Diversity of transgene integration and gene-editing events in wheat (Triticum aestivum L.) transgenic plants generated using Agrobacterium-mediated transformation.

IF 4.9 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY
Frontiers in genome editing Pub Date : 2023-12-19 eCollection Date: 2023-01-01 DOI:10.3389/fgeed.2023.1265103
Louie Cris Lopos, Natalia V Bykova, Janeen Robinson, Susan Brown, Kerry Ward, Andriy Bilichak
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Abstract

Improvement in agronomic traits in crops through gene editing (GE) relies on efficient transformation protocols for delivering the CRISPR/Cas9-coded transgenes. Recently, a few embryogenesis-related genes have been described, the co-delivery of which significantly increases the transformation efficiency with reduced genotype-dependency. Here, we characterized the transgenic and GE events in wheat (cv. Fielder) when transformed with GROWTH-REGULATING FACTOR 4 (GRF4) and its cofactor GRF-INTERACTING FACTOR 1 (GIF1) chimeric gene. Transformation efficiency in our experiments ranged from 22% to 68%, and the editing events were faithfully propagated into the following generation. Both low- and high-copy-number integration events were recovered in the T0 population with various levels of integrity of the left and right T-DNA borders. We also generated a population of wheat plants with 10 different gRNAs targeting 30 loci in the genome. A comparison of the epigenetic profiles at the target sites and editing efficiency revealed a significant positive correlation between chromatin accessibility and mutagenesis rate. Overall, the preliminary screening of transgene quality and GE events in the T0 population of plants regenerated through the co-delivery of GRF-GIF can allow for the propagation of the best candidates for further phenotypic analysis.

利用农杆菌介导转化产生的小麦(Triticum aestivum L.)转基因植株中转基因整合和基因编辑事件的多样性。
通过基因编辑(GE)改善农作物的农艺性状有赖于高效的转化方案来传递 CRISPR/Cas9 编码的转基因。最近,一些与胚胎发生相关的基因被描述出来,这些基因的联合传递大大提高了转化效率,降低了基因型依赖性。在这里,我们描述了小麦(cv. Fielder)在转入生长调节因子 4(GRF4)及其辅助因子 GRF-INTERACTING FACTOR 1(GIF1)嵌合基因后的转基因和 GE 事件。在我们的实验中,转化效率从 22% 到 68%不等,而且编辑事件能忠实地传播到下一代。在 T0 群体中,低拷贝数和高拷贝数整合事件都得到了恢复,T-DNA 左右边界的完整性程度各不相同。我们还利用基因组中针对 30 个基因位点的 10 种不同 gRNA 生成了小麦植株群体。通过比较目标位点的表观遗传学特征和编辑效率,发现染色质可及性和诱变率之间存在显著的正相关。总之,在通过联合递送 GRF-GIF 再生的 T0 植株群体中对转基因质量和 GE 事件进行初步筛选,可以繁殖出最佳候选者,以便进一步进行表型分析。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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CiteScore
7.00
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0.00%
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审稿时长
13 weeks
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