Impaginato 205 Adv. Hort. Sci., 2017 31(3): 205-214 DOI: 10.13128/ahs-21959 Effect of different physio-chemical factors on sex expression and fruit yield in greenhouse cucumber M. Golabadi 1, P. Golkar 2 (*), A.-R. Eghtedari 3 1 Department of Agronomy and Plant Breeding, College of Agriculture, Isfahan (Khorasgan) Branch, Islamic Azad University, 81595158 Isfahan, Iran. 2 Research Institute for Biotechnology and Bioengineering, Isfahan University of Technology, 8415683111 Isfahan, Iran. 3 Department of Agronomy and Plant Breeding, College of Agriculture, Isfahan (Khorasgan) Branch, Islamic Azad University, 81595158 Isfahan, Iran. Key words: cucumber, flowering, male, sex expression, silver ions. Abstract: Male flower expression is considered an important aim in greenhouse cucumber breeding for creating paternal lines as a base for hybrid progeny. The study was carried out to evaluate the effects of different treatments on sex expression and fruit yield of cucumber in two different season (autumn-winter and spring-summer), in particular this research focuses on the influence of 1) usage of two chemical agents: silver thiosulphate [Ag(S2O3)2 -3] and silver nitrate (AgNO3) at different concentration, respectively 200 and 500 ppm and 100, 200 and 300 ppm 2) plant development stages at the moment of the treatment (5, 10, and 15-leaf growth stages) and 3) application of single or double sprayings. Analysis of variance showed that season, chemical applications and number of spaying had significant effect on the induction of a higher number of male flow- ers. A positive significant effect of season suggested that longer days and high- er temperature promote the formation of male flowers in cucumber. This study showed that male flower production was induced by all concentrations of silver ions, especially high concentrations. Important traits related to change of sex expression such as the number of days to male flowering and the number of male flowers are more affected by different interactions of studied factors in contrast to vegetative and yield related traits. Also, the quadruple interaction effects indicated that silver ion could change sex expression at higher tempera- tures and longer days (second season in summer) with high concentration when applied in 15 leaf stage for AgNO3 and 5 and 15 leaf growth stage for Ag(S2O3)2 -3 with double spraying. Consequently, female lines can be induced to male flowering with silver ions, thus increasing the feasibility of large scale seed production of gynoecious × gynoecious cucumber hybrid. 1. Introduction Commercial cucumber (Cucumis sativus L., 2n= 2x= 14) is a member of cucurbitaceae that is indigenous to India (Renner et al., 2007). It is one of (*) Corresponding author: golkar@cc.iut.ac.ir Citation: GOLABADI M., GOLKAR P., EGHTEDARI A.-R., 2017 - Effect of different physio-chemical factors on sex expression and fruit yield in greenhouse cucumber. - Adv. Hort. Sci., 31(3): 205-214. Copyright: © 2017 Golabadi M., Golkar P., Eghtedari A.-R. This is an open access, peer reviewed article published by Firenze University Press (http://www.fupress.net/index.php/ahs/) and distribuited under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Competing Interests: The authors declare no competing interests. Received for publication 9 November 2016 Accepted for publication 19 June 2017 AHS Advances in Horticultural Science Adv. Hort. Sci., 2017 31(3): 205-214 206 the most economically important cucurbit vegetable p l a n t s ( T a t l i o g l u , 1 9 9 3 , R o b i n s o n a n d D e c k e r - Walters, 1997) and among the most widely-grown vegetable crop in the world, after tomato, onion and cabbage (Plader et al., 2007). Cucumber is used in different types (Harvesting, slicing and fresh eating) that are used as fresh or processed vegetable (Shetty and Wehner, 2002). The cucumber is a thermophilic and frost- susceptible crop, usually cultivated in fields during the spring-summer period. Its high demand has also made it an important crop to be widely grown in glasshouses or plastic houses (Sarkar and Sirohi, 2011). Sex expression is an important factor that has a positive effect on yield and that consti- tutes a major component of cucumber improvement programs (Yamasaki et al., 2003). In gynoecious culti- vars, all flowers are female, so, male flowers are required for new line production via female flowers crossing. The sex expression of Cucumis sativus L. is determined by