely randomized design with three replicates. healthy taro seedlings obtained from the national root crops research institute (nrcri), umudike, were planted in plastic pots (5000 cm3) containing sterilized soil enriched with poultry manure. the plant extracts were applied as foliar spray on taro leaves with a manually operated hand sprayer at 7 weeks after planting and continued at four days intervals for 28 days. positive check was maintained with the fungicide ridomil (a.i. mefenoxam) applied at the rate of 0.67 mg.ml-1 while zero concentration in distilled water served as negative control. data were subjected to one-way analysis of variance, and means were separated using f-lsd. results showed a very high reduction of disease with plant extracts (p < 0.05) and ridomil compared to the control. citrus aurantifolia juice was more efficient in reducing the incidence and severity of tlb compared to x. aethiopica extracts and was highly significant (p<0.05). the overwhelmingly fungitoxic effects of c. aurantifolia and x. aethiopica extracts on p. colocasiae, as expressed in the reduction of disease, suggests that these extracts can serve as alternative bio-fungicide for the control of tlb. hence, further studies under field conditions are required to reestablish their efficacy. keywords: disease, fungitoxic, plant extracts, phytophthora colocasiae before plant extracts introduction plant disease epidemics have influenced the course of history in countries where they have had a devastating effect, and continue to be of great importance especially for those people whose day-to-day survival depends on their crops. the phytophthora leaf blight epidemic in nigeria in 2009 almost wiped out subsistence and large scale cocoyam production (ugwuja and chiejina, 2011) within two months of its incidence. farmers in southeast nigeria, where the epidemic started, were alarmed at the swift destruction of their taro crops and the onset of severe scarcity and exorbitant prices of taro commodities in all the markets. similar situations attributed to the same cocoyam leaf blight were reported in cameroon's southwest and northwest districts (mbong et al., 2013) and ghana (omane et al., 2012) where farmers could not produce enough cocoyam to eat or sell. taro (colocasia esculenta (l.) schott), known as cocoyam, is one of the oldest tropical crops grown primarily for its edible starchy corms and highly nutritive leaves (ekanem et al., 2008; ukpabi et al., 2011) as well as for its ornamental and traditional uses (manner and taylor, 2011). it is an important traditional staple food for millions of people in developing countries and is a good source of carbohydrates, wax, and therapeutics. ethno-botanical evidence suggests that taro originated in south central asia, probably in india or the malaya peninsula (onwueme and charles, 1994). from its center of origin, taro spread eastward to the rest of south east asia, china, japan and the pacific islands (manner and taylor, 2011). from asia, taro spread westward to arabia and the mediterranean region. it arrived on the east coast of africa over 2,000 years ago. voyagers took it, first across the continent to west africa, and journal of tropical crop science vol. 10 no. 1, february 2023 www.j-tropical-crops.com 18 ugwuja, f. n., offor, v.n., akanwa, f.e., achi, n.k. and onifade, a. k. later on slave ships to the caribbean. today taro is pan-tropical in its distribution and cultivation due to its wide adaptability, large-scale acceptability and high return per unit area. taro also thrives well on heavy soils on which flooding and waterlogging can occur. from time immemorial, taro has been an essential daily diet for many ethnic groups in nigeria, creating a relatively constant year-round demand as it provides an alternative source of carbohydrates to supplement yams and cassava (echebiri, 2004; nwachukwu and osuji, 2008; okereke, 2020). the scarcity of this important food crop occasioned by the recurring epidemic of taro leaf blight has tremendously increased costs beyond the reach of most people and overall demand. phytophthora colocasiae, an oomycete phytopathogen (cabi, 2020; otieno, 2020), has been known for several decades as the causal agent of the most infectious and devastating disease of colocasiae esculenta commonly referred to as “taro leaf blight” (fullerton and tyson, 2004; brooks 2005; brook, 2008; misra et al., 2008; misra et al., 2011; ugwuja and chiejina, 2011; omeje et al., 2016; padmaja et al., 2017). ugwuja et al. (2020) noted that the swift development of taro leaf blight infections has catastrophic consequences for both small, medium and large-scale production of taro due to the ubiquitous nature of the pathogen and the rapid rate of spread by wind-blown rain. the pathogen is also soilborne and favored by field flooding conditions (grade and joshi, 2003). it is believed to have originated in south east asia and is widely distributed throughout tropical regions (cmi, 1997). it attacks mainly the foliage, first appearing as small, brown, water-soaked spots that rapidly enlarge to form large, dark brown lesions. the lesions often coalesce to destroy a large portion of the leaf, sometimes with numerous droplets of orange or reddish exudates (ugwuja et al., 2021). depending on the aggressiveness of the infection, rapid and complete defoliation of leaves may occur within 14 days against 40 days normal longevity in the absence of disease (brooks, 2008; misra et al., 2008; nath et al., 2015), leading to a strong hindrance of photosynthesis, stunted corms and a yield loss up to 80% (adomako et al., 2017). yield losses can also vary from 25-30% in some areas when the infection is mild (miyasaka et al., 2001; misra et al., 2011), up to 50% in severe cases (brunt et al., 2001; misra, 2008) and over 70% in extremely severe cases (nelson et al., 2011; adomako et al., 2017). leaf yield losses can be up to 95%. the epidemic of taro leaf blight may occur throughout the year during the continuous rainy season over vast weather where night temperatures are 20 – 22oc and daily temperatures of 25 28oc with little seasonal variation (mbong et al., 2013).the pathogen also causes pre-harvest and post-harvest deterioration of corms (omeje et al., 2016) leading to the depreciation of their market value. significant depletion in the proximate and phytochemical composition of infected corms has also been reported (ugwuja et al., 2020). several measures of integrated pest management have been adopted to control this disease. these include the use of cultural methods, synthetic chemicals, biological control methods, and natural plant use of natural plant extracts (botanicals). synthetic chemicals such as sodium ortho-phenyl phenate, borax, captan, thiabendazole, sodium hypochlrorite (bleach), nordox, benomyl and mancozeb have been found to significantly reduce tlb (mishra et al., 2008). however, the high costs of these chemicals and obvious pollution of nontarget organisms have prompted investigations on exploiting pesticides of plant origin to control fungal pathogens. there has been increasing interest in using bio-pesticides to manage many plant diseases due to their relative accessibility and availability at little or no cost. these bio-pesticides are also specific, biodegradable and environmentally friendly. on the other hand, the conventional synthetic chemicals used in managing of plant diseases have continually received criticismsdue to the associated ecological and health hazards (komarek et al., 2010; dhaliwal and koul 2011). moreover, researchers have shown that a repeated exposure of fungal pathogens to fungicides can lead to resistance in the pathogen (adomako et al., 2017). given the many demerits of synthetic chemicals and the need to boost taro production through a sustainable disease control strategy against taro leaf blight, there should be no limit to the exploration/exploitation of readily available and biodegradable products of nature in our environment. therefore, this study aimed to evaluate the antifungal potentials of lime, ethanol, and aqueous extracts of xylopia aethopica on taro leaf blight disease under screenhouse conditions. materials and methods study location the study was carried out in a screenhouse at the national root crops research institute (ncri) umudike, nigeria (05o 211n and 07o 331e). sources of materials healthy taro corms were obtained from the research farm at the ncri, umudike, nigeria. materials for effects of citrus aurantifolia linn and xylopia aethopica (dunal) a. rich extracts .......... journal of tropical crop science vol. 10 no. 1, february 2023 www.j-tropical-crops.com 19 plant extracts: xylopia aethopica (locally known as “uda”) fruit and lime (citrus aurantifolia) fruit were obtained from the umuahia town market. the identity of these plant materials was authenticated by the herbarium section of the department of plant science and biotechnology, michael okpara university of agriculture, umudike. soil preparation loam soil used in this work was collected from a fallow field at the nrcri and sterilized by heating in metallic drums at temperatures above 160ºc for 2 hours to destroy soil-borne pathogens. the sterilized soil was allowed to cool for three days and mixed with dry poultry dung at the rate of 4 g per kg of soil in order to increase the soil fertility status. the enriched soil was transferred into plastic pots (5000 cm3) in the screen house where the corms were to be planted. thereafter, the soil was left for 7 days before the corms were planted so that heat generated during the decomposition of the poultry manure does not suffocate the corm (fokunang et al., 2016). experimental design and planting the experiment was a completely randomized design comprising three plant extracts applied in four levels (control, 10%, 20%, 30%). healthy seedlings of a highly susceptible variety, nce001 were planted one per plastic pot (5000 cm3) containing ¾ filled enriched loam soils in july during the 2019 planting seasons. the plants were maintained in a screenhouse and watered twice daily. preparation of the plant extracts the aqueous and ethanolic plant extracts were obtained following the soaking method described by onaebi et al. (2019) with slight modifications. preparation of ethanolic extract of xylopia fifty grams of pulverized xylopia fruits were measured into a glass bottle with a lid and soaked with 500 ml of 95 % ethanol. the mixture was stirred thoroughly and allowed to stand for three days. during this time, the mixture was stirred at least twice each day. after the third day, it was filtered into a beaker through four layers of sterile muslin cloth and covered with a perforated foil in order for the ethanol to evaporate and form a paste-like end product. preparation of aqueous extract of xylopia in preparing the aqueous extract of xylopia, hot distilled water at 40ºc was used in soaking the milled sample of xylopia fruits. the mixture thus obtained was shaken vigorously and allowed to stand for only 24 hours to prevent microbial growth and later on sieved through four layers of sterile muslin cloth. the filtrate was concentrated under a vacuum at 20-40ºc using a rotary evaporator (model: re – 52a) supplied by union laboratory, england. the crude extracts (stocks) were put into sterile screw-capped bottles, labeled accordingly, and stored in the refrigerator as long as the experiments lasted. preparation of the lime extract fresh lime fruits were washed, sliced into four pieces, and the juice was squeezed out into a beaker which was later sieved to remove debris and stored. reconstitution of crude extracts one gram portion of each crude extract was mixed with 1 ml of 25% of dimethyl sulfur oxide (dmso) in a sterile bijou bottle; dmso served as a binding agent to make the extract adhere or stick to the foliage surface when it was being sprayed. the mixture was stirred thoroughly and an additional 9 ml of sterile distilled water was added to form 1a 100 mg/ml stock solution of the particular extract. by means of a sterile pipette 10ml of the stock was taken and added to 90 ml of sterile distilled water in a conical flask to form 10%. another 20 ml of the stock was added to 80 ml of sterile water to form 20%, and the last concentration 30%, was formed by adding 30 ml of stock to 70 ml of the sterile water. fungal cultures the inoculum used in this study was obtained from previously isolated stock cultures of phytophthora colocasiae maintained in the phytopathology laboratory of ncri, umudike. inoculation inoculation of taro plants was done artificially with inocula obtained from a 7-day-old actively growing cultures of p. colocasiae. the cultures were grown on cornmeal agar (21 g of cornmeal in 1 liter of distilled water, autoclaved under pressure at 121oc for 15 minutes) and incubated at room temperature for seven days. thereafter spore suspension was made by pouring 10 ml of sterile distilled water to each culture plate and carefully dislodging the spore with sterile carmel’s -hair brush and chilling at 10oc for 30 minutes to allow zoospore release (brooks, 2008). the spore suspension was diluted and two drops were applied to the third-oldest leaves of the taro plant to cause infection. journal of tropical crop science vol. 10 no. 1, february 2023 www.j-tropical-crops.com 20 ugwuja, f. n., offor, v.n., akanwa, f.e., achi, n.k. and onifade, a. k. application of treatment the plant extract treatments were applied when the first symptoms of tlb were observed on the inoculated plants as foliage and petioles spray with a manually operated hand sprayer at seven weeks after planting (7 wap) and continued at four days intervals for up to 28 days. application of treatment was carried out early in the morning or evening to avoid the effect of wind which might drift extracts from their intended targets. distilled water was sprayed on the leaves and petioles of control plants. disease monitoring the crops were closely monitored for initiation of disease symptoms. tlb incidence and severity were measured on the leaves of treated plants starting from the day the first symptom was noticed and continued consecutively at 4-day intervals for 28 days. disease incidence percent blight incidence was worked out according to the expression of ganie et al. (2013). disease incidence was calculated based on the total number of infected plants per total number of observed plants. pbi = pbi = x100%   x100% where pbi = percentage blight incidence tnip = total number of infected plants per treatment tnp = total number of plants per treatment disease severity a six-point numerical scale (0, 1, 2, 3, 4, 5) adopted from ganie et al. (2013) was used for the assessment of disease severity. these numerical values denote the percent (%) leaf area infected as follows: 0 = no disease (0%), 2 = 1-10%, 3 = 1125%, 4 = 26 50%, 5 = 5175 %, 6 = > 76 %. percent disease intensity (pdi) was calculated following the formula given by ganie et al. (2013): pdi = σ (ni x vi) × 100 n x s where, σ = summation; ni = number of leaves in each category; vi = score of leaves observed, and s = maximum score. statistical analysis data obtained from the experiment were subjected to one-way analysis of variance, and treatment means were separated at 5% level of significance. results effect of citrus aurantifolia extracts on tlb leaf blight incidence plants treated with extracts of c. aurantifolia had significantly lower (p < 0.05) incidence than that treated with distilled water, the negative control (table 1). disease incidence gradually reduced as the concentration of citrus juice extract increased. no disease was recorded on the plants treated with ridomil, the standard fungicide and c. aurantifolia extract at 30% concentration, while the highest disease incidence 38.9% was obtained with the distilled water (zero strength of the extract). there was a remarkable reduction in disease incidence from the 12 dai (day after infection) to the 24th day reaching zero levels at 20 and 30% concentrations. effect of citrus aurantifolia extract on tlb severity table 2 shows the effect c. aurantifolia extract on percent leaf blight severity days after infection. mean disease severity varied amongst the concentrations and assessment periods and was statistically (p<0.05) significant. no disease was equally observed on the plants treated with the c. aurantifolia extract at 30 % concentration as it was with the incidence while the table 1. effect of citrus aurantifolia extract on tlb incidence (%) time (dai) concentration (%) 4 8 12 16 20 24 0 38.9 36.1 16.7 16.1 11.1 10.0 10 33.3 25.0 20.2 10.0 6.7 3.3 20 33.3 19.4 0.0 0.0 0.0 0.0 30 16.7 6.1 0.0 0.0 0.0 0.0 lsd0.05 (treatment): 9.21* lsd0.05 (time): 11.28** lsd0.05 (interaction): 22.57 ns effects of citrus aurantifolia linn and xylopia aethopica (dunal) a. rich extracts .......... journal of tropical crop science vol. 10 no. 1, february 2023 www.j-tropical-crops.com 21 highest severities (16.0) was observed on the plants treated with the distilled water. disease severity equally decreased with increasing concentration and time as was observed earlier. effect of aqueous extract of xylopia aethopica on tlb incidence the effect of aqueous extract of xylopia aethopica revealed less incidence on application of high concentration (30%) than others comparing favourably with that of ridomil (table 3). the results showed that taro plants treated with distilled water (control) expressed significantly (p<0.05) higher disease ranging from 66.7% to 44.4% as time increased compared to the ones that received the xylopia extract treatments at 10, 20 and 30% which had disease incidence range of 16.70.0%. reduction of disease with time observed with xylopia extract was not as remarkable as that of the citrus extract which had 0.0% incidences from the 12th -24th dai under 20 and 30% concentrations. effect of aqueous extract of xylopia on tlb severity results showed that plants treated with aqueous extract of x. aethopica recorded zero (0%) disease at 30 and 20% concentrations from 1624 dai and 24 dai, respectively. this result was compared favourably with that of the ridomil but significantly differed (p< 0.05) from the ones obtained from plants treated with distilled water and extract at 10% concentration (table 4). effect of ethanol extract of xylopia aethopica on tlb incidence results presented in table 5 showed that ethanol extract of xylopia aethopica significantly (p< 0.05) reduced the incidence of tlb compared to the control and also had a better effect than the aqueous extract in reducing disease. similarly, in a dosage dependent pattern, disease declined as the concentration of the extract increased. there was a drastic reduction of disease amongst concentrations 10-30 % of the extract from the 12-24 dai and a complete elimination of disease (zero incidence) at 30% concentration from12-24 dai. this suggests that 30% concentration is the most efficient and the best concentration for the ethanol extract. effect of ethanol extract of xylopia aethopica on tlb