Industrialized breeding method of undaria pinnatifida seedling
By propagating the male and female gametophytes of wakame and controlling light, water temperature, nutrient addition, and stirring, the problems of high cost and long cycle in high-temperature areas in traditional methods have been solved, realizing the industrialized propagation of high-quality seedlings, which is suitable for wakame seedling cultivation in high-temperature areas in summer and autumn.
Patent Information
- Application Number
- CN202610405530.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2026-03-30
- Publication Date
- 2026-05-29
AI Technical Summary
Traditional methods of propagating wakame seedlings require prolonged refrigeration in areas with high seawater temperatures during summer and autumn, resulting in high costs and making them unsuitable for industrial propagation. Furthermore, the seedling cultivation cycle is long.
The female and male parthenophytes of Wakame seaweed were propagated separately and then mixed and inoculated on a seedling tray. By controlling the light intensity and water temperature gradient, adding nitrogen and phosphorus nutrients, and stirring, the marine environment was simulated to accelerate the development of rhizoids of the young sporophytes.
It shortens the seedling cycle, reduces costs, and improves seedling quality. It is suitable for industrial breeding in high-temperature areas during summer and autumn, forming a complete industrial chain system of seedling breeding, aquaculture and processing with southern characteristics.
Smart Images
Figure CN122095982A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of seedling propagation technology, and in particular relates to an industrial propagation method for wakame seedlings. Background Technology
[0002] Wakame seaweed is a typical warm-water, large, economically important brown algae, named for its skirt-like shape. With its delicious flavor and high nutritional value, wakame has seen a year-on-year increase in market demand in recent years, fueling a wakame farming boom in coastal areas. Currently, collecting zoospores is the core method of traditional wakame seedling propagation. This process relies on the release and attachment of zoospores during their natural life cycle. In coastal areas where seawater temperatures do not exceed 22°C during summer and autumn, the seedling production process does not require cooling of the seedling water, but the entire seedling cycle takes more than three months. However, wakame is a cold-water algae, suitable for winter cultivation. Applying traditional wakame seedling propagation methods to areas with higher seawater temperatures (22-32°C) during summer and autumn requires prolonged cooling of the seedling water, resulting in high costs and hindering industrial-scale propagation. Therefore, there is an urgent need to establish an industrial-scale wakame seedling propagation method suitable for areas with higher seawater temperatures in summer and autumn. Summary of the Invention
[0003] The purpose of this invention is to provide an industrial propagation method for wakame seedlings, which is suitable for industrial propagation of wakame seedlings in areas with higher temperatures.
[0004] This invention provides an industrial propagation method for wakame seedlings, comprising the following steps: The female and male parthenogenetic gametophytes of Wakame seaweed were propagated separately to obtain female and male parthenogenetic gametophytes for inoculation. The female and male parthenogenetic gametophytes used for inoculation are mixed and then inoculated onto a seedling tray for seedling cultivation. The seedling cultivation procedure includes: days 0-3, 12 hours of darkness followed by 12 hours of light, with a light intensity of 1-8 μmol photons·m⁻¹. -2 ·s -1 The water temperature was 15-18 ℃; from day 4 until the end of the seedling stage, the light intensity was increased every 3-4 days, with each increase being 6-10 μmol photons·m⁻². -2 ·s -1 The water temperature is increased once every 8 to 12 days, with each increase being 0.5 to 1 ℃. The seedling cultivation procedure further includes: from day 4 to 18, adding nitrogen and phosphorus nutrients to the water body for seedling cultivation; the nitrogen and phosphorus nutrients include NaNO3 and KH2PO4, the concentration of NaNO3 in the water body is 3-4 ppm, the concentration of KH2PO4 in the water body is 0.3-0.4 ppm, and changing the water during the seedling cultivation process, with a frequency of once every two days, and a single water change volume of 50% of the total water volume; from day 19 to 30, adding nitrogen and phosphorus nutrients to the water body for seedling cultivation at the same concentration as from day 4 to 18, and changing the water during the seedling cultivation process, with a frequency of once every two days, and a single water change volume of 75% of the total water volume; From day 31 until the end of the seedling cultivation period, nitrogen and phosphorus nutrients are added to the water used for seedling cultivation. The nitrogen and phosphorus nutrients include NaNO3 and KH2PO4. The concentration of NaNO3 in the water is 4-5 ppm, and the concentration of KH2PO4 in the water is 0.4-0.5 ppm. During the seedling cultivation process, the water is changed every two days, and the volume of water changed each time is 100% of the total water volume. The seedling raising procedure also includes: intermittent stirring from day 19 to day 10 before the end of seedling raising, stirring for 40 minutes every 20 minutes; and continuous stirring from day 9 before the end of seedling raising to the end of seedling raising.
[0005] Preferably, the light intensity at the end of the seedling stage is 70~120 μmol photons·m -2 ·s -1 .
[0006] Preferably, the water temperature at the end of the seedling raising process is 18~20℃.
[0007] Preferably, the flow rate of the seedling water during the intermittent stirring is 0.3~0.4 m / s.
[0008] Preferably, the flow rate of the seedling water during continuous stirring is 0.5~0.6 m / s.
[0009] Preferably, the inoculation concentrations of the female parthenogenetic gametes and the male parthenogenetic gametes used for inoculation are 0.03 g / L to 0.035 g / L, respectively.
[0010] Preferably, the propagation of female and male parthenogenetic gametophytes of wakame seaweed includes: Female and male parthenogenetic gametophytes were mixed with sterilized seawater and then crushed to obtain female and male parthenogenetic gametophyte suspensions, respectively. After the female and male parthenophore algal fragment suspensions were cultured in the dark for recovery, they were then propagated separately.
