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http://journals.tubitak.gov.tr/biology/

Turkish Journal of Biology Turk J Biol(2016) 40: 571-579© TÜBİTAKdoi:10.3906/biy-1502-55

Induction and development of anther-derived gametic embryos in cucumber (Cucumis sativus L.) by optimizing the macronutrient and agar concentrations in culture medium

Mohammad Reza ABDOLLAHI1,*, Samereh NAJAFI1, Hasan SARIKHANI2, Sayyed Saeed MOOSAVI1

1Department of Agronomy and Plant Breeding, Faculty of Agriculture, Bu-Ali Sina University, Hamedan, Iran2Department of Horticultural sciences, Faculty of Agriculture, Bu-Ali Sina University, Hamedan, Iran

* Correspondence: m.abdollahi@basu.ac.ir

1. IntroductionCucumber, a plant belonging to the family Cucurbitaceae, is among the top ten vegetables produced globally (Plader et al., 2007). This species is believed to have been domesticated in India for 3000 years and in eastern Iran and China for probably 2000 years (Aydemir, 2009). In cucumber, the development of homozygous parental lines using the traditional self-pollination method takes from 6 to 8 years (Gémes-Juhász et al., 2002). Therefore, the development of an efficient production system of doubled haploids and its further application in breeding programs could reduce the time required for cultivar development. In the family Cucurbitaceae, haploid plants have recently been obtained by different methods such as in vitro gynogenesis in summer squash (Cucurbita pepo L.) (Shalaby, 2007), in situ induction of haploid embryos via irradiated pollen in winter squash (Cucurbita maxima Duchesne ex Lam.) (Kurtar and Balkaya, 2010), parthenogenesis method using gamma-irradiated pollen in snap melon (Cucumis melo L.)

(Godbole and Murthy, 2012), and anther culture method in watermelon (Citrullus lanatus L.) (Abdollahi et al., 2015). Several methods including spontaneous parthenogenesis (Aalders, 1958), in vitro gynogenesis (Diao et al., 2009; Li et al., 2013), pollination with irradiated pollen (Claveria et al., 2005; Lotfi and Salehi, 2008), and in vitro androgenesis (Ashok Kumar et al., 2003, 2004; Ashok Kumar and Murthy, 2004; Song et al., 2007; Chen et al., 2008) were also used to obtain haploid and double haploid (DH) plants in cucumber. Anther culture was reported as an efficient method for haploid production in many plants species (Ferrie et al., 1995). This method would allow breeders to release new cucumber lines, especially for resistance to disease, more quickly and efficiently (Ashok Kumar et al., 2003). The production of haploids in cucumber through anther culture has been reported by several researchers. The first report of cucumber anther culture was limited to the production of calli without any plantlet regeneration (Lazarte and Sasser, 1982). Ashok Kumar et

Abstract: Anther culture response of a cucumber cultivar (Beta Alpha) and three Iranian landraces of cucumber (Basmenj, Isfahani, and Korki) on MS culture media with an altered macronutrient strength (half, full, 1.5-fold, and double-strength) or various concentrations of agar (0, 3.5, 7, and 14 g L–1) was evaluated in two separate experiments. The effect of different concentrations of agar on the development of callus-derived embryos was also assessed. The androgenic potentials of various cucumbers were significantly (P < 0.01) influenced by the different macronutrient strengths and agar concentrations, although anther culture responses could vary according to the cucumber type. A doubled strength of macronutrients in culture medium resulted in the highest percentage of callogenesis (100%) in Isfahani cucumber, while use of full strength of macronutrients produced the highest percentage of embryogenic calli (83.33%) in the mentioned landrace compared to other tested forms. The maximum number of gametic embryos (1.26 and 1.23 embryos/anther) differentiated from Isfahani anther cultures on full- and half-strength MS medium, respectively. In the second experiment, the highest percentages of callogenesis were obtained in MS culture media solidified with 14 g L–1 and 7 g L–1 agar for Basmenj, with 3.5 g L–1 agar or liquid medium for Isfahani, with 7 g L–1 agar for Beta Alpha, and with 14 g L–1 agar for Korki. Culture media solidified with 7 g L–1 agar produced the highest percentage of embryogenic calli compared to other treatments. Anther culture of Korki cucumber on culture medium with 14 g L–1 agar produced the maximum number of embryos per anther compared to other cucumbers. Improved production of mature somatic embryos (torpedo and cotyledonary embryos) was achieved with higher agar concentrations (7 and 14 g L–1) for the cucumber cultivar and all landraces.

