Method for creating and evaluating new germplasm of suspended culture of spermatozoid of sargassum fusiforme

Abstract

The application discloses a kind of Sargassum fusiforme fertilized egg suspension culture new germplasm creation and evaluation method, including wild Sargassum fusiforme geographical parent selection, isolation breeding and unit mass evaluation;Parent high-value character strain female and male plant screening, isolation breeding and enemy biological control;High-value character strain female and male plant epiphytic enemy biological removal;High-value character strain female and male plant lateral branch indoor temporary nursing optimum environmental factor creation;Parent high-value character strain female and male plant lateral branch indoor temporary nursing processing and temporary nursing time;New germplasm fertilized egg classification collection, impurity removal and cleaning, new germplasm fertilized egg suspension system creation;New germplasm embryo to young sporophyte suspension growth and development stage suspension system typical enemy biological control;New germplasm young sporophyte is clamped and indoor temporary nursing;New germplasm young sporophyte is put in sea area;Parent and offspring new germplasm evaluation method;Can be Sargassum fusiforme new germplasm creation, new germplasm quality evaluation and new variety cultivation provide scientific, standard operation and evaluation method.

Description

TECHNICAL FIELD

[0001] The present application relates to a kind of Sargassum fulvum fertilized egg suspension culture new germplasm creation and evaluation method, which is suitable for Sargassum fulvum new germplasm creation, Sargassum fulvum new germplasm genetic stability evaluation, Sargassum fulvum new germplasm high-quality strain screening, Sargassum fulvum hybrid breeding and molecular breeding, Sargassum fulvum new variety creation and other scientific research and agricultural production fields. BACKGROUND

[0002] Sargassum fulvum is the special food and medicine Sargassum thunbergii in the northwest coast of Pacific Ocean, and its medicinal value is recorded in "Shennong Bencao Jing". Dongtou District of Wenzhou is the largest Sargassum fulvum breeding, breeding, processing, export and domestic sales area in China, with more than 12,000 mu of breeding area and more than 61 million yuan of total breeding output value per year.

[0003] Practical research shows that after wild Sargassum fulvum parent is artificially domesticated and bred, the mature algal body air bag shows diversity characteristics, and different plants show morphological adaptability under the same environmental conditions. This habitat adaptation characteristic seriously affects the uniformity and stability evaluation between strains, seriously restricts the development of new varieties and large-scale agricultural production application, and this scientific problem has been reported in relevant literatures.

[0004] At present, how to efficiently solve the Sargassum fulvum new germplasm creation technology and accurately grasp the morphological stability characteristics of different new germplasm offspring, implement the large-scale creation of Sargassum fulvum high-value strain seed parent, and ensure the efficient production and application of high-value strain, has become an urgent problem of Sargassum fulvum scientific researchers, breeders and processing enterprises.

[0005] In the process of systemically preparing Sargassum fulvum sexual reproduction new germplasm creation and evaluation innovation technology development, the present inventors properly integrate the innovation technology obtained, realize the Sargassum fulvum sexual reproduction new germplasm creation and evaluation technology innovation, and the production pilot test proves that the present application effectively solves the problems of Sargassum fulvum new germplasm creation and stability evaluation. SUMMARY

[0006] Based on the technical field and technical background, the purpose of the present application is to propose a method for Sargassum fulvum fertilized egg suspension culture new germplasm creation and Sargassum fulvum new germplasm evaluation. The method is suitable for Sargassum fulvum new germplasm creation, Sargassum fulvum new germplasm genetic stability evaluation, Sargassum fulvum new germplasm high-quality strain screening, Sargassum fulvum hybrid breeding and molecular breeding, Sargassum fulvum new variety creation and other scientific research and agricultural production fields.

[0007] Based on the purpose of the present application, the present application provides a kind of Sargassum fulvum fertilized egg suspension culture new germplasm creation and evaluation method,

[0008] including the following steps:

[0009] S1 Selection, isolation breeding and unit mass evaluation of wild Sargassum fusiforme parent;

[0010] S2 Selection, isolation breeding and pest control of wild Sargassum fusiforme parent high-value trait line female and male plants;

[0011] S3 Removal of epiphytic pests of wild Sargassum fusiforme high-value trait line female and male plants;

[0012] S3.1 Temporary indoor cultivation of wild Sargassum fusiforme high-value trait line female and male plants

[0013] S3.1.1 Removal of epiphytic macroalgae

[0014] Manual removal;

[0015] S3.1.2 Removal of epiphytic animals

[0016] Room temperature fresh water soaking for 20 min to 25 min, low osmotic killing of epiphytic animals, fresh, filtered, sterilized seawater flushing 3 to 5 times;

[0017] S3.1.3 Removal of epiphytic microalgae

[0018] The mass (kg) of algal fresh weight and citric acid (C6H8O7) solution volume (L) is 1:3, the concentration of citric acid is 1.5 mg / ml, the soaking time is 5 min to 8 min, after taking out, room temperature fresh water flushing 3 to 5 times, fresh, filtered, sterilized seawater flushing 3 to 5 times;

[0019] S3.2 Treatment of wild Sargassum fusiforme high-value trait line female and male plants

[0020] S3.2.1 Selection of lateral branches and removal of stems

[0021] Remove fresh algal body primary or secondary lateral branch stems, and retain secondary or tertiary lateral branches densely growing on the receptacle;

[0022] S3.2.2 Lateral branch placement of female and male plants and mass ratio

[0023] According to the actual area of the culture tray, reasonably allocate the lateral branches, the allocation standard is that the lateral branches do not overlap each other, so as to ensure uniform illumination, and the mixed mass ratio (kg:kg) of female plants and male plants is 1:1;

[0024] S4 Creation of optimal environmental factors for indoor temporary cultivation of wild Sargassum fusiforme high-value trait line female and male plant lateral branches

[0025] S4.1 Optimal water environment

[0026] Fresh: take the clear seawater of the same day rising tide, natural sedimentation for 3 h to 4 h;

[0027] Filtration: the supernatant seawater is filtered through a 280-300 mesh gauze net;

[0028] High-temperature sterilization: the filtered seawater is heated to 80-90℃, high-temperature sterilized for 30-40 min, and then naturally cooled to room temperature, so as to kill animal eggs, microorganisms, microalgae and large seaweed seedlings;

[0029] pH value: the pH value of the sterilized seawater is adjusted to 7.8-8.3 by using 0.05 mol / L NaOH solution and 0.05 mol / L HCl solution;

[0030] Salinity: 28-33;

[0031] Nutrient salt: NaNO3 and KH2PO3 are used to prepare a seawater solution with N:P=10:1, and the concentration is 10 (N) g / L or 1 (P) g / L. The volume ratio (V:V) of nutrient salt to culture seawater is (0.6-0.8) ml:1 L;

[0032] S4.2 Culture room, light culture rack and temporary culture device

[0033] The main structure of the light culture rack includes carbon steel left and right side frames, tempered glass panels, glass panel bottom frames, crossbars, switch boxes and LED lighting lamps. The temporary culture conditions in the seawater biological room are set to ensure 0-350 µmol•m -2 •s -1 -350 µmol•m -2 •s -1 light intensity, and an external control power supply timing converter can realize photoperiod regulation. The culture room is an indoor darkroom equipped with air conditioning equipment to regulate the space temperature in the culture room. The main structure of the temporary culture device group includes water storage barrels, culture trays, peristaltic pumps, oxygen supply pumps, water supply hoses and sand heads, which can realize centralized oxygen dissolution, water supply and circulation of seawater, and can ensure sufficient supply of oxygen and nutrients for temporary culture of S. polycystum seedlings;

[0034] S4.3 Setting the optimum temperature, light intensity and photoperiod

[0035] Temperature: 23-25℃;

[0036] Light intensity: 200-280 µmol•m -2 •s -1 -280 µmol•m -2 •s -1 ;

[0037] Photoperiod: 14 h:10 h-12 h:12 h;

[0038] S5. Treatment and temporary culture time of wild S. polycystum parent high-value trait line female and male plants in lateral branch room

[0039] S5.1 In-lab temporary cultivation of side shoots

[0040] The total volume of seawater in the culture group was replaced with 1 / 2 volume of seawater every day, and the side shoots were turned over once every 6-8 hours to ensure that each side shoot receptacle received uniform light;

[0041] S5.2 In-lab temporary cultivation time of side shoots

[0042] The side shoots were temporarily cultivated in-lab for 5-7 days;

[0043] S6 Classification and collection of fertilized eggs of new germplasm of Sargassum fusiforme, removal of impurities, and cleaning

[0044] The fertilized eggs were collected using a 260-280 mesh gauze, and the gas sacs, receptacles, and other impurities were removed using a 30-50 mesh gauze. The fertilized eggs were placed in a 2000 mL beaker, 1500 mL of fresh filtered seawater was added, and after stirring, they were allowed to settle for 2-3 minutes. The upper suspension was discarded to remove suspended impurities. This operation was repeated 5-7 times to obtain fresh pure fertilized eggs. Different strains required separate collection and removal of impurities.

[0045] S7 Creation of a suspension system for fertilized eggs of new germplasm of Sargassum fusiforme

[0046] S7.1 Quantitative dispensing of fertilized eggs of new germplasm of Sargassum fusiforme

[0047] The excess water in the fresh pure fertilized eggs was removed, and the volume density of the fertilized eggs was statistically determined. 8000-10000 fertilized eggs were quantitatively dispensed into each 3000 ml or 5000 ml triangular flask, and fresh filtered seawater was added. The ratio of the number of fresh pure fertilized eggs (pieces) to the volume of seawater (ml) was 10:1-10:3. Different strains required separate dispensing and labeling.

[0048] S7.2 Creation of optimal environment for suspension culture of fertilized eggs of new germplasm of Sargassum fusiforme

[0049] S7.2.1 Optimal water environment

[0050] Fresh, filtered, high-temperature sterilized, pH, salinity, and nutrient salt configurations were prepared according to the method in S4.1. The volume ratio of nutrient salt to culture seawater (V:V) was (0.5-0.7) ml:1 L-(0.6-0.8) ml:1 L. The optimal water environment conditions remained unchanged during the suspension culture period.

