Rapid acquisition method of purple menu spore seedlings

Abstract

The invention discloses a method for rapidly acquiring spore seedlings of meniscus violaceum, and relates to the field of asexual reproduction and breeding of laver. The method comprises the following steps: crushing laver to different degrees by using a wall breaking machine, crushing thalli into fragments with certain sizes, collecting the fragments, culturing the fragments for several days, and adding seedling ropes after the fragments develop into monosporangiums and start to diffuse monospores. The seedling ropes and the fragments are mixed and subjected to aerobic culture, and a large number of monospore seedlings can be obtained. According to the mechanical crushing method, a manual chopping crushing method can be replaced, the crushing efficiency of the laver is greatly improved, and large-scale induction and diffusion of the purple menu spores can be realized. Meanwhile, by conducting joint aerobic culture on the fragments and the seedling ropes, the step of collecting the monospore seedlings can be simplified, a large number of monospores are effectively attached to the seedling ropes and rapidly develop into the monospore seedlings, and large-scale monospore seedling culture becomes possible.

Description

Technical Field

[0001] The invention relates to asexual reproduction and breeding technology of laver, and in particular to a method for quickly obtaining laver monospore seedlings. Background Art

[0002] China is the world's largest laver producer, and laver farming plays a pivotal role in my country's aquaculture economic system. As people pursue higher nutritional quality of food, the laver consumer market is growing rapidly at a rate of more than 10% per year as a healthy food with high nutritional value. The laver seedling raising process is one of the key links in laver production, which can directly determine the quality and yield of cultivated laver.

[0003] At present, the common method of sexual reproduction is used to grow seedlings of laver, that is, using conchospores released by conchospores to provide seedling sources. However, there are three main problems with conchospore seedling cultivation: first, the seedling cultivation period is as long as 6-8 months, the maintenance cost is high, and diseases are prone to occur during this period; second, a large number of conchospores need to be cultivated, which consumes manpower and material resources; third, the seedling cultivation effect is unstable, and the cultivation rate of high-quality seedling nets is usually only 30% or even less, and the economic benefits are unstable.

[0004] Monospores originate from the vegetative cells of the thallus of Porphyra and reproduce asexually. They can directly develop into the thallus of Porphyra without going through the filamentous stage. Compared with shell spores, monospores grow rapidly and have a high germination rate. Monospores can be released from frozen Porphyra after recovery, and are not restricted by conditions such as filamentous shell drilling and swelling. After appropriate stimulation, monospores can be formed and released within 6 to 8 days, which can greatly reduce the time for Porphyra seedling cultivation. At the same time, the asexual reproduction characteristics of monospores can also highly maintain the excellent traits of Porphyra varieties. Since the release of monospores is affected by multiple factors such as light, tides and abiotic stress, the production of monospore seedlings is unstable. Compared with traditional shell filamentous seedlings, monospore seedling cultivation lacks efficient and rapid methods for large-scale acquisition of seedlings.

[0005] Currently, the main method used to break up laver is manual chopping to make the laver fragments reach a certain size to release single spores. This method can only be used to process a small amount of laver samples. Processing a large number of samples is too time-consuming and labor-intensive, and it is difficult to achieve large-scale single spore seedling production. Summary of the invention

[0006] Purpose of the invention: In order to solve the above problems, the present invention provides a method for quickly obtaining purple monospore seedlings to make up for the shortcomings of the prior art.

[0007] Technical solution: A method for quickly obtaining purple monospore seedlings, comprising the following steps:

[0008] (1) Preparation of laver fragments: selecting laver with good growth, breaking it with a wall breaking machine, and obtaining laver fragments;

[0009] (2) Collection of fragments: Filter the laver fragments using sieve silk, discard the filtrate, and collect the laver fragments;

[0010] (3) Fragment culture: The collected laver fragments are subjected to aeration culture to allow the fragment cells to develop into single spore sacs;

[0011] (4) Inoculating the seedling rope: When the monosporangium begins to release monospores, add the seedling rope and mix and culture for several days to obtain monosporic seedlings.

[0012] The method for quickly obtaining porphyra monospore seedlings comprises the following steps: in step (1), the porphyra is broken into pieces for 1-7 minutes by a wall-breaking machine, and ice is added every 0.3-0.7 minutes during the breaking process to prevent the water temperature from being overheated. Preferably, in step (1), the porphyra is broken into pieces for 1-5 minutes by a wall-breaking machine, and ice is added every 0.5 minutes during the breaking process.

[0013] In the method for quickly obtaining monospore seedlings of Porphyra, the Porphyra selected in step (1) is a Porphyra thallus.

[0014] In the method for quickly obtaining laver monospore seedlings, the sieve silk used for collecting laver fragments in step (2) is 300-600 mesh, and the collected fragments are rinsed with clean seawater.

[0015] The method for rapidly obtaining purple monospore seedlings, in step (3), the culture illumination of the fragments is 50-70 μmol m - 2 s -1 , the temperature is 10-15℃, and the photoperiod is light:dark = 10-14h:10-14h.

