CN111316916A - Novel variety breeding method for inducing mutation of malus baccata anther - Google Patents
Novel variety breeding method for inducing mutation of malus baccata anther Download PDFInfo
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- A—HUMAN NECESSITIES
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- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/008—Methods for regeneration to complete plants
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
- A01H1/04—Processes of selection involving genotypic or phenotypic markers; Methods of using phenotypic markers for selection
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
- A01H1/06—Processes for producing mutations, e.g. treatment with chemicals or with radiation
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Abstract
The invention belongs to the technical field of plant propagation, and particularly relates to a new variety cultivation method for inducing the anther mutation of fructus viticis. The novel artificial cultivation variety of the malus baccata, which has the characteristics of strong independence of each individual plant, high purity of individual genetic information, clear individual plant source and the like, is obtained by taking the malus baccata anther as a material, soaking the malus baccata in ethyl methanesulfonate with proper concentration for a certain time, combining with a plant tissue culture technology and adopting methods of embryogenic callus induction, redifferentiation, embryoid development and the like, is genetically stable, and can be used for strain cultivation and construction of a malus baccata mutant library.
Description
Technical Field
The invention belongs to the technical field of new plant varieties, and particularly relates to a new variety breeding method for inducing the anther mutation of fructus viticis.
Background
Fructus Viticis negundo (academic name: Malus baccata (L.) Borkh.): is deciduous tree of the genus Malus of the family Rosaceae, which is also called as follows: the tree height of the tree can reach 4-5 meters, and can reach more than 10 meters at most, and the tree is grey brown, smooth and not easy to crack; young shoots are yellow brown and hairless; the tender tips are green and slightly reddish brown. Elliptic shape with tip tapered, wedge-shaped base, sharp saw teeth at blade edge, and umbrella-shaped general inflorescence. White in color, with flower column 5 or 4, and long and soft hair at the base; 4-6 flowers are gathered at the top of the short branch. The malus baccata blossoms in 6 months, the fruit is approximately spherical, the diameter is 0.8-1 cm, the fruit is red or yellow, calyx is removed, round embroidery spots are formed in calyx depressions, and the length of a fruit stem is 3-4 times that of the fruit. The fruit ripens in the middle and last ten months in 9 months. The elevation of the mountain chaste tree is 50-1500 m in the mountain slope forest and the bush in valley yin, and the mountain chaste tree is distributed in Mongolia, Korean, Russian Siberian and other places. It is good for light, strong in cold resistance (some types can resist low temperature of-50 deg.C), barren-resistant, salt-free, deep-rooted and long in service life. The tree has elegant and beautiful appearance, luxuriant flowers and leaves, white flowers, green leaves and red branches are mutually reflected and beautiful and bright, and the tree is an excellent ornamental tree species. Besides the ornamental value, the seedlings can be used as grafting stocks of apples, malus asiatica and Chinese flowering crabapple fruits; is a good dense source plant; the wood has straight grains and fine structure and is used for printing and engraving plates, jointers, tool holders and the like; the tender leaves can be used as tea, and can also be used as livestock feed, and the fruits have certain curative effect on dysentery and vomiting and diarrhea; can also be used as an original material for cultivating cold-resistant apple varieties.
The wild fructus viticis has the problems of small fruit, sourness, poor taste, low quality, poor disease resistance, low edible value, low yield, common damping off, various insect pests and the like, so that the development and the utilization of the wild fructus viticis are greatly limited.
In the prior art, most researches on the malus baccata are focused on the aspects of artificial breeding of seed seedlings, greening modeling, stock grafting and the like, and no related reports on variety improvement and new variety (line) cultivation are found. And the fruits are small and sour, so that the method is not widely applied to the field of food, and the artificial large-scale cultivation production is delayed.
