A method for cultivating kelp seedlings by utilizing diploid female gametophyte parthenogenesis

Abstract

The present invention discloses a method for cultivating kelp seedlings using parthenogenesis of diploid female gametophytes, belonging to the field of genetic breeding in aquatic engineering. Female and male single-cell clones of induced conchoderma are hybridized to obtain tetraploid sporophytes. The tetraploid sporophytes mature and release zoospores, which germinate to form diploid gametophytes. Male and female gametophytes are then separated and batches of diploid female gametophyte clones are obtained. Under the culture conditions provided by the present invention, seedlings are cultivated using parthenogenesis of the diploid female gametophyte clones. These diploid female gametophytes begin to ovulate within 6 to 7 days, significantly earlier than the ovulation time of haploid gametophytes under the same conditions during the same period. The ovulation rate reaches 86.5% on day 12, and the sporophyte conversion rate reaches 98.3% on day 24. Most of the sporophytes have normal morphology. During the seedling cultivation period, the diploid female gametophytes complete parthenogenesis. The diploid female gametophytes can be used as a new kelp germplasm material for kelp seedling cultivation.

Description

Technical Field

[0001] The present invention relates to a method for raising kelp seedlings, and belongs to the field of aquatic engineering genetic breeding. Background Art

[0002] Parthenogenesis refers to a reproductive method in which the eggs produced by the female gametophyte develop directly into sporophytes without fertilization. Parthenogenesis offers unique technical advantages in breeding, such as efficiently fixing superior genotypes, simplifying the breeding process, and precisely controlling offspring traits. The genotype of offspring produced by parthenogenesis is entirely inherited from the maternal parent, allowing for the direct production of individuals with superior traits. The highly consistent genetic background of the offspring facilitates rapid screening for target traits such as disease resistance and stress tolerance. The genotype of parthenogenetic offspring is identical to that of the maternal parent, preventing trait segregation caused by genetic recombination during sexual reproduction through female-male hybridization and ensuring the stable transmission of desirable traits (such as economically desirable traits and stress resistance). Parthenogenesis can fix the homozygous genotype of a superior maternal parent and then cross it with the paternal parent, allowing for the rapid development of new hybrid varieties with outstanding traits. Parthenogenesis also plays a positive role in rescuing endangered species or overcoming reproductive barriers. With the maturation of bioengineering technologies (such as gene editing and epigenetic regulation), parthenogenesis will become an indispensable innovative tool in modern breeding systems.

[0003] China's kelp aquaculture is a massive industry, with nearly all cultivated kelp coming from genetically improved strains. Seedling quality determines product quality and yield, and is fundamental to the sustainable development of the seaweed industry. Early kelp breeding methods typically involved selecting the best strains from multiple generations of self-pollination to create pure lines with relatively stable inheritance of desired traits. In the 1970s, Chinese researchers utilized haploid hybridization and hybrid vigor to develop new varieties. However, these new varieties are often subject to widespread production due to extensive hybridization, resulting in trait variation and loss of desirable traits, ultimately diminishing their value as improved varieties. The severe situation of strain degeneration urgently requires the development of new breeding technologies.

[0004] Laminaria is a large seaweed with a distinct alternation of generations: a haploid gametophyte generation (n) and a diploid sporophyte generation (2n). Laminaria sporophytes release zoospores at maturity, which germinate to produce female and male gametophytes. The female gametophyte ovulates, while the male gametophyte spermates. Fertilization forms a zygote, which develops into a new sporophyte, completing the alternation of generations. Under certain conditions, kelp gametophytes can be transformed into sporophytes through parthenogenesis. However, according to those skilled in the art, the vast majority of sporophytes produced by parthenogenesis of haploid (n) female gametophytes in Laminaria are morphologically deformed and cannot meet the requirements of seedling cultivation.

[0005] For example, a domestic research institute studied parthenogenesis in filamentous female gametophytes of kelp and published the paper "Preliminary Study on Parthenogenesis in Filamentous Female Gametophytes of Kelp (Bai Fengwei et al., Marine Science, Issue 6, 1998)." This paper used haploid female gametophytes for parthenogenesis, but the experiments only covered the initial sporophyte stage, approximately 35 days old. The material was not cultured to seedling size, nor was the morphology of the parthenogenetic sporophyte seedlings normal.

