CN111316915B - New variety cultivation method for inducing hypocotyl mutation of actinidia arguta embryo by EMS - Google Patents
New variety cultivation method for inducing hypocotyl mutation of actinidia arguta embryo by EMS Download PDFInfo
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Abstract
The invention belongs to the technical field of plant propagation, and particularly relates to a novel variety cultivation method for inducing mutation of hypocotyl of actinidia arguta embryo by EMS. According to the novel variety cultivation method for inducing the sub-embryonal axis mutation of the actinidia arguta embryo by using the sub-embryonal axis of the actinidia arguta embryo as a material, combining Ethyl Methylsulfonate (EMS) with a plant tissue culture technology, adopting methods of embryogenic callus induction, redifferentiation, embryoid rejuvenation and the like, obtaining novel actinidia arguta artificial cultivars with the characteristics of strong independence of each individual plant, high purity of the genetic information of the individual plant, clear individual plant sources and the like, and having important protection and ecological significance for the amplification of actinidia arguta population, and the method can be further applied to the construction of a actinidia arguta mutant library and cultivation and variety approval directly serving as a strain.
Description
Technical Field
The invention belongs to the technical field of new plant varieties, and particularly relates to a novel variety cultivation method for inducing mutation of hypocotyl of actinidia arguta embryo by EMS.
Background
Actinidia arguta (sieb.et zucc.) plane.ex miq.), the Chinese Actinidia arguta, fructus actinidiae chinensis, fructus kochiae, fructus actinidiae chinensis, radix actinidiae chinensis, fructus actinidiae chinensis, and fructus actinidiae chinensis are vine fallen leaves of Actinidia of actinidiaceae. The fruit is edible, has high nutritive value, contains a large amount of vitamin C, starch, pectin and the like, and can be processed into jam, fruit juice, preserved fruit, can, wine or used for making various foods such as cakes, candies and the like. Can also be used for treating diseases, such as strengthening body constitution, relieving fever, invigorating stomach, and stopping bleeding; the root and root bark have certain treatment and inhibition effects on digestive tract cancer; the seed oil content is 35.62%, which is a better drying oil with extremely high economic and medicinal value; also good honey source and ornamental plant, at present, actinidia arguta is mainly wild and artificially cultivated (variety) is auxiliary.
In the prior art, actinidia arguta is bred by adopting a hybridization mode, the characters of the offspring of the propagation of the hybridized seeds are separated, and then plants with excellent characters are selected and bred in the seedlings for cultivation. By observing the genetic stability of the seedlings, the actinidia arguta is found to have genetic diversity, and the good strain bred after hybridization of the actinidia arguta has cultivation polymorphism of regional fruit shapes and quality with different temperatures, geographies, climates and the like, so that the same species is cultivated in different regions to have fruit shapes and quality differences, the uncertainty of the species is caused, and disputes between a grower and a species authority are caused.
Disclosure of Invention
In order to solve the problems that the actinidia arguta is bred in a hybridization mode, genetic diversity and cultivation polymorphism are easy to occur, the fruit shape, quality and other differences are caused in cultivation of the same variety in different areas, the variety uncertainty is caused, disputes between a grower and a variety authority are caused, and the like, the invention provides a novel variety cultivation method for inducing the hypocotyl mutation of the actinidia arguta embryo by using EMS.
