Mutagenesis breeding method for obtaining bamboo reed cold-resistant mutant based on EMS

Abstract

The invention belongs to the technical field of bamboo reed breeding, and discloses a mutation breeding method for obtaining a bamboo reed cold-resistant mutant based on EMS (Enhanced Mass Spectrometry), which comprises the following steps: step 1, inducing embryogenic callus; step 2, carrying out EMS mutagenesis treatment; step 3, performing EMS stress screening; 4, differentiation and rooting culture of adventitious buds; 5, screening the tissue culture seedlings at low temperature; the mutation obtained through mutagenesis treatment is a permanent change of a genetic structure of an organism and may appear in germ cells and somatic cells of a plant. Physical or chemical mutagenic agents are used to increase the frequency of mutations. In chemical mutagens, EMS is considered to be the most effective mutagen, the EMS can generate a large number of non-lethal point mutations in the whole genome, the mutation frequency is high, the chromosome abnormality frequency is low, the EMS is used for treating embryogenic calluses of the bamboo reeds, Hyp is used for screening, the low-temperature-resistant bamboo reed mutants are obtained, the planting area of the bamboo reeds can be enlarged, and the survival rate of the bamboo reeds is increased. And waste land in a low-temperature region is fully utilized.

Description

Technical Field

[0001] The present invention relates to the technical field of Phragmites australis breeding, and in particular to a mutagenesis breeding method for obtaining cold-resistant mutants of Phragmites australis based on EMS. Background Art

[0002] Arundo donax, a perennial plant of the Poaceae family and genus Arundo, has tall stems and a strong tillering ability. Arundo donax is resilient to adverse conditions and can be cultivated on a large scale on abandoned marginal land. Its harvested stems can replace coal in thermal power generation and can also be processed downstream to produce hydrogen, natural gas, biochar, and other products.

[0003] Large areas of abandoned land in northern my country await development and utilization. Planting Phragmites australis can increase its utilization, boost economic returns, and improve the ecological environment. However, low temperatures are a major factor limiting its growth and distribution. Breeding new cold-tolerant Phragmites australis varieties is the most effective solution. Phragmites australis reproduces asexually, making hybridization difficult. Through mutation breeding, Phragmites australis varieties with tolerance to biotic and abiotic stress factors can be developed, enriching the existing germplasm resources.

[0004] Ethyl methanesulfonate (EMS) is currently the most widely used and effective chemical mutagen in crop mutation breeding. Its mutagenic effects are characterized by low cost, ease of operation, high mutation rate, minimal damage to materials, and a broad mutation spectrum. The accumulation of osmotic agents after low-temperature stress in plants enhances their cold tolerance and freezing resistance. Proline is the most widespread of these agents, retaining water within cells under conditions of osmotic stress. It also stabilizes cell membranes, cytoplasmic proteins, and the cell membrane itself. After recovery from stress, proline becomes a source of carbon, nitrogen, energy, and reducing equivalents, facilitating the restoration of normal plant metabolism. Free hydroxyproline (Hyp) is a phytotoxic analog of proline in plants. The negative effects of adding Hyp to culture media can be mitigated by increasing intracellular proline levels. Therefore, this suggests that regenerating cells surviving on Hyp culture media may have elevated proline levels, thereby improving freezing tolerance. Summary of the Invention

[0005] In order to solve the above problems existing in the prior art, the present invention aims to provide an external-use composition and a preparation method and application thereof.

[0006] The technical solution adopted in the present invention is:

[0007] A mutagenesis breeding method for obtaining a cold-resistant mutant of Phragmites australis based on EMS, comprising the following steps:

[0008] Step 1: Induction of embryonic callus;

[0009] Select young inflorescences of Phragmites australis that are one year old or older and cut them into several small segments of 3 to 5 cm in length. After surface disinfection, insert them into callus induction medium of inflorescences and culture them in a culture room at a temperature of 25°C ± 2°C and a humidity of 70 to 80% for 90 days in the dark to form callus tissue.

