CN105638473B - A kind of complete set of culture medium of Chinese scholar tree cotyledon somatic embryo inducement - Google Patents
A kind of complete set of culture medium of Chinese scholar tree cotyledon somatic embryo inducement Download PDFInfo
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- CN105638473B CN105638473B CN201610018015.XA CN201610018015A CN105638473B CN 105638473 B CN105638473 B CN 105638473B CN 201610018015 A CN201610018015 A CN 201610018015A CN 105638473 B CN105638473 B CN 105638473B
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/001—Culture apparatus for tissue culture
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Abstract
The invention discloses a kind of complete set of culture medium of Chinese scholar tree cotyledon somatic embryo inducement, including somatic embryo inducement culture medium, adventitious bud induction culture base, strong seedling culture base and root media.Using the present invention culture medium breed Chinese scholar tree seedling, can save first establish Chinese scholar tree soon numerous regenerating system the step for, greatly save the time.Regeneration rate is high, budding is more, and plantlet of transplant survival rate can reach more than 90%, and seedling early growth is healthy and strong, not only can effectively solve good seed germplasm degenerate problem, can also provide a preferable receptor system for Chinese scholar tree genetic transformation or mutation breeding.
Description
Technical field
The present invention relates to a kind of complete set of culture medium of Chinese scholar tree cotyledon somatic embryo inducement, belong to plant biotechnology field.
Background technology
Chinese scholar tree (Sophora japonica Linn), alias man Chinese scholartree, middle Chinese scholar tree belong to Papillionoideae, Sophora, are preferable
Avenue tree planting and courtyard greening seeds.Moreover, Chinese scholar tree material is excellent, flower, fruit, root skin, bark can be used as medicine, and seed can extract oil
Soapmaking, has higher economic value, and application prospect is very wide.Meanwhile Chinese scholar tree be grafting Chinese pagoda tree again, it is Jin Zhihuai, merely red
Unique stock seeds of the fancy breeds such as Chinese scholartree, butterfly Chinese scholartree, nursery stock demand are big.Chinese scholar tree is mainly based on seminal propagation, but it is bloomed
As a result there is biennial bearing phenomenon, and seed is easily endangered by pest and disease damage, shrivelled, cavitation is serious.Conventional breeding method relies on
Seed or cutting propagation, it is difficult to meet the needs of production.
In recent years, cultivated using biotechnology resistant if the new kind such as degeneration-resistant, pest-resistant, disease-resistant is Chinese scholar tree breeding
New trend.Up to the present, to the forming layer culture of Chinese scholartree, stem culture, leaf culture, embryo culture, the culture of Unpollinated ovules
Complete plant has been obtained, plasmic culture has also been made some progress.Yuan Xiuyun etc. utilizes the cotyledon of Chinese scholar tree
Generate adventitious bud with hypocotyl, the grade of king's Zhe using Anther Culture form haplobiont, Han K.H etc. (1993,
1997) after the branch of a diameter of 0.5~1.0cm is sterilized, callus induction in forming layer access calli induction media is taken out, then
Callus is transferred in differential medium, obtains Multiple Buds, seedling is transferred in root media, can induce and take root.On
The method stated there are the shortcomings that：Since Chinese scholar tree is perennial woody plant, regeneration rate is generally relatively low, and budding is few, directly affects
The genetic transformation of Chinese scholar tree.It can not only be reached using plant embryonal induction technology and preserve maternal plant excellent genes, the mesh of fast breeding
Or improve gene genetic conversion or mutation breeding important channel.At present, the somatic embryo inducement technology in relation to Chinese scholar tree is ground
That studies carefully is less, and the prior art that may be referred to is fewer.Consulting literatures, it can be seen that also someone lures doing body embryo other seeds
It leads, Chinese scholar tree is perennial arbor, and genome is huge, in view of the limitation of genotype, without reference to value.
Excised leaf, tip of a root of Chinese scholar tree etc. can induce and generate embryoid.Its precondition is to first have to establish one surely
Fixed high regeneration frequency system only in this way, could provide sufficient sterile body material.In Shandong District, the danger of Chinese scholar tree small peak
Evil is very serious, to gather Chinese scholar tree seed, it is necessary to which just having been formed during beginning forms fruit pod to pollination in Chinese scholar tree inflorescence will adopt
The state Chinese scholartree plucked and its periphery are intended to spray insecticide, and Chinese scholar tree chalcid fly is prevented to be deposited in seed.After fruit pod expands maturation, can both it adopt
Sowing.102499086 A of patent CN disclose a kind of propagation method of locust tree, since Chinese scholar tree from locust tree adheres to different categories separately,
Species differential is larger, it is impossible to obtain the Somatic Cell Culture of Chinese scholar tree cotyledon with reference to the Somatic Cell Culture method of locust tree pod.