In order to achieve the above purpose, the technical scheme adopted by the invention is as follows: a method for cultivating a new variety of EMS induced hypocotyl mutation of actinidia arguta embryo comprises the following steps:
(1) Material selection and pretreatment: taking the mature fruit of the wild actinidia arguta in late 9 months, cleaning the seeds in the fruit with clear water, and obtaining the mature seeds with water bottom; mixing the mature seeds with river sand at a ratio of 1:3, keeping the relative humidity of the mixture of the seeds and the river sand at 75%, and placing the mixture in a climatic chamber with the temperature of 18-20 ℃ and the relative humidity of 78% for dark treatment for 30 days;
separating and cleaning seeds from river sand after the embryo is further developed and mature, washing the seeds with 75% ethanol for 30s in a sterile ultra-clean workbench, then transferring the seeds to a 2-5% sodium hypochlorite solution for soaking for 10min, washing the seeds with sterile water for 10 times, and sucking surface moisture with sterile filter paper; peeling off seed coats with a sterile scalpel and forceps, breaking off two cotyledons, taking out seed embryos, separating hypocotyls, putting the hypocotyls into EMS solutions with different concentrations in batches, and soaking for 2-6h; finally, the hypocotyl is washed by sterile water for 10 times for inoculation;
(2) Induction culture of hypocotyl embryogenic callus: transferring the hypocotyl soaked by EMS in the step (1) into embryogenic callus induction culture medium, and placing in a lighting period of 7h.d -1 Culturing for 55 days at the illumination intensity of 800lx and the temperature of 26+/-2 ℃, completely dedifferentiating the hypocotyl into yellow-green embryogenic callus, and carrying out secondary proliferation culture on the embryogenic callus;
(3) And (3) carrying out sub-differentiation culture on the hypocotyl embryogenic callus: cutting the embryogenic callus in the step (2) into small blocks, transferring the small blocks into embryogenic callus redifferentiation medium, and placing the small blocks in an illumination period of 12 h.d -1 Continuously culturing for 45 days under the conditions of illumination intensity of 1200lx and temperature of 26+/-2 ℃, and differentiating embryoid callus to obtain independent, bipolar and separable embryoids;
(4) And (3) rejuvenating, growing and culturing embryoid: peeling the embryoid in the step (3), transferring to embryoid rejuvenation growth medium, and placing in a lighting period of 10h.d -1 Rejuvenating and growing the embryoid at the illumination intensity of 1000lx and the temperature of 26+/-2 ℃ until the embryoid grows in two polarizations and grows into plantlets containing roots and stems and leaves after 30 days of complete development;
(5) Transplanting and field planting and strain determination: planting the acclimatized and acclimatized plantlets of the small plants in the step (4) in a nutrition pot, planting and cultivating the plantlets in a germplasm garden when the plantlets grow to 15-20cm, observing the growth conditions of the plantlets, and classifying and labeling the plantlets; then breeding large seedlings by grafting different mutants, and cultivating in different areas; and finally, screening to determine the genetically stable excellent character mutant as a strain, and numbering.
Further, in the step (1), EMS was used at a concentration of 0.1 mol.L -1 Phosphate buffer with ph=7 was prepared as a solvent into EMS solution with a volume percentage concentration of 0.1-1.5%.
Further, the EMS solutions with different concentrations are respectively 0.1%, 0.3%, 0.5%, 0.7%, 0.9%, 1.1%, 1.3% and 1.5% of the volume percentage.
Further, the culture medium comprises a basic culture medium, and the basic culture medium comprises the following components: 25 g.L of potato supernatant -1 The method comprises the steps of carrying out a first treatment on the surface of the Iron salt: 6.95 mg.L -1 FeSO 4 ·7H 2 O，9.325mg·L -1 Na 2 ·EDTA·2H 2 O; trace elements: 1.25 mg.L -1 MnSO 4 ·4H 2 O，0.25mg·L -1 ZnSO 4 ·7H 2 O，0.095mg·L -1 KI。
Further, the culture medium in the step (2) is a basic culture medium added with 1.75 mg.L -1 Brassinolide and 0.5 mg.L -1 2,4-D and 7.0 g.L -1 Agar powder, 50 g.L -1 Sucrose.
Further, the pH of the medium in step (2) was 6.0.
Further, the culture medium in the step (3) is a basic culture medium added with 1.5 mg.L -1 Brassinolide and 0.2 mg.L -1 NAA and 7.0 g.L -1 Agar powder, 30 g.L -1 Sucrose.
Further, the pH of the medium in step (3) was 6.0.
Further, the culture medium in the step (4) is a basic culture medium added with 0.08 mg.L -1 Brassinolide, 0.12 mg.L -1 Gibberellin GA 3 And 0.01 mg.L -1 IBA and 7.8g.L -1 Agar strips, 10 g.L -1 Sucrose, and adjusting the pH value of the culture medium to 6.0.
Further, the classification of mutants is differentially classified according to the fruit shape of the mutants; the strain is determined and screened according to one or more factors of mutant fruit shape, resistance and quality.
The invention provides a novel variety cultivation method for inducing mutation of hypocotyl of actinidia arguta embryo by EMS, which has the following beneficial effects:
(1) The characteristics of the Ethyl Methylsulfonate (EMS) are that the Ethyl Methylsulfonate (EMS) acts on the induction points to cause the induction point mutation, so that chromosome deformity is not caused, the dominant mutation is high, the genetically stable mutant can be directly obtained without further genetic transformation, the cultivation polymorphism is extremely small, and the excellent character mutant can be directly selected as strain cultivation and variety approval, so that the breeding period is obviously shortened;
(2) The method has the advantages of simplicity, multiple effects, high mutation rate of the induced offspring, wide mutation range, good genetic stability of mutants, diversity of the mutants and the like;
(3) The method takes the hypocotyl of the actinidia arguta embryo as a material, combines a mutagen EMS (energy management system) with a plant tissue culture technology, adopts methods of embryogenic callus induction, redifferentiation, embryoid rejuvenation and the like, has the characteristics of strong independence of each individual plant, high purity of the genetic information of the individual plant, clear individual plant sources and the like, and can be further applied to the construction of actinidia arguta mutant libraries.
