| Peach,as an important temperate fruit tree in the world,belongs to the rose family.The yield and cultivated area of peach in China rank first in the world.Therefore,the innovation of peach germplasm resources has always been given great significance by Chinese breeding experts.At present,traditional cross breeding is still the main method of peach tree breeding in China,which is easy to operate and is beneficial to concentrate some of the good traits of parents in the offspring,but it also has the disadvantages of longer breeding cycle and difficulty in integrating multiple desirable traits in a directional way.Current gene editing techniques developed in crops can achieve targeted improvement of germplasm resources,and this technique relies on a complete transgenic system.So far,no transgenic system of peach has been reported.Therefore,in this study,peach hypocotyls were used as explants to induce callus formation through induction medium,and callus were treated with different cytokinin types and concentrations in order to differentiate into adventitious buds.As bud generation is still difficult,the expression characteristics of WOX gene family members related to induction of root and bud formation under cytokinin induction were further studied,and the causes of bud generation difficulties in peach genetic transformation were preliminarily analyzed from the molecular point of view,so as to provide new ideas and tools for subsequent bud generation induced by peach genetic transformation.At the same time,the induced callus was used as the experimental material for the agrobacterium tumefaciens mediated genetic transformation experiment,and GUS gene was used as the reporter gene to carry out the peach genetic transformation by agrobacterium tumefaciens mediated method.A genetic transformation method of peach callus was explored,which laid a foundation and provided a technical method for gene function verification and molecular breeding of peach in the future.The main research results are as follows:1.Induction and subculture of peach callus.Cotyledons were taken as explants and inoculated on the surface of solid callus induction medium after disinfection.The callus can be subcultured to further expand the growth after the callus is observed to grow bright yellow clumpy.The callus induction medium was formulated as MS+1.0 mg/L 6-BA+1.0 mg/L TDZ+0.5 mg/L IBA+20 g/L SUC+6-8 g/L Agar+0.5 g/L MES.The medium formula of callus subculture was MS+1.0～2.0 mg/L 6-BA+0.2～0.6mg/L IBA+20 g/L sucrose+6～8 g/L AGAR+0.5 g/L MES,p H 5.6～5.8.2.Six members of peach WOX gene family were screened out,which may closely related to the regulation of bud formation.It was found that the expression of Pp WOX4 increased significantly on the4th day when the concentration of cytokinin was 1 mg/L,while At WOX4,the homologous gene of Pp WOX4 in Arabidopsis thaliana,did not have the function of bud induction.Therefore,it was speculated that the reason for the difficulty of peach genetic transformation in bud formation was that the cytokinin in the bud formation medium could not activate the expression of WOX gene family members which could effectively induce bud formation.3.Preliminary construction of peach genetic transformation system.Peach callus with good regeneration ability can be used as the acceptor of genetic transformation after three times of subculture.When the concentration of Agrobacterium infection solution was adjusted to OD600≈0.3,the conversion efficiency was higher.The callus was immersed in MS infection solution at 25℃,50 to 80 r/min for10 to 20 min,and the dark culture time was 3 to 5 d.GUS staining showed that the transformation efficiency of Agrobacterium tumefaciens GV3101 was higher than that of EHA105 and LBA4404,which could reach about 3%.The contamination rate of solid medium was lower than that of liquid medium. |
