Production of marker-free transgenic plants from mature tissues of navel orange using a Cre/loxP site-recombination system  

作　　者：Yongrui He Lanzhen Xu Aihong Peng Tiangang Lei Qiang Li Lixiao Yao Guojin Jiang Shanchun Chen Zhengguo Li Xiuping Zou 

机构地区：[1]Key Laboratory of Plant Hormones and Development Regulation of Chongqing,School of Life Sciences,Chongqing University,Chongqing 401331,China [2]National Citrus Engineering Research Center(NCERC),Citrus Research Institute,Southwest University/Chinese Academy of Agricultural Sciences,Chongqing 400712,China

出　　处：《Horticultural Plant Journal》2023年第3期473-480,共8页园艺学报（英文版）

基　　金：supported by the Fundamental Research Funds for the Central Universities (Grant No. XDJK 2018B016);the National Natural Sciences Foundation of China (Grant No. 31972393);he earmarked fund for China Agriculture Research System (Grant No. CARS-26);the Natural Science Foundation of Chongqing (Grant No. cstc2020jcyj-msxmX1064)。

摘　　要：Genetic transformation with mature material as the explants could shorten the transgenic period and avoid seed dependence compared with genetic transformation using the epicotyl seedling stem segments as the receptor. Here, we constructed an Agrobacterium tumefaciensmediated transformation for generation of marker-free transgenic plants from navel orange(Citrus sinensis Osbeck) mature stems using a CreloxP recombination system. To efficiently recover the regenerated buds from mature tissues, five recovery methods were compared: in vitro micrografting of 0.1-0.5(1-2 weeks), > 0.5 cm(3-4 weeks) and > 1 cm long lignified bud and in vitro micrografting of explants with a bud and rooting regenerated bud. The data showed that in vitro micrografting of > 1 cm long regenerated bud with expanded leaves after one month of continuous culture for lignification was the optimal solution for plant recovery from mature tissues. Transgenic plants without selectable marker genes were created from navel orange(Citrus sinensis Osbeck) tissue using a transformation vector PLI-35SPR1aCB containing a Cre/loxP system recombination together with genes encoding the selectable marker isopentenyl transferase(IPT) and an anti-bacterial peptide(PR1aCB).Using IPT positive selection, the transformation efficiency determined by PCR was 0.9%, and in total, 20 transgenic plants were obtained.Southern blotting confirmed further their transgenicity. PCR and sequencing analysis demonstrated that both the Cre and IPT genes had been successfully removed from the transgenic plants(deletion efficiency 100%). Over all, using Cre/loxP system recombination together with the IPT positive selection, marker-free transgenic plants can be recovered efficiently from mature tissues of navel orange(Citrus sinensis Osbeck), which provides a potential method for production of transgenic plants from citrus mature tissue.

关 键 词：Citrus sinensis Osbeck Mature stem segment MARKER-FREE TRANSGENE Bud micrografting 

分 类 号：S666.4[农业科学—果树学]