Porphyra haitanensis pure line seedling cultivation method

Abstract

The invention relates to the technical field of porphyra haitanensis cultivation, in particular to a porphyra haitanensis pure line seedling cultivation method. The method comprises the following steps: selecting porphyra haitanensis samples, classifying the porphyra haitanensis samples according to expression characters, hybridizing the porphyra haitanensis samples with different characters, carrying out induced culture on a hybrid product, and carrying out multi-generation selfing to improve the homozygous degree of the porphyra haitanensis so as to obtain the porphyra haitanensis homozygous strain with stable genetic background and uniform characters. According to the method, genetic recombination and genetic variation creation of the porphyra haitanensis are achieved in an initial sexual propagation mode of the porphyra haitanensis, then porphyra haitanensis individuals with comprehensive excellent characters are screened out, then excellent genotypes of the porphyra haitanensis are rapidly amplified in a vegetative propagation amplification mode, character separation is avoided, and finally, a secondary sexual propagation mode is adopted. Recessive harmful mutation accumulated in the asexual propagation amplification process of the porphyra haitanensis is eliminated, and the porphyra haitanensis homozygote with genetic stability is obtained, so that the yield of the porphyra haitanensis is ensured.

Description

Technical Field

[0001] The present invention relates to the technical field of cultivation of Porphyra haitanensis, and specifically, to a method for cultivating pure-line seedlings of Porphyra haitanensis. Background Art

[0002] Porphyra haitanensis is a kind of seaweed rich in nutrients, with extremely high iodine content, which has a significant effect on treating "goiter" caused by iodine deficiency. Its unique function of softening hard lumps also shows practical value in relieving other stagnant masses.

[0003] Genetically stable seedlings of Porphyra haitanensis usually have stronger disease resistance and stress resistance, and can better resist the invasion of pests and diseases, thereby improving the success rate of aquaculture and economic benefits. Therefore, in order to ensure the genetic stability of seedlings and increase the yield, it is necessary to cultivate pure-line seedlings of Porphyra haitanensis. For example, CN105993909B involves a method for cultivating pure-line seedlings of Porphyra haitanensis. This method selects single fronds with known excellent traits, takes single cells from their minute tissues for single-cell cloning culture to directly germinate into filaments, and then through step-by-step propagation of the filaments until they replace carpospores and are transplanted onto shells for the cultivation of pure-line seedlings.

[0004] In the above cultivation method, single cells of fronds with excellent traits are selected, and then the obtained single cells are used for purebred cultivation. This process of separating and cultivating purebreds from single cells of fronds essentially uses the totipotency of somatic cells for asexual reproduction. However, this cultivation method by asexual reproduction makes the genes of the offspring of Porphyra haitanensis exactly the same as those of the parent. During the cultivation process of Porphyra haitanensis, harmful genes accumulate generation by generation, resulting in low genetic stability of Porphyra haitanensis seedlings, affecting key physiological functions such as photosynthesis efficiency and cell division speed, further leading to slow growth, thinner or fragile thalli, and the decline of cell division ability and the reduction of photosynthetic pigment content due to long-term asexual reproduction, which affects the nutrient accumulation and growth rate of Porphyra haitanensis. Summary of the Invention

[0005] The purpose of the present invention is to avoid the genetic instability of Porphyra haitanensis seedlings caused by the accumulation of harmful genes generation by generation due to long-term asexual reproduction, thereby affecting the yield of Porphyra haitanensis.

[0006] The purpose of the present invention is to provide a method for cultivating pure-line seedlings of Porphyra haitanensis. By combining sexual reproduction and asexual reproduction, on the premise of ensuring genetic stability, homozygous excellent individuals are selected, and then large-scale production of pure-line seedlings is carried out to avoid hybrid degradation, thereby avoiding the decline in the yield of Porphyra haitanensis.

[0007] To achieve the above purpose, the present invention aims to provide a method for cultivating pure-line seedlings of Porphyra haitanensis, including the following steps:

[0008] Step S1: First, collect the germplasm resources of wild Pyropia haitanensis. After selecting Pyropia haitanensis samples and cleaning them, isolate single thalli from the Pyropia haitanensis samples, and then cultivate and amplify the thalli to form a Pyropia haitanensis population.