genetics as well as environment (e.g. photoperiod, temperature) (Yamasaki et al., 2003; Bano and Khokhar, 2009). Change from vegetative growth to reproductive stages is a complex process regulated by many factors, and could be influenced b y t h e a p p l i c a t i o n o f p l a n t g r o w t h r e g u l a t o r s (Ainsworth, 1999; Sure et al., 2013). Growth regula- tors have tremendous effects on sex modification and flowering in cucurbits that leading to either sup- pression of male flowers or an increase in the num- ber of female flowers (Al-Masoum and Al-Masri, 1999). Some researchers have already reported the effects of plant growth regulators on modification of sex expression in cucumber flowers (Vadigeri et al., 2001; Rafee kher et al., 2002; Bano and Khokhar, 2009). Line production is important in breeding pro- grams, because many cultivars in cucumber are hybrids (Golabadi et al., 2015). In gynoecious cucum- bers, male flower induction is necessary for produc- tion of F1 hybrid seeds (Yamasaki et al., 2003; Wang et al., 2011). Therefore, change of sex expression in gynoecious cucumber is necessary. In general, auxin, ethylene and cytokinins pro- mote female sex expression in various monoecious and dioecious systems (Mohan Ram and Sett, 1982). Exogenous application of plant growth regulators could alter the sex ratio if applied at the two- or four- leaf stage, which is the critical stage at which the sup- pression or proportion of either sex is possible (Hossain et al., 2006). The effect of ethylene can be antagonized by specific ethylene inhibitors (Hirayama and Alonso, 2000). Silver ions (Ag+) restrain the physi- ological effect of the ethylene by blocking the ethyl- ene receptors (Hirayama and Alonso, 2000). It is known that silver ions (Ag+) applied as silver nitrate (AgNO3) or as silver thiosulfate [Ag(S2O3)2 -3] replace copper ions (Cu+) which is a part of the ethylene receptor preventing the receptor from responding to ethylene (Abeles et al., 1992). Another inhibitor of the ethylene action, AgNO3, suppresses the develop- ment of female flowers and induces the male ones (Kumar et al., 2009; Stankovic and Prodanovic, 2002). Male flower formation increased proportionally with the concentration of AgNO3 applied (Stankovic and Prodanovic, 2002). According to previous reports, AgNO3 induced more male flowers than GA3 on gynoecious breeding lines of cucumber (Kalloo and Franken, 1978) and summer squash (Yongan et al., 2002). The positive effects of AgNO 3 on male flower production in cucumber have reported by Karakaya and Padem (2011). Thappa et al. (2011) used different plant growth regulators (ethephon, naphthalene acetic acid and maleic hydrazide) at the two, four and six- leaf and full-bloom stages in cucumber to induce male flowers. Law et al. (2002) reported that silver thiosulfate, an ethylene inhibitor, enhanced stamen development in female white campion (Silene latifo- lia). However, little effort seems to have been direct- ed toward the study of the effects of silver ions on male flower production sprayed at different growth stages and if the numbers of spray applications could improve the effectiveness of the induction. On the other hand, it seems that there are few studies that correlate the effect of environmental conditions, especially season, to the number of spraying and leaf-stage growth on male flower induction of plant treated with silver ions. Although silver is a toxicity ion, however the amount of its toxicity depends on species, silver con- centration, times of application and plant growth stage spraying and exposure media. For exmple Fuente et al. (2014) applied different concentrations of AgNO3 (0, 30, 60, 90, 200 mg l -1) at intervals of 8 days throughout the crop cycle (90 days) in water- melon. Their results showed that silver accumulate in root more than other parts of plants and antioxidant in fruits increased in plants exposed to 30 mg l-1 AgNO3, but lycopene content decreased. Therefore the main objectives of this study was to investigate the effects of chemical application [AgNO3 and Ag(S2O3)2 -3], different leaf growth stages and number of spraying on flower and fruit charac- teristics of greenhouse cucumber in two different Golabadi et al. - Effect of different physio-chemical factors on sex expression and fruit yield in greenhouse cucumber 207 seasons to identify the best condition and the opti- mal level of chemical application to boost the forma- tion of male flowers. Our findings will help to develop practical recommendations on these issues for improve male flowers production in cucumber breed- ing programs. Furthermore, this work aimed to eval- uate the stability of sex expression of a gynoecious cultivar Adrian in different seasons. The present investigation was done as a first step to a hybridiza- tion program of cucumber. 