Genotip, Tomurcuk Uygulamaları, İnkübasyon Koşulları ve Besion Ortamlarının Biber Anther Kültüründe Embriyo ve Bitkicik Oluşumuna Etkisi
Öz
Amaç: Bu çalışmanın amacı, biberde anther kültüründe embriyoid ve bitkicik oluşumuna etki eden çeşitli kültür koşullarını araştırmaktır. Bu amaçla, ortam bileşimleri, vitamin B₁₂, aktif kömür, tomurcuk uygulamaları ve inkübasyon sıcaklıklarının etkileri değerlendirilmiştir.
Materyal ve Yöntem: Çalışma, Gaziosmanpaşa Üniversitesi’nde tül sera ve doku kültürü laboratuvarlarında yürütülmüştür. Üç biber (Capsicum annuum L.) genotipi—Belissa F₁, Bafra F₁ ve İstek F₁—ve bunların F₂ popülasyonları kullanılarak, farklı kültür koşullarının embriyoid oluşumuna etkileri araştırılmıştır. Donör bitkiler, Hoagland besin çözeltisi ve entegre zararlı yönetimi yaklaşımıyla kontrollü ortamda yetiştirilmiştir. Antherler, vitamin B₁₂’nin farklı konsantrasyonları (kontrol, 0.03 mg L⁻¹, 0.05 mg L⁻¹) ile modifiye edilen DDVX ve 0.10% ve 0.20% oranında aktif kömür eklenmiş Double Layer (DL) ortamlarında kültüre alınmıştır. Embriyoid oluşumunu teşvik etmek amacıyla tomurcuklara 4°C’de 24 veya 48 saat süreyle soğuk şoku uygulanmış ve inkübasyon sıcaklıkları olarak 9°C ile 35°C arasında değişen koşullar denenmiştir.
Bulgular: DDVX ortamında 702 embriyoid ve 124 haploid bitkicik elde edilmiştir. En yüksek embriyoid oluşumu (51), Belissa F₂ genotipinde, 4°C’de 24 saatlik ön uygulama, vitamin B₁₂ eklenmemesi ve 35°C inkübasyon koşullarında elde edilmiştir. Bafra ve İstek genotiplerinde, vitamin B₁₂ (0.03–0.05 mg·L⁻¹) ve 35°C inkübasyon, embriyoid oluşumunu anlamlı derecede artırmıştır. 4°C’de 24 saatlik ön işlem en iyi sonuçları verirken, 48 saatlik ön işlem herhangi bir ek fayda sağlamamıştır. DL ortamında ise 274 embriyoid ve 49 haploid bitkicik elde edilmiştir. En yüksek embriyoid sayısı (34), Belissa F₂ genotipinde, kontrol koşullarında %0.20 aktif kömür ve 9°C inkübasyon ile elde edilmiştir. Bafra ve İstek genotiplerinde, %0.10–0.20 aktif kömür ve 9°C inkübasyon en verimli sonuçları sağlamıştır. DDVX ortamının aksine, DL ortamında tomurcuk ön işlemleri belirgin bir avantaj sağlamamıştır. DDVX ortamı, DL ortamına kıyasla embriyoid oluşumunda %256 daha başarılı olmuştur. F₂ popülasyonları F₁ popülasyonlarına kıyasla daha yüksek embriyoid oluşturmuştur. DDVX ortamında 35°C, DL ortamında 9°C inkübasyon daha etkili olmuştur.
Sonuç: Bu çalışmada, genotip, besin ortamı ve inkübasyon koşullarının biberde embriyoid oluşumunu belirgin şekilde etkilediği anlaşılmıştır. DDVX ortamı, DL ortamına göre önemli düzeyde daha fazla embriyoid ve bitkicik üretmiştir.
Anahtar Kelimeler
DDVX ortamı double layer ortamı aktif kömür vitamin B₁₂ haploid
Kaynakça
- Arı, E., Bedir, H., Yıldırım, S., & Yıldırım, T. (2016). Androgenic responses of ornamental pepper (Capsicum annuum L.) genotypes to shed-microspore culture in the autumn season. Turkish Journal of Biology, 40(3).