[0009] Harvest the Pyropia haitanensis population, and classify the harvested Pyropia haitanensis according to the leaf morphology, stress resistance, and growth rate shown by the Pyropia haitanensis to obtain a trait sample library.

[0010] Step S2: Use Pyropia haitanensis with different traits in the trait sample library as parents, cultivate the parental filaments to obtain parental thalli.

[0011] Then select male and female thalli with different traits for hybridization. After completing the hybridization treatment, continue to cultivate the female thalli until the female thalli produce carpospores.

[0012] Step S3: Continuously culture the above-mentioned carpospores. After the carpospores pass through the filamentous and conchospore stages to form thalli, first perform surface sterilization on the thalli, then quickly freeze the thalli with liquid nitrogen, and then obtain thallus single cells through cell separation.

[0013] Induce the culture of thallus single cells to the stage of regenerated bud differentiation to form cell clusters, and use the cell clusters for asexual reproduction to obtain a large number of homologous Pyropia haitanensis.

[0014] Step S4: Treat the thalli of the homologous Pyropia haitanensis obtained in Step S3 above by ultrasonic wave to induce the combination of male and female gametes of the same plant, and improve the homozygosity through multiple generations of self-crossing to obtain a homozygous strain with a stable genetic background and uniform traits.

[0015] As a further improvement of this technical solution, in Step S1, collect Pyropia haitanensis thalli in the high tide zone, middle tide zone, and low tide zone of the intertidal zone, and the cleaning treatment is to wash the Pyropia haitanensis thalli with sterilized seawater to remove epiphytic organisms.

[0016] As a further improvement of this technical solution, in Step S1, the leaf morphology is the length-width ratio, edge serration degree, thickness, and pigment content of the leaf; the stress resistance includes high temperature test and low salt test. The high temperature test is to treat the Pyropia haitanensis at 32 °C for 48 hours, and then measure the decline rate of the maximum photochemical efficiency of PSII. The growth rate test method is to regularly measure the leaf expansion area.

[0017] As a further improvement of this technical solution, in Step S2, the cultivation method of the parental filaments is to promote the formation of sporangia through temperature oscillation, collect free spores and inoculate them onto the shell matrix, and then trigger spore release through light and temperature regulation and physical stimulation, collect the spores and spray the spores onto the seedling curtain for attachment cultivation until the parental thalli are obtained.

[0018] As a further improvement of the technical solution, the light and temperature regulation is to culture under continuous light for 12 - 18 days, then add a solution containing calcium ion solution at 15°C, and synchronously perform physical stimulation during the light and temperature regulation. The physical stimulation is to periodically apply pulsed water flow with a flow rate of 10 - 20 cm / s to the seedling curtain.

[0019] As a further improvement of the technical solution, in step S2, the hybridization treatment method is to cut the male thallus into multiple fragments with equal areas, then mix the fragments with the intact female thallus and let it stand for treatment. After 24 hours, use sterilized seawater to rinse and remove the sperm and fragments not bound to the female thallus;

[0020] After the female thallus produces carpospores, use a negative pressure adsorption device to treat the female thallus to obtain carpospores.

[0021] As a further improvement of the technical solution, the area of the fragment is 30 - 40% of the area of the intact female thallus.

[0022] As a further improvement of the technical solution, in step S3, the method of cell separation is to perform low-temperature grinding on the thallus, and then obtain intact thallus single cells through density gradient centrifugation, and inoculate the thallus single cells into the culture medium.

[0023] As a further improvement of the technical solution, the grinding frequency during low-temperature grinding is 40 - 55 Hz.

[0024] As a further improvement of the technical solution, in step S4, the ultrasonic treatment parameters are 30 - 50 KHz, and the treatment time is 3 - 8 min.

[0025] In the present invention, genetic recombination of Porphyra haitanensis is achieved through the initial sexual reproduction method to create genetic variation, then individuals of Porphyra haitanensis with comprehensive excellent traits are screened out, and then the excellent genotypes are rapidly amplified by asexual reproduction amplification to avoid trait segregation. Finally, the secondary sexual reproduction method is adopted to eliminate the recessive harmful mutations accumulated during the asexual reproduction amplification process and obtain genetically stable Porphyra haitanensis homozygotes, thereby ensuring the yield of Porphyra haitanensis.

