A method for culturing asparagus aphyllus spore

Abstract

This invention belongs to the technical field of marine algae cultivation methods, specifically relating to a method for cultivating carposporophytes of *Asparagus cylindrica*. This invention discloses for the first time a method for cultivating carposporophytes of *Asparagus cylindrica*, comprising the following steps: (1) field collection; (2) algal body treatment; (3) induction of spore release; (4) germination and cultivation of carposporophytes; and (5) cultivation of carposporophytes. Research has shown that preserving the collected female gametophytes in low-temperature seawater is beneficial for subsequent spore induction; conversely, drying and rehydrating the collected female gametophytes resulted in spore failure. The release of the obtained spores and the subsequent cultivation of carposporophytes require specific culture media, photoperiods, and light intensities to achieve better spore release and carposporophyte growth.

Description

Technical Field

[0001] This invention belongs to the technical field of marine algae cultivation methods. More specifically, it relates to a method for culturing the sporophytes of *Asparagus officinalis*. Background Technology

[0002] Asparagopsis taxiformis is a red alga with unique biological characteristics and economic value. Its carposporophyte, as a crucial stage in reproduction, is of great significance for the preservation, propagation, and subsequent aquaculture research of its germplasm resources. However, the induction and further cultivation of Asparagopsis taxiformis carposporophytes face numerous challenges. The optimal culture conditions remain unclear, making it difficult to meet the needs of large-scale cultivation. Asparagopsis taxiformis carposporophytes have high requirements for culture conditions, including light intensity, photoperiod, temperature, salinity, and nutrient concentration. Inappropriate culture conditions can lead to slow sporophyte growth, high mortality, and even failure to complete the life cycle. These difficulties limit in-depth research and the development of its potential applications.

[0003] While some studies have reported on algal spore release and cultivation, methods for cultivating *Asparagus officinalis* sporophytes are still incomplete. For example, Chinese patent application CN110106134A discloses a method for releasing green algal spores, emphasizing that drying and rehydrating under dark conditions improves spore release rates. However, this method is not suitable for cultivating *Asparagus officinalis* sporophytes. Additionally, Chinese patent application CN105684879A discloses a method for suspending *Chlorophytum comosum*, which includes steps such as field sampling, algal body treatment, spore release, spore collection, and algal seedling suspension cultivation. Although this method has achieved some success in *Chlorophytum comosum* cultivation, due to the differences in biological characteristics and cultivation requirements between *Asparagus officinalis* and *Chlorophytum comosum*, it is not suitable for cultivating *Asparagus officinalis* sporophytes.

[0004] Therefore, there is an urgent need to provide an effective method to improve the release and germination of sporophytes of *Asparagus setaceus*. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to overcome the shortcomings and deficiencies of existing methods for releasing and culturing sporophytes of *Asparagus officinalis*, which are still in the initial stage and whose sporophyte release rate and germination rate need to be improved. The present invention provides a method for culturing sporophytes of *Asparagus officinalis*.

[0006] The above-mentioned objective of this invention is achieved through the following technical solution:

[0007] This invention protects a method for culturing the sporophyte of *Asparagus officinalis*, comprising the following steps:

[0008] S1. Field collection: Collect female gametophytes with mature carposporangia and preserve the obtained female gametophytes in seawater at a temperature 5°C lower than the air temperature;

[0009] S2. Algal treatment: Remove the epiphytes from the female gametophytes obtained in step S1;

[0010] S3. Inducing Spore Release: The female gametophytes obtained in step S2 (with epiphytes removed) were placed in sterilized seawater and incubated at a temperature of 18–22°C, a photoperiod of 12L:12D, and a light intensity of 20–40 μmol photons / m². -2 s -1 Cultured under specific conditions to induce the release of carpospores from female gametophytes;

[0011] S4. Germination and culture of carpospores: Collect the carpospores obtained in step S3 and transfer them to 1 / 2 PES medium. Incubate at a temperature of 18–22℃, a photoperiod of 12L:12D, and a light intensity of 20–40 μmol photons / m². -2 s -1 Under the conditions of cultivation, carposporophytes were obtained;

[0012] S5. Culture of carposporophytes: The carposporophytes obtained in step S3 were transferred to fresh 1 / 2 PES medium and cultured at a temperature of 18–22℃, a photoperiod of 12L:12D, and a light intensity of 20–40 μmol photons / m². -2 s -1 Under specific conditions, after the carposporophyte length reached ≥0.5 cm, it was transferred for large-scale culture. The carposporophyte density was set at 0.8–1.2 g / L, the temperature at 18–22℃, the photoperiod at 12 L:12 D, and the light intensity at 140–330 μmol photons / m². -2 s -1 The culture medium was 1 / 2 PES medium, and the culture was continued.

[0013] A carposporophyte is a specialized filamentous spore-bearing structure observed in true red algae. For example, in most species of the Nemalionales order, after fertilization, cells branching from the fertilized nucleus elongate to form a group of short, filamentous cells. All or only the apical cells of these cells transform into carpospores, which then detach from the parent plant. This filamentous structure is the carposporophyte. The purpose of these stages is to obtain a stable source of carposporophytes and to acquire as many carposporophytes as possible. Through carposporophyte culture, asexual reproduction of the plant can be achieved later, rapidly obtaining a large number of plants.

[0014] Collecting carposporangia from the wild to cultivate carpospores, then inducing carposporophyte cultivation, and finally cultivating the carposporophyte into gametophyte seedlings in a nursery before transplanting them back into the wild, thus achieving full life-cycle cultivation of red algae, is a feasible technical path for the commercial production of red algae. However, each stage of algal growth is affected by a combination of factors, including light intensity, photoperiod, and temperature, and different algal species respond differently to these factors. For example, some mid-tidal algae require desiccation stress to promote spore release (such as *Porphyra yezoensis*), while some subtidal algae die after prolonged desiccation (such as *Solanum lyratum*); some algae are demanding in terms of light conditions, and even slight fluctuations in light can lead to death; some are particularly sensitive to nutrient concentrations, requiring low or high salt concentrations to grow, thus making it impossible to refer to the cultivation conditions of other algae for the cultivation of *Asparagus cochinchinensis* carposporophytes. Therefore, it is essential to explore suitable culture conditions for the carposporophytes of *Asparagus purpurea* to maximize their formation, development, and growth, which is crucial for achieving large-scale seaweed cultivation.

[0015] Furthermore, in step S2, the disinfected seawater is the filtered and boiled seawater, which is FSW.

[0016] Furthermore, in step S2, the salinity of the disinfected seawater is 28–32 ppt. Salinity can be controlled by adding water (such as deionized water, distilled water, or other non-seawater sources) or artificial seawater, and monitored with a salinity meter.

[0017] Artificial seawater, also known as man-made seawater, refers to a mixed salt solution used to raise marine animals or to preserve their organs and tissues in a normal state. The preparation of artificial seawater is a conventional, existing technology.

[0018] Preferably, in step S2, the salinity of the disinfected seawater is 32 ppt.

[0019] Furthermore, the full name of the PES medium is Provasoli's ES Medium, or simply PES medium, which is a commonly used culture medium for seaweed cultivation.

[0020] The PES medium formulation is as follows:

[0021] Mother liquor N: NH4NO3, 23.5 g / L;

[0022] Mother liquor P: NaH2PO4·2H2O 3.89 g / L;

[0023] Mother liquor II: Fe(NH4)2(SO4)·6H2O 0.701 g / L, Na2EDTA 0.66 g / L;

[0024] Mother liquor III: Na2EDTA 1.00g / L, H3BO3 1.14g / L, FeCl3·6H2O 0.049g / L, MnCl2·4H2O0.146g / L, ZnSO4·7H2O 0.0022g / L, CoCl2·6H2O 0.004g / L.

[0025] The above four stock solutions are mixed in a ratio of 1.4:1.4:5.0:5.0 to prepare a total stock solution. Then, add 10 mL of the total stock solution to 1 L of disinfected seawater to prepare a PES solution. 1 / 2 PES is prepared by adding 5 mL of the total stock solution to 1 L of disinfected seawater. 1 / 4 PES is prepared by adding 2.5 mL of the total stock solution to 1 L of disinfected seawater.

[0026] Furthermore, the salinity of the 1 / 2 PES culture medium is 28–32 ppt, preferably 32 ppt.

[0027] Furthermore, the mature carposporangia are red in color (dark reddish-brown, slightly purplish-red, etc. are all classified as red), spherical in shape, and 0.4-0.6 mm in diameter.

[0028] Furthermore, in step S2, the epiphyte that removes the female gametophyte obtained in step S1 is the female gametophyte that has been rinsed with naturally disinfected seawater.

[0029] Preferably, in step S3, the temperature for inducing spore release is 19–21°C, more preferably 20°C.

[0030] Preferably, in step S3, the light intensity for inducing spore release is 22–30 μmol photons / m². -2 s -1 More preferably, 24 μmol photons m -2 s -1 Light conditions below or above the aforementioned range will result in poor release or even no release at all.

[0031] Preferably, in step S4, the temperature for germination and cultivation of the carpospores is 19–21°C, more preferably 20°C.

[0032] Preferably, in step S4, the light intensity for the germination and cultivation of the carpospores is 22–30 μmol photonsm. -2 s -1 More preferably, 24 μmol photons m -2 s -1 .

[0033] Preferably, in step S5, before expanding the culture, the temperature for culturing the caryophytes is 19–21°C, more preferably 20°C.

[0034] Preferably, in step S5, before scaling up the culture, the light intensity for culturing the carposporophytes is 22–30 μmol photons / m². -2 s -1 More preferably, 24 μmol photons m -2 s -1 .

[0035] Preferably, in step S5, after the expansion culture, the density of the carposporocytes is 0.9–1.1 g / L, more preferably 1 g / L. This density refers to the wet weight of the carposporocytes divided by the volume of the culture medium, where the wet weight refers to the mass of the carposporocytes when they normally contain water, generally referring to the mass after wiping off surface moisture.

[0036] Preferably, in step S5, after the expansion culture, the light intensity for the carposporophyte culture is 140–200 μmol photons / m². -2 s -1 .

[0037] More preferably, in step S5, after the expansion culture, the light intensity for the carposporophyte culture is 150 μmol photons / m². -2 s -1 .

[0038] Furthermore, in step S5, after the expansion culture is carried out, the culture medium is changed once a week.

[0039] Furthermore, the expanded culture involves using a larger culture container, including flasks, and the appropriate size of the culture container can be selected according to the actual situation.

[0040] Compared with the prior art, the present invention has the following beneficial effects: The present invention discloses for the first time a method for culturing carposporophytes of *Asparagus setaceus*, comprising the following steps: (1) field collection, (2) algal body treatment, (3) induction of spore release, (4) germination and culture of carposporites, and (5) carposporophyte culture. The present invention has found that preserving the collected female gametophytes in low-temperature seawater is beneficial for subsequent spore induction; conversely, drying and rehydrating the collected female gametophytes resulted in spores not being released. The release of the obtained spores and the subsequent culture of carposporophytes require specific culture medium, photoperiod, and light intensity to achieve better spore release and carposporophyte growth effects. Attached Figure Description

[0041] Figure 1This is a statistical graph showing the release rate of Asparagus purpureus spores under different photoperiods and culture medium conditions. Different letters above the columns represent significant differences; success indicates the percentage of gametophytes that released ≥50 spores out of the total number of gametophytes, and failure indicates the percentage of gametophytes that released <50 spores out of the total number of gametophytes; the value n below the column, such as n=17, indicates that the total number of gametophytes to be tested is 17.

[0042] Figure 2 This is a statistical graph showing the germination rate of *Asparagus purpurea* spores under different culture media. Different letters above the columns represent significant differences.

[0043] Figure 3 This is a statistical graph showing the changes in carposporophyte growth rate under different treatments. The three density groups were analyzed for significance separately. At the same density, different letters above the bars represent significant differences. The three numbers above are p-values, indicating the results of the significance comparison of the average SCG values ​​of the three different density groups. For example, 0.95 means p(0.5, 2) = 0.95, indicating no significant difference in the average SCG values ​​between the 0.5 g / L and 2 g / L density groups; 0.00034 means p(0.5, 1) = 0.00034, indicating a significant difference in the average SCG values ​​between the 0.5 g / L and 1 g / L groups; and 9.5e-7 means p(1, 2) = 9.5e-7, indicating an extremely significant difference in the average values ​​between the 1 g / L and 2 g / L groups. Detailed Implementation

[0044] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0045] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0046] Provasoli's ES Medium (PES) formulation:

[0047] Mother liquor N: NH4NO3, 23.5 g / L;

[0048] Mother liquor P: NaH2PO4·2H2O 3.89 g / L;

[0049] Mother liquor II: Fe(NH4)2(SO4)·6H2O 0.701 g / L, Na2EDTA 0.66 g / L;

[0050] Mother liquor III: Na2EDTA 1.00g / L, H3BO3 1.14g / L, FeCl3·6H2O 0.049g / L, MnCl2·4H2O

[0051] 0.146g / L, ZnSO4·7H2O 0.0022g / L, CoCl2·6H2O 0.004g / L.