[0011] Preferably, the sterilized seawater is sterilized seawater in a mixture of ice and water.
[0012] Preferably, the number of crushing operations is ≥2 times, and the time for each crushing operation is 20~30 s; after crushing, the lengths of the female parthenogenetic gametophyte segments and the male parthenogenetic gametophyte segments are 100~200 μm, respectively.
[0013] Preferably, the light-protected recovery culture time is 2-3 days; the light intensity for the propagation culture is increased by 3-4 μmol photons·m³ per day. -2 ·s -1 The value was gradually increased from 0 to 20 μmol photons·m -2 ·s -1 The propagation and culture procedure includes: a temperature of 18-20 °C, a photoperiod of 12L:12D, and a light intensity of 20-22 μmol photons·m⁻¹. -2 ·s -1 The duration is 30-35 days.
[0014] This invention provides an industrial propagation method for wakame seedlings, comprising the following steps: The female and male parthenogenetic gametophytes of Wakame seaweed were propagated separately to obtain female and male parthenogenetic gametophytes for inoculation. The female and male parthenogenetic gametophytes used for inoculation are mixed and then inoculated onto a seedling tray for seedling cultivation. The seedling cultivation procedure includes: days 0-3, 12 hours of darkness followed by 12 hours of light, with a light intensity of 1-8 μmol photons·m⁻¹. -2 ·s -1 The water temperature was 15-18 ℃; from day 4 until the end of the seedling stage, the light intensity was increased every 3-4 days, with each increase being 6-10 μmol photons·m⁻². -2 ·s -1 The water temperature is increased once every 8 to 12 days, with each increase being 0.5 to 1 ℃. The seedling cultivation procedure further includes: from day 4 to 18, adding nitrogen and phosphorus nutrients to the water body for seedling cultivation; the nitrogen and phosphorus nutrients include NaNO3 and KH2PO4, the concentration of NaNO3 in the water body is 3-4 ppm, the concentration of KH2PO4 in the water body is 0.3-0.4 ppm, and changing the water during the seedling cultivation process, with a frequency of once every two days, and a single water change volume of 50% of the total water volume; from day 19 to 30, adding nitrogen and phosphorus nutrients to the water body for seedling cultivation at the same concentration as from day 4 to 18, and changing the water during the seedling cultivation process, with a frequency of once every two days, and a single water change volume of 75% of the total water volume; From day 31 until the end of the seedling cultivation period, nitrogen and phosphorus nutrients are added to the water used for seedling cultivation. The nitrogen and phosphorus nutrients include NaNO3 and KH2PO4. The concentration of NaNO3 in the water is 4-5 ppm, and the concentration of KH2PO4 in the water is 0.4-0.5 ppm. During the seedling cultivation process, the water is changed every two days, and the volume of water changed each time is 100% of the total water volume. The seedling raising procedure also includes: intermittent stirring from day 19 to day 10 before the end of seedling raising, stirring for 40 minutes every 20 minutes; and continuous stirring from day 9 before the end of seedling raising to the end of seedling raising.
[0015] The present invention provides an industrial propagation method for wakame seedlings. By separately propagating the female and male parthenogenetic gametophytes of wakame, a sufficient number of gametophytes are obtained, laying the foundation for industrialized production through gametophyte cloning. From day 4 until the end of seedling cultivation, the light intensity is increased every 3-4 days, with each increase being 6-10 μmol photons·m². -2 ·s -1 The water temperature is increased by 0.5-1°C every 8-12 days to allow the wakame seedlings to gradually adapt to the light intensity and water temperature conditions of the sea area. In the industrial propagation method of wakame seedlings of this invention, nitrogen and phosphorus nutrients are added to the water during the 4th-18th day of seedling cultivation. These nutrients include NaNO3 and KH2PO4, with the NaNO3 concentration at 3-4 ppm and the KH2PO4 concentration at 0.3-0.4 ppm. Water is changed every two days during the seedling cultivation process, with each change replacing 50% of the total water volume. During this stage, the gametophytes complete attachment, fertilization, and early sporophyte development. In the industrial propagation method of wakame seedlings of this invention, continuous stirring is performed from 9 days before the end of seedling cultivation until the end of the cultivation period. During this stage, the young sporophytes of wakame grow rapidly. Continuous stirring can simulate the marine environment, accelerate the development of rhizoids of the young sporophytes, and enable them to firmly attach to the seedling curtain. Attached Figure Description
[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.
