CN103125398B - A kind of method for tissue culture improving head cabbage in-vitro regeneration efficiency - Google Patents
A kind of method for tissue culture improving head cabbage in-vitro regeneration efficiency Download PDFInfo
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- CN103125398B CN103125398B CN201310089116.2A CN201310089116A CN103125398B CN 103125398 B CN103125398 B CN 103125398B CN 201310089116 A CN201310089116 A CN 201310089116A CN 103125398 B CN103125398 B CN 103125398B
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Abstract
The invention discloses a kind of method for tissue culture improving head cabbage in-vitro regeneration efficiency, first get the explant that regeneration inductivity is high; Sterilizing is carried out with clorox; Tender leaf first carries out the light culture of 16-20h on inducing culture, then carries out light cultivation; Tender stem section with axillalry bud directly carries out light cultivation on inducing culture; By the bud that induces or Multiple Buds, transfer on proliferated culture medium, carry out shoot proliferation; The simple bud of getting robust growth carries out culture of rootage; At room temperature remove sealed membrane after taking root to carry out practicing seedling, after then root medium being washed, transplant in the dish of ready cave.The present invention solves the problem of different explants disinfecting time, solves the problem that gain factor is low in head cabbage Regeneration in Vitro Induction Process.The present invention is from disinfecting time, and draw materials position, hormon proportioning aspect of explant establishes head cabbage regenerating system the most efficiently, for head cabbage germ plasm resource preservation and carry out head cabbage genetic engineering breeding and lay the foundation.
Description
Technical field
The invention belongs to Plant Biotechnology and head cabbage technical field of cultivation, particularly relate to a kind of method for tissue culture improving head cabbage in-vitro regeneration efficiency.
Background technology
Head cabbage belongs to 2 years raw herbaceous plant of Cruciferae (Cruciferae) Brassica genus (Brassica), is one of our important vegetables.Within 2 years, namely raw herbaceous plant forms leaf-head through vegetative growth phase then.Leaf-head is by preserving the impact of survive the winter low temperature and long-day in the coming year, and enter reproductive stage, bolting is blossomed and had seeds, and completes life process.The features such as head cabbage has that strong adaptability, disease-resistant, cold-resistant output are high, quality better, and easily storing, Salt And Alkali Tolerance, cultivation are easy.But affect by Changes in weather when germ plasm resource breed conservation larger, utilize tissue cultures to preserve the adverse effect that can alleviate environmental stress conditions and head cabbage Germ-plasma resources protection is reserved seed for planting.Setting up stable wild cabbage Tissue Culture Regeneration System is carry out important plasm resource protection and engineered basis.The regeneration capacity of cabbage plant is strong, but different genotype, different explants and its regeneration frequency of different seedling age have larger difference.The research of the tissue culture technique of head cabbage Regeneration in Vitro has many reported success, but most adopt flower pesticide microspore, the cotyledon of indefinite bud blade and aseptic seedling and lower shaft to be explant, in different explants draws materials position, different sterilization time, hormon proportioning etc., there is no more concrete research.This research adopts the tender stem of head cabbage tender leaf and band axillalry bud as outer planting donor, establishes a kind of more simple, high-frequency plant regeneration system of being more suitable for genetic transformation.
Summary of the invention
The invention provides a kind of method for tissue culture improving head cabbage in-vitro regeneration efficiency, establish head cabbage high-efficiency regeneration system.Be intended to solve different explants and draw materials the hormone combination of the disinfecting time at position, indefinite bud and root induction medium to change the low problem of gain factor.
The embodiment of the present invention is achieved in that a kind of method for tissue culture improving head cabbage in-vitro regeneration efficiency, and this method for tissue culture comprises the following steps:
To choose the high explant of Regeneration in Vitro inductivity, bloom at head cabbage bolting and get the side shoot of sprouting latter stage;
Clorox with 7% carries out sterilizing, and tender leaf and tender stem sterilization time are respectively 10min and 20min; A3, blade first carries out the dark culturing of 16-20h in Induction Process, then carries out light cultivation, cultivates after 15 days and carries out transferring once again;
The induction of tender stem section, each little stem section is induced with axillalry bud;
By the bud that induces or Multiple Buds, carry out shoot proliferation; Time indefinite bud grows to about 3.0cm, the simple bud of getting robust growth carries out culture of rootage; Select the plantlet in vitro of well developed root system, first at room temperature remove sealed membrane and carry out practicing seedling about 7d, after root medium is washed, transplant in the dish of ready cave.
