A precise observation method for the development process of Undaria pinnatifida juveniles in seedling production

Abstract

The present invention discloses a method for accurately observing the development process of kelp juveniles in seedling production, comprising the following steps: taking kelp juvenile spore liquid in seedling production, diluting and shaking it with sterilized seawater, and injecting it into the counting chamber of a hemocytometer covered with a cover glass within 5 minutes; after the spores in the counting chamber develop into embryospores, observing the embryospores under a microscope, and using the grids in the large squares of the counting chamber to locate and number the embryospores; removing the cover glass, suspending the hemocytometer in the pool water of kelp juveniles from the same batch as the embryospores on the hemocytometer, observing the growth and development status and development stage of the kelp juveniles marked on the hemocytometer every day, and conducting production management accordingly. The present invention can continuously and accurately track and observe the development and growth of multiple kelp embryospores, and accurately manage the kelp juveniles in the seedling pond accordingly.

Description

Technical Field

[0001] The present invention relates to the technical field of kelp cultivation, and in particular to a method for accurately observing the development process of kelp juveniles in seedling production. Background Art

[0002] Undaria pinnatifida WFR Suringar, 1873, also known as Wabu or Wakabu, belongs to the genus Undaria, class Phaeophyceae, order Laminariales, family Alariaceae, and the phylum Phaeophyta. Popularly known as the "vegetable of longevity," kelp is nutritious and economically valuable. Its cultivation plays a key role in maintaining the ecological health of coastal waters. It is one of the three major cultivated economic seaweeds in my country, with an annual production of approximately 200,000 tons of dry product. Currently, the cultivation method for kelp seedlings is fully artificial. During seedling production, the sporangium, the reproductive organ of kelp, releases zoospores in the nursery pond. Use a microscope to check the spore density in the nursery pond water. When 100-200 spores are observed per field of view at 100x magnification, remove the kelp sporangium net bag and spread the seed curtain flat in the pond water, while suspending a glass slide in the water. When the spore density on the slide reaches 60-120 per field of view at 100x magnification, move the seed curtain to another nursery pond and begin nursery management.

[0003] During seedling management, small sections of seedling curtain rope are cut daily and the status of the embryospores, or the gametophytes or juvenile sporophytes that develop from them, is observed under a microscope or dissecting microscope. This information is then used to manage the seedlings, including adjusting light intensity, nutrient levels, and water flow. However, when observing juvenile Undaria pinnae on the seedling curtain ropes under a microscope, due to the ropes' opacity and rough surface, juvenile Undaria pinnae in the embryospore or gametophyte stage are difficult to observe clearly, resulting in only a rough assessment of their development. Furthermore, the dissecting microscope provides insufficient magnification, suitable only for observing larger juvenile Undaria pinnae at the juvenile sporophyte stage, and unsuitable for observing the status and development of earlier stage juveniles. Furthermore, the seedling curtain ropes are discarded after each observation, and a new one is cut and observed the following day. Consequently, different embryospores, gametophytes, or juvenile sporophytes are observed each day. This makes it difficult to accurately assess the development of juvenile Undaria pinnae at the embryospore and gametophyte stages during production.

[0004] Accurately observing the process of embryos developing into gametophytes, then into oocysts or spermatocysts, and finally releasing germ cells, as well as the timing of reaching certain developmental milestones, is crucial for controlling seedling management, adjusting lighting and fertilization, and ensuring successful seedling production. Therefore, methods for accurately observing the development of young Undaria pinnatifida are urgently needed in artificial seedling production. Summary of the Invention

[0005] In response to the above-mentioned prior art, the present invention aims to provide a method for accurately observing the development process of kelp juveniles during seedling production. The present invention adds a spore solution to a hemocytometer covered with a glass slide, allowing a limited number of spores to enter a counting tank and develop into embryospores. The embryospores are located and numbered using the hemocytometer, the counting tank, and the large square grid within the tank. The hemocytometer is then suspended in the seedling tank, allowing the growth and development of the numbered embryospores to be continuously and accurately observed using a microscope. The lighting, nutrients, running water, and other conditions for seedling production are determined based on the growth and development of the embryospores.

[0006] To achieve the above object, the present invention adopts the following technical solutions:

[0007] A first aspect of the present invention provides a method for accurately observing the development process of juvenile Undaria pinnatifida in seedling production, comprising the following steps:

[0008] (1) Take the spore solution of Undaria pinnatifida in seedling production, dilute it with sterilized seawater and shake it evenly, then inject it into the counting chamber of a hemocytometer covered with a cover glass within 5 minutes;

[0009] (2) After the spores in the counting pool develop into embryospores, observe them under a microscope and use the blood cell counting plate number, the counting pool number and the grid position of the large square in the counting pool to locate and number the embryospores; remove the cover glass and suspend the blood cell counting plate in the pool water of the same batch of kelp juveniles as the embryospores on the blood cell counting plate; observe the development status and development stage of the kelp juveniles marked on the blood cell counting plate every day, and carry out production management accordingly.