Drawings
FIG. 1 is a block diagram of embryogenic callus of the hypocotyl of actinidia arguta containing embryogenic cells;
line 1 (line code: c 2004-001);
b. line 2 (line code: c 2004-032);
c. line 3 (line code: c 2004-22);
d. line 4 (line code: c 2004-31);
e. line 5 (line code: c 2005-29);
f. line 6 (line code: c 2005-9);
g. line 7 (line code: c 2007-34);
h. line 8 (line code: c 2007-8);
i. line 9 (line code: c 2007-36);
j. line 10 (line code: c 2008-33);
k. line 11 (line code: c 2008-23);
line 12 (line code: c 2009-12);
line 13 (line code: c 2010-13);
strain 14 (strain code: x 2009-10);
line 15 (line code: x 2010-11).
Detailed Description
The following description of the embodiments of the present invention will be made clearly and fully with reference to the accompanying drawings, in which it is evident that the embodiments described are only some, but not all embodiments of the invention.
As shown in fig. 1 and 2, a new variety cultivation method for inducing mutation of hypocotyl of actinidia arguta embryo by EMS comprises the following steps:
1 materials and methods
1.1 Material selection and pretreatment
Selecting mature fruit of wild actinidia arguta (fruit is full and soft without deformity) in middle and late 9 months, cleaning the seeds in the fruit with clear water to obtain mature seeds at the bottom of the water, uniformly mixing the mature seeds with river sand in a ratio of 1:3, keeping the relative humidity of the mixture of the seeds and the river sand to be 75%, and carrying out dark treatment in a climatic chamber with the temperature of 18-20 ℃ and the relative humidity of 78% for about 30 days. After the embryo is further developed and matured, the seeds are separated from river sand and washed for standby.
1.2 method
1.2.1 preparation of Ethyl Methylsulfonate (EMS) solution
EMS at a concentration of 0.1 mol.L -1 Phosphate buffer solution with pH=7 is used as solvent to prepare EMS solution with volume percentage concentration of 0.1-1.5%;
namely 0.1 mol.L at pH 7.0 -1 Ethyl Methylsulfonate (EMS) was formulated to a volume percent concentration (v.v.) -1 ) The concentrations of the solutions are respectively 0.1%, 0.3%, 0.5%, 0.7%, 0.9%, 1.1%, 1.3% and 1.5%, and phosphate buffer is used as a control solution (CK), and filtration sterilization is needed before use.
1.2.2 preparation of Medium
The basic culture medium comprises the following components in percentage by weight: 25 g.L of potato supernatant -1 The method comprises the steps of carrying out a first treatment on the surface of the Iron salt: 6.95 mg.L -1 FeSO 4 ·7H 2 O，9.325mg·L -1 Na 2 ·EDTA·2H 2 O; trace elements: 1.25 mg.L -1 MnSO 4 ·4H 2 O，0.25mg·L - 1 ZnSO 4 ·7H 2 O，0.095mg·L -1 KI。
Hypocotyl embryogenic callus induction medium and culture conditions: 1.75mg.L of the minimal medium is added -1 Brassinolide and 0.5 mg.L -1 2,4-D，7.0g·L -1 Agar powder, adding 50g.L -1 Sucrose, and adjusting the pH value of the culture medium to 6.0; culturing the inoculated hypocotyl in a culture flask with 7 h.d illumination period -1 Culturing at a temperature of 26+ -2deg.C under 800lx illumination intensity.
Embryogenic callus redifferentiation medium and culture conditions: 1.5 mg.L of the minimal medium was added -1 Brassinolide and 0.2 mg.L -1 NAA，7.0g·L -1 Agar powder, adding 30g.L -1 Sucrose, and adjusting the pH of the medium to 6.0. Placing the culture flask in an illumination period of 12 h.d after the embryogenic callus is inoculated -1 Culturing at temperature of 26+ -2deg.C under illumination intensity of 1200 lx.
Embryoid rejuvenation growth medium and culture conditions: the basic culture medium is added with 0.08 mg.L -1 Brassinolide, 0.12 mg.L -1 Gibberellin GA 3 And 0.01 mg.L -1 Indolebutyric acid IBA,7.8 g.L -1 Agar strips, 10 g.L added -1 Sucrose, and adjusting the pH value of the culture medium to 6.0. The culture flask is placed in an illumination period of 10 h.d after embryoid inoculation -1 Culturing at 26+ -2deg.C under 1000lx illumination intensity.