- Barany, I., Gonzalez-Melendi, P., Fadon, B., Mityko, J., Risueno, M. C., & Testillano, P. S. (2005). Microspore-derived embryogenesis in pepper (Capsicum annuum L.): Subcellular rearrangements through development. Biological Cell, 97(9), 709–722.
- Başay, S., & Ellialtıoğlu, Ş. (2013). Effect of genotypical factors on the effectiveness of anther culture in eggplant (Solanum melongena L.). Turkish Journal of Biology, 37, 499–505.
- Dias, J. S., & Correia, M. C. (2002). Effect of medium renovation and incubation temperature regimes on tronchuda cabbage microspore culture embryogenesis. Scientia Horticulturae, 93, 205–214.
- Dolcet-Sanjuan, R., Claveira, E., & Huerta, A. (1997). Androgenesis in Capsicum annuum L.: Effects of carbohydrate and carbon dioxide enrichment. Journal of the American Society for Horticultural Science, 122, 468–475.
- Dumas de Vaulx, R., Chambonnet, D. ve Pochard, E. (1981). In vitro anther culture in red pepper (Capsicum annuum L.): improvement of the rate of plant production in different genotypes by treatments at 35 C. Agronomie 1:859–864.
- Elçi, Ş. (1982). Sitogenetikte Gözlemler ve Araştırma Yöntemleri. Fırat Üniversitesi Fen-Edebiyat Fakültesi Yayınları. Biyoloji: 3. Elazığ. 165s.
- Ercan, N., Sensoy, F., & Sensoy, A. S. (2006). Influence of growing season and donor plant age on anther culture response of some pepper cultivars (Capsicum annuum L.). Scientia Horticulturae, 110(1), 16-20.
- Grozeva, S., & Nankar, A. N. (2020). Effect of incubation period and culture medium on pepper anther culture. Indian Journal of Biotechnology, 19, 53-59.
- Grozeva, S., Todorova, V., Cholakov, T., & Rodeva, V. (2013). Effect of temperature and growth period of donor plants on pepper anther culture. Acta Physiologiae Plantarum, 35(7), 2365–2372.
- Irikova, T. P., Kintzios, S., Grozeva, S., & Rodeva, V. (2016). Pepper (Capsicum annuum L.) anther culture: Fundamental research and practical applications. Turkish Journal of Biology, 40(4), 618-624.
- Irikova, T., Grozeva, S., Popov, P., Rodeva, V., & Todorovska, E. (2011). In vitro response of pepper anther culture (Capsicum annuum L.) depending on genotype, culture medium and duration of cultivation. Biotechnology & Biotechnological Equipment, 25, 2604–2609.
- Irikova, T., & Rodeva, V. (2004). Anther culture of pepper (Capsicum annuum L.): The effect of nutrient media. Capsicum and Eggplant Newsletter, 23, 101–104.
- İlhan, M., & Kurtar, E. S. (2022). Double-haploidization efficiency of selected pepper genotypes via in vitro anther culture. Selcuk Journal of Agriculture and Food Sciences, 36(2), 253-259.
- Jha, K., Choudhary, P. K., & Agarwal, A. (2021). Doubled haploid production in Capsicum annuum L. using anther culture: A review. Plant Archives, 21(1), 168–173.
- Keleş, D., Pınar, H., Ata, A., Taşkın, H., Yıldız, S., & Büyükalaca, S. (2015). Effect of pepper types on obtaining spontaneous doubled haploid plants via anther culture. HortScience, 50(11), 1671–1676.
- Kim, M., Kim, J., Yoon, M., Choı, D.-Iı., & Lee, K.-M. (2004). Origin of multicellular pollen and pollen embryos in cultured anthers of pepper (Capsicum annuum). Plant Cell, Tissue and Organ Culture, 77, 63–72.
- Koleva-Gudeva, L., Spasenoski, M., & Trajkova, F. (2007). Somatic embryogenesis in pepper anther culture: The effect of incubation treatments and different media. Scientia Horticulturae, 111, 114–119.
