CN103125398A - Tissue culture method for improving in-vitro regeneration efficiency of common head cabbage - Google Patents
Tissue culture method for improving in-vitro regeneration efficiency of common head cabbage Download PDFInfo
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- CN103125398A CN103125398A CN2013100891162A CN201310089116A CN103125398A CN 103125398 A CN103125398 A CN 103125398A CN 2013100891162 A CN2013100891162 A CN 2013100891162A CN 201310089116 A CN201310089116 A CN 201310089116A CN 103125398 A CN103125398 A CN 103125398A
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Abstract
The invention discloses a tissue culture method for improving the in-vitro regeneration efficiency of a common head cabbage. The tissue culture method comprises the steps of at first, taking explant with high regeneration and induction rate; sterilizing by using sodium hypochlorite; carrying out dark culture on a tender leaf on an induction culture medium for 16-20 hours, and then carrying out light culture; directly carrying out the light culture on a tender stem section with an axillary bud on the induction culture medium; transferring induced bud or cluster buds on an enrichment culture medium for enrichment culture; taking a strong single bud for rooting culture; removing a sealing film at room temperature for plantlet hardening after the strong single bud is rooted; washing off the culture medium from the root; and transplanting the bud in a prepared hole plate. With the adoption of the tissue culture method provided by the invention, the problem that explant sterilization time is different can be solved, and the problem that the regeneration coefficient in an in-vitro regeneration and induction process of the common head cabbage is low can be solved. With the adoption of the tissue culture method provided by the invention, the most efficient regeneration system of the common head cabbage can be established on the aspects of the sterilization time, an explant taking part, and different hormone combinations, and lays the foundation for the storage of genetic resources of the common head cabbage and the development of breeding of common head cabbage genetic engineering.
Description
Technical field
The invention belongs to Plant Biotechnology and head cabbage culture technique field, relate in particular to a kind of method for tissue culture that improves head cabbage Regeneration in Vitro efficient.
Background technology
Head cabbage belongs to 2 years these plants of sward that Cruciferae (Cruciferae) rape belongs to (Brassica), is one of our important vegetables.This plant of sward was namely then through forming leaf-head vegetative growth phase in 2 years.Leaf-head enters reproductive stage by storage survive the winter low temperature and the impact of long-day in the coming year, and bolting is blossomed and had seeds, and completes life process.The characteristics 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 it is larger affected by Changes in weather when the germ plasm resource breed conservation, utilizes tissue to cultivate and preserve to alleviate the environment-stress condition adverse effect that preservation is reserved seed for planting to head cabbage germ plasm resource.Setting up stable wild cabbage Tissue Culture Regeneration System is to 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 much successfully report, but cotyledon and the lower shaft of most employing flower pesticide microspores, indefinite bud blade and aseptic seedling are explant, and the aspects such as position, different sterilization time, the hormon proportioning of drawing materials in different explants do not have more concrete research.This research adopt the head cabbage tender leaf and with the tender stem of axillalry bud as the outer planting donor, set up 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 that improves head cabbage Regeneration in Vitro efficient, set up the head cabbage high-efficiency regeneration system.Be intended to solve the draw materials hormone combination of disinfecting time, indefinite bud and root induction medium at position of different explants and change the low problem of gain factor.
