The Optimization Of Xinjiang Saussurea Plant Regeneration System And Agrobacterium-mediated Genetic Transformation

Saussurea involucrata Kar.et Kir.ex Maxim is a valuable traditional Chinese medicinal herb of the family Asteraceae.Owing to overexploitation of wild plants and difficulty of cultivation,S.involucrata is now almost extinct in China.Plant biotechnology will help to conserve the rare and endangered plant resources.In this study,a stable and effective plant regeneration system was established.Meanwhile,a genetic transformation system mediated by Agrobacterium tumefaciens was investigated.The results were as follows:Healthy and thick leaves from vigorous micropropagated plant were effective and easily available explants.Nearly 100%of leaf fragments could produced callus on medium containing suitable content of plant grow regulators.The texture and regeneration ability of callus depended greatly on the combination of grow regulators,light and culture time.On medium of MS+6-BA2.0mg/L+NAA0.5mg/L,70%percent of leave fragments could produced healthy adventitious shoot clusters directly when they were cultured in darkness for 3 weeks followed by 3 weeks under light.On MS+NAA 0.5 mg/L+TDZ1.0mg/L medium,leaf fragments produced callus with a lot of shoot buds on the surface when they were put in darkness for 2 weeks followed by 2 weeks under light.The shoot buds could develop into complete shoots after transferred to MS+6-BA2.0mg/L+NAA0.5mg/L for 3 weeks.Adventitious shoots could grow vigorously and proliferate more shoots on medium of MS+6-BA2.0mg/L+NAA0.5mg/L,and hereafter,could root at nearly 100%percent on medium of 1/2MS+IBA0.5mg/L+l%activated carbon for 4 weeks.These regenerated plants could survival well after being transplanted to soil.An expression-vector p3301-rd29b-TaCBF1 containing stress response TaCBFl gene was constructed and transformed into Agrobacterium tumefaciens.With the bacterium,a preliminary system for S.involucrata transformation was established.That is:leaf fragments that pre-cultured in darkness for 7d were steeped for 4min in the suspension of LBA4404(OD600=0.4)made of MS liquid medium,then were co-cultured with the bacterium in darkness for 3d.The infected explants were transferred to new medium without Basta for two weeks to recover and then transferred to the medium containing 2mg/L Basta and 300mg/L Cef to screen resistant callus or shoots.With this system,some resistant callus were obtained.