CN115299338A - Method for synchronously promoting maturity of Chinese variety parents and preserving seedlings of Sargassum hemiphyllum - Google Patents
Method for synchronously promoting maturity of Chinese variety parents and preserving seedlings of Sargassum hemiphyllum Download PDFInfo
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Abstract
The invention relates to a method for synchronously accelerating maturity of Chinese variety parents of gulfweed semiloba and storing seedlings, which comprises the following steps: planting the collected parent of the Chinese variety of the half-leaf gulfweed under the sea surface by using a cultivation raft frame, carrying out algae cultivation and ripening, collecting the reproductive bracket of the synchronously mature male and female algae strain, co-culturing in a water tank filled with filtered seawater, filtering and collecting the fertilized eggs. The seedling preservation method comprises the following steps: washing collected fertilized eggs with sterilized seawater, culturing in a container with an opening, sealing with a gas-permeable sealing film, placing in an illumination incubator at 18 + -1 deg.C under illumination intensity of 30-50 μmol photons m ‑2 s ‑1 The photoperiod is 12L:12D, replacing the sterilized seawater every 5 to 10 days. The invention can save the field sea area investigation and sampling for many times, and can also effectively improve the collection efficiency of the fertilized eggs; can effectively promote the synchronous maturation of male and female parents of the gulfweed Chinese variety and the preservation of seedling seeds, and has convenient operation and low cost.
Description
Technical Field
The invention belongs to the field of large-scale seaweed research, and particularly relates to a method for synchronously accelerating maturity of Chinese variety parents and preserving seedlings of Sargassum hemiphyllum.
Background
The Sargassum hemiphyllum Chinese variety (Sargassum hemiphylum var. Chinensis) belongs to Phaeophyta, phaeophyceae, fucales, sargassaceae and Sargassum, is a common large-scale seaweed in the east sea and the south sea of China, is naturally distributed in the area from the chamois of the south Zhejiang province to the coastal sea of Wenchang, and grows on rocks near the low tide line or in the sub-tidal zone. The sargassum semilobayense Chinese variety has yellow brown algae body, branches and leaves usually 80-100 cm high, and is divided into holdfast, main branch, leaf and air sac, the holdfast is in the form of rhizoid, and is divided into sexual reproduction and vegetative reproduction with rhizoid regenerated plant. The high algae and huge biomass of the Sargassum hemiphyllum in the blooming period can provide food sources and inhabitation sheltering places for other organisms, is an important colonizing species of an ecological system of a seaweed field, and has important ecological value; meanwhile, the Sargassum hemiphyllum Chinese variety is rich in various active substances, such as polysaccharide, phenols, fucoxanthin and the like, and is an important seaweed resource for developing marine foods and marine medicines. However, due to the influence of global climate change and human activity interference, the resource amount of the gulfweed Chinese varieties in the sea area in south China is gradually reduced, the distribution and maintenance of the seaweed field in the sea area are influenced, and resource protection and seaweed field repair work needs to be carried out on the gulfweed Chinese varieties. Because the Chinese variety of the gulfweed distributed in different areas in the natural habitat has the phenomenon of asynchronous development and maturity due to the difference of the environmental factors, and the alga body withers and fades in summer with high temperature. Therefore, the technology for synchronously accelerating the maturity of the parents of the gulfweed semilobar Chinese variety and storing the seedlings has important value for developing artificial seedling culture, hybrid breeding and seed storage and is a key technology for constructing the gulfweed semilobar Chinese variety seaweed field.
The problem that the growth and the maturation of the Chinese variety of the gulfweed are asynchronous in natural habitat is a technical problem for developing artificial seedling. When the artificial breeding of the Chinese variety of the Sargassum hemiphyllum is carried out, parent collection is needed to be carried out in advance, when wild parent collection is carried out, the parent with higher maturity can not be collected smoothly due to asynchronous development and maturity of algae, and sufficient developed parent can be obtained only by carrying out field investigation for many times; or when the hybrid seedling culture of different geographical populations is carried out, the hybrid seedling culture can not be carried out because one parent is mature and the other parent is not mature. The sex identification of the mature reproductive support of the algae, the collection and culture of fertilized eggs and the seedling storage are necessary technologies for developing the artificial breeding of Chinese variety of gulfweed, the summer-fall of seedlings and the seedling storage. For example, a patent of 2015103241502 discloses a summer cultivation method for Sargassum hemiphyllum seedlings, which comprises treating an alga body with a mature pessary by drying in the shade and stimulating to make fertilized eggs drop in a water body, and adding phosphorus nutritive salt into the cultivation water body to strengthen the Sargassum hemiphyllum seedlings and prevent and control miscellaneous algae. The patent selects the algae with mature pessary as parent, the collected fertilized eggs are the fertilized suspension-pessary eggs before the algae are collected, and the seedling culture needs to allocate the nutrient salt concentration of the culture water body. The patent does not relate to the synchronous ripening of male and female algae, has high requirements on parent selection, easily causes damage to fertilized eggs hung on the parents in the collection and transportation process, reduces the survival rate of seedlings and ensures that the seedling collection efficiency is low.