2. Materials and Methods Planting material and field evaluation The seeds of cv. Adrian as a gynoecious genotype were sown at the mid autumn of 2014 and mid spring of 2015 at Research Greenhouse of Agriculture Department at Islamic Azad University Isfahan Branch, Isfahan, Iran (51° 36´ longitude and 32°63 lat- itude). The soil used was loam with pH 7.7 and Electrical conductivity (EC) of 4.1 (ds/m). Seeds were planted to soil directly and covered with peat moss and coco peat. Plants were disposed in two rows, where the spaces between cucumber seedling was 50 cm and within the rows were 90 cm respectively, and 180 cm was left between every couple rows. The greenhouse air temperature at the growing period was maximum 29°C/18°C (day/night) in first season (autumn-winter) and 34°C/20°C (day/night) in second season (spring-summer) with a relative humidity of about 55% and 60%, respectively. Nutrient levels in the irrigation solution water were N:216 (mg l-1), P:58 (mg l-1), K:286 (mg l-1), Ca:185 (mg l-1), Mg:185 (mg l- 1), S:43 (mg l-1), Fe:5.59 (mg l-1), Mn:1.97 (mg l-1), B:0.7 (mg l-1), Zn:0.2 (mg l-1), Cu:0.07 (mg l-1) and Mo:0.05 (mg l-1). Different fertilizers were used based on soil analysis that included: potassium nitrate, ammonium nitrate, magnesium nitrate, iron and other mineral elements such as sulphate dissolved in the irrigation water. Dichlorvos, Trigard, Abamectin and organic neem oil were applied for insect control. The same fertilizers and pest management were used for all plants in the same time. Drip irrigation was applied when needed. The source of water was urban water with electrical conductivity (EC) of 0.4 (ds/m). The pH of the irrigation water was adjusted to 6.5 by adding nitric acid. Silver application Different chemical treatments including AgNO3 [h1=100 ppm (0.59 mM), h2=200 ppm (1.18 mM) and h3=300 ppm (1.77 mM)], and Ag(S2O3)2 -3 [h4=200 ppm (0.6 mM) and h5=500 ppm (1.5 mM)] and control (h6) were applied to induce changes in sex expression. A g N O 3 w a s p u r c h a s e d f r o m M e r c k C h e m i c a l Company and Ag(S2O3)2 -3 was synthesized according to Law et al. (2002). The solutions were applied as a spray on the whole plant. Water was sprayed on the control plants. Spraying was accomplished at 5, 10, and 15-leaf growth stages (five leaves stage: LS1, ten leaves stage: LS2, and fifteen leaves stage: LS3). Chemical treatments were employed in single or double spraying at one week interval. AgNO3 and Ag(S2O3)2 -3 applications were conducted early in the morning (before sunrise) to avoid plant sunburn. Each treatment was applied with adequate amounts of the solutions to assure that all the leaves were completely wetted in each spray event. The total time of experiment in every season was about 4 months after sowing and measurement of traits were done in this period. Studied traits The traits of interest were measured on eight plants per replication and included: days to male flower expression (days to first male flower appear- ance after sowing) (DMF); the node number that the first male flower was appear (NNMF); the mean of internode length (the mean of five internode from node number 15 to 20) (IL); the number of male flower (total male flower that opened in whole plant) (NMF), male flowering period (days from observation the first male flowering until the last male flowering observation (MFP); male flower diameter (was recorded at the widest points) (MFD); single fruit weight (from dividing total fruit weight to total fruit number in every harvesting) (SFH); fruit number per harvesting (FNH); fruit weight per harvesting (FWH) (the numbers and the weights of all the fruits har- vested in each plot in each harvesting). Statistical analysis The experiment was conducted as a combined analysis of variance with three different factors (chemical treatments, leaf growth stage, and number of spraying) under two different seasons. The data collected were subjected to analysis of variance (ANOVA) based on a completely randomized block design with three replications using the general lin- ear model (GLM). Statistical analysis system program (SAS Ver. 9) was used for data analysis. The differ- ences between applications were decided on the basis of the Least Significant Difference (LSD) test Adv. Hort. Sci., 2017 31(3): 205-214 208 (P<0.05) according to their importance at the 0.05 confidence level. 