CN114027170B - Method for inducing large-amount centralized discharge of reef membrane gametes - Google Patents
Method for inducing large-amount centralized discharge of reef membrane gametes Download PDFInfo
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G33/00—Cultivation of seaweed or algae
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/80—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in fisheries management
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/80—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in fisheries management
- Y02A40/81—Aquaculture, e.g. of fish
Abstract
The invention relates to a method for inducing a large amount of reef membrane gametes to be intensively discharged, belonging to the technical field of seaweed reproduction regulation and control; putting immature, clean and robust reef membrane gametophyte into a seedling collecting container filled with 1/2 volume of culture solution; the culture solution is prepared by adding 6.4-16mg/L potassium dihydrogen phosphate into sterilized and filtered seawater, and adding 3.75 +/-0.33 g of gametophyte into each liter of culture solution; at the temperature of 20 ℃ and the illumination intensity of 80 mu mol.m ‑2 s ‑1 Photoperiod 12L: standing and culturing under 12D condition, regularly stirring the water body 3 times every day, and changing the culture solution 1 time every 2 days; after continuous culture for 9-10 days, a large amount of reef gamete is intensively discharged, wherein the culture solution is formed by adding 8mg/L monopotassium phosphate into disinfected and filtered seawater>10 10 Per liter of culture solution,>10 10 The algae/g are discharged in a large amount and intensively. The method has the advantages of saving the using amount of the seed algae, improving the utilization rate of the seed algae, the gamete yield and the gamete discharge amount, being easy to operate and low in cost, and being capable of meeting the requirements of large-scale seedling production.
Description
The technical field is as follows:
the invention belongs to the technical field of reproduction regulation and control in a large-scale economic seaweed seedling process, and particularly relates to a method for inducing a large amount of reef membrane gametes to be intensively discharged.
Background art:
reef diaphragmMonostroma nitidumIs an economic green algae species widely distributed along the coast of the east sea and the south sea in China, and belongs to the Monostroma of the ulva family. The algae is in the shape of a membrane, the length of the membrane can reach nearly 20cm, and the algae grows on high and medium tide reefs. The Monostroma nitidum is nutritious, and can be eaten fresh or washed and dried to obtain a dry product, and a green laver cake or sauce is prepared; the reef is salty and cold in nature, and has effects of clearing heat, eliminating phlegm, promoting diuresis, removing toxic substance, softening and resolving hard mass; the Monostroma nitidum polysaccharide has the effects of anticoagulation, antivirus,anti-tumor, anti-oxidation and anti-X-ray radiation. Under the action of benefit drive and natural factors, the natural resources of the reef membranes are obviously influenced, the resources are nearly exhausted sometimes, and the development of artificial breeding is a great trend.
Relevant research on the reef membranes starts late in China, basic biological research starts in the nineties of the last century, the relation between reef membrane gametophytes and zygote growth and development and ecological factors is researched, reef membrane morphological observation and molecular identification, reef membrane protoplast separation and culture and the like are realized; researches on cultivation practices are developed at the beginning of the century, and indoor large-scale artificial seedling raising, sporophyte cultivation devices and the like are explored. At present, the research of the basic physiology of the reef is still weak in China, the basic theories and technologies related to artificial culture of the reef and artificial breeding of the reef are still in the initial and experimental stages, and the reproductive regulation and control related to the reef breeding are still quite lacking.
At present, the seed algae for artificial breeding of the reef membranes are from wild mature reef membranes, but under natural conditions, the maturity seasonality of the reef membrane gametophytes is strong, the maturity is asynchronous, the mixed phenomena of maturity, immaturity, different mature areas and the like exist, the requirement of the breeding production on the mature seed algae amount is difficult to meet, and the phenomenon of excessive fishing is often generated in practice.
