Method for semi-artificial breeding of solieria seedlings

Abstract

The invention discloses a semi-artificial breeding method for solieria seedlings, and relates to the technical field of solieria cultivation. According to the method, an efficient and stable denseleaf crazyweed seedling breeding technical system is systematically established for the first time, the provided seedling breeding method comprises the steps of algae seed selection, algae seed treatment, spore diffusion, indoor seedling breeding, medium-term culture and the like, wild mature algae are used as algae seeds, and the genetic stability of germplasm is guaranteed; by means of treatment such as shade-drying stimulation, spore diffusion and seedling collecting culture, concentration and mass diffusion of fruit spores are achieved, the method has the advantages of being short in seedling collecting period and high in seedling attachment rate, the overall seedling obtaining efficiency can be greatly improved, and the problems that seedling sources are unstable and the yield is low are effectively solved. The solieria seedlings cultivated by the method can be directly used for circulating water factory-like large-scale cultivation and do not need to be put into sea area cultivation, so that the risk of uncontrollable factors in the sea area is reduced, the utilization rate of carpospores and seedlings is increased, and the labor cost is reduced.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of red seaweed cultivation, in particular to a method for semi-artificial breeding of red seaweed seedlings. BACKGROUND

[0002] Solieriaceae seaweed is a kind of tropical economic seaweed with very wide application prospect, which has important application value in the fields of food, medicine, biotechnology, etc. Its representative species include Betaphycus, Kappaphycus, Eucheuma and Solieria. However, China is still in the primary stage in the development and utilization and large-scale cultivation of Solieriaceae seaweed, and the seaweed industry has been stagnant. According to the 2025 China Fishery Yearbook, the production of Eucheuma in China in 2024 was only 562 tons of dry weight, and the annual production has been maintained at the level of hundreds of tons. According to the data of the Food and Agriculture Organization (FAO) in previous years, the total production of Eucheuma in China is negligible compared to the global production (million tons of fresh weight) and has been dependent on imports. The main reasons for this result include: (1) changes in the local cultivation environment in China, with a decrease in the original suitable sea area; (2) lack of representative germplasm resources of Solieriaceae seaweed suitable for local cultivation conditions; (3) lack of mature seedling breeding methods and cultivation techniques. Therefore, China needs to develop Solieriaceae seaweed germplasm suitable for local seawater environment cultivation and establish effective seedling cultivation techniques and cultivation techniques to break through the difficulties in the development of China's Solieriaceae seaweed industry.

[0003] China is a large country in the cultivation industry of macroalgae. At present, other macroalgae such as kelp and seaweed in China have stable and mature seedling breeding methods. However, the breeding of red seaweed seedlings is still in the experimental stage, and no breeding technology system suitable for large-scale cultivation has been formed, which seriously hinders the development of the industrialization of this seaweed. Therefore, in view of the technical bottleneck of the current development of Solieriaceae seaweed industry, it is necessary to develop a Solieriaceae seaweed germplasm resource suitable for China's local cultivation, and to develop a method for breeding red seaweed seedlings and establish a stable seedling acquisition technology. SUMMARY

[0004] The main purpose of the present application is to provide a method for semi-artificial breeding of red seaweed seedlings, which aims to solve the problem of lack of effective means for obtaining Solieria seaweed seedlings in the existing seedling cultivation technology.

[0005] To achieve the above-mentioned purpose, the present application provides a method for semi-artificial breeding of red seaweed seedlings, comprising the following steps:

[0006] S1, collecting algae: from May to July every year, collect Solieria female gametophyte cystocarp plants as algae in coastal areas where Solieria is distributed;

[0007] S2, algal seed pretreatment: the red wing female gametophyte capsule plant is washed with seawater, and then the surface moisture is wiped dry and stored in a water-free environment at 0-10°C;

[0008] S3, algal seed air-drying stimulation: the pretreated red wing algae seed is subjected to air-drying stimulation to obtain the air-drying stimulated red wing algae seed;

[0009] S4, spore release: the air-drying stimulated red wing algae seed is subjected to spore release in filtered seawater to obtain spore water;

[0010] S5, spore seedling collection: the spore water is filtered, and then a seedling collector is placed in the filtered spore water, so that spores adhere to the seedling collector, to obtain a seedling collector after seedling adhesion;

[0011] S6, spore germination culture: the seedling collector after seedling adhesion is subjected to spore germination culture in a culture room, and the spore seedlings are grown to 2-5 mm to obtain a seedling collector with seedlings;

[0012] S7, mid-term culture: the seedling collector with seedlings is subjected to suspension and aeration culture in a land-based seedling culture pool.

