Multiplex CRISPR/Cas9‐mediated mutagenesis of alfalfa FLOWERING LOCUS Ta1 (MsFTa1) leads to delayed flowering time with improved forage biomass yield and quality

Abstract

Summary 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.

Mutating alfalfa COUMARATE 3-HYDROXYLASE using multiplex CRISPR/Cas9 leads to reduced lignin deposition and improved forage quality

“…Alfalfa is grown for hay, silage, dry pellets, or used directly for grazing as a quality source of proteins for livestock ( Zhou et al., 2011 ; Barros et al., 2019b ). The fast-growing perennial growth habit of alfalfa makes it ideal for multiple harvests within one growing season ( Singer et al., 2018 ; Wolabu et al., 2023 ). The herbaceous nature of alfalfa plants with abundant lignocellulosic biomass yields and the ability to thrive in a variety of soil conditions make it a promising next-generation source of feedstock for biofuels ( Hisano et al., 2009 ; Tong et al., 2015 ).…”

“…The herbaceous nature of alfalfa plants with abundant lignocellulosic biomass yields and the ability to thrive in a variety of soil conditions make it a promising next-generation source of feedstock for biofuels ( Hisano et al., 2009 ; Tong et al., 2015 ). Alfalfa leaves are more nutritious than stems, since high lignification in stems is a limiting factor to cell wall digestion, and any improvement that helps to liberate nutrients embedded in stems during digestion results in overall benefits for forage animals ( Lacefield, 2004 ; Shadle et al., 2007 ; Getachew et al., 2018 ; Wolabu et al., 2023 ). Therefore, lignin is one of the most important limiting factors for forage nutritional quality ( Getachew et al., 2018 ; Singer et al., 2018 ; Wolabu et al., 2023 ).…”

“…Alfalfa leaves are more nutritious than stems, since high lignification in stems is a limiting factor to cell wall digestion, and any improvement that helps to liberate nutrients embedded in stems during digestion results in overall benefits for forage animals ( Lacefield, 2004 ; Shadle et al., 2007 ; Getachew et al., 2018 ; Wolabu et al., 2023 ). Therefore, lignin is one of the most important limiting factors for forage nutritional quality ( Getachew et al., 2018 ; Singer et al., 2018 ; Wolabu et al., 2023 ). Forage digestibility is negatively correlated with lignin content, which limits the nutritional value of forage crops ( Li et al., 2008 ).…”

“…Genome editing approaches, such as CRISPR/Cas9, have revolutionized the field of plant biotechnology through genome engineering, facilitating important trait-driven improvement in major crop species ( Chen et al., 2020 ; Wolabu et al., 2020a , 2020b ; Bottero et al., 2021 ; Zheng et al., 2021 ; Bao et al., 2022 ; Kumar et al., 2022 ; Nerkar et al., 2022 ; Singer et al., 2022 ; Wolabu et al., 2023 ). CIRSPR/Cas9 has been employed to manipulate lignin content in various plant species to improve the efficiency of lignocellulose processing and enhance forage quality, digestibility, and saccharification efficiency ( Park et al., 2017 ; Jang et al., 2021 ; Afifi et al., 2022 ; Roy et al., 2023 ; Wolabu et al., 2023 ; Laksana et al., 2024 ). A recent study that altered flowering time via multiplex-CRISPR/Cas9 edition of a florigen in alfalfa generated promising lines with improved biomass yield and quality ( Wolabu et al., 2023 ).…”

“…CIRSPR/Cas9 has been employed to manipulate lignin content in various plant species to improve the efficiency of lignocellulose processing and enhance forage quality, digestibility, and saccharification efficiency ( Park et al., 2017 ; Jang et al., 2021 ; Afifi et al., 2022 ; Roy et al., 2023 ; Wolabu et al., 2023 ; Laksana et al., 2024 ). A recent study that altered flowering time via multiplex-CRISPR/Cas9 edition of a florigen in alfalfa generated promising lines with improved biomass yield and quality ( Wolabu et al., 2023 ). Editing the switchgrass 4CL gene has also been shown to generate mutant lines with reduced lignin content and improved sugar release ( Park et al., 2017 ).…”