CN112715355A - Safe and efficient mutation method for bluegrass - Google Patents
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- A01H1/06—Processes for producing mutations, e.g. treatment with chemicals or with radiation
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- A—HUMAN NECESSITIES
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Abstract
The invention discloses a safe and efficient mutation method of bluegrass. Through variety screening, pretreatment and induced breeding, the morphology and polyploidy identification of the cultivated variety is carried out, and a new variety of the bluegrass with an excellent shape is obtained. The method has wide applicability, obtains the mutagenesis concentration and the processing time suitable for the cultivation of the new variety of the meadow bluegrass by the colchicine mutagenesis and tissue culture method, is not only suitable for obtaining the new variety of the meadow bluegrass, but also reduces the cultivation cost of the new variety, shortens the breeding time, reduces the cultivation time of the new variety, has high mutagenesis rate and high later germination rate, and can be widely applied to the large-scale cultivation of the new variety of the meadow bluegrass.
Description
Technical Field
The invention belongs to the field of plant breeding, and mainly relates to a meadow bluegrass60A breeding method of Co-gamma ray radiation mutagenesis and colchicine induced polyploidy.
Background
The world of Poa (A)Poa) About 500 plants, 1055 plants including varieties in China. The grass of Poa pratensis (Poa pratensis) (now, much research worldwide)Poa pratensisL., annual bluegrass (L.), annual bluegrass (C.), (Poa annuaL. and (D) Poa pratensis (L.) KuntzePoa crymophilaKeng), hard bluegrass (Keng)Poa sphondylodesTrin.), and the like. Poa pratensis is a perennial herb of Poa pratensis of GramineaeThe material, native to europe, north asia and north africa, is now spread over temperate and partial frigid regions of the world. The green grass is glary and resistant to yin, strong in cold resistance, long in green period, bright in color, soft and resistant to trampling, strong in adaptability, known as 'king of lawn', widely used for planting of lawn or green land and is a precious resource of lawn grass and herbage plant germplasm.
Commercial varieties of poa annua, which are introduced from foreign countries and mainly introduced in the market of China, are mainly 'velvet', 'Apollo', 'Madison', 'emerald', 'jadeite', 'blackish', 'blue tune', 'warrior', 'grace', 'wild horse', and the like. The cool-season lawn grass which is used most widely in cool and humid climatic regions has good cold resistance and high lawn quality. The developed rhizome improves the strength of the turf, and has strong trampling resistance and recovery capability, but the disadvantages of poor drought resistance and high temperature resistance and the like generally exist. The commercial excellent variety is taken as a test material for variety improvement, so that the defect of the excellent variety is compensated while the excellent property is kept.
The traditional breeding method of the bluegrass comprises single plant selection, intraspecific hybridization, interspecific hybridization and the like, and obtains remarkable achievements in the aspect of breeding new varieties, but has obvious limitations, such as: the breeding period is long, the mutation frequency is small, the amplitude is narrow, and the like, which all become problems to be solved urgently in the breeding work. With the rapid development of modern biotechnology, more ways are applied to plant breeding work. For example, molecular breeding is carried out, specific characters are improved through a transgenic method, the method has the characteristics of strong pertinence, high breeding efficiency and the like, but related molecular regulation and control machines are deeply understood and researched, and the breeding period is long.
The mutation breeding comprises physical mutagenesis and chemical mutagenesis, and the physical mutagenesis method mainly comprises radiation mutagenesis, including ray radiation, ultraviolet radiation or microwave radiation, and the like,60co-gamma ray radiation mutagenesis is widely applied. Chemical mutagenesis is mainly mutation and polyploid induction. Chemical mutagens mainly include alkylating agents, nucleic acid base analogs, antibiotics, etc., and change phenotypic traits by mutation selectively acting on a specific gene or nucleic acid. The polyploid mutagenesis method mainly adopts chemical reagents such as colchicine, oryzanol and the likeBy destroying the spindle body, inhibiting mitosis, stopping the chromosome in metaphase, and finally doubling the chromosome to form polyploid. Mutation breeding plays an important role in breeding new varieties and developing new germplasm resources, and a large number of valuable mutants can be obtained in a short time. Furthermore, mutations and polyploidization are ubiquitous in the angiosperm kingdom and are a major intrinsic factor in plant evolution. Compared with molecular breeding, mutation breeding is lack of artificial editing and insertion of exogenous fragments, and has higher biological safety. In addition, mutant materials can also be used as parents, and the traditional breeding method and biotechnology are combined to improve varieties, so that the mutation breeding plays an important role in both basic research and application research.
