Establishment Of A Genetic Transformation System For Malus Domestica Based On Somatic Embryogenesis

Apple is a perennial fruit tree,genetically highly heterozygous,using traditional hybrid breeding methods to breed a good variety is difficult and has a long cycle.The rapid development of modern biotechnology,especially transgenic technology,provides a new way for the selection and breeding of apples.However,the current genetic transformation of apples mainly uses leaf organogenesis as the regeneration system,and its transformation efficiency is low,which limits the practical application of this technology in breeding work.The method of obtaining regenerated plants through somatic embryogenesis has the advantages of large number,fast speed,high seedling rate,good genetic stability,etc.,and genetic transformation based on direct somatic embryogenesis can reduce the incidence of transgenic mosaicism.Therefore,we established an apple leaf direct somatic embryogenesis system,and carried out genetic transformation based on this system.The results of the existing research are as follows:1.The establishment of somatic embryogenesis system: We take the tissue culture seedlings of’Gala’ apple as the object.After the selection of explant materials,the adjustment of medium hormones,the optimization of culture conditions and other processes,we obtain the best apple directly somatic embryogenesis system is: select the unfolded leaves at the top2-4 of the tissue culture seedlings subcultured for 30 days,cut the leaf pieces near the petiole as explants,and first apply MS+TDZ 0.5 mg/L+ NAA 6 mg/L+2,4-D 0.6 mg/L+sucrose 30g/L embryogenic cell induction medium cultured in the dark for 8 days,then transferred to MS+6-BA 1.0 mg/L+sucrose 30 g/L The embryogenesis medium was cultured in the dark for25 days.After 30 days of exposure to strong seedlings,the leaf somatic embryogenesis rate was 82.3%,and the average number of regenerated somatic embryos per leaf was 9.1.2.Establishment of genetic transformation system: based on the leaf direct somatic embryogenesis system,established by adjusting the pre-culture time,Agrobacterium liquid concentration,infection time,co-cultivation time,selection pressure,and GUS instantaneous transformation efficiency as an indicator The effective genetic transformation system is as follows: the leaves are pre-cultured in embryogenic cell induction medium for 6 days,the concentration of Agrobacterium solution during infection is OD600=0.4,the infection time is10 min,and the co-cultivation is 2 days before embryogenesis.Sterilize and screen in the culture medium.3.Obtaining transgenic plants: In order to visually demonstrate the feasibility of the apple genetic transformation system based on somatic embryogenesis,we constructed a CRISPR-Cas9 vector to mutate the apple PDS gene and successfully obtained transgenic albino plants.