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

文献类型： 外文期刊

第一作者： He, Yongrui

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

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关键词： Citrus sinensis Osbeck; Mature stem segment; Marker-free; Transgene; Bud micrografting

期刊名称：HORTICULTURAL PLANT JOURNAL （ 影响因子：5.7； 五年影响因子：5.4 ）

ISSN： 2095-9885

年卷期： 2023 年 9 卷 3 期

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收录情况： SCI

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 tumefaciens-mediated transformation for generation of marker-free transgenic plants from navel orange (Citrus sinensis Osbeck) mature stems using a Cre-loxP 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.

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