Genetic Transformation System Of Dioscorea Zingiberensis C. H. Wright Mediated By Agrobacterium Tumfaciens

Dioscorea zingiberensis C.H.Wright is a native species in China,which is one of the most important medicinal medicinal resource plants,and has the highest content of diosgenin among all the species in the word.However,it has been so extremely harvested that there are few wild plants of this species in recent years.While the artificial plantings suffer from the decline of diosgenin concentration in them and the degeneration of the germplasm.Conventional methods,such as hybriding breeding,autotetraploid breeding and so on,to improve the artificial plantings quality have not solve this difficulty.With the development of the biotechnology,genetic transformation mediated by Agrobacterium provides a new efficient way to solve the matters.The studies explored and optimized the factors that may influence Agribacterium-mediated transformation of Dioscorea zingiberensis C.H.Wright based on the rate of GFP transient expression.The transformation protocol was established.The main results are as follows:一.Preparation of transformation receptors1.Genotypes selection.By callus induction from the immatural embryos of four genotypes,the tissue culture capability of different genotype was different.The rates of callus induction were respectively 95.3%in 4-73,91.2%in 4-61 and 76.3% in 5-88.Almost no callus was induced in 4-72.4-73 was selected as used to the following transformation study.2.Embryogenic callus obtained and subculture.The initiated calli from the immatural embryos were non- globular,growth slow and watery.By formulating the optimum combination and concentration of plant growth regulators,organic supplements, components of basic medium,subculture methods and so on.Embryogenic calliwhitish yellow,compact and globular in apperace were obtained.3.Calli from inflorescences were induced on MS medium supplemented with 0.5 mg/L 2,4-D,1.0 mg/L 6-BA or 0.2 mg/L 2,4-D,1.0 mg/L 6-BA.二.Main factors influencing the transformation1.Explant types.Embryogenic calli from immatural embryos,calli from inflorescences and inflorescences were infected with Agrobactium respectively,GFP transient expression was detected after co-cultivation.GFP transient expration rate of embryogenic calli from immatural embryos was highest.Inflorescences could not used as receptors directly.2.Pre-cultivation.Comparying GFP transient expression rates when embryogenic calli and inflorescence calli were transformated after pre-cultivation or not,pre-cultivation was beneficial to transformation.3.Components of basic medium in pre-cultivation and co-cultivation.MS,1/4 MS and MS without CaCl₂ were used as pre-cultivation and co-cultivation medium.The results indicated MS without CaCl₂ was the optimal basic medium for pr-culture and co-culture.4.AS.Camparying GFP transient expression rates between with or not 200μmol/L AS,results showed that pre-cultivation and co-cultivation medium with 200μmol/ L AS was beneficial to transformation.5.Concentraion of Agribacterium solution.GFP transient expression was detected by infecting with OD₆₀₀为0.3.,0.5,0.8 Agribacterium solution.The results showed OD₆₀₀=0.5 was the optimal concentration.6.Co-cultivation days.Comparying GFP transient expression rates when embryogenic calli were co-cultured fro 1,2,3 and 4 days,the results showed 3 days was the optimal co-cultivation time.7.Validation of the transformation conditions.By the conditions as the discribed above, embryogenic calli and inflorescence calli were infected with agrobacterium with GUS gene.The results showed the conditions for trasformation were benificial.三.Selection of transgenic cells,transgenic plants regeneration and PCR identyfication.1.Determination of hygromycin concentration.Embryogenic calli were plated on the medium with different concentration of hygromycin.The concentration of hygromycin 70 mg/L was determined as the selection concentration.2.The transgenic cells selection and transgenic plants regeneration.The three explants were reprectedly planted on the selection medium.After 1-2 months' selection,the hygromycin-resistant calli on the selection medium were planted on a regeneration medium without antibiotics and eight putative transgenic plants were obtained. 3.GFP detection of putative transgenic plants.The putative transgenic plants obtained were propagated.Faint green fluorescence was observed in the leaves.4.PCR identyfication of the transgenic plants.The hygromycin gene was detected by PCR in the genome of them.The results showed the transgenic plants were obtained.