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Journal Article Epublish 6 citations

PubMed: 38504900 DOI: 10.3389/fpls.2024.1363182

Mutating alfalfa using multiplex CRISPR/Cas9 leads to reduced lignin deposition and improved forage quality.

Front Plant Sci 15:1363182

Authors: Tezera W Wolabu, Kashif Mahmood, Fang Chen, Ivone Torres-Jerez, Michael Udvardi, Million Tadege, Lili Cong, Zengyu Wang, Jiangqi Wen

Affiliations

  • Institute for Agricultural Bioscience, Oklahoma State University, Ardmore, OK, United States.
  • Center for Biotechnology and Genomics, Texas Tech University, Lubbock, TX, United States.
  • Queensland Alliance for Agriculture and Food Innovation, The University of Queensland, St. Lucia, QLD, Australia.
  • College of Grassland Science, Qingdao Agricultural University, Qingdao, Shandong, China.

Abstract

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

MeSH

Rna, Clustered Regularly Interspaced Short Palindromic Repeats, Detergents, Mutation, Genes, Nutritive Value, Lignin

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