- Matsubara, S., Yamamoto, M., Man-Hyun, J., Murakami, K., & Man, H. J. (1998). Embryoid and callus formation from microspores by anther culture from July to November in pepper (Capsicum annuum L.). Sci Rep Faculty Agric Okayama Univ, 87, 117–112.
- Mityko, J., Andrásfalvy, A., Csilléry, G., & Fári, M. (1995). Anther culture response in different genotypes and F1 hybrids of pepper (Capsicum annuum L.). Plant Breeding, 114, 78–80.
- Nervo, G., Carannante, G., Azzimonti, M. T., & Rotino, G. L. (1995). Use of anther culture method in pepper breeding: Factors affecting plantlets production. In Curren tissues in plant molecular and cellular biology (pp. 155–160). Springer Netherlands.
- Nowaczyk, L., Nowaczyk, P., & Olszewska, D. (2015). Genetic analysis of anther culture-derived diploids of Capsicum spp. The Journal of Horticultural Science and Biotechnology, 90(6), 747–752.
- Nowaczyk, P., & Kisiała, A. (2006). Effect of selected factors on the effectiveness of Capsicum annuum L. anther culture. Journal of Applied Genetics, 47, 113–117.
- Ozsan, T., & Onus, A. (2017). In vitro pepper (Capsicum annuum L.) anther culture: can be affected via vitamins B. Biotechnology Journal International, 20(1), 1-13.
- Özkum, D., & Tıpırdamaz, R. (2002). The effects of cold treatment and charcoal on the in vitro androgenesis of pepper (Capsicum annuum L.). Turkish Journal of Biology, 26, 131–139.
- Parra-Vega, V., Renau-Morata, B., Sifres, A., & Seguí-Simarro, J. M. (2013). Stress treatments and in vitro culture conditions influence microspore embryogenesis and growth of callus from anther walls of sweet pepper (Capsicum annuum L.). Plant Cell, Tissue and Organ Culture, 112, 353–360.
- Popova, T., Grozeva, S., Todorova, V., Stankova, G., Anachkov, N., & Rodeva, V. (2016). Effects of low temperature, genotype and culture media on in vitro androgenic response of pepper (Capsicum annuum L.). Acta Physiologiae Plantarum, 38, 1–11.
- Rodeva, V. N., Irikova, T. P., & Todorova, V. J. (2004). Anther culture of pepper (Capsicum annuum L.): Comparative study on effect of the genotype. Biotechnology & Biotechnological Equipment, 18(3), 34–38.
- Rodeva, V., Grozeva, S., & Todorova, V. (2006). In vitro response of Bulgarian pepper (Capsicum annuum L.) varieties. Genetica, 38(2), 129–136.
- Rodeva, V., Gudeva, L. K., Grozeva, S., & Trajkova, F. (2007). Obtaining haploids in anther culture of pepper (Capsicum annuum L.) and their inclusion in the breeding process. Journal of Agriculture and Plant Sciences, 7(1), 7–18.
- Sangwan, R. S., & Sangwan-Norrell, B. S. (1990). Anther and pollen culture. In S. S. Bhojwani (Ed.), Plant Tissue Culture: Applications and Limitations (pp. 220–242). Elsevier Science Publishers.
- Seguí-Simarro, J. M., Corral-Martínez, P., Parra-Vega, V., & González-García, B. (2011). Androgenesis in recalcitrant solanaceous crops. Plant Cell Reports, 30(5), 765–778.
- Supena, E. D., Suharsono, S., Jacobsen, E. ve Custers, J. B, (2006). Successful development of a shed-microspore culture protocol for doubled haploid production in Indonesian hot pepper (Capsicum annuum L.). Plant Cell Rep 25:1– 10.
- Thomas, T. D. (2008). The role of activated charcoal in plant tissue culture. Biotechnology advances, 26(6), 618-631.
- Xynias, I.N., Gouli-Vavdinoudi, E. ve Roupakias, D.G. (2001). Effect of Cold Pretreatment and Incubation Temperature on Bread Wheat (Triticum aestivum L.) Anther Culture. Cereal Research Communications, 29 (3-4): 331-338.