[0010] The formula of the callus induction medium is as follows: 4.73 g of MS medium powder, 2.0-4.0 mg / L of 2,4-dichlorophenoxyacetic acid, 0.5-2.0 mg / L of kinetin, 30 g / L of sucrose, and 7 g / L of agar, and the pH value of the mixed solution is maintained at 5.8, and high-temperature sterilization is performed;

[0011] Step 2, EMS mutagenesis treatment;

[0012] The embryonic callus induced from the inflorescence was selected to have a diameter of 0.5 cm and immersed in a 0.6-1.0% EMS solution. The solution was shaken at 25°C and 80 rpm for 5-8 hours. After the treatment, the callus was washed three times with sterile water. The washed callus was blotted dry with sterile absorbent paper. At the same time, the embryonic callus treated with sterile water without EMS was used as a control callus.

[0013] Step 3, hydroxyproline (EMS) stress screening;

[0014] The callus tissue after EMS induction treatment was inoculated into callus proliferation medium for recovery culture for 30 days, and dark cultured in a constant temperature and humidity culture room. At the same time, the control callus tissue in step S2 was inoculated into callus proliferation medium without Hyp as a control, and 20-40mM Hyp was added to the callus proliferation medium;

[0015] The formula of the callus proliferation medium is as follows: 4.73 g of MS medium powder, 0.5-1.0 mg / L of 2,4-dichlorophenoxyacetic acid, 0.5 mg / L of 6-benzylaminopurine, 30 g / L of sucrose, and 7 g / L of agar, the pH value of the mixed solution is maintained at 5.8, and high-temperature sterilization is performed;

[0016] Step 4: differentiation and rooting of adventitious buds;

[0017] The callus tissue that survived the screening in steps S2 and S3 was inoculated into a medium for adventitious bud differentiation, and the cells were placed in a culture room for 60 days to obtain adventitious buds. The differentiated adventitious buds were inoculated into a rooting medium, and the cells were placed in a culture room for 30 days under a light intensity of 2000 lx and a light duration of 12 h / d to induce rooting, thereby obtaining complete tissue culture seedlings of Phragmites australis and their clones.

[0018] The formula of adventitious bud differentiation medium is: 4.73g MS medium powder, 4.0-8.0mg / L 6-benzylaminopurine, 0.4-0.8mg / L naphthaleneacetic acid, 30g / L sucrose, and 7g / L agar.

[0019] The formula of the rooting medium is: 4.73g of MS medium powder, 0.5-1.0mg / L of naphthaleneacetic acid, 30g / L of sucrose, and 7g / L of agar;

[0020] The pH value of the mixed solution was maintained at 5.8 and sterilized at high temperature;

[0021] Step 5, low temperature screening of tissue culture seedlings;

[0022] The complete plants of the tissue culture seedlings of Phragmites australis obtained in step 4 and their asexual lines were subjected to low-temperature acclimation screening, and the control callus tissue was used as a control for low-temperature acclimation treatment. Starting from 20°C in a low-temperature incubator, the temperature was lowered at a rate of 1°C per hour until the temperature dropped to 1°C. Subsequently, the plants were acclimated and cultured for 14 days at a periodic temperature of 14h / 3°C during the day and 10h / 1°C at night. After 14 days, the plants were taken out for observation and the dehydration and wilting status of the plants were recorded to obtain a cold-resistant mutant of Phragmites australis.

[0023] Furthermore, in step 1, the formula of the callus induction medium includes: 3.0 mg / L of 2,4-dichlorophenoxyacetic acid and 1.0 mg / L of kinetin.

[0024] Furthermore, in step 3, 30 mM Hyp is added to the callus proliferation medium, and the formula of the callus proliferation medium contains 1.0 mg / L of 2,4-dichlorophenoxyacetic acid.

[0025] Furthermore, in step 4, the formula of the adventitious bud differentiation medium includes 6.0 mg / L of 6-benzylaminopurine and 0.6 mg / L of naphthaleneacetic acid.

[0026] Furthermore, in step 4, the formula of the rooting medium contains 0.8 mg / L of naphthaleneacetic acid.

[0027] Furthermore, the culture conditions of the constant temperature and humidity culture room are: temperature of 25°C ± 2°C, humidity of 70-80%.

[0028] Furthermore, in step 2, a mass of 0.5 cm in diameter of the embryonic callus induced from the inflorescence is selected and immersed in a 0.8% EMS solution, and shaken at 25° C. and 80 rpm for 6 hours.

