Establishment Of Rapid Propagation And Callus Genetic Transformation Systems In Three Malus Cultivars

Apple（Malus×domestica）is an important cultivated fruit tree in China.The total cultivation area,total output and export of Chinese apple production are ranked first in the world after several year development.Development the rapid propagation system and genetic transformation system will be used to discover the gene resource,and provide materials for apple breeding,due to the late flowering,strong adaptability and long fruit storage in wild apple germplasm.The objective of this research is establish the rapid propagation system and callus genetic transformation system of three Malus species:Malus×domestica‘Red Fuji’,Malus×domestica‘Granny Smith’and Malus orientalis.We studied the effects of different concentrations of plant growth regulators on the primary generation,subgeneration and rooting culture of three Malus species,and to obtain the optimal culture medium for callus induction and proliferation of three Malus species.Finally,Md MPK4 gene was transformed into apple callus to establish a stable genetic transformation system in three apple cultivars and provide technical basis for future studies.The main results are as follows:1.To study the effects of different concentrations of plant growth regulators on the primary culture,secondary culture and rooting culture of three Malus cultivars.The results showed that the optimal medium for the first generation of three Malus species was 0.5 mg/L 6-BA+0.5 mg/L NAA.The survival rate reached 80%.The optimal subculture medium for‘Red Fuji’and‘Granny Smith’was 0.3 mg/L 6-BA+0.3 mg/L NAA,and the proliferation coefficient was 2.8.The optimal subculture medium for oriental apple was 0.3 mg/L 6-BA+0.05 mg/L NAA,with a proliferation coefficient of 2.5 and rapid growth.The optimal rooting medium for three Malus species was1/2MS+0.5 mg/L IBA.2.To study the effects of different plant growth regulators concentrationon callus induction and subculture of three Malus species.The results showed that the optimal medium for callus induction was TDZ 2 mg/L+6-BA 0.5 mg/L,and the induction rate reached 80%.In subculture,it was found that 1.5 mg/L 2,4-D+0.3 mg/L 6-BA medium had the best growth and the proliferation coefficient was 2.5.3.To establish the stable transformation system in Malus plants,we transformed Md MPK4gene in these three Malus cultivars.The results showed that the optimal concentration OD₆₀₀was0.8,the infection time was 8 min,the optimal screening concentration of kanamycin resistance was 10 mg/L and the transformation rate was 70%.Compared with the wild-type callus,the transgenic callus turned red after 14 days high light and low temperature treatment,suggesting that the transgenic Md MPK4 gene could positively regulate anthocyanin accumulation in apple.PCR results showed that a 1135 bp fragment was detected in transgenic callus,which indicating that Md MPK4 gene had been transferred into the callus.In this study,the efficient regeneration system and genetic transformation system of three Malus plants were established,which laid a good foundation for the production of virus-free seedlings,conservation and exploration of germplasm resources,and gene function study of Malus plants.