1.2.3 method of operation
(1) And (3) material treatment: in a sterile ultra-clean workbench, washing seeds separated and cleaned from river sand in 1.1 for 30 seconds, then immersing the seeds in a sodium hypochlorite solution (mass concentration) of 3.0 percent, washing the seeds for 10 minutes with sterile water, sucking the surface moisture by sterile filter paper, peeling off seed coats by a sterile surgical knife and forceps, breaking off cotyledons, taking out embryo, separating hypocotyls to a certain number, and then immersing the embryo in a phosphate buffer (CK, 0%) of 0.1 percent, 0.3 percent, 0.5 percent, 0.7 percent, 0.9 percent, 1.1 percent, 1.3 percent, 1.5 percent of Ethyl Methanesulfonate (EMS) and serving as a control solution for 2h, 2.5h, 3h, 3.5h, 4h, 4.5h, 5.5h and the control solution (CK, 6 h) (the embryo is obtained through experiments according to the hypocotyl mortality). Washing the hypocotyl with sterile water for 10 times after soaking for inoculation;
(2) Induction culture of hypocotyl embryogenic callus: transferring the hypocotyl soaked in EMS into embryogenic callus induction medium (medium composition 1.2.2), and placing in illumination period 7h.d -1 Culturing at temperature of 26+ -2deg.C at illumination intensity of 800lx for 55 days to obtain embryogenic callus (shown in figure 1) with hypocotyl completely dedifferentiated and changed into yellowish green, and performing embryogenic callus subculture for proliferation;
(3) And (3) carrying out differential culture on embryogenic callus: embryogenic callus was cut to 0.3cm 3 Is transferred to embryogenic callus redifferentiation medium (medium composition see 1.2.2) and is placed in a light cycle of 12 h.d -1 Continuously culturing for 45 days under the conditions of illumination intensity of 1200lx and temperature of 26+/-2 ℃, and differentiating embryoid callus to obtain independent, bipolar and separable embryoids;
(4) And (3) rejuvenating, growing and culturing embryoid: the embryoid is peeled off and transferred to embryoid rejuvenation growth medium (medium composition is 1.2.2) and placed in a light cycle of 10h.d -1 Rejuvenating and growing the embryoid at the illumination intensity of 1000lx and the temperature of 26+/-2 ℃ until the embryoid grows in two polarizations and grows into plantlets containing roots and stems and leaves after 30 days of complete development;
(5) Transplanting and field planting and strain determination: and (3) domesticating and hardening seedlings of the plantlets, planting the plantlets in a nutrition pot, planting and cultivating the plantlets in a planting pot until the plantlets grow to 15-20cm, observing the growth condition of the plantlets, classifying and labeling the plantlets according to morphological characteristic differences such as fruit shapes and the like after the plantlets grow, breeding large seedlings of proper quantity by a grafting method for regional cultivation, observing the fruit shapes, cold resistance, quality and the like of the mutants, and screening and determining excellent mutants with stable inheritance as strains for numbering (strain codes). The strain can be further subjected to new variety approval.
In order to ensure the rationality of matching the EMS product percentage concentration and the soaking time and reduce the experiment times, the experiment is designed by a uniform design method, and U is selected 8 (8 2 ) And (3) uniformly inoculating 30 hypocotyls for each treatment, repeating for 3 times, and screening to determine the optimal proportion of EMS volume percentage concentration and soaking time of the hypocotyl embryogenic callus redifferentiation mutants affecting actinidia arguta. Mutation rate = number of mutants/plantlet formed by embryoid body and cultivated to survive the plant total number x 100%.
2 results and analysis
2.1 Influence of EMS volume percentage concentration and soaking time on the hypocotyl embryogenic callus redifferentiation mutant of actinidia arguta
TABLE 1U affecting screening of factors of tender stem slice embryogenic callus differentiation mutants of actinidia arguta 8 (8 2 ) Test design and results
The regression equation of the data (Table 1) obtained by the test after analysis and treatment by the uniform design software is Y= -5.91+6.18X 1 +1.48X 2 Significance level α=0.05, complex correlation coefficient r= 0.9654, residual standard deviation s=0.976, test value F t =34.25 > critical value F (0.05,2,5) The regression equation has significance, and the ethyl methylsulfonate volume percent concentration and the soaking time have significant effects on actinidia arguta mutation and have certain regular mutation. By calculating the volume percentage concentration of the ethyl methylsulfonateAnd the contribution value and the contribution rate of the soaking time to mutation are known, U 1 ＝58.9，U 1 /U＝90.3％；U 2 ＝21.2，U 2 /u=32.5%, indicating that EMS contributed significantly more than soak time to the mutation. The regression equation shows that the volume percent concentration and the soaking time of the EMS are positively correlated with the mutation rate, and the survival rate of the hypocotyl is less than 20% because the hypocotyl is soaked for 5.5 hours by 1.5% of the EMS in the pre-experiment, although the volume percent concentration and the soaking time of the EMS are positively correlated with the mutation rate, the reduction of the survival rate of the hypocotyl caused by the increase of the volume percent concentration and the soaking time of the EMS is also meaningless.
