Method for cultivating undaria pinnatifida triploid sporophyte by inducing homozygous diploid gametophyte
By inducing the reproduction and hybridization of homozygous diploid spores of wakameng, the technical bottleneck of wakameng triploid spore sports species was solved, and efficient cultivation of high-yield and high-quality triploid spores was achieved, and the genetic diversity of wild populations was protected.
Patent Information
- Application Number
- CN202510725767.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-06-03
- Publication Date
- 2025-08-29
AI Technical Summary
The prior art is difficult to isolate the diploid gametophytes of the single gender of wakame, making it difficult to obtain triploid sporophytes through hybridization, affecting breeding efficiency and quality.
By inducing the spore-free propagation of homozygous diploid sporophytes of wakana, homozygous diploid gametophytes are produced and hybridized with haploid gametophytes of the opposite gender, wakana triplophytes are obtained.
The cultivation process of wakame triploid sporophytes has been simplified, breeding efficiency has been improved, breeding cycle has been shortened, high-yield and high-quality new varieties have been obtained, and the genetic diversity of wild populations has been protected.
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Figure CN120548982A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to genetic breeding, in particular to a method for cultivating triploid sporophytes of Undaria pinnatifida by inducing homozygous diploid gametophytes. Background Art
[0002] Undaria pinnatifida is the second largest economic brown algae in my country, second only to Laminaria japonica in importance. It belongs to the Laminariales order and has a typical life history of Laminaria japonica brown algae. In nature, the sporophyte of Undaria pinnatifida is diploid (2n) and the gametophyte is haploid (n). The main methods for Undaria pinnatifida breeding at present are continuous directional selection and haploid gametophyte cloning hybrid breeding, and the sporophyte offspring cultivated are all 2n sporophytes. Triploid (3n) is usually high in yield and good in quality because it is sterile or has low fertility, and the energy consumed by reproduction is allocated to the vegetative growth stage. Therefore, the cultivation of 3n sporophytes has important potential value for the creation of new germplasm of Undaria pinnatifida.
[0003] Undaria pinnatifida has diverse reproductive methods. Besides the most common spore reproduction method, 2n sporophytes can also reproduce directly through somatic cells asexually, forming filamentous gametophytes. Because heterozygous 2n sporophytes possess both U and V chromosomes, the 2n gametophytes they produce asexually have the same genotype as the parent sporophyte, possessing both U and V chromosomes. Consequently, their sexual phenotype is complex, manifesting as a mixture of males and females, or hermaphrodites. This makes it difficult to isolate single-sex 2n gametophytes for hybridization and triploid breeding. Consequently, there is an urgent need to overcome the technical bottleneck of heterozygous 2n gametophytes, which presents a complex sexual phenotype and makes it difficult to isolate single-sex 2n gametophytes for hybridization and obtaining triploid sporophytes. Summary of the Invention
[0004] The present invention aims to provide a method for cultivating Undaria pinnatifida triploid sporophyte by inducing homozygous diploid gametophyte.
[0005] To achieve the above-mentioned purpose, the technical solution adopted by the present invention is:
[0006] The invention discloses a method for cultivating Undaria pinnatifida triploid sporophyte by inducing homozygous diploid gametophyte. The method comprises the following steps: inducing homozygous 2n Undaria pinnatifida sporophyte to reproduce aposporously to produce homozygous 2n gametophyte, and then hybridizing the homozygous 2n gametophyte with a common haploid (n) gametophyte to obtain Undaria pinnatifida triploid (3n) sporophyte.
[0007] Further,
[0008] Using the young sporophytes produced by parthenogenesis of Undaria pinnatifida as starting materials, homozygous diploid (2n) female gametophytes were induced by tissue culture.
[0009] Using the young sporophytes produced by self-pollination of hermaphroditic gametophytes as starting materials, homozygous 2n male gametophytes were induced by tissue culture.
[0010] The homozygous 2n gametophytes obtained above were then hybridized with haploid (n) gametophytes of the opposite sex to obtain triploid (3n) sporophytes of Undaria pinnatifida.
