Porphyra haitanensis-based mutation breeding method

Abstract

The invention belongs to the technical field of breeding, and particularly provides a porphyra haitanensis-based mutation breeding method, which comprises the following steps: 1) carrying out ultraviolet radiation on a porphyra haitanensis filament; the invention discloses a method for breeding thalli through mutagenesis, which comprises the following steps of: 1) preparing protonema, 2) treating and irradiating the protonema with a mutagenic agent which comprises nitrosoguanidine and a slow-release solution which comprises bleomycin and pingyangmycin, and 3) screening the protonema subjected to mutagenesis treatment to obtain a mutant strain, and continuously culturing to obtain the thalli. The breeding method has the advantages of high efficiency, high mutation rate and good specificity.

Description

Technical Field

[0001] This application belongs to the field of breeding technology, and particularly relates to a mutagenic breeding method based on Pyropia haitanensis. Background Art

[0002] Porphyra belongs to Rhodophyta, Protoflorideae, Bangiales, Bangiaceae in taxonomy, and its distribution range covers frigid zones, temperate zones, subtropical zones and tropical seas. Porphyra is rich in nutrients, including protein, polysaccharide, lipid, vitamin and mineral. Due to its high content of polysaccharide and phycoerythrin and unique biological functions, it has broad application prospects in the fields of medicine, food, cosmetics and animal nutrition.

[0003] Due to the influence of natural climate change and sea conditions, coupled with human over-harvesting and the predation of algae-eating fish and shellfish, its natural resources are decreasing day by day. And so far, the large-scale artificially cultivated Pyropia haitanensis is still mainly self-retained seeds, showing obvious phenomena such as germplasm degradation, yield and quality decline, which affect the healthy and sustainable development of the Porphyra cultivation industry. Therefore, screening new germplasms of Pyropia haitanensis with high quality and strong stress resistance is still an important requirement for the industrial development.

[0004] The acquisition of Porphyra genetic variants is one of the important ways to carry out Porphyra breeding work. Based on physical mutagenesis and chemical mutagenesis methods, phycologists have prepared a large number of Porphyra color mutants, providing extensive research materials for promoting scientific progress in the research fields of Porphyra genetics, physiology, etc. The artificial color mutants of Porphyra are mainly obtained through physical and chemical mutagenesis approaches. Mutagenic breeding is one of the most widely used artificial mutagenesis methods. Different from traditional breeding with a long breeding cycle and unstable traits, it can effectively improve the breeding efficiency and can cultivate new varieties directionally according to the breeding goal.

[0005] At present, using ray irradiation can achieve a relatively fast mutagenic effect, but it is easily absorbed by the chromosome set, has great destructiveness to the chromosome structure, and the mutation sites are relatively random. Chemical mutagenesis has a high mutation rate and site specificity, but the mutation range is wide and the efficiency is slow. Therefore, it is necessary to find a breeding method for Pyropia haitanensis with high efficiency and excellent strains. Summary of the Invention

[0006] In view of the above problems, in order to further improve the mutagenic breeding effect of Pyropia haitanensis, this application provides a mutagenic breeding method based on Pyropia haitanensis.

[0007] This application provides a mutagenic breeding method based on Pyropia haitanensis, including the following steps: 1) Ultraviolet irradiation is carried out on the filaments of Pyropia haitanensis; 2) Treat the filaments after ultraviolet irradiation in step 1) with a mutagen, where the mutagen includes nitrosoguanidine and a sustained-release solution, and the sustained-release solution includes bleomycin and pingyangmycin; 3) Screen the filaments after mutagenic treatment to obtain mutant strains, and continue culturing to obtain thalli.

[0008] Further, in step 1), the filaments are statically cultured with MES culture medium before ultraviolet irradiation, and the MES culture medium contains 5 - 15 μg / L of copper sulfate.

[0009] Further, in step 1), the irradiation dose of ultraviolet irradiation is 20 - 50 J / m 2 。

[0010] Further, in step 2), the mutagenic time is 20 - 60 min.

[0011] Further, in step 2), the concentration of nitrosoguanidine in the mutagen is 15 - 60 μg / mL.