Key words: Cucumis sativus L., anther culture, macronutrients, agar concentration, embryo maturation

Received: 22.02.2015 Accepted/Published Online: 26.06.2015 Final Version: 18.05.2016

Research Article

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al. (2003) studied the effect of medium composition and cold pretreatments on the response of cultured cucumber anthers. These researchers obtained the optimal rate of embryogenic calli/embryo production on B5 medium supplemented with 2.0 mM 2,4-dichlorophenoxyacetic acid (2,4-D) and 1.0 mM 6-benzylaminopurine (BAP) following a 2-day cold shock (4 °C) and 87% and 70% haploid plants were obtained in Calypso and Green Long cultivars, respectively. Other researchers (Ashok Kumar and Murthy, 2004; Ashok Kumar et al., 2004) used various kinds of sugars, amino acids, and polyamines in induction medium and improved the anther culture method for cucumber. Their experiments resulted in a low anther culture efficiency of 1.6 embryos/anther (Ashok Kumar et al., 2004). Furthermore, Song et al. (2007) evaluated the effect of pretreatment, embryonic callus induction media, preculture conditions, embryo induction media, embryo germination media, and genotype on efficiency of DH plant production in cucumber and, as a result, developed a protocol for anther culture that gave the highest efficiency (three embryos per anther and 0.9 DH plants per anther). The genotype plays an important role in androgenic response of plant species in vitro. In anther culture studies, both species within a genus and cultivars of the same species have been shown to have different responses (Góralski et al., 1999). Thus, anther culture procedures should be optimized for each genotype (Atanassov et al., 1995). The success of plant tissue culture is greatly influenced by the composition of the culture medium (George et al., 2008). Further improvements in embryogenic callus growth and embryo production could be expected by optimizing inorganic salts in the basal medium. However, information on the mineral nutrition of embryogenic callus, especially in anther cultures, is insufficient (Ammirato, 1983). Agar, the most common gelling agent, has traditionally been used in anther and plant tissue culture studies (Calleberg and Johansson, 1996). For cucumber anther culture, the most commonly used solidifying agent has been agar at a concentration of 8 g L–l (Ashok Kumar et al., 2003; Ashok Kumar and Murthy, 2004; Song et al., 2007); however, no studies have determined the effects of agar concentration on response of cucumber anther cultures. Embryo maturation is a key factor affecting plant conversion in somatic embryogenesis systems (Ammirato, 1987). Many researchers have been previously studied the maturation of induced somatic embryos in various plant species (Groll et al., 2002; Márquez-Martín et al., 2011; Yadollahi et al., 2011). In cucumber anther culture, the majority of induced embryos in direct as well as callus-mediated embryogenesis remain in the first stages (globular and heart stages) of embryo development and the low conversion rate of these embryos into plants is the main bottleneck in plant regeneration (Ashok Kumar et

al., 2003). Therefore, an embryo maturation treatment is necessary for maturation and subsequent development of embryos into normal plantlets. The objective of this study was to determine the effect of macronutrient and agar concentrations on callogenesis and embryogenesis from cultured anthers of different cultivars and landraces of cucumber. In the second part of this study, a further experiment was conducted to compare the effect of agar concentrations on the maturation of formed embryos from cultured anthers of a cucumber cultivar and landraces.

