Ahmed Shahbaz, Hulbert Anna K, Xin Xin, Neff Michael M
The Department of Crop and Soil Sciences, Washington State University, Pullman, WA, United States.
Graduate Program in Molecular Plant Sciences, Washington State University, Pullman, WA, United States.
Front Plant Sci. 2024 Oct 16;15:1408230. doi: 10.3389/fpls.2024.1408230. eCollection 2024.
After successfully performing mediated CRISPR-Cas9-based gene editing in plants, isolation of the T-DNA is essential for the stable inheritance of induced mutations. Here, we report a simple technique that allows the isolation of -free mutants, eliminating the need for outcrossing or other intricate methods. This method is based on the ability of Basta-sensitive seedlings, which generally perish, to recover and grow once transplanted to Basta-free growth media. By growing gene-edited heterozygous populations of single-locus insertion Basta-resistant plants on Basta selection media, plants lacking the T-DNA can be identified. These pale-looking plants lacking Cas9 are then rescued on media lacking the Basta to recover -free plants. The ability of seedlings to recover from Basta selection was also studied in camelina, canola, and wheat. All three crops showed different recovery rates, with wheat demonstrating the highest recovery once transplanted from Basta to normal growth media. In summary, our findings demonstrate that by harnessing the recovery capability of Basta-sensitive seedlings, we can effectively identify and rescue plants lacking the Cas9 T-DNA, enabling the isolation of Cas9-free mutants in and potentially extending to other crops.
在植物中成功进行基于CRISPR-Cas9的介导基因编辑后，分离T-DNA对于诱导突变的稳定遗传至关重要。在此，我们报告了一种简单的技术，该技术可分离无T-DNA的突变体，无需进行杂交或其他复杂方法。该方法基于对Basta敏感的幼苗（通常会死亡）一旦移植到无Basta的生长培养基中就能恢复生长的能力。通过在Basta选择培养基上培养单基因座插入的抗Basta基因编辑杂合群体，可以鉴定出缺乏T-DNA的植株。然后将这些看起来苍白且缺乏Cas9的植株在不含Basta的培养基上拯救培养，以获得无T-DNA的植株。还在亚麻荠、油菜和小麦中研究了幼苗从Basta选择中恢复的能力。所有这三种作物都表现出不同的恢复率，其中小麦从Basta移植到正常生长培养基后恢复率最高。总之，我们的研究结果表明，通过利用对Basta敏感的幼苗的恢复能力，我们可以有效地鉴定和拯救缺乏Cas9 T-DNA的植株，从而在拟南芥中分离出无Cas9的突变体，并有可能推广到其他作物。