60Co-gamma ray radiation mutagenesis is a mutagenesis method for generating gene mutation or chromosome deformity by ionizing radiation, and can improve the mutation frequency and the mutation range and create various mutants. The gene recombination rate is improved by breaking gene linkage, a new genotype is obtained, and a new type and a new variety of variation are obtained. This mutagenesis approach often changes only individual gene loci, thereby improving the trait while substantially maintaining the original genetic background. Because the influence on chromosome is small, the mutation character is stable and fast, and the breeding period can be obviously shortened. The generation of mutants is not facilitated by excessively high or excessively low radiation dose, so the survival rate and the mutation rate are considered in actual breeding, a semi-lethal dose or a critical dose is usually selected as a more appropriate dose, and a valuable new variety is screened by phenotype observation and molecular identification of a survival strain after mutagenesis.
Polyploid artificial inducing agents include colchicine, oryzanol, trifluralin, propionamide, and amiprihos methyl, etc., and double chromosomes by resisting mitosis. Colchicine is the most effective mutagenic reagent, the method has little influence on the structure of chromosome, has few adverse variation and is widely applied to the character improvement of crops, fruit trees, medicinal plants and ornamental flowers. Polyploidy is usually expressed as the whole plant is enlarged and the stress resistance is enhanced due to the doubling of chromosomes and the doubling of genetic materials. It can also be used as parent to cross with diploid to obtain triploid variety and improve plant characteristics. However, colchicine itself is toxic and has a toxic effect on the material, and improper treatment time and concentration may cause the test material to be poisoned and dead in the treatment process, and simultaneously cause damage to the body of operators. Therefore, the selection of suitable treatment materials, pretreatment methods, colchicine treatment concentrations and treatment times, post-treatment culture, and the like are particularly important. The inducing treatment usually leads to the weakening of plant vitality, and the methods of water culture, tissue culture and the like can provide support of nutrient elements and reduce interference of adverse factors, thereby having good effect on improving the survival rate.
Disclosure of Invention
The main object of the present invention is to provide a simple and novel method which is useful for a mutagenic breeding test or production direction of a plant. The physical mutagenesis method is "60Co-gamma-ray radiation mutagenesis ". The chemical induction method is 'colchicine mutagenesis grass seeds', and three culture methods, namely a soil culture method, a water culture method and a tissue culture method, are matched and compared, and varieties which meet the requirements and have excellent characters are screened out through specific conditions.
The method comprises the following steps: and (3) variety screening, namely selecting seeds of the poa annua variety as materials, carrying out seed germination rate and drought resistance and rehydration tests, and selecting a variety with good effect according to the test result for induction.
The specific germination rate, drought resistance and rehydration test is as follows: poa pratensis varieties include but are not limited to nine varieties of 'velvet', 'Apollo', 'Madison', 'jadeite', 'ink jade', 'blue tune', 'Wushi', 'grace', 'wild horse', shelled seeds are used as test materials, germination dates are recorded, drought lasts for 5 days after seedling formation, and withering ratios are recorded; and (5) carrying out room temperature rehydration culture for the next 15 d, recording the recovery condition, and continuing to dry for 10 d after recovery. By comparing the germination rates, drought resistance and rehydration capacity of different varieties, the variety more suitable for mutation breeding is screened.
Step two: pretreating varieties, namely performing batch pretreatment on the shelled seeds screened in the step one before radiation, wherein the pretreatment modes include but are not limited to:
untreated shelled dry seeds;
soaking in water for 24 h; soaking in water for 48 h; fourthly, soaking the sodium hypochlorite solution with the concentration of 5% in water for 24 hours after 30 min; fifthly, soaking the 5 percent sodium hypochlorite solution in water for 48 hours after 30 minutes.
