Good seedling raising method for Porphyra haitanensis

Abstract

The present invention relates to the technical field of laver cultivation, and particularly to a method for breeding fine varieties of Pyropia haitanensis. Through the improvement of the seedling cultivation device, since the two ends of the hanging rope are respectively wound and connected between the first support rod and the second support rod, and are suspended in a U shape, and since shells are hung on the hanging rope, in the first state, the third gear meshes with the first gear and the second gear respectively. By driving the third gear to rotate, the first support rod and the second support rod can be driven to rotate synchronously and in the same direction, thereby controlling the movement of the hanging rope. When the first support rod and the second support rod rotate synchronously and in the same direction, the movement position of the shells on the hanging rope can be controlled. By regularly controlling the forward and reverse rotation of the third gear, the automatic turning of the shells can be realized, and there is no need to manually turn the hanging rope regularly, ensuring that each shell can receive uniform light intensity, thereby further improving the seedling cultivation quality of the seedlings.

Description

Technical Field

[0001] The present invention relates to the technical field of laver cultivation, and particularly relates to a method for breeding high-quality seedlings of Pyropia haitanensis. Background Art

[0002] Pyropia haitanensis is one of the two main species of artificially cultivated laver in China. It is native to the coastal areas of Fujian in China and is a unique warm-temperate species in China. Since the successful full artificial cultivation in the 1960s of the last century, the cultivation area of Pyropia haitanensis has been continuously expanding. Currently, it has become one of the main objects of seawater aquaculture in the southern coastal areas of China, and its output accounts for more than 75% of the total laver output in the country, creating extremely considerable economic benefits. With the development of the Pyropia haitanensis industry, it is necessary to breed high-quality seedlings of Pyropia haitanensis and improve the quality of seedling cultivation. Summary of the Invention

[0003] The technical problem to be solved by the present invention is: it is urgent to improve the method for breeding high-quality seedlings of Pyropia haitanensis and enhance the quality of seedling cultivation of Pyropia haitanensis.

[0004] To solve the above technical problem, the technical solution adopted by the present invention is as follows:

[0005] A method for breeding high-quality seedlings of Pyropia haitanensis, comprising the following steps:

[0006] S1: Select wild purebred algae from the open sea and conduct pretreatment;

[0007] S2: Enzymatically hydrolyze the pretreated parent algae to obtain single cells;

[0008] S3: Conduct somatic cell culture on the single cells to obtain filamentous somatic cell seedlings;

[0009] S4: Purify and rejuvenate the filamentous somatic cell seedlings to obtain pure filamentous Pyropia haitanensis;

[0010] S5: The obtained pure filamentous Pyropia haitanensis can be used for primary preservation and secondary preservation respectively after primary amplification and secondary amplification, and a germplasm resource bank of Pyropia haitanensis is established;

[0011] S6: Arrange shells in the seedling pond, cut the homogenate of filamentous somatic cell seedlings of Pyropia haitanensis into algal segments with a size of 0.045 - 0.055 mm, dilute with seawater, and evenly spray it in the pond with a sprayer. The algal segments will attach to the bottom of the pond or the hanging shells to form conchospores;

[0012] S7: Collect conchospores for seeding, transplant them to the raft frame, and conduct thallus culture. During the thallus cultivation period, select the second-generation high-quality carpospores and prepare carpospore water;

[0013] S8: Arrange shells in the seedling pond, spray the carpospore water on the shells, and the carpospores will attach to the shells and grow;

[0014] S9: Collect carpospores for seeding, transplant them to the raft frame, and conduct thallus cultivation. During the thallus cultivation period, select the second-generation high-quality carpospores and prepare carpospore water;

[0015] S10: Arrange shells in the nursery pond, spray the carpospore water on the shells, and the carpospores will attach to the shells and grow;

[0016] Repeat S9 and S10 until high-quality carpospores are screened out;

[0017] The seeding method is based on a seeding device, which includes the nursery pond and shells mentioned in S6, S8, and S10, and also includes multiple groups of support mechanisms; the support mechanism includes a first support rod, a second support rod, a hanging rope, a first gear, a second gear, a U-shaped support, a base, a third gear, and a driving motor;

[0018] The shape of the nursery pond is rectangular;

[0019] The shapes of the first support rod and the second support rod are both cylindrical;

[0020] Each group of the support mechanisms includes 4 U-shaped supports, which are arranged on the upper parts of the two side walls in the width direction of the nursery pond. The U-shaped supports are used to support the first support rod and the second support rod. The first gear is coaxially sleeved at one end of the first support rod, and the second gear is coaxially sleeved at one end of the second support rod;

[0021] The third gear is connected to the base in a liftable and rotatable manner, and the driving motor is in transmission connection with the third gear for driving the third gear to rotate;

[0022] One end of the hanging rope is wound and connected to the first support rod, and the other end is wound and connected to the second support rod. The shells are hung on the hanging rope at equal intervals along the extension direction of the hanging rope;

[0023] In the first state, the first support rod and the second support rod are horizontally arranged on the U-shaped supports on both sides of the nursery pond in parallel. The third gear meshes with the first gear and the second gear respectively, and the hanging rope is suspended in a U shape between the first support rod and the second support rod;

[0024] In S6, S8, and S10, the steps all include: in the first state, regularly control the driving motor to rotate forward, drive the first gear and the second gear to rotate in the same direction through the third gear, so that the hanging rope between the first support rod and the second support rod moves, and make the shells all located on the X-direction side of the hanging rope; after a preset time period, control the driving motor to rotate backward, drive the first gear and the second gear to rotate in the same direction through the third gear, so that the hanging rope between the first support rod and the second support rod moves, and make the shells all located on the reverse side of the X-direction of the hanging rope.

[0025] Further, in the above-mentioned method for breeding high-quality seedlings of Porphyra haitanensis, the first support rod and the second support rod are both made of fiberglass.

[0026] Further, in the above-mentioned method for breeding high-quality seedlings of Porphyra haitanensis, the first gear, the second gear and the third gear are all made of nylon.

[0027] Further, in the above-mentioned method for breeding high-quality seedlings of Porphyra haitanensis, the support mechanism further includes a lifting cylinder and a lifting seat. The lifting seat is connected with a bearing seat, and the third gear is connected to the bearing seat through a rotating shaft; the driving motor is connected to the lifting seat, and the driving cylinder is connected between the base and the lifting seat for driving the lifting seat to move in the vertical direction.