[0026] Compared with the prior art, the beneficial effects of the present invention:

[0027] In this method for cultivating pure-line seedlings of Porphyra haitanensis, first, the gene pool is expanded by batch selection of Porphyra haitanensis, classified according to the phenotypic traits of Porphyra haitanensis, and Porphyra haitanensis with phenotypic traits of high leaf length-width ratio and high pigment content is preferentially selected as the parent. Then, genetic recombination of Porphyra haitanensis is achieved through the initial sexual reproduction method to create genetic variation, and then individuals of Porphyra haitanensis with good comprehensive phenotypic traits, that is, high growth rate and high pigment content, are screened out;

[0028] Then, the selected individuals of Porphyra haitanensis are used as female parents, and the cell clusters induced from the female parents are used for asexual reproduction to rapidly amplify excellent genotypes and avoid trait segregation. Finally, ultrasonic treatment is used to promote the combination of male and female gametes of the same thallus plant, and a pure line is cultivated through sexual reproduction by multi-generation self-crossing, so as to eliminate the recessive harmful mutations accumulated during the asexual reproduction amplification process and obtain a genetically stable homozygote of Porphyra haitanensis, thereby ensuring the yield of Porphyra haitanensis. Brief Description of the Drawings

[0029] Figure 1 This is the overall flow chart of the present invention. Detailed Embodiment

[0030] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the drawings in the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0031] In the existing method of cultivating pure-line seedlings by selecting single thallus with known excellent traits, taking single cells of its tiny tissues for single-cell cloning culture to directly germinate into filaments, and then gradually expanding the filaments until replacing the carpospores and transplanting them onto shells, the essence is to use the totipotency of somatic cells for asexual reproduction, so that the genes of the offspring of Porphyra haitanensis produced are exactly the same as those of the parent. However, long-term asexual reproduction will accumulate harmful genes, resulting in a decrease in the genetic stability of Porphyra haitanensis seedlings, and further leading to a decline in cell division ability and a decrease in photosynthetic pigment content, affecting the nutrient accumulation and growth rate of Porphyra haitanensis.

[0032] Therefore, please refer to Figure 1 As shown, the purpose of the embodiment of the present invention is to provide a method for cultivating pure-line seedlings of Porphyra haitanensis, including the following steps:

[0033] Step S1: First, collect wild germplasm resources of Porphyra haitanensis. Batch-select and collect at least 30 wild Porphyra haitanensis thalli in the high tide zone, middle tide zone, and low tide zone of the intertidal zone as Porphyra haitanensis samples. After cleaning treatment, separate single thalli from the Porphyra haitanensis samples, and then induce the thalli to form filaments and cultivate and amplify them to form a Porphyra haitanensis population. Among them, the cleaning treatment is to wash the Porphyra haitanensis thalli with sterilized seawater to remove epiphytic organisms and prevent the interference of miscellaneous bacteria in the subsequent treatment process;

[0034] Harvest the Porphyra haitanensis population, and classify the harvested Porphyra haitanensis according to the leaf morphology, stress resistance, and growth rate shown by the Porphyra haitanensis to obtain a trait sample library;

[0035] It should be noted that the leaf morphology includes the length-width ratio, edge serration degree, thickness, and pigment content of the leaf. Among them, in order to improve the working efficiency of the entire process, the ImageJ software + AI edge recognition technology is selected to analyze the length-width ratio and edge serration degree according to the leaf image, and the acetone extraction method is used to measure the pigment content in the leaf by measuring chlorophyll a and phycoerythrin;

[0036] The stress resistance includes high-temperature test and low-salt test. The high-temperature test is to treat Porphyra haitanensis at 32 °C for 48 hours, and then measure the decline rate of the maximum photochemical efficiency (Fv / Fm) of PSII (PSII is an important component in the conversion of light energy in photosynthesis. It is the second photosynthetic system, mainly responsible for the photolysis of water and the conversion of light energy into chemical energy. PSII is located on the inner membrane of the chloroplast, can absorb the energy from sunlight, and promote the synthesis of ATP through a series of electron transfer processes). If the decline rate of the maximum photochemical efficiency < 15%, it indicates that Porphyra haitanensis is less affected by high temperature and has good high-temperature resistance. The growth rate test method is to regularly measure the leaf expansion area and then calculate the growth rate (growth rate = (leaf area after growth - leaf area before growth) / time interval between two measurements);