The content of the invention
The purpose of the present invention is exactly to solve the above-mentioned problems, to provide a kind of complete training of Chinese scholar tree cotyledon somatic embryo inducement
Base is supported, to solve the problems, such as that Chinese scholar tree regeneration rate is low.
To achieve these goals, the present invention adopts the following technical scheme that：
A kind of complete set of culture medium of Chinese scholar tree cotyledon somatic embryo inducement, including
Somatic embryo inducement culture medium：A+3.0~5.0mg/L 2,4-D (2,4- dichlorphenoxyacetic acid)+0.5~1.0mg/
L BA+0.5~1.0mg/L NAA (methyl α-naphthyl acetate)+0.1~0.5mg/L TDZ+0.5~1.0mg/L glutamine+0.5~
1.0mg/L CH (caseinhydrolysate)+5.0g/L agar+30g/L sucrose, pH value 5.8~6.0；
Adventitious bud induction culture base：A+0.1~0.5mg/L BA+0.1~0.5mg/L NAA+0.5~1.0mg/L paddy ammonia
Amide+0.5~1.0mg/L CH (caseinhydrolysate)+5.0g/L agar+30g/L sucrose, pH value 5.8~6.0；
Test tube seedling proliferation culture medium formula:MS+1.0~2.0mg/L BA (6-benzyladenine)+1.0~2.0mg/LIBA
(indolebutyric acid)+5.0g/L agar+30g/L sucrose, pH value 5.8~6.0；
Strong seedling culture base：MS+5.0g/L agar+30g/L sucrose, pH value 5.8~6.0；
Root media：1/2MS+0.1~0.5mg/L IBA+0~0.05mg/L NAA+5.0g/L agar+20g/L sugarcanes
Sugar, pH value 5.8~6.0, the 1/2MS are that MS full doses halve；
It is described：A is formulated organic examination of the grand nutrition element included in MS culture mediums, micronutrient element and B5 medium
Agent.
It is preferred that：MS culture mediums include grand nutrition element, micronutrient element and organic reagent.
It is preferred that：Component concentration corresponding with its of the grand nutrition element of the MS culture mediums is as follows：Potassium nitrate 1900mg/
L, ammonium nitrate 1650mg/L, epsom salt 370mg/L, anhydrous potassium dihydrogenphosphate 170mg/L, calcium chloride dihydrate 440mg/L, second
Edetate disodium 37.3mg/L, ferrous sulfate heptahydrate 27.8mg/L.
It is preferred that：Component concentration corresponding with its of the micronutrient element of the MS culture mediums is as follows：Four water manganese sulfates
22.3mg/L, zinc sulfate 8.6mg/L, boric acid 6.2mg/L, potassium iodide 0.83mg/L, sodium molybdate 0.25mg/L, copper sulphate
0.025mg/L, cobalt chloride 0.025mg/L.
It is preferred that：Component concentration corresponding with its of the A formulas B5 organic reagents is as follows：Thiamine hydrochloride 100mg/L,
Niacin 10mg/L, puridoxine hydrochloride 10mg/L, inositol 1000mg/L.
Beneficial effects of the present invention：
The factor of somatic embryo occur includes internal cause and external cause, and internal cause includes species and genotype, and external cause relates generally to train
Support the species and concentration of supplementary element in base.Even the plant of same category, since genotype is different, somatic embryo occur frequency
Difference is very big, the reason is as follows that:First, different genotype somatic embryo occur frequency is different, second is that the most suitable induction of different genotype
Condition is different.The physiological status and development degree of explant all directly affect the generation of somatic embryo, and general physiological metabolism is vigorous
And the relatively low tissue of differentiation degree is conducive to the induction of somatic embryo.
For required auxin, the basic element of cell division, gibberellin, abscisic acid in culture medium, reproducibility nitrogen salt and external source ammonia
Other factors, their action direction, effect degrees such as base acid, pH value are different from.Induce plant somatic embryo occur because
Element is numerous, but their effect degree is not quite similar, and the various factors are accurately regulated and controled by transcribing with translation skill complexity, are made
The related gene of somatic embryo occur is temporally and spatially able to selectively activate and express, and only various factors cooperation makes
Used time could fast and efficiently induce somatic embryo.