At present, in the conventional breeding method of sargassum algae, an attaching matrix is provided for attaching algae seedlings, the attaching matrix is made of materials such as a net curtain, a cloth curtain, bricks, a cement board and the like, the seedlings need to be preserved and cultivated in large water bodies such as a water tank, a water tank or a water pool after being attached, the space occupied in the cultivation process is large, a large amount of water needs to be changed or running water is adopted, and the seedlings are easily polluted by other algae.
Disclosure of Invention
The invention aims to provide a method for synchronously promoting maturity of a Sargassum hemiphyllum Chinese variety parent and preserving seedlings, which is characterized in that the Sargassum hemiphyllum Chinese variety parent is cultivated in a raft frame in a sea area, an alga body is planted underwater on the sea surface, the alga body is always in the water and is not dried out under the influence of tide, the growth and development speed is higher than that under a natural environment, male and female alga bodies can be promoted to be synchronously mature, and fertilized eggs discharged by the collected mature alga body are cultivated in an illumination incubator for seedling preservation, thereby overcoming the defects of asynchronous maturity of parent from a field source, low fertilized egg collection efficiency and difficult alga seed preservation.
A method for synchronously promoting maturity of Chinese variety parents and preserving seedlings of gulfweed semiloba comprises the following steps: planting the collected parent of the Chinese variety of the half-leaf gulfweed under the sea surface by using a cultivation raft frame, carrying out algae cultivation and ripening, collecting the reproductive bracket of the synchronously mature male and female algae strain, co-culturing in a water tank filled with filtered seawater, filtering and collecting the fertilized eggs.
Further, the method for synchronously maturing the gulfweed Chinese variety parents comprises the following steps of:
(1) Parent collection and cleaning: collecting Chinese variety parents of gulfweed semiloba in sea area when the tide falls in the heavy tide period from 12 months to 2 months in the next year, and bringing back as soon as possible to avoid damage and dryness; scrubbing collected Chinese variant parents of gulfweed in half leaves in filtered seawater, and placing the washed Chinese variant parents in the filtered seawater for temporary culture; during collection, the whole algae is collected from the rhizoid, and a hairbrush is preferably used for filtering seawater to remove silt and impurities of the algae, attached animals and miscellaneous algae;
(2) Parent selection: selecting Chinese variety parents of the gulfweed with the length of 15-70cm, good growth state and strong rhizoid as cultivation and ripening parents;
(3) Planting and synchronous ripening: inserting the rhizoid of the parent strain selected in the step (2) and cultured to promote maturity between strands of the seedling rope, hanging the rhizoid of the parent strain on a raft frame in the sea area, wherein the seedling rope is 20-40cm below the water surface, and culturing the parent strain in the sea area for 70-90 days to ensure that male and female algae strains can synchronously mature and grow out of a reproductive support;
(4) Identifying male and female algae strains: identifying the gulfweed Chinese variety male and female strain through appearance shape observation and slice microscopic observation; preferably, a stainless steel blade is used for transversely cutting the mature and healthy genital support to manufacture a water-sealed free-hand section with a cross section, a 10 multiplied by 10 optical microscope is used for observation, and the male and female genital supports can be conveniently identified according to the structural difference of the cross section;
(5) Collecting fertilized eggs: collecting mature male and female parents of the gulfweed Chinese variety on a culture raft frame according to male plants: female strain =1:1-5 co-culturing in a water tank filled with filtered seawater, filtering after 10-12 hours, and collecting fertilized eggs released in the water body.
Experiments show that after the male and female reproductive trays of the gulfweed Chinese variety develop and mature, egg cells and sperms can be discharged in seawater successively, and male and female parents are subjected to the following steps: female strain =1:1-5, the female and male parents can be co-cultured in the filtered seawater for multiple times to collect the fertilized eggs. The male and female parents are as follows: female strain =1: the fertilization effect is best when the proportion of 1-5 is co-cultured, partial egg cells of female plants can not be fertilized when the number of male plants is too small, and the water quality can be influenced and the fertilization of the egg cells can not be facilitated when the number of male plants is too large.