Disclosure of Invention
In order to solve the problems that the development and utilization of the malus baccata are greatly limited due to small fruit size, sourness, poor taste, low quality, poor disease resistance, small edible value and low yield of the malus baccata, common damping off and various insect pests of the malus baccata and the like, the invention provides a new variety cultivation method for inducing the anther mutation of the malus baccata.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows: a new variety breeding method for inducing the flower drug mutation of fructus viticis includes the following steps:
(1) material selection and pretreatment: in the first ten days of 6 months, selecting full buds after the wild fructus viticis shows buds, washing the buds with 75% ethanol for 10s, transferring the buds to a saturated sodium hypochlorite solution for soaking for 2min, washing with sterile water for 10 times, and sucking off surface water with sterile filter paper; then, the flower buds are stripped, anthers on stamens are picked off, and the anthers are put into EMS solutions with different concentrations in batches and soaked for 40-120 min; finally, the anther is flushed by sterile water for 10 times for inoculation;
(2) induction culture of anther embryonic callus: transferring the anther soaked in EMS in the step (1) into an embryonic callus induction culture medium, and placing the embryonic callus induction culture medium in a light cycle of 6 h.d-1Culturing at the illumination intensity of 800lx and the temperature of 26 +/-2 ℃ for 80 days, and completely dedifferentiating the anther to convert the anther into a white and yellow embryonic callus;
(3) and (3) redifferentiation culture of the embryogenic callus: cutting the embryogenic callus in the step (2) into small pieces, transferring to embryogenic callus redifferentiation culture medium, and culturing while placing in light cycle for 10 h.d-1Culturing for 60 days under the conditions of illumination intensity of 1000lx and temperature of 25 +/-2 ℃, and differentiating embryoid with independence, bipolarity and separability from the embryogenic callus;
(4) and (3) development and culture of embryoid: continuously culturing the embryogenic callus containing the embryoid in the step (3) for 25 days, and gradually separating the embryoid from the embryogenic callus; then the embryoid is peeled off and transferred into an embryoid development culture medium and placed in a light cycle of 12 h.d-1Carrying out development culture under the conditions of illumination intensity of 1200lx and temperature of 24 +/-2 ℃, and culturing for 45 days until embryoids are subjected to double polarization development to form small plants containing roots, stems and leaves;
(5) transplanting, field planting and strain determination: when the plantlets in the step (4) grow to 2.0cm in a culture bottle, taking out the plantlets from the bottle, planting the plantlets in a nutrition pot after domestication and hardening, planting the plantlets in a field after the plantlets grow to 20cm, and observing the growth condition of the plantlets; then labeling the different single plants and grafting the labels to different suitable regions for cultivation; and finally, screening and determining the excellent character mutants with stable heredity as strains, and numbering.
Further, EMS in the step (1) is used in a concentration of 0.1 mol.L-1And a phosphate buffer solution with the pH value of 7 is used as a solvent to prepare an EMS solution with the volume percentage concentration of 0.08-0.24%.
Further, EMS solutions of different concentrations are EMS solutions of 0.08%, 0.10%, 0.12%, 0.14%, 0.16%, 0.18%, 0.20%, 0.22%, 0.24% by volume, respectively.
Further, the culture medium comprises a minimal medium, and the components of the minimal medium comprise the following substances: potato supernatant 60 g.L-1(ii) a Macroelements: 20.0 mg. L-1NH4NO3，7.5mg·L-1CaCl2·2H2O，8.9mg·L-1MgSO4·7H2O; iron salt: 1.5 mg. L-1FeSO4·7H2O，1.8mg·L-1Na2·EDTA·2H2O。
Further, in the step (2), the culture medium is a minimal medium supplemented with 2.45 mg.L-1Isopentenyladenine and 0.35 mg.L-1NAA, and 6.8 g.L-1Agar powder, 50 g.L-1Sucrose.
Further, the pH of the medium in the step (2) was 6.0.
Further, in the step (3), the culture medium is a minimal medium supplemented with 3.2 mg.L-1Isopentenyladenine and 7.0 g.L-1Agar powder, 40 g.L-1Sucrose.
Further, the pH of the medium in step (3) was 5.9.
Further, in the step (4), the culture medium is a minimal medium supplemented with 0.02 mg.L-1KT, and 6.8 g. L-1Agar strips, 30 g.L-1Sucrose, and adjusting the pH value of the culture medium to 6.0.
Further, the strain determination is screened according to one or more factors of the growth vigor, morphological characteristics, fruit shape, fruit size, acid sweetness, taste, adaptability, yield, disease resistance and seed seedling genetic condition of the mutant.