[0013] In an embodiment, in step S3:

[0014] the pretreated red wing algae seed is subjected to air-drying stimulation in a dark environment; and / or,

[0015] the pretreated red wing algae seed is subjected to air-drying stimulation at a temperature of 20-25°C; and / or,

[0016] the air-drying stimulation treatment time is 6-12 h.

[0017] In an embodiment, in step S3, the water loss rate of the pretreated red wing algae seed after air-drying stimulation is 30%-50%.

[0018] In an embodiment, the step S4 comprises:

[0019] the air-drying stimulated red wing algae seed is subjected to spore release in filtered seawater in the period from 8 am to 12 pm to obtain spore water.

[0020] In an embodiment, in step S4:

[0021] the release time is 3-6 h; and / or,

[0022] the amount of filtered seawater is calculated by adding 2-5 L of filtered seawater per 100 g of air-drying stimulated red wing algae seed; and / or,

[0023] The conditions of the spore dissemination include: water temperature 25-33℃, salinity 25-30, natural light intensity 60-120μmol*m -2 *s -1 .

[0024] In an embodiment, in step S5, the seedling collector comprises an oxygen pump pipe or a cloth net curtain; and / or,

[0025] The attachment time is 2-3h; and / or,

[0026] The spore water is filtered with a 200-500 mesh screen.

[0027] In an embodiment, in step S6:

[0028] The spore germination culture time is 10-15 days; and / or,

[0029] The culture solution of the spore germination culture comprises F / 2 medium and filtered seawater, wherein the volume ratio of the F / 2 medium and the filtered seawater is (0.5-1.5):1000, and the culture solution is replaced every 2-3 days; and / or,

[0030] The environmental conditions of the spore germination culture include: temperature 27-30℃, salinity 25-30, artificial light intensity 30-60μmol*m -2 *s -1 .

[0031] In an embodiment, in step S6:

[0032] When yellow attachments are observed on the surface of the seedlings or yellow spots appear in the culture container, the seedlings are treated with fresh water for 10-30s.

[0033] In an embodiment, the step S7 comprises:

[0034] The seedling collector with seedlings is placed in a land-based seedling culture pool and is suspended and fixed at a distance of 30-50cm from the water surface for suspension aeration culture.

[0035] In an embodiment, in step S7:

[0036] The environmental conditions of the suspension aeration culture include: temperature 20-33℃, salinity 25-30, natural light intensity 60-120μmol*m -2 *s -1 ; and / or

[0037] The suspension aeration culture time is 25-35 days.

[0038] The technical scheme of the present application provides a method for semi-artificial breeding of red wing algae seedlings, and for the first time, a high-efficiency and stable red wing algae seedling breeding technical system is systematically established, which provides an effective technical scheme for the large-scale development and utilization of algae of the red wing algae genus. The seedling breeding method provided by the present application includes the key steps of selecting red wing algae spores, treating red wing algae spores, spore dispersal, indoor seedling cultivation, and mid-term culture. In the technical scheme of the present application, first, the mature algal body is used as the algal spore, the natural sexual reproduction pathway of the seaweed is followed, the seedling propagation is carried out through the fruiting spore, the genetic stability of the germplasm is ensured, and compared with the large-scale economic seaweed (such as the Gracilaria) which has long relied on vegetative reproduction, the risk of germplasm degeneration caused by the accumulation of asexual reproduction can be significantly reduced; secondly, through the treatment of shade drying stimulation, spore dispersal and seedling cultivation, combined with the spore dispersal law, suitable spore adhesion material and suitable spore germination and seedling culture conditions, the concentrated and massive dispersal of the fruiting spore is realized, which has the advantages of short seedling collection cycle and high seedling adhesion rate, so that the overall seedling acquisition efficiency is greatly improved, and the problems of unstable seedling source and low yield in the process of artificial seedling cultivation of red wing algae are effectively solved. The red wing algae seedlings cultivated by the present application can be directly used for the industrialized scale cultivation in the circulating water, and the artificial controllability is high, so compared with other economic seaweeds (such as Porphyra), the present application does not need to be cultivated in the sea area, and therefore the risk of uncontrollable factors in the sea area is avoided, the utilization rate of the fruiting spore and the seedling is improved, and the artificial cost is reduced. BRIEF DESCRIPTION OF DRAWINGS

[0039] In order to more clearly illustrate the technical scheme in the embodiments of the present application or the prior art, the drawings needed to be used in the following embodiment or prior art description will be briefly introduced. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can be obtained by those skilled in the art without any creative effort on the basis of the drawings shown.

[0040] Figure 1 It is the real object picture of the field red wing algae in the embodiment 1 of the present application;

[0041] Figure 2 It is the real object picture of the red wing algae female gametophyte sac fruit plant in the embodiment 1 of the present application;

[0042] Figure 3 It is the real object picture of the red wing algae spore before shade drying stimulation in the embodiment 1 of the present application;

[0043] Figure 4 It is the real object picture of the red wing algae female gametophyte sac fruit plant in the embodiment 1 of the present application;

[0044] Figure 5 It is the real object picture of the red wing algae spore after shade drying stimulation in the embodiment 1 of the present application;

[0045] Figure 6It is a section picture of the female gametophyte sac fruit of red seaweed in the embodiment 1 of the present application;

[0046] Figure 7 It is a real picture of spore water in the embodiment 1 of the present application;

[0047] Figure 8 It is a result picture of spore water under 10*10 times field microscope in the embodiment 1 of the present application;

[0048] Figure 9 It is a microscope examination result picture of red seaweed seedling after 15 days of germination culture in the embodiment 1 of the present application;

[0049] Figure 10 It is a real picture of red seaweed seedling after 10 days of mid-term culture in the cement pool in the embodiment 1 of the present application;

[0050] Figure 11 It is a real picture of red seaweed seedling after 30 days of mid-term culture in the cement pool in the embodiment 1 of the present application.

[0051] The implementation, functional features and advantages of the present application will be further described with reference to the accompanying drawings in combination with the embodiments. DETAILED DESCRIPTION

[0052] The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments of the present application. Based on the embodiments in the present application, all the other embodiments obtained by those skilled in the art without creative work belong to the protection scope of the present application.

[0053] It should be noted that if the embodiments of the present application involve directional indications (such as up, down, left, right, front, back, etc.), the directional indications are only used to explain the relative position relationship, movement condition, etc. between the components in a certain posture, and if the certain posture changes, the directional indications also change accordingly.

[0054] In addition, if the description of "first", "second" and the like is involved in the embodiments of the present application, the description of "first", "second" and the like is only for the purpose of description, and cannot be understood as indicating or implying the relative importance of the indicated technical features or implicitly indicating the number of the indicated technical features. Therefore, the features limited by "first", "second" can be explicitly or implicitly included at least one of the features. In addition, "and / or" or "and / or" appearing throughout the text means that the three parallel schemes include the A scheme, or the B scheme, or the A and B schemes. In addition, the technical solutions of each embodiment can be combined with each other, but it must be based on the realization of the ordinary skilled in the art, when the combination of technical solutions appears contradictory or unachievable, it should be considered that the combination of technical solutions does not exist, nor within the scope of protection required by the present application.

[0055] At present, there are only related researches on classification identification, nutrition analysis, carrageenan preparation and active ingredient separation and extraction of red seaweed, and the seedling breeding and culture technology of red seaweed is in the blank stage. The red seaweed reported in China includes two species of Pacific red seaweed and fine weak red seaweed, which has great development value. But its seedling acquisition technology is in the stage of development, so it is necessary to develop an effective method to obtain Chinese native red seaweed seedlings.

[0056] Most of the life history and reproductive mode of seaweed are basically the same, and the seedling propagation of large seaweed has two means of asexual and sexual reproduction. For example, the life history of kelp and seaweed is three generations, and the existing technology is relatively mature, basically relying on sexual reproduction, while the cultivation of Gracilaria lemaneiformis currently depends on asexual reproduction. Although there are some mature seedling breeding methods for some algae, different algae have different characteristics and breeding methods in the actual process, different seaweed stimulation methods (such as flow stimulation, light stimulation, shade stimulation, etc.), different algae treatment methods, different spore dispersal time, method and rule, different spore attachment materials, different spore germination conditions, and different seedling culture environment conditions and methods. Whether the seedling can be bred depends on the life history characteristics of the breeding object. For example, seaweed, after spore collection, spore stimulation, spore dispersal and indoor seedling cultivation, only shell filamentous body is formed, and after a period of cultivation, shell spore needs to be further stimulated and dispersed for re-seeding, while Gracilaria has no filamentous body stage. Some seaweed seedling breeding technology is not mature, and spores cannot be dispersed, which cannot achieve the effect of seedling breeding. For example, the current effective method for spore dispersal of Gracilaria lemaneiformis has not been found, and the seedling breeding technology has not been substantially improved. Therefore, for the problem of seedling breeding of red seaweed, the skilled in the art cannot directly realize the breeding method of other algae.