Further, the distance between the parent plants cultivated and ripened in the step (3) is 30-50cm, the distance between the seedling ropes is 1.5-2.5m, and the two ends of the seedling ropes are tied and hung on a cultivation raft frame in the sea area. The blended kelp seedling rope with the diameter of 3cm is preferably used as the seedling rope, and the length of the seedling rope is 4m.
Further, the method for identifying the male and female algae strains comprises the following steps: observing appearance and making a bare-handed slice, and performing microscopic examination under an optical microscope of 10 x 10 times to judge whether the male and female genital supports are cylindrical and relatively slender, wherein the cross section of the bare-handed slice is observed by microscopic observation to see that the genital support is internally provided with an intensive short rod-shaped structure; the female genital support is cylindrical and relatively thick and short, small branches are arranged at the tail ends of parts of the female genital support, egg cells or fertilized eggs can be seen to hang on the female genital support sometimes, and the cross section of the female genital support is observed by bare-handed microscopic slicing, so that oval egg cells are seen in the female genital support.
Further, the filtration and collection method of the fertilized eggs comprises the following steps: the seawater in the water tank is filtered by 80-mesh bolting silk to remove the fallen leaves, the genital tray and the impurities, and then is filtered by 260-mesh bolting silk to collect the fertilized eggs in the water body. If the number of the collected fertilized eggs is not enough, the parents with the male and female genital trays are placed in the filtered seawater again for culturing, and after the egg cells are discharged from the male and female genital trays and hung on the female genital trays for fertilization, the water body is stirred every morning, and the fertilized eggs are collected by filtration. Debris of algae and other unremoved organisms fall in the seawater, the large-size impurities can be intercepted and removed by using 80-mesh bolting silk, and the egg cells can be left in the seawater through the 80-mesh bolting silk; because the egg cells can not pass through the 260-mesh bolting silk, the 260-mesh bolting silk can intercept the egg cells. If 260 mesh silk is used directly, the impurities and egg cells will be mixed together.
Further, the filtered seawater in the step (5) is natural seawater which is subjected to sand filtration and has salinity of 29-33.
A method for preserving Sargassum hemiphyllum Chinese variety seedling comprises washing collected fertilized ovum with sterilized seawater, culturing in a container with an opening, sealing with a gas-permeable sealing film, placing in an illumination incubator at 18 + -1 deg.C with illumination intensity of 30-50 μmol photons m -2 s -1 The photoperiod is 12L:12D, replacing the sterilized seawater every 5 to 10 days.
Further, the sterilized seawater is seawater which is obtained by filtering seawater through a mixed fiber filter membrane with the aperture of 0.45 mu m, heating and boiling the seawater, and then placing the seawater at normal temperature. The filtering with 0.45 μm pore size is to remove impurities in seawater, including zooplankton, phytoplankton, particle debris, etc., and the purpose of heating and boiling seawater is to sterilize seawater, including killing most of harmful organisms such as microorganisms and microalgae spores, etc.
A cultivation raft frame for synchronously promoting maturity of half-leaf gulfweed Chinese variety parents comprises a mooring rope, a seabed iron anchor, a floating ball and an interval traction rope, wherein the mooring rope is fixed through the seabed iron anchor, the floating ball is connected with the mooring rope through a buoyancy rope, the seabed iron anchor, the floating ball and the interval traction rope are pulled to enable the mooring rope to form two parallel mooring ropes with an interval of 5m and 20-40cm under the sea surface, and a seedling rope is arranged between the two parallel mooring ropes.
Furthermore, the two ends of the seedling rope are tied with polyethylene ropes as fixing ropes, and the fixing ropes are bound on two parallel mooring ropes, so that the seedling rope and the mooring ropes are perpendicular to each other. Preferably, the polyethylene cord is about 50cm in length and 0.5cm in diameter.
Further, the mooring rope and the spacing hauling rope are polyethylene ropes with the diameter of 3 cm; the floating ball is a cylindrical PVC inflatable floating ball with the diameter of 40cm and the height of 60cm, and rope tying holes are formed in two ends of the floating ball and connected with the buoyancy rope; the buoyancy rope is a polyethylene rope with the diameter of 0.5cm.