2. Materials and methods2.1. Plant materialA cultivar of C. sativus L., Beta Alpha, and three Iranian landraces of cucumber (Isfahani, Basmenj, and Korki) were used in this experiment. The seeds of Beta Alpha were procured from Delta Green South Agricultural Company, Tehran, Iran. The seeds of the three cucumber landraces of Isfahani, Basmenj, and Korki were obtained from the Agricultural Research Center of Isfahan, Tabriz, and Kermanshah, respectively. These landraces are cultivated throughout the mentioned regions due to their qualitative features and taste of the fruit. Therefore, the production of DH plants in these landraces helps to accelerate the breeding programs for cultivar development. Plants were grown in the experimental plot of the Department of Agronomy and Plant Breeding, Bu Ali Sina University, Hamedan, Iran, using standard agronomic practices.2.2. Anther culture procedureMale flower buds (10–15 mm in length) containing microspores at the middle to late uninucleate stages were collected from healthy mother plants of 35–45 days old between 0900 and 1000 hours on sunny days. The suitable stage of the microspores was determined by microscopic observation of anthers squashed in acetocarmine (1 g of carmine in 100 mL of glacial acetic acid 45%) (Dunwell, 1985). For surface disinfection, the male flower buds were first immersed in 70% (v/v) alcohol for 1 min followed by 2.5% (v/v) sodium hypochloride solution for 5 min. The anthers were then separated and placed in 100 × 15 mm petri dishes (10 anthers per dish) containing 20 mL of culture medium. Anther cultures were incubated at 25 ± 2 °C under a 16-h photoperiod with a light source providing 60 µmol m–2 s–1 light intensity. Calli, 30–35 days after culture initiation, were transferred onto fresh media for embryo induction. Callus and embryo induction media used in our experiments were MS (Murashige and Skoog, 1962) supplemented with growth regulators of 2,4-D (2.26 µM), BAP (4.44 µM), and kinetin (4.64 µM) for callus induction and with 0.54 µM α-naphthalene acetic acid (NAA) and 13.32 µM BAP for embryo induction on calli. Media were adjusted to pH 5.7 prior to autoclaving at 121 °C for 20 min.

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2.2.1. Experiment I: Effect of macronutrient concentration on callus and embryo induction In this experiment, anthers from the tested cultivar and landraces of cucumber were placed on an induction medium containing either half, full, 1.5-fold, or double-strength MS macronutrients and supplemented with normal strength MS micronutrients and vitamins, 7 g L–l agar, and 30 g L–l sucrose. 2.2.2. Experiment II: Effect of agar concentration on callogenesis and embryo induction and maturationIn this experiment, the effect of gelling agent of agar at different concentrations was studied on induction and maturation of gametic embryos in anther culture of different cucumber cultivar and landraces. Callus and embryo induction MS medium supplemented with 30 g L–l sucrose was solidified with agar (A-1296, Sigma-Aldrich) at 0, 3.5, 7, and 14 g L–l and used in this experiment. The maturation of induced embryos was assessed on the same embryo induction medium containing various concentrations of agar (0, 3.5, 7, and 14 g L–l). Counts of embryos at the pretorpedo, torpedo, and cotyledonary stages of development were made after 60 days of culture.2.3. Cytological analysisChromosome counts were carried out on root tip cells of cucumber diploid seedlings and callus-derived embryos according to the method Darlington and La Cour (1976). Briefly, both root tips (1.0 ± 0.2 cm in length) and callus-derived embryos were collected, treated with 0.05% (w/v) colchicine for 3 h in the dark at room temperature, and rinsed three times with distilled water. The samples were then fixed in 3:1 ethanol/glacial acetic acid for 24 h and stored in 70% ethanol at 4 °C prior to chromosome number count. Fixed root tips and callus-derived embryos were rinsed in double-distilled water for 15 min and then strained in 1% (w/v) acetocarmine for 12 h. Sample tissues were squashed in a drop of 45% acetic acid. Chromosome spreads in the metaphase stage were photographed with a Canon digital camera attached to a Motic binocular microscope (100×).2.4. Statistical analysisBoth experiments were conducted factorially based on a completely randomized design with 3 replications (each petri dish containing 10 cultured anthers was considered as one replication). In each experiment, four forms of cucumber (Beta Alpha, Isfahani, Basmenj, and Korki) were considered as the first factor and different concentrations of macronutrients (experiment I) and agar (experiment II) were the second factors. The traits of callogenesis percentage (number of anthers producing callus), embryogenic callus percentage (number of anthers producing calli with embryos, EC%), and number of embryo per anther (number of induced embryos per each petri dish/10, ME/A) were measured in each experiment.