[0037] Step S2: Use Porphyra haitanensis with different traits in the trait sample library as parents, culture the parental filaments to obtain parental thalli. Specifically, the formation of sporangia of the parental filaments is promoted by temperature oscillation (15 °C → 25 °C cycle for 3 days). Subsequently, free spores are collected and inoculated onto the shell matrix, and then the release of spores is triggered by light and temperature regulation and physical stimulation. The spores are collected and sprayed onto the seedling curtain for attachment cultivation until the parental thalli are obtained;

[0038] Furthermore, the light and temperature regulation is to continuously culture under continuous light at 20 °C for 12 - 18 days, and then add a solution containing calcium ions at 15 °C to activate the signal pathway of the spores for induced growth. During the light and temperature regulation, physical stimulation is simultaneously carried out. The physical stimulation is to regularly apply a pulsed water flow with a flow rate of 10 - 20 cm / s to the seedling curtain to simulate tidal scouring and continuously trigger the release of spores.

[0039] Select male and female thalli with different traits for hybridization. The hybridization treatment method is to cut the male thalli into multiple fragments with equal area, and then mix the fragments with the intact female thalli and let them stand. After 24 hours, use sterilized seawater to rinse and remove the unbound sperm and fragments on the female thalli. The area of the fragments is 30-40% of the area of the intact female thalli. Fragmentation can increase the surface area of the male tissue, promote the sperm diffusion efficiency, avoid uneven fertilization caused by the occlusion of the intact male thalli, and the fragmented male thalli are more likely to move with the water flow, reducing the phenomenon of non-fertilization in the "dead corner" area. When the area of the male fragments accounts for 30%-40% of the female thalli, the fertilization rate can reach the peak;

[0040] After completing the hybridization treatment, continue to culture the female thalli until the female thalli produce carpospores. After the female thalli produce carpospores, use a negative pressure adsorption device to treat the female thalli to obtain carpospores. Specifically, the negative pressure adsorption device forms a pressure difference of -5 kPa on the surface of the female thalli to adsorb the carpospores;

[0041] Step S3: Continuously culture the above-mentioned carpospores. The carpospores form thalli after passing through the filamentous and conchospore stages. First, perform surface sterilization on the thalli, then quickly freeze the thalli with liquid nitrogen, and then obtain single thallus cells through cell separation. The method of cell separation is to perform cryogenic grinding on the thalli at a grinding frequency of 40-55 Hz, and then obtain complete single thallus cells through density gradient centrifugation, and inoculate the single thallus cells into the culture medium. Here, using the property that the brittleness of substances increases at low temperatures and is easier to break can reduce the difficulty of extracting single thallus cells. Too high a grinding frequency will also cause the ground material, that is, the thalli, to overheat and affect the extraction of single cells;

[0042] Induce the single thallus cells to be cultured until the stage of regenerated bud differentiation to form cell clusters, and use the cell clusters for asexual reproduction to obtain a large number of homologous Porphyra haitanensis;

[0043] Step S4: Treat the thalli of the homologous Porphyra haitanensis obtained in the above step S3 by ultrasonic treatment. The ultrasonic treatment parameters are 30-50 KHz, and the treatment time is 3-8 min. Break the spatio-temporal isolation of the release of male and female gametes, make the gametes of the same thallus mature synchronously, and then facilitate the combination of male and female gametes of the same plant. Improve the homozygosity through multiple generations of self-crossing to obtain a pure line with a stable genetic background and uniform traits, and reduce the trait segregation in subsequent breeding.

[0044] In the present invention, genetic recombination of Pyropia haitanensis is achieved through initial sexual reproduction to create genetic variation, and then individuals of Pyropia haitanensis with comprehensive excellent traits are selected. Subsequently, the excellent genotypes are rapidly amplified by asexual propagation to avoid trait segregation. Finally, the secondary sexual reproduction method is adopted to eliminate the recessive harmful mutations accumulated during the asexual propagation amplification process and obtain homozygous Pyropia haitanensis with genetic stability, thereby ensuring the yield of Pyropia haitanensis.

