| Chinese yam（Dioscorea opposita Thunb.）is a perennial herbaceous vine of Dioscoreaceace Dioscorea,tube of which is food and medicine.Due to asexual reproduction,its quality and yield was degradated.We have been cloned DoSERK2 gene and construct its expression vector.It is of significant value for Chinese yam breeding to study its somatic embryogenesis and genetic transformation.The main results are as follows:1.Somatic embryogenesis of D.opposita.（1）Picloram is a key factor affecting the induction of somatic embryos.Stems with axillary buds can induce the formation of somatic embryos in culture medium containing different concentrations of picloram.The best induction effect was obtained when the medium is consists of PIC 5 mg·L-1 and CuSO42 μmol·L-1.After 8 weeks of induction,the rate of somatic embryogenesis and the number of somatic embryos in a single nodal segment were 91.67% and 6,respectively.The efficient of embryogenesis induction were compared among stem with axillary buds,stem with nodal,and leaf,and stems with axillary buds has the most highest rate of induction.（2）In somatic embryo multiplication experiment,the optimal culture medium is MS + 6-BA 0.5mg·L-1 + PIC 1 mg·L-1 + CH 0.6 g·L-1+ Pro 1 g·L-1,coefficient of which reached 3.25.The somatic embryo has not browning and death phenomenon.Under the light condition,the surface of embryo generated yellow or white compact callus,and they turned green after a period of time.When transferring them to medium congtaining 6-BA 0.4 mg·L-1,they form regenerated plants after 6-7 weeks.（3）The results from double staining with acetocarmine and Evans blue showed that the globular body grown from the axillary buds is embryoids consist of embryogenic tissue polymerization.The globular body developed into globular embryo,shield shaped embryo and abnormal embryo,and abnormal embryo can not form regenerated plants.In callus proliferation process,we found that only dense yellow or white tissue is embryogenic callus.2.Establishment of genetic transformation system in Chinese yam（1）The construction of silencing vector pART27-SERK.The interference fragment was cloned into intermediate vector pHANNIBAL,and the interference fragment with 35 S promoter and NOS terminator was cut and insert the vector pART27 to generate the silence vector.（2）The antibiotic sensitivity of different explants is different.The selection pressure of Hyg and Kan on Dioscorea opposita PLB were 15 and 160 mg·L-1;the two selection pressure of micro tubers were 15 and 120 mg·L-1;the two selection pressure of rooting were 20 and 160 mg·L-1;concentration of Tim was determined to be 500 mg·L-1.（3）The Agrobacterium-mediated genetic transformation system of Chinese yam has established.Agrobacterium OD600 was 0.5,the infection of micro tuber slices of 30 min,and then transferred to the co-culture medium containing AS 100 μmol·L-1,after 3 d,transferred to the regenerated plantlets culture containing Hyg 15 mg·L-1 medium and rooting medium containing Hyg 20 mg·L-1.Transgenic plants of DoSERK2 overexpressing were identified,by PCR and GUS.In the same method,the regenerated plantlets were selected as Kan mg L-1 and Kan160 mg·L-1.Transgenic plants of DoSERK2 silencing were identified,by PCR.（4）Agrobacterium mediated transient expression system of Chinese yam has established.The beta glucuronidase（GUS）and green fluorescent protein（GFP）gene as reporter genes,the concentration OD6000.6 of bacteria injected,3 d of co-culture,GUS gene and GFP gene began to express.The suitable time for observation of the co-culture were 5 d and 4 d. |