[0051] S7.2.2 Optimal light intensity, photoperiod, temperature, aeration, and other conditions

[0052] Optimal light intensity: 50 µmol•m -2 •s -1 - 60 µmol•m -2 •s -1 ;

[0053] Light cycle: 14 h:10 h~12 h:12 h;

[0054] Temperature: 23 ℃~24 ℃;

[0055] Aeration: auxiliary aeration;

[0056] S7.2.3 Suspension culture mode of fertilized eggs of new germplasm of Sargassum fusiforme

[0057] The fertilized eggs of Sargassum fusiforme are suspended and cultured by magnetic stirring + aeration at the initial stage (1 d~10 d) of suspension culture, and the fertilized eggs of Sargassum fusiforme have grown into embryos after being cultured for 8 d~10 d, and the embryo growth and development period begins, and the suspension culture device is used for suspension culture, and aeration suspension culture is implemented;

[0058] S7.3 Low-density quantitative sub-packaging of suspension growth and development of new germplasm of Sargassum fusiforme from embryo to young sporophyte

[0059] At the stage of growth and development of embryo to young sporophyte, 1 / 2 equal volume density is sub-packaged once every 19 d~21 d per bottle, and with the gradual extension of the culture time, the low-density quantitative sub-packaging is gradually reduced, the first low-density sub-packaging is reduced from 8000~10000 to 4000~5000, the second low-density is reduced from 4000~5000 to 2000~2500, the third low-density is reduced from 2000~2500 to 1000~1200, and finally the low-density is reduced from 1000~1200 to 400~500;

[0060] S8 Typical enemy biological control of suspension system at the stage of growth and development of new germplasm of Sargassum fusiforme from embryo to young sporophyte

[0061] S8.1 Enemy animal control method

[0062] If enemy animals are found in the suspension culture solution, use ultrapure water to soak for 20 min~25 min every 10 d~12 d of culture period, and kill the enemy animals by hypotonicity;

[0063] S8.2 Enemy cyanobacteria control method

[0064] During the S7 culture period, the embryos or young sporophytes are regularly examined by microscopy, and if the embryos or young sporophytes have signs of epiphytic blue algae, use 300,000~600,000 / L penicillin seawater liquid to soak the culture for 24 h~48 h, kill the blue algae, and then filter the seawater for 3~5 times, and continue to use the filtered seawater for suspension culture;

[0065] S8.3 Enemy green algae control method

[0066] During the S7 culture period, the embryos or young sporophytes were observed regularly under a microscope. If the embryos or young sporophytes showed signs of epiphyte green algae, they were soaked in 1.5 mg / ml citric acid solution for 6-8 min, rinsed with fresh water for 3-5 times, rinsed with filtered seawater for 2-3 times, and then suspended in filtered seawater for continuous culture;

[0067] S9. Clamping of new germplasm young sporophytes of Sargassum fusiforme and temporary indoor cultivation

[0068] The young sporophytes with base leaves of 1.5-2.0 cm were clamped on cotton ropes or fiber ropes with a diameter of 0.4 cm at a spacing of 3-5 cm. The young sporophytes with base leaves less than 1.5 cm were continuously suspended in culture until the base leaves grew to 1.5-2.0 cm, and then clamped on ropes. The young sporophytes clamped on ropes were temporarily cultivated indoors for 15-20 days.

[0069] S10. Sea area release of new germplasm young sporophytes of Sargassum fusiforme

[0070] S11. Evaluation method of wild Sargassum fusiforme parents and offspring new germplasms

[0071] S11.1. Evaluation of wild Sargassum fusiforme parents and offspring traits

[0072] S11.2. Evaluation of wild Sargassum fusiforme parents and offspring yield

[0073] S11.3. Evaluation of processing ratio of wild Sargassum fusiforme parents, offspring dry raw materials and crude processed products

[0074] S11.4. Evaluation of rehydration rate of wild Sargassum fusiforme parents and offspring crude processed products

[0075] S11.5. Evaluation of wild Sargassum fusiforme parents and offspring algae and main nutritional components of products

[0076] S11.6. Analysis of genetic stability of wild Sargassum fusiforme offspring-new germplasms.

[0077] The culture seawater comprises the following preparation steps: fresh filtered seawater in the wild area is stored in a 1000 L chemical barrel, transported to the factory area, then moved to the indoor device in a 25 L chemical barrel, the seawater is heated to 80-90 ℃ using a water heater, high temperature for 30-40 min to kill animal eggs, microorganisms, microalgae and large seaweed young, naturally cooled to room temperature, and then 4-5 L of seawater is added per 100 g of Sargassum fusiforme to match the ratio of water placement in the water storage barrel of the temporary cultivation device; the nutrient salt is added at a volume ratio (V:V) of 0.6-0.8 ml:1 L of culture seawater; the light intensity is adjusted to 200-280 µmol•m -2 •s -1 -2 80 µmol•m -2 •s -1, the photoperiod is 14 h:10 h~12 h:12 h, and the temperature is 24 DEG C~25 DEG C;the parent female and male quality ratio of the vegetable is 2:1~1:1.

[0078] As a further setting of the above scheme, in the S3.1.1 step, the epiphytic large seaweed removed manually includes stone pure, enteromorpha, fresh vegetable and the like which are epiphytic on the new germplasm parent of the saccharina japonica;in the S3.1.2 step, the epiphytic animals removed by low permeability include hook shrimp, bamboo-like insects, fish eggs and the like which are epiphytic on the new germplasm parent of the saccharina japonica.

[0079] As a further setting of the above scheme, in the S7.2.1 step, the volume ratio (V:V) of the nutrient salt and the culture seawater of the saccharina japonica fertilized egg to the young sporophyte (trilobes to multilobes) suspended culture growth stage is (0.5-0.6) ml:1L.

[0080] As a further setting of the above scheme, in the S7 step, the main structure of the saccharina japonica fertilized egg suspended culture device includes a 5000 ml glass triangular cone bottle, a glass tube, an air control valve, a gas supply pump and a hose, and the low-density quantitative dispensing operation is implemented in the suspended culture process.

[0081] As a further setting of the above scheme, in the S10 step, the raft device for the natural sea area isolation and culture of the new germplasm seedling of the saccharina japonica adopts a soft raft design, the whole frame is 100 m*100 m in length*width, and nine equal hanging areas are arranged in the frame, so that the new germplasm seedling of the saccharina japonica is hung at the four corners of the raft and the surrounding net is arranged to ensure the isolation culture effect.

[0082] The method for creating and evaluating new germplasm of saccharina japonica provided by the application is suitable for the fields of scientific research and agricultural production such as creation of new germplasm of saccharina japonica, evaluation of genetic stability of new germplasm of saccharina japonica, screening of high-quality strains of new germplasm of saccharina japonica, hybrid breeding and molecular breeding of saccharina japonica, and creation of new varieties of saccharina japonica, and can provide scientific and standard operation and evaluation methods for creation of new germplasm of saccharina japonica, quality evaluation of new germplasm, and breeding of new varieties of saccharina japonica.

[0083] Fig. 1 The saccharina japonica new germplasm fertilized egg is suspended cultured in a room.

[0084] Fig. 2 The saccharina japonica new germplasm young sporophyte is temporarily cultured in a room and released in a sea area.

[0085] Fig. 3 The raft is used for isolation culture of seedlings of different strains (wild germplasm) of saccharina japonica. Specific embodiments

[0086] In order to embody the technical solutions and operation processes of the present application, the present application is further described in detail below in combination with specific implementation examples.

[0087] Reference Figs. 1-3 The method for creating and evaluating new germplasm of Hizikia fusiformis zygote suspension culture of the present application shown includes the following structures and operation steps:

[0088] a. The parent and offspring seedling quality evaluation, new germplasm parent and offspring trait evaluation method, new germplasm parent and offspring yield evaluation method, processing ratio evaluation of dry raw materials and rough processing products, rehydration rate evaluation of rough processing products, algal and product main nutrient component evaluation, and new germplasm genetic stability analysis of the present application are implemented by the method of the authorized invention patent “Unit mass evaluation and difference comparison method of Hizikia fusiformis young sporophyte cultivation” (ZL 202110213367.1); the new germplasm parent and offspring trait evaluation of Hizikia fusiformis is implemented by the method of the authorized invention patent “Hizikia fusiformis strain identification and classification method” (ZL201511004442.4); the new germplasm parent and offspring yield evaluation of Hizikia fusiformis is implemented by the method of the authorized invention patent “Evaluation and comparison method of unit fresh / dry weight yield and water content of cultured Hizikia fusiformis primary product” (ZL201911080353.6); the processing ratio evaluation of Hizikia fusiformis dry raw materials and rough processing products is implemented by referring to the relevant content of the local technical specification “Hizikia fusiformis primary dry product quality grading specification” (DB330305 / T 24-2023) of Dongtou District, Wenzhou City, which is drafted by the inventors and has been published; the rehydration rate evaluation of Hizikia fusiformis rough processing products is implemented by referring to the relevant content of the local technical specification “Hizikia fusiformis primary dry product quality grading specification” (DB330305 / T 24-2023) of Dongtou District, Wenzhou City, which is drafted by the inventors and has been published; the Hizikia fusiformis algal and product main nutrient component evaluation is implemented by the method of the authorized invention patents “Hizikia fusiformis primary product dry product quality grade classification method” (ZL201911080356.X) and “Breeding Hizikia fusiformis gas bag outlet product quality grade classification method” (ZL201911132779.1); and the new germplasm genetic stability analysis of Hizikia fusiformis is implemented by referring to the scientific research paper “Strain screening research of Zhejiang Dongtou cultivated Hizikia fusiformis (Sargassaceae) based on clustering and simplified genome combined analysis”, which is published by the inventors, and the technical process of the present application is systematically programmed. Sargassum fusiforme

[0089] ​b.The illumination culture rack and the temporary breeding device group are independently designed and built by the inventor, and can meet the regulation of optimal temperature, optimal light intensity, optimal light cycle and optimal seawater, etc. The main structure of the illumination culture rack includes carbon steel left and right side frames, tempered glass panels, glass panel bottom frames, crossbars, switch boxes and LED lighting lamps, and is placed in a controllable darkroom that meets the temporary breeding conditions in the seawater biological room, which can guarantee 0 µmol•m -2 •s -1 ~350 µmol•m -2 •s -1 The light intensity can be regulated by an external power supply timing converter; the culture room is an indoor darkroom, and air conditioning equipment is provided to regulate the space temperature in the culture room.

[0090] The carbon steel left and right side frames cooperate with the glass panel bottom frames arranged therebetween to form a main frame, and the tempered glass panels are fixed in the frame layer by layer. It is worth noting that the glass panel bottom frame in the middle of the frame is detachably arranged relative to the left and right side frames for regulating the height. The LED lighting lamps are fixed on the glass panel bottom frame below the tempered glass panels, and the LED lighting lamps are downwardly irradiated. The switch box is provided to control the independent switch of the LED lighting lamps. The culture trays of the temporary breeding device group are placed above the tempered glass panels. The tempered glass can also improve light transmission and light utilization based on the light transmission characteristics.

[0091] The main structure of the temporary breeding device group includes a water storage bucket, a culture tray, a peristaltic pump, an oxygen supply pump, a water supply hose and a sand head, which can realize seawater centralized oxygenation, water supply and circulation, and can guarantee sufficient oxygen and nutrient supply for the temporary breeding of Sargassum fusiforme.