Pretreatment is carried out before colchicine induction, and the pretreatment mode comprises but is not limited to: the pretreatment mode of water culture is that the seeds with the shells are soaked or disinfected by 5 percent of sodium hypochlorite by mass fraction, and then are transferred to a germination disk after being soaked overnight or disinfected by the sodium hypochlorite, and the influence of the two pretreatment methods on the germination efficiency of the bluegrass is compared by comparing the pollution rate, the germination rate and the growth condition.
The pretreatment mode of tissue culture is that seeds with shells are used as materials, the seeds are sterilized by a sodium hypochlorite solution with the mass fraction of 5% for 30min on a superclean workbench, and then the seeds are washed by sterile water for 3 times and dried for standby.
Further, in the tissue culture method pretreatment, the optimal treatment time of sodium hypochlorite is firstly screened, 4 different treatment times are designed within 15-60 min, and the optimal pretreatment conditions of different varieties are obtained by comparing the pollution rate, the germination rate and the growth condition.
Step three: and (3) induced breeding: selecting seeds with shells of the variety of the bluegrass for induction cultivation, and respectively carrying out physical induction and chemical induction; wherein the physical inducing method comprises60And (3) performing Co-gamma ray radiation mutagenesis, performing irradiation treatment on the seeds in the step two, uniformly sowing the irradiated seeds, observing the germination condition of each treatment after the seeds completely germinate after sowing for one week, and counting the germination rate of each treatment after culturing for 30 days, thereby determining the semi-lethal dose of the material and obtaining the optimal mutagenesis dose rate and dose of the Poa annua.
The chemical induced breeding method selects colchicine for breeding, and cultures the colchicine by a colchicine water culture method and a tissue culture method, wherein the water culture method comprises the following steps: selecting shell-carrying seeds of a Poa annua variety, soaking the seeds in water or disinfecting the seeds with sodium hypochlorite, transferring the treated seeds into a colchicine solution with the mass percentage of 0.2-1% and treating the seeds for 0-72 h, performing colchicine induction through a random block test with different concentrations and different treatment times, cleaning the treated seeds for a plurality of times, and transferring the seeds to a water culture germination tray for culture to obtain the variety with high induction rate.
The tissue culture method comprises the following steps: taking the seeds of the preferred variety in the first step as a material, sterilizing the seeds by using a sodium hypochlorite solution and washing the seeds by using sterile water, airing the seeds after sterilization and placing the seeds into a centrifugal tube, treating the seeds for 0 to 72 hours by using a colchicine solution with the mass percentage of 0.05 to 0.5 percent, and performing a random block test by using different concentrations and different treatment times under the treatment condition of the colchicine-induced seeds at the temperature of 25 ℃ and in the dark; the treated seeds were sown under aseptic conditions in prepared and sterilized MS +30g/L sucrose +6g/L agar, pH =5.8 medium for cultivation, and the seeds germinated after 3 d of cultivation. When the material is cultured for about 40 days and grows to 7-10 cm, the germination condition, the germination rate and the morphological indexes of the seeds are compared, so that the half-lethal dose, the survival rate and the mutagenesis efficiency suitable for the mutation breeding of the colchicine of the bluegrass are obtained, and the optimal mutagenesis conditions of the bluegrass of different varieties are obtained.
Step four: and (3) determining whether the induced variety is a mutagenic strain or not by observing the surface state change and a flow cytometer according to the cultured variety, and establishing a good variety screening library.