The invention cultivates half-leaf gulfweed Chinese variety parents in the raft frame of the sea area, and the alga body on the raft frame of the sea area is always in the shallow layer below the surface of the seawater, and is not subject to dry out and wind wave scouring caused by tide. The natural wild algae grows on the reefs in the low tide zone, the algae are dried out of the water surface during the ebb tide, the algae are subjected to drying stress during the period of the algae being dried out of the water surface, and the sea waves can wash the algae to a greater extent during the ebb tide. The wild algae are dried out for a period of time every day due to tides, the dried algae are in a state of stagnant growth in the period of time, and the algae are damaged by wave washing, so that longer branches are broken; on the sea raft frame, the algae are always in water and can grow continuously, and the impact of sea waves on the algae in the water is smaller than that of the wild algae, so that the algae are branched more and the biomass is larger. The algae of the sea raft frame are matured synchronously, while the wild algae have different tolerance degrees to the desiccation stress to influence the development steps, so that some algae mature early and some mature late.
Compared with the prior art, the invention has the following advantages:
1. the method is simple, the effect is obvious, the synchronously mature sargassum semilobata Chinese variety fronds can be conveniently collected, the field sea area investigation sampling for many times is omitted, and the situation that the artificial seedling culture work can not be carried out due to the fact that the parents with synchronous maturity can not be collected is avoided;
2. the sea area cultivation raft frame is simple in structure and convenient to operate, algae bodies are not dried out when being positioned in the surface layer of seawater, and are uniform in light receiving, so that the development and maturation of parents are facilitated, and the sea area cultivation raft frame is also suitable for cultivation and maturation promotion of other sargassum seaweeds;
3. the invention effectively improves the collection efficiency of the fertilized eggs, the cultivated male and female parents can collect the fertilized eggs for many times, and the proportion of the collected fertilized eggs with activity is high;
4. the seedling preservation method is simple, small in occupied space, convenient to operate, low in pollution risk and long in preservation time, and can be used as a germplasm resource for long-term preservation.
Drawings
FIG. 1 is a schematic view of a half-leaf gulfweed Chinese variety parent synchronous maturity promoting cultivation raft;
wherein: 1-mooring rope, 2-seabed iron anchor, 3-floating ball, 4-buoyancy rope, 5-spacing hauling rope, 6-seedling rope and 7-half-leaf gulfweed Chinese variety parent;
FIG. 2 is a drawing of a female genital support of a Chinese variety of Sargassum hemiphyllum;
FIG. 3 is a drawing of the female reproductive hanging eggs of the Chinese variety gulfweed;
FIG. 4 is a drawing of a male germ support of a Chinese variety of Sargassum hemiphyllum;
FIG. 5 is a cross-sectional microscopic view of a female genital base of a Chinese variety of Sargassum hemiphylum, showing that there are ovate egg cells in the genital base;
FIG. 6 is a cross-sectional microscopic view of a male germ support of a Chinese variety of Sargassum hemiphylum, showing dense short rod-like structures in the germ support;
FIG. 7 is a graph of dead eggs that have fallen off when the parent is placed back in water after being dried in the shade;
FIG. 8 is an unfertilized multinucleated egg cell of a Sargassum hemiphyllum Chinese variety;
FIG. 9 is a diagram of fertilized eggs of a Chinese variety of disrupted Sargassum hemiphyllum;
FIG. 10 is a diagram of seedlings of the Chinese variety Sargassum hemiphyllum preserved for 11 weeks;
FIG. 11 is a graph of seedlings of a Chinese variety of Sargassum hemiphyllum preserved for 38 weeks;
FIG. 12 shows the average weight difference between the parent algal bodies of the Chinese variety of Sargassum hemiphyllum collected in the field and cultivated in a raft;
FIG. 13 shows the average length difference between the parent algal bodies of the Chinese variety of Sargassum hemiphyllum harvested in the field and cultivated in raft racks.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention will be described in further detail with reference to the accompanying drawings.
Example 1
Raft cultivation
The embodiment provides a method for synchronously accelerating maturity of Chinese variety parents and storing seedlings of gulfweed semiloba, which comprises the following steps:
1. collecting and cleaning algae: collecting the algae of the Sargassum hemiphyllum Chinese variety once from the low tide zone of the deep Australian gulf sea area of the Shannan Australian island in 1 month during the ebb tide in 2021 year, collecting the whole algae from the rhizoid by using an iron shovel, quickly taking the sample back to the Australian experiment station, brushing the algae in filtered seawater by using a brush, and temporarily culturing in a water tank containing the filtered seawater;
2. preparing a cultivation raft frame: two mooring ropes are fixed on the seabed through four seabed iron anchors, a plurality of cylindrical PVC inflatable floating balls with the diameter of 40cm and the height of 60cm are connected with the mooring ropes through buoyancy ropes, the seabed iron anchors, the floating balls and the interval hauling ropes are pulled to enable the two mooring ropes to be in a parallel state with the interval of 5m and 20-40cm under the sea surface, seedling ropes are arranged between the two parallel mooring ropes, polyethylene ropes with the length of about 50cm and the diameter of 0.5cm are tied at the two ends of the seedling ropes to serve as fixing ropes, and the fixing ropes are tied on the two parallel mooring ropes to enable the seedling ropes and the mooring ropes to be perpendicular to each other, as shown in figure 1.
