A method for evaluating the kelp breeding performance based on sporangium spore dissemination parameters and application thereof

Abstract

The application provides a kelp breeding performance evaluation method based on sporangium spore dissemination parameters and application, and belongs to the technical field of seaweed cultivation. The method comprises the following steps: S1, taking mature kelp samples, washing, and air-drying treatment to obtain pretreated kelp; S2, taking kelp tissue blocks from the pretreated kelp obtained in step S1, loading the kelp tissue blocks into a mesh bag, immersing the kelp tissue blocks in seawater, promoting the kelp tissue blocks to disseminate, and collecting zoospore liquid; S3, measuring sporangium spore dissemination parameters in step S2; S4, calculating dissemination capacity according to the sporangium spore dissemination parameters obtained in step S3, and evaluating the breeding performance of the kelp according to the dissemination capacity. The above method can quantify the sporangium spore dissemination characteristics of kelp, systematically evaluate the breeding potential of kelp, and improve the breeding efficiency and yield of kelp.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of seaweed cultivation, and relates to a method for evaluating the breeding performance of laminaria based on spore sac spore dispersal parameters and application thereof. BACKGROUND

[0002] Laminaria is a nutrient-rich brown algae rich in various minerals and has high economic value in industry and food processing. As a perennial macroscopic brown algae, laminaria has a life cycle that includes a sporophyte and a gametophyte stage. This alternation of generations reproductive mode provides a basis for the propagation and genetic diversity of laminaria and has a decisive influence on its cultivation and artificial breeding. The ability of spore sacs to release zoospores and the motility of zoospores in the sporophyte stage of laminaria have a crucial influence on the formation of the gametophyte stage. The motility of zoospores directly affects the formation and development of gametophytes and is related to the genetic quality and diversity of gametophytes, which is crucial for the adaptability and survival ability of laminaria populations.

[0003] The spore sac groups on the surface of mature laminaria blades can effectively mature and release a large number of zoospores under suitable environmental conditions such as suitable water temperature, light intensity and nutrient salt concentration, ensuring the transmission of genetic material and the production of new individuals. This process not only ensures the continuation of the laminaria population, but also provides a stable source of seedlings for the laminaria aquaculture industry. In addition, the spore release ability of spore sac groups also affects the genetic diversity of laminaria. Through natural or artificial selection, laminaria varieties with excellent traits can be screened, thereby improving the economic value and adaptability of cultivated laminaria. Therefore, the study of the spore release ability of spore sac groups of laminaria is of great significance for improving the breeding efficiency and quality of laminaria.

[0004] The zoospores released by spore sac dispersal have a limited swimming time during which they can move freely and then attach to suitable substrates to further develop into new laminaria individuals. Although the swimming time of zoospores is short, their motility during this period is crucial for the success of algal breeding. First, the motility of zoospores is directly related to the success rate of laminaria breeding, and healthy zoospores are a prerequisite for the success of laminaria breeding. In addition, the motility of zoospores is closely related to the yield of laminaria. When the released zoospores are highly motile and abundant, it is conducive to the smooth progress of seedling harvesting, laying a foundation for obtaining healthy seedlings in the later stage.

[0005] Chinese invention patent CN101822206A provides a method for breeding kelp seedlings, including four breeding stages of spring breeding, summer breeding, autumn breeding and winter breeding of kelp. Autumn breeding, winter breeding and spring breeding include seed selection, seedling collection, indoor cultivation and seedling release. The seed selection refers to selecting individuals that have not entered the mature stage from the kelp breeding population as seed in April-May each year, and placing the selected kelp individuals in a sea area with deep water, large flow and low temperature for further cultivation and summering. During the cultivation and summering process, when the seawater temperature reaches about 13℃, the cultivation water layer is controlled to be 2-3m away from the sea surface; when the seawater temperature reaches about 15℃, the cultivation water layer is controlled to be 3-3.5m away from the sea surface; 20-30 days before autumn breeding, winter breeding and spring breeding, the seedlings are lifted to a water layer about 0.5m away from the sea surface in turn for maturation cultivation.

