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CN-121970678-A - Method for distant sexual hybridization of plants

CN121970678ACN 121970678 ACN121970678 ACN 121970678ACN-121970678-A

Abstract

Selecting a good variety of a desired sexual propagation plant type as a hybrid female parent, emasculating male flowers before maturity, treating female flowers before insemination and removing reproductive isolation, selecting sperm of any organism among varieties with the properties of the desired variety and further, adding a distant hybridization agent, stirring uniformly to form a sperm-giving solution, inseminating the female parent, carrying out embryo rescue and culturing to form seedlings after the fertilized embryos are retrieved, carrying out chromosome doubling on the obtained seedlings, and then transplanting to soil. Harvesting each true hybrid by single plant in the mature period, and cultivating each future substitution pedigree method F 2 until the true hybrid is homozygous. The invention breaks through the distant sexual hybridization bottleneck, can cultivate high-yield and high-quality varieties, and has wide application prospect.

Inventors

  • HE JUEMIN
  • DAI PENG
  • LU LI
  • DAI CHAOHUI
  • HE YUFENG
  • ZENG SONGTING
  • HE RONG
  • HE YI
  • ZHOU JIANWEI
  • TANG LINHUA
  • HE JUEXING
  • HE YUAN
  • LI YU

Assignees

  • 深圳前海觉民科技有限公司

Dates

Publication Date
20260505
Application Date
20210204

Claims (10)

  1. 1. A method for sexual hybridization of plants is characterized in that the method comprises the following steps: (1) Selecting a good variety of a desired sexual propagation plant type as a hybrid female parent, emasculating male flowers before maturity, treating female flowers before insemination, and removing reproductive isolation; (2) Selecting sperm or pollen of a species or more distant organism having the trait of the desired breed, adding a distant hybridization agent and stirring to obtain semen, inseminating the female parent with the inseminating liquid, or inseminating the female parent stigmas with the distant hybridization agent and the sperm or pollen, respectively, preferably sealing with a sheepskin paper bag, further preferably, re-pollinating the bag in the next day, repeating for 2-12 days; (3) Harvesting fertilized eggs after hybridization success, preferably harvesting fertilized eggs after 7-70d hybridization success; (4) Embryo rescue is carried out on fertilized eggs, seedlings are cultivated, and the fertilized eggs are transplanted to soil after chromosome doubling; Further still include: (5) False hybrids are identified and removed.
  2. 2. The method of claim 1, wherein the sexual hybridization is a sexual hybridization comprising an intergranular, an interocular, an interpedical, an intergeneric, and an intergeneric.
  3. 3. The method according to claim 1, wherein in step (1) the female flowers are treated prior to insemination, in particular by removing the coating outside the ovary, leaving it exposed, such as the calyx, petals, ovary walls and integuments.
  4. 4. The method of claim 1, wherein the reproductive isolation is removed in step (1), specifically by dripping 1 to several drops of enzymatic hydrolysate into the ovary to wet the ovary, wherein the enzymatic hydrolysate is an animal glycoprotein enzymatic hydrolysate.
  5. 5. The method of claim 1, wherein the distant hybridization agent comprises 100-300mg/L gibberellin, 10-100mg/L auxin, 1500-5000ml/L enzymatic hydrolysate and 1500-5000mg/L compound amino acid, more preferably comprises 150-250mg/L gibberellin, 40-70mg/L auxin, 2500-4000. Mu.l/L enzymatic hydrolysate and 2500-4000mg/L compound amino acid, and preferably the enzymatic hydrolysate is an animal glycoprotein enzymatic hydrolysate complete glycosyl decomposition enzymatic hydrolysate.
  6. 6. The method of claim 1, wherein said inseminating the female parent with the insemination liquid in step (3) is performed by applying the ovary with the insemination liquid, sealing with a bag, preferably, repeating for 2 to 12 days once a day.
  7. 7. The method according to claim 1, wherein in step (4) the fertilized eggs are embryo-rescued and cultivated to shoots, in particular with MS medium.
  8. 8. The method of claim 1, wherein the chromosome doubling method is to double the chromosome with colchicine, then transplant the chromosome into soil, harvest each true hybrid from a single plant in the mature period, and culture each surrogate pedigree method for the generation F 2 and later until the chromosome is homozygous.
  9. 9. The method of claim 1, wherein the method of identifying and removing a false hybrid in step (5) is selected from one or more of the following three: 1) identifying and removing false hybrids close to the female parent by morphology, 2) identifying and removing individuals close to the female parent by composition, 3) identifying and removing individuals close to the female parent by genetic analysis.
  10. 10. The method of claim 1, further comprising the step of: The method for detecting typical individual metabolites in the variety by adopting the broad-range targeting metabolome technology is used for assisting in purifying and rejuvenating, wherein the method for breeding the variety is that on the basis of field primary selection and indoor check of individuals with typical characters of the variety, individuals without specific metabolites of the male parent in the final selection hybrid are detected by adopting the broad-range targeting metabolome technology and are used for breeding the variety.

