Sargassum fusiforme fertilized egg suspension culture new germplasm creation and evaluation method

Abstract

The invention discloses a sargassum fusiforme fertilized egg suspension culture new germplasm creation and evaluation method. The method comprises the steps of wild sargassum fusiforme geographical parent selection, isolated culture and unit quality evaluation. Screening male and female plants of parent high-value character strains, performing isolated culture, and preventing and treating harmful organisms; removing epiphytic harmful organisms of male and female plants of the high-value character strain; creating an optimal environmental factor for indoor temporary rearing of lateral branches of male and female plants of the high-value character strain; indoor temporary rearing treatment and temporary rearing time of lateral branches of male and female plants of the parent high-value character strain; classifying and collecting new germplasm fertilized eggs, removing impurities, cleaning, and creating a suspension system of the new germplasm fertilized eggs; preventing and controlling typical harmful organisms of a suspension system in a suspension growth and development stage from new germplasm embryos to juvenile sporophytes; carrying out rope clamping and indoor temporary rearing on new germplasm juvenile sporophytes; stocking the new germplasm juvenile sporophytes in a sea area; an evaluation method of new germplasms of parents and filial generations; a scientific and standard operation and evaluation method can be provided for new sargassum fusiforme germplasm creation, new germplasm quality evaluation and new variety cultivation.

Description

Technical Field

[0001] The present invention relates to a method for creating and evaluating new germplasms by suspending the fertilized eggs of Sargassum fusiforme, which is applicable to scientific research and agricultural production fields such as the creation of new germplasms of Sargassum fusiforme, the evaluation of genetic stability of new germplasms of Sargassum fusiforme, the screening of high-quality strains of new germplasms of Sargassum fusiforme, the cross-breeding and molecular breeding of Sargassum fusiforme, and the creation of new varieties of Sargassum fusiforme. Background Art

[0002] Sargassum fusiforme is a medicinal and edible Sargassum unique to the northwest coast of the Pacific Ocean. Its medicinal value was first recorded in "Shennong Ben Cao Jing". Dongtou District of Wenzhou City is currently the largest area for breeding, farming, processing, exporting and domestic sales of Sargassum fusiforme in China, with an annual farming area of more than 12,000 mu and an annual total farming output value of more than 61 million yuan. Modern research shows that China leads Japan and South Korea in innovative technologies for sexual and asexual reproduction breeding of Sargassum fusiforme, and is world-leading in innovative technologies such as the identification and classification of Sargassum fusiforme strains, the quality evaluation of primary and rough processed products of Sargassum fusiforme, the evaluation of the unit mass of Sargassum fusiforme seedlings, and the artificial use of sexual reproduction cross-breeding of Sargassum fusiforme. Practical research shows that after the wild-derived Sargassum fusiforme parents are artificially domesticated and cultured, the air bladders of their mature thalli show diverse characteristics, highlighting the multi-morphological adaptability of different plants under the same environmental conditions. This habitat adaptation characteristic seriously affects the uniformity and stability evaluation among strains, severely restricts the development of new varieties and large-scale agricultural production applications, and this scientific problem has been reported in relevant literature. At present, how to efficiently solve the technology for creating new germplasms of Sargassum fusiforme and accurately master the morphological stability characteristics of different new germplasm progenies, implement the large-scale creation of high-value strain breeding parents of Sargassum fusiforme, and ensure the high-efficiency input of high-value strains into production applications has now become an urgent concern for scientific researchers, farmers and processing enterprises of Sargassum fusiforme. During the systematic compilation of the innovative technology for creating and evaluating new germplasms by sexual reproduction of Sargassum fusiforme, the inventor appropriately integrated the obtained innovative technologies and achieved the innovation of the technology for creating and evaluating new germplasms by sexual reproduction of Sargassum fusiforme. The production pilot test proves that the technology of the present invention effectively solves the problems of creating new germplasms of Sargassum fusiforme and stability evaluation. Summary of the Invention

[0003] Based on the technical field and technical background, the purpose of the present invention is to propose a method for creating new germplasms by suspending the fertilized eggs of Sargassum fusiforme and evaluating new germplasms of Sargassum fusiforme. This method is applicable to scientific research and agricultural production fields such as the creation of new germplasms of Sargassum fusiforme, the evaluation of genetic stability of new germplasms of Sargassum fusiforme, the screening of high-quality strains of new germplasms of Sargassum fusiforme, the cross-breeding and molecular breeding of Sargassum fusiforme, and the creation of new varieties of Sargassum fusiforme.

[0004] Based on the invention purpose, the present invention provides a method for creating and evaluating new germplasms by suspending the fertilized eggs of Sargassum fusiforme,

[0005] including the following steps:

[0006] S1 Selection, isolated cultivation and unit mass evaluation of the wild Sargassum fusiforme geographical parent;

[0007] S2 Screening of male and female plants of high-value trait lines of wild Sargassum fusiforme parents, isolated cultivation and control of harmful organisms;

[0008] S3 Removal of epiphytic harmful organisms on male and female plants of high-value trait lines of wild Sargassum fusiforme;

[0009] S3.1 Indoor temporary cultivation of male and female plants of high-value trait lines of wild Sargassum fusiforme

[0010] S3.1.1 Removal of epiphytic macroalgae

[0011] Manual removal;

[0012] S3.1.2 Removal of epiphytic animals

[0013] Soak in fresh water at room temperature for 20 min - 25 min, hypotonic to kill epiphytic animals, and rinse 3 - 5 times with fresh, filtered and sterilized seawater;

[0014] S3.1.3 Removal of epiphytic microalgae

[0015] The mass (kg) of the fresh weight of the thallus to the volume (L) of the citric acid (C6H8O7) solution is 1:3, the concentration of citric acid is 1.5 mg / ml, the soaking time is 5 min - 8 min, after taking out, rinse 3 - 5 times with fresh water at room temperature, and rinse 3 - 5 times with fresh, filtered and sterilized seawater;

[0016] S3.2 Treatment of male and female plants of high-value trait lines of wild Sargassum fusiforme

[0017] S3.2.1 Screening of lateral branches and removal of stems

[0018] Remove the stems of the first- or second-order lateral branches of the fresh thallus, and retain the second- or third-order lateral branches with dense receptacles;

[0019] S3.2.2 Placement of lateral branches of male and female plants and mass ratio

[0020] 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 with each other, so as to ensure uniform light. The mixing mass ratio (kg:kg) of female plants to male plants is 1:1;

[0021] S4 Creation of the optimal environmental factors for indoor temporary cultivation of lateral branches of male and female plants of high-value trait lines of wild Sargassum fusiforme

[0022] S4.1 Optimal water environment

[0023] Fresh: Take the clear seawater at high tide on the same day and let it settle naturally for 3 h - 4 h;

[0024] Filtration: The supernatant seawater is filtered through a 280 - 300 - mesh silk screen.

[0025] High - temperature sterilization: The filtered seawater is heated to 80°C - 90°C and sterilized at high temperature for 30 min - 40 min to kill animal eggs, microorganisms, microalgae and young macroalgae, and then cooled naturally to room temperature.

[0026] pH value: The pH value of the sterilized seawater is adjusted to 7.8 - 8.3 with 0.05 mol / L NaOH solution and 0.05 mol / L HCl solution.

[0027] Salinity: 28 - 33.

[0028] Nutrient salts: 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, and the volume ratio (V:V) of nutrient salts to culture seawater is (0.6 - 0.8)ml:1L.

[0029] ]>S4.2 Culture room, light - incubating rack and temporary - rearing device

[0030] The main structure of the light - incubating rack includes left and right side frames made of carbon steel, tempered - glass laminates, glass - laminate bottom frames, cross - bars, switch boxes, and LED lighting lamps. It is set to meet the conditions for indoor temporary rearing of seawater organisms, and can ensure a light intensity of 0 μmol·m -2 ·s -1 ~350 μmol·m -2 ·s -1 The external - control power - supply timing converter can realize photoperiod regulation; the culture room is set as an inner dark room and is equipped with air - conditioning equipment to regulate the indoor space temperature; the main structure of the temporary - rearing device group includes a water - storage bucket, a culture tray, a peristaltic pump, an oxygen - supply pump, a water - delivery hose and a diffuser head, which can realize centralized oxygen dissolution, water supply and circulation of seawater, and can ensure sufficient supply of oxygen and nutrient nutrients for temporarily rearing Sargassum fusiforme seedlings.

[0031] S4.3 Set the optimal temperature, light intensity and photoperiod

[0032] Temperature: 23°C - 25°C.

[0033] Light intensity: 200 μmol·m -2 ·s -1 ~280 μmol·m -2 ·s -1 ;

[0034] Photoperiod: 14h:10h - 12h:12h.

[0035] S5 Treatment and temporary - rearing time for indoor temporary rearing of male and female lateral branches of high - value - trait strains of wild Sargassum fusiforme parents

[0036] S5.1 Indoor temporary cultivation treatment of lateral branches

[0037] Replace 1 / 2 of the total culture seawater in the culture group every day, and turn the lateral branches once every 6h - 8h to ensure that the receptacles of each lateral branch receive uniform light;

[0038] S5.2 Indoor temporary cultivation time of lateral branches

[0039] The lateral branches are temporarily cultivated indoors for 5d - 7d;

[0040] S6 Classification collection, impurity removal and cleaning of fertilized eggs of new germplasm of Sargassum fusiforme

[0041] Use a 260 - 280 - mesh silk screen to collect fertilized eggs, and a 30 - 50 - mesh silk screen to remove impurities such as air bladders and receptacles. Place the fertilized eggs in a 2000mL beaker, add 1500mL of freshly filtered seawater, stir well and let it settle naturally for 2min - 3min. Discard the upper suspension to remove suspended minute impurities. Repeat this operation 5 - 7 times to obtain pure fresh fertilized eggs. For different strains, the collection of fertilized eggs and impurity removal need to be carried out separately;

[0042] S7 Establishment of suspension system for fertilized eggs of new germplasm of Sargassum fusiforme

[0043] S7.1 Quantitative packaging of fertilized eggs of new germplasm of Sargassum fusiforme

[0044] Remove the excess water from the pure fresh fertilized eggs, technically calculate the volume density of the fertilized eggs, and quantitatively package 8000 - 10000 fertilized eggs in each 3000ml or 5000ml Erlenmeyer flask. Add freshly filtered seawater, and the ratio of the number of pure fresh fertilized eggs (pieces) to the volume of seawater (ml) is 10:1 - 10:3. For different strains, they need to be classified and packaged and labeled;

[0045] S7.2 Creation of the optimal environment for suspension culture of fertilized eggs of new germplasm of Sargassum fusiforme

[0046] S7.2.1 Optimal water environment

[0047] The fresh, filtered, high - temperature sterilized, pH value, salinity and nutrient salts are configured according to the method of S4.1. The volume ratio (V:V) of nutrient salts to culture seawater is (0.5 - 0.7)ml:1L - (0.6 - 0.8)ml:1L, and the optimal water environment conditions remain unchanged during the suspension culture period;

[0048] S7.2.2 Optimal light intensity, photoperiod, temperature, aeration and other conditions

[0049] Optimal light intensity: 50μmol·m -2 ·s -1 ~60μmol·m -2 ·s -1 ;