[0016] The method for rapidly obtaining purple monospore seedlings, step (4) specifically comprises adding seedling ropes after the fragmented cells develop into monospore sacs and begin to release monospores, mixing and ventilating and culturing for several days to obtain monospore seedlings attached to the seedling ropes.

[0017] The method for quickly obtaining purple monospore seedlings comprises the following steps: the mixed aeration culture is performed for 3 to 6 days.

[0018] The method for quickly obtaining monospore seedlings of Porphyra, wherein the Porphyra is selected from Porphyra yezoensis.

[0019] In the method for quickly obtaining purple monospore seedlings, the ventilation is air.

[0020] In the method for quickly obtaining purple mesospore seedlings, the power of the wall breaking machine is 2000-2500W.

[0021] Preferably, in (1), the wall breaking machine performs breaking for 1-5 minutes, and seawater ice is added once every half minute of breaking.

[0022] Preferably, the fragments are collected using a 500-mesh sieve in (2), and the fragments are rinsed at least three times with sterilized seawater.

[0023] Preferably, the culture conditions of the fragments in (3) are: the culture medium is PES, the culture temperature is 10-15°C, and the light intensity is 50-70 μmol m -2 s -1 , aeration culture.

[0024] Preferably, the fragments and seedling ropes in (4) are co-cultured for 3-6 days under the following culture conditions: the culture medium is PES, the culture temperature is 10-15°C, and the light intensity is 50-70 μmol m -2 s -1 , aeration culture.

[0025] Beneficial effects: Compared with the prior art, the present invention has the following advantages: (1) Compared with the traditional manual crushing method, the present invention adopts mechanized means to greatly save manpower and crushing time, improve crushing efficiency, and is more conducive to large-scale production of monospore seedlings. (2) Compared with the previous method of collecting single spores after releasing the fragments and then collecting seedlings, the present invention co-cultivates the fragments with the seedling rope, which can simplify the single spore seedling collection process. (3) Compared with the static culture method, the ventilation culture is conducive to the attachment of single spores to the seedling rope, increasing the amount of seedlings collected. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is the result of microscopic observation after the wall breaking machine was broken for 1 minute;

[0027] Figure 2 This is the result of microscopic observation after the wall was broken by a wall breaking machine for 2 minutes;

[0028] Figure 3 This is the result of microscopic observation after the wall was broken by a wall breaking machine for 3 minutes;

[0029] Figure 4 This is the result of microscopic observation after the wall was broken by a wall breaking machine for 4 minutes;

[0030] Figure 5 This is the result of microscopic observation after the wall was broken by a wall breaking machine for 5 minutes;

[0031] Figure 6 This is a comparison chart of cell death rates after different fragmentation times;

[0032] Figure 7 The purple monospore seedlings grown for 28 days after inoculation in the embodiment;

[0033] Figure 8This is a comparison chart of the number of seedlings inoculated under different ventilation and static treatments. DETAILED DESCRIPTION

[0034] The technical solution of the present invention is further described below in conjunction with embodiments.

[0035] The wall breaking machine is kormes model WK-05, 2200W. The ventilation equipment is a 35W ventilation pump equipped with a 6-hole diverter.

[0036] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources. Example 1, aeration culture steps:

[0037] (1) Weigh 1 g of Porphyra yezoensis as one group, and weigh five groups in total. Put one group of Porphyra yezoensis into a wall-breaking machine filled with 500 mL of sterilized seawater for crushing. Add ice every half minute during the crushing process to prevent the Porphyra from being damaged by overheating. Treat the five groups of Porphyra yezoensis with the wall-breaking machine for 1, 2, 3, 4, and 5 minutes, respectively, to obtain Porphyra yezoensis fragments with different crushing times;

[0038] (2) Collect each group of fragments using a 500-mesh silk sieve and rinse them several times with clean seawater. Weigh 0.2 g of laver fragments from each group.

[0039] (3) placing the weighed fragments into a ventilation bottle filled with culture medium and culturing them in an incubator to allow them to develop into single spore sacs;

[0040] (4) On the sixth day of culture, the fragments began to release single spores one after another. At this time, sterilized cotton ropes with a length of 5 cm and a diameter of 0.1 cm were added, 6 in each group, and aerated and cultured together with the fragments to allow the released single spores to adhere to the seedling ropes;

[0041] (5) On the tenth day of cultivation, the fragments were removed and the seedling ropes were retained, and the seedling ropes were cultured separately with ventilation.

[0042] The culture conditions in step (3-5) are as follows: the culture medium is sterile seawater with PES culture medium added, the temperature is 10°C, the light intensity is 60 μmol m -2 s -1 , photoperiod 12h / 12h (day / night), and culture medium was replaced every three days.

