The Transformation Of 'Manicure Finger' Grapevine With RolC Gene

An effective system regeneration system of 'Manicure Finger' grapevinevine was developed in this study.Agrobacterium-mediated transformation and pollen-tube pathway were adopted to introduce the role gene into 'Manicure Finger' grapevine to improve the ability of abloom.The detailed results were Listed as follows:1.Plant regeneration were induced from leaf,petiole and stem explants of 'Manicure Finger' grapevine with difference media supplemented with different combinations of 6-BA,TDZ and IBA through organogenesis.The results indicated that the basic media MS,B₅ and NN₆₉ all induce adventitious buds from leaf,petiole and stem explants.The highest regeneration rate of adventitious buds was 6.00%,when explants were incubated on MS medium with 6-BA 3.0 mg/L and IBA 0.1 mg/L.The regeneration rate of adventitious buds was 4.00%when explants were incubated on MS+TDZ1.0mg/L+IBA0.2 mg/L.2.Transgenic plants were generated from leaves,petiole and stem explants of 'Manicure Finger' grapevine by Agrobacterium-mediated transformation with rolC gene,and the following factors were investigated:Agrobacterium dipping time,co=culture time,concentration of Kanamycin,and concentration of Cefotaxine.The optimal conditions included that infected 4 min by Agrobacterium,co-cultured 3d,Km3.0mg/L,and Cef 250 mg/L.3.Twelve Km-resistant shoots of 'Manicure Finger' grapevine were obtained by Agrobacterium-mediated transformation.Histochemical GUS assay on these resistant shoots showed that two shoots were GUS positive plants;one of them was confirmed that rolC gene was integrated into the host genome of 'Manicure Finger' grapevine by both PCR and RT-PCR analysis.4.Agrobacterium-mediated transformation was adopted to introduce the rolC gene into the inflorescence of 'Manicure Finger' grapevine via the pollen-tube pathway.40 planflets were obtained from 'Manicure Finger' seeds.By GUS histochemistry assay,PCR amplifiation and RT-PCR analysis,five plants showed GUS positive with a 12.5%positive rate,and two shoots showed PCR and RT-PCR positive,which initially demonstrated that the rolC gene was integrated into the genome of 'Manicure Finger' grapevine.