3. Selecting algae, planting and synchronously accelerating maturity: selecting an alga body which is good in growth condition, developed in rhizoid, 44.4 +/-13.1 cm in average alga body length and 8.0 +/-3.1 g in average alga body weight as a seedling in 20 days 1 month, clamping the rhizoid of the alga body between strands of a kelp seedling rope with the length of 4m, wherein the seedling clamping interval is 30-50cm, the seedling rope is hung on a designed sea area raft frame, the seedling rope interval is 2m, the alga body is fixed on the raft frame and is 20-40cm below the water surface, the alga body is always kept in seawater and does not dry out in the whole cultivation process, after 76 days of sea area cultivation, the average alga plant length reaches 154.9 +/-55.9 cm in 6 days 4 month, the average alga body weight is 817.5 +/-399.5 g, and the male and female alga plants synchronously mature and grow out of a peston;
4. and (3) identifying male and female parents: collecting mature Chinese variety parent of Sargassum hemiphyllum from the cultivation raft frame, taking off 3-5 clusters of reproductive support, cross-cutting the mature reproductive support by stainless steel blade to make bare-handed slice by appearance observation, observing by 10 × 10 times optical microscope, and identifying male and female reproductive support according to appearance form and cross section structure, the specific method comprises: the male genital support is cylindrical and relatively slender, as shown in fig. 4, microscopic observation of a bare-handed section shows that the inner part of the genital support has a dense short rod-shaped structure, as shown in fig. 6, the female genital support is cylindrical and relatively thick and short, small branches are arranged on the part, sometimes egg cells or fertilized eggs are hung on the support, as shown in fig. 2 and fig. 3, and microscopic observation of a bare-handed section shows that the inner part of the genital support has oval egg cells, as shown in fig. 5.
5. Collecting fertilized eggs: mature half-leaf gulfweed Chinese variety parents are treated according to male strains: female strain =1:5, placing the mixture into a water tank containing filtered seawater, stirring the water body in the next morning, turning over algae bodies to enable fertilized eggs on female genital trays to fall into the water, taking out the algae bodies, filtering out fallen leaves, genital trays and impurities by using 80-mesh bolting silk, retaining the seawater, filtering the seawater by using 260-mesh bolting silk, collecting the fertilized eggs in the water body, and washing the fertilized eggs on the 260-mesh bolting silk by using filtered seawater for multiple times as shown in a figure 9 to remove the impurities; wherein the filtered seawater is natural seawater with salinity of 29-33 and filtered by sand filtration; the temperature of the seawater is proper to the water temperature of natural seawater in the mature period of the algae, and the temperature range is 18.5-22.5 ℃; the number of fertilized eggs collected in this embodiment is about 44 ten thousand; the proportion of fertilized eggs with activity is about 80 percent;
6. and (3) seedling preservation: the fertilized egg collected is washed with the disinfection seawater for several times and then cultured in the conical flask with the disinfection seawater, the conical flask is placed in the illumination incubator, and the parameters of the illumination incubator are set as follows: the temperature is 18 +/-1 ℃, and the illumination intensity is 30-50 mu mol phosns m -2 s -1 The photoperiod is 12L:12D, replacing the sterilized seawater every 7 days, and performing long-term preservation on the Chinese variety seedlings of the gulfweed semiloba, as shown in figures 10 and 11, wherein the fertilized eggs develop into seedlings under the condition, and the seedlings can survive for a long time under the condition, so that the purpose of seed preservation is achieved. The seed protection method has the advantages of small occupied space, convenient operation and small pollution risk. The sterilized seawater is the seawater which is obtained by filtering the filtered seawater through a mixed fiber filter membrane with the aperture of 0.45 mu m, heating and boiling the filtered seawater, and then placing the filtered seawater at normal temperature.
This example shows that mature female pessaries can shed eggs many times in seawater culture, and the eggs hang on the pessaries after shedding. The male and female genital trays are cultured in the same water body together, and the discharged egg cells are found to be hung on the female genital trays to be fertilized and then are divided into multicellular fertilized eggs. Then, the male and female algae parents with mature reproductive trays are placed in a water tank filled with filtered seawater together for overnight culture, the water body is stirred in the next morning, the algae are turned over to enable fertilized eggs hung on the trays to fall into the water, and the fertilized eggs are collected by filtration. In the method of the embodiment, the parent is damaged to a low degree, the parent can be used for multiple times, fertilized eggs can be collected continuously for multiple days, and the proportion of the collected fertilized eggs with activity is high.
