Establishment Of Agrobacterium-mediated Transformation Of Somayic Embrys In Quercus Variabilis

Quercus variabilis is the most important softwood resource tree species in China,which has important ecological and economic value.Forest tree genetic improvement is an important way to improve productivity and resistance to diseases and insect pests,which could be accelerated by conventional breeding methods combining plant genetic engineering techniques,one of which is Agrobacterium-mediated genetic transformation is a powerful biotechnology tool for plant transgenesis.Therefore,it is of great significance to carry out research on the genetic transformation system of Quercus variabilis.In this study,immature zygotic embryos of Q.variabilis were used as explants to induce somatic embryogenesis.The effects of different factors on embryogenic clusters proliferation for cell suspension culture were examined.The effect of low temperature drying maturtes somatic embryos on germination was investigated.Transgenic receptor system of plant regeneration by somatic embryogenesis in Q.variabilis was optimized.On this basis,the effects of bacterial solution concentration,acetosyringone concentration,pre-culture time,infection time and co-cultivation time on genetic transformation were studied.A preliminary protocol for genetic transformation basied somatic embryogenesis in Q.variabilis was developed.The main results were as follows:1.Quercus variabilis is propagated through secondary somatic embryogenesis.In the temporary immersion culture,the embryonic clusters were immersed in the liquid medium for 1 min at intervals of 48 h,and the proliferation rate of 363.8%was obtained.In the process of cell suspension culture,the initial inoculation amount of 0.1 g per 50 ml of liquid medium is cultured at 110 r·min^-1rotation speed for 28 days,and the highest proliferation rate of 638.5%is obtained.2.In germination culture,using MS as the basic medium,the combination of 0.5mg·L^-16-BA and 0.2 mg·L^-1NAA can promote the germination of somatic embryos,and the germination rate is 40.0%.After maturation culture,the somatic embryos are refrigerated or semi-dried for a certain period of time,which is conducive to the germination of somatic embryos.When refrigerated at 4?for 2 weeks,the germination rate of somatic embryos reached 47.8%.3.In the antibiotic sensitivity test,the cefotaxime concentration of 300 mg·L^-1can significantly inhibit the growth of embryogenic clusters,and the survival rate of embryogenic clusters is only 1.7%when the concentration of hygromycin is 30 mg·L^-1.Therefore,in the screening stage of transformed clusters,the appropriate concentrations of cefotaxime and hygromycin were 300 mg·L^-1and 30 mg·L^-1,respectively.4.Agrobacterium tumefaciens EHA105-mediated genetic transformation obtains embryogenic clusters of transformed GUS gene:the embryogenic clusters was pre-cultured for 15 days,and was inoculated with bacterial suspension of OD₆₀₀=0.5 for 20 min;the infected clusters were co-cultured for 3 days in medium added with 200?mol·L^-1acetosyringone,and the GUS transformation rate is up to 100%.5.Twelve clusters were selected from the transformed clusters verified by GUS staining,and DNA was extracted for PCR detection.Ten clusters expanded the target band,which further proved that the GUS gene has been integrated into the Q.variabilis genome.The other two clusters did not amplify any bands,which were false positive tissues.