Abstract: Disclosed is a method for obtaining multileaflet Medicago sativa materials by means of MsPALM1 artificial site-directed mutants. The method comprises: selecting a target site from an exon region of a compound leaf developmental regulatory gene MsPALM1 of Medicago sativa and constructing a plant CRISPR/Cas9 editing recombinant vector MsCRISPR/Cas9::PALM1 and introducing the vector into Medicago sativa cells and regenerating into plants, cutting and repairing to cause a loss-of-function mutation in the MsPALM1 gene of Medicago sativa cells, and then screening the mutant plants by restriction endonuclease digestion and/or targeted deep sequencing of the target sites of the regenerated plants to obtain lines carrying four MsPALM1 allelic genes with simultaneous loss of function mutation. After phenotypic identification, it was confirmed that the compound leaves of the regenerated plants changed from three leaflets to five leaflets.

Abstract: A method for site-specific mutagenesis of Medicago sativa genes by using a CRISPR/Cas9 system. The method comprises: first constructing a binary expression vector MsCRISPR/Cas9 that can be used for transforming Medicago sativa by Agrobacterium tumefaciens; then designing a target site for a target gene, and ligating the DNA fragment containing the guide sequence of the target site into MsCRISPR/Cas9 to construct a vector MsCRISPR/Cas9::target; and then transforming the Medicago sativa by Agrobacterium tumefaciens, and generating, by screening, a mutant transformed plant with the target gene mutated. According to the method, an MtU6 promoter is used for driving sgRNA transcription in the Medicago sativa.

Abstract: Disclosed is a method for obtaining multileaflet Medicago sativa materials by means of MsPALM1 artificial site-directed mutants. The method comprises: selecting a target site from an exon region of a compound leaf developmental regulatory gene MsPALM1 of Medicago sativa and constructing a plant CRISPR/Cas9 editing recombinant vector MsCRISPR/Cas9::PALM1 and introducing the vector into Medicago sativa cells and regenerating into plants, cutting and repairing to cause a loss-of-function mutation in the MsPALM1 gene of Medicago sativa cells, and then screening the mutant plants by restriction endonuclease digestion and/or targeted deep sequencing of the target sites of the regenerated plants to obtain lines carrying four MsPALM1 allelic genes with simultaneous loss of function mutation. After phenotypic identification, it was confirmed that the compound leaves of the regenerated plants changed from three leaflets to five leaflets.

Abstract: Disclosed is a method for obtaining multileaflet Medicago sativa materials by means of MsPALM1 artificial site-directed mutants. The method comprises: selecting a target site from an exon region of a compound leaf developmental regulatory gene MsPALM1 of Medicago sativa and constructing a plant CRISPR/Cas9 editing recombinant vector MsCRISPR/Cas9::PALM1 and introducing the vector into Medicago sativa cells and regenerating into plants, cutting and repairing to cause a loss-of-function mutation in the MsPALM1 gene of Medicago sativa cells, and then screening the mutant plants by restriction endonuclease digestion and/or targeted deep sequencing of the target sites of the regenerated plants to obtain lines carrying four MsPALM1 allelic genes with simultaneous loss of function mutation. After phenotypic identification, it was confirmed that the compound leaves of the regenerated plants changed from three leaflets to five leaflets.

Abstract: A method for site-specific mutagenesis of Medicago sativa genes by using a CRISPR/Cas9 system. The method comprises: first constructing a binary expression vector MsCRISPR/Cas9 that can be used for transforming Medicago sativa by Agrobacterium tumefaciens; then designing a target site for a target gene, and ligating the DNA fragment containing the guide sequence of the target site into MsCRISPR/Cas9 to construct a vector MsCRISPR/Cas9::target; and then transforming the Medicago sativa by Agrobacterium tumefaciens, and generating, by screening, a mutant transformed plant with the target gene mutated. According to the method, an MtU6 promoter is used for driving sgRNA transcription in the Medicago sativa.