[0052] The above four stock solutions are mixed in a ratio of 1.4:1.4:5.0:5.0 to prepare a total stock solution. Then, add 10 mL of the total stock solution to 1 L of disinfected seawater to prepare a PES solution. To prepare 1 / 2 PES, add 5 mL of the total stock solution to 1 L of disinfected seawater. To prepare 1 / 4 PES, add 2.5 mL of the total stock solution to 1 L of disinfected seawater to prepare a 1 / 4 PES solution.

[0053] Disinfected seawater is seawater that has been filtered and then boiled, also known as FSW.

[0054] Example 1: Method for inducing the release of spores from *Asparagus officinalis*

[0055] 1. Experimental Methods

[0056] The method for inducing the release of spores from *Asparagus purpurea* includes the following steps:

[0057] S1. Field Sampling: *Asparagus purpureus* grows in the low-tide to subtidal zone. During sampling, female gametophytes with mature cysts are identified by their color (pink), shape (spherical), and size (approximately 0.5 mm). The female gametophytes are then plucked from the stolons. During transport, the female gametophytes are placed in seawater at a temperature 5°C lower than the air temperature to prevent premature spore release due to temperature increases.

[0058] S2. Algal Treatment: To eliminate the influence of epiphytes on cystospore release, the female gametophytes collected in step S1 were placed in a 2L polyethylene beaker and rinsed 10 times with 2L of sterilized natural seawater. After the female gametophytes settled naturally, the seawater was poured out one by one, which removed almost all epiphytes. The female gametophytes were then transferred to a clean petri dish containing sterilized seawater, and any remaining epiphytes were removed with tweezers under an inverted microscope. During the transfer, the ends of the female gametophytes were held with tweezers, avoiding contact with the cystospore distribution area to prevent damage to the cystospores.

[0059] S3. Spore Release and Counting: Individual gametophytes were transferred to 100 mm culture dishes containing sterilized seawater (FSW, filtered and boiled seawater, salt concentration 32 ppt), 1 / 2 PES medium (prepared from sterilized seawater and PES medium, salt concentration 32 ppt), and 1 / 4 PES medium (salt concentration 32 ppt), respectively. Two photocycles were set: 12 L: 12 D and 8 L: 16 D. The light conditions were 24 μmol photons / m². -2 s -1 After being treated in darkness, the release of carpospores from the gametophytes was observed and recorded every hour using an inverted microscope.

[0060] Because the number of cysts per gametophyte varies, the theoretical spore release data from a gametophyte exhibits significant variability. Therefore, a successful spore release event is defined as "a single gametophyte releasing ≥50 spores," and the opposite is considered an unsuccessful event. The number of successful spore release events under each treatment is counted and divided by the total number of events under a single treatment, representing the proportion of successful spore release from gametophytes under each treatment. A chi-square test is used to examine whether there are significant differences in the proportion of successful spore release under different treatments.

[0061] 2. Experimental Results

[0062] The spore release under each treatment was statistically analyzed, and the results are shown below:

[0063] (1) The peak of spore release occurs around 3 hours after exposure to light, lasts for 3-5 hours, and there is no spore release after 30 hours.

[0064] (2) The release of spores from gametophytes under different culture media and photoperiods was statistically analyzed, and the results are as follows: Figure 1 As shown, the spore release was best in the 12L:12D photoperiod treatment group using FSW as the initial medium, with each gametophyte releasing more than 50 spores (100%). This was followed by the FSW and 8L:16D treatment group (67%), then the 1 / 4 PES medium and 12L:12D treatment group (50%) and the 1 / 4 PES medium and 8L:16D treatment group (25%). Neither treatment group using 1 / 2 PES medium as the initial medium released spores under either of the above two photoperiods. This may be because higher nutrient levels inhibit spore release.

[0065] It is evident that FSW and a photoperiod of 12L:12D can effectively induce spore release.

[0066] Example 2 Germination and culture of *Asparagus purpurea* spores

[0067] Using a 1 mL sterile pipette, transfer the released spores to petri dishes containing FSW, 1 / 4 PES (PES / 4), 1 / 2 PES (PES / 2), and PES, respectively. Place 2-3 glass slides at the bottom of the petri dishes for spore attachment, ensuring the transferred spores are evenly distributed on the slides. At 20℃, 12L:12D, 24μmol photons m -2 s -1 Cultured under suitable conditions, with the culture medium changed weekly. Observe the germination of spores under a microscope:

[0068] (1) Before germination, the spores swell to almost twice their original size, and after germination they divide into multicellular carpophytes.

[0069] (2) Spores generally germinate within 3 days. Slides that do not germinate within 3 days or are heavily contaminated should be discarded.

[0070] (3) Divide the number of germinating spores by the number of spores on the glass slide to obtain the germination probability of spores under different culture media. Figure 2 The results showed that 1 / 2 PES had the highest germination rate (64.3%), followed by PES (41.2%), 1 / 4 PES (37.5%), and FSW (12.5%).

[0071] It is evident that 1 / 2 PES can effectively promote spore germination.

[0072] Example 3: Culture of caryophytes

[0073] The slides with attached carposporocytes were transferred to new petri dishes and cultured under the original conditions, with weekly transfers to new petri dishes. Once the carposporocytes on the slides reached a length of at least 0.5 cm, they were detached from the slides using a sterile blade and transferred to flasks for further culture. The culture medium was 1 / 2 PBS, and the culture temperature was 20℃. Experimental optimization of the culture conditions was conducted. Three initial carposporocyte culture densities (0.5, 1, and 2 g / L (wet weight)) and corresponding density values ​​(24, 44, 96, 150, 220, 330, and 510 μmol photons m) were established. -2 s -1 Seven light cycles were applied in 500mL flasks with a photoperiod of 12L:12D for 7 days. After cultivation, the culture medium was filtered through a 200μm sieve to obtain carposporophytes. The carposporophytes were dried and weighed. The specific growth rate (SGR) of the carposporophytes was determined based on the difference in weight before and after cultivation. The calculation formula is: SGR = ln(m t / m o ), m t The density of caryophytes after culture time t, m oThis refers to the initial culture density. Additionally, wet weight refers to the mass of the carposporium when it contains normal water, generally after wiping away surface moisture; culture density is the wet weight divided by the volume of the culture medium.

[0074] Changes in the growth rate of caryophytes under different treatments are as follows: Figure 3 As shown:

[0075] (1) Both culture density and light intensity have a significant effect on the growth rate of carposporophytes. The culture rate of carposporophytes at medium density (1 g / L) is significantly higher than that at high density (2 g / L) and low density (0.5 g / L).

[0076] (2) The effect of light on the growth rate of carposporophytes showed a trend of first increasing and then decreasing, with the values ​​of 150 and 220 μmol photons m -2 s -1 The growth rates of caryophytes under the two light intensities were 8.30±0.70% / d and 8.13±0.66% / d at 1 g / L, respectively, which were higher than those under other light intensities.

[0077] Overall, using a density of 1 g / L and 150–330 μmol photons m -2 s -1 All light conditions effectively promoted the growth of caryophytes; when the density remained constant, the light conditions were 150–220 μmol photons / m². -2 s -1 The growth rate of carposporophytes can be further improved.

[0078] Comparative Example 1: Method for Inducing the Release of Spores from *Asparagus purpurea*

[0079] 1. Experimental Methods

[0080] The method for inducing the release of spores from *Asparagus purpurea* includes the following steps:

[0081] S1. Field Sampling: *Asparagus purpureus* grows in the low-tide to subtidal zone. During sampling, female gametophytes with mature cysts are identified by their color (pink), shape (spherical), and size (approximately 0.5 mm). The female gametophytes are then plucked from the stolons. During transportation, the female gametophytes are placed in a waterless, low-temperature environment to prevent premature spore release due to temperature increases.

[0082] S2. Algal Treatment: To eliminate the influence of epiphytes on cystospore release, the female gametophytes collected in step S1 were placed in a 2L polyethylene beaker and rinsed 10 times with 2L of sterilized natural seawater. After the female gametophytes settled naturally, the seawater was poured out one by one, which removed almost all epiphytes. The female gametophytes were then transferred to a clean petri dish containing sterilized seawater, and any remaining epiphytes were removed with tweezers under an inverted microscope. During the transfer, the ends of the female gametophytes were held with tweezers, avoiding contact with the cystospore distribution area to prevent damage to the cystospores.

[0083] S3. Spore release and counting: Individual gametophytes were transferred to 100 mm culture dishes containing sterilized seawater (FSW) medium, with two photocycles of 12 L: 12 D, and a light condition of 24 μmol photons / m². -2 s -1 After being treated in darkness, the release of carpospores from the gametophytes was observed and recorded every hour using an inverted microscope.

[0084] Because the number of cysts per gametophyte varies, the theoretical spore release data from a gametophyte exhibits significant variability. Therefore, a successful spore release event is defined as "a single gametophyte releasing ≥50 spores," while the opposite is considered an unsuccessful event. The number of successful spore release events under each treatment is counted and divided by the total number of events under each treatment, representing the proportion of successful spore release from gametophytes under each treatment. A chi-square test is used to examine whether there are significant differences in the proportion of successful spore release under different treatments.