Compared with the main differences of auxiliary soil culture, water culture and tissue culture technologies, the tissue culture aims at improving the germination rate of the material and simultaneously carrying out asexual propagation on an excellent strain or ploidy material, thereby achieving the possibility of obtaining an excellent new variety in a short time. Through comparison of the germination rates of the nine varieties, the following results are found: under normal soil cultivation conditions, 'Apollo' has the lowest germination rate of only 25%. However, under tissue culture conditions, the germination rate of the seeds of 'Apollo' is improved to 90%. The best method is a colchicine tissue culture method: treating the seeds for 30min by 5% sodium hypochlorite, washing with sterile water, drying, selecting a proper colchicine semi-lethal dose, inoculating into a sterile culture medium, and screening variant strains according to phenotypic changes of leaves, roots and the like for flow cytometry identification. The tissue culture technology can greatly improve the effect of exogenous factors on seed growth in adverse environment after colchicine treatment; meanwhile, the production cost of colchicine mutagenesis can be reduced, and the breeding time of new species can be shortened.
Compared with the prior art, the method can further determine the mode of colchicine breeding matched with tissue culture by comparing the processes of pretreatment mode, mutation breeding method, later-stage culture, screening and identification of mutation materials and the like, has simple treatment mode, short culture period and obvious later-stage phenotype of the mutation materials, is more suitable for breeding new varieties of bluegrass and is more suitable for large-scale breeding and culture of new varieties of other grasses. Meanwhile, higher mutagenesis rate can be obtained, higher germination rate can be obtained, the breeding time of a new variety is shortened, and the method has good demonstration effect on large-scale mutagenesis breeding of the new variety.
Drawings
FIG. 1 is an exemplary diagram of flow cytometry detection of polyploids.
Detailed Description
The foregoing invention is described in further detail with reference to specific embodiments thereof, but it should not be construed that the scope of the above-described subject matter is limited to the following examples.
Example 1
Nine varieties of bluegrass, namely velvet, Apollo, Madison, emerald, black, blue, Wushi, beautiful and wild horse, are selected and commonly used in the market. And (4) carrying out primary determination on the germination rate and the drought resistance of the variety. The specific operation is as follows: firstly, selecting 100 seeds of each variety, and respectively carrying out a and water immersion for 24 h; b. soaking seeds in a sodium hypochlorite solution with the mass fraction of 5% for 30min and then soaking in water for 24 h. The treated seeds were evenly sown in soil to observe the germination rate. Secondly, after sprouting and culturing the materials treated by the varieties a for 15 d, transferring the materials into an environment with the following conditions: culturing for 30d under illumination conditions of 8h, 3000 Lx illumination intensity, 48 deg.C illumination and 16 h dark at 30 deg.C dark; and respectively counting the states of various varieties after 5 d of culture, recovering the culture state for 15 d at room temperature and drying the varieties for 10 d after recovery.
TABLE 1 germination rates and drought resistance of different varieties
The germination time of the treated seeds is effectively shortened after the seeds are pretreated, wherein the germination time required by the seeds for water culture is effectively shortened by the treatment of materials such as velvet, emerald, bluetone, warrior and the like through a sodium hypochlorite solution, which shows that the germination time can be effectively shortened after the shells of the seeds are subjected to proper damage treatment. Combining the withering rate and recovery condition of the material, the material is divided into three drought resistance grades: first-order drought-and heat-tolerant varieties (2): apollo 'and velvet'; second-level general cultivars (3): 'jadeite', 'china jade', 'blue tone'; three-level drought and heat resistant varieties (5): 'wildhorse', 'warrior', 'madison' and 'grace'.
Example 2
Nine varieties of bluegrass, namely swan velvet, Apollo, Madison, jadeite, black jade, blue tone, warrior, beautiful and wild horse, are selected as test materials. Five different treatment methods are adopted to pretreat various seeds, and the influence of different pretreatment conditions on the germination capacity and the radiation resistance of the material is compared. The five pretreatment methods are respectively as follows:
shelled dry seeds.
Soaking in water for 24 hr.
Soaking in water for 48 hr.
5% sodium hypochlorite solution 30Soaking the seeds with the shells in water for 24h after min.