[0017] Figure 1 This describes the cell development and transformation process during the clonal seedling stage of *Wakame seaweed* gametophyte; where a. female gametophyte; b. male gametophyte; c. female gametophyte forming an egg-like protuberance after fertilization of male and female gametes; d. all gametophytes transform into sporophytes 9 days after inoculation; e. sporophytes 13 days after inoculation; g. sporophytes 18 days after inoculation; h. sporophytes 30 days after inoculation. Figure 2 This image depicts the growth process of young sporophytes attached to seedling curtains during the gametophyte seedling stage of *Wakame seaweed*. The images show: a. Gametophyte segments evenly attached to vinylon rope; b. Seedling curtain 13 days after inoculation; c. Seedling curtain 18 days after inoculation; d. Seedling curtain 30 days after inoculation; e. Seedling curtain 41 days after inoculation; f. Gametophyte segments on freshly inoculated seedling curtains; g. Young sporophytes 13 days after inoculation; h. Young sporophytes 18 days after inoculation; i. Young sporophytes 30 days after inoculation; j. Young sporophytes 41 days after inoculation. Figure 3 A curtain for propagating wakame seedlings. Detailed Implementation
[0018] This invention provides an industrial propagation method for wakame seedlings, comprising the following steps: The female and male parthenogenetic gametophytes of Wakame seaweed were propagated separately to obtain female and male parthenogenetic gametophytes for inoculation. The female and male parthenogenetic gametophytes used for inoculation are mixed and then inoculated onto a seedling tray for seedling cultivation. The seedling cultivation procedure includes: days 0-3, 12 hours of darkness followed by 12 hours of light, with a light intensity of 1-8 μmol photons·m⁻¹. -2 ·s -1 The water temperature was 15-18 ℃; from day 4 until the end of the seedling stage, the light intensity was increased every 3-4 days, with each increase being 6-10 μmol photons·m⁻². -2 ·s -1 The water temperature is increased once every 8 to 12 days, with each increase being 0.5 to 1 ℃. The seedling cultivation procedure further includes: from day 4 to 18, adding nitrogen and phosphorus nutrients to the water body for seedling cultivation; the nitrogen and phosphorus nutrients include NaNO3 and KH2PO4, the concentration of NaNO3 in the water body is 3-4 ppm, the concentration of KH2PO4 in the water body is 0.3-0.4 ppm, and changing the water during the seedling cultivation process, with a frequency of once every two days, and a single water change volume of 50% of the total water volume; from day 19 to 30, adding nitrogen and phosphorus nutrients to the water body for seedling cultivation at the same concentration as from day 4 to 18, and changing the water during the seedling cultivation process, with a frequency of once every two days, and a single water change volume of 75% of the total water volume; From day 31 until the end of the seedling cultivation period, nitrogen and phosphorus nutrients are added to the water used for seedling cultivation. The nitrogen and phosphorus nutrients include NaNO3 and KH2PO4. The concentration of NaNO3 in the water is 4-5 ppm, and the concentration of KH2PO4 in the water is 0.4-0.5 ppm. During the seedling cultivation process, the water is changed every two days, and the volume of water changed each time is 100% of the total water volume. The seedling raising procedure also includes: intermittent stirring from day 19 to day 10 before the end of seedling raising, stirring for 40 minutes every 20 minutes; and continuous stirring from day 9 before the end of seedling raising to the end of seedling raising.
[0019] This invention involves propagating the female and male parthenogenetic gametophytes of wakame seaweed to obtain female and male parthenogenetic gametophytes for inoculation.
[0020] As one implementation method, the industrialized propagation method for wakame seedlings is applicable to coastal areas where seawater temperatures exceed 22°C in summer and autumn, further extending to warm temperate-subtropical coastal areas globally, and even more specifically, coastal areas in southern China. This invention provides a new application technology for developing the wakame seedling cultivation and aquaculture industry in warm temperate-subtropical coastal areas worldwide. The industrialized propagation method for wakame seedlings of this invention has advantages such as short cycle, low cost, and high seedling quality. It not only completely eliminates the reliance on the traditional "north-to-south seedling transport" model but also plays a significant role in promoting the formation of a complete industrial chain system for wakame seedling cultivation, aquaculture, and processing with southern characteristics.
[0021] In one embodiment, the wakame seaweed includes male and female parthenocarpic bodies formed by the release of zoospores from mature wakame spores.
[0022] As one implementation method, the propagation of female and male parthenocarps of Wakame seaweed includes: mixing female and male parthenocarps with sterilized seawater, crushing them separately to obtain female and male parthenocarp fragment suspensions, respectively; and then performing light-protected recovery culture on the female and male parthenocarp fragment suspensions, followed by propagation culture.
[0023] In one implementation, the sterilized seawater is sterilized seawater in an ice-water mixture state. This is because, during the crushing process described in the above scheme, the crushing equipment heats up, and using sterilized seawater in an ice-water mixture state can lower the temperature, avoiding damage to the cells of female and male parthenogenetic gametophytes.
[0024] As one implementation method, the equipment used for the disruption includes a tissue disruptor; the disruption is performed ≥2 times, with each disruption lasting 20-30 seconds. Multiple disruptions can avoid cell damage caused by a sudden and prolonged increase in temperature and shear force. After disruption, the lengths of the female and male parthenocarpy algal segments are 100-200 μm, which meets the requirements for attaching seedling ropes and is also suitable for ovulation and sperm release, thus facilitating fertilization.
[0025] As one implementation method, the light-protected recovery culture time is 2-3 days; the light intensity for the propagation culture is increased by 3-4 μmol photons·m³ per day. -2 ·s -1 The value was gradually increased from 0 to 20 μmol photons·m -2 ·s -1 The propagation and culture procedure includes: a temperature of 18-20 °C, a photoperiod of 12 L:12 D, and a light intensity of 20-22 μmol photons·m⁻¹. -2 ·s -1 The time is 30-35 days, which is calculated from the time after the culture is restored in the dark.
[0026] After obtaining the female and male parthenogenetic gametophytes for inoculation, the present invention mixes the female and male parthenogenetic gametophytes for inoculation and inoculates them onto a seedling tray for seedling cultivation.
[0027] In one implementation method, after obtaining the female and male parthenogenetic gametophytes for inoculation, the present invention places the female and male parthenogenetic gametophytes for inoculation into a low-temperature constant temperature chamber and transports them to a standardized cement seedling bed within 6 hours for inoculation and seedling cultivation; the specifications of the cement seedling bed are: 8.7 m long × 2.4 m wide × 0.4 m high.