- Yang, B. Z., Zhou, S. D., Zhang, Z. Q., Dai, X. Z., Li, L. H. Ve Xie, D. P. (2009). Effects of different medium and hormone on cultured anther of hot pepper [J]. Journal of Hunan Agricultural University (Natural Sciences), 1, 018.
Effect of Genotype, Cold Pre-Treatment, Incubation Conditions and Media on Embryo and Plantlet Formation in Pepper Anther Culture
Öz
Objectives: The aim of this study was to investigate the effects of various culture conditions on embryoid and plantlet formation in pepper anther cultures. To achieve this, the effects of media compositions, vitamin B₁₂, activated charcoal, bud treatments, and incubation temperatures were evaluated.
Materials and Methods: The study was conducted at the screenhouse and tissue culture laboratory of Gaziosmanpaşa University. Three pepper (Capsicum annuum L.) genotypes—Belissa F₁, Bafra F₁, and İstek F₁—and their F₂ populations were used to examine the effects of different culture conditions on embryoid formation. Donor plants were grown in a controlled environment using Hoagland’s nutrient solution and an integrated pest management approach. Anthers were cultured in DDVX medium, modified with different concentrations of vitamin B₁₂ (control, 0.03 mg L⁻¹, 0.05 mg L⁻¹), and in Double Layer (DL) medium with 0.10% and 0.20% activated charcoal. To stimulate embryoid formation, buds were subjected to cold shock at 4°C for 24 or 48 hours, and incubation temperatures ranging from 9°C to 35°C were tested.
Results: In DDVX medium, 702 embryoids and 124 haploid plantlets were obtained. The highest embryoid formation (51) was observed in the Belissa F₂ genotype under conditions of 24-hour pre-treatment at 4°C, no vitamin B₁₂ supplementation, and incubation at 35°C. In the Bafra and İstek genotypes, vitamin B₁₂ (0.03–0.05 mg·L⁻¹) and 35°C incubation significantly enhanced embryoid formation. A 24-hour pre-treatment at 4°C yielded the best results, while a 48-hour pre-treatment did not provide any additional benefit. In DL medium, 274 embryoids and 49 haploid plantlets were produced. The highest number of embryoids (34) was observed in the Belissa F₂ genotype under control conditions with 0.20% activated charcoal and incubation at 9°C. In the Bafra and İstek genotypes, 0.10–0.20% activated charcoal and 9°C incubation provided the most effective results. Unlike DDVX, bud pre-treatments in DL medium did not show a clear advantage. DDVX medium was 256% more successful in embryoid formation compared to DL medium. F₂ populations produced higher embryoid numbers compared to F₁ populations. In DDVX, 35°C incubation was more effective, while in DL, 9°C incubation yielded better results.
Conclusion: The study demonstrated that genotype, nutrient medium, and incubation conditions significantly influence embryoid formation in pepper. DDVX medium produced significantly more embryoids and plantlets compared to DL medium.
Anahtar Kelimeler
DDVX medium double layer medium activated charcoal vitamine B₁₂ haploidy
Kaynakça
- Arı, E., Bedir, H., Yıldırım, S., & Yıldırım, T. (2016). Androgenic responses of ornamental pepper (Capsicum annuum L.) genotypes to shed-microspore culture in the autumn season. Turkish Journal of Biology, 40(3).
- Barany, I., Gonzalez-Melendi, P., Fadon, B., Mityko, J., Risueno, M. C., & Testillano, P. S. (2005). Microspore-derived embryogenesis in pepper (Capsicum annuum L.): Subcellular rearrangements through development. Biological Cell, 97(9), 709–722.
- Başay, S., & Ellialtıoğlu, Ş. (2013). Effect of genotypical factors on the effectiveness of anther culture in eggplant (Solanum melongena L.). Turkish Journal of Biology, 37, 499–505.
- Dias, J. S., & Correia, M. C. (2002). Effect of medium renovation and incubation temperature regimes on tronchuda cabbage microspore culture embryogenesis. Scientia Horticulturae, 93, 205–214.