[0012] Further, in step 2), the concentration of bleomycin in the sustained-release solution is 300 - 500 ppm.

[0013] Further, in step 2), the concentration of pingyangmycin in the sustained-release solution is 100 - 200 ppm.

[0014] Further, in step 2), the sustained-release solution is prepared by a method including the following steps: S1: Mix cyclodextrin, polyethylene glycol, and deionized water evenly to obtain a base solution; S2: Place amino polystyrene particles in domestic sewage, statically culture, then wash with ultrapure water, sterilize, and dry to obtain biofilm@amino polystyrene particles; S3: Add biofilm@amino polystyrene particles, bleomycin, and pingyangmycin to the base solution, and mix evenly.

[0015] Further, in step S2, the average particle size of the amino polystyrene particles is 15 - 80 μm.

[0016] Further, in step S3, the content of biofilm@amino polystyrene particles in the sustained-release solution is 0.5 - 0.85 wt%.

[0017] Compared with the prior art, the present application has the following beneficial effects: 1. In the present application, the filaments are first irradiated with low-dose ultraviolet rays to a certain extent, and the filament cells are fully activated, and then treated with a mutagen, which can improve the mutation efficiency and quantity of the filament cells.

[0018] 2. Based on the induction by nitrosoguanidine, the present application introduces a bleomycin and pingyangmycin sustained-release system, which can further improve the mutation efficiency and specificity. At the same time, the biofilm@aminopolystyrene particles can adsorb / control the release of bleomycin and pingyangmycin, increase the mutation survival rate, and the biofilm@aminopolystyrene particles themselves can also provide a certain induction effect, thereby enhancing the effective induction effect and promoting the formation of conchospore branches by mutant filaments in the subsequent stage. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 It is a schematic diagram of the development of mutant filaments in the method for mutagenic breeding of Porphyra haitanensis in Examples 1-2 and Control Groups 1-2 of the present application. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0020] Next, the technical solutions in the embodiments of the present application will be clearly and completely described in conjunction with the drawings in the embodiments of the present application. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without making creative efforts belong to the scope of protection of the present application.

[0021] Through a large number of experimental studies, the present application explores various induction breeding methods and finds that under low-dose ultraviolet irradiation treatment, it can provide a mutation basis for chemical induction, and bleomycin and pingyangmycin are assisted in induction under the environment of a sustained-release solution to further improve the induction effect.

[0022] Specifically, the present application provides a method for mutagenic breeding of Porphyra haitanensis, including the following steps: 1) Ultraviolet irradiate the filaments of Porphyra haitanensis; 2) Treat the filaments after ultraviolet irradiation in step 1) with a mutagen, and the mutagen includes nitrosoguanidine and a sustained-release solution, and the sustained-release solution includes bleomycin and pingyangmycin; 3) Screen the filaments after mutagenic treatment to obtain mutant strains, and continue to culture them to obtain thalli.

[0023] Furthermore, in step 1), the filaments are statically cultured with MES culture solution before ultraviolet irradiation, and the MES culture solution contains 5-15 μg / L of copper sulfate.

[0024] In some specific embodiments, in step 1), the filamentous body is statically cultured in MES culture medium before ultraviolet irradiation. The MES culture medium may contain copper sulfate at concentrations of 5 μg / L, 6 μg / L, 7 μg / L, 8 μg / L, 9 μg / L, 10 μg / L, 11 μg / L, 12 μg / L, 13 μg / L, 14 μg / L, 15 μg / L. More preferably, under normal circumstances, in step 1), when the MES culture medium contains 10 μg / L of copper sulfate, better experimental results can be obtained.

[0025] Further, in step 1), the irradiation dose of ultraviolet irradiation is 20 - 50 J / m 2 .

[0026] In some specific embodiments, in step 1), the irradiation dose of ultraviolet irradiation can be 20 J / m 2 , 25 J / m 2 , 30 J / m 2 , 35 J / m 2 , 40 J / m 2 , 45 J / m 2 , 50 J / m 2 . More preferably, under normal circumstances, in step 1), when the irradiation dose of ultraviolet irradiation is 30 J / m 2 , better experimental results can be obtained.