Example 2
Raft culture
The embodiment provides a method for synchronously accelerating maturity of Chinese variety parents and storing seedlings of gulfweed semiloba, which comprises the following steps:
1. collecting and cleaning algae: collecting algae of half-leaf gulfweed Chinese variety from the low tide zone of Shenzhen Dapeng waxberry pit in the 1 st-month flood period of 2021, collecting the whole algae from rhizoid by using an iron shovel, putting the sample in a plastic sample bag with separated water to return to the south Australia experiment station, washing the algae for several times by using filtered seawater, brushing the algae in the filtered seawater by using a brush, and putting the algae in a water tank containing the filtered seawater for temporary culture;
2. the preparation of the cultivation raft is the same as in example 1.
3. Algae body selection, field planting and synchronous ripening: selecting an alga body which is good in growth condition, free of damage, developed in rhizoid, 20.3 +/-2.4 cm in average alga body length and 6.2 +/-1.7 g in average alga body weight as a seedling in 12 days in 1 month, clamping the rhizoid of the alga body between strands of a kelp seedling rope, wherein the seedling clamping distance is 30-50cm, the seedling rope distance is 2m, and the alga body is tied and hung on a designed raft frame in a sea area, wherein the seedling rope is fixed on the raft frame and is 20-40cm below the water surface, the alga body is not dried out in seawater all the time in the whole cultivation process, the alga body is cultivated in the sea area for 84 days, the average alga body length reaches 168.8 +/-18.7 cm in 6 days in 4 months, the average alga body weight is 694.9 +/-268.1 g, and male and female alga plants synchronously grow out of a reproductive tray at maturity;
4. and (3) identifying male and female parents: the identification was performed in the same manner as in example 1;
5. collecting fertilized eggs: mature half-leaf gulfweed Chinese variety parents are treated according to male strains: female strain =1:3, placing the mixture in a water tank containing filtered seawater, stirring the water body in the next morning, turning algae bodies to enable fertilized eggs which do not fall from the female genital organs to fall into the water, taking out the algae bodies, filtering the seawater in the water tank by using 80-mesh bolting silk to filter out fallen leaves, genital organs and other impurities, retaining the seawater, filtering the seawater by using 260-mesh bolting silk to collect fertilized eggs in the water body, and washing the fertilized eggs on the 260-mesh bolting silk for multiple times by using filtered seawater to remove the impurities; culturing the algae in the water tank containing clean filtered seawater again, stirring the water body in the next morning, turning the algae, collecting fertilized eggs again, and flushing the fertilized eggs; the filtered seawater is natural seawater with salinity of 29-33 and is filtered by sand filtration; in this embodiment, the algae are cultured again to make the parents release the fertilized eggs for multiple times, and collect more fertilized eggs, so as to achieve the purpose of utilizing the parents for multiple times, the number of the collected fertilized eggs is about 35 ten thousand, and the ratio of the fertilized eggs with activity is about 80%;
6. and (3) seedling preservation: the fertilized egg collected is washed with the disinfection seawater for several times and then cultured in the conical flask with the disinfection seawater, the conical flask is placed in the illumination incubator, and the parameters of the illumination incubator are set as follows: the temperature is 18 +/-1 ℃, and the illumination intensity is 30-50 mu mol phosns m -2 s -1 The photoperiod is 12L:12D, changing the sterilized seawater every 7 days to achieve the purpose of preserving the gulfweed half-leaf Chinese variety seedlings for a long time. The sterilized seawater is the seawater which is obtained by filtering the filtered seawater through a mixed fiber filter membrane with the aperture of 0.45 mu m, heating and boiling the filtered seawater, and then placing the filtered seawater to the normal temperature.
