Journal of Applied Botany and Food Quality 90, 68 - 75 (2017), DOI:10.5073/JABFQ.2017.090.010 1 Department of Agronomy and Plant Breeding, College of Agriculture, Isfahan (Khorasgan) Branch, Islamic Azad University, Isfahan, Iran 2 Ataturk University, Agricultural Faculty, Department of Horticulture, Erzurum, Turkey Embryo and callus induction by different factors in ovary culture of cucumber Maryam Golabadi1*, Samaneh Ghanbari1, Kourosh Keighobadi1, Sezai Ercisli2 (Received June 23, 2016) * Corresponding author Summary Haploid and doubled haploid lines can be obtained in a short time using in vitro methods. In this study, unfertilized ovaries of cucum- ber were harvested and placed on semi-solid MS media as explants in Petri dishes. The thermal shock pretreatment, cucumber genotypes, hormonal combinations and AgNO3 were evaluated as experimental factors in three consecutive experiments. The results of first experi- ment revealed that the thermal shock pretreatment had a significant influence on embryo induction, and the highest rate of embryoge- nesis was produced in presence of thermal shock pretreatment and the NAA+2,4-D+KIN+BAP (0.5+0.7+1+1.8) mg/L hormonal combi- nation. The highest rate of callus induction was recorded in com- bination of absence thermal shock and the NAA+2,4-D+KIN+BAP (1.5+0.7+1+1.8) mg/L hormonal combination. According to the sec- ond experiment results, genotypes and the other hormonal combi- nations in media culture had highly significant effects on embryo and callus induction. The NBDC6*6/32441 genotype had the high- est effects on these traits. A combination of BAP (4 mg/L) and 2,4- D (1.5 mg/L) was found to be optimal for embryogenesis. In third experiment, the highest level of embryogenesis (24.93 %) and callus (24.19 %) induction were found in the local Iranian cultivar in com- parison to NBDC6*6/32441 genotype. Silver nitrate treatment had significant effects on the embryo and callus induction. The highest rate of embryo induction was recorded in presence of silver nitrate; however the absence of AgNO3 had a positive effect on the callus in- duction. In conclusion, the thermal shock pretreatment, silver nitrate, genotype and hormonal combination factors could play key roles in embryo and callus production, independently and simultaneously. Keywords: Ovary culture, hormone, AgNO3, genotype, cucumber Introduction Cucumber (Cucumis sativus L.) is one of the world’s most important vegetable crops belonging to the family Cucurbitaceae and the fruit is rich in phosphorus, potassium and oxalic acid. It is also called “Khiar” and has been used for 3,000 years in particular Asia and Africa (KHAN et al., 2015). One of important genetic materials in vegetable breeding programs is doubled haploid (DH) line. The use of completely homozygous DH lines would be considerably shortened breeding program. One of the most significant biotechnological advances in plant breeding has been the development of in vitro techniques to produce haploid plants that can be used to generate homozygous lines (EVANS et al., 2003). There are several processes leading to haploid and DH cucurbits. These include primarily haploid parthenogenesis (induced primari- ly by pollination with irradiated pollen), in vitro gynogenesis (during in vitro culture of ovules and ovaries) and in vitro androgenesis (during in vitro culture of microspores and anthers) (GALAZKA and NIEMIROWICZ-SZCZYTT, 2013). DH lines have been used successfully in plant breeding programs for many crops such as barley, wheat, maize, pepper, rice, and tobacco (THOMAS et al., 2003). The main advantage of double haploid lines is their complete homozygosis. This facilitates the phenotypic selection for qualitative and quantitative characters much easier. Thus, DHs can improve the efficiency and it is well known as time-consuming and labor-intensive. DH systems are widely applied in breeding (THOMAS et al., 2003) and genetic mapping (FORSTER and THOMAS, 2005). Ovary culture has been one of the approaches used to generate ho- mozygous lines for commercial production of F1 hybrid varieties and genetic studies (DIAO et al., 2009). It has been pointed out in many experiments. On the other hand, successful regeneration of haploids and doubled haploids in ovary or ovule culture depend on several fac- tors, i.e., pretreatment, genotype, female gametophyte development stage, and culture media composition (DIAO et al., 2009). DIAO et al. (2009) examined the effect of several factors in detail, i.e. the duration of thermal shock pretreatment at 35 ºC, the concen- trations of Thidiazuron (TDZ) and the presence of silver nitrate and investigated their effects on embryo formation in ovary culture of cucumber (Cucumis sativus L.). Their results showed that a thermal shock for 3 days at 35 °C at the start of the culture in contrast to 2 or 4 days would result in higher frequency of embryo formation. Thus the TDZ has a positive effect on embryo formation. The highest embryo formation frequency (72.7 %) was recorded when 0.04 mg/L TDZ into the induction medium. The results also revealed that was added to AgNO3 to the induction medium had no significant effect on frequency of embryo formation, however embryo germination period was shortened and the number of embryos increased in each ovary slice. MALIK et al. (2011) observed that thermal pre-treatment (4 °C) for 4 days increased embryo formation frequency (63.3 %) significantly in melon (Cucumis melo L.). Their experiment showed that TDZ (0.04 and 0.02) mg/L to induction medium significantly increased the number of responding ovaries (46.6 % and 65.83 %, respective- ly). The maximum number of plantlet regeneration (22.5 %) was achieved by culturing the ovary derived embryos on Murashige and Skoog medium (MS medium) supplemented with 0.6 mg/L 6-benzyl- aminopurine. The objectives of this study were 1) to evaluate the effect of different concentrations of hormones and also hormone combinations on cal- lus and embryo induction, 2) the influences of low and high tempera- ture pre-treatment on callus and embryo induction, 3) the effect of silver nitrate on embryo and callus formation and 4) the influence of genotypes on callus and embryo induction, after all, as a local variety of cucumber in Iran in terms of taste and aroma, it is highly regarded and highly marketable for the Iranian consumers. Material and methods Plant material The present experiment was carried out at the Plant Improvement and Seed Production Research Center of the Islamic Azad Univer- sity, Isfahan (Khorasgan) Branch, Iran, (51° 36´ longitude and 32° Ovary culture in cucumber 69 63´ latitude) during 2013-2014. In this experiment one local variety of open field cucumber and two Breeding lines (NBDC20*15/53211 and NBDC6*6/32441) of greenhouse cucumbers, that have been re- leased in this center, were used. All three genotypes were planted in a greenhouse and managed using standard agronomic practices. During the growing period, the diurnal greenhouse air temperature was kept within 25-30 °C and the nocturnal one within 19-21 °C with a relative humidity of about 60 %. Ovary culture The female flowers were picked one day before opening and trans- ferred to laboratory in the ice box. Most of the flowers were selected from first nodes (SHALABI, 2007). Unfertilized ovaries were isolated and surface sterilized by soaking in water for 3 min, followed by a 3 min washing in a water and detergent solution, and then rinsed three times in sterile distilled water. Ovaries were then placed in 10 % sodium hypochlorite for 10 min, rinsed three or four times in sterile distilled water and finally, rinsed in 70 % Ethanol for 5 min. The ovaries were sliced into 1 mm cross section under sterile con- ditions and placed carefully in a semi-solid medium in a petri dish. MS medium (MOQBELI et al., 2013) was used as basal medium in all experiments. All media were supplemented with 3 % (w/v) sucrose and solidified with 0.8 % (w/v) agar. The pH of the medium was ad- justed to 5.8 before autoclaving at 121 °C, 1.1 kg/cm2 for 20 min. All cultures were incubated at 24±2 °C under a 16/8 h (light/dark) photoperiod with light provided using cool-white fluorescent lamps with 3000 Lux. Experimental factors In this study, consecutive experiments were conducted with the indi- vidual results used for the preceding ones. Experiment 1: This experiment was performed with respect to the two factors; factor I consisted of thermal shock pretreatment (35 °C pretreatment for 4 days and without pretreatment), and factor II con- sisted of various concentrations of cytokinins and auxins (2, 4-D, Kin, IBA, NAA, BAP, IAA from 0.1 to 3 mg/L). Immediately after placing the ovary slices on induction medium, cultures were exposed to thermal shock pretreatment at 35 °C for 4 days. After the thermal pretreatment, all cultures were transferred to the 25 °C growth cham- ber. Control petri dishes (without pretreatment) had been transferred to the 25 °C growth chamber from the beginning. Experiment 2: According to the results of thermal shock pretreat- ment, the effect of genotype and different concentrations of hor- mones were investigated in the second experiment. The first factor consisted of two greenhouse inbred lines (NBDC515*20/3211 and NBDC6*6/32441) and the second factor consisted of 20 treatments of different concentrations and combinations of cytokinin and auxin plant growth regulators (2, 4-D, Kin, IBA, NAA, BAP, IAA from 0.1 to 3 mg/L). Experiment 3: The most suitable greenhouse inbred line (NBDC6*6/ 32441) was selected and compared with the cucumber local culti- var as the first factor. The experiment carried out in presence and absence of silver nitrate in the culture medium as the second factor. Finally, seven selected concentrations and combinations of different hormones (2, 4-D, Kin, IBA, NAA, BAP, TDZ from 0.1 to 0.8 mg/L) were considered as the third factor. After thermal shock pretreat- ment, all treatments were transferred to the 25 °C growth chamber. Statistical analysis All cultures were observed when one month had elapsed from the first day of ovary slices incubation. The frequency of embryo and callus formation was recorded based on the ratio of the number of ovaries responding to the total number of treated ovaries. The results of all three experiments were analyzed based on the factorial experiment in a completely randomized design in three replications and three petri dishes in every replicate. Analysis of variance was performed by SAS (Ver. 9) software, and mean comparisons were assayed by an LSD (Least Significant Differences) test at p≤0.05 Proportion values were transformed with arcsine transformation before they were used in the statistical analysis. Interaction mean comparisons were carried out by MSTATC software. Results and discussion Effect of thermal pretreatment and growth regulators on callus and embryo induction (first experiment) In the first experiment, analysis of variance indicated that thermal shock pretreatment (2 levels) and various concentrations of hormones (6 levels) had significant effects on callus induction and embryoge- nesis. Data presented in Tab. 1 showed that the most embryogenesis (38.98 %) occurred during the thermal shock pretreatment. DUBAS et al. (2014) revealed that the distribution of auxin changes during embryo development and this innovation depends on the tempera- ture-inducible in vitro culture conditions. Many factors effect on successful ovary culture. For example, thermal treatment, (i.e. high or low temperature) applied to ovary or during in vitro culture, may have a strong influence on embryo formation (YANG, 1982). In cu- cumber, 35 ºC thermal shock pretreatment was effective on ovary culture (GEMES et al., 2002). Specific changes that occur in cellular architecture during the switching from gametophytic to embryogenic pathway period following a heat shock treatment in Brassica napus had been reported earlier (SATPUTE et al., 2005). The DIAO et al. (2009) results showed that with high temperature (35 °C) treatments, the amount of embryogenesis increased and the most embryogenesis observed in 3-day treatments. The main objec- tive of the KOLEVA-GUDEVA et al. (2007) study was to evaluate the response of anthers from different genotypes to various media and heat and cold-shock pre-treatments regarding the direct somatic em- bryogenesis. Their findings indicated that the cold and heat shocks (at 7 °C and 35 °C respectively, both in darkness) as a precondition will push the anther cultures towards direct somatic embryogenesis. SHALABY (2007) found that ovules exposed to 32 °C for 4 days pro- duced the greatest number of gynogenic ovules in squash. KURT and EVANS (1998) revealed that the pretreatment of flower buds (at 4 °C for three days) significantly lowered the number of calli induced in solid medium but, it could not have that statistically significant effect in the liquid medium. The fact that temperature can control the development of sexual explants such as isolated microspores has been well documented in Brassica napus. In this plant lower temperatures favor asymmetric divisions, while heat treatment favors symmetric divisions (SEGUL- SIMARRO et al., 2003). The BUENO et al. (1997) results were revealed that stress, particularly heat shock, sucrose starvation or a combina- tion of both treatments could be the major and general sign of the inhibition of the normal gametophytic development of microspores and for the induction of the