[0027] Further, in step 2), the mutagenesis time is 20 - 60 min.

[0028] In some specific embodiments, in step 2), the mutagenesis time can be 20 min, 25 min, 30 min, 35 min, 40 min, 45 min, 50 min, 55 min, 60 min. More preferably, under normal circumstances, in step 2), when the mutagenesis time is 25 min, better experimental results can be obtained.

[0029] Further, in step 2), the concentration of nitrosoguanidine in the mutagen is 15 - 60 μg / mL.

[0030] In some specific embodiments, in step 2), the concentration of nitrosoguanidine in the mutagen can be 15 μg / mL, 20 μg / mL, 25 μg / mL, 30 μg / mL, 35 μg / mL, 40 μg / mL, 45 μg / mL, 50 μg / mL, 55 μg / mL, 60 μg / mL. More preferably, under normal circumstances, in step 2), when the concentration of nitrosoguanidine in the mutagen is 20 μg / mL, better experimental results can be obtained.

[0031] Further, in step 2), the concentration of bleomycin in the sustained-release solution is 300 - 500 ppm.

[0032] In some specific embodiments, in step 2), the concentration of bleomycin in the sustained-release solution can be 300 ppm, 310 ppm, 320 ppm, 330 ppm, 340 ppm, 350 ppm, 360 ppm, 370 ppm, 380 ppm, 390 ppm, 400 ppm, 410 ppm, 420 ppm, 430 ppm, 440 ppm, 450 ppm, 460 ppm, 470 ppm, 480 ppm, 490 ppm, 500 ppm. More preferably, under normal circumstances, when the concentration of bleomycin in the sustained-release solution in step 2) is 380 ppm, better experimental results can be obtained.

[0033] Further, in step 2), the concentration of pingyangmycin in the sustained-release solution is 100 - 200 ppm.

[0034] In some specific embodiments, in step 2), the concentration of pingyangmycin in the sustained-release solution can be 100 ppm, 110 ppm, 120 ppm, 130 ppm, 140 ppm, 150 ppm, 160 ppm, 170 ppm, 180 ppm, 190 ppm, 200 ppm. Under normal circumstances, when the concentration of pingyangmycin in the sustained-release solution in step 2) is 120 ppm, better experimental results can be obtained.

[0035] Further, in step 2), the sustained-release solution is prepared by a method comprising the following steps: S1: Mix cyclodextrin, polyethylene glycol, and deionized water evenly to obtain a base solution; S2: Place amino polystyrene particles in domestic sewage, statically culture, then wash with ultrapure water, sterilize, and dry to obtain biofilm@amino polystyrene particles; S3: Add biofilm@amino polystyrene particles, bleomycin, and pingyangmycin to the base solution and mix evenly.

[0036] Further, in step S2, the average particle size of the amino polystyrene particles is 15 - 80 μm.

[0037] In some specific embodiments, in step S2, the average particle size of the amino polystyrene particles can be 15μm, 20μm, 25μm, 30μm, 35μm, 40μm, 45μm, 50μm, 55μm, 60μm, 65μm, 70μm, 75μm, 80μm. More preferably, under normal circumstances, in step S2, when the average particle size of the amino polystyrene particles is 60μm, better experimental results can be obtained.

[0038] Further, in step S3, the content of biofilm@amino polystyrene particles in the sustained-release solution is 0.5 - 0.85 wt%.

[0039] In some specific embodiments, in step S3, the content of biofilm@amino polystyrene particles in the sustained-release solution can be 0.5 wt%, 0.55 wt%, 0.6 wt%, 0.65 wt%, 0.7 wt%, 0.75 wt%, 0.8 wt%, 0.85 wt%. More preferably, under normal circumstances, in step S3, when the content of biofilm@amino polystyrene particles in the sustained-release solution is 0.8 wt%, better experimental results can be obtained.

