Tef is a staple food and a valuable cash crop for millions of people in Ethiopia. Lodging is a major limitation to tef production, and for decades, the development of lodging resistant varieties proved difficult with conventional breeding approaches. We used CRISPR/Cas9 to introduce knockout mutations in the tef orthologue of the rice SEMIDWARF-1 (SD-1) gene to confer semidwarfism and ultimately lodging resistance. High frequency recovery of transgenic and SD-1 edited tef lines was achieved in two tef cultivars by Agrobacterium-mediated delivery into young leaf explants of gene editing reagents along with transformation and regeneration enhancing morphogenic genes, BABY BOOM (BBM) and WUSCHEL2 (WUS2). All of the 23 lines analyzed by next-generation sequencing had at least two or more alleles of SD-1 mutated. Of these, 83% had tetra-allelic frameshift mutations in the SD-1 gene in primary tef regenerants, which were inherited in subsequent generations. Phenotypic data generated on T₁ and T₂ generations revealed that the sd-1 lines have reduced culm and internode lengths with no reduction in either panicle or peduncle lengths. These characteristics are comparable with rice sd-1 plants. Measurements of lodging, in greenhouse-grown plants, showed that sd-1 lines have significantly higher resistance to lodging at the heading stage compared with the controls. This is the first demonstration of the feasibility of high frequency genetic transformation and CRISPR/Cas9-mediated genome editing in this highly valuable but neglected crop. The findings reported here highlight the potential of genome editing for the improvement of lodging resistance and other important traits in tef.

中文翻译：

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CRISPR/Cas9 介导的“绿色革命”SEMIDWARF-1 (SD-1) 基因的四等位基因突变赋予 tef (Eragrostis tef) 倒伏抗性

Tef 是埃塞俄比亚数百万人的主食和宝贵的经济作物。倒伏是 tef 生产的主要限制，几十年来，用传统育种方法开发抗倒伏品种很困难。我们使用 CRISPR/Cas9 在水稻SEMIDWARF-1 ( SD-1 ) 基因的 tef 直系同源物中引入敲除突变，以赋予半矮化和最终抗倒伏性。通过农杆菌介导的基因编辑试剂与转化和再生增强形态发生基因BABY BOOM ( BBM) 和WUSCHEL2 ( WUS2 )。通过下一代测序分析的所有 23 个品系都具有至少两个或多个SD-1等位基因突变。其中，83% 在初级 tef 再生体中的SD-1基因中具有四等位基因移码突变，这些突变在后代中遗传。_{T 1}和T ₂代产生的表型数据表明，sd-1系的茎秆和节间长度减少，而圆锥花序或花序梗长度没有减少。这些特性与水稻sd-1植物相当。对温室种植植物的倒伏测量表明，sd-1与对照相比，线在抽穗阶段具有显着更高的抗倒伏性。这是首次证明高频遗传转化和 CRISPR/Cas9 介导的基因组编辑在这种极具价值但被忽视的作物中的可行性。这里报道的研究结果强调了基因组编辑在提高 tef 抗倒伏性和其他重要性状方面的潜力。