Optimization of Tissue Culture and Gene Transfer in Lettuce (Lactuca sativa L.)

Lettuce (Lactuca sativa L.) is a major leafy vegetable and belongs to the Asteraceae family (Compositae). The genetic manipulation of lettuce requires a reliable and efficient regeneration method. Since lettuce is a suitable bioreactor for production of recombinant proteins especially edible vaccines. This study was conducted to identify an appropriate method for lettuce gene transformation. At first, the effects of explants age and seven different combinations of plant growth regulators on callus induction and direct shoot regeneration of lettuce were examined. The experiment was factorial based on a completely randomized design. The sensitivity of lettuce cotyledons to hygromycin was assayed by culturing the cotyledons without co-cultivation with Agrobacterium tumefaciens on selection medium containing different concentrations of hygromycin. For gene transformation, Agrobacterium tumefaciens (LBA4404 with pCAMBIA1304) was used and transgenic plants were analyzed with PCR method. The highest percentage of callus induction was obtained using 0.54 µM NAA and 0.44 µM BA on 7 days old cotyledon explants. The highest number of direct shoot regenerations was also obtained using M1 (0.54 µM NAA and 0.44 µM BA) and M7 (0.1 µM NAA and 0.44 µM BA) media. Hygromycin with a concentration of 15 mg.l-1, completely inhibited the regeneration from untransformed explants and it was therefore used in selection medium. Optimal transformation conditions were obtained by co-culturing cotyledon explants with Agrobacterium tumefaciens in MS medium without any plant growth regulators for 72 hours. Primary analysis of regenerated plants in DNA level was conducted by specific primers for hph gene and transgenic plants were screened for further studies.