[0028] Further, in the above-mentioned method for breeding high-quality seedlings of Porphyra haitanensis, in S1, the steps for pretreatment are specifically as follows: The parent thallus is immersed in disinfected seawater, healthy thalli are selected, the marginal and apical carposporangia and holdfast parts are cut off, the cut leaves are disinfected, decontaminated, and added with NO 3- N 10PPM, PO4 3- -P 1PPM., the water is changed 1-2 times a day to allow the thalli to fully recover; then the leaves are cut into small pieces of 1-2 mm, washed in disinfected seawater until the supernatant is colorless, the water is blotted dry, then rinsed twice with 1 mol / L glucose solution, the water is blotted dry, and stored at -20 °C;

[0029] The parent thallus is taken out from the -20 °C environment and immediately put into disinfected seawater with a temperature of 10-20 °C. After the leaves are unfolded, healthy thalli with normal color are selected, washed with disinfected seawater, and the miscellaneous algae and attached mud on the surface of the thalli are removed again. While being cleaned, the parts with carpospores, spermatangia and rhizoids at the upper end of the Porphyra are cut off cleanly with a blade, then soaked in antibiotics for 5-7 min, washed 3 times with disinfected seawater, and then put into disinfected seawater containing nitrogen and phosphorus nutrient solution for static culture for 4 d. First, illuminate with a light intensity of 1000 Lx for 1 d, then increase the light intensity to 2000 Lx, the light cycle is 12L∶12D, and the culture temperature is set at 18-20 °C. Stir the seawater during the culture process to increase gas exchange.

[0030] Further, in the above-mentioned method for breeding high-quality seedlings of Porphyra haitanensis, in S2, the steps for obtaining single cells by enzymolysis of the pretreated parent thallus are specifically as follows:

[0031] Fresh corolla moon snails were selected as extraction materials. The material animals were first rinsed with seawater, starved for 2 days, the shells were gently broken, the digestive tract and internal organs were removed, and a mixed solution 4 times heavier than the tissue was added, wherein the mixed solution contained 0.3M KC1:0.15MMgSO4:pH7.0 phosphate buffer, and the mixture was homogenized in a pounder, and the homogenate was centrifuged at 0°C using a refrigerated centrifuge, and the supernatant, i.e., the crude enzyme solution, was taken and stored at -20°C; the -20°C conch enzyme solution was thawed, divided into round-bottom flasks, first frozen at -20°C, and then continuously dried at -27°C using a freeze dryer for 24 hours until it became dry powder, and stored at -20°C;

[0032] Prepare enzyme solution in the proportion of 10 ml for 1g of treated algae to weigh enzyme powder, put the weighed enzyme powder into the measured enzyme solvent and stir it thoroughly to dissolve the enzyme powder, adjust the pH value to 5.8-6.2; filter and disinfect the prepared enzyme solution using a microporous filter with a pore size of 0.22μm;

[0033] Select the algae from the previously treated seed algae and wash them with sterilized seawater. After filtering out the seawater, use a sterilized knife or scissors to cut the algae into 1-2 mm pieces. 2 The small pieces were washed with seawater, and then washed and filtered with 1 mol / L glucose solution to remove the chopped cytoplasm in order to avoid affecting the enzymatic hydrolysis effect. The small pieces were dried with washing paper and set aside for later use.

[0034] Place the treated algae in the prepared enzyme solution on a shaker at 31-32°C and 100 rpm / min in the dark.

[0035] When no algae blocks can be seen under the fluorescence microscope, the proportion of single cells has increased significantly, and the cell clusters are 2-3 cells connected together with a low proportion, the enzymatic hydrolysis is stopped.

[0036] Furthermore, in the above-mentioned method for raising improved varieties of Porphyra haitanensis seedlings, in said S3, single cells are subjected to somatic cell culture to obtain conchoidal somatic cell seedlings specifically by:

[0037] After the enzymatic hydrolysis is completed, add MES culture medium 4 times the amount of enzyme solution, mix and filter with 80um sieve silk, centrifuge the filtrate, discard the supernatant after 2 minutes, add MES culture medium with salt, shake well and centrifuge, repeat this washing 3 times, finally collect the cells at the bottom of the centrifuge tube and divide them into 3 50ml conical flasks to obtain culture medium No. 1, culture medium No. 2 and culture medium No. 3 respectively;

[0038] The MES culture medium is obtained by diluting the MES stock solution 50 times with seawater of the corresponding specific gravity; the MES stock solution is prepared from the following raw materials: 1.4 g of sodium nitrate; 0.2 g of sodium glycerophosphate; 2 g of tris (hydroxymethyl) aminomethane; 100 ml of MPII; 400 ml of H2O; pH 7.6;

[0039] The MPII stock solution is prepared from the following raw materials by weight: 1.5 g of EDTA-Na2; 1.25 g of boric acid; 0.175 g of MnCl2·4H2O; 62.5 mg of FeCl3·6H2O; 12.5 mg of ZnCl2; 5 mg of CoCl·6H2O; 300 mg of ferric citrate; 500 ml of H2O;

[0040] The collected somatic cells are allowed to stand still for about 2 hours and then 3# culture medium is added for culturing in a constant temperature incubator;

[0041] The freshly enzymolyzed somatic cells are placed in the dark for 1 day, the light intensity is 10001x on the second day, the light intensity increases to 2000 lx on the third day, and the light intensity remains at 2500 lx after the fourth day. The photoperiod is 12L:12D;

[0042] For 1 to 6 days of somatic cell culture, 3# MES culture medium is used. On the 7th day after culture, it is changed to MES culture medium, and then the culture medium is changed every 7 - 10 days. At the beginning of culture, following the principle of gradually decreasing density, the initial culture density is maintained at about 1.030 g / cm 3 After about 10 h, the density decreases to about 1.027 g / cm 3 After another 12 h, the density drops to about 1.025 g / cm 3 After another 12 h, the density remains at about 1.023 g / cm 3 or so;

[0043] The density of cell culture: 1.0 - 1.2×10 4 cells / ml.