The invention provides a new variety breeding method for inducing the flower drug mutation of fructus viticis negundo, which has the following beneficial effects:
(1) the characteristic of the Ethyl Methane Sulfonate (EMS) is acting on an induction point, so that the induction point mutates, chromosome deformity cannot be caused, dominant mutation is high, further genetic transformation is not needed, a genetic stable mutant can be directly obtained, the cultivation polymorphism is extremely small, a good character mutant can be directly selected as strain cultivation and variety approval, and the breeding period is obviously shortened;
(2) the method has the advantages of multiple effects, high mutation rate of induced offspring, wide variation range, good genetic stability of mutants, diversity of mutation and the like;
(3) the method uses the malus baccata anther as a material, combines a mutagen EMS with a plant tissue culture technology, adopts culture approaches such as embryogenic callus induction, embryogenic callus redifferentiation, embryoid development and the like to obtain the genetic stable mutant, has the characteristics of strong independence of each individual plant, high purity of individual genetic information, clear individual plant source and the like, and can be further applied to the construction and variety culture of a malus baccata mutant library.
Drawings
FIG. 1 is a diagram of the structure of anther embryogenic callus containing embryogenic cells of malus baccata;
FIG. 2 a. the fruit of line 1 (line code: 2013-;
b. line 2 (line code: 2013-;
c. line 3 (line code: 2013-;
d. fruit of wild fructus Viticis negundo.
Detailed Description
The technical solutions in the present invention will be described clearly and completely with reference to the accompanying drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only some embodiments of the present invention, not all embodiments.
As shown in fig. 1 and 2, a method for breeding a new variety inducing the anther mutation of fructus viticis includes the following steps:
1 materials and methods
1.1 Material selection and pretreatment
In the first ten days of 6 months, after the wild fructus viticis shows buds, selecting full buds (before the wild fructus viticis opens and white leaves do not appear), taking off the buds, washing the buds with 75% ethanol for 10s, transferring the buds to a saturated sodium hypochlorite solution, soaking for 2min, washing with sterile water for 10 times, and sucking surface water with sterile filter paper for later use.
1.2 methods
1.2.1 preparation of Ethyl Methylsulfonate (EMS) solution
EMS concentration is 0.1 mol.L-1Preparing EMS solution with the volume percentage concentration of 0.08-0.24% by taking phosphate buffer solution with the pH value of 7 as a solvent;
i.e., at 0.1 mol. L of pH 7.0-1Using the phosphate buffer solution of (2) as a solvent, and preparing Ethyl Methanesulfonate (EMS) into a volume percentage concentration (v.v.. v.)-1) The concentration of the solution is 0.08%, 0.10%, 0.12%, 0.14%, 0.16%, 0.18%, 0.20%, 0.22%, 0.24%, respectively, and phosphate buffer is used as control solution (CK), the concentration range is obtained by pre-experiment according to anther survival rate, and filtration sterilization is needed before use.
1.2.2 preparation of the culture Medium
The basic culture medium comprises the following components in percentage by weight: potato supernatant 60 g.L-1(ii) a Macroelements: 20.0 mg. L-1NH4NO3，7.5mg·L-1CaCl2·2H2O，8.9mg·L-1MgSO4·7H2O; iron salt: 1.5 mg. L-1FeSO4·7H2O，1.8mg·L- 1Na2·EDTA·2H2O。
Anther embryogenic callus induction medium and culture conditions: adding 2.45 mg.L to the minimal medium-1Isopentenyladenine (2-ip) and 0.35 mg. L-1NAA，6.8g·L-1Adding 50 g/L agar powder-1Sucrose and adjusting the pH value of the culture medium to 6.0; placing the culture bottle in illumination cycle 6 after anther inoculationh·d-1Culturing under the conditions of illumination intensity of 800lx and temperature of 26 +/-2 ℃.
Redifferentiation culture medium and culture conditions for embryogenic callus: adding 3.2 mg.L to the minimal medium-1Isopentenyladenine (2-ip), 7.0 g.L-1Adding 40 g/L agar powder-1Sucrose, and adjusting the pH of the medium to 5.9. Placing the culture flask in a light cycle for 10 h.d after the embryogenic callus is inoculated-1Culturing under the conditions of illumination intensity of 1000lx and temperature of 25 +/-2 ℃.