[0045] A homozygous line is cultivated according to a method for cultivating pure lines of Pyropia haitanensis provided by an embodiment of the present invention. Then, some individuals are randomly selected from the cultivated homozygous lines for continuous cultivation for five generations, and the coefficient of variation (CV < 5% is considered qualified) of key traits (such as growth rate and pigment content) is recorded. (In the embodiments of the present invention, the coefficient of variation is used to quantify the degree of dispersion of traits such as growth rate and pigment content within a population, reflecting genetic stability or environmental response consistency), and the breeding effect of the homozygous lines is evaluated.

[0046] Among them, the growth rate calculation method is to cultivate the homozygous lines in the wild in a rough manner under the conditions of a temperature of 18 - 22°C, a salinity of 28 - 32‰, and a light intensity of 50 - 100 μmol / (m 2 ·s). Select healthy thalli, wash the surface attachments with sterile seawater, and calculate the growth rate according to the change in thallus area. The growth rate = (A2 - A1) / T, where T is the time, A1 is the initial area of the thallus, and A2 is the area of the thallus after T days.

[0047] The pigment content measurement method is to detect the mass ratio of chlorophyll a in the thallus by real-time fluorescence quantitative PCR technology.

[0048] The coefficient of variation measurement method is to select multiple groups of samples for detection, with at least 30 individuals in each group. Detect the growth rate of the samples, calculate the mean and standard deviation of the growth rate of each group of samples, and then calculate the coefficient of variation. The coefficient of variation = (standard deviation of growth rate / mean of growth rate) × 100% (if the coefficient of variation is greater than 20%, it indicates genetic admixture and genetic instability).

[0049] Record the above test results in Table 1.

[0050] Table 1 Growth rate, pigment content, and coefficient of variation of homozygous lines

[0051] <![CDATA[Growth rate / (cm 2 / d)]]> Pigment content / (mg / g) Coefficient of variation / % 1.1 7.76 5.5

[0052] It can be seen from Table 1 that after continuous cultivation for five generations of the cultivated homozygous lines, the growth rate is 1.1 cm 2 / d, the pigment content was 7.76 mg / g, and the coefficient of variation was 5.5%. The growth rate and pigment content were both maintained at a high level. The yield of large-scale cultured Porphyra hainanensis was high, and the coefficient of variation was maintained at a low level, indicating that the homozygous strain was genetically stable, trait separation would not occur, and the breeding evaluation was qualified.

[0053] In summary, the present invention cleverly combines the advantages of genetic recombination and cloning through the three-stage cycle of "sexual-asexual-sexual". Specifically, the gene pool is first expanded by batch selection of Porphyra haitanensis, and the Porphyra haitanensis is classified according to its performance traits, and Porphyra haitanensis with performance traits of high leaf aspect ratio and high pigment content are preferentially selected as parents. Then, the gene recombination of Porphyra haitanensis is achieved through initial sexual reproduction to create genetic variation, and then Porphyra haitanensis individuals with good comprehensive performance traits, i.e., high growth rate and high pigment content are screened out;

[0054] The screened Porphyra haitanensis individuals are then used as mother parents, and asexual reproduction is carried out using the cell clusters induced by the mother parents to quickly amplify excellent genotypes and avoid trait separation. Finally, ultrasonic treatment is used to promote the fusion of male and female gametes in the same thallus plant, and homozygous strains are cultivated through sexual reproduction through multiple generations of self-pollination, thereby eliminating recessive harmful mutations accumulated during asexual reproduction and amplification, and obtaining genetically stable Porphyra haitanensis homozygotes, thereby ensuring the yield of Porphyra haitanensis.

[0055] The above shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited by the above embodiments. The above embodiments and descriptions are only preferred examples of the present invention and are not intended to limit the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, which fall within the scope of the present invention. The scope of protection of the present invention is defined by the attached claims and their equivalents.