[0040] Example 1 Preparation of MES culture medium: Accurately weigh 1.4 g of sodium nitrate, 0.2 g of sodium glycerophosphate, 2 g of tris(hydroxymethyl)aminomethane, 0.3 g of EDTA-Na2, 0.25 g of boric acid, 0.035 g of MnCl 2· 4H2O, 12.5 mg of FeCl3·6H2O, 2.5 mg of ZnCl2, 1 mg of CoCl2·6H2O, and 60 mg of ferric citrate, add them to a sterile cup, and make up the volume to 500 mL with ultrapure water to obtain the mother liquor of MES culture medium; add 20 mL of the MES culture medium mother liquor to 1000 mL of seawater (specific gravity 1.022), and then add copper sulfate and mix evenly. Control the content of copper sulfate in the MES culture medium to be 10 μg / L.

[0041] Preparation of inducer: Add nitrosoguanidine and 20 mL of the sustained-release solution to 1000 mL of sterilized seawater (specific gravity 1.022), and make up the volume after mixing. Control the concentration of nitrosoguanidine in the inducer to be 20 μg / mL.

[0042] The sustained-release solution is prepared by a method including the following steps: S1: Take 3 g of cyclodextrin, 15 mL of polyethylene glycol, and 200 mL of deionized water, and mix them evenly to obtain the base solution; S2: Add bleomycin and pingyangmycin to the base solution and mix evenly. Control the concentration of bleomycin in the sustained-release solution to be 380 ppm; the concentration of pingyangmycin in the sustained-release solution to be 120 ppm.

[0043] The mutagenesis breeding method of Porphyra haitanensis in this embodiment includes the following steps: 1) Preserve the filaments of wild Porphyra haitanensis (NH, collected from Pingtan County, Fujian Province) in the laboratory for later use. The preservation conditions are: MES culture solution, temperature 23 ± 1°C, light intensity 4 μmol photons / (m 2 ·s), photoperiod 12L︰12D, and replace 1 / 2 of the MES culture solution every 180d; Chop the filaments with a crusher to a length of about 100 μm, then inoculate them into a culture dish and culture for 10d. Then use a short-wave ultraviolet lamp tube as the irradiation source for ultraviolet irradiation, and control the irradiation dose of ultraviolet irradiation to be 20 - 50 J / m 2 ; 2) Add 5 mL of mutagen to the culture dish, and treat the filaments after ultraviolet irradiation in step 1) with the mutagen for 25 min. Then wash the culture dish with MES culture solution to remove the residual mutagen, and then supplement the MES culture solution. Incubate at a temperature of 20°C, light intensity of 2500 Lux, photoperiod 12L︰12D, and replace the culture solution every 3d; 3) Cut the filaments after mutagenesis treatment into small segments with a tissue homogenizer, select and place them in a 48-well plate, and culture them in the dark at 20°C for 3d. Then gradually transfer them to normal light of 50 μmol photons / (m 2 ·s) for culture. After 1 month, count the survival amount of the filaments, and transfer the surviving filaments to a cell culture flask. Select the samples with more and larger thalli as experimental materials for screening to obtain mutant strains, and continue to culture to obtain thalli.

[0044] Example 2 Configuration of MES culture solution: Accurately weigh 1.4 g of sodium nitrate, 0.2 g of glycerophosphate, 2 g of tris(hydroxymethyl)aminomethane, 0.3 g of EDTA-Na2, 0.25 g of boric acid, MnCl 2· 4H2O 0.035 g, FeCl3·6H2O 12.5 mg, ZnCl2 2.5 mg, CoCl2·6H2O 1 mg, and 60 mg of ferric citrate, add them to a sterile cup, and make up the volume to 500 mL with ultrapure water to obtain the mother liquor of MES culture solution; Add 20 mL of the mother liquor of MES culture solution to 1000 mL of seawater (specific gravity 1.022), and then add copper sulfate and mix evenly. Control the content of copper sulfate in the MES culture solution to be 10 μg / L.

[0045] Configuration of inducer: Add nitrosoguanidine and 20 mL of slow-release solution to 1000 mL of sterilized seawater (specific gravity 1.022), and make up the volume after fixing. Control the concentration of nitrosoguanidine in the inducer to be 20 μg / mL.

