| As a source of raw materials for Industry and energy,Castor(Ricinus Communis.L)is regarded as one of the most valuable special oil crops and has high economic value and wide application prospect.At present,the increasing castor demand at home and abroad has led to the shortage of castor raw materials,which seriously restricts the development of castor industry.Molecular breeding technology can greatly accelerate the breeding process,which is of great significance to solve the bottleneck of castor production advancement.Efficient and stable regeneration system in vitro and genetic transformation efficiency are the decisive factors for molecular breeding.This paper aims to research the problem that low efficiency of genetic transformation and difficulty of regeneration for in vitro culture of castor molecular breeding.We used the GUS transient expressive technique to study of influence factors on genetic transformation efficiency of agrobacterium mediated castor,and in the process of transformation,optimize the system by taking auxiliary measures in order to improve genetic transformation efficiency of castor.We adopt Quantitative Real-time PCR and bioinformatics analysis to study the expression of regeneration candidate genes in tissue culture response of castor,which provided reference to improve the regeneration rate of casto in tissue culture.The concrete results are as following.(1)The optimum concentration of Agrobacterium was different for different strains.The optimum concentration of Agrobacterium EHA105 and Agrobacterium LBA4404 was OD600=0.8,The optimum concentration of Agrobacterium GV3301 was OD600=1.0;Agrobacterium strains have significant influence on genetic transformation efficiency of castor that has the same genotype,Agrobacterium EHA105 transformation ability is best,LBA4404 second,andGV3301 poor;The sensitivity of different genotypes to Agrobacterium was different,in ten varieties used in this experiment,HP003 and Jia xiang 2 which were sensitive to Agrobacterium infection were screened.(2)In genetic transformation of embryonic tips of castor,minimally invasive and surfactant added have significant effect on improvement of genetic transformation efficiency.We determined that the best mode of infection was ultrasonic assisted infection for 3min,the optimal concentration of surfactant Silwet-77 is 0.05%,and the best time of co-culture was 3 days.(3)Using the optimized castor embryo genetic transformation system to transformat castor of Jiaxiang 2,The results showed that 30 resistant plants were obtained and the probability is 4.65%.7 transgenic castor plants were obtained by PCR detection,and the transformation rate was 1.08%,and 13.68%higher than the original system.(4)Using Quantitative Real-time PCR technology to study the expression of RcSERK1,RcSERK2,RcWUS and RcNiR gene in organs and during tissue culture process,results showed that RcSERK1,RcSERK2,RcWUS and RcNiR genes are involved in the growth and development of castor,and play a role in the pathway of somatic embryogenesis in castor.(5)Bioinformatics analysis was used to predict the basic physicochemica properties,hydrophilicity,Stability,signal peptide,transmembrane structure and Secondary structure of RcSERK1 gene,The results showed that RcSERKl gene and AtSERK1 gene had similarity in protein physicochemical properties and structure,and RcSERK1 gene expression was related to somatic embryogenesis. |
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