Vigna caracalla L. Verdc. (İzmir Sarmaşığı) bitkisinde sıvı ortamlarda in vitro rejenerasyonun gerçekleştirilmesi
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Dosyalar
Tarih
2021
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Yayıncı
Ege Üniversitesi, Fen Bilimleri Enstitüsü
Erişim Hakkı
info:eu-repo/semantics/openAccess
Özet
Bu tezde Vigna caracalla L. Verdc. (İzmir Sarmaşığı) bitkisinin sıvı kültürlerde in vitro meristematik nodül rejenerasyon potansiyeli incelenmiştir. Çalışmanın ilk aşamasında, temin edilen tohumların yüzey sterilizasyonunun gerçekleştirilmesinin ardından, yarı katı MS besin ortamında in vitro steril bitkicikler elde edilmiştir. Kallus rejenerasyonu için MS temelli K1, K5 ve K6 gövde eksplantlarında %100; K5 besin ortamı ise yaprak eksplantlarında %100 en iyi sonucu vermiştir. Çalışmada, yaprak kökenli yeşil kalluslar daha gevşek yapıda oldukları için bu kallus tipinin sıvı kültürlere daha uygun olduğu belirlenmiştir. Sıvı kültürlerde gerçekleştirilen denemelerde kalluslardan meristematik nodüllerin gelişimi için bitki büyüme düzenleyicileri ve temel besin ortamları (MS, MS tuzları + B5 vitaminleri (MSB5), ½ MS, ½ MS tuzları + ½ B5 vitaminleri (1/2 MSB5) test edilmiştir. Bu denemelerde; S33Z (4 mg/L BAP içeren ½ MSB5) besin ortamında en yüksek meristematik nodül oluşum yüzdesi %100 ve yaş ağırlık ortalaması ise 2.06 g olarak elde edilmiştir. Meristematik nodüllerdeki kararma sorunu 100 mg/L Askorbik asit eklenmesiyle tamamen giderilmiştir. Çalışmada, en uygun besin ortamı kompozisyonu belirlendikten sonra sekonder çoğaltım denemeleri başlatılmıştır. 100 mg/L L-Glutamin ve 100 mg/L Askorbik asit içeren ½ MSB5 temelli S33Z, (Ç3) olarak kodlanan bu besin ortamında en fazla biyokütle artışı haftalık ortalama 0.37 kat olarak kaydedilmiştir. Araştırmada, Vigna caracalla bitkisine ait nodül kültürlerinde ölçek büyütme çalışmaları da başarıyla optimize edilmiştir. Çalışma sırasında kitlesel üretimi gerçekleştirmek üzere sürekli daldırma sistemine dayalı, kesikli operasyon modunda, pnömatik olarak havalandırılan otoklavlanabilir 2 L'lik şişe biyoreaktörler kullanılmıştır. Büyük ölçek denemelerinde inokulüm miktarının etkisi incelendiğinde, 6 g/L meristematik nodül inokulümü, haftalık ortalama 0.52 kat biyokütlenin artmasına ve en yüksek ortalama yaş ağırlığın (27.19 g) elde edilmesine yol açmıştır. Hasat edilen meristematik nodüllerin, rejenerasyon denemeleri için farklı konsantrasyonlarda BAP, GA3 ve NAA ilave edilen Ç3 besin ortamında kültüre alındıklarında kallusa dönüştükleri belirlenmiştir.