Percentage data were transformed using square root transformation prior to analysis. Analysis of variance was carried out using SPSS 16.0 and mean comparisons were accompanied by Duncan’s multiple range test at the 5% probability level.

3. Results and discussionMale flower buds (10–15 mm, Figure 1A) containing anthers (Figure 1B) with microspores at the middle to late uninucleate stages (Figure 1C) became swollen in 2–3 weeks on MS culture medium and induced calli in the next 2 weeks (Figures 1D and 1E). Globular embryos were induced on embryogenic calli in another 2 weeks (Figure 1F). The next stages of embryo development such as torpedo (Figure 1G) and cotyledonary (Figure 1H) embryos started to appear in 3–4 weeks from embryo induction. We performed chromosome counts in root tip cells of cucumber seedlings (2n = 2x = 14) as a control, observing cells with 14 chromosomes (Figure 1I). Ten randomly selected cotyledonary embryos were used for cytological analysis in each cucumber cultivar or landrace. In Beta Alpha, 8 of 10 embryos showed the haploid chromosome number (n = 7, Figure 1J) and the remaining two embryos were diploid, whereas in Basmenj, 6 of 10 embryos were haploid and 4 embryos were diploid. In the other two cultivars, Isfahani and Korki, 7 of 10 embryos were haploid for each cultivar while the remaining three embryos were diploid.3.1. The effects of macronutrients on callus and embryo inductionThe androgenic potential of the four cucumber types was evaluated in MS culture media with different macronutrient compositions. Results showed significant differences (P < 0.01) among various cultivars or landraces of cucumber for callogenesis (%), EC (%), and ME/A. This is in agreement with the observations of other researchers (Ashok Kumar et al., 2003; Song et al., 2007), who proved genotypic effects on the ability of cucumber anthers to be cultured in vitro. In earlier studies on haploid production via androgenesis techniques, significant difference in callus formation using varieties or crosses was observed. In some species, only a few genotypes have responded of the many that were tested (Sopory and Munshi, 1996; Custódio et al., 2005). Thus, culture procedure should be optimized for each genotype so that this genotypic effect can be decreased or even minimized (Atanassov et al., 1995). The traits of callogenesis (%), EC (%), and ME/A were found to be significantly (P < 0.01) influenced also by different concentrations of macronutrients in culture medium. The interaction effects of cultivar × macronutrient were also significantly different (P < 0.01) for all traits studied in this experiment. An increase in the strength of macronutrients from half to double in the MS

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medium resulted in a significant enhancing of callogenesis percentage in the landraces of Korki and Isfahani, while this increment was obtained with 1.5-fold macronutrient strength in Beta Alpha (Table 1). In landrace Basmenj, a reduction in callogenesis percentage was observed when double-strength macronutrients were used in MS medium. In the last landrace, MS medium containing half-strength macronutrients produced the highest percentage of callogenesis (90%, Table 1). Full-strength macronutrients

in MS medium revealed the highest percentages of EC in the landraces of Isfahani and Korki (83.33% and 70%, respectively), whereas the highest EC% (70%) in Basmenj was obtained with 1.5-fold strength of macronutrients in culture medium (Table 1). The highest frequency of embryos was formed on cultured anthers of Isfahani with full and half strengths of macronutrients in culture medium (1.26 and 1.23, respectively). This result in Isfahani cucumber is in agreement with the findings of Groll et

Figure 1. Callus induction and gametic embryogenesis in cultured anthers of Cucumis sativus L. (A) A suitably sized (10–15 mm) male flower bud for cucumber anther culture. (B) A swollen anther of cucumber on induction medium. (C) A cucumber microspore in late uninucleate stage proper for anther culture. (D and E) Callus induction from anther of cucumber. (F) Globular stage embryo of cucumber. (G) Torpedo stage embryo of cucumber. (H) Cotyledonary stage embryo of cucumber. (I) Chromosome preparation of a root tip cell of a diploid cucumber seedling (2n = 2x = 14). (J) A cucumber haploid cell prepared from callus-derived embryo with 7 chromosomes (n = x = 7).