Soaking the seeds with the shells in water for 48h after 30min by using a 5% sodium hypochlorite solution. The materials of various types are divided into five types according to different pretreatment methods, the materials of each type are divided into seven parts, and the seven parts are respectively filled into mesh bags with the same mesh number, so that the normal breathing of the seeds is ensured. And respectively carrying out irradiation treatment on the subpackaged seeds with seven doses of 0 Gy, 30 Gy, 50 Gy, 80 Gy, 100 Gy, 150 Gy and 200 Gy according to test requirements, wherein the irradiation treatment dose rates are all 2.34 Gy/min. After the radiation is finished, uniformly sowing the radiated seeds in grass carbon: perlite 2:1 mixed matrix. Culturing the irradiated seeds under greenhouse conditions of about 25 ℃ of culture temperature and about 60% of environmental humidity; ② seeding culture is carried out respectively under the conditions of culture room temperature of 23 ℃ and environment humidity of 30 percent. And counting the germination condition and the semi-lethal dose of each treatment after complete emergence of seedlings. The optimal mutagenesis dose rate and dose of the bluegrass are determined.
TABLE 2 radiation mutagenesis pretreatment and culture
Remarking: A. preprocessing (I); B. preprocessing is performed; C. preprocessing (III); D. preprocessing to obtain a product; E. pretreating.
It was found that 'velvet' is optimally treated to 150 Gy; 'Madison' optimal treatment 200 Gy; optimal treatment of 'jadeite' 150 Gy; 'sepia' optimal treatment 150 Gy; optimal treatment 150 Gy for 'blue toning'; optimal treatment 150 Gy for 'warrior'; 'grace' best treatment 150 Gy; 'wild horse' was optimally treated to 150 Gy. Due to the influence of culture environment conditions, the germination rate of the variety Apollo is seriously interfered, and the variety Apollo is considered to be optimally treated to 150 Gy according to the existing results. Therefore, when the radiation breeding technology is selected, the treatment dose with the irradiation dose rate higher than 150 Gy and the irradiation dose rate of 2.34 Gy/min can be selected to treat the test material.
Example 3
Selecting wild horses, soaking overnight or 5% by mass of sodium hypochlorite, soaking for 24h or 72 h, transferring the seeds into colchicine solutions with different concentrations (the mass fractions are 0.3%, 0.4% and 0.6%), treating for 24h, 48h and 72 h respectively, cleaning the seeds for a plurality of times, and transferring the seeds into a water culture germination tray for culture. The optimal pretreatment method, the colchicine use concentration and time are evaluated by comparing the difference of the induction efficiency of the colchicine under different concentrations and treatment time and comparing the pollution rate, the germination rate and the growth condition.
TABLE 3 'Himalayan' colchicine hydroponic induction method
The best treatment for ` tram ` colchicine was 0.4% treatment for 48 h. During the water culture treatment of colchicine, the change of the phenotype of the treated material, including the difference of plant height and the change of the width of new leaves, can be obviously observed at the initial germination stage of the treated material. As the material passes through the juvenile period, the phenotype change gradually has no obvious difference, the root system has no obvious difference, and the material grows slowly under the influence of the environmental conditions. The suspected doubled plant is detected by a flow cytometer, and the accuracy of judging the suspected doubled plant is only 60%.
Example 4
Selecting 'Mocui', soaking overnight or 5% by mass of sodium hypochlorite, soaking for 24h or 72 h, transferring the seeds into colchicine solutions with different concentrations (the mass fractions are 0.3%, 0.4% and 0.6%), treating for 24h, 48h and 72 h respectively, cleaning the seeds for a plurality of times, and transferring the seeds into a water culture germination tray for culture. The optimal pretreatment method, the colchicine use concentration and time are evaluated by comparing the difference of the induction efficiency of the colchicine under different concentrations and treatment time and comparing the pollution rate, the germination rate and the growth condition.
TABLE 4 hydroponic induction method of "Cui" colchicine
The optimal treatment for 'sepia' colchicine was 0.4% treatment for 48 h. The treated material has obvious phenotypic change at the initial stage of germination, which is shown as the change of plant height and new leaf width. Along with the growth of the material, the phenotype gradually has no obvious difference, the root system also has no obvious change, and the material is slowly grown under the influence of environmental conditions. Through the flow cytometry detection of the suspected doubled plant line, the judgment accuracy of the suspected doubled plant is lower than 50%.