[0092] c.The indoor temporary breeding of the new Sargassum fusiforme germplasm parent includes seawater storage, seawater sterilization, seawater replacement, optimal light intensity, light cycle and temperature setting, and matching of female and male Sargassum fusiforme, etc. The culture seawater for seawater storage includes the following preparation steps: fresh and filtered seawater in the wild is stored in a 1000 L chemical bucket, transported to the factory area, then divided into 25 L chemical buckets and transported to the indoor room, the seawater is heated to 80 ℃-90 ℃ by using a water heater, high temperature for 30 min-40 min, and the animal eggs, microorganisms, microalgae and large seaweed larvae are killed, and then naturally cooled to room temperature. 4 L-5 L seawater is matched with every 100 g of Sargassum fusiforme, and then added to the water storage bucket of the temporary breeding device group; the volume ratio (V:V) of nutrient salt to culture seawater is (0.6-0.8) ml:1 L; the light intensity is adjusted to 200 µmol•m -2 •s -1 ~280 µmol•m -2 •s -1The photoperiod is 14 h:10 h to 12 h:12 h, and the temperature is 24 ℃ to 25 ℃; the male-female ratio of the parent plants is 2:1 to 1:1.

[0093] d. The pest control methods involved in this invention include manual removal of large epiphytic seaweed, freshwater soaking to remove epiphytic animals, and chemical soaking to remove epiphytic microalgae. Large seaweeds such as *Leptochloa crus-galli*, *Ulva prolifera*, and *Vegetaria stenoptera* epiphytic on the new germplasm parent of *Sargassum fusiforme* are removed by manual removal. Epiphytic shrimp, stick insects, and fish eggs on the new germplasm parent of *Sargassum fusiforme* are removed by soaking in freshwater for 15-20 minutes to kill them with low osmotic pressure. Microalgae epiphytic on the new germplasm parent of *Sargassum fusiforme* are removed by soaking in citric acid (C6H8O7) solution at a mass (kg): volume (L) ratio of 1:3, with a citric acid concentration of 1.5 mg / ml, for 5-8 minutes. After removal, the microalgae are rinsed 3-5 times with room temperature fresh water and then rinsed 3-5 times with fresh, filtered, and sterilized seawater. The progeny young sporophytes of epiphytic cyanobacteria (cyanobacteria) were cultured by soaking in seawater solution containing 300,000 to 600,000 penicillin per L for 24 to 48 hours to disinfect the cyanobacteria. Afterward, they were washed with filtered seawater 3 to 5 times and then cultured in filtered seawater again to ensure no interference from harmful organisms.

[0094] e. The optimal nutrients involved in this invention include nitrogen (N) and phosphorus (P) elements. The optimal nutrient ratio is a seawater solution prepared with NaNO3 and KH2PO3, where N:P = 10:1, and the concentration is 10 (N) g / L or 1 (P) g / L. The volume ratio (V:V) of nutrients to culture seawater during the suspension culture growth and development stage of Sargassum fusiforme fertilized eggs to young sporophytes (trifoliolate to multifoliolate) is (0.5–0.7) ml:1 L to (0.6–0.8) ml:1 L.

[0095] f.The present application relates to the Sargassum fusiforme zygote suspension culture device group is independently designed and built by the inventor, which can guarantee the indoor suspension growth and development environment of Sargassum fusiforme zygote. The main structure of the Sargassum fusiforme zygote suspension culture device group includes a 5000 ml glass triangular flask, a glass tube, an air control valve, a gas supply pump, a hose and the like (all are standard parts). The glass triangular flask is opened in the darkroom environment, one end of the glass tube is connected with the hose, and the other end is inserted into the water level of the glass triangular flask. The other end of the hose is connected with the gas supply pump, and the air control valve is arranged on the hose to control the air inlet. When the device is used, the gas supply pump pressurizes the gas into the glass triangular flask through the hose. The glass tube is inclined relative to the triangular flask, and the outlet forms an angle with the bottom of the bottle. The gas stream drives the water to form a vortex, which drives the water in the bottle and forms a stirring force to drive the zygote at the bottom of the bottle to move in the water, forming a suspension culture basis, which guarantees that the zygote will not deposit at the bottom of the bottle. During the suspension culture process, low-density quantitative dispensing operation is also carried out. The density is gradually reduced from 8000 to 10000 zygotes per 5000 ml seawater to 400 to 500 young sporophytes per 5000 ml seawater, which guarantees normal growth and development.

[0096] g.The raft device group for isolating and raising the new germplasm seedlings of Sargassum fusiforme in a natural sea area is independently designed and built, which can guarantee the purity of the new germplasm. The raft device for isolating and raising the new germplasm seedlings of Sargassum fusiforme in a natural sea area is designed as a soft raft, and the whole raft has a length of 100 m and a width of 100 m. Nine equal hanging areas are arranged in the raft. In order to guarantee the isolation and breeding effect, the new germplasm seedlings of Sargassum fusiforme are hung at the four corners of the raft, and a purse seine is arranged to guarantee the purity of the germplasm.

[0097] Example 1: New germplasm creation and evaluation of Shandong wild Sargassum fusiforme parent to artificial domestication and breeding in Wenzhou Dongtou District, Zhejiang Province. The wild Sargassum fusiforme parent in Wenzhou Dongtou District is used for comparison.

[0098] In this example, the geographical position difference and ecological factor difference of the original growth place and the domestication and breeding place of the wild Sargassum fusiforme plant are not significant.

[0099] As the first embodiment of the present application, the method is suitable for the new germplasm creation and evaluation of wild Sargassum fusiforme parent with sexual reproduction of “north to south migration”. The method for creating and evaluating the new germplasm of Sargassum fusiforme zygote suspension culture includes the following steps:

[0100] 1. Selection, isolation and breeding and unit mass evaluation of wild Sargassum fusiforme parent

[0101] The present application takes Wenzhou Dongtou District, which is known as the “hometown of Sargassum fusiforme in China”, as the geographical center, and takes Shandong wild Sargassum fusiforme (north to south migration) as the wild parent.

[0102] Every October, a suitable amount of wild Sargassum fusiforme parent plants are collected from an island in Shandong, and then ecologically isolated and cultured in the Dongtou sea area after being secured with ropes.

[0103] The quality evaluation of wild Sargassum fusiforme parent (new germplasm parent) was carried out with reference to the method of the authorized invention patent "A method for unit quality evaluation and difference comparison of Sargassum fusiforme larval sporophyte cultivation" (ZL202110213367.1).

[0104] 2. Screening, isolation, and biological control of male and female plants of high-value parental strains of wild Sargassum fusiforme

[0105] The screening of male and female plants of high-value parental traits of wild Sargassum fusiforme was based on the inventor's published research paper, "Symplocos fusiforme cultivated in Dongtou, Zhejiang Province, based on clustering and simplified genome joint analysis". Sargassum fusiforme The relevant content of the "Research on Strain Screening" and the authorized invention patent "A Method for Strain Identification and Classification of Sargassum" (ZL201511004442.4) shall be implemented; the isolation breeding and pest biological control shall be implemented in accordance with the relevant content of the Wenzhou City Dongtou District Local Technical Specifications "Technical Specifications for High-Quality Seedling Breeding of Sargassum" (DB330305 / T22-2023) and "Technical Specifications for Sargassum Cultivation and Management" (DB330305 / T23-2023) drafted and published by the inventor.

[0106] The quality evaluation of the offspring of wild Sargassum fusiforme parents was carried out in accordance with the method of the authorized invention patent "A method for evaluating the unit quality and comparing differences in the cultivation of Sargassum fusiforme larval sporophytes" (ZL202110213367.1).

[0107] 3. Removal of epiphytic pests from male and female plants of high-value parental strains of wild Sargassum fusiforme.

[0108] 3.1 High-value trait strains of wild Sargassum fusiforme parent plants, male and female plants temporarily cultured indoors

[0109] 3.1.1 Removal of epiphytic macroalgae

[0110] Removed manually.

[0111] 3.1.2 Removal of Epiphytes

[0112] Soak in room temperature fresh water for 20 minutes to kill epiphytes using hypotonic methods, then rinse three times with fresh, filtered, and sterilized seawater.

[0113] 3.1.3 Removal of epiphytic microalgae

[0114] The ratio of fresh algal body weight to citric acid (C6H8O7) solution mass (kg): volume (L) was 1:3, the concentration of citric acid was 1.5 mg / ml, the soaking time was 5 min, and after removal, the algae were rinsed 3 times with room temperature fresh water, and then rinsed 3 times with fresh, filtered, and sterilized seawater.

[0115] 3.2 Wild parent high-value trait line of S. japonica female and male plant treatment

[0116] 3.2.1 Lateral branch screening and stem removal

[0117] Remove the primary or secondary lateral branch stems of fresh algal bodies, and retain the secondary or tertiary lateral branches densely growing on the receptacles.

[0118] 3.2.2 Lateral branch placement of female and male plants and mass ratio

[0119] According to the actual area of the culture tray, the lateral branches are reasonably distributed, and the distribution standard is that the lateral branches do not overlap each other, so as to ensure uniform illumination. The mixed mass ratio (kg: kg) of female plants and male plants is 1:1.

[0120] 4 Optimal environmental factors for indoor temporary cultivation of wild parent high-value trait line of S. japonica female and male plant lateral branches

[0121] 4.1 Optimal water environment

[0122] Fresh: Take the clear seawater of the same day rising tide, and naturally settle for 3 h;

[0123] Filter: The supernatant seawater is filtered through a 280-mesh gauze net;

[0124] High-temperature sterilization: The filtered seawater is heated to 80℃, high-temperature sterilized for 30 min, and the animal eggs, microorganisms, microalgae and large seaweed larvae are killed, and then naturally cooled to room temperature;

[0125] pH value: Adjust the pH value of the sterilized seawater to 7.8 with 0.05 mol / L NaOH solution and 0.05 mol / L HCl solution;

[0126] Salinity: 28;

[0127] Nutrient salt: NaNO3 and KH2PO3 are used to configure seawater solution with N:P=10:1, and the concentration is 10 (N) g / L or 1 (P) g / L. The volume ratio (V:V) of nutrient salt to culture seawater is 0.6 ml:1 L.

[0128] 4.2 Culture room, light culture shelf and temporary cultivation device

[0129] The culture room used in the present application is an indoor darkroom. The culture room is based on a light culture shelf for light intensity control, and the temporary cultivation device is placed on the layer plate of the light culture shelf. The lower side of the plate body between layers is provided with LED lamp tubes to provide light;

[0130] 4.3 Optimal temperature, light intensity and photoperiod

[0131] Temperature: 23℃;

[0132] Light intensity: 200 µmol•m -2 •s -1

[0133] Photoperiod: 14 h: 10 h.

[0134] 5. Treatment and duration of indoor temporary cultivation of lateral branches from male and female plants of high-value trait parental lines of wild Sargassum fusiforme.