[0046] The sustained-release solution is prepared by a method including the following steps: S1: Take 3 g of cyclodextrin, 15 mL of polyethylene glycol, and 200 mL of deionized water, mix them evenly to obtain a base solution; S2: Place 20 g of amino polystyrene particles (the average particle size of the amino polystyrene particles is 60 μm) into 500 mL of domestic sewage (pH value 7.31, COD Cr 98.25 mg / L, TN 9.82 mg / L, NH4 + -N 8.58 mg / L), statically culture for 30 d at a stirring speed of 150 rpm and a temperature of 25 °C. After the culture ends, filter, then wash with ultrapure water, sterilize, and dry to obtain biofilm@amino polystyrene particles; S3: Add biofilm@amino polystyrene particles, bleomycin, and pingyangmycin to the base solution, mix them evenly. Control the concentration of bleomycin in the sustained-release solution to be 380 ppm; the concentration of pingyangmycin in the sustained-release solution to be 120 ppm; the content of biofilm@amino polystyrene particles in the sustained-release solution to be 0.8 wt%.

[0047] The method for mutagenic breeding of Porphyra haitanensis in this example includes the following steps: 1) Preserve the filaments of wild Porphyra haitanensis (NH, collected from Pingtan County, Fujian Province) in the laboratory for later use. The preservation conditions are: MES culture solution, temperature 23 ± 1 °C, light intensity 4 μmol photons / (m 2 ·s), light cycle 12L︰12D, and replace 1 / 2 of the MES culture solution every 180 d; Use a pulverizer to cut the filaments into pieces about 100 μm in length, then inoculate them into a culture dish and culture for 10 d. Then use a short-wave ultraviolet lamp tube as the irradiation source for ultraviolet irradiation, and control the irradiation dose of the ultraviolet irradiation to be 20 - 50 J / m 2 ; 2) Add 5 mL of mutagen to the culture dish, treat the filaments after ultraviolet irradiation in step 1) with the mutagen for 25 min, then wash the culture dish with MES culture solution to remove the residual mutagen, then supplement the MES culture solution, and culture at a temperature of 20 °C, light intensity of 2500 Lux, light cycle 12L︰12D, and replace the culture solution every 3 d; 3) Use a tissue homogenizer to cut the filaments after mutagenic treatment into small segments, select and place them into a 48-well plate, culture in the dark at 20 °C for 3 d, and then gradually transfer them to normal light at 50 μmol photons / (m 2 ·s) for culture. After 1 month, count the survival amount of the filaments, and transfer the surviving filaments to a cell culture flask. Select the samples with more and larger thalli as experimental materials for screening to obtain mutant strains, and continue to culture to obtain thalli.

[0048] Control Group 1 Preparation of MES culture medium: Weigh accurately 1.4 g of sodium nitrate, 0.2 g of sodium glycerophosphate, 2 g of tris(hydroxymethyl)aminomethane, 0.3 g of EDTA-Na2, 0.25 g of boric acid, 0.035 g of MnCl 2· 4H2O, 12.5 mg of FeCl3·6H2O, 2.5 mg of ZnCl2, 1 mg of CoCl2·6H2O, and 60 mg of ferric citrate, add them to a sterile cup, and make up the volume to 500 mL with ultrapure water to obtain the stock solution of MES culture medium; add 20 mL of the stock solution of MES culture medium to 1000 mL of seawater (specific gravity 1.022), then add copper sulfate and mix evenly. Control the content of copper sulfate in the MES culture medium to be 10 μg / L.

[0049] Preparation of the inducer: Add nitrosoguanidine to 1000 mL of sterilized seawater (specific gravity 1.022), and make up the volume after dissolution. Control the concentration of nitrosoguanidine in the inducer to be 20 μg / mL.