Example 5
Choose to useNine varieties of Poa pratensis 'velvet', 'Apollo', 'Madison', 'emerald', 'black jade', 'blue tune', 'Wu' and shi ', ' Youmei ', ' wild horse 'Are test materials. On a sterile ultra-clean workbench, four sterilization time treatments of 15 min, 30min, 45 min and 60 min are respectively carried out on each variety of seeds by using a 5% sodium hypochlorite solution, and then the seeds are washed by sterile water for 3 times and dried for standby. And uniformly sowing the aired seeds in a prepared and sterilized MS +30g/L sucrose +6g/L agar and a culture medium with pH =5.8, culturing at the temperature of 25 ℃ for 12 h/12 h (light/dark), and observing the germination condition and the germination rate.
TABLE 5 optimal sterilization method for colchicine tissue culture
The results of the comparative tests show that when the treatment time reaches 15 min,the material 3 d of the 'wild horse', the 'warrior' and the like Germinates and buds, but has little bacterial pollution and the germination rate is lower than 30 min; except for Apollo, Madison, jadeite, The 'blue tone' and the likeBesides the variety, other varieties are best in the sterilization time of 30min, and have short sterilization time, short germination time and long germination time, The germination rate is high and the pollution rate is low; under the condition of 30min treatment, the four varieties have obvious data except for longer germination time The method is better. Therefore, the method for sterilizing by using 5% sodium hypochlorite for 30min is a suitable sterilization method in the tissue culture breeding process of the colchicine.
Example 6
Selecting and using 'wild horse' on a sterile super-clean workbench, sterilizing the seeds for 30min by using a 5% sodium hypochlorite solution, washing the seeds for 3 times by using sterile water, and airing the seeds for later use. Placing the sterilized and air-dried seeds in a sterile centrifuge tube, and respectively treating with colchicine solutions with concentrations of 0.05%, 0.1%, 0.2% and 0.3% for 0 h, 6 h, 12 h, 24h, 48h and 72 h at 25 deg.C under dark condition. And (3) moving the material processed according to the test requirements on an ultra-clean workbench to an aseptic culture dish for airing, uniformly sowing the aired seeds in a prepared and sterilized MS +30g/L sucrose +6g/L agar and a culture medium with pH =5.8, culturing for 3-4 d at the temperature of 25 ℃ for 12 h/12 h (light/dark), and then, beginning to count the germination time. And when the material grows to 7-10 cm after about 40 days of culture, counting the germination condition and the germination rate of the seeds.
TABLE 6 tissue culture induction method of colchicine of Himala's Himalayan
When the concentration of colchicine is 0.05%, the germination rate of each treatment time is not less than 70%, when the treatment concentration is 0.1% -0.2%, the germination rate is obviously reduced along with the extension of the treatment time, when the treatment time is less than 24h, the germination rate of the material is over 65%, the germination rate of the material is obviously reduced after 48h and above, and the minimum value is only 30%; when the treatment concentration is 0.3%, the minimum value of the germination rate of the seeds is reduced to 10%. The half-lethal dose of colchicine treatment 'wild horse' was 0.2% of the treatment concentration, and the treatment time was 48h, at which point the germination rate of the material was 30%. By comparing with a control plant, selecting a plant with increased leaf width, reduced leaf number and increased or shortened root system as a detection material to carry out flow cytometry detection, referring to the detection result of the flow cytometer, treating the detection material to be lower than the semi-lethal concentration, and changing the plant accuracy rate by only 80% according to phenotype screening ploidy; the accuracy of the plant change by the treatment of the semi-lethal concentration is up to 95 percent according to the phenotype screening ploidy. Namely, the processing concentration of the semi-lethal dose is selected, and ploidy-changed plants can be effectively screened out by combining the change of the phenotypic indexes of the materials.