[0006] Chinese invention patent CN102771394A provides a method for breeding kelp gametophyte clones. The seed kelp block with mature sporangia is cut; the seed kelp block is scrubbed with sterilized and degreased cotton dipped in boiled and sterilized seawater; the pretreated seed kelp block is soaked in sterile double-antibiotic seawater for 30 minutes, 1.5% concentration of potassium iodide sterile seawater solution for 10 minutes, and sterile seawater for 20 minutes respectively; the zoospore dispersion and filtration are carried out in a serum bottle; after the zoospores are attached to the slide to form embryonic spores, the spore water is poured out, sterile double-antibiotic seawater is added, and the single female and male gametophytes are separated into the serum bottle by micro pipette, and then cultured with sterile double-antibiotic seawater, so that single-cell kelp gametophyte clones are obtained. The kelp gametophyte clones obtained by using the technical method have stable characteristics, high survival rate, and can effectively avoid pollution and be suitable for long-term preservation.

[0007] Chinese invention patent CN112616646A provides a transportation method for inhibiting spore dispersion of seed kelp. The selected seed kelp is washed and trimmed, and then hung in a water tank, with the kelp body immersed in seawater in the water tank, and the seawater temperature maintained at 10-12℃ for transportation. The technical method has the beneficial effects that the seed kelp can inhibit spore dispersion and reduce damage during transportation, realizes smooth transition of the seed kelp from the growth environment to the water body in the breeding field, ensures the spore dispersion quality of the seed kelp in the water body of the breeding field, and greatly improves the breeding quality of the kelp.

[0008] However, the prior art has not yet provided a better method for evaluating the breeding performance of kelp. SUMMARY

[0009] Therefore, aiming at the problems of the prior art, the present application aims to provide a kelp breeding performance evaluation method based on sporangium spore dissemination parameters and application.

[0010] The first aspect of the present application provides a kelp breeding performance evaluation method based on sporangium spore dissemination parameters, comprising the following steps:

[0011] S1, taking mature kelp samples, washing, drying in the shade, and obtaining pretreated kelp;

[0012] S2, taking kelp tissue blocks from the pretreated kelp obtained in step S1, putting them into a mesh bag, immersing them in seawater, allowing the kelp tissue blocks to disseminate, and collecting the zoospore liquid;

[0013] S3, measuring the sporangium spore dissemination parameters in step S2;

[0014] S4, calculating the dissemination capacity according to the sporangium spore dissemination parameters obtained in step S3, and evaluating the kelp breeding performance according to the dissemination capacity.

[0015] Preferably, in step S1, the mature kelp is kelp with sporangia; the washing is specifically washing with sterile seawater at 8-12℃; and the drying time is 3-5h.

[0016] Preferably, in step S2, the size of the kelp tissue is 8-10cm 2 , more preferably 9cm 2 , and the mesh number of the mesh bag is 250-350, more preferably 300.

[0017] Preferably, in step S2, the temperature of the dissemination is 4-10℃, more preferably 6℃, and the light intensity of the dissemination is 1000-2000lux, more preferably 1500lux.

[0018] Preferably, in step S2, the process of collecting the zoospore liquid comprises the following steps:

[0019] From the timing of immersing in seawater, the mesh bag is lifted after 10min-6h, and sampling is performed.

[0020] More preferably, the sampling is specifically stirring the seawater in step S2 of the dissemination uniformly, and using a pipette to take the liquid.

[0021] Preferably, in step S3, the sporangium spore dissemination parameters are obtained through the number of zoospores, the total number of spores, and the video of the movement of zoospores under a microscope.

[0022] More preferably, in step S3, the measurement method of the sporangium spore dissemination parameters comprises the following steps:

[0023] The zoospore liquid obtained in step S2 is quantified, and the number of zoospores and the total number of spores are counted using a hemocytometer;

[0024] A video of the movement of the zoospores under a microscope is recorded;

[0025] The spore dispersal parameters of the sporangia are obtained based on the number of zoospores, the total number of spores, and the video.

[0026] Preferably, in step S3, the spore dispersal parameters of the sporangia include the unit sporangium area spore release rate, the total amount of spore dispersal of the sporangium, the proportion of zoospores, and the zoospore speed.

[0027] Preferably, in step S3, the calculation method of the spore dispersal parameters of the sporangia includes:

[0028] V = (n x L) ÷ (s x a);

[0029] K = V x S x a;

[0030] R = b ÷ c x 100%;

[0031] v = l ÷ t;

[0032] wherein V is the unit sporangium area spore release rate, with the unit of cells / cm 2 ; n is the spore density, with the unit of cells / mL; L is the total volume of seawater used for the dispersal of the kelp, with the unit of mL; s is the area of the kelp tissue block, with the unit of cm 2 ; a is the sporangium area proportion;

[0033] K is the total amount of spore dispersal of the sporangium, with the unit of cells, and S is the total area of the kelp blade, with the unit of cm 2 ;

[0034] R is the proportion of zoospores, with the unit of dimensionless quantity, b is the number of zoospores in the field of view, with the unit of cells, and c is the total number of spores in the field of view, with the unit of cells;

[0035] v is the zoospore movement speed, with the unit of μm / s, l is the length of the zoospore movement curve, with the unit of μm, and t is the zoospore movement time, with the unit of s.

[0036] In another aspect, the present application provides the use of the above-mentioned kelp breeding performance evaluation method in the evaluation of the breeding performance of kelp.

[0037] Compared with the prior art, the present application has the following beneficial effects:

[0038] The application provides a kelp breeding performance evaluation method based on sporangium spore dissemination parameters, and through quantitative detection of the sporangium spore dissemination capacity and zoospore activity of kelp, more accurate prediction of the kelp breeding capacity is realized, which is helpful for better guiding the production of kelp and improving the breeding efficiency. DETAILED DESCRIPTION

[0039] Terms and statements of the application:

[0040] 1. The articles 'a', 'an', and 'the': Unless otherwise expressly specified, include plural objects.

[0041] 2. Numerical ranges: Unless otherwise expressly indicated, all ranges or ratios disclosed herein are to be understood to be inclusive of any and all sub-ranges or sub-ratios subsumed therein. For example, a stated range or ratio of 1 to 30 should be considered to include any and all sub-ranges or sub-ratios between the minimum value of 1 and the maximum value of 30, including by way of example only sub-ranges or sub-ratios ranging from and including essentially every integer and fraction between the minimum and maximum values.

[0042] The following non-limiting examples can make those skilled in the art more fully understand the present application, but in no way limit the present application. The following content is only an exemplary description of the scope of the present application, and those skilled in the art can make various changes and modifications to the present application according to the disclosed content, and it should also belong to the scope of the present application.

[0043] The application will be further described below in the form of specific examples. The various chemical reagents used in the examples of the application are obtained by conventional commercial routes unless otherwise specified. If not specified, the content described below is the mass content. If not specified, it is understood to be carried out at room temperature.

[0044] In the following examples and comparative examples, the information of the kelp samples used is shown in Table 1 as follows:

[0045] Table 1

[0046]

[0047]

[0048] Example 1

[0049] S1, take the mature kelp sample of the collection time from August to October, wash with 10℃ sterile seawater, dry for 4h, and get the pretreated kelp.

[0050] S2, a 3cm x 3cm square kelp tissue block was taken from the pre-processed kelp obtained from step S1, packed into a mesh bag made of 300-mesh silk screen, immersed in sterilized seawater (200mL, placed in a 250mL beaker), and dispersed under the conditions of temperature 6℃ and light intensity 1500lux. The mesh bag was lifted at 10min, 30min, 1h, 2h, 3h, 4h, 5h, 6h, respectively, and the culture solution in the beaker was thoroughly stirred. 1mL of zoospore solution was sampled using a pipette.

[0051] S3, microscopic observation, counting using a blood cell counting plate, recording spore density, zoospore number, and total spore number. Under a microscope with a 10x objective lens, the culture solution was dropped onto a blood cell counting plate, and the zoospore movement field was video recorded. The video was analyzed by a long-base medical sperm analysis software to obtain the proportion of zoospores and the zoospore swimming speed.

[0052] S4, the dispersal capacity was calculated according to the spore sac spore dispersal parameters obtained in step S3, and the formula was as follows:

[0053] V = (n x L) ÷ (s x a);

[0054] K = V x S x a;

[0055] R = b ÷ c x 100%;

[0056] v = l ÷ t;

[0057] wherein V is the unit spore sac group area spore release rate, with units of cells / cm 2 ; n is the spore density, with units of cells / mL; L is the total volume of seawater used for dispersal kelp, with units of mL; s is the kelp tissue block area, with units of cm 2 ; a is the spore sac group area proportion;

[0058] K is the total amount of spore sac group spore dispersal, with units of cells, S is the total area of kelp blade, with units of cm 2 ;

[0059] R is the proportion of motile spores, with dimensionless quantity, b is the number of zoospores in the field, with units of cells, c is the total number of spores in the field, with units of cells;

[0060] v is the zoospore swimming speed, with units of pm / s, l is the zoospore swimming curve length, with units of pm, t is the zoospore swimming time, with units of s.

[0061] wherein the spore density (n) is obtained by the blood cell counting plate method.