[0029] The beneficial effects of the present invention are:

[0030] The present invention can cultivate plant varieties that are tolerant to biotic and abiotic stress factors through mutagenesis breeding. The mutation obtained by mutagenesis is a permanent change in the genetic structure of an organism, which may appear in the reproductive cells and somatic cells of the plant. Physical or chemical mutagens are used to increase the frequency of mutations. Among chemical mutagens, EMS is considered to be the most effective mutagen. EMS can produce a large number of non-lethal point mutations throughout the genome, and it causes a high mutation frequency in random regions and a low frequency of chromosomal abnormalities. The present method uses EMS to treat the embryonic callus of Phragmites australis and simultaneously uses Hyp, an analog of proline, for screening to obtain low-temperature-resistant Phragmites australis mutants, which can expand the planting area of Phragmites australis and make full use of abandoned land in low-temperature areas. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 is a flow chart of the method of the present invention;

[0032] Figure 2 These are photos of the tissue culture seedlings obtained by low temperature treatment mutagenesis Example 2 and the control group. DETAILED DESCRIPTION

[0033] The present invention will be further described below with reference to the accompanying drawings and specific embodiments.

[0034] Example

[0035] A mutagenesis breeding method for obtaining a cold-resistant mutant of Phragmites australis based on EMS, comprising the following steps:

[0036] Step 1: Induction of embryonic callus;

[0037] Select young inflorescences of Phragmites australis that are one year old or older and cut them into several small segments of 3 to 5 cm in length. After surface disinfection, insert them into callus induction medium of inflorescences and culture them in a culture room at a temperature of 25°C ± 2°C and a humidity of 70 to 80% for 90 days in the dark to form callus tissue.

[0038] The formula of the callus induction medium is as follows: 4.73 g of MS medium powder, 2.0-4.0 mg / L of 2,4-dichlorophenoxyacetic acid, 0.5-2.0 mg / L of kinetin, 30 g / L of sucrose, and 7 g / L of agar, and the pH value of the mixed solution is maintained at 5.8, and high-temperature sterilization is performed;

[0039] Step 2, mutagenesis treatment with ethyl methanesulfonate (EMS);

[0040] The embryonic callus induced from the inflorescence was selected to have a diameter of 0.5 cm and immersed in a 0.6-1.0% EMS solution. The solution was shaken at 25°C and 80 rpm for 5-8 hours. After the treatment, the callus was washed three times with sterile water. The washed callus was blotted dry with sterile absorbent paper. At the same time, the embryonic callus treated with sterile water without EMS was used as a control callus.

[0041] Step 3, EMS stress screening;

[0042] The callus tissue after EMS induction treatment was inoculated into callus proliferation medium for recovery culture for 30 days, and then cultured in the dark in a constant temperature and humidity culture room. At the same time, the control callus tissue in step S2 was inoculated into callus proliferation medium without Hyp (free hydroxyproline) as a control, and 20-40mM Hyp was added to the callus proliferation medium;

[0043] The formula of the callus proliferation medium is as follows: 4.73 g of MS medium powder, 0.5-1.0 mg / L of 2,4-dichlorophenoxyacetic acid, 0.5 mg / L of 6-benzylaminopurine, 30 g / L of sucrose, and 7 g / L of agar, the pH value of the mixed solution is maintained at 5.8, and high-temperature sterilization is performed;

[0044] Step 4: differentiation and rooting of adventitious buds;

[0045] The callus tissue that survived the screening in steps S2 and S3 was inoculated into a medium for adventitious bud differentiation, and the cells were placed in a culture room for 60 days to obtain adventitious buds. The differentiated adventitious buds were inoculated into a rooting medium, and the cells were placed in a culture room for 30 days under a light intensity of 2000 lx and a light duration of 12 h / d to induce rooting, thereby obtaining complete tissue culture seedlings of Phragmites australis and their clones.

[0046] The formula of adventitious bud differentiation medium is: 4.73g MS medium powder, 4.0-8.0mg / L 6-benzylaminopurine, 0.4-0.8mg / L naphthaleneacetic acid, 30g / L sucrose, and 7g / L agar.

[0047] The formula of the rooting medium is: 4.73g of MS medium powder, 0.5-1.0mg / L of naphthaleneacetic acid, 30g / L of sucrose, and 7g / L of agar;

[0048] The pH value of the mixed solution was maintained at 5.8 and sterilized at high temperature;

[0049] Step 5, low temperature screening of tissue culture seedlings;

[0050] The intact plants of the tissue culture seedlings of Phragmites australis obtained in step 4 and their clones were subjected to low-temperature acclimation screening, and the control callus tissue was used as a control for low-temperature acclimation treatment. The temperature was lowered from 20°C in a low-temperature incubator at a rate of 1°C per hour until the temperature dropped to 1°C. The plants were then acclimated and cultured for 14 days at a cycle temperature of 14h / 3°C during the day and 10h / 1°C at night. After 14 days, the plants were taken out for observation and recording of dehydration and wilting status, thereby obtaining cold-resistant mutants of Phragmites australis.

