In the world, rice (Oryza sativa L) is the major source of of food for more than 2 Bangladesh Agron. J. 2016, 19(2): 115-123 ANKURI TECHNOLOGY FOR SEED GERMINATION AND SEEDLING EMERGENCE OF RICE IN COLD ENVIRONMENT Tahmid Hossain Ansari and Montasir Ahmed Bangladesh Rice Research Institute, Regional Station, Rajshahi Corresponding author: tahmidhansari@yahoo.com Key words: Ankuri germination, rice seed germination, seedling emergence, tray seedling, Zag- method Abstract Rice seed germination as well as seedling emergence in cold environment is a challenge for successful rice cultivation during dry (Boro) season in northern Bangladesh Experiments were conducted in two consecutive Boro seasons in Rajshahi to develop techniques for improving germination and seedling emergence under cold conditions.. Seeds of rice var. BRRI dhan29 and BRRI dhan50 were germinated following the traditional “Zag-method” and separately in a frame covered with air tight heat insulating plastic tripal (called Ankuri- germinator). Then vapor therapy was given inside Ankuri and maintained 25- 350C with high humidity. External and internal temperatures of Ankuri were recorded. Seed germination was checked at 12 h interval. In another experiment, germinated seeds of BRRI dhan29 were sown on soil in trays followed by light irrigation. One set of trays was placed in Ankuri. Another two sets of trays were placed in the field with polythene covered and uncovered. Similar experiment using non-germinated dry seed was also conducted. Vapor therapy was applied in Ankuri only in and trays were checked regularly to observe the plumule and radicle growth. The germination rate (%) of the tested varieties in Ankuri ranged from 82-96% which was 64- 92% in Zag-method. High variation of germination in Zag-method was due to improper temperature and humidity during the Zag period. Seed germination was faster in Ankuri which was observed within 2.5 days. This was also 2.5 days earlier in comparison with the Zag-method. Soaking period ranged 22-24 h outside in cold water/weather. But 18 h soaking in Ankuri resulted good germination. Irrespective of varieties, the growth was 5.4 and 1.7 times more respectively for plumule and radicle in Ankuri than Zag-method. Seedling emergence was 2-3 days earlier in both dry and soaked/germinated seeds sown in trays of Ankuri than the outside polythene covered trays. Seedling emergence failed in open-trays. Average air temperature from 18:00-9:00 was cooler (150C) than 9:00-18:00 period (150C). Ankuri maintained 25-350C and high humidity which favored seed germination and seedling growth. Therefore, Ankuri could be an effective seed germination technique with faster germination, radicle and plumule growth. Introduction Rice is considered as the staple food crops in Bangladesh, a sub-tropical country producing 34.7 million metric tons covering from 80% of the total cultivated area (BBS, 2015). Yield loss due to low temperature are well documented in Japan (Shimono et al., 2007), Korea, India, Nepal, Bangladesh and other countries (Kaneda and Beachell, 1974; Lee 2001). In the dry season, a large part of Bangladesh especially in the northern part is generally affected with low temperature. This cold environment affects seed germination, seedling raising and transplanted seedling establishment. Low temperature reduces germination (Basnayake et al., 2003; Ali et al., 2006) and cause poor establishment (Sasaki 1979 1981; Lewin and Maccaffery 116 Ansari et al. 1985; Shimono et al., 2002, 2004, 2007). Ali et al. (2006) reported the effects of genotypes and physiological age of rice seeds on germination under low temperature in Bangladesh. A high percentage of seed germination is an important criterion for successful rice cultivation. In Bangladesh, farmers obtain their seed from a wide range of sources, because of the unavailability of adequate quantities of good quality seed for raising seedling. Therefore, the quality of seed generally varies considerably, which results in poor or no germination in low temperature or cold environment. Farmers are germinating these kinds of seed following traditional Zag-method. Poor germination or even germination failure is a general scenario in this practiced method because of improper temperature and humidity during Zag (incubation)-period in dry season. Thus the Zag-method in association with low temperature during dry season hampers or affects seed germination. Therefore, attempts have been taken to develop a germination technique for better rice seed germination as well as seedling emergence in tray during cold period. Material and Methods The experiments were conducted at Bangladesh Rice Research Institute (BRRI), Regional Station, Rajshahi during the dry (Boro) seasons 2012-13 and 2013-14. A seed germination chamber was developed where