[0011] Going further,
[0012] Homozygous diploid (2n) sporophytes were obtained by selecting single-ploid (n) female gametophyte clones of A. truncatum through induced parthenogenesis;
[0013] Single-fold (n) hermaphroditic gametophyte clones of Azalea were selected and homozygous 2n sporophytes were obtained by self-pollination;
[0014] The 2n sporophytes obtained above were cultured to a length of 1 cm, the rhizoctonia were cut off, and the somatic cells were induced to reproduce aposporously by tissue culture to produce homozygous 2n female and male gametophytes, respectively;
[0015] The 2n female gametophytes obtained above were hybridized with n male gametophytes, and the 2n male gametophytes obtained above were hybridized with n female gametophytes to obtain triploid (3n) sporophytes of Undaria pinnatifida.
[0016] The obtained Undaria pinnatifida triploid (3n) sporophyte is sterile, that is, cannot form zoospores.
[0017] The process of inducing parthenogenesis and self-pollination of hermaphrodite gametophytes to obtain 2n sporophytes is as follows:
[0018] The female and hermaphrodite gametophytes were cultured in PES seawater medium at a temperature of 18-20°C and a light intensity of 50-80 μmol photons m -2 s -1 The photoperiod was 12 hours day and 12 hours night. The culture was continued until the sporophytes reached 1 mm. The PES seawater medium was replaced every 3-5 days.
[0019] The young sporophytes were cultured to 1 mm in PES seawater medium at a temperature of 18-20°C and a light intensity of 50-80 μmol photons m -2 s -1 , photoperiod 12h day: 12h night, and culture under air-filled conditions until the length of the young sporophyte reaches 1 cm, and the PES seawater medium is replaced every 2-3 days.
[0020] The process of inducing somatic cells to reproduce aposporously by tissue culture to produce homozygous 2n female and male gametophytes is as follows:
[0021] The sporophyte with 1 cm of rhizoid removed was cultured in PES seawater medium at a temperature of 17-18 °C and a light intensity of 40-50 mol photons m-2 s -1 , photoperiod 12h day: 12h night; static culture for 1-2 months; and half the amount of PES seawater culture medium was replaced every 15-20 days.
[0022] The 2n female gametophytes are hybridized with n male gametophytes, and the 2n male gametophytes are hybridized with n female gametophytes:
[0023] The 2n gametophytes were mixed with n gametophytes of the opposite sex in equal mass and cultured in PES seawater medium at a temperature of 18°C and a light intensity of 50-80 μmol photons m -2 s -1 , photoperiod: 12h day: 12h night; and PES seawater culture medium was replaced every 3-5 days.
[0024] The PES culture medium is prepared by adding 20 mL of preparation liquid to every 1 L of sterilized seawater; wherein the preparation liquid is prepared by adding 3.5 g of NaNO3, 0.5 g of KH2PO4, 250 mL of iron metal liquid and 250 mL of metal liquid to every 1 L of distilled water; after dissolution and disinfection, the medium is cooled for use; the iron metal liquid is prepared by adding 0.702 g of Fe(NH4)2(SO4)2·6H2O and 0.6 g of Na2EDTA to every 1 L of distilled water; and the metal liquid is prepared by adding 1 g of Na2EDTA, 1.14 g of H3BO3, 49 mg of FeCl3·6H2O, 164 mg of MnSO4·H2O, 22 mg of ZnSO4·7H2O and 4.8 mg of CoSO4·7H2O to every 1 L of distilled water.
[0025] The advantages of the present invention are:
[0026] 1. The present invention induces homozygous 2n gametophytes with the same genotype as the parent sporophyte through homozygous 2n sporophytes. Compared to the complex sex phenotype of heterozygous 2n gametophytes, the homozygous 2n gametophytes of the present invention have a simpler sex phenotype, making them more suitable for obtaining 3n sporophytes through hybridization with n gametophytes of the opposite sex.