alternative embryogenic pathway. Tab. 1: Effects of thermal pretreatment on embryo and callus induction in experiment 1. Traits Thermal shock Control pretreatment Embryogenesis (%) 38.98 a 7.20 b Callus induction (%) 28.60 b 35.73 a Means followed by a common letter are not significantly different at 5 % level according to LSD test 70 M. Golabadi, Y. Ghanbari, K. Keighobadi, S. Ercisli Plant growth regulators such as cytokinin and auxin are the principal hormones in the plants involved in cell division and regulation of the differentiation (FEHER et al., 2003). Hormone combinations had a significant effect on callus induction and embryogenesis. Accord- ing to Fig. 1, combination of 2,4-D+KIN (1.5+1) mg/L showed the highest level of embryogenesis (52.76 %). However, this combination had no significant difference with that of NAA+2,4-D+KIN+BAP (0.8+0.8+0.8+2.5) mg/L. Among the similar somatic embryogenesis production factors, the 2,4-D is one of the highest efficiency, and thus it is used in the ma- jority of embryonic cell and tissue culture procedures (FEHER et al., 2003). Mean comparison results showed that the highest rate of callus in- duction (47.76 %) is observed in combination of 2,4-D+NAA+KIN+ BAP (0.5+0.5+0.5+1.5) mg/L, however it didn’t have any significant difference with NAA+2,4-D+KIN+BAP (1.5+0.7+1+1.8) mg/L com- bination (Fig. 1). The least embryogenesis and callus induction was observed in IBA+NAA+KIN+BAP (0.4+0.1+1+0.5) mg/L. In gene- ral, the medium containing 2,4-D and KIN had the highest rate of embryogenesis, and the medium containing NAA had the highest rate of callus induction. However, with increased BAP, the amount of callus induction decreases. Both auxins and cytokinins are essential for cell division and play a significant role in the meristem’ establish- ment and activity, however fluctuation of cytokinin and auxin ratios favor development of either root or shoot meristems (SUGIMOTO et al., 2011). In general, increasing the ratio of cytokinin to auxin results in a shift from root to shoot organogenesis (HILL and SCHALLER, 2013). The KOLI and MURTHY (2013) results showed that the maximum cal- lus induction observed in NAA+BAP (2+1) mg/L, 2,4-D+TDZ (2+2) mg/L and NAA+KIN (5+2) mg/L combinations and also reported that the more cytokinins and the less auxins the more callus induc- tion occurs. The analysis of variance indicated that there was a significant inter- action between the thermal shock pretreatment and the hormonal combinations of the culture medium. The highest rate of embryoge- nesis was obtained in the T4 hormone combination with thermal shock pretreatment, which is indicative of the fact that there is a significant difference between this treatment and other treatments (Tab. 2). The lowest rate of embryogenesis was observed in hormonal combinations IBA+NAA+KIN+BAP (0.4+0.1+1+0.5) mg/L and IAA+KIN+BAP (0.7+1.5+3) mg/L without thermal shock pretreatment. Hormonal combination of NAA+2,4-D+KIN+BAP (1.5+0.7+1+1.8) mg/L with- out thermal shock pretreatment showed the maximum callus produc- tion induction, although it did not have any significant difference with 2,4-D+NAA+KIN+BAP (0.5+0.5+0.5+1.5) mg/L hormonal combination (Tab. 2). Effect of genotypes and growth regulators on callus and embryo induction (second experiment) According to the results of the first experiment, the thermal shock pretreatment compared to no heat treatment had a better effect on the traits in question. Therefore, thermal shock pretreatment was ap- plied to all ovary cultures in this experiment. On the other hand, the second experimental hormonal compounds were designed on the basis of the best combination and concentration of PGRs in the first experiment. As the results of ANOVA showed, genotypes in terms of embryo- genesis and callus induction were significantly different. A com- parison of means between genotypes (Tab. 3) showed that genotype 6*6/32441 resulted in the highest rate of embryogenesis and callus production induction. In a similar study RAKHI et al. (2010) cultured cotyledon explants from seven varieties of pumpkin. According to their results, percentage of callus production induction was different among various genotypes. The separate analyses of the KURT and EVANS (1998) demonstrated that cultivar significantly influenced cal- lus production induction in both solid and liquid media. Mean comparison for hormonal composition showed that combi- nation of 2,4-D+BAP (1.5+4) mg/L had the highest rate of embryo- genesis (i.e. 37.5 %), although the difference among the four hor- monal combinations NAA+2,4-D+BAP (0.7+0.1+2.5) mg/L, NAA+ Fig. 1: Effects of different hormonal combinations on embryogenesis and callus induction in experiment 1. For every trait, letters compared separately. Means followed by a common letter are not significantly different at 5 % level according to LSD test T1: 2,4-D+KIN (1.5+1 mg·l-1); T2: IBA+NAA+KIN+BAP (0.4+0.1+1+0.5 mg·l-1); T3: IAA+KIN+BAP (0.7+1.5+3 mg·l-1); T4: NAA+2,4-D+KIN+BAP (0.5+0.5+0.5+1.5 mg·l-1); T5: NAA+2,4-D+KIN+BAP (0.8+0.8+0.8+2.5 mg·l-1); T6: NAA+2,4-D+KIN+BAP (1.5+0.7+1+1.8 mg·l-1) Tab. 2: Effects of thermal pretreatment and different hormonal combinations on embryogenesis and callus induction in experiment 1. Growth regulators Embryogenesis (%) Callus induction (%) Treatment Concentration Thermal shock Control Thermal shock Control (mg/L) pretreatment pretreatment 2,4-D+KIN 1.5+1 57.20 b 48.33 b 0.00 e 26.66 bc IBA+NAA+KIN+BAP 0.4+0.1+1+0.5 25.00 c 0.00 d 0.00 e 0.00 e IAA+KIN+BAP 0.7+1.5+3 51.66 b 0.00 d 0.00 e 50.00 b NAA+2,4-D+KIN+BAP 0.5+0.5+0.5+1.5 70.53 a 23.33 c 43.33 bc 52.20 ab NAA+2,4-D+KIN+BAP 0.8+0.8+0.8+2.5 23.33 c 25.00 c 0.00 e 21.66 de NAA+2,4-D+KIN+BAP 1.5+0.7+1+1.8 51.66 b 29.43 c 0.00 e 63.86 a For every trait, letters compared separately. Means followed by a common letter are not significantly different at 5 % level according to LSD test Ovary culture in cucumber 71 Tab. 3: Effects of genotypes on callus and embryo induction in experiment 2. Traits Genotype NBDC6*6 / 32441 NBDC20*15 / 53211 Embryogenesis (%) 17.44 a 12.30 b Callus induction (%) 70.91a 38.91b Means followed by a common letter are not significantly different at 5 % level according to LSD test BAP+KIN (1+1.5+2.5) mg/L, IBA+BAP+KIN (1+1.5+2.5) mg/L and NAA+2,4-D+BAP (1+2+1.75) mg/L was not significant (Fig. 2). The most callus induction was observed in hormone combinations of IAA+KIN (1+1.5) mg/L (T2) and 2,4-D+BAP (0.5+2) mg/L (T3), although their difference with KIN+BAP+IAA+IBA (0.5 mg/L) (T5) and 2,4-D (3 mg/L) (T4) hormonal combinations was not significant (Fig. 2). These results show that the presence of auxin in the culture medium increased the callus formation rate. Auxins have several effects including cell enlargement, cell division, root initiation and apical dominance. In the culture medium high concentration of auxin inhibits root formation and leads to callus formation. Culture me- dium is a principal factor controlling the induction and development of intact plants (MUKHAMBETZHANOV, 1997). In some culture media, the presence of low levels of auxin is essential for stimulating ex- plants to the embryos formation. This result is consistent with those of UGANDHAR et al. (2011), on the culturing of cucumber cotyledons. They showed that the presence of 0.5 mg/L IAA and 3 mg/L BAP or 3 mg/L KIN, helped to increase accountability and induce root formation from explants. Effect of genotype, absence or presence of AgNO3 and growth regulators on callus and embryo induction (third experiment) Based on the results of ANOVA and comparison of the hormones and genotypes in the second experiment, seven hormonal combina- tions and genotype NBDC6*6.32441 were selected, optimized and prepared for the third experiment. In the third experiment, one local and one greenhouse genotypes of cucumber (NBDC6*6/32441) were compared. The results showed that the genotypes used in this experiment (the local open field and greenhouse cucumber) made a significant dif- ference in the percentage of embryogenesis and callus induction (Tab. 4). According to Tab. 4, the local genotype with the 24.93 % had the highest rate of embryogenesis. It also had the highest cal- lus induction rate with 24.19 %. Therefore, the Iranian local variety was more successful in producing embryos and plantlets compared to the other genotype. SHALABY (2007) stated that differences among genotypes indicated that the genotype-specific results can be ob- tained depending on the genetic structure of the individually used hybrids. This agreed with the results obtained by DUMAS-DE-VALUX and CHAMBONNET (1986) in squash, LUX et al. (1990) in sugar beet, KOBAYASHI et al. (1993) in sweet potato and ALAN et al. (2004) in onion. Therefore, if the aim of tissue culture is to produce pure lines of local varieties, the results of this experiment will be applicable. The inbred line production in indigenous varieties could be used in order to transfer desirable traits such as fruit