The embodiment of the present invention is achieved in that a kind of method for tissue culture that improves head cabbage Regeneration in Vitro efficient, and this method for tissue culture comprises the following steps:
To choose the high explant of Regeneration in Vitro inductivity, bloom at the head cabbage bolting and get the side shoot of sprouting latter stage;
Clorox with 7% is sterilized, and tender leaf and tender stem sterilization time are respectively 10min and 20min; A3, blade first carry out the dark culturing of 16-20h in Induction Process, then carry out light and cultivate, and cultivate and transfer once after 15 days again;
Inducing of tender stem section, each little stem section is induced with axillalry bud;
Bud or Multiple Buds with inducing carry out shoot proliferation; When indefinite bud grew to the 3.0cm left and right, the simple bud of getting robust growth carried out culture of rootage; Select the group training seedling of well developed root system, first at room temperature remove sealed membrane and practice seedling 7d left and right, after the 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 the 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, gets with the handle tender leaf and removes tender stem on the spray of blade;
Sterilization: the field explant of choosing is placed under running water rinses 20-30min, then use afterwards the blotting paper suck dry moisture 3-4 time with distilled water flushing; The separately sterilization of tender leaf and tender stem, each is with 75% alcohol disinfecting 1min, then carries out disinfection with 7%Naclo, and tender leaf and tender stem sterilization time are respectively 10min and 20min, have sterilized rear with aquae destilIata sterilis flushing 3-4 time, are seeded on medium;
Differentiation: the sterilization rear blade is cut into the square of 0.5cm left and right, is seeded in MS+6-BA2mg.L
-1+ NAA0.2mg.L
-1On medium; Sheet leaf 8-12 sheet/bottle; Process 10 bottles, the dark cultivation of 16-20h will be first carried out in inducing of blade, is carrying out the light cultivation, and blade is transferred once after light is cultivated 15d, the blade that increases is cut into small pieces cultivates, and can directly induce indefinite bud about 30d; After tender stem section stem section sterilization after blade is removed, be cut into stems with bud, be cut into the long segment of 0.5-1.0cm; 5-10 segment of every bottle graft kind; Directly cultivate under light is cultivated, just can see the bud that induces after 15d;
The second, shoot proliferation: with bud or the Multiple Buds that induces, switching is at MS+6-BA2mg.L
-1On, every bottle of 4-6 indefinite bud, 25-30d transfers once the left and right, and through the 3-4 cultivation in generation, the multiple of bud reaches the growth of 200 times of left and right;
Three, inducing of root: when simple bud or Multiple Buds grew into the 3.0cm left and right, the simple bud of getting robust growth carried out culture of rootage, medium MS+NAA0.3mg.L
-1, each processing connects 10 bottles, every bottle graft 5-6 simple bud, and 15d " Invest, Then Investigate " record root induction rate reaches 95% left and right, and the 25-30d root system is healthy and strong flourishing;
Four, transplanting and field planting: select the group training seedling of well developed root system, first remove sealed membrane and practice seedling 7d left and right under normal room temperature, after the root medium is washed, transplant in the dish of ready cave; Matrix is selected seedling medium.Adopt moist medium in tray, agglomerating to grab with hand one, a pine and loose being advisable; In order to make the abundant combination of root system and cultivation matrix, after transplanting, the cave dish is placed in the pond during transplanting, suction naturally is put in the cool after suctioning water, 1 week the left and right can survive, the cave dish keeps doing wet, survival rate reaches 100%.
Further, explant branch topmost is near the 2nd, 3 tender leaf of growing point, and the band petiole takes off, and removes the side shoot of blade and gets tender stem.
Further, the tender leaf inducing culture is MS+6-BA2mg.L
-1+ NAA0.2mg.L
-1Get stems with bud 0.5-1.0cm, directly at MS+6-BA2mg.L
-1On induce cultivation; The bud that induces, switching is at proliferated culture medium MS+6-BA2mg.L
-1Upper cultivation, through the 3-4 cultivation in generation, the multiple of bud reaches the growth of 200 times of left and right; Root media is selected MS+NAA0.3mg.L
-1, the seedling of taking root of acquisition robust growth well developed root system.
Further, temperature is controlled at 22-25 ℃, illumination every day 16/8h, and intensity of illumination 2000-2500Lx adds 30g.L in medium
-1Sucrose and 7g.L
-1Agar powder, PH5.8-5.9.
Further, agglomerating to grab with hand one with moist medium in tray, a pine and loose being advisable; In order to make the abundant combination of root system and cultivation matrix, after transplanting, the cave dish is placed in the pond during transplanting, naturally absorbs water, put in the cool after suctioning water, survival rate reaches 100%.
This research is take head cabbage as material; with optimization draw materials position, sterilization time and culture medium prescription; reduce pollution rate; improve and induce differentiation rate; improve inducing of gain factor and root system; set up the tender leaf blade and with the stripped high-efficiency regeneration system of the tender stem section of axillalry bud, for the protection of head cabbage germ plasm resource with carry out the 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.
Description of drawings
Fig. 1, figure A are the performance that blade is cultivated the 15d left and right; Figure B is that blade is cultivated the indefinite bud that induces;
Fig. 2, with the bud that induces between axillary bud stem;
The bud of Fig. 3, the regeneration of indefinite bud subculture;
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 purpose of the present invention, technical scheme and advantage clearer, 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, is not intended to limit the present invention.
The embodiment of the present invention is achieved in that a kind of method for tissue culture that improves head cabbage Regeneration in Vitro efficient, and this method for tissue culture comprises the following steps:
1 induces
1.1 draw materials: bloom at the 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, gets with the handle tender leaf and removes tender stem on the spray of blade.
