Effect of Genotype, Cold Pre-Treatment, Incubation Conditions and Media on Embryo and Plantlet Formation in Pepper Anther Culture

Abstract

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.

Keywords

• DDVX medium
• double layer medium
• activated charcoal
• vitamine B₁₂
• haploidy

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

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