Efficient Androgenic Embryo Induction and Plant Regeneration in Different Genotypes of Sweet Pepper via Anther Culture

Creation of pure lines is one of the basic requirements in plant breeding. Doubled haploid (DH) lines are completely homozygous lines and anther culture is an efficient method for DH line production. Induction of androgenic embryos in anther culture is the main restricting factor. In this study we investigated androgenic embryo induction in different F1 hybrid peppers including Cadia, Magno, Plato, King Arthur and Maratus. Excised anthers were cultured in C medium containing 2 mg L-1 2-4D and 2 mg L-1 Kinetin and incubated at 35°C for eight days and followed by four days at 25°C in darkness, then anthers sub cultured to R medium containing 0.1 mg L-1 Kinetin and incubated at 25°C and 16h photoperiod. The effect of genotype, cold pretreatment (4°C, 24 h) and heat shock on the efficiency of anther culture in pepper (Capsicum annuum L.) was evaluated. Cold pretreatment applied to excised buds improved microspore embryogenesis efficiency as compared to control. Among genotypes tested, Cadia, Magno, and Maratus were shown to be more responsive than King Arthur and Plato. Plants produced in responsive genotypes were approximately three-fold higher (2.5, 2.33 and 2.33 regenerated plant per petri dish, respectively) than King Arthur and Plato (0.83 and 0.66, respectively). Results showed that heat shock (35°C) applied to cultured anthers was effective on microspore embryogenesis, as heat shock (35°C) treatment for eight days had the best results in Cadia with 0.16 plant per petri dish.

Pepper (Capsicum annuum L.) because of its nutritional effects and pharmaceutical application is one of the most important vegetables around the world.Nowadays, F1 hybrids demonstrating unique characteristics are mainly applied seeds, especially in greenhouses.Conventional breeding methods to produce inbred lines through selfing is time and labor consuming.DH technology sheered disadvantages of traditional breeding and made possible achievement to parental lines in one generation.This technology consists of stimulation and germination of haploid plants via anther or microspores culture.Haploids are sporophytes containing gametophytic chromosomes so are valuable for not only inbreeding programs but also cytogenetic and genetic studies (Touraev et al. 1997).Gaining to haploids was made possible through application of female and male gametic cells.Androgenesis (anther or microspores culture) is the most interested method because of its higher efficiency and number of male gametes existing in each bud.As reported in the literature, pepper is one of the recalcitrant crops in haploid and DH production because of its various nature (Nowaczyk & KisiaBa 2006;Kothari et al. 2010).At the first time, Wang et al., (1973) and George and Narayanaswamy (1973) Abdollahi et al., (2015) studied the anther culture response of Citrullus lanatus L. to evaluate the effect of growth regulators and wheat ovary-conditioned medium in Charleston Gray cultivar and the effect of temperature pretreatment in Crimson sweet cultivar.Their results showed that the highest frequency of mean number of embryos per anther was obtained from cultured anthers incubated at 32°C for 2 days.Embryos germinated into plantlets on MS medium containing 2.22 µM BAP, 3 % sucrose, and 0.8 % agar.The root tips of 12 regenerated plantlets were analyzed for ploidy level, of which 10 have been haploids (Abdollahi et al. 2015).So, in this study, we examined effects of different genotypes and temperature pretreatments on pepper anther culture.

Anther culture process
Buds with equal sepal and petal in size that were containing microspores at mid to late uninucleate developmental stages were harvested at early morning and pretreated at 4°C for 24 h.Pretreated buds were surface disinfected by immersion in 70% ethanol for 30 seconds followed by 2.5 %( v/v) sodium hypochlorite solution for 10 min and three times rinsing with cold sterile distilled water.The anthers were detached and placed in 80 mm petri dishes containing 12 ml C culture medium(Dumas de Vaulx et al. 1981) supplemented by 2mgL -1 2,4-D, 2mgL -1 kinetin, 30 gL -1 sucrose, 0.7% plant agar and pH adjusted on 5.8.Each petri dish containing six anthers from a bud.Anther cultures were incubated at 35°C and darkness for eight days followed by incubation for four days at 25°C and darkness.Then anthers sub-cultured to R medium (Dumas de Vaulx et al. 1981) supplemented with 0.1 mgL -1 kinetin, 30 gL -1 sucrose, 0.7% plant agar and pH adjusted on 5.8 and incubated at 25°C and 16h photoperiod.In a separate part of the experiment, we examined the effect of different duration of heat stress on the efficiency of anther culture of the most responsive genotype, Cadia.So that anthers of two buds cultured in 80 mm petri dishes containing C medium (according to the previous experiment) (   of different genotypes and cold pretreatment applied to buds for pepper microspore embryogenesis efficiency during anther culture.