[0028] In one embodiment, the volume ratio of the female parthenogenetic gametophytes to the male parthenogenetic gametophytes used for inoculation is 1:1. After mixing the female and male parthenogenetic gametophytes, the invention further includes diluting the mixed gametophytes with seawater, which facilitates more even distribution of the gametophyte segments on the seedling curtain during inoculation. The inoculation includes spraying onto the seedling curtain; the spraying is performed using a spray bottle; the spraying is carried out evenly; the seedling curtain is a vinylon seedling curtain; based on a volume of 100 L of seawater used for dilution, the dimensions of the seedling curtain are 58 cm long × 28 cm wide; the diameter of the seedling rope on the seedling curtain is 2 mm.
[0029] As one implementation method, the seedling raising procedure includes: days 0-3, 12 hours of darkness followed by 12 hours of light, with a light intensity of 1-8 μmol photons·m⁻¹. -2 ·s -1 Water temperature 15-18℃; from day 4 until the end of seedling cultivation, increase light intensity every 3-4 days, with each increase being 6-10 μmol photons·m -2 ·s -1 The water temperature is increased by 0.5-1℃ every 8-12 days. The seedling raising procedure also includes: from day 4 to 18, adding nitrogen and phosphorus nutrients to the seedling water; the nitrogen and phosphorus nutrients include NaNO3 and KH2PO4, with NaNO3 concentration of 3-4 ppm and KH2PO4 concentration of 0.3-0.4 ppm. Water is changed every two days during the seedling raising process, with each water change replacing 50% of the total water volume. From day 19 to 30, nitrogen and phosphorus nutrients are added to the seedling water at the same concentration as from day 4 to 18. Water is changed every two days during the seedling raising process, with each water change replacing 75% of the total water volume. From day 31... From day 19 to day 10 before the end of the seedling stage, nitrogen and phosphorus nutrients are added to the water used for seedling cultivation. The nitrogen and phosphorus nutrients include NaNO3 and KH2PO4. The concentration of NaNO3 in the water is 4-5 ppm, and the concentration of KH2PO4 in the water is 0.4-0.5 ppm. During the seedling cultivation process, the water is changed every 2 days, and the volume of water changed each time is 100% of the total water volume. The seedling cultivation procedure also includes: from day 19 to day 10 before the end of the seedling cultivation, intermittent stirring is performed, stirring for 40 minutes every 20 minutes; from day 9 before the end of the seedling cultivation to the end of the seedling cultivation, continuous stirring is performed.
[0030] As one implementation method, with a total water storage capacity of 70 t in the wakame seedling pond, the total amount of NaNO3 added is 210 g and the total amount of KH2PO4 added is 25 g.
[0031] As one implementation method, the light intensity at the end of the seedling stage is 70~120 μmol photons·m -2 ·s -1 .
[0032] In one implementation method, the water temperature at the end of the seedling cultivation is 18~20 ℃.
[0033] In one implementation, the flow rate of the seedling water during the intermittent stirring is 0.3~0.4 m / s.
[0034] In one embodiment, the flow rate of the seedling water during continuous stirring is 0.5~0.6 m / s.
[0035] In one embodiment, the inoculation concentration of the female and male parthenogenetic gametes used for inoculation is 0.03 g / L to 0.035 g / L.
[0036] To further illustrate the present invention, the following detailed description of an industrial propagation method for wakame seedlings provided by the present invention is provided in conjunction with the accompanying drawings and embodiments, but these descriptions should not be construed as limiting the scope of protection of the present invention.
[0037] Example 1 Gametophyte cloning and propagation were carried out in the algae culture room of Fujian Provincial Fisheries Research Institute. The outdoor temperature range was 26~32℃, and the indoor temperature was controlled at 20℃ using a low-temperature air conditioner.
[0038] 1. Gametophyte cloning and propagation On September 17, 2025, clonal propagation of *Wakame* gametophytes was carried out under indoor experimental conditions using both male and female parthenophores. The selected male and female gametophytes were placed in a tissue homogenizer, and sterilized seawater in an ice-water mixture was added for multiple homogenization cycles (each cycle lasting 30 seconds), cutting the gametophytes into 100–200 μm segments. The homogenized suspension was placed in culture flasks for 3 days of recovery in the dark, after which the light intensity was gradually increased (4 μmol photons·m² per day). -2 ·s -1 ), ultimately maintained at 20 μmol photons·m -2 ·s -1 The culture conditions for the propagation stage were set as follows: temperature 20 ℃, photoperiod 12L:12D, light intensity 20 μmol photons·m -2 ·s -1 After 30 days of propagation, a sufficient number of gametes were obtained.
[0039] 2. Gamete cloning inoculation On October 17, 2025, the propagated gametophytes were placed in a low-temperature constant-temperature incubator and transported to a standardized cement seedling pond (8.7 m long × 2.4 m wide × 0.4 m high) within 6 hours for inoculation. The inoculation amount of both female and male gametophytes was 30-35g. Before inoculation, the female and male gametophytes were mixed in a 1:1 ratio, diluted in 100 L of seawater, and sprayed evenly onto the vinylon seedling curtain (58 cm long × 28 cm wide, seedling rope diameter 2 mm) using a sprayer.