Embryoid development culture medium and culture conditions: 0.02 mg.L is added into the minimal medium-1KT, and 6.8 g. L-1Agar strips, adding 30 g.L-1Sucrose, and adjusting the pH value of the culture medium to 6.0. Placing the culture flask in a light cycle of 12 h.d after the embryoid is inoculated-1Culturing under the conditions of illumination intensity of 1200lx and temperature of 24 +/-2 ℃.
1.2.3 operating method
(1) Material treatment: in an ultraclean workbench, 1.1 disinfected and sterilized flower buds are stripped, anthers on stamens are picked off, the anthers are placed into a filtration and sterilization phosphate buffer solution (CK, 0%) with the volume percentage concentration of 0.08%, 0.10%, 0.12%, 0.14%, 0.16%, 0.18%, 0.20%, 0.22%, 0.24% and Ethyl Methanesulfonate (EMS) as a control solution for soaking for 40min, 50min, 60min, 70min, 80min, 90min, 100min, 110min and 120min and a control solution (CK, 120min) (the soaking time is obtained by pre-experiments according to the anther survival rate), and the anthers are washed for 10 times by sterile anthers after soaking for inoculation;
(2) induction culture of anther embryonic callus: the anther soaked in EMS is inoculated into embryogenic callus induction culture medium (culture medium components are shown in 1.2.2) in a flat-laying manner, and the illumination period is 6 h.d-1Culturing at the illumination intensity of 800lx and the temperature of 26 + -2 deg.C for 80 days to completely dedifferentiate anther and convert into white and yellow embryogenic callus (as shown in FIG. 1);
(3) and (3) redifferentiation culture of the embryogenic callus: when anther is completely dedifferentiated to form embryogenic callus, the embryogenic callus is cut into suitable small pieces and transferred to embryogenic callus for redifferentiationCulturing in culture medium (medium components are shown in 1.2.2), and standing in light cycle for 10h d-1Culturing for 60 days under the conditions of illumination intensity of 1000lx and temperature of 25 +/-2 ℃, and differentiating the embryogenic callus into conical embryoid with independence, bipolarity and separability;
(4) and (3) development and culture of embryoid: continuously culturing the embryogenic callus containing the conical embryoid for 25 days, and gradually separating the embryoid from the embryogenic callus; then the embryoid is peeled off and transferred into an embryoid development culture medium and placed in a light cycle of 12 h.d-1Carrying out development culture under the conditions of illumination intensity of 1200lx and temperature of 24 +/-2 ℃, and culturing for 45 days until embryoids are subjected to double polarization development to form small plants containing roots, stems and leaves;
(5) transplanting, field planting and strain determination: when the plantlets grow to about 2.0cm in the culture bottles, taking out the plantlets from the bottles, domesticating and hardening the plantlets, planting the plantlets into a nutrition pot, planting the plantlets into a field when the plantlets grow to 20cm, and observing the growth condition of each single plant; and then labeling the different individual plants, grafting the labeled individual plants to different suitable regions for cultivation, screening and determining the excellent character mutants with stable heredity as strains according to factors such as growth vigor, morphological characteristics, fruit shape, fruit size, acid sweetness, taste, adaptability, yield, disease resistance, seed seedling heredity and the like, and numbering (strain codes).
In order to reduce the experiment times and ensure the reasonability of matching two factors of the volume ratio concentration of the ethyl methanesulfonate and the soaking time, the experiment is designed by a uniform design method, and U is selected9(92) In the table, 100 anthers are inoculated in each treatment, repeated for 3 times, and the optimal proportion of EMS volume specific concentration and soaking time influencing the differentiation mutant of the embryogenic callus of the malus baccata anther is screened and determined, wherein the mutation rate is × 100 percent of the number of mutants/plantlet formed by the development of the embryoid and the total number of cultivated survived plants.