[0057] By combining natural algal species resources and artificial control culture conditions, both wild excellent germplasm can be effectively utilized and spore dispersal rate and seedling survival rate can be improved, which is an effective way to realize sustainable supply of red fringe algae seedlings. However, there is still a lack of systematic and efficient semi-artificial breeding process in the prior art, especially lacking mature technical solutions in key links such as algal species preservation, spore induction and mid-term culture.

[0058] Based on the above background, the present application provides a method for semi-artificial breeding of red fringe algae seedlings, comprising the following steps:

[0059] S1, algal species collection: in May to July every year, collect red fringe algae female gametophyte fruiting bodies as algal species in coastal areas where red fringe algae is distributed;

[0060] S2, algal species pretreatment: rinse the red fringe algae female gametophyte fruiting bodies with seawater, then wipe off the surface moisture and store them in a water-free environment at 0-10℃;

[0061] S3, algal species shade drying stimulation: shade drying stimulation is performed on the pretreated red fringe algae seedlings to obtain shade dried and stimulated red fringe algae seedlings;

[0062] S4, spore dispersal: spore dispersal is performed on the shade dried and stimulated red fringe algae seedlings in filtered seawater to obtain spore water;

[0063] S5, spore seedling collection: the spore water is filtered, then a seedling collector is placed in the filtered spore water, so that spores adhere to the seedling collector, and a seedling collector with adhered seedlings is obtained;

[0064] S6, spore germination culture: the seedling collector with adhered seedlings is subjected to spore germination culture in a culture room, and when the spore seedlings grow to 2-5mm, a seedling collector with seedlings is obtained;

[0065] S7, mid-term culture: the seedling collector with seedlings is subjected to suspension and aeration culture in a land-based seedling culture pond.

[0066] The technical scheme of the present application provides a method for semi-artificial breeding of red wing algae seedlings, which first systematically establishes an efficient and stable red wing algae seedling breeding technology system, and provides an effective technical scheme for the large-scale development and utilization of algae of the red wing algae genus. The seedling breeding method provided by the present application includes the key steps of selecting red wing algae spores, treating red wing algae spores, spore dispersal, indoor seedling cultivation, and mid-term culture. In the technical scheme of the present application, first, the mature algal body is used as the algal spore, the natural sexual reproduction pathway of the seaweed is followed, the seedling propagation is carried out through the fruiting spore, the genetic stability of the germplasm is ensured, compared with the large economic seaweed (such as Gracilaria) which has long relied on vegetative reproduction, the risk of germplasm degeneration caused by long-term vegetative reproduction can be significantly reduced; secondly, through the treatment of shade drying stimulation, spore dispersal and seedling cultivation, combined with the spore dispersal law, suitable spore attachment material and suitable spore germination and seedling culture conditions, the concentrated and massive dispersal of fruiting spores is realized, which has the advantages of short seedling collection cycle and high seedling attachment rate, greatly improving the overall seedling acquisition efficiency, and effectively solving the problems of unstable seedling source and low yield in the process of artificial seedling cultivation of red wing algae. The red wing algae seedlings cultivated by the present application can be directly used for industrial-scale cultivation in circulating water, which has high artificial controllability, and compared with other economic seaweeds (such as Porphyra), it does not need to be cultivated in the sea, so it also avoids the risk of uncontrollable factors in the sea, improves the utilization rate of fruiting spores and seedlings, and reduces the artificial cost.

[0067] Please refer to Figure 1 and Figure 2 In the present application, the female gametophyte cystocarp plant of wild germplasm is selected as the algal spore, Figure 2 The arrow in the figure is the cystocarp of red wing algae. It can be understood that the wild germplasm of red wing algae is obtained through the natural sexual reproduction pathway of seaweed, which ensures the genetic stability of the seedlings.

[0068] It needs to be further explained that there are various stimulation methods for seaweed (such as cutting stimulation, flowing water stimulation, light stimulation, shade drying stimulation, etc.), and the stimulation method for different seaweeds is different, and it is necessary to select which method according to the actual effect. Shade drying stimulation can make cells lose water, and after rehydration, the cells swell and can release spores through osmotic pressure. The inventors found that the method of shade drying stimulation is beneficial to the release of spores from red wing algae spores.

[0069] In the embodiment of the present application, the pre-treated red wing algae spores are subjected to shade drying stimulation in a dark environment in step S3. During shade drying, the dark environment is maintained as much as possible to avoid the influence of light stimulation on the concentrated release of seaweed spores.

[0070] In the embodiment of the present application, the pre-treated red wing algae spores are subjected to shade drying stimulation at a temperature of 20-25℃ in step S3. The temperature of shade drying stimulation should not be too high, and the present application selects a temperature of 20-25℃ for shade drying stimulation.

