CN101595846B - Method for establishing high-frequency regeneration system of euscaphis konishii hayata leaves and quickly reproducing euscaphis konishii hayata leaves - Google Patents
Method for establishing high-frequency regeneration system of euscaphis konishii hayata leaves and quickly reproducing euscaphis konishii hayata leaves Download PDFInfo
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- CN101595846B CN101595846B CN200910304603XA CN200910304603A CN101595846B CN 101595846 B CN101595846 B CN 101595846B CN 200910304603X A CN200910304603X A CN 200910304603XA CN 200910304603 A CN200910304603 A CN 200910304603A CN 101595846 B CN101595846 B CN 101595846B
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Abstract
The invention provides a method for establishing a high-frequency regeneration system of euscaphis konishii hayata leaves and quickly reproducing euscaphis konishii hayata leaves, and belongs to the technique of culturing euscaphis konishii hayata embryo. The method solves the problems existing in the prior art that the seed germination rate of the euscaphis konishii hayata embryo is low, and well purified tissue culture seedlings cannot be provided in large amount. In the method, partial tender leaves removed during the sub-transplanting in the stem reproduction or seed reproduction are selected as materials, and the tender leaves undergo induced culture for 15 to 20 days to grow strong seedlings; after proliferation culture for 20 to 30 days and induced rooting for 20 to 25 days, the seedlings are grown and transplanted, and the survival rate is as high as more than 95 percent; and the propagation factor is higher, and the survived seedlings are tall and strong, and well grown.
Description
Technical field
The invention belongs to the culture technique of euscaphis konishii embryo, more specifically relate to a kind of euscaphis konishii blade high frequency regenerating system and set up and method for quickly breeding.
Background technology
Euscaphis konishii is the wild crow Toona of Staphyleaceae; Another name flower bromine wood, wooden, light Chinese toon of the arm beam of steelyard, opening green pepper; The wild crow Chinese toon (Euscaphis fukienensis Hsu) in a novel species Fujian that the sixties are found by professor Xu Ping Sheng of biology department of Xiamen University; Being this kind also also later on, is the distinctive ornamental tree species of China.Bloom summer, winter the result, the fruit phase is long, ftractures along ventral suture during red fruit maturation, with the seed of its black, just as a lifelike red butterfly of beauty, has ornamental value, can do specimen tree or street tree.Fruit-growing time is very long, comes off from the exocarp pericarp that reddens, for up to half a year.Therefore, can supply the time of viewing and admiring long, be good sight fruit tree kind.The fatty oily 25%-30% of its seed, but soapmaking also can be made other iundustrial oil; Bark can carry baking vanish and utensil is used material.In addition, but its root or root skin, flower and dry fruit hyoscine all, property is hot, sweet, flat, and root is dispelled rheumatism, and invigorating the spleen is controlled dysentery, is had loose bowels, colic, rheumatalgia pain, traumatic injury; The flower analgesia, it is dizzy to control headache; Dry fruit has that Wen Chong regulates the flow of vital energy, the effect of swelling and pain relieving, cures mainly stomachache, cold hernia, rushes down disease, prolapse of the anus, and is excellent to coating with lacquer irritated curative effect, is a kind of good Chinese medicinal herbs.But because the seed of euscaphis konishii has hard, the deep dormancy of shell, cause that seedling raise period is long, emergence rate is low, therefore be difficult to obtain good germination rate and more healthy seedling.Units such as forest seedling station, Ganzhou City have carried out seeding growing seedlings method research; To the seed husky storing and germination accelerating that wets, make seed germination rate bring up to 51%-65%, but still can't satisfy needs of production and large tracts of land is promoted requirement; When particularly it being carried out the medical value exploitation; Then require the tissue cultivating seedling of good purifying, this just requires when group training test, to reduce and pollute; Improve success rate; And the blade that utilizes seed or stem section breeding nursery stock carries out the plant high frequency regeneration and be able to satisfy this condition with breeding fast, but patent openly before the report of the relevant euscaphis konishii blade of the neither one high frequency regenerating system tissue culture of setting up and breeding fast, more do not have successful examples.
Summary of the invention
The purpose of this invention is to provide the method for tissue culture that a kind of euscaphis konishii blade high frequency regenerating system is set up and bred fast; The sub-germination rate of euscaphis konishii semina is low in the solution prior art, the problem of the tissue cultivating seedling that good purifying is provided that can not be a large amount of, and the bud that this method is cultivated is healthy, healthy and strong, generation time short, become the survival rate height after seedling is transplanted.