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al. (2002), who obtained the greatest number of cassava somatic embryos from nodular embryogenic calli on either half- or full-strength macronutrients in MS culture media. In the landraces of Basmenj and Korki, increasing the macronutrient strength of the culture medium from half to 1.5-fold enhanced the ME/A, while use of MS culture medium with double-strength macronutrients markedly reduced the ME/A in landraces Basmenj, Isfahani, and Korki. These results are in agreement with the results of Choi et al. (1998), whereas increment in the concentration of total macrosalt in culture medium up to 2 times resulted in the highest somatic embryo formation in Panax ginseng, while use of 4, 6, and 8 times concentrations of macrosalts reduced the frequency of somatic embryos in this species. There were no significant differences (P < 0.05) among the different strengths of macronutrients for ME/A in Beta Alpha.

It has been reported that high salt level in the medium can affect plant growth in three ways: ion toxicity, ionic imbalances, and depressed water potential (Flowers et al., 1977). Macronutrient salts in MS medium provide six

major elements, N, P, K, Ca, Mg, and S. It also has very high nitrogen content in the form of nitrate and ammonium. In a previous study (Choi et al., 1998), to investigate the effects of macrosalt stress on somatic embryogenesis in Panax ginseng, macrosalts (NH4NO3, KNO3, KH2PO4, MgSO4.7H2O, or CaCl2.2H2O) were added individually at 3, 6, 12, and 24 times the standard salt concentration in MS medium. In these experiments, the highest frequency of somatic embryo formation was obtained when 61.8 mM NH4NO3 (3 times the standard concentration in MS culture medium) was used in the culture medium. It has been reported that ammonium compounds as nitrogen sources are highly effective for somatic embryogenesis (Walker and Sato, 1981). 3.2. Effect of agar concentrations on callus and embryo inductionSignificant differences (P < 0.001) in the capacity of callus, embryogenic callus, and embryo formation were observed among the cucumber cultivar and the landraces and different concentrations of agar in MS medium. There was also a significant interaction (P < 0.001) between the

Table 1. Effect of macronutrient concentration on callus and embryo induction parameters in anther culture of a cultivar and different landraces of cucumber (Cucumis sativus L.).

Anther culture efficiencyMacronutrient concentration (×)Cultivar Mean number of embryos

per anther (ME/A) ± SEEmbryogenic callipercentage (EC%)Callogenesis (%)

0.17 ± 0.07 e10.00 e50.00 f0.5

Beta Alpha0.13 ± 0.03 e13.33 e83.33 a–d1.0

0.17 ± 0.03 e13.33 e90.00 a–c1.5

0.13 ± 0.03 e13.33 e76.67 c–e2.0

0.50 ± 0.06 d43.33 d90.00 a–c0.5

Basmenj0.67 ± 0.12 b–d43.33 d83.33 a–d1.0

0.83 ± 0.03 a–d70.00 ab83.33 a–d1.5

0.13 ± 0.03 e10.00 e70.00 de2.0

1.23 ± 0.19 a63.33 bc90.00 a–c0.5

Isfahani1.26 ± 0.12 a83.33 a96.67 ab1.0

1.03 ± 0.08 ab73.33 ab93.33 a–c1.5

0.20 ± 0.06 e20.00 e100.00 a2.0

0.63 ± 0.18 cd50.00 cd63.33 ef0.5

Korki0.93 ± 0.24 a–c70.00 ab80.00 b–d1.0

1.00 ± 0.17 a–c53.33 cd90.00 a–c1.5

0.13 ± 0.03 e13.33 e93.33 a–c2.0

Means followed by the same letter(s) indicate nonsignificant differences at P = 0.05.  