Inventor considers the various factors for influencing Chinese scholar tree cotyledon somatic embryo occur, around raising regeneration rate, increases
The purpose of budding, raising plantlet of transplant survival rate, a set of culture medium of the present invention is devised, which trains compared to others
Foster base regeneration rate is high, budding is more, and plantlet of transplant survival rate can reach more than 90%, and seedling early growth is healthy and strong, not only can effectively solve
Good seed germplasm degenerate problem can also provide a preferable receptor system for Chinese scholar tree genetic transformation or mutation breeding
Using the present invention culture medium breed Chinese scholar tree seedling, can save first establish Chinese scholar tree soon numerous regenerating system the step for,
Greatly save the time.
Description of the drawings
Fig. 1 cotyledons scratch mouth；
Fig. 2 forms embryo callus；
Fig. 3 callus inductions go out Multiple Buds.
Specific embodiment
The invention will be further described with embodiment below in conjunction with the accompanying drawings.
A formulas are combined with B5 medium for improved MS, including in MS culture mediums grand nutrition element, micronutrient
The organic reagent of element and B5 medium.
MS culture mediums include grand nutrition element, micronutrient element and organic reagent.
Component concentration corresponding with its of the grand nutrition element of the MS culture mediums is as follows：Potassium nitrate 1900mg/L, nitre
Sour ammonium 1650mg/L, epsom salt 370mg/L, anhydrous potassium dihydrogenphosphate 170mg/L, calcium chloride dihydrate 440mg/L, ethylenediamine
Tetraacethyl disodium 37.3mg/L, ferrous sulfate heptahydrate 27.8mg/L.
Component concentration corresponding with its of the micronutrient element of the MS culture mediums is as follows：Four water manganese sulfate 22.3mg/
L, zinc sulfate 8.6mg/L, boric acid 6.2mg/L, potassium iodide 0.83mg/L, sodium molybdate 0.25mg/L, copper sulphate 0.025mg/L, chlorine
Change cobalt 0.025mg/L.
It is preferred that：Component concentration corresponding with its of the A formulas B5 organic reagents is as follows：Thiamine hydrochloride 100mg/L,
Niacin 10mg/L, puridoxine hydrochloride 10mg/L, inositol 1000mg/L.
It is preferred that：Component concentration corresponding with its of the organic reagent of the MS culture mediums is as follows：Glycine 20mg/L, hydrochloric acid
Thiamine 10mg/L, niacin 1.0mg/L, puridoxine hydrochloride 1.0mg/L, inositol 100mg/L
Embodiment one：
Robust growth, the Chinese scholar tree mature seed raw then of no disease and pests harm are chosen, fruit pod is removed, is carefully cleaned with liquid detergent
Afterwards, when flowing water flushing 1 is small, excessive moisture outside filter paper exhaustion seed prepares disinfection.
Examination material is placed on superclean bench, with 70% alcohol disinfecting 30s, aseptic water washing 3 times, then with 0.1% liter
Mercury sterilizing 8min, aseptic water washing 5 times with sterilizing filter paper suck dry moisture, gently peel off kind of a skin, by son with knife blade and tweezers
Leaf takes out.Two panels cotyledon tweezers and blade are gently separated, retain plumule, then by that more flat face-down, convex surface of cotyledon
It is placed on upward in culture dish, 3~4 wounds is drawn perpendicular to cotyledon convex surface face master pulse with knife blade, be careful not to draw to son
Limb edge.
Above-mentioned cotyledon is seeded in body embryo inducing culture (A+3.0mg/L 2,4-D+0.5mg/L BA+0.5mg/L NAA
+ 0.1mg/L TDZ+0.5mg/L glutamine+0.5mg/L CH (caseinhydrolysate)+5.0g/L agar+30g/L sucrose, pH value
5.8) in.Light culture 30d induces the formation of body embryo.Start, blade is slowly hardened thickening, gradually forms callus, passes through
The light culture of 45d, explant become light yellow glutinous shape callus.