Example 7
Selecting 'Mocui' on a sterile super clean workbench, sterilizing the seeds for 30min by using a 5% sodium hypochlorite solution, washing with sterile water for 3 times, and drying for later use. Placing the sterilized and air-dried seeds in a sterile centrifuge tube, and respectively treating with colchicine solutions with concentrations of 0.05%, 0.1%, 0.2% and 0.3% for 0 h, 6 h, 12 h, 24h, 48h and 72 h at 25 deg.C under dark condition. And (3) moving the material processed according to the test requirements on an ultra-clean workbench to an aseptic culture dish for airing, uniformly sowing the aired seeds in a prepared and sterilized MS +30g/L sucrose +6g/L agar and a culture medium with pH =5.8, culturing for 3-4 d at the temperature of 25 ℃ for 12 h/12 h (light/dark), and then, beginning to count the germination time. And when the material grows to 7-10 cm after about 40 days of culture, counting the germination condition and the germination rate of the seeds.
TABLE 7 tissue culture induction method of "Cui" colchicine
When the concentration of colchicine is 0.05%, the germination rate of each treatment time is not less than 70%, when the treatment concentration is 0.1% -0.2%, the germination rate is obviously reduced along with the extension of the treatment time, when the treatment time of each concentration is less than 24 hours, the germination rate of the material is more than 60%, and the germination rate of the material after 48 hours or more is obviously reduced to about 50%; when the treatment concentration is 0.3%, the minimum value of the germination rate of the seeds is reduced to 30%. The semi-lethal dose of colchicine mutagenized in "sepia" was 0.3% of the treatment concentration, and the treatment time was 72 h, at which the germination rate of the material was 30%. Compared with a control leaf, the selection is widened, the total number of the leaves is relatively reduced, and the plant with the increased root system is used as a detection material for flow cytometry detection. Compared with the detection result of a flow cytometer, the accuracy of screening ploidy-changed plants according to phenotypes is only 75% when the concentration of colchicine is lower than 0.2%; the accuracy rate of the ploidy change plant is up to 90% according to the treatment of 0.3% colchicine concentration and phenotype screening. Therefore, 0.3 percent of colchicine is selected, and the ploidy-changed plants can be effectively screened out according to the change of the phenotypic indexes of the materials after the treatment for more than 24 hours.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.
Claims (9)
1. A safe and efficient mutation method of bluegrass is characterized by comprising the following steps:
(1) the method comprises the following steps: selecting seeds of the annual bluegrass variety as materials, carrying out seed germination rate and drought resistance and rehydration tests, and selecting a variety with high survival rate and good resistance for induction according to the test result;
(2) step two: pretreating the seeds with shells screened in the step one in batches before radiation or colchicine induction, wherein the pretreatment mode comprises water immersion or sodium hypochlorite treatment;
(3) step three: and (3) induced breeding: selecting seeds with shells of the variety of the bluegrass for induction cultivation, and respectively carrying out physical induction and chemical induction; the physical induction method is 60 Co-gamma ray radiation mutagenesis, the seeds in the step two are subjected to irradiation treatment, the irradiated seeds are uniformly sown, the initial germination time is recorded, after one week of sowing, the germination conditions of each treatment are observed when the seeds germinate, the germination rates of each treatment are counted after 30 days of cultivation, the semi-lethal dose of the material is further determined, and the optimal mutagenesis dose rate and dose of the Poa pratensis are obtained;
the chemical induced breeding method selects colchicine for breeding, and culture is carried out by colchicine water culture or tissue culture method, wherein the water culture method comprises the following steps: selecting shell-carrying seeds of a Poa annua variety, soaking the seeds in water or sterilizing the seeds with sodium hypochlorite, transferring the treated seeds into a colchicine solution with the mass percentage of 0.2-1% and treating the seeds for 0-72 h, performing a random block test by colchicine induction for different concentrations and different treatment times, cleaning the treated seeds for a plurality of times, transferring the seeds into a germination disc for culture, recording the initial germination time, and comparing the germination condition, the germination rate and the survival rate of the seeds when the material grows to about 10 cm after about 40 days; performing primary screening of a mutagenic strain according to morphological indexes, and calculating the mutagenic efficiency through the detection result of a flow cytometer so as to obtain a variety with high induction rate, pretreatment conditions and time, and mutagenic concentration and time;