[0050] The mutagenesis breeding method of Porphyra haitanensis in this control group includes the following steps: 1) Preserve the filaments of wild Porphyra haitanensis (NH, collected from Pingtan County, Fujian Province) in the laboratory for future use. The preservation conditions are: MES culture medium, temperature 23 ± 1 °C, light intensity 4 μmol photons / (m 2 ·s), photoperiod 12L︰12D, and replace 1 / 2 of the MES culture medium every 180 d; Chop the filaments with a pulverizer to a length of about 100 μm, then inoculate them into a culture dish and culture for 10 d, and then use a short-wave ultraviolet lamp tube as the irradiation source for ultraviolet irradiation. Control the irradiation dose of ultraviolet irradiation to be 20 - 50 J / m 2 ; 2) Add 5 mL of the mutagen to the culture dish, treat the filaments after ultraviolet irradiation in step 1) with the mutagen for 25 min, then wash the culture dish with MES culture medium to remove the residual mutagen, and then supplement the MES culture medium. Incubate at a temperature of 20 °C, light intensity of 2500 Lux, photoperiod 12L︰12D, and replace the culture medium every 3 d; 3) Cut the filaments after mutagenesis treatment into small segments with a tissue homogenizer, select and place them in a 48-well plate, culture them in the dark at 20 °C for 3 d, and then gradually transfer them to normal light at 50 μmol photons / (m 2 ·s) for culture. After 1 month, count the survival amount of the filaments, and transfer the surviving filaments to a cell culture flask. Select the samples with more and larger thalli as experimental materials for screening to obtain mutant strains, and continue to culture to obtain thalli.

[0051] Control Group 2 Preparation of MES culture medium: Accurately weigh 1.4 g of sodium nitrate, 0.2 g of sodium glycerophosphate, 2 g of tris (hydroxymethyl) aminomethane, 0.3 g of EDTA-Na2, 0.25 g of boric acid, 0.035 g of MnCl 2· 4H2O, 12.5 mg of FeCl3·6H2O, 2.5 mg of ZnCl2, 1 mg of CoCl2·6H2O, and 60 mg of ferric citrate, add them to a sterile cup, and make up the volume to 500 mL with ultrapure water to obtain the stock solution of MES culture medium; add 20 mL of the stock solution of MES culture medium to 1000 mL of seawater (specific gravity 1.022), then add copper sulfate and mix evenly. Control the content of copper sulfate in the MES culture medium to be 10 μg / L.

[0052] Preparation of the inducer: Add nitrosoguanidine to 1000 mL of sterilized seawater (specific gravity 1.022), and make up the volume after dissolution. Control the concentration of nitrosoguanidine in the inducer to be 20 μg / mL.

[0053] The method for mutagenic breeding of Porphyra haitanensis in this control group includes the following steps: 1) Preserve the filaments of wild Porphyra haitanensis (NH, collected from Pingtan County, Fujian Province) in the laboratory for later use. The preservation conditions are: MES culture medium, temperature 23 ± 1°C, light intensity 4 μmol photons / (m 2 ·s), light cycle 12L:12D, and replace 1 / 2 of the MES culture medium every 180 days; 2) Cut the filaments into pieces about 100 μm in length with a pulverizer, then inoculate them into a culture dish and culture for 10 days; then add 5 mL of the mutagen to the culture dish, treat with the mutagen for 25 minutes, then wash the culture dish with MES culture medium to remove the residual mutagen, then supplement the MES culture medium, and culture at a temperature of 20°C, light intensity of 2500 Lux, light cycle 12L:12D, and replace the culture medium every 3 days; 3) Cut the mutagen-treated filaments into small segments with a tissue homogenizer, select and place them in a 48-well plate, and culture them in the dark at 20°C for 3 days, and then gradually transfer them to normal light of 50 μmol photons / (m 2 ·s) for culture. After 1 month, count the survival amount of the filaments, and transfer the surviving filaments to a cell culture flask. Select the samples with more and larger thalli as experimental materials for screening to obtain mutant strains, and continue to culture to obtain thalli.

