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Optimisation of agrobacterium mediated gene transfer and micrografting systems in lentil (lens culinaris medik)

Kamçı, Hamdi
In this work Agrobacterium (KYRT1::pTJK136) mediated gene transfer to lentil (Lens culinaris Medik.) embriyo apex and regeneration through micro-grafting in lentil was studied. In micro-grafting two different types root stock stem height and root stock preparations were optimized. According to the results half stem length was found to be more successful then the full. Also lentil root stock was more successful then the chickpea root stock. The types of root stock preparations studied were designated as Z and M. The Z type root stock was superior then the M type, when the micro-grafting, hardening and green-house stages were concerned. In study of Agrobacterium mediated gene transfer to lentil embryo apex the effect of the following parameters on the transformation efficiency were addressed; type and intensity of injury, type of pre-incubation media for injured explants, effect of evacuation, effect of L-cysteine during co-cultivation and Agrobacterium incubation duration. According to the results crushing type of injury was superior over the poking and sonication type of injuries. Following the injury Hoglands solution was used as pre-incubation media prior to infection. The effect of evacuation parameter was found to be insignificant whereas the effect of L-cysteine during co-cultivation was negative on the transformation efficiency. According to the Agrobacterium incubation duration studies, 240 minutes, followed by 120 minutes of Agrobacterium incubation were the most efficient in terms of transformation efficiency. However, since there was no significant difference among the two 120 minutes chosen to be the optimum bacterial incubation duration.