[0050] Photoperiod: 14h:10h to 12h:12h;

[0051] Temperature: 23°C to 24°C;

[0052] Aeration: Auxiliary aeration;

[0053] S7.2.3 Suspension culture method of fertilized eggs of new germplasm of Sargassum fusiforme

[0054] In the initial stage (1d - 10d) of suspension culture of fertilized eggs of Sargassum fusiforme, magnetic stirring + aeration suspension culture is adopted. After culturing for 8d - 10d, the fertilized eggs of Sargassum fusiforme have grown and developed into embryos. When the embryo growth and development stage begins, a suspension culture device is used for suspension culture, and aeration suspension culture is implemented;

[0055] S7.3 Low - density quantitative sub - packaging during the suspension growth and development stage from embryo to young sporophyte of new germplasm of Sargassum fusiforme

[0056] During the growth and development stage from embryo to young sporophyte, sub - packaging is carried out at a density of 1 / 2 equal volume per bottle every 19d - 21d. As the culture time gradually extends, low - density quantitative sub - packaging is carried out step by step. The first low - density sub - packaging decreases from 8000 - 10000 to 4000 - 5000, the second low - density sub - packaging decreases from 4000 - 5000 to 2000 - 2500, the third low - density sub - packaging decreases from 2000 - 2500 to 1000 - 1200, and the final low - density sub - packaging decreases from 1000 - 1200 to 400 - 500;

[0057] S8 Control of typical harmful organisms in the suspension system during the suspension growth and development stage from embryo to young sporophyte of new germplasm of Sargassum fusiforme

[0058] S8.1 Control method for harmful animals

[0059] If harmful animals are found in the suspension culture solution, soak it in ultrapure water for 20min - 25min every 10d - 12d of the culture period to kill the harmful animals by hypotonic treatment;

[0060] S8.2 Control method for harmful cyanobacteria

[0061] During the S7 culture period, regularly examine the embryo or young sporophyte under a microscope. If signs of epiphytic cyanobacteria are found on the embryo or young sporophyte, soak and culture it in a seawater solution containing 300,000 - 600,000 units / L of penicillin for 24h - 48h to kill the cyanobacteria, then filter the seawater and wash it 3 - 5 times, and continue the suspension culture with filtered seawater;

[0062] S8.3 Control method for harmful green algae

[0063] During the S7 culture period, the embryos or young sporophytes were regularly examined under a microscope. If any signs of epiphytic green algae were found on the embryos or young sporophytes, they were soaked in 1.5 mg / ml citric acid solution for 6 - 8 minutes, rinsed 3 - 5 times with fresh water, rinsed 2 - 3 times with filtered seawater, and then continuously suspended and cultured with filtered seawater;

[0064] Clamping the Rope and Indoor Temporary Cultivation of Young Sporophytes of the New Germplasm of Sargassum fusiforme

[0065] The young sporophytes with a basal leaf length of 1.5 - 2.0 cm were clamped onto cotton ropes or fiber ropes with a diameter of 0.4 cm, with a spacing of 3 - 5 cm. The young sporophytes with a basal leaf length less than 1.5 cm were continuously cultured in suspension until the basal leaf grew to 1.5 - 2.0 cm and then clamped onto the rope. The clamped young sporophytes were temporarily cultured indoors for 15 - 20 days;

[0066] Sea Area Release of Young Sporophytes of the New Germplasm of Sargassum fusiforme

[0067] Evaluation Methods for Parents and Offspring of New Germplasm of Wild Sargassum fusiforme

[0068] S11.1 Trait Evaluation of Parents and Offspring of Wild Sargassum fusiforme

[0069] S11.2 Yield Evaluation of Parents and Offspring of Wild Sargassum fusiforme

[0070] S11.3 Processing Ratio Evaluation of Parents, Offspring, Dried Raw Materials and Rough - processed Products of Wild Sargassum fusiforme

[0071] S11.4 Rehydration Rate Evaluation of Rough - processed Products of Parents and Offspring of Wild Sargassum fusiforme

[0072] S11.5 Evaluation of Main Nutritional Components of Adult Algae and Products of Parents and Offspring of Wild Sargassum fusiforme

[0073] S11.6 Genetic Stability Analysis of Offspring of Wild Sargassum fusiforme - New Germplasm.

[0074] The culture seawater includes the following preparation steps: Store the freshly filtered seawater from the wild area in a 1000 L chemical bucket and transport it to the factory area, then transfer it to 25 L chemical buckets and carry it indoors. Use a water heater to heat the seawater to 80℃ - 90℃ for 30 - 40 minutes to kill animal eggs, microorganisms, microalgae and young large algae. Let it cool naturally to room temperature, and inject water into the storage buckets of the temporary culture device group according to the ratio of 4 - 5 L of seawater per 100 g of seaweed seedlings; Add nutrients according to the volume ratio (V:V) of nutrients to culture seawater of (0.6 - 0.8) ml:1 L; Adjust the light intensity to 200 μmol·m -2 ·s -1 ~280 μmol·m -2 ·s -1, the photoperiod is 14h:10h to 12h:12h, and the temperature is 24°C to 25°C; the mass ratio of male to female of the vegetable-growing parent is 2:1 to 1:1.

[0075] As a further setting of the above solution, in step S3.1.1, the epiphytic large seaweeds manually removed include Sargassum, Enteromorpha, and fresh vegetables attached to the parents of the new germplasm of Sargassum fusiforme; in step S3.1.2, the epiphytic animals removed by hypotonic treatment include Gammarus, phasmids, and fish eggs attached to the parents of the new germplasm of Sargassum fusiforme.

[0076] As a further setting of the above solution, in step S7.2.1, the volume ratio (V:V) of nutrients to culture seawater during the suspension culture growth and development stage of the fertilized eggs of Sargassum fusiforme to young sporophytes (trilobate to multi-lobate) is (0.5 - 0.6) ml: 1 L.

[0077] As a further setting of the above solution, in step S7, the main structure of the suspension culture device for the fertilized eggs of Sargassum fusiforme new germplasm includes a 5000 ml glass Erlenmeyer flask, a glass tube, an air control valve, an air supply pump, and a hose, and low-density quantitative packaging operation is implemented during the suspension culture process.

[0078] As a further setting of the above solution, in step S10, the raft device for isolating and releasing the new germplasm seedlings of Sargassum fusiforme in the natural sea area adopts a soft raft design, with the overall length × width of 100 m × 100 m, and 9 equal hanging culture areas are arranged inside. To ensure the isolation and culture effect, the new germplasm seedlings of Sargassum fusiforme are respectively hung at the four corners of the raft, and a surrounding net is set up.

[0079] The method for creating new germplasm of Sargassum fusiforme and evaluating new germplasm of Sargassum fusiforme proposed by the present invention is applicable to scientific research and agricultural production fields such as the creation of new germplasm of Sargassum fusiforme, the evaluation of genetic stability of new germplasm of Sargassum fusiforme, the screening of high-quality strains of new germplasm of Sargassum fusiforme, cross-breeding and molecular breeding of Sargassum fusiforme, and the creation of new varieties of Sargassum fusiforme, and can provide scientific and standard operation and evaluation methods for the creation of new germplasm of Sargassum fusiforme, the evaluation of new germplasm quality, and the cultivation of new varieties.

[0080] Figure description

[0081] Figure 1 For the indoor suspension culture of the fertilized eggs of the new germplasm of Sargassum fusiforme of the present invention.

[0082] Figure 2 For the indoor temporary culture and sea area release of the young sporophytes of the new germplasm of Sargassum fusiforme of the present invention.

[0083] Figure 3 For the schematic diagram of the isolation culture raft of different strains (wild germplasm) seedlings of Sargassum fusiforme of the present invention. Detailed implementation method

[0084] To clearly illustrate the technical solution and operation process of the present invention, the present invention will be further described in detail below in conjunction with specific implementation examples.

[0085] Reference Figures 1-3 A method for creating and evaluating a new germplasm of floating culture of Sargassum fusiforme fertilized eggs according to the present invention is shown as follows, including the following structures and operation steps:

[0086] a. For the evaluation of the quality of parents and offspring seedlings, the evaluation methods for the traits of parents and offspring of the new germplasm, the evaluation methods for the yields of parents and offspring of the new germplasm, the processing ratio evaluation of dry product raw materials and rough processing products, the rehydration rate evaluation of rough processing products, the evaluation of the main nutritional components of adult algae and products, and the genetic stability analysis of the new germplasm of Sargassum fusiforme, refer to the method of "A Method for Unit Quality Evaluation and Difference Comparison of Cultivation of Juvenile Sporophytes of Sargassum fusiforme" (ZL 202110213367.1) for implementation; the evaluation of the traits of parents and offspring of the new germplasm of Sargassum fusiforme is implemented according to the method of referring to the authorized invention patent "A Method for Strain Identification and Classification of Sargassum fusiforme" (ZL201511004442.4); the evaluation of the yields of parents and offspring of the new germplasm of Sargassum fusiforme is implemented according to the method of referring to the authorized invention patent "A Method for Evaluating and Comparing the Yields per Unit Fresh / Dry Weight and Water Content of Primary Products of Cultured Sargassum fusiforme" (ZL201911080353.6); the evaluation of the processing ratio of dry product raw materials and rough processing products of Sargassum fusiforme is implemented by referring to the relevant content of the local technical specification of Dongtou District, Wenzhou City, "Specification for Quality Grading of Primary Dried Products of Sargassum fusiforme" (DB330305 / T 24-2023) drafted by the inventor; the evaluation of the rehydration rate of rough processing products of Sargassum fusiforme is implemented by referring to the relevant content of the local technical specification of Dongtou District, Wenzhou City, "Specification for Quality Grading of Primary Dried Products of Sargassum fusiforme" (DB330305 / T 24-2023); the evaluation of the main nutritional components of adult algae and products of Sargassum fusiforme is implemented by referring to the methods of the authorized invention patents "A Method for Classifying the Quality Grades of Primary Dried Products of Sargassum fusiforme" (ZL201911080356.X) and "A Method for Classifying the Quality Grades of Air Bladder Export Products of Cultured Sargassum fusiforme" (ZL201911132779.1); the genetic stability analysis of the new germplasm of Sargassum fusiforme refers to the scientific research paper "Research on the Screening of Strains of Cultivated Sargassum fusiforme in Dongtou, Zhejiang Based on Cluster and Reduced Representation Genome Joint Analysis" published by the inventor, and the technical process of the present invention is systematically compiled.