[0006] Chinese invention patent application publication number CN117296702A discloses a "method and application for the cultivation of tetraploid hybrid kelp." This method involves cultivating tetraploid kelp. Diploid filaments of different varieties (lines) of kelp are obtained through tissue culture using young sporophytes. Microscopic selection is performed, and at least 95% of the diploid filaments within the diameter range of 9 μm ≤ φ ≤ 20 μm are selected as female parents. At least 95% of the diploid filaments within the diameter range of 4 μm ≤ φ < 9 μm are selected as male parents. The female and male parents are then hybridized to produce tetraploid hybrid kelp. Mature sporangia of the tetraploid hybrid kelp are then released into zoospores, which are then cultured to produce diploid gametophytes. While the patent discloses the possibility of obtaining diploid gametophytes, the culture conditions only cover developmental conditions and do not disclose the growth conditions of the diploid gametophytes or whether male and female gametophytes can be separated. Summary of the Invention

[0007] The technical problem to be solved by the present invention is to provide a method for cultivating kelp seedlings using parthenogenesis of diploid female gametophytes. First, diploid (2n) kelp male and female gametophytes are obtained in batches. Then, a specific culture medium is used to cultivate 2n female gametophyte parthenogenetic seedlings to obtain morphologically normal sporophyte seedlings.

[0008] The technical solution adopted in the present invention is as follows:

[0009] A method for cultivating kelp seedlings by parthenogenesis using diploid female gametophytes, comprising the following steps:

[0010] (1) Obtaining batches of diploid female gametophytes of Laminaria japonica;

[0011] (1.1) Establishment of diploid induced conchoderma single-cell clones: The induced conchoderma of Laminaria japonica, which is the source of somatic cells, was ground and filtered; the culture medium was incubated at 10 ± 1 °C and 10-20 μmol·m -2 ·s -1 The cells were cultured under white light for 24 hours per day. After 3-5 days, the single cells were aspirated and transferred to a container for culture. The culture medium was kept at 10 ± 1 °C and 25-35 μmol·m -2 ·s -1The cells are cultured under white light for 24 hours per day; when the cell clusters grow to a diameter of 2 to 5 mm, the cell clusters are ground again, collected into another container, and cultured to the desired amount;

[0012] (1.2) Cultivation and offshore aquaculture of tetraploid kelp: A single-cell clone of a phenotypically female Laminaria induced conchocelis was used as the maternal parent, and a single-cell clone of a phenotypically male Laminaria induced conchocelis was used as the paternal parent. The maternal and paternal clones were mixed at a mass ratio of 2:1 and sprayed onto a seedling curtain woven from white vinyl rope. Seedlings were cultivated at an ambient temperature of 8 ± 1°C. When the seedlings reached 1.5–2.0 cm in length, they were temporarily raised at sea and aquacultured by clamping.

[0013] (1.3) Obtaining diploid male and female gametophytes of Laminaria japonica in batches: Take the mature sporangium blocks of the tetraploid Laminaria japonica grown in step (1.2), sterilize and rinse them, and place them in a light incubator at 6-8°C, away from light and dry for 2-5 hours; stop releasing the spores when the density reaches 5-10 per field of view under a 100× microscope, remove the sporangium blocks, and culture them in a light incubator at 10 ± 1°C and 20-30 μmol·m -2 ·s -1 The cells were cultured under white light and 24-hour illumination for 20 to 30 days;

[0014] The females were aspirated under a microscope and transferred to a container for culture at 10 ± 1 °C and 30-40 μmol·m -2 ·s -1 The cells are cultured under white light for 24 hours a day; when the cell segments grow into cell clusters with a diameter of 3 to 5 mm, the cell clusters are ground again, collected into containers and cultured to the required amount.

[0015] Preferably, the culture medium used in step (1.1) is N / P + Fe 3+ culture medium.

[0016] Preferably, the method further comprises the following steps:

[0017] (2) Cultivation of diploid female gametophyte parthenogenetic seedlings of kelp;

[0018] (2.1) Parthenogenesis: Disintegrate the diploid female gametophyte, filter it and culture it in 2% PES medium. The temperature, light intensity and photoperiod should all be 10 ± 1°C, 40-50 μmol photons·M -2 · s -1 , 10L:14D;

[0019] (2.2) Parthenogenetic seedling cultivation: Diploid female gametophytes are used as parents for parthenogenetic seedling cultivation in the seedling cultivation bank. The parthenogenetic seedling cultivation medium is 2% PES medium, and the light source is white light.

[0020] Further preferably, the 2% PES medium is mainly prepared from H3BO3, FeCl3, MnSO4, ZnSO4, CoSO4, EDTA, vitamin B12, vitamin B1, biotin, tris(hydroxymethyl)aminomethane, ferrous ammonium sulfate, sodium nitrate and sodium glycerophosphate.