Further, this method for tissue culture specifically comprises the following steps:
The first, induce
Draw materials: bloom at head cabbage bolting and get explant latter stage, get the tender lateral branch without scab and insect pest of sprouting, the 2-3 sheet leaf on branch top near lobus cardiacus is tender leaf, the tender stem got band handle tender leaf and remove on the spray of blade;
Sterilizing: rinse 20-30min under the field explant chosen is placed on running water, then use blotting paper suck dry moisture afterwards 3-4 time with distilled water flushing; Tender leaf and tender stem are separated sterilizing, each with 75% alcohol disinfecting 1min, then carry out disinfection with 7%Naclo, tender leaf and tender stem sterilization time are respectively 10min and 20min, and rear aquae destilIata sterilis of having sterilized flushing 3-4 time, is seeded on medium;
Differentiation: sterilizing rear blade is cut into the square of about 0.5cm, is seeded in MS+6-BA2mg.L
-1+ NAA0.2mg.L
-1on medium; Sheet leaf 8-12 sheet/bottle; Process 10 bottles, the induction of blade first will carry out the light culture of 16-20h, is carrying out light cultivation, and blade carries out transferring once after light cultivates 15d, and the blade increased is cut into small pieces and cultivates, about 30d directly can induce indefinite bud; After tender stem section stem section sterilizing after blade is removed, be cut into the stem section of band axillalry bud, be cut into the segment that 0.5-1.0cm is long; Every bottle graft kind 5-10 segment; Directly cultivate under light is cultivated, after 15d, just can see the bud induced;
The second, shoot proliferation: by the bud that induces or Multiple Buds, transfer at MS+6-BA2mg.L
-1on, every bottle of 4-6 indefinite bud, about 25-30d carries out transferring once, and through the cultivation in 3-4 generation, the multiple of bud reaches the growth of about 200 times;
Three, the induction of root: time simple bud or Multiple Buds grow into about 3.0cm, the simple bud of getting robust growth carries out culture of rootage, medium MS+NAA0.3mg.L
-1, each process connects 10 bottles, every bottle graft 5-6 simple bud, and 15d " Invest, Then Investigate " is recorded root induction rate and reached the healthy and strong prosperity of about 95%, 25-30d root system;
Four, transplanting and field planting: the plantlet in vitro selecting well developed root system, first removes sealed membrane at nominal room temperature and carries out practicing seedling about 7d, after root medium is washed, transplants in the dish of ready cave; Matrix selects seedling medium.Adopt moist medium in tray, to grab agglomerating with hand one, a pine and to be loosely advisable; In order to make root system fully be combined with cultivation matrix during transplanting, after transplanting, cave dish being placed in pond, naturally absorbing water, put in the cool, within about 1 week, can survive after suctioning water, cave dish keeps dry wet, survival rate 100%.
Further, explant branch topmost is near the 2nd, 3 tender leaf of growing point, and band petiole takes off, and the side shoot removing blade gets tender stem.
Further, tender leaf inducing culture is MS+6-BA2mg.L
-1+ NAA0.2mg.L
-1; Get the stem section 0.5-1.0cm of band axillalry bud, directly at MS+6-BA2mg.L
-1upper Fiber differentiation; The bud induced, transfers at proliferated culture medium MS+6-BA2mg.L
-1upper cultivation, through the cultivation in 3-4 generation, the multiple of bud reaches the growth of about 200 times; Root media selects MS+NAA0.3mg.L
-1, obtain the seedling of taking root of robust growth well developed root system.
Further, temperature controls at 22-25 DEG C, and illumination every day 16/8h, intensity of illumination 2000-2500Lx, adds 30g.L in medium
-1sucrose and 7g.L
-1agar powder, PH5.8-5.9.
Further, with the medium in tray of humidity, to grab agglomerating with hand one, a pine and loose to be advisable; In order to make root system fully be combined with cultivation matrix during transplanting, after transplanting, cave dish is placed in pond, naturally absorb water, put after suctioning water in the cool, survival rate is to 100%.