[0087] b. The light cultivation rack and the temporary cultivation device group involved in the present invention are both independently innovatively designed and built by the inventor, which can meet the regulation of environmental factors such as the optimal temperature, light intensity, photoperiod, and seawater. The main structure of the light cultivation rack includes left and right side frames made of carbon steel, toughened glass laminates, glass laminate bottom frames, crossbars, switch boxes, and LED lighting lamps, and is placed in a controllable dark room that meets the conditions for temporary cultivation in a seawater biology laboratory, which can ensure 0 μmol·m -2 ·s -1 ~350 μmol·m -2 ·s -1 light intensity. The external control power supply timing converter can achieve photoperiod regulation; the cultivation room is set as an inner dark room and is equipped with air conditioning equipment to regulate the temperature of the space in the cultivation room;

[0088] The left and right side frames made of carbon steel and the glass laminate bottom frames arranged between them form the main framework. The toughened glass laminates are laid and fixed in the layers of the framework. It should be noted that the glass laminate bottom frame in the middle of the framework is detachably engaged with the left and right side frames for regulating the floor height. LED lighting lamps are fixed on the glass laminate bottom frame below the toughened glass laminate, and the LED lighting lamps irradiate downward. A switch box is set to control the independent switch of the LED lighting lamps. The cultivation trays of the temporary cultivation device group are placed above the toughened glass laminates. When in use, based on the light transmission characteristics of the toughened glass, it can also improve light transmission and utilization rate;

[0089] The main structure of the temporary cultivation device group includes a water storage bucket, a cultivation tray, a peristaltic pump, an oxygen supply pump, a water delivery hose, and a sand head, which can achieve centralized oxygen dissolution, water supply and circulation of seawater, and can ensure sufficient supply of oxygen and nutrient nutrients for the temporarily cultivated Sargassum fusiforme seedlings;

[0090] c. The indoor temporary cultivation of the new germplasm parents of Sargassum fusiforme involved in the present invention includes seawater storage, seawater sterilization, seawater replacement, setting of the optimal light intensity, photoperiod, and temperature, matching of male and female for seeding on the support, etc. The cultivation seawater for seawater storage includes the following preparation steps: Store the freshly filtered seawater in the wild area in a 1000L chemical barrel and transport it to the factory area, then transfer it to 25L chemical barrels and carry them indoors. Use a water heater to heat the seawater to 80℃~90℃ for 30min~40min at high temperature to kill animal eggs, microorganisms, microalgae, and large seaweed larvae, and naturally cool it to room temperature. Inject water into the water storage bucket of the temporary cultivation device group according to the ratio of 4L~5L of seawater per 100g of seedlings; Add nutrients according to the volume ratio (V:V) of nutrients to the cultivation seawater of (0.6~0.8) ml:1L; Adjust the light intensity to 200 μmol·m -2 ·s -1 ~280 μmol·m -2 ·s -1, the photoperiod is 14h:10h to 12h:12h, and the temperature is 24°C to 25°C; the mass ratio of male to female of the vegetable-growing parent is 2:1 to 1:1;

[0091] d. The pest control method involved in the present invention includes manually removing epiphytic macroalgae, soaking in fresh water to remove epiphytic animals, soaking in chemical liquid to remove epiphytic microalgae, etc. The macroalgae such as Ulva lactuca, Enteromorpha prolifera, and fresh vegetables attached to the parents of the new germplasm of Sargassum fusiforme are removed by manual picking; the amphipods, phasmid-like insects, fish eggs, etc. attached to the parents of the new germplasm of Sargassum fusiforme are removed by the method of low-osmosis disinfection by soaking in fresh water for 15 min to 20 min; for the epiphytic microalgae on the parents of the new germplasm of Sargassum fusiforme, the mass (kg): volume (L) ratio of fresh algae to citric acid (C6H8O7) solution is 1:3, the concentration of citric acid is 1.5 mg / ml, the soaking time is 5 min to 8 min, and after taking out, it is rinsed 3 to 5 times with fresh water at room temperature and 3 to 5 times with fresh, filtered, and sterilized seawater for removal. The epiphytic cyanobacteria (cyanobacteria) in the offspring young sporophyte suspension system are soaked and cultured in a penicillin seawater solution of 300,000 to 600,000 per liter for 24 h to 48 h to kill the cyanobacteria, then filtered seawater is used to wash 3 to 5 times, and then placed in filtered seawater for continuous suspension culture to ensure no interference from pest organisms.

[0092] e. The optimal nutrients involved in the present invention include nitrogen (N) and phosphorus (P) elements, and the ratio of the optimal nutrients is a seawater solution prepared by NaNO3 and KH2PO3 with N:P = 10:1, and the concentration is 10 (N) g / L or 1 (P) g / L. The volume ratio (V:V) of the nutrients to the culture seawater during the suspension culture growth and development stage from the fertilized eggs of Sargassum fusiforme to the young sporophytes (trilobate to multi-lobate) is (0.5 to 0.7) ml:1 L to (0.6 to 0.8) ml:1 L;

[0093] f. The suspension culture device group of Sargassum fusiforme fertilized eggs involved in the present invention was independently innovatively designed and built by the inventor, which can ensure the environmental conditions for the indoor suspension growth and development of Sargassum fusiforme fertilized eggs. The main structure of the suspension culture device group of Sargassum fusiforme fertilized eggs includes a 5000 ml glass conical flask, a glass tube, an air control valve, an air supply pump, a hose, etc. (all used are standard parts). Among them, the glass conical flask is set with an opening under the condition of ensuring the environment in the dark room. One end of the glass tube is connected to the hose, and the other end is inserted below the water level in the glass conical flask. The other end of the hose is connected to the air supply pump. The air control valve is arranged on the hose to control the air intake. When the above device is used, the air supply pump presses the air into the glass conical flask through the hose. The glass tube is inclined relative to the conical flask, and the air outlet forms an angle with the bottom of the bottle. The ejected air flow drives the water liquid to form a vortex, which rolls the water liquid in the bottle and forms a stirring force to drive the fertilized eggs at the bottom of the bottle to move continuously in the water liquid, forming the basis of suspension culture, ensuring that the fertilized eggs will not deposit at the bottom of the bottle. During the suspension culture process, low-density quantitative sub-packaging operations also need to be carried out. The density gradually decreases from 8000 - 10000 fertilized eggs / 5000 ml of seawater to 400 - 500 young sporophytes / 5000 ml of seawater to ensure normal growth and development;

[0094] g. The raft device group for the natural sea area isolation and stocking of new germplasm seedlings of Sargassum fusiforme involved in the present invention was independently innovatively designed and built, which can ensure the purity of the new germplasm strain. The raft device for the natural sea area isolation and stocking of new germplasm seedlings of Sargassum fusiforme adopts a soft raft design. The overall length × width of the raft is 100 m × 100 m, and it is internally provided with 9 equal hanging culture areas. To ensure the isolation and culture effect, the new germplasm seedlings of Sargassum fusiforme are respectively hung at the four corners of the raft, and a surrounding net is set to ensure the strain purity.

[0095] Example 1: The creation and evaluation of new germplasm through sexual reproduction of wild Sargassum fusiforme parents from Shandong Moye Island (Yellow Sea and Bohai Sea area) to artificial domestication and cultivation in Dongtou District, Wenzhou City, Zhejiang Province, and comparison with wild Sargassum fusiforme parents in Dongtou District, Wenzhou City.

[0096] In this example, the geographical location differences and ecological factor differences between the original growth areas and domestication and cultivation areas of wild Sargassum fusiforme plants are not significant.

[0097] As the first example of the present invention, applicable to the creation and evaluation of new germplasm through sexual reproduction of "north-south transplanted" wild Sargassum fusiforme parents, the method for creating and evaluating new germplasm by suspension culture of Sargassum fusiforme fertilized eggs includes the following steps:

[0098] 1. Selection, isolation culture, and unit mass evaluation of wild Sargassum fusiforme geographical parents

[0099] The present invention takes Dongtou District, Wenzhou City, known as the "Hometown of Sargassum fusiforme in China", as the geographical center, and wild Sargassum fusiforme from the Yellow Sea and Bohai Sea (north-south transplanted) as the geographical wild parents.

[0100] Collect wild Sargassum fusiforme parents moderately from October every year to a certain island in the Yellow Sea and Bohai Sea, and culture them in an ecological isolation manner in the Dongtou sea area after tying them with ropes.

[0101] The quality evaluation of wild Sargassum fusiforme parents (new germplasm parents) is carried out with reference to the method of the authorized invention patent "A Method for Unit Quality Evaluation and Difference Comparison of Cultivation of Sargassum fusiforme Juvenile Sporophytes" (ZL202110213367.1).

[0102] 2. Screening, isolated cultivation and control of enemy organisms of male and female plants of high-value trait lines of wild Sargassum fusiforme parents

[0103] The screening of male and female plants of high-value trait lines of wild Sargassum fusiforme parents is carried out with reference to the relevant content of the scientific research paper "Research on the Screening of Cultivated Sargassum fusiforme (Sargassum fusiforme) Lines in Dongtou, Zhejiang Based on the Joint Analysis of Clustering and Reduced Genome" published by the inventor and the authorized invention patent "A Method for Strain Identification and Classification of Sargassum fusiforme" (ZL201511004442.4); the isolated cultivation and control of enemy organisms are carried out with reference to the relevant content of the local technical specifications of Dongtou District, Wenzhou City, "Technical Regulations for Cultivating High-quality Seedlings of Sargassum fusiforme" (DB330305 / T22-2023) and "Technical Regulations for Cultivation Management of Sargassum fusiforme" (DB330305 / T23-2023) drafted and released by the inventor.

[0104] The quality evaluation of the offspring of wild Sargassum fusiforme parents is carried out with reference to the method of the authorized invention patent "A Method for Unit Quality Evaluation and Difference Comparison of Cultivation of Sargassum fusiforme Juvenile Sporophytes" (ZL202110213367.1).

[0105] 3. Removal of epiphytic enemy organisms of male and female plants of high-value trait lines of wild Sargassum fusiforme parents

[0106] 3.1 Indoor temporary cultivation of male and female plants of high-value trait lines of wild Sargassum fusiforme parents

[0107] 3.1.1 Removal of epiphytic macroalgae

[0108] Manually remove.

[0109] 3.1.2 Removal of epiphytic animals

[0110] Soak in fresh water at room temperature for 20 min, kill epiphytic animals by hypotonic treatment, and rinse 3 times with fresh, filtered and sterilized seawater.

[0111] 3.1.3 Removal of epiphytic microalgae

[0112] The mass (kg) of the fresh weight of the thallus to the volume (L) of the citric acid (C6H8O7) solution is 1:3, the concentration of citric acid is 1.5 mg / ml, the soaking time is 5 min, take it out and rinse 3 times with fresh water at room temperature, and rinse 3 times with fresh, filtered and sterilized seawater.

[0113] 3.2 Treatment of male and female strains of high-value trait strains of wild Sargassum fusiforme parents

[0114] 3.2.1 Screening of lateral branches and removal of stems

[0115] Remove the stems of the first- or second-order lateral branches of the fresh thalli, and retain the second- or third-order lateral branches with dense receptacles attached.

[0116] 3.2.2 Placement of lateral branches of male and female strains and mass ratio

[0117] According to the actual area of the culture tray, reasonably distribute the lateral branches. The distribution standard is that the lateral branches do not overlap with each other to ensure uniform light. The mixed mass ratio (kg:kg) of female and male strains is 1:1.

[0118] 4 Creation of the optimal environmental factors for indoor temporary cultivation of lateral branches of male and female strains of high-value trait strains of wild Sargassum fusiforme parents

[0119] 4.1 Optimal water environment

[0120] Fresh: Take the clear seawater at high tide on the same day and let it settle naturally for 3 h;

[0121] Filtration: Filter the supernatant seawater through a 280-mesh silk screen;

[0122] High-temperature sterilization: Heat the filtered seawater to 80 °C and sterilize it at high temperature for 30 min to kill animal eggs, microorganisms, microalgae, and larvae of large seaweeds, and then let it cool naturally to room temperature;

[0123] 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;

[0124] Salinity: 28;

[0125] Nutrient salts: 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 nutrient salts to culture seawater is 0.6 ml:1 L.