[0027] 2. The present invention hybridizes 2n gametophytes with n gametophytes of the opposite sex to obtain 3n sporophytes that are infertile and exhibit the characteristics of late aging and inability to release spores during the breeding season. Sterility has three important advantages: (1) The organism allocates more energy to nutritional growth, which is more conducive to the creation of high-yield, high-quality new varieties; (2) 3n sporophytes cannot be used to directly reproduce the next generation, which is beneficial for the protection of intellectual property rights; (3) During the breeding process, genetic introgression will not cause genetic contamination to nearby wild populations, thus protecting the genetic diversity of wild populations.
[0028] 3. This invention allows the establishment of numerous homozygous 2n gametophyte clones with diverse genotypes. These clones can then be hybridized with various n gametophyte clones to yield numerous 3n hybrid lines, from which superior 3n hybrid lines can be selected. Therefore, using this invention, high-quality new 3n hybrid lines can be screened and obtained within two years, significantly shortening the breeding cycle and improving breeding efficiency.
[0029] 4. The 2n gametophytes induced by the present invention can be continuously expanded and cultured through vegetative growth until the biomass required for seedling cultivation is reached. Therefore, large-scale production of 3n sporophytes can be achieved by hybridizing a large number of 2n gametophytes with n gametophytes.
[0030] 5. The 2n gametophytes induced by the present invention can be stored indoors for a long time, which is particularly beneficial for the long-term maintenance of new varieties in breeding practice. BRIEF DESCRIPTION OF THE DRAWINGS
[0031] Figure 1 This is the technical roadmap of the present invention.
[0032] Figure 2a This is the chromosome ploidy detection diagram of the common 2n sporophyte as a reference;
[0033] Figure 2b This is the chromosome ploidy detection diagram of the sporophyte obtained by hybridizing the homozygous 2n gametophyte with the n gametophyte;
[0034] It can be seen from the figure that the nuclear DNA content of the sporophyte obtained by hybridization of the present invention is 1.5 times that of the 2n sporophyte ( Figure 2b ), proving that they are triploid (3n) sporophytes.
[0035] Figure 3 These are photos of the appearance of three 3n sporophyte hybrid lines after four months of marine culture (mid-December to mid-April of the following year). DETAILED DESCRIPTION
[0036] The specific embodiments of the present invention are further described below with reference to examples. It should be noted that the specific embodiments described here are only for illustrating and explaining the present invention, and are not intended to limit the present invention.
[0037] The present invention selects haploid (n) female gametophyte clones and induces parthenogenesis to obtain homozygous diploid (2n) sporophytes; selects haploid (n) hermaphrodite gametophyte clones and obtains homozygous 2n sporophytes through self-pollination; uses the homozygous 2n sporophytes as materials, and induces aposporous reproduction through tissue culture to produce homozygous 2n female and male gametophytes, respectively; and hybridizes the 2n female gametophytes with n male gametophytes, or hybridizes the 2n male gametophytes with n female gametophytes, to obtain triploid (3n) sporophytes. The relative nuclear DNA content of the hybrid sporophytes is measured using flow cytometry to identify their chromosome ploidy. The 3n sporophytes are then transplanted to sea for cultivation, and their economic traits are measured during the main harvest season. During the kelp seedling (reproduction) season, they are identified as infertile, i.e., unable to release zoospores. This method can be used to cultivate new high-yield and high-quality kelp varieties, and is of great significance for conducting genetic breeding and new variety creation research in kelp and even Laminariales brown algae.
[0038] Example 1
[0039] See also Figure 1 , homozygous diploid (2n) sporophytes were obtained by parthenogenesis of haploid (n) female gametophyte clones and self-fertilization of haploid (n) hermaphroditic gametophyte clones, specifically:
[0040] The female and hermaphrodite gametophytes were cultured in PES seawater medium at a temperature of 18°C and a light intensity of 50 μmolphotons m -2 s -1 The photoperiod was 12 hours day and 12 hours night. The culture was continued until the sporophytes reached 1 mm. The PES seawater medium was replaced every 3 days.