[0021] More preferably, the 2% PES medium is prepared according to the following steps:

[0022] 1. Preparation of stock solution

[0023] Prepare stock solution 1: 1.9 g H3BO3, 0.05 g FeCl3, 0.273 g MnSO4·H2O, 0.0367 g ZnSO4·7H2O, 0.008 g CoSO4·7H2O, 11.4 mL 0.5 M EDTA solution (pH 8), dissolve in distilled water and dilute to 1000 mL; prepare stock solution 2: 3.35 mg vitamin B12, 165 mg vitamin B1, 1.65 mg biotin, 166.5 g TRIS, dissolve in distilled water and dilute to 500 mL; prepare stock solution 3: 1.7 g ferrous ammonium sulfate hexahydrate, 6.8 mL 0.5 mol / L EDTA solution (pH 8), dissolve in distilled water and dilute to 1000 mL mL; and prepare stock solution 4: weigh 23 g of sodium nitrate and dissolve it in distilled water to 1000 mL; and prepare stock solution 5: weigh 3.33 g of sodium glycerophosphate pentahydrate and dissolve it in distilled water to 1000 mL; and prepare 0.5 M EDTA solution with pH 8: weigh 6.5 g of sodium hydroxide and add it to 75 mL of distilled water, stir until completely dissolved, add 14.6 g of EDTA and continue stirring until completely dissolved, adjust the pH to 8.0 with sodium hydroxide, and dilute the solution to 100 mL with distilled water.

[0024] 2. Preparation of 2% PES medium

[0025] First, prepare PES stock solutions: Dissolve 100 mL of each of stock solutions 1, 3, 4, and 5, and 10 mL of stock solution 2 in distilled water, adjust the pH to 7.8, and dilute to 1000 mL with distilled water. Then, prepare 2% PES medium: Dissolve 2 mL of PES stock solution in autoclaved seawater to 100 mL, and sterilize again for later use.

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] First, the classic haploid gametophyte (n) develops from zoospores undergoing meiosis. Induced conchoceles are formed by inducing somatic cells from the sporophyte using tissue culture techniques. They are diploid conchoceles (2n) structures resembling gametophytes. The present invention hybridizes female and male single-cell clones of the induced conchoceles to produce tetraploid (4n) sporophytes. Upon maturation, the tetraploid sporophyte releases zoospores, which germinate to form diploid (2n) gametophytes. Males and females are then separated to obtain batches of diploid female gametophyte clones. Under the culture conditions provided by the present invention, diploid (2n) female gametophyte clones are used for parthenogenesis and seedling cultivation. These diploid female gametophytes begin ovulating at 6-7 days old, significantly earlier than haploid gametophytes under the same conditions and at the same time. The ovulation rate reaches 86.5% on day 12, and the sporophyte conversion rate reaches 98.3% on day 24. Most sporophytes exhibit normal morphology. During the seedling cultivation period, the diploid female gametophyte completed parthenogenesis. Diploid female gametophytes can be used as a new type of kelp germplasm for kelp seedling cultivation. The successful parthenogenesis of diploid female gametophytes provides a new approach for the cultivation of new kelp varieties.

[0028] The present invention involves a two-stage expansion culture process, the goal of which is to achieve optimal growth at a reasonable growth density and improve the utilization of culture space. Expansion culture is the process of continuously increasing biomass under suitable conditions. Cultivation to the appropriate density refers to culturing to the required amount, allowing stable batch production of gametophyte clones. In the nursery, the present invention completes parthenogenetic seedling cultivation with 2n female gametophytes.

[0029] Second, the present invention establishes diploid induced coniferous phenotype female and phenotype male single cell clones, and then conducts male-female hybridization to form tetraploid kelp. Establishing single cell clones can effectively ensure that there is no contamination from other genotypes, thereby improving the accuracy of seedling cultivation.

[0030] Third, the present invention uses a specially formulated 2% PES medium, which achieves a 91.3% development rate for diploid female gametophytes at 12 days, a peak at 15 days, and a 98.3% sporophyte conversion rate at 24 days. Experiments conducted by the present invention show that the development rate for haploid female gametophytes is only 34.1% at 12 days, peaks at 21 days, and a mere 82.9% sporophyte conversion rate at 24 days.

[0031] In conventional seedling cultivation, N / P + Fe 3+ In the culture medium, the 24-day development rate of diploid female gametophytes is less than 50%, while the 24-day development rate of haploid gametophytes is even lower, less than 7%. Experiments have shown that, under other conditions unchanged, the specially formulated PES culture medium of the present invention is more conducive to the occurrence of female gametophyte parthenogenesis, and the parthenogenetic development rate of diploid female gametophytes is faster than that of haploid female gametophytes.