[0040] 3. Control of light and water temperature during the seedling stage On October 17th, the male and female gametophytes were mixed and inoculated onto the seedling tray. For three days after inoculation, the seedling room was shaded to reduce light exposure, with daytime light intensity controlled at 1–8 μmol photons·m⁻¹. -2 ·s -1 When the water temperature in the seedling tank naturally rises to 18℃, turn on the external water pump and slowly inject 10℃ cooling water to lower the temperature of the seedling tank. Stop pumping cold water when the temperature drops to 15℃, and maintain the water temperature at 15~18℃. Repeat this process for 3 days. From October 20th to November 26th, when the seedling cultivation ends, adjust the light intensity every 3~4 days, increasing the light intensity by 6~10 μmol photons·m⁻¹ each time. -2 ·s -1 At the end of the seedling stage, the light intensity can be increased to 70~120 μmol photons·m -2 ·s -1 Meanwhile, the water temperature is increased by 1°C every 10 days to allow the wakame seedlings to adapt to the light intensity and water temperature conditions of the sea area in advance.
[0041] 4. Control of water exchange rate, nutrient salt addition amount, and mixer running time. The total water storage capacity of the wakame seedling pond is about 70 t, and the total amount of nitrogen and phosphorus nutrients added is 210 g NaNO3 and 25 g KH2PO4. Different stirring times are set for different development stages.
[0042] (1) Gamete fertilization and early development of sporophytes: From October 22 to November 8, that is, within 18 days after inoculation, the water exchange rate in the seedling pond is 50% of the total water volume, and 50% of the required total nutrients are added simultaneously (nitrogen and phosphorus nutrients are NaNO3 and KH2PO4, the concentration of NaNO3 in the water is 3~4 ppm, and the concentration of KH2PO4 in the water is 0.3~0.4 ppm). After that, the water exchange rate is increased to 75% of the total water volume, and nutrients are added simultaneously. Nitrogen and phosphorus nutrients are NaNO3 and KH2PO4, the concentration of NaNO3 in the water is 3~4 ppm, and the concentration of KH2PO4 in the water is 0.3~0.4 ppm. The turbine does not run during this period. During this stage, the gametophytes complete the attachment, fertilization process and early development of sporophytes.
[0043] (2) Sporophyte growth period: From November 8th, 22 days after inoculation, when the sporophytes are visible to the naked eye, the turbine is started daily to increase the seawater flow rate in the seedling tank for hardening. At the same time, the light intensity and nutrient application are gradually increased to promote the development of rhizoids and enhance the adhesion of the seedlings. The water exchange ratio in the seedling tank is increased from 75% to 100%, and nutrients are added simultaneously. The nitrogen and phosphorus nutrients are NaNO3 and KH2PO4. The concentration of NaNO3 in the water is 4~5 ppm, and the concentration of KH2PO4 in the water is 0.4~0.5 ppm. The turbine runs for 40 minutes every 20 minutes, and a 24-hour continuous operation mode is set. Nine days before the seedlings are removed from the warehouse, the turbine operation time is increased to 24 hours. During this stage, the wakame sporophytes grow rapidly, and the mixer runs continuously to simulate the marine environment, accelerate the development of rhizoids of the sporophytes, and make them firmly attached to the seedling rope.
[0044] 5. Observation and analysis of seedling development during the seedling stage The attachment of gametophytes, fertilization of gametes, development and transformation, and the developmental degree of young sporophytes during the seedling cultivation process were observed using an inverted microscope, and photographic records and data analysis were performed.
[0045] 6. Results 6.1 Gametophyte Development and Sporophyte Growth Three days after inoculation, the pulverized filamentous male and female gametophytes gradually adhered to the surface of the seedling rope, and no large-scale shedding was observed. Figure 1 a, b; Figure 2 (a, f in the original text). Subsequently, the female gametophyte forms an egg process that combines with the sperm released from the male gametophyte to complete fertilization. Figure 1 (c) From the female gametophyte, young sporophytes gradually emerge. Nine days after inoculation, all gametophyte vegetative cells complete the transformation into sporophytes and grow rapidly. The average length of the young sporophytes is 39.35 μm and the average width is 13.08 μm (Table 1). Figure 1 (d) 13 days after inoculation, the young sporophytes were in the early developmental stage, with an average length of 100.17 μm and an average width of 44.05 μm (Table 1, d). Figure 1 e~f in; Figure 2 (b, g); 18 days after inoculation, the young sporophytes significantly increased in size, with uniform growth morphology, averaging 227.45 μm in length and 91.04 μm in width (Table 1, g). Figure 1 g in Figure 2 (c, h); 30 days after inoculation, the young sporophytes were uniformly dense, the algal strains were brightly colored, and the growth was good, with an average length of 2029.30 μm and an average width of 625.45 μm (Table 1, ...). Figure 1 h in; Figure 2 (d, i in the text). At 41 days of cultivation, the seedlings reached the specifications for shipment, with an average length of 9260.18 μm and an average width of 3835.06 μm (Table 1, ...). Figure 2 (e, j in the text). No obvious growth inhibition or disease was observed throughout the development process, and algal contamination was effectively controlled.
[0046] Table 1. Growth indicators of sporophytes during gametophyte cloning and seedling raising.
[0047] 6.2 Seedlings leaving the warehouse like Figure 3 (The image above shows multiple pictures of seedling trays, and the image below shows a single picture of a seedling tray.) As shown, after 41 days of indoor cultivation, the wakame seedlings reached the size suitable for sea aquaculture. Testing revealed that the number of seedlings attached to each 1 cm length of seedling rope remained stable at 185-220, with 40%-50% being robust seedlings longer than 0.2 cm. The seedlings exhibited firm root attachment, healthy algae, uniform density, intact morphology, and normal color.