taste and quality of the commercial cultivars. Fig. 2: Effects of different hormonal combinations on embryogenesis and callus induction in experiment 2. For every trait, letters compared separately. Means followed by a common letter are not significantly different at 5 % level according to LSD test T1: KIN+2,4-D (1.25+0.5 mg·l-1); T2: KIN+IAA (1.5+1 mg·l-1); T3: BAP+2,4-D (2+0.5 mg·l-1); T4: 2,4-D (3 mg·l-1); T5: KIN+BAP+IAA+IBA (0.5+0.5+ 0.5+0.5 mg·l-1); T6: BAP+2,4-D+NAA (1.75+2+1 mg·l-1); T7: BAP+NAA (3+1.5 mg·l-1); T8: KIN+BAP+IAA (2.5+1.5+1 mg·l-1); T9: KIN+BAP+2,4-D (2.5+ 1.5+1 mg·l-1); T10: KIN+BAP+NAA (2.5+1.5+1 mg·l-1); T11: KIN+BAP+IBA (2.5+1.5+1 mg·l-1); T12: BAP+2,4-D (4+1.5 mg·l-1); T13: BAP+IBA (1+1.5 mg·l-1); T14: KIN+BAP+2,4-D (0.75+1.5+0.5 mg·l-1); T15:KIN+BAP+IBA (1.3+0.6+0.5 mg·l-1); T16: KIN+BAP+2,4-D (0.5+2+1.8 mg·l-1); T17: KIN+BAP+2,4-D (0.5+3.5+1.8 mg·l-1); T18: BAP+2,4-D+NAA (2.5+0.1+0.7 mg·l-1); T19: KIN+2,4-D+IAA+IBA (0.3+1.1+0.7+0.5 mg·l-1); T20: KIN+ BAP+IAA (3+0.75+0.5 mg·l-1) Tab. 4: Effects of genotypes on embryogenesis and callus induction in experiment 3. Traits Genotype NBDC6*6 / 32441 Local cultivar Embryogenesis (%) 11.93 b 24.93 a Callus induction (%) 3.57 b 24.19 a Means followed by a common letter are not significantly different at 5 % level according to LSD test 72 M. Golabadi, Y. Ghanbari, K. Keighobadi, S. Ercisli Few characteristics of AgNO3 such as stability, availability, water- solubility and specificity make it very useful for several applications in regulation used in the plant growth and morphogenesis in vivo and in vitro (KUMAR et al., 2009). In this research, silver nitrate had the significant effect on embryogenesis and induction of callus produc- tion. The highest rate of embryogenesis (15.71 %) was observed in the medium with silver nitrate, but the highest rate of the callus produc- tion induction (18.34 %) was obtained in the medium without silver nitrate (Tab. 5). Thus, the application of silver nitrate had a positive effect on the regeneration of ovary culture. Silver ions in the form of nitrate, such as AgNO3, play a major role in somatic embryogenesis efficient shoot and root formation (BAIS et al., 2001). AgNO3 is used in plant tissue culture as an ethylene antagonist. (BEYER, 1976a). The results of the KUMAR et al. (2009) were revealed that AgNO3 promotes embryogenesis by blocking the inhibitory effect of endo- genous ethylene on embryo formation (KUMAR et al., 2009). Embryo production were significantly increased in the majority of the mor- photypes by addition of silver nitrate to the medium, and AgNO3 treatment produced embryos in what would otherwise have been un- responsive morphotypes (DIAS and MARTINS, 1999). LI et al. (2013) conducted a study on the culturing of cucumber ovaries in two inbred lines cucumbers, four TDZ and three silver nitrate concentrations (0.5 and 10 mg/L). Their results showed that the biggest number of the regenerated plants produced in the presence of silver nitrate at a concentration of 10 mg/L. Their results also showed that the presence of silver nitrate in the culture media prolongs the time needed for the formation of the embryo. They stated that the presence of silver nitrate increased the number of the embryos, though it did not make a significance difference with culture without silver nitrate. There are various opinions and experimental documents on the silver subject. According to this hypothesis, the ethylene role in plants is inhibited by weak antagonists like CO2 and strong antagonists such as Ag com- pounds. These procedures are possibly due to oxidation of ethylene by a metal-ion enzyme system. Another theory is that silver nitrate inhibits the ethylene role by means of silver ions by decreasing the receptor capacity to bind ethylene, thus inhibiting the earlier steps of its own pathway (KUMAR et al., 2009). The culture media with different hormone concentrations and com- binations used in this experiment made a significant difference with respect to the measured traits. The highest rate of embryogenesis was observed in 2,4-D+KIN+BAP (1.5+1+1.8) mg/L (i.e. 20.83 %), and the difference between this treatment and other hormonal com- pounds like IBA+2,4-D+TDZ+KIN+BAP (0.8+1+1+0.5+1.5) mg/L and 2,4-D+TDZ+KIN (1.5+0.5+1.5 mg/L) was significant (Tab. 6). The highest rate of callus induction (i.e. 20.83 %) was detected in the hormonal compound NAA+2,4-D+TDZ+BAP (0.5+0.5+0.5+1.5) mg/L. However, the difference with hormonal compounds