Therefore, according to test results and regression analysis, in order to ensure the survival rate of the tender hypocotyl and higher mutation rate, verification tests are carried out by selecting EMS (electromagnetic induction system) with volume percentage concentration of 1.40% and soaking time of 4.5 hours, and the result shows that the survival rate of the hypocotyl after inoculation reaches more than 78.59%, and after 3 years of cultivation observation, the mutation rate reaches 11.48%, and 7 genetically stable mutants are obtained in 61 single plants cultivated.
Transferring the hypocotyl soaked by ethyl methylsulfonate into embryogenic callus induction medium, and culturing for about 55 days to completely dedifferentiate the hypocotyl into yellowish green embryogenic callus. Cutting embryogenic callus into small blocks (according to the situation), transferring to embryogenic callus redifferentiation culture medium for continuous culture, culturing embryogenic callus for about 45 days, and differentiating embryogenic callus into independent, bipolar and separable embryoid. The embryoid is transferred into an embryoid rejuvenation growth culture medium for rejuvenation growth culture after being stripped, the embryoid grows in two polarizations, and the embryoid grows to about 30 days, and fully develops into a plantlet containing roots and stems. According to the series of experiments, the method of combining the method of soaking the undershaft of the actinidia arguta embryo with the plant tissue culture technology through ethyl methylsulfonate can be used for obtaining higher mutation rate, and the mutant character is genetically stable, so that the actinidia arguta embryo can be used as a strain for cultivating and constructing a actinidia arguta mutant library.
At present, 106 genetically stable mutants have been obtained by the method after 8 years of cultivation in 5 suitable areas, wherein 15 mutants have extremely strong economical excellent properties, 13 are female strains, 2 are male strains, and the genetically stable mutants can be used as strains for artificial cultivation. The following are characteristic profiles for 15 lines (see figure 2 for details of each line):
strain 1 (strain code: c 2004-001): in 2004, the single plant selected from the mutant induced by EMS to generate hypocotyl of wild actinidia arguta embryo has oval fruit shape, average single fruit weight of 25g, and maximum single fruit weight of more than 40 g. The soluble solids content was 12.8%, total sugar 6.7 g/100 g, vitamin C432.3 mg/100 g. The yield of the 8-year-old single plant shed frame is about 110kg, and the high yield and the good stability are achieved. Has strong resistance, good fruit quality and better storage resistance. Is suitable for cultivation in southeast area of Jilin province. Generally, the plant begins to mature in the last 10 months and belongs to late-maturing strains.
Line 2 (line code: c 2004-032): in 2004, the single plant selected from the mutant induced by EMS to generate hypocotyl of wild actinidia arguta embryo has oval shape, average single fruit weight of 18 g, and maximum single fruit weight of more than 20 g. The soluble solids content was 13.1%, total sugar 6.9 g/100 g, vitamin C407.6 mg/100 g. The yield of the 8-year-old single plant shed frame is about 100kg, and the high yield and the good stability are achieved. Has strong resistance, good fruit quality and better storage resistance. Is suitable for cultivation in southeast area of Jilin province. Maturing is generally started in 9 middle ten days, and belongs to a medium-maturing strain.
Strain 3 (strain code: c 2004-022): in 2004, the individual plant selected from the mutant induced by EMS to generate hypocotyl of wild actinidia arguta embryo was long, the average single fruit weight was 18 g, and the maximum single fruit weight was 25g or more. The soluble solids content was 13.6%, total sugar 6.8 g/100 g, vitamin C418.9 mg/100 g. The yield of the 8-year-old single plant shed frame is about 110k, and the high yield and the good stability are achieved. Has strong resistance, good fruit quality and better storage resistance. Is suitable for cultivation in southeast area of Jilin province. Maturing is generally started in 9 middle ten days, and belongs to a medium-maturing strain.
