| In the present study, a high efficient transgenic system via particle bombardment was established and optimized using the long-term maintained embryogenic calli (EC) induced from immature embryos of Litchi chinensis Sonn. cv. Xiafanzhi. The main results were described as follows:1) A high efficient plant regeneration system was established through optimization of parameters affecting somatic embryogenesis in litchi. The optimal medium for inducing somatic embryogenesis was MS medium supplemented with 5mg/L zeatin (ZT), 6% sucrose and 1.0% agar. Somatic embryos matured on MS medium supplemented with 6% sucrose, 1.0% agar and 10% coconut water (CW), and germinated on MS medium without plant growth regulators but supplemented with 2% sucrose and 0.7 % agar.2) An appropriate recipient system for litchi genetic transformation was established. Calli pre-cultured for 4-5 days were proved at their rapid-growth stage through the measurement of EC fresh weight increase and suitable for transgenics. The sensitivities of litchi EC to hygromycin B and kanamycin were evaluated through the method of the measurement of fresh weight increase combined with qualitative observation, which showed that litchi EC were not sensitive to kanamycin but sensitive to hygromycin B. Hygromycin B obviously depressed the growth of litchi EC. which was mortal to them. The concentration of 50 mg/L for hygromycin B was proved to be suitable for litchi EC transgenic selection through further experiments.3) The optimal parameters used in litchi EC bombardment were obtained. Litchi EC were bombarded using the plastmd pCAMBI01301 including gus and hpt genes, and the parameters affecting bombardment were optimized. The results showed that the best transient expression of GUS was obtained when litchi EC were bombarded with the following procedures: litchi EC pre-cultured for 5-10 days were transferred onto the osmotic medium supplemented with 0.25 mol/L mannitol for 4 hour-long pre-treatment; and then they were bombarded one time after the preparation of particle-plasmid compounds was performed using 0.5 Ugplasmid DNA and 600 ug gold microparticies per shoot under the conditions of 1100 psi helium pressure and 6 cm shooting distance; and finally cultured on the same osmotic medium as above for another 16 hours.4) A whole technical system of litchi resistant calli selection, somatic embryogenesis from resistant cell lines, transgenic plant regeneration and transgenic assay was established. After the bombarded litchi EC were selected on MS medium supplemented with 50 mg/L hygromycin B, some 80 resistant cell lines were obtained, and finally 7 cell lines were maintained for further studies through the optimization of culture conditions and further selecting procedures. Gus and hpt gene had both integrated into the genome of the 7 resistant cell lines proved by PCR assays and among them, 4 resistant cell lines steadily expressed the GUS enzyme proved by GUS histochemical assays. About 30 plantlets regenerated via somatic embryogenesis from theresistant cell lines. Among them, about 20 plantlets regenerated from the maintained resistant cell lines steadily expressing the GUS enzyme, and they were proved to be transgemc plantlets by GUS histocherrucal assay; and the other were non-GUS expressing plantlets which regenerated from the resistant cell lines without GUS expression.Additionally, a preliminary study onAgrobacterium-mediated litchi EC transformation was also performed, from which 4 resistant cell lines were obtained, and finally 2 resistant cell lines were maintained for further studies, from which transgenic plantlets expressing the GUS enzyme were obtained.All the above 9 resistant cell lines were well maintained in our laboratory.The establishment and optimization of EC transformation system in litchi would be of great importance for genetic improvement in litchi; and finally some problems about litchi genetic transformtion to be solved were put forward in this thesis.
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