Comparative example 1
Open-air collection
Collecting fertilized eggs and preserving seedlings by taking mature fronds of wild Sargassum hemiphyllum Chinese varieties as parents, wherein the steps comprise:
1. parent acquisition: collecting Sargassum hemiphyllum Chinese varietal algae with more mature reproductive trays as parent from the lower tide zone of deep Australian sea area of Shantou south Australian island 3 times during the period of 5 month big tide in 2021 year, rapidly bringing back to south Australian experiment station, and temporarily culturing in filtered seawater, wherein the average algae length is 103.7 + -41.4 cm, and the average algae weight is 49.0 + -8.5 g;
2. the method for picking the seedling of the parent comprises the following specific steps: fishing out the parents from seawater, drying in the shade for 2 hours, then putting the parents into a water tank filled with filtered seawater, stirring a water body and algae overnight in the next morning to make fertilized eggs fall into water, taking out the parents, filtering the seawater in the water tank by using 80-mesh bolting silk to filter out the falling leaves, reproductive trays and impurities, retaining the seawater, filtering the seawater by using 260-mesh bolting silk to collect the fertilized eggs in the water body, washing the fertilized eggs on the 260-mesh bolting silk for multiple times by using the filtered seawater, wherein the ratio of the collected fertilized eggs with activity is about 25 million, and the fertilized egg ratio is about 10%;
the optical microscope examination shows that the collected egg cells are mostly black or deformed dead eggs, as shown in fig. 7, and are partially unfertilized multinucleated egg cells, as shown in fig. 8, and only a few divided fertilized eggs, so that the fertilized egg collection efficiency is low. In addition, the parents are put into the filtered seawater after being dried in the shade, and the parents are not favorable for seedling collection for many times due to easy decay and easy odor of water.
3. And (3) seedling preservation: the fertilized egg collected is washed with the disinfection seawater for several times and then cultured in the conical flask with the disinfection seawater, the conical flask is placed in the illumination incubator, and the parameters of the illumination incubator are set as follows: the temperature is 18 +/-1 ℃, and the illumination intensity is 30-50 mu mol phosns m -2 s -1 The photoperiod is 12L:12D, picking the germinated seedlings one week later, removing dead eggs, replacing the sterilized seawater every 7 days, and performing long-term storage.
Comparative example 2
Field collection
Collecting fertilized eggs and preserving seedlings by taking mature fronds of wild Sargassum hemiphyllum Chinese varieties as parents, wherein the steps comprise: parent acquisition: in the 4-5 month flood tide period in 2021, the gulfweed Chinese varietal algae with more mature germ cells are collected from the low tide zone of the Shenzhen Dapeng waxberry pit sea area 2 times to be used as parents, are rapidly brought back to the Nanao laboratory station and are temporarily cultivated in filtered seawater, the average algae length is 126.3 +/-18.6 cm, and the average algae weight is 43.0 +/-12.3 g. The other steps were similar to those of comparative example 1. About 26 million fertilized eggs are obtained, and the proportion of the fertilized eggs with activity is about 10%.
The method for collecting mature algae as parents from the field is adopted in the comparative examples 1 and 2, the field algae are not mature synchronously, sufficient female algae with fertilized eggs are difficult to collect by one field collection operation, the field operation needs to be carried out for many times, and the consumption of manpower and material resources is high.
In addition, the comparative examples 1 and 2 adopt a conventional shade-drying treatment method, that is, the algae with more mature pessaries are collected as parents, the parents are put into seawater after being cleaned and stimulated by shade-drying so that the fertilized eggs on the pessaries are separated from the seawater and fall into the water, and the fertilized eggs in the water are filtered and collected. According to the method, after the parent subjected to shade drying is placed into seawater, the algal bodies of the parent are easily damaged and rotted, the seawater is easily smelled, the proportion of the fertilized eggs with activity collected after shade drying stimulation is low, and a plurality of damaged and dead egg cells (figure 7) and unfertilized eggs (figure 8) exist. The parent is difficult to be used for many times, the number of collected active fertilized eggs is small, and the seedling picking efficiency is low.
Meanwhile, it is found that the algae cultured on the raft racks in the sea areas of the examples 1 and 2 generally have more branches and larger biomass than the algae cultured in the field environments of the comparative examples 1 and 2, as shown in fig. 12 and 13, and the mature algae parents needed by seedling collection can be obtained more conveniently due to synchronous maturity of the algae.
Claims (10)
1. A method for synchronously accelerating maturity of Sargassum hemiphyllum Chinese variety parents is characterized by comprising the following steps: planting the collected parent of the Chinese variety of the half-leaf gulfweed under the sea surface by using a cultivation raft frame, carrying out algae cultivation and ripening, collecting the reproductive bracket of the synchronously mature male and female algae strain, co-culturing in a water tank filled with filtered seawater, filtering and collecting the fertilized eggs.