the tissue culture method comprises the following steps: taking the seeds of the preferred variety in the first step as a material, sterilizing the seeds by using a sodium hypochlorite solution and washing the seeds by using sterile water, airing the seeds after sterilization and placing the seeds into a centrifugal tube, treating the seeds for 0 to 72 hours by using a colchicine solution with the mass percentage of 0.05 to 0.5 percent, and performing a random block test by using different concentrations and different treatment times under the treatment condition of the colchicine-induced seeds at the temperature of 25 ℃ and in the dark; sowing the treated seeds in a prepared and sterilized MS +30g/L sucrose +6g/L agar and a culture medium with pH =5.8 under an aseptic condition for culturing, germinating the seeds after culturing for 3 days, and comparing the germination condition and the germination rate of the seeds when the materials grow to 7-10 cm after culturing for about 40 days to obtain the survival rate suitable for the colchicine mutation breeding of the poa pratensis; after primary screening of morphological indexes, evaluating the mutagenesis efficiency by using a flow cytometry detection method to obtain the optimal mutagenesis conditions of different varieties of bluegrass;
(4) step four: and determining whether the induced variety is a mutagenic strain according to the phenotype change of the cultivated variety and the detection result of the flow cytometry, and screening the variety with excellent properties.
2. The method of claim 1, wherein the method comprises the steps of: the germination rate, drought resistance and rehydration test method comprises the following steps: taking the shelled seeds of the annual bluegrass variety as a test material, recording the germination date, continuously drying for 5 days after seedling formation, and recording the dry-dry ratio; carrying out room temperature rehydration culture for the next 15 d, recording the recovery condition, and continuing to dry for 10 d after recovery; by comparing the germination rates, drought resistance and rehydration capacity of different varieties, the variety more suitable for mutation breeding is screened.
3. A safe and efficient method for mutagenizing bluegrass as claimed in claim 2, wherein said method comprises the steps of: the variety of the bluegrass comprises velvet, Apollo, Madison, emerald, blackish, bluetone, warrior, beautiful and wild horse.
4. A safe and efficient method for mutagenizing bluegrass as claimed in claim 3, wherein said method comprises the steps of: performing batch pretreatment on the shelled seeds screened in the first step before irradiation, wherein the pretreatment mode comprises but is not limited to:
untreated shelled dry seeds;
soaking in water for 24 h; soaking in water for 48 h; fourthly, soaking the sodium hypochlorite solution with the concentration of 5% in water for 24 hours after 30 min; fifthly, soaking the 5 percent sodium hypochlorite solution in water for 48 hours after 30 minutes.
5. The method of claim 4, wherein the method comprises the steps of: pretreating before colchicine induction, wherein the pretreatment mode of water culture is that seeds with shells are soaked or disinfected by 5 percent of sodium hypochlorite by mass fraction, and then are transferred to a germination disk after being soaked overnight or disinfected by sodium hypochlorite, and the influence of the two pretreatment methods on the germination efficiency of the bluegrass is compared by comparing the pollution rate, the germination rate and the growth condition.
6. The method of claim 5, wherein the method comprises the steps of: the pretreatment mode of group banks up for with the shell seed for the material, on superclean bench, sterilizes 30min with 5% mass fraction sodium hypochlorite solution to the seed, and later washes 3 times with aseptic water, dries for subsequent use.
7. The method of claim 6, wherein the method comprises the steps of: in the tissue culture method pretreatment, the optimal treatment time of sodium hypochlorite is firstly screened, 4 different treatment times are designed within 15-60 min, and the optimal pretreatment conditions of different varieties are obtained by comparing the pollution rate, the germination rate and the growth condition.
8. The method of claim 7, wherein the method comprises the steps of: the irradiation dose in the third step is seven irradiation treatments with the dose of 0-200 Gy, and the irradiation treatment dose rate is 2.34 Gy/min.
9. The method of claim 8, wherein the method comprises the steps of: the culture medium is MS +30g/L sucrose +6g/L agar, and the pH = 5.8.
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