severity ethanol extract of xylopia aethopica significantly (p < 0.05) reduced disease severity compared to the control and also had a better effect than the aqueous extract in reducing disease (table 6). similarly, in a dosage-dependent manner, the disease was drastically eliminated to zero values as the table 2. effect of citrus aurantifolia juice extract on tlb severity (%) time (dai) concentration (%) 4 8 12 16 20 24 0 16.0 14.9 11.0 10.1 10.6 11.6 10 9.5 5.2 0.4 0.2 0.0 0.0 20 9.0 0.7 0.0 0.0 0.0 0.0 30 2.7 0.1 0.0 0.0 0.0 0.0 lsd0.05 (treatment): 2.89 ** lsd0.05 (time): 3.53** lsd0.05 (interaction): 7.07 ns table 3. effect of aqueous extract of xylopia aethiopica on tlb incidence (%) time (dai) concentration (%) 4 8 12 16 20 24 0 66.7 41.7 48.9 50.0 55.6 44.4 10 19.4 19.1 18.3 11.7 8.3 3.3 20 16.7 14.4 8.3 6.7 5.0 1.7 30 16.7 12.0 6.7 3.3 3.3 0.0 lsd0.05 (treatment): 9.92 ** lsd0.05 (time): 12.14 ns lsd0.05 (interaction): 24.29 ns journal of tropical crop science vol. 10 no. 1, february 2023 www.j-tropical-crops.com 22 ugwuja, f. n., offor, v.n., akanwa, f.e., achi, n.k. and onifade, a. k. concentration of the extract increased at 20 and 30 % on day 12 dai and 1224 dai, respectively. this suggests that 30% concentration is the most efficient and the best concentration for the ethanol extract. discussion in several laboratory and field trials, botanical-based antifungal products are effective in the control of pathogenic fungi (ahmed, 1998; carabet et al., 2005; sealy et al., 2007; shakywar et al., 2007; shakywa et al., 2012; jimoh et al., 2013; balamurugam, 2014; sesan et al., 2017). findings from the present study have also demonstrated the ability of extracts from the test plants to control an oomycete pathogenic fungus, phytophthora colocasiae through vivid reduction of incidence and severity of phytophthora leaf blight under screen house conditions. the disease was markedly reduced in the extract treated plants than in control. this reflects the ability of the extracts to reduce the amount of inocula (infective propagules) which would have caused more infection within the screen house. moreover, the character exhibited by the plant extracts also reflects their direct fungistatic or fungicidal effect on the causal pathogen phytophthora colocasiae likely due to the antifungal principles reported to be present in them (fleischer 2003; nata’ala et al., 2018; oikeh et al., 2015; aibinu et al., 2007). the results are comparable to those previously obtained by earlier workers who studied the effects of these plant species on pathogenic organisms in different experiments (balamurugam, 2014; okigbo and nmeka, 2005; amadioha and obi, 1998; aibinu et al., 2007). table 4. effect of aqueous extract of xylopia aethiopica on tlb severity (%) time (dai) concentration (%) 4 8 12 16 20 24 0 12.0 6.3 9.2 9.9 5.8 5.8 10 0.4 1.7 1.0 0.3 0.3 0.1 20 3.8 2.0 1.0 0.2 0.1 0.0 30 0.1 0.1 0.1 0.0 0.0 0.0 lsd0.05 (treatment): 2.4** (**) highly significant lsd0.05 (time): 2.94** lsd0.05 (interaction): 5.88 ns table 5. effect of ethanol extract of xylopia aethopica on tlb incidence (%) time (dai) concentration (%) 4 8 12 16 20 24 0 33.3 25.0 44 .4 44.4 38.9 41.7 10 20.1 19.4 13.3 6.7 5.0 5.0 20 16.7 11.1 5.0 5.0 5.0 5.0 30 8.3 11.1 0.0 0.0 0.0 0.0 lsd0.05 (treatment): 10.03 ** lsd0.05 (time): 12.28 ns lsd0.05 (interaction): 24.57 ns table 6. effect of ethanol extract of xylopia aethopica on tlb severity (%) time (dai) concentration (%) 4 8 12 16 20 24 0 30.6 26.5 22.3 21.3 19.7 20.5 10 0.4 1.6 1.7 0.8 0.4 0.2 20 2.4 2.7 0.2 0.0 0.0 0.0 30 2.4 0.1 0.0 0.0 0.0 0.0 lsd0.05 (treatment): 4.6** lsd0.05 (time): 5.6** lsd0.05 (interaction): 11.3 ns effects of citrus aurantifolia linn and xylopia aethopica (dunal) a. rich extracts .......... journal of tropical crop science vol. 10 no. 1, february 2023 www.j-tropical-crops.com 23 the results illustrated in the tables show that disease incidence and severity decreased with increasing concentration of extracts indicating that the antifungal capacity of the extracts is dependent on the amount of active principle in each given concentration of crude extract. this corroborates the findings of many workers (sagar et al., 2007; balamurugam, 2014; tegang et al., 2018) who reported marked concentration-dependent inhibition of phytopathogenic fungi in different studies. although the extracts exhibited effective control of tlb, c. aurantifolia extract was the most efficient in reducing disease given the lowest (zero) percentage of disease at a 30% concentration. this suggests that 30% concentration is the absolute fungicidal dose. the differential capacity of the extracts to reduce disease could be attributed to differences in their quantitative and qualitative phytochemical composition as opined by edeoga et al. (2005). plants of different species vary in the type and amount of bioactive principles and their biological activities are bound to be different. the observed variation in the performance of the extracts could also be attributed to the type of solvent used for extraction (okwu, 2006). the antifungal activity of c. aurantifolia could be ascribed to some components such as limonene and geranial which were reported to have antimicrobial activity. two studies by pasqua et al. (2006 and 2007) reported that limonene, the major component in citrus essential oil, had high antimicrobial activity against pathogens when applied separately. this result agrees with the results of tchameni et al. (2017) who reported the highest inhibition of sporangia production (72.84%) by lime oil at 400 ppm. although the aqueous and ethanol extracts of xylopia aethiopica showed a significant reduction of disease they were not as efficient as that of c. aurantifolia in the present study. however, there are reports of the overwhelming potency of x. aethiopica against many pathogenic fungi and bacteria in different studies (amadioha and obi, 1998; fleischer, 2003; okigbo and nmeka, 2005; fleischer et al., 2008, nweze and onyishi, 2010; tegang et al., 2018). amadioha and obi 1998 reported that hot water and oil extracts of xylopia aethiopica significantly reduced spore germinatsion and growth of colletotricum lindemuthianum in vitro. test with cowpea (vigna unguiculata) indicated that the extracts applied before or after infection of plant with c. lindemuthianum were also effective in reducing the size of pathogen induced lesions greater than that of benomyl. conclusion our present study indicated that fruit juice of c. aurantifolia, aqueous and ethanol extracts of x. aethiopica significantly exhibited antifungal potency in controlling leaf blight disease of c. esculenta caused by p. colocasiae. the results revealed that among the extracts used, an aqueous extract of c. aurantifolia (lime juice) was the best alternative for biocontrol of taro leaf blight. this finding is the first report of these extracts in controlling phytophthora leaf blight disease of colocasia. however, the mechanism of action of these extracts still needs further investigation in both in-vitro and field trials. having observed the efficacy of citrus aurantifolia aqueous extract as well as aqueous and ethanol extracts of xylopia aethiopica on the reduction of phytophthora leaf blight disease of taro, we therefore, recommend that farmers should adopt these extracts because of their bio-friendly and availability status. further research should be carried out in this area to isolate the specific antifungal fractions of these extracts and make them available in commercial quantities. acknowledgement we immensely express our gratitude to the national roots crops research institute (nrcri) umudike who provided the colocasia species and the screenhouse used for this research. references adomako, j., kwoseh, c. and moses, e. 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(2018). essential oil of xylopia aethiopica from cameroon: chemical composition, antiradical and in-vitro antifungal activity against some mycotoxigenic fungi. journal of king saud university science 30, 466-471. journal of tropical crop science vol. 10 no. 1, february 2023 www.j-tropical-crops.com 26 ugwuja, f. n., offor, v.n., akanwa, f.e., achi, n.k. and onifade, a. k. tehameni, s.n., mbiakeu, s.n., sameza, m. l., jazet, p.m.d and tchoumbougnang, n.c.(2017). using citrus aurantifolia essential oil for the potential biocontrol of colocasia esculenta (taro) leaf blight caused by phytophthora colocasiae. environmental science pollution research doi://doi.org/10.1007/s11356-0170506-0. ugwuja, f.n., onyeka, t.j., chiejina, n.v. and ugwuoke, k. i. (2020). effect of phytophthora leaf blight disease of taro (colocasia esculenta (l.) schott on proximate and phytochemical constituents of infected taro corms. nigerian agricultural journal 51,142-150. ugwuja, f.n., oti, c.p., onaebi, c.n. and oti, v. o. (2021). comparative antifungal potency of some botanical-fungicides obtained with different extraction solvents against taro leaf blight (phytophthora colocasiae) in south east nigeria. nigerian agricultural journal 52, 24725. ugwuja, f. n. and chiejina, n.v. (2011). preliminary investigation of the causes of leaf rots of cocoyam in nsukka zone of nigeria. nigerian journal of plant protection 25, 203-213. ukpabi, u.j., chijioke, u. and mbanaso, e. n. a. (2013). feasibility of producing acceptable carotene and energy rich taro crisps with deep palm-oil frying in nigeria. pakistan journal of nutrition 12, 811-814. effect of nutrient management and weeds on incidence of fungal diseases in rice .......... journal of tropical crop science vol. 10 no. 2, june 2023 www.j-tropical-crops.com 101 effect of nutrient management and weeds on incidence of fungal diseases in rice t.d.c. priyadarshani* 0000-0003-2482-7582, w.m.d.m. wickramasinghe 0000-0002-0267-9067, w.c.p. egodawatta 0000-0003-3934-6351, p. tharsinia 0000-0002-8600-1826, d.i. benaragamab 0000-0002-5878-5432, d.a.u.d. devasinghe 0000-0002-3785-9594 a department of plant science, faculty of agriculture, rajarata university of sri lanka, anuradhapura, sri lanka. b department of plant science, faculty of agriculture and food science, university of manitoba, canada. *corresponding author; email: cpchamarika@gmail.com abstract high-input, modern agriculture uses large amounts of energy, water, fertilizers, and pesticides to produce high crop yields. one of the major bottlenecks of the modern agriculture in the tropics is substantial yield losses due to fungal diseases including rice blast, leaf spots and leaf scald. the aim of this study was to compare the incidence of fungal diseases in judicious nutrient management systems, including organic, integrated, and conventional, under different weed categories during dry season (may to september 2020) and wet season (november 2020 to march 2021). rice disease incidence were collected for both seasons from 48-84 days after sowing. additionally, disease incidences on grasses and sedges weeds were also calculated. brown spot, narrow brown leaf spot, leaf scald, and rice blast incidences were substantial in wet season, while the disease incidences during dry season in 2020 were negligible. the disease incidences were significantly higher (p<0.05) in organic and conventional input systems compared to the integrated input system. disease incidences of brown spot and leaf scald were found in the dry season. higher disease incidences were recorded in the wet season than in the dry season. the incidences of the brown spot were higher on sedges than in grasses and vice versa were observed for narrow brown leaf spot disease. leaf scald incidences were positively correlated with the significant nitrogen status of the rice crop. disease incidence was low in integrated input system compared to conventional and organic input systems, while weeds were reported as alternative hosts. it can be concluded that the integrated nutrient management with recommended dosage of nitrogen application with proper weed management can lead to low disease incidents, hence is ecologically more sustainable. keywords: brown spot, narrow brown leaf spot, nutrient management, rice, weed management. introduction the major challenges of rice cultivations include insect pests, diseases, and weeds. microbial pathogens like fungi, viruses, and bacteria are responsible for diseases like rice blast, rice yellow mottle virus disease, and bacterial blight on rice (skamnioti and gurr, 2009). according to irri (year) rice pests and diseases caused around 37% of yield losses in rice, whereas in the most severe condition, yield losses due to fungal disease can reach 80% (godfray et al., 2016). however, depending on the production environment, these yield losses can vary between 24% 41% (sparks et al., 2012). weeds may become alternative host habitats to disease-causing pathogens (seneviratne and jeyanandarajah, 2004). therefore, effective fungal rice disease control strategies are essential to reduce yield losses. during the past few decades, synthetic chemicals have been used to control rice diseases (khoury and makkouk, 2010). unfortunately, chemical disease control in agriculture has resulted in numerous hazardous effects, including environmental pollution, fungal resurgence, the emergence of resistant fungal strains, and secondary fungal infections (singh et al., 2021). therefore, research scientists have started focusing on more efficient and environment-friendly disease control in rice cultivations (zibaee, 2013). a need for strategic studies on disease management in rice has already been stated. nutrient-based disease management approaches are now evolving to meet this research needs (walters and bingham, 2007). plant diseases can be controlled by, among others, managing the supply of mineral nutrients to plants at journal of tropical crop science vol. 10 no. 2, june 2023 www.j-tropical-crops.com 102 t.d.c. priyadarshani, w.m.d.m. wickramasinghe, w.c.p. egodawatta, p. tharsini, d.i. benaragama, d.a.u.d. devasinghe the appropriate time, while also reducing pesticide application. optimal nutrition management for crops can enhance plant health and resistance to diseases. minerals including nitrogen (n), sulphur (s), phosphorus (p), potassium (k), and silicon (si) directly affect pathogens and plant resistance or tolerance against pathogens (walters and bingham, 2007). nitrogen is essential for plant growth and development, and plants obtain it from the soil in various forms such as nitrate, ammonium, and urea. managing the supply of nitrogen to crops is crucial for their health; excessive use of nitrogen fertilizers can lead to environmental issues. n levels in plants has been correlated to high infections and sporulation of fungal pathogens (walters and bingham, 2007). either high or low n may result in significantly higher foliar fungal diseases incidences (robert et al., 2002); high n fertilizer inputs increases the incidence of foliar diseases narrow brown leaf spot (simón et al., 2003; walters and bingham, 2007). high n in leaf tissues creates a favourable environment for fungal disease development. in low external input systems, such as organically grown crops, little knowledge is available on the relationship between nutrient management and disease development. in organic systems, a limited supply of nitrogen reduces the k+ utilization by the crops and may also limit other nutrient supplies (fortune et al., 2004). this also causes a reduction in plant tissue concentrations and vigour, which may increase disease development through reducing the plant’s immune strength. therefore, adjusting fertilizers judiciously can minimize disease development and spread (walters and bingham, 2007). this concept of integrative plant nutrition management is a key element in a sustainable agriculture. incorporation of nutrients through amendments or modifications of the soil environment is important for the management of rice plant diseases (huber and haneklaus, 2007). the target of this research was to assess the incidence of fungal rice diseases in organic, integrated, and conventional nutrient management systems in wet and dry seasons, along with disease incidence on weeds and the nitrogen status of the crops. materials and methods the field experiments were conducted during the 2020 srilanka’s yala season (dry season; may 2020 to september 2020) and 2020/2021 maha season (wet season; november 2020 to march 2021) in the research field of the faculty of agriculture, rajarata university of sri lanka, puliyankulama, anuradhapura. the site has an agro-ecological region of dl1b. the study area consisted of imperfectly drained reddish brown earth soils (dassanayake et al., 2020). rainfall, relative humidity, and temperature were collected from at the meteorological station, anuradhapura (table 1). the laboratory studies were conducted at the plant pathology laboratory, faculty of agriculture, rajarata university of sri lanka. experimental design and treatments three main nutrient input systems, conventional input system (conv), integrated input system (int) and organic (org) input systems, were tested in this study. these systems were different from each other based on the sources of n fertilizer applied. conv uses 100 % inorganic fertilizer application as recommended by the department of agriculture (doa) 2013; int uses 50% inorganic and 50% organic fertilizers; org did not apply inorganic fertilizers and the soil fertility was managed through pre-determined organic manure application at a half rate to equate n application in table 1. treatments and the nutrient content nutrients source (kg.ha-1) mineral fertilizer rate (doa 2013) (kg.ha-1) nutrients from organic fertilizer (kg.ha-1) organic fertilizer (kg.ha-1) conventional nutrient management (conv) n 103.5 (urea) 225 (urea) 0 0 p 3.9 (p2o5) 55 (tsp) 0 0 k 30.0 (k2o) 60 (mop) 0 0 integrated nutrient management (inm) n 51.8 (urea) 112.5 (urea) 25.9 6 p 1.9 (p2o5) 27.5 (tsp) 41.52 6 k 15 (k2o) 30 (mop) 52.5 6 organic nutrient management (onm) n 0 0 51.8 12 p k 0 0 0 0 83.03 0 12 0 note: urea contains 46% n, p2o5 contains 43.7% p, k2o contains 60% k, mop contains 62% k2o. effect of nutrient management and weeds on incidence of fungal diseases in rice .......... journal of tropical crop science vol. 10 no. 2, june 2023 www.j-tropical-crops.com 103 the conventional system. organic fertilizers were prepared by incorporating buffalo manures, poultry manures, gliricidia and crotalaria leaves, which had the n content of 0.2%. the nutrient management systems were defined based on the elemental n supply and sources as described in table 2. the phosphorus (p) and potassium (k) rates were not standardized; the amount of these two elements were dependent on the quality of materials used to supply n (table 2). the three treatments were laid-out as a randomized complete block design with six replicates in dry season and 12 replicates in wet season. the size of a single plot was 90 m2 (15 m × 6 m). crop establishment and management pre-germinated seeds of the “bg300” rice variety were broadcasted at a rate of 