The invention content is as follows:
in order to solve the problems, the maturation quantity and gamete discharge quantity of the reef gametophyte are improved on the basis of not destroying the mature algae seed resource, and the requirement on the mature algae seed quantity in production is met, the invention provides a method for inducing the massive concentrated discharge of reef gametes, and the specific technical scheme is as follows:
a method for inducing mass concentrated discharge of reef membrane gametes comprises the following steps:
(1) Putting immature, clean and robust hermatypic gametophytes into a seedling collection container, wherein the seedling collection container is filled with 1/2 volume of culture solution, 3.75 +/-0.33 g of gametophytes are put into each liter of culture solution, and the culture solution is prepared by adding 6.4-1695g/L of monopotassium phosphate into sterilized and filtered seawater;
(2) At the temperature of 20 ℃ and the illumination intensity of 80 mu mol.m -2 s -1 Photoperiod 12L: performing static culture under 12D condition, regularly stirring the water body for 3 times every day, and replacing the culture solution for 1 time every 2 days;
(3) Continuously culturing for 9 to 10 days, and intensively discharging a large amount of reef film gametes.
Preferably, the gametophyte in the step (1) is pretreated, wherein the pretreatment method comprises the steps of collecting wild or artificially cultured immature and strong reef membrane gametophyte, shearing off irregular parts on the edge of the wild or artificially cultured immature and strong reef membrane gametophyte by using sterilized surgical scissors, scrubbing the upper surface and the lower surface of the wild or artificially cultured immature and strong reef membrane gametophyte for 6 times in sterilized and filtered seawater by using a soft brush pen, temporarily culturing the wild or artificially cultured immature and strong reef membrane gametophyte in the sterilized and filtered seawater for 24 hours, and then removing water.
Preferably, the condition of temporary culture is at 20 deg.C and 60 μmol · m light intensity -2 s -1 Photoperiod 12L: and (5) carrying out static culture under the condition of 12D.
Preferably, the step of dewatering treatment is to take out the temporarily-cultured reef gametophyte and place the reef gametophyte on 4 layers of sterile medical gauze and sterile filter paper to absorb water for 3 times respectively so that the reef gametophyte does not have watermarks any more.
Preferably, the seawater subjected to disinfection and filtration is 28-32 of salinity, is filtered by silk screen cloth with the aperture of 48 mu m, is disinfected in a high-pressure steam sterilization pot, and is cooled to room temperature.
Preferably, the background nitrite nitrogen content in the seawater is 0.012 plus or minus 0.003mg/L, and the inorganic phosphorus content is 0.077 plus or minus 0.003mg/L.
Preferably, the concentration of the potassium dihydrogen phosphate in the step (1) is 8mg/L.
Preferably, the culture medium is replaced in step (2) at 20 ℃ and the concentration of potassium dihydrogen phosphate is the same.
Preferably, the seedling collecting container is a white plastic, a colorless glass container or a cement pond internally paved with white ceramic tiles.
Compared with the prior art, the invention has the beneficial effects that:
under the condition of a certain quantity of algae, the immature and robust reef membrane gametophyte is trimmed, scrubbed and temporarily cultured, and the components of the culture solution are improved to match with the culture strips such as temperature, illumination conditions, stirring frequency, solution changing frequency and the likeModification of the culture medium to allow it to grow for several days>10 10 Per liter of culture solution,>10 10 Gamete is intensively discharged in a large amount at the level of each gram of algae, the using amount of the algae, the utilization rate of the algae, the yield and the discharge amount of gamete are saved, the operation is easy, the cost is low, and the requirement of large-scale seedling production can be met.
The specific implementation mode is as follows:
the present invention will be further described with reference to the following examples, but the present invention is not limited to these examples.
Example 1:
preparation of disinfected filtered seawater: taking a plurality of seawater, wherein the seawater background nitrite nitrogen content is required to be 0.012 +/-0.003 mg/L, and the inorganic phosphorus content is required to be 0.077 +/-0.003 mg/L. Filtering the seawater by using silk screen cloth with the pore size of 48 mu m, sterilizing the seawater in a high-pressure steam sterilization pot, and cooling the seawater to room temperature, wherein the salinity of the seawater after sterilization and filtration is 28-32 for later use.