- Dolcet-Sanjuan, R., Claveira, E., & Huerta, A. (1997). Androgenesis in Capsicum annuum L.: Effects of carbohydrate and carbon dioxide enrichment. Journal of the American Society for Horticultural Science, 122, 468–475.
- Dumas de Vaulx, R., Chambonnet, D. ve Pochard, E. (1981). In vitro anther culture in red pepper (Capsicum annuum L.): improvement of the rate of plant production in different genotypes by treatments at 35 C. Agronomie 1:859–864.
- Elçi, Ş. (1982). Sitogenetikte Gözlemler ve Araştırma Yöntemleri. Fırat Üniversitesi Fen-Edebiyat Fakültesi Yayınları. Biyoloji: 3. Elazığ. 165s.
- Ercan, N., Sensoy, F., & Sensoy, A. S. (2006). Influence of growing season and donor plant age on anther culture response of some pepper cultivars (Capsicum annuum L.). Scientia Horticulturae, 110(1), 16-20.
- Grozeva, S., & Nankar, A. N. (2020). Effect of incubation period and culture medium on pepper anther culture. Indian Journal of Biotechnology, 19, 53-59.
- Grozeva, S., Todorova, V., Cholakov, T., & Rodeva, V. (2013). Effect of temperature and growth period of donor plants on pepper anther culture. Acta Physiologiae Plantarum, 35(7), 2365–2372.
- Irikova, T. P., Kintzios, S., Grozeva, S., & Rodeva, V. (2016). Pepper (Capsicum annuum L.) anther culture: Fundamental research and practical applications. Turkish Journal of Biology, 40(4), 618-624.
- Irikova, T., Grozeva, S., Popov, P., Rodeva, V., & Todorovska, E. (2011). In vitro response of pepper anther culture (Capsicum annuum L.) depending on genotype, culture medium and duration of cultivation. Biotechnology & Biotechnological Equipment, 25, 2604–2609.
- Irikova, T., & Rodeva, V. (2004). Anther culture of pepper (Capsicum annuum L.): The effect of nutrient media. Capsicum and Eggplant Newsletter, 23, 101–104.
- İlhan, M., & Kurtar, E. S. (2022). Double-haploidization efficiency of selected pepper genotypes via in vitro anther culture. Selcuk Journal of Agriculture and Food Sciences, 36(2), 253-259.
- Jha, K., Choudhary, P. K., & Agarwal, A. (2021). Doubled haploid production in Capsicum annuum L. using anther culture: A review. Plant Archives, 21(1), 168–173.
- Keleş, D., Pınar, H., Ata, A., Taşkın, H., Yıldız, S., & Büyükalaca, S. (2015). Effect of pepper types on obtaining spontaneous doubled haploid plants via anther culture. HortScience, 50(11), 1671–1676.
- Kim, M., Kim, J., Yoon, M., Choı, D.-Iı., & Lee, K.-M. (2004). Origin of multicellular pollen and pollen embryos in cultured anthers of pepper (Capsicum annuum). Plant Cell, Tissue and Organ Culture, 77, 63–72.
- Koleva-Gudeva, L., Spasenoski, M., & Trajkova, F. (2007). Somatic embryogenesis in pepper anther culture: The effect of incubation treatments and different media. Scientia Horticulturae, 111, 114–119.
- Matsubara, S., Yamamoto, M., Man-Hyun, J., Murakami, K., & Man, H. J. (1998). Embryoid and callus formation from microspores by anther culture from July to November in pepper (Capsicum annuum L.). Sci Rep Faculty Agric Okayama Univ, 87, 117–112.
- Mityko, J., Andrásfalvy, A., Csilléry, G., & Fári, M. (1995). Anther culture response in different genotypes and F1 hybrids of pepper (Capsicum annuum L.). Plant Breeding, 114, 78–80.
- Nervo, G., Carannante, G., Azzimonti, M. T., & Rotino, G. L. (1995). Use of anther culture method in pepper breeding: Factors affecting plantlets production. In Curren tissues in plant molecular and cellular biology (pp. 155–160). Springer Netherlands.
