A method for obtaining multi-leaf alfalfa materials using mspalm1 artificial site-directed mutants

Abstract

The present invention provides a method for obtaining multi-leaf alfalfa materials by using MsPALM1 artificial site-directed mutants. First, a target fragment is selected in the exon region of the alfalfa compound leaf development regulatory gene MsPALM1 and a plant CRISPR / Cas9 targeting recombinant vector MsCRISPR / Cas9 is constructed. ::PALM1, introduced into alfalfa cells and regenerated into shoots, the loss-of-function mutation of the MsPALM1 gene in alfalfa cells was caused by post-cutting repair, and then through restriction endonuclease digestion and / or targeting of the target fragment of the regenerated line genome Mutant plants were screened by spot deep sequencing, and lines carrying four alleles of MsPALM1 gene with simultaneous loss-of-function mutations were obtained. After phenotypic identification, it was confirmed that the compound leaves of the regenerated plants had changed from three leaflets to five leaflets. Experiments show that this method can quickly obtain multi-leaf alfalfa materials, the breeding cycle is short, and the obtained materials have no change in other agronomic traits except that the five-pinnate compound leaves are stable, and the four alleles can be quickly obtained. Mutated stably inheritable homozygous mutants.

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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