Study On Genetic Transformation System Of Mulberry Mediated By Agrobacterium

Mulberry is a perennial dicotyledonous woody plant that is the main material basis for sericulture production.Mulberry is one of the earliest cultivated tree species in China,it is rich in germplasm resources and has a cultivation history of more than 4,000 years.Mulberry is also a traditional medicinal plant in China containing a large number of secondary metabolites.Flavonoids have significant medicinal value,and have the effects inhibiting thrombosis and lowering blood glucose,blood lipids,and blood pressure,in addition to antiaging,antitumor,and anticancer properties.Therefore,mulberry has very important economic value and research value,it is an important economic tree species in China.Among the previous studies on genetic transformation of mulberry,the most reported is the transformation of leaf disc mediated by Agrobacterium tumefaciens,while the transformation of cotyledon node of mulberry is less reported.In addition to Agrobacterium tumefaciens mediated method,Ri plasmid from wild-type Agrobacterium rhizogenes or Ri plasmid and exogenous plasmid together form a dual vector system to induce transgenic hairy roots of mulberry trees.Most of the studies on transgenic hairy roots are based on tissue culture instead of directly inoculating mulberry seedlings in the open air,inducing transgenic hairy roots without tissue culture technology have not been reported.From the perspective of genetic modification,this study strives to break through the common converted varieties of mulberry such as “sinyichinose”,“fengchi”,and “K2” using “the hybridization generation 1(YH1)of Yun7 and Huasang”,“Guiyou 12”,and “Guiyou 62” as experimental materials.The leaves of cotyledon segments of mulberry were transformed by A.tumefaciens GV3101,and the whole mulberry seedlings were transformed by A.rhizogenes K599 to induce transgenic hairy roots.The main research contents and results are as follows:1.A.tumefaciens GV3101 was used to infect the cotyledon node,and the explants were observed by histochemical staining.Histochemical staining showed that the expression sites ofβ-glucosidase(GUS)gene were different in different time periods after infection.GUS gene expression was concentrated in the base of the cotyledon,the main stem,leaf of transformed seedling,while the positive plants formed chimeras.GUS staining of 30 transformed seedlings showed a 36.7% infection rate.2.Leaf adventitious buds were induced by regeneration medium MS524 + sucrose 30g/L+p H 5.5+ Agar 7 g/L +TDZ 8 mg/L on Guiyou 62,Chuan Sang,and YH1,and the leaf regeneration systems of different varieties of mulberry were established.After transformation by A.tumefaciens,the best leaf transformation receptor was YH1.The infection rate of petiole incision and adventitious bud regeneration rate after transformation were calculated.The infection rate of petiole decreased from 100% to 32% with the extension of infection time,and the regeneration rate of petiole after transformation was 5.56%–13.33%.Leaves of 22 YH1 transformed seedlings were stained with GUS and showed no blue color.PCR detection of the hypopycin phosphotransferase(HYG)gene showed no bands.3.A.rhizogenes K599,carrying the endogenous plasmid p Ri2659 and the exogenous vector pLGNL(containing GUS and NPTII genes),was injected into the cotyledon nodes of mulberry seedlings,and adventitious roots were formed after 20 days.Fifty-five adventitious roots were selected for PCR detection of rol B genes,and three strains were screened to be amplified into the target bands of rol B.These results indicated that the three adventitious roots were all hairy roots induced by A.rhizogenes,and the rol B genes were successfully integrated into the plant genome.Specific primers were designed for pLGNL,and PCR was used to detect the hairy roots of three strains.The results showed that the target bands of 2,800 bp could be amplified by PCR for the hair roots of three strains.It was further verified that these three hairy roots were transgenic hairy roots.4.The expression vectors pLGNL and pLGNL-e GFP were introduced into A.rhizogenes K599,and the expression of GUS gene was obvious in hairy roots,the enhanced green fluorescent protein(e GFP)gene was strongly expressed in root tip meristem under fluorescence microscope.Overexpression of the exogenous NPTII gene and e GFP gene in new adventitious roots were successfully identified.5.The positive rate of hairy roots was improved by setting different seedling ages,different preparation methods of bacterial liquid and different injection sites.The optimal conditions for inducing hairy roots were as follows: 2 mulberry seedlings with incomplete true leaves were used,and inoculated against cotyledon nodes of mulberry seedlings for about 3 times through plate bacteria production,which significantly increased the positive rate of hairy roots.In this experiment,the highest induction rate of hairy roots was 2.9%.