ISSN: 0973-7510
E-ISSN: 2581-690X
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 2mg L-1 2-4D and 2mg 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, 24h) 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.
Androgenesis; breeding; Capsicum annuum L.; cold pretreatment; heat shock.
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) reported plant regeneration from C. annuum anthers (Wang et al. 1973; George & Narayanaswamy 1973). Afterward, many other researchers presented lots about the different methods of androgenesis including anther, shed-microspore and isolated microspore cultures (Sibi et al. 1979; Dumas de Vaulx et al. 1981; E. D. J. Supena et al. 2006b; Kim et al. 2008). The genotype has the primary role and often restricting aspect in the pepper androgenesis (Buyukalaca et al. 2004; Wang & Zhang 2001; Rodeva et al. 2004; Koleva-Gudeva et al. 2007; Liu et al. 2007). It cannot be overcome by controlling of growth conditions of parental plants and androgenic cultures (Kristiansen & Andersen 1993). So finding responsive genotypes for androgenic programs in plant breeding is one of the crucial factors. Embryo induction both in plants and animals is more or less equal and requires stress treatment(Zimmerman & Cohill 1991). To deviate from the gametophytic to the sporophytic pathway, stresses such as low or high temperature, osmotic pressure or starvation stress are needed (Maraschin et al. 2005; Shariatpanahi et al. 2006; Koleva-Gudeva et al. 2009). Olszewska et al., (2014) investigated the combined effect of anther incubation time on CP induction medium (12, 14, and 16 days) and 2 concentrations of kinetin in R1 regeneration medium (0.1 and 0.3 mg L-1) on the effectiveness of androgenesis in 17 genotypes of Capsicum grown in Poland(Olszewska et al. 2014). 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.
Plant materials
Seeds of Five F1 hybrids including Cadia, Magno, Maratus (Enza-Zaden Co.), Plato and King-Arthur (Seminis Co.) were sown in the greenhouse of Agricultural Biotechnology Research Institute of Iran (ABRII) at November of 2015. They were within plastic pots containing a mixture of soil: peat: perlite (1:1:1). Plants grew at 25-30/18-22°C (day/night temperature) and natural light condition. Fertilization and irrigation were done according to standard procedure (Bosland et al. 2012).
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 24h. Pretreated buds were surface disinfected by immersion in 70% ethanol for 30 seconds followed by 2.5% (v/v) sodium hypochlorite solution for 10min and three times rinsing with cold sterile distilled water. The anthers were detached and placed in 80 mm petri dishes containing 12ml C culture medium(Dumas de Vaulx et al. 1981) supplemented by 2mgL-1 2,4-D, 2mgL-1 kinetin, 30gL-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 80mm petri dishes containing C medium (according to the previous experiment)(Dumas de Vaulx et al. 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 16h light.
Plant regeneration
After 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 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).
Fig. 1. Anther culture of pepper. (A) Embryogenesis (embryoid indicated with arrow) (B) callusing (C) Cotyledonary embryoid (D) Regenerated embryoid (E) Regenerated plantlet ( F) Adapted plants
Effects of genotype on microspore embryogenesis induction and plant regeneration
The genotype had a significant effect on callusing and embryogenesis (Table 1). According to the results obtained, Cadia showed as the most responsive genotype among tested ones in which 9.5% of cultured anthers succeeded to embryo formation and approximately 0.57 embryo was produced per bud. This genotype had the best results in the number of regenerated plantlets per bud (0.15 plantlet), though there was no significant difference between Cadia, Magno, and Maratus. A similar phenomenon has been reported by other research groups (Dumas de Vaulx et al. 1981; Dolcet-Sanjuan et al. 1997; Ltifi & Wenzel 1994; Rodeva et al. 2004; E. D. J. Supena et al. 2006b). Recalcitrant nature of some genotypes in microspore embryogenesis could be referred to the ability of formation of the competent microspores that is under the control of cytoplasmic and genomic genes and are under the influence of the environment (Testillano et al. 2000). Working on Solanum tuberosum, demonstrated that microspore embryogenesis capacity was a heritable trait controlled by several recessive genes (Chupeau et al. 1998; Rudolf et al. 1999; Smýkal 2000).
Table (1):
Effect of genotype on the embryo induction and development of embryos in anther culture of pepper (Capsicum annuum L.).
Genotype |
Embryogenesis (%) |
No. embryos (per bud) |
Callusing (%) |
Regenerated plantlets (per bud) |
Plant regeneration (%) |
|---|---|---|---|---|---|
Cadia |
9.5a |
0.57a |
90.0a |
0.15a |
2.5a |
Plato |
3.6b |
0.22b |
69.3c |
0.04b |
0.66b |
Maratus |
9.3a |
0.56a |
82.5b |
0.14a |
2.33a |
King Arthur |
2.0c |
0.12c |
53.0d |
0.05b |
0.83b |
Magno |
8.8a |
0.53a |
65.3c |
0.14a |
2.33a |
*Column with same letter had no significant difference (p>0.5).
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) pre-treatment 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 100h before anther culture provoked an androgenic response(Morrison et al. 1986; E. D J Supena et al. 2006b).
Table (2):
Effect of cold pre-treatment on the embryo induction and development of embryos in anther culture of pepper (Capsicum annuum L.).
Genotype |
Embryogenesis (%) |
No. embryos (per bud) |
Callusing (%) |
Regenerated plantlets (per bud) |
Plant regeneration (%) |
|---|---|---|---|---|---|
4ᵒC |
8.0a |
0.48a |
74.8a |
013a |
2.2a |
25ᵒC |
5.33b |
0.32b |
69.26b |
0.076b |
1.26b |
*Column with same letter had no significant difference (p>0.5).
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 J Supena et al. 2006a).
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.
Table (3):
Effect of heat shock duration on the embryo induction and development
of embryos in anther culture of pepper (Capsicum annuum L.).
Heat shock duration (days) |
Embryogenesis (%) |
No. embryos (per bud) |
Callusing (%) |
Regenerated plantlets (per bud) |
Plant regeneration (%) |
|---|---|---|---|---|---|
0 |
0c |
0b |
87.75a |
0c |
0b |
4 |
3.25b |
0.17b |
81.00b |
0c |
0b |
8 |
11.62a |
0.59a |
89.50a |
0.16a |
2.42a |
12 |
2.75b |
0.06b |
64.00c |
0.04b |
0b |
16 |
0c |
0b |
27.50d |
0c |
0b |
*Column with same letter had no significant difference (p>0.5).
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.
ACKNOWLEDGMENTS
This research was supported by grants from Agricultural Biotechnology Research Institute of Iran (ABRII) Project No. 14-05-05-9154-91002.
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