Agrobacterium-Mediated Transformation Of Malus Robusta With IRT1 Gene

Based on previous shoot regeneration system of Malus Robusta,we optimized the transformation system ofMalus Robusta,established a high-efficient transformation system, obtained four transgenic plants of Malus robusta with IRT1 gene,and also studied the effects of different subculture times on isolated culture and transformation of Malus robusta.The research results were as follows:1.The Malus Robusta shoots and young leaves in vitro were used as materials for studying the effects of different subculture times on isolated culture and genetic transformation.The results indicated:with subculturing times increasing,the isolated culture and transformation abilities of the shoots subcultured in vitro for 10 and 48 times were significantly higher than that for 5 timesin terms of its shoot multiplication,rooting, adventitious bud regeneration rate and GUS transient expression from leaves.Subculturing 10 times were superior to 48 times on the aspects of shoot multiplication and GUS transient expression,but on the others,there were no significant differences.It is indicated that the shoots subcultured in vitro for 10 times were suitable for shoot multiplication,and its young leaves were the ideal materials in the transformation of Malus Robusta.2.A high-efficientcy transformation system of Malus robusta was developed by researching the effects of the different type of Agrobacterium strains,selective pressure, time of adding kanmycin,washing time with MS0 mixed with cefotaxime and the manners of adding AS.We chose LBA4404 as infected strain,and leaves of Malus Robusta in vitro as infected explants which were cultivated for 35d in subculture medium and then cultured for 10d on another medium.The leaves were been dippedthen co-cultivated on regeneration culture medium with 75μmol/L AS in the dark for 3d.We washed the leaves with MS0 added cefotaxime before transfer them to regeneration medium added 250mg/L cb(MS + BA4.0mg/L + NAA0.2mg/L+cb 250mg/L)and cultivate 3d,then transfer them to selective medium(MS + BA4.0mg/L + NAA0.2mg/L+km25 mg/L +cb 250mg/L).When seedling height was 2 cm,transfer them to selective medium(MS + BA0.5mg/L + NAA0.1mg/L + Km50mg/L).3.In this study,GV3101 and LBA4404 strains were used to transform the Malus robusta.We obtained Malus robusta transgenetic plants from both of these two srains carrying 1RT1 gene.The strain GV3101 obtained 7 transgenic plants which had km resistance,compared with this,the strain LBA4404 obtained transgenic 17 plants.The rate of transformation was 1.86%and 6.58%respectively.The plants were been identified by the expression of gus gene,PCR and RT-PCR.The result indicated were successfully been transferred into 4 transgene lines.Further analysis of the the iron deficiency tolerance of these 3 transgene lines has improved in different degree compared with control and another one has not.