2 results and analysis
2.1 Effect of EMS volume ratio concentration and soaking time on Vitex negundo anther embryogenic callus redifferentiation mutant
TABLE 1 screening of U affecting the embryogenic callus redifferentiation mutant factors of the tender stem section of fructus Viticis negundo9(92) Experimental design and results
The data (table 1) obtained in the experiment were analyzed and processed by the uniform design software to obtain the regression equation of-0.168 +4.05X1+0.00763X2Significance level α -0.05, complex correlation coefficient R-0.9272, residual standard deviation S-0.142, test value FtMore than 18.39 & gt critical value F(0.05,2,6)5.143, the regression equation has significance, and the influence of the ethyl methanesulfonate volume ratio concentration and the soaking time on the mutation of the malus baccata is significant and has certain regular mutation. The U can be obtained by calculating the contribution value and the contribution rate of the volume ratio concentration of the ethyl methanesulfonate and the soaking time to the mutation1＝0.394，U1/U＝53.0％；U2＝0.350，U247.0% of/U, which shows that ethyl methanesulfonate and soaking time contribute equally to the vitex anther embryogenic callus differentiation mutant. According to a regression equation, the EMS volume ratio concentration and the soaking time are positively correlated with the mutation rate, the anther survival rate is less than 5 percent because 0.24 percent of EMS is soaked for 120min in a pre-experiment, and although the EMS volume ratio concentration and the soaking time are positively correlated with the mutation rate, the anther death can not be tested due to the increase of the EMS volume ratio concentration and the soaking time.
Therefore, according to the test results and regression analysis, in order to ensure the anther survival rate and higher mutation rate, the EMS volume ratio concentration of 0.22% and the soaking time of 110min are selected for verification test, and the result shows that the anther survival rate after inoculation reaches more than 70%, the mutation rate reaches 1.83% through 4-year cultivation observation, and 4 genetically stable mutants are obtained from 219 cultivated single plants.
The anther soaked by the ethyl methane sulfonate is inoculated into an embryonic callus induction culture medium and is completely dedifferentiated and converted into a white and yellow embryonic callus after about 80 days of culture. Cutting the embryonic callus into small blocks, transferring the small blocks into an embryonic callus redifferentiation culture medium, continuously culturing for about 60 days, separating an embryoid with independence, bipolarity and separability on the surface of the embryonic callus, continuously culturing for 25 days, gradually separating the embryoid from the embryonic callus, peeling the embryoid, transferring the embryoid into an embryoid development culture medium for development culture for about 45 days, and carrying out bipolarization development on the embryoid to obtain small plants containing roots and stems and leaves. Through the series of tests, the vitex negundo linn anther is used as a material, is soaked in ethyl methanesulfonate with a proper concentration for a certain time, and is induced by a plant tissue culture technology to obtain the mutant, and the mutant has stable hereditary character and can be used as a strain for cultivating and constructing a vitex negundo linn mutant library.
At present, all the single plants are cultivated in 7 suitable regions for 8 years, 87 genetically stable single plants are obtained by the method, and 3 of the 87 genetically stable single plants are excellent lines with large fruits, good taste, moderate sweet and sour taste, good fresh eating property, high yield, stability and strong resistance. The following is a profile of the traits of the elite mutant lines (see figure 2 for a detailed line picture):
line 1 (line code: 2013-: in 2013, a single plant with excellent characteristics is selected from a mutant produced by inducing wild fructus viticis anther by EMS, and the strain has strong growth vigor and multiple branches; the fruits are oblate, large, the transverse diameter is about 2.4-2.6cm, the height is about 2.0-2.2cm, the fruits are dark red after being mature, the average single fruit weight is 7.4 g, and the maximum single fruit weight is more than 8.3 g; the taste is good, the sour and sweet taste is moderate, the fresh eating performance is good, the content of soluble solid is 6.2 percent, the total sugar is 3.3 g/100 g, and the vitamin C is 203.5 mg/100 g; the yield is high, about 55kg of the yield of an 8-year-old single plant, high yield stability is good, and the resistance is strong. Is suitable for wide cultivation in southeast areas of Jilin province. The mature of the strain begins in 10 months and belongs to a late-maturing strain.
Line 2 (line code: 2013-: in 2013, a single plant with excellent characteristics is selected from a mutant produced by inducing wild fructus viticis anther by EMS, and the strain has strong growth vigor and more branches; the fruits are elliptic and columnar, the fruits are large, the longitudinal length is about 1.8-2.0cm, the transverse width is about 1.5-1.7cm, the fruits are purplish red after being mature, the average single fruit weight is 6.2 g, and the maximum single fruit weight is more than 6.8 g; the sugar-free sweet potato has good mouthfeel, moderate sweet and sour taste and good fresh eating property, the content of soluble solid matters is 5.8 percent, the total sugar is 3.2 g/100 g, and the vitamin C is 197.3 mg/100 g; the yield is high, about 55kg of the yield of an 8-year-old single plant, high yield stability is good, and the resistance is strong. Is suitable for wide cultivation in southeast areas of Jilin province. It begins to mature in late 9 months and belongs to middle and late maturing lines.