IBA+2,4- D+TDZ+KIN+BAP (0.8+1+1+0.5+1.5) mg/L, NAA+KIN+BAP (0.1+ 1.5+1.5) mg/L and IBA+2,4-D+BAP (0.8+1+0.8) mg/L was not sig- nificant (Tab. 9). The least rate of callus induction (i.e. 3.33 %) was observed in the hormonal compound 2,4-D+TDZ+KIN (1.5+0.5+1.5) mg/L. The interaction between genotype and silver nitrate was not signifi- cant for embryogenesis and callus induction traits. This means that regardless of what genotype has been used (local or commercial), silver nitrate is able to increase the embryogenesis. The interactive effect of genotype and hormonal combinations for these traits was significant. The local cultivar with combination of NAA+2,4-D+TDZ+BAP (0.1+0.3+1+2) mg/L, had the highest rate of embryogenesis, even though its difference with some other hormonal combinations and local and inbred line genotypes was not statisti- cally significant (Tab. 7). Tab. 5: Effects of presence or absence of silver nitrate on embryogenesis and callus induction in experiment 3. Traits Silver nitrate presence absence Embryogenesis (%) 15.71a 12.24 b Callus induction (%) 11.19 b 18.34 a Means followed by a common letter are not significantly different at 5 % level according to LSD test Tab. 6: Effects of different hormonal combinations on embryogenesis and callus induction in experiment 3. Growth regulators Embryogenesis (%) Callus induction (%) T1 4.17 c 20.83 a T2 15.00 ab 12.50 c T3 12.50 b 3.33 d T4 16.66 ab 20.83 a T5 20.83 a 10.83 cd T6 15.16 ab 20.16 ab T7 13.63 ab 14.54 ab Means followed by a common letter are not significantly different at 5 % level according to LSD test T1: IBA+2,4-D+TDZ+KIN+BAP (0.8+1+1+0.5+1.5 mg·l-1); T2: NAA+2,4- D+TDZ+BAP (0.1+0.3+1+2 mg·l-1); T3: 2,4-D+TDZ+KIN (1.5+0.5+1.5 mg·l-1); T4: NAA+2,4-D+TDZ+BAP (0.5+0.5+0.5+1.5 mg·l-1); T5: 2,4-D+ KIN+BAP (1.5+1+1.8 mg·l-1); T6: NAA+KIN+BAP (0.1+1.5+1.5 mg·l-1), T7: IBA+2,4-D+BAP (0.8+1+0.8 mg·l-1) The inbred line genotype showed the lowest embryogenesis rate with 3.33 % in hormonal treatment of IBA+2,4-D+TDZ+KIN+BAP (0.8+1+1+0.5+1.5) mg/L, the highest rate of callus induction was observed in local cultivar treated with NAA+2,4-D+TDZ+BAP (0.5+0.5+0.5+1.5) mg/L, although their difference from the two dif- ferent hormone and genotype treatments was not significant (Tab. 7). The interactive effect of silver nitrate and hormone combinations was significant for embryogenesis and callus induction. The biggest per- centage of embryogenesis (i.e. 25.88 %) was observed in the presence of silver nitrate and hormonal combination NAA+2,4-D+TDZ+BAP (0.5+0.5+0.5+1.5) mg/L (Fig. 3). This treatment made a significant difference with some other treatments. Silver nitrate has been used in several studies for its similar hormonal activity and transgender cucumber, which indicates the key role of this material in plant physiological changes in the vegetative and reproductive phases (STANKOVIC and PRODANOVIC, 2002; NAGAR et al., 2014; LAW et al., 2002). The lowest rate of embryogenesis (i.e. 1.65 %) was observed in the absence of silver nitrate and hormonal combination IBA+2,4- D+TDZ+KIN+BAP (0.8+1+1+0.5+1.5) mg/L. The highest rate of cal- lus induction was observed in the hormonal combination IBA+2,4- D+TDZ+KIN+BAP (0.8+1+1+0.5+1.5) mg/L without silver nitrate, although it did not make any statistically significant difference with A1T6, A2T4 and A2T7 treatments (Fig. 3). In many crop species, TDZ stimulated embryogenesis with a higher efficiency and frequency than other cytokinins or the combined treatments of auxins and cy- tokinins (VISSER et al., 1992; ZHANG et al., 2001). According to the analysis of variance results, interaction of genotype×silver nitrate×hormonal combinations was also significant for the rate of embryogenesis and callus induction. The highest rate of embryogenesis was observed in the treatment of local cultivar Ovary culture in cucumber 73 with 2,4-D+TDZ+KIN (1.5+0.5+1.5) mg/L in the presence of sil- ver nitrate. However, the difference among treatments with C1A1T2, C1A1T4, C1A2T5, C1A2T6, C2A1T2, C2A1T4, C2A1T5, C2A1T7, C2A2T2, C2A2T5 and C2A2T7 was not statistically significant (Tab. 8). Callus production induction in the local cultivar with hormonal com- bination of IBA+2,4-D+TDZ+KIN+BAP (0.8+1+1+0.5+1.5) mg/L in the absence of silver nitrate (C2A2T1) resulted in the highest rate (i.e. 13.56 %) of callus production which is significantly different from all of the other treatments with the exception of C2A2T5 and C2A1T6. Conclusion Haploids can be obtained using