Abstract:【Objective】Using biotechnology to improve and innovate apple germplasm can further improve the production efficiency and quality of apple. The transformation method of Agrobacterium rhizogenes 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 apple (Malus domestica) seedlings. 【Methods】 Using apple seedlings of different ages as materials, A. rhizogenes K599 carrying GFP and GUS overexpression plasmids was used to infect the apple rhizome by injection (use a 2 ml sterile syringe to draw the strain resuspension and inject it at the rhizome of the apple seedlings), activating bacteria solution infiltration method (cut off the main root system at the rhizome ofapple seedlings, and then directly invade 30 min in the activated bacteria solution) and strain smearing method (cut off the main root system at the rhizome of apple seedlings, and then the strains on the plate were collected by aseptic spreader and applied to the wounds of apple 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 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 hairy roots of apple genetic transformation lines, the GFP signal of hairy roots was identified by portable fluorescent protein excitation light source and photographed. To identify whether the target gene was integrated into the apple root genome, 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.【Results】The hairy root induction rate of apple seedlings of different ages was induced by injection, in which the rooting rate of seedlings in 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 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 different ages of apple seedlings and it was found that there were differences in the hairy roots induction number. Among them, the hairy roots induction rate of apple seedlings at the three-leaf stage was 84%, and the induction rates of apple seedlings at the five-leaf stage and eight-leaf stage are 47% and 52.5% respectively. Therefore, apple seedlings at the three-leaf stage were more suitable as transformation materials for the activating bacteria solution infiltration method. Different ages of apple seedlings could successfully induce hairy roots, among which 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 apple 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 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 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 strain with GUS signal detected in leaves was selected as material, and the expression of GUS in root, stem and leaf samples was detected. The results showed that weak expression of GUS gene was detected in stems and leaves. Then the total proteins of roots and leaves were further extracted. Western blotting showed that the expression of 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 migrated 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 apple seedlings infected by A. rhizogenes. Using strain smearing method to infect three-leaf seedlings, the positive hairy root plants with high uniformity can 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. rhizogenes transformation technology.
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