[0126] 4.2 Culture room, light culture rack, and temporary cultivation device

[0127] The culture room used in the present invention is an indoor dark room. Based on the light culture rack, the light intensity is controlled, and the temporary cultivation device is placed on the shelf of the light culture rack. LED lamps are installed on the lower side of the board between layers to provide light;

[0128] 4.3 Optimal temperature, light intensity, and photoperiod

[0129] Temperature: 23 °C;

[0130] Light intensity: 200 μmol·m -2 ·s -1

[0131] Photoperiod: 14h:10h.

[0132] Treatment and temporary cultivation time of lateral branches of male and female plants of high-value trait lines of wild Sargassum fusiforme parents in the greenhouse

[0133] 5.1 Treatment of lateral branches in greenhouse temporary cultivation

[0134] Replace 1 / 2 of the total culture seawater in the culture group every day, and turn the lateral branches once every 6 h to ensure that the receptacles of each lateral branch receive uniform light.

[0135] 5.2 Temporary cultivation time of lateral branches in greenhouse

[0136] Temporarily cultivate the lateral branches in the greenhouse for 5 d.

[0137] 6. Classification collection, impurity removal and cleaning of fertilized eggs of new germplasm of Sargassum fusiforme

[0138] Collect the fertilized eggs with a 260-mesh silk screen, and remove impurities such as air sacs and receptacles with a 30-mesh silk screen. Place the fertilized eggs in a 2000 mL beaker, add 1500 mL of freshly filtered seawater, stir well and let it settle naturally for 2 min. Discard the upper suspension to remove suspended minute impurities, and repeat this operation 5 times to obtain fresh and pure fertilized eggs. The fertilized eggs of different lines need to be collected and the impurities removed separately.

[0139] 7. Establishment of suspension system for fertilized eggs of new germplasm of Sargassum fusiforme

[0140] 7.1 Quantitative packaging of fertilized eggs of new germplasm of Sargassum fusiforme

[0141] Remove the excess water from the fresh and pure fertilized eggs, technically count the volume density of the fertilized eggs, quantitatively package 8000 fertilized eggs in each 3000 ml Erlenmeyer flask, and add freshly filtered seawater. The ratio of the number of fresh and pure fertilized eggs (pieces) to the volume of seawater (ml) is 10:1. For different lines, they need to be classified and packaged and labeled.

[0142] 7.2 Creation of the optimal environment for suspension culture of fertilized eggs of new germplasm of Sargassum fusiforme

[0143] 7.2.1 Optimal water environment

[0144] The fresh, filtered, high-temperature sterilized, pH value, salinity and nutrient salts are configured according to the method of Example 4.1 of this embodiment, and the volume ratio (V:V) of nutrient salts to culture seawater is 0.5 ml:1 L. The optimal water environment conditions remain unchanged during the suspension culture period.

[0145] 7.2.2 Optimal conditions such as light intensity, photoperiod, temperature, aeration, etc.

[0146] Optimal light intensity: 50 μmol·m -2 ·s -1 ;

[0147] Photoperiod: 14h:10h;

[0148] Temperature: 23°C;

[0149] Aeration: Auxiliary aeration.

[0150] 7.2.3 Suspension culture method for fertilized eggs of new Sargassum fusiforme germplasm

[0151] In the initial stage of suspension culture of Sargassum fusiforme fertilized eggs, magnetic stirring + aeration suspension culture is adopted. After 8 days of culture, the fertilized eggs of Sargassum fusiforme have grown and developed into embryos. Since the beginning of the embryo growth and development stage, a self-developed suspension culture device group is used for suspension culture ( Figure 1 ), and only aeration suspension culture is implemented.

[0152] 7.3 Low-density quantitative sub-packaging during the suspension growth and development stage of embryos to young sporophytes of new Sargassum fusiforme germplasm

[0153] During the growth and development stage of embryos to young sporophytes, sub-packaging is carried out at a density of 1 / 2 equal volume per bottle every 19 days. As the culture time gradually extends, sub-packaging is carried out step by step at a decreasing low density, that is: the first low-density sub-packaging decreases from 8000 to 4000, the second low-density sub-packaging decreases from 4000 to 2000, the third low-density sub-packaging decreases from 2000 to 1000, and the last low-density sub-packaging decreases from 1000 to 400.

[0154] 8. Control of typical harmful organisms in the suspension system during the suspension growth and development stage of embryos to young sporophytes of new Sargassum fusiforme germplasm

[0155] 8.1 Control method for harmful animals

[0156] If harmful animals are found in the suspension culture solution, soak it in ultrapure water for 20 minutes every 10-day culture period to kill the harmful animals by hypotonic treatment.

[0157] 8.2 Control method for harmful cyanobacteria

[0158] Regularly examine embryos or young sporophytes under a microscope. If signs of epiphytic cyanobacteria are found on embryos or young sporophytes, soak and culture them in a seawater solution of 300,000 units of penicillin for 24 hours to kill the cyanobacteria, then wash them 3 times with filtered seawater, and continue suspension culture with filtered seawater.

[0159] 8.3 Control method for harmful green algae

[0160] Regularly microscopically examine the embryo or young sporophyte. If signs of epiphytic green algae are found on the embryo or young sporophyte, soak it in 1.5 mg / ml citric acid solution for 6 min, rinse it 3 times with fresh water, rinse it 2 times with filtered seawater, and then continue to suspend and culture it with filtered seawater.

[0161] 9. Clamping the ropes and indoor temporary cultivation of young sporophytes of the new germplasm of Sargassum fusiforme (the structure of the clamped ropes and young sporophytes, such as Figure 2 )

[0162] Clamp the young sporophytes with a basal leaf length of 1.5 cm onto cotton ropes or fiber ropes with a diameter of 0.4 cm, with a spacing of 3 cm. For young sporophytes with a basal leaf length less than 1.5 cm, continue to suspend and culture them until the basal leaf length ≥ 1.5 cm and then clamp the ropes. The clamped young sporophytes are temporarily cultivated indoors for 15 d.

[0163] 10. Sea area stocking of young sporophytes of the new germplasm of Sargassum fusiforme

[0164] This content is implemented with reference to the relevant content of the local technical specification "Technical Regulations for Cultivation and Management of Sargassum fusiforme" (DB330305 / T 23-2023) drafted and published by the inventor.

[0165] 11. Evaluation methods for wild Sargassum fusiforme parents and new germplasms of their offspring

[0166] This item refers to the methods of authorized invention patents, drafted and published local standards, and published scientific research papers, and implements item by item the evaluation of the quality of new germplasm seedlings, trait evaluation, yield evaluation, processing ratio evaluation, rehydration rate evaluation, evaluation of the main nutritional components of the adult algae and products (including the valence state and composition of arsenic), and evaluation of the genetic stability of new germplasms, as follows:

[0167] 11.1 Trait evaluation of wild Sargassum fusiforme parents and their offspring

[0168] This item is evaluated with reference to the method of the inventor's authorized invention patent "A Method for Strain Identification and Classification of Sargassum fusiforme" (ZL201511004442.4).

[0169] 11.2 Yield evaluation of wild Sargassum fusiforme parents and their offspring

[0170] This item is evaluated with reference to the method of the inventor's authorized invention patent "A Method for Evaluating and Comparing the Yield per Unit Fresh / Dry Weight and Water Content of Primary Products of Cultured Sargassum fusiforme" (ZL201911080353.6).

[0171] 11.3 Processing ratio evaluation of wild Sargassum fusiforme parents, offspring dry product raw materials and rough processing products

[0172] This content is evaluated by referring to the relevant content of the local technical specification of Dongtou District, Wenzhou City, "Quality Grading Specification for Semi-dried Sargassum fusiforme" (DB330305 / T24-2023) drafted and published by the inventor.

[0173] 11.4 Evaluation of the rehydration rate of wild Sargassum fusiforme parent and offspring rough processing products

[0174] This content is evaluated by referring to the relevant content of the local technical specification of Dongtou District, Wenzhou City, "Quality Grading Specification for Semi-dried Sargassum fusiforme" (DB330305 / T24-2023) drafted and published by the inventor.

[0175] 11.5 Evaluation of the main nutritional components of wild Sargassum fusiforme parent, offspring adult algae and products

[0176] This item is evaluated by referring to the methods of the inventor's authorized invention patents "A Method for Classifying the Quality Grade of Semi-dried Primary Products of Sargassum fusiforme" (ZL201911080356.X) and "A Method for Classifying the Quality Grade of the Air-bladder Export Products of Cultured Sargassum fusiforme" (ZL201911132779.1).

[0177] 11.6 Genetic stability analysis of wild Sargassum fusiforme offspring - new germplasm

[0178] This item is qualitatively analyzed by referring to the method of the inventor's published scientific research paper "Research on the Screening of Cultivated Sargassum fusiforme Strains in Dongtou, Zhejiang Based on Cluster and Reduced-representation Genome Joint Analysis".

[0179] Example 2 of implementation: Creation and evaluation of new germplasm by artificial domestication and cultivation of wild Sargassum fusiforme parents from Mount Moye Island in Shandong (Yellow Sea and Bohai Sea) and wild Sargassum fusiforme parents from Nan'ao Island in Guangdong (South China Sea) at the same time, and comparison with wild Sargassum fusiforme parents in Dongtou District, Wenzhou City.

[0180] In this example, the geographical location differences and ecological factor differences of the original growth areas and domestication and cultivation areas of each wild Sargassum fusiforme plant are not significant, and the differences in the original growth areas, geographical locations and ecological factors among the three are significant.

[0181] As the first implementation example of the present invention, it is applicable to the creation and evaluation of new germplasm through sexual reproduction of wild Sargassum fusiforme parents of "northward species transplantation to the south" + "southward species transplantation to the north". The method for creating and evaluating new germplasm by suspension culture of Sargassum fusiforme fertilized eggs includes the following steps:

[0182] 1. Selection, isolated cultivation and unit mass evaluation of wild Sargassum fusiforme geographical parents

[0183] The present invention takes Dongtou District, Wenzhou City, known as the "Hometown of Sargassum fusiforme in China", as the geographical center, and uses wild Sargassum fusiforme from the Yellow Bohai Sea (northward transplantation to the south) and wild Sargassum fusiforme from the South China Sea (southward transplantation to the north) as wild parents with geographical differences. In November each year, appropriate amounts of wild Sargassum fusiforme parents are collected from certain islands in the Yellow Bohai Sea and the South China Sea respectively, and after being tied with ropes, they are ecologically isolated and cultured in the sea area of Dongtou. The quality evaluation of wild Sargassum fusiforme parents (new germplasm parents) is implemented with reference to the method of the inventor's authorized invention patent "A Method for Unit Quality Evaluation and Difference Comparison of Cultivation of Sargassum fusiforme Juvenile Sporophytes" (ZL202110213367.1).

