Kelp breeding performance evaluation method based on sporangium spore diffusion parameters and application

Abstract

The invention provides a kelp breeding performance evaluation method based on sporangium spore diffusion parameters and application, and belongs to the technical field of seaweed cultivation. The method comprises the following steps: S1, taking a mature kelp sample, cleaning, and drying in the shade to obtain pretreated kelp; s2, taking kelp tissue blocks from the pretreated kelp obtained in the step S1, putting the kelp tissue blocks into a mesh bag, immersing the mesh bag into seawater to promote diffusion, and collecting zoospores liquid; s3, determining diffusion parameters of the sporangium spores in the step S2; and S4, calculating the diffusion capacity according to the diffusion parameters of the sporangium spores obtained in the step S3, and evaluating the breeding performance of the kelp according to the diffusion capacity. By utilizing the method to quantify the diffusion characteristics of the sporangium spores of the kelp, the breeding potential of the kelp can be systematically evaluated, and the breeding efficiency and yield of the kelp are improved.

Description

Technical Field

[0001] The invention belongs to the technical field of seaweed cultivation, and relates to a method for evaluating kelp breeding performance based on sporangium spore release parameters and an application thereof. Background Art

[0002] Kelp is a nutritious brown algae rich in various minerals and has extremely high economic value in industry and food processing. As a perennial large brown algae, kelp's life cycle includes two stages: sporophyte and gametophyte. This alternating generation reproduction mode provides the basis for the reproduction and genetic diversity of kelp, and has a decisive influence on its cultivation and artificial breeding. The ability of sporangium in the sporophyte stage of kelp to release zoospores and the swimming activity of zoospores have a crucial influence on the formation of the gametophyte stage of kelp. The activity of zoospores directly affects the formation and development of gametophytes, and is also related to the genetic quality and diversity of gametophytes, which is crucial to the adaptability and survival of kelp populations.

[0003] Under suitable environmental conditions such as water temperature, light intensity and nutrient concentration, the sori on the surface of mature kelp leaves can effectively mature and release a large number of zoospores, ensuring the spread of genetic material and the generation of new individuals. This process not only ensures the continuation of the kelp population, but also provides a stable source of seedlings for the kelp farming industry. In addition, the spore release ability of the sori also affects the genetic diversity of kelp. Through natural or artificial selection, kelp varieties with excellent traits can be selected, thereby improving the economic value and adaptability of cultured kelp. Therefore, the study of the spore release ability of kelp sori is of great significance for improving the efficiency and quality of kelp breeding.

[0004] The zoospores released from the sporangium have a limited swimming time, during which they can move freely, and then attach to a suitable substrate to further develop into new kelp individuals. Although the swimming time of the zoospores is short, their mobility during this period is crucial to the success of algae breeding. First of all, the vitality of the zoospores is directly related to the success rate of kelp seedling cultivation. Healthy zoospores are the prerequisite for the success of kelp seedling cultivation. In addition, the vitality of the zoospores is closely related to the yield of kelp. When the released zoospores are highly active and sufficient in number, it is conducive to the smooth progress of seedling collection and lays the foundation for obtaining healthy seedlings in the later stage.

[0005] Chinese invention patent CN101822206A provides a method for multi-crop seedling cultivation of kelp, including four seedling cultivation stages of spring seedling cultivation, summer seedling cultivation, autumn seedling cultivation and winter seedling cultivation of kelp. Autumn seedling cultivation, winter seedling cultivation and spring seedling cultivation include vegetable cultivation and conservation, seedling collection, indoor cultivation and seedling delivery. The vegetable cultivation and conservation refers to selecting individuals that have not yet entered the mature stage from the kelp breeding group as vegetable cultivation in April and May each year, and releasing the selected kelp individuals in a sea area with deep water, large current and low temperature to continue to cultivate and survive the summer. During the cultivation and summering process, the seawater temperature reaches about 13°C, and the cultivation water layer is controlled to be 2-3m away from the sea surface; the seawater temperature reaches about 15°C, and the cultivation water layer is controlled to be 3-3.5m away from the sea surface; 20-30 days before the autumn seedling cultivation, winter seedling cultivation and spring seedling cultivation, the vegetables for cultivation and summering are successively lifted to a water layer about 0.5m away from the sea surface for ripening cultivation.