This research take head cabbage as material; to draw materials position, sterilization time and culture medium prescription with optimization; carry out decreasing pollution rate; improve differentiation-inducing rate; improve the induction of gain factor and root system; establish tender leaf blade and and the in vitro high-efficiency regeneration system of tender stem section of band axillalry bud, for head cabbage germ plasm resource protection and carry out head cabbage genetic engineering breeding and lay the foundation.The invention provides a kind of method of simple Regeneration in Vitro plant, compared with prior art have the following advantages and good effect.
Accompanying drawing explanation
Fig. 1, figure A are the performance of leaf culture about 15d; Figure B is the indefinite bud that leaf culture induces;
The bud induced between Fig. 2, band axillary bud stem;
The bud of Fig. 3, indefinite bud subculture regeneration;
Fig. 4, the root induction on root media of figure A regeneration bud; Figure B is that little transplantation of seedlings is in nutritive cube;
C survives seedling.
Embodiment
In order to make object of the present invention, technical scheme and advantage clearly understand, below in conjunction with drawings and Examples, the present invention is further elaborated.Should be appreciated that specific embodiment described herein only in order to explain the present invention, be not intended to limit the present invention.
The embodiment of the present invention is achieved in that a kind of method for tissue culture improving head cabbage in-vitro regeneration efficiency, and this method for tissue culture comprises the following steps:
1 induction
1.1 draw materials: bloom at head cabbage bolting and get explant latter stage, and get the tender lateral branch without scab and insect pest of sprouting, the 2-3 sheet leaf on branch top near lobus cardiacus is tender leaf, the tender stem got band handle tender leaf and remove on the spray of blade.
1.2 sterilizings: rinse 20-30min under the field explant chosen is placed on running water, then use blotting paper suck dry moisture afterwards 3-4 time with distilled water flushing; Tender leaf and tender stem are separated sterilizing, each with 75% alcohol disinfecting 1min, then carry out disinfection with 7%Naclo, tender leaf and tender stem sterilization time are respectively 10min and 20min, and rear aquae destilIata sterilis of having sterilized flushing 3-4 time, is seeded on medium;
1.3 differentiation: sterilizing rear blade is cut into the square of about 0.5cm, is seeded in MS+6-BA2mg.L
-1+ NAA0.2mg.L
-1on medium; Sheet leaf 8-12 sheet/bottle; Process 10 bottles, the induction of blade first will carry out the light culture of 16-20h, is carrying out light cultivation, and blade after light cultivates 15d (as Figure 1A) carries out transferring once, the blade increased is cut into small pieces cultivate, about 30d directly can induce indefinite bud (as Figure 1B); After tender stem section stem section sterilizing after blade is removed, be cut into the stem section of band axillalry bud, be cut into the segment that 0.5-1.0cm is long; Every bottle graft kind 5-10 segment.Directly cultivate under light is cultivated, after 15d, just can see the bud induced;
2 shoot proliferations: by the bud that induces or Multiple Buds, transfer at MS+6-BA2mg.L
-1on, every bottle of 4-6 indefinite bud, about 25-30d carries out transferring once, and through the cultivation in 3-4 generation, the multiple of bud reaches the growth of about 200 times.
The induction of 3: time simple bud or Multiple Buds grow into about 3.0cm, the simple bud of getting robust growth carries out culture of rootage, medium MS+NAA0.3mg.L
-1, each process connects 10 bottles, every bottle graft 5-6 simple bud, and 15d " Invest, Then Investigate " is recorded root induction rate and reached about 95%, 25-30d root system healthy and strong flourishing (as Fig. 4 A, B).
4 transplant and field planting: the plantlet in vitro selecting well developed root system, first remove sealed membrane at nominal room temperature and carry out practicing seedling about 7d, after root medium is washed, transplant in the dish of ready cave.Matrix selects seedling medium.Adopt moist medium in tray, to grab agglomerating with hand one, a pine and to be loosely advisable.In order to make root system fully be combined with cultivation matrix during transplanting, after transplanting, cave dish being placed in pond, naturally absorbing water, put in the cool, within about 1 week, can survive after suctioning water, cave dish keeps dry wet, survival rate 100%.
