Regeneration and cultivation method of high frequency somatic embryos for overcoming shamrock variety genotypic disorder

Abstract

The invention discloses a regeneration and cultivation method of high frequency somatic embryos for overcoming shamrock variety genotypic disorder, which comprises the following steps: 1. material selection: selecting three explants, i.e. an epicotyl, a hypocotyls and a regeneration plant leaf of an aseptic seedling after a mature seed of the shamrock is flushed, sterilized, disinfected and dried;2. inoculating the selected explants onto an embryogenic callus inducing culture medium for cultivation; 3. inoculating the explants cultivated through the embryogenic callus inducement onto an embryo successive formation culture medium for cultivation; 4. inoculating the explants cultivated through the embryo successive formation cultivation onto an embryo maturation germination seedling culturemedium for cultivation to form seedlings; 5. carrying out rooting cultivation on strong seedlings; and 6. transplanting a regeneration plant for survival. The invention can effectively overcome the problems of different varieties and genotypic disorder in the regeneration and cultivation of the high frequency somatic embryos of the shamrock, greatly improve the cell embryogenesis frequency and the regeneration plant frequency, and improve the breeding efficiency.

Description

technical field [0001] The invention relates to a method for high-frequency somatic embryo regeneration and cultivation of leguminous herbage, and more specifically relates to a method for overcoming the genotype barrier of clover varieties and belongs to the field of botany. Background technique [0002] Clover is an excellent leguminous forage that is distributed and cultivated worldwide. It is the preferred grass species for artificial grassland in temperate regions, and it is also the main component of various ornamental lawns and green spaces. , nitrogen fixation and fertilization of orchard trees, soil and water conservation of road embankments, etc. all play an irreplaceable important role. In view of the important economic value of clover, many developed countries have used biotechnology methods to study and improve clover to solve the problems existing in clover production. [0003] Clover is a heterozygote with self-incompatibility. At present, more than 80% of th...

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Problems solved by technology

[0003] Clover is a heterozygote, self-incompatible. At present, more than 80% of the varieties used in the world are comprehensive varieties, and the genotypes of the varieties vary greatly. Some important breeding goals are difficult to achieve through conventional breeding methods, such as clover drought and salt tolerance, disease and insect resistance, increased seed and grass yield, nitrogen fixation ability, etc. can not achieve the desired results. Therefore, in recent years, people have tried to use modern genes Engineering technology, that is, cultivating new varieties of clover by obtaining resistant plants through genetic modification, and improving the adaptability of clover to various ecological environments, but the prerequisite for the successful application of this technology is that clover has high-frequency body Embryo regeneration ability, which has been an insurmountable problem

[0004] The research on tissue culture regeneration technology of Clover plants began in the 1970s. Some reports have shown that the tissue culture regeneration rates of most Clover plants are extremely low. Only 1% or less, and the regeneration process is long, generally takes about 4 to 6 months, the regeneration and regeneration frequency are easily affected by the genotype, which has become a bottleneck restricting the transgenic of different genotypes, especially good varieties

The establishment of the clover regeneration system is mostly through the organogenesis pathway, and there are few reports on the establishment of the somatic embryogenesis pathway, and there are no reports on the high-frequency somatic embryo regeneration tissue culture technology system suitable for clover gene transformation

[0005]Most of the previous researchers only selected 1~3 varieties of 1 species as materials. Due to the small number of species studied and not representative, these studies are difficult Solve the problem of different varieties and genotypes of clover

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