[0051] In the above steps, the culture conditions of the constant temperature and humidity culture room are: temperature of 25°C ± 2°C, humidity of 70-80%.

[0052] Example 1

[0053] Step 1: Induction of embryonic callus

[0054] Young inflorescences of Phragmites australis that are one year old or older are selected, cut into 3-5 cm pieces, surface-sterilized, and inoculated into an inflorescence callus induction medium. The inflorescences are cultured in a culture room for 90 days to form callus tissue. The culture conditions are as follows: temperature of 25°C ± 2°C, humidity of 70-80%, dark culture, induction medium formula of 4.73 g MS medium powder, 4.0 mg / L of 2,4-dichlorophenoxyacetic acid, 0.5 mg / L of kinetin, 30 g / L of sucrose, 7 g / L of agar, pH 5.8, and high temperature sterilization.

[0055] Step 2: EMS mutagenesis treatment

[0056] Embryogenic calli induced from inflorescences were selected and 0.5 cm in diameter. They were immersed in 0.6% EMS solution and shaken at 25°C and 80 rpm for 8 h. After treatment, they were washed three times with sterile water and blotted dry with sterile absorbent paper. Embryogenic calli treated with sterile water without EMS were used as controls.

[0057] Step 3: Hyp stress screening

[0058] The callus tissue after EMS induction treatment was inoculated into a callus proliferation medium for recovery culture for 30 days. The culture conditions were as follows: temperature of 25°C ± 2°C, humidity of 70-80%, and dark culture. At the same time, the control callus tissue in step 2 was inoculated into a culture medium without Hyp as a control. 20 mM Hyp was added to the callus proliferation medium. The proliferation medium formula was 4.73 g of MS medium powder, 0.5 mg / L of 2,4-dichlorophenoxyacetic acid, 0.5 mg / L of 6-benzylaminoadenine, 30 g / L of sucrose, and 7 g / L of agar. The pH was 5.8 and the culture medium was sterilized at high temperature.

[0059] Step 4: Differentiation and rooting of adventitious buds

[0060] The callus tissue that survived EMS mutagenesis and Hyp screening was inoculated into an adventitious bud differentiation medium, and cultured in a culture room for 60 days to obtain adventitious buds. The adventitious bud differentiation medium formula was 4.73 g of MS medium powder, 4.0 mg / L of 6-benzylaminoadenine, 0.4 mg / L of naphthaleneacetic acid, 30 g / L of sucrose, and 7 g / L of agar, with a pH of 5.8, and was sterilized at high temperature. The differentiated adventitious buds were inoculated into a rooting medium, and cultured in a culture room for 30 days to induce rooting, thereby obtaining complete tissue culture seedlings of Phragmites australis and its asexual lines. The rooting medium formula was 4.73 g of MS medium powder, 0.5 mg / L of naphthaleneacetic acid, 30 g / L of sucrose, and 7 g / L of agar, with a pH of 5.8, and was sterilized at high temperature. The culture conditions were: temperature 25°C ± 2°C, humidity 70-80%, light intensity 2000 lx, and photoperiod 12 h / d.

[0061] Step 5: Low temperature screening of tissue culture seedlings

[0062] Robust Phragmites australis tissue culture seedling clones formed by callus differentiation that survived EMS mutagenesis and Hyp stress treatment were selected for low-temperature acclimation screening. At the same time, tissue culture rooted seedlings formed by callus differentiation that were not mutagenized or Hyp stressed were used as controls for low-temperature acclimation treatment. The temperature in a low-temperature incubator was set to start at 20°C and decrease by 1°C every hour until the temperature dropped to 1°C. The low-temperature acclimation culture was continued for 14 days using a temperature change cycle of 14 hours (3°C) during the day and 10 hours (1°C) at night. After 14 days, the plants were taken out for observation and the dehydration and wilting status of the plants were recorded.