1.2 sterilization: the field explant of choosing is placed under running water rinses 20-30min, then use afterwards the blotting paper suck dry moisture 3-4 time with distilled water flushing; The separately sterilization of tender leaf and tender stem, each is with 75% alcohol disinfecting 1min, then carries out disinfection with 7% Naclo, and tender leaf and tender stem sterilization time are respectively 10min and 20min, have sterilized rear with aquae destilIata sterilis flushing 3-4 time, are seeded on medium;
1.3 differentiation: the sterilization rear blade is cut into the square of 0.5cm left and right, is seeded in MS+6-BA2mg.L
-1+ NAA 0.2mg.L
-1On medium; Sheet leaf 8-12 sheet/bottle; Process 10 bottles, the dark cultivation of 16-20h will be first carried out in inducing of blade, is carrying out the light cultivation, and blade after light is cultivated 15d (as Figure 1A) is transferred once, the blade that increases is cut into small pieces cultivates, can directly induce indefinite bud (as Figure 1B) about 30d; After tender stem section stem section sterilization after blade is removed, be cut into stems with bud, be cut into the long segment of 0.5-1.0cm; 5-10 segment of every bottle graft kind.Directly cultivate under light is cultivated, just can see the bud that induces after 15d;
2 shoot proliferations: with bud or the Multiple Buds that induces, switching is at MS+6-BA 2mg.L
-1On, every bottle of 4-6 indefinite bud, 25-30d transfers once the left and right, and through the 3-4 cultivation in generation, the multiple of bud reaches the growth of 200 times of left and right.
3 induce: when simple bud or Multiple Buds grew into the 3.0cm left and right, the simple bud of getting robust growth carried out culture of rootage, medium MS+NAA0.3mg.L
-1, each processing connects 10 bottles, every bottle graft 5-6 simple bud, and 15d " Invest, Then Investigate " record root induction rate reaches 95% left and right, 25-30d root system healthy and strong flourishing (as Fig. 4 A, B).
4 transplant and field planting: select the group training seedling of well developed root system, first remove sealed membrane and practice seedling 7d left and right under normal room temperature, after the root medium is washed, transplant in the dish of ready cave.Matrix is selected seedling medium.Adopt moist medium in tray, agglomerating to grab with hand one, a pine and loose being advisable.In order to make the abundant combination of root system and cultivation matrix, after transplanting, the cave dish is placed in the pond during transplanting, suction naturally is put in the cool after suctioning water, 1 week the left and right can survive, the cave dish keeps doing wet, survival rate reaches 100%.
In embodiments of the present invention, induce the proportioning with proliferated culture medium: inducing culture forms, on the basis that MS solid culture medium medium forms, and by NAA and the 6-BA of interpolation variable concentrations, following processing, 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, and the MS solid culture medium adds the 6-BA of two kinds of concentration gradients and the NAA of five kinds of variable concentrations gradients, and 6-BA concentration is 1.0mg.L
-1And 2.0mg.L
-1, add respectively 0,0.05,0.1,0.2,0.4mg.L
-1NAA.Filtered out in research process, the 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 the 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 the present invention is further illustrated by concrete experimental program.
1. material
Be field inbred line head cabbage for the examination material, spring bolting bloom and get the side shoot without damage by disease and insect of its sprouting latter stage, tender leaf (comprising petiole), Lao Ye, the tender stem got on branch are standby as explant.
2. method
2.1 disinfect
The branch topmost is near the 2nd, 3 tender leaf of growing point, and the band petiole takes off; Branch foot the 2nd, 3 Lao Ye are not with petiole to take off; Go the branch of lower blade to get tender stem.On superclean bench, the blade and the tender stem that clean up are respectively charged in aseptic container, alcohol disinfecting 1min with 75%, carry out disinfection with 7%NaClO again, if 8,10,15,20,25, a 30min6 disinfecting time, sterilized rear with aquae destilIata sterilis flushing 3-4 time, be seeded on medium.Each processes 10 bottles, 6-10 explant of every bottle graft kind, statistics primary pollution rate after inoculation 7d, statistics one-shot rate after 15d.Disinfecting time 30min inoculates rear blade, stem section, shows as withered shape, and the short 8min pollution rate of disinfecting time is high.Illustrate that disinfecting time is too short or oversize the head cabbage explant induction all exerted an influence.The short pollution rate of disinfecting time is high, and the time, long the pollution reduced, but the death of plant cell has lost regeneration capacity, and starting rate has also reduced 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 of pollution/inoculation) * 100%
Starting rate=(uncontaminated explant starts number/uncontaminated explant sum) * 100%
2.2 adventitious bud inducing
Test is carried out inducing of indefinite bud respectively to the different positions of drawing materials: Lao Ye, tender leaf, tender leaf petiole, stems with bud on the medium of 9 kinds of proportionings.Medium is by adding NAA and the 6-BA of variable concentrations, following processing on the basis that MS solid culture medium medium forms.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.