Collecting immature and strong reef gametophyte cultured by wild or artificial culture, cutting off irregular part of the reef gametophyte with sterilized surgical scissors, and scrubbing the upper and lower surfaces with soft brush for 6 times in sterilized and filtered seawater. Temporarily culturing in sterilized and filtered seawater for 24h under the conditions: at a temperature of 20 ℃ and an illumination intensity of 60 mu mol.m -2 s -1 Photoperiod 12L: and (5) carrying out static culture under the condition of 12D. Taking out the temporarily cultured reef membrane gametophyte, placing on 4 layers of sterilized medical gauze and sterilized filter paper, and respectively absorbing water for 3 times to make the algae have no watermark any longer, thereby obtaining immature, clean and robust reef membrane gametophyte.
Putting the immature, clean and robust reef membrane gametophyte obtained in the step into a seedling collection container such as white plastic, colorless glass or a cement pond internally paved with white ceramic tiles, and filling 1/2 volume of culture solution into the container, wherein the culture solution is prepared by adding 8mg/L potassium dihydrogen phosphate into sterilized and filtered seawater, and 3.75 +/-0.33 g of gametophyte is put into each liter of culture solution; at the temperature of 20 ℃ and the illumination intensity of 80 mu mol.m -2 s -1 Photoperiod 12L: standing and culturing under 12D condition, stirring water body 3 times every day, changing culture solution 1 time every 2 days, continuously culturing for 10 days, discharging large amount of gamete of Monostroma nitidum concentratedly, and continuing for 2 daysThe radioactive density reaches (8.96 +/-0.36) x 10 10 The number of the discharged gametophytes per liter reaches (2.93 +/-0.12) multiplied by 10 10 Per gram.
Example 2:
preparing sterilized filtered seawater: taking a plurality of seawater, wherein the seawater background nitrite nitrogen content is required to be 0.012 +/-0.003 mg/L, and the inorganic phosphorus content is required to be 0.077 +/-0.003 mg/L. Filtering the seawater by using a silk screen cloth with the aperture of 48 mu m, disinfecting the seawater in a high-pressure steam sterilization pot, and cooling the seawater to room temperature, wherein the salinity of the disinfected and filtered seawater is 28 to 32 for later use.
Collecting immature and strong reef gametophyte cultured by wild or artificial culture, cutting off irregular part of the reef gametophyte with sterilized surgical scissors, and scrubbing the upper and lower surfaces with soft brush for 6 times in sterilized and filtered seawater. Temporarily culturing in sterilized and filtered seawater for 24h under the conditions: at the temperature of 20 ℃ and the illumination intensity of 60 mu mol.m -2 s -1 Photoperiod 12L: and (5) carrying out static culture under the condition of 12D. And taking out the temporarily-cultured reef gametophyte, putting the reef gametophyte on 4 layers of sterile medical gauze and sterile filter paper, and absorbing water for 3 times respectively to ensure that the reef gametophyte does not have a watermark any more, thereby obtaining an immature, clean and robust reef gametophyte.
Putting the immature, clean and robust reef membrane gametophyte obtained in the step into a seedling collection container such as white plastic, colorless glass or a cement pond internally paved with white ceramic tiles, and filling 1/2 volume of culture solution into the container, wherein the culture solution is sterilized and filtered seawater, 6.4mg/L potassium dihydrogen phosphate is added into the seawater, and 3.75 +/-0.33 g of gametophyte is put into each liter of culture solution; at a temperature of 20 ℃ and an illumination intensity of 80 mu mol.m -2 s -1 Photoperiod 12L: standing and culturing under 12D condition, stirring water body 3 times every day at regular time, changing culture solution 1 time every 2 days, continuously culturing for 10 days, intensively discharging large amount of gamete of Monostroma nitidum, and maintaining for 1 day, wherein gamete discharge density reaches (1.77 + -0.50) × 10 9 The number of the discharged gametophytes per liter reaches (7.75 +/-0.98) multiplied by 10 8 Per gram.
