Laminaria asexual propagation breeding techniques

In China, the cultivation of kelp seedlings by the summer seedlings cultivation method is commonly used. It has two major shortcomings: (1) Long seedlings, high costs, and high risks. 2 Due to mixed mining and mixed breeding during nursery, it is easy to cause degradation of good traits. At present, some kelp breeding farms have started to use clonal propagation techniques, that is, the use of kelp gametophytes for the cultivation of kelp seedlings. The seedlings can be released from the sea for 50 to 60 days before being released to the sea. This not only saves nearly half of the cost of nursery seedlings, but also maintains the quality of the seaweed varieties. ,High Yield. The nursery method is summarized as follows:

First, the clonal propagation

1. The methods and procedures for preparing seaweeds for the selection of seaweeds are basically the same as those for the traditional method of picking seedlings. Individuals with long, dark, shiny, and healthy algae are selected, and the sporangia on the algae are free of attachments and have a good maturity. . Cut about 5 cm (5 cm) of sporangia with scissors, cool the seawater with boiled cotton boil, and wipe the dirt on it. Then rinse it several times with sterile seawater, dry it, and put it in water at 10 °C. In it, it makes the sporades spawn.

2. When the number of spores attached to the sporulation reaches 2 to 5/160*, the kelp is taken out, and the spore water is filtered with a gauze to remove the mucus and the slide is put into the spore. When the spores adhered to the slide at a density of about 10/160, the slides were removed and incubated in low-temperature sterile seawater. Culture conditions: water temperature 10 ~ 15 °C; light intensity 1500 ~ 2500 meters candle; illumination time 24 hours a day light. Culture medium: Boiling cooling disinfected seawater, Sodium nitrate-N: 5 parts per million, Potassium dihydrogen phosphate-P: 1 parts per million. The culture solution needs to be replaced at regular intervals, and it is usually changed once a week.

3. Primary culture of asexual reproduction When the gametophyte grows to distinguish between male and female, the female and male cells are removed by capillary tubes and placed in test tubes for culture. The culture method is the same as above. With the rapid growth of male and female gametophyte, it will soon become a spherical filamentous algae ball. Since the internal cells of the algae bulbs cannot receive light, the growth rate is affected. When the algae balls grow to a diameter of 1 to 2 mm, they can be crushed using a sterile slide and transferred to a 250-ml Erlenmeyer flask. Each filamentous cell soon afterwards. The segment can be re-grown into algae balls, which can be crushed again for rapid growth.

4. Asexual propagation system expansion culture

(1) Culture container: As the weight of the clonal propagation system increases, it can be transferred to a large container for culture. Due to the fragile and generally high price of glassware, mineral water tanks can be used as a substitute for light, durable, affordable, and good culture. However, the light transmittance is about 50%, and the light intensity is needed to convert the light intensity according to the growth needs.

(2) Breakage of cell mass: When expanding the culture, due to the large workload, it is not suitable to continue manual crushing. The tissue crusher can be used to break the cell mass into cell segments. Generally, it takes 10 to 20 seconds.

(3) Culture conditions: As the culture volume increases, the upper cells will block the culture light and affect the growth of the underlying cells. In order to accelerate the growth rate and reduce the production cost, it is possible to continuously inflate the culture fluid so that all cell clusters can receive sufficient light, and since the culture fluid around the cell cluster is fluid, it has better nutrient conditions and can increase the growth rate. As the density increases, the light intensity can be increased appropriately.

(4) Daily management: Change the water 1 or 2 times per week, increase the number of water changes when the growth status is poor and the concentration is high. When the water was changed, the inflation was stopped 1 hour earlier. When the cell pellet settled, the supernatant was aspirated and fresh medium was added. Microscopic examination of poor growth conditions with attached matter, the cell pellet can be poured into a 300 mesh cage, repeatedly washed with a spray until no attachment. In order to avoid contamination of the culture fluid when inflated, it is necessary to regularly light the ultraviolet lamp for disinfection of air for half an hour every day.

Second, picking seedlings

Cell cluster separation

(1) One-time separation: To reach a certain density of seedlings, each nursery curtain requires 3 grams of a female vegetative propagation line and 1.5 grams of a male vegetative propagation line. According to the amount of seedlings, the required male and female clonal propagation lines are mixed together, and a small amount of the culture liquid is placed into a tissue comminutor. The cutting time is 10 to 20 seconds, and the length of the cut cells is about 200 microns, that is, there are about 10 cells per cell segment. .