In embodiments of the present invention, the proportioning of inducation and proliferation medium: inducing culture forms, on the basis of MS solid culture medium medium composition, by adding NAA and 6-BA of variable concentrations, process as follows, 1. adding concentration is 0.4mgL
-1nAA, and 0,0.5,1.0,1.5,2.0mgL
-16-BA; 2. adding concentration is 2.0mgL
-16-BA, and 0,0.05,0.1,0.2,0.4mgL
-1nAA.Proliferated culture medium forms, MS solid culture medium, and add the NAA of 6-BA and five kind of variable concentrations gradient of two kinds of concentration gradients, 6-BA concentration is 1.0mg.L
-1and 2.0mg.L
-1, add 0 respectively, 0.05,0.1,0.2,0.4mg.L
-1nAA.Filtered out in research process, tender leaf inducing culture is MS+6-BA2mg.L
-1+ NAA0.2mg.L
-1, the inducing culture of stem segment with axillary bud is all MS+6-BA2mg.L with increment medium
-1, inductivity and growth coefficient are improved;
In embodiments of the present invention, root media proportioning: adopt medium 1/2MS and MS+NAA0.3mg.L
-1take root, filter out MS+NAA0.3mg.L
-1medium Proportion is best, obtains the healthy seedling (as Fig. 4) of well developed root system.
Below by way of concrete experimental program, the present invention is further illustrated.
1. material
Be field inbred line head cabbage for examination material, spring bolting bloom and get its side shoot without damage by disease and insect sprouted latter stage, get the tender leaf (comprising petiole) on branch, Lao Ye, tender stem be for subsequent use as explant.
2. method
2.1 disinfect
Branch topmost is near the 2nd, 3 tender leaf of growing point, and band petiole takes off; Branch foot the 2nd, 3 Lao Ye, are not with petiole to take off; The branch of lower blade is gone to get tender stem.On superclean bench, the blade cleaned up and tender stem are respectively charged in aseptic container, with the alcohol disinfecting 1min of 75%, carry out disinfection with 7%NaClO again, if 8,10,15,20,25, a 30min6 disinfecting time, rear aquae destilIata sterilis of having sterilized rinses 3-4 time, is seeded on medium.Each process 10 bottles, every bottle graft kind 6-10 explant, adds up primary pollution rate after inoculation 7d, adds up one-shot rate after 15d.Disinfecting time 30min inoculates rear blade, stem section, shows as withered shape, and disinfecting time short 8min pollution rate is high.Illustrate that disinfecting time is too short or oversizely all have an impact to head cabbage explant induction.The short pollution rate of disinfecting time is high, and the time, long pollution reduced, but the death of plant cell, lose regeneration capacity, starting rate also reduces simultaneously, so the Best Times of sterilization: tender leaf 10min, Lao Ye 15min, tender stem section 20min.
Pollution rate=(the explant sum of the explant number/inoculation of pollution) × 100%
Starting rate=(uncontaminated explant starts number/uncontaminated explant sum) × 100%
2.2 adventitious bud inducing
Test to the stem section of different positions of drawing materials: Lao Ye, tender leaf, tender leaf petiole, band axillalry bud, the medium of 9 kinds of proportionings carries out the induction of indefinite bud respectively.Medium is NAA and 6-BA by interpolation variable concentrations on the basis of MS solid culture medium medium composition, processes as follows.1. adding concentration is 0.4mgL
-1nAA, and 0,0.5,1.0,1.5,2.0mgL
-16-BA; 2. adding concentration is 2.0mgL
-16-BA, and 0,0.05,0.1,0.2,0.4mgL
-1nAA.
Sterilizing rear blade is cut into the square of about 0.5cm, and petiole is cut into the long segment of 0.5-1cm; After the sterilizing of tender stem section stem section, be cut into the stem section of band axillalry bud, be cut into the segment that 0.5-1.0cm is long; Be inoculated in and be added with on the calli induction media of hormon.Each process connects 10 bottles altogether, 8-12 sheet/bottle, and the induction of blade first will carry out the light culture of 16-20h, carrying out light cultivation, cultivation 15d rear blade obviously increases and carries out transferring once again, and the blade increased is cut into small pieces and cultivates, about 30d directly can induce indefinite bud.The every bottle graft kind 5-10 segment of tender stem section with axillalry bud.To draw materials position from explant, the speed of the tender stem section induction indefinite bud of band axillalry bud is fast and inductivity is the highest, is secondly tender leaf.Tender leaf petiole, Lao Ye inductivity are very low.Tender leaf evoking adventive bud directly produces from blade differentiation without callus phase.From the induction of medium, the different requirement to medium in position of drawing materials is different, and adopt the optimal medium that have selected induction, tender leaf is MS+6-BA2.0mg.L
-1+ NAA0.2mg.L
-1, the tender stem of band axillalry bud is MS+6-BA2.0mg.L
-1+ NAA0mg.L
-1.