120 kg.ha-1 in both wet and dry seasons. the application of inorganic fertilizer was based on the doa recommendation in 2013. organic fertilizer was applied with basal dressing and at the third top dressing of inorganic fertilizers. irrigation was started one week after the seed sowing and a water depth of 5 cm was impounded above the soil level to retain sufficient moisture throughout the cultivation period. the flood irrigation method was practiced, and bund were used to separate each plot to avoid water flow from one to another plot. pest and weed management the insect pest incidences were low during the crop cycles; thus, agrochemicals were not applied to control insect pests. for both conventional and integrated systems, pretilachlor (a.i. 2‐chloro‐2′, 6′‐ diethyl‐n‐(2‐propoxyethyl) acetanilide) + safener 300 ec was applied to the plots at a rate of 1.6 l.ha-1 three days after crop establishment and 14 days after sowing. bispyribac sodium was applied as an organic weed controller at a rate of 225 g.ha-1. hand weeding was done to remove visible weeds in the organic input system. analogous to the herbicide application to conventional and reduced, weeds were thinned out by removing visible weeds during the period of 3 days to 14 days. thereafter, in the organic system, weeds were not controlled. table 2. rainfall, temperatures, and relative humidity during 2020 yala (dry) and 2020/2021 maha (wet season) in srilanka. yala maha mean rainfall (mm) 94.7 150.22 minimum temperature (oc) 25.08 23.39 maximum temperature (oc) 32.70 30.82 relative humidity (%) 79.79 83.94 data collection quadrats with dimensions of 50 cm × 50 cm were used to collect data. two quadrat samplings were done for each plot with three days interval between two sampling days. the data were collected for 12 sampling days (commencing from the disease) from 48 days after sowing (das) to 84 das. disease incidence plots were allowed for natural rice disease incidence. the number of infected plants and the total number of plants were counted within a quadrat to obtain disease incidence. data collection for disease incidence was started from 48 das. to calculate the disease incidence the following equation was used:   5    pest and weed management the insect pest incidences were low during the crop cycles; thus, agrochemicals were not applied to control insect pests. for both conventional and integrated systems, pretilachlor (a.i. 2-chloro-2′, 6′-diethyl-n-(2-propoxyethyl) acetanilide) + safener 300 ec was applied to the plots at a rate of 1.6 l.ha-1 three days after crop establishment and 14 days after sowing. bispyribac sodium was applied as an organic weed controller at a rate of 225 g.ha-1. hand weeding was done to remove visible weeds in the organic input system. analogous to the herbicide application to conventional and reduced, weeds were thinned out by removing visible weeds during the period of 3 days to 14 days. thereafter, in the organic system, weeds were not controlled. data collection quadrats with dimensions of 50 cm × 50 cm were used to collect data. two quadrat samplings were done for each plot with three days interval between two sampling days. the data were collected for 12 sampling days (commencing from the disease) from 48 days after sowing (das) to 84 das. disease incidence plots were allowed for natural rice disease incidence. the number of infected plants and the total number of plants were counted within a quadrat to obtain disease incidence. data collection for disease incidence was started from 48 das. to calculate the disease incidence the following equation was used: disease incidence number of infected plants per quadrattotal number of plants per quadrat accordingly, disease incidence was calculated for brown spot, narrow brown leaf spot, rice blast and leaf scald by observing the symptoms. diseased leaf samples with moderately large lesions were randomly collected from the field and were cultured after disinfection with 10% clorox solution. standard protocol was followed for the culturing process (delves-broughton, 2007). isolation and purification of the responsible fungal pathogens by using the samples were done at the plant pathology laboratory. first, the infected leaves were surface sterilized using 1% sodium hypochlorite solution and 70% ethanol for 1 minute followed by a washing step with distilled water. potato dextrose agar (pda) medium was used for culturing the microorganisms. surface sterilized samples were dried after placing between two sterile filter papers. then the samples were placed on pda medium under the aseptic conditions. subsequently, the samples were incubated at 28 ± 1°c for 72 h. identification of fungi associated with accordingly, disease incidence was calculated for brown spot, narrow brown leaf spot, rice blast and leaf scald by observing the symptoms. diseased leaf samples with moderately large lesions were randomly collected from the field and were cultured after disinfection with 10% clorox solution. standard protocol was followed for the culturing process (delves-broughton, 2007). isolation and purification of the responsible fungal pathogens by using the samples were done at the plant pathology laboratory. first, the infected leaves were surface sterilized using 1% sodium hypochlorite solution and 70% ethanol for 1 minute followed by a washing step with distilled water. potato dextrose agar (pda) medium was used for culturing the microorganisms. surface sterilized samples were dried after placing between two sterile filter papers. then the samples were placed on pda medium under the aseptic conditions. subsequently, the samples were incubated at 28 ± 1°c for 72 h. identification of fungi associated with symptoms was done based on spore morphology, cultural characteristics, and microscopic observations. journal of tropical crop science vol. 10 no. 2, june 2023 www.j-tropical-crops.com 104 t.d.c. priyadarshani, w.m.d.m. wickramasinghe, w.c.p. egodawatta, p. tharsini, d.i. benaragama, d.a.u.d. devasinghe 1. brown spot disease (bs): cochliobolus miyabeanus 2. narrow brown leaf spot disease (nbls): cercospora janseana 3. rice blast (rb): magnaporthe oryzae 4. leaf scald (ls): monographella albescens after observing the symptoms related to each disease, the koch postulation was tested on the samples taken from the field to confirm the disease (koch, 1891). weed count total number of weeds categorized into grasses, sedges and broadleaves were taken within a quadrat. then, the number of disease-infected weeds were also counted to calculate the disease incidence. the diseases were also noted down separately. quadrat samplings were done randomly at two different locations within each plot. weed counts were done starting from 48 das. leaf nitrogen status nitrogen status analysis was conducted on leaf samples collected from a 50 cm × 50 cm quadrant at 40 and 60 das. relative leaf chlorophyll content was measured as spad (soil plant analysis development) value using spad-502 plusleaf chlorophyll meter (konica minolta sensing inc., japan). total nitrogen content of plant samples was determined by the kjeldhal procedure (bremner and mulvaney, 1982) and extractable chlorophyll content was analyzed using arnon’s method (arnon, 1949). data analysis the disease incidence variation with seasons, input systems and weed abundance were analysed by anova (analysis of variance) using the repeated mixed model in sas statistical software program version 9.0. the treatments (input systems) were considered as fixed factors and the replicates (blocks) were considered as random factors. all means were separated using the lsd method at 5% probability level. the correlation between disease incidence and foliar nitrogen content was assessed using the pearson correlation coefficient at p<0.05. results and discussion effect of seasons on disease incidences fungal disease incidences of narrow brown leaf spot (nbls), brown spot (bs), rice blast (rb) and leaf scald (ls) were observed in the rice field during wet season of 2020/2021. incidences of bs and ls disease were limited to dry season of 2020. disease incidences of bs (p<0.0001), nbls (p<0.0001) and rb (p<0.0001) were significantly different between the dry season (may 2020 september 2020) and the wet season (november 2020 march 2021), except for ls (p=0.06). the disease incidences were higher in the wet season than in dry season. the seasonal differences were limited to bs and ls, while nbls and rb were recorded only in the wet season (figure 1). wet season is more humid than the dry season. in comparison, the amount, frequency, and incidences of precipitation are higher in wet season, than in the dry season (table 1). the biological cycles of pathogens are affected by climatic changes (mcmichael et al., figure 1. rice disease incidence at heading and dough stages of rice plant in 2020 yala (dry) and 2020/2021 maha (wet) seasons. means values followed by same letter in each growth stages in each disease are not significantly different at p<0.05 (lsd). effect of nutrient management and weeds on incidence of fungal diseases in rice .......... journal of tropical crop science vol. 10 no. 2, june 2023 www.j-tropical-crops.com 105 2006) and germination of fungal spores is favoured in high moisture (sathischandra et al., 2014). dry environments have a negative impact on the incidence and progress of fungal diseases whereas wet conditions favour pathogen development (jain et al., 2019). disease incidence at heading and dough stages heading and dough stages are the most critical stages of rice crop as the panicles emerge after the booting stage with transitional changes of the reproductive stage (heading) and to grain filling afterward. detrimental factors may limit the yield formation during reproductive and ripening phases and that can reduce the final yield substantially. correct monitoring of crops for diseases during those two stages is helpful in mitigating possible economic losses (moldenhauer and slaton, 2001). at heading, the disease incidences of bs and nbls were significantly higher in the wet season. the disease incidences of rb and ls were similar and substantially low in both dry and wet seasons (figure 2). an increasing trend of bs and nbls were observed and peaked at the reproductive phase, while same trend is well documented (irri, 2002). rice blast starts appearing between seedling and late tillering stages and ls occurs on maturing leaves at the latter stages (groth et al., 1991). at the dough stage, the disease incidences bs and nbls were substantial, but rb was low. yet, disease incidences were greater than those in the dry season figure 2. effect of seasons on the disease incidence of (a) brown spot, (b) leaf scald, (c) narrow brown leaf spot and (d) rice blast. (figure 2). the development of fungal diseases is common under high humid conditions, relatively long dew periods and cool nights, which are typical in wet season. furthermore, rb was a significant disease in the early days, thus most of the late improved varieties were bred to be resistant (groth et al., 1991). the rice variety “bg300” is moderately resistant to rb (rrdi), therefore it had a low incidence and less economic loss due to rb. the bs disease incidence was increased around four folds at the dough stage, compared to heading in wet season (figure 2). brown spot is the most prevalent disease during the maturity stages of rice (groth et al., 1991), however, incidences were not severe during the life cycle of the crop in dry season. the disease incidence of nbls at the dough stage was also more than three folds higher than heading stage in the wet season, whereas incidences during dry season were negligible. similarly, incidence of rb was also low in both seasons, while it remained below one (figure 2). disease incidences in 2020/2021 wet season the lower of disease occurrences in the dry season resulted in them being excluded from further analysis. the interaction between sampling time and input system was significant for bs (p<0.0001) and nbls (p<0.0001), while for rb (p=0.4) and ls (p=0.97), it was not significant. the incidence of ls (p<0.0001) was significant only due to the impact of input system. the incidence of rb was only affected by the time (p=0.04). diseases like bs, nbls and ls were dependent on crop rotation and nitrogen fertilizer journal of tropical crop science vol. 10 no. 2, june 2023 www.j-tropical-crops.com 106 t.d.c. priyadarshani, w.m.d.m. wickramasinghe, w.c.p. egodawatta, p. tharsini, d.i. benaragama, d.a.u.d. devasinghe management, while rb incidence was more related to weather conditions during cropping season (groth et al., 1991). the disease incidences of bs and nbls were increasing with crop growth and development irrespective of input systems (figure 3). the disease incidences of bs in organic and conventional input systems were higher and increased at a higher rate than the integrated system (figure 3). the incidence of bs showed higher infestation under insufficient nitrogen conditions in organic input system; however, past studies suggested that bs can be managed by proper fertilization (groth et al., 1991). the disease incidence of nbls was higher in conventional input system than the integrated nutrient management system, but lower than that of the organic input system. at 57 das when rice crops were at the panicle initiation stage, and 63 das at the heading stage, the incidence of nbls disease in conventional input system was significantly higher than that of both integrated and organic input systems. figure 3. brown spot (a) and narrow brown leaf spot (b) disease incidence in rice on different cultural systems. figure 4. incidences of narrow brown leaf spot (a) and brown leaf spot (c) on sedges; narrow brown leaf spot (b) and brown leaf spot (d) on grasses in different cultural systems. effect of nutrient management and weeds on incidence of fungal diseases in rice .......... journal of tropical crop science vol. 10 no. 2, june 2023 www.j-tropical-crops.com 107 however, at the milking stage (75 das) and at the soft dough stage (81 das), the disease incidence of nbls in integrated input system was significantly higher than that of the conventional input system and lower than that of organic system (figure 4). the disease incidence of rb also recorded an increasing trend. according to growth et al. (1991), rice blast disease incidence was affected only by the seasonal variations, while no impact from input systems and fertilizer applications were reported. the ls disease incidence was significantly higher in conventional input system with 0.39 disease incidence than that of integrated and organic input systems, which reported disease incidence less than 0.01. this difference might be associated with the dependence of incidence of ls on nitrogen fertilization. the development of ls disease increases with high rates of nitrogen application and split applications have higher effects than single dosages (singh and gupta, 1983). disease incidence on weeds weeds act as alternative host plants by harbouring fungal pathogens and transmitting them to the crops via wind and insects when conditions are favourable (seneviratne and jeyanandarajah, 2004). sedges (cyperus spp.) and grasses are commonly reported weed types in the field experiment as alternative host plants for rice diseases. grasses and sedges are commonly affected by leaf spot diseases like bs and nbls. brown spot disease affects grasses comparatively higher than the sedges (adur-okello et al., 2020). the two-way interaction of time and treatment on disease incidence of bs on grasses (p=0.002) and sedges (p=0.02) was significant. however, the same two-way interaction nbls disease incident was significant only for sedges (p<0.0001). the disease incidence of nbls on grasses was only significant with the sampling time (p<0.0001). the disease incidence of bs on sedges and grasses showed an increasing trend with time in all three input systems (figure 4). the bs disease incidence was higher in the organic input system compared to reduced and conventional systems (figure 4). the incidences of bs disease on grasses were lower than that on sedges. the disease incidence of nbls on sedges and grasses showed an increasing trend with time in all three input systems (figure 4). the disease incidence on sedges was higher in the organic input system compared to reduced and conventional systems (figure 4). the incidences of nbls on grasses were higher at the beginning than that on sedges, and later the incidents were similar. grasses and broadleaves were effectively controlled in all systems, but the population of sedges were not effectively controlled. further, even after herbicide application, sedges were emerged, thus the incidences of sedges were higher proportionate to their presence within systems. the incidence of bs disease on the rice crop, grasses and sedges showed an increasing trend in organic, integrated, and conventional input systems with time. however, the sedges showed a dissimilar figure 5. brown spot disease (a) and narrow brown leaf spot (b) incidence on rice, sedges, and grasses. journal of tropical crop science vol. 10 no. 2, june 2023 www.j-tropical-crops.com 108 t.d.c. priyadarshani, w.m.d.m. wickramasinghe, w.c.p. egodawatta, p. tharsini, d.i. benaragama, d.a.u.d. devasinghe trend, as rice and grass weeds belong to the family poaceae and while sedge weeds belong to the family cyperaceae (perera and dahanayake, 2015). the disease incidence of bs on sedges was substantially higher than that of both rice and grasses from the beginning (figure 9). this can be due to higher presence of sedges in the systems. even under low fertilizer systems it can be cause due to seed bank of sedges of previous seasons, slow growth at early days so hand weeding might not have effective, manual weeding, by pulling the entire plant may not have been uprooted. at the heading stage (65 das) disease incidences appearance started increase on rice and grasses, however, the rate was higher on grasses. grass weeds act as alternative host plants of bs disease. the incidence of nbls disease on rice showed an increasing trend that is similar to bs, while nbls progression on grasses and sedges were different (figure 5). at 65 das (the heading stage), the disease incidences were increased at 65 das, while recorded a higher increasing rate than rice and sedges at the beginning. the incidence of rice was the lowest (figure 5). the disease incidence of nbls on rice in the organic system was the highest and incidence in the integrated system was the lowest while a similar trend was observed on grasses and sedges too (figure 5). correlation between disease incidence and leaf nitrogen status the disease incidence of bs and nbls were negatively and significantly correlated to spad value and extractable chlorophyll content, while no correlation was recorded to n content (table 3). disease incidence of rb was negatively correlated to spad value only. the correlations of disease incidence of ls were positive and significant (p<0.05) to all three factors (table 3). foliar leaf spot diseases like bs and nbls affect the foliar nitrogen status of rice leaves (julião et al., 2020). with seasonal differences, rb disease incidents are not prominent, thus correlations were weak, while no impact of input systems and fertilizer applications were recorded (groth et al., 1991). the negative correlations of bs and nbls were due to the changes in the number of lesions on rice leaf, table 3. associated probability values and the correlation between the disease incidences of brown spot, narrow brown leaf spot, rice blast, leaf scald and nitrogen status of rice in 2020/2021 maha (wet) season. disease incidence relative leaf chlorophyll content (spad value) extractable chlorophyll leaf n brown spot -0.60 ** -0.51 ** -0.11 ns narrow brown leaf spot -0.70 ** -0.57 ** 0.004 ns rice blast -0.34 ** -0.23 ns no leaf scald 0.41 ** 0.37 ** 0.70 ** ** significant at p<0.05; ns=not significant; no= not observed. figure 6. correlation between diseases incidence and rice leaf spad values (a), and between disease incidence and extractable chlorophyll content (b). effect of nutrient management and weeds on incidence of fungal diseases in rice .......... journal of tropical crop science vol. 10 no. 2, june 2023 www.j-tropical-crops.com 109 the nitrogen content in leaf tissues decrease. the inverse relationship of bs and nbls were possible (carvalho et al., 2010), yet an increase in nitrogen status of crop, in this study is indicated by the spad values, generally increases the disease incidence of rice ls (babu, 2016). the leaf chlorophyll content indicates the nitrogen status of the rice crop indirectly (marschner, 1995). the disease incidence of rice blast did not significantly correlate with the chlorophyll content (figure 6 b). conclusions brown spot (cochliobolus miyabeanus) and narrow brown leaf spot (cercospora janseana) diseases were the most recorded fungal diseases during the study. the disease incidence of brown spot and narrow brown leaf spot diseases developed progressively with the development of the crop and peaked around 55 – 65 das irrespective of the input system. the highest disease incidences were recorded in the organic input system, while the integrated system recorded the lowest. nutrient deficiency and imbalance increased the crop’s vulnerability to biological stresses such as diseases when microclimate is much more favourable for fungal disease development. rice nitrogen status affected the incidence of brown spot, narrow brown leaf spot and leaf scald. brown spot and narrow brown leaf spot disease incidences on weeds were higher in the organic input system. the incidences of brown spot and narrow brown leaf spot diseases on sedges were the highest. the disease incidences were higher on weeds; thus, these diseases can prevail even during the fallow by continuing the life cycle. hence, judicious management of nitrogen via alternative sources can suppress the disease susceptibility, while without toppling the production expectations. therefore, an integrated nutrient management system with mineral and alternative sources minimizes the disease incidence and weeds management reduced the persistence of fungal rice 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(2013). rice: importance and future. journal of rice research 1, 102. https://doi. org/10.4172/jrr.1000e102 journal of tropical crop science vol. 10 no. 2, june 2023 www.j-tropical-crops.com 124 belangie tuahte gundala, eny widajati, satriyas ilyas the development of germination tests and breaking dormancy techniques of bitter seeds (andrographis paniculata (burm. f.) wall. ex nees) belangie tuahte gundalaa, eny widajatib * 0000-0002-3586-6395, satriyas ilyasb 0000-0002-8173-3817 a graduate school of seed science and technology, ipb university, bogor 16680, indonesia b department of agronomy and horticulture, faculty of agriculture, ipb university, bogor 16680, indonesia *corresponding author; email: eny_widajati@apps.ipb.ac.id abstract bitter seed (andrographis paniculata (burm. f.) wall. ex nees) is a medicinal species used in phyto pharmacy and known as ‘the king of bitterness’. bitter seeds contain andrographolide, a diterpenoid lactone which is widely used in indian medicine for snake bites and for the treatment of hepatitis. the research was conducted at the seed management unit experimental field of the indonesian spice and medicinal plants research institute (imacri) and at the seed quality testing laboratory of ipb university, indonesia, from january until may 2021. the objectives of the study is to determine the best germination test and effective breaking dormancy techniques to increase the germination percentage of bitter seeds. the study was arranged in a completely randomized design with two-factors. the first factor was seed treatments, i.e., control/untreated, chemical scarification with 5% h2so4 for 10 minutes, and 5% h2so4 followed by a 100 ppm ga3 soaking treatment. the second factor conducted was germination test methods according to international seed testing association, i.e., germinating the seeds on the top of paper and in pleated paper. our study demonstrated that seed treatment with h2so4 can break seed dormancy and speed up seed germination. bitter seed germination on the pleated paper was higher than with the top-of-paper method. bitter seed germination with the top-of-paper method was at 65 das and the final germination was at 80 days after sowing (das), and the final germination was 55.2%. with the pleated paper method, the seeds germinated at 30 das and the final germination was at 70 das, and the final the germination of 59.2%. keywords: chemical scarification, ga3, pleated paper and top of paper germination, seed viability, seed vigor. introduction bitter seed (andrographis paniculata (burm. f.) wall. ex nees) is originated from india (valdiani et al., 2012) and it is one of the important medicinal plants in the tropics. bitter seed in indonesia has been long used as a traditional and medicinal ingredient because it contains andrographolide compounds which have various properties, such as an immune stimulator, anti-inflammatory, anti-cancer, hepatitis drug, and a covid-19 inhibitor (rajagopal et al., 2020). bitter seed production in indonesia has increased in the last five years (2017-2021), with an average annual production reaching 1,754705 kg per year (cas, 2021). one of the limitations in the cultivation of bitter seed is the limited availability of quality seeds. the seeds, including the freshly harvested seeds, generally have low germination rates. the bitter seeds have a dormancy period (rusmin et al., 2007), and germination rate is highly affected by the environment. according to rouhi et al., (2015), seed dormancy may also be influenced by pre-chilling after harvest and seed desiccation. apart from the low germination rates, the seeds have rather low growth potentials, which can lead to misinterpretation in the evaluation of seed quality. bitter seed dormancy is classified as an ‘exogenous,’ or a physical dormancy due to the hard seed coat (talei et al., 2012), resulting in a long period of germination that can reach 5-6 months (rusmin et al., 2007). physical dormancy was caused by one or more water-impermeable layers of palisade cells in the seed coat. physiological dormancy is a complex, and can be classified as deep, intermediate, and nondeep dormancy (baskin and baskin, 2004). chemical or mechanical scarification can promote germination of seeds with a non-deep physiological dormancy (baskin and baskin, 2004). the development of germination tests and breaking dormancy techniques of bitter .......... journal of tropical crop science vol. 10 no. 2, june 2023 www.j-tropical-crops.com 125 information regarding the germination test methods of bitter seeds is still limited. based on ista (2018), germination tests for small size seeds can use papers. a laboratory analysis is important to determine the inhibitory agent and the type of dormancy in selecting the best dormancy-breaking solution (liu et al., 2011). identification of the molecular mechanisms underlying the induction and release of dormancy after ripening in arabidopsis thaliana seeds through cloning and characterization of the rdo2 mutant has been reported (liu et al., 2011). understanding of the molecular mechanisms are important information to further study other species with long seed dormancy. dormancy-breaking treatment of bitter seeds through the scarification of the seed coat and soaking in h2so4 solution can potentially overcome the problem of seed dormancy. however, this technique still needs to be improved as the germination rate was still low (58%; kataky and handique, 2010). a study by talei et al. (2012) reported that the soaking treatment of bitter seed in 5% (v/v) h2so4 for 10 minutes resulted in 50% seed germination compared to 30% in the untreated seeds (control). therefore, it is necessary to further study the dormancy mechanism of bitter seeds as a basis for determining an effective method of breaking dormancy and to obtain a higher germination percentage. this study aims to determine an optimum germination test method and effective breaking-dormancy techniques to increase bitter seed germination. material and methods time and location the study was conducted from january to may 2021 at the seed management unit experimental field of the indonesian spice and medicinal plants research institute (imacri) in bogor, and the seed quality testing and storage laboratory, faculty of agriculture, ipb university. materials bitter seeds “sambina 1” variety were harvested from the experimental station of the seed management unit, imacri when the pods are uniformly in brown color. chemical scarification treatment was implemented using h2so4, ga3, kno3, and kinetin. development of germination test of bitter seed the experiment was arranged using a two-factor completely randomized design. the first factor was seed treatment, which consisted of three levels: no treatment (control), chemical scarification, and chemical scarification followed by ga3 immersion. the second factor is the germination test methods: the top-of-paper (tp) and the pleated-paper (pp) methods. each treatment was repeated four times, so there were 24 experimental units. chemical scarification treatment was conducted by soaking the seeds in 5% h2so4 for 10 minutes (talei et al., 2012). the seeds were rinsed thoroughly using running distilled water, and air-dried at room environment (26-32 ºc, rh 61-79%) for one hour. the treatment with ga3 (100 ppm) was conducted seeds 24 hours after chemical scarification (cipcigan et al., 2020), followed by air drying for 12-18 hours (powell et al., 2000). germination was carried out using two methods according to ista (2018): top-of-paper (tp), i.e., the seeds were placed on paper; and on pleated paper (pp), i.e., the seeds are placed between folds of a paper strip folded. for each method, one-hundred seeds were germinated on two-ply moist filter papers in a germination box set at 25 ºc and relative humidity 80-90%. measurement and data analysis seed viability was measured based on seed viability and seed vigor. data was analyzed using anova at α = 5% using statistical analysis system (sas) 9.4. significant differences between means were further analyzed using duncan multiple range test at α=5%. observation and measurement seed vigor, seed viability, speed of germination (sg), germination percentage (gp), and maximum growth potential (mgp) were measured. seed germination is defined as the emergence of a radicle of >5 mm (twala and fisher, 2022). seed vigor and seed viability was measured twice when the first-time seeds germinated, and on the final germination. the highest percentage of the normal seedling was determined as the first count; the final count was determined by the highest percentage of cumulative normal seedlings. the data of the first and final count were presented in a curve analysis. the speed of germination (sg) was measured every etmal, i.e., every 24 hours, from the first day the seeds were germinated to the last day (final count) of measurement. the speed of germination was calculated using the following formula: sg (%ns/etmal) = observation and measurement seed vigor, seed viability, speed of germination (sg), germination percentage (gp), and maximum growth potential (mgp) were measured. seed germination is defined as the emergence of a radicle of >5 mm (twala and fisher, 2022). seed vigor and seed viability was measured twice when the first-time seeds germinated, and on the final germination. the highest percentage of the normal seedling was determined as the first count; the final count was determined by the highest percentage of cumulative normal seedlings. the data of the first and final count were presented in a curve analysis. the speed of germination (sg) was measured every etmal, i.e., every 24 hours, from the first day the seeds were germinated to the last day (final count) of measurement. the speed of germination was calculated using the following formula: sg (%ns/etmal) = ∑ � %��������� � sg : speed of germination (%ns/etmal) %ns : percentage of normal germination at each observation time etmal : observation time every 24 hours t : observation time the germination percentage (gp) was carried out after the first and the final count of bitter seeds were determined. observations were made on seedlings that grew normally (ista, 2018). the normal seedling criteria of bitter seeds on paper were determined based on the presence of radicle, hypocotyl, and cotyledon. the criteria of healthy seedlings were the presence of a healthy root of a minimum 5 mm in length, a minimum hypocotyl length of 20 mm, and exhibiting no damage. the germination percentage was calculated using the following formula: gp �%� � number of ns i � number of ns iitotal seeds germinated x 100% where: ns i: normal seedlings on the first count ns ii: normal seedlings on the final count maximum growth potential (mgp) is calculated based on the proportion of seeds germinating normally and abnormally until the last day of observation (final count). the mgp formula is as follows: mgp �%� � number of seeds germinated �normal and abnormal �total seeds germinated x 100 result and discussion the bitter seed consists of testa (seed coat), embryo, and a radicle candidate (figure 2). the testa is the maternal tissue, maternally inherited. in an angiosperm seed type, the embryo is surrounded by two layers: the testa (seed coat) and the endosperm (savage and matzger, 2006). the seed coat of bitter seed is separate from the cotyledons (figure 2). cotyledon, radicle and hypocotyl are important structures of the healthy seedlings (figure 2c) journal of tropical crop science vol. 10 no. 2, june 2023 www.j-tropical-crops.com 126 belangie tuahte gundala, eny widajati, satriyas ilyas sg : speed of germination (%ns/etmal) %ns : percentage of normal germination at each observation time etmal : observation time every 24 hours t : observation time the germination percentage (gp) was carried out after the first and the final count of bitter seeds were determined. observations were made on seedlings that grew normally (ista, 2018). the normal seedling criteria of bitter seeds on paper were determined based on the presence of radicle, hypocotyl, and cotyledon. the criteria of healthy seedlings were the presence of a healthy root of a minimum 5 mm in length, a minimum hypocotyl length of 20 mm, and exhibiting no damage. the germination percentage was calculated using the following formula: observation and measurement seed vigor, seed viability, speed of germination (sg), germination percentage (gp), and maximum growth potential (mgp) were measured. seed germination is defined as the emergence of a radicle of >5 mm (twala and fisher, 2022). seed vigor and seed viability was measured twice when the first-time seeds germinated, and on the final germination. the highest percentage of the normal seedling was determined as the first count; the final count was determined by the highest percentage of cumulative normal seedlings. the data of the first and final count were presented in a curve analysis. the speed of germination (sg) was measured every etmal, i.e., every 24 hours, from the first day the seeds were germinated to the last day (final count) of measurement. the speed of germination was calculated using the following formula: sg (%ns/etmal) = ∑ � %��������� � sg : speed of germination (%ns/etmal) %ns : percentage of normal germination at each observation time etmal : observation time every 24 hours t : observation time the germination percentage (gp) was carried out after the first and the final count of bitter seeds were determined. observations were made on seedlings that grew normally (ista, 2018). the normal seedling criteria of bitter seeds on paper were determined based on the presence of radicle, hypocotyl, and cotyledon. the criteria of healthy seedlings were the presence of a healthy root of a minimum 5 mm in length, a minimum hypocotyl length of 20 mm, and exhibiting no damage. the germination percentage was calculated using the following formula: gp �%� � number of ns i � number of ns iitotal seeds germinated x 100% where: ns i: normal seedlings on the first count ns ii: normal seedlings on the final count maximum growth potential (mgp) is calculated based on the proportion of seeds germinating normally and abnormally until the last day of observation (final count). the mgp formula is as follows: mgp �%� � number of seeds germinated �normal and abnormal �total seeds germinated x 100 result and discussion the bitter seed consists of testa (seed coat), embryo, and a radicle candidate (figure 2). the testa is the maternal tissue, maternally inherited. in an angiosperm seed type, the embryo is surrounded by two layers: the testa (seed coat) and the endosperm (savage and matzger, 2006). the seed coat of bitter seed is separate from the cotyledons (figure 2). cotyledon, radicle and hypocotyl are important structures of the healthy seedlings (figure 2c) where: ns i: normal seedlings on the first count ns ii: normal seedlings on the final count maximum growth potential (mgp) is calculated based on the proportion of seeds germinating normally and abnormally until the last day of observation (final count). the mgp formula is as follows: observation and measurement seed vigor, seed viability, speed of germination (sg), germination percentage (gp), and maximum growth potential (mgp) were measured. seed germination is defined as the emergence of a radicle of >5 mm (twala and fisher, 2022). seed vigor and seed viability was measured twice when the first-time seeds germinated, and on the final germination. the highest percentage of the normal seedling was determined as the first count; the final count was determined by the highest percentage of cumulative normal seedlings. the data of the first and final count were presented in a curve analysis. the speed of germination (sg) was measured