Above-mentioned callus is transferred to adventitious bud induction culture base (A+0.3mg/L BA+0.1mg/L NAA+0.5mg/L
Glutamine+0.5mg/L CH (caseinhydrolysate)+5.0g/L agar+30g/L sucrose, pH value 5.8) in.It is put into temperature daytime
25 ± 2 DEG C, 20 ± 2 DEG C, 1000~1500lx of intensity of illumination of night is cultivated in the culturing room of light application time 16h/d, callus
Gradually multiplication.Cultivate 98d in the environment, centre with culture medium subculture of the same race twice.Subculture once after, callus occur it is green
The bud point of color.It is further continued for subculture once, green bud point gradually grows up to the seedling of 1~2cm, forms Multiple Buds seedling, will grow thickly
Bud seedling is cut into callus and is transferred to test tube seedling proliferated culture medium progress fast culture, the test tube seedling proliferation culture medium formula soon:
MS+1.0~2.0mg/L BA (6-benzyladenine)+1.0~2.0mg/LIBA (indolebutyric acid)+5.0g/L agar+30g/L sugarcanes
Sugar, pH value 5.8~6.0.
The seedling of growing thickly of Multiplying culture from base portion is cut, is transferred to strong seedling culture base (MS+5.0g/L agar+30g/L sugarcanes
Sugar, pH value 5.8) in, carry out strong seedling culture.Culture environment is same as above.About cultivate 25d, seedling length to 3~4cm high.
Seedling is cut from base portion, the blade of removal seedling the latter half, insertion root media (1/2MS+0.1mg/L
IBA+0.05mg/L NAA+5.0g/L agar+20g/L sucrose, pH value 5.8) in.12d or so, seedling base portion have the root restriction long
Go out, 15d has new root to grow, when new root long is to more than 0.5cm, you can acclimatization and transplants.
Test tube seedling sealed membrane tied rope is unclamped first, 1~2d is adapted in culturing room, is transferred in the greenhouse of obscurity 50%
(not divulge information), removes sealed membrane, 2~3d of hardening, independently after folding, gently can be pressed from both sides out seedling with tweezers after seedling stomata,
Clear water washes away the remaining culture medium of base portion, is transplanted in the flowerpot equipped with mixing seedling medium, utilizes intermittent spraying device or people
Work water spray keeps relative humidity after so taming 5~10d of hardening, to grow new root, sprouting eruption gradually decreases more than 80%
Irrigation times simultaneously carry out Routine Management.
The ingredient of flowerpot mixing seedling medium using volume basis as：Perlite:Sawdust:Fertile soil=30:30:40.
Developmental state in the reproductive process is as shown in Figure 1-Figure 3.
One experimental data of example：
Embodiment two：
Robust growth, the Chinese scholar tree mature seed raw then of no disease and pests harm are chosen, fruit pod is removed, is carefully cleaned with liquid detergent
Afterwards, when flowing water flushing 1 is small, excessive moisture outside filter paper exhaustion seed prepares disinfection.
Examination material is placed on superclean bench, with 70% alcohol disinfecting 30s, aseptic water washing 3 times, then with 0.1% liter
With sterilizing filter paper suck dry moisture, kind of a skin is gently peelled off with knife blade and tweezers for mercury sterilizing 10min, aseptic water washing 5 times, will
Cotyledon takes out.Two panels cotyledon tweezers and blade are gently separated, retain plumule, it is then by more flat that of cotyledon down, convex
It is placed on up in culture dish, draws 3~4 wounds perpendicular to cotyledon convex surface face master pulse with knife blade, be careful not to draw extremely
Cotyledon margin.
Above-mentioned cotyledon is seeded in body embryo inducing culture (A+4.0mg/L 2,4-D+0.7mg/L BA+0.7mg/L NAA
+ 0.4mg/L TDZ+0.8mg/L- glutamine+0.8mg/L CH (caseinhydrolysate) ++ 5.0g/L agar+30g/L sucrose,
PH value 5.8) in.By the light culture of 41d, the blade of explant from green becomes light yellow glutinous shape callus.
Above-mentioned callus is transferred to adventitious bud induction culture base (A+0.4mg/L BA+0.2mg/L NAA+0.8mg/L
Glutamine+0.8mg/L CH (caseinhydrolysate)+5.0g/L agar+30g/L sucrose, pH value 5.8) in.It is put into temperature daytime
25 ± 2 DEG C, 20 ± 2 DEG C, 1000~1500lx of intensity of illumination of night is cultivated in the culturing room of light application time 16h/d, callus
Gradually multiplication.Cultivate 93d in the environment, centre with culture medium subculture of the same race twice.Subculture once after, callus occur it is green
The bud point of color.It is further continued for subculture once, green bud point gradually grows up to the seedling of 1~2cm, forms Multiple Buds seedling, will grow thickly
Bud seedling is cut into callus and is transferred to test tube seedling proliferated culture medium progress fast culture, the test tube seedling proliferation culture medium formula soon:
MS+1.0~2.0mg/L BA (6-benzyladenine)+1.0~2.0mg/LIBA (indolebutyric acid)+5.0g/L agar+30g/L sugarcanes
Sugar, pH value 5.8~6.0.
