Method for obtaining multifoliaolate alfalfa material by using MsPALM1 artificial site-directed mutant

Abstract

The invention provides a method for obtaining a multifoliaolate alfalfa material by using an MsPALM1 artificial site-directed mutant. Firstly, a target fragment is selected in an exon region of a alfalfa compound leaf development regulatory gene MsPALM1, a plant CRISPR / Cas9 targeting recombinant vector MsCRISPR / Cas9:PALM1 is constructed, alfalfa cells are introduced and regenerated into seedlings,functional deletion mutation in the MsPALM1 gene of the alfalfa cells is induced by repair after shearing, mutant plants are screened by restriction endonuclease digestion and / or target deep sequencing of the target fragment of a regenerated strain genome to obtain the strain carrying allelic MsPALM1 genes and with the functional deletion mutation, and after phenotypic identification, compound leaves of the regenerated plants are confirmed to be changed from three leaflets to five leaflets. Experiments show that the method can rapidly obtain the multifoliaolate alfalfa material, the breedingcycle is short, and the obtained material is unchanged in other agronomic traits except for the five pinnate compound leaves, is stable in the traits, and can quickly obtain a stable genetically homozygous mutant with four allele mutations.

Description

technical field [0001] The invention belongs to the field of biotechnology breeding of alfalfa, and in particular relates to a method for obtaining multi-leaf alfalfa material by using an artificial site-directed mutant of MsPALM1. Background technique [0002] Alfalfa (Medicago sativa L.) is a high-quality perennial leguminous forage with a long history of cultivation. It has the characteristics of strong adaptability, high nutritional value, wide planting area, complete ecological functions, and high economic benefits. It is a perennial leguminous forage with the largest planting area in the world. It is known as the "king of forage". Alfalfa has been cultivated in my country for more than 2,000 years. At present, the area of ​​alfalfa planted in my country is about 1.467 million hectares. Current production level: the average hay yield is 13-17t / hm2, and the average seed yield is 375-600kg / hm 2 . Therefore, China's grass industry is mainly the industrialization of alfa...

AI Technical Summary

Problems solved by technology

The multi-leaf traits of alfalfa are also found in nature. The research results have shown that compared with the three-leaf alfalfa, the photosynthetic efficiency, specific leaf weight, stem-to-leaf ratio and other growth characteristics of multi-leaf alfalfa are significantly increased; in addition, alfalfa has more Leaf traits can increase the leaf area of ​​alfalfa, and the grass yield of alfalfa is positively correlated with the leaf area; the grass yield of multi-leaf alfalfa lines is significantly higher than that of three-leaf alfalfa varieties; Hidden phenomenon

[0004] Although multi-leaf material lines have broad application prospects in the cultivation of high-quality new varieties of alfalfa, natural genetic mutation resources are limited, and alfalfa is a cross-pollinated autotetraploid plant with self-incompatibility characteristics , to obtain homozygous and stably genetic new alfalfa varieties through traditional hybridization technology, the breeding cycle is long and the feasibility is not high

After retrieval, the patent No. is CN104737899B-a patent for a method for breeding multi-leaf alfalfa induced by spaceflight mutagenesis, and the first new variety of alfalfa multi-leaf alfalfa induced by spaceflight has been bred in my country. The requirements are very strict, multi-generation hybrid breeding, repeated screening, and unstable, it takes a long time, and the reproducibility is not strong

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