[0184] 2. Screening, isolated cultivation and control of enemy organisms for male and female plants of high-value trait strains of wild Sargassum fusiforme parents

[0185] The screening of male and female plants of high-value trait strains of wild Sargassum fusiforme parents is implemented with reference to the relevant content of the scientific research paper "Research on the Screening of Cultivated Sargassum fusiforme Strains in Dongtou, Zhejiang Based on the Joint Analysis of Clustering and Reduced Genome" published by the inventor and the authorized invention patent "A Method for Strain Identification and Classification of Sargassum fusiforme" (ZL201511004442.4); the isolated cultivation and control of enemy organisms are implemented with reference to the relevant content of the local technical specifications of Dongtou District, Wenzhou City, "Technical Regulations for Cultivation of High-quality Seedlings of Sargassum fusiforme" (DB330305 / T22 - 2023) and "Technical Regulations for Cultivation Management of Sargassum fusiforme" (DB330305 / T23 - XXXX) drafted and issued by the inventor. The quality evaluation of the offspring of wild Sargassum fusiforme parents is implemented with reference to the method of the inventor's authorized invention patent "A Method for Unit Quality Evaluation and Difference Comparison of Cultivation of Sargassum fusiforme Juvenile Sporophytes" (ZL202110213367.1).

[0186] 3. Removal of epiphytic enemy organisms from male and female plants of high-value trait strains of wild Sargassum fusiforme parents

[0187] 3.1 Indoor temporary cultivation of male and female plants of high-value trait strains of wild Sargassum fusiforme parents

[0188] 3.1.1 Removal of epiphytic macroalgae

[0189] Manually remove.

[0190] 3.1.2 Removal of epiphytic animals

[0191] Soak in fresh water at room temperature for 23 minutes, hypotonic to kill epiphytic animals, and rinse 4 times with fresh, filtered and sterilized seawater.

[0192] 3.1.3 Removal of epiphytic microalgae

[0193] The fresh weight of the thallus and the mass (kg) of the citric acid (C6H8O7) solution: volume (L) is 1:3, the concentration of citric acid is 1.5 mg / ml, the soaking time is 7 min, and after taking out, it is rinsed 4 times with fresh water at room temperature and 4 times with fresh, filtered, and sterilized seawater.

[0194] 3.2 Treatment of male and female plants of the high-value trait strain of wild Sargassum fusiforme parents

[0195] 3.2.1 Screening of lateral branches and removal of stems

[0196] Remove the stems of the first- or second-order lateral branches of the fresh thallus, and retain the second- or third-order lateral branches with dense receptacles.

[0197] 3.2.2 Placement of lateral branches of male and female plants and mass ratio

[0198] According to the actual area of the culture tray, reasonably distribute the lateral branches. The distribution standard is that the lateral branches do not overlap with each other to ensure uniform light. The mixing mass ratio (kg:kg) of female plants to male plants is 1:1.

[0199] 4 Creation of the optimal environmental factors for indoor temporary cultivation of lateral branches of male and female plants of the high-value trait strain of wild Sargassum fusiforme parents

[0200] 4.1 Optimal water environment

[0201] Fresh: Take the clear seawater at high tide on the same day and let it settle naturally for 3.5 h;

[0202] Filtration: The supernatant seawater is filtered through a 290-mesh silk screen;

[0203] High-temperature sterilization: Heat the filtered seawater to 85 °C and sterilize it at high temperature for 35 min to kill animal eggs, microorganisms, microalgae, and the larvae of large seaweeds, and then let it cool naturally to room temperature;

[0204] pH value: Adjust the pH value of the sterilized seawater to 8.0 with 0.05 mol / L NaOH solution and 0.05 mol / L HCl solution;

[0205] Salinity: 30;

[0206] Nutrient salts: 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 nutrient salts to culture seawater is 0.7 ml:1 L.

[0207] 4.2 Culture room, light culture rack, and temporary cultivation device

[0208] Same as Example 1

[0209] 4.3 Optimal temperature, light intensity, and photoperiod

[0210] Temperature: 24°C;

[0211] Light intensity: 240 μmol·m -2 ·s -1 ;

[0212] Photoperiod: 13h:11h.

[0213] Treatment and temporary culture time of lateral branches of male and female plants of high-value trait lines of wild Sargassum fusiforme parental plants

[0214] 5.1 Indoor temporary culture treatment of lateral branches

[0215] Replace 1 / 2 of the total culture seawater volume of the culture group every day, and turn the lateral branches once every 7 hours to ensure that the receptacles of each lateral branch receive uniform light.

[0216] 5.2 Indoor temporary culture time of lateral branches

[0217] The lateral branches are temporarily cultured indoors for 6 days.

[0218] 6. Classification collection, impurity removal and cleaning of fertilized eggs of new germplasm of Sargassum fusiforme

[0219] Collect fertilized eggs using a 270-mesh silk screen, and remove impurities such as air bladders and receptacles with a 40-mesh silk screen. Place the fertilized eggs in a 2000 mL beaker, add 1500 mL of freshly filtered seawater, stir well and let it settle naturally for 2.5 minutes. Discard the upper suspension to remove suspended minute impurities, and repeat this operation 6 times to obtain a fresh and pure product of fertilized eggs. For different strains, fertilized eggs need to be collected and impurities removed separately.

[0220] 7. Creation of suspension system for fertilized eggs of new germplasm of Sargassum fusiforme

[0221] 7.1 Quantitative packaging of fertilized eggs of new germplasm of Sargassum fusiforme

[0222] Remove the excess water from the fresh and pure product of fertilized eggs, technically calculate the volume density of fertilized eggs, quantitatively package 9000 fertilized eggs in each 5000 ml Erlenmeyer flask, and add freshly filtered seawater. The ratio of the number of fresh and pure products of fertilized eggs (pieces) to the volume of seawater (ml) is 10:2. For different strains, they need to be classified and packaged and labeled.

[0223] 7.2 Creation of the optimal environment for suspension culture of fertilized eggs of new germplasm of Sargassum fusiforme

[0224] 7.2.1 Optimal water environment

[0225] For fresh, filtered, high-temperature sterilized water, the pH value, salinity and nutrient salts are configured according to the method of Example 4.1 of this embodiment. The volume ratio (V:V) of nutrient salts to culture seawater is 0.55 ml: 1 L. The optimal water environment conditions remain unchanged during suspension culture.

[0226] 7.2.2 Optimal light intensity, photoperiod, temperature, aeration and other conditions

[0227] Optimal light intensity: 55 μmol·m -2 ·s -1 ;

[0228] Photoperiod: 13h:11h;

[0229] Temperature: 23.5 °C;

[0230] Aeration: Auxiliary aeration.

[0231] 7.2.3 Suspension culture method of fertilized eggs of new Sargassum fusiforme germplasm

[0232] In the initial stage of suspension culture of Sargassum fusiforme fertilized eggs, magnetic stirring + aeration suspension culture is adopted. After 9 days of culture, the fertilized eggs of Sargassum fusiforme have grown and developed into embryos. Since the beginning of the embryo growth and development stage, a self-created suspension culture device group is used for suspension culture ( Figure 1 ), and only aeration suspension culture is implemented.

[0233] 7.3 Low-density quantitative sub-packaging of suspension culture during the suspension growth and development stage of embryos to young sporophytes of new Sargassum fusiforme germplasm

[0234] During the growth and development stage of embryos to young sporophytes, sub-packaging is carried out at a density of 1 / 2 equal volume per bottle every 20 days. As the culture time gradually extends, sub-packaging is carried out step by step at a decreasing low density, that is: the first low-density sub-packaging decreases from 9000 to 4500, the second low-density sub-packaging decreases from 4500 to 2250, the third low-density sub-packaging decreases from 2250 to 1125, and the last low-density sub-packaging decreases from 1125 to 562.

[0235] 8. Control of typical harmful organisms in the suspension system during the suspension growth and development stage of embryos to young sporophytes of new Sargassum fusiforme germplasm

[0236] 8.1 Control methods for harmful animals

[0237] If harmful animals are found in the suspension culture solution, soak it in ultrapure water for 23 minutes every 11-day culture period to kill the harmful animals by hypotonic treatment.

[0238] 8.2 Control methods for harmful cyanobacteria

[0239] Regularly examine embryos or young sporophytes under a microscope. If signs of epiphytic cyanobacteria are found on the embryos or young sporophytes, soak and culture them in a seawater solution containing 500,000 units / L of penicillin for 36 hours to kill the cyanobacteria, then wash them 4 times with filtered seawater, and continue suspension culture with filtered seawater.

[0240] 8.3 Control methods for harmful green algae

[0241] Regularly microscopically examine the embryo or young sporophyte. If signs of epiphytic green algae are found on the embryo or young sporophyte, soak it in 1.5 mg / ml citric acid solution for 7 min, rinse it 4 times with fresh water, rinse it 3 times with filtered seawater, and then continue to suspend and culture it with filtered seawater.

[0242] 9. Clamping the ropes with young sporophytes of the new germplasm of Sargassum fusiforme and indoor temporary cultivation

[0243] Clamp the young sporophytes with a basal leaf length of 1.5 cm - 2.0 cm onto cotton ropes or fiber ropes with a diameter of 0.4 cm, with a spacing of 4 cm. For young sporophytes with a basal leaf length less than 1.5 cm, continue to suspend and culture them until the basal leaf growth length ≥ 1.8 cm and then clamp the ropes. The clamped young sporophytes are temporarily cultivated indoors for 18 d.

[0244] 10. Sea area stocking of young sporophytes of the new germplasm of Sargassum fusiforme

[0245] This content is implemented according to the relevant content of the local technical specification of Dongtou District, Wenzhou City, "Technical Regulations for Cultivation and Management of Sargassum fusiforme" (DB330305 / T23 - 2023) drafted and published by the first inventor of the present invention.

[0246] 11. Evaluation methods for wild Sargassum fusiforme parents and new germplasms of their offspring

[0247] This item of content refers to the methods of the inventor's authorized invention patent, the drafted and published local standards, and the published scientific research papers, and implements item by item the evaluation of the quality of new germplasm seedlings, trait evaluation, yield evaluation, processing ratio evaluation, rehydration rate evaluation, evaluation of the main nutritional components of adult algae and products (including the valence state and composition of arsenic), and evaluation of the genetic stability of new germplasms, etc.

[0248] 11.1 Trait evaluation of wild Sargassum fusiforme parents and their offspring

[0249] This item of content is evaluated by referring to the method of the inventor's authorized invention patent "A Method for Strain Identification and Classification of Sargassum fusiforme" (ZL201511004442.4).

[0250] 11.2 Yield evaluation of wild Sargassum fusiforme parents and their offspring

[0251] This item of content is evaluated by referring to the method of the inventor's authorized invention patent "A Method for Evaluating and Comparing the Yield per Unit Fresh / Dry Weight and Water Content of Primary Products of Cultured Sargassum fusiforme" (ZL201911080353.6).

[0252] 11.3 Processing ratio evaluation of wild Sargassum fusiforme parents, offspring dry product raw materials and rough processing products

[0253] This content is evaluated by referring to the relevant content of the local technical specification "Quality Grading Specification for Semi-dried Sargassum fusiforme" (DB330305 / T24-2023) drafted and published by the inventor in Dongtou District, Wenzhou City.

[0254] 11.4 Evaluation of the rehydration rate of the rough processed products of wild Sargassum fusiforme parents and offspring

[0255] This content is evaluated by referring to the relevant content of the local technical specification "Quality Grading Specification for Semi-dried Sargassum fusiforme" (DB330305 / T24-2023) drafted and published by the inventor in Dongtou District, Wenzhou City.