3. Results and Discussion The result of analysis of variance based on a facto- rial experiment is presented in Table 1. According to Table 1, the season (S) had significant effect on all of the studied traits, except for single fruit weight. This result is logical, since phenotypic expression of sex is strongly modified by environmental and hormonal factors (Mohan Ram and Sett, 1982). Long days, high temperature, and silver ion promote formation of male flowers, whereas short days and low tempera- ture promote the formation of female flowers (Perl- Treves, 1999). Clearly, chemical treatments (CH) had significant effects on all the traits studied, except for fruit num- ber per harvesting (Table 1). Number of spraying (sin- gle or double) also had significant effects on the days to male flowering, the node number of first male flower, the mean of internode length, the number of male flower and male flowering period (Table 1). Hallidri (2004) reported that increased number of spraying events had significant effects on male flower production. He suggested that doses of 400 and 500 ppm of AgNO3 produced the highest number of male flowers. The leaf growth stage (LGS) of spray- ing showed significant differences on days to male flowering, the node number of first male flower, male flowering period, male flower diameter and fruit weight per harvesting (Table 1). The interaction effect of environment × silver application had a significant effect on all traits with the exception of male flower period and fruit number per harvesting (Table 1). Interaction of season × leaf stage was significant for days to male flowering, the node number of first male flower, the number of male flower and male flower diameter. Interaction of number of spray events × chemical application had a significant effect on days to male flowering, the node number of first male flower, the number of male flower and single fruit weight (Table 1). Interaction of chemical application × leaf stage of spraying showed significant differences on days to male flowering and the node number of first male flower (Table 1). The interaction of environment × chemical application × leaf stage of spraying showed significant difference on days to male flowering, the node number of first male flower, the number of male flower and period of male flowering. Study of these interactions DF= Degree of freedom; S= season; CH= Chemical treatments [Silver nitrate (AgNO3) and Silver thiosulphate Ag(S2O3)2 -3); SP= number of spraying (single or double); LGS= Leaf growth stage (5, 10 and 15). DMF= days to male flowering; NNMF= the node number of first male flower; IL= the mean of internode length (cm); NMF= the number of male flower; MFP= male flowering period; MFD= male flower diameter (cm); SFW= single fruit weight (g); FNH= fruit number per har- vesting; FWH= fruit weight per harvesting (g). NS= non significant. *, ** significant at P<0.05 and P<0.01, respectively. Table 1 - Analysis of variance for single and combined effects of different studied traits in greenhouse cucumber Source of variation Mean squares of studied traits DF DMF NNMF IL NMF MFP MFD SFW FNP FNH Season (S) 1 ** ** ** * ** ** NS ** ** Replication (season) 4 NS NS ** NS NS NS ** NS NS Chemical treatments (CH) 5 ** ** ** ** ** ** ** NS ** Number of spraying (S0) 1 ** * ** ** ** NS NS NS Leaf growth stage (LGS) 2 ** 9** NS NS ** ** NS NS NS** SP × CH 5 ** ** * ** NS ** ** NS NS S×SP 1 NS NS NS * NS * NS NS ** S×LGS 2 ** ** NS * NS ** NS NS NS CH×SP 5 * * NS ** NS NS * NS NS CH ×LGS 10 * ** NS NS NS NS NS NS NS SP×LGS 2 NS NS NS NS * NS NS NS NS S×CH×SP 5 NS NS NS NS NS NS NS NS NS S×CH×LGS 10 ** ** NS ** * NS NS NS NS CH×SP×LGS 10 NS NS NS NS NS NS * NS NS S×SP×LGS 2 NS NS NS NS * NS NS NS NS S×CHSP×LGS 10 NS NS NS NS NS NS NS NS NS Residual 140 NS NS NS NS NS NS NS NS NS Golabadi et al. - Effect of different physio-chemical factors on sex expression and fruit yield in greenhouse cucumber 209 showed that important traits related to change of sex expression such as days to male flowering, node number of first male flower, and number of male flower more affected by different interactions in con- trast to