OPTIMISATION OF TISSUE CULTURE CONDITIONS AND GENE TRANSFER STUDIES IN LENTIL

Mahmoudian, Mehrzad
Ph.D., Department of Biotechnology
Supervisor: Prof. Dr. Hüseyin Avni Öktem
Co-Supervisor: Prof. Dr. Haluk Hamamcı

August 2000, 140 pages

This work describes an attempt to optimise the tissue culture conditions for in vitro regeneration of Turkish cultivars of lentil (Lens culinaris), Sultan-1 and Fırat-87, in association with the transformation systems, vacuum infiltration based Agrobacterium mediated and microprojectile particle bombardment. Direct and indirect organogenesis were the two main avenues of in vitro regeneration followed in this study. Throughout the regeneration studies in this work, the effect of three crucial factors, genotype, explant source, and growth regulators, which are known to influence the fate of the regeneration of plants, were taken into consideration. In the indirect organogenesis, upon their cultivation in three different media, the regeneration response of three different explant sources, were investigated. The nodal segments of both cultivar were evidenced to be the most responsive explant source for callogenesis and shoot regeneration, in a medium containing 0.1 mg/L GA and 10 mg/L Kinetin. In the direct organogenesis, cotyledonary nodes cultivated on 1 mg/L BAP containing medium were used as the regeneration system, and shown to be of high potential to regenerate multiple shoots, therefore assigned to be used in the transformation studies. The best rooting of the regenerated shoots was achieved mainly through either rhizogenesis in 0.1 mg/L IAA or 0.1 mg/L IBA, as well as through micrografting. Agrobacterium tumefaciens GV2260:pGUSINT and pBSGUSINT transformation vector, were used in transformation experiments, where the efficiency of the system could be assessed using transient gene expression assay, via histochemical GUS staining. These assays indicated that, cotyledonary node of lentil, subjected to either of the transformation systems, was amenable to the genetic transformation, with a high transient gene expression of the foreign gene.
Keywords: Lentil, Organogenesis, Agrobacterium, Vacuum Infiltration, Microprojectile Particle Bombardment, GUS

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