[0256] 11.5 Evaluation of the main nutritional components of the adult algae and products of wild Sargassum fusiforme parents and offspring

[0257] This item is evaluated by referring to the methods of the invention patents authorized to the inventor, "A method for classifying the quality grades of semi-dried primary products of Sargassum fusiforme" (ZL201911080356.X) and "A method for classifying the quality grades of the air bladder export products of cultured Sargassum fusiforme" (ZL201911132779.1).

[0258] 11.6 Analysis of the genetic stability of the offspring of wild Sargassum fusiforme - new germplasms

[0259] This item is qualitatively analyzed by referring to the method of the scientific research paper "Research on the screening of strains of cultivated Sargassum fusiforme in Dongtou, Zhejiang based on combined analysis of clustering and reduced-representation genome" published by the inventor.

[0260] Example 3 of implementation: The creation and evaluation of new germplasms of wild Sargassum fusiforme parents from Mount Moye Island in Shandong (Yellow Sea and Bohai Sea), wild Sargassum fusiforme parents from Nan'ao Island in Guangdong (South China Sea), and wild Sargassum fusiforme parents from Nanji Island in Wenzhou City (adjacent area) through artificial domestication and cultivation in Dongtou District, Wenzhou City, and comparison with wild Sargassum fusiforme parents in Dongtou District, Wenzhou City. In this example, the geographical location differences and ecological factor differences between the original growth areas and domestication and cultivation areas of each wild Sargassum fusiforme plant are not significant, and the differences in the original growth areas, geographical locations, and ecological factors among the four are significant.

[0261] As the first implementation example of the present invention, it is applicable to the creation and evaluation of new germplasms through sexual reproduction of wild Sargassum fusiforme parents of "northward species transfer to the south" + "southward species transfer to the north" + "local", and the method for creating and evaluating new germplasms by suspension culture of Sargassum fusiforme fertilized eggs includes the following steps:

[0262] 1. Selection, isolated cultivation, and unit mass evaluation of wild Sargassum fusiforme geographical parents

[0263] The present invention takes Dongtou District, Wenzhou City, known as the "Hometown of Sargassum fusiforme in China", as the geographical center, and uses wild Sargassum fusiforme from the Yellow Bohai Sea (northward transplantation to the south), the East China Sea (adjacent areas), and the South China Sea (southward transplantation to the north) as wild parents with geographical differences. In December each year, appropriate amounts of wild Sargassum fusiforme parents are collected from certain islands in the Yellow Bohai Sea, the East China Sea, and the South China Sea respectively, and after being tied with ropes, they are ecologically isolated and cultured in the sea area of Dongtou. The quality evaluation of the wild Sargassum fusiforme parents (new germplasm parents) is carried out according to the method of the inventor's authorized invention patent "A Method for Unit Quality Evaluation and Difference Comparison of Cultivation of Sargassum fusiforme Juvenile Sporophytes" (ZL202110213367.1).

[0264] 2. Screening, isolated cultivation and pest control of male and female plants of high-value trait strains of wild Sargassum fusiforme parents

[0265] The screening of male and female plants of high-value trait strains of wild Sargassum fusiforme parents is carried out with reference to the relevant content of the scientific research paper "Research on the Screening of Cultivated Sargassum fusiforme Strains in Dongtou, Zhejiang Based on the Joint Analysis of Clustering and Reduced Representation Genome" published by the inventor and the authorized invention patent "A Method for Strain Identification and Classification of Sargassum fusiforme" (ZL201511004442.4); the isolated cultivation and pest control are carried out with reference to the relevant content of the local technical specification of Dongtou District, Wenzhou City, "Technical Regulations for Cultivating High-quality Seedlings of Sargassum fusiforme" (DB330305 / T22 - 2023) and "Technical Regulations for Cultivation Management of Sargassum fusiforme" (DB330305 / T23 - 2023) drafted and issued by the inventor. The quality evaluation of the offspring of wild Sargassum fusiforme parents is carried out according to the method of the inventor's authorized invention patent "A Method for Unit Quality Evaluation and Difference Comparison of Cultivation of Sargassum fusiforme Juvenile Sporophytes" (ZL202110213367.1).

[0266] 3. Removal of epiphytic pests on male and female plants of high-value trait strains of wild Sargassum fusiforme parents

[0267] 3.1 Indoor temporary cultivation of male and female plants of high-value trait strains of wild Sargassum fusiforme parents

[0268] 3.1.1 Removal of epiphytic macroalgae

[0269] Manually remove.

[0270] 3.1.2 Removal of epiphytic animals

[0271] Soak in fresh water at room temperature for 25 minutes to kill epiphytic animals by hypotonic treatment, and then rinse 5 times with fresh, filtered and sterilized seawater.

[0272] 3.1.3 Removal of epiphytic microalgae

[0273] The fresh weight of the thallus and the mass (kg) of the citric acid (C6H8O7) solution: volume (L) is 1:3, the concentration of citric acid is 1.5 mg / ml, the soaking time is 8 min, and after taking out, it is rinsed 5 times with fresh water at room temperature and 5 times with fresh, filtered and sterilized seawater.

[0274] 3.2 Treatment of male and female plants of the high-value trait strain of the wild Sargassum fusiforme parent

[0275] 3.2.1 Screening of lateral branches and removal of stems

[0276] Remove the stems of the first- or second-order lateral branches of the fresh thallus, and retain the second- or third-order lateral branches with densely growing receptacles.

[0277] 3.2.2 Placement of lateral branches of male and female plants and mass ratio

[0278] According to the actual area of the culture tray, reasonably distribute the lateral branches. The distribution standard is that the lateral branches do not overlap with each other to ensure uniform light. The mixing mass ratio (kg:kg) of female plants to male plants is 1:1.

[0279] 4 Creation of the optimal environmental factors for indoor temporary cultivation of lateral branches of male and female plants of the high-value trait strain of the wild Sargassum fusiforme parent

[0280] 4.1 Optimal water environment

[0281] Fresh: Take the clear seawater at high tide on the same day and let it settle naturally for 4 h;

[0282] Filtration: The supernatant seawater is filtered through a 300-mesh silk screen;

[0283] High-temperature sterilization: Heat the filtered seawater to 90 °C and sterilize it at high temperature for 40 min to kill animal eggs, microorganisms, microalgae and the larvae of large seaweeds, and then cool it naturally to room temperature;

[0284] pH value: Adjust the pH value of the sterilized seawater to 8.3 with 0.05 mol / L NaOH solution and 0.05 mol / L HCl solution;

[0285] Salinity: 33;

[0286] Nutrient salts: 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 nutrient salts to culture seawater is 0.8 ml:1 L.

[0287] 4.2 Culture room, light culture rack and temporary cultivation device

[0288] 4.3 Optimal temperature, light intensity and photoperiod

[0289] Temperature: 23 °C - 25 °C;

[0290] Light intensity: 280 μmol·m -2 ·s -1

[0291] Photoperiod: 12h:12h.

[0292] Treatment and temporary cultivation time of high-value trait strains of male and female lateral branches of wild Sargassum fusiforme parents in indoor

[0293] 5.1 Indoor temporary cultivation treatment of lateral branches

[0294] Replace 1 / 2 of the total culture seawater volume in the culture group every day, and turn the lateral branches once every 8 hours to ensure that the receptacles of each lateral branch receive uniform light.

[0295] 5.2 Indoor temporary cultivation time of lateral branches

[0296] The lateral branches are temporarily cultivated indoors for 7 days.

[0297] 6. Classification collection, impurity removal and cleaning of fertilized eggs of new germplasm of Sargassum fusiforme

[0298] Use a 280-mesh silk screen to collect fertilized eggs, and a 50-mesh silk screen to remove impurities such as air bladders and receptacles. Place the fertilized eggs in a 2000 mL beaker, add 1500 mL of freshly filtered seawater, stir well and let it settle naturally for 3 minutes, discard the upper suspension to remove suspended minute impurities, and repeat this operation 7 times to obtain a fresh and pure product of fertilized eggs. For different strains, the collection of fertilized eggs and impurity removal need to be carried out separately.

[0299] 7. Creation of suspension system of fertilized eggs of new germplasm of Sargassum fusiforme

[0300] 7. Quantification and sub-packaging of fertilized eggs of Sargassum fusiforme

[0301] Remove the excess water from the fresh and pure product of fertilized eggs, technically calculate the volume density of fertilized eggs, quantitatively sub-package 10,000 fertilized eggs in each 5000 ml Erlenmeyer flask, add freshly filtered seawater, and the ratio of the number of fresh and pure products of fertilized eggs (pieces) to the volume of seawater (ml) is 10:3. For different strains, classification sub-packaging and labeling are required.

[0302] Creation of the optimal environment for suspension culture of fertilized eggs of new germplasm of Sargassum fusiforme

[0303] 7.2.1 Optimal water environment

[0304] Fresh, filtered, high-temperature sterilized, pH value, salinity and nutrient salts are configured according to the method of Example 4.1 of this embodiment, and the volume ratio (V:V) of nutrient salts to culture seawater is 0.6 ml:1 L. The optimal water environment conditions remain unchanged during the suspension culture period.

[0305] 7.2.2 Optimal conditions such as light intensity, photoperiod, temperature, aeration, etc.

[0306] Optimal light intensity: 60 μmol·m -2 ·s -1 ;

[0307] Photoperiod: 12h:12h;

[0308] Temperature: 24 °C;

[0309] Aeration: Auxiliary aeration.

[0310] 7.2.3 Suspension culture method of fertilized eggs of new Sargassum fusiforme germplasm

[0311] In the initial stage of suspension culture of Sargassum fusiforme fertilized eggs, magnetic stirring + aeration suspension culture is adopted. After 10 days of culture, the fertilized eggs of Sargassum fusiforme have grown and developed into embryos. Since the beginning of the embryo growth and development period, a self-created suspension culture device group is used for suspension culture ( Figure 1 ), and only aeration suspension culture is implemented.

[0312] 7.3 Low-density quantitative sub-packaging of suspension culture during the growth and development stage from embryo to young sporophyte of new Sargassum fusiforme germplasm

[0313] During the growth and development stage from embryo to young sporophyte, sub-packaging is carried out at a density of 1 / 2 equal volume per bottle every 21 days. As the culture time gradually extends, sub-packaging is carried out step by step at a lower density, that is: the first low-density sub-packaging is reduced from 10,000 to 5,000, the second low-density is reduced from 5,000 to 2,500, the third low-density is reduced from 2,500 to 1,250, and the last low-density is reduced from 1,250 to 625.

[0314] 8. Control of typical harmful organisms in the suspension system during the suspension growth and development stage from embryo to young sporophyte of new Sargassum fusiforme germplasm

[0315] 8.1 Control methods for harmful animals

[0316] If harmful animals are found in the suspension culture solution, soak it in ultrapure water for 25 minutes every 12-day culture period to kill the harmful animals by hypotonic treatment.

[0317] 8.2 Control methods for harmful cyanobacteria

[0318] Regularly examine the embryo or young sporophyte under the microscope. If signs of epiphytic cyanobacteria are found on the embryo or young sporophyte, soak and culture it in a seawater solution of 600,000 units / L penicillin for 48 hours to kill the cyanobacteria, then wash it 5 times with filtered seawater, and continue suspension culture with filtered seawater.