[0010] The blood cell counting chamber used in the present invention was purchased from Shanghai Qiujing Biochemical Reagent Instrument Co., Ltd., with a model specification of XB-K-25, 0.10 mm, 1 / 400 mm 2 .

[0011] Preferably, in step (1), when the concentration of the zoospore liquid reaches 100 to 200 zoospores per visual field under a 100× microscope, the zoospore liquid is diluted with boiled and cooled sand-filtered seawater.

[0012] Preferably, in step (1), the disinfected seawater is diluted 10 to 20 times.

[0013] Preferably, in step (2), the culturing of the zoospores to develop into embryospores is performed by placing a hemocytometer in an environment with a pressure lower than 1000 Lx and a temperature of 16-18° C. for 1-2 hours.

[0014] Preferably, in step (2), two blood cell counting plates are hung in each seedling pool.

[0015] The second aspect of the present invention provides the application of the above-mentioned precise observation method in the production of kelp seedlings.

[0016] Beneficial effects of the present invention:

[0017] The present invention solves the problem of difficulty in accurately observing the early developmental state of Undaria pinnae during seedling production. Conventional seedling management involves cutting a small section of seedling curtain rope daily and observing the state of the embryospores, or the gametophytes or sporophytes that develop from them, under a microscope or dissecting microscope. Seedling management, including adjusting light intensity, nutrient usage, and water flow, is then conducted accordingly. However, when observing Undaria pinnae on the seedling curtain rope under a microscope, due to the rope's opacity and rough surface, the Undaria pinnae in the embryospore or gametophyte stage attached to the rope are difficult to observe clearly, resulting in only a rough assessment of the developmental state of the Undaria pinnae. Furthermore, the dissecting microscope provides insufficient magnification, suitable only for observing larger Undaria pinnae that have reached the sporophyte stage, and unsuitable for observing the state and developmental stage of early stage juveniles. Furthermore, the seedling curtain rope is discarded after each observation, and a new one is cut and observed the following day. Consequently, different embryospores, gametophytes, or sporophytes are observed each day. Therefore, it is difficult to accurately grasp the development of kelp juveniles during the embryospore and gametophyte stages during production, making it difficult to precisely manage kelp seedling production. In the present invention, the kelp juveniles positioned on the hemocytometer are from the same batch as the kelp juveniles on the seedling curtain ropes during production, and grow and develop under the same conditions. Therefore, their growth and development are synchronized and can represent the kelp juveniles on the kelp seed curtain ropes. The kelp juveniles positioned on the hemocytometer facilitate accurate observation under a continuous microscope during the seedling period, providing accurate information for seedling management.

[0018] (2) In the present invention, the kelp embryospores are attached to the grid surface of the counting pool of the hemocytometer, and the attachment points are mostly located on the grid lines. The embryospores and the gametophytes and sporophytes that subsequently develop are stable on the hemocytometer and are not easy to fall off, which is convenient for long-term continuous observation.

[0019] (3) The method of the present invention is simple and effective, and can efficiently realize the growth observation of kelp juveniles, solving the problem of being difficult to accurately grasp the development of kelp juveniles in production, and has promotion significance in kelp cultivation. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 :Microscopic photograph of 7-day-old gametophyte of Undaria pinnatifida;

[0021] Figure 2 :Microscopic photograph of the gametophyte of Undaria pinnatifida at 68 days old;

[0022] Figure 3 :Microscopic photograph of the gametophyte of Undaria pinnatifida at 70 days old;

[0023] Figure 4:Microscopic photograph of the gametophyte of Undaria pinnatifida at 73 days old;

[0024] Figure 5 :Microscope photo of 82-day-old kelp larvae

[0025] Note: The circle shows the juvenile stage of kelp. DETAILED DESCRIPTION

[0026] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present application belongs.

[0027] In order to enable those skilled in the art to more clearly understand the technical solution of the present application, the technical solution of the present application will be described in detail below with reference to specific embodiments.

[0028] The test materials used in the examples of the present invention are all conventional test materials in the art and can be purchased through commercial channels.

[0029] Example

[0030] 1. On June 18, 2021, take 6 blood cell counting slides and mark them as 1, 2, 3, 4, 5, and 6. Mark the two counting pools of each blood cell counting slide as A and B, and cover them with cover slips.