Line 3 (line code: 2013-: in 2013, a single plant with excellent characteristics is selected from a mutant produced by inducing wild fructus viticis anther by EMS, and the strain has strong growth vigor and extremely many branches; the fruit is cone-shaped, the fruit is large, the diameter of the cone bottom is about 2.0-2.5cm, the cone height is about 2.5-2.8cm, the fruit is purplish red after being mature, the average single fruit weight is 7.0g, and the maximum single fruit weight is more than 7.7 g; the taste is good, the taste is sweet, the fresh eating performance is good, the content of soluble solid matters is 7.1 percent, the total sugar is 3.6 g/100 g, and the vitamin C is 183.7 mg/100 g; the yield is high, about 60kg of the yield of an 8-year-old single plant, high yield stability is good, and the resistance is strong. Is suitable for wide cultivation in southeast areas of Jilin province. It begins to mature in the middle of 9 months and belongs to an early maturing strain.
The invention takes the malus baccata anther as a material, the malus baccata anther is soaked for a certain time by ethyl methane sulfonate with proper concentration, and then the method of embryogenic callus induction, redifferentiation, embryoid development and the like is adopted by combining with the plant tissue culture technology to obtain a novel artificial malus baccata cultivar which has the characteristics of strong independence of each individual plant, high purity of individual plant genetic information, clear individual plant source and the like, and the cultivar is stable in heredity and can be used as a strain for cultivating and constructing a malus baccata mutant library.
The above description is only a preferred embodiment of the present invention, but the design concept of the present invention is not limited thereto, and any insubstantial modifications made by using the design concept should fall within the scope of infringing on the protection scope of the present invention.
Claims (10)
1. A new variety breeding method for inducing the flower drug mutation of fructus viticis negundo is characterized by comprising the following steps:
(1) material selection and pretreatment: in the first ten days of 6 months, selecting full buds after the wild fructus viticis shows buds, washing the buds with 75% ethanol for 10s, transferring the buds to a saturated sodium hypochlorite solution for soaking for 2min, washing with sterile water for 10 times, and sucking off surface water with sterile filter paper; then, the flower buds are stripped, anthers on stamens are picked off, and the anthers are put into EMS solutions with different concentrations in batches and soaked for 40-120 min; finally, the anther is flushed by sterile water for 10 times for inoculation;
(2) induction culture of anther embryonic callus: transferring the anther soaked in EMS in the step (1) into an embryonic callus induction culture medium, and placing the embryonic callus induction culture medium in a light cycle of 6 h.d-1Culturing at the illumination intensity of 800lx and the temperature of 26 +/-2 ℃ for 80 days, and completely dedifferentiating the anther to convert the anther into a white and yellow embryonic callus;
(3) and (3) redifferentiation culture of the embryogenic callus: cutting the embryogenic callus in the step (2) into small pieces, transferring to embryogenic callus redifferentiation culture medium, and culturing while placing in light cycle for 10 h.d-1Culturing for 60 days under the conditions of illumination intensity of 1000lx and temperature of 25 +/-2 ℃, and differentiating embryoid with independence, bipolarity and separability from the embryogenic callus;
(4) and (3) development and culture of embryoid: continuously culturing the embryogenic callus containing the embryoid in the step (3) for 25 days, and gradually separating the embryoid from the embryogenic callus; then the embryoid is peeled off and transferred into an embryoid development culture medium and placed in a light cycle of 12 h.d-1Carrying out development culture under the conditions of illumination intensity of 1200lx and temperature of 24 +/-2 ℃, and culturing for 45 days until embryoids are subjected to double polarization development to form small plants containing roots, stems and leaves;
(5) transplanting, field planting and strain determination: when the plantlets in the step (4) grow to 2.0cm in a culture bottle, taking out the plantlets from the bottle, planting the plantlets in a nutrition pot after domestication and hardening, planting the plantlets in a field after the plantlets grow to 20cm, and observing the growth condition of the plantlets; then labeling the different single plants and grafting the labels to different suitable regions for cultivation; and finally, screening and determining the excellent character mutants with stable heredity as strains, and numbering.