[0032] Furthermore, when parthenogenetic seedling production is performed on diploid (2n) female gametophyte clones, the present invention utilizes a specially formulated 2% PES medium. These diploid female gametophytes begin ovulating within 6-7 days, earlier than haploid gametophytes under the same conditions and during the same period. The ovulation rate reaches 86.5% by day 12. In the nursery, the diploid female gametophytes complete parthenogenetic seedling production, with the yield and size of seedlings comparable to those from conventional male-female haploid hybrids.

[0033] Fourth, the present invention utilizes diploid female gametophytes for parthenogenesis and uses 2% PES culture medium, which is different from the materials and culture medium in the paper described in the background technology section. Using the optimal conditions in the paper, the sporophyte formation rate was only 60% at 35 days. Using the materials and 2% PES culture medium used in the present invention, the sporophyte formation rate of diploid female gametophytes parthenogenetic reached 98.3% at 24 days, and the sporophyte morphology was normal; while under the same conditions, the sporophyte formation rate of haploid female gametophytes parthenogenetic was only 82.9%, and most of the sporophytes were deformed and could not grow into morphologically normal sporophyte seedlings. The present invention uses diploid female gametophytes for parthenogenesis in 2% PES culture medium, and it took more than two months to obtain morphologically normal sporophyte seedlings, achieving unexpected technical effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 Haploid and diploid female gametophytes reproduce in the conventional N / P + Fe 3+ Schematic diagram comparing the individual development rate over time in the culture medium and the 2% PES culture medium of the present invention. Figure 1 A in the middle corresponds to haploid gametophyte / N / P + Fe 3+ culture medium; Figure 1 B corresponds to the diploid female gametophyte / N / P + Fe 3+ culture medium; Figure 1 Middle C corresponds to haploid gametophytes / 2% PES medium; Figure 1 Middle D corresponds to diploid female gametophytes / 2% PES medium.

[0035] Figure 2 This is a schematic diagram comparing the parthenogenetic development process of haploid and diploid female gametophytes.

[0036] Figure 3 Schematic diagram of the seedling raising situation involved in the examples of the present invention. Figure 3 Middle a corresponds to the diploid female gametophyte parthenogenetic seedlings (experimental group); Figure 3 Middle b corresponds to the seedlings of the haploid male-female gametophyte hybrid combination (control group); Figure 3 Middle c corresponds to parthenogenetic seedlings with haploid female gametophytes (control group). DETAILED DESCRIPTION

[0037] The present invention is further described below with reference to examples and experimental comparison results. It should be noted that the specific embodiments described herein are only for the purpose of illustrating and explaining the present invention, and the scope of protection of the present invention is not limited to these examples.

[0038] The following are examples of specific implementation processes of the present invention:

[0039] 1. Obtaining diploid female gametophytes of kelp in batches

[0040] 1.1 Establishment of single-cell clones of diploid-induced condyles

[0041] The induced conchoderma of Dongfang No. 6 and Dongfang No. 7 kelp, which were used as somatic cell sources, were fully ground using the slide cross-grinding method, filtered through a 300-mesh silk sieve, and the filtrate was allowed to stand for 2 h before the culture medium was replaced. The filtrate was transferred to a culture dish (φ9 cm) and diluted to allow 10-20 cells and cell segments to be observed in each field of view under a 100× microscope. The cells were then incubated at 10°C with 10 μmol·m -2 ·s -1 After 3 days, single cells were gently aspirated with a capillary glass tube under a microscope and placed in a 0.5-1.0 cm 2 The coverslip was placed on the droplet and examined again under a microscope to confirm that only single cells were present. The slide was transferred to a 15 mL test tube and cultured at 10°C with 30 μmol·m -2 ·s -1 The cells were cultured under white light with a 24:0 (L: D) photoperiod, and the culture medium was changed every 30 days. After half a year, the cells grew to a cell mass with a diameter of 2-5 mm. These cell mass clusters were ground again and collected into 100 mL glass Erlenmeyer flasks and cultured for 6 months. The culture conditions remained unchanged, and the culture medium was changed every 15 days. The cells and cell segments increased their biomass through asexual reproduction. The culture medium used throughout the process was N / P + Fe 3+ culture medium.

[0042] This step is used to establish single-cell clones to ensure the same ploidy and the absence of contamination by other genotypes.