vegetative and yield related traits. Therefore selection of best treatments should be done based on other treatments. For example numbers of male flower are affected by silver application, leaf stage, number of spraying and all their interactions. There was no significant difference for other triple interac- tions and environment × chemical application × leaf stage of spraying × number of spray events interac- tion in all of the studied traits. Effects of season on the studied traits The comparison between two seasons showed that the first season had superior mean on days to male flowering, the node number of first male flower, male flower diameter and single fruit weight. It could be concluded that in the first season male flowers expressed later than second season and therefore DMF and NNMF were increased (Table 2). On the other hand, in second season the number of male flower was increased (from 36 to 100 male flowers) and also the time of male flowering was ear- lier that is related to high temperature and long days. This result showed that environmental condi- tions, especially low temperature, could retard male flower formation and confirmed also that cucumber is a thermophilic plant. Accordingly, Stankovic and Prodanovic (2002) reported that sowing season affected the number of male flowers. The interaction effect of season × chemical application × leaf stage was significant for number of male flower, days to male flowering and period of male flowering that all of them are related to sex expression, even though this interaction was not significant for vegetative related traits (Table 2). Effects of silver application on the studied traits In this study, different doses of chemical treat- ments [AgNO3 and Ag(S2O3)2 -3] were used. The mean comparisons showed that chemical application increased the mean values of days to male flowering (Table 3). There was a narrow variation in days to male flowering between different chemical treat- ments. The least day to male flowering was at a high concentrations of AgNO3 and Ag(S2O3)2 -3 (Table 3). This result showed that high concentrations of differ- ent chemicals agent caused earlier male flowering. DMF= days to male flowering; NNMF= the node number of first male flower; IL= the mean of internode length (cm); NMF= the number of male flower; MFP= male flowering period; MFD= male flower diameter (cm); SFW= single fruit weight (g); FNH= fruit number per har- vesting; FWH= Fruit weight per harvesting (g). Means followed by the same letter in each column were not significantly different at 0.05 level using LSD test. Table 2 - The mean comparison of different studied traits in cucumber in different seasons Season Traits DMF NNMF IL NMF MFP MFD SFW FNH FWH S1 23.08 a 20.17 a 7.13 b 36.24 b 12.20 b 5.46 a 73.17 a 1.93 b 138.80 b S2 19.89 b 11.96 b 7.63 a 99.78 a 13.35 a 4 74.95 a 3.60 a 272.96 a Table 3 - The effects of various chemical treatments application on studied traits in cucumber Means followed by the same letter in each row were not significantly different at 0.05 level using LSD test. Traits Control Chemical treatments AgNO3 Ag(S2O3)2 -3 100 ppm 200 ppm 300 ppm 200 ppm 500 ppm Days to male flowering 0.23 c 26.41 a 26.08 a 25.04 b 26.02 a 25.17 b Node number of male flower 0.13 b 20.18 a 19.11 a 19.23 a 19.08 a 18.68 a The mean of internode length 6.25 b 7.38 a 7.8 a 7.64 a 7.28 a 7.65 a The number of male flower 0.18 d 20.89 cd 68.07 b 129.76 a 44.79 bc 144.4 a Male flowering period 0.074 e 10.16 d 16.13 bc 17.366 ab 14.71 c 18.21 a Male flower diameter 3.29 d 5.36 c 5.45 bc 5.65 ab 5.36 c 5.71 a Single fruit weight 84.7 a 73.9 b 70.95 b 72.15 b 70.68 b 71.89 b Fruit number per harvesting 2.69 a 2.71 a 2.65 a 2.88 a 2.84 a 2.84 a Fruit weight per harvesting 219.74 a 205.88 a 190.87 a 212.9 a 199.72 a 206.22 a Adv. Hort. Sci., 2017 31(3): 205-214 210 The highest value for male flower diameter observed at 500 (ppm) of Ag(S2O3)2 -3 and 300 (ppm) of AgNO3 (Table 3). Therefore these concentrations of Ag (S2O3)2 -3 and AgNO3 could affect both vegetative and reproductive growth of flower. However there was no significant difference between all treatments for internode length. Hallidri (2004) reported that the greatest number of staminate nodes was produced on plants sprayed two or three times with 400 to 500 (ppm) of AgNO3. Rafee Kher et al. (2002) reported that 200 (ppm) of GA increased the length of intern- ode in cucumber. According our results the highest