2. The method for breeding a new variety inducing an anther mutation of fructus viticis as claimed in claim 1, wherein: EMS concentration in the step (1) is 0.1 mol.L-1Phosphate buffer solution with pH 7 as solvent is prepared into the concentration of volume percentageEMS solution of 0.08-0.24%.
3. The method for breeding a new variety inducing an anther mutation of fructus viticis as claimed in claim 2, wherein: the EMS solutions with different concentrations are EMS solutions with the volume percentages of 0.08%, 0.10%, 0.12%, 0.14%, 0.16%, 0.18%, 0.20%, 0.22% and 0.24%, respectively.
4. The method for breeding a new variety inducing an anther mutation of fructus viticis as claimed in claim 1, wherein: the culture medium comprises a minimal medium, and the components of the minimal medium comprise the following substances: potato supernatant 60 g.L-1(ii) a Macroelements: 20.0 mg. L-1NH4NO3，7.5mg·L-1CaCl2·2H2O，8.9mg·L-1MgSO4·7H2O; iron salt: 1.5 mg. L- 1FeSO4·7H2O，1.8mg·L-1Na2·EDTA·2H2O。
5. The method for breeding a new variety inducing an anther mutation of fructus viticis as claimed in claim 1 or 4, wherein: the culture medium in the step (2) is a basic culture medium with the addition of 2.45 mg.L-1Isopentenyladenine and 0.35 mg.L-1NAA, and 6.8 g.L-1Agar powder, 50 g.L-1Sucrose.
6. The method for breeding a new variety inducing an anther mutation of fructus viticis as claimed in claim 5, wherein: the pH value of the culture medium in the step (2) is 6.0.
7. The method for breeding a new variety inducing an anther mutation of fructus viticis as claimed in claim 1 or 4, wherein: the culture medium in the step (3) is a basic culture medium with the addition of 3.2 mg.L-1Isopentenyladenine and 7.0 g.L-1Agar powder, 40 g.L-1Sucrose.
8. The method for breeding a new variety inducing an anther mutation of fructus viticis as claimed in claim 7, wherein: the pH value of the culture medium in the step (3) is 5.9.
9. The method for breeding a new variety inducing an anther mutation of fructus viticis as claimed in claim 1 or 4, wherein: the culture medium in the step (4) is a basic culture medium with 0.02 mg.L-1KT, and 6.8 g. L-1Agar strips, 30 g.L-1Sucrose, and adjusting the pH value of the culture medium to 6.0.
10. The method for breeding a new variety inducing an anther mutation of fructus viticis as claimed in claim 1, wherein: the strain is determined to be screened according to one or more factors of the growth vigor, morphological characteristics, fruit shape, fruit size, acid sweetness, mouthfeel, adaptability, yield, disease resistance and genetic condition of the seedlings.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2007121518A1 (en) * | 2006-04-21 | 2007-11-01 | The State Of Western Australia Through Its Department Of Agriculture | Improved plant culture methods using a modified auxin treatment step |
| CN101185421A (en) * | 2007-12-14 | 2008-05-28 | 中国农业科学院果树研究所 | Method of 'hanfu' apple anther culture plant strain |
| CN109548649A (en) * | 2017-11-01 | 2019-04-02 | 岭南生态文旅股份有限公司 | A kind of M. laxiflora tissue cultures inducer substance and its application method |
-
2020
- 2020-04-13 CN CN202010286188.6A patent/CN111316916A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2007121518A1 (en) * | 2006-04-21 | 2007-11-01 | The State Of Western Australia Through Its Department Of Agriculture | Improved plant culture methods using a modified auxin treatment step |
| CN101185421A (en) * | 2007-12-14 | 2008-05-28 | 中国农业科学院果树研究所 | Method of 'hanfu' apple anther culture plant strain |
| CN109548649A (en) * | 2017-11-01 | 2019-04-02 | 岭南生态文旅股份有限公司 | A kind of M. laxiflora tissue cultures inducer substance and its application method |
Non-Patent Citations (3)
| Title |
|---|
| 左伟勇等: "《细胞工程技术》", 31 August 2014, 重庆大学出版社 * |
| 穆平: "《作物育种学》", 31 July 2017, 中国农业大学出版社 * |
| 蔡庆生: "《植物生理学实验》", 28 February 2013, 中国农业大学出版社 * |
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