Technology contents of the present invention is following:
1) method of drawing material: choosing the euscaphis konishii young leaflet tablet is that explant is inoculated;
2) culture medium preparation: at minimal medium is several kinds that add respectively among the WPM among somatotropin: ZE, NAA or the IBA; Be configured to bud inducing culture, clump bud inducing culture, bud proliferated culture medium and root media respectively; Also adding Su in said these medium is 20g/L, Ag
+Be 7.5g/L, pH value is 5.8, and additives is active carbon Ac; The thickness of said these medium is 1.4～1.6cm;
3) material processed: the part young leaflet tablet in that the stem section is bred or seminal propagation is carried out removing when subculture is transferred is directly inoculated as explant.
4) inducing culture: select blade, blade is cut into 1.5cm * 1.5cm size, be seeded in the described bud inducing culture; 3 embryos of bud inducing culture inoculation of 100～150g; The condition of culture of said inducing culture: culturing room's temperature is 25 ± 2 ℃, light application time 12h/d, intensity of illumination 1500～2000Lx; 15～20 days time of inducing culture;
5) clump bud inducing culture: the induced bud that will pass through the robust growth of step 4) inducing culture is cut into the long fritter of 0.5cm, is seeded in the said clump bud inducing culture; 3 embryos of clump bud inducing culture inoculation of 100～150g; The condition of culture of said inducing culture: culturing room's temperature is 25 ± 2 ℃, light application time 12h/d, intensity of illumination 1500～2000Lx; 15～20 days time of clump bud inducing culture;
6) enrichment culture: will pass through well-grown clump of bud of step 5) clump bud inducing culture and cut open, and be seeded in the bud proliferated culture medium; 3 embryos of bud proliferated culture medium inoculation of 100～150g; The condition of culture of said enrichment culture: culturing room's temperature is 25 ± 2 ℃, light application time 12h/d, intensity of illumination 1500～2000Lx; 20～30 days time of enrichment culture;
7) root induction is cultivated: when the bud of growing thickly grows to 2cm when high, bud is divided into individual plant, transfers in the said root media; 3 embryos of root media inoculation of 100～150g; The condition of culture of said culture of rootage: culturing room's temperature is 25 ± 2 ℃, light application time 12h/d, intensity of illumination 1500～2000Lx; 20～25 days time of culture of rootage;
8) test-tube plantlet is accomplished: when test-tube plantlet grows to 3～4cm height, 3～5 roots are arranged, 5～7 blades during length 2～3cm, are accomplished the foundation of euscaphis konishii blade high frequency regenerating system and breeding test-tube plantlet cultivation fast.
9) little transplantation of seedlings: the euscaphis konishii of the completion test-tube plantlet in the said step 8) is transplanted, and test-tube plantlet is placed on the natural daylight lower refining seedling opens bottle cap after 3～5 days again and refined seedling earlier 2～4 days, to strengthen the adaptive capacity of test-tube plantlet to outdoor environment; From blake bottle, take out then, flush away root medium moves in the matrix of coconut palm chaff and (mass ratio) humous mixing in 1: 2, the cultivation of sheltering from heat or light of preserving moisture, and the euscaphis konishii test-tube plantlet survival rate of said transplanting reaches more than 95%.
Remarkable advantage of the present invention is: adopt the inventive method to carry out, vanes is crossed inducing culture, can sprout the sprouting of robust growth in 15～20 days; Through 20～30 days enrichment culture, can become seedling, transplanting after the root induction in 20～25 days again, survival rate is up to more than 95%; Expand numerous coefficient more than 18 times, robust growth after nursery stock becomes to live grows fine; Basically it is low to have solved in the prior art the sub-germination rate of euscaphis konishii semina, and the problem of the tissue cultivating seedling that good purifying is provided that can not be a large amount of has remarkable economic efficiency.
Description of drawings
Fig. 1 is the indefinite bud that the direct dedifferentiation of stripped mesophyll palisade tissue produces.
Fig. 2 is the indefinite bud that callus produces.
Fig. 3 is the embryoid that forms after the dedifferentiation of blade table chrotoplast.
Fig. 4 is the plant soma fast asexual propagation system that leaf cell is set up.
Fig. 5 is the clump bud of the dedifferentiation of mesophyll palisade tissue, embryoid, callus formation.
Fig. 6 is the bud seedling behind the long root.
Embodiment
Below in conjunction with embodiment the present invention is described further.
Embodiment 1
Method of drawing material: choose the part young leaflet tablet of removing when the subculture switching is carried out in breeding of stem section or seminal propagation and directly inoculate as explant;
Medium:
The MS minimal medium adopts 1986, and Beijing Higher Education Publishing House publishes, and the prescription in Chen Zhenghua chief editor " woody plant tissure is cultivated and used " is prepared.