[0043] Experimental results:

[0044] 1. Microscopic observation of cell death rate in debris

[0045] In order to explore the optimal time for the wall-breaking machine to break the thallus, the present invention respectively carried out five different time (1, 2, 3, 4, 5 minutes) breaking treatment on laver. After the breaking process of step (1) was completed, the fragments were fully mixed, a small amount of fragments were randomly sampled, and the images under a fixed field of view were recorded using a microscope (in order of Figures 1 to 5 ), and finally the mortality rate of fragment cells in each group was statistically analyzed. Figure 6 As shown: As the fragmentation time increases, the mortality rate of fragmented cells also increases.

[0046] Death rate = (number of dead cells / total number of cells) × 100%

[0047] 2. Development of seedlings inoculated on seedling ropes

[0048] On the 28th day, the seedlings on the seedling ropes were observed under a microscope and it was found that the seedlings inoculated into the seedling ropes were growing and developing healthily (e.g. Figure 7 ).

[0049] 3. Number of seedlings inoculated on the seedling rope

[0050] On the 28th day of cultivation, the seedlings on the seedling ropes were counted. The statistical data showed that the seedling ropes inoculated with fragments treated by the wall-breaking machine for 1 to 5 minutes had an inoculation density of approximately 117.2 seedlings / cm 2 , 268.2 seedlings / cm 2 , 270.4 seedlings / cm 2 , 150.3 seedlings / cm 2 , 50.7 seedlings / cm 2 (like Figure 8 ).

[0051] Comparative Example 1 A method similar to Example 1, the only difference being that the culture method of the fragments and the seedling ropes in step (3-4) is static culture.

[0052] Experimental results:

[0053] 1. Number of seedlings inoculated on the seedling rope

[0054] On the 28th day of cultivation, the seedlings on the seedling ropes were counted. The statistical data showed that the seedling ropes inoculated with fragments treated by the wall-breaking machine for 1 to 5 minutes had an inoculation density of approximately 2.12 seedlings / cm 2 , 2.33 seedlings / cm 2 , 1.91 seedlings / cm 2 , 1.27 seedlings / cm 2 , 1.06 seedlings / cm 2 (like Figure 8 ).

[0055] After comprehensive analysis of the results, it was found that compared with the static culture in Comparative Example 1, the aeration culture in Example 1 significantly increased the number of seedlings inoculated (e.g. Figure 8 ). Therefore, the method for quickly obtaining monospore seedlings is as follows: after the thallus of Porphyra yezoensis is broken by a wall breaking machine for 2-3 minutes, the fragments are collected and washed several times, and then the fragments and seedling ropes are aerated and cultured to obtain a large number of monospore seedlings.

Claims

1. A method for quickly obtaining monospore seedlings of purple violet, characterized in that: The following steps are involved: (1) Preparation of laver fragments: selecting laver with good growth, breaking it with a wall breaking machine, and obtaining laver fragments; (2) Collection of fragments: Filter the laver fragments using sieve silk, discard the filtrate, and collect the laver fragments; (3) Fragment culture: The collected laver fragments are subjected to aeration culture to allow the fragment cells to develop into single spore sacs; (4) Inoculating the seedling rope: When the monosporangium begins to release monospores, add the seedling rope and mix and culture for several days to obtain monosporic seedlings.

2. The rapid acquisition method of purple monospore seedlings as claimed in claim 1, characterized in that, In step (1), the laver is broken into pieces by a wall-breaking machine for 1-7 minutes, and ice is added every 0.3-0.7 minutes during the breaking process to prevent the water temperature from being overheated.

3. The rapid acquisition method of purple monospore seedlings as claimed in claim 1, characterized in that, The laver selected in step (1) is laver thallus.

4. The rapid acquisition method of purple monospore seedlings as described in claim 1 is characterized in that, In step (2), the laver fragments are collected using a sieve of 300-600 mesh, and the collected fragments are rinsed with clean seawater.

5. The rapid acquisition method of purple monospore seedlings as described in claim 1 is characterized in that, Step (3) The fragments were cultured at an illumination of 50-70 μmol m -2 s -1 , the temperature is 10-15℃, and the photoperiod is light:dark = 10-14h:10-14h.

6. The rapid acquisition method of purple monospore seedlings as described in claim 1 is characterized in that, Step (4) specifically includes adding a seedling rope after the fragmented cells develop into a monospore sac and begin to release monospores, mixing and aerating and culturing for several days to obtain monospore seedlings attached to the seedling rope.

7. The rapid acquisition method of purple monospore seedlings as described in claim 6 is characterized in that, The mixed aeration culture is carried out for 3-6 days.

8. The rapid acquisition method of purple monospore seedlings as described in claim 1 is characterized in that, The laver is selected from Porphyra yezoensis.

9. The rapid acquisition method of purple monospore seedlings as described in claim 1 is characterized in that, The ventilation is the ventilation of air.

10. The rapid acquisition method of purple monospore seedlings as described in claim 1 is characterized in that, The power of the wall breaking machine is 2000-2500W.

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