[0006] Chinese invention patent CN102771394A provides a method for cloning and cultivating kelp gametophytes. Cut the seed kelp block with mature spore sacs; use sterilized absorbent cotton to dip in boiled and sterilized seawater to scrub the seed kelp block; soak the pretreated seed kelp block in sterile double-resistance seawater for 30 minutes, 1.5% potassium iodide sterile seawater solution for 10 minutes, and sterile seawater for 20 minutes; release and filter the spores in a serum bottle: pour the filtered spore water into a vertical staining tank equipped with a slide, and when the spores attach to the slide to form embryos, pour out the spore water, add sterile double-resistance seawater, and then use a micropipette to separate the single male and female gametophytes into the serum bottle, and then culture with sterile double-resistance seawater to obtain a single-cell kelp gametophyte clone. The kelp gametophyte clones cultivated using this technical method have stable traits, high survival rate, can effectively avoid contamination, and are suitable for long-term storage.

[0007] Chinese invention patent CN112616646A provides a transportation method for inhibiting the release of spores of seed kelp. The selected seed kelp is rinsed and trimmed, and the kelp is hung in a water tank, and the kelp body is immersed in the seawater in the water tank, and the seawater temperature is maintained at 10-12°C for transportation. The beneficial effects of this technology are: by adopting the above-mentioned transportation method, the spore release of the seed kelp can be inhibited during transportation, and the damage to the seed kelp can be reduced, and a smooth transition of the seed kelp from the growth environment to the water body of the nursery is achieved, ensuring the spore release quality of the seed kelp in the water body of the nursery, and greatly improving the quality of kelp seedling.

[0008] However, the existing technology has not yet provided a better method for evaluating the breeding performance of kelp. Summary of the invention

[0009] In view of this, and in view of the above-mentioned problems of the prior art, the object of the present invention is to provide a method and application for evaluating the breeding performance of kelp based on sporangiospore release parameters.

[0010] The first aspect of the present invention provides a method for evaluating the breeding performance of kelp based on sporangium spore release parameters, comprising the following steps:

[0011] S1. Take a mature kelp sample, wash it, and dry it in the shade to obtain a pretreated kelp;

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

[0013] S3, determining the sporangium spore release parameters in step S2;

[0014] S4. Calculate the release capacity according to the sporangium spore release parameters obtained in step S3, and evaluate the kelp breeding performance according to the release capacity.

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

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

[0017] Preferably, it is characterized in that, in step S2, the temperature of the emitted light is 4-10°C, more preferably 6°C, and the intensity of the emitted light is 1000-2000 lux, more preferably 1500 lux.

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

[0019] The time is counted from the time of immersion in seawater, and the net bag is lifted up after 10 minutes to 6 hours to take samples.

[0020] More preferably, the sampling is specifically to stir the seawater in the step S2 of releasing evenly and take the liquid using a pipette.

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

[0022] More preferably, in step S3, the method for determining the sporangium spore release parameters comprises the following steps:

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

[0024] Record videos of spore movement downstream of the microscope;

[0025] The sporangium spore release parameters are obtained through the obtained number of zoospores, total number of spores and video.

[0026] Preferably, in step S3, the sporangium spore release parameters include the spore release rate per unit sori area, the total amount of spore release per sori, the zoospore ratio and the zoospore rate.

[0027] Preferably, in step S3, the method for calculating the sporangium spore release parameters comprises:

[0028] V = (n × L) ÷ (s × a);

[0029] K = V × S × a;

[0030] R = b ÷ c × 100%;

[0031] v = l ÷ t;

[0032] Where V is the spore release rate per unit sori area, expressed in cells / cm 2 ; n is the spore density, in cells / mL; L is the total volume of seawater used to release the kelp, in mL; s is the area of ​​the kelp tissue block, in cm 2 ; a is the proportion of sori area;

[0033] K is the total amount of spores released by the sporangium, in cells, and S is the total area of ​​the kelp leaf, in cm 2 ;

[0034] R is the zoospore ratio, a dimensionless quantity, b is the number of zoospores in the field of view, in cells, and c is the total number of spores in the field of view, in cells;

[0035] v is the swimming velocity of the zoospores, in μm / s, l is the length of the zoospore swimming curve, in μm, and t is the swimming time of the zoospores, in s.