The seedling of growing thickly of Multiplying culture from base portion is cut, is transferred to strong seedling culture base (MS+5.0g/L agar+30g/L sugarcanes
Sugar, pH value 5.8) in, carry out strong seedling culture.Culture environment is same as above.About cultivate 25d, seedling length to 3~4cm high.
Seedling is cut from base portion, the blade of removal seedling the latter half, insertion root media (1/2MS+0.3mg/L
IBA+0.03mg/L NAA+5.0g/L agar+20g/L sucrose, pH value 5.8) in.10d or so, seedling base portion have the root restriction long
Go out, 13d has new root to grow, when new root long is to more than 0.5cm, you can acclimatization and transplants.
Test tube seedling sealed membrane tied rope is unclamped first, 1~2d is adapted in culturing room, is transferred in the greenhouse of obscurity 50%
(not divulge information), removes bottle cap, 2~3d of hardening, independently after folding, gently can be pressed from both sides out seedling with tweezers, clearly after seedling stomata
The remaining culture medium of base portion is removed in washing, is transplanted in the flowerpot equipped with mixing seedling medium, using intermittent spraying device or manually
Water spray keeps relative humidity after so taming 5~10d of hardening, to grow new root, sprouting eruption is gradually decreased and poured more than 80%
Waterside number simultaneously carries out Routine Management.
In flowerpot mix seedling medium ingredient using volume basis as：Perlite:Sawdust:Fertile soil=30:30:40.
According to the result such as following table of the embodiment：
Embodiment three：
Robust growth, the Chinese scholar tree mature seed raw then of no disease and pests harm are chosen, fruit pod is removed, is carefully cleaned with liquid detergent
Afterwards, when flowing water flushing 1 is small, excessive moisture outside filter paper exhaustion seed prepares disinfection.
Examination material is placed on superclean bench, with 70% alcohol disinfecting 30s, aseptic water washing 3 times, then with 0.1% liter
With sterilizing filter paper suck dry moisture, kind of a skin is gently peelled off with knife blade and tweezers for mercury sterilizing 12min, aseptic water washing 5 times, will
Cotyledon takes out.Two panels cotyledon tweezers and blade are gently separated, retain plumule, it is then by more flat that of cotyledon down, convex
It is placed on up in culture dish, draws 3~4 wounds perpendicular to cotyledon convex surface face master pulse with knife blade, be careful not to draw extremely
Cotyledon margin.
Above-mentioned cotyledon is seeded in body embryo inducing culture (A+5.0mg/L 2,4-D+1.0mg/L BA+1.0mg/L NAA
+ 0.5mg/L TDZ+1.0mg/L glutamine+1.0mg/L CH (caseinhydrolysate)+5.0g/L agar+30g/L sucrose, pH value
5.8) in.By the light culture of 35d, explant becomes light yellow glutinous shape callus.
Above-mentioned callus is transferred to adventitious bud induction culture base (A+0.5mg/L BA+0.1mg/L NAA+1.0mg/L
Glutamine+1.0mg/L CH (caseinhydrolysate)+5.0g/L agar+30g/L sucrose, pH value 5.8) in.It is put into temperature daytime
25 ± 2 DEG C, 20 ± 2 DEG C, 1000~1500lx of intensity of illumination of night is cultivated in the culturing room of light application time 16h/d.In this environment
Middle culture 85d, centre with culture medium subculture of the same race twice.Subculture once after, callus occur green bud point.Be further continued for after
In generation, once green bud point gradually grew up to the seedling of 1~2cm, forms Multiple Buds seedling, Multiple Buds seedling is cut into callus fast-turn construction
It is connected to test tube seedling proliferated culture medium and carries out fast culture, the test tube seedling proliferation culture medium formula:MS+1.0~2.0mg/L BA
(6-benzyladenine)+1.0~2.0mg/LIBA (indolebutyric acid)+5.0g/L agar+30g/L sucrose, pH value 5.8~6.0.