[0135] 5.1 Indoor Temporary Care Treatment of Lateral Branches

[0136] Half the total volume of seawater in the culture group is replaced daily, and the lateral branches are turned over once every 6 hours to ensure that the reproductive receptacles of each lateral branch receive uniform light.

[0137] 5.2 Indoor temporary cultivation time of lateral branches

[0138] Lateral branches were temporarily kept indoors for 5 days.

[0139] 6. Classification, collection, impurity removal, and cleaning of fertilized eggs from new Sargassum fusiforme germplasm.

[0140] Fertilized eggs were collected using a 260-mesh silk screen, and impurities such as air sacs and reproductive receptacles were removed using a 30-mesh silk screen. The fertilized eggs were placed in a 2000 mL beaker, and 1500 mL of fresh, filtered seawater was added. After stirring, the mixture was allowed to settle naturally for 2 minutes. The supernatant was discarded, and any remaining small suspended impurities were removed. This process was repeated 5 times to obtain a pure sample of fertilized eggs. Different strains require separate fertilized egg collection and impurity removal procedures.

[0141] 7. Creation of a suspension system for fertilized eggs of new Sargassum fusiforme germplasm

[0142] 7.1 Quantitative dispensing of fertilized eggs from new Sargassum fusiforme germplasm

[0143] Excess moisture was removed from the fresh fertilized eggs. The volume density of the fertilized eggs was statistically determined. 8000 fertilized eggs were quantitatively dispensed into each 3000 ml Erlenmeyer flask. Fresh filtered seawater was added, and the ratio of the number of fresh fertilized eggs to the volume of seawater was 10:1. Different strains need to be classified, packaged, and labeled.

[0144] 7.2 Establishment of the optimal environment for suspension culture of new Sargassum fusiforme germplasm fertilized eggs

[0145] 7.2.1 Optimal Water Environment

[0146] Freshness, filtration, high-temperature sterilization, pH value, salinity, and nutrient salt preparation were performed according to the method in Example 4.1. The volume ratio (V:V) of nutrient salt to culture seawater was 0.5 ml:1 L. The optimal aquatic environment conditions remained unchanged during suspension culture.

[0147] 7.2.2 Optimal light intensity, photoperiod, temperature, aeration, and other conditions

[0148] Optimum light intensity: 50 pmol m -2 •s -1 ;

[0149] Photoperiod: 14 h:10 h;

[0150] Temperature: 23 °C;

[0151] Aeration: assisted aeration.

[0152] 7.2.3 Suspension culture mode of Sargassum fusiforme new germplasm zygote

[0153] The Sargassum fusiforme zygote was initially suspended in culture using magnetic stirring + aeration suspension culture, and after 8 days of culture, the Sargassum fusiforme zygote had grown and developed into an embryo. At the beginning of the embryo growth and development period, a self-made suspension culture device was used for suspension culture (Fig. 7.2.3), and only aeration suspension culture was performed. Fig. 1

[0154] 7.3 Low-density quantitative sub-packaging of Sargassum fusiforme new germplasm embryo to young sporophyte suspension growth and development stage suspension culture

[0155] At the embryo to young sporophyte growth and development stage, 1 / 2 equal volume density sub-packaging was performed every 19 d per bottle. As the culture time gradually extended, the low-density quantitative sub-packaging was gradually reduced, i.e., the first low-density sub-packaging was reduced from 8000 to 4000, the second low-density was reduced from 4000 to 2000, the third low-density was reduced from 2000 to 1000, and the final low-density was reduced from 1000 to 400.

[0156] 8. Typical enemy biological control of Sargassum fusiforme new germplasm embryo to young sporophyte suspension growth and development stage suspension system

[0157] 8.1 Enemy animal control method

[0158] If enemy animals are found in the suspension culture solution, soak in ultrapure water for 20 min every 10 d of culture period, and perform low-osmotic killing of enemy animals.

[0159] 8.2 Enemy cyanobacteria control method

[0160] Periodically examine the embryos or young sporophytes under a microscope, and if the embryos or young sporophytes have signs of epiphytic blue-green algae, soak the culture in 300,000 count / L penicillin seawater solution for 24 h to kill the blue-green algae, then filter the seawater to wash 3 times, and continue the suspension culture using the filtered seawater.

[0161] 8.3 Enemy green algae control method

[0162] ​Periodically check the embryos or young sporophytes under a microscope. If the embryos or young sporophytes show signs of epiphyte growth, immerse them in 1.5 mg / ml citric acid solution for 6 minutes, rinse them with fresh water three times, rinse them with filtered seawater twice, and continue to suspend them in filtered seawater for culture.

[0163] 9. Clamping and indoor temporary cultivation of new germplasm young sporophytes of Sargassum fusiforme (Clamping and structure of young sporophytes, as shown in Fig. 2 )

[0164] Clamp young sporophytes with 1.5 cm long basal leaves onto cotton or fiber ropes with a diameter of 0.4 cm at a spacing of 3 cm. Young sporophytes with basal leaves less than 1.5 cm long continue to be suspended for culture until the basal leaves grow to a length of ≥1.5 cm before clamping. Clamp the young sporophytes and temporarily cultivate them indoors for 15 days.

[0165] 10. Sea area release of new germplasm young sporophytes of Sargassum fusiforme

[0166] This content is implemented in reference to the relevant content of the Local Technical Specification "Sargassum fusiforme Cultivation and Management Technical Regulations" (DB330305 / T23-2023) drafted and published by the inventors in Wenzhou Dongtou District.

[0167] 11. Evaluation method of wild Sargassum fusiforme parent and offspring new germplasm

[0168] This content refers to the methods of authorized invention patents, drafted and published local standards, and published scientific research papers. The new germplasm seed quality evaluation, trait evaluation, yield evaluation, processing ratio evaluation, rehydration rate evaluation, algal formation and product main nutrient component evaluation (including arsenic valence and composition), and new germplasm genetic stability evaluation are implemented item by item, as follows:

[0169] 11.1 Trait evaluation of wild Sargassum fusiforme parent and offspring

[0170] This content refers to the method of the authorized invention patent "Sargassum fusiforme strain identification and classification method" (ZL201511004442.4) of the inventors for evaluation.

[0171] 11.2 Yield evaluation of wild Sargassum fusiforme parent and offspring

[0172] This content refers to the method of the authorized invention patent "Evaluation and comparison method of unit fresh / dry weight yield and water content of cultured Sargassum fusiforme primary product" (ZL201911080353.6) of the inventors for evaluation.

[0173] 11.3 Processing ratio evaluation of wild Sargassum fusiforme parent and offspring dry product raw materials and rough processing products

[0174] The content refers to the relevant content of the local technical specification "Quality Grading Specification for Primary Dried Sargassum Fusiforme" (DB330305 / T24-2023) drafted and published by the inventor of the invention.

[0175] 11.4 Wild Sargassum fusiforme parent and offspring crude product rehydration rate evaluation

[0176] The content refers to the relevant content of the local technical specification "Quality Grading Specification for Primary Dried Sargassum Fusiforme" (DB330305 / T24-2023) drafted and published by the inventor of the invention.

[0177] 11.5 Wild Sargassum fusiforme parent and offspring algal and product main nutrient component evaluation

[0178] This content refers to the methods of the inventor's authorized invention patents "Method for Classifying Quality Grades of Primary Dried Sargassum Fusiforme Products" (ZL201911080356.X) and "Method for Classifying Quality Grades of Cultivated Sargassum Fusiforme Airbag Export Products" (ZL201911132779.1) for evaluation.

[0179] 11.6 Wild Sargassum fusiforme offspring-new germplasm genetic stability analysis

[0180] This content refers to the method of the inventor's published scientific paper "Study on Sargassum fusiforme (Harv.) Setch. Cultivar Screening in Zhejiang Dongtou Based on Clustering and Simplified Genome Joint Analysis" for qualitative analysis. Sargassum fusiforme

[0181] Example 2: Shandong wild Sargassum fusiforme parent and Guangdong wild Sargassum fusiforme parent are domesticated and cultivated in the same period in Wenzhou Dongtou District to create and evaluate new germplasm, and wild Sargassum fusiforme parent in Wenzhou Dongtou District is compared.

[0182] The geographical location and ecological factor differences of the original growth place, domestication and cultivation place of each wild Sargassum fusiforme plant in this example are not significant, and the differences among the original growth place, geographical location and ecological factor are significant.

[0183] As the first implementation example of the invention, it is suitable for "north to south" + "south to north" wild Sargassum fusiforme parent sexual reproduction new germplasm creation and evaluation. The Sargassum fusiforme zygote suspension culture new germplasm creation and evaluation method comprises the following steps:

[0184] 1. Selection, isolation and unit mass evaluation of wild Sargassum fusiforme parent

[0185] ​This invention centers on Dongtou District of Wenzhou City, known as the "Hometown of Sargassum fusiforme in China," and uses wild Sargassum fusiforme from Shandong (northern species migrated south) and wild Sargassum fusiforme from Guangdong (southern species migrated north) as geographically differentiated wild parent plants. Every November, appropriate quantities of wild Sargassum fusiforme parent plants are collected from islands in Shandong and Guangdong, and then ecologically isolated and cultured in the Dongtou sea area after being secured with ropes. The quality evaluation of the wild Sargassum fusiforme parent plants (new germplasm parent plants) is carried out in accordance with the method outlined in the inventor's authorized invention patent, "A Method for Unit Quality Evaluation and Difference Comparison of Sargassum fusiforme Larval Sporophyte Cultivation" (ZL202110213367.1).

[0186] 2. Screening, isolation, and biological control of male and female plants of high-value parental strains of wild Sargassum fusiforme.

[0187] The screening of male and female plants of high-value parental traits of wild Sargassum fusiforme was conducted in accordance with the inventor's published research paper, "Sargassum fusiforme cultivated in Dongtou, Zhejiang Province, based on clustering and simplified genome joint analysis". Sargassum fusiforme The research on strain selection and the relevant content of the authorized invention patent "A Method for Strain Identification and Classification of Sargassum fusiforme" (ZL201511004442.4) were implemented. Isolation breeding and biological control of pests were implemented in accordance with the relevant content of the Wenzhou City Dongtou District local technical specifications "Technical Regulations for High-Quality Seedling Breeding of Sargassum fusiforme" (DB330305 / T22-2023) and "Technical Regulations for Cultivation and Management of Sargassum fusiforme" (DB330305 / T23-2023), drafted and published by the inventor. The quality evaluation of the offspring of wild Sargassum fusiforme parents was implemented in accordance with the method of the inventor's authorized invention patent "A Method for Unit Quality Evaluation and Difference Comparison of Sargassum fusiforme Larval Sporophyte Cultivation" (ZL202110213367.1).

[0188] 3. Removal of epiphytic pests from male and female plants of high-value parental strains of wild Sargassum fusiforme.

[0189] 3.1 High-value trait strains of wild Sargassum fusiforme parent plants, male and female plants temporarily cultured indoors

[0190] 3.1.1 Removal of epiphytic macroalgae

[0191] Removed manually.