[0031] 2. When the kelp seedlings are collected using seedling curtains, the zoospore liquid is collected and the zoospore density is tested under a 100× microscope, with 160 zoospores per field of view.

[0032] 3. Take 10 ml of the zoospore solution and dilute it 16-fold with sterile, cooled, sand-filtered seawater. Shake well. Within 2 minutes, use a pipette to inject the zoospore solution into the H-shaped groove of each hemocytometer covered with a slide. The zoospore solution should enter the counting chamber between the coverslip and the hemocytometer. Use filter paper to absorb any excess liquid around the coverslip.

[0033] 4. Place the hemocytometer flat on the workbench in a laboratory under a light source of 220 lx and a temperature of 17°C. After 1 hour, observe the hemocytometer under a microscope. Select embryos attached to the grid lines within the large squares of the A and B counting pools of hemocytometers No. 1, 2, 3, 4, 5, and 6 and mark and number them. 1.5 hours after collecting zoospores, the seed curtains in the seedling pools are harvested and transferred to the incubation pools. At this time, remove the coverslips from hemocytometers No. 1, 2, 3, 4, 5, and 6. Suspend two hemocytometers from the bamboo poles used to suspend the seed curtains in each of the three seedling pools. Place both the seed curtains and hemocytometers in the seedling pools for incubation. Observe the growth and development of the kelp larvae marked on the hemocytometer daily under a microscope. Use the general growth and development of the kelp larvae observed under a microscope on the seed curtain ropes as a reference to accurately determine the developmental stage and status of the larvae and adjust lighting and other conditions in a timely manner to achieve optimal kelp seedling cultivation results.

[0034] The developmental status of Undaria pinnatifida No. 1 in the large square in the middle of the counting pool of the first hemocytometer A is as follows:

[0035] Figure 1 : On June 25, 7 days later, the gametophyte of kelp was in the gametophyte growth stage.

[0036] Figure 2 : August 25, 68 days, gametophytes in mature stage.

[0037] Figure 3 : August 27, 70 days, gametophytes in mature stage.

[0038] Figure 4 : August 30, 73 days, gametophytes in mature stage.

[0039] The developmental status of the kelp larvae in the large square of the second hemocytometer A counting chamber at day 82: Figure 5 .

[0040] The above description is merely a preferred embodiment of the present application and is not intended to limit the present application. Various modifications and variations are possible for those skilled in the art. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present application shall be included within the scope of protection of the present application.

Claims

1. A method for accurately observing the development process of kelp juveniles in seedling production, characterized in that: The following steps are involved: (1) Take 6 blood cell counting chambers and mark them as 1, 2, 3, 4, 5, and 6 respectively. Mark the two counting chambers of each blood cell counting chamber as A and B respectively, and cover them with cover slips. (2) When the seedlings of Undaria pinnatifida were harvested using seedling curtains, zoospore fluid was collected and the zoospore density was measured under a 100× microscope, with 160 zoospores per field of view. (3) Take 10 ml of the zoospore solution, dilute it 16 times with sterilized cooled sand-filtered seawater, shake well, and within 2 minutes, use a pipette to take the zoospore solution and inject it into the H-shaped groove of each hemocytometer covered with a slide. The zoospore solution enters the counting pool space between the cover glass and the hemocytometer. Use filter paper to absorb the excess liquid around the cover glass. (4) The blood cell counting plate was placed flat on the operating table of the laboratory with an illumination of 220 lx and an air temperature of 17 ℃. After 1 hour, the blood cell counting plate was observed under a microscope. The embryos spores attached to the grid lines in the large squares of the counting pools A and B of blood cell counting plates No. 1, 2, 3, 4, 5, and 6 were selected and marked and numbered. 1.5 hours after the collection of zoospores, the seedling curtain in the seedling pool was completed and transferred to the cultivation pool. At this time, the cover glass of blood cell counting plates No. 1, 2, 3, 4, 5, and 6 was removed. Two blood cell counting plates were hung on the bamboo poles of the seedling curtains in the three seedling cultivation pools respectively. The seedling curtains and blood cell counting plates were placed in the seedling cultivation pool for cultivation. The growth and development of the kelp juveniles marked on the blood cell counting plate were observed under a microscope every day. At the same time, the growth and development of the kelp juveniles observed under a microscope on the seedling curtain rope was used as a reference to accurately determine the development stage and status of the kelp juveniles and adjust the light intensity, nutrient salt usage, and water flow in time.

2. Application of the method for precise observation of the development process of kelp juveniles in seedling production according to claim 1 in kelp seedling production.

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