Establishment Of The Shoot Regeneration System Of Apple Dwarfing Self-Rootstock

In apple planting production,dwarfing and dense planting is not only an effective way to improve land utilization efficiency,but also convenient for management.Currently,the most effective way to achieve dwarfing and dense planting of apples is to use dwarfing rootstocks.The dwarfing rootstocks are beneficial to the size of fruit trees and for getting the earlier fruit and high yield.Apple dwarfing self-rooted rootstocks are propagated mainly by cutting,beading and branched in production.Establishing a set of highly efficient regenerating shoot system for apple dwarfing self-rooted rootstocks is of great theoretical and practical significance in apple production and breeding.Apple dwarfing self-rooted rootstocks ‘T337' is a widely used stock variety.There are many advantages in ‘T337' such as smaller tree size and convenient for daily management.But its shoot regenetaion system has not been established.In this study,‘T337' leaves were used as explants to establish their regenerative bud system by inducing calli to differentiate adventitious buds,and through the Agrobacterium tumefaciens-mediated genetic transformation method,the MdWUS1 gene was successfully introduced into apple dwarfing self-rooted rootstock ‘T337',which further increased the frequency of ‘T337' regenerated shoots.The research progress is as follows:‘T337' efficient regeneration bud system had been established: Using ‘T337' leaves as explants,the effects of hormone concentration ratio,dark culture time,seedling age,and leaf growth nodes on leaf bud regeneration were studied.The results showed that the optimal medium for inducing the bud regeneration system of ‘T337' leaves was: MS + 2 mg / L 6-BA+ 0.5 mg / L NAA + 30 g / L sucrose + 8g / L agar(pH5.8),The best dark culture time is 14 days,the best seedling age is 45 days,and the best leaf growth nodes are the leaves from top to bottom,sections 2-3,The frequency of regenerated buds and the average number of regenerated buds reached more than 90% and more than 3.Arabidopsis WUSCHEL(AtWUS)is a stem cell regulatory gene in stem meristem,and its overexpression can promote the occurrence of Arabidopsis buds.To further improve theefficiency of ‘T337' bud regeneration,we transformed the homologous gene MdWUS1 of AtWUS into ‘T337' by genetic transformation.The specific results are as follows: Using‘T337' leaves as explants,after pre-cultivating for 2 days in the dark,agrobacterium was cultivated to inoculate the explants for 8 min when the bacterial solution concentration was OD600 = 0.4-0.6,in order to fully contacted the cut cells.After 20 mg/L AS(acetylsyringone)was added to the co-culture medium for 2 to 3 days,Tm(termetine)200 mg / L bacteriostatic culture for 2 days followed by HYG(hygromycin)3 mg / L selective pressure screening culture.After 5-7 generations of culturing,resistant seedlings were obtained.In the experiment,it was found that the bacteriostatic culturing with 200mg/L Tm and the screening culturingwith 3 mg/L HYG had the best bacteriostatic effect and screening effect.With 10 mg/ E2 as the inducing agent concentration in the induction medium,the expression vector was the best for 7-days cultivation.The total DNA of the leaves of the resistant seedlings after screening was extracted for PCR detection,and the results proved that the ‘T337' transgenic plants had been successfully obtained.By qRT-PCR analysis,it was found that the transcription level of MdWUS1 in transgenic plants was up-regulated.Phenotype observation showed that the frequency of regenerated buds of transgenic ‘T337' increased significantly after induction.In conclusion,this study successfully established the bud regeneration system of apple dwarfing self-rooted rootstock ‘T337' and successfully transferred the MdWUS1 gene into‘T337',further increasing the frequency of bud regeneration.