Example 3:
preparing sterilized filtered seawater: taking a plurality of seawater, wherein the seawater background nitrite nitrogen content is required to be 0.012 +/-0.003 mg/L, and the inorganic phosphorus content is required to be 0.077 +/-0.003 mg/L. Filtering the seawater by using silk screen cloth with the pore size of 48 mu m, sterilizing the seawater in a high-pressure steam sterilization pot, and cooling the seawater to room temperature, wherein the salinity of the seawater after sterilization and filtration is 28-32 for later use.
Collecting immature and strong reef gametophyte cultured by wild or artificial culture, cutting off irregular part of the reef gametophyte with sterilized surgical scissors, and scrubbing the upper and lower surfaces with soft brush for 6 times in sterilized and filtered seawater. Temporarily culturing in sterilized and filtered seawater for 24h under the conditions: at the temperature of 20 ℃ and the illumination intensity of 60 mu mol.m -2 s -1 Photoperiod 12L: and (5) carrying out static culture under the condition of 12D. And taking out the temporarily-cultured reef gametophyte, putting the reef gametophyte on 4 layers of sterile medical gauze and sterile filter paper, and absorbing water for 3 times respectively to ensure that the reef gametophyte does not have a watermark any more, thereby obtaining an immature, clean and robust reef gametophyte.
Putting the immature, clean and robust reef membrane gametophyte obtained in the step into a seedling collection container such as white plastic, colorless glass or a cement pond internally paved with white ceramic tiles, and filling 1/2 volume of culture solution into the container, wherein the culture solution is sterilized and filtered seawater, 9.6mg/L potassium dihydrogen phosphate is added into the seawater, and 3.75 +/-0.33 g of gametophyte is put into each liter of culture solution; at the temperature of 20 ℃ and the illumination intensity of 80 mu mol.m -2 s -1 Photoperiod 12L: standing and culturing under 12D condition, stirring water body 3 times every day at regular time, changing culture solution 1 time every 2 days, continuously culturing for 10 days, intensively discharging large amount of gamete of Monostroma nitidum, and maintaining for 1 day, wherein gamete discharge density reaches (3.31 + -0.13) x 10 9 The number of the discharged gametophytes per liter reaches (1.40 +/-0.36) multiplied by 10 9 Per gram.
Example 4:
preparing sterilized filtered seawater: taking a plurality of seawater, wherein the seawater background nitrite nitrogen content is required to be 0.012 +/-0.003 mg/L, and the inorganic phosphorus content is required to be 0.077 +/-0.003 mg/L. Filtering the seawater by using a silk screen cloth with the aperture of 48 mu m, disinfecting the seawater in a high-pressure steam sterilization pot, and cooling the seawater to room temperature, wherein the salinity of the disinfected and filtered seawater is 28 to 32 for later use.
Collecting immature and strong reef gametophyte cultured by wild or artificial culture, cutting off irregular part of the reef gametophyte with sterilized surgical scissors, and scrubbing the upper and lower surfaces with soft brush for 6 times in sterilized and filtered seawater. In the disinfection and filtration of seawaterTemporarily culturing for 24 hours under the conditions that: at the temperature of 20 ℃ and the illumination intensity of 60 mu mol.m -2 s -1 Photoperiod 12L: and (5) carrying out static culture under the condition of 12D. Taking out the temporarily cultured reef membrane gametophyte, placing on 4 layers of sterilized medical gauze and sterilized filter paper, and respectively absorbing water for 3 times to make the algae have no watermark any longer, thereby obtaining immature, clean and robust reef membrane gametophyte.