[0062] Specifically, 10 μL of the obtained sample was taken by a pipette to a hemocytometer for counting. According to the counting principle of the hemocytometer, the cell density (number / mL) = (total number of four large squares ÷ 4) × 10000 × dilution multiple, and the spore density was obtained.

[0063] The total volume (L) of seawater used for the release of the spores of the sporangium of the kelp was controlled by quantitative feeding of seawater; and the area (s) of the kelp tissue block was obtained by controlling the length and width of the rectangle when the kelp was cut.

[0064] The area ratio (a) of the sporangium group was obtained by a visual estimation method. The base of the sporangium region of the mature kelp had dark brown cyst spots, which were the sporangium group area, and the green other kelp region was the non-sporangium region. By visual estimation, the area ratio of the sporangium group was obtained.

[0065] The total area (S) of the kelp blade was obtained by trapezoidal integration.

[0066] Specifically, the kelp was laid flat to stretch it as much as possible, and a ruler with an accuracy of 0.1 cm was used to measure the length and width of the kelp blade. The width of the blade was measured every 10 cm from the base of the algal body, and was recorded as A1, A2, A3, …, An, and the last interval was recorded as H (<10 cm); the base was considered as a triangle. The following calculation formula was followed:

[0067] S blade surface area = A1 × 10 ÷ 2 + (A1 + A2) × 10 ÷ 2 + (A2 + A3) × 10 ÷ 2 + … + (An-1 + An) × H ÷ 2.

[0068] Among them, the number of motile spores (b) and the total number of spores (c) in the field of view were obtained by counting with a hemocytometer; the motile spore swimming curve length (l) and the motile spore swimming time (t) were obtained according to the video under the microscope by video analysis software.

[0069] Effect evaluation

[0070] 1. Spore release ability of kelp sporangium spores.

[0071] The method provided in Example 1 was used to detect the spore dispersal ability of spore masses of different samples of Laminaria. The results of the spore dispersal ability of spore masses of different samples of Laminaria are shown in Tables 2 and 3. As the dispersal time increased, the spore release rate per unit area of the spore mass showed an overall upward trend. The spore release rate per unit area of the spore mass of Sample 1 increased as the dispersal time increased, and there was no significant difference in the dispersal rate after 4 h. The spore release rate per unit area of the spore mass of Sample 2 and Sample 3 increased first and then decreased as the dispersal time increased. The spore release rate per unit area of the spore mass of Sample 4 and Sample 5 increased as the dispersal time increased, and there was no significant difference in the spore dispersal rate after 4 h. At 4 h, the spore release rate per unit area of the spore mass of Sample 1 was significantly higher than that of Samples 2 to 4. In terms of the total amount of spore release, although the total amount of spore release of Sample 1 was significantly higher than that of Samples 2 to 4 in the same batch of samples, the total amount of spore release of Samples 2 to 4 was higher than that of Sample 1 in the two batches in September. It is thus inferred that 4 h is more suitable than 6 h for guiding the collection of Laminaria seedlings.

[0072] Table 2 Spore release rate per unit area of the spore mass of Laminaria (unit: 10 5 cells / cm 2 )

[0073]

[0074] Note: Different letters (a, b, c, d) indicate significant differences (p < 0.05); “--” indicates no data.

[0075] Table 3 Total amount of spore release of the spore mass of Laminaria after 4 h of dispersal (unit: 10 9 cells)

[0076]

[0077] Note: “--” indicates no data.

[0078] 2. Viability of spores released from spore masses of Laminaria.

[0079] The method provided in Example 1 was used to detect the spore release activity of spore sac groups of different samples of Laminaria. The spore release activity of spore sac groups of different samples of Laminaria is shown in Table 4 and Table 5. With the extension of the release time, the proportion of motile spores first increased and then decreased. The proportion of motile spores released by spore sac groups of Laminaria sample 1 and Laminaria sample 5 was close, about 40%. The proportion of motile spores released by spore sac groups of Laminaria sample 3 and Laminaria sample 4 was higher than that of Laminaria sample 4. With the extension of the release time, the motility rate of spores showed a fluctuating trend. The motility rate of spores released by spore sac groups of Laminaria sample 1 was the fastest at 30 min (122.33±9.24 μm / s), while the motility rate of spores released by spore sac groups of Laminaria sample 5 was the fastest at 3 h (128.80±4.58 μm / s). The motility rate of spores released by spore sac groups of Laminaria sample 3 and Laminaria sample 4 was the fastest at 2 h.