The seedling of growing thickly of Multiplying culture from base portion is cut, is transferred to strong seedling culture base (MS+5.0g/L agar+30g/L sugarcanes
Sugar, pH value 5.8) in, carry out strong seedling culture.Culture environment is same as above.About cultivate 25d, seedling length to 3~4cm high.
Seedling is cut from base portion, the blade of removal seedling the latter half, insertion root media (1/2MS+0.5mg/L
IBA+0.05mg/L NAA+5.0g/L agar+20g/L sucrose, pH value 5.8) in.8d or so, seedling base portion have root restriction to grow,
11d has new root to grow, when new root long is to more than 0.5cm, you can acclimatization and transplants.
Test tube seedling sealed membrane tied rope is unclamped first, 1~2d is adapted in culturing room, is transferred in the greenhouse of obscurity 50%
(not divulge information), removes sealed membrane, 2~3d of hardening, independently after folding, gently can be pressed from both sides out seedling with tweezers after seedling stomata,
Clear water washes away the remaining culture medium of base portion, is transplanted in the flowerpot equipped with mixing seedling medium, utilizes intermittent spraying device or people
Work water spray keeps relative humidity after so taming 5~10d of hardening, to grow new root, sprouting eruption gradually decreases more than 80%
Irrigation times simultaneously carry out Routine Management.
In flowerpot mix seedling medium ingredient using volume basis as：Perlite:Sawdust:Fertile soil=30:30:40.
According to the result such as following table of the embodiment：
Above-mentioned, although the foregoing specific embodiments of the present invention is described with reference to the accompanying drawings, not protects model to the present invention
The limitation enclosed, those skilled in the art should understand that, based on the technical solutions of the present invention, those skilled in the art are not
Need to make the creative labor the various modifications or changes that can be made still within protection scope of the present invention.
Claims (3)
1. a kind of complete set of culture medium for inducing Chinese scholar tree cotyledon somatic embryo, it is characterized in that：Including
Somatic embryo inducement culture medium：A+3.0~5.0mg/L 2,4-D+0.5~1.0mg/L BA+0.5~1.0mg/L NAA+
0.1~0.5mg/L TDZ+0.5~1.0mg/L glutamine+0.5~1.0mg/L CH+5.0g/L agar+30g/L sucrose, pH
Value 5.8~6.0；
Adventitious bud induction culture base：A+0.1~0.5mg/L BA+0.1~0.5mg/L NAA+0.5~1.0mg/L glutamine+
0.5~1.0mg/L CH+5.0g/L agar+30g/L sucrose, pH value 5.8~6.0；
Test tube seedling proliferation culture medium formula:MS+1.0~2.0mg/L BA+1.0~2.0mg/L IBA+5.0g/L agar+30g/L
Sucrose, pH value 5.8~6.0；
Strong seedling culture base：MS+5.0g/L agar+30g/L sucrose, pH value 5.8~6.0；
Root media：1/2MS+0.1~0.5mg/L IBA+0~0.05mg/L NAA+5.0g/L agar+20g/L sucrose, pH
Value 5.8~6.0, the 1/2MS are that MS full doses halve；
After the formula of the A includes the grand nutrition element in MS culture mediums, the micronutrient element in MS culture mediums and improvement
B5 medium organic reagent；
The component of the organic reagent of B5 medium after improvement concentration corresponding with its is as follows：Thiamine hydrochloride 100mg/L,
Niacin 10mg/L, puridoxine hydrochloride 10mg/L, inositol 1000mg/L.
2. complete set of culture medium as described in claim 1, it is characterized in that：The component of the grand nutrition element of the MS culture mediums and
Its corresponding concentration is as follows：Potassium nitrate 1900mg/L, ammonium nitrate 1650mg/L, epsom salt 370mg/L, anhydrous phosphoric acid dihydro
Potassium 170mg/L, calcium chloride dihydrate 440mg/L, disodium ethylene diamine tetraacetate 37.3mg/L, ferrous sulfate heptahydrate 27.8mg/L.
3. complete set of culture medium as described in claim 1, it is characterized in that：The component of the micronutrient element of the MS culture mediums and
Its corresponding concentration is as follows：Four water manganese sulfate 22.3mg/L, zinc sulfate 8.6mg/L, boric acid 6.2mg/L, potassium iodide 0.83mg/L,
Sodium molybdate 0.25mg/L, copper sulphate 0.025mg/L, cobalt chloride 0.025mg/L.
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