- Nowaczyk, L., Nowaczyk, P., & Olszewska, D. (2015). Genetic analysis of anther culture-derived diploids of Capsicum spp. The Journal of Horticultural Science and Biotechnology, 90(6), 747–752.
- Nowaczyk, P., & Kisiała, A. (2006). Effect of selected factors on the effectiveness of Capsicum annuum L. anther culture. Journal of Applied Genetics, 47, 113–117.
- Ozsan, T., & Onus, A. (2017). In vitro pepper (Capsicum annuum L.) anther culture: can be affected via vitamins B. Biotechnology Journal International, 20(1), 1-13.
- Özkum, D., & Tıpırdamaz, R. (2002). The effects of cold treatment and charcoal on the in vitro androgenesis of pepper (Capsicum annuum L.). Turkish Journal of Biology, 26, 131–139.
- Parra-Vega, V., Renau-Morata, B., Sifres, A., & Seguí-Simarro, J. M. (2013). Stress treatments and in vitro culture conditions influence microspore embryogenesis and growth of callus from anther walls of sweet pepper (Capsicum annuum L.). Plant Cell, Tissue and Organ Culture, 112, 353–360.
- Popova, T., Grozeva, S., Todorova, V., Stankova, G., Anachkov, N., & Rodeva, V. (2016). Effects of low temperature, genotype and culture media on in vitro androgenic response of pepper (Capsicum annuum L.). Acta Physiologiae Plantarum, 38, 1–11.
- Rodeva, V. N., Irikova, T. P., & Todorova, V. J. (2004). Anther culture of pepper (Capsicum annuum L.): Comparative study on effect of the genotype. Biotechnology & Biotechnological Equipment, 18(3), 34–38.
- Rodeva, V., Grozeva, S., & Todorova, V. (2006). In vitro response of Bulgarian pepper (Capsicum annuum L.) varieties. Genetica, 38(2), 129–136.
- Rodeva, V., Gudeva, L. K., Grozeva, S., & Trajkova, F. (2007). Obtaining haploids in anther culture of pepper (Capsicum annuum L.) and their inclusion in the breeding process. Journal of Agriculture and Plant Sciences, 7(1), 7–18.
- Sangwan, R. S., & Sangwan-Norrell, B. S. (1990). Anther and pollen culture. In S. S. Bhojwani (Ed.), Plant Tissue Culture: Applications and Limitations (pp. 220–242). Elsevier Science Publishers.
- Seguí-Simarro, J. M., Corral-Martínez, P., Parra-Vega, V., & González-García, B. (2011). Androgenesis in recalcitrant solanaceous crops. Plant Cell Reports, 30(5), 765–778.
- Supena, E. D., Suharsono, S., Jacobsen, E. ve Custers, J. B, (2006). Successful development of a shed-microspore culture protocol for doubled haploid production in Indonesian hot pepper (Capsicum annuum L.). Plant Cell Rep 25:1– 10.
- Thomas, T. D. (2008). The role of activated charcoal in plant tissue culture. Biotechnology advances, 26(6), 618-631.
- Xynias, I.N., Gouli-Vavdinoudi, E. ve Roupakias, D.G. (2001). Effect of Cold Pretreatment and Incubation Temperature on Bread Wheat (Triticum aestivum L.) Anther Culture. Cereal Research Communications, 29 (3-4): 331-338.
- Yang, B. Z., Zhou, S. D., Zhang, Z. Q., Dai, X. Z., Li, L. H. Ve Xie, D. P. (2009). Effects of different medium and hormone on cultured anther of hot pepper [J]. Journal of Hunan Agricultural University (Natural Sciences), 1, 018.
Ayrıntılar
| Birincil Dil | İngilizce |
|---|---|
| Konular | Sebze Yetiştirme ve Islahı |
| Bölüm | Makaleler |
| Yazarlar | |
| Yayımlanma Tarihi | 30 Haziran 2025 |
| Gönderilme Tarihi | 20 Mart 2025 |
| Kabul Tarihi | 2 Mayıs 2025 |
| Yayımlandığı Sayı | Yıl 2025 Cilt: 14 Sayı: 1 |