every etmal, i.e., every 24 hours, from the first day the seeds were germinated to the last day (final count) of measurement. the speed of germination was calculated using the following formula: sg (%ns/etmal) = ∑ � %��������� � sg : speed of germination (%ns/etmal) %ns : percentage of normal germination at each observation time etmal : observation time every 24 hours t : observation time the germination percentage (gp) was carried out after the first and the final count of bitter seeds were determined. observations were made on seedlings that grew normally (ista, 2018). the normal seedling criteria of bitter seeds on paper were determined based on the presence of radicle, hypocotyl, and cotyledon. the criteria of healthy seedlings were the presence of a healthy root of a minimum 5 mm in length, a minimum hypocotyl length of 20 mm, and exhibiting no damage. the germination percentage was calculated using the following formula: gp �%� � number of ns i � number of ns iitotal seeds germinated x 100% where: ns i: normal seedlings on the first count ns ii: normal seedlings on the final count maximum growth potential (mgp) is calculated based on the proportion of seeds germinating normally and abnormally until the last day of observation (final count). the mgp formula is as follows: mgp �%� � number of seeds germinated �normal and abnormal �total seeds germinated x 100 result and discussion the bitter seed consists of testa (seed coat), embryo, and a radicle candidate (figure 2). the testa is the maternal tissue, maternally inherited. in an angiosperm seed type, the embryo is surrounded by two layers: the testa (seed coat) and the endosperm (savage and matzger, 2006). the seed coat of bitter seed is separate from the cotyledons (figure 2). cotyledon, radicle and hypocotyl are important structures of the healthy seedlings (figure 2c) result and discussion the bitter seed consists of testa (seed coat), embryo, and a radicle candidate (figure 1). the testa is the maternal tissue, maternally inherited. in an angiosperm seed type, the embryo is surrounded by two layers: the testa (seed coat) and the endosperm (savage and matzger, 2006). the seed coat of bitter seed is separate from the cotyledons (figure 2). cotyledon, radicle and hypocotyl are important structures of the healthy seedlings (figure 1c) the first count of germination was determined based on the highest daily percentage of normal seedlings. the final count was determined on the day the highest cumulative normal seedlings were added or when the germination rate decreased until no more seeds germinated (ningsih et al., 2021). the value when seedlings no longer grow was typically determined as the final count (rusmin et al., 2016). figure 1. morphology of bitter seed and seedlings; an entire seed (a), a cross section of bitter seed (b), normal seedlings (c), and abnormal seedlings (d). based on the curve analysis results of the tp and pp methods (figures 2 and 3), the first bitter seedlings appeared in 5-10 days after sowing (das). the number of germinated seeds with the tp method and the first count of untreated seeds was determined based on the highest daily normal seedling at 65 das. the final count was determined based on the highest cumulative normal seedling count at 80 das (figure 2a). the first count of seeds treated with chemical scarification using 5% h2so4 for 10 minutes was set at 60 das, and the final count was 75 das (figure 2b). observations of the first and final count of seeds treated with chemical scarification followed by soaking in 100 ppm ga3 were determined at 55 and 75 das, respectively (figure 2c). contrary to the pp method, the first and final count of the untreated seeds were set at 35 and 70 das, respectively (figure 3a), while the first and final count of seeds treated with chemical scarification were conducted at 35 and 65 das (figure 3b). the chemical scarification was followed by a 100 ppm ga3 soaking treatment and resulted in the first and final count observations at 30 and 70 das (figure 3c). chemical scarification with 5% h2so4 for 10 minutes resulted in a shorter first and final count observation period than the untreated seeds in each tp and pp method (figures 2 and 3). there are two main categories of seed dormancy, exogenous and endogenous, i.e., the presence of inhibitors in the seed embryo, which inhibit the emergence of radicles in fully imbibed seeds the development of germination tests and breaking dormancy techniques of bitter .......... journal of tropical crop science vol. 10 no. 2, june 2023 www.j-tropical-crops.com 127 figure 2. the curves for determining bitter seed germination percentage using the top-of-paper (tp) method: (a) control, (b) chemical scarification, and (c) chemical scarification followed by ga3 treatment. y = -4e-05x3 + 0,0023x2 + 0,1116x 0,7537 r² = 0,7154 y = -0,0002x3 + 0,0326x2 0,5038x + 2,1544 r² = 0,9972 0 10 20 30 40 50 60 70 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 n or m al s ee dl in gs (% ) days after sowing (das) y = -9e-05x3 + 0,0077x2 0,0533x + 0,9412 r² = 0,8267 y = -0,0003x3 + 0,0422x2 0,7354x + 4,875 r² = 0,9946 0 10 20 30 40 50 60 70 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 n or m al s ee dl in gs (% ) days after sowing (das) y = -7e-05x3 + 0,0053x2 + 0,057x + 0,0515 r² = 0,7701 y = -0,0004x3 + 0,053x2 1,0114x + 6,614 r² = 0,9942 0 10 20 30 40 50 60 70 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 n or m al s ee dl in gs ( % ) days after sowing (das) normal seedlings cumulative seedlings poly. (normal seedlings) poly. (cumulative seedlings) a b figure 2. the curves for determining bitter seed germination percentage using the top-of-paper (tp) method: (a) control, (b) chemical scarification, and (c) chemical scarification followed by ga3 treatment. journal of tropical crop science vol. 10 no. 2, june 2023 www.j-tropical-crops.com 128 belangie tuahte gundala, eny widajati, satriyas ilyas figure 3. the curves for determining bitter seed germination count using the pleated-paper (pp) method. (a) control, (b) chemical scarification, and (c) chemical scarification followed by ga3 treatment. y = 0,0002x3 0,0227x2 + 0,9078x 3,5216 r² = 0,7623 y = -0,0003x3 + 0,0193x2 + 0,8389x 5,4103 r² = 0,99 0 10 20 30 40 50 60 70 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 n or m al s ee dl in gs ( % ) days after sowing (das) y = 0,0001x3 0,0215x2 + 0,8707x 2,6729 r² = 0,8579 y = -0,0003x3 + 0,0218x2 + 0,8862x 4,8864 r² = 0,9962 0 10 20 30 40 50 60 70 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 n or m al s ee dl in gs ( % ) days after sowing (das) y = 0,0002x3 0,026x2 + 0,9937x 3,0712 r² = 0,9086 y = -0,0002x3 + 0,0096x2 + 1,3615x 7,8652 r² = 0,9938 0 10 20 30 40 50 60 70 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 n or m al s ee dl in gs ( % ) days after sowing (das) normal seedlings cumulative seedlings poly. (normal seedlings) poly. (cumulative seedlings) b a figure 3. the curves for determining bitter seed germination count using the pleated-paper (pp) method: (a) control, (b) chemical scarification, and (c) chemical scarification followed by ga3 treatment. the development of germination tests and breaking dormancy techniques of bitter .......... journal of tropical crop science vol. 10 no. 2, june 2023 www.j-tropical-crops.com 129 (baskin and baskin, 2004). bitter seed dormancy is exogenous or physical, due to the thick and hard seed coat; the seed coat is the primary modulator of the interaction between the internal structure of the seed and the surrounding environment, as well as keeping the embryo from germinating (baskin and baskin, 2004). tieu et al. (2001) stated that the specific metabolic pathways in seeds can be blocked by several inhibitory compounds, such as cutin and suberin, found in seeds with a hard-shell structure. breaking seed dormancy that is caused by hard seed coat can be done mechanically and chemically (ertekin, 2011). table 1 shows that seed treatment and test methods highly significantly affected speed of germination. the speed of germination in the control group was significantly lower than the chemical scarification with 5% h2so4 for 10 minutes and the chemical scarification followed by 100 ppm ga3 soaking treatment (table 1). however, the speed of germination between 5% h2so4 with and without ga3 were not significantly different. table 1 also shows that the germination using the pp method were twice as fast as the tp method. this is likely due to differences in the humidity in the germination media which affect the acceleration of the germination of bitter seeds. seeds that were germinated with tp method were laid flat on the filter paper, so the evaporation occurred quickly. in contrast, the seeds with the pp method were laid between damp papers; the moist environment with this method accelerated germination as hard and thick seed testa require high humidity to germinate. regardless of the chemical scarification treatment, the germination percentage (gp) and maximum growth potential (mgp) in the pp method were not significantly different from the tp method (table 2). this might be related to the optimal light condition that allow seeds to germinate in the tp method. in line with the baskin and baskin (2004) report, in addition to moisture, light is one of the important factors to break dormancy and to allow morphogenesis (muhar et al., 2015). phytochromes and other light receptors regulate various morphogenesis, from seed germination, seedling development, the formation of flowers, fruits, and seeds (muhar et al., 2015). differences in the time to germinate can be caused by the interaction of various environmental factors including water, light, and temperature (gupta and chakrabarty, 2013). light and temperature can affect ga biosynthesis and signaling, hence, affecting seed germination (skubacz and golec, 2017). a suitable germination substrate is characterized by adequate porosity that can supply oxygen and retain water which is required for seed germination (santos et al., 2022). the low bitter seed germination is also caused by the hard seed coat, which makes it impermeable to gas and water (rusmin et al., 2007). a study by sun et al. (2018) demonstrated that glycyrrhiza uralensis hard seed coat contains hydrophobic chemicals such as cutin, suberin, cellulose, hemicellulose, and a waxy coating, which prevented the seeds from imbibing water. chemical scarification treatment can effectively accelerates the germination by creating cracks and cavities in the seed coat tissue, hence improved water imbibition and increased seed metabolism (yuniarti and djaman, 2015). the chemical scarification followed by 100 ppm ga3 soaking for 24 hours resulted in a shorter first count observation period (± 5 days) than the control and chemical scarification treatments (figure 3). gibberellic acid is a natural regulator of the seed germination; it stimulates the production of the hydrolytic enzyme α-amylase in the aleurone layer of germinating seeds (gupta and chakrabakti, 2013). gibberellic acid can exert its influence in two ways, by increasing the growth potential of the embryo, and by stimulating the production of hydrolytic enzymes (gupta and chakrabakti, 2013). table 2 showed that the germination of the untreated seeds were significantly higher in the pleated paper method (59.2%) than those in the top of paper method (55.2%). seed germination with 5% h2so4 with or without ga3 were similar (63.5% – 64%) in both test method (pp and tp). seeds treated with chemical scarification followed by 100 ppm ga3 soaking showed that the pp method produced significantly higher mean growth potential (mgp) than those in the tp method. according to the indonesian ministry of agriculture (2019), bitter seeds fulfill the criteria as an extension seed class if the gp reached 60%, therefore, the seed treatment developed in this study has met the germination percentage of bitter seeds met this criteria. journal of tropical crop science vol. 10 no. 2, june 2023 www.j-tropical-crops.com 130 belangie tuahte gundala, eny widajati, satriyas ilyas table 1. effect of seed treatment and test methods on germination speed seed treatment germination speed (%ns per day) control 2.06 b 5% h2so4 2.40 a 5% h2so4 + 100 ppm ga3 2.51 a test methods top-of-paper 1.69 b pleated paper 2.96 a note: values in the same column followed by the same letters are not significantly different based on the dmrt at α=0.05. ns = normal seedlings. table 2. the interaction effects of seed treatments and test methods on germination percentage (gp) and maximum growth potential (mgp) seed treatment gp (%) mgp (%) tp pp tp pp control 55.2 bb 59.2 ab 74.0 ab 73.5 ab 5% h2so4 65.5 aa 64.0 aa 77.2 aa 79.0 aa 5% h2so4 + 100 ppm ga3 63.7 aa 66.5 aa 76.7 ba 81.0 aa cv (%) 3.34 2.30 note: at the same variable, values followed by the same lowercase letter in a column or capital letter in a row show not significant difference based on the dmrt at α=0.05. * = significant at p< 0.05, ** = significant at p < 0.01, ns = not significant. tp = top-of-paper, pp = pleated-paper, cv = coefficient of variation. conclusion our study demonstrated that treatment with h2so4 can break seed dormancy and speed up germination. bitter seed germination on the pleated paper was higher than with the top-of-paper method. the first and final count of germination with 5% h2so4 with the top-of-paper method was at 60 and 75 das with germination percentage of 65.5%, whereas with the pleated paper method it was at 35 and 65 das with germination percentage of 64%. the first and final germination with the 5% h2so4 + 100 ppm ga3 with the top-of-paper method was at 55 and 75 das with germination percentage of 63.7%, whereas with the pleated paper method at 35 and 70 das with germination percentage of 66.5%. acknowledgement the authors thanked aceh human resources development agency for the scholarship, supports, and funding provided for this study. references baskin, j.m., and baskin, c.c. 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(2015). dormancy breaking technique to accelerate courbaril seed germination (hymenaea coubaril) in “proceedings of the national seminar on indonesian biodiversity society” pp. 14331437. https://doi.org/10.13057/psnmbi/ m010629 biomulch treatment effects on weed control and soil properties in cassava .......... journal of tropical crop science vol. 10 no. 2, june 2023 www.j-tropical-crops.com 111 biomulch treatment effects on weed control and soil properties in cassava plantation bayu suwitonoac 0000-0003-2955-0121, muhamad achmad chozinb 0000-0003-3820-2639, dwi guntorob, suwartob 0000-0002-1767-6299 a department of agronomy and horticulture graduate school, faculty of agriculture, ipb university. jl. meranti, ipb darmaga campus, bogor 16680, west java, indonesia. b department of agronomy and horticulture, faculty of agriculture, ipb university. jl. meranti ipb darmaga campus, bogor 16680, west java, indonesia. c research center for food crops, national research and innovation agency cibinong science center, cibinong, bogor 16915, indonesia. *corresponding author; email:bayusuwitono@yahoo.com; ma_chozin@yahoo.com email second author abstract legume cover crop (lcc) are plants grown as live mulch so that it is beneficial to the agro-ecosystem and can be included in cropping systems to regulate soil fertility and suppress weed growth. this study aims to determine the benefits of using arachis pintoi as biomulch applied at different times on weed composition and soil fertility. the experiment was carried out at the teaching farm of ipb, jonggol, west java, indonesia; the soil type is ultisol. this study used two factors and was designed in a factorial randomized block design. the first factor was four accessions of cassava “ketan malang”, “genjah bayam”, “ir jonggol”, and “manggu”; the second factor is the time of the biomulch planting: 4, 8, and 12 weeks before planting the cassava, at cassava planting, and without biomulch + manual weeding as the control. weed vegetation was analyzed before land preparation and before cassava harvest. the physical and chemical properties of the soil were analyzed before and after the biomulch treatment. our study demonstrated that melastoma malabathricum, tetracera indica, oxalis barrelieri, mimosa invisa, ottochloa nodosa, ipomoea sp and cyperus sp. are the dominant weeds in the cassava plantation. biomulch planted four and eight weeks before cassava was able to reduce weed dry weight. all biomulch treatments were able to improve soil density and total pore space. soil with biomulch planted eight weeks before cassava planting had the highest c-organic (7.59%) and total nitrogen (0.41%). keywords: legumes cover crop, live mulch, soil fertility, ultisol, weed control. introduction cassava (manihot esculenta crantz) is a native american species that has become an important food crop in africa and asia, including indonesia. cassava is a high calorie food source with the most affordable price. cassava carbohydrate contents are 20% higher than those in corn and 40% higher than those in rice (tonukari, 2004). cassava has high tolerance to drought and highly acidic environment. in indonesia, cassava is an important raw material for the food industry which comes from processing starch and its derivatives (howeler, 2012). the increasing population and the development of cassava-based industries will further increase the demand for cassava in the future (nintania et al., 2021). according to widyatmoko et al. (2018), several processed cassava products such as tapioca flour, modified cassava flour (mocaf), and cassava rice, are developing into industrial products for the domestic as well as international markets. cassava is widely grown on alfisol, ultisol, entisol, and inceptisol soil types, and it can grow on soils with low nutrient and organic matter or marginal land, where the soil’s physical, chemical, and mineral content is sub-optimal. therefore, cassava can be one of the crops that can utilize marginal lands (saidi, 2020). ultisol develops under forest vegetation containing argillic horizons, and like alfisol, have a low alkaline cation content so it tends to be acidic (yaalon, 1996). ultisols are indonesia’s second largest soil area after inceptisols, with a total area of 24.3% or equivalent to 45.8 million ha (nursyamsi, 2006). the fertility of ultisol is affected by erosion as the organic matter in the top layer is prone to erosion, thereby reducing its organic matter and nutrients (prasetyo and suriadikarta, 2014). the low fertility of ultisols is reflected in their low ph, low soil c-organic, cec and journal of tropical crop science vol. 10 no. 2, june 2023 www.j-tropical-crops.com 112 bayu suwitono, muhamad achmad chozin, dwi guntoro,suwarto base saturation (aswiguna et al., 2022). according to ma’ruf et al. (2017), one of the determinants of a successful land improvement is the selection of the suitable legume cover crops (lcc), especially those that have fast growth to suppress weeds and produce high decomposed biomass. ultisol