Establishment Of Agrobacterium Rhizogenes Mediated Hairy Root Genetic Transformation System In Malus Domestica

The apple(Malus domestica)is one of the most productive fruits in the world and is known as the “king of temperate fruits”.The apples produced and consumed in our country make up half of the total amount in the world every year.At present,the common genetic transformation system of apple at home and abroad is mainly “leaf disc transformation method”.The “leaf disc transformation method” is to infect the leaves of apple explants with Agrobacterium tumefacien,co-culture them for a short period,and obtain apple transgenic lines after induction under different conditions in the later period.The application of Agrobacterium rhizogenes in apple genetic transformation technology system is mainly aimed at solving the problem that some rare apple varieties cannot take root with the existing tissue culture system and indirectly analyzing gene function by using Agrobacterium rhizogenes to induce transgenic hairy roots.So far,the study on apple hairy roots obtained by inoculation with Agrobacterium rhizogenes and redifferentiation to obtain apple transgenic regenerated plants has not been reported.In this study,a new regeneration and genetic transformation system of wild type apple ‘GL-3’ was established by optimizing tissue culture technology from the perspective of transgenic.The main test results are as follows:1.Agrobacterium rhizogenes induced hairy root formation in apple ‘GL-3’ stem.The results showed that hairy roots could be obtained when the rooting medium was4.43g/LMS+30g/Lsucrose +7.5g/LAgar or 4.43g/L1/2MS+30g/Lsucrose+7.5g/L Agar powder and 500mg/L cefotaxime sodium was added after high temperature sterilization.There was no significant difference in the growth status of hairy roots between the two rooting media.2.Optimization of regeneration system for adventitious buds obtained from hairy root redifferentiation.In this regeneration system,hairy roots were inoculated into bud induction medium,dark culture for 14 days and light culture for 30 days.Through the exploration and optimization of hairy root re-differentiation system,it was found that the optimal bud induction medium for adventitious buds obtained by hairy root re-differentiation was as follows.4.43g/LMS + 2.0mg/L6-BA + 0.2mg/LNAA + 30g/Lsucrose + 2.5g/Lphytagel.3.Agrobacterium rhizogenes mediated genetic transformation of apple.Agrobacterium rhizogenes containing modified vector p Cambia1301 was used for plant infection transformation.The vector combined with the GFP reporter gene and Md PGT2,a key gene for trilobatin synthesis.The transgenic hairy roots obtained by transformation experiment obtained adventitious buds through the hairy root regeneration system,and the obtained adventitious buds were first identified by fluorescence for preliminary screening.Then a series of molecular tests were performed to identify the transgenic plant as its transgene,and the content of trilobatin in transgenic plants was analyzed by high performance liquid chromatography.By this method,a complete genetic transformation system of Agrobacterium rhizogenes mediated apple hairy roots was established.