Statistical analysis
The present study performed in three replicates and each replication consisted of four petri dishes (each petri dish containing six anthers taken from a bud).ANOVA conducted via SAS 9 for Windows and means were compared using Duncan's multiple-range test (p>0.05).

Effects of genotype on microspore embryogenesis induction and plant regeneration
The genotype had a significant effect on callusing and embryogenesis (Table 1 Recalcitrant nature of some genotypes in microspore embryogenesis could be referred to the ability of formation of the competent

Effects of cold pretreatment on the induction of microspore embryogenesis in anther culture of pepper
Application of cold pretreatment (4°C) on excised flower buds for 24 h causes to a significant improvement in microspore embryogenesis efficiency in genotypes tested (Table 2).It has also been reported that physical pretreatment applied to harvested buds or whole inflorescences before culture stimulated sporophytic divisions in microspores (Sunderland & Roberts 1977;Lazar et al. 1985;Armstrong et al. 1987;Henry & Buyser 1990).Chilling pretreatment is one of the most effective practices in microspore embryogenesis.Sibi et al (1979) reported that cold (4°C) pretreatment for 48h applied to flower buds enhanced embryogenesis and 1-3 plants per 100 anthers were obtained (Sibi et al. 1979).Cold pretreatment of flower buds from 24 to 100 h before anther culture provoked an androgenic response (Morrison et al. 1986

Effects of heat shock duration on microspore embryogenesis in anther culture of pepper
The responses to heat shock duration on anther cultures were different (Table 3).Incubation of anther cultures at 25°C (without thermal shock), caused to no embryoid production.Heat shock treatment of anther cultivations from 4 to 12 days stimulated androgenesis response and showed the best results at eight days (Table 3).Increasing the duration of heat shock by more than 12 days had a detrimental effect and suppressed microspore embryogenesis as 16 days decreased callusing dramatically.
Stress treatment is the vital requirement for microspore embryogenesis (Shariatpanahi et al. 2006).Heat shock treatment has been used as a trigger to induce embryogenesis in pepper (Dumas de Vaulx et al. 1981;Gudeva 2003;Prayantini et al. 2006).Heat shock has been demonstrated to be an effective stimulating factor on microspore embryogenesis; it caused to change in microtubule and cytoskeleton (Hause et al. 1993;Cordewener et al. 1994;Simmonds 1994).Also, HSPs were reported to be synthesized in heat shock treated microspores (Segu1-Simarro et al. 2003) among which HSP70 was suggested to inhibit apoptosis (Jaattela et al. 1998).Application of proper period of heat stress is vital because a short period of heat temperature could not trigger enough ultra-structural changes and microspores tend to follow gametophytic pathway and longer duration had a detrimental effect.
As a result, during this study we achieved to an efficient protocol for production of pure lines by anther culture.Assessment of attained doubled haploid lines under greenhouse condition can help us to find appropriate lines for being applied in breeding programs as valuable plant materials.

Fig. 1 .
Fig. 1.Anther culture of pepper.(A) Embryogenesis (embryo indicated with arrow) (B) callusing (C) Cotyledonary embryo (D) Regenerated embryo (E) Regenerated plantlet ( F) Adapted plants ; E. D JSupena et al. 2006b).Working on wheat,Lazar et al., (1985) expressed callus yield and a frequency of spontaneous chromosome doubling increased in regenerated plants obtained with cold pretreatment.It assumed that cold pre-treatment slows down metabolism so suppresses normal gametophytic pathway and triggers sporophytic divisions.Another hypothesis suggests that starvation effects under low temperature had the primary role in anther culture response(Zheng 2003;Ziauddin et al. 1992;Kasha et al. 1990).Cold pretreatment of flower buds from 24 to 100 h before excising anthers for culture stimulated the androgenic response(Morrison et al. 1986; E. D JSupena et al. 2006a).

Table 1 .
(Gamborg et al. 1968.1981) were incubated at 35°C for 0, 4, 8, 12 and 16 days and darkness.Then they transferred to R medium (Dumas de Vaulx et al. 1981) and incubated at 25°C and 16 h light.Plant regenerationAfter five weeks, embryos were transferred to hormone-free B5 medium(Gamborg et al. 1968) with 20 gL -1 sucrose and solidified with 0.7% plant agar.In this study, we compared effects HEIDARI et al.: EFFICIENT ANDROGENIC EMBRYO INDUCTION Effect of genotype on the embryo induction and development of embryos in anther culture of pepper (Capsicum annuum L.) *Column with same letter had no significant difference (p≥0.5).

Table 2 .
Effect of cold pre-treatment on the embryo induction and development of embryos in anther culture of pepper (Capsicum annuum L.) *Column with same letter had no significant difference (p≥0.5).

Table 3 .
Effect of heat shock duration on the embryo induction and development of embryos in anther culture of pepper (Capsicum annuum L.) *Column with same letter had no significant difference (p≥0.5)