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agar concentration in culture medium and genotype of cucumber for all callus and embryo formation parameters studied in this experiment. The highest percentages of callogenesis in the Basmenj landrace were obtained in MS culture media solidified with 7 and 14 g L–1 agar (96.67% and 93.33%, respectively), while in landrace Isfahani, cultured anthers in liquid medium and half-strength medium (3.5 g L–1 agar) resulted in a higher percentage of callogenesis (90%) compared to other agar concentrations (Table 2). Using of 7 g L–1 agar in anther culture medium of the Beta Alpha cultivar and 14 g L–1 agar in Korki anther culture medium produced the highest frequency of callogenesis (86.67%, Table 2). The frequency of calli producing embryos (EC%) was enhanced in Beta Alpha and Basmenj cucumbers when 14 g L–1 agar was used in culture medium, whereas in the other two landraces, Isfahani and Korki, the higher EC% was observed with 7 g L–1 agar in culture medium (70% and 66.67%, respectively, Table 2). The maximum numbers of embryos per anther were obtained in landraces Korki and Isfahani from

culture media solidified with 7 g L–1 agar (1.33 and 0.83 embryos, respectively, Table 2). This result is consistent with a study of Landyman et al. (1992) that yielded more somatic embryos in cucumber on a medium gelled with 0.7% agar rather than media solidified with 1.4% or 0.35% agar. In Basmenj and Beta Alpha cucumbers, use of 14 g L–1 in MS culture medium resulted in maximum ME/A (0.87 and 0.25 embryos, respectively, Table 2), compared to other concentrations of agar. In agreement with this result, in a study by Grewal et al. (2009) on anther culture of chickpea (Cicer arietinum L.), embryo induction and development was obtained in culture media solidified with 16 g L–1 agar. Results of this study show that optimum concentration of agar in culture medium is different for each cucumber cultivar. Similarly, Jaramillo and Summers (1990) showed that tomato cultivars respond differently to anther culture in culture media containing various concentrations of agar. Agars contain small amounts of macro- and microelements, particularly calcium, sodium, potassium, phosphate (Beruto et al., 1995), carbohydrates,

Table 2. Effect of agar concentration on callus and embryo induction parameters in anther culture of a cultivar and different landraces of cucumber (Cucumis sativus L.).

Anther culture efficiencyAgarconcentration(g L–1)

Cultivar Mean number of embryo per anther (ME/A) ± SE

Embryogenic calli percentage (EC%)Callogenesis (%)

0.00 ± 0.00 h0.00 i0.00 f0

Beta Alpha0.00 ± 0.00 h0.00 i0.00 f3.5

0.13 ± 0.03 gh13.33 gh86.67 a7

0.25 ± 0.05 fg25.00 ef45.00 e14

0.40 ± 0.06 ef20.00 fg53.33 de0

Basmenj0.53 ± 0.09 e33.33 de63.33 cd3.5

0.80 ± 0.06 bc50.00 b–d96.67 a7

0.87 ± 0.07 bc63.33 a–c93.33 a14

0.50 ± 0.06 e43.33 d90.00 a0

Isfahani0.73 ± 0.09 cd46.67 cd90.00 a3.5

0.83 ± 0.03 bc70 a83.33 ab7

0.13 ± 0.03 gh10 h70.00 bc14

0.57 ± 0.09 de36.67 d40.00 e0

Korki0.20 ± 0.06 g20.00 f–h40.00 e3.5

1.33 ± 0.09 a66.67 ab70.00 bc7

0.93 ± 0.07 b40 d86.67 a14

Means followed by the same letter(s) indicate nonsignificant differences at P = 0.05.

