Tricoli David M, Debernardi Juan M
Plant Transformation Facility, University of California, Davis, CA 95616, USA.
Hortic Res. 2023 Dec 13;11(1):uhad266. doi: 10.1093/hr/uhad266. eCollection 2024 Jan.
CRISPR-Cas technologies allow for precise modifications in plant genomes and promise to revolutionize agriculture. These technologies depend on the delivery of editing components into plant cells and the regeneration of fully edited plants. In vegetatively propagated plants, such as grape, protoplast culture provides one of the best avenues for producing non-chimeric and transgene-free genome-edited plants. However, poor regeneration of plants from protoplasts has hindered their implementation for genome editing. Here, we report an efficient protocol for regenerating plants from protoplasts from multiple grape varieties. By encapsulating the protoplasts in calcium alginate beads and co-culturing them with feeder cultures, the protoplasts divide to form callus colonies that regenerate into embryos and ultimately plants. This protocol worked successfully in wine and table grape () varieties, as well as grape rootstocks and the grapevine wild relative . Moreover, by transfecting protoplasts with CRISPR-plasmid or ribonucleoprotein (RNP) complexes, we regenerated albino plants with edits in gene in three varieties and in . The results reveal the potential of this platform to facilitate genome editing in species.
CRISPR-Cas技术能够对植物基因组进行精确修饰，并有望给农业带来变革。这些技术依赖于将编辑组件导入植物细胞以及再生出完全编辑的植物。在葡萄等无性繁殖植物中，原生质体培养为生产非嵌合且无转基因的基因组编辑植物提供了最佳途径之一。然而，原生质体再生植株能力较差阻碍了其在基因组编辑中的应用。在此，我们报告了一种从多个葡萄品种的原生质体再生植株的高效方案。通过将原生质体包裹在海藻酸钙珠中并与饲养培养物共培养，原生质体分裂形成愈伤组织菌落，进而再生为胚胎并最终发育成植株。该方案在酿酒葡萄和鲜食葡萄品种、葡萄砧木以及葡萄野生近缘种中均成功奏效。此外，通过用CRISPR质粒或核糖核蛋白（RNP）复合物转染原生质体，我们在三个品种以及[具体品种未给出]中再生出了在[具体基因未给出]基因中有编辑的白化植株。结果揭示了该平台在促进[具体物种未明确给出]物种基因组编辑方面的潜力。