[0071] In the embodiment of the present application, in step S3, the treatment time of the dry stimulation is 6-12 hours. The stimulation time can be adjusted according to the state of the algae, and the dry shrinkage of the algae skin can be used as a reference, or the weight of the algae before and after the stimulation can be compared to control the water loss rate.

[0072] In the embodiment of the present application, in step S3, the water loss rate of the pre-processed red wing algae after the dry stimulation is 30%-50%. Different seaweeds have different water loss rates when they are dry stimulated, and the effect of spore release is also different. If the dry degree is too high or too low, the germplasm will be wasted, and the utilization rate will be low. If the dry degree is too high, the spores will die due to excessive water loss, and if the dry degree is insufficient, the spores will not be easily concentrated and dispersed. By setting the water loss rate of the algae after the dry stimulation to be 30%-50%, the survival rate of the algae is high, and the spore dispersion effect is good.

[0073] In the embodiment of the present application, step S4 comprises:

[0074] The dry stimulated red wing algae is placed in the filtered seawater for spore dispersion from 8 am to 12 pm to obtain spore water.

[0075] Different seaweeds have different stimulation methods and spore release times. Some seaweeds are suitable for spore dispersion in the morning, some seaweeds are suitable for spore dispersion in the afternoon or evening, and some seaweeds cannot be concentrated and dispersed, and need to be stimulated repeatedly, for example, Porphyra yezoensis, which needs to be stimulated repeatedly for half a month to continuously release spores, increasing the labor cost. The inventors found that the dispersion amount of red wing algae is higher from 8 am to 10 am, and the dispersion amount decreases sharply after 10 am. Therefore, the technical solution of the present application selects the spore dispersion period from 8 am to 12 pm.

[0076] It should be noted that the spore dispersion in the filtered seawater is selected on the one hand to reduce pollutants and facilitate microscopic spore counting, and on the other hand to avoid the influence of other suspended matters on the survival rate and adhesion rate of spores.

[0077] In the embodiment of the present application, in step S4, the dispersion time is 3-6 hours. After the dehydrated algae is rehydrated, the algae cells swell due to the osmotic pressure, which stimulates the algae to release spores. By controlling the dispersion time to be 3-6 hours, the problem of algae waste caused by spore adhesion or inactivation and degradation can be avoided.

[0078] In the embodiment of the present application, the filtered seawater is injected into the spore dispersion tank, and then the dry stimulated red wing algae is placed in the filtered seawater for spore dispersion. During the dispersion period, the seawater in the spore dispersion tank is stirred every 20-30 minutes by hand or using a water pump to promote the spore dispersion of the algae and make the spores evenly dispersed.

[0079] In the embodiment of the present application, in step S4, the amount of filtered seawater is 2-5 L per 100 g of dried and stimulated red seaweed spores. Too little water is not conducive to the complete soaking of the spores in the water, and too much water is not conducive to the recovery of spores, resulting in waste of spores. Setting the amount of filtered seawater in the above range can ensure uniform dispersion of the spores, avoid overcrowding or aggregation, improve the success rate of subsequent culture steps, and at the same time help maintain the osmotic pressure balance inside and outside the algal body to ensure normal dispersion of the spores.

[0080] In the embodiment of the present application, in step S4, the conditions for spore dispersion include: water temperature 25-33℃, salinity 25-30, natural light intensity 60-120 μmol*m -2 *s -1 Water temperature, salinity and light parameters affect spore dispersion efficiency, especially light and temperature control. Different seaweeds have different requirements for light and temperature. Too high a temperature can also cause spore death. Appropriate light and temperature are also factors affecting the concentrated dispersion of spores.

[0081] In the embodiment of the present application, in step S5, the spore collector includes an oxygen pump pipe or a cloth strip net curtain. The selection of the material of the spore collector is very critical. Different seaweed spores have different attachment efficiencies on different materials, and the same seaweed spores also have significant differences in attachment efficiency on different materials. For example, porphyra is suitable for nylon rope, and gracial is suitable for stone or other calcareous materials. The inventor found that red seaweed spores are easy to attach to oxygen pump pipes or cloth strip net curtains, so they are selected. Before using the spore collector, the spore collector can be soaked in fresh water overnight to avoid the adverse effects of other impurities.