[0043] 1.2 Cultivation and offshore growth of tetraploid kelp

[0044] A single-cell clone of the induced coniferous filaments of the phenotypically female Dongfang No. 6 kelp was used as the maternal parent, and a single-cell clone of the induced coniferous filaments of the phenotypically male Dongfang No. 7 kelp was used as the paternal parent. After filtering through a 300-mesh sieve, the parental material was collected and weighed separately. The maternal and paternal clones were mixed in a 2:1 ratio (mass ratio) and sprayed onto seedling screens woven from white vinyl rope with a diameter of 3 mm. A fixed weight of the parental clone was mixed into each seedling screen, depending on the size of the screen. Seedlings were cultivated in a refrigerated nursery (8°C). When the seedlings reached approximately 2 cm in length, they were temporarily raised in the sea and then grown using clamps.

[0045] In this step, the parents used are phenotypically female and phenotypically male induced coniferous single-cell clones as the maternal and paternal parents, which can ensure the accuracy of tetraploid kelp sporophytes.

[0046] 1.3 Obtaining Laminaria japonica diploid male and female gametophytes in batches

[0047] Before the kelp matures, the ploidy of the tetraploid kelp is detected by a third-party flow cytometer, and the relationship between the tetraploid kelp and its parents is identified by molecular biological methods. Take the mature sporangium mass of the tetraploid kelp grown in step 1.2, rinse and wipe it thoroughly with boiled and sterilized and pre-cooled (to 8°C) seawater, soak it in 1.5% potassium iodide solution and double antibody solution (two antibiotics mixed in equal proportions, with a concentration of 80 mg / L) for 15 minutes respectively to sterilize and disinfect, and then rinse with sterilized seawater. Place it in a light incubator at 7°C, avoid light and dry it for 4 hours until there is no moisture on the surface of the sporangium mass. After adding N / P + Fe 3+ The zoospores were released from a culture dish (φ9 cm) filled with culture medium and observed regularly under a microscope (OLYMPUS CX22LED). When the zoospore density reached 5-10 per field of view under a 100× microscope, the release was stopped. The sporangium mass was removed, and the spore solution was filtered through a nylon mesh with a pore size of 40 μm and then distributed into a culture dish (φ9 cm). The spores were cultured in a light incubator at 10°C and 25 μmol·m -2 ·s -1 The culture medium was changed every 10 days under white light with a 24:0 (L:D) photoperiod. The zoospores attached to the embryos, which germinated and developed into diploid gametophytes.

[0048] Under these growth conditions, the diploid gametophytes formed by zoospore development differentiated morphologically, with female gametophytes being stout and male gametophytes being elongated, with obvious morphological differences. Under a microscope (OLYMPUS CX22LED), the female and male gametophytes were gently aspirated using a capillary glass tube (the male gametophyte was removed for later use as a control for sex identification) and placed in a 0.5-1.0 cm 2The coverslip was placed on the plate and examined again under a microscope to confirm that only a single cell segment existed in the droplet. The slide was transferred to a 15 mL test tube and cultured at 10°C and 35 μmol·m -2 ·s -1 The cells were cultured under white light with a 24:0 (L: D) photoperiod, and the culture medium was fully replaced every 30 days. After about 6 months of culture, the cell segments grew into cell clusters with a diameter of about 5 mm. These cell clusters were ground again and collected into 100 mL glass triangular flasks and cultured to the required amount. The culture conditions remained unchanged and the culture medium was replaced every 30 days. These cell segments increased their biomass through asexual reproduction. The culture medium used throughout the process was N / P + Fe 3+ Culture medium. Diploid gametophytes were now obtained in batches. PCR amplification with sex-specific primers was used to identify the genetic sex of the diploid female or male gametophytes, and the results were all single-sex, consistent with the phenotypic sex.

[0049] The growth conditions of the present invention are more conducive to the formation of gametophyte clones. This involves two stages of expansion: the first stage is 15mL test tube expansion, and the second stage is 100 Erlenmeyer flask expansion. The goal is to achieve optimal growth at a reasonable growth density and improve the utilization of culture space. Expansion is the process of continuous increase in biomass under appropriate conditions. Cultivation to the appropriate density means culturing to the required amount, which can stably obtain gametophyte clones in batches.