[0192] 3.1.2 Removal of Epiphytes

[0193] Soak in room temperature fresh water for 23 minutes to kill epiphytes using hypotonic methods, then rinse four times with fresh, filtered, and sterilized seawater.

[0194] 3.1.3 Removal of epiphytic microalgae

[0195] Fresh weight of algal body and mass (kg) : volume (L) of citric acid (C6H8O7) solution was 1:3, the concentration of citric acid was 1.5 mg / ml, the soaking time was 7 min, and after taking out, it was washed with fresh water at room temperature for 4 times, and fresh, filtered, and sterilized seawater for 4 times.

[0196] 3.2 Wild parent high-value trait line of S. polyidium, female and male treatment

[0197] 3.2.1 Lateral branch screening and stem removal

[0198] Remove the primary or secondary lateral branch stems of fresh algal body, and retain the secondary or tertiary lateral branches densely growing on the receptacle.

[0199] 3.2.2 Lateral branch placement of female and male plants and mass ratio

[0200] According to the actual area of the culture tray, the lateral branches are reasonably distributed, and the distribution standard is that the lateral branches do not overlap each other, so as to ensure uniform illumination. The mixed mass ratio (kg:kg) of female and male plants is 1:1.

[0201] 4. Optimal environmental factors for indoor temporary cultivation of wild S. polyidium parent high-value trait line of female and male lateral branches

[0202] 4.1 Optimal water environment

[0203] Fresh: Take the clear seawater of the same day when the tide rises, and naturally settle for 3.5 h;

[0204] Filtration: The supernatant seawater is filtered through a 290-mesh gauze net;

[0205] High-temperature sterilization: The filtered seawater is heated to 85 ℃, high-temperature sterilized for 35 min, and the animal eggs, microorganisms, microalgae, and large seaweed larvae are killed, and the temperature is naturally lowered to room temperature;

[0206] pH value: 0.05 mol / L NaOH solution and 0.05 mol / L HCl solution are used to adjust the pH value of the seawater after sterilization to 8.0;

[0207] Salinity: 30;

[0208] Nutrient salt: NaNO3 and KH2PO3 are used to configure seawater solution with N:P=10:1, and the concentration is 10 (N) g / L or 1 (P) g / L. The volume ratio (V:V) of nutrient salt to culture seawater is 0.7 ml:1 L.

[0209] 4.2 Culture room, illumination culture rack and temporary cultivation device Embodiment

[0210] 4.3 Optimal temperature, light intensity and photoperiod

[0211] Temperature: 24 ℃;

[0212] Light intensity: 240 µmol•m -2 •s -1 ;

[0213] Photoperiod: 13 h:11 h.

[0214] 5 Wild parent high-value traits of Sargassum siliquastrum strain female and male lateral branches indoor temporary treatment and temporary time

[0215] 5.1 Lateral branch indoor temporary treatment

[0216] Replace 1 / 2 capacity of seawater in the culture group every day, and turn the lateral branches once every 7 hours to ensure that each lateral branch receptacle receives uniform light.

[0217] 5.2 Lateral branch indoor temporary time

[0218] Indoor temporary culture of lateral branches for 6 days.

[0219] 6. Classification and collection of fertilized eggs, impurity removal and washing of Sargassum siliquastrum new germplasm

[0220] Use 270 gauze to collect fertilized eggs, and 40-mesh gauze to remove impurities such as air sacs and receptacles. Add 1500 mL of fresh filtered seawater to a 2000 mL beaker, stir well, and let it settle naturally for 2.5 minutes. Discard the upper suspension to remove suspended impurities. Repeat the operation 6 times to obtain fresh pure fertilized eggs. Different strains need to be collected and impurities removed separately.

[0221] 7. Creation of a suspension system for Sargassum siliquastrum new germplasm fertilized eggs

[0222] 7.1 Quantitative dispensing of Sargassum siliquastrum new germplasm fertilized eggs

[0223] Remove excess water from the fresh pure fertilized eggs, and statistically determine the volume density of the fertilized eggs. Quantitatively dispense 9000 fertilized eggs in each 5000 ml triangular flask, and add fresh filtered seawater. The ratio of the number of fresh pure fertilized eggs (pieces) to the volume of seawater (ml) is 10:2. Different strains need to be classified and labeled.

[0224] 7.2 Creation of the optimal environment for Sargassum siliquastrum new germplasm fertilized egg suspension culture

[0225] 7.2.1 Optimal water environment

[0226] Fresh, filtered, high-temperature sterilized, pH, salinity and nutrient salt configuration refer to the method of this embodiment 4.1. The volume ratio (V:V) of nutrient salt to culture seawater is 0.55 ml:1 L. The optimal water environment remains unchanged during the suspension culture period.

[0227] 7.2.2 Optimum light intensity, photoperiod, temperature, aeration, etc.

[0228] Optimum light intensity: 55 pmol m2s -2 •s -1 ;

[0229] Photoperiod: 13 h:11 h;

[0230] Temperature: 23.5 °C;

[0231] Aeration: auxiliary aeration.

[0232] 7.2.3 Suspension culture method of fertilized eggs of new germplasm of Sargassum fusiforme

[0233] The fertilized eggs of Sargassum fusiforme were initially cultured by magnetic stirring + aeration suspension culture, and after 9 days of culture, the fertilized eggs of Sargassum fusiforme had grown and developed into embryos. At the beginning of the embryo growth and development period, the self-made suspension culture device group was used for suspension culture ( Fig. 1 ), and only aeration suspension culture was implemented.

[0234] 7.3 Low-density quantitative sub-packaging of suspension culture of new germplasm of Sargassum fusiforme embryos to young sporophytes at the suspension growth and development stage

[0235] At the embryo to young sporophyte growth and development stage, 1 / 2 equal volume density sub-packaging was performed every 20 d per bottle. With the gradual extension of the culture time, the low-density quantitative sub-packaging was gradually reduced, i.e., the first low-density sub-packaging was reduced from 9000 to 4500, the second low-density was reduced from 4500 to 2250, the third low-density was reduced from 2250 to 1125, and the last low-density was reduced from 1125 to 562.

[0236] 8. Typical enemy biological control of suspension lines of new germplasm of Sargassum fusiforme embryos to young sporophytes at the suspension growth and development stage

[0237] 8.1 Enemy animal control method

[0238] If enemy animals are found in the suspension culture solution, use ultrapure water to soak for 23 min every 11 d of culture period to reduce the osmotic pressure and kill the enemy animals.

[0239] 8.2 Enemy cyanobacteria control method

[0240] Periodically examine the embryos or young sporophytes under a microscope, and if signs of epiphytic blue algae are found on the embryos or young sporophytes, use 500,000 counts / L of penicillin seawater solution to soak the culture for 36 h to kill the blue algae, and then filter the seawater to wash it 4 times, and continue to use the filtered seawater for suspension culture.

[0241] 8.3 Enemy green algae control method

[0242] Periodically check the embryos or young sporophytes under a microscope. If the embryos or young sporophytes show signs of epiphyte green algae, immerse them in 1.5 mg / ml citric acid solution for 7 minutes, rinse them with fresh water four times, rinse them with filtered seawater three times, and continue to suspend them in filtered seawater for culture.

[0243] 9. Clamping of new germplasm young sporophytes of Sargassum fusiforme and temporary indoor cultivation

[0244] Clamp the young sporophytes with basal leaves 1.5 cm to 2.0 cm in length onto cotton ropes or fiber ropes with a diameter of 0.4 cm at an interval of 4 cm. The young sporophytes with basal leaves less than 1.5 cm in length continue to be suspended for culture until the length of the basal leaves is greater than or equal to 1.8 cm, and then clamp the ropes. The young sporophytes after clamping are temporarily cultivated indoors for 18 days.

[0245] 10. Sea area release of new germplasm young sporophytes of Sargassum fusiforme

[0246] This content is implemented according to the relevant content of the Local Technical Specification “Sargassum fusiforme Cultivation and Management Technical Regulations” (DB330305 / T23-2023) of Dongtou District, Wenzhou City, which is drafted by the first inventor of the present invention and has been published.

[0247] 11. Evaluation method of wild Sargassum fusiforme parent and new germplasm offspring

[0248] This content refers to the methods of the inventor's authorized invention patents, drafted and published local standards, and published scientific research papers. The quality evaluation of new germplasm seedlings, trait evaluation, yield evaluation, processing ratio evaluation, rehydration rate evaluation, algal formation and product main nutrient component evaluation (including arsenic valence and composition), and new germplasm genetic stability evaluation are implemented item by item.

[0249] 11.1 Trait evaluation of wild Sargassum fusiforme parent and offspring

[0250] This content refers to the method of the inventor's authorized invention patent “A Method for Identifying and Classifying Strains of Sargassum fusiforme” (ZL201511004442.4) for evaluation.

[0251] 11.2 Yield evaluation of wild Sargassum fusiforme parent and offspring

[0252] This content refers to the method of the inventor's authorized invention patent “Method for Evaluating and Comparing Fresh / Dry Weight Yield and Moisture Content of Cultivated Sargassum fusiforme Primary Products” (ZL201911080353.6) for evaluation.

[0253] 11.3 Processing ratio evaluation of wild Sargassum fusiforme parent, offspring dry product raw materials and rough processing products

[0254] This content is evaluated with reference to the relevant content of the Wenzhou City Dongtou District Local Technical Specification "Quality Grading Specification for Initially Dried Seaweed" (DB330305 / T24-2023), which was drafted and published by the inventor.

[0255] 11.4 Evaluation of the rehydration rate of primary processed products from wild Sargassum fusiforme parent and offspring

[0256] This content is evaluated with reference to the relevant content of the Wenzhou City Dongtou District Local Technical Specification "Quality Grading Specification for Initially Dried Seaweed" (DB330305 / T24-2023), which was drafted and published by the inventor.

[0257] 11.5 Evaluation of the main nutritional components of wild Sargassum fusiforme parent plants, offspring algae, and products

[0258] This evaluation is conducted with reference to the methods in the inventor's authorized invention patents "A method for classifying the quality grade of primary dried Sargassum fusiforme products" (ZL201911080356.X) and "A method for classifying the quality grade of exported Sargassum fusiforme airbag products" (ZL201911132779.1).

[0259] 11.6 Analysis of the genetic stability of wild Sargassum fusiforme offspring—new germplasm

[0260] This section references the inventor's published research paper, "Cultivation of Sargassum fusiforme in Dongtou, Zhejiang Province Based on Clustering and Simplified Genome Joint Analysis" Sargassum fusiforme Qualitative analysis was conducted using the methods described in "Strain Screening Research".

