Application of CRISPR/Cas9 technology in forages

Bao, Qinyan; Wolabu, Tezera W.; Zhang, Qian; Zhang, Tian; Liu, Zhipeng; Sun, Juan; Wang, Zeng‐Yu; Zhou, Chuanen

doi:10.1002/glr2.12036

Cited by 5 publications

(5 citation statements)

References 53 publications

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“…Alfalfa ( Medicago sativa L.) is cultivated worldwide due to its high forage yield and quality, perennial growth habit, adaptability, nitrogen fixation ability and related agricultural and economic benefits (Bao et al ., 2022 ; Chen et al ., 2020 ; Gao et al ., 2018 ; Lorenzo et al ., 2020 ; Singer et al ., 2018 ; Wolabu et al ., 2020a ; Zhou et al ., 2011 ). Alfalfa is a fast‐growing forage, both before and after cutting, making it ideal for multiple harvests within one growing season (Du et al ., 2021 ; Kang et al ., 2019 ; Lorenzo et al ., 2020 ; Singer et al ., 2018 ).…”

Section: Introductionmentioning

confidence: 99%

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

Wolabu

Mahmood

Jerez

et al. 2023

Plant Biotechnology Journal

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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.

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Section: Introductionmentioning

confidence: 99%

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

Wolabu

Mahmood

Jerez

et al. 2023

Plant Biotechnology Journal

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“…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 ).…”

Section: Introductionmentioning

confidence: 99%

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

Wolabu,

Mahmood,

Chen

et al. 2024

Front. Plant Sci.

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Alfalfa (Medicago sativa L.) 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 the COUMARATE 3-HYDROXYLASE (MsC3H) gene, which encodes a key enzyme in lignin biosynthesis. Four guide RNAs (gRNAs) targeting the first exon of MsC3H were designed and clustered into a tRNA-gRNA polycistronic system and introduced into tetraploid alfalfa via Agrobacterium-mediated transformation. Out of 130 transgenic lines, at least 73 lines were confirmed to contain gene-editing events in one or more alleles of MsC3H. Fifty-five lines were selected for lignin content/composition analysis. Amongst these lines, three independent tetra-allelic homozygous lines (Msc3h-013, Msc3h-121, and Msc3h-158) with different mutation events in MsC3H were characterized in detail. Homozygous mutation of MsC3H in 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, and in vitro true dry matter digestibility. Taken together, these results showed that CRISPR/Cas9-mediated editing of MsC3H successfully 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.

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“…Alfalfa ( Medicago sativa L.), widely grown globally, is favored for its exceptional yield, superior quality, and perennial growth characteristics [ 1 , 2 , 3 , 4 , 5 ]. Improving agricultural production heavily depends on the efficient optimization of the flowering time in forage crops, which remains a prominent objective within breeding programs.…”

Section: Introductionmentioning

confidence: 99%

Functional Characterization of the MsFKF1 Gene Reveals Its Dual Role in Regulating the Flowering Time and Plant Height in Medicago sativa L.

Jiang,

Zhang,

et al. 2024

Plants

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Alfalfa (M. sativa), a perennial legume forage, is known for its high yield and good quality. As a long-day plant, it is sensitive to changes in the day length, which affects the flowering time and plant growth, and limits alfalfa yield. Photoperiod-mediated delayed flowering in alfalfa helps to extend the vegetative growth period and increase the yield. We isolated a blue-light phytohormone gene from the alfalfa genome that is an ortholog of soybean FKF1 and named it MsFKF1. Gene expression analyses showed that MsFKF1 responds to blue light and the circadian clock in alfalfa. We found that MsFKF1 regulates the flowering time through the plant circadian clock pathway by inhibiting the transcription of E1 and COL, thus suppressing FLOWERING LOCUS T a1 (FTa1) transcription. In addition, transgenic lines exhibited higher plant height and accumulated more biomass in comparison to wild-type plants. However, the increased fiber (NDF and ADF) and lignin content also led to a reduction in the digestibility of the forage. The key genes related to GA biosynthesis, GA20OX1, increased in the transgenic lines, while GA2OX1 decreased for the inactive GA transformation. These findings offer novel insights on the function of MsFKF1 in the regulation of the flowering time and plant height in cultivated M. sativa. These insights into MsFKF1’s roles in alfalfa offer potential strategies for molecular breeding aimed at optimizing flowering time and biomass yield.

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Application of CRISPR/Cas9 technology in forages

Cited by 5 publications

References 53 publications

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

Multiplex CRISPR/Cas9‐mediated mutagenesis of alfalfa FLOWERING LOCUS Ta1 (MsFTa1) leads to delayed flowering time 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

Functional Characterization of the MsFKF1 Gene Reveals Its Dual Role in Regulating the Flowering Time and Plant Height in Medicago sativa L.

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