[0050] 2. Cultivation of kelp 2n female gametophyte parthenogenetic seedlings

[0051] 2.1 Parthenogenesis Experiment

[0052] The haploid (n) female gametophytes of Oriental No. 6 were used as the control group, and the diploid (2n) female gametophytes obtained in step 1.3 were used as the experimental group. The n and 2n female gametophytes were broken up by cross-grinding with a glass slide, and filtered into the culture medium using a nylon mesh with a pore size of 40 μm. Cell suspensions of 2 to 5 cells were obtained, and the density was adjusted to 20 to 30 cell segments per field of view under a 100× microscope. The suspensions were inoculated into culture dishes (φ9 cm) and replicated three times. The control and experimental groups were treated with N / P + Fe 3+ The culture medium was replaced every 3 days with 2% PES seawater culture medium. The light source for both the control and experimental groups was provided by a 21W white light emitting diode, and the light quantum flux density was set to 45 μmol photons·M -2 · s -1The temperature and photoperiod were 10°C, 10 L:14 D. Observation and imaging were performed every three days using an OLYMPUS 1X51 inverted microscope. Ten 100× fields of view were randomly observed in each dish, and the percentage of three developmental stages (vegetative body, egg release, and sporophyte formation) in the female group was counted. Developmental rate was calculated as the sum of the number of ovulated eggs and sporophytes relative to the total cell count.

[0053] The following is an example of a comparative experiment of the present invention:

[0054] The present invention is to treat haploid and diploid female gametophyte parthenogenesis in conventional N / P + Fe 3+ A comparative experiment was conducted on the individual development rate of the culture medium and the 2% PES culture medium of the present invention over time. The results are as follows Figure 1 .

[0055] Depend on Figure 1 It can be seen that in the 2% PES medium of the present invention, the diploid gametophytes ( Figure 1 Middle D) The development rate reached 91.3% on day 12, reached the extreme value on day 15, and the sporophyte conversion rate reached 98.3% on day 24. Figure 1 Middle C) The development rate was only 34.1% on day 12, and reached its peak on day 21. The sporophyte conversion rate was 82.9% on day 24. 3+ In the culture medium, diploid female gametophytes ( Figure 1 Middle B) The parthenogenetic development rate is less than 50%, while the haploid gametophyte ( Figure 1 The development rate of medium (A) is even lower, less than 7%. Experiments have shown that, under other conditions remaining unchanged, 2% PES medium is more conducive to the occurrence of female gametophyte parthenogenesis.

[0056] Furthermore, in N / P + Fe 3+ On the 24th day of culture, the haploid female gametophytes rarely ovulate and transform into sporophytes, and most of the branches grow into vegetative filaments ( Figure 1 In A); in N / P + Fe 3+ At 24 days in culture, the diploid female gametophyte parthenogenesis showed significant differences in ovulation and sporophyte transformation ( Figure 1 Middle B) is higher than haploid female gametophyte parthenogenesis ( Figure 1 Middle A); Ovulation and transsporophyte rates of haploid female gametophytes at 24 days in 2% PES medium ( Figure 1 C) is much higher than that in N / P + Fe 3+ Culture medium ( Figure 1 A) in the middle; the rate of parthenogenesis and ovulation of diploid female gametophytes and the rate of transsporophyte in 2% PES medium ( Figure 1D) were higher than those of haploid female gametophyte reproduction ( Figure 1 C), and also higher than the N / P + Fe 3+ The situation in the culture medium ( Figure 1 Middle B).

[0057] The present invention conducted a comparative experiment on the parthenogenetic development process of haploid and diploid female gametophytes, and the results are shown in Figure 2 .

[0058] Depend on Figure 2 It can be seen that after 7 days of culture in 2% PES medium, the diploid gametophyte has ovulated, while the haploid gametophyte has not yet ovulated. On the 30th to 40th day of culture, most of the diploid gametophyte parthenogenetic sporophytes have normal morphology and continue to grow, while most of the haploid gametophyte parthenogenetic sporophytes are deformed. In 2% PES medium, diploid gametophyte parthenogenetic ovulation is earlier than haploid female gametophyte, and the young sporophyte has normal morphology. Haploid parthenogenetic can be transformed into sporophyte, but most of the sporophytes are deformed. The comparison of development rates during the same period shows that the diploid gametophyte parthenogenetic development speed is faster. At the same time, the experiment confirms that the use of 2% PES medium for parthenogenetic seedling cultivation is faster than the conventional N / P + Fe 3+ The culture medium can improve the sporophyte transformation rate.

[0059] 2.2 Parthenogenetic seedling cultivation

[0060] Parthenogenetic reproduction of female gametophytes of Dongfang No. 6 (n-FG) and the combination of female gametophytes of Dongfang No. 6 and male gametophytes of Dongfang No. 7 (n-FG×n-MG) were used as controls. Diploid (2n) female gametophytes were used as parents for parthenogenetic reproduction in the seedling bank. The seedlings were raised in the seedling bank according to the method and conditions for the hybrid male-female combination of Li et al. (2007). Laminaria longissima (Laminariales,Phaeophyta) and a female one of L. japonica J Appl phycol 19:139-151), the parthenogenetic culture medium was 2% PES medium, and the combined culture medium was conventional N / P + Fe 3+Culture medium and light source are all white light.