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traces of amino acids, and vitamins (Day, 1942), that affect the osmotic and nutrient characteristics of a gel and also can influence plant tissue culture process.3.3. Effect of agar concentrations on embryo maturationCucumber cultivar or landraces, agar concentrations, and the interaction of cultivar × agar concentration significantly (P < 0.01) affected embryo growth during the maturation phase (Figure 2). The higher concentration of agar (14 g L–1) in MS medium produced a greater percentage of developed embryos (torpedo and cotyledonary stages) in landraces Korki and Basmenj (90% and 85%, respectively, Figure 2A). In cultivar Beta Alpha, 75% of induced embryos developed towards the torpedo and cotyledonary stages, while no developed embryos were observed in liquid culture medium and medium containing 3.5 g L–1 agar (Figure 2A). Use of 7 and 14 g L–1 agar in culture medium similarly enhanced the percentage of mature embryos in landrace Isfahani (75.92% and 75%, respectively, Figure 2A). The larger number of mature embryos in anther cultures of Basmenj and Beta Alpha

cucumbers were observed in culture media containing 14 g L–1 agar (0.73 and 0.15 embryos per anther, respectively, Figure 2B), whereas the highest number of mature embryos in other cucumbers, Korki and Isfahani, was obtained with 7 g L–1 agar in culture medium (0.93 and 0.63 embryos per anther, respectively, Figure 2B). According to Ashok Kumar et al. (2003), in anther cultures of C. sativus L., the embryos that were transferred to maturation medium (B5 medium supplemented with 0.09 M sucrose and 5.0 mM ABA) developed normally, but without this treatment they were abnormal and did not grow further. In the present study, culture media with higher concentrations of agar promoted the development of globular embryos toward progressed stages, torpedo and cotyledonary embryos, without any other embryo maturation treatment. This result is consistent with previous investigations, which also reported an improvement in the development and maturation of somatic embryos of avocado (Márquez-Martín et al., 2011) and Pinus strobus by increasing gelling agent concentration (Klimaszewska et al., 2000).

Figure 2. Effect of agar concentration on embryo maturation traits in anther culture of different cultivars of cucumber (Cucumis sativus L.). (A) Effect of agar concentrations on embryo maturation percentage. (B) Effect of agar concentrations on mean number of mature embryos per each anther. Means followed by the same letter(s) indicate nonsignificant differences at P = 0.05.

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In a study by Song et al. (2007) on cucumber anther culture, the best embryo germination was obtained on MS germination medium solidified with 1.2% (12 g L–1) agar. In another study (Thompson and Harrington, 2001), various maturation treatments including different nutrient media, hormone combinations, and different desiccation methods were tested to mature and germinate the oak somatic embryos. It was found that the use of a higher agar concentration (1%) in culture medium was the most effective method for maturation of oak somatic embryos. Spomer and Smith (1996) reported that the effect of medium gelling agent concentration on plant response may be caused by changes in some of the gel properties affecting water availability or plant water relations. Water relations between the embryo and its environment play an important role in embryo development, particularly during the maturation phase (Adams and Rinne, 1980). In a study by Márquez-Martín et al. (2011) on maturation of avocado somatic embryos, to decrease water availability of the culture medium the concentration of agar was increased, resulting in a noticeable increase in embryo maturation percentage confirming results of Witjaksono and Litz (1999) and Perán-Quesada et al. (2004) in avocado cultivars and the results of the present study in a cucumber cultivar and landraces.

In conclusion, various genotypes of cucumber differentially responded to anther culture efficiency in culture media containing different strengths of macronutrients and agar concentrations. Depending on the genotype, some forms show higher callogenesis and embryo induction in culture media containing the higher concentrations of macronutrients or agar, while some other cucumber cultivars greatly respond to anther culture in culture media with lower macronutrient strength or agar concentration. In this research, the highest number of embryos per anther (1.26 ME/A) was obtained in the Isfahani landrace with full-strength macronutrients in the culture medium. Although this efficiency of androgenic embryo induction is not very high when compared with previous studies with 1.6 embryos/anther (Ashok Kumar et al., 2004) and 3 embryos/anther (Song et al., 2007), in contrast with previous studies, the current results were obtained with some cucumber landraces. However, the main problem in cucumber anther culture is turning on the embryo development and maturation program to enhance the efficiency of plant regeneration. In this study, use of higher concentrations of agar in culture media shows promise in helping to overcome this bottleneck and enhance the percentage of mature embryos in cucumber anther culture.

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