Genome editing and cis‐gene breeding have rapidly accelerated crop improvement efforts, but their impacts are limited by the number of species capable of being genetically transformed. Many dicot species, including some vital potato relatives being used to accelerate breeding and genetics efforts, remain recalcitrant to standard Agrobacterium tumefaciens‐based transformation. Hairy root transformation using Agrobacterium rhizogenes (A. rhizogenes) provides an accelerated approach to generating transgenic material but has been limited to analysis of hairy root clones. In this study, strains of A. rhizogenes were tested in the wild diploid potato relative Solanum chacoense, which is recalcitrant to infection by Agrobacterium tumefaciens. One strain of A. rhizogenes MSU440 emerged as being capable of delivering a T‐DNA carrying the GUS marker and generating transgenic hairy root clones capable of GUS expression and regeneration to whole plants. CRISPR/Cas9 reagents targeting the potato PHYTOENE DESATURASE (StPDS) gene were expressed in hairy root clones and regenerated. We found that 64%–98% of transgenic hairy root clones expressing CRISPR/Cas9 reagents carried targeted mutations, while only 14%–30% of mutations were chimeric. The mutations were maintained in regenerated lines as stable mutations at rates averaging at 38% and were capable of germ‐line transmission to progeny. This novel approach broadens the numbers of genotypes amenable to Agrobacterium‐mediated transformation while reducing chimerism in primary events and accelerating the generation of edited materials.

中文翻译：

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使用毛状根转化技术进行马铃薯的第一代基因组编辑

基因组编辑和顺式基因育种已迅速加速了作物改良工作，但其影响受到能够进行遗传转化的物种数量的限制。许多双子叶植物物种，包括一些重要的马铃薯亲戚，被用于加速育种和遗传工作，仍然对基于根癌农杆菌的标准转化持顽固态度。使用发根土壤杆菌（A. rhizogenes）进行毛根转化提供了一种加速的方法来生成转基因材料，但仅限于分析毛根克隆。在这项研究中，在野生二倍体马铃薯亲戚茄中测试了发根农杆菌菌株。，它抵抗根癌农杆菌的感染。一种发根农杆菌MSU440的出现是因为它能够传递带有GUS标记的T-DNA，并产生能够GUS表达和再生到整个植物的转基因毛状根克隆。针对马铃薯PHYTOENE DESATURASE（StPDS）的CRISPR / Cas9试剂）基因在毛状根克隆中表达并再生。我们发现，表达CRISPR / Cas9试剂的转基因毛状根克隆中有64％–98％具有目标突变，而只有14％–30％的突变是嵌合的。这些突变以稳定的突变形式保持在再生品系中，平均发生率为38％，并且能够通过种系传递给后代。这种新颖的方法拓宽了土壤杆菌介导的转化基因型的数量，同时减少了主要事件中的嵌合现象，并加速了编辑材料的产生。