[0063] Example 2

[0064] Step 1: Callus induction

[0065] Young inflorescences of Phragmites australis that are one year old or older are selected, cut into 3-5 cm pieces, surface-sterilized, and inoculated into an inflorescence callus induction medium. The inflorescences are then cultured in a culture room for 90 days to form callus tissue. The culture conditions are: temperature of 25°C ± 2°C, humidity of 70-80%, dark culture, and the induction medium is 4.73 g of MS medium powder, supplemented with 3 mg / L of 2,4-dichlorophenoxyacetic acid, 1.0 mg / L of kinetin, 30 g / L of sucrose, and 7 g / L of agar, pH 5.8, and sterilized at high temperature.

[0066] Step 2: EMS mutagenesis treatment

[0067] Embryogenic callus induced from inflorescences was selected and 0.5 cm in diameter. The clumps were immersed in 0.8% EMS solution and shaken at 25°C and 80 rpm for 6 h. After treatment, the callus was washed three times with sterile water and blotted dry with sterile absorbent paper. Embryogenic callus treated with sterile water without EMS was used as a control.

[0068] Step 3: Hyp stress screening

[0069] The callus tissue after EMS induction treatment was inoculated into a callus proliferation medium for recovery culture for 30 days. The culture conditions were as follows: temperature of 25°C ± 2°C, humidity of 70-80%, and dark culture. At the same time, the control callus tissue in step 2 was inoculated into a culture medium without Hyp as a control. 30 mM Hyp was added to the callus proliferation medium. The proliferation medium formula was 4.73 g of MS medium powder, 1.0 mg / L of 2,4-dichlorophenoxyacetic acid, 0.5 mg / L of 6-benzylaminoadenine, 30 g / L of sucrose, and 7 g / L of agar. The pH was 5.8 and the culture medium was sterilized at high temperature.

[0070] Step 4: Differentiation and rooting of adventitious buds

[0071] The callus tissue that survived EMS mutagenesis and Hyp screening was inoculated into an adventitious bud differentiation medium, and cultured in a culture room for 60 days to obtain adventitious buds. The adventitious bud differentiation medium formula was 4.73 g of MS medium powder, 6.0 mg / L of 6-benzylaminoadenine, 0.6 mg / L of naphthaleneacetic acid, 30 g / L of sucrose, and 7 g / L of agar, with a pH of 5.8, and was sterilized at high temperature. The differentiated adventitious buds were inoculated into a rooting medium, and cultured in a culture room for 30 days to induce rooting, thereby obtaining complete tissue culture seedlings of Phragmites australis and its asexual lines. The rooting medium formula was 4.73 g of MS medium powder, 0.8 mg / L of naphthaleneacetic acid, 30 g / L of sucrose, and 7 g / L of agar, with a pH of 5.8, and was sterilized at high temperature. The culture conditions were: temperature 25°C ± 2°C, humidity 70-80%, light intensity 2000 lx, and photoperiod 12 h / d.

[0072] Step 5: Low temperature screening of tissue culture seedlings

[0073] Robust Phragmites australis tissue culture seedling clones formed by callus differentiation that survived EMS mutagenesis and Hyp stress treatment were selected for low-temperature acclimation screening. At the same time, tissue culture rooted seedlings formed by callus differentiation that were not mutagenized or Hyp stressed were used as controls for low-temperature acclimation treatment. The temperature in a low-temperature incubator was set to start at 20°C and decrease by 1°C every hour until the temperature dropped to 1°C. The low-temperature acclimation culture was continued for 14 days using a temperature change cycle of 14 hours (3°C) during the day and 10 hours (1°C) at night. After 14 days, the plants were taken out for observation and the dehydration and wilting status of the plants were recorded.

[0074] Example 3

[0075] Step 1: Callus induction

[0076] Young inflorescences of Phragmites australis that are one year old or older are selected, cut into 3-5 cm pieces, surface-sterilized, and inoculated into an inflorescence callus induction medium. The inflorescences are then cultured in a culture room for 90 days to form callus tissue. The culture conditions are as follows: temperature of 25°C ± 2°C, humidity of 70-80%, dark culture, and the induction medium is 4.73 g of MS medium powder, supplemented with 2 mg / L of 2,4-dichlorophenoxyacetic acid, 2.0 mg / L of kinetin, 30 g / L of sucrose, and 7 g / L of agar, pH 5.8, and sterilized at high temperature.