rice var. BRRI dhan29 and BRRI dhan50 seeds were tested for germination in both the years. Further, seedling emergence in trays was also tried in the second season only. Development of germination chamber In the first season, a germination chamber was developed using angle-bar, screw, plastic tripal, foam and electric appliances like electric ware, plug etc. in such a way that seed can be germinated and seedlings in trays can be raised through vapor therapy with controlled temperature and high humidity. The hypothesis was that the seed germination and raising seedlings could be possible through continuous supply of vapor therapy inside the chamber maintaining proper temperature and humidity. Investigation on seed germination After the development of the seed germination chamber called “Ankuri”, the performance of Ankuri for seed germination was tested in two consecutive seasons. Seeds were soaked for 22-24 h in first season and 18 h in second season. Then the seeds were divided into two parta and kept in the moist cloth bag separately and rapped with moist sac. One part was preserved following the Zag-method and another part in the Ankuri. Then vapor therapy was given to boost up the temperature and humidity inside Ankuri in such a way that the temperature could be maintained 25- 350C with high humidity. Temperature of both outside and inside Ankuri was recorded at three h interval starting from 6 h to 21/24 h. Germinating seed samples were checked at 24 h interval for 2 days and then 12 h interval until the sprouting observed. Seed sampling was done from each replicated samples of RCB design keeping consideration of both inner and outer side effect of the seed bag. The germinated and non germinated seeds were counted and percent germination was calculated. The length of plumule and radicle was also measured from randomly selected 10 seedlings in each replication. The germination percentage was calculated using the formula- Germination (%)= (Number of seed germinated/Total number seeds tested) × 100 117 Ankuri Technology for Seed Germination and Seedling Emergence of Rice in Cold Environment Seedling vigor index= (average of plumule length + average of radicle length) × germination percentage. Raising seedling in trays Ankuri germinated seeds were shown on the pulverized soil in trays following tray seeding method. The seeds were sown in the trays at 120 g per tray for BRRI dhan29. Fifteen trays were prepared and soaked by sprinkler. One set of three trays covered with polythene was kept outside (open field) and another set of three trays was kept open at field adjacent to the polythene covered trays. Moreover, nine trays were kept in the Ankuri where vapor therapy was given in such a way that the temperature could be maintained 25-350C with high humidity. The trays were checked every 12 h interval to observe the seedling emergence. The trays were taken out from Ankuri and kept outside with polythene cover when the sprouted young seedling was attained 2-3 mm in length. Statistical analysis All the collected data were analyzed by using CROPSTAT package developed by IRRI. Results and Discussion Development of Ankuri The design of the germination chamber Ankuri is shown in Figure 1. The length was 120 cm., the width was 90 cm and the height was 120 cm. There were few tray holding levels or stares each differed by 25 cm. Two electric heaters were prepared using different sizes nut screws in an electric fiber board (7.5 cm × 2.5 cm inch). Two screws were used in each electric heater. The sizes of the screws were 0.35 cm / -2.5 cm and 1 / -4 cm, respectively for the energy expenditure 200 W/h and 275 W/h. Both the screws were connected with electric wares and attached with electric power by a plug. The mini heater was placed in a bowl filled with 15 L water inside the Ankuri. All the heaters tested separately for 24 hours. Vapor generation was investigated and the temperature inside the Ankuri was recorded during testing period (Table 1). Table 1. Specification of screws and their electric consumption in Ankuri Sl. No. Screw size Screw type Power consumption (Wh) Temperature range in Ankuri (0C) Vapour generation 1. 0.35 cm 2.5 cm Nut screw 200 21.2 - 37.3 Observed very slow 2. 1-4 cm- Nut screw 275 25.0 - 40.0 Observed slow The mini electric heater (200 W/h) made of 0.35 cm/ -2.5 cm screw was considered good for seed germination and tested further for temperature during seed germination. During seed germination both outside and inside temperatures of Ankuri was recorded at an interval of 3 h from 6:00 am to 9:00 pm. The temperature inside the Ankuri ranged from 21.2-37.30C and the outside temperature was recorded as 9.0-30.00C (Table 2). Using this technique, seed germination was done successfully as compared to the Zag method (Fig. 2, Table 2). 