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首页> 外文期刊>African Journal of Biotechnology >Agrobacterium-mediated transformation of Jatropha curcas leaf explants with a fungal chitinase gene
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Agrobacterium-mediated transformation of Jatropha curcas leaf explants with a fungal chitinase gene

机译：农杆菌介导的真菌几丁质酶基因转化麻疯树叶片外植体

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Jatropha curcas L. oil has been shown to be suitable for the production of biodiesel. However, this species has not been domesticated yet. Genetic breeding through conventional methods is time consuming and costly, hence, genetic transformation could contribute positively to the improvement of interesting traits. Although in vitro regeneration and stable genetic transformation has been pursued for several years, variation in transformation efficiency remains strongly genotype-dependent and indicates that protocols optimization is still needed. Thus, this study was carried out to introduce a chitinase gene from the Trichoderma viride fungus into the genome of a J. curcas superior genotype by inoculating leaf explants with Agrobacterium tumefaciens EHA 105 strain in axenic conditions. Some key parameters such as pre-culture period and antibiotic doses were optimized with 500 mg.L-1 cefotaxime and 100 mg.L-1 kanamycin concentrations being suitable for A. tumefaciens inhibition and explant selection, respectively. The best transformation efficiency (50%) was obtained when leaf explants were incubated on a culture medium promoting shoot regeneration at 15 days before the induction of the transformation process. Plants where chitinase gene amplicons could be detected were assessed for transgene copy number and expression levels by quantitative real-time polymerase chain reaction (PCR). One, two and three copies of the introduced gene were confirmed in nine transgenic lines with two of them that were assessed for gene expression and showed quantitative variation for this variable. These results bring valuable information for further gene insertions in breeding programs of J. curcas for fungal disease resistance.
机译：麻风树麻疯树油已被证明适合生产生物柴油。但是，该物种尚未被驯化。通过常规方法进行遗传育种既费时又费钱，因此遗传转化可以为改善性状做出积极贡献。尽管已经进行了几年的体外再生和稳定的遗传转化，但是转化效率的变化仍然强烈依赖于基因型，这表明仍然需要优化方案。因此，通过在农杆菌条件下用根癌农杆菌EHA 105菌株接种叶外植体，从而将来自木霉木霉菌真菌的几丁质酶基因引入到优良的J.curcas基因型中。用500 mg.L-1头孢噻肟和100 mg.L-1卡那霉素的浓度分别优化了一些关键参数，如培养前期和抗生素剂量，分别适用于抑癌农杆菌和外植体选择。在诱导转化过程之前的第15天，将叶片外植体在促进芽再生的培养基上孵育时，可获得最佳转化效率（50％）。通过定量实时聚合酶链反应（PCR）评估可以检测到几丁质酶基因扩增子的植物的转基因拷贝数和表达水平。在9个转基因品系中确认了导入基因的1、2和3拷贝，其中2个对基因表达进行了评估，并显示了该变量的定量变化。这些结果为J. curcas的真菌抗病性育种程序中的进一步基因插入提供了有价值的信息。

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