Construction and optimization of transformation system mediated by Agrobacterium rhizogenes in Malus hupehensis var.mengshanensis G.Z.Qian & W.H.Shao
[Objective]Using biotechnology to improve and innovate apple germplasm can further im-prove the production efficiency and quality of apple.The transformation method of Agrobacterium rhi-zogenes has the advantages of high transformation efficiency and simple operation steps,especially in the mining and functional verification of soil stress-related resistance genes.However,A.rhizogenes transformation system for apples is still time-consuming with complicated steps and low efficiency.Therefore,we established and optimized an efficient and rapid A.rhizogenes transformation system for Malus hupehensis var.mengshanensis G.Z.Qian & W.H.Shao seedlings.[Methods]The seedlings at different ages were used as materials,A.rhizogenes K599 carrying GFP and GUS overexpression plas-mids was used to infect M.hupehensis var.mengshanensis rhizome by injection(a 2 mL sterile syringe was used to draw the strain resuspension and inject it at the rhizome of the the seedlings),activating bac-teria solution infiltration method(the main root system at the rhizome of the seedlings was cut off,and then the seedlings were directly invaded 30 min in the activated bacteria solution)and strain smearing method(the main root system at the rhizome of the seedlings was cut off,and then the strains on the plate were collected by aseptic spreader and applied to the wounds of the seedlings),then the infected seedlings were planted in sterilized nutrient soil and kept in high humidity environment,co-cultured in dark for 2 days,and the hairy root induction was detected one month later.The lines with successfully induced hairy roots were selected and the DNA was extracted.In order to identify the effectiveness of the hairy roots of the genetic transformation lines,the GFP signal of the hairy roots was identified by portable fluorescent protein excitation light source and photographed.The hairy root DNA was further extracted,the GUS tag gene was cloned by PCR,and the GUS staining of the roots and leaves of the transformed lines were analyzed to identify whether the target gene was integrated into M.hupehensis var.mengshanensis root genome.[Results]The hairy root induction rate of the seedlings at different ag-es was induced by injection,and the rooting rate of seedlings at eight-leaf stage was 39％,and wilting death occurred in three-leaf stage seedlings after injection,and the overall hairy root induction rate was 35％.After excluding the dead lines,the hairy root induction rate was 67.9％,indicating that the hairy root induction ability of the three-leaf stage seedlings was high,but the overall hairy root induction rate was low due to weak growth.The activating bacteria solution infiltration method was used to infect the seedlings at different ages and it was found that there were differences in the hairy roots induction num-ber.Among them,the hairy roots induction rate of the seedlings at the three-leaf stage was 84％,and the induction rates of the seedlings at the five-leaf stage and eight-leaf stage were 47％and 52.5％,respec-tively.Therefore,the seedlings at the three-leaf stage would be more suitable as transformation materi-als for the activating bacteria solution infiltration method.The hairy roots could be successfully induced in the seedlings at different ages,and the hairy roots induced by direct smearing of strains in three-leaf stage and eight-leaf stage were 96％and 60％,respectively.In addition,after comparing the hairy roots induced by the above ways,the roots induced by strain smearing method of the seedlings at the three-leaf stage were evenly distributed at the base of the stem segment,and the roots were more abundant,so the hairy root distribution and hairy root induction rate were the best.In order to identify the expression pattern of the target gene in A.rhizogenes transformed plants,the DNA of the roots,stems and leaves of transformed plants were extracted.Through the PCR cloning of GUS gene,it was found that there were GUS signals in the roots,stems and leaves.The GUS staining analysis of the transformed roots and leaves showed that there were GUS signals in the petiole and main leaf vein of some lines.The strains with GUS signal detected in the leaves were selected as material,and the expression of GUS in the root,stem and leaf samples was detected.The results showed that weak expression of the GUS gene was de-tected in the stems and leaves.Then the total proteins of the roots and leaves were further extracted.Western blotting showed that the expression of the GFP protein was detected in the roots of transformed positive lines,but no obvious expression of GFP fluorescent protein was detected in leaves.The above results showed that in the transgenic lines obtained by A.rhizogenes transformation,A.rhizogenes mi-grated randomly to the shoot through vascular tissue.[Conclusion]A rapid,simple and efficient apple transformation system mediated by A.rhizogenes K599 was established in this study.Seedling age is the key factor affecting the transformation efficiency of the seedlings infected by A.rhizogenes.Using strain smearing method to infect three-leaf seedlings,the positive hairy root plants with high uniformity could be obtained in about 45 days,and the induction rate is as high as 96％.The migration of A.rhizogenes in transformed plants was explored to provide a theoretical basis for the further utilization of A.rhizo-genes transformation technology.
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