Agrobacterium tumafaciens aracılığıyla nohut bitkisi (Cicer arietinum L.)’ne gen aktarımı

Bu çalışmada çeşitli eksplantlar (kotiledon nodları ve embriyo eksenleri), çeşitli büyüme düzenleyicileri ve bu düzenleyicilerin farklı konsantrasyonları gibi parametrelerin rejenerasyon ve transformasyona etkileri araştırılmıştır. Kotiledon nodlarının Agro-vakum infiltrasyonu, çimlendirilmiş nohut tohumlarının Agroinokülasyonu, embriyonun Agroenjeksiyonu ve yüzey sterilizasyonu yapılmış kuru nohut tohumlarının Agrobacterium solusyonu ile imbibisyonu (şişirme, emdirme) olmak üzere dört farklı transformasyon stratejisinin etkinliği denenmiştir. Transformasyon etkinliğine Agrobacterium suşları, kokültüvasyon süresi, eksplantın gelişme evresi, yaralama, infiltrasyon ve kültüvarlar gibi çeşitli parametrelerin etkisi araştırılmıştır. Transformasyon etkinliği geçici GUS aktivitesine bakarak veya selektif ortamda yaşayan eksplantlara dayanarak belirlenmiştir. Transgenlerin varlığı, ayda bir alt kültüre alınan selektif ortamda 3-4 ay seleksiyona tabi tutulmuş bitkilerde PCR analizleri ile belirlenmiştir. Test edilen Agrobacterium tumefaciens suşları arasında (KYRT1, C58C1 ve EHA 101) en iyi yanıt EHA 101 suşundan elde edilmiştir. Selektif ortamda elde edilen sonuca göre transformasyona ise en iyi cevabı Er çeşidi vermiştir.

Anahtar Kelimeler:

nohut, Agrobacterium, transformasyon, enjeksiyon, infiltrasyon

Agrobacterium tumefaciens mediated transformation in chickpea (Cicer arietinum L.)

In this study, the effect of various parameters such as different explants (cotyledonary nodes and embryo axes), different growth regulators (Benzylaminopurine and thidiazuron) and their various concentrations have been investigated. Four different transformation strategies namely; Agro-vacuum infiltration of cotyledonary nodes, Agroinoculation of germinating chickpea seedlings, Agro-injection of seedlings and imbibitions of surface sterilized dry chickpea seeds were also investigated. The effect of various parameters such as, Agrobacterium strains, infection durations, explant developmental stage, injury, infiltration and cultivars, on transformation efficiency were investigated. Transformation efficiency was determined based on transient GUS activity or survival on selective media. The presence of transgene was determined by PCR analysis on green plants subcultured monthly on selective media for 3-4 times. The best response were obtained from EHA 101 among the tested strains of Agrobacterium tumefaciens (KYRT1, C58C1 and EHA 101). Er was determined to be best responding cultivar according to the results obtained in selective medium.

Keywords:

chickpea, Agrobacterium, transformation, injection, infiltration,

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