[0080] Table 4 Proportion of motile spores released by spore sac groups of Laminaria (%)

[0081]

[0082]

[0083] Note: Different letters (a, b, c) represent significant differences (p<0.05); “--” represents no data.

[0084] Table 5 Motility rate of motile spores released by spore sac groups of Laminaria (μm / s)

[0085]

[0086] Note: Different letters (a, b, c) represent significant differences (p<0.05); “--” represents no data.

[0087] In summary, the evaluation method can effectively improve the breeding success rate of Laminaria by precisely controlling and adjusting the spore release parameters of Laminaria spore sacs, and provides a scientific basis for the sustainable development of Laminaria culture. The implementation of the patent helps to better grasp the sampling time in production practice, saves manpower, material resources and financial resources, and improves the overall breeding efficiency.

[0088] Finally, it should be noted that the above content is only used to illustrate the technical solutions of the present application, and is not a limitation on the protection scope of the present application. Simple modifications or equivalent replacements of the technical solutions of the present application made by ordinary skilled persons in the art do not deviate from the essence and scope of the technical solutions of the present application.

Claims

1. A method for evaluating the performance of kelp breeding based on the spore dispersal parameters of sporangia, characterized in that, The method comprises the following steps: S1, taking mature kelp samples, washing, and air-drying to obtain pretreated kelp; S2, taking kelp tissue blocks from the pretreated kelp obtained in step S1, placing them in a mesh bag, and immersing them in seawater to allow the kelp tissue blocks to release zoospores, and collecting the zoospore liquid; S3, measuring the parameters of sporangium spore release of the zoospore liquid obtained in step S2; S4, calculating the release capacity according to the sporangium spore release parameters obtained in step S3, and evaluating the kelp breeding performance according to the release capacity; In step S3, the sporangium spore release parameters include unit sporangium area spore release rate, sporangium spore release total amount, zoospore proportion, and zoospore swimming speed; The calculation method of the sporangium spore release parameters comprises: V=(n×L)÷(s×a); K=V×S×a; R =b÷c×100%; v=l÷t; wherein V is the spore release rate per sporangia area unit, in number of cells / cm 2 ; n is the spore density, in number of cells / mL; L is the total volume of seawater used for the release of the seaweed, in mL; s is the area of the seaweed tissue piece, in cm 2 ; a is the sporangia area ratio K is the total amount of spore dispersal of a sori, unit is cell, S is the total area of the kelp blade, unit is cm 2 ; R is the proportion of zoospores, which is a dimensionless quantity, b is the number of zoospores in the field of view, and c is the total number of spores in the field of view; v is the zoospore swimming speed, l is the length of the zoospore swimming curve, t is the zoospore swimming time, and the unit is μm / s.

2. The method for evaluating the performance of kelp breeding according to claim 1, wherein, In step S1, the mature kelp is mature kelp with sporangia; the washing is specifically washing with sterile seawater at 8-12℃; and the air-drying time is 3-5h.

3. The method for evaluating the performance of kelp breeding according to claim 1, wherein In step S2, the size of the kelp tissue block is 4-16 cm 2 , the mesh number of the net bag is 200-400 mesh, and the seawater is disinfected seawater.

4. The method for evaluating the performance of kelp breeding according to claim 1, wherein, In step S2, the release temperature is 4-10℃, and the light intensity is 1000-2000lux.

5. The method for evaluating the performance of kelp breeding according to claim 1, wherein, In step S2, the process of collecting the zoospore liquid comprises the following steps: After immersing in seawater for timing, the mesh bag is lifted after 10min-6h, and the sample is taken.

6. The method for evaluating the performance of kelp breeding according to claim 5, wherein, The sampling is specifically stirring the seawater in step S2 for release, and using a pipette to take the liquid.

7. The method of evaluating the performance of kelp cultivation according to claim 1, wherein In step S3, the sporangium spore release parameters are obtained by the number of zoospores, the total number of spores, and the video of zoospore movement under a microscope.

8. The method for evaluating the performance of kelp breeding according to claim 7, wherein, The determination method of the sporangium spore release parameters comprises the following steps: Quantitatively taking the zoospore liquid obtained in step S2, and using a blood cell counting plate to count the number of zoospores and the total number of spores; Recording the video of zoospore movement under a microscope; The sporangium spore release parameters are obtained by the number of zoospores, the total number of spores, and the video.

9. The application of the kelp breeding performance evaluation method in claim 1-8 in evaluating the breeding performance of kelp.

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