physical and chemical properties can be improved by adding organic matter. the use of lcc is an alternative to providing organic matter in the soil (suwarto and asih, 2021), as well as increasing soil nitrogen supply (whittaker et al., 2022). lcc can improve soil physical properties including bulk density, soil porosity, soil moisture content (crotty and stoate, 2019). lcc increased the soil organic matter, n, p, and k, and improve the soil’s biological properties including enhancing earthworms and mesofauna growth (crotty and stoate, 2019). the ornamental bean arachis pintoi is a legume species that can add the soil nitrogen, reduce erosion, suppress weeds, and has ornamental values due to their attractive bright flowers. arachnis pintoi was reported to reduce rate of soil erosion by 80.45% (sarjono et al., 2019) and maintain soil water levels (yuniarti et al., 2018). according to muddarisna et al. (2009) growing a. pintoi combined with raising chicken, cows, and goats can increase soil organic c and improve soil porosity. a study by kartika et al. (2009) reported that arachnis pintoi as a ground cover can reduce water loss from evaporation and improve soil structure, soil porosity and density. our current study aimed to determine the optimal time to plant biomulch a. pintoi to suppress weed growth and to improve soil fertility in the cassava plantation. material and methods experimental site the experiment was conducted in the experimental field of the faculty of agriculture, ipb university, at singasari village, jonggol district, bogor regency, west java. the land in the study area is dry ultisol with a slope of 15o to ‎‎26.79%. the experiment was performed from november 2021 to september 2022. the study used stem cuttings of the ornamental bean a. pintoi and four cassava accessions, “ketan malang”, “genjah” “bayam”, “ir jonggol”, and “manggu”. the planting medium for a. pintoi cuttings consisted of soil and rice husks with a volume ratio of 2:1. other materials used were cow manure, urea, sp-36, and kcl fertilizers, herbicide (a.i. glyphosate), insecticide (a.i. carbofuran), 10x15 cm polybags, 0.5 m x 0.5 m2 square frames for weed sampling, and a digital scale. the herbicide was applied before land preparation, insecticides were applied only when necessary. experimental design the experimental design used was a factorial randomized block design. the first factor was the cassava varieties: “ketan malang”, “genjah” “bayam”, “ir jonggol”, and “mangu”. the second factor was the planting time of the arachis pintoi, i.e., b1 = planted at the time of cassava planting, b2 = four weeks, b3 = eight weeks, and b4 = 12 weeks before planting cassava, and without a. pintoi /b0, as a control. there were 20 treatments repeated four times to obtain 80 experimental units. each experimental unit consisted of 25 cassava plants and 80 of the 4-weekold a. pintoi cuttings. prior to planting in the field, a stem cutting of a. pintoi per polybag was prepared; the a. pintoi were planted in the field with a 20 cm distance between plants when they had 3-4 shoots. cassava cuttings 25 cm in length were planted with a spacing of 100 x 100 cm, one cutting per planting hole. c-organic, total n, available p, k2o, and cec, were determined prior to planting. vegetation analysis weed vegetation was analyzed before land preparation, and six months after planting cassava. weed vegetation was analyzed using the quadratic method measuring 0.5 m x 0.5 m with three random throws in the experimental plots. weed vegetation analysis data includes relative density (rds), relative frequency (rf), relative dominance (rd), and sum dominance ratio (sdr) ((marianan and warso, 2016). the weed dominance ratio is calculated using the formula described in listyowati et al. (2022): relative density (rds) = density of one species x 100% density of all species relative frequency (rf) = frequency of one species x 100% frequency of all species relative dominance (rd) = dominance of one species x 100% dominance of all species sum dominance ratio (sdr) = rd + rf + rd 3 soil analysis the soil sample for chemical and physical property analysis were collected twice, before the land preparation, and the after the cassava was harvested. the soil chemical properties tested include c-organic (walkey and black method), total n (kjedahl method), biomulch treatment effects on weed control and soil properties in cassava .......... journal of tropical crop science vol. 10 no. 2, june 2023 www.j-tropical-crops.com 113 available p (bray method), k2o (morgan method), and cec (basic percolation method) as described in eviati and sulaeman (2009). soil sampling (1000 g each) was collected diagonally at five sample points with a depth of 0-30 cm from the soil surface. the soil samples were homogenized and placed in containers for later analysis in the soil chemistry laboratory. soil samples for the soil physical property analysis were collected using the sample ring with a diameter of 7.35 cm and a height of 4 cm. the ring has a cylindrical shape and was inserted to a certain soil depth then dismantled carefully so the volume of the soil remains constant. the sample points were determined randomly according to the ground cover treatments. soil physical property analysis was conducted on soil bulk density, soil-specific gravity, and the total pore space at the soil physics laboratory at the soil research institute, bogor. ‎ result and discussion the growth of a. pintoi that were planted 12 weeks before cassava (b4) was even and covered almost the entire surface of the beds (85-90%). arachis pintoi planted at eight weeks before cassava (b3) had a coverage of 60-75%, whereas those planted four weeks before cassava (b2) had the coverage of 30-45%. arachis pintoi planted at the time of cassava planting (b1) grew very slowly and did not have much coverage (figure 1). arachis pintoi planted at 12 weeks (b4) had a greater fresh weight (232.6 g.m-2) and dry weight (65.1 g.m-2) compared to those planted at the other times (table 1). arachis pintoi planted at cassava planting (b1) had the lowest weight, likely because they grew slower due to competition with the weeds and the cassavas. arachnis pintoi planted at 8 or 12 weeks before planting cassava was able to suppress weed growth (table 2). at 6 map, the weed dry weight with a. pintoi planted at 8 weeks before planting cassava was 15.55 g per 0.25 m2, whereas with a. pintoi planted 12 weeks before cassava was 14.65 g per 0.25m2 (table 2). summed dominance ratio vegetation analysis before land preparation and after cassava harvest are described in table 3. before planting cassava, 11 weed species were identified, nine of which were broadleaf weeds, one species of grass, and one species of sedge. broadleaf weeds appeared to be more dominant at the beginning (before planting cassava) with an sdr value of 51.18% compared to the grass weeds (11.41%) and the sedge weeds (37.41%) (table 3). there was a shift in the weed dominance at six months after cassava planting, especially the broadleaf weeds, and this was influenced by biomulch treatments (figure 2). our results agreed with silmi and chozin (2014) who reported a shift in the weed figure 1. arachis pintoi biomulch cover in the cassava plantation: control (without a. pintoi, b0), a. pintoi planted at the time of cassava planting (b1), and planted at four weeks (b2), eight weeks (b3), and 12 weeks (b4) before planting cassava. journal of tropical crop science vol. 10 no. 2, june 2023 www.j-tropical-crops.com 114 bayu suwitono, muhamad achmad chozin, dwi guntoro,suwarto table 1. fresh weight and dry weight of a. pintoi biomulch at six months after planting treatment fresh weight (g.m-2) dry weight (g.m-2) b1 100.0 c 38.9 b2 161.2 b 55.3 b3 171.7 b 54.0 b4 232.6 a 65.1 note: values followed by different letters within the same column are significantly different according to the honestly different test at α=0.05. map: months after planting. b0: without biomulch application, b1: simultaneously with cassava planting, b2: four weeks before cassava planting, b3: eight weeks before cassava planting, b4: twelve weeks before cassava planting table 2. the average weed dry weight at two and six months after cassava planting. treatment weed dry weight (g per 0.25 m2) 2 map 6 map b0 11.07 19.27 b1 25.83 23.60 b2 19.23 22.90 b3 15.93 15.55 b4 17.27 14.65 note: values followed by different letters within the same column are significantly different according to the honestly different test at α=0.05. map: months after planting cassava. b0: without biomulch, b1: biomulch was planted at the time of cassava planting, b2: biomulch was planted four weeks before cassava planting, b3: biomass was planted eight weeks before cassava planting, b4: biomass was planted twelve weeks before cassava planting. honestly different test at α=0.05. map: months after planting cassava. b0: without biomulch, b1: biomulch was planted at the time of cassava planting, b2: biomulch was planted four weeks before cassava planting, b3: biomass was planted eight weeks before cassava planting, b4: biomass was planted twelve weeks before cassava planting. figure 2. rainfall at the research location from november 2021 to september 2022 summed dominance ratio vegetation analysis before land preparation and after cassava harvest are described in table 3. before planting cassava, 11 weed species were identified, nine of which were broadleaf weeds, one species of grass, and one species of sedge. broadleaf weeds appeared to be more dominant at the beginning (before planting cassava) with an sdr value of 51.18% compared to the grass weeds (11.41%) and the sedge weeds (37.41%) (table 3). table 3. summed dominance ratio (%) of weeds at different biomulch treatments at six months after cassava planting. no weeds species morphology summed dominance ratio (%) preplanting b0 b1 b2 b3 b4 1. melastoma malabatrichum l. bl 0 10.16 0 11.28 21.54 5.66 2. mimosa invisa l. bl 5.20 8.07 11.81 14.75 0 8.29 3. oldenladia corymbosa l. bl 0 11.20 0 0 0 0 4. oxalis barrelieri l. bl 2.35 0 0 5.64 16.30 4.15 5. ottochloa nodosa (kunth) dandy g 11.41 21.61 18.77 13.66 21.03 26.08 6. tetracera indica (l). merr bl 2.10 0 7.01 4.55 0 4.15 7. solanum sp. bl 0 10.94 10.34 9.11 0 4.15 8. ipomoea sp bl 10.66 0 4.07 8.02 0 8.29 9. cyperus sp s 37.41 18.49 14.36 12.37 16.03 11.32 10. ageratum conyzoides l. bl 0 0 5.54 0 0 0 11. panicum repens l. g 0 0 0 6.73 0 0 12. desmodium turtuosum (sw). dc bl 11.56 6.51 4.80 0 0 0 13. cleome rutidosperma d.c bl 0 0 0 6.94 0 0 14. melochia corchorifolia l. bl 0 0 13.68 0 0 0 15. mimosa pudica l. bl 10.41 0 0 0 12.69 8.29 219 413 227 109 158 305,5 203,5 191 125 73 181 0 50 100 150 200 250 300 350 400 450 r ai nf al l ( m m p er m on th ) rainfall 2021-2022 (mm) figure 2. rainfall at the research location from november 2021 to september 2022 types on sweet corn plantations due to biomulch treatment. based on the summed dominance ratio (sdr), broadleaf weeds were more dominant than weeds and grasses in each treatment. the sdr values of each weed groups can be seen in table 2, which clearly demonstrated that planting biomulch a. pintoi is effective to suppress sedge weeds but is less effective to suppress grass and broadleaf weeds. broadleaf weeds grow fast and tall, so they become cassava’s competitor for sunlight. weed competitiveness is also affected by the leaf morphology; the wider the leaf, the more it can capture light for photosynthesis. the slow growing nature of a. pintoi allows broadleaf weeds to grow faster. according to ciaccia et al. (2014) it is crucial to plant the ground cover at the correct time, and the selection of living mulch is important for suppressing the weeds. combination of ground cover crops and herbicides can effectively control broadleaf weeds biomulch treatment effects on weed control and soil properties in cassava .......... journal of tropical crop science vol. 10 no. 2, june 2023 www.j-tropical-crops.com 115 (majidi et. al., 2020), and planting season, planting time, and tillage can affect the levels of weed control by the ground cover (osipitan et al., 2019). planting a. pintoi biomulch 8 and 12 weeks before planting cassava was able to suppress weed growth, demonstrated by a decrease in weed dry weight compared to without biomulch treatment (table 2). this shows that biomulch a. pintoi can compete with weeds. living mulch suppress weed growth by competing for water, light, and nutrients in the soil (mohammadi, 2012). according to mckenzie-gopsill et al. (2022), cover crops can produce large amounts of biomass so they can effectively suppress weeds. biomulch planted 12 weeks before planting cassava had the highest biomass weight (table 1), showing that a longer time is required for the biomass to cover the soil surface. in addition, the cassava canopy also reduces weed growth under the cassava plants. according to mzabri et al., (2022) 70% of shade can inhibit weed growth, although this depends on the weed species and their tolerance to environmental stress condition. the dominant weed species from the broadleaf group include melastoma malabathricum, mimosa invisa, oxalis barrelieri, dioscorea hispida, tertacera indica, ipomoea obscura, and from the grass group ottochloa nodosa (figure 3). it is suspected that these weed species are adaptable and tolerant to the environmental stress, making it difficult to be suppressed by ground cover plants. according to a table 3. summed dominance ratio (%) of weeds at different biomulch treatments at six months after cassava planting. no weeds species morphology summed dominance ratio (%) preplanting b0 b1 b2 b3 b4 1. melastoma malabatrichum l. bl 0 10.16 0 11.28 21.54 5.66 2. mimosa invisa l. bl 5.20 8.07 11.81 14.75 0 8.29 3. oldenladia corymbosa l. bl 0 11.20 0 0 0 0 4. oxalis barrelieri l. bl 2.35 0 0 5.64 16.30 4.15 5. ottochloa nodosa (kunth) dandy g 11.41 21.61 18.77 13.66 21.03 26.08 6. tetracera indica (l). merr bl 2.10 0 7.01 4.55 0 4.15 7. solanum sp. bl 0 10.94 10.34 9.11 0 4.15 8. ipomoea sp bl 10.66 0 4.07 8.02 0 8.29 9. cyperus sp s 37.41 18.49 14.36 12.37 16.03 11.32 10. ageratum conyzoides l. bl 0 0 5.54 0 0 0 11. panicum repens l. g 0 0 0 6.73 0 0 12. desmodium turtuosum (sw). dc bl 11.56 6.51 4.80 0 0 0 13. cleome rutidosperma d.c bl 0 0 0 6.94 0 0 14. melochia corchorifolia l. bl 0 0 13.68 0 0 0 15. mimosa pudica l. bl 10.41 0 0 0 12.69 8.29 16. stachytapherta indica bl 0 0 0 0 0 4.15 17. hyptis brevipes poit. bl 0 0 0 0 5.51 5.66 18. portulaca oleracea l. bl 4.45 3.65 0 0 0 0 19. emilia sonchifolia (l).dc bl 0 2.86 4.80 3.47 0 0 20. dioschorea hispida dennst bl 4.70 2.86 0 0 7.18 5.66 21. phyllantus niruri l. bl 4.95 3.65 4.80 3.47 0 4.15 sdr (%) bl 51.18 59.90 66.86 67.24 62.95 62.60 g 11.41 21.61 18.77 20.39 21.03 26.08 s 37.41 18.49 14.36 12.37 16.03 11.32 note: bl= broad leaf weeds; g = grasses; s = sedges. journal of tropical crop science vol. 10 no. 2, june 2023 www.j-tropical-crops.com 116 bayu suwitono, muhamad achmad chozin, dwi guntoro,suwarto study by listyowati et al. (2022), the dominance of broadleaf weeds is due to by the growth of weed seeds after tillage; soil tillage brings the weed seeds to the soil surface and exposed them to light required for germination. the dominant weed species has vine and creeping growth habits, such as tetracera indica, ottochloa nodosa, dioscorea hispida, and ipomoea obscura. according to sudira et al. (2017) root system greatly affects plant growth, those with shallow and creeping roots get more nutrients and water, whereas those with taproot can grow deep into the soil to access water. the important characters for selecting cover crop are fast growth and tolerance to shades and drought, and these characters are very important for a long-term weed control (schappert et al., 2019). using a. pintoi as biomulch can be an option in environmentally friendly weed management because it can reduce the use of chemicals from herbicides. according to monteiro and santos (2022), using herbicides in weed control often causes weed resistance and ecosystem imbalance. environmental pollution, weed resistance, and increased production costs are the effects of an excessive herbicide use (pradhan et al., 2022). long-term weed control for sustainable agriculture must be safe and efficient; it is important to select ground cover that can increase soil fertility, pore space (pavlovic et al., 2022), and soil moisture (yuniarti et al., 2018). according to kaluba et al., (2021), intercropping of cassava and legumes can help suppress weed growth, use less fertilizer, and reduce labor input. cassava productivity can increase if weeded at least twice during cassava growing season (kintché et al., 2017). weed control by application of glyphosate herbicide is equivalent to five times manual weeding but is more expensive than the manual weeding conducted twice (chikoye et al., 2002). therefore, the use of biomulch can potentially reduce the use of herbicides and labor. soil chemical quality the soil analysis before tillage showed that it had an acidic ph (4.12), low total nitrogen (0.14%), and low cation exchange capacity (28.53 cmol.kg-1), and the soil has a dominant clay texture (51.79%), 25.34% sand, and 22.87% silt. the results of the soil chemical analysis are presented in table 2. the soil chemical analysis six months after planting cassava showed an increase in c-organic, n-total, available p, and cec (table 3). figure 3. dominant weed species in cassava plantation at jonggol, west java, indonesia. melastoma malabatrichum tetracera indica ipomoea sp mimosa invisa l. oxalis barrelieri l. cyperus sp figure 3. dominant weed species in cassava plantation at jonggol, west java, indonesia. according to mckenzie-gopsill et al. (2022), cover crops can produce large amounts of biomass so they can effectively suppress weeds. biomulch planted 12 weeks before planting cassava had the highest biomass weight (table 1), showing that a longer time is required for the biomass to cover the soil surface. in addition, the cassava canopy also reduces weed growth under the cassava plants. according to mzabri et al., (2022) 70% of shade can inhibit weed growth, although this depends on the weed species and their tolerance to environmental stress condition. the dominant weed species from the broadleaf group include melastoma malabathricum, mimosa invisa, oxalis barrelieri, dioscorea hispida, tertacera indica, ipomoea obscura, and from the grass group ottochloa nodosa (figure 3). it is suspected that these weed species are adaptable and tolerant to the environmental stress, making it difficult to be suppressed by ground cover plants. according to a study by listyowati et al. (2022), the dominance of broadleaf weeds is due to by the growth of weed seeds after tillage; soil tillage brings the weed seeds to the soil surface and exposed them to light required for germination. the dominant weed species has vine and creeping growth habits, such as tetracera indica, ottochloa nodosa, dioscorea hispida, and ipomoea obscura. according to sudira et