[0082] In the embodiment of the present application, in step S5, the spore collector has a spore collection area of 1-2 billion spores: 100 m 2 During spore dispersion, spore water is sampled every 1-2 h for microscopic examination, and a blood cell counting plate is used to calculate the amount of spore dispersion. The amount of spore collection (i.e. the spore collection area) is determined according to the amount of spore dispersion, the purpose being to control the spore collection density and avoid waste of spores. The more spores are dispersed, the larger the spore collection area should be. In the technical solution of the present application, the spore collection area (the amount of spore collection) of the spore collector is set to 1-2 billion spores: 100 m 2 .

[0083] In the embodiment of the present application, in step S5, the attachment time is 2-3 h. Setting the attachment time in the above range ensures sufficient spore attachment and a high attachment success rate, while avoiding unnecessary resource consumption or the effects of environmental changes caused by too long an attachment time.

[0084] In an embodiment of the present invention, in step S5, the spore water is filtered through a 200-500 mesh screen. To avoid contamination by algal debris and other impurities and to improve spore utilization, the spore water is filtered through a 200-500 mesh silk screen before subsequent spore germination culture.

[0085] In an embodiment of the present invention, in step S6, the spore germination culture time is 10 to 15 days.

[0086] In an embodiment of the present invention, in step S6, the culture medium for spore germination culture includes F / 2 medium and filtered seawater, wherein the volume ratio of F / 2 medium to filtered seawater is (0.5-1.5):1000, and the culture medium is changed every 2-3 days.

[0087] In an embodiment of the present invention, the environmental conditions for spore germination culture in step S6 include: temperature 27–30°C, salinity 25–30, and artificial light intensity 30–60 μmol*m. -2 *s -1 Setting temperature, salinity, and light intensity within the above ranges helps promote successful spore germination and improve seedling quality and yield.

[0088] In an embodiment of the present invention, in step S6:

[0089] When yellow deposits are observed on the surface of the seedlings or yellow spots appear in the culture container, the seedlings should be treated with fresh water for 10–30 seconds.

[0090] During spore germination culture, samples were taken daily for microscopic examination of spore attachment, germination status, and algal contamination. When a large amount of algae was observed, seawater rinsing was ineffective in removing them; even after seawater rinsing, the algae quickly re-emerged. To improve seedling germination and survival rates, when yellow deposits were observed on the seedling surface or yellow spots appeared in the culture container, the seedlings could be soaked or rinsed with fresh water for 10–30 seconds to remove the algae. These yellow deposits were generally mainly diatom contamination. For algae that remained after freshwater treatment, manual removal was necessary.

[0091] In an embodiment of the present invention, step S7 includes:

[0092] The seedling collection device is placed in a land-based seedling culture pond and suspended and fixed at a height of 30-50 cm above the water surface for suspension and aeration culture.

[0093] Suspending the seedling collector in the water at a certain height above the water surface allows for suspended growth, eliminating spatial limitations and facilitating adjustment of light requirements. Low water temperature and weak light at the bottom mean that if the seedling collector is placed too shallowly, the temperature and light become too intense, easily killing the red pea seedlings. Conversely, if the collector is too deep, the weak light results in slow growth. This invention sets the seedling collector at a height of 30-50 cm above the water surface, providing suitable light and temperature conditions, which is beneficial for the growth of the red pea seedlings.

[0094] In an embodiment of the present invention, the environmental conditions for the suspension aeration culture in step S7 include: temperature 20–33°C, salinity 25–30, and natural light intensity 60–120 μmol*m. -2 *s -1 .

[0095] In an embodiment of the present invention, in step S7, the suspension aeration culture time is 25 to 35 days. The culture time can be adjusted according to the growth and contamination situation.

[0096] In an embodiment of the invention, mid-term cultivation is carried out in a land-based recirculating aquaculture tank, which includes a land-based seedling cultivation tank, a water storage and sedimentation tank, a sand filtration system, a microfilter, a piping system, and a light-regulating shading net. The land-based seedling cultivation tank provides aquaculture space; the water storage and sedimentation tank allows larger particles to settle to the bottom of the tank through settling, thereby ensuring the cleanliness and stability of the water quality; the sand filtration system can remove large particulate matter and miscellaneous algae from seawater; the microfilter includes a microfiltration membrane, during which seawater passes through a porous microfiltration membrane (i.e., a microfiltration membrane), which can effectively trap particulate matter, bacteria, microorganisms, and certain macromolecules in the solution, but allows water and small molecule solutes to pass through; the light-regulating shading net is used to regulate the intensity of natural light.

[0097] The technical solution of the present invention will be further described in detail below with reference to specific embodiments and accompanying drawings. It should be understood that the following embodiments are only used to explain the present invention and are not intended to limit the present invention.