118 Ansari et al. Fig. 1. Ankuri seed germination chamber Fig. 2. Zag-method for seed germination Table 2. Air and Ankuri temperature during seed germination Hour Temp. (0C) 2013 Temp. (0C) 2014 Air Ankuri Air Ankuri 6:00 9.0 21.2 15.3 25.0 9:00 14.1 27.7 23.1 30.0 12:00 22.4 31.3 34.6 37.3 15:00 21.3 29.1 30.0 33.8 18:00 15.1 27.3 - - 21:00 - - 18.3 35.2 Figures are averages of five observations Investigation on seed germination The germination percentages of the tested varieties are shown in Table 3. The percentage of germination ranged from 72-92% in Zag-method which was 85-96% in Ankuri in the first season. Here, significant difference in the percentage of seed germination was observed in the case of BRRI dhan50 but insignificant in BRRI dhan29.However, both the varieties showed a large difference in seed germination in the second season that ranged from 64-75% in Zag-method in compare with the Ankuri-method (82-91%). Table 3. Germination (%) of dry (Boro) season varieties in Zag and Ankuri methods. Variety* Year (January) Germination (%) LSD (0.05) Zag-method Ankuri-method BRRI dhan29 2013 92 96 4.56 BRRI dhan50 72 85 8.23 119 Ankuri Technology for Seed Germination and Seedling Emergence of Rice in Cold Environment BRRI dhan29 2014 75 91 8.26 BRRI dhan50 64 82 15.04 *Seed source- BRRI, Rajshahi During the germination period, temperatures of both outside and inside was measured which indicated that air temperature was lowest at 6:00 am and highest at 12:00 noon (Table 2). Air temperature increased gradually from morning to noon and then decreased to 150C at 6:00 pm in 2013. On the other hand, the temperature of Ankuri ranged from 21.20C to 31.30C in 2013 and 25.00C to 37.30C in 2014. In 2014, air temperature at 6:00 am and 9:00 pm were 15.30C and 18.30C corresponding to the Ankuri temperature 25.00C and 37.30C, respectively. The effect of treatment i.e., germination method on sprouting period was highly significant. On the other hand, both plumule and radicle lengths was also different (Table 4). Table 4. Effect of germination methods on sprouting period, plumule and radicle growth irrespective of varieties in the dry season, 2014. Germination method aSprouting time (hour) Plumule length (mm) Radicle length (mm) Zag-method 120 0.43 4.66 Ankuri-method 60 2.32 7.93 LSD (0.05) 0.36 1.40 2.98 Figures are averages of 6 observations aAll the replicated samples germinated in the same period in each germination method The effect of variety irrespective of methods was similar (Table 5). Data shows that the radicle growth of BRRI dhan29 was better (1.4 times) than the BRRI dhan50 with almost same growth of plumule (1.30-1.46). Table 5. Effect of varieties on sprouting period, plumule and radicle growth irrespective of germination methods in the dry season, 2014. Variety aSprouting time (hour) Plumule length (mm) Radicle length (mm) BRRI dhan29 90 1.30 7.43 BRRI dhan50 90 1.46 5.16 LSD (0.05) ns ns ns Figures are averages of six observations aAll the replicated samples germinated in the same period in each germination method The interaction effect of variety and germination methods shows a great difference in sprouting period irrespective of variety and germination methods (Table 6). Again, significant different in plumule and radicle length was also found. The seedling vigor index value was high in Ankuri germinated seeds compared to Zag-method (Table 7). Table 6. Interaction effect of varieties and germination methods on sprouting period, plumule and radicle growth in the dry season, 2014. Variety Germination method aSprouting time (hour) Plumule length (mm) Radicle length (mm) BRRI dhan29 Ankuri 60 2.13 10.05 Zag 120 0.46 4.80 BRRI dhan50 Ankuri 60 2.52 5.80 120 Ansari et al. Zag 120 0.39 4.52 LSD (0.05) 0.51 1.98 4.21 Figures are averages of 3 replications aAll the replicated samples germinated in the same period in each variety and germination method Table 7. Interaction effect of seedling vigor of Boro varieties in the dry season, 2014. Variety Treatment Seedling Vigor Index BRRI dhan29 Ankuri 1108.7 Zag 394.8 BRRI dhan50 Ankuri 682.0 Zag 314.2 LSD (0.05) Variety × treatment ns Raising seedling in trays Figure 3 and Figure 4 shows the scenario of seedling emergence in trays in Ankuri. Seedling emergence was the best in Ankuri followed by polythene covered trays kept outside in the field. But seed germination was failed in the trays kept in open field. Seedling emergence was faster in Ankuri compare to polythene covered outside trays (PCOT). The growth of seedlings was better in Ankuri compare to the PCOT. The seedlings were not affected with disease in Ankuri. Qualitative evaluation of Ankuri germinated seeds showed faster germination as well as good quality seedling (Table 8). 