Putting the immature, clean and robust reef membrane gametophyte obtained in the step into a seedling collection container such as white plastic, colorless glass or a cement pond internally paved with white ceramic tiles, and filling 1/2 volume of culture solution into the container, wherein the culture solution is prepared by adding 16mg/L potassium dihydrogen phosphate into sterilized and filtered seawater, and 3.75 +/-0.33 g of gametophyte is put into each liter of culture solution; at a temperature of 20 ℃ and an illumination intensity of 80 mu mol.m -2 s -1 Photoperiod 12L: standing and culturing under 12D condition, stirring water body 3 times per day at regular time, changing culture solution 1 time every 2 days, continuously culturing for 9 days, discharging a large amount of gamete of Monostroma nitidum concentratedly, and continuing for 1 day, wherein gamete discharge density reaches (5.73 + -1.12) × 10 9 The number of the discharged gametophytes per liter reaches (1.81 +/-0.16) multiplied by 10 9 Per gram.
TABLE A result of mass concentrated discharge of reef gametes in examples 1 to 4
As can be seen from the data in Table I, under the culture conditions of example 1, the duration of concentrated gametophyte discharge was longest, the gametophyte discharge density was greatest, and the number of gametophyte discharge gametophyte was highest, so that>10 10 Per liter of culture solution,>10 10 Gametes are intensively discharged in a large quantity at the level of one/g of algae.
The above description is only an embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be able to cover the technical scope of the present invention and the equivalent alternatives or modifications according to the technical solutions and the inventive concepts of the present invention within the technical scope of the present invention.
Claims (4)
1. A method for inducing a large amount of reef membrane gametes to be intensively discharged is characterized in that: the method comprises the following steps of (a) carrying out,
preparation of disinfected filtered seawater: taking seawater, wherein the seawater background nitrite nitrogen content is required to be 0.012 +/-0.003 mg/L, and the inorganic phosphorus content is required to be 0.077 +/-0.003 mg/L; filtering the seawater by using silk screen cloth with the pore size of 48 mu m, sterilizing the seawater in a high-pressure steam sterilization pot, and cooling the seawater to room temperature, wherein the salinity of the seawater after sterilization and filtration is 28 to 32 for later use;
pretreatment of gametophytes: collecting immature and strong reef gametophytes which are wild or artificially cultured, cutting irregular parts of the edges of the immature and strong reef gametophytes by using disinfected surgical scissors, brushing the upper surface and the lower surface of the immature and strong reef gametophytes for 6 times by using a soft brush pen in the disinfected and filtered seawater, and temporarily culturing the upper surface and the lower surface of the reef gametophytes in the disinfected and filtered seawater for 24 hours under the condition that: at the temperature of 20 ℃ and the illumination intensity of 60 mu mol.m -2 s -1 Photoperiod 12L: carrying out static culture under the condition of 12D;
water removal treatment: taking out the temporarily-cultured reef membrane gametophyte, placing on 4 layers of sterilized medical gauze and sterilized filter paper, and respectively absorbing water for 3 times to ensure that the algae does not have watermarks any more, thereby obtaining an immature, clean and robust reef membrane gametophyte;
culturing: putting the immature, clean and robust reef membrane gametophyte into a seedling collection container, wherein the seedling collection container is filled with 1/2 volume of culture solution, each liter of culture solution is added with 3.75 +/-0.33 g of gametophyte, and the culture solution is prepared by adding 6.4 to 16mg/L of monopotassium phosphate into sterilized and filtered seawater; at a temperature of 20 ℃ and an illumination intensity of 80 mu mol.m -2 s -1 Photoperiod 12L: performing static culture under 12D condition, regularly stirring the water body for 3 times every day, and replacing the culture solution for 1 time every 2 days; continuously culturing for 9 to 10 days, and intensively discharging a large amount of reef gametes.
2. The method of claim 1, wherein: the concentration of the potassium dihydrogen phosphate is 8mg/L.
3. The method of claim 1, wherein: the replacement culture solution is a culture solution with the temperature of 20 ℃ and the same concentration of potassium dihydrogen phosphate.
4. A method according to claim 1,2 or 3, characterized in that: the seedling collecting container is a white plastic, colorless glass container or a cement pond internally paved with white ceramic tiles.
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