[0048] In addition, the traditional seed production method for wakame is long, generally taking 3 to 5 months from seed selection in the sea area to the end of seed production, while this application only requires 41 days.
[0049] Comparative Example 1 On October 17, 2025, crushed single female and single male gametophytes were evenly sprayed onto single vinylon seedling curtains (58 cm long × 28 cm wide, with a seedling rope diameter of 2 mm) using a spray bottle. After inoculation, they were placed in separate glass seedling tanks. For three days after inoculation, the seedling tanks were shaded to reduce light exposure, with daytime light intensity controlled at 1–8 μmol photons·m⁻¹. -2 ·s -1When the water temperature naturally rises to 18℃, slowly inject 10℃ cooling water to lower the temperature of the seedling tank, maintaining the water temperature at 15~18℃. Repeat this process for 3 days. Starting from October 20th, adjust the light intensity every 3~4 days, increasing it by 6~10 μmol photons·m⁻² each time. -2 ·s -1 By October 25th, microscopic examination of the seedling curtains after 9 days of propagation revealed numerous deformed sporophyte seedlings on curtains inoculated with a single female gametophyte, while no sporophyte seedlings were observed on curtains inoculated with a single male gametophyte; all seedlings remained gametophytes and had not developed into sporophytes. Inoculation of vinylon seedling curtains with either single female or single male gametophytes failed to achieve seedling production, and its effectiveness was inferior to Example 1.
[0050] Comparative Example 2 On September 17, 2025, the selected female and male gametophytes were placed in a tissue homogenizer, and sterilized seawater at room temperature (24 ℃) was added. The homogenizers were then subjected to multiple homogenization cycles (each cycle lasting 30 s) to cut the gametophytes into algal segments of 100–200 μm. Microscopic examination revealed that a small number of gametophyte segments showed cytochrome leakage, giving the algae a green color. The algal cells lacked vitality and could not be used for subsequent inoculation and seedling production. Therefore, the use of sterilized seawater in a non-ice-water mixed state during the gametophyte pulverization process was less effective than in Example 1.
[0051] Comparative Example 3 On September 17, 2025, selected female and male gametophytes were placed in a tissue homogenizer, and sterilized seawater at room temperature was added. The homogenizer was then subjected to a single, 240-second long-duration homogenization process, cutting the gametophytes into algal segments of 100-200 μm. The homogenized algal solution was viscous and hot to the touch; the homogenizer walls were noticeably warm. Microscopic examination revealed that a large amount of cytochrome from the gametophyte segments had leaked out, giving the algae a green color. The algal cells lacked vitality and could not be used for subsequent inoculation and seedling production. Using a single, long-duration homogenization process instead of multiple homogenizations was less effective than in Example 1.
[0052] Comparative Example 4 On October 17, 2025, unmixed, uncrushed gametophytes were evenly sprayed onto single vinylon seedling curtains (58 cm long × 28 cm wide, with a seedling rope diameter of 2 mm) using a spray bottle. After inoculation, the seedlings were placed in the same seedling bed and tagged. For three days after inoculation, the seedling room was shaded to reduce light exposure, with daytime light intensity controlled at 1–8 μmol photons·m⁻¹. -2 ·s -1When the water temperature in the seedling tank naturally rose to 18°C, an external water pump was turned on to slowly inject 10°C cooling water to lower the temperature of the seedling tank. Pumping was stopped when the temperature reached 15°C, maintaining the water temperature between 15°C and 18°C. This process was repeated for 3 days. From October 20th to November 26th, when seedling cultivation ended, the light and water temperature regulation, water exchange rate, nutrient salt addition, and mixer operation time in the seedling tank were exactly the same as in Example 1. After 41 days of cultivation, a large number of sporophyte seedlings fell off the seedling curtain, and the sporophyte seedlings were not firmly attached. A large amount of diatoms adhered to the blank areas of the seedling curtain, failing to meet the requirements for commercially viable seedling curtains. Inoculating seedlings with undiluted mixed gametophytes yielded worse results than in Example 1.
[0053] Comparative Example 5 On September 17, 2025, clonal propagation of *Wakame* gametophytes was carried out under indoor experimental conditions using both male and female parthenophores. The selected male and female gametophytes were placed in a tissue homogenizer, and sterilized seawater in an ice-water mixture was added for multiple homogenization cycles (each cycle lasting 30 seconds), cutting the gametophytes into 100–200 μm segments. The homogenized suspension was placed in culture flasks for 3 days of recovery in the dark, after which the light intensity was gradually increased (5 μmol photons·m² per day). -2 ·s -1 ), ultimately maintained at 40 μmol photons·m -2 ·s -1 The culture conditions for the propagation stage were set as follows: temperature 24 ℃, photoperiod 12L:12D, light intensity 40 μmol photons·m -2 ·s -1 After 11 days of propagation, the gametophytes all showed a dull color and a small amount of diatom contamination.
[0054] The above propagation method is not as effective as Example 1.
[0055] Comparative Example 6 On September 17, 2025, clonal propagation of *Wakame* gametophytes was carried out under indoor experimental conditions using both male and female parthenophores. The selected male and female gametophytes were placed in a tissue homogenizer, and sterilized seawater in an ice-water mixture was added for multiple homogenization cycles (each cycle lasting 30 seconds), cutting the gametophytes into 100–200 μm segments. The freshly homogenized suspension was directly placed in culture flasks for suspension culture, with an initial light intensity of 4 μmol photons·m⁻¹. -2 ·s -1 Then gradually increase the light intensity (4 μmol photons·m² per day). -2 ·s -1 ), ultimately maintained at 20 μmol photons·m-2 ·s -1 The culture conditions for the propagation stage were set as follows: temperature 20 ℃, photoperiod 12L:12D, light intensity 20 μmol photons·m -2 ·s -1 After 5 days of propagation, the gametophyte cells were hollow, the algal filaments turned white, and the gametophytes lacked vitality. Without light-protected recovery culture of the freshly crushed gametophytes, the results were not as good as in Example 1.