Line 4 (line code: c 2004-031): in 2004, the single plant selected from the mutant induced by EMS to generate hypocotyl of wild actinidia arguta embryo has long fruit, average single fruit weight of 16 g, and maximum single fruit weight of more than 18 g. The soluble solids content was 13.9%, total sugar 6.8 g/100 g, vitamin C413.7 mg/100 g. The yield of the 8-year-old single plant shed frame is about 90kg, and the high yield and the good stability are achieved. Has strong resistance, good fruit quality and better storage resistance. The dormancy period is short, and the greenhouse cultivation method is suitable for greenhouse cultivation in southeast areas of Jilin provinces. Generally, the seeds begin to mature in the last 9 months and belong to early maturing strains.
Line 5 (line code: c 2005-29): in 2005, the single plant selected from the mutant induced by EMS to generate the hypocotyl of the wild actinidia arguta embryo has oblate fruit, and the fruit peel and the fruit core are red after the fruit is ripe, the average single fruit weight is 16.5 g, and the maximum single fruit weight is more than 19 g. The soluble solids content was 13.4%, total sugar 6.5 g/100 g, vitamin C441.4 mg/100 g. The yield of the 8-year-old single plant shed frame is about 95kg, and the high yield and the good stability are achieved. Has strong resistance, good fruit quality and better storage resistance. Is suitable for cultivation in southeast area of Jilin province. Maturing is generally started in the middle and late 9 months, and belongs to a middle and late maturing strain.
Strain 6 (strain code: c 2005-9): in 2005, the single plant selected from the mutant induced by EMS to generate the hypocotyl of the wild actinidia arguta embryo has oval shape, the average single fruit weight is 18 g, and the maximum single fruit weight is more than 20 g. The soluble solids content was 13.6%, total sugar 6.7 g/100 g, vitamin C438.5 mg/100 g. The yield of the 8-year-old single plant shed frame is about 105kg, and the high yield and the good stability are achieved. Has strong resistance, good fruit quality and better storage resistance. Is suitable for cultivation in southeast area of Jilin province. Generally, the seeds begin to mature in the last 9 months and belong to early maturing strains.
Strain 7 (strain code: c 2007-34): in 2007, the single plant selected from the mutant induced by EMS to generate the hypocotyl of the wild actinidia arguta embryo has conical fruit shape, the average single fruit weight is 17 g, and the maximum single fruit weight is more than 20 g. The soluble solids content was 13.8%, total sugar 6.9 g/100 g, vitamin C427.9 mg/100 g. The yield of the 8-year-old single plant shed frame is about 100kg, and the high yield and the good stability are achieved. Has strong resistance, good fruit quality and better storage resistance. Is suitable for cultivation in southeast area of Jilin province. Generally, the seeds begin to mature in the last 9 months and belong to early maturing strains.
Strain 8 (strain code: c 2007-8): in 2007, the single plant selected from the mutant induced by EMS to generate the hypocotyl of the wild actinidia arguta embryo has oval shape, the pericarp is red after the fruit is ripe, the average single fruit weight is 13.5 g, and the maximum single fruit weight is more than 15 g. The soluble solids content was 14.2%, total sugar 7.1 g/100 g, vitamin C429.0 mg/100 g. The yield of the 8-year-old single plant shed frame is about 90kg, and the high yield and the good stability are achieved. Has strong resistance, good fruit quality and better storage resistance. Is suitable for cultivation in southeast area of Jilin province. Maturing is generally started in the middle and late 9 months, and belongs to a middle and late maturing strain.
Strain 9 (strain code: c 2007-36): in 2007, individual plants selected from mutants induced by EMS to generate hypocotyl of wild actinidia arguta embryo have long cylindrical fruits, average single fruit weight of 16.8 g, and maximum single fruit weight of more than 18 g. The soluble solids content was 13.9%, total sugar 6.6 g/100 g, vitamin C430.7 mg/100 g. The yield of the 8-year-old single plant shed frame is about 95kg, and the high yield and the good stability are achieved. Has strong resistance, good fruit quality and better storage resistance. Is suitable for cultivation in southeast area of Jilin province. Generally, the plant begins to mature in the last 10 months and belongs to late-maturing strains.
Line 10 (line code: c 2008-33): in 2008, the single plant selected from the mutant induced by EMS to generate the hypocotyl of the wild actinidia arguta embryo has long cylindrical fruit shape, the ripe fruit has red peel and pulp, the average single fruit weight is 15.6 g, and the maximum single fruit weight is more than 18 g. The soluble solids content was 14.3%, total sugar 7.2 g/100 g, vitamin C422.6 mg/100 g. The yield of the 8-year-old single plant shed frame is about 90kg, and the high yield and the good stability are achieved. Has strong resistance, good fruit quality and better storage resistance. Is suitable for cultivation in southeast area of Jilin province. Maturing is generally started in 9 middle ten days, and belongs to a medium-maturing strain.