2.3 Multiplying culture
The indefinite bud induced or clump bud are taken off, transfer on proliferated culture medium, proliferated culture medium is selected: MS solid culture medium, and add the NAA of 6-BA and 5 kind of variable concentrations gradient of 2 kinds of concentration gradients, 6-BA concentration is 1.0mg.L
-1and 2.0mg.L
-1, add 0 respectively, 0.05,0.1,0.2,0.4mg.L
-1nAA.The each process inoculation of the indefinite bud induced 10 bottles, every bottle of 4-6 indefinite bud, 15-20d institutes an inquiry record, and find that the regeneration of indefinite bud and the hormone combination of medium have direct relation, 6-BA concentration is 2.0mg.L
-1, NAA concentration is only at 0mg.L
-1time, growth coefficient reaches more than 3.0, does not have root to generate, and grows up healthy and strong; All the other value-added coefficients between 2.0-3.0, and have root to generate.Filter out MS+6-BA2mg.L
-1+ NAA0mg.L
-1for optimum multiplication medium, the indefinite bud induced, growth coefficient is high and do not have root system to generate, and each indefinite bud on average can breed 4-5 indefinite bud at every turn, improves gain factor.
The induction of 2.4 and transplanting
Reach the quantity of needs through propagation, select the aseptic seedling of robust growth to carry out culture of rootage, medium selects conventional 1/2MS and MS+NAA0.3mg.L
-1two kinds, with MS+NAA0.3mg.L
-1root induction rate is high, and obviously, inductivity can up to about 95%, and root growth is healthy and strong in performance.After obtaining the seedling of taking root of robust growth well developed root system, first remove sealed membrane at nominal room temperature and carry out practicing seedling about 7d, after root medium being washed during transplanting, with the medium in tray of humidity, to grab agglomerating with hand one, a pine and to be loosely advisable; In order to make root system fully be combined with cultivation matrix, after transplanting, cave dish is placed in pond, naturally absorb water, put after suctioning water in the cool, survival rate is to 100%.
3, conclusion
By regulating the disinfecting time of different explants, and correct method of operating, inducing effect can be improved by decreasing pollution.Having optimized head cabbage in-vitro inducing and regenerated ideal explant, is the stem section of tender leaf and band axillalry bud; Having optimized best induction, propagation and root media, is MS+6-BA2mg.L respectively
-1+ NAA0.2mg.L
-1, MS+6-BA2mg.L
-1, MS+NAA0.3mg.L
-1.
Note: MS minimal medium forms
The foregoing is only preferred embodiment of the present invention, not in order to limit the present invention, all any amendments done within the spirit and principles in the present invention, equivalent replacement and improvement etc., all should be included within protection scope of the present invention.
Claims (1)
1. improve a method for tissue culture for head cabbage in-vitro regeneration efficiency, it is characterized in that, this method for tissue culture comprises the following steps:
To choose the high explant of Regeneration in Vitro inductivity, bloom at head cabbage bolting and get the side shoot of sprouting latter stage;
Clorox with 7% carries out sterilizing, and tender leaf and tender stem sterilization time are respectively 10min and 20min; Blade first carries out the dark culturing of 16-20h in Induction Process, then carries out light cultivation, cultivates after 15 days and carries out transferring once again;
The induction of tender stem section, each little stem section is induced with axillalry bud;
By the bud that induces or Multiple Buds, carry out shoot proliferation; Time indefinite bud grows to about 3.0cm, the simple bud of getting robust growth carries out culture of rootage; Select the plantlet in vitro of well developed root system, first at room temperature remove sealed membrane and carry out practicing seedling about 7d, after root medium is washed, transplant in the dish of ready cave;
This method for tissue culture specifically comprises the following steps:
The first, induce
Draw materials: bloom at head cabbage bolting and get explant latter stage, get the tender lateral branch without scab and insect pest of sprouting, the 2-3 sheet leaf on branch top near lobus cardiacus is tender leaf, the tender stem got band handle tender leaf and remove on the spray of blade;
Sterilizing: rinse 20-30min under the field explant chosen is placed on running water, then use blotting paper suck dry moisture afterwards 3-4 time with distilled water flushing; Tender leaf and tender stem are separated sterilizing, each with 75% alcohol disinfecting 1min, then carry out disinfection with 7%NaClO, tender leaf and tender stem sterilization time are respectively 10min and 20min, and rear aquae destilIata sterilis of having sterilized flushing 3-4 time, is seeded on medium;
Differentiation: sterilizing rear blade is cut into the square of about 0.5cm, is seeded in MS+6-BA2mg.L
-1+ NAA0.2mg.L
-1on medium; Blade 8-12 sheet/bottle; Process 10 bottles, the induction of blade first will carry out the light culture of 16-20h, then carries out light cultivation, and blade carries out transferring once after light cultivates 15d, and the blade increased is cut into small pieces and cultivates, about 30d directly can induce indefinite bud; After tender stem section sterilizing after blade is removed, be cut into the stem section of band axillalry bud, be cut into the segment that 0.5-1.0cm is long; Every bottle graft kind 5-10 segment; Directly cultivate under light is cultivated, after 15d, just can see the bud induced;
The second, shoot proliferation: by the bud that induces or Multiple Buds, transfer at MS+6-BA2mg.L
-1on, every bottle of 4-6 indefinite bud, 25-30d carries out transferring once, and through the cultivation in 3-4 generation, the multiple of bud reaches the growth of about 200 times;
Three, the induction of root: time simple bud or Multiple Buds grow into about 3.0cm, the simple bud of getting robust growth carries out culture of rootage, medium MS+NAA0.3mg.L
-1, each process connects 10 bottles, every bottle graft 5-6 simple bud, and 15d " Invest, Then Investigate " is recorded root induction rate and reached the healthy and strong prosperity of about 95%, 25-30d root system;
Four, transplanting and field planting: the plantlet in vitro selecting well developed root system, first removes sealed membrane at nominal room temperature and carries out practicing seedling about 7d, after root medium is washed, transplants in the dish of ready cave; Matrix selects seedling medium; Adopt moist medium in tray, to grab agglomerating with hand one, a pine is namely loose is advisable; In order to make root system fully be combined with cultivation matrix during transplanting, after transplanting, cave dish being placed in pond, naturally absorbing water, put in the cool, within about 1 week, can survive after suctioning water, cave dish keeps dry wet, survival rate 100%;
Explant branch topmost is near the 2nd, 3 tender leaf of growing point, and band petiole takes off, and the side shoot removing blade gets tender stem;
Tender leaf inducing culture is MS+6-BA2mg.L
-1+ NAA0.2mg.L
-1; Get the stem section 0.5-1.0cm of band axillalry bud, directly at MS+6-BA2mg.L
-1upper Fiber differentiation; The bud induced, transfers at proliferated culture medium MS+6-BA2mg.L
-1upper cultivation, through the cultivation in 3-4 generation, the multiple of bud reaches the growth of about 200 times; Root media selects MS+NAA0.3mg.L-1, obtains the seedling of taking root of robust growth well developed root system;
Temperature controls at 22-25 DEG C, and illumination every day 16/8h, intensity of illumination 2000-2500Lx, adds 30g.L in medium
-1sucrose and 7g.L
-1agar powder, pH5.8-5.9;
With the medium in tray of humidity, in order to make root system fully be combined with cultivation matrix during transplanting, after transplanting, cave dish is placed in pond, naturally absorb water, put after suctioning water in the cool, survival rate is to 100%.
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| CN103609437B (en) * | 2013-10-23 | 2016-08-17 | 青岛文创科技有限公司 | A kind of cabbage somatic embryogenesis plant induction method |
| CN106818481A (en) * | 2017-01-25 | 2017-06-13 | 厦门市园林植物园 | A kind of kohlrabi quick breeding method for tissue culture |
| CN108719062A (en) * | 2018-05-11 | 2018-11-02 | 广州禾立田生物科技有限公司 | A kind of quick breeding method for tissue culture of brussels sprout |
| CN109169279B (en) * | 2018-09-18 | 2022-02-15 | 江苏省农业科学院 | Method for efficiently obtaining regenerated plants by culturing common head cabbage seed pods |
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| CN101564009B (en) * | 2009-06-03 | 2011-12-28 | 西北农林科技大学 | Propagation method for keeping cabbage RGMS male sterile line |
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