Department of Agronomy, University of Wisconsin, Madison, WI, USA.
Wisconsin Crop Innovation Center, University of Wisconsin, Middleton, WI, USA.
Plant Biotechnol J. 2023 Sep;21(9):1860-1872. doi: 10.1111/pbi.14098. Epub 2023 Jun 26.
The ability of plant somatic cells to dedifferentiate, form somatic embryos and regenerate whole plants in vitro has been harnessed for both clonal propagation and as a key component of plant genetic engineering systems. Embryogenic culture response is significantly limited, however, by plant genotype in most species. This impedes advancements in both plant transformation-based functional genomics research and crop improvement efforts. We utilized natural variation among maize inbred lines to genetically map somatic embryo generation potential in tissue culture and identify candidate genes underlying totipotency. Using a series of maize lines derived from crosses involving the culturable parent A188 and the non-responsive parent B73, we identified a region on chromosome 3 associated with embryogenic culture response and focused on three candidate genes within the region based on genetic position and expression pattern. Two candidate genes showed no effect when ectopically expressed in B73, but the gene Wox2a was found to induce somatic embryogenesis and embryogenic callus proliferation. Transgenic B73 cells with strong constitutive expression of the B73 and A188 coding sequences of Wox2a were found to produce somatic embryos at similar frequencies, demonstrating that sufficient expression of either allele could rescue the embryogenic culture phenotype. Transgenic B73 plants were regenerated from the somatic embryos without chemical selection and no pleiotropic effects were observed in the Wox2a overexpression lines in the regenerated T0 plants or in the two independent events which produced T1 progeny. In addition to linking natural variation in tissue culture response to Wox2a, our data support the utility of Wox2a in enabling transformation of recalcitrant genotypes.
植物体细胞的去分化能力、形成体细胞胚并在体外再生整株植物的能力，已被用于克隆繁殖和作为植物遗传工程系统的关键组成部分。然而，在大多数物种中，胚胎发生培养反应受到植物基因型的显著限制。这阻碍了基于植物转化的功能基因组学研究和作物改良工作的进展。我们利用玉米自交系之间的自然变异，在组织培养中对体细胞胚发生潜力进行遗传作图，并鉴定出潜在全能性的候选基因。利用一系列源自可培养亲本 A188 和非响应亲本 B73 杂交的玉米品系，我们确定了与胚胎发生培养反应相关的 3 号染色体上的一个区域，并根据遗传位置和表达模式，集中研究该区域内的三个候选基因。两个候选基因在 B73 中外源表达时没有效果，但发现 Wox2a 基因可诱导体细胞胚发生和胚胎发生愈伤组织增殖。具有 B73 和 A188 Wox2a 编码序列强组成型表达的转基因 B73 细胞被发现以相似的频率产生体细胞胚，表明任一等位基因的充分表达都可以挽救胚胎发生培养表型。从体细胞胚再生出的转基因 B73 植物无需化学选择，并且在再生 T0 植物或产生 T1 后代的两个独立事件中，在 Wox2a 过表达系中未观察到多效性效应。除了将组织培养反应的自然变异与 Wox2a 联系起来外，我们的数据还支持 Wox2a 在使难转化基因型转化方面的应用。