Line 11 (line code: c 2008-23): in 2008, the single plant selected from the mutant induced by EMS to generate the hypocotyl of the wild actinidia arguta embryo has oval fruits, the ripe fruit peel and pulp are red, the average single fruit weight is 18.8 g, and the maximum single fruit weight is more than 20 g. The soluble solids content was 13.9%, total sugar 7.1 g/100 g, vitamin C429.7 mg/100 g. The yield of the 8-year-old single plant shed frame is about 110kg, and the high yield and the good stability are achieved. Has strong resistance, good fruit quality and better storage resistance. Is suitable for cultivation in southeast area of Jilin province. Maturing is generally started in the middle and late 9 months, and belongs to a middle and late maturing strain.
Line 12 (line code: c 2009-12): in 2009, a single plant selected from mutants induced by EMS to generate hypocotyls of wild actinidia arguta embryo has long cylindrical fruits, and the ripe fruits have purple peel and pulp, and the average single fruit weight is 17.7 g, and the maximum single fruit weight is more than 20 g. The soluble solids content was 13.8%, total sugar 7.3 g/100 g, vitamin C411.3 mg/100 g. The yield of the 8-year-old single plant shed frame is about 105kg, and the high yield and the good stability are achieved. Has strong resistance, good fruit quality and better storage resistance. Is suitable for cultivation in southeast area of Jilin province. Maturing is generally started in the middle and late 9 months, and belongs to a middle and late maturing strain.
Line 13 (line code: c 2010-13): in 2010, a single plant selected from mutants generated by inducing wild actinidia arguta embryo hypocotyl by EMS has oval fruit shape, ripe fruit and reddish peel, and the average single fruit weight is 17.5 g, and the maximum single fruit weight is more than 20 g. The soluble solids content was 13.7%, total sugar 7.2 g/100 g, vitamin C413.9 mg/100 g. The yield of the 8-year-old single plant shed frame is about 110kg, and the high yield and the good stability are achieved. Has strong resistance, good fruit quality and better storage resistance. Is suitable for cultivation in southeast area of Jilin province. Maturing is generally started in the middle and late 9 months, and belongs to a middle and late maturing strain.
Strain 14 (strain code: x 2009-10): in 2009, the single plant selected from mutants induced by EMS to generate hypocotyl of wild actinidia arguta embryo is male, petals are white, flower buds are more, pollen quantity is large, pollen germination force is extremely strong, corolla is green, flowering period is long, and resistance is strong. Is suitable for cultivation in southeast area of Jilin province. Flowering is generally started in the last ten days of 6 months, and the male strain belongs to middle-colored flowers.
Line 15 (line code: x 2010-11): in 2011, the single plant selected from mutants generated by inducing the hypocotyl of the wild actinidia arguta embryo by EMS is male, has white petals, more flower buds, large pollen quantity, extremely strong pollen germination force, red brown corolla, long flowering phase and strong resistance. Is suitable for cultivation in southeast area of Jilin province. Flowering is generally started in the middle and late 5 months, and belongs to early-flowering male lines.
The invention uses the hypocotyl of actinidia arguta embryo as the material, combines the Ethyl Methylsulfonate (EMS) and plant tissue culture technology, adopts methods of embryogenic callus induction, redifferentiation, embryoid rejuvenation and the like to obtain novel actinidia arguta artificial cultivars with the characteristics of strong independence of each individual plant, high purity of the individual plant genetic information, clear individual plant sources and the like, has important protection and ecological significance for the amplification of actinidia arguta population, and can be further applied to the construction of actinidia arguta mutant libraries and the direct cultivation and variety approval as strains.
The foregoing is merely a preferred embodiment of the present invention, but the design concept of the present invention is not limited thereto, and any insubstantial modification of the present invention by using the concept shall fall within the scope of the present invention.
