Screening for recombinants of Crambe abyssynica after transformation by the pMF1 marker-free vector based on chemical selection and meristematic regeneration

文献类型： 外文期刊

第一作者： Qi, Weicong

作者： Qi, Weicong;Zhang, Xiaolin;Zhao, Han;Qi, Weicong;Tinnenbroek-Capel, Iris E. M.;Salentijn, Elma M. J.;Schaart, Jan G.;Cheng, Jihua;Denneboom, Christel;Visser, Richard G. F.;Van Loo, Eibertus N.;Krens, Frans A.;Zhang, Zhao;Huang, Bangquan

作者机构：

期刊名称：SCIENTIFIC REPORTS （ 影响因子：4.379； 五年影响因子：5.133 ）

ISSN： 2045-2322

年卷期： 2015 年 5 卷

页码：

收录情况： SCI

摘要： The T-DNA region of pMF1 vector of marker-free system developed by Wageningen UR, has Recombinase R-LBD gene fusion and nptII and codA gene fusion between two recombination sites. After transformation applying dexamethasone (DEX) can activate the recombinase to remove the T-DNA fragment between recombination sites. The recombinant ought to be selected on 5-fluorocytocine (5-FC) because of codA converting 5-FC into 5-fluorouracil the toxic. A PMF1 vector was transformed into hexaploid species Crambe abyssinica. Two independent transformants were chosen for DEX-induced recombination and later 5-FC selection. In contrast to earlier pMF1 experiments, the strategy of stepwise selection based on meristematic regeneration was engaged. After a long period of 5-FC selection, recombinants were obtained successfully, but most of the survivors were wildtype and non-recombinant. The results revealed when applying the PMF1 marker-free system on C. abyssinica, 1) Increasing in the DEX concentration did not correspondingly enhance the success of recombination; 2) both of the DEX-induced recombination and 5-FC negative selection were apparently insufficient which was leading to the extremely high frequency in chimerism occurring for recombinant and non-recombinant cells in tissues; 3) the strategy of stepwise selection based on meristem tissue regeneration was crucial for successfully isolating the recombinant germplasm from the chimera.

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