Establishment and optimization of peach hairy root transformation system
[Objective]In order to solve the problems of chimera,time-consuming and heavy workload in peach genetic transfor-mation,this experiment established and optimized the hairy root transformation system mediated by Agrobacterium rhizogenes.[Methods]The hypocotyl,cotyledons and upper parts of 3-week-old sterile tissue-cultured'Zhongtao 24'peach seedlings were used as explants,and the transformation was mediated by Agrobacterium rhizogenes MSU440 transformed with pCAMBIAsu-per1300-GFP plasmid.The infected explants were transferred to the medium containing antibiotics(cephalosporins)and cultured for about 7 d.The formation of callus and hairy roots was observed.The explants from hairy roots were detected by stereo fluorescence microscope to observe the green fluorescence signal.The total RNA of hairy roots with fluorescence signal was extracted,and the positive hairy roots were detected by PCR,and the optimum transformation varieties,infection concentra-tion and infection time were screened.[Results]Agrobacterium rhizogenes MSU440 was used to infect the hypocotyl of peach,and hairy roots were induced.Transgenic hairy roots were quickly screened by GFP expressed in the transformation vector,and RolB and eGFP gene fragments were amplified by PCR.The results of optimization showed that soaking in the infection solu-tion of Agrobacterium rhizogenes with OD value of 0.8 for 30 min was the best transformation condition,and the highest transformation rate was 63.6％.[Conclusion]The hairy root transformation system mediated by Agrobacterium rhizogenes was established in this experiment,and the transformation conditions were optimized,which provided a technical basis for verifying the function of peach genes and obtainingstable transgenic peach plants.
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