[0319] 8.3 Control methods for harmful green algae

[0320] Regularly microscopically examine the embryo or young sporophyte. If signs of epiphytic green algae are found on the embryo or young sporophyte, soak it in 1.5 mg / ml citric acid solution for 8 min, rinse it with fresh water 5 times, rinse it with filtered seawater 3 times, and then continue to suspend and culture it with filtered seawater.

[0321] 9. Clamping the Rope and Indoor Temporary Cultivation of Young Sporophytes of New Germplasm of Sargassum fusiforme

[0322] Clamp the young sporophytes with a basal leaf length of 1.5 cm - 2.0 cm onto a cotton rope or fiber rope with a diameter of 0.4 cm, with a spacing of 3 cm - 5 cm. For young sporophytes with a basal leaf length less than 1.5 cm, continue to carry out suspension culture until the basal leaf growth length ≥ 2.0 cm and then clamp the rope. The clamped young sporophytes are temporarily cultured indoors for 20 d and then transferred to the natural sea area for stocking.

[0323] 10. Sea Area Stocking of Young Sporophytes of New Germplasm of Sargassum fusiforme

[0324] This content is implemented according to the relevant content of the local technical specification of Dongtou District, Wenzhou City, "Technical Regulations for the Cultivation and Management of Sargassum fusiforme" (DB330305 / T23 - 2023) drafted and published by the first inventor of the present invention.

[0325] 11. Evaluation Methods for Wild Sargassum fusiforme Parents and Offspring New Germplasms

[0326] This item of content refers to the methods of the inventor's authorized invention patent, the drafted and published local standards, and the publicly published scientific research papers, and implements item by item the evaluation of the quality of new germplasm seedlings, character evaluation, yield evaluation, processing ratio evaluation, rehydration rate evaluation, evaluation of the main nutritional components of the adult algae and products (including the valence state and composition of arsenic), and evaluation of the genetic stability of new germplasms, etc.

[0327] 11.1 Character Evaluation of Wild Sargassum fusiforme Parents and Offspring

[0328] This item of content is evaluated by referring to the method of the inventor's authorized invention patent "A Method for Identifying and Classifying Strains of Sargassum fusiforme" (ZL201511004442.4).

[0329] 11.2 Yield Evaluation of Wild Sargassum fusiforme Parents and Offspring

[0330] This item of content is evaluated by referring to the method of the inventor's authorized invention patent "A Method for Evaluating and Comparing the Yield per Unit Fresh / Dry Weight and Water Content of Primary Products of Cultured Sargassum fusiforme" (ZL201911080353.6).

[0331] 11.3 Processing Ratio Evaluation of Wild Sargassum fusiforme Parents, Offspring Dried Product Raw Materials and Rough Processing Products

[0332] This content is evaluated by referring to the relevant content of the local technical specification "Quality Grading Specification for Semi-dried Sargassum fusiforme" (DB330305 / T24-2023) drafted and published by the inventor in Dongtou District, Wenzhou City.

[0333] 11.4 Evaluation of the rehydration rate of semi-processed products of wild Sargassum fusiforme parents and offspring

[0334] This content is evaluated by referring to the relevant content of the local technical specification "Quality Grading Specification for Semi-dried Sargassum fusiforme" (DB330305 / T24-2023) drafted and published by the inventor in Dongtou District, Wenzhou City.

[0335] 11.5 Evaluation of the main nutritional components of adult algae and products of wild Sargassum fusiforme parents and offspring

[0336] This item is evaluated by referring to the methods of the inventor's authorized invention patents "A Method for Classifying the Quality Grade of Dried Products of Primary Sargassum fusiforme Products" (ZL201911080356.X) and "A Method for Classifying the Quality Grade of Pneumocystis Exports of Cultivated Sargassum fusiforme" (ZL201911132779.1).

[0337] 11.6 Genetic stability analysis of offspring of wild Sargassum fusiforme - new germplasm

[0338] This item is qualitatively analyzed by referring to the method of the inventor's published scientific research paper "Research on the Screening of Cultivated Sargassum fusiforme Strains in Dongtou, Zhejiang Based on the Combined Analysis of Clustering and Reduced Representation Genome".

[0339] Technical literature cited and referenced in the above technical solutions:

[0340] 1. Lin Lidong, Wu Mingjiang. 2021. Research on the Screening of Cultivated Sargassum fusiforme Strains in Dongtou, Zhejiang Based on the Combined Analysis of Clustering and Reduced Representation Genome [J]. Oceanologia et Limnologia Sinica, 52(5): 1224-1235;

[0341] 2. Lin Lidong, Wu Mingjiang, Zheng Ying. Patent Name: A Method for Evaluating and Comparing the Unit Mass of Cultivated Sargassum fusiforme Juvenile Sporophytes. Authorized Patent Number: ZL202110213367.1. Authorization Announcement Date: September 23, 2022. Approved Country: State Intellectual Property Office of the People's Republic of China. Patentee: Wenzhou Dongtou Marine and Fishery Development Research Center, Wenzhou University;

[0342] 3. Lin Lidong, Wu Mingjiang, Yan Xiufeng, Ma Zengling, Zhang Xu, Zheng Ying, Yu Ping. Patent Name: A Method for Strain Identification and Classification of Sargassum fusiforme. Authorized Patent Number: ZL201511004442.4. Authorization Announcement Date: January 9, 2018. 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] 4. Lin Lidong, Wu Mingjiang, Ma Zengling, Chen Binbin, Zheng Ying. Patent Name: A Method for Evaluating and Comparing the Yield per Unit Fresh / Dry Weight and Water Content of Primary Products of Cultured Sargassum fusiforme. Authorized Patent Number: ZL201911080353.6. Authorization Announcement Date: June 7, 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;

[0344] 5. Lin Lidong, Ma Zengling, Chen Binbin, Wu Mingjiang, Zheng Ying. Patent Name: A Method for Classifying the Quality Grades of Dried Primary Products of Sargassum fusiforme. Authorized Patent Number: ZL201911080356.X. Authorization Announcement Date: May 24, 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;

[0345] 6. Wu Mingjiang, Lin Lidong, Ma Zengling, Chen Binbin, Yu Ping, Wang Shengqin. Patent Name: A Method for Classifying the Quality Grades of Inflated-air Products of Cultured Sargassum fusiforme. Authorized Patent Number: ZL201911132779.1. Authorization Announcement Date: April 9, 2024. Approval Country: State Intellectual Property Office of the People's Republic of China. Patentee: Wenzhou University, Wenzhou Dongtou District Marine and Fishery Development Research Center;

[0346] 7. Lin Lidong, Yan Xiufeng, Zhang Peng, Wang Tiegan, Jia Yang, Liang Jia, Wu Fengmiao, Zhu Mingyi. Technical Regulation Name: Technical Regulation for Cultivating High-quality Seedlings of Sargassum fusiforme. Regulation Release Number: DJG330305 / T22—2023. Regulation Release Date: July 11, 2023. Specification Implementation Date: October 1, 2023. Regulation Drafting Units: Wenzhou Dongtou District Marine and Fishery Development Research Center, Wenzhou University, Zhejiang Ocean Aquaculture Research Institute, Zhejiang Ocean University, Zhejiang Ishihara Jinniu Biotechnology Co., Ltd., Wenzhou Dongtou District Fishery and Agriculture Technology Promotion Center;

[0347] 8. Lin Lidong, Yan Xiufeng, Zhang Peng, Wang Tiegan, Jia Yang, Liang Jia, Wu Fengmiao, Zhu Mingyi. Name of Technical Regulation: Technical Regulation for Cultivation and Management of Sargassum fusiforme. Regulation Issuance Number: DJG330305 / T23—2023. Regulation Issuance Date: July 11, 2023. Regulation Implementation Date: October 1, 2023. Regulation Drafting Units: Wenzhou Dongtou Marine and Fishery Development Research Center, Wenzhou University, Zhejiang Ocean Aquaculture Research Institute, Zhejiang Ocean University, Zhejiang Ishihara Jinniu Biotechnology Co., Ltd., Wenzhou Dongtou Fishery and Agriculture Technology Extension Center;

[0348] 9. Lin Lidong, Liang Jia, Yan Xiufeng, Wang Tiegan, Zhang Peng, Jia Yang, Wu Fengmiao, Zhu Mingyi. Name of Technical Specification: Quality Grading Specification for Semi-dried Sargassum fusiforme. Specification Issuance Number: DB330305 / T24—2023. Specification Issuance Date: July 11, 2023. Specification Implementation Date: October 1, 2023. Specification Drafting Units: Wenzhou Dongtou Marine and Fishery Development Research Center, Zhejiang Ocean University, Wenzhou University, Zhejiang Ocean Aquaculture Research Institute, Zhejiang Ishihara Jinniu Biotechnology Co., Ltd., Wenzhou Dongtou Fishery and Agriculture Technology Extension Center.