[0098] Example 1

[0099] 1. A method for semi-artificial propagation of red pea shoot seedlings, comprising the following steps:

[0100] (1) Algal strain collection: In May 2025, female gametophyte cysts of *Lycopodium clavatum* were collected as algal strains in Nanji Village, Xuwen County, Zhanjiang City, Guangdong Province (109°55′25″E, 20°13′13″N).

[0101] (2) Pretreatment of algal strains: The strains were cleaned with seawater, dried, stored at low temperature (4℃) and without water, and transported back to the laboratory. 938g of female gametophyte cysts of *Lysimachia nummularia* were selected as algal strains.

[0102] (3) Shade-drying stimulation of algal seeds: The *Pterocarya spp.* algal seeds were spread out in a 25°C air-conditioned room for shade-drying stimulation. After 12 hours of treatment, wrinkling of the algal seed surface was observed, and the weight of the algal seeds decreased to 578g, with a water loss rate of 41.2%. Please refer to the attached images and slices of the *Pterocarya spp.* algal seeds before shade-drying. Figure 3 and Figure 4 Please refer to the actual photos and slice results of the air-dried *Pterocarya spp.* Figure 5 and Figure 6 .

[0103] (4) Spore release: At 8:00 AM, the air-dried and stimulated algae seeds were placed in a 6L storage tank containing filtered seawater for rehydration. The storage tank was then placed outdoors for 6 hours to release the fruit spores, yielding spore water. Figure 7 and Figure 8 The conditions for spore release are: water temperature 25–33℃, salinity 25–30, and natural light intensity 60–120 μmol*m. -2 *s -1 During the release period, the seawater was stirred every 20 minutes, and water samples were taken at 10:00, 12:00, and 14:00. The water samples were examined under a microscope in 10 fields of view, and the amount of carpospores released was counted using a hemocytometer. The results are shown in Table 1. The formula for calculating the amount of carpospores released is: Carpospore release amount = Average release amount of spores * 100 divisions * 6L of water volume / 100μL of water volume in the hemocytometer; The formula for calculating the average release amount of carpospores is: Average release amount of carpospores = Carpospore release amount / Fresh weight of algae.

[0104] (5) Spore collection: The spore water from step (4) was filtered and collected using a 500-mesh silk screen. A plastic tube was used as a spore collection device to collect the spores for 2 hours to obtain the spore collection device after spore attachment.

[0105] (6) Spore germination culture: Place the seedling collection device after seedling attachment in step (5) in the seaweed culture room for spore germination culture for 15 days. Figure 9 A seedling collection device with seedlings was obtained; the cultivation temperature was 27–30℃, the salinity was 25–30, and the artificial light intensity was 30–60 μmol*m. -2 *s -1 The culture medium was changed every two days during this period.

[0106] (7) Mid-term cultivation: The seedling collection device from step (6) was transferred to a land-based recirculating water cement tank for suspension aeration cultivation for 10 days and 30 days. Figure 10 and Figure 11The cultivation temperature was 20–33℃, the salinity was 25–30, and the natural light intensity was 60–120 μmol*m. -2 *s -1 .

[0107] Table 1 Results of spore release from *Lysimachia nummularia*

[0108]

[0109] Monitoring showed that the water temperature at 10:00 AM was 27.5℃, and the average number of carpospores released per square in the hemocytometer was 23.9. Based on the formula above, the calculated carpospore release rate was 1.4 * 10⁻⁶. 8 The average release rate of algal carpospores was 1.5 x 10^10 per gram of fresh algae. 5 The water temperature at 12:00 was 28.6℃. The average number of carpospores released per square in the hemocytometer was 7.9, which calculates to a total release of 4.7 x 10^12 carpospores. 7 The average release rate of algal carpospores was 5.0 x 10^10 per gram of fresh algae. 4 At 2 PM, the water temperature was 34.1℃. The average number of carpospores released per square in the hemocytometer was 8.1, which calculates to a total release of 4.9 x 10^12 carpospores. 7 The average release rate of carpospores from the algal species was 5.2 x 10^6 spores per gram of fresh algae. 4 indivual.

[0110] As shown in Table 1, the total release of spores from 938g of *Rhizophora rubiginosa* seeds was 2.4 x 10⁻⁶. 8 The average release rate of carpospores from the algal species was 2.6 x 10^6 per gram of fresh algae. 5 Furthermore, Table 1 shows that there are significant differences in the release of carpospores at different times. The highest spore release occurs between 8 and 10 a.m., while the release rate drops sharply after 10 a.m.

