Endosperm-trait related genes are associated with grain yield or quality in maize. There are vast numbers of these genes whose functions and regulations are still unknown. The biolistic system, which is often used for transient gene expression, is expensive and involves complex protocol. Besides, it cannot be used for simultaneous analysis of multiple genes. Moreover, the biolistic system has little physiological relevance when compared to cell-specific based system. Plant protoplasts are efficient cell-based systems which allow quick and simultaneous transient analysis of multiple genes. Typically, PEG-calcium mediated transfection of protoplast is simple and cost-effective. Notably, starch granules in cereal endosperm may diminish protoplast yield and integrity, if the isolation and transfection conditions are not accurately measured. Prior to this study, no PEG-calcium mediated endosperm protoplast system has been reported for cereal crop, perhaps, because endosperm cells accumulate starch grains. Here, we showed the uniqueness of maize endosperm-protoplast system (EPS) in conducting endosperm cell-based experiments. By using response surface designs, we established optimized conditions for the isolation and PEG-calcium mediated transfection of maize endosperm protoplasts. The optimized conditions of 1% cellulase, 0.75% macerozyme and 0.4 M mannitol enzymolysis solution for 6 h showed that more than 80% protoplasts remained viable after re-suspension in 1 ml MMG. The EPS was used to express GFP protein, analyze the subcellular location of ZmBT1, characterize the interaction of O2 and PBF1 by bimolecular fluorescent complementation (BiFC), and simultaneously analyze the regulation of ZmBt1 expression by ZmMYB14. The described optimized conditions proved efficient for reasonable yield of viable protoplasts from maize endosperm, and utility of the protoplast in rapid analysis of endosperm-trait related genes. The development of the optimized protoplast isolation and transfection conditions, allow the exploitation of the functional advantages of protoplast system over biolistic system in conducting endosperm-based studies (particularly, in transient analysis of genes and gene regulation networks, associated with the accumulation of endosperm storage products). Such analyses will be invaluable in characterizing endosperm-trait related genes whose functions have not been identified. Thus, the EPS will benefit the research of cereal grain yield and quality improvement.

中文翻译：

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玉米胚乳原生质体分离转染条件的优化

胚乳性状相关基因与玉米的产量或品质有关。有大量这些基因的功能和调节仍然未知。通常用于瞬时基因表达的基因枪系统价格昂贵且涉及复杂的协议。此外，它不能用于同时分析多个基因。此外，与基于细胞的系统相比，基因枪系统几乎没有生理相关性。植物原生质体是高效的基于细胞的系统，可以对多个基因进行快速和同时的瞬时分析。通常，PEG-钙介导的原生质体转染简单且具有成本效益。值得注意的是，如果没有准确测量分离和转染条件，谷物胚乳中的淀粉颗粒可能会降低原生质体的产量和完整性。在这项研究之前，没有报道过用于谷类作物的 PEG-钙介导的胚乳原生质体系统，这可能是因为胚乳细胞积累了淀粉粒。在这里，我们展示了玉米胚乳原生质体系统 (EPS) 在进行基于胚乳细胞的实验中的独特性。通过使用响应面设计，我们为玉米胚乳原生质体的分离和PEG-钙介导的转染建立了优化条件。1% 纤维素酶、0.75% macerozyme 和 0.4 M 甘露醇酶解溶液 6 h 的优化条件表明，在 1 ml MMG 中重新悬浮后，超过 80% 的原生质体仍然存活。EPS用于表达GFP蛋白，分析ZmBT1的亚细胞定位，通过双分子荧光互补（BiFC）表征O2和PBF1的相互作用，同时分析ZmMYB14对ZmBt1表达的调控。所描述的优化条件证明对于从玉米胚乳中合理产量的可行原生质体以及原生质体在快速分析胚乳性状相关基因中的效用是有效的。优化原生质体分离和转染条件的开发，允许在进行基于胚乳的研究（特别是与胚乳储存积累相关的基因和基因调控网络的瞬态分析中）利用原生质体系统相对于基因枪系统的功能优势产品）。这种分析对于鉴定功能尚未确定的胚乳性状相关基因将是无价的。因此，EPS将有利于谷物产量和品质改良的研究。