[0261] Example 3: Creation and evaluation of new germplasm from wild Sargassum fusiforme parents from Shandong, Guangdong, and Nanji Island (near the same area) in Wenzhou City, during the same period, through artificial domestication and cultivation in Dongtou District, Wenzhou City. Wild Sargassum fusiforme parents from Dongtou District, Wenzhou City were used for comparison. In this example, the geographical location and ecological factors of the original growth sites and domestication and cultivation sites of the wild Sargassum fusiforme plants were not significant, but the differences in original growth sites, geographical locations, and ecological factors among the four groups were significant.

[0262] As the first embodiment of the present invention, it is applicable to the creation and evaluation of new germplasm from sexual reproduction of wild Sargassum fusiforme parents that combine "northern species transplanted to the south" + "southern species transplanted to the north" + "local" species. The method for creating and evaluating new germplasm from Sargassum fusiforme fertilized egg suspension culture includes the following steps:

[0263] 1. Selection, isolated culture, and unit quality evaluation of wild Sargassum fusiforme parent plants

[0264] This invention centers on Dongtou District of Wenzhou City, known as the "Hometown of Sargassum fusiforme in China," and uses three geographically distinct wild parent plants: Shandong wild Sargassum fusiforme (northern species migrated south), Wenzhou wild Sargassum fusiforme (from a similar region), and Guangdong wild Sargassum fusiforme (southern species migrated north). Every December, appropriate quantities of wild Sargassum fusiforme parent plants are collected from islands in Shandong, Guangdong, and Wenzhou, and then ecologically isolated and cultured in the Dongtou sea area after being secured with ropes. The quality evaluation of the wild Sargassum fusiforme parent plants (new germplasm parent plants) is carried out in accordance with the method outlined in the inventor's authorized invention patent, "A Method for Unit Quality Evaluation and Difference Comparison of Sargassum fusiforme Larval Sporophyte Cultivation" (ZL202110213367.1).

[0265] 2. Screening, isolation, and biological control of male and female plants of high-value parental strains of wild Sargassum fusiforme.

[0266] The screening of male and female plants of high-value parental traits of wild Sargassum fusiforme was conducted in accordance with the inventor's published research paper, "Sargassum fusiforme cultivated in Dongtou, Zhejiang Province, based on clustering and simplified genome joint analysis". Sargassum fusiforme The research on strain selection and the relevant content of the authorized invention patent "A Method for Strain Identification and Classification of Sargassum fusiforme" (ZL201511004442.4) were implemented. Isolation breeding and biological control of pests were implemented in accordance with the relevant content of the Wenzhou City Dongtou District local technical specifications "Technical Regulations for High-Quality Seedling Breeding of Sargassum fusiforme" (DB330305 / T22-2023) and "Technical Regulations for Cultivation and Management of Sargassum fusiforme" (DB330305 / T23-2023), drafted and published by the inventor. The quality evaluation of the offspring of wild Sargassum fusiforme parents was implemented in accordance with the method of the inventor's authorized invention patent "A Method for Unit Quality Evaluation and Difference Comparison of Sargassum fusiforme Larval Sporophyte Cultivation" (ZL202110213367.1).

[0267] 3. Removal of epiphytic pests from male and female plants of high-value parental strains of wild Sargassum fusiforme.

[0268] 3.1 High-value trait strains of wild Sargassum fusiforme parent plants, male and female plants temporarily cultured indoors

[0269] 3.1.1 Removal of epiphytic macroalgae

[0270] Removed manually.

[0271] 3.1.2 Removal of Epiphytes

[0272] Soak in room temperature fresh water for 25 minutes to kill epiphytes using hypotonic methods, then rinse 5 times with fresh, filtered, and sterilized seawater.

[0273] 3.1.3 Removal of epiphytic microalgae

[0274] Fresh weight of algal body and mass (kg) : volume (L) of citric acid (C6H8O7) solution was 1:3, the concentration of citric acid was 1.5 mg / ml, the soaking time was 8 min, and after taking out, it was washed with fresh water at room temperature for 5 times, and fresh, filtered, and sterilized seawater for 5 times.

[0275] 3.2 Wild parent high-value trait line of S. polyidium, female and male treatment

[0276] 3.2.1 Lateral branch screening and stem removal

[0277] Remove the primary or secondary lateral branch stems of fresh algal body, and retain the secondary or tertiary lateral branches densely growing on the receptacle.

[0278] 3.2.2 Lateral branch placement and mass ratio of female and male

[0279] According to the actual area of the culture tray, reasonably allocate the lateral branches, the allocation standard is that the lateral branches do not overlap each other, so as to ensure uniform illumination. The mixed mass ratio (kg: kg) of female and male is 1:1.

[0280] 4. Optimal environmental factors for indoor temporary cultivation of wild parent high-value trait line of S. polyidium, female and male lateral branches

[0281] 4.1 Optimal water environment

[0282] Fresh: Take the clear seawater of the same day rising tide, and naturally settle for 4 h;

[0283] Filtration: The supernatant seawater is filtered through a 300-mesh gauze net;

[0284] High-temperature sterilization: The filtered seawater is heated to 90℃, high-temperature sterilized for 40 min, and the animal eggs, microorganisms, microalgae and large seaweed larvae are killed, and naturally cooled to room temperature;

[0285] pH value: 0.05 mol / L NaOH solution and 0.05 mol / L HCl solution are used to adjust the pH value of the seawater after sterilization to 8.3;

[0286] Salinity: 33;

[0287] Nutrient salt: NaNO3 and KH2PO3 are used to configure seawater solution with N:P=10:1, and the concentration is 10 (N) g / L or 1 (P) g / L. The volume ratio (V:V) of nutrient salt and culture seawater is 0.8 ml: 1 L.

[0288] 4.2 Culture room, illumination culture rack and temporary cultivation device

[0289] 4.3 Optimal temperature, light intensity and photoperiod

[0290] Temperature: 23 ℃~25 ℃;

[0291] Light intensity: 280 µmol•m -2 •s -1

[0292] Photoperiod: 12 h:12 h.

[0293] 5 Wild parent high-value trait line of Sargassum siliquastrum indoor treatment and temporary time of lateral branch

[0294] 5.1 Indoor treatment of lateral branch

[0295] The total culture seawater of the culture group was replaced by 1 / 2 capacity of seawater every day, and the lateral branches were turned over once every 8 hours to ensure that each lateral branch receptacle received uniform light.

[0296] 5.2 Indoor temporary time of lateral branch

[0297] The lateral branches were temporarily cultured indoors for 7 days.

[0298] 6. Classification and collection of fertilized eggs, removal of impurities, and washing of new germplasm of Sargassum siliquastrum

[0299] The fertilized eggs were collected using a 280-mesh gauze screen, and the air sacs, receptacles, and other impurities were removed using a 50-mesh gauze screen. The fertilized eggs were placed in a 2000-mL beaker, 1500 mL of fresh filtered seawater was added, and after stirring, they were allowed to settle naturally for 3 minutes. The upper suspension was discarded to remove suspended impurities, and the operation was repeated 7 times to obtain fresh pure fertilized eggs. Different lines need to be collected and impurities removed separately.

[0300] 7. Creation of a suspension system for fertilized eggs of new germplasm of Sargassum siliquastrum

[0301] 7.1 Quantitative dispensing of fertilized eggs of Sargassum siliquastrum

[0302] The excess water in the fresh pure fertilized eggs was removed, and the volume density of the fertilized eggs was statistically calculated. 10000 fertilized eggs were quantitatively dispensed into each 5000-ml triangular flask, and fresh filtered seawater was added. The ratio of the number of fresh pure fertilized eggs (pieces) to the volume of seawater (ml) was 10:3. Different lines need to be classified and labeled.

[0303] 7.2 Creation of optimal environment for suspension culture of fertilized eggs of new germplasm of Sargassum siliquastrum

[0304] 7.2.1 Optimal water environment

[0305] Fresh, filtered, high-temperature sterilized, pH, salinity, and nutrient salt configurations refer to the methods in Example 4.1. The volume ratio (V:V) of nutrient salt to culture seawater was 0.6 ml:1 L. The optimal water environment conditions remained unchanged during the suspension culture period.

[0306] 7.2.2 Optimal light intensity, photoperiod, temperature, aeration, and other conditions

[0307] Optimum light intensity: 60 pmol m -2 •s -1 ;

[0308] Photoperiod: 12 h:12 h;

[0309] Temperature: 24℃;

[0310] Aeration: assisted aeration.

[0311] 7.2.3 Suspension culture mode of Sargassum fusiforme new germplasm zygote

[0312] The Sargassum fusiforme zygote was initially suspended and cultured by magnetic stirring + aeration, and after 10 days of culture, the zygote had grown and developed into an embryo. At the beginning of the embryo growth and development period, the self-made suspension culture device group was used for suspension culture (see Fig. 1 ), and only aeration suspension culture was implemented.

[0313] 7.3 Low-density quantitative sub-packaging of Sargassum fusiforme new germplasm embryo to young sporophyte suspension growth and development stage

[0314] At the embryo to young sporophyte growth and development stage, 1 / 2 equal volume density sub-packaging was performed every 21 days per bottle. With the gradual extension of the culture time, the low-density quantitative sub-packaging was gradually reduced, i.e., the first low-density sub-packaging was reduced from 10,000 to 5,000, the second low-density was reduced from 5,000 to 2,500, the third low-density was reduced from 2,500 to 1,250, and the last low-density was reduced from 1,250 to 625.

[0315] 8. Typical enemy biological control of Sargassum fusiforme new germplasm embryo to young sporophyte suspension growth and development stage suspension system

[0316] 8.1 Enemy animal control method

[0317] If enemy animals are found in the suspension culture solution, immerse in ultrapure water for 25 min every 12 d culture period to kill the enemy animals by hypotonicity.

[0318] 8.2 Enemy cyanobacteria control method

[0319] Periodically examine the embryos or young sporophytes under a microscope, and if there are signs of cyanobacteria attached to the embryos or young sporophytes, immerse in 600,000 U / L penicillin seawater solution for 48 h to kill the cyanobacteria, then wash 5 times with filtered seawater, and continue to suspend and culture in the filtered seawater.

[0320] 8.3 Enemy green algae control method

[0321] Periodically check the embryos or young sporophytes under a microscope. If the embryos or young sporophytes show signs of epiphyte green algae, immerse them in 1.5 mg / ml citric acid solution for 8 minutes, rinse them with fresh water for 5 times, rinse them with filtered seawater for 3 times, and continue to suspend them in filtered seawater for culture.

[0322] 9. Clamping and indoor temporary cultivation of new germplasm young sporophytes of Sargassum fusiforme

[0323] Clamp the young sporophytes with base leaves 1.5 cm to 2.0 cm in length to cotton ropes or fiber ropes with a diameter of 0.4 cm, with a spacing of 3 cm to 5 cm. The young sporophytes with base leaves less than 1.5 cm in length continue to be suspended for culture until the length of the base leaves is greater than or equal to 2.0 cm, and then the young sporophytes are clamped to the ropes. After clamping, the young sporophytes are temporarily cultured indoors for 20 days, and then are transferred to natural sea areas for release.