[0036] On the other hand, the present invention provides the application of the above-mentioned method for evaluating the breeding performance of kelp in evaluating the breeding performance of kelp.

[0037] Compared with the prior art, the beneficial effects of the present invention include:

[0038] The present invention provides a method for evaluating the breeding performance of kelp based on sporangiospore release parameters. By quantitatively detecting the sporangiospore release ability and zoospore vitality of kelp, a more accurate prediction of the breeding ability of kelp can be achieved, which is helpful to better guide the production of kelp and improve the breeding efficiency. DETAILED DESCRIPTION

[0039] Terms and Claims of the Present Invention:

[0040] 1. The articles “a”, “an” and “the” include plural referents unless expressly limited to one or more referents otherwise.

[0041] 2. Numerical range: Unless otherwise expressly stated, all ranges or ratios disclosed herein will be understood to include any and all sub-ranges or sub-ratios contained therein. For example, a range or ratio of 1 to 30 stated should be considered to be included between a minimum value of 1 and a maximum value of 30, and any sub-range or sub-ratio, integer, decimal, or sub-range or sub-ratio consisting of integers or decimals including the end points.

[0042] The following non-limiting examples can enable those skilled in the art to more fully understand the present invention, but do not limit the present invention in any way. The following content is merely an exemplary description of the scope of the present invention, and those skilled in the art can make various changes and modifications to the present invention based on the disclosed content, which should also fall within the scope of the present invention.

[0043] The present invention is further described below by way of specific examples. The various chemical reagents used in the examples of the present invention are obtained through conventional commercial channels unless otherwise specified. Unless otherwise specified, the contents described below are all weight contents. Unless otherwise specified, it is understood that the process is 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 below:

[0045] Table 1

[0046]

[0047]

[0048] Example 1

[0049] S1. Take mature kelp samples harvested from August to October, wash them with 10°C sterile seawater, and dry them in the shade for 4 hours to obtain pretreated kelp.

[0050] S2. Take a 3cm×3cm square kelp tissue block from the pretreated kelp obtained in step S1, put it into a mesh bag made of 300-mesh sieve silk, immerse it in disinfected seawater (200mL, placed in a 250mL beaker), and release it under the conditions of temperature 6°C and light intensity 1500lux. Timing is carried out from the immersion of the kelp in seawater, and the mesh bag is lifted at 10min, 30min, 1h, 2h, 3h, 4h, 5h, and 6h, respectively. The culture solution in the beaker is fully stirred, and 1mL of spore solution is sampled using a pipette.

[0051] S3. Observe under a microscope and count using a hemocytometer to record the spore density, number of zoospores, and total number of spores. Under a 10x objective lens, drop the culture solution on the hemocytometer, record the visual field of zoospore movement, and analyze the video using the Changji Medical Sperm Analysis Software to obtain the zoospore percentage and zoospore movement rate.

[0052] S4, calculating the release capacity according to the sporangium spore release parameters obtained in step S3, the formula is as follows:

[0053] V = (n × L) ÷ (s × a);

[0054] K = V × S × a;

[0055] R = b ÷ c × 100%;

[0056] v = l ÷ t;

[0057] Where V is the spore release rate per unit sori area, expressed in cells / cm 2 ; n is the spore density, in cells / mL; L is the total volume of seawater used to release the kelp, in mL; s is the area of ​​the kelp tissue block, in cm 2 ; a is the proportion of sori area;

[0058] K is the total amount of spores released by the sporangium, in cells, and S is the total area of ​​the kelp leaf, in cm 2 ;

[0059] R is the zoospore ratio, a dimensionless quantity, b is the number of zoospores in the field of view, in cells, and c is the total number of spores in the field of view, in cells;

[0060] v is the swimming velocity of the zoospores, in μm / s, l is the length of the zoospore swimming curve, in μm, and t is the swimming time of the zoospores, in s.

[0061] The spore density (n) was obtained by the hemocytometer method.