Description

Method for distant sexual hybridization of plants Technical Field The invention belongs to the field of plant improvement. In particular to a method for distant sexual hybridization of plants. Background Scientific researches prove that each revolutionary evolution of plants is initiated by a heteropolyploid formed by distant hybridization, so that the plant evolution can be effectively promoted only by the distant hybridization, and breakthrough plant varieties are cultivated. However, because of the reproductive isolation between species, hybridization between species and beyond is difficult to succeed. However, modern scientific research has shown that the same gene sequence exists between different species of organisms, although the profiles are far apart (Mao Li, the same gene sequence exists for different plant species, science and technology journal, 4/11/2012, 1 st edition). Therefore, if the parent and parent chromosomes meet, the chromosomes can be paired or partially paired no matter the relativity is far or near, and even if the chromosomes cannot be paired, the chromosomes can be doubled. Thus, the inability of the parent chromosome to be located at the same corner is the root cause of the distant species to cross infertility. The distant hybrid parent and parent chromosomes cannot be distinguished because of the presence of recognition proteins, i.e., glycoproteins, on the plant stigma and pollen/sperm surfaces, which act like a lock, outside the door, which repels distant pollen/sperm. Therefore, by only degrading glycoprotein on the stigma and the outer layer of the sperm, genetic materials such as chromosomes of distant parents can meet, and distant parents can obtain distant hybrid seeds by pairing/partially pairing/combining the chromosomes/genetic materials of distant parents. The zymolyte of the complete glycosyl decomposition kit for glycoprotein provided by biotechnology company can degrade the recognition protein-glycoprotein, so that distant hybridization is possible. Disclosure of Invention The invention aims to overcome the defects of the prior art, breaks through the bottleneck of distant sexual hybridization of plants by adopting effective measures, realizes distant sexual hybridization among any organisms taking plants as female parents, and breeds and obtains expected plant varieties. Accordingly, the present invention provides a method for sexual crossing of plants, characterized in that said method comprises the steps of: (1) Selecting a good variety of a desired sexual propagation plant type as a hybrid female parent, emasculating male flowers before maturity, treating female flowers before insemination, and removing reproductive isolation; (2) Selecting animal sperm or plant flowers of organisms having traits of the desired breed or more, adding a hybridization agent and stirring to obtain semen, inseminating the female parent with the inseminating liquid, or smearing the stigma of the female parent with the hybridization agent and said sperm or pollen, respectively, for insemination, preferably sealing with a parchment paper bag, repeating pollination the next day, untangling the bag again, repeating this for 2-11 days; (3) Inseminating female parent with inseminating liquid, and harvesting fertilized egg after hybridization; (4) Harvesting fertilized eggs preferably for 7-70 days after fertilization, performing embryo rescue and culturing to form seedlings, and transplanting the seedlings to soil after chromosome doubling; Further still include: (5) False hybrids are identified and removed. The plant distant sexual hybridization of the present invention uses plant as female parent, including sexual hybridization between boundary, phylum, class, mesh, family, genus and species. In a specific embodiment, the insemination-pre-treatment female flowers in step (1), in particular, removing the coating outside the ovary, leaving it exposed, e.g., removing the calyx, petals, ovary walls and integuments. And removing reproductive isolation before insemination, namely dripping 1 to a plurality of drops of enzymolysis liquid into an ovary to wet the ovary thoroughly, wherein the enzymolysis liquid is an animal glycoprotein enzymolysis complete glycosyl decomposition enzymolysis liquid. The formula of the distant hybridization agent comprises 100-300mg/L gibberellin, 10-100mg/L auxin, 1500-5000ml/L enzymolysis liquid and 1500-5000mg/L compound amino acid, preferably 150-250mg/L gibberellin, 40-70mg/L auxin, 2500-4000 mu L enzymolysis liquid and 2500-4000mg/L compound amino acid, preferably the enzymolysis liquid is animal glycoprotein enzymolysis complete glycosyl decomposition enzymolysis liquid, and can be purchased through commercial paths. Wherein the compound amino acid is a compound amino acid in a conventional meaning, and can be prepared by a method described in CN200610031424.X, for example, and can be obtained commercially. In a specific embodiment, the insemination method