[0061] Depend on Figure 3 It can be seen that after about 2 and a half months of cultivation, the number of 2n gametophyte parthenogenetic seedlings ( Figure 3 Middle a) and the conventional combination of Dongfang No. 6 kelp female gametophyte × Dongfang No. 7 kelp male gametophyte ( Figure 3 The emergence of seedlings in b) is similar, while the haploid female gametophyte of Dongfang No. 6 reproduces parthenogenetically ( Figure 3 In the nursery room, parthenogenetic seedlings from 2n female gametophytes were successfully cultivated.

[0062] The following are examples of the method for preparing the culture medium in the examples of the present invention.

[0063] N / P + Fe 3+ The culture medium was prepared as follows:

[0064] (1) Weigh 121 g of sodium nitrate, dissolve it in sterile seawater, and then continue to dilute the volume to 500 mL with sterile seawater to obtain a sodium nitrate solution.

[0065] (2) Weigh 17.5 g of potassium dihydrogen phosphate, dissolve it in sterile seawater, and then dilute the volume to 1000 mL with sterile seawater to obtain a potassium dihydrogen phosphate solution.

[0066] (3) Weigh 3 g of ferric citrate pentahydrate, dissolve it in distilled water, let it cool to room temperature, and then dilute it to 1000 mL with distilled water to obtain a ferric citrate solution.

[0067] (4) 1 mL of the sodium nitrate solution, 1 mL of the potassium dihydrogen phosphate solution, and 4 mL of the ferric citrate solution were respectively added to 4000 mL of sterilized seawater and shaken to obtain the N / P + Fe 3+ culture medium.

[0068] The preparation method of 2% PES culture medium of the present invention is as follows:

[0069] 1. Preparation of stock solution

[0070] (1) Preparation of stock solution 1: Dissolve 1.9 g of H3BO3, 0.05 g of FeCl3, 0.273 g of MnSO4·H2O, 0.0367 g of ZnSO4·7H2O, 0.008 g of CoSO4·7H2O, and 11.4 mL of EDTA solution (0.5 M, pH 8) in distilled water and adjust the volume to 1000 mL.

[0071] (2) Preparation of stock solution 2: Weigh vitamin B12 (Vitamin B 12) 3.35 mg, vitamin B1 (Thiamine) 165 mg, biotin (Biotin) 1.65 mg, tris (hydroxymethyl) aminomethane (TRIS) 166.5 g, dissolve in distilled water and make up to 500 mL.

[0072] (3) Preparation of stock solution 3: Weigh 1.7 g of ammonium ferrous sulfate hexahydrate and 6.8 mL of EDTA solution (0.5 mol / L, pH 8), dissolve them in distilled water and adjust the volume to 1000 mL.

[0073] (4) Preparation of stock solution 4: Weigh 23 g of sodium nitrate and dissolve it in distilled water to make up to 1000 mL.

[0074] (5) Preparation of stock solution 5: Weigh 3.33 g of sodium glycerophosphate pentahydrate and dissolve it in distilled water to make up to 1000 mL.

[0075] (6) Preparation of EDTA solution (0.5 M, pH 8): Weigh 6.5 g of sodium hydroxide, add it to 75 mL of distilled water, and stir until completely dissolved. Add 14.6 g of EDTA and continue stirring until completely dissolved. Adjust the pH to 8.0 with sodium hydroxide and dilute the solution to 100 mL with distilled water.

[0076] The prepared stock solutions were sterilized with high pressure steam and stored at 4°C in glass bottles. Stock solution 2 was stored in brown bottles.

[0077] 2. Preparation of 2% PES medium

[0078] (1) Preparation of PES stock solution: Take 100 mL of stock solution 1, stock solution 3, stock solution 4, stock solution 5 and 10 mL of stock solution 2, dissolve them in distilled water, adjust the pH to 7.8, and make up to 1000 mL with distilled water.

[0079] (2) Preparation of 2% PES culture medium: Take 2 mL of PES stock solution and dissolve it to 100 mL with high-pressure steam sterilized seawater. Then filter and sterilize it again with a 0.22 μm needle filter.

