Optimization of regeneration and transformation conditions for chickpea (Cicer arietinum L.)

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Tezin Türü: Doktora

Tezin Yürütüldüğü Kurum: Orta Doğu Teknik Üniversitesi, Fen Edebiyat Fakültesi, Biyolojik Bilimler Bölümü, Türkiye

Tezin Onay Tarihi: 2003

Tezin Dili: İngilizce

Öğrenci: MİKAİL AKBULUT

Danışman: HÜSEYİN AVNİ ÖKTEM

Özet:

This study was conducted to develop a reproducible and reliable regeneration and transformation system for chickpea. In tissue culture studies; different explants (cotyledonary nodes and embryo axes), different microsalt concentrations (1X, 2X, 4X), different growth regulators for shooting (Benzyl amino purine (BAP) and thidiazuron (TDZ)) and rooting (Napthalenacetic acid and Indole butyric acid) have been investigated. Best regeneration response was obtained from cotyledonary nodes (CNs) cultured on modified Murashige and Skoog medium(MSSG) amended with 3 mg/L BAP (3.7 shoots/explant) and subsequent rooting in MS medium amended with 0.1 mg/L indole butyric acid (84% rooting). Three different transformation strategies namely, in planta electroporation of axillary nodes of intact one week old plants, Agro-vacuum infiltration of CNs and embryo axes and Agroinoculation of germinating seeds with one cotyledon was evaluated. To optimize in planta electroporation system, effect of various voltage (V) and current (uF) on duration of electroporation and viability of axillary meristem were determined. Electroporation duration was adjusted to 100-1 10 msec at 100 V and 975 uF using exponential decay type of pulse. Survival of axillary meristem derived shoot was 89 and 36% at 1 00 V for non-injected and injected axillary nodes, respectively. In the presence or absence of DNA protecting agents (spermidine and lipofectin®), none of the axillary-meristem- derived shoots exhibited B-glucuronidase activity under the above mentioned electroporation conditions. Agro-vacuum infiltration experiments were carried out on CNs and embryo axis. Vacuum infiltration of Agrobacterium cells at 200 mmHg for 20 or 40 minutes was found to be the optimum for transient GUS expression in both explants. Compared to CNs, embryo axes demonstrated higher transient GUS activity (42 GUS foci/explant). Effect of Agrobacterium tumefaciens strains (KYRT1, C58C1, EHA105) containing the piasmid pTJK136, explant developmental stage, infection time (2, 8, 16 and 24 hours), injury, infiltration and cultivars on transient GUS activity were investigated. Highest transient GUS activity was obtained from 16 hours imbibed explants injured with a needle and incubated for 24 hour with KYRT1 strain (226 GUS foci/explant). Stable transformation events were also verified by histochemical GUS assays and PCR amplification of npt-ll gene in one month old transformed plants.Keywords. Chickpea, regeneration, Agrobacterium, transformation, electroporation, infiltration