[0062] Specifically, 10 μL of the obtained sample was taken by a pipette and counted on a hemacytometer. According to the counting principle of the hemacytometer, the spore density was obtained by calculating the cell density (cells / mL) = (the total number of four large grids ÷ 4) × 10000 × dilution factor.

[0063] The total volume (L) of seawater used for releasing kelp was controlled by quantitatively administering seawater; the area of ​​kelp tissue block (s) was obtained by controlling the length and width of the rectangle when cutting kelp.

[0064] The sori area ratio (a) was obtained by visual estimation. There are dark brown spots at the base of the sporangium area of ​​mature kelp, which are the sori area, while the other green kelp areas are non-sporangium areas. The sori area ratio can be obtained by visual estimation.

[0065] The total leaf area (S) of kelp was measured by the trapezoidal integration method.

[0066] Specifically, the kelp was spread out as far as possible, and the length and width of the kelp blade were measured with a ruler with an accuracy of 0.1 cm. The blade width was measured every 10 cm from the base of the algae, and recorded as A1, A2, A3...An, and the last interval was recorded as H (<10 cm); the base was regarded as a triangle. The following calculation formula was used:

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

[0068] The number of zoospores in the field of view (b) and the total number of spores (c) were obtained by counting with a hemocytometer; the length of the zoospore swimming curve (l) and the swimming time of the zoospores (t) were obtained by video analysis software based on the video under the microscope.

[0069] Effect evaluation

[0070] 1. The ability of kelp sporangium to release spores.

[0071] The method provided in Example 1 was used to detect the spore release ability of the kelp sori of different kelp samples. The results of the spore release ability of the kelp sample sori are shown in Tables 2 and 3. As the release time is prolonged, the spore release rate per unit area of ​​the kelp sori generally shows an increasing trend as the release time is prolonged. The spore release rate per unit sori area of ​​kelp sample 1 increases as the release time is prolonged, and there is no significant difference in the release rate after 4h. In the process of sporangium release of kelp samples 2 and kelp sample 3, the spore release rate per unit sori area increases first and then decreases as the release time is prolonged. The spore release rate per unit sori area of ​​kelp samples 4 and kelp sample 5 increases as the release time is prolonged, and there is no significant difference in the spore release rate after 4h. At 4 hours, the spore release rate per unit sori area of ​​Laminaria japonica sample 1 was significantly higher than that of Laminaria japonica sample 2-4. In terms of the total spore release, although the total spore release of Laminaria japonica sample 1 was significantly higher than that of Laminaria japonica sample 2-4 in the same batch of kelp samples, the opposite was true for the two batches in September. It can be inferred that 4 hours is more suitable for guiding kelp seedling collection than 6 hours.

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

[0073]

[0074] Note: Different letters (a, b, c, d) indicate significant differences (p<0.05); “--” means data were not measured.

[0075] Table 3 Total amount of spores released from kelp sori after 4 hours of release (unit: 10 9 cells)

[0076]

[0077] Note: “--” means data not measured.

[0078] 2. Kelp sporangium releases zoospore activity.

[0079] The method provided in Example 1 was used to detect the activity of zoospores released by kelp sori of different kelp samples. The activity of spores released by the kelp sample sori is shown in Tables 4 and 5. As the release time increases, the proportion of zoospores first increases and then decreases. The proportion of zoospores released by kelp sori of kelp sample 1 and kelp sample 5 sampled in the same period is close, about 40%; the proportion of zoospores released by kelp sori of kelp sample 3 and kelp sample 4 is higher than that of kelp sample 4. As the release time prolonged, the spore swimming rate showed a fluctuating trend. The spore swimming rate of the spore sori of kelp sample 1 sampled during the same period was the fastest at 30 minutes (122.33±9.24μm / s), while the spore swimming rate of the spore sori of kelp sample 5 was the fastest at 3 hours (128.80±4.58μm / s); the spore swimming rate of the spore sori of kelp sample 3 and kelp sample 4 was the fastest at 2 hours.

[0080] Table 4 Proportion of zoospores released by kelp spore sori (%)

[0081]

[0082]

[0083] Note: Different letters (a, b, c) indicate significant differences (p<0.05); “--” means data were not measured.