Claims

1. A method for cultivating kelp seedlings by parthenogenesis using diploid female gametophytes, characterized in that Here are the steps: (1) Obtaining batches of diploid female gametophytes of Laminaria japonica; (1.1) Establishment of diploid induced conchoderma single-cell clones: The induced conchoderma of Laminaria japonica, which is the source of somatic cells, was ground and filtered; the culture medium was incubated at 10 ± 1 °C and 10-20 μmol·m -2 ·s -1 The cells were cultured under white light for 24 hours per day. After 3-5 days, the single cells were aspirated and transferred to a container for culture. The culture medium was kept at 10 ± 1 °C and 25-35 μmol·m -2 ·s -1 The cells are cultured under white light for 24 hours per day; when the cell clusters grow to a diameter of 2 to 5 mm, the cell clusters are ground again, collected into another container, and cultured to the desired amount; (1.2) Cultivation and offshore aquaculture of tetraploid kelp: A single-cell clone of a phenotypically female Laminaria induced conchocelis was used as the maternal parent, and a single-cell clone of a phenotypically male Laminaria induced conchocelis was used as the paternal parent. The maternal and paternal clones were mixed at a mass ratio of 2:1 and sprayed onto a seedling curtain woven from white vinyl rope. Seedlings were cultivated at an ambient temperature of 8 ± 1°C. When the seedlings reached 1.5–2.0 cm in length, they were temporarily raised at sea and aquacultured by clamping. (1.3) Obtaining diploid male and female gametophytes of Laminaria japonica in batches: Take the mature sporangium blocks of the tetraploid Laminaria japonica grown in step (1.2), sterilize and rinse, and place them in a light incubator at 6-8°C, away from light and dry for 2-5 hours; stop releasing the gametospores when the density reaches 5-10 per field of view under a 100× microscope, remove the sporangium blocks, and place them in a light incubator for incubation at 10 ± 1 °C and 20-30 μmol·m -2 ·s -1 The cells were cultured under white light and 24-hour illumination for 20 to 30 days; The females were aspirated under a microscope and transferred to a container for culture at 10 ± 1 °C and 30-40 μmol·m -2 ·s -1 The cells are cultured under white light for 24 hours per day; when the cell segments grow to cell clusters with a diameter of 3 to 5 mm, the cell clusters are ground again, collected into containers, and cultured to the required amount; (2) Cultivation of diploid female gametophyte parthenogenetic seedlings of kelp; (2.1) Parthenogenesis: Disintegrate the diploid female gametophyte, filter it and culture it in 2% PES medium; The temperature, light intensity, and photoperiod were all 10±1°C, 40-50 μmol photons·M -2 · s -1 , 10L:14D; (2.2) Parthenogenetic seedling cultivation: Diploid female gametophytes are used as parents for parthenogenetic seedling cultivation in a seedling cultivation bank. The parthenogenetic seedling cultivation medium is 2% PES medium, and the light source is white light. The 2% PES medium was prepared according to the following steps:

1. Preparation of stock solution Prepare stock solution 1: 1.9 g H3BO3, 0.05 g FeCl3, 0.273 g MnSO4·H2O, 0.0367 g ZnSO4·7H2O, 0.008 g CoSO4·7H2O, 11.4 mL 0.5 M EDTA solution (pH 8), dissolve in distilled water and dilute to 1000 mL; prepare stock solution 2: 3.35 mg vitamin B12, 165 mg vitamin B1, 1.65 mg biotin, 166.5 g TRIS, dissolve in distilled water and dilute to 500 mL; prepare stock solution 3: 1.7 g ferrous ammonium sulfate hexahydrate, 6.8 mL 0.5 mol / L EDTA solution (pH 8), dissolve in distilled water and dilute to 1000 mL; prepare stock solution 4: 23 g sodium nitrate, 100 mg ... g, dissolve in distilled water and make up to 1000 mL; prepare stock solution 5: weigh 3.33 g sodium glycerophosphate pentahydrate, dissolve in distilled water and make up to 1000 mL; prepare 0.5M EDTA solution with pH 8: weigh 6.5 g sodium hydroxide, add to 75 mL distilled water, stir until completely dissolved, add 14.6 g EDTA, continue stirring until completely dissolved, adjust the pH to 8.0 with sodium hydroxide, and make up to 100 mL with distilled water.

2. Preparation of 2% PES medium First, prepare PES stock solutions: Dissolve 100 mL of each of Stock Solution 1, Stock Solution 3, Stock Solution 4, and Stock Solution 5, as well as 10 mL of Stock Solution 2, in distilled water, adjust the pH to 7.8, and dilute to 1000 mL with distilled water. Then, prepare 2% PES medium: Dissolve 2 mL of PES stock solution in autoclaved seawater to 100 mL, and sterilize again for later use.

2. The method for cultivating kelp seedlings by parthenogenesis using diploid female gametophytes as claimed in claim 1, wherein: The culture medium used in step (1.1) is N / P + Fe 3+ culture medium.

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