Alfalfa (Medicago sativa L.) is a perennial flowering plant in the legume family that is widely cultivated as a forage crop for its high yield, forage quality and related agricultural and economic benefits. Alfalfa is a photoperiod sensitive long-day (LD) plant that can accomplish its vegetative and reproductive phases in a short period of time. However, rapid flowering can compromise forage biomass yield and quality. Here, we attempted to delay flowering in alfalfa using multiplex CRISPR/Cas9-mediated mutagenesis of FLOWERING LOCUS Ta1 (MsFTa1), a key floral integrator and activator gene. Four guide RNAs (gRNAs) were designed and clustered in a polycistronic tRNA–gRNA system and introduced into alfalfa by Agrobacterium-mediated transformation. Ninety-six putative mutant lines were identified by gene sequencing and characterized for delayed flowering time and related desirable agronomic traits. Phenotype assessment of flowering time under LD conditions identified 22 independent mutant lines with delayed flowering compared to the control. Six independent Msfta1 lines containing mutations in all four copies of MsFTa1 accumulated significantly higher forage biomass yield, with increases of up to 78% in fresh weight and 76% in dry weight compared to controls. Depending on the harvesting schemes, many of these lines also had reduced lignin, acid detergent fibre (ADF) and neutral detergent fibre (NDF) content and significantly higher crude protein (CP) and mineral contents compared to control plants, especially in the stems. These CRISPR/Cas9-edited Msfta1 mutants could be introduced in alfalfa breeding programmes to generate elite transgene-free alfalfa cultivars with improved forage biomass yield and quality.

中文翻译：

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苜蓿开花基因座 Ta1 (MsFTa1) 的多重 CRISPR/Cas9 介导的诱变导致开花时间延迟，同时提高饲料生物量产量和质量

苜蓿（Medicago sativa L.）是豆科植物中的一种多年生开花植物，因其产量高、饲料品质好以及相关的农业和经济效益而被广泛种植作为饲料作物。苜蓿是一种光周期敏感的长日照（LD）植物，可以在短时间内完成营养和繁殖阶段。然而，快速开花会损害饲料生物量产量和质量。在这里，我们尝试使用多重 CRISPR/Cas9 介导的FLOWERING LOCUS Ta1 ( MsFTa1 )（一种关键的花整合子和激活基因）诱变来延迟苜蓿的开花。设计了四种引导 RNA (gRNA)，并将其聚集在多顺反子 tRNA-gRNA 系统中，并通过农杆菌介导的转化引入苜蓿。通过基因测序鉴定了九十六个假定的突变株系，并对其开花时间延迟和相关的理想农艺性状进行了表征。在 LD 条件下对开花时间进行表型评估，鉴定出 22 个独立的突变株系，与对照相比，其开花延迟。全部四个MsFTa1拷贝均含有突变的6 个独立的Msfta1品系积累了显着更高的草料生物量产量，与对照相比，鲜重增加了 78%，干重增加了 76%。根据收获方案，与对照植物相比，其中许多品系的木质素、酸性洗涤纤维（ADF）和中性洗涤纤维（NDF）含量也降低，粗蛋白（CP）和矿物质含量显着升高，尤其是在茎中。这些 CRISPR/Cas9 编辑的Msfta1突变体可以被引入苜蓿育种计划中，以产生具有改善的饲料生物量产量和质量的优良无转基因苜蓿品种。