[0324] 10. Release of new germplasm young sporophytes of Sargassum fusiforme in sea areas

[0325] This content is implemented according to the relevant content of the Local Technical Specification “Sargassum fusiforme Cultivation and Management Technical Regulations” (DB330305 / T23-2023) of Dongtou District, Wenzhou City, which is drafted by the first inventor of the present invention and has been published.

[0326] 11. Evaluation method of wild Sargassum fusiforme parent and new germplasm offspring

[0327] This content refers to the methods of the inventor's authorized invention patents, drafted and published local standards, and published scientific research papers. The quality evaluation, trait evaluation, yield evaluation, processing ratio evaluation, rehydration rate evaluation, algal formation and product main nutrient component evaluation (including arsenic valence and composition), and new germplasm genetic stability evaluation of new germplasm seedlings are implemented item by item.

[0328] 11.1 Trait evaluation of wild Sargassum fusiforme parent and offspring

[0329] This content refers to the method of the inventor's authorized invention patent “Line Identification and Classification Method of Sargassum fusiforme” (ZL201511004442.4) for evaluation.

[0330] 11.2 Yield evaluation of wild Sargassum fusiforme parent and offspring

[0331] This content refers to the method of the inventor's authorized invention patent “Evaluation and Comparison Method of Fresh / Dry Weight Yield and Water Content of Cultivated Sargassum fusiforme Primary Product” (ZL201911080353.6) for evaluation.

[0332] 11.3 Processing ratio evaluation of wild Sargassum fusiforme parent, offspring dry product and rough processing product

[0333] This content refers to the content of the invention drafted and published by the inventor Wenzhou Dongtou District Local Technical Specification "Quality Grading Specification for Sargassum Fusiforme Primary Dried Products" (DB330305 / T24-2023).

[0334] 11.4 Wild Sargassum Fusiforme Parent, Offspring Rough Processing Product Rehydration Rate Evaluation

[0335] This content refers to the content of the invention drafted and published by the inventor Wenzhou Dongtou District Local Technical Specification "Quality Grading Specification for Sargassum Fusiforme Primary Dried Products" (DB330305 / T24-2023).

[0336] 11.5 Wild Sargassum Fusiforme Parent, Offspring Algae and Product Main Nutrient Component Evaluation

[0337] This content refers to the content of the invention drafted and published by the inventor Wenzhou Dongtou District Local Technical Specification "Quality Grading Specification for Sargassum Fusiforme Primary Dried Products" (DB330305 / T24-2023).

[0338] 11.6 Wild Sargassum Fusiforme Offspring - New Germplasm Genetic Stability Analysis

[0339] This content refers to the content of the invention drafted and published by the inventor Wenzhou Dongtou District Local Technical Specification "Quality Grading Specification for Sargassum Fusiforme Primary Dried Products" (DB330305 / T24-2023). Sargassum fusiforme The above technical solutions refer to and refer to technical documents:

[0340] 1. Lin Lidong, Wu Mingjiang. 2021. Based on clustering and simplified genome combined analysis of Zhejiang Dongtou cultivated Sargassum fusiforme (S. fusiforme) strain screening research [J]. Ocean and lake, 52(5): 1224-1235;

[0341] Sargassum fusiforme 2. Lin Lidong, Wu Mingjiang, Zheng Jing. Patent name: A unit mass evaluation and difference comparison method for Sargassum fusiforme young sporophyte cultivation. Authorized patent number: ZL202110213367.1. Authorized announcement date: September 23, 2022. Approval country: State Intellectual Property Office of the People's Republic of China. Patentee: Wenzhou Dongtou District Marine and Fishery Development Research Center, Wenzhou University;

[0342] 2. Lin Lidong, Wu Mingjiang, Zheng Jing. Patent name: A unit mass evaluation and difference comparison method for Sargassum fusiforme young sporophyte cultivation. Authorized patent number: ZL202110213367.1. Authorized announcement date: September 23, 2022. Approval country: State Intellectual Property Office of the People's Republic of China. Patentee: Wenzhou Dongtou District Marine and Fishery Development Research Center, Wenzhou University;

[0343] ​3. Lin Lidong, Wu Mingjiang, Yan Xiufeng, Ma Zengling, Zhang Xu, Zheng Ying, Yu Ping. Patent name: A method for identifying and classifying Sargassum fusiforme strains. Patent number: ZL201511004442.4. Patent announcement date: January 9, 2018. Approved country: State Intellectual Property Office of the People's Republic of China. Patentee: Wenzhou Dongtou Marine and Fisheries Development Research Center, Wenzhou University;

[0344] 4. Lin Lidong, Wu Mingjiang, Ma Zengling, Chen Binbin, Zheng Ying. Patent name: A method for evaluating and comparing the fresh / dry weight yield and water content of Sargassum fusiforme primary products. Patent number: ZL201911080353.6. Patent announcement date: June 7, 2022. Approved country: State Intellectual Property Office of the People's Republic of China. Patentee: Wenzhou Dongtou Marine and Fisheries Development Research Center, Wenzhou University;

[0345] 5. Lin Lidong, Ma Zengling, Chen Binbin, Wu Mingjiang, Zheng Ying. Patent name: A method for classifying the quality grades of Sargassum fusiforme primary products. Patent number: ZL201911080356.X. Patent announcement date: May 24, 2022. Approved country: State Intellectual Property Office of the People's Republic of China. Patentee: Wenzhou Dongtou Marine and Fisheries Development Research Center, Wenzhou University;

[0346] 6. Wu Mingjiang, Lin Lidong, Ma Zengling, Chen Binbin, Yu Ping, Wang Shengqin. Patent name: A method for classifying the quality grades of Sargassum fusiforme gas sac outlet products. Patent number: ZL201911132779.1. Patent announcement date: April 9, 2024. Approved country: State Intellectual Property Office of the People's Republic of China. Patentee: Wenzhou University, Wenzhou Dongtou Marine and Fisheries Development Research Center;

[0347] 7. Lin Lidong, Yan Xiufeng, Zhang Peng, Wang Tiegan, Jia Yang, Liang Jia, Wu Fengmiao, Zhu Mingyi. Technical regulation name: Sargassum fusiforme high-quality seedling cultivation technology regulation. Regulation release number: DJG330305 / T22-2023. Regulation release date: July 11, 2023. Specification implementation date: October 1, 2023. Regulation drafting unit: Wenzhou Dongtou Marine and Fisheries Development Research Center, Wenzhou University, Zhejiang Provincial Marine and Aquaculture Institute, Zhejiang Ocean University, Zhejiang Shiyuan Jin Niu Biological Technology Co., Ltd., Wenzhou Dongtou Fisheries and Agriculture Technology Extension Center;

[0348] 8. Lin Lidong, Yan Xiufeng, Zhang Peng, Wang Tielv, Jia Yang, Liang Jia, Wu Fengmei, Zhu Mingyi. Technical Regulation Name: Sargassum fusiforme Cultivation and Management Technical Regulation. Regulation Release Number: DJG330305 / T23-2023. Regulation Release Date: July 11, 2023. Regulation Implementation Date: October 1, 2023. Regulation Drafting Unit: Wenzhou Dongtou District Marine and Fisheries Development Research Center, Wenzhou University, Zhejiang Provincial Marine Aquaculture Research Institute, Zhejiang Ocean University, Zhejiang Shiyuan Jin Niu Biological Technology Co., Ltd., Wenzhou Dongtou District Fisheries Technology Extension Center;

[0349] 9. Lin Lidong, Liang Jia, Yan Xiufeng, Wang Tielv, Zhang Peng, Jia Yang, Wu Fengmei, Zhu Mingyi. Technical Specification Name: Sargassum fusiforme Primary Dry Product Quality Grading Specification. Specification Release Number: DB330305 / T24-2023. Specification Release Date: July 11, 2023. Specification Implementation Date: October 1, 2023. Specification Drafting Unit: Wenzhou Dongtou District Marine and Fisheries Development Research Center, Zhejiang Ocean University, Wenzhou University, Zhejiang Provincial Marine Aquaculture Research Institute, Zhejiang Shiyuan Jin Niu Biological Technology Co., Ltd., Wenzhou Dongtou District Fisheries Technology Extension Center.

Claims

1. A method for creating and evaluating new germplasm from Sargassum fusiforme fertilized eggs via suspension culture, characterized in that: Includes the following steps: Selection, isolated culture and unit quality evaluation of wild Sargassum fusiforme parent plants; Screening of male and female plants of high-value parental traits of S2 wild Sargassum fusiforme; isolated culture and biological control of pests; The removal of epiphytic pests from male and female plants of the S3 wild Sargassum fusiforme high-value trait strain; S3.1 High-value wild Sargassum fusiforme strain, male and female plants temporarily housed indoors. S3.1.1 Removal of epiphytic macroalgae Remove manually; S3.1.2 Removal of Epiphytes Soak in room temperature fresh water for 20 to 25 minutes to kill epiphytes using hypotonic methods, then rinse 3 to 5 times with fresh, filtered, and sterilized seawater. S3.1.3 Removal of epiphytic microalgae The ratio of fresh algal body weight to citric acid (C6H8O7) solution (kg:L) was 1:3, the concentration of citric acid was 1.5 mg / ml, the soaking time was 5 min to 8 min, and after removal, the algae were rinsed 3 to 5 times with room temperature fresh water, and then rinsed 3 to 5 times with fresh, filtered, and sterilized seawater. S3.2 Treatment of male and female plants in high-value wild Sargassum fusiforme cultivars S3.2.1 Selection of lateral branches and removal of stems Manually cut and remove the primary or secondary lateral branches of the fresh algae, retaining the secondary or tertiary lateral branches with densely packed reproductive receptacles; S3.2.2 Placement and weight ratio of lateral branches from male and female plants Lateral branches are allocated according to the actual area of ​​the cultivation tray, and the allocation standard is that the lateral branches do not overlap with each other to ensure uniform light. The mixed mass ratio (kg:kg) of female plants to male plants is 1:

1. Creating the optimal environmental factors for indoor temporary cultivation of S4 wild Sargassum fusiforme high-value trait strains from male and female plants and lateral branches. S4.1 Optimal Water Environment for Indoor Temporary Holding Cultivating seawater: ① Fresh: Take clear seawater from the day's high tide and allow it to settle naturally for 3-4 hours. ② Filtration: Supernatant seawater is filtered through a 280-300 mesh silk screen. ③ High-temperature sterilization: Filtered seawater is heated to 80 ℃~90 ℃ and sterilized at high temperature for 30 min~40 min to disinfect animal eggs, microorganisms, microalgae and large seaweed larvae, and then naturally cooled to room temperature; ④ pH value: Adjust the pH value of the sterilized seawater to 7.8-8.3 with 0.05 mol / L NaOH solution and 0.05 mol / L HCl solution; ⑥ Salinity: 28–33; ⑦ Nutrients: Prepare a seawater solution with N:P = 10:1 using NaNO3 and KH2PO3, with a concentration of 10 (N) g / L or 1 (P) g / L. The volume ratio (V:V) of the nutrients to the culture seawater is (0.6~0.8) ml:L. S4.2 Culture room, light-illuminated culture rack and temporary incubation device S4.3 sets the optimal temperature, light intensity, and photoperiod. Temperature: 23℃~25℃; Light intensity: 200 µmol•m -2 •s -1 ~280µmol•m -2 •s -1 ; Photoperiod: 14 h:10 h~12 h:12 h; Treatment and duration of indoor temporary cultivation of lateral branches from male and female plants of the S5 wild Sargassum fusiforme parental high-value trait line. S5.1 Lateral branch indoor temporary care treatment Replace half the total volume of seawater in the culture group daily, and turn the lateral branches over every 6 to 8 hours to ensure that the reproductive receptacles of each lateral branch receive uniform light. S5.2 Lateral branch indoor temporary care time Lateral branches were temporarily kept indoors for 5 to 7 days; Classification, collection, impurity removal, and cleaning of fertilized eggs from the new germplasm of Sargassum fusiforme (S6). Fertilized eggs are collected using a 260-280 mesh silk screen, and air sacs and reproductive receptacles are removed using a 30-50 mesh silk screen. The fertilized eggs are placed in a 2000 mL beaker, and 1500 mL of fresh filtered seawater is added. After stirring, the eggs are allowed to settle naturally for 2-3 minutes. The supernatant is discarded, and small suspended impurities are removed. This process is repeated 5-7 times to obtain fresh and pure fertilized eggs. Different strains require separate collection of fertilized eggs and removal of impurities. Creation of a suspension system for fertilized eggs of new S7 Sargassum fusiforme germplasm S7.1 Quantitative dispensing of fertilized eggs from new Sargassum fusiforme germplasm Remove excess water from the fresh fertilized eggs, determine the volume density of the fertilized eggs, and quantitatively dispense 8,000 to 10,000 fertilized eggs into 3,000 ml or 5,000 ml standard Erlenmeyer flasks. Add fresh filtered seawater, with the ratio of the number of fresh fertilized eggs to the volume of seawater (ml) ranging from 10:1 to 10:

3. Classify, package, and label the eggs according to different strains. S7.2 Optimal Environment for Suspension Culture of New Sargassum Germplasm Zygophyllum S7.2.1 Optimal Aqueous Environment for Suspension Culture Freshness, filtration, high-temperature sterilization, pH value, salinity and nutrient salt preparation are in accordance with method S4.

1. The volume ratio (V:V) of nutrient salt to culture seawater is (0.5~0.7) ml:L~(0.6~0.8) ml:1L, and the optimal water environment conditions are kept constant during suspension culture. S7.2.2 Optimal light intensity, photoperiod, temperature, and aeration conditions for suspension culture Optimal light intensity: 50 µmol•m -2 •s -1 ~60µmol•m -2 •s -1 ; Photoperiod: 14 h:10 h~12 h:12 h; Temperature: 23℃~24℃; Aeration: Assisted aeration; S7.2.3 Suspension Culture Method for Fertilized Eggs of New Sargassum Germplasm In the initial stage of suspension culture of Sargassum fusiforme fertilized eggs, magnetic stirring and aeration suspension culture were used. After 8 to 10 days of culture, the Sargassum fusiforme fertilized eggs had grown and developed into embryos. At the beginning of the embryo growth and development period, suspension culture device was used for suspension culture and aeration suspension culture was implemented. S7.3 Low-density quantitative dispensing of suspension culture of Sargassum fusiforme germplasm from embryo to sporophyte stage during its suspension growth and development. During the embryo to sporophyte growth and development stage, the cells were dispensed at half volume density every 19 to 21 days. As the culture time gradually increased, the cells were dispensed at lower density in stages. The first low-density dispensing was reduced from 8,000 to 10,000 cells to 4,000 to 5,000 cells, the second low-density dispensing was reduced from 4,000 to 5,000 cells to 2,000 to 2,500 cells, the third low-density dispensing was reduced from 2,000 to 2,500 cells to 1,000 to 1,200 cells, and the final low-density dispensing was reduced from 1,000 to 1,200 cells to 400 to 500 cells. S8 Sargassum fusiforme germplasm, embryo-to-sporophyte suspension growth and development stage, typical pests and biological control in suspension systems. S8.1 Methods for controlling pests During S7 culture, the embryos or young sporophytes are examined under a microscope regularly. If harmful animals are found in the suspension culture medium, the culture medium is soaked in ultrapure water for 20 to 25 minutes every 10 to 12 days to disinfect the harmful animals. S8.2 Control methods for harmful cyanobacteria During S7 culture, the embryos or young sporophytes are examined under a microscope regularly. If signs of epiphytic cyanobacteria are found in the embryos or young sporophytes, they are soaked in seawater solution containing 300,000 to 600,000 penicillin per L for 24 to 48 hours to disinfect the cyanobacteria. After that, they are washed with filtered seawater 3 to 5 times and then suspended in filtered seawater for continued culture under the same conditions. S8.3 Methods for controlling harmful green algae During S7 culture, the embryos or young sporophytes are examined under a microscope regularly. If signs of epiphytic green algae are found in the embryos or young sporophytes, they are soaked in 1.5 mg / ml citric acid solution for 6 min to 8 min, rinsed with fresh water 3 to 5 times, then rinsed with filtered seawater 2 to 3 times, and then suspended in filtered seawater for continued culture under the same culture conditions. The larval sporozoites of the new S9 Sargassum fusiforme variety were bound together and temporarily housed indoors. Clamp the young sporophytes with basal leaves of 1.5 cm to 2.0 cm in length onto cotton or fiber ropes with a diameter of 0.4 cm, with a spacing of 3 cm to 5 cm. Continue to suspend the young sporophytes with basal leaves less than 1.5 cm in length until the basal leaves grow to 1.5 cm to 2.0 cm, then clamp them onto the ropes. After clamping, temporarily incubate the young sporophytes indoors for 15 to 20 days. Sea-based release of larval sporozoites of the new S10 Sargassum fusiforme germplasm. Evaluation Methods for New Germplasm of Wild Sargassum fusiforme Parents and Offspring (S11) S11.1 Evaluation of the traits of wild Sargassum fusiforme parents and offspring S11.2 Evaluation of the yield of wild Sargassum fusiforme parents and offspring S11.3 Evaluation of the processing ratio of dried raw materials and primary processed products of wild Sargassum fusiforme parent and offspring S11.4 Evaluation of rehydration rate of primary processed products of wild Sargassum fusiforme parent and offspring Evaluation of the main nutritional components of wild Sargassum fusiforme parent plants, offspring algae, and products (S11.5) Analysis of genetic stability of S11.6 wild Sargassum fusiforme offspring – new germplasm.

2. The method for creating and evaluating new germplasm through suspension culture of Sargassum fusiforme fertilized eggs according to claim 1, characterized in that: In step S4.2, the light-incubation rack structure includes carbon steel left and right side frames, tempered glass shelves, a glass shelf bottom frame, crossbars, a switch box, LED lighting, and a controllable darkroom that meets the indoor temporary rearing conditions for marine organisms. The LED lighting of the main body of the light-incubation rack, in conjunction with the darkroom control, can ensure 0 µmol•m -2 •s -1 ~350µmol•m -2 •s -1 Light intensity adjustment is achieved by setting an external power supply timer converter to control the light cycle; the culture room of the controllable darkroom is set as an internal darkroom and is equipped with air conditioning equipment to regulate the temperature of the culture room. The temporary incubation device set on the light-incubation rack includes a water storage tank, a culture tray, a peristaltic pump, an oxygen supply pump, a water delivery hose, and a sand head. It is used for centralized dissolved oxygenation of seawater and circulating water supply, which can ensure sufficient oxygen and nutrient supply for the temporarily incubated Sargassum fusiforme.

3. The method for creating and evaluating new germplasm through suspension culture of Sargassum fusiforme fertilized eggs according to claim 1, characterized in that: The preparation of the culture seawater includes the following steps: Fresh filtered seawater from the field is stored in 1000 L chemical tanks and transported to the factory area. It is then transferred to 25 L chemical tanks and moved indoors. The seawater is heated to 80℃~90℃ using a water heater for 30-40 minutes to disinfect animal eggs, microorganisms, microalgae, and large seaweed larvae. It is then allowed to cool naturally to room temperature. Seawater is added to the storage tank of the temporary rearing device at a ratio of 4-5 L per 100 g of seed vegetables. Nutrients are added at a volume ratio (V:V) of (0.6-0.8) ml:1 L of culture seawater. The light intensity is adjusted to 200 µmol•m. -2 •s -1 ~280µmol•m -2 •s -1 The photoperiod is 14 h:10 h to 12 h:12 h, and the temperature is 24 ℃ to 25 ℃; the male-female ratio of the parent plants is 2:1 to 1:

1.

4. The method for creating and evaluating new germplasm through suspension culture of Sargassum fusiforme fertilized eggs according to claim 1, characterized in that: In step S3.1.1, the large epiphytic seaweeds removed by hand include *Sargassum fusiforme*, *Ulva prolifera*, and *Vegetaria spp.* that are epiphytic to the new germplasm parent of *Sargassum fusiforme*; in step S3.1.2, the epiphytic animals removed by hypotonic treatment include *Hylocereus undatus*, *Cladosporium*, and fish eggs that are epiphytic to the new germplasm parent of *Sargassum fusiforme*.

5. The method for creating and evaluating new germplasm through suspension culture of Sargassum fusiforme fertilized eggs according to claim 1, characterized in that: In step S7, the structure of the Sargassum fusiforme fertilized egg suspension culture device includes a 5000 ml glass triangular conical flask, glass tube, air control valve, air supply pump, and tubing. During the suspension culture process, low-density quantitative dispensing is performed based on the suspension culture device.

6. The method for creating and evaluating new germplasm through suspension culture of Sargassum fusiforme fertilized eggs according to claim 1, characterized in that: In step S7.2.1, the volume ratio (V:V) of nutrients to culture seawater during the suspension culture growth and development stage of Sargassum fusiforme fertilized eggs to young sporophytes is (0.5-0.6) ml: 1 L.

7. The method for creating and evaluating new germplasm through suspension culture of Sargassum fusiforme fertilized eggs according to claim 1, characterized in that: In step S10, the raft frame device for the isolation and release of new Sargassum fusiforme germplasm seedlings in natural sea areas adopts a soft raft frame design. The entire soft raft frame is 100 m long × 100 m wide and has 9 equal hanging areas. The new Sargassum fusiforme germplasm seedlings are hung at the four corners of the raft frame and surrounded by netting.

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