(2) Short-day culture: A vegetative propagation system after separation was placed in an aerated suspension culture in a culture flask. The daily illumination time was 10 hours.

(3) Secondary separation: The cells gradually increased about 10 days after the first separation, pigments deepened, and gradually entered the developmental state. A second separation can take place at this point. The cells were again crushed and cut with a tissue grinder, and then the cells that had been crushed to an appropriate size were filtered using a 500-mesh sieve to be used for the next step of seedling collection. Generally, there were 1 to 4 cells per cell segment. The cells were not filtered out and continued to be crushed and filtered until they all reached the harvesting requirement.

2. Picking method

(1) Attachment base: The brown cord seedling curtain used in the conventional production is used. After conventional treatment, a 500-mesh sieve box with a size of 1 cm and 3 cm is used for daily inspection in the early stage of nursery.

(2) Attachment base treatment: The prepared seedling curtains are washed, straightened, and pre-cooled in low-temperature seawater soaking, and the single-layer flatness is placed neatly in the pool that has been filled with low-temperature water.

(3) Spraying: Dilute the cell fluid to 50 pieces/160 or less with low-temperature seawater and spray evenly on the surface of the pool where the attachment base is located, so that it will naturally settle evenly on the surface of the nursery curtain.

Third, nursery

1. The flowing water cell segment does not have the ability of active attachment and is easily detached from the attachment base. Therefore, hydrostatic culture is first adopted after the seedlings are collected, and micro-flowing water (surface flow velocity is 5 cm/s or less) after 24 hours, and normal flow after 72 hours (surface layer). Flow rate 5 to 10 cm/s). In this way, the cells can both grow normally and avoid large losses with water.

2. Although the pseudo-roots were found in the 4 to 8 rows of cells that were commonly used to wash seedlings, some of them were not transformed into sporozoites and some of the sporozoites were in 1 to 2 rows of cells. The false roots were not obvious. If scrubbed at this time, gametophyte and microspores must fall off. If the sporophyte body is washed to a length of 1 mm, some seedlings will not be cleared in time, which will affect the light and make the stem weak and gradually fall off, and affect the robustness of other seedling stems. Therefore, the best time to start scrubbing is when the sporophyte body grows to 8 to 16 rows of cells. When the length of the seedling is about 0.5 mm, the false root is obvious and firmly attached. Washing at this time will not only reduce the unnecessary loss of gametophyte and young spores, but will also promote the normal healthy growth of seedlings.

When the sporozoites grow to 8 to 16 rows of cells, they begin to wash with a smaller pressure of about 0.5 kg/cm2, gradually increasing the intensity and frequency of washing as the growth and adhesion of young sporophytes increases.

3. Keep the temperature of the water at 5~6°C during the seedling raising period, and do not exceed 15°C during the still water period, and 7～8°C during the flowing period.

4. Light photo gametes before 8 rows of cells, high light 3000m candle, average light 1700-1800m candle; 8 cells to 0.3mm, high light 3300m candle, average light 1800-1900m candle; 0.3-3mm, high light 3600 Meter candle, average light 1900 to 2000 meters candle; 3 to 5 mm, high light 4000 meter candle, average light 2100 to 2200 meters candle; 5 to 10 mm, high light 4500 m candle, average light 2200 to 2400 meters candle; 10 mm or more , high light more than 5,000 meters candle.

5. Before the new water seedling size is 5 millimeters, add 1/3 of new water daily. After the seedling size is 5 millimeters, add 1/2 of fresh water daily.

6. Nutrient salt seedling size 5 mm before, sodium nitrate - nitrogen: 3 parts per million, potassium dihydrogen phosphate - phosphorus: 3 parts per million, ferric citrate - iron: 2 parts per million; seedling size 5 mm after , Sodium Nitrate-N: 4ppm, Potassium Dihydrogen Phosphate-Phosphorus: 40%.

IV. Summary and discussion

Compared with traditional nursery methods, asexual propagation techniques have not been able to harvest seedlings because of a number of techniques. However, this does not mean that the effect of the nursery will not reach the effect of the spore sport seedlings. The production potential of kelp seedlings is relatively large and there is a certain margin for the quality of seedlings. Corresponding technical measures after seedling collection are crucial and can compensate for the lack of quality of seedlings. Under the implementation of the above-mentioned series of technical measures, there are two There is no significant difference in the seedling raising effect.

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