The sterilization rear blade is cut into the square of 0.5cm left and right, and petiole is cut into the long segment of 0.5-1cm; After tender stem section stem section sterilization, be cut into stems with bud, be cut into the long segment of 0.5-1.0cm; Be inoculated on the calli induction media that is added with hormon.Each is processed and connects altogether 10 bottles, 8-12 sheet/bottle, and the dark cultivation of 16-20h will be first carried out in inducing of blade, carrying out the light cultivation, cultivate the obvious increase of 15d rear blade and transfer again once, the blade that increases is cut into small pieces cultivates, can directly induce indefinite bud about 30d.5-10 segment of the every bottle graft kind of tender stem section with axillalry bud.From the explant position of drawing materials, fast and inductivity is the highest with the speed of the tender stem section induction indefinite bud of axillalry bud, be secondly tender leaf.Tender leaf petiole, Lao Ye inductivity are very low.The tender leaf evoking adventive bud directly produced from blade differentiation without the callus stage.From inducing of medium, the position difference of drawing materials is different to the requirement of medium, and selecting and purchasing has gone out the optimal medium of inducing, and 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 propagation is cultivated
The indefinite bud or the clump bud that induce are taken off, and switching is on proliferated culture medium, and proliferated culture medium is selected: the MS solid culture medium, add the 6-BA of 2 kinds of concentration gradients and the NAA of 5 kinds of variable concentrations gradients, and 6-BA concentration is 1.0mg.L
-1And 2.0mg.L
-1, add respectively 0,0.05,0.1,0.2,0.4mg.L
-1NAA.Each processes 10 bottles of inoculations the indefinite bud that induces, every bottle of 4-6 indefinite bud, and 15-20d institutes an inquiry record, finds that the regeneration of indefinite bud and the hormone combination of medium have direct relation, and 6-BA concentration is 2.0mg.L
-1, NAA concentration is only at 0mg.L
-1The time, 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 and have root to generate between 2.0-3.0.Filtered out MS+6-BA2mg.L
-1+ NAA0mg.L
-1Be optimum multiplication medium, the indefinite bud that induces, growth coefficient is high and do not have root system to generate, and each indefinite bud on average can be bred 4-5 indefinite bud at every turn, has improved gain factor.
2.4 the inducing and transplanting of root
Reach the quantity that needs through propagation, select the aseptic seedling of robust growth to carry out culture of rootage, medium is selected 1/2MS commonly used and MS+NAA0.3mg.L
-1Two kinds, with MS+NAA0.3mg.L
-1The root induction rate is high, performance obviously, inductivity can be up to 95% left and right, and root growth is healthy and strong.After obtaining the seedling of taking root of robust growth well developed root system, first remove sealed membrane and practice seedling 7d left and right under normal room temperature, agglomerating to grab with hand one with moist medium in tray after during transplanting, the root medium being washed, a pine and loose being advisable; In order to make the abundant combination of root system and cultivation matrix, after transplanting, the cave dish is placed in the pond, naturally absorb water, to put in the cool after suctioning water, survival rate reaches 100%.
3, conclusion
By regulating the disinfecting time of different explants, reach correct method of operating, can reduce to pollute to improve and induce effect.Having optimized the ideal explant of head cabbage in-vitro inducing regeneration, is tender leaf and stems with bud; Optimized that the best is induced, propagation and root media, be respectively MS+6-BA2mg.L
-1+ NAA0.2mg.L
-1, MS+6-BA2mg.L
-1, MS+NAA0.3mg.L
-1
Note: the MS minimal medium forms
The above is only preferred embodiment of the present invention, not in order to limiting the present invention, all any modifications of doing within the spirit and principles in the present invention, is equal to and replaces and improvement etc., within all should being included in protection scope of the present invention.
Claims (6)
1. a method for tissue culture that improves head cabbage Regeneration in Vitro efficient, 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 the head cabbage bolting and get the side shoot of sprouting latter stage;
Clorox with 7% is sterilized, and tender leaf and tender stem sterilization time are respectively 10min and 20min; A3, blade first carry out the dark culturing of 16-20h in Induction Process, then carry out light and cultivate, and cultivate and transfer once after 15 days again;
Inducing of tender stem section, each little stem section is induced with axillalry bud;
Bud or Multiple Buds with inducing carry out shoot proliferation; When indefinite bud grew to the 3.0cm left and right, the simple bud of getting robust growth carried out culture of rootage; Select the group training seedling of well developed root system, first at room temperature remove sealed membrane and practice seedling 7d left and right, after the root medium is washed, transplant in the dish of ready cave.