2. The method for synchronously maturing sargassum semilobata var chinese parents according to claim 1, comprising the steps of:
(1) Parent collection and cleaning: collecting Chinese variety parents of gulfweed semiloba in sea area when the tide falls in the heavy tide period from 12 months to 2 months in the next year, and bringing back as soon as possible to avoid damage and dryness; scrubbing collected half-leaf gulfweed Chinese variety parents in filtered seawater, and placing the half-leaf gulfweed Chinese variety parents in the filtered seawater for temporary culture;
(2) Parent selection: selecting Chinese variety parents of the gulfweed with the length of 15-70cm, good growth state and strong rhizoid as cultivation and ripening parents;
(3) Planting and synchronous ripening: inserting the rhizoid of the parent strain selected in the step (2) and cultured to promote maturity between strands of the seedling rope, hanging the rhizoid of the parent strain on a raft frame in the sea area, wherein the seedling rope is 20-40cm below the water surface, and culturing the parent strain in the sea area for 70-90 days to ensure that male and female algae strains can synchronously mature and grow out of a reproductive support;
(4) Identifying male and female algae strains: identifying the gulfweed Chinese variety male and female strain through appearance shape observation and slice microscopic observation;
(5) Collecting fertilized eggs: collecting mature male and female parents of the gulfweed Chinese variety on a culture raft frame according to male plants: female strain =1:1-5 co-culturing in a water tank filled with filtered seawater, filtering after 10-12 hours, and collecting fertilized eggs released in the water body.
3. The method for synchronously maturing half-leaf gulfweed Chinese variety parents according to claim 2, wherein the distance between the matured half-leaf gulfweed parents cultivated in the step (3) is 30-50cm, the distance between seedling ropes is 1.5-2.5m, and two ends of the seedling ropes are tied and hung on a sea area cultivation raft frame.
4. The method for synchronously maturing half-leaf gulfweed Chinese variety parents according to claim 2, wherein the method for identifying male and female strains comprises the following steps: observing appearance and making a bare-handed slice, and performing microscopic examination under an optical microscope of 10 x 10 times to judge whether the male and female genital supports are cylindrical and relatively slender, wherein the cross section of the bare-handed slice is observed by microscopic observation to see that the genital support is internally provided with an intensive short rod-shaped structure; the female genital support is cylindrical and relatively thick and short, small branches are arranged at the tail ends of parts of the female genital support, egg cells or fertilized eggs can be seen to hang on the female genital support sometimes, and the cross section of the female genital support is observed by bare-handed microscopic slicing, so that oval egg cells are seen in the female genital support.
5. The method for synchronously maturing half-leaf gulfweed Chinese variety parents according to claim 2, wherein the method for filtering and collecting fertilized eggs comprises the following steps: the seawater in the water tank is filtered by 80-mesh bolting silk to remove fallen leaves, genital trays and impurities, and then is filtered by 260-mesh bolting silk to collect the fertilized eggs in the water body.
6. The method for synchronously maturing sargassum semilaevis var chinese parents according to claim 2, wherein said filtered seawater in step (5) is natural seawater filtered by sand filtration and having salinity of 29-33.
7. A method for preserving the young seedlings of Chinese variety of Sargassum hemiphyllum includes such steps as washing the fertilized ova with sterilized seawater, culturing in a container with an opening, sealing with a gas-permeable sealing film, and culturing in an illumination incubator at 18 +/-1 deg.C and 30-50 microns of illumination intensity -2 s -1 The photoperiod is 12L:12D, replacing the sterilized seawater every 5 to 10 days.
8. The method for preserving seedling of Sargassum hemiphylum var chinensis as claimed in claim 7, wherein the said sterilized seawater is seawater obtained by filtering natural seawater with mixed fiber filter membrane with pore size of 0.45 μm, heating to boil, and standing to room temperature.
9. A cultivation raft frame for synchronously promoting maturity of half-leaf gulfweed Chinese variety parents is characterized by comprising a mooring rope, a seabed iron anchor, a floating ball and a spacing traction rope, wherein the mooring rope is fixed through the seabed iron anchor, the floating ball is connected with the mooring rope through a buoyancy rope, the seabed iron anchor, the floating ball and the spacing traction rope are pulled to enable the mooring rope to form two parallel mooring ropes with the spacing of 5m and 20-40cm under the sea surface, and a seedling rope is arranged between the two parallel mooring ropes.
10. The cultivation raft frame for synchronously maturing half-leaf gulfweed Chinese variety parents according to claim 9, wherein polyethylene ropes are tied to two ends of the seedling rope to be used as fixing ropes, and the fixing ropes are bound on two parallel mooring ropes so that the seedling rope and the mooring ropes are perpendicular to each other.
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| CN117136833A (en) * | 2023-08-18 | 2023-12-01 | 生态环境部华南环境科学研究所(生态环境部生态环境应急研究所) | Sargassum periwinkle germplasm preservation method |
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