Culture medium preparation: minimal medium is WPM, and adding somatotropin is ZE, NAA, IBA, and adding Su (sucrose) in the medium is 20g/L, Ag
+Be 7.5g/L, pH value is 5.8, and additives is active carbon Ac etc.; Medium thickness is generally 1.4～1.6cm;
Bud inducing culture → WPM+ZE (1.0mg/L)+NAA (1.0mg/L)+Ag
+(7.5g/L)+Su (20g/L)+Ac (2～2.5g/L)
Clump bud inducing culture → WPM+ZE (1.0mg/L)+NAA (0.5mg/L)+Ag
+(7.5g/L)+Su (20g/L)+Ac (2～2.5g/L)
Bud proliferated culture medium → WPM+ZE (2.0mg/L
-1)+NAA (0.3mg/L)+Ag
+(7.5g/L)+Su (20g/L)+Ac (2～2.5g/L)
Root media → 1/2WPM+NAA (0.1mg/L)+IBA (0.5mg/L)+Ag
+(7.5g/L)+Su (20g/L)+Ac (2～2.5g/L)
Ag in these medium
+Derive from AgNO
3
Minimal medium 1/2 WPM is meant: macroelement reduces by half among the WPM, and all the other are constant.
Inducing culture: select blade, blade is cut into 1.5cm * 1.5cm size, be seeded in the described bud inducing culture; 3 embryos of bud inducing culture inoculation of 100～150g; The condition of culture of said inducing culture: culturing room's temperature is 25 ± 2 ℃, light application time 12h/d, intensity of illumination 1500～2000Lx; 15～20 days time of inducing culture;
Clump bud inducing culture: the induced bud that will pass through the robust growth of inducing culture is cut into the long fritter of 0.5cm, is seeded in said from the bud inducing culture; 3 embryos of bud inducing culture inoculation of 100～150g; The condition of culture of said inducing culture: culturing room's temperature is 25 ± 2 ℃, light application time 12h/d, intensity of illumination 1500～2000Lx; 15～20 days time of clump bud inducing culture;
Enrichment culture: will pass through clump well-grown clump of bud of bud inducing culture and cut open, and be seeded in the bud proliferated culture medium; 3 embryos of bud inducing culture inoculation of 100～150g; The condition of culture of said enrichment culture: culturing room's temperature is 25 ± 2 ℃, light application time 12h/d, intensity of illumination 1500～2000Lx; 20～30 days time of enrichment culture;
Root induction is cultivated: when the bud of growing thickly grows to 2cm when high, bud is divided into individual plant, transfers in the said root media; 3 embryos of bud inducing culture inoculation of 100～150g; The condition of culture of said enrichment culture: culturing room's temperature is 25 ± 2 ℃, light application time 12h/d, intensity of illumination 1500～2000Lx; 20～25 days time of culture of rootage;
Little transplantation of seedlings: when test-tube plantlet grows to 3～4cm height, 3～5 roots are arranged, 5～7 blades during length 2～3cm, are accomplished the foundation of euscaphis konishii blade high frequency regenerating system and breeding fast.
Accomplish the euscaphis konishii of test-tube plantlet and transplant, test-tube plantlet is placed on the natural daylight lower refining seedling refined seedling earlier 2～4 days opening bottle cap after 3～5 days, to strengthen the adaptive capacity of test-tube plantlet to outdoor environment; From blake bottle, take out then, flush away root medium moves in the matrix of coconut palm chaff and (mass ratio) humous mixing in 1: 2, the cultivation of sheltering from heat or light of preserving moisture, and the euscaphis konishii test-tube plantlet survival rate of said transplanting reaches more than 95%.
Carry out according to above method, vanes is crossed inducing culture, can sprout the sprouting of robust growth in 15～20 days; Through 20～30 days enrichment culture, can become seedling, transplanting after the root induction in 20～25 days again, survival rate is up to more than 95%; Expand numerous coefficient more than 18 times, robust growth after nursery stock becomes to live grows fine.