121 Ankuri Technology for Seed Germination and Seedling Emergence of Rice in Cold Environment Fig 3. Seedling emergence of BRRI dhan29 in Ankuri trays Fig. 4. Seedling emergence of BRRI dhan29 in Polythene covered trays. Table 8. Qualitative score of emerged seedling in trays in the dry season, 2014 Treatment Emergence Germination rate Seedling quality Tray seedling (Ankuri) Complete emergence Faster germination Good (++) Tray seedling (Polythene covered) Partial emergence Slow germination Moderate (+) Tray seedlings (Open field) No or very poor emergence No or very late germination Bad (-) ++ indicates good growth of seedling + indicates moderate growth of seedlings compare to Ankuri treated seedlings - indicates very poor or no growth of seedlings Development of simple seed germination chamber is important in the changing climate environment. Seed germination is poor in low temperature (Sipaseuth et al., 2007) and the farmers are practicing traditional Zag-method that cannot be ensured good seed germination. Because, farmers prepared the Zag-environment in different ways with their own experience with available materials they find e.g., rice straw, jute sac, bamboo made basket etc. This method cannot maintain proper temperature and humidity during germination period. As a result, farmers failed to get good germination in the used seed even if it is good quality seed. The low quality seed is again at high risk for germination in Zag-method as this cannot maintain proper temperature and humidity. In some cases, farmers keep their seed for germination in rice straw-mass which sometimes produce high vapor with higher temperature that cause the seed even boiled and finally failure of germination. Therefore, climate resilient and farmers’ friendly seed germination chamber could be an alternative for good seed germination in low temperature during dry season. Ankuri maintained the temperature ranged from 250C to 350C with a few exceptions that could be minimized. This can also generate high humidity (around 98%). The temperature and high humidity mentioned above in the Ankuri is favorable for rice germination. Therefore, Ankuri could be the alternative option for good seed germination. A comparative advantages and disadvantages between Ankuri and Zag-method are listed in the Table 9. 122 Ansari et al. Table 9. Comparative results of Ankuri and Zag- methods for seed germination. Sl. No. Ankuri- Method Zag- Method 1. Soaking period 22-24 h (18h is enough if seeds are soaked in Ankuri). Soaking period 22-24 h. 2. Seeds are sprouted first in 24 h after soaking. Seeds are sprouted first in 72 h after soaking. 3. Seeding can be done after 60 h i.e., 2.5 days. Seeding can be done after 120 h i.e., 5.0 days. 4. Germination rate is always higher. Germination rate is lower. 5. Germination is ensured. Not so reliable. 6. Possibility of seed infection is less due to less time needed in preservation. Possibility of seed infection is comparatively high. 7. Easy to follow the method. Difficult to follow the method. 8. Temperature and humidity can be controlled. Difficult to control temperature and humidity. 9. Helpful for tray seedling raising. Not applicable. 10. Cost is comparatively high. No extra cost is required. 11. Seed can be saved (av. 13% in the expt.) that will mitigate seed crisis a lot. No such advantage. Both the varieties showed higher percentage of seed germination in Ankuri as compared with the Zag-methodbecause of the maintenance of proper temperature and humidity in the Ankuri. The probability of receiving low temperature during December- January in the northern and south-western part of Bangladesh has been prevailing in the last few years. Seedling mortality or failure of raising seedling at that time has become a general scenario in those areas during dry season. The results of this study showed that Zag-method produced lower germination especially due to improper lower temperature. This result is inconsistent with the others who reported that temperature below 110C reduce the rate of germination by 10-22% (Ali et al., 2006) and 100C caused germination failure in most Asian rice growing areas (Matsushima et al., 1968; Tajima et al., 1983; Lee, 2001; Ali et al., 2006). In 2013, there was no difference in percent seed germination in BRRI dhan29 which showed the significant difference in 2014. The Zag-method applied in these experiments was done inside the building room. Probably the temperature was maintained well during Zag period but the building room environment is not available at farmers’ level. Therefore, risk in Zag-method at farmers’ level is always inevitable. The plumule and radicle length which is the component of seed vigor are associated with seed germination also. Plumule length was much higher in Ankuri than Zag- method at p=0.05and the radicle length was 70% more in the same Ankuri method. These indicate that the germination method