[0085] 2. Experimental Results

[0086] The results showed that gametophytes preserved in an anhydrous, low-temperature environment did not successfully release spores. This indicates that freshly collected female gametophytes need to be preserved in a low-temperature seawater environment to induce spore release.

[0087] As can be seen from the above, the culture conditions for sporophyte culture of *Asparagus officinalis* vary at different stages, and different culture conditions are required at different stages. Specifically, in this application, preserving the collected female gametophytes in low-temperature seawater is beneficial for subsequent spore induction; conversely, drying and rehydrating the collected female gametophytes resulted in spores not being released. During the spore release induction stage, FSW is required as a culture medium for better release efficiency. For spore germination and culture, 1 / 2 PES is required as a culture medium for better germination efficiency; conversely, FSW results in very poor germination efficiency. In both the early release and germination stages, spores are cultured under low light intensity (e.g., 24 μmol photons m). -2 s -1After the spores form carposporophytes, they require suitable density (1 g / L) and high light intensity (150–330 μmol photons / m²) for cultivation. -2 s -1 Only under specific conditions can the growth of carposporophytes be facilitated. Therefore, the release of spores obtained in this application and the subsequent cultivation of carposporophytes require cultivation under specific culture media, photoperiods, and light intensities to achieve good spore release and carposporophyte growth effects.

[0088] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A method for culturing the sporophyte of *Asparagus officinalis*, characterized in that, Includes the following steps: S1. Field collection: Collect female gametophytes with mature carposporangia and preserve the obtained female gametophytes in seawater at a temperature 5°C lower than the air temperature; S2. Algal treatment: Remove the epiphytes from the female gametophytes obtained in step S1; S3. Inducing Spore Release: The female gametophytes obtained in step S2 (with epiphytes removed) were placed in sterilized seawater and incubated at a temperature of 18–22°C, a photoperiod of 12L:12D, and a light intensity of 20–40 μmol photons / m². -2 s -1 Cultured under specific conditions to induce the release of carpospores from female gametophytes; S4. Germination and culture of carpospores: Collect the carpospores obtained in step S3 and transfer them to 1 / 2 PES medium. Incubate at a temperature of 18–22℃, a photoperiod of 12L:12D, and a light intensity of 20–40 μmol photons / m². -2 s -1 Under the conditions of cultivation, carposporophytes were obtained; S5. Culture of carposporophytes: The carposporophytes obtained in step S3 were transferred to fresh 1 / 2 PES medium and cultured at a temperature of 18–22℃, a photoperiod of 12L:12D, and a light intensity of 20–40 μmol photons / m². -2 s -1 Under specific conditions, after the carposporophyte length reached ≥0.5 cm, it was transferred for large-scale culture. The carposporophyte density was set at 0.8–1.2 g / L, the temperature at 18–22℃, the photoperiod at 12 L:12 D, and the light intensity at 140–330 μmol photons / m². -2 s -1 The culture medium was 1 / 2 PES medium, and the culture was continued.

2. The method according to claim 1, characterized in that, In step S2, the epiphyte that removes the female gamete obtained in step S1 is the female gamete that has been rinsed with disinfected seawater.

3. The method according to claim 1, characterized in that, In step S3, the temperature for inducing spore release is 19–21°C.

4. The method according to claim 1, characterized in that, In step S3, the light intensity for inducing spore release is 22–30 μmol photons / m². -2 s -1 .

5. The method according to claim 1, characterized in that, In step S4, the temperature for germination and culture of the carpospores is 19–21°C.

6. The method according to claim 1, characterized in that, In step S4, the light intensity for the germination and cultivation of the carpospores is 22–30 μmol photons / m². -2 s -1 .

7. The method according to claim 6, characterized in that, In step S4, the light intensity for the germination and cultivation of the carpospores is 24 μmol photons / m². -2 s -1 .

8. The method according to claim 1, characterized in that, In step S5, after the expansion culture, the density of the caryophytes is 0.9–1.1 g / L.

9. The method according to claim 1, characterized in that, In step S5, after the expansion culture, the light intensity for the carposporophyte culture is 140–200 μmol photons / m². -2 s -1 .

10. The method according to claim 9, characterized in that, In step S5, after the expansion culture, the light intensity for the carposporophyte culture is 150 μmol photons / m². -2 s -1 .

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