Biberde rejenerasyon ve Na-H antiportır ve izopentenil transferaz genlerinin aktarılması üzerine araştırmalar
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Dosyalar
Tarih
2019
Yazarlar
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Yayıncı
Ege Üniversitesi, Fen Bilimleri Enstitüsü
Erişim Hakkı
info:eu-repo/semantics/openAccess
Özet
Bu tez çalışmasında, dört farklı biber hattına (sivri, çarliston, kapya ve dolma) Agrobacterium tumefaciens aracılığıyla kuraklığa ve tuzluluğa tolerans sağlayan PSARKIPT ve AtNHX1 genlerinin aktarılarak, transformasyon ve rejenerasyon protokollerinin geliştirilmesi amaçlanmıştır. Araştırmada, biber hatlarının rejenerasyon ve transformasyon protokolleri geliştirilmiştir. Rejenerasyon denemelerinde somatik embriyogenesis ve organogenesis için farklı protokoller araştırılmıştır. En iyi bitki rejenerasyonları, BA ve IAA'nın farklı kombinasyonlarında, organogenesis denemelerinde elde edilmiştir. En yüksek sürgün gelişimleri ise sivri biberin hipokotil eksplantından %80 oranında 4 mg/l BA+0.5 mg/l IAA'da, çarliston biberin hipokotil eksplantından %80 oranında 3 mg/l BA+0.5 mg/l IAA'da, kapya biberin hipokotil eksplantından %80 oranında 5 mg/l BA+0.5 mg/l IAA'da ve dolma biberin hipokotil eksplantından %84 oranında 5 mg/l BA+0.5 mg/l IAA'da belirlenmiştir. Geliştirilen sürgünler köklendirilmiş ve bitkicikler elde edilmiştir. Transformasyon denemelerinde toplam 3857 eksplanttan 119 sürgün gelişirken, bunların 87 adedi PCR'da pozitif sonuç vermiştir. Transformasyon frekansları ise AtNHX1 geni için kapya biberde %2.44, çarliston biberde %2.53, sivri biberde %2.92 ve dolma biberde %1.18 iken, PSARKIPT için ise kapya biberde %1.73, çarliston biberde %3.74, sivri biberde %2.30 ve dolma biber için ise %1.34 olarak belirlenmiştir. PCR ile doğrulaması yapılan aday transgenik biber hatlarının mikroçoğaltımları gerçekleştirilmiştir. AtNHX1'lı transgenik biber hatlarının mikroğaltım oranları, %81-87 arasında değişirken PSARKIPT'li aday transgenik hatlar ise %80-84 arasında değişim göstermiştir.
In this thesis, transformation of PSARKIPT and AtNHX1 genes causing tolerance to drought and salinity by Agrobacterium tumefaciens to four different lines of pepper (Green, banana, capia and bell) was aimed. In the present study, regeneration and transformation protocols of pepper lines were developed. Different protocols for somatic embryogenesis and organogenesis were investigated in regeneration experiments. Optimum plant regeneration was obtained in different combinations of BA and IAA in organogenesis assays. The highest shoot development was determined as 80% from hypocotyl explant of green pepper in 4 mg/l BA+0.5 mg/l and IAA, 80% from hypocotyl explant of banana pepper in 3 mg/l BA+0.5 mg/l IAA, 80% from hypocotyl explant of capia pepper in 5 mg/l BA+0.5 mg/l and 84% from hypocotyl explant of bell pepper in 5 mg/l BA+0.5 mg/l IAA. Then, shoots were rooted and a complete plant forms were obtained. In the transformation experiments, 119 shoots were developed from 3857 explants and 87 of them were produced positive results in PCR analysis. The transformation frequencies for the AtNHX1 gene as 2.44% for capia pepper, 2.53% for banana pepper, 2.92% for green pepper and 1.18% for bell pepper while PSARKIPT gene as 1.73% for capia pepper, 3.74% for banana pepper, 2.30% for green pepper and 1.34% for bell pepper were determined. Micropropagation of the putative transgenic pepper lines confirmed by PCR was performed. While the micropropagation rates of the AtNHX1 putative transgenic pepper lines ranged from 81-87%, the putative transgenic lines with PSARKIPT ranged from 80-84%.
In this thesis, transformation of PSARKIPT and AtNHX1 genes causing tolerance to drought and salinity by Agrobacterium tumefaciens to four different lines of pepper (Green, banana, capia and bell) was aimed. In the present study, regeneration and transformation protocols of pepper lines were developed. Different protocols for somatic embryogenesis and organogenesis were investigated in regeneration experiments. Optimum plant regeneration was obtained in different combinations of BA and IAA in organogenesis assays. The highest shoot development was determined as 80% from hypocotyl explant of green pepper in 4 mg/l BA+0.5 mg/l and IAA, 80% from hypocotyl explant of banana pepper in 3 mg/l BA+0.5 mg/l IAA, 80% from hypocotyl explant of capia pepper in 5 mg/l BA+0.5 mg/l and 84% from hypocotyl explant of bell pepper in 5 mg/l BA+0.5 mg/l IAA. Then, shoots were rooted and a complete plant forms were obtained. In the transformation experiments, 119 shoots were developed from 3857 explants and 87 of them were produced positive results in PCR analysis. The transformation frequencies for the AtNHX1 gene as 2.44% for capia pepper, 2.53% for banana pepper, 2.92% for green pepper and 1.18% for bell pepper while PSARKIPT gene as 1.73% for capia pepper, 3.74% for banana pepper, 2.30% for green pepper and 1.34% for bell pepper were determined. Micropropagation of the putative transgenic pepper lines confirmed by PCR was performed. While the micropropagation rates of the AtNHX1 putative transgenic pepper lines ranged from 81-87%, the putative transgenic lines with PSARKIPT ranged from 80-84%.
Açıklama
Anahtar Kelimeler
Capsicum Annuum L., Transgenik, Agrobacterium Tumefaciens, Genetik Transformasyon, Rejenerasyon, Transgenic, Agrobacterium Tumefaciens, Genetic Transformation, Regeneration
