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Alfalfa (Medicago sativaL.) forage quality is adversely affected by lignin deposition in cell walls at advanced maturity stages. Reducing lignin content through RNA interference or antisense approaches has been shown to improve alfalfa forage quality and digestibility. We employed a multiplex CRISPR/Cas9-mediated gene-editing system to reduce lignin content and alter lignin composition in alfalfa by targeting theCOUMARATE 3-HYDROXYLASE (MsC3H)gene, which encodes a key enzyme in lignin biosynthesis. Four guide RNAs (gRNAs) targeting the first exon ofMsC3Hwere designed and clustered into a tRNA-gRNA polycistronic system and introduced into tetraploid alfalfa viaAgrobacterium-mediated transformation. Out of 130 transgenic lines, at least 73 lines were confirmed to contain gene-editing events in one or more alleles ofMsC3H. Fifty-five lines were selected for lignin content/composition analysis. Amongst these lines, three independent tetra-allelic homozygous lines (Msc3h-013, Msc3h-121, andMsc3h-158) with different mutation events inMsC3Hwere characterized in detail. Homozygous mutation ofMsC3Hin these three lines significantly reduced the lignin content and altered lignin composition in stems. Moreover, these lines had significantly lower levels of acid detergent fiber and neutral detergent fiber as well as higher levels of total digestible nutrients, relative feed values, andin vitrotrue dry matter digestibility. Taken together, these results showed that CRISPR/Cas9-mediated editing ofMsC3Hsuccessfully reduced shoot lignin content, improved digestibility, and nutritional values without sacrificing plant growth and biomass yield. These lines could be used in alfalfa breeding programs to generate elite transgene-free alfalfa cultivars with reduced lignin and improved forage quality. 

Summary Alfalfa (Medicago sativaL.) 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 ofFLOWERING 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 byAgrobacterium‐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 independentMsfta1lines containing mutations in all four copies ofMsFTa1accumulated 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‐editedMsfta1mutants could be introduced in alfalfa breeding programmes to generate elite transgene‐free alfalfa cultivars with improved forage biomass yield and quality. 

Abstract Plant organ size is an important agronomic trait tightly related to crop yield. However, the molecular mechanisms underlying organ size regulation remain largely unexplored in legumes. We previously characterized a key regulator F‐box protein MINI ORGAN1 (MIO1)/SMALL LEAF AND BUSHY1 (SLB1), which controls plant organ size in the model legumeMedicago truncatula. In order to further dissect the molecular mechanism, MIO1 was used as the bait to screen its interacting proteins from a yeast library. Subsequently, a KIX protein, designated MtKIX8, was identified from the candidate list. The interaction between MIO1 and MtKIX8 was confirmed further by Y2H, BiFC, split‐luciferase complementation and pull‐down assays. Phylogenetic analyses indicated that MtKIX8 is highly homologous toArabidopsisKIX8, which negatively regulates organ size. Moreover, loss‐of‐function ofMtKIX8led to enlarged leaves and seeds, while ectopic expression ofMtKIX8inArabidopsisresulted in decreased cotyledon area and seed weight. Quantitative reverse‐transcription PCR and in situ hybridization showed thatMtKIX8is expressed in most developing organs. We also found that MtKIX8 serves as a crucial molecular adaptor, facilitating interactions with BIG SEEDS1 (BS1) and MtTOPLESS (MtTPL) proteins inM. truncatula. Overall, our results suggest that the MIO1‐MtKIX8 module plays a significant and conserved role in the regulation of plant organ size. This module could be a good target for molecular breeding in legume crops and forages.