The Solanaceae family includes the largest flowering crops such as tomatoes, potatoes, and eggplants. Consumer demand has led to massive development of plants in the Solanum genus, and many different Solanum varieties are now available on the market. The recent advances in Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)-based genome editing have allowed laboratories and smaller crop production companies to utilize the technology in various crops. The traditional transformation method in crops involves the use of Agrobacterium, which is considered the most efficient method for introducing exogenous genetic materials in target plants. The Agrobacterium-mediated transformation method has been also established in the Solanaceae family, enabling CRISPR/Cas-based genome editing in crops like tomatoes, potatoes, and eggplants. However, the Agrobacterium-mediated approach inevitably accompanies the insertion of exogenous DNA into the plant genome and often causes the formation of chimera that require further propagation steps. Alternatively, the CRISPR/Cas components can be introduced into protoplasts in the form of DNA for transient expression or a mixture of protein and RNA to avoid genomic insertion of foreign materials. The protoplast transformation approach involves processes including protoplast preparation, transfection, and regeneration, which require a comprehensive understanding and greater technical mastery of the tissue culture phase. Here we highlight the current research advances in protoplast transformation and discuss how to optimize the procedures of protoplast isolation, transfection, and regeneration for efficient and reproducible CRISPR/Cas-based genome editing in the genus Solanum.

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用于 CRISPR/Cas 介导的非转基因基因组编辑的茄属原生质体分离、转染和再生的优化方案

茄科包括最大的开花作物，如西红柿、土豆和茄子。消费者的需求导致了茄属植物的大规模开发，现在市场上有许多不同的茄属品种。基于成簇规则间隔短回文重复序列 (CRISPR)/CRISPR 相关蛋白 9 (Cas9) 的基因组编辑的最新进展使实验室和小型作物生产公司能够在各种作物中利用该技术。作物的传统转化方法涉及使用农杆菌，这被认为是将外源遗传物质引入目标植物的最有效方法。农杆菌介导的转化方法也已在茄科家族中建立，从而能够在西红柿、马铃薯和茄子等作物中进行基于 CRISPR/Cas 的基因组编辑。然而，农杆菌介导的方法不可避免地伴随着外源DNA插入植物基因组，并且常常导致嵌合体的形成，需要进一步的繁殖步骤。或者，可以将 CRISPR/Cas 组件以 DNA 的形式引入原生质体进行瞬时表达，或者以蛋白质和 RNA 的混合物的形式引入原生质体，以避免外来物质的基因组插入。原生质体转化方法涉及原生质体制备、转染和再生等过程，需要对组织培养阶段有全面的了解和更高的技术掌握。在这里，我们重点介绍当前原生质体转化的研究进展，并讨论如何优化原生质体分离、转染和再生程序，以在茄属植物中实现高效、可重复的基于 CRISPR/Cas 的基因组编辑。