[0084] Table 5 Swimming speed of spores released by kelp spore sori (μm / s)

[0085]

[0086] Note: Different letters (a, b, c) indicate significant differences (p<0.05); “--” means data were not measured.

[0087] In summary, this evaluation method can effectively improve the success rate of kelp breeding by accurately controlling and adjusting the release parameters of kelp sporangium spores, and provide a scientific basis for the sustainable development of kelp farming. The implementation of this patent helps to better grasp the time of seedling collection in production practice, save manpower, material and financial resources, and improve the overall farming efficiency.

[0088] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, rather than to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions of the technical solution of the present invention by ordinary technicians in this field do not deviate from the essence and scope of the technical solution of the present invention.

Claims

1. A method for evaluating the breeding performance of kelp based on sporangiospore release parameters, characterized in that: The following steps are involved: S1. Take a mature kelp sample, wash it, and dry it in the shade to obtain a pretreated kelp; S2, taking the kelp tissue blocks from the pretreated kelp obtained in step S1, putting them into a mesh bag, immersing them in seawater, releasing the kelp tissue blocks, and collecting the zoospore liquid; S3, measuring the sporangium spore release parameter of the zoospore liquid obtained in step S2; S4. Calculate the release capacity according to the sporangium spore release parameters obtained in step S3, and evaluate the kelp breeding performance according to the release capacity.

2. The method for evaluating the breeding performance of kelp according to claim 1, characterized in that: In step S1, the mature kelp is a mature kelp with spore sacs; the washing is specifically washing with sterile seawater at 8-12° C.; and the shade drying time is 3-5 hours.

3. The method for evaluating the breeding performance of kelp according to claim 1, characterized in that: In step S2, the size of the kelp tissue block is 4-16 cm 2 The mesh number of the mesh bag is 200-400 meshes, and the seawater is sterilized seawater.

4. The method for evaluating the breeding performance of kelp according to claim 1, characterized in that: In step S2, the emitted temperature is 4-10°C, and the emitted light intensity is 1000-2000 lux.

5. The method for evaluating the breeding performance of kelp according to claim 1, characterized in that: In step S2, the process of collecting the zoospore liquid comprises the following steps: The time is counted from the time of immersion in seawater, and the net bag is lifted up after 10 minutes to 6 hours to take samples.

6. The method for evaluating the breeding performance of kelp according to claim 5, characterized in that: The sampling specifically includes stirring the seawater in the step S2 for dispersion evenly and taking the liquid using a pipette.

7. The method for evaluating the breeding performance of kelp according to claim 1, characterized in that: In step S3, the sporangium spore release parameters are obtained by the number of zoospores, the total number of spores and the video of spore movement under the microscope.

8. The method for evaluating the breeding performance of kelp according to claim 7, characterized in that: The method for determining the sporangium spore release parameters comprises the following steps: The zoospore liquid obtained in step S2 is quantitatively measured, and the number of zoospores and the total number of spores are counted using a hemocytometer; Record videos of spore movement downstream of the microscope; The sporangium spore release parameters are obtained through the obtained number of zoospores, total number of spores and video.

9. The method for evaluating the breeding performance of kelp according to claim 1, characterized in that: In step S3, the sporangium spore release parameters include the spore release rate per unit sori area, the total amount of spore release per sori, the zoospore ratio and the zoospore swimming rate; 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; Where V is the spore release rate per unit sori area, expressed in cells / cm 2 ; n is the spore density, in cells / mL; L is the total volume of seawater used to release the kelp, in mL; s is the area of ​​the kelp tissue block, in cm 2 ; a is the proportion of sori area; K is the total amount of spores released by the sporangium, in cells, and S is the total area of ​​the kelp leaf, in cm 2 ; R is the zoospore ratio, a dimensionless quantity, b is the number of zoospores in the field of view, in cells, and c is the total number of spores in the field of view, in cells; v is the swimming velocity of the zoospores, in μm / s, l is the length of the zoospore swimming curve, in μm, and t is the swimming time of the zoospores, in s.

10. Use of the method for evaluating the breeding performance of kelp according to any one of claims 1 to 9 in evaluating the breeding performance of kelp.

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