Using A Protoplast Transformation System to Enable Functional Studies in Mangifera indica L.

文献类型： 外文期刊

第一作者： Adjei, Mark Owusu

作者： Adjei, Mark Owusu;Zhao, Huan;Tao, Xiaoguang;Yang, Li;Deng, Shuyue;Li, Xiyan;Mao, Xinjing;Ma, Jun;Adjei, Mark Owusu;Li, Shujiang;Huang, Jianfeng;Luo, Ruixiong;Gao, Aiping

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关键词： Mangifera indica L.; protoplast isolation; transformation; polyethylene glycol mediated; transient expression

期刊名称：INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES （ 影响因子：5.6； 五年影响因子：6.2 ）

ISSN： 1661-6596

年卷期： 2023 年 24 卷 15 期

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收录情况： SCI

摘要： Mangoes (Mangifera indica L.) are an important kind of perennial fruit tree, but their biochemical testing method and transformation technology were insufficient and had not been rigorously explored. The protoplast technology is an excellent method for creating a rapid and effective tool for transient expression and transformation assays, particularly in plants that lack an Agrobacterium-mediated plant transformation system. This study optimized the conditions of the protoplast isolation and transformation system, which can provide a lot of help in the gene expression regulation study of mango. The most beneficial protoplast isolation conditions were 150 mg/mL of cellulase R-10 and 180 mg/mL of macerozyme R-10 in the digestion solution at pH 5.6 and 12 h of digestion time. The 0.16 M and 0.08 M mannitol in wash solution (WI) and suspension for counting (MMG), respectively, were optimal for the protoplast isolation yield. The isolated leaf protoplasts (similar to 5.4 x 10(5) cells/10 mL) were transfected for 30 min mediated by 40% calcium-chloride-based polyethylene glycol (PEG)-4000-CaCl2, from which 84.38% of the protoplasts were transformed. About 0.08 M and 0.12 M of mannitol concentration in MMG and transfection solutions, respectively, were optimal for protoplast viability. Under the florescence signal, GFP was seen in the transformed protoplasts. This showed that the target gene was successfully induced into the protoplast and that it can be transcribed and translated. Experimental results in this paper show that our high-efficiency protoplast isolation and PEG-mediated transformation protocols can provide excellent new methods for creating a rapid and effective tool for the molecular mechanism study of mangoes.

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