[0077] Step 2: EMS mutagenesis treatment

[0078] Embryogenic calli induced from inflorescences were selected and 0.5 cm in diameter. They were immersed in 1.0% EMS solution and shaken at 25°C and 80 rpm for 5 h. After treatment, they were washed three times with sterile water and blotted dry with sterile absorbent paper. Embryogenic calli treated with sterile water without EMS were used as controls.

[0079] Step 3: Hyp stress screening

[0080] The callus tissue after EMS induction treatment was inoculated into a callus proliferation medium for recovery culture for 30 days. The culture conditions were as follows: temperature of 25°C ± 2°C, humidity of 70-80%, and dark culture. At the same time, the control callus tissue in step 2 was inoculated into a culture medium without Hyp as a control. 40 mM Hyp was added to the callus proliferation medium. The proliferation medium formula was 4.73 g of MS medium powder, 1.0 mg / L of 2,4-dichlorophenoxyacetic acid, 0.5 mg / L of 6-benzylaminoadenine, 30 g / L of sucrose, and 7 g / L of agar. The pH was 5.8 and the culture medium was sterilized at high temperature.

[0081] Step 4: Differentiation and rooting of adventitious buds

[0082] The callus tissue that survived EMS mutagenesis and Hyp screening was inoculated into a medium for adventitious bud differentiation, and cultured in a culture room for 60 days to obtain adventitious buds. The adventitious bud differentiation medium formula was 4.73 g of MS medium powder, 8.0 mg / L of 6-benzylaminoadenine, 0.8 mg / L of naphthaleneacetic acid, 30 g / L of sucrose, and 7 g / L of agar, with a pH of 5.8, and was sterilized at high temperature. The differentiated adventitious buds were inoculated into a rooting medium, and cultured in a culture room for 30 days to induce rooting, thereby obtaining complete tissue culture seedlings of Phragmites australis and its asexual lines. The rooting medium formula was 4.73 g of MS medium powder, 1.0 mg / L of naphthaleneacetic acid, 30 g / L of sucrose, and 7 g / L of agar, with a pH of 5.8, and was sterilized at high temperature. The culture conditions were: temperature 25°C ± 2°C, humidity 70-80%, light intensity 2000 lx, and photoperiod 12 h / d.

[0083] Step 5: Low temperature screening of tissue culture seedlings

[0084] Robust Phragmites australis tissue culture seedling clones formed by callus differentiation that survived EMS mutagenesis and Hyp stress treatment were selected for low-temperature acclimation screening. At the same time, tissue culture rooted seedlings formed by callus differentiation that were not mutagenized or Hyp stressed were used as controls for low-temperature acclimation treatment. The temperature in a low-temperature incubator was set to start at 20°C and decrease by 1°C every hour until the temperature dropped to 1°C. The low-temperature acclimation culture was continued for 14 days using a temperature change cycle of 14 hours (3°C) during the day and 10 hours (1°C) at night. After 14 days, the plants were taken out for observation and the dehydration and wilting status of the plants were recorded.

[0085] In summary, the rationality of the ratio of each key parameter in the embodiment and the comprehensive analysis of breeding logic show that the ratio of Example 2 is better, and the specific basis is as follows:

[0086] 1. Balance of EMS mutagenesis parameters

[0087] Example 1: EMS concentration 0.6% + treatment for 8h, the concentration is low but the treatment time is long, which may lead to insufficient mutagenesis efficiency;

[0088] Example 3: EMS concentration 1.0% + treatment for 5h. Too high a concentration can easily cause a large number of callus tissues to die;

[0089] Example 2: EMS concentration of 0.8% + treatment for 6 hours is within the commonly used mutagenic concentration range (0.5% to 1.0%). The treatment time and concentration form a synergistic effect, which can not only ensure the mutation rate but also reduce cytotoxic damage.

[0090] 2. Adaptability of Hyp Screening Pressure

[0091] Example 1: The low concentration of 20 mM Hyp may result in insufficient screening pressure for cold-tolerant mutants;

[0092] Example 3: 40mM Hyp concentration is too high and can easily inhibit the growth of normal cells and mutant cells;

[0093] Example 2: The concentration of 30 mM Hyp is an intermediate value, which complies with the principle of "sublethal dose" in plant stress screening and can effectively enrich cold-resistant mutant cells while avoiding an overly strict screening system.