value for the number of male flower was denoted at 300 (ppm) of AgNO3 and 500 (ppm) of Ag(S2O3)2 -3 about 130 and 144 male flower, respectively. Law et al. (2002) reported that application of Ag2S2O3 pro- duced stamens in female flowers of white campion (Silene Latifolia) with longer filaments and larger anther locules. Yongan et al. (2002) reported that AgNO3 had more significant effects than GA3 on male flower production in summer squash. Stankovic and Prodanovic (2002) reported that increasing concen- trations of AgNO3 from 0.01% to 0.04% enhanced the number of male flowers in gynoecious lines, which is similar with the results obtained in the present study. Jadav et al. (2010) reported that ethrel (200 ppm) had the greatest effect on male flower production among the hormones they investigated (GA3, ethrel, naphthalene acetic acid, and absisic acid). Jutamanee et al. (1994) reported that the effects of different doses of GA3 and AgNO3 depended on the genotype and photoperiod. Similar to their results, AgNO3 in the present study induced the formation of male flowers in all concentration. Hallidri (2004) reported the concentration of 100 (ppm) of AgNO3 was inef- fective on male flower induction. Kalloo and Franklen (1978) reported that different doses of AgNO3 (50, 200, and 500 mg l-1) led to non-significant effects on male flower production, fruit weight and fruit num- ber. They showed similar trends in all the treatments. However in this study all concentrations of AgNO3 (100, 200, 500 ppm) produced male flower with sig- nificant differences. One explanation which might account for disparities observed between experi- ments was the difference in average quanta of solar radiation received by the plants during two different environmental conditions in two seasons. This result confirms the inducing effects of different chemical compounds used on male flower production. The highest value for single fruit weight was observed at control. Mean comparison showed that chemical application has reduced the mean of single fruit weight in comparison to control, because production of male flower in every treated plant will reduce female flower number in contrast to control. There were no significant differences between all treat- ment for fruit number per Harvesting and fruit weight per harvesting (Table 3). So, this result could demonstrated that chemical application has only effects on flower number and its sex expression. Effects of number of sprays on the traits studied Number of sprays (single or double) showed sig- nificant effects on days to male flowering, the node number of first male flower, the number of male flower and period of male flowering (Table 4). According to Table 4, double spraying treatment gave rise to higher mean values for days to male flower- ing, the node number of first male flower, the num- ber of male flower and period of male flowering. On the other hand, double spray treatment was found to affect only sex expression while it had no effect on the traits related to morphology and fruit yield. Effects of leaf stage on the studied traits The comparison of the mean values for the stud- ied traits at different stages of leaf growth [Five leaf DMF= days to male flowering; NNMF= the node number of first male flower; IL= the mean of internode length (cm); NMF= the number of male flower; MFP= male flowering period; MFD= male flower diameter (cm); SFW= single fruit weight (g); FNH= fruit number per har- vesting; FWH= fruit weight per harvesting (g). Means followed by the same letter in each column were not significantly different at 0.05 level using LSD test. Table 4 - The effects of single and double spraying on studied traits in cucumber Number of Spraying Traits DMF NNMF IL NMF MFP MFD SFW FNH FWH Single spraying (SP) 20.85 b 15.49 b 7.31 a 49.15 b 11.61 b 5.11 a 74.04 a 2.76 a 205.96 a Double spraying (DP) 22.13 a 16.64 a 7.44 a 86.86 a 13.94 a 5.16 a 74.08 a 2.77 a 205.82 a Golabadi et al. - Effect of different physio-chemical factors on sex expression and fruit yield in greenhouse cucumber 211 stage: LS1, Ten leaf stage: LS2, and Fifteen leaf stage: LS3] is presented at Table 5. The highest mean for DMF and NNMF were observed at LS3 stage (Table 5).This logical result is resulted from the most dis- tance (days) between planting time to fifteen leaf stage. On the other hand, the highest DMF at 15 leaf growth stage belonged to 200 ppm Ag(S2O3)2 -3 treat- ment (Fig. 1). El-Ghamriny et