Claims

1. A method for cultivating purebred seedlings of Porphyra haitanensis, characterized in that, It includes the following steps: Step S1: After selecting the Porphyra haitanensis samples and cleaning them, isolate single thalli from the Porphyra haitanensis samples, and then cultivate and amplify the thalli to form a Porphyra haitanensis population; Harvest the Porphyra haitanensis population, and classify the harvested Porphyra haitanensis according to the leaf morphology, stress resistance and growth rate shown by the Porphyra haitanensis to obtain a trait sample library; Step S2: Use the Porphyra haitanensis in the trait sample library as parents, and culture the parental filaments to obtain parental thalli; Then select male and female thalli with different traits for hybridization. After completing the hybridization treatment, continue to culture the female thalli until the female thalli produce carpospores; Step S3: Continuously culture the above-mentioned carpospores. After the carpospores pass through the filamentous and conchospore stages to form thalli, first perform surface sterilization treatment on the thalli, then quickly freeze the thalli with liquid nitrogen, and then obtain thallus single cells through cell separation; Induce the culture of thallus single cells to the stage of regeneration bud differentiation to form cell clusters, and use the cell clusters for asexual reproduction to obtain homologous Porphyra haitanensis; Step S4: Induce the combination of male and female gametes of the homologous Porphyra haitanensis thalli through ultrasonic treatment, and improve the homozygosity through multiple generations of self-crossing to obtain a homozygous strain with a stable genetic background and uniform traits.

2. The method for cultivating pure-line seedlings of Porphyra haitanensis according to claim 1, characterized in that: In the said step S1, collect Porphyra haitanensis thalli in the high tide zone, middle tide zone and low tide zone of the intertidal zone respectively, and the cleaning treatment is to wash the Porphyra haitanensis thalli with sterilized seawater to remove epiphytic organisms.

3. The method for cultivating pure-line seedlings of Pyropia haitanensis according to claim 1, characterized in that: In the said step S1, the leaf morphology is the length-width ratio, edge serration degree, thickness and pigment content of the leaf; The stress resistance includes high temperature test and low salt test. The high temperature test is to treat the Porphyra haitanensis at 32 °C for 48 hours, and then measure the decline rate of the maximum photochemical efficiency of PSII. The growth rate test method is to regularly measure the leaf expansion area.

4. The method for cultivating pure line seedlings of Porphyra haitanensis according to claim 1, characterized in that: In the said step S2, the culture method of the parental filaments is to promote the formation of sporangia through temperature oscillation, collect free spores and inoculate them onto the shell matrix, and then trigger the release of spores by means of light and temperature regulation and physical stimulation. Collect the spores and spray the spores onto the nursery curtain for attachment cultivation until the parental thalli are obtained.

5. The method for cultivating pure-line seedlings of Porphyra haitanensis according to claim 4, characterized in that: The said light and temperature regulation is continuous light culture for 12-18 days, and then add a solution containing calcium ion solution at 15 °C. Physical stimulation is carried out synchronously during the light and temperature regulation; The physical stimulation is to regularly apply a pulsed water flow with a flow rate of 10-20 cm / s to the nursery curtain.

6. The method for cultivating pure-line seedlings of Pyropia haitanensis according to claim 1, characterized in that: In the said step S2, the hybridization treatment method is to cut the male thalli into multiple fragments with equal areas, and then mix the fragments with the intact female thalli and let them stand. After 24 hours, use sterilized seawater to wash and remove the unbound sperm and fragments on the female thalli; After the female thalli produce carpospores, use a negative pressure adsorption device to treat the female thalli to obtain carpospores.

7. The method for cultivating pure line seedlings of Porphyra haitanensis according to claim 6, characterized in that: The area of the said fragment is 30-40% of the area of the intact female thalli.

8. The method for cultivating pure line seedlings of Porphyra haitanensis according to claim 1, characterized in that: In the said step S3, the way of cell separation is to perform low-temperature grinding on the thalli, and then obtain complete thallus single cells through density gradient centrifugation, and inoculate the thallus single cells into the culture medium.

9. The method for cultivating pure-line seedlings of Porphyra haitanensis according to claim 8, characterized in that: The grinding frequency during the said low-temperature grinding is 40-55 Hz.

10. The method for cultivating pure line seedlings of Porphyra haitanensis according to claim 1, characterized in that: In the step S4, the ultrasonic treatment parameters are 30 - 50KHz, and the treatment time is 3 - 8 minutes.

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