METHOD OF OBTAINING MULTI-LEAF ALFALFA MATERIAL BY MEANS OF MSPALM1 ARTIFICIAL SITE-DIRECTED MUTANT

摘要

Disclosed is a method for obtaining multi-leaf alfalfa material by means of a MsPALM1 artificial site-directed mutant. The method comprises: selecting a target fragment from an exon region of a compound leaf developmental regulatory gene MsPALM1 of alfalfa and constructing a plant CRISPR/Cas9 targeting recombinant vector MsCRISPR/Cas9::PALM1 and introducing the same into alfalfa cells and reproducing into sprouts, cutting and repairing to cause a loss-of-function mutation in the MsPALM1 gene of alfalfa cells, and then screening the mutant plants by restriction endonuclease digestion and/or targeted deep sequencing of the target fragments of the reproduced strain genome to obtain lines carrying four allelic MsPALM1 genes with simultaneous loss of function mutation. After phenotypic identification, it was confirmed that the compound leaves of the reproduced plants changed from three leaflets to five leaflets. The method can quickly obtain a multi-leaf alfalfa material with a short breeding period and stable traits.