به كمك اگروباكتريوم با استفاده از ژن كيتينلز لوبيا )GOSSYPIUM HIRSUTUM L( تراريزش گياه پنبه

Agro bacterium- mediated Transformation of Cotton (ossypium hirsutum L.) using Bean Chitinase Gene

Optimization of chitinase gene transformation (from bean) into cotton (Gossypium hirsutum L.) to confer resistance against fungal diseases, verticilliosis and fusariosis was studied using Agro bacterium and vector pB1121. In order to construct a plasmid containing chitinase gene (pB1121-BCH), a 1.0 kbp bean chitinase gene (chti) was sub cloned into EcoRI site of lacZʹ operon in pGEM-7Zf(+). The insert fragment was then digested with two different restriction enzymes, Xbal and Sad, and subsequently ligated into the homologous sites in pB1121. In this case, the chti gene expression was placed under the control of a constitutive CaMV35S promoter. Gene orientation was verified by double restriction digestion pattern. Cotton hypocotyl tissue of Coker and Sahil varieties were inoculated with three A. tumefaciens strains; EHA101, LBA4404 and C58. All Agrobacterium strains contained a virulent Ti plasmid harboring with binary vector pB1121 containing the chitinase gene from bean, driven by the CaMV35S promotor. Explants (Hypocot1) were also inoculated with A. tumefaciens and co cultivated on the callus induction medium for 48h. The most Kanamycin-resistant calli (%87) and plants (%68) were obtained from LBA4404 strain. Expression of GUS in leaves of Coker variety of confirmed transformation. PCR analysis of putative transgenic and control plants showed the integration of at least one copy of Chitinase and nptII genes into the transgenic cotton plant genome.