[0056] Comparative Example 7 Propagation was carried out using traditional methods of collecting zoospores for seedling cultivation.
[0057] For traditional propagation methods, please refer to [Pang Shaojun, Shan Danfeng, Liu Mingtai, et al. Indoor ambient temperature artificial seedling cultivation of Wakame seaweed in Liaodong Peninsula: dual high light control, temperature change and gametophyte development [J]. Advances in Fisheries Science, 2011, 32(5):74-83, DOI:10.3969 / j.issn.1000-7075.2011.05.011.]. The results showed that the traditional method of propagation using zoospores took 90 days, and its effect was not as good as that of Example 1.
[0058] Comparative Example 8 1. Gametophyte cloning and propagation On September 17, 2025, clonal propagation of *Wakame* gametophytes was carried out under indoor experimental conditions using both male and female parthenophores. The selected male and female gametophytes were placed in a tissue homogenizer, and sterilized seawater in an ice-water mixture was added for multiple homogenization cycles (each cycle lasting 30 seconds), cutting the gametophytes into 100–200 μm segments. The homogenized suspension was placed in culture flasks for 3 days of recovery in the dark, after which the light intensity was gradually increased (4 μmol photons·m² per day). -2 ·s -1 ), ultimately maintained at 20 μmol photons·m -2 ·s -1 The culture conditions for the propagation stage were set as follows: temperature 20 ℃, photoperiod 12L:12D, light intensity 20 μmol photons·m -2 ·s -1 After 30 days of propagation, a sufficient number of gametes were obtained.
[0059] 2. Gamete cloning inoculation On October 17, 2025, the propagated gametophytes were placed in a low-temperature constant-temperature incubator and transported to a standardized cement seedling pond (8.7 m long × 2.4 m wide × 0.4 m high) within 6 hours for inoculation. The inoculation amount of both female and male gametophytes was 30-35g. Before inoculation, the female and male gametophytes were mixed in a 1:1 ratio, diluted in 100 L of seawater, and sprayed evenly onto the vinylon seedling curtain (58 cm long × 28 cm wide, seedling rope diameter 2 mm) using a sprayer.
[0060] 3. Control of light and water temperature during the seedling stage On October 17th, the female and male gametophytes were mixed and inoculated onto the seedling curtain in another seedling bed. For three days after inoculation, the seedling room was shaded to reduce light exposure, with daytime light intensity controlled at 1–8 μmol photons·m⁻¹. -2 ·s -1 When the water temperature in the seedling tank naturally rises to 18℃, turn on the external water pump and slowly inject 10℃ cooling water to lower the temperature of the seedling tank. Stop pumping cold water when the temperature drops to 15℃, and maintain the water temperature at 15~18℃. Repeat this process for 3 days. From October 20th to November 26th, when the seedling cultivation ends, adjust the light intensity every 3~4 days, increasing the light intensity by 6~10 μmol photons·m⁻¹ each time. -2 ·s -1 At the end of the seedling stage, the light intensity can be increased to 70~120 μmol photons·m -2 ·s -1 Meanwhile, the water temperature is increased by 1°C every 10 days to allow the wakame seedlings to adapt to the light intensity and water temperature conditions of the sea area in advance.
[0061] 4. Water exchange rate, nutrient salt addition amount, and mixer operation control The total water storage capacity of the wakame seedling pond is 70 t, and the total amount of nitrogen and phosphorus nutrients added is 140 g NaNO3 and 15 g KH2PO4. Throughout the entire seedling process, the mixer is not turned on, and a seedling production method combining static and micro-flow rate is adopted.
[0062] (1) Gamete fertilization and early development of young sporophytes: From October 17 to November 8, that is, within 22 days after inoculation, the water exchange rate in the seedling pond is 50% of the total water volume, and 25% of the total amount of nutrients required is added simultaneously. After that, the water exchange rate is increased to 50% of the total water volume, and nutrients are added simultaneously. Nitrogen and phosphorus nutrients include NaNO3 and KH2PO4. The concentration of NaNO3 in the water is 1.5~2 ppm, and the concentration of KH2PO4 in the water is 0.15~0.2 ppm. The turbine is turned off to keep the seedling water static. During this stage, the gametophytes complete the attachment, fertilization process and early development of sporophytes.
[0063] (2) Sporophyte growth period: From November 8th, when the sporophytes are visible to the naked eye 22 days after inoculation, the turbine is still turned off, and a water pump is used to create a micro-flow rate (0.1~0.15 m / s) water body for hardening off. However, the light intensity and nutrient application are gradually increased to promote seedling growth. The water exchange ratio in the seedling pond is increased from 50% to 100%, and nutrients are added simultaneously. The nitrogen and phosphorus nutrients include NaNO3 and KH2PO4. The concentration of NaNO3 in the water body is 2~2.5 ppm, and the concentration of KH2PO4 in the water body is 0.2~0.25 ppm.
