CN106234226A - A kind of in-vitro culture method of Ligularia rumicifolia (Drumm.) S.W. Liu. - Google Patents
A kind of in-vitro culture method of Ligularia rumicifolia (Drumm.) S.W. Liu. Download PDFInfo
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- CN106234226A CN106234226A CN201610710556.9A CN201610710556A CN106234226A CN 106234226 A CN106234226 A CN 106234226A CN 201610710556 A CN201610710556 A CN 201610710556A CN 106234226 A CN106234226 A CN 106234226A
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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
- A01H4/008—Methods for regeneration to complete plants
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Abstract
The invention discloses the in-vitro culture method of a kind of Ligularia rumicifolia (Drumm.) S.W. Liu..The in-vitro culture method that the present invention provides comprises the steps (a1) and (a2): (a1) takes the adventitious bud of Ligularia rumicifolia (Drumm.) S.W. Liu., strengthens at adventitious bud and cultivates in bud culture medium；(a2), after completing step (a1), take adventitious bud, root media is cultivated, obtain regrowth.Utilize Ligularia rumicifolia (Drumm.) S.W. Liu. in-vitro culture method of the present invention can be successfully obtained Ligularia rumicifolia (Drumm.) S.W. Liu. regeneration plant, regeneration plant is transplanted, survival rate 90%.The present invention, for effectively protecting and utilize wild Ligularia rumicifolia (Drumm.) S.W. Liu. resource significant, may advantageously facilitate the large-scale production of Ligularia rumicifolia (Drumm.) S.W. Liu. simultaneously.
Description
Technical field
The present invention relates to the in-vitro culture method of a kind of Ligularia rumicifolia (Drumm.) S.W. Liu..
Background technology
Ligularia rumicifolia (Drumm.) S.W. Liu. [Ligularia rumicifolia (Drumm.) S.W.Liu] is that Compositae Ligularia From Sichuan, China perennial herb is planted
Thing.It is distributed in China's Southeastern Tibet to northeast, is born in the lakeside of height above sea level 3700-4500m, sylvan life, shrubbery and hillside, Buddhist nun
Bo Er also has distribution.
Ligularia rumicifolia (Drumm.) S.W. Liu. is the folk medicinal plants of Tibet region, has the highest medical value.In " Chinese herbal medicine is commonly used in Tibet "
Record, Ligularia rumicifolia (Drumm.) S.W. Liu., hide name Xiao, be used as medicine with root, bitter in the mouth, warm in nature.Described in Tibetan medicine grand ceremony " Jingzhubencao ", day Xiao Gongxiao is
Telling Baconic, red bar closes complication, changes cool in nature, and more skin ulcer, removing summer-heat is difficult to the old plague fever dissipated." Chinese medicine resource will will " record, hides
Farfugium kaemferi may be used for treating cough due to wind and cold, bronchitis, pulmonary tuberculosis hemoptysis, pharyngolaryngitis.
Summary of the invention
It is an object of the invention to provide the in-vitro culture method of a kind of Ligularia rumicifolia (Drumm.) S.W. Liu..
The invention provides the in-vitro culture method of a kind of Ligularia rumicifolia (Drumm.) S.W. Liu., comprise the steps (a1) and (a2):
(a1) take the adventitious bud of Ligularia rumicifolia (Drumm.) S.W. Liu., strengthen at adventitious bud and cultivate in bud culture medium；
(a2), after completing step (a1), take adventitious bud, root media is cultivated, obtain regrowth.
The preparation method of described adventitious bud is following (b1) or (b2):
(b1) take the organogenesis of Ligularia rumicifolia (Drumm.) S.W. Liu., adventitious bud induction culture base (adventitious bud induction culture base A) is trained
Support, obtain adventitious bud；
(b2) take the organogenesis of Ligularia rumicifolia (Drumm.) S.W. Liu., callus inducