CN105557525A - Breeding method for somaclonal variation of caladium bicolor - Google Patents
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Abstract
The invention discloses a breeding method for somaclonal variation of caladium bicolor, and belongs to the technical field of genetic breeding of flowers. The method comprises the steps that a tissue culture rapid propagation system of a parent is built in a callus induction way; variant plants with special characters are screened out of offspring groups and numbered, the content of relative nuclear DNA of the variant plants is detected, and when the peak value position of the variant plants is 1.6 to 2.2 times that of the parent, the corresponding plants are determined as preferable plants of the caladium bicolor; a clonal offspring group of the preferable plants is built, multi-year multi-point experimental research is carried out on the main characters of the offspring group, the specificity, consistency and stability of the preferable plant offspring group are verified, and finally the preferable plants are determined as a new variety of the caladium bicolor. According to the method, good variant plants of the caladium bicolor can be effectively obtained, the variant plants can be stabilized fast, an operating method is simple and convenient, the breeding cycle is shortened, and breeding efficiency is improved.
Description
Technical field
The invention belongs to flowers Biotechnology in Genetic Breeding field, be specifically related to a kind of selection of Caladium bicolor somaclonal variation.
Background technology
Caladium bicolor (
caladium × hortulanum) be Araeceae perennial herb, originate in torrid areas, South America, the change of leaf look very abundant, there are redness, pink colour, white, yellow, purple etc., bright in luster and unique, very popular, be widely used in garden and Landscape in western countries, also can be used as potted plant and cut leaf production, is important colored ornamental foliage plant.
Somaclonal variation (SomaclonalVariation) makes a general reference the genetic variation or phenogenetics variation that produce in plant cell, tissue and organ culture process.Utilize the method to obtain much to have the variation proterties of economic worth, as disease resistance, dwarfing, resistance and yielding ability etc., now bring out the new varieties such as many High quality agricultural products and flowers.For example, be under the jurisdiction of Araeceae conjunction fruit taro platymiscium (
syngonium), close fruit taro for parent with sulphur butterfly, select 22 new varieties by the method.Somaclonal variation seed selection closes fruit taro to belong to the of paramount importance selection of cultivation new varieties, but close the method that fruit taro variation Single-plant selection mainly adopts artificial selection, and the meristic variation individual plant of acquisition still exists the problem of proterties Genomic instability.
Current Caladium bicolor breeding mainly adopts conventional cross breeding method, there is breeding cycle longer, the problems such as process is slow, and the research of its somaclonal variation seed selection is just at the early-stage, bibliographical information also not relevant at home.
Summary of the invention
The present invention improves the technological process of somaclonal variation Single-plant selection, and there is cycle long, the problem such as process is slow for the breeding of Caladium bicolor conventional hybridization, the new method of a kind of Caladium bicolor somaclonal variation seed selection is provided, it can effectively obtain can the improved Varieties of genetic stability, and shortening the breeding cycle.
To achieve these goals, the technical scheme taked of the present invention is as follows:
A medium for Caladium bicolor somaclonal variation selection, is made up of following three parts:
1) inducing culture: on the basis of MS medium, add the 6-BA of 0.5 ~ 2mg/L, 0.1 ~ 2mg/L 2,4-D, 20 ~ 40g/L sucrose and 6 ~ 8g/L carragheen, pH5.6 ~ 6.0;
2) differential medium: on the basis of MS medium, the 6-BA of interpolation 1 ~ 3mg/L, the NAA of 0.1 ~ 1mg/L, 20 ~ 40g/L sucrose and 6 ~ 8g/L carragheen, pH5.6 ~ 6.0;
3) Rooting and hardening-off culture base: on the basis of MS medium, adds the NAA of 0.1 ~ 1mg/L, 20 ~ 40g/L sucrose and 6 ~ 8g/L carragheen, pH5.6 ~ 6.0.
Preferably, described medium is made up of following three parts:
1) inducing culture: on the basis of MS medium, add the 6-BA of 0.5 ~ 2mg/L, 0.1 ~ 1mg/L 2,4-D, 25 ~ 35g/L sucrose and 6 ~ 8g/L carragheen, pH5.8 ~ 6.0;
2) differential medium: on the basis of MS medium, the 6-BA of interpolation 1 ~ 2mg/L, the NAA of 0.1 ~ 0.5mg/L, 25 ~ 35g/L sucrose and 6 ~ 8g/L carragheen, pH5.8 ~ 6.0;
3) Rooting and hardening-off culture base: on the basis of MS medium, adds the NAA of 0.1 ~ 0.5mg/L, 25 ~ 35g/L sucrose and 6 ~ 8g/L carragheen, pH5.8 ~ 6.0.
A selection for Caladium bicolor somaclonal variation, comprises the following steps:
(1) preparation of medium: configure medium described above;
(2) explant selection and disinfect: choose the young leaflet tablet that not yet launches and petiole is explant, to its disinfection;
(3) induction of callus: the blade after disinfecting is cut into 1 ~ 2cm segment, is inoculated in dark culturing in inducing culture;
(4) differentiation is cultivated: be forwarded in differential medium by the callus that produces of induction, differentiation-inducingly sprout;
(5) Rooting and hardening-off culture: when bud grows to 1.8 ~ 2.5cm, be cut into fritter, is transferred to root induction in Rooting and hardening-off culture base, grows up to seedling;
(6) transplant hardening: when seedling grows to 4 ~ 5cm, blake bottle is moved to hardening in green house and, after 5 ~ 8 days, test-tube plantlet is taken out, rinses well, plant in the dish of cave; When plant to be planted has grown to 3-4 sheet leaf, by basin in transplantation of seedlings;
(7) selection of variation individual plant: from the clonal offspring colony of parent, filter out the variation individual plant of proterties uniqueness, and adopt the cell DNA content of flow cytometer to variation individual plant to measure and analyze, the peak of variation individual plant is 1.6 ~ 2.2 times of parent, definitive variation individual plant is doubled to cause by parental set of chromosome, can genetic stability, be the Superior line of Caladium bicolor;
(8) foundation of the clonal offspring colony of Superior line: the young leaflet tablet getting Superior line is explant, carry out tissue culture expanding propagation, and be contrast with parent, to major traits such as plant strain growth, blade, stem tuber and growth potentials, carry out multiple years pilot survey, verify the specificity of Superior line progeny population, uniformity and stability;
(9) cultivation of new varieties: through varietal yield test investigation, progeny population plant has unique fancy points, forms the colony that proterties is consistent with performance, meets breeding objective, be decided to be new varieties.
Preferably, the step of disinfecting of the explant in step (2) is 70% alcohol immersion 30s, aseptic water washing 2 times, and 0.1% mercuric chloride soaks 8-10min, finally uses rinsed with sterile water 5 times.
Preferably, the condition that in step (4), differentiation is cultivated is intensity of illumination is 800 ~ 1000lux, and light application time is 11 ~ 13 hours every days.
Preferably, the matrix in step (6) in the dish of cave is the high perlitic mass ratio of Ni Tan ︰ of wanting is the mixed-matrix of 3 ︰ 1.
Preferably, the standard of screening variation individual plant comprises in step (7): blade becomes circular, quality is hardening, the thicker strong and/or plant type of petiole becomes short.
Preferably, in step (7), variation individual plant adopts the cell DNA content of flow cytometer to variation individual plant to measure and analyze, and the peak of variation individual plant is 1.8 ~ 2 times of parent, is defined as the Superior line of Caladium bicolor.
Preferably, Caladium bicolor kind is selected from ' rosebud ' (' Rosebud '), ' welfare reaches ' (' FriedaHemple '), ' egression ' (' Candidum '), ' Catherine ' (' Katheleen ') ' are mini white ' (' MiniWhite '), ' Jin Zhelan ' (' Gingerland ').
Preferably, Caladium bicolor kind is ' rosebud ' or ' welfare reaches '.
The invention has the beneficial effects as follows:
(1) improve existing somaclonal variation individual plant selection technology, the stability of variation individual plant significantly improves:
The aroid somaclonal variation individual plant selection method in the past reported, being the method only adopting artificial selection when making a variation Single-plant selection, there is lability in the variation individual plant of acquisition.The present invention is on the basis of artificial selection, in conjunction with flow cytomery method, can obtain the preferred variation individual plant of genetic stability fast, thus substantially increase the stability of variation individual plant, the method is that one stable, speed fast and the new method of the somaclonal variation seed selection that efficiency is high.
(2) shortening the breeding cycle, breeding efficiency is improved:
The inventive method seed selection cycle is 5 ~ 6 years, needs 1 year comprising carrying out tissue cultures to parent to emerging; The transplantation of seedlings of parent's bottle is planted, and carries out variation Single-plant selection, and utilizes flow cytometer to the mensuration of the cell DNA content of variation individual plant and analysis, determines that Superior line needs 1 year; Tissue cultures is carried out to Superior line and needs 1 year to emerging; Variation individual plant bottle transplantation of seedlings plantation, carries out the investigation of multiple years variety trial, finally selects new varieties and need 2 ~ 3 years.The Caladium bicolor conventional cross-breeding cycle is 7 ~ 8 years, and the inventive method approximately shortens 2 years.
(3) the Comprehensive Traits new varieties more excellent than parent can effectively be obtained:
The present invention improves traditional category, and the new varieties plant type cultivated becomes short, and blade becomes circular, and leaf look more bright-coloured, and blade quality is harder, and petiole is more sturdy, and Comprehensive Traits is obviously better than parent.
Accompanying drawing explanation
Fig. 1 is that parent ' rosebud ' (left side) compares with variation individual plant ' C2-0 ' (right side) plant;
Fig. 2 is parent's ' rosebud ' and variation individual plant ' C2-0 ' relatively core DNA content comparison diagram;
Fig. 3 is the individual plant the Characters comparison diagram of parent's ' rosebud ' and variation individual plant ' C2-0 ' Caladium bicolor;
Fig. 4 is that parent's ' rosebud ' shows comparison diagram with the population characteristics of variation individual plant ' C2-0 ' Caladium bicolor;
Fig. 5 is parent's ' welfare reaches ' and variation individual plant ' C3-0 ' relatively core DNA content comparison diagram;
Fig. 6 is the blade comparison diagram of parent's ' welfare reaches ' and variation individual plant ' C3-0 ' Caladium bicolor;
Fig. 7 is the individual plant the Characters comparison diagram of parent's ' welfare reaches ' and variation individual plant ' C3-0 ' Caladium bicolor;
Fig. 8 is that parent's ' welfare reaches ' shows comparison diagram with the population characteristics of variation individual plant ' C3-0 ' Caladium bicolor.
Embodiment
English initialism:
6-benzylamino adenine is 6-BA, 2,4-dichlorphenoxyacetic acids are 2,4-D.
Below in conjunction with specific embodiment, the present invention is further illustrated, but be not limited thereto.
embodiment 1
With Caladium bicolor kind ' rosebud ' for parent material, obtain the selection of variation individual plant C2-0 new varieties.
The method is carried out according to the following steps:
(1) preparation of medium:
Medium is made up of following three parts:
A) inducing culture: on the basis of MS medium, add the 6-BA of 0.5 ~ 2mg/L, 0.1 ~ 2mg/L 2,4-D, 20 ~ 40g/L sucrose and 6 ~ 8g/L carragheen, pH5.6 ~ 6.0;
B) differential medium: on the basis of MS medium, the 6-BA of interpolation 1 ~ 3mg/L, the NAA of 0.1 ~ 1mg/L, 20 ~ 40g/L sucrose and 6 ~ 8g/L carragheen, pH5.6 ~ 6.0;
C) Rooting and hardening-off culture base: on the basis of MS medium, adds the NAA of 0.1 ~ 1mg/L, 20 ~ 40g/L sucrose and 6 ~ 8g/L carragheen, pH5.6 ~ 6.0;
(2) explant selection and disinfect: choosing the young leaflet tablet that parent's ' rosebud ' not yet launches is explant, with 70% alcohol immersion 30s, aseptic water washing 2 times, 0.1% mercuric chloride soaks 8 ~ 10min, finally uses rinsed with sterile water 5 times;
(3) induction of callus: the blade blade of disinfecting is cut into 1 ~ 2cm segment, is inoculated in dark culturing in inducing culture;
(4) differentiation is cultivated: be transferred in differential medium by the callus that produces of induction, cultivate under illumination condition, intensity of illumination is 800 ~ 1000lux, and every day, light application time 12 hours, differentiation-inducingly sprouted;
(5) Rooting and hardening-off culture: when bud grows to 2cm, be cut into fritter, is transferred to root induction in Rooting and hardening-off culture base, grows up to seedling;
(6) transplant hardening: when seedling grows to 4 ~ 5cm, blake bottle is moved to hardening in green house, after 1 week, taken out by test-tube plantlet, after rinsing well with water, plant in the dish of cave, matrix is that height wants Ni Tan ︰ perlite to equal in the mixed substrate of 3 ︰ 1,1 strain/cave.After 1 first quarter moon, when growing to 3 ~ 4 leaves, basin in transplanting;
(7) selection of variation individual plant: from the clonal offspring colony of parent, filter out that blade becomes circular, quality is hardening and the thicker strong variation individual plant of petiole, be numbered C2-0(and see Fig. 1), and by flow cytometer cell DNA content measured and analyze, the results are shown in Figure 2: parent only relative intensity of fluorescence value be about 400K position occur one unimodal; Variation individual plant C2-0 occur in about 800K position one unimodal, peak is about 2 times of parent, infer thus, variation individual plant C2-0 occurs obviously to change leaf and blade quality etc., doubled to cause by parental set of chromosome, can genetic stability, thus determine that C2-0 is the Superior line of Caladium bicolor;
(8) foundation of Superior line C2-0 clonal offspring colony: the young leaflet tablet getting Superior line C2-0 is explant, carry out tissue culture expanding propagation, method is the same, and with parent in contrast, to major traits such as growth of seedling, blade, stem tuber and growth potentials, carry out multiple years pilot survey, verify the specificity of variation individual plant progeny population, uniformity and stability;
(9) cultivation of new varieties: investigate through comparative trial, the plant type of progeny population plant, leaf more consistent with leaf look etc., define the colony that proterties is consistent with performance, compare with parent (Fig. 3 ~ Fig. 4), the plant type of strain becomes short, and blade becomes circular, blade quality is harder, petiole is more sturdy, meets breeding goal, determines that C2-0 is Caladium bicolor new varieties.
embodiment 2
With Caladium bicolor kind ' welfare reaches ' for parent material, obtain the selection of variation individual plant C3-0 new varieties.The method is carried out according to the following steps:
(1) preparation of medium:
Medium is made up of following three parts:
A) inducing culture: on the basis of MS medium, add the 6-BA of 0.5 ~ 2mg/L, 0.1 ~ 1mg/L 2,4-D, 25 ~ 35g/L sucrose and 6 ~ 8g/L carragheen, pH5.8 ~ 6.0;
B) differential medium: on the basis of MS medium, the 6-BA of interpolation 1 ~ 2mg/L, the NAA of 0.1 ~ 0.5mg/L, 25 ~ 35g/L sucrose and 6 ~ 8g/L carragheen, pH5.8 ~ 6.0;
C) Rooting and hardening-off culture base: on the basis of MS medium, adds the NAA of 0.1 ~ 0.5mg/L, 25 ~ 35g/L sucrose and 6 ~ 8g/L carragheen, pH5.8 ~ 6.0;
(2) explant selection and disinfect: choosing the young leaflet tablet that parent's ' welfare reaches ' not yet launches is explant, with 70% alcohol immersion 30s, aseptic water washing 2 times, 0.1% mercuric chloride soaks 8 ~ 10min, finally uses rinsed with sterile water 5 times;
(3) induction of callus: the blade blade of disinfecting is cut into 1 ~ 2cm segment, is inoculated in dark culturing in inducing culture;
(4) differentiation is cultivated: be transferred in differential medium by the callus that produces of induction, cultivate under illumination condition, intensity of illumination is 800 ~ 1000lux, and every day, light application time 12 hours, differentiation-inducingly sprouted;
(5) Rooting and hardening-off culture: when bud grows to 2cm, be cut into fritter, is transferred to root induction in Rooting and hardening-off culture base, grows up to seedling;
(6) transplant hardening: when seedling grows to 4 ~ 5cm, blake bottle is moved to hardening in green house, after 1 week, taken out by test-tube plantlet, after rinsing well with water, plant in the dish of cave, matrix is that height wants Ni Tan ︰ perlite to equal in the mixed substrate of 3 ︰ 1,1 strain/cave.After 1 first quarter moon, when growing to 3-4 sheet leaf, basin in transplanting;
(7) make a variation the selection of individual plant: from the clonal offspring colony of parent, filters out that blade becomes circular, quality is hardening, leaf look reddens and the thicker strong variation individual plant of petiole, is numbered C3-0; By flow cytometer cell DNA content measured and analyze, the results are shown in Figure 5: parent only relative intensity of fluorescence value be about 500K position occur one unimodal; ' C3-0 ' occur in about 900K position one unimodal, peak is about 1.8 times of parent, infers thus, ' C3-0 ' occurs obviously to change leaf and blade quality etc., doubled to cause by parental set of chromosome, can genetic stability, thus determine that C3-0 is the Superior line of Caladium bicolor;
(8) foundation of Superior line C3-0 clonal offspring colony: the young leaflet tablet getting preferred strain individual plant C3-0 is explant, carry out tissue culture expanding propagation, method is the same, and with parent in contrast, to major traits such as growth of seedling, blade, stem tuber and growth potentials, carry out multiple years pilot survey, checking variation individual plant progeny population specificity, uniformity and stability;
(9) cultivation of new varieties: investigate through comparative trial, the plant type of progeny population plant, leaf more consistent with leaf look etc., define the colony that proterties is consistent with performance, compare with parent (Fig. 6 ~ 8), the plant type of strain becomes short, and leaf look reddens, and blade becomes circular, blade quality is harder, and petiole is more sturdy; Meet breeding goal, determine that C3-0 is Caladium bicolor new varieties.
Above-described embodiment is the present invention's preferably embodiment; but embodiments of the present invention are not restricted to the described embodiments; change, the modification done under other any does not deviate from Spirit Essence of the present invention and principle, substitute, combine, simplify; all should be the substitute mode of equivalence, be included within protection scope of the present invention.
Claims (10)
1. a medium for Caladium bicolor somaclonal variation selection, is made up of following three parts:
1) inducing culture: on the basis of MS medium, add the 6-BA of 0.5 ~ 2mg/L, 0.1 ~ 2mg/L 2,4-D, 20 ~ 40g/L sucrose and 6 ~ 8g/L carragheen, pH5.6 ~ 6.0;
2) differential medium: on the basis of MS medium, the 6-BA of interpolation 1 ~ 3mg/L, the NAA of 0.1 ~ 1mg/L, 20 ~ 40g/L sucrose and 6 ~ 8g/L carragheen, pH5.6 ~ 6.0;
3) Rooting and hardening-off culture base: on the basis of MS medium, adds the NAA of 0.1 ~ 1mg/L, 20 ~ 40g/L sucrose and 6 ~ 8g/L carragheen, pH5.6 ~ 6.0.
2. medium according to claim 1, is characterized in that: be made up of following three parts:
1) inducing culture: on the basis of MS medium, add the 6-BA of 0.5 ~ 2mg/L, 0.1 ~ 1mg/L 2,4-D, 25 ~ 35g/L sucrose and 6 ~ 8g/L carragheen, pH5.8 ~ 6.0;
2) differential medium: on the basis of MS medium, the 6-BA of interpolation 1 ~ 2mg/L, the NAA of 0.1 ~ 0.5mg/L, 25 ~ 35g/L sucrose and 6 ~ 8g/L carragheen, pH5.8 ~ 6.0;
3) Rooting and hardening-off culture base: on the basis of MS medium, adds the NAA of 0.1 ~ 0.5mg/L, 25 ~ 35g/L sucrose and 6 ~ 8g/L carragheen, pH5.8 ~ 6.0.
3. a selection for Caladium bicolor somaclonal variation, is characterized in that, comprises the following steps:
(1) preparation of medium: the medium of configuration described in claim 1 or 2;
(2) explant selection and disinfect: choose the young leaflet tablet that not yet launches and petiole is explant, to its disinfection;
(3) induction of callus: the blade after disinfecting is cut into 1 ~ 2cm segment, is inoculated in dark culturing in inducing culture;
(4) differentiation is cultivated: be forwarded in differential medium by the callus that produces of induction, differentiation-inducingly sprout;
(5) Rooting and hardening-off culture: when bud grows to 1.8 ~ 2.5cm, be cut into fritter, is transferred to root induction in Rooting and hardening-off culture base, grows up to seedling;
(6) transplant hardening: when seedling grows to 4 ~ 5cm, blake bottle is moved to hardening in green house and, after 5 ~ 8 days, test-tube plantlet is taken out, rinses well, plant in the dish of cave; When plant to be planted has grown to 3-4 sheet leaf, by basin in transplantation of seedlings;
(7) selection of variation individual plant: from the clonal offspring colony of parent, filter out the variation individual plant of proterties uniqueness, and adopt the cell DNA content of flow cytometer to variation individual plant to measure and analyze, the peak of variation individual plant is 1.6 ~ 2.2 times of parent, definitive variation individual plant is doubled to cause by parental set of chromosome, can genetic stability, be the Superior line of Caladium bicolor;
(8) foundation of the clonal offspring colony of Superior line: the young leaflet tablet getting Superior line is explant, carry out tissue culture expanding propagation, and be contrast with parent, to major traits such as plant strain growth, blade, stem tuber and growth potentials, carry out multiple years pilot survey, verify the specificity of Superior line progeny population, uniformity and stability;
(9) cultivation of new varieties: through varietal yield test investigation, progeny population plant has unique fancy points, forms the colony that proterties is consistent with performance, meets breeding objective, be decided to be new varieties.
4. selection according to claim 3, is characterized in that: the step of disinfecting of the explant in step (2) is 70% alcohol immersion 30s, aseptic water washing 2 times, and 0.1% mercuric chloride soaks 8-10min, finally uses rinsed with sterile water 5 times.
5. selection according to claim 3, is characterized in that: the condition that in step (4), differentiation is cultivated is intensity of illumination is 800 ~ 1000lux, and light application time is 11 ~ 13 hours every days.
6. selection according to claim 3, is characterized in that: the matrix in step (6) in the dish of cave is the high perlitic mass ratio of Ni Tan ︰ of wanting is the mixed-matrix of 3 ︰ 1.
7. selection according to claim 3, is characterized in that: the standard of screening variation individual plant comprises in step (7): blade becomes circular, quality is hardening, the thicker strong and/or plant type of petiole becomes short.
8. selection according to claim 3, it is characterized in that: in step (7), variation individual plant adopts the cell DNA content of flow cytometer to variation individual plant to measure and analyze, the peak of variation individual plant is 1.8 ~ 2 times of parent, is defined as the Superior line of Caladium bicolor.
9. selection according to claim 3, is characterized in that: Caladium bicolor kind is selected from ' rosebud ', ' welfare reaches ', ' egression ', ' Catherine ', ' mini white ', ' Jin Zhelan '.
10. selection according to claim 3, is characterized in that: Caladium bicolor kind is ' rosebud ' or ' welfare reaches '.
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| CN113951137A (en) * | 2021-10-28 | 2022-01-21 | 中国热带农业科学院海口实验站 | High-concentration cytokinin-mediated high-frequency banana clone mutagenesis method |
| CN115024224A (en) * | 2022-06-28 | 2022-09-09 | 广东省农业科学院环境园艺研究所 | Method for obtaining polyploid colocasia esculenta by tuber thin-layer culture |
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