[0064] 5. Observation and analysis of seedling development during the seedling stage Inverted microscopes were used to observe gametophyte attachment, gamete fertilization, development and transformation, and the developmental stage of young sporophytes during the seedling cultivation process. Results showed that gametophyte attachment, fertilization, and development were unaffected; however, sporophyte growth was slow in the later stages, and the rhizoid attachment was noticeably weak. By November 26th, after 41 days of large-scale indoor seedling cultivation, the seedlings on the seedling curtains were relatively small, with an average length of 2373.39 μm and an average width of 823.86 μm. Seedling attachment density was low, with only 21-38 seedlings attached per 1 cm length of seedling rope. Only 10%-20% of these were robust seedlings with a body length greater than 0.2 cm. Sporophyte seedlings easily detached from the seedling curtains, indicating poor seedling adhesion, which did not meet the requirements for commercially viable seedling curtains. Therefore, a decrease in the amount of nutrients added to the seedling water, a decrease in the water exchange ratio, and failure to turn on the mixer to increase the flow rate of the seedling water for hardening off cannot produce wakame clone seedlings that meet the requirements for sea aquaculture.
[0065] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.
Claims
1. An industrialized method for propagating wakame seedlings, characterized in that, Includes the following steps: The female and male parthenogenetic gametophytes of Wakame seaweed were propagated separately to obtain female and male parthenogenetic gametophytes for inoculation. The female and male parthenogenetic gametophytes used for inoculation are mixed and then inoculated onto a seedling tray for seedling cultivation. The seedling cultivation procedure includes: days 0-3, 12 hours of darkness followed by 12 hours of light, with a light intensity of 1-8 μmol photons·m⁻¹. -2 ·s -1 The water temperature was 15-18 ℃; from day 4 until the end of the seedling stage, the light intensity was increased every 3-4 days, with each increase being 6-10 μmol photons·m⁻². -2 ·s -1 The water temperature is increased once every 8 to 12 days, with each increase being 0.5 to 1 ℃. The seedling cultivation procedure further includes: from day 4 to 18, adding nitrogen and phosphorus nutrients to the water body for seedling cultivation; the nitrogen and phosphorus nutrients include NaNO3 and KH2PO4, the concentration of NaNO3 in the water body is 3-4 ppm, the concentration of KH2PO4 in the water body is 0.3-0.4 ppm, and changing the water during the seedling cultivation process, with a frequency of once every two days, and a single water change volume of 50% of the total water volume; from day 19 to 30, adding nitrogen and phosphorus nutrients to the water body for seedling cultivation at the same concentration as from day 4 to 18, and changing the water during the seedling cultivation process, with a frequency of once every two days, and a single water change volume of 75% of the total water volume; From day 31 until the end of the seedling cultivation period, nitrogen and phosphorus nutrients are added to the water used for seedling cultivation. The nitrogen and phosphorus nutrients include NaNO3 and KH2PO4. The concentration of NaNO3 in the water is 4-5 ppm, and the concentration of KH2PO4 in the water is 0.4-0.5 ppm. During the seedling cultivation process, the water is changed every two days, and the volume of water changed each time is 100% of the total water volume. The seedling raising procedure also includes: intermittent stirring from day 19 to day 10 before the end of seedling raising, stirring for 40 minutes every 20 minutes; and continuous stirring from day 9 before the end of seedling raising to the end of seedling raising.
2. The industrial propagation method for wakame seedlings according to claim 1, characterized in that, The light intensity at the end of the seedling stage was 70~120 μmol photons·m -2 ·s -1 .
3. The industrial propagation method for wakame seedlings according to claim 1, characterized in that, The water temperature at the end of the seedling stage is 18-20℃.
4. The industrial propagation method for wakame seedlings according to claim 1, characterized in that, The flow rate of the seedling water during the intermittent stirring is 0.3~0.4 m / s.
5. The industrial propagation method for wakame seedlings according to claim 1, characterized in that, The flow rate of the seedling water during continuous stirring is 0.5~0.6 m / s.
6. The industrial propagation method for wakame seedlings according to claim 1, characterized in that, The inoculation concentrations of the female parthenogenetic gametes and the male parthenogenetic gametes used for inoculation were 0.03 g / L to 0.035 g / L, respectively.
7. The industrial propagation method for wakame seedlings according to claim 1, characterized in that, Propagation of female and male parthenogenetic gametophytes of wakame seaweed includes: Female and male parthenogenetic gametophytes were mixed with sterilized seawater and then crushed to obtain female and male parthenogenetic gametophyte suspensions, respectively. After the female and male parthenophore algal fragment suspensions were cultured in the dark for recovery, they were then propagated separately.
8. The industrial propagation method for wakame seedlings according to claim 7, characterized in that, The sterilized seawater is sterilized seawater in a mixture of ice and water.
9. The industrial propagation method for wakame seedlings according to claim 7, characterized in that, The number of crushing operations is ≥2 times, and the time for each crushing operation is 20~30 s; after crushing, the lengths of the female parthenogenetic gametophyte segments and the male parthenogenetic gametophyte segments are 100~200 μm, respectively.
10. The industrial propagation method for wakame seedlings according to claim 7, characterized in that, The light-protected recovery culture time is 2-3 days; the light intensity for the propagation culture is increased by 3-4 μmol photons·m³ per day. -2 ·s -1 The value was gradually increased from 0 to 20 μmol photons·m -2 ·s -1 ; The propagation and culture procedure includes: a temperature of 18–20 °C, a photoperiod of 12 L:12 D, and a light intensity of 20–22 μmol photons·m⁻¹. -2 ·s -1 The duration is 30-35 days.