influence the seed germination even with same seed vigor. Therefore, Zag-method can affect the quality seed for germination, plumule and radicle growth and thereby seed vigor. In the case of Ankuri, vapor induced temperature and humidity successfully overcome the low seed germination, plumule and radicle growth i.e., quality seed and even farmers’ saved seed can successfully be used for germination in Ankuri comparatively with Zag-method. The Ankuri technique could also be used irrespective of the variety. All the parameters like percent germination, plumule growth and radicle growth showed similar results irrespective of variety with few exceptions. These were observed when no interaction effect was found in radicle and plumule growth irrespective of variety and germination method except for BRRI dhan50 in Zag- method. In the raising of seedling in trays, the better growth was also observed in the Ankuri treated trays seedling. The seed vigor index was higher in Ankuri treated seed due to higher germination percentage, plumule and radicle growth at p=0.05. The variation in temperature might have influence in good germination and the growth of plumule and radicle. However, 123 Ankuri Technology for Seed Germination and Seedling Emergence of Rice in Cold Environment there is no interaction effect of variety and treatment in this method. It means, Ankuri significantly contribute to promote seed germination with higher vigorous seedlings. Conclusion The experiments clearly demonstrated the efficacy of Ankuri in rice seed germination as well as the weakness of the traditional Zag-method. Therefore, Ankuri could be an effective germination technique which influenced faster germination and higher plumule as well as radicle growth. It can also be used for raising seedling in trays as well. Commercial exploitation and adoption of this technology at farmers’ level could minimize seed loss ensuring germination and economic benefit. References Ali, M. G., R. E. I. Naylor and S. Mathews 2006. Distinguishing the effect of genotype and seed physiological age on low temperature tolerance of rice (Oryza sativa L.) Expt. Agric. 42: 337-349. Basnayake, J., V. Sihathep, P. Sonekham, M. Senthonghae, V. Sibounheuang, V. Phamixay, M. Chanphengxay and S. Fukai. 2003. Effects of time of planting on agronomic and yield performance of several rice cultivars under various temperature condition in Lao PDR. In: Proceedings of the 11th Agronomy conference. Melbourne, 1-6 February (www.regional .org.au/au/asa/2003/). BBS (Bangladesh Bureau of Statistics). 2015. Statistical Pocketbook Bangladesh 2015. Bangladesh Bureau of Statistics, Statistics and Informatics Division (SID), Ministry of Planning, Govt. of the People's Republic of Bangladesh. April 2016. Pp. 499 Kaneda, C. and H. M. Beachell. 1974. Response of indica-japonica rice to low temperature. SABRO J, 6: 17-32. Lee, M. H. 2001. Low temperature tolerance in rice: the Korean experience In: Fukai S, Basnayake J (Eds), ACAIR Proceedings 101: Increased low–land rice production in the mekong Region. Australian Centre for International Agricultural Research, GPO Box 1571, Canbera, ACT 2601, pp. 138-146 Lewin, L. And D. Maccaffery. 1985. Rice seedlings growth is influenced by water depth and temperature. Farmers’ Newsl. Large Area 127. Shimono, H., T. Hasegawa, K. Iwama. 2002. Response of growth and grain yield in paddy rice to cool water at different growth stages. Field Crops Res, 73: 67-79 Shimono, H., T. Hasegawa, S. Fujimura, K. Iwama. 2004. Response of leaf photosynthesis and plant water status in rice to low water temperature at different growth stages. Field Crops Res, 89: 71-83 Shimono, H., H. Okada, E. Kanda, I. Arakawa. 2007. Low temperature induced sterility in rice: evidence for the effects of temperature before panicle initiation. Field Crops Res, 101: 221-231. Sipaseuth, J. Basnayake, S. Fukai, T.C. Farrell, M. Senthonghae, Sengkeo, S. Phamixay, B. Linquist and M. Chanphengsay. 2007. Opportunities to increasing dry season rice productivity in low temperature affected areas. Field Crops Res, 102: 87–97. 124 Ansari et al. Sympson, G. M. 1990. Seed dormancy in grasses. Cambridge University Press, U.K. Tarima, K., A. Amemiya, N. Kabaki. 1983. Physiological study of growth inhibition in rice plant as affected by low temperature II. Physiological mechanism and varietal difference of chilling injury in rice plant. Bull National Institute of Agricultural Science, vol 34, pp. 69-111. Wu, R. and A. Garg. 2003. Engineering rice plant with trehalose producing genes improve tolerance to drought, salt and low temperature. Seed quest, ISB News Report, March 2003. Department of Molecular Biology and Genetics, Cornell University, USA. (www.seedquest.com/News/release/2003/march/5456 htm). http://www.seedquest.com/News/release/2003/march/5456