Claims

1. A method for creating and evaluating a new germplasm of floating culture of Sargassum fusiforme fertilized eggs, characterized in that: It includes the following steps: S1 Selection, isolated cultivation and unit mass evaluation of wild Sargassum fusiforme geoparents; S2 Screening of male and female plants of high-value trait strains of wild Sargassum fusiforme parents, isolated cultivation and control of pest organisms; S3 Removal of epiphytic pest organisms on male and female plants of high-value trait strains of wild Sargassum fusiforme; S3.1 Indoor temporary cultivation of male and female plants of high-value trait strains of wild Sargassum fusiforme S3.1.1 Removal of epiphytic macroalgae Manual removal; S3.1.2 Removal of epiphytic animals Soak in fresh water at room temperature for 20 min to 25 min, hypotonic to kill epiphytic animals, and rinse 3 to 5 times with fresh, filtered, and sterilized seawater; S3.1.3 Removal of epiphytic microalgae The mass (kg): volume (L) ratio of the fresh weight of the thallus to the citric acid (C6H8O7) solution is 1:3, the concentration of citric acid is 1.5 mg / ml, the soaking time is 5 min to 8 min, take out and rinse 3 to 5 times with fresh water at room temperature, and rinse 3 to 5 times with fresh, filtered, and sterilized seawater; S3.2 Treatment of male and female plants of high-value trait strains of wild Sargassum fusiforme S3.2.1 Screening of lateral branches and removal of stems Manually shear and remove the first- or second-order lateral branch stems of fresh thalli, and retain the second- or third-order lateral branches with dense receptacles; S3.2.2 Placement of lateral branches of male and female plants and mass ratio Allocate lateral branches according to the actual area of the culture tray. The allocation standard is that the lateral branches do not overlap with each other to ensure uniform light. The mixing mass ratio (kg:kg) of female plants to male plants is 1:1; S4 Creation of the optimal environmental factors for indoor temporary cultivation of lateral branches of male and female plants of high-value trait strains of wild Sargassum fusiforme S4.1 Optimal water environment for indoor temporary cultivation Culture seawater: ① Fresh: Take the clear seawater at high tide on the same day and let it settle naturally for 3 h to 4 h ② Filtration: Filter the supernatant seawater through a 280- to 300-mesh silk screen ③ High-temperature sterilization: Heat the filtered seawater to 80°C to 90°C and sterilize it at high temperature for 30 min to 40 min to kill animal eggs, microorganisms, microalgae, and young macroalgae, and let it cool naturally to room temperature; ④ pH value: Adjust the pH value of the sterilized seawater to 7.8 to 8.3 with 0.05 mol / L NaOH solution and 0.05 mol / L HCl solution; ⑥ Salinity: 28 - 33; ⑦ Nutrient salts: Prepare a seawater solution with N:P = 10:1 using NaNO3 and KH2PO3, with a concentration of 1 g / L for N or 1 g / L for P, and the volume ratio (V:V) of nutrient salts to culture seawater is (0.6 - 0.8) ml:L; S4.2 Culture room, light culture rack and temporary cultivation device S4.3 Set the optimal temperature, light intensity and photoperiod Temperature: 23°C to 25°C; Light intensity: 200 μmol·m -2 ·s -1 ~280 μmol·m -2 ·s -1 ; Photoperiod: 14 h:10 h to 12 h:12 h; S5 Treatment and temporary cultivation time of lateral branches of male and female plants of high-value trait strains of wild Sargassum fusiforme parents S5.1 Indoor temporary cultivation treatment of lateral branches Replace 1 / 2 of the total volume of the culture seawater in the culture group every day, and turn the lateral branches once every 6 h to 8 h to ensure that the receptacles of each lateral branch receive uniform light; S5.2 Indoor temporary cultivation time of lateral branches Indoor temporary cultivation of lateral branches for 5 d to 7 d; Classification collection, impurity removal and cleaning of fertilized eggs of new Sargassum fusiforme germplasm Use a 260-mesh to 280-mesh silk screen to collect fertilized eggs, and a 30-mesh to 50-mesh silk screen to remove impurities such as air bladders and receptacles. Place the fertilized eggs in a 2000 mL beaker, add 1500 mL of freshly filtered seawater, stir well and let it settle naturally for 2 to 3 minutes. Discard the upper suspension to remove suspended minute impurities. Repeat this operation 5 to 7 times to obtain fresh and pure fertilized eggs. For different strains, the collection of fertilized eggs and impurity removal need to be carried out separately; Creation of suspension system for fertilized eggs of new Sargassum fusiforme germplasm Quantitative sub-packaging of fertilized eggs of new Sargassum fusiforme germplasm Remove the excess water from the fresh and pure fertilized eggs, technically calculate the volume density of the fertilized eggs, and quantitatively sub-package 8000 to 10000 fertilized eggs in a 3000 ml or 5000 ml standard conical flask. Add freshly filtered seawater. The ratio range of the number of fresh and pure fertilized eggs (pieces) to the volume of seawater (ml) is 10:1 to 10:

3. Classify and sub-package according to different strains and mark them; Creation of the optimal environment for suspension culture of fertilized eggs of new Sargassum fusiforme germplasm Optimal water environment for suspension culture For fresh, filtered, high-temperature sterilized water, the configuration of pH value, salinity and nutrients refers to the method of S4.

1. The volume ratio (V:V) of nutrients to culture seawater is (0.5 - 0.7) ml:L to (0.6 - 0.8) ml:1L, and the optimal water environment conditions remain unchanged during suspension culture; Optimal light intensity, photoperiod, temperature and aeration conditions for suspension culture Optimal light intensity: 50 μmol·m -2 ·s -1 ~60 μmol·m -2 ·s -1 ; Photoperiod: 14h:10h to 12h:12h; Temperature: 23°C to 24°C; Aeration: Auxiliary aeration; Suspension culture method for fertilized eggs of new Sargassum fusiforme germplasm In the initial stage (8 to 10 days) of suspension culture of Sargassum fusiforme fertilized eggs, magnetic stirring + aeration suspension culture is adopted. After 8 to 10 days of culture, the Sargassum fusiforme fertilized eggs have grown and developed into embryos. When the embryo growth and development stage begins, a suspension culture device is used for suspension culture, and aeration suspension culture is implemented; Low-density quantitative sub-packaging during the suspension growth and development stage from embryo to young sporophyte of new Sargassum fusiforme germplasm During the growth and development stage from embryo to young sporophyte, sub-package at an equal volume density of 1 / 2 per bottle every 19 to 21 days. As the culture time gradually extends, gradually carry out low-density quantitative sub-packaging. The first low-density sub-packaging decreases from 8000 to 10000 to 4000 to 5000, the second low-density decreases from 4000 to 5000 to 2000 to 2500, the third low-density decreases from 2000 to 2500 to 1000 to 1200, and the final low-density decreases from 1000 to 1200 to 400 to 500; Prevention and control of typical harmful organisms in the suspension system during the suspension growth and development stage from embryo to young sporophyte of new Sargassum fusiforme germplasm S8.1 Prevention and control methods for harmful animals During the S7 culture period, regularly examine the embryo or young sporophyte under a microscope. If harmful animals are found in the suspension culture solution, soak it in ultrapure water for 20 to 25 minutes every 10 to 12 days of the culture period to kill harmful animals by hypotonic treatment; Prevention and control methods for harmful cyanobacteria During the S7 culture period, the embryos or young sporophytes were regularly examined under a microscope. If any signs of epiphytic blue-green algae were found on the embryos or young sporophytes, they were soaked in a seawater solution containing 300,000 to 600,000 units / L of penicillin for 24 to 48 hours. After disinfecting the blue-green algae, they were washed 3 to 5 times with filtered seawater and then continuously suspended in filtered seawater for culture under the same conditions. S8.3 Control method for harmful green algae During the S7 culture period, the embryos or young sporophytes were regularly examined under a microscope. If any signs of epiphytic green algae were found on the embryos or young sporophytes, they were soaked in a 1.5 mg / ml citric acid solution for 6 to 8 minutes, rinsed 3 to 5 times with fresh water, then rinsed 2 to 3 times with filtered seawater, and then continuously suspended in filtered seawater for culture under the same conditions; S9 Clamping the ropes and indoor temporary cultivation of young sporophytes of the new germplasm of Sargassum fusiforme The young sporophytes with a basal leaf length of 1.5 to 2.0 cm were clamped onto a cotton rope or fiber rope with a diameter of 0.4 cm at intervals of 3 to 5 cm. The young sporophytes with a basal leaf length less than 1.5 cm were continuously suspended in culture until the basal leaf grew to 1.5 to 2.0 cm and then the ropes were clamped. The clamped young sporophytes were temporarily cultivated indoors for 15 to 20 days. S10 Sea area stocking of young sporophytes of the new germplasm of Sargassum fusiforme S11 Evaluation methods for wild Sargassum fusiforme parents and new germplasms of their offspring S11.1 Evaluation of the traits of wild Sargassum fusiforme parents and their offspring S11.2 Evaluation of the yields of wild Sargassum fusiforme parents and their offspring S11.3 Evaluation of the processing ratios of the raw materials and roughly processed products of the dried products of wild Sargassum fusiforme parents and their offspring S11.4 Evaluation of the rehydration rates of the roughly processed products of wild Sargassum fusiforme parents and their offspring S11.5 Evaluation of the main nutritional components of the adult algae and products of wild Sargassum fusiforme parents and their offspring S11.6 Analysis of the genetic stability of the offspring of wild Sargassum fusiforme - new germplasm.

2. A method for creating and evaluating a new germplasm for suspension culture of Sargassum fusiforme fertilized eggs according to claim 1, characterized in that: In the step S4.2, the light cultivation rack structure includes left and right frames made of carbon steel, tempered glass laminates, glass laminate bottom frames, crossbars, switch boxes, LED lighting lamps, and a controllable darkroom configured to meet the indoor temporary cultivation conditions of marine organisms. The LED lighting lamps of the light cultivation rack main body are controlled in cooperation with the darkroom, which can ensure 0 μmol·m -2 ·s -1 ~350 μmol·m -2 ·s -1 light intensity adjustment. An external control power supply timing converter is set to control the light cycle; the cultivation room of the controllable darkroom is an inner darkroom, and an air conditioning device is provided to adjust the temperature of the space in the cultivation room; The structure of the temporary cultivation device group set on the light culture rack includes a water storage bucket, a culture tray, a peristaltic pump, an oxygen supply pump, a water delivery hose and a diffuser head, which are used for centralized oxygen dissolution and circulating water supply of seawater, and can ensure the sufficient supply of oxygen and nutrient nutrients for temporarily cultivating the Sargassum fusiforme seedlings.

3. A method for creating and evaluating new germplasms of floating culture of Sargassum fusiforme fertilized eggs according to claim 1, characterized in that: The preparation of the cultured seawater includes the following steps: storing the freshly filtered seawater in the wild area in a 1000L chemical barrel and transporting it to the factory area, then transferring it to 25L chemical barrels and carrying them indoors. Use a water heater to heat the seawater to 80°C - 90°C for 30 - 40 minutes to kill animal eggs, microorganisms, microalgae, and large seaweed larvae. Naturally cool it to room temperature, and inject water into the storage bucket of the temporary cultivation device group according to the ratio of 4L - 5L of seawater per 100g of the planted vegetables. Add nutrients according to the volume ratio (V:V) of nutrients to the cultured seawater of (0.6 - 0.8) ml:1L. Adjust the light intensity to 200 μmol·m -2 ·s -1 ~280 μmol·m -2 ·s -1 , with a photoperiod of 14h:10h - 12h:12h and a temperature of 24°C - 25°C. The mass ratio of male to female of the planted vegetable parents is 2:1 - 1:

1.

4. A method for creating and evaluating a new germplasm of floating culture of Sargassum fusiforme fertilized eggs according to claim 1, characterized in that: In step S3.1.1, the epiphytic large algae manually removed include Ulva lactuca, Enteromorpha prolifera, Scytosiphon lomentaria, etc. attached to the parents of the new germplasm of Sargassum fusiforme; in step S3.1.2, the epiphytic animals removed by hypotonic treatment include Gammarus spp., phasmids, fish eggs, etc. attached to the parents of the new germplasm of Sargassum fusiforme.

5. A method for creating and evaluating a new germplasm for suspension culture of Sargassum fusiforme fertilized eggs according to claim 1, characterized in that: In step S7, the main structure of the suspension culture device for Sargassum fusiforme fertilized eggs includes a 5000 ml glass Erlenmeyer flask, a glass tube, an air control valve, an air supply pump, and a hose. During the suspension culture process, low-density quantitative sub-packaging operations are carried out based on the suspension culture device.

6. The new germplasm creation and evaluation method for suspended culture of Sargassum fusiforme fertilized eggs according to claim 1, characterized in that: In step S7.2.1, the volume ratio (V:V) of the nutrient salts to the culture seawater during the suspension culture growth and development stage from Sargassum fusiforme fertilized eggs to young sporophytes (trilobate to multi-lobate) is (0.5 to 0.6) ml: 1 L.

7. A method for creating and evaluating new germplasms of suspended culture of Sargassum fusiforme fertilized eggs according to claim 1, characterized in that: In step S10, the raft device for natural sea area isolation and stocking of the new germplasm seedlings of Sargassum fusiforme adopts a soft raft design. The overall length × width of the soft raft is 100 m × 100 m, with 9 equal hanging culture areas inside. The new germplasm seedlings of Sargassum fusiforme are respectively hung at the four corners of the raft, and a surrounding net is set.

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