ovary as well as other sexual ex- plants. The current study reveals different factors for the efficient ovary embryogenesis induction in cucumber using various treat- ments. Our findings demonstrated that thermal shock (at 35 °C) as a pretreatment favors an embryogenesis pathway. Other experiments in the present study showed that rate of embryo production induction was different among genotypes, and that silver nitrate had a signifi- cant effect on the embryogenesis and callus production induction. In a nutshell, the results of the present assessment can be described and summarized as follows: A- The thermal shock pretreatment compared to no heat treatment produced a better effect on embryo and callus production induc- tion. Therefore, thermal shock pretreatment was applied to all ovary cultures. B- The results showed that the genotype 6*6/32441 produced the highest rate of embryogenesis and callus production induction, and combination of 2,4-D+BAP (1.5+4 mg · l-1) produced the highest rate of embryogenesis. C- The application of silver nitrate had a positive effect on the rege- neration of ovary culture. References ALAN, A.R., BRANTS, A., COBB, E., GOLDSCHNIED, P.A., 2004: Fecund gynogenic lines from onion (Allium cepa L.) breeding materials. 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Means followed by a common letter are not significantly different at 5 % level according to LSD test T1: IBA+2,4-D+TDZ+KIN+BAP (0.8+1+1+0.5+1.5 mg·l-1); T2: NAA+2,4- D+TDZ+BAP (0.1+0.3+1+2 mg·l-1), T3: 2,4-D+TDZ+KIN (1.5+0.5+1.5 mg·l-1); T4: NAA+2,4-D+TDZ+BAP (0.5+0.5+0.5+1.5 mg·l-1), T5: 2,4-D+ KIN+BAP (1.5+1+1.8 mg·l-1); T6: NAA+KIN+BAP (0.1+1.5+1.5 mg·l-1), T7: IBA+2,4-D+BAP (0.8+1+0.8 mg·l-1) Tab. 7: Interaction effects of different hormonal combinations and genotype on embryogenesis and callus induction in experiment 3. Growth regulators Embryogenesis (%) Callus induction (%) Treatment Concentration (mg/L) Greenhouse line Local cultivar Greenhouse line Local cultivar BA+2,4-D+TDZ+KIN+BAP 0.8+1+1+0.5+1.5 3.33 f 6.16 ef 1.65 d 38.33 a NAA+2,4-D+TDZ+BAP 0.1+0.3+1+2 11.21 b-f 23.68 a 0.00 d 25.55 b 2,4-D+TDZ+KIN 1.5+0.5+1.5 6.10 ef 19.28 abc 0.00 d 6.60 cd NAA+2,4-D+TDZ+BAP 0.5+0.5+0.5+1.5 15.71 a-e 18.01 a-d 0.00 d 42.66 a 2,4-D+KIN+BAP 1.5+1+1.8 20.08 ab 18.18 a-d 8.46 cd 11.53 cd NAA+KIN+BAP 0.1+1.5+1.5 21.48 a 9.81 c-f 1.66 d 39.25 a IBA+2,4-D+BAP 0.8+1+0.8 8.15 d-f 20.10 ab 8.85 cd 17.60 bc For every trait, letters compared separately. Means followed by a common letter are not significantly different at 5 % level according to LSD test 74 M. Golabadi, Y. Ghanbari, K. Keighobadi, S. Ercisli FORSTER, B.P., THOMAS, W.T.B., 2005: Doubled haploids in genetics and plant breeding. In: Janick, J. (ed.), Plant Breeding Reviews, 57-88, Vol- ume 25. 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Embryogenesis (%) Callus induction (%) Greenhouse line (C1) Local cultivar (C2) Greenhouse line (C1) Local cultivar (C2) Presence Absence Presence Absence Presence Absence Presence Absence Ag (A1) Ag (A2) Ag (A1) Ag (A2) Ag (A1) Ag (A2) Ag (A1) Ag (A2) T1 6.66 e-g 0.00 g 9.03 d-g 3.30 fg 0.00 h 3.30 gh 23.43 c-f 56.13 a T2 22.43 a-d 0.00 g 19.43 a-c 27.93 ab 0.00 h 0.00 h 22.13 c-g 28.96 cd T3 3.16 fg 9.03 d-g 31.43 a 7.13 e-g 0.00 h 0.00 h 3.13 gh 9.80 e-h T4 28.10 ab 3.33 fg 23.66 a-c 12.36 c-g 0.00 h 0.00 h 34.56 bc 50.93 a T5 12.23 c-g 27.93 ab 19.03 a-e 17.33 a-f 0.00 h 16.93 c-h 5.53 f-h 17.53 ch T6 14.53 b-f 28.43 ab 10.13 c-g 9.50 d-g 0.00 h 3.16 gh 50.10 ab 28.40 c-e T7 6.46 e-g 9.83 cg 20.16 a-e 20.00 a-e 0.00 h 17.70 c-h 15.58 d-h 20.20 c-e For every trait, letters compared separately. Means followed by a common letter are not significantly different at 5 % level according to LSD test. T1: IBA+2,4-D+TDZ+KIN+BAP (0.8+1+1+0.5+1.5 mg·l-1); T2: NAA+2,4-D+TDZ+BAP (0.1+0.3+1+2 mg·l-1); T3: 2,4-D+TDZ+KIN (1.5+0.5+1.5 mg·l-1); T4: NAA+2,4-D+TDZ+BAP (0.5+0.5+0.5+1.5 mg·l-1); T5: 2,4-D+KIN+BAP (1.5+1+1.8 mg·l-1); T6: NAA+KIN+BAP (0.1+1.5+1.5 mg·l-1); T7: IBA+2,4-D+BAP (0.8+1+0.8 mg·l-1) Growth regulators Ovary culture in cucumber 75 Address of the corresponding author: Maryam Golabadi, Department of Agronomy and Plant Breeding, College of Agriculture, Isfahan (Khorasgan) Branch, Islamic Azad University, Isfahan, Iran E-Mail: golabadim@gmail.com, m.golabadi@khuisf.ac.ir © The Author(s) 2017. This is an Open Access article distributed under the terms of the Creative Commons Attribution Share-Alike License (http://creative- commons.org/licenses/by-sa/4.0/).