[0054] Performance detection 1. According to the mutagenic breeding method of Porphyra haitanensis in Examples 1-2 and Control Groups 1-2, the mutation rate and mortality rate of the conchocelis were statistically analyzed. The mutation rate = S1 / S2, where S1 is the number of conchocelis at the beginning of the experiment and S2 is the number of conchocelis that mutated at the end of the experiment; the mortality rate = S3 / S4, where S3 is the number of conchocelis at the beginning of the experiment and S4 is the number of conchocelis that died at the end of the experiment. The results are shown in Table 1. It can be seen that the breeding method of this application has a more efficient and specific induction effect and can obtain stable mutants.

[0055] Table 1 Mutagenesis data of conchocelis in the mutagenic breeding method of Porphyra haitanensis in Examples 1-2 and Control Groups 1-2 of this application Serial number Mortality rate / % Mutation rate / % Example 1 49.36 15.37 Example 2 50.58 18.12 Control group 1 38.64 10.93 Control group 2 20.15 7.11

[0056] 2. The mutant conchocelis filaments of Examples 1-2 and Control Groups 1-2 were broken up, and 0.07 g (fresh weight) was taken and cultured at 20 °C for 6 weeks. The development of the conchocelis filaments was observed under a microscope. The results are as Figure 1 shown, where a is Example 1, b is Example 2, c is Control Group 1, and d is Control Group 2. It can be seen that the mutant conchocelis filaments obtained by the breeding method of this application have a better growth and development state and are more likely to form carposporangial branches than the control groups.

[0057] Although the present application has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or perform equivalent replacements for some of the technical features. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present application shall be included within the protection scope of the present invention.

Claims

1. A mutagenesis breeding method based on Pyropia haitanensis, characterized in that: It includes the following steps: 1) Ultraviolet irradiation is carried out on the conchocelis of Pyropia haitanensis; 2) The conchocelis after ultraviolet irradiation in step 1) is treated with a mutagen. The mutagen includes nitrosoguanidine and a slow-release solution, and the slow-release solution includes bleomycin and pingyangmycin; 3) The conchocelis after mutagenesis treatment is screened to obtain mutant strains, and the mutant strains are continuously cultured to obtain thalli.

2. The mutagenesis breeding method of Porphyra haitanensis according to claim 1, wherein: In step 1), the conchocelis is statically cultured with MES culture medium before ultraviolet irradiation, and the MES culture medium contains 5 - 15 μg / L of copper sulfate.

3. The method for induced mutation breeding of Porphyra haitanensis according to claim 1, characterized in that: In the said step 1), the irradiation dose of ultraviolet irradiation is 20 - 50 J / m 2 .

4. The mutagenesis breeding method of Porphyra haitanensis according to claim 1, characterized in that: In step 2), the mutagenesis time is 20 - 60 min.

5. The method for mutagenic breeding of Porphyra haitanensis according to claim 1, characterized in that: In step 2), the concentration of nitrosoguanidine in the mutagen is 15 - 60 μg / mL.

6. The mutagenesis breeding method of Porphyra haitanensis according to claim 1, characterized in that: In step 2), the concentration of bleomycin in the slow-release solution is 300 - 500 ppm.

7. The mutagenesis breeding method of Porphyra haitanensis according to claim 1, characterized in that: In step 2), the concentration of pingyangmycin in the slow-release solution is 100 - 200 ppm.

8. The mutagenesis breeding method of Porphyra haitanensis according to claim 1, wherein: In step 2), the slow-release solution is prepared by a method including the following steps: S1: Cyclodextrin, polyethylene glycol, and deionized water are mixed evenly to prepare a base solution; S2: Amino polystyrene particles are placed in domestic sewage, statically cultured, then washed with ultrapure water, sterilized, and dried to obtain biofilm@amino polystyrene particles; S3: Biofilm@amino polystyrene particles, bleomycin, and pingyangmycin are added to the base solution and mixed evenly.

9. The mutagenesis breeding method of Porphyra haitanensis according to claim 8, characterized in that: In step S2, the average particle size of the amino polystyrene particles is 15 - 80 μm.

10. The method for mutagenic breeding of Porphyra haitanensis according to claim 8, characterized in that: In step S3, the content of biofilm@amino polystyrene particles in the slow-release solution is 0.5 - 0.85 wt%.

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