Claims (3)
1. the foundation and the method for quickly breeding of an euscaphis konishii blade high frequency regenerating system, it is characterized in that: the concrete steps of said method comprise:
1) method of drawing material: choosing the euscaphis konishii young leaflet tablet is that explant is inoculated;
2) culture medium preparation: at minimal medium is several kinds that add respectively among the WPM among somatotropin: ZE, NAA or the IBA; Be mixed with bud inducing culture, clump bud inducing culture, bud proliferated culture medium and root media respectively; Also adding Su in said these medium is 20g/L, Ag
+Be 7.5g/L, pH value is 5.8, and additives is active carbon Ac; The thickness of said these medium is 1.4～1.6cm;
3) material processed: the part young leaflet tablet in that the stem section is bred or seminal propagation is carried out removing when subculture is transferred is directly inoculated as explant;
4) inducing culture: select blade, blade is cut into 1.5cm * 1.5cm size, be seeded in the described bud inducing culture; 3 embryos of bud inducing culture inoculation of 100～150g; The condition of culture of said inducing culture: culturing room's temperature is 25 ± 2 ℃, light application time 12h/d, intensity of illumination 1500～2000 Lx; 15～20 days time of inducing culture;
5) clump bud inducing culture: the induced bud that will pass through the robust growth of step 4) inducing culture is cut into the long fritter of 0.5cm, is seeded in the said clump bud inducing culture; 3 embryos of clump bud inducing culture inoculation of 100～150g; The condition of culture of said inducing culture: culturing room's temperature is 25 ± 2 ℃, light application time 12h/d, intensity of illumination 1500～2000 Lx; 15～20 days time of clump bud inducing culture;
6) enrichment culture: will pass through well-grown clump of bud of step 5) clump bud inducing culture and cut open, and be seeded in the bud proliferated culture medium; 3 embryos of proliferated culture medium inoculation of 100～150g; The condition of culture of said enrichment culture: culturing room's temperature is 25 ± 2 ℃, light application time 12h/d, intensity of illumination 1500～2000 Lx; 20～30 days time of enrichment culture;
7) root induction is cultivated: when the bud of growing thickly grows to 2cm when high, bud is divided into individual plant, transfers in the said root media; 3 embryos of root media inoculation of 100～150g; The condition of culture of said culture of rootage: culturing room's temperature is 25 ± 2 ℃, light application time 12h/d, intensity of illumination 1500～2000 Lx; 20～25 days time of culture of rootage;
8) little transplantation of seedlings: when test-tube plantlet grows to 3～4cm height, 3～5 roots are arranged, 5～7 blades during length 2～3cm, are accomplished the foundation of euscaphis konishii blade high frequency regenerating system and breeding test-tube plantlet cultivation fast;
Said step 2) in the medium preparation:
Said bud inducing culture is: add among the minimal medium WPM: ZE 1.0mg/L again; NAA 1.0mg/L; Ag
+7.5g/L; Su 20g/L and Ac 2～2.5 g/L;
Said clump bud inducing culture is: add among the minimal medium WPM: ZE 1.0mg/L again; NAA 0.5mg/L; Ag
+7.5g/L; Su 20g/L and Ac 2～2.5 g/L;
Said bud proliferated culture medium is: add among the minimal medium WPM: ZE 2.0mg/L again; NAA 0.3mg/L; Ag
+7.5g/L; Su 20g/L and Ac 2～2.5 g/L;
Said root media is: add among the minimal medium 1/2WPM: NAA 0.1mg/L again; IBA 0.5mg/L; Ag
+7.5g/L; Su 20g/L and Ac 2～2.5 g/L;
Said minimal medium 1/2 WPM is meant: macroelement reduces by half among the WPM, and all the other are constant.
2. the foundation and the method for quickly breeding of euscaphis konishii blade high frequency regenerating system according to claim 1; It is characterized in that: the euscaphis konishii of the completion test-tube plantlet in the said step 8) is transplanted; Test-tube plantlet is placed on the natural daylight lower refining seedling opens bottle cap after 3～5 days again and refined seedling earlier 2～4 days, to strengthen the adaptive capacity of test-tube plantlet outdoor environment; From blake bottle, take out then, flush away root medium moves in the matrix of coconut palm chaff and mass ratio humous=mixing in 1: 2, the cultivation of sheltering from heat or light of preserving moisture, and the euscaphis konishii test-tube plantlet survival rate of said transplanting reaches more than 95%.
3. the foundation and the method for quickly breeding of euscaphis konishii blade high frequency regenerating system according to claim 1 is characterized in that: Ag in the said medium
+Derive from AgNO
3
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| CN104857036B (en) * | 2014-08-23 | 2018-01-12 | 福建农林大学 | A kind of extracting method and product of euscaphis konishii active material |
| CN109601213A (en) * | 2018-11-24 | 2019-04-12 | 福建农林大学 | A kind of open country crow Chinese toon container seedling culture method |
| CN110506632B (en) * | 2019-09-05 | 2020-12-22 | 厦门市园林植物园 | Tissue culture method of euscaphis konishii hayata |
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| CN101263789A (en) * | 2008-05-08 | 2008-09-17 | 福建农林大学 | Tissue culture method for stem segment of euscaphis konishii |
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