Study On Agrobacterium Mediated Cucumis Melo L. Transformation Of Antisense PG Gene

Cucumis melon is of typically climacteric fruits and attains the maturity appears to breathe the high peak soon during the post harvest period.That leads to softening and rotting of fruit flesh. Therefore,the effective way to solve the difficult problem is to breed varieties that can endure storage transportation.Polygalacturonases,a kind of cell wall hydrze enzyme,is highly expressed in the fruit of melon and plays a key role in fruit softening.Genetic engineering can provide traditional breeding method a new rapid approach on melon for reaching the aim.The research for introducing antisence PG gene into melon mediated by Agrobacterium tumefaciens was done. A regeneration protocol for"GT-1"melon has been established,and this protocol was combined with Agrobacterium tumefaciens transformation to produce transgenic melon plants. Cotyledon explants the five days cotyledon pieces of melon as explants were cocultivated 15 minute with A.tumefaciens pBI-aPG/ LBA4404 and pCA-aPG/ LBA4404,which carrying a Ti plasmid in which an antisence PG coding sequence as well as the neomycin phosphotxansferaseâ…¡(NPTâ…¡) coding sequnence were fused to the Cauliflower Mosaic Virus( CaMV )35S promoter.After co-cultivation of melon cotyledon discs with agrobacterium 72 hours, The explant were transfer to regeneration medium(Murashinge and Skoog medium containing 1.0mg 6-benzylaminopurine, kanamycin at 100mg and carbincillin at 500mg,per liter medium).Shoot regenerated at 29.64%and 35.90%,respectively.On the medium MS supplemented with 6-BA0.05mg,Kan 75mg and Carb 500mg per liter,payter of the shoots elongated.However,only about 20% elongated shoots of"GT-1"can rooting on Kan 37.5mg/L medium, Rooting rat was 1.386%and 1.958%.PCR specific gene amplification was used to detect the integration of the purpose genes. The results showed that there were melon plants transferred with plasmid pBI-aPG and plasmid pCA-aPG appeared special lane.GUS lying of different organize in the transgenic plant of melon displayed blue.All these results indicated that the foreign gene had integrated within the genome of the regenerated plants and was expressed normally.