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The Establishment of an Agrobacterium-Mediated Transformation Platform for the non-embryogenic calli of Vitis vinifera L.

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Agricultural Sciences in China 2011年第5期10卷 686-694页

作者： ZHAO Feng-xia CHEN Shang-wu Perl Avihai DAI Ru XU Hai-ying MA Hui-qin College of Food Science and Nutrition Engineering China Agricultural University Beijing 100063 P.R. China Department of Fruit Tree Sciences Volcani Center Bet Dagan 50250 Israel College of Agriculture and Biotechnology China Agricultural University Beijing 100193 P.R.China Institute of Forestry and Fruit Tree Sciences Beijing Academy of Agricultural Sciences Beijing 100093 P.R.China

non-embryogenic calli （NEC） was inevitably and heavily produced when grape embryogenic calli （EC） was induced from explants or during the subculture of EC.A stable and highly efficient NEC transformation platform is required to further sort out and verify key genes which determine/switch the identity of NEC and *** this research,a vector pA5 containing a chitinase signal sequence fused to gfp （green fluorescent protein） and an HDEL motive was used to target and immobilize into Agrobacterium strain EHA105 to establish a transformation platform for Vitis vinifera *** *** was determined that NEC 10 d after subculture was the best target tissue;30 min for inoculation followed by 3 d co-cultivation with the addition of 200 μmol L-1 acetosyringone （AS） was optimized as *** use of bacterial densities as 1.0 at OD600 did not result in serious tissue hypersensitive reaction and it had higher *** at 200 mg L-1 was picked for positive expression *** stable transformation of NEC was proved by reverse transcription-polymerase chain reaction techniques （RT-PCR） and fluorescent microscopy after three sub-cultures of the selected cell *** efficient genetic transformation protocol of grape NEC was achieved and some of the optimized parameters were different from that reported for *** transformation platform could facilitate the verification of candidate somatic embryogenesis （SE） decisive genes,and the successfully transformed NEC with certain genes can also be used as bioreactors for the production of functional products,as NEC not only proliferates fast,but also keeps in a rather stable condition.

关键词： Agrobacterium tumefaciens non-embryogenic calli transformation Vitis vinifera L. RT-PCR

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