[0041] The young sporophytes were cultured to 1 mm in PES seawater medium at a temperature of 18 °C and a light intensity of 80 μmolphotons m -2 s -1 , a photoperiod of 12h day: 12h night, and air was supplied to culture until the young sporophyte reached 1 cm in length, and the PES seawater medium was replaced every 2 days; thus, homozygous 2n sporophytes were obtained.
[0042] The homozygous 2n sporophyte was used as material to induce aposporous reproduction by tissue culture to produce homozygous 2n female and male gametophytes respectively; specifically:
[0043] After the two 2n sporophytes obtained above were cultured to 1 cm, the rhizoctonia were cut off and the somatic cells were induced to reproduce aposporously by tissue culture. That is, the somatic cells were cultured in PES seawater medium at a temperature of 18°C and a light intensity of 40 mol photons m -2 s -1The cells were cultured under a 12-hour daytime and 12-hour nighttime photoperiod for 2 months, and the PES seawater medium was replaced with half the amount every 15 days to produce homozygous 2n female and male gametophytes.
[0044] 2n female gametophytes are hybridized with n male gametophytes, and 2n male gametophytes are hybridized with n female gametophytes to obtain 3n sporophytes, specifically:
[0045] The above 2n female gametes and n male gametes, and the above 2n male gametes and n female gametes were mixed in equal mass, and incubated in PES seawater medium at a temperature of 18°C and a light intensity of 50 μmol photons m -2 s -1 The cells were cultured statically for 30 days under the conditions of 12 h day and 12 h night photoperiod and changing the culture medium every 3 days until the 3n sporophytes reached more than 1 mm. The cells were then transferred to a 2 L beaker and cultured at 18 °C with a light intensity of 50 μmol photons m -2 s -1 The hybrid sporophytes were cultured under a 12-hour daytime:12-hour nighttime photoperiod, with PES seawater medium replaced every two days and continuously aerated with air until the sporophytes reached 10 cm in length. The relative nuclear DNA content of the hybrid sporophytes was determined by flow cytometry, confirming their chromosome ploidy to be 3n (see Figure 2).
[0046] The 3n sporophytes were then transferred to sea culture (December) and their morphological characteristics were observed in April of the following year (see Figure 3 In June of the following year, although the 3n sporophytes formed sporophylls, microscopic observation revealed that they were unable to release spores and were sterile.
[0047] As shown in Figure 2 above, compared with the common 2n sporophyte ( Figure 2a ) compared to the sporophyte obtained by hybridization, the nuclear DNA content is 1.5 times that of the sporophyte ( Figure 2b ), proving that they are triploid (3n) sporophytes. Figure 3 It can be seen that the three 3n sporophyte lines have a growth period of 2 months less than that of ordinary cultivation varieties (ordinary varieties start cultivation at 10 copies), but their total length and leaf length are longer than those of ordinary varieties, and the leaf pinnae are well developed. They are still in a state of vigorous growth in late April, while ordinary varieties have obviously begun to age.
Claims
1. A method for cultivating triploid sporophytes of Undaria pinnatifida by inducing homozygous diploid gametophytes, characterized in that: The homozygous 2n Undaria sporophyte was induced to reproduce aposporously to produce homozygous 2n gametophyte, which was then hybridized with the normal haploid (n) gametophyte to obtain the Undaria triploid (3n) sporophyte.
2. The method for cultivating triploid sporophytes of Undaria pinnatifida by inducing homozygous diploid gametophytes according to claim 1, characterized in that: Using the young sporophytes produced by parthenogenesis of Undaria pinnatifida as starting materials, homozygous diploid (2n) female gametophytes were induced by tissue culture. Using the young sporophytes produced by self-pollination of hermaphroditic gametophytes as starting materials, homozygous 2n male gametophytes were induced by tissue culture. The homozygous 2n gametophytes obtained above were then hybridized with haploid (n) gametophytes of the opposite sex to obtain triploid (3n) sporophytes of Undaria pinnatifida.