[0094] 3. Scientificity of hormone ratio in culture medium

[0095] 1. Callus induction medium

[0096] Example 1: 2,4-dichlorophenoxyacetic acid 4.0 mg / L + kinetin 0.5 mg / L. The high concentration of 2,4-dichlorophenoxyacetic acid may lead to excessive proliferation of callus tissue and decreased differentiation ability.

[0097] Example 3: 2,4-dichlorophenoxyacetic acid 2.0 mg / L + kinetin 2.0 mg / L. Too high a kinetin concentration may promote direct organ differentiation and be detrimental to embryonic callus formation.

[0098] Example 2: 2,4-dichlorophenoxyacetic acid 3.0 mg / L + kinetin 1.0 mg / L, the ratio of 2,4-dichlorophenoxyacetic acid to cytokinin (kinetin) is close to 3:1, which is in line with the optimal ratio for embryonic callus induction of grass plants and can take into account both callus induction rate and embryonic cell quality.

[0099] 2. Adventitious bud differentiation medium

[0100] Example 1: 6-benzylaminoadenine 4.0 mg / L + naphthaleneacetic acid 0.4 mg / L, the cytokinin concentration is low, which may affect the differentiation efficiency of buds;

[0101] Example 3: 6-benzylaminoadenine 8.0 mg / L + naphthaleneacetic acid 0.8 mg / L. Too high a concentration of 6-benzylaminoadenine can easily lead to weak and vitrified buds.

[0102] Example 2: 6-benzylaminoadenine 6.0 mg / L + naphthaleneacetic acid 0.6 mg / L, the ratio of 6-benzylaminoadenine to naphthaleneacetic acid is 10:1, which is in line with the classic ratio of "high cytokinin + low auxin" in the bud differentiation stage and can promote the differentiation of strong buds.

[0103] 3. Rooting medium

[0104] Example 1: 0.5 mg / L naphthaleneacetic acid, a low concentration may result in insufficient rooting rate;

[0105] Example 3: 1.0 mg / L naphthaleneacetic acid. A high concentration can easily cause short, thick, and bifurcated roots.

[0106] Example 2: 0.8 mg / L of naphthaleneacetic acid is within the appropriate concentration range for rooting culture of grass plants (0.5-1.0 mg / L), and can induce balanced development of root quantity and length.

[0107] 4. Consistency of low temperature acclimation conditions

[0108] The low-temperature acclimation procedures (20°C → 1°C cooling rate + 3°C / 1°C diurnal cycle) in the three examples were identical, so the differences in screening results were primarily due to the initial mutagenesis and screening parameters. The parameter combination in Example 2 was more likely to achieve a higher mutant yield and plant survival rate throughout the entire "mutagenesis-screening-tissue culture" process.

[0109] In Example 2, the control graph (Control) is compared with the experimental results of the other culture groups (EH-12, EH-17, EH-38, and EH-57) induced by this method. Figure 2 As shown, if "high mutagenesis efficiency, moderate screening pressure, and high tissue culture seedling rate" are taken as the optimization goals, the ratio of Example 2 (EMS 0.8% / 6h+Hyp 30mM+various culture medium hormone ratios) has the best comprehensive performance.

[0110] The present invention is not limited to the above-mentioned optional implementation modes. Anyone can derive other forms of products under the inspiration of the present invention. However, no matter what changes are made in the shape or structure, any technical solution that falls within the scope defined by the claims of the present invention falls within the scope of protection of the present invention.