In this thesis, Vigna caracalla L. Verdc. (İzmir Ivy) plant in vitro meristematic nodule regeneration potential in liquid cultures was investigated. In the first stage of the study, in vitro sterile plantlets were obtained in semi-solid MS nutrient medium after surface sterilization of the seeds provided. 100% in MS-based K1, K5 and K6 stem explants for callus regeneration; on the other hand, K5 nutrient medium gave the best results with 100% in leaf explants. In the study, it was determined that this type of callus is more suitable for liquid cultures, since green calli of leaf origin are more loose.Plant growth regulators and basal nutrient media (MS, MS salts + vitamins B5 (MSB5), ½ MS, ½ MS salts + ½ B5 vitamins (1/2 MSB5) were tested for the development of meristematic nodules from calli in experiments performed in liquid cultures. In these trials; The highest percentage of meristematic nodule formation was obtained as 100% and the average fresh weight was 2.06 g in S33Z (½ MSB5 containing 4 mg/L BAP) nutrient medium. The blackening problem in meristematic nodules was completely eliminated by adding 100 mg/L Ascorbic acid. In the study, secondary propagation experiments were started after the most suitable nutrient medium composition was determined. The highest increase in biomass was recorded in this nutrient medium, ½ MSB5-based S33Z containing 100 mg/L L-Glutamine and 100 mg/L Ascorbic acid, coded as (Ç3), with a weekly average of 0.37 times. In the study, after the formation of meristematic nodules from callus from leaf explants and secondary propagation successfully, scale-up studies in nodule cultures of Vigna caracalla were successfully optimized. During the study, pneumatically agitated, continuous immersion system in batch mode autoclavable 2 L bottle bioreactors were used to realize mass production. In large-scale trials, when the effect of the amount of inoculum was examined, 6 g/L meristematic nodule inoculum led to an increase in weekly average biomass of 0.52 times and the highest average fresh weight (27.19 g). It was determined that the harvested meristematic nodules converted into callus when cultured in Ç3 nutrient medium with different concentrations of BAP, GA3 and NAA added for regeneration experiments.
In this thesis, Vigna caracalla L. Verdc. (İzmir Ivy) plant in vitro meristematic nodule regeneration potential in liquid cultures was investigated. In the first stage of the study, in vitro sterile plantlets were obtained in semi-solid MS nutrient medium after surface sterilization of the seeds provided. 100% in MS-based K1, K5 and K6 stem explants for callus regeneration; on the other hand, K5 nutrient medium gave the best results with 100% in leaf explants. In the study, it was determined that this type of callus is more suitable for liquid cultures, since green calli of leaf origin are more loose.Plant growth regulators and basal nutrient media (MS, MS salts + vitamins B5 (MSB5), ½ MS, ½ MS salts + ½ B5 vitamins (1/2 MSB5) were tested for the development of meristematic nodules from calli in experiments performed in liquid cultures. In these trials; The highest percentage of meristematic nodule formation was obtained as 100% and the average fresh weight was 2.06 g in S33Z (½ MSB5 containing 4 mg/L BAP) nutrient medium. The blackening problem in meristematic nodules was completely eliminated by adding 100 mg/L Ascorbic acid. In the study, secondary propagation experiments were started after the most suitable nutrient medium composition was determined. The highest increase in biomass was recorded in this nutrient medium, ½ MSB5-based S33Z containing 100 mg/L L-Glutamine and 100 mg/L Ascorbic acid, coded as (Ç3), with a weekly average of 0.37 times. In the study, after the formation of meristematic nodules from callus from leaf explants and secondary propagation successfully, scale-up studies in nodule cultures of Vigna caracalla were successfully optimized. During the study, pneumatically agitated, continuous immersion system in batch mode autoclavable 2 L bottle bioreactors were used to realize mass production. In large-scale trials, when the effect of the amount of inoculum was examined, 6 g/L meristematic nodule inoculum led to an increase in weekly average biomass of 0.52 times and the highest average fresh weight (27.19 g). It was determined that the harvested meristematic nodules converted into callus when cultured in Ç3 nutrient medium with different concentrations of BAP, GA3 and NAA added for regeneration experiments.
Açıklama
Anahtar Kelimeler
Vigna Caracalla L. Verdc., In Vitro, Sıvı Kültür, Meristematik Nodül, Rejenerasyon, Büyük Ölçek, Vigna Caracalla L. Verdc., In Vitro, Liquid Culture, Meristematic Nodule, Regeneration, Large Scale
