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目的:建立起丹参的转基因毛状根诱导及离体培养体系。方法:考察了不同外植体、不同农杆菌侵染时间和共培养时间诱导丹参毛状根的效率,共培养外植体用400 g.L-1Cef水除菌5 min,接种在MS+400 g.L-1Cef+2.5 g.L-1Hyg的固体培养基上,完全除菌后转接入6,7-V+2.5 g.L-1Hyg液体培养基继代培养,GFP荧光检测阳性毛状根,PCR检测农杆菌特征基因rolC,并测定不同生长时期的毛状根干重和二氢丹参酮I的积累。结果:用丹参叶片基部诱导毛状根,成功率可达到93.3%;农杆菌侵染10 min诱导效率最高为63.3%;共培养2～3 d诱导效果最好;PCR结合GFP荧光检测的方法鉴定阳性转基因材料具有较高的可信性;丹参毛状根生物量变化与次生代谢物积累之间有着紧密联系。结论:成功建立起丹参转基因毛状根离体培养体系,为进一步的基因工程应用打下基础。
Objective: To establish a transgenic hairy root induction and in vitro culture system of salvia miltiorrhiza. Methods: The efficiency of inducing the hairy root of Salvia miltiorrhiza Bunge by different explants, different Agrobacterium tumefaciens infection times and co-culture time were investigated. The co-cultured explants were sterilized with 400 g L-1Cef water for 5 min and inoculated on MS + 400 g L- 1Cef +2.5 gL-1Hyg solid medium, completely sterilized and then transferred to 6,7-V +2.5 gL-1Hyg liquid medium subculture, GFP fluorescence detection of hairy roots, PCR detection of Agrobacterium tumefaciens characteristic genes rolC, and determined the dry weight of hairy roots and the accumulation of dihydrotanshinone I in different growth stages. Results: The root of hairy root of Salvia miltiorrhiza was induced with the rate of 93.3%. The highest induction efficiency of Agrobacterium tumefaciens was 63.3% after 10 min of infection. The best induction effect was obtained after 2 ~ 3 days of co-culture. PCR and GFP fluorescence detection Positive transgenic material has a high credibility; Salvia hairy root biomass changes and secondary metabolites have a close relationship between the accumulation. Conclusion: The transgenic hairy root culture system of Salvia miltiorrhiza Bge. Was successfully established, laying the foundation for further genetic engineering application.