2. the method for claim 1, is characterized in that, this method for tissue culture specifically comprises the following steps:
The first, induce
Draw materials: bloom at the 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, gets with the handle tender leaf and removes tender stem on the spray of blade;
Sterilization: the field explant of choosing is placed under running water rinses 20-30min, then use afterwards the blotting paper suck dry moisture 3-4 time with distilled water flushing; The separately sterilization of tender leaf and tender stem, each is with 75% alcohol disinfecting 1min, then carries out disinfection with 7%Naclo, and tender leaf and tender stem sterilization time are respectively 10min and 20min, have sterilized rear with aquae destilIata sterilis flushing 3-4 time, are seeded on medium;
Differentiation: the sterilization rear blade is cut into the square of 0.5cm left and right, is seeded in MS+6-BA2mg.L
-1+ NAA0.2mg.L
-1On medium; Sheet leaf 8-12 sheet/bottle; Process 10 bottles, the dark cultivation of 16-20h will be first carried out in inducing of blade, is carrying out the light cultivation, and blade is transferred once after light is cultivated 15d, the blade that increases is cut into small pieces cultivates, and can directly induce indefinite bud about 30d; After tender stem section stem section sterilization after blade is removed, be cut into stems with bud, be cut into the long segment of 0.5-1.0cm; 5-10 segment of every bottle graft kind; Directly cultivate under light is cultivated, just can see the bud that induces after 15d;
The second, shoot proliferation: with bud or the Multiple Buds that induces, switching is at MS+6-BA2mg.L
-1On, every bottle of 4-6 indefinite bud, 25-30d transfers once the left and right, and through the 3-4 cultivation in generation, the multiple of bud reaches the growth of 200 times of left and right;
Three, inducing of root: when simple bud or Multiple Buds grew into the 3.0cm left and right, the simple bud of getting robust growth carried out culture of rootage, medium MS+NAA0.3mg.L
-1, each processing connects 10 bottles, every bottle graft 5-6 simple bud, and 15d " Invest, Then Investigate " record root induction rate reaches 95% left and right, and the 25-30d root system is healthy and strong flourishing;
Four, transplanting and field planting: select the group training seedling of well developed root system, first remove sealed membrane and practice seedling 7d left and right under normal room temperature, after the root medium is washed, transplant in the dish of ready cave; Matrix is selected seedling medium; Adopt moist medium in tray, agglomerating to grab with hand one, a pine and loose being advisable; In order to make the abundant combination of root system and cultivation matrix, after transplanting, the cave dish is placed in the pond during transplanting, suction naturally is put in the cool after suctioning water, 1 week the left and right can survive, the cave dish keeps doing wet, survival rate reaches 100%.
3. method for tissue culture as claimed in claim 1, is characterized in that, explant branch topmost is near the 2nd, 3 tender leaf of growing point, and the band petiole takes off, and removes the side shoot of blade and gets tender stem.
4. method for tissue culture according to claim 1, is characterized in that, the tender leaf inducing culture is MS+6-BA2mg.L
-1+ NAA0.2mg.L
-1Get stems with bud 0.5-1.0cm, directly at MS+6-BA2mg.L
-1On induce cultivation; The bud that induces, switching is at proliferated culture medium MS+6-BA2mg.L
-1Upper cultivation, through the 3-4 cultivation in generation, the multiple of bud reaches the growth of 200 times of left and right; Root media is selected MS+NAA0.3mg.L
-1, the seedling of taking root of acquisition robust growth well developed root system.
5. method for tissue culture as claimed in claim 1, is characterized in that, temperature is controlled at 22-25 ℃, illumination every day 16/8h, and intensity of illumination 2000-2500Lx adds 30g.L in medium
-1Sucrose and 7g.L
-1Agar powder, PH5.8-5.9.
6. method for tissue culture as claimed in claim 1, is characterized in that, with moist medium in tray, in order to make the abundant combination of root system and cultivation matrix, after transplanting, the cave dish is placed in the pond, naturally suction during transplanting, put in the cool after suctioning water, survival rate reaches 100%.
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| 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 |
| CN109169279A (en) * | 2018-09-18 | 2019-01-11 | 江苏省农业科学院 | A method of culture common head cabbage seed pods efficiently obtain regeneration plant |
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| CN103609437A (en) * | 2013-10-23 | 2014-03-05 | 张亚丽 | Induction method of common-head-cabbage embryoid regenerated plant |
| 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 |
| CN109169279A (en) * | 2018-09-18 | 2019-01-11 | 江苏省农业科学院 | A method of culture common head cabbage seed pods efficiently obtain regeneration plant |
| 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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