Establishment of Agrobacteriumtumefaciens-Mediated Transformation System for Phytophthora infestans
ZHAO Dong-mei
HE Jia-yu
YANG Zhi-hui
ZHU Jie-hua
XU Jin
Abstract：The aim of this study was to establish a low-cost,rapid and efficient Agrobacterium tumefaciens-mediated transformation(ATMT )system of Phytophthora infestans.The transformation conditions of acetosyringone concentration,coculture temperature,coculture time and membrane-transferring method were optimized using P.infestans isolate HK09-19.The optimized conditions for this ATMT system included 1.0 μg/mL hygromycin B for transformant-screening,using cellophane as transferring membrane,with 200μmol/L acetosyringone added,and taking 6 d for incubation at 22 ℃ at coculture stage.Under this condition,the transformation efficiency was 50-60 transformants per 106 zoospores.The obtained transformants showed high resistance to hygromycin B after five generations for propagation.An 800 bp band was amplified from ten transformants randomly selected by PCR using specific primers which were designed for the hph gene.Meantime,the vir gene of A.tumefaciens was tested by PCR in colonies of P.infestans transformants in order to eliminate the false-positivity caused by A.tumefaciens contamination.The results showed that exogenous T-DNA was successfully integrated into the genome of P.infestans and able to stably inherit.An efficient and time-saving ATMT system of P.infestans was established,which provided a convenient method for the P.infestans mutant library construction and gene function analysis.
Keywords：Phytophthora infestansAgrobacterium tumefaciens-mediated transformation(ATMT)T-DNAtransformation efficiency
Publication Date：2014-01-01
Online Publishing Date：2026-09-12(First online date of this platform, not the publication date of the document)
Pages：5( 83-87 )