Agrobacterium Tumefaciens-mediated Genetic Transformation Of Litchi (Litchi Chinensis Sonn.) With LEAFY Gene

In this study, the division and growth of embryogenic callus of litchi (Litchi chinensis Sonn.) were observed by slice , which provided the basis of selecting target cells of transformation. Then, the protocol of Agrobacterium tumefac2 ens-mediated transformation of litchi was built by optimizing the factors affecting gene transfer efficiency and the selection and regeneration of transformed cells. A plant expression vector inserting LEAFYgene of Arabidopsis thaliana was constructed. LFY gene was introduced into litchi cv. Yuanhong, and regenerated plants were obtained. This work will benefit understanding the influnce of LFY constitutive expression to flowering time of litchi seedlings.1. The structure , division and growth of embryogenic callus of litchi and longan were observed by paraffin method. Litchi embryogenic callus was composed of embryogenic cells and nonembryogenic cells, embryogenic cells outside the callus and nonembryogenic cells inside. A piece of new callus derived from a single cell on the surface of the old callus. So, litchi embryogenic calli were chosen as transformation material. Longan callus was also composed of embryogenic cells and nonembryogenic cells. But differently, embryogenic cells of longan spread over the callus and the division centers were inside the callus.2. The factors affecting gene transfer efficiency during early transformation steps were studied using uidA , the GUS gene with intron. The results showed that (1) Agrobacterium strain EHA105 had the strongest virulence to litchi among threestrains LBA4404, AGL-1 and EHA105. (2)Cocultivation for two days not only had higher GUS transient expression but also could avoid Agrobacterium staining. (3)The bacterial density of 0.5 X 10s cells/ml was the best inoculum desity. ㏕he embryogenic calli subculturing for 15 days and dried treament before transformation improved the transformation efficiency.3. The protocol of Agrobacterium tumefaciens-mediated transformation of litchi was established, nptil gene was used as selectable marker gene and G418 as antibiotics. Resistant calli regenerated via somatic embryogenesis. Chimeric plantlets were avoided followed by three steps selection, including resistant calli, somatic embryogenesis and embryoids' germination.4. A plant expression vector, pBILFY, was constructed by inserting LFY gene into pBI121 ,with a CaMV35S promoter. pBILFY was introduced into AgrobacteriuoiEtiAlQ5 by triparental cross.5. LFY gene was introduced into litchi cv. Yuanhong and transgenic plantlets were obtained. Three transgenic plantlets were identified by PCR-Southern analysis.