Claims (5)
1. A novel variety cultivation method for inducing hypocotyl mutation of actinidia arguta embryo by EMS is characterized by comprising the following steps:
(1) Material selection and pretreatment: taking the mature fruit of the wild actinidia arguta in late 9 months, cleaning the seeds in the fruit with clear water, and obtaining the mature seeds with water bottom; mixing the mature seeds with river sand at a ratio of 1:3, keeping the relative humidity of the mixture of the seeds and the river sand at 75%, and placing the mixture in a climatic chamber with the temperature of 18-20 ℃ and the relative humidity of 78% for dark treatment for 30 days;
separating and cleaning seeds from river sand after the embryo is further developed and mature, washing the seeds with 75% ethanol for 30s in a sterile ultra-clean workbench, then transferring the seeds to a 2-5% sodium hypochlorite solution for soaking for 10min, washing the seeds with sterile water for 10 times, and sucking surface moisture with sterile filter paper; removing seed coat with sterile scalpel and forceps, separating two cotyledons, collecting embryo, separating hypocotyl, placing hypocotyl into EMS solution with different concentration, soaking for 2-6 hr, and collecting EMS solution with concentration of 0.1 mol.L -1 Phosphate buffer solution with pH=7 is used as solvent to prepare EMS solution with volume percentage concentration of 0.1-1.5%; finally, the hypocotyl is washed by sterile water for 10 times for inoculation;
(2) Induction culture of hypocotyl embryogenic callus: the warp yarn in the step (1)Transferring the hypocotyl soaked by EMS into embryogenic callus induction culture medium, and placing in illumination period of 7h.d -1 Culturing for 55 days at the illumination intensity of 800lx and the temperature of 26+/-2 ℃, completely dedifferentiating the hypocotyl into yellow-green embryogenic callus, and carrying out secondary proliferation culture on the embryogenic callus; the embryogenic callus induction culture medium is a basic culture medium added with 1. mg.L -1 Brassinolide and 0.5 mg.L -1 2,4-D and 7.0 g.L -1 Agar powder, 50 g.L -1 Sucrose;
(3) And (3) carrying out sub-differentiation culture on the hypocotyl embryogenic callus: cutting the embryogenic callus in the step (2) into small blocks, transferring the small blocks into embryogenic callus redifferentiation medium, and placing the small blocks in an illumination period of 12 h.d -1 Continuously culturing for 45 days under the conditions of illumination intensity of 1200lx and temperature of 26+/-2 ℃, and differentiating embryoid callus to obtain independent, bipolar and separable embryoids; the embryogenic callus redifferentiation culture medium is a basic culture medium added with 1. mg.L -1 Brassinolide and 0.2 mg.L -1 NAA and 7.0 g.L -1 Agar powder, 30 g.L -1 Sucrose;
(4) And (3) rejuvenating, growing and culturing embryoid: peeling the embryoid in the step (3), transferring to embryoid rejuvenation growth medium, and placing in a lighting period of 10h.d -1 Rejuvenating and growing the embryoid at the illumination intensity of 1000lx and the temperature of 26+/-2 ℃ until the embryoid grows in two polarizations and grows into plantlets containing roots and stems and leaves after the embryoid grows for 30 days; the embryoid rejuvenation growth medium is a basic medium added with 0.08 mg.L -1 Brassinolide, 0.12 mg.L -1 Gibberellin GA 3 And 0.01 mg.L -1 IBA and 7.8g.L -1 Agar strips, 10 g.L -1 Sucrose and adjusting the pH value to 6.0;
(5) Transplanting and field planting and strain determination: planting the acclimatized and acclimatized plantlets of the small plants in the step (4) in a nutrition pot, planting and cultivating the plantlets in a germplasm garden when the plantlets grow to 15-20cm, observing the growth conditions of the plantlets, and classifying and labeling the plantlets; then breeding large seedlings by grafting different mutants, and cultivating in different areas; finally, screening and determining the excellent character mutant with stable heredity as a strain, and numbering;
the minimal medium consists of the following substances: 25 g.L of potato supernatant -1 The method comprises the steps of carrying out a first treatment on the surface of the Iron salt: 6.95 mg.L -1 FeSO 4 ·7H 2 O，9.325mg·L -1 Na 2 ·EDTA·2H 2 O; trace elements: 1.25 mg.L -1 MnSO 4 ·4H 2 O，0.25mg·L -1 ZnSO 4 ·7H 2 O，0.095mg·L -1 KI。
2. The method for breeding new species of EMS-induced hypocotyl mutation of actinidia arguta embryo according to claim 1, wherein: the EMS solutions with different concentrations are respectively 0.1%, 0.3%, 0.5%, 0.7%, 0.9%, 1.1%, 1.3% and 1.5% of the volume of the EMS solution.
3. The method for breeding new species of EMS-induced hypocotyl mutation of actinidia arguta embryo according to claim 1, wherein: the pH value of the embryogenic callus induction medium is 6.0.
4. The method for breeding new species of EMS-induced hypocotyl mutation of actinidia arguta embryo according to claim 1, wherein: the pH value of the embryogenic callus re-differentiation medium is 6.0.
5. The method for breeding new species of EMS-induced hypocotyl mutation of actinidia arguta embryo according to claim 1, wherein: the classification of mutants is carried out according to the shape of the mutants; strain determination is selected according to one or more factors of mutant fruit shape, resistance and quality.
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