Studies On Transformation Of Anti-Phospholipase D_γ(Anti-PLD_γ) Gene Into Strawberry (Fragaria×ananassa Duch.)

Strawberry( Fragaria ananassa Duch.) is a kind of small,perennial berry with high economic worth .Because the mature period is concentrate, its conveyance with the large-scale production are limited. The leaves of strawberry were used as the material and several factors such as hormone concentration, physiological conditions of explant, illumination of culture , and so on were investigated to optimize the regeneration system in vitro. A high frequency regeneration system in culture has been etablished. This culture system was adapted to Agrobacerium-mediated transformation. The young leaf of strawberry were inoculated with Agrobacerium tumefaciens strains harboring binary vectors, which contained the neomycin phosphotransferase Ⅱ gene and the anti-phospholipase Dγ(PLDγ)gene. The establishing of strawberry heredity converts system plays an important role in improving strawberry species by making use of the genetic engineering.The research result expresses the different genotype, leaf's age , browness, darkness to culture, MS medium , different hormone , Ag+ and so on have influenced leave regeneration. The results showed that 6-BA and 2,4-D in plant initiation medium played an important role in improving plant regeneration. The most effective concentration for 6-BA and 2,4-D was 1.6-1.8mg/L, 0.1mg/L, which increased the regeneration frequency of Tudla(Fragaria×ananassa) to 60%. The regeneration frequency of Tudla wasdecreased by applying AgNO3 to the medium. The best explants are those apical leaves which from the 18-21 days cultured plantlets. Leaves were placed on the medium which is mentioned above for shoot induction. After 14 days cultured , the explants began to directly regenerate adventitious buds. The maximum of the adventitious buds were observed within 20 to 30 days. The shoot differentiation frequency was more than 60%. On the basis of regeneration system, transformation system was established by screening transformation conditions with antibiotics selection. Diluted the Agrobacterium until the OD(600nm) ranging from 0.3 to 0.5 with the same volume of 0.5% sucrose solution (contain concentration detergent), inoculated the strawberry leaves for 10 minutes, then co-cultivated the explants on sucrose liquid medium for 3 days and placed on the regeneration medium with 300mg/L Cef for 7 days. The resistant shoots were obtained on regeneration medium with 300mg/L Cef and 20mg/L Kanamycin.The presence and integration of the antisense PLDγ gene was confirmed by polymerase chain reaction (PCR) and Southern blot analysis.