al. (1988) confirmed that sex differentiation in cucumber takes place at the 2-true leaf stage and that this was the best time for studying the effects of growth regulators on sex expression. There was no-significant difference between three leaf growth stages for number of male flower (Table 5). Therefore use of silver ions at different three growth stages had similar effects on m o d i f i c a t i o n o f f l o w e r s e x t y p e i n c u c u m b e r . Therefore, if it is necessary to separate monoecious or androecious from gynodioecious plant types in breeding programs (for example in line production), breeder could apply these chemical agents at 15 leaf stage, when sex form of plant has been expressed. Raymond (2004) proposed that 1000 ppm of GA3 at the 2-leaf stage had the best effect on male flower production with 3 hormone applications at 2-week intervals in cucumber. Also, the highest values for period of male flowering and male flower diameter were obtained at the five leaf stage; that is logical, since the period between male flower observation and end of male flowering in LS1 was higher than LS2 and LS3. As already mentioned, male flower diameter was greater at the 5-leaf stage, which might be caused by the higher production of pollen and the increased capacity for crossing in the breeding pro- grams. Effect of different factor interactions on number of male flower As the number of male flower trait is the most important trait related to male flower induction in cucumber, the different significant interaction effects in this trait was assayed (Table 2). The highest num- ber of male flower was recorded at double spraying in 500 ppm of AgS2O3 and double spraying at 300 ppm of AgNO3, respectively of 187.36 and 179 flow- ers (Fig. 2 a). In the two chemical treatments at high- er concentration, double spraying caused increasing o f m a l e f l o w e r n u m b e r , a l t h o u g h d i f f e r e n c e s between one and two spraying in low concentrations of chemical agents was not significant. The highest (119.2) and the least (24) number of male flower was observed at second season and the first season at fif- teen leaf stage, respectively (Fig. 2 b). In all stages, high number of male flower was observed in second season and pointed out significant differences with first season. Also, the most number of male flowers was obtained in high concentration of chemical treat- ments at the second season, however in all chemical treatments the number of male flowers was more than at second season (Fig. 2 c). Again, the second season showed the highest number of male flower with double spraying in contrast to first season, although double spraying in two seasons was more than single spraying (Fig. 2 d). The triple interaction effects of season, leaf growth stage and chemical treatment on male flower number showed that male flower number in all three leaf growth stages and all chemical treatments under second season were more than first season, specially high concentration DMF= days to male flowering; NNMF= the node number of first male flower; IL= the mean of internode length (cm); NMF= the number of male flower; MFP= male flowering period; MFD= male flower diameter (cm); SFW= single fruit weight (g); FNH= fruit number per har- vesting; FWH= fruit weight per harvesting (g). Means followed by the same letter in each column were not significantly different at 0.05 level using LSD test. Table 5 - The mean comparison for some of the studied traits in different leaf growth stages in cucumber Developmental stages Traits DMF NNMF IL NMF MFP MFD SFW FNH FWH Five leaf stage (LS1) 19.44 c 8.73 c 7.55 a 68.94 a 14.21 a 5.40 a 74.63 a 2.88 a 216.06 a Ten leaf stage (LS2) 21.40 b 17.10 b 7.40 a 63.47 a 12.40 b 5.05 b 73.00 a 2.69 a 195.85 a Fifteen leaf stage (LS3) 23.62 a 22.38 a 7.18 a 71.60 a 11.71 b 4.95 b 74.55 a 2.73 a 205.76 a Fig. 1 - The interaction effect of chemical treatment × leaf growth stage on the days to male flowering in cucumber Adv. Hort. Sci., 2017 31(3): 205-214 212 of chemical treatments in fifteen leaf growth stage under second season (Table 6). These results demon- strated that the second season had progressive effect on induction and development of male flower and environmental condition could be having a significant effect on this phenomenon. As mentioned before, in second season the temperature of greenhouse was higher than first season and also days were longer. These two reasons changed sex expression in second season more than first season. 