Claims

1. A mutagenesis breeding method for obtaining cold-resistant mutants of Phragmites australis based on EMS, characterized in that: The following steps are involved: Step S1, induction of embryonic callus; Select young inflorescences of Phragmites australis that are one year old or older and cut them into several small segments of 3 to 5 cm in length. After surface disinfection, insert them into callus induction medium of inflorescences and culture them in a culture room at a temperature of 25°C ± 2°C and a humidity of 70 to 80% for 90 days in the dark to form callus tissue. The formula of the callus induction medium is as follows: 4.73 g of MS medium powder, 2.0-4.0 mg / L of 2,4-dichlorophenoxyacetic acid, 0.5-2.0 mg / L of kinetin, 30 g / L of sucrose, and 7 g / L of agar, and the pH value of the mixed solution is maintained at 5.8, and high-temperature sterilization is performed; Step S2, EMS mutagenesis treatment; The embryonic callus induced from the inflorescence was selected to have a diameter of 0.5 cm and immersed in a 0.6-1.0% EMS solution. The solution was shaken at 25°C and 80 rpm for 5-8 hours. After the treatment, the callus was washed three times with sterile water. The washed callus was blotted dry with sterile absorbent paper. At the same time, the embryonic callus treated with sterile water without EMS was used as a control callus. Step S3, EMS stress screening; The callus tissue after EMS induction treatment was inoculated into callus proliferation medium for recovery culture for 30 days, and dark cultured in a constant temperature and humidity culture room. At the same time, the control callus tissue in step S2 was inoculated into callus proliferation medium without Hyp as a control, and 20-40mM Hyp was added to the callus proliferation medium; The formula of the callus proliferation medium is as follows: 4.73 g of MS medium powder, 0.5-1.0 mg / L of 2,4-dichlorophenoxyacetic acid, 0.5 mg / L of 6-benzylaminopurine, 30 g / L of sucrose, and 7 g / L of agar, the pH value of the mixed solution is maintained at 5.8, and high-temperature sterilization is performed; Step S4, differentiation and rooting culture of adventitious buds; The callus tissue that survived the screening in steps S2 and S3 was inoculated into a medium for adventitious bud differentiation, and the cells were placed in a culture room for 60 days to obtain adventitious buds. The differentiated adventitious buds were inoculated into a rooting medium, and the cells were placed in a culture room for 30 days under a light intensity of 2000 lx and a light duration of 12 h / d to induce rooting, thereby obtaining complete tissue culture seedlings of Phragmites australis and their clones. The formula of adventitious bud differentiation medium is: 4.73g MS medium powder, 4.0-8.0mg / L 6-benzylaminopurine, 0.4-0.8mg / L naphthaleneacetic acid, 30g / L sucrose, and 7g / L agar. The formula of rooting medium is: 4.73g MS medium powder, 0.5-1.0mg / L naphthaleneacetic acid, 30g / L sucrose, and 7g / L agar; The pH value of the mixed solution was maintained at 5.8 and sterilized at high temperature; Step S5, low temperature screening of tissue culture seedlings; The complete plants of the tissue culture seedlings of Phragmites australis obtained in step S4 and their clones were subjected to low-temperature acclimation screening, and the control callus tissue was used as a control for low-temperature acclimation treatment. The temperature was lowered in a low-temperature incubator starting from 20°C at a rate of 1°C per hour until the temperature dropped to 1°C. Subsequently, the plants were acclimated and cultured for 14 days at a periodic temperature of 14h / 3°C during the day and 10h / 1°C at night. After 14 days, the plants were taken out for observation and the dehydration and wilting status of the plants were recorded to obtain a cold-resistant mutant of Phragmites australis.

2. The method for obtaining cold-resistant mutants of Phragmites australis based on EMS according to claim 1, characterized in that: In step S1, the formula of the callus induction medium includes: 3.0 mg / L of 2,4-dichlorophenoxyacetic acid and 1.0 mg / L of kinetin.

3. The method for obtaining cold-resistant mutants of Phragmites australis based on EMS according to claim 1, characterized in that: In step S3, 30 mM Hyp is added to the callus proliferation medium, and the formula of the callus proliferation medium contains 1.0 mg / L of 2,4-dichlorophenoxyacetic acid.

4. The method for obtaining cold-resistant mutants of Phragmites australis based on EMS according to claim 1, characterized in that: In step S4, the adventitious bud differentiation culture medium contains 6.0 mg / L of 6-benzylaminopurine and 0.6 mg / L of naphthaleneacetic acid.

5. The method for obtaining cold-resistant mutants of Phragmites australis based on EMS according to claim 1, characterized in that: In step S4, the formula of the rooting medium contains 0.8 mg / L of naphthaleneacetic acid.

6. The method for obtaining cold-resistant mutants of Phragmites australis based on EMS according to claim 1, characterized in that: The culture conditions of the constant temperature and humidity culture room are: temperature of 25℃±2℃, humidity of 70-80%.

7. The method for obtaining cold-resistant mutants of Phragmites australis based on EMS according to claim 1, characterized in that: In the step S2, a mass of embryogenic callus with a diameter of 0.5 cm is selected from the inflorescence-induced embryogenic callus and immersed in a 0.8% EMS solution, and shaken at 25° C. and 80 rpm for 6 hours.

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