Studies On Optimization Of Genetic Transformation System Of Immature Embryonic Cotyledons And Mature Embryonic Hypocotyls In Prunus Mume

Since many Mei flower cultivars are difficult to grow in the cold and dry climate of north China,most can only be cultivated as a potted plants for appreciation.Therefore,it is essential to breed new cultivars with stronger cold resistance.The traditional breeding ways are costly,time-consuming,and usually cannot obtain the target traits with certainty.However,method of genetic transformation can change Mei flower traits directly to meet with breeding objectives.This experiment takes Mei flower immature cotyledons and mature embryonic hypocotyls as the material.Based on the optimization of regeneration and genetic transformation system,two Pm CBFgenes are introduced by Agrobacterium,and 30 transformed plants are obtained.The results are as follows:1.Optimization of regeneration system and genetic transformation of immature cotyledonsUsing the immature embryo cotyledons of ’Xue Mei’ × ‘Fenpi Gongfen’ as explants,the systems of regeneration and genetic transformation are optimized.The results show that:(1)Immature embryo cotyledons about 55d-65 d after fully flowering in the 1 / 2MS + TDZ 0.2mg / L + BA 2.0 mg / L + IBA 0.5 mg / L under no dark pre-culture have the highest regeneration rate up to 32.5%.The influence of TDZ,IBA and dark culture time on the regeneration is significant,while BA had smaller effect on the regeneration.(2)The best culture medium selected from the previous experiment is used for genetic transformation.The best transformation parameters are 3d pre-culture,15 min infection,5d co-culture,no delay-screening and 20 mg / L Km by orthogonal experiment with 20% highest rate of transformation and the target genes are Pm CBFa and Pm CBFc.Then regeneration plant and transformed plants cultured for 40 d are transferred to the elongation medium(1/2MS + IBA 0.1mg / L + BA 0.1mg / L).After 20 d,the regeneration plant are moved to medium(1/2QL + NAA 0.5 mg / L)for rooting,and transplanted to the potted medium successfully.Finally,total 110 shoots with Km resistance are produced,but target gene is amplified by PCR from only 25 ones.2.Regeneration and genetic transformation of mature embryonic hypocotylsWe compare the influence of different parts of mature embryonic hypocotyls of ’Xue Mei’ on regeneration and find that the part near radicle has higher regeneration rate,so it is chosen as the explants for optimizing the regeneration and genetic transformation system.The results show that:(1)Mature embryonic about 120d-150 d after fully flowering are optimal for plant regeneration.Under no dark pre-culture,those in 1 / 2MS+ TDZ 2.0mg / L + BA 2.0 mg / L + NAA 0.2 mg / L have the highest regeneration rate up to 51.39%.Four factors designed in the orthogonal experiment all show significant differences on the regeneration of mature embryonic hypocotyls.(2)the best culture medium selected from the immature cotyledons hypocotyls regeneration experiment is used for the transformation of mature embryonic hypocotyls.By orthogonal experiment,we find the optimal transformation parameters: 5d pre-culture,10 min infection,2d co-culture,10 d delay-screening and 15mg/ L Km.The target genes are Pm CBFa and Pm CBFc.Then regeneration plants and transformed plants cultured for 40 d are then transferred to the elongation medium(1/2MS + IBA 0.1mg / L + BA 0.1mg / L).After20 d,the regeneration plant are moved to medium(1/2QL + NAA 0.5 mg / L)for rooting,and transplanted to the potted medium successfully.Total 55 shoots with Km resistance are produced,but target gene is amplified by PCR from only 5 ones.