al. (2017) root system greatly affects plant growth, those with shallow and creeping roots get more nutrients and water, whereas those with taproot can grow deep into the soil to access water. the important characters for selecting cover crop are fast growth and tolerance to shades and drought, and these characters are very important for a long-term weed control (schappert et al., 2019). using a. pintoi as biomulch can be an option in environmentally friendly weed management because it can reduce the use of chemicals from herbicides. according to monteiro and santos (2022), using herbicides in weed control often causes weed resistance and ecosystem imbalance. environmental pollution, weed resistance, and increased production costs are the effects of an excessive herbicide use (pradhan et al., 2022). long-term weed control for sustainable agriculture biomulch treatment effects on weed control and soil properties in cassava .......... journal of tropical crop science vol. 10 no. 2, june 2023 www.j-tropical-crops.com 117 there was a decrease of c-organic content in the control and in the biomulch b1 treatment (planted at the same time as cassava planting). the initial c-organic value was 3.69% (moderate), in the treatment without biomulch it was 1.97% (low), and in the b1 treatment it was 1.79% (low). with treatment b2 the c-organic value was 5.12% (very high), b3 was 7.54% (very high), and treatment b4 was 2.60% (moderate). these results show that the c-organic content, which is abundant in the topsoil, can be easily leached by soil erosion. on the other hand, with the biomulch treatment b2 and b3 the amount of specific c-organic increased (table 5). according to nariratih et al. (2013), the amount of carbon in the soil is influenced by several factors including evapotranspiration, activity of soil microorganisms in breaking down organic matter, soil erosion, and carbon removal at harvest. high residual biomass from the ground cover increases soil organic matter, and the biomass production is affected by rainfall. lower rainfall reduces growth, hence lower the biomass production (blanco-canqui et al., 2015). ‎ the soil total-n value before tillage is 0.14% which is in the low n category (table 4), and this value is similar to the value before the cassava harvest. the total-n in the control (without biomulch) was 0.18%, and with biomulch b1 was 0.16% (also in the low category, table 5). with the biomulch treatments b2, b3, and b4, there was an increase of 0.22-0.41% in the total n, which is in the moderate category. this shows that the biomulch treatment four, eight, and twelve weeks before planting cassava can increase nitrogen in the soil. increased nitrogen levels in the table 4. physicochemical properties of the topsoil (0–30 cm depth) of the experimental sites before tillage. soil properties values cassava suitability levels ph (h2o) 4.12 4.5-7.0 total nitrogen 0.14 0.20-0.50 organic-c (%) 3.69 2.0-4.0 c/n 12 cec (cmol.kg-1) 28.53 bs (%) 47.65 p2o5 -bray (ppm) 8.6 k2o (ppm) 170 exchangeable bases (cmol.kg-1) k 0.37 0.15-2.5 ca 4.92 1.0-5.0 mg 8.14 0.4-1.0 na 0.15 sand (%) 25.34 clay (%) 51.79 silt (%) 22.87 textural class clay note: cassava suitability level according to howeler (2012) and kaluba et al. (2022). table 5. soil chemical properties at six month after planting soil properties b0 b1 b2 b3 b4 c-organic (%) 1.97(l) 1.79(l) 5.12(vh) 7.54(vh) 2.60(m) total n (%) 0.18(l) 0.16(l) 0.36(m) 0.41(m) 0.22(m) p2o5 (ppm) 16.6(m) 9.8(vl) 17.9(m) 12.9(l) 4.0(vl) k2o (ppm) 119.7(vh) 194.5(vh) 271.8(vh) 374.1(vh) 173.8(vh) cec (cmol.kg-1) 18.88(l) 20.47(l) 48.70(vh) 68.81(vh) 26.47(m) notes: b0: without biomulch application, b1: simultaneously with cassava planting, b2: four weeks before cassava planting, b3: eight weeks before cassava planting, b4: twelve weeks before cassava planting, cec: capacity cation exchange, vl: very low, l: low, m: moderate, h: high, vh: very high journal of tropical crop science vol. 10 no. 2, june 2023 www.j-tropical-crops.com 118 bayu suwitono, muhamad achmad chozin, dwi guntoro,suwarto soil with a.pintoi biomulch treatment might be due to nitrogen fixation by rhizobium bacteria. according to a study by parvin et al., (2022) the increase in total n-was influenced by the activity of ‎rhizobium, and the use of legume cover crops under optimal growth conditions was able to fix up to 50 kg n/ha-1. according to de notaris et al., (2020), using legume plants as ground cover increases soil carbon and nitrogen. increasing the organic matter content in the soil may also increase the n content in the soil (asbur et al., 2018; plaza-bonilla et al., 2015). using a. pintoi cover crops can increase the availability of soil nitrogen and maintain soil moisture (chozin et al., 2015). the use of cover crops can minimize n loss due to climate change ‎‎(kaye and quemada, 2017), and reduce no3 leaching regardless of the type of soils ‎‎(lapierre et al., 2022)‎. the available soil p before the study was classified as very low (8.6 ppm, table 4) but it increased after the biomulch treatment, except for the b4 treatment. this shows that a. pintoi biomulch can increase the available phosphorus in the soil. according to lizcano toledo et al. (2022), the use of cover crops for years can increase the soil available p. however, the increase in phosphorus does not guarantee that the plants can use the phosphorus because of the high levels of fe, which may bind p and make p unavailable to the plants. the solubility of fe and al in acidic soils is relatively high and form strong bonds of al-p and fe-p, making them unavailable to the plants (daras et al., 2012). according to syahputra et al., (2015), ultisols’ high and low p content depends on the p content in the parental materials. the unavailability of p for plants is also affected by the presence of p bonds in al and fe minerals. this is in line with a study by mardamootoo et al. (2021), which stated that phosphorus is present in most soils, but it is not necessarily available for plants. the cec value before tillage was 28.53 cmol. kg-1, which is classified as high. the high cec values may be due to the soil texture at the study site which is dominated by clay fraction (table 4). at the time of harvest, the results of soil analysis showed an increase in cec compared to without cover crop treatment, whereas soil cec values increased in all biomulch treatments (table 4). the biomulch b2 and b3 treatments showed the highest cec values, i.e., 48.7 cmol.kg-1 and 28.8 cmol.kg-1 (table 5). ‎ this shows that cover crops can increase the soil cec value. according to husni et al. (2016) and sari et al. (2022), soils that predominantly has the clay fraction tend to have a high capacity for ion exchange and bind water, so they have highly stable soil aggregates. in addition, these soils contain ca, mg, k, and na, which also affects the cec values (husni et al. 2016, sari et al., 2022). soils with high cec values can hold more nutrients in the form of cations (taisa et al., 2019). soil physical quality ‎ our study showed that the biomulch treatment reduced soil bulk density (table 6). at the beginning of planting, treatment b1 to b4 had a bulk density value of 0.77 g.cm-3 to 0.81 g.cm-3, lower than without biomulch treatment (table 6). at the time of harvest, the soil with biomulch treatment had a lower soil bulk density than those without the biomulch treatment (table 6). the lowest bulk density was in the b2 treatment of 0.65 g.cm-3 or decreased by about 35% by weight. this shows that a. pintoi biomulch increased the soil organic matter and reduced soil bulk density. study by muddarisna et al. (2009) reported that the use of manures and green manure a. pintoi reduced the soil weight, increased soil porosity, and increased soil permeability and soil aggregates. soil with low bulk density makes it easier for plant roots to penetrate the deeper soil layers (maysarah et al., 2021), whereas high bulk density reduces soil pores hence reduces aeration (meli et al., 2018). biomulch treatment b1, b2, and b3 reduced the specific gravity of the soil by 2.12 g.cm-3, 2.14 g.cmtable 6. effect of biomulch treatments on the soil bulk density, specific gravity, and total pore space at the beginning of planting (0 map) and at cassava harvest (6 months after planting). biomulch treatment bulk density (g.cm-3) specific gravity (g.cm-3) total pore space (%) 0 map 6 map 0 map 6 map 0 map 6 map b0 0.85 1.00 2.03 2.23 58.2 55.3 b1 0.81 0.87 1.87 2.12 56.7 59.2 b2 0.79 0.65 2.20 2.14 63.8 69.8 b3 0.80 0.80 2.04 2.15 60.8 62.7 b4 0.77 0.95 1.97 2.23 60.9 57.5 notes: results of soil testing in the soil research center laboratory. b0: without biomulch application, b1: simultaneously with cassava planting, b2: four weeks before cassava planting, b3: eight weeks before cassava planting, b4: twelve weeks before cassava planting. map: months after planting cassava. biomulch treatment effects on weed control and soil properties in cassava .......... journal of tropical crop science vol. 10 no. 2, june 2023 www.j-tropical-crops.com 119 3, and 2.15 g.cm-3, respectively, whereas the control had 2.23 g.cm-3 (table 6). these values show that the use of biomulch increases the soil organic matter, which presumably reduce the soil-specific gravity and bulk density as according to safitri et al. (2018), the higher the organic matter applied to the soil, the lower the soil specific gravity. increasing organic matter content improves soil physical properties, resulting in a more stable soil structure, and increased soil n and k (yuliani ‎ and rahayu, ‎‎2016). with the optimal crop and soil management, ultisols can be utilized as agricultural land for food crops (syahputra et al., 2015). the total pore space at the study site was significantly increased by the a. pintoi biomulch treatment (table 6), and treatment b2 had a total pore space increase of 20.7% ‎(table 6). this indicates that the decrease in bulk density increases the total pore space. our results agree with khair et al. (2017) in that soil bulk density is inversely proportional to the total pore space and is affected by the soil organic matter. the increase in the soil total pore space is related to the formation of soil aggregates by soil organic matter, hence increasing the soil volume and pores (ardiansyah et al., 2015). the soil pores formed between soil particles affects the growth of plant roots; the high soil porosity allows the plant roots to penetrate deeper soil and access more water and nutrients (taisa et al., 2019). conclusion arachnis pintoi planted four and eight weeks before planting cassava controlled the weed growth, demonstrated by the lowest weed dry weight of 15.55 g per 0.25 m-2 and 14.65 g per 0.25 m-2, respectively at six months after cassava planting. all biomulch treatments increased the soil density and pore spaces. the soil with biomulch planted eight weeks before cassava had the highest c-organic (7.59%) and total n (0.41%) compared to the control/without a. pintoi biomulch. the dominant weeds in the cassava study site are melastoma malabathricum, tetracera indica, oxalis barrelieri, mimosa invisa, ipomoea sp, ottochloa nodosa and cyperus sp. acknowledgment the author would like to thank the agricultural research and development agency and the national research and innovation agency for funding the authors through the agricultural research and development agency scholarship and the national innovation research agency scholarship. the author greatly appreciates mr. anton nialek, mr. ardi, mr. rafi, and ms. dian for their generous helps during the study. references adiele, j. g., schut, a. g. t., ezui, k. s., pypers, p., and giller, k. e. 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(2015). composition, structure, physicochemical properties, and modifications of cassava starch. carbohydrate polymers 122, 456–480. https://doi.org/10.1016/j. carbpol.2014.10.063 citrus is a multivitamin treasure trove: a review .......... journal of tropical crop science vol. 10 no. 1, february 2023 www.j-tropical-crops.com 57 citrus as a multivitamin treasure trove: a review rahmat budiartoa, syariful mubaroka, nursuhuda, and bayu pradana nur rahmatb a department of agronomy, faculty of agriculture, universitas padjadjaran, sumedang, 45363, indonesia b master of agronomy study program, faculty of agriculture, universitas padjadjaran, sumedang, 45363, indonesia *corresponding author email: rahmat.budiarto@unpad.ac.id abstract citrus is popularly known as the source of beneficial and essential nutrients for human health, including vitamins. the current review revealed the content of multivitamins, not only vitamin c but also vitamins a, b, and e that are not widely acknowledged within citrus. numerous citrus genotypes contain vitamin c, with the grapefruit (citrus paradisi) being the richest, and citron (c. medica) the poorest. vitamin a in the form of β-carotene, α-carotene, and β-cryptoxanthin is commonly found within citrus, especially in several coloured flesh species such as grapefruit, mandarin (c. reticulate), and orange (c. sinensis). in terms of vitamin b, orange and grapefruit are proven to contain b-complex, including thiamine (b1), riboflavin (b2), niacin (b3), pantothenic acid (b5), pyridoxine (b6), biotin (b7), inositol (b8) and folate (b9). vitamin e in the form of α-tocopherol was detected in leaf kaffir lime (c. hystrix) and orange (c. sinensis), lemon (c. limon), mandarin (c. reticulate), and tangerine (c. nobilis) fruit. this review summarizes the nutritional content of citrus: citrus contains not only vitamin c but also other vitamins beneficial to human health, therefore citrus consumption is highly recommended. keywords: antioxidant, grapefruit, mandarin, orange, tangerine. introduction fruit consumption is reported to reduce the risk of cardiovascular disease (lapuente et al., 2019), in contrast with fast-food consumption (auestad et al., 2015). among numerous fruits, citrus has become one of the most economically important and popular horticulture produce worldwide (fao, 2016). total citrus (tangerines/mandarin) production worldwide increased yearly, from 30,346,000 tons in 2016/2017 to 37,933,000 tons in 2021/2022 (usda, 2022). increased citrus production is due to increasing demand. similarly, in indonesia there is a significant increase in citrus consumption from 1995 to 2020 due to population growth and better health awareness (hanif et al., 2021). although citrus fruits are highly demanded worldwide, the production area is centered in only a few countries. citrus was reported to spread worldwide, with a route from southeast asia to the mediterranean (langgut, 2017). similarly, the latest genomic study revealed that citrus originated from the southeastern himalayan valley, including western yunnan, northern myanmar, and eastern assam, before it eventually spread worldwide (wu et al., 2018). an earlier study by (liu et al., 2012) also confirmed that citrus is native to tropical and subtropical asian regions, i.e., china, the malay archipelago, and south asia. citrus is split into two groups based on their market popularity, i.e., major and minor citrus. kaffir lime is an example of minor citrus from southeast asian countries (araujo et al., 2003; mabberley, 2004), with the leaf used as a cooking spice (budiarto et al., 2022a, 2022b, 2022c, 2021a, 2021b, 2019a, 2019b, efendi et al., 2021). rough lemon (c. jambhiri), citron, flying dragon (c. trifoliata), japansche citroen (c. limonia), kasturi (c. madurensis), khasi papeda (c. latipes) and limau (c. amblycarpa) are other examples of minor citrus (budiarto et al., 2017; efendi and budiarto, 2022). orange (c. sinensis), however, one of the most popular citrus species cultivated (by about 80% worldwide), is categorized as major citrus (turner and burri, 2013). mandarin, tangerine, and pummelo are three major citrus popularly consumed as table fruit (budiarto et al., 2018; efendi and budiarto, 2022; hanif et al., 2021). lime (c. aurantifolia) and lemon are also members of the major citrus group, and both are actively traded on the world market (budiarto and pratita, 2022). citrus trading activity is highly affected by the balance of supply and demand. citrus demand is associated with the role of this fruit as a functional food due to its rich nutritional content (lu et al., 2021; saini et al., 2022; zhang journal of tropical crop science vol. 10 no. 1, february 2023 www.j-tropical-crops.com 58 rahmat budiarto, syariful mubarok, nursuhud, and bayu pradana nur rahmat et al., 2021). various beneficial phytochemicals found in citrus have beneficial bioactivities such as cancer prevention (kaur and kaur, 2015), antioxidant (adenaike and abakpa, 2021), antibacterial, antifungal, and antiviral activities (abobatta, 2019). previous review studies have concluded that citrus consumption could increase the body’s antioxidant status, immune function, and cardiovascular health (saeid and ahmed, 2021; turner and burri, 2013). healthier lifestyles are currently on the increase due to the covid-19 outbreak (rothan and byrareddy, 2020). uncertain living conditions amidst the covid-19 lockdown prompted society to adapt, with increased fruit consumption as one such adaptation, especially vitamin-rich fruit. numerous reports have highlighted the importance of a vitamin-rich diet to improve the immune system during the covid-19 pandemic era and to boost the post-infection period’s recovery process (galanakis, 2020). therefore, a healthy habit of fresh fruit, such as citrus, is important. as a functional fruit, citrus is often seen only as a source of vitamin c. it is not widely acknowledged that citrus is multivitamin fruit, containing not only vitamin c but also vitamin a, b, and e (zhou, 2012). multivitamins are required to boost the immune system (pantelidis et al., 2007), especially during the pandemic of covid-19 (bae and kim, 2020; galanakis, 2020). thus, vitamins are one of the important aspects of nutritional quality in fruit (vittori et al., 2018). however, there is still limited information regarding the multivitamin content of citrus. therefore, this paper is aimed at reviewing the multivitamin content found in numerous citrus species. vitamin c it is important to increase fruit and vegetable consumption by more than 90% to fulfill the daily requirement of vitamin c (citak and sonmez, 2010). vitamin c, also known as ascorbic acid in the human diet is mainly derived from fruit commodities (fenech et al., 2019). the content of vitamin c in citrus is lower than in guava, i.e., 140 to 146 mg per 100 g (widyastuti, et al., 2022; widyastuti, et al., 2022); however, it is higher than other fruit commodities, namely soursop, banana, grape, and rose apple (silva and sirasa, 2018). therefore, citrus and its derivative products are still well known as the source of vitamin c in the human diet (ting, 1980). vitamin c is often an important indicator of citrus nutritional quality (chen et al., 2016, 2019; gambetta et al., 2014; ghorbani et al., 2018; lado et al., 2018; magwaza