[0111] The above description is merely an exemplary embodiment of the present invention and does not limit the patent scope of the present invention. Any equivalent structural transformations made using the contents of the present invention specification and drawings under the technical concept of the present invention, or direct / indirect applications in other related technical fields, are included within the patent protection scope of the present invention.

Claims

1. A method for semi-artificial propagation of *Gnaphalium affine* seedlings, characterized in that, Includes the following steps: S1. Algal Seed Collection: Every year from May to July, female gametophyte cysts of *Lycopodium clavatum* are collected as algal seeds in coastal areas where *Lycopodium clavatum* is distributed. S2. Pretreatment of algae: Rinse the female gametophyte cysts of the red pheasant with seawater, then wipe off the surface moisture and store them in an environment of 0-10℃ and no water. S3. Shade-drying stimulation of algae: The pretreated *Rhododendron simsii* algae seeds are shade-dried to obtain shade-dried *Rhododendron simsii* algae seeds. S4. Spore release: The dried and stimulated *Pterocarya spp.* seeds are released into filtered seawater to obtain spore water. S5. Spore collection: Filter the spore water, and then place the spore collector in the filtered spore water to allow the spores to attach to the spore collector, thus obtaining a spore collector with attached spores. S6. Spore germination culture: The seedling collection device with attached seedlings is placed in a culture room for spore germination culture. When the spore seedlings grow to 2-5 mm, the seedling collection device with seedlings is obtained. S7. Mid-term cultivation: The seedling collection device with seedlings is used for suspension and aeration cultivation in a land-based seedling culture pond.

2. The method for semi-artificial propagation of *Gnaphalium affine* seedlings as described in claim 1, characterized in that, In step S3: The pretreated *Pterocarya spp.* algae were subjected to air-drying stimulation in a dark environment; and / or... The pretreated *Pterocarya spp.* strain was subjected to shade-drying stimulation at a temperature of 20–25°C; and / or, The treatment time for the air-drying stimulation is 6 to 12 hours.

3. The method for semi-artificial propagation of *Gnaphalium affine* seedlings as described in claim 1, characterized in that, In step S3, the water loss rate of the pretreated *Pterocarya spp.* after air-drying stimulation is 30%–50%.

4. The method for semi-artificial propagation of *Gnaphalium affine* seedlings as described in claim 1, characterized in that, Step S4 includes: Between 8 a.m. and 12 p.m., the air-dried and stimulated *Pterocarya spp.* seeds were released into filtered seawater to obtain spore water.

5. The method for semi-artificial propagation of *Gnaphalium affine* seedlings as described in claim 1, characterized in that, In step S4: The release time is 3–6 hours; and / or, The amount of filtered seawater used is calculated based on adding 2-5L of filtered seawater to every 100g of shade-dried and stimulated *Pterocarya stenoptera* seed; and / or, The conditions for spore release include: water temperature 25–33℃, salinity 25–30, and natural light intensity 60–120 μmol*m. -2 *s -1 .

6. The method for semi-artificial propagation of *Gnaphalium affine* seedlings as described in claim 1, characterized in that, In step S5, the seedling collector includes an oxygen pump tube or a cloth mesh curtain; and / or, The attachment time is 2-3 hours; and / or, The spore water was filtered through a 200-500 mesh sieve.

7. The method for semi-artificial propagation of *Gnaphalium affine* seedlings as described in claim 1, characterized in that, In step S6: The spore germination culture time is 10–15 days; and / or, The spore germination culture medium comprises F / 2 medium and filtered seawater, wherein the volume ratio of F / 2 medium to filtered seawater is (0.5–1.5):1000, and the culture medium is changed every 2–3 days; and / or, The environmental conditions for spore germination culture included: temperature 27–30℃, salinity 25–30, and artificial light intensity 30–60 μmol*m. -2 *s -1 .

8. The method for semi-artificial propagation of *Gnaphalium affine* seedlings as described in claim 1, characterized in that, In step S6: When yellow deposits are observed on the surface of the seedlings or yellow spots appear in the culture container, the seedlings should be treated with fresh water for 10–30 seconds.

9. The method for semi-artificial propagation of *Gnaphalium affine* seedlings as described in claim 1, characterized in that, Step S7 includes: The seedling collection device is placed in a land-based seedling culture pond and suspended and fixed at a height of 30-50 cm above the water surface for suspension and aeration culture.

10. The method for semi-artificial propagation of *Gnaphalium affine* seedlings as described in claim 1, characterized in that, In step S7: The environmental conditions for the suspension aerated culture include: temperature 20–33℃, salinity 25–30, and natural light intensity 60–120 μmol*m. -2 *s -1 ; and / or The suspension aeration culture time is 25 to 35 days.

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