4. Conclusions Phenotypic expression of sex determining loci in cucumber is strongly modified by environmental, chemical and hormonal factors. The results of the experiment revealed that AgNO3 and Ag(S2O3)2 -3 have similar and positive effects on the male expression of cucumber. This study showed that male flower induction was induced by all concentrations of silver ions. These two chemical agents could induce a high number of male flowers in 300 and 500 ppm concen- t r a t i o n [ A g N O 3 a n d A g ( S 2O 3) 2 - 3] , r e s p e c t i v e l y . Chemical treatments with silver ions as AgNO3 and Ag(S2O3)2 -3 application can, therefore, be recom- mended for enhancing male flower production in cucumber, if further study will confirm the accumula- tions of silver in the fruit is negligible for the human health. As the time of AgNO3 spraying in this experi- ment was only three times, so the toxicity of this ion was low in a short time of period. On the other hand, as this chemical agent only was applied in experi- mental greenhouse, therefore the fruits of treated Chemical treatment Seasons Season 1 Season 2 LS1 LS2 LS3 LS1 LS2 LS3 Control 0.00 n 0.00 n 0.00 n 0.00 n 0.00 n 0.00 n AgNO3 (100 ppm) 6.83 mn 8.00 mn 9.17 mn 42.42 i-n 31.75 j-n 27.17 k-n AgNO3 (200 ppm) 43.00 i-n 20.33 lmn 14.67 mn 64.33 h-m 123.33 d-h 142.75 d-g AgNO3 (300 ppm) 122.33 d-h 91.00 e-j 19.33 lmn 145.33 def 147.42 cde 253.17 a Ag(S2O3)2 -3 (200 ppm) 16.331 mn 26.00 k-n 19.00 lmn 84.58 f-k 38.00 i-n 84.67 f-k Ag(S2O3)2 -3 (500 ppm) 77.33 h-l 97.17 e-i 81.83 g-k 224.83 ab 178.75 bcd 206.5 abc LS1= Five leaf growth stage; LS2= Ten leaf growth stage; LS3= Fifteen leaf growth stage. Means followed by the same letter were not significantly different at 0.05 level using LSD test. Table 6 - The effects of interaction between chemical treatment, seasons and leaf growth stage on male flower number in cucumber Fig. 2 - The effect of different interactions (a, b, c and d) on the number of male flower in cucumber. Golabadi et al. - Effect of different physio-chemical factors on sex expression and fruit yield in greenhouse cucumber 213 plants were disposed from toxic effects of Ag ion. Also, the morphologic appearance of the cucumbers showed no abnormality after ion application. Overall, this result indicates that silver ion could change sex expression in higher temperatures and longer days with high concentration and when applied in 15 leaf stage for AgNO 3 and in 5 and 15 leaf stage for Ag(S2O3)2 -3, although this ion is able to modify sex form in lower concentration and earlier growth stage in cucumber. Although double spraying led to late ma l e f l o w eri n g, h o wever t h i s t rea t men t c o u l d i n c r e a s e n u m b e r o f m a l e f l o w e r s i g n i f i c a n t l y . Therefore, the double spray treatment is proposed for male flower production in greenhouse conditions. This can be beneficial for breeding programs that need high numbers of male flowers and flowering periods, especially for parents that have different periods of growth stage. The interaction effect showed that the highest number of male flowers was induced by applying Ag(S2O3)2 -3 (500 ppm) in 5 and 15 leaf stage in high t e m p e r a t u r e a b o u t m a x i m u m 3 5 ° C a t d a y a n d minimum 20˚C at night. Another finding of this study was that the number of male flowers showed signifi- cant increase with increasing doses of AgNO3 from 100 ppm to 300 ppm and Ag(S2O3)2 -3 from 200 ppm to 500 ppm. Therefore, high dosages of AgNO3 and Ag(S2O3)2 -3 should be selected for inducing male flow- ers in cucumber. The highest dosage of chemical agents led to decrease in days to male flowering and the mean of internode length that is related to the effect of silver ion on sex form earlier than low dosage of this ion. Leaf stage of the spray event was found to have a toxic effect on fruit. Over all, this result indicates that high concentration of silver ion could change sex expression at higher temperatures and longer days in 15 leaf stage for AgNO3 and in 5 and 15 leaf stage for Ag(S2O3)2 -3. Because the highest level of number of male flower and male flower peri- od and the lowest level of days to male flowering were obtained in these treatments. Finally, male flowering started about 3 weeks after treatment and lasted for a period of up to 3 weeks thereafter. 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