3. The method for cultivating triploid sporophytes of Undaria pinnatifida by inducing homozygous diploid gametophytes according to claim 2, characterized in that: Homozygous diploid (2n) sporophytes were obtained by selecting single-ploid (n) female gametophyte clones of A. truncatum through induced parthenogenesis; Single-fold (n) hermaphroditic gametophyte clones of Azalea were selected and homozygous 2n sporophytes were obtained by self-pollination; The 2n sporophytes obtained above were cultured to a length of 1 cm, the rhizoctonia were cut off, and the somatic cells were induced to reproduce aposporously by tissue culture to produce homozygous 2n female and male gametophytes, respectively; The 2n female gametophytes obtained above were hybridized with n male gametophytes, and the 2n male gametophytes obtained above were hybridized with n female gametophytes to obtain triploid (3n) sporophytes of Undaria pinnatifida.
4. The method for cultivating triploid sporophytes of Undaria pinnatifida by inducing homozygous diploid gametophytes according to any one of claims 1 to 3, characterized in that: The obtained Undaria pinnatifida triploid (3n) sporophyte is sterile, that is, cannot form zoospores.
5. The method for cultivating triploid sporophytes of Undaria pinnatifida by inducing homozygous diploid gametophytes according to claim 3, characterized in that: The process of inducing parthenogenesis and self-pollination of hermaphrodite gametophytes to obtain 2n sporophytes is as follows: The female and hermaphrodite gametophytes were cultured in PES seawater medium at a temperature of 18-20°C and a light intensity of 50-80 μmolphotons m -2 s -1 The photoperiod was 12 hours day and 12 hours night. The culture was continued until the sporophytes reached 1 mm. The PES seawater medium was replaced every 3-5 days. The young sporophytes were cultured to 1 mm in PES seawater medium at a temperature of 18-20 °C and a light intensity of 50-80 μmolphotons m -2 s -1 , photoperiod 12h day: 12h night, and culture under air-filled conditions until the length of the young sporophyte reaches 1 cm, and the PES seawater medium is replaced every 2-3 days.
6. The method for cultivating triploid sporophytes of Undaria pinnatifida by inducing homozygous diploid gametophytes according to claim 3, characterized in that: The process of inducing somatic cells to reproduce aposporously by tissue culture to produce homozygous 2n female and male gametophytes is as follows: The sporophyte with 1 cm of rhizoid removed was cultured in PES seawater medium at a temperature of 17-18 °C and a light intensity of 40-50 molphotons m -2 s -1 , photoperiod 12h day: 12h night; static culture for 1-2 months; and half the amount of PES seawater culture medium was replaced every 15-20 days.
7. The method for cultivating triploid sporophytes of Undaria pinnatifida by inducing homozygous diploid gametophytes according to claim 3, characterized in that: The 2n female gametophytes are hybridized with n male gametophytes, and the 2n male gametophytes are hybridized with n female gametophytes: The 2n gametophytes were mixed with n gametophytes of the opposite sex in equal mass and cultured in PES seawater medium at a temperature of 18°C and a light intensity of 50-80 μmol photons m -2 s -1 , photoperiod: 12h day: 12h night; and PES seawater culture medium was replaced every 3-5 days.
8. The method for cultivating triploid sporophytes of Undaria pinnatifida by inducing homozygous diploid gametophytes according to any one of claims 5 to 7, characterized in that: The PES culture medium is prepared by adding 20 mL of preparation liquid to every 1 L of sterilized seawater; wherein the preparation liquid is prepared by adding 3.5 g of NaNO3, 0.5 g of KH2PO4, 250 mL of iron metal liquid and 250 mL of metal liquid to every 1 L of distilled water; after dissolution and disinfection, it is cooled and set aside for use; the iron metal liquid is prepared by adding 0.702 g of Fe(NH4)2(SO4)2·6H2O and 0.6 g of Na2EDTA to every 1 L of distilled water; and the metal liquid is prepared by adding 1 g of Na2EDTA, 1.14 g of H3BO3, 49 mg of FeCl3·6H2O, 164 mg of MnSO4·H2O, 22 mg of ZnSO4·7H2O and 4.8 mg of CoSO4·7H2O to every 1 L of distilled water.