et al., 2017). vitamin c is commonly found in the peel and flesh of citrus fruit, which is biosynthesized through both l-galactose and d-galacturonic acid pathways (zhang et al., 2015). vitamin c is also the most popular water-soluble antioxidant (maggini et al., 2010; martí et al., 2009) for reducing oxidative stress (zou et al., 2016). the lack of vitamin c supplementation is associated with weakened immune systems and a slower body recovery during the post-infection period (maggini et al., 2010). additional vitamin c intake can significantly shorten the duration of the common cold (ran et al., 2018). multiple studies have concluded that a vitamin c-rich diet could enhance the body’s immune systems during malaria (qin et al., 2019), covid-19 (bae and kim, 2020; hiedra et al., 2020), influenza (kim et al., 2016) and any other virusinduced respiratory infections (gorton and jarvis, 1999). due to its importance, the recommended dietary allowance (rda) for vitamin c is 60 mg per day (carr and frei, 1999). more specific to adult women and men, the rda is determined as much as 75 mg/day and 90 mg/day respectively (institute of medicine panel on dietary antioxidants and related compounds, 2000). the latest study on the effects of high vitamin c dosage (24 g day-1) medication on covid-19 patients for seven days revealed that a high dosage of vitamin c accelerated the covid-19 patient’s recovery process (carr, 2020, j. zhang et al., 2021, zhao et al., 2021). numerous studies have also previously reported the success of vitamin c therapy for covid-19 patients (carr and rowe, 2020; farjana et al., 2020; holford et al., 2020; kumari et al., 2020). moreover, less adequate vitamin c intake is associated with severe pneumonia (patterson et al., 2021). vitamin c content within citrus fruit is strongly influenced by genetic factors (escobedo-avellaneda et al., 2014; magwaza et al., 2017; sdiri et al., 2012) with multigenic inheritance (fanciullino et al., 2006) leading to multiple genes being involved in vitamin c biosynthesis (alós et al., 2014). numerous studies reported the vitamin c content variation found within different citrus genera species (krehl and cowgill, 1950; kumar et al., 2019; sharma et al., 2006). the vitamin c content of seven citrus species ranked from lowest to highest per 100 ml of juice is as follows; citron (20.65 mg), sweet orange (25.13 mg), mandarin (30.42 mg), lemon (34.67 mg), lime (38.70 mg), pomelo (40.50 mg) and grapefruit (53.64 mg) (kumar et al., 2019). moreover, the variation in vitamin c content is found not only at the inter-species level but also within the intra-species level. different varieties of the same orange species (c. sinensis) could have different vitamin c content (proteggente et al., 2003). aside from the genetic factors, vitamin c is also known to be affected by climate, growing location and the culture practices. an earlier review highlighted the effects of temperature on the fruit vitamin c; fruits in citrus is a multivitamin treasure trove: a review .......... journal of tropical crop science vol. 10 no. 1, february 2023 www.j-tropical-crops.com 59 the cooler temperatures had higher vitamin c than those in the warmer regions (nagy, 1980). mudambi and rajagopal (1977) also reported that the nigerian sweet oranges grown in high temperatures had lower vitamin c. growing and cultural practices in the field, such as growing cycle and crop nutrition (caruso et al., 2011), application of plant growth regulator (pgr), and harvesting season, fruit maturity, and fruit positions on the tree, affect the fruit vitamin c content. exogenous application of pgr significantly decreased fruit drop and increased vitamin c (nawaz et al., 2008). previous research has shown that gibberellic acid (ga3) application correlates with kumquat (c. japonica) fruit vitamin c (cai et al., 2021). a similar finding was reported by rokaya et al. (2016) : ga3treated mandarin citrus has higher vitamin c content than control fruit. these findings followed previous studies on ‘baramasi’ lemon (sindhu and singhrot, 1993) and ‘balady’ mandarin (el-shereif et al., 2017). in addition, the application of 1-methylcyclopropene (1-mcp) solely or combined with ga3 successfully prevented vitamin c breakdown metabolism (taş et al., 2021). the fruit should be cultured under organic farming systems to produce higher vitamin c content (conti et al., 2014; oliveira et al., 2013) or treated with plant growth-promoting rhizobacteria (pgpr) (erturk et al., 2012), and excessive nitrogen fertilization should be avoided since it could reduce fruit vitamin c content (rupp and tränkle, 2000). continuous decline of vitamin c content, specifically ascorbic acid, as the fruit ripens (alvarez-suarez et al., 2014; nagy, 1980) is caused by an enzymatic process that converts l-ascorbic acid to 2-3-deoxy–l-gluconic acid (mapson, 1970). the decline of vitamin c was also observed in the late harvest season (rokaya et al., 2016). late-season oranges have lower vitamin c than early and mid-season oranges (ting, 1980). these findings confirmed the opinion (caruso et al., 2011) that the growing season influences vitamin c content within fruits. lastly, the fruit position in the tree canopy can influence vitamin c content. sunexposed fruits have higher vitamin c than those in the shade (lee and kader, 2000). vitamin a in addition to vitamin c, citrus is also a remarkable source of vitamin a. citrus can be an alternative solution for vitamin a deficiency, which is still the most prevalent disorder worldwide (priyadarshani, 2017). an earlier report by the institute of medicine panel on micronutrients (2001) showed the rda for women and men to be about 700 and 900 μg retinol activity equivalents (rae) per day respectively. additionally, the consumption of 100 g of tangerine helps to meet 72% of the vitamin a rda of children (turner, 2012). vitamin a is a fat-soluble organic phytochemical, as are several carotenoids with provitamin a activity, retinal retinol, and retinoic acid (amitava, 2014). vitamin a is highly beneficial for maintaining healthy vision, the body’s immune system, and bone formation (turner and burri, 2013). vitamin a or its precursor (provitamin a) is known to be varied within a fruit depending on the plant’s genetic material. citrus is reported to be the richest carotenoid fruit species (kato, 2012). numerous studies have reported the pathway of carotenoid biosynthesis and its related gene in several citrus genotypes (lu et al., 2016, 2018; ma et al., 2018; quian-ulloa and stange, 2021; rodrigo et al., 2019; wei et al., 2014; zeng et al., 2013; zhu et al., 2017). those produced carotenoids are then transferred and accumulated in certain sites, especially in the fruit’s juice sac and flavedo (hermanns et al., 2020; li and yuan, 2013; yuan et al., 2015). one of the important carotenoids for the human diet is provitamin a carotenoid (ikoma et al., 2016). in more detail, a previous study has revealed 16 carotenoids with provitamin a activity in citrus, with β-carotene, α-carotene, and β-cryptoxanthin as the most numerous (silalahi, 2002). β-carotene can be converted to zeaxanthin via β-cryptoxanthin, while α-carotene can be converted into lutein (ikoma et al., 2016). an earlier study reported that β-carotene content varied in different citrus genotypes. orange, pomelo, grapefruit, and lemon have 345, 120, 98, and 50 μg.g-1 of β-carotene, respectively (paul and shaha, 2004). in addition to genotypes, fruit maturity stage also plays an important role in determining carotenoids containing provitamin-a, like β-carotene. numerous studies on tomatoes revealed that as the fruit matured, there was an increase in β-carotene content within the fruit (mubarok et al., 2015, 2019, 2021). aside β -carotene, citrus fruit is believed to contain more zeaxanthin than other food products, such as green leafy vegetables (garcía-closas et al., 2004). genotype also influences the content of zeaxanthin within the fruit, i.e., mandarin, pomelo, calamansi, orange, and lemon have 6.46 μg.g-1, 0.51 μg.g-1, 36.4 μg.g-1, 27.7 μg.g-1, 0.81 μg.g-1 of zeaxanthin, respectively (wang et al., 2008). in addition, citrus fruit is also believed to be a source of β-cryptoxanthin, as well as numerous other foods (liu et al., 2012). β-cryptoxanthin is the major carotenoid found in mandarin (kato, 2012; zhu et al., 2017). moreover, matsumoto et al., (2007) classified not only mandarin but also oranges to the group of β-cryptoxanthin rich citrus. the richness level of carotenoids in citrus can be preliminary monitored by the fruit colour. the red and pink flesh of grapefruit contained more provitamin a carotenoids than the white (ting, 1980). journal of tropical crop science vol. 10 no. 1, february 2023 www.j-tropical-crops.com 60 rahmat budiarto, syariful mubarok, nursuhud, and bayu pradana nur rahmat additionally, the white flesh of lime, lemon, pomelo, and citron varieties are also reported to have low provitamin a carotenoids (kato, 2012; matsumoto et al., 2007). future study to breed β-cryptoxanthinrich varieties should be conducted to solve the aforementioned problem (burri et al., 2011). vitamin b like vitamin a, vitamin b is an essential micronutrient that only our diet can provide. this water-soluble vitamin is associated with energy production and the physiological biosynthesis of important cell molecules (schellack et al., 2019). institute of medicine (us) standing committee on the scientific evaluation of dietary reference intakes and its panel on folate, other b vitamins, and choline (1998) revealed the daily rda of numerous vitamin b as follows: (i) 1.2 mg/day and 1.1 mg/day of vitamin b1 for men and women, respectively; (ii) 1.1 mg/day and 1.3 mg/day of vitamin b2 for adult women and men, respectively; (iii) 16 mg/day and 14 mg/day of vitamin b3 equivalents for men and women, respectively; (iv) 1.3 mg/day of vitamin b6 for young adults; (v) 2.4 μg/day of vitamin b12 for adults; (vi) 400 μg/day of vitamin b9 equivalents for both men and women; and (vii) adequate intake (ai) of vitamin b5 and b7 for an adult was 5 mg/day and 30 μg/day, respectively. daily fruit consumption may help provide the daily vitamin b that our body needs. various fruits are reported to be a good source of several types of vitamin b, namely mango (maldonado-celis et al., 2019), hass avocado (dreher and davenport, 2013), papaya (vij and prashar, 2015), kiwi berry (latocha, 2017), mulberry (yuan and zhao, 2017), date (khalid et al., 2017), tomato (raiola et al., 2014), and jujube (fard et al., 2015; pareek, 2013; rashwan et al., 2020). numerous types of vitamin b, such as b1, b2, b3, b5, b6, b7, b8, and b9, were reported to be derived from citrus and its derivative products, placing citrus as one of the vitamin b-rich agri-food products (liu et al., 2012). vitamin b1 or thiamine is abundant in citrus (ting, 1980) and is relatively stable to heat exposure (godswill et al., 2020). thiamine plays an important role in glucose metabolism (ramsey and muskin, 2013) and maintaining brain, gastrointestinal, heart, muscular function, and nervous system health (abobatta, 2019). different genotypes caused variations in vitamin b1 content among citrus. citrus ranking from the highest to lowest in vitamin b1 are orange, grapefruit, pomelo, and lemon for about 0.12, 0.12, 0.03, and 0.02 mg in 100 g fruits, respectively (paul and shaha, 2004). abobatta (2019) reported that mandarin contains more thiamine (40 mg per 100g) than grapefruit (0.02 mg per 100g), lime (0.02 mg per 100g), or lemon (0.02 mg per 100 ml juice). vitamin b2 or riboflavin, vitamin b3 or niacin, and vitamin b5 or pantothenic acid are also found in citrus juice (ting, 1980). riboflavin and niacin are unstable to heat exposure, while pantothenic acid displays opposite results (godswill et al., 2020). these vitamins are responsible for energy production by converting food to energy (bellows et al., 2012). different citrus genotypes may contain different amounts of said vitamins. an earlier study reported that the content of riboflavin in orange was 0.05 mg per 100 g, and was higher than pomelo, grapefruit, and even lemon by about 0.03, 0.02, and 0.01 mg per 100 g, respectively (paul and shaha, 2004). niacin content was higher in grapefruit (0.3 mg per 100 g) than in lime and lemon (< 0.1 mg in 100 g fruits; abobatta, 2019). vitamins b6, b7, and b8, known as pyridoxine, biotin, and inositol, are also involved in human metabolic functions (godswill et al., 2020). pyridoxine is wellknown for its role in serotonin synthesis (stover and field, 2015), while biotin is involved in the energy release process (bellows et al., 2012) with a coenzyme function (schellack et al., 2019). in addition, inositol is needed by our bodies to maintain healthy hair (schellack et al., 2019). an earlier study by (krehl and cowgill, 1950) reported the content of pyridoxine, biotin, and inositol in orange, grapefruit, and tangerine fruits. pyridoxine content in tangerine, orange, and grapefruit ranged from 22.6-33.4, 1632.4, and 8.0-18.2 μg per 100 ml, respectively. orange, grapefruit, and tangerine contain 0.42-1.5, 0.36-0.97, and 0.45-0.46 μg of biotin in 100ml of juice, respectively. grapefruit contains less inositol (88-112 mg per 100 ml) than orange (104-178 mg per 100 per ml) and tangerine (135-147 mg per 100 ml). this finding supports the idea that differences in citrus genotypes cause differences in the content of these multivitamins. folic acid, also known as folate or vitamin b9, is also frequently reported to be derived from citrus and its derivative products, such as fruit juice. the earlier study determined that orange juice has a higher folate content than other fruit juices (ting, 1980). moreover, folate was fully bioavailable and highly stable in orange juice (öhrvik and witthöft, 2008). folate is important due to its ability to reduce the risk of coronary heart illness (bellows et al., 2012). moreover, folate is known to be beneficial for a baby’s brain development during pregnancy (silalahi, 2002). folate content in orange juice is higher than in tangerine and grapefruits, i.e. 1.3-3.2, 1.2-1.8, and 0.8-2.2 μg , respectively, in 100ml of juice (krehl and cowgill, 1950). due to its health importance, folate has become the most fortified vitamin in 62 countries (godswill et al., 2020). the consumption of 100 g citrus is a multivitamin treasure trove: a review .......... journal of tropical crop science vol. 10 no. 1, february 2023 www.j-tropical-crops.com 61 citrus can supply up to 20% of daily folate needs (saeid and ahmed, 2021) specifically in nine year-old or younger children, while for adults, it covers up to 10% (turner and burri, 2013). consumption of 750 ml orange juice daily for four weeks increased folate levels by 18% (kurowska et al., 2000). vitamin e vitamin ε, also known as tocochromanols (fritsche et al., 2017), is a liposoluble essential micronutrient (colombo, 2010; g. lee and han, 2018) that are found in eight natural isoforms, for instance, α-, β-, γ-, δ-tocotrienol and α -, β-, γ-, δ-tocopherol (peh et al., 2016; poiroux-gonord et al., 2010; zou et al., 2016). this vitamin acts as an antioxidant (amitava, 2014; zingg, 2019) that prevents lipid peroxidation damage (zou et al., 2016). alpha-tocopherol is the major compound among other chemical vitamin e forms that has strong antioxidant activities to reduce lipid peroxidation (niki and abe, 2019) and maintain cell membranes’ stability (munné-bosch and falk, 2004). vitamin e can support healthy aging, and prevent cardiovascular and neurological diseases (rizvi et al., 2014; shahidi et al., 2021). since an earlier study by (salinthone et al., 2013) reported the association between vitamin e and human immunity, the administration of this vitamin as a food supplement is believed to help covid-19 patient recovery (erol et al., 2021; tavakol and seifalian, 2022). a previous report by the institute of medicine panel on dietary antioxidants and related compounds (2000) revealed the rda of vitamin e is 15 mg (35 µmol)/ day of α-tocopherol daily, irrespective of gender. in general, the main source of vitamin e in a plantderived product can be found in seed oil (ahsan et al., 2015; mène-saffrané, 2017), for instance, olive oil and sunflower oil (cayuela and garcía, 2017; garcía-closas et al., 2004). vitamin e can also be found in green leafy vegetables (cruz and casal, 2013), fruit peels, and seeds (zhou, 2012). an earlier study (ornelas-paz et al., 2017) reported the reduction of vitamin e in seedless mandarin as the effect of phytosanitary irradiation during postharvest handling. vitamin e content can also vary between citrus genotypes; for example, orange, tangerine, and lemon have 5.60, 4.50, and 11.40 mg.kg-1 of vitamin e, respectively (zhou, 2012). previous studies recommended raw consumption of citrus fruits to obtain the maximum amount of vitamin e since both tocopherol and tocotrienol levels can decline in response to processing treatment (knecht et al., 2015). other than fruits, leaves of kaffir lime contain 66.00 and 39.83 mg per 100 g of vitamin e per dry weight and fresh weight basis, respectively (ching and mohamed, 2001). vitamin e content is influenced by the genetics and the environmental factors. under certain abiotic stresses, plants may boost vitamin e production, such as toco-chromanol, which is an antioxidant to adapt to the stress (bao et al., 2020). xiang et al., (2019) reported the gradual accumulation of vitamin e in sweet corns treated with low-temperature stress. a study by kruk et al. (2005) highlighted the accumulation of tocopherols, especially α-tocopherol, under high light and heat stress environment to protect photosystem. however, information on the effect of environmental factors, including applied culture practice, on multivitamin content in citrus are still limited; thus, future research should be directed to these aspects. conclusion citrus has long been lauded as a functional agrifood famous for its vitamin c content. this review highlighted not only vitamin c but also vitamins a, b, and e, found within numerous citrus species, illustrating this plant as a vitamin treasure box. the variation of vitamin c content was reported in citron, orange, mandarin, tangerine, lemon, lime, kumquat, pomelo, and grapefruit. in terms of vitamin a, the red and pink flesh variety of grapefruit and yellow colored flesh of calamansi, orange, pomelo, lemon, and mandarin are rich in carotenoid-provitamin a. orange and grapefruits are reported to contain high amounts of vitamin b, whereas tangerine, mandarin, pomelo, lemon, and lime contain small amounts. the fruits of orange, mandarin, tangerine, lemon, and the leaf of kaffir lime contain vitamin e. those multivitamins are essential as antioxidants to enhance our immune system. acknowledgement this research was funded by universitas padjadjaran through a scheme of online library data research grant, number 2203/un6.3.1/pt.00/2022. references abobatta, f. w. 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