[0044] Furthermore, in the above-mentioned method for breeding improved varieties of Porphyra haitanensis, in S5, the obtained pure Porphyra haitanensis filaments can be used for primary preservation and secondary preservation respectively after primary amplification and secondary amplification. The specific method for establishing the Porphyra haitanensis germplasm resource bank is as follows:

[0045] Primary amplification: The pure filaments are chopped into pieces of 100 - 500 μm with a tissue grinder sterilized at high temperature, and then placed in a 500 - ml conical flask for static culture in the dark for 1 day. On the second day, 200 - 300 ml of the culture medium is replaced and then cultured under normal light with static culture. The culture concentration is about 1 g of wet - weight free filaments per 100 ml of the culture medium. During the culture period, 50 - 100 ml of the culture medium is replaced every 30 days;

[0046] Secondary amplification: The pure filamentous bodies obtained after primary amplification are chopped into pieces of 100 - 500 μm with a tissue grinder sterilized at high temperature, and then placed in a 2000 ml conical flask for static culture in the dark for 1 day. On the second day, after replacing 1200 - 1500 ml of the culture medium, normal light is restored for static culture. After 3 - 5 days, an aeration tube is added for aeration culture. The air needs to be filtered through three processes to ensure that the air entering the flask is pollution-free. The culture concentration is 2 - 3 g of wet-weight free filamentous bodies per 100 ml of the culture medium. If the density of the filamentous bodies exceeds 3 g / 100 ml of the culture medium, sub-culture is carried out in a timely manner. During the culture period, 1000 - 1500 ml of the culture medium is replaced every 10 days. The culture medium and culture conditions are the same as those in the primary culture.

[0047] The beneficial effects of the present invention are as follows: Through the pretreatment, enzymatic hydrolysis treatment, and somatic cell culture of the pure seaweed of Yetan in the open sea, filamentous somatic cells are obtained. Then, through primary amplification and secondary amplification, they are respectively used for primary preservation and secondary preservation to establish a germplasm resource bank of Pyropia haitanensis. Through a specific seedling-raising device for seedling culture to form conchospores, and then the conchospores are collected and transplanted onto the raft frame for thallus culture. During the thallus culture period, the second-generation excellent gonidia are selected, gonidia water is prepared, and sprayed on the shells in the seedling-raising pond for seedling culture. The excellent gonidia are continuously screened, so as to improve the seedling-raising quality of Pyropia haitanensis seedlings and avoid the decline of the excellent traits of the improved seeds.

[0048] Through the improvement of the seedling-raising device, since the two ends of the hanging rope are respectively wound and connected between the first support rod and the second support rod and are suspended in a U shape, and since the shells are hung on the hanging rope, in the first state, the third gear meshes with the first gear and the second gear respectively. By driving the third gear to rotate, the first support rod and the second support rod can be driven to rotate synchronously and in the same direction, thereby controlling the movement of the hanging rope. When the first support rod and the second support rod rotate synchronously and in the same direction, the moving position of the shells on the hanging rope can be controlled. By timing the forward and reverse rotation of the third gear, the automatic turning of the shells can be realized. For example, within a certain period of time, by controlling the third gear to drive the first gear and the second gear to rotate in the same direction, the hanging rope between the first support rod and the second support rod moves, so that the shells are all located on the X-side of the hanging rope; after a preset period of time, the driving motor is controlled to reverse, and through the third gear, the first gear and the second gear are driven to rotate in the same direction, so that the hanging rope between the first support rod and the second support rod moves, so that the shells are all located on the reverse X-side of the hanging rope, and there is no need for manual regular turning of the hanging rope, ensuring that each shell can receive uniform light intensity; thereby further improving the seedling-raising quality of the seedlings. Description of the Drawings

[0049] Figure 1 It is a schematic structural diagram of the seedling-raising device related to the specific embodiment of the present invention;

[0050] Figure 2 isFigure 1 Enlarged view of part A;

[0051] Label description:

[0052] 1. Seedling-raising pond;

[0053] 2. Shell;

[0054] 3. Support mechanism; 31. First support rod; 32. Second support rod; 33. Hanging rope; 34. First gear; 35. Second gear; 36. U-shaped support; 37. Third gear; 38. Driving motor; 39. Lifting cylinder; 310. Lifting seat. Specific implementation mode

[0055] To describe in detail the technical content, achieved purpose and effect of the present invention, the following is described in conjunction with the implementation mode and with reference to the drawings.

[0056] The specific implementation mode of the present invention relates to a method for breeding high-quality seedlings of Porphyra haitanensis, including the following steps:

[0057] S1: Select wild Porphyra haitanensis from the open sea for pretreatment;

[0058] S2: Enzymatically hydrolyze the pretreated parent algae to obtain single cells;

[0059] S3: Carry out somatic cell culture on the single cells to obtain filamentous somatic cell seedlings;

[0060] S4: Purify and rejuvenate the filamentous somatic cell seedlings to obtain pure filamentous Porphyra haitanensis;

[0061] S5: The obtained pure filamentous Porphyra haitanensis can be used for primary preservation and secondary preservation respectively after primary amplification and secondary amplification, and a germplasm resource bank of Porphyra haitanensis is established;

[0062] S6: Arrange the shells 2 in the seedling-raising pond 1, cut the homogenate of the somatic filament seedlings of Porphyra haitanensis into algal segments of 0.045 - 0.055 mm, dilute with seawater and evenly spray it in the pool with a sprayer. The algal segments will attach to the bottom of the pool or the hanging shells 2 to form conchospores;

[0063] S7: Collect conchospores for seeding, transplant them to the raft frame for thallus culture. During the thallus cultivation period, select the second-generation high-quality carpospores and prepare carpospore water;

[0064] S8: Arrange the shells 2 in the seedling-raising pond 1, spray the carpospore water on the shells 2, and the carpospores will attach to the shells 2 and grow;

[0065] S9: Collect carpospores for seeding, transplant them to the raft frame for thallus culture. During the thallus cultivation period, select the second-generation high-quality carpospores and prepare carpospore water;

[0066] S10: Arrange the shells 2 in the seedling-raising pond 1, spray the carpospore water on the shells 2, and the carpospores will attach to the shells 2 and grow;

[0067] Repeat S9 and S10 until excellent carpospores are screened out;

[0068] Refer to Figures 1 to 2 , the seedling-raising method is based on a seedling-raising device, the seedling-raising device includes the seedling-raising pond 1 and the shells 2 mentioned in S6, S8 and S10, and also includes multiple groups of support mechanisms 3; the support mechanism 3 includes a first support rod 31, a second support rod 32, a hanging rope 33, a first gear 34, a second gear 35, a U-shaped support 36, a base, a third gear 37 and a driving motor 38;

[0069] The shape of the seedling-raising pond 1 is rectangular;

[0070] The shapes of the first support rod 31 and the second support rod 32 are both cylindrical;

[0071] Each group of the support mechanisms 3 includes 4 U-shaped supports 36, the U-shaped supports 36 are arranged on the upper parts of the two side walls in the width direction of the seedling-raising pond 1, the U-shaped supports 36 are used to support the first support rod 31 and the second support rod 32, the first gear 34 is coaxially sleeved on one end of the first support rod 31, and the second gear 35 is coaxially sleeved on one end of the second support rod 32;

[0072] The third gear 37 is connected to the base in a liftable and rotatable manner, and the driving motor 38 is in transmission connection with the third gear 37 for driving the third gear 37 to rotate;

[0073] One end of the hanging rope 33 is wound and connected to the first support rod 31, and the other end is wound and connected to the second support rod 32, and the shells 2 are hung on the hanging rope 33 at equal intervals along the extending direction of the hanging rope 33;

[0074] In the first state, the first support rod 31 and the second support rod 32 are horizontally arranged on the U-shaped supports 36 on both sides of the seedling-raising pond 1 in parallel, the third gear 37 meshes with the first gear 34 and the second gear 35 respectively, and the hanging rope 33 is suspended in a U shape between the first support rod 31 and the second support rod 32;

[0075] In S6, S8, and S10, the steps are as follows: In the first state, the driving motor 38 is periodically controlled to rotate forward, driving the first gear 34 and the second gear 35 to rotate in the same direction through the third gear 37, causing the hanging rope 33 between the first support rod 31 and the second support rod 32 to move, so that the shells 2 are all located on the X-direction side of the hanging rope 33; after a preset time period, the driving motor 38 is controlled to rotate reversely, driving the first gear 34 and the second gear 35 to rotate in the same direction through the third gear 37, causing the hanging rope 33 between the first support rod 31 and the second support rod 32 to move, so that the shells 2 are all located on the reverse X-direction side of the hanging rope 33.

[0076] On the one hand, by performing pretreatment, enzymatic hydrolysis treatment, and somatic cell culture on the wild purebred algae in the open sea, filamentous somatic cells are obtained. Then, through primary amplification and secondary amplification, they are respectively used for primary preservation and secondary preservation to establish a germplasm resource bank of Porphyra haitanensis. Through a specific seedling-raising device, seedlings are cultured to form conchospores, and then the conchospores are collected and transplanted to a raft frame for thallus culture. During the thallus culture period, the second-generation superior conchocelis are selected, conchospore water is prepared, and sprayed on the shells 2 in the seedling-raising pond 1 for seedling-raising culture, and superior conchospores are continuously screened, thereby being able to improve the seedling-raising quality of Porphyra haitanensis seedlings.

[0077] On the other hand, because Porphyra spores or filaments have the habit of "penetrating the shell" during the seedling-raising process, in actual production, shells 2 are mostly used as the breeding carrier for Porphyra original seeds. For example, in the three-dimensional hanging breeding method, bamboo poles are placed on the upper part of a pool filled with water, and multiple hanging ropes 33 with strings of shells 2 are suspended from the bamboo poles. By cutting the somatic filamentous seedlings of Porphyra haitanensis into algal segments of 0.045 - 0.055 mm and spraying them into the pool, the algal segments will attach to the bottom of the pool or hang on the shells 2.

[0078] Since the light intensity is an important factor affecting the growth and development of Porphyra filaments during the seedling-raising process, in the prior art, in order to improve the seedling-raising quality, it is necessary to periodically turn around the hanging ropes 33 with strings of shells 2 suspended from the bamboo poles, so that the light and oxygen received by each shell 2 on the hanging ropes 33 are close to balance. During the seedling-raising process of Porphyra filaments, it is necessary to turn the hanging ropes 33 back and forth many times, which requires a large amount of labor costs.

[0079] In the seedling raising method of the present invention, through the improvement of the seedling raising device, since both ends of the hanging rope 33 are respectively wound and connected between the first support rod 31 and the second support rod 32 and are suspended in a U shape, and since the shell 2 is hung on the hanging rope 33, in the first state, the third gear 37 meshes with the first gear 34 and the second gear 35 respectively. By driving the third gear 37 to rotate, the first support rod 31 and the second support rod 32 can be driven to rotate synchronously and in the same direction, thereby controlling the movement of the hanging rope 33. When the first support rod 31 and the second support rod 32 rotate synchronously and in the same direction, the position of the shell 2 on the hanging rope 33 can be controlled. By timing the positive and negative rotation of the third gear 37, the automatic turning of the shell 2 can be realized. For example, within a certain period of time, by controlling the third gear 37 to drive the first gear 34 and the second gear 35 to rotate in the same direction, the hanging rope 33 between the first support rod 31 and the second support rod 32 moves, so that the shells 2 are all located on the X-side of the hanging rope 33; after a preset period of time, the driving motor 38 is controlled to reverse, and the third gear 37 drives the first gear 34 and the second gear 35 to rotate in the same direction, so that the hanging rope 33 between the first support rod 31 and the second support rod 32 moves, so that the shells 2 are all located on the opposite side of the X-direction of the hanging rope 33. There is no need to manually turn the hanging rope 33 regularly, ensuring that each shell 2 can receive uniform light intensity; thereby further improving the seedling raising quality of the seedlings.

[0080] As an optional implementation manner, the materials of the first support rod 31 and the second support rod 32 are both fiberglass.

[0081] The fiberglass material has the advantages of large structural strength and seawater corrosion resistance. Moreover, the surfaces of the first support rod 31 and the second support rod 32 made of fiberglass are flatter than those of bamboo poles, making it easier for them to rotate on the U-shaped support 36 and facilitating the sleeving of the first gear 34 or the second gear 35, and their meshing transmission with the third gear 37 is more stable.

[0082] As an optional implementation manner, the materials of the first gear 34, the second gear 35 and the third gear 37 are all nylon, which can resist seawater corrosion.

[0083] As an optional implementation manner, the support mechanism 3 further includes a lifting cylinder 39 and a lifting seat 310. The lifting seat 310 is connected with a bearing seat, and the third gear 37 is connected to the bearing seat through a rotating shaft; the driving motor 38 is connected to the lifting seat 310, and the driving cylinder is connected between the base and the lifting seat 310 for driving the lifting seat 310 to move in the vertical direction.

[0084] In the above embodiments, the structures of the lifting seat 310 and the lifting cylinder 39 are designed, which can control the lifting of the third gear 37, so as to control the engagement or separation of the third gear 37 with the first gear 34 and the second gear 35, facilitating the loading or disassembly of the first support rod 31 and the second support rod 32.

[0085] As an alternative embodiment, in the step S1, the specific steps of the pretreatment are as follows: Take out the algal seeds 4 - 5 days in advance, soak them in sterilized seawater, select the healthy algal bodies with a length of 1 - 2 cm, cut off the marginal and apical carposporangia and holdfast parts, and brush the cut leaves with a sterilized writing brush more than three times to remove dirt, miscellaneous algae, etc., and add NO 3- N 10 PPM, PO4 3- -P 1 PPM. Change the water 1 - 2 times a day to allow the algal bodies to fully recover; then cut the leaves into small pieces of 1 - 2 mm, wash them in sterilized seawater until the supernatant is colorless, blot dry the water, then rinse them twice with 1 mol / L glucose solution, blot dry the water, and store them at -20 °C;

[0086] Take out the algal seeds from the -20 °C environment and immediately put them into the sterilized seawater with a temperature of 10 - 20 °C. After the leaves unfold, select the healthy algal bodies with normal color, wash them with sterilized seawater, and remove the miscellaneous algae and attached mud on the surface of the algal bodies again. While cleaning them, cut off the parts with carpospores, spermatangia and rhizoids at the upper end of the laver with a blade, then soak them in antibiotics for 5 - 7 minutes, wash them 3 times with sterilized seawater, and then put them into the sterilized seawater containing nitrogen and phosphorus nutrient solution for static culture for 4 days. First, irradiate them with a light intensity of 1000 Lx for 1 day, then increase the light intensity to 2000 Lx, with a light cycle of 12L∶12D, and set the culture temperature to 18 - 20 °C. Stir the seawater during the culture process to increase gas exchange; in addition, during the culture process, start from the third day, add 1 drop of CuSO4 solution to the culture solution every day to eliminate the harmful organisms (especially ciliates) attached to the algal seeds.

[0087] As an alternative embodiment, in the step S2, the specific steps of obtaining single cells by enzymolysis of the pretreated algal seeds are as follows:

[0088] Fresh corolla lunella was selected as the extraction material. The material animals were first rinsed with seawater, starved for 2 days, the outer shells were gently knocked to break, the digestive tracts and internal organs were taken, and a mixed solution 4 times heavier than the tissue was added. The mixed solution contained 0.3M KC1: 0.15M MgSO4: pH 7.0 phosphate buffer solution. It was homogenized in a blender and centrifuged at 5000 rpm and 10000 rmp for 20 minutes respectively at 0 °C using a refrigerated centrifuge. The supernatant was taken, which was the crude enzyme solution, and it was stored at -20 °C; the -20 °C conch enzyme solution was thawed, aliquoted into 250 ml round-bottom flasks, first frozen at -20 °C, and then continuously dried at -27 °C using a freeze dryer for 24 hours until it became a dry powder, and it was stored at -20 °C;

[0089] Determination of enzyme activity:

[0090] Preparation of the standard curve: Weigh 100 mg of glucose dried to a constant weight, dissolve it and make up the volume to 100 ml. This solution contains 1000 μg / ml of glucose. 0.2, 0.4, 0.6, 0.8, and 1.0 ml of glucose were respectively pipetted into 5 test tubes, all diluted to 1 ml with distilled water, 3 ml of 3,5-dinitrosalicylic acid color reagent was added, and it was boiled and developed color in boiling water for 15 minutes, cooled, and 21 ml of distilled water was added and shaken well. Use 1 ml of distilled water instead of glucose as the blank tube, and colorimetric at 550 nm. With the optical density as the ordinate and the microgram number of glucose as the abscissa, draw the standard curve, referring to Table 1:

[0091] Table 1

[0092]

[0093]

[0094] Take 1 ml of 0.1% substrate (agar), 1 ml of disodium hydrogen phosphate-citric acid buffer solution at pH 6.4, and 1 ml of enzyme solution respectively, mix well and incubate in a 35 °C water bath for 1 h, boil, centrifuge at 4000 rpm for 15 minutes, then take 0.2 ml of the supernatant, develop color with DNS, and measure its OD550 with a 721-100 type spectrophotometer. Under these conditions, the amount of enzyme required to produce 1 μg of reducing sugar per hour is one enzyme unit. Use the boiled enzyme solution as the blank control.

[0095] The activity of the crude enzyme solution agarase is shown in Table 2:

[0096] Table 2

[0097]

[0098] Weigh the enzyme powder according to the ratio of 10 ml of enzyme solution for every 1 g of the treated algal seeds. After weighing the enzyme powder, put it into the measured enzyme solvent and stir well to fully dissolve the enzyme powder. Adjust the pH value to 5.8 - 6.2; filter and disinfect the prepared enzyme solution using a microporous filter with a pore size of 0.22 μm.

[0099] From the pre - treated algal seeds, select the algal seeds, wash them with disinfected seawater, filter off the seawater, and then cut the algal seeds into small pieces of 1 - 2 mm with a disinfected knife or scissors. 2 Wash the small pieces with seawater again, then wash and filter them with 1 mol / L glucose solution to remove the chopped cytoplasm to avoid affecting the enzymatic hydrolysis effect. After blotting the water from the small pieces with blotting paper, set them aside for use.

[0100] Put the treated algal seeds into the prepared enzyme solution, place them on a shaker, at a temperature of 31 - 32 °C, and oscillate at a speed of 100 rpm / min under dark conditions.

[0101] After 1 h of enzymatic hydrolysis, use a dropper to take a small amount of the enzyme solution and drop it on a glass slide, then examine it under a fluorescence microscope. It is observed that the 3 - 5 rows of cells at the edge of the algal pieces in both enzyme solutions become significantly rounder. The smaller the algal pieces, the more round cells there are. At the same time, it can be observed that the common gelatinous membrane on the upper and lower surfaces of the algal pieces starts to loosen, and the edge part starts to separate from the cell group.

[0102] After 1.5 h of enzymatic hydrolysis, observe under fluorescence. The common wall of the algal cells forms a film - like structure, and some have even turned up as a whole piece. The cells in the center of the algal pieces start to loosen. Most of the cells are round, and the free single cells are ellipsoidal, emitting white fluorescence under the fluorescence microscope.

[0103] After 1.75 h of enzymatic hydrolysis, observe under the fluorescence microscope. Outside the large and small cell clusters and even outside single cells, only traces of incomplete green fibrous cell walls remain, and in some cases, nothing can be observed. This indicates that the cell walls between cells after 1.75 h of enzymatic hydrolysis have been basically enzymatically hydrolyzed, while the common membranes rich in fibrous substances on the upper and lower layers of the cell layer are not easily digested and only detach from the cell layer as a whole. The cells are enzymatically hydrolyzed into small clumps, with two or three cells together or single cells. Therefore, more free cell clusters and single cells are observed, and the cells in the center of the algal pieces are more loose.

[0104] After 2 h of enzymatic hydrolysis, the cells in the center of the algal pieces are very loose, there are many free single cells, and in some cases, the algal pieces can no longer be seen.

[0105] After 2.5 h of enzymatic hydrolysis, the common membranes on the upper and lower surfaces of most algal pieces have almost completely detached from the cell group. What is observed under the fluorescence microscope are mostly single cells and cell clusters with 2 - 5 cells connected together.

[0106] After enzymatic hydrolysis for 2.75 h, no algal clumps were visible under the fluorescence microscope. The proportion of single cells increased significantly, and the cell clusters were also composed of 2-3 cells connected together with a relatively low proportion. At this time, enzymatic hydrolysis was stopped, and cell collection and cultivation were started. The results are shown in Table 1.

[0107] Table 1

[0108]

[0109] When no algal clumps were visible under the fluorescence microscope, the proportion of single cells increased significantly, and the cell clusters were also composed of 2-3 cells connected together with a relatively low proportion. At this time, enzymatic hydrolysis was stopped.

[0110] As an alternative implementation, in S3, the somatic cell culture of single cells to obtain filamentous somatic cell seedlings is specifically as follows:

[0111] After the enzymatic hydrolysis is completed, add MES culture medium four times the volume of the enzyme solution, mix and filter with an 80-μm sieve silk. The filtrate is centrifuged at 2000 rpm in a centrifuge. After 2 minutes, discard the supernatant, then add MES culture medium with salt, shake well and centrifuge. Repeat this washing process 3 times. Finally, collect the cells at the bottom of the centrifuge tube and dispense them into 3 50-ml Erlenmeyer flasks to obtain No. 1 culture medium, No. 2 culture medium and No. 3 culture medium respectively;

[0112] The MES culture medium is obtained by diluting the MES stock solution 50 times with seawater of the corresponding specific gravity; the MES stock solution is prepared from the following raw materials: sodium nitrate 1.4 g; glycerophosphate 0.2 g; tris(hydroxymethyl)aminomethane 2 g; MPII 100 ml; H2O 400 ml; pH 7.6;

[0113] The MPII stock solution is prepared from the following raw materials by weight: EDTA-Na2 1.5 g; boric acid 1.25 g; MnCl2·4H2O 0.175 g; FeCl3·6H2O 62.5 mg; ZnCl2 12.5 mg; CoCl·6H2O 5 mg; ferric citrate 300 mg; H2O 500 ml;

[0114] The collected somatic cells are allowed to stand still for about 2 hours and then added with No. 3 culture medium and cultured in a constant temperature incubator;

[0115] The freshly enzymatically hydrolyzed somatic cells are placed in the dark for 1 day, exposed to a light intensity of 10001x on the second day, the light intensity is increased to 2000lx on the third day, and the light intensity is maintained at 2500lx after the fourth day. The light cycle is 12L:12D;

[0116] The somatic cells are cultured with No. 3 MES culture medium for 1 to 6 days, and then changed to MES culture medium on the 7th day after cultivation. After that, the culture medium is changed every 7-10 days. At the beginning of cultivation, following the principle of gradually decreasing density, the initial cultivation density is maintained at 1.030 g / cm3 or so, the density decreased to 1.027 g / cm after 10 h 3 or so. After another 12 h, the density dropped to 1.025 g / cm 3 or so. After another 12 h, the density remained at 1.023 g / cm 3 or so;

[0117] Density of cell culture: 1.0 - 1.2×10 4 cells / ml.

[0118] As an alternative embodiment, in S5, the obtained pure Porphyra haitanensis filaments can be used for primary preservation and secondary preservation respectively after primary amplification and secondary amplification. The specific steps for establishing the germplasm resource bank of Porphyra haitanensis are as follows:

[0119] Primary amplification: Chop the pure filaments into pieces of 100 - 500 μm with a tissue grinder sterilized by high temperature, then place them in a 500 ml conical flask and incubate statically in the dark for 1 day. On the 2nd day, replace 200 - 300 ml of the culture medium and then resume static incubation under normal light. The culture concentration is about 1 g of wet weight free filaments per 100 ml of the culture medium. During the culture period, replace 50 - 100 ml of the culture medium every 30 days.

[0120] Secondary amplification: Chop the pure filaments after primary amplification into pieces of 100 - 500 μm with a tissue grinder sterilized by high temperature, then place them in a 2000 ml conical flask and incubate statically in the dark for 1 day. On the 2nd day, replace 1200 - 1500 ml of the culture medium and then resume static incubation under normal light. After 3 - 5 days, add an air pipe for aerated culture. The air needs to be filtered through three layers to ensure that the air entering the flask is pollution-free. The culture concentration is 2 - 3 g of wet weight free filaments per 100 ml of the culture medium. If the filament density exceeds 3 g / 100 ml of the culture medium, subculture in time. During the culture period, replace 1000 - 1500 ml of the culture medium every 10 days. The culture medium and culture conditions are the same as those in the primary culture.

[0121] In S9, the seeding season is selected before the obvious cold air comes around the White Dew. If seeding in the hot sun and high temperature environment before the White Dew, the time should be set on the fifth and twentieth days of Dashuiwei to avoid the raft frame being exposed to the sun at noon, which can greatly improve the seeding effect.

[0122] The raft frame should be of a modern flip type or set at a shallow tidal level with about three hours of dry exposure. The weaving lines of the seeding curtain must be of the same length. It is better to have 8 - 10 layers in the concentrated layer. When setting up the seeding raft frame, first remove the floating stem rope to straighten all the lines to the top layer of the water, which is beneficial for uniform seeding.

[0123] The containers for transporting seedlings must be absolutely clean, free of oil, dirt, acid, alkali and other dirt. Water should be poured frequently during dry transportation to keep the seedling shells moist and prevent them from drying out. They must be handled gently.

[0124] After the seedlings are transported to the destination, it is best to keep them away from water, moisturize them, and protect them from the sun. They can be put into the sea for stimulation after 2 pm that day, and the sea hanging time should be prolonged as much as possible. The stimulation sea area should be selected in a place with clear water, fast current, and not drying out.

[0125] The seedling shells must be rinsed clean in sea water before 6 a.m. and lifted into the ship's hold. Sea water should be added at a ratio of 200 jin per acre to release the spore water. The release time is from 8 a.m. to 12 p.m. every day, and it will be postponed to 1 a.m. on hot days. It is better to spray the seedlings once every 1-2 hours, and 2-3 times in one morning.

[0126] After the laver shell spores are released, the spores will sink to the bottom of the ship in a calm sea area, and the spores will float to the surface of the water in a turbulent situation. Therefore, the water should be stirred constantly when pouring spore water to promote the uniformity of the spore content poured out front and back.

[0127] When pouring spore water, pour more water in the direction of wind and water flow to let the spores drift with the wind, waves and tides to promote uniform attachment of seedlings.

[0128] The above descriptions are merely embodiments of the present invention and are not intended to limit the patent scope of the present invention. Any equivalent transformations made using the contents of the present invention's specification and drawings, or directly or indirectly applied in related technical fields, are also included in the patent protection scope of the present invention.

Claims

1. A method for breeding high-quality seedlings of Porphyra haitanensis, characterized in that, The following steps are involved: S1: Select pure strains of Porphyra oleracea from the open sea for pre-treatment; S2: The pre-treated algae are enzymatically hydrolyzed to obtain single cells; S3: Somatic cell culture of single cells to obtain filamentous somatic cell seedlings; S4: purifying and rejuvenating the conidia somatic cell seedlings to obtain pure conidia of Porphyra haitanensis; S5: The obtained pure line conidia of Porphyra haitanensis can be used for primary preservation and secondary preservation respectively after primary amplification and secondary amplification to establish a germplasm resource bank of Porphyra haitanensis; S6: Place shells in the nursery pond, homogenize the somatic cell conidia of Porphyra haitanensis and cut them into 0.045-0.055 mm algae segments, dilute with seawater and spray evenly in the pond with a spray bottle. The algae segments will cling to the bottom of the pond or the hanging shells to form conchospores; S7: collecting seedlings of conchospores, transplanting them to rafts, and carrying out thallus cultivation. During the thallus cultivation period, selecting second-generation superior fruit spores, and preparing fruit spore water; S8: Arrange shells in the nursery pond and spray fruit spore water on the shells. Fruit spores will cling to the shells and grow; S9: fruit spores are collected and transplanted to rafts for thallus culture. During the thallus culture period, second-generation superior fruit spores are selected and fruit spore water is prepared; S10: Arrange shells in the nursery pond and spray fruit spore water on the shells. Fruit spores will cling to the shells and grow; Repeat S9 and S10 until superior fruit spores are screened; The seedling raising method is based on a seedling raising device, which includes the seedling raising pool and shells mentioned in S6, S8 and S10, and also includes multiple groups of supporting mechanisms; the supporting mechanism includes a first support rod, a second support rod, a hanging rope, a first gear, a second gear, a U-shaped support, a base, a third gear and a driving motor; The shape of the nursery pond is rectangular; The first support rod and the second support rod are both cylindrical in shape; Each group of the support mechanisms includes four U-shaped supports, which are arranged on the upper part of the two side walls in the width direction of the nursery pool, and are used to support the first support rod and the second support rod, the first gear is coaxially sleeved on one end of the first support rod, and the second gear is coaxially sleeved on one end of the second support rod; The third gear is liftable and rotatably connected to the base, and the driving motor is transmission-connected to the third gear to drive the third gear to rotate; One end of the hanging rope is wound and connected to the first support rod, and the other end is wound and connected to the second support rod, and the shells are hung on the hanging rope at equal intervals along the extension direction of the hanging rope; In the first state, the first support rod and the second support rod are parallel to each other and are erected on the U-shaped supports on both sides of the nursery pool, the third gear is meshed with the first gear and the second gear respectively, and the hanging rope is suspended between the first support rod and the second support rod in a U shape; In S6, S8, and S10, each includes the steps of: in the first state, periodically controlling the driving motor to rotate forward, driving the first gear and the second gear to rotate in the same direction through the third gear, causing the hanging rope between the first support rod and the second support rod to move, so that the shells are all located on the X-side of the hanging rope; after a preset time period, controlling the driving motor to rotate in reverse, driving the first gear and the second gear to rotate in the same direction through the third gear, causing the hanging rope between the first support rod and the second support rod to move, so that the shells are all located on the reverse X-side of the hanging rope; The support mechanism further includes a lifting cylinder and a lifting seat. The lifting seat is connected with a bearing seat, and the third gear is connected to the bearing seat through a rotating shaft; the driving motor is connected to the lifting seat, and the lifting cylinder is connected between the base and the lifting seat for driving the lifting seat to move in the vertical direction.

2. The method for breeding fine strains of Porphyra haitanensis according to claim 1, characterized in that, The materials of the first support rod and the second support rod are both fiberglass.

3. The method for breeding high-quality seedlings of Porphyra haitanensis according to claim 1, wherein, The materials of the first gear, the second gear, and the third gear are all nylon.

4. The breeding method of good varieties of Porphyra haitanensis according to claim 1, characterized in that, In S1, the pre-treatment steps are as follows: soaking the algae in sterilized seawater, selecting healthy algae, cutting off the edges and top fruit spores and the holdfast, disinfecting and decontaminating the cut algae leaves, and adding NO 3- N 10PPM, PO4 3- -P 1PPM, change the water 1-2 times a day to allow the algae to fully recover; then cut the leaves into 1-2mm small pieces, wash them in sterilized seawater until the supernatant is colorless, absorb the water, rinse them twice with 1mol / L glucose solution, absorb the water, and store them at -20℃; The algal species are taken out from the -20°C environment and immediately placed in sterilized seawater with a temperature of 10 - 20°C. After the blades are unfolded, select the algal bodies with normal color and health, wash them with sterilized seawater, and remove the miscellaneous algae and attached mud on the surface of the algal bodies again. While cleaning them, cut off the parts with carpospores, spermatangia, and rhizoids at the upper end of the laver with a blade, then soak them in antibiotics for 5 - 7 minutes, wash them 3 times with sterilized seawater, and then place them in sterilized seawater containing nitrogen and phosphorus nutrient solution for static culture for 4 days. First, irradiate them with light intensity of 1000 Lx for 1 day, then increase the light intensity to 2000 Lx, with a light cycle of 12L∶12D, and set the culture temperature to 18 - 20°C. Stir the seawater during the culture process to increase gas exchange.

5. The method for breeding high-quality seedlings of Porphyra haitanensis according to claim 4, characterized in that, In S2, the step of obtaining single cells from the pretreated algal species by enzymatic hydrolysis is specifically as follows: Select fresh Littorina coronata as the extraction material. First, rinse the material animal with seawater, starve it for 2 days, gently tap to break the outer shell, take the digestive tract and internal organs, add a mixture 4 times heavier than the tissue. The mixture contains 0.3M KCl: 0.15M MgSO4: pH7.0 phosphate buffer solution. Homogenize it in a homogenizer, centrifuge the homogenate separately with a refrigerated centrifuge at 0°C, take the supernatant, which is the crude enzyme solution, and store it at -20°C; thaw the crude enzyme solution of the conch stored at -20°C, dispense it into a round-bottom flask, first freeze it at -20°C, and then continuously dry it with a freeze dryer at -27°C for 24 hours until it becomes a dry powder, and store it at -20°C; Weigh the enzyme powder according to the ratio of 10 ml of enzyme solution for every 1 g of pretreated algal species. After weighing the enzyme powder, put it into the measured enzyme solvent and stir well to fully dissolve the enzyme powder, and adjust the pH value to 5.8 - 6.2; filter and disinfect the prepared enzyme solution with a microporous filter with a pore size of 0.22 μm; From the pre-treated algal seeds, select the algal seeds, wash them with sterilized seawater, filter off the seawater, and then cut the algal seeds into small pieces of 1-2 mm with a sterilized knife. 2 Wash the small pieces with seawater again, then wash and filter them with 1 mol / L glucose solution to remove the chopped cytoplasm so as not to affect the enzymolysis effect. After drying the water of the small pieces with blotting paper, they are ready for use. Place the treated algal species in the prepared enzyme solution, put it on a shaker, at a temperature of 31 - 32°C, and oscillate at a speed of 100 rpm / min under dark conditions; When no algae blocks can be seen under the fluorescence microscope, the proportion of single cells has increased significantly, and the cell clusters are 2-3 cells connected together with a low proportion, the enzymatic hydrolysis is stopped.

6. The method for breeding improved varieties of Pyropia haitanensis according to claim 5, wherein, In S3, the single cell is subjected to somatic cell culture to obtain the mycelial somatic cell seedlings as follows: After the enzymatic hydrolysis is completed, add MES culture medium 4 times the amount of enzyme solution, mix and filter with 80um sieve silk, centrifuge the filtrate, discard the supernatant after 2 minutes, add MES culture medium with salt, shake well and centrifuge, repeat this washing 3 times, finally collect the cells at the bottom of the centrifuge tube and divide them into 3 50ml conical flasks to obtain culture medium No. 1, culture medium No. 2 and culture medium No. 3 respectively; The MES culture solution is obtained by diluting the MES stock solution 50 times with seawater of corresponding specific gravity; the MES stock solution is prepared from the following raw materials: 1.4 g sodium nitrate; 0.2 g sodium glycerophosphate; 2 g tris(hydroxymethyl)aminomethane; 100 ml MPII; 400 ml H2O; pH 7.6; The MPII is prepared from the following raw materials in parts by weight: 1.5 g EDTA-Na2; 1.25 g boric acid; 0.175 g MnCl2.4H2O; 62.5 mg FeCl3.6H2O; 12.5 mg ZnCl2; 5 mg CoCl6H2O; 300 mg ferric citrate; 500 ml H2O; The collected somatic cells were placed in a static state for about 2 hours and then added with No. 3 culture medium and cultured in a constant temperature incubator; The somatic cells just enzymatically hydrolyzed were placed in the dark for 1 day, and exposed to 10001x light intensity on the second day, and the light intensity was increased to 2000lx on the third day. After the fourth day, the light intensity was maintained at 2500lx, and the photoperiod was 12L:12D. The somatic cells were cultured in Medium No. 3 for 1 to 6 days, and then changed to MES medium on the 7th day after culture. After that, the culture medium was changed every 7 - 10 days. At the beginning of the culture, following the principle of gradually decreasing density, the initial culture density was maintained at 1.030 g / cm 3 or so. After 10 h, the density decreased to 1.027 g / cm 3 or so. After another 12 h, the density dropped to 1.025 g / cm 3 or so. After another 12 h, the density remained at 1.023 g / cm 3 or so.

7. The method for breeding high-quality seedlings of Pyropia haitanensis according to claim 1, characterized in that, In S5, the obtained pure line conidia of Porphyra haitanensis can be used for primary preservation and secondary preservation respectively after primary amplification and secondary amplification. The establishment of the Porphyra haitanensis germplasm resource bank is specifically as follows: Primary amplification: mince the pure line filaments into 100-500 μm with a high temperature sterilized tissue grinder, then place them in a 500 ml conical flask and culture them in the dark for 1 day. On the second day, replace 200-300 ml of culture medium and then restore normal lighting for static culture. The culture concentration is about 1 g wet weight of free filaments per 100 ml of culture medium. During the culture period, replace 50-100 ml of culture medium every 30 days. Secondary amplification: The pure lineage filaments that have undergone primary amplification are chopped into 100-500μm using a high-temperature sterilized tissue grinder, and then placed in a 2000ml conical flask for static culture in the dark for 1 day. On the second day, 1200-1500ml of culture medium is replaced and normal lighting is restored for static culture. After 3-5 days, an aeration tube is added for aeration culture. The air needs to be filtered three times to ensure that the air entering the bottle is pollution-free. The culture concentration is 2-3g wet weight of free filaments per 100ml of culture medium. If the density of filaments exceeds 3 g / 100ml of culture medium, it is promptly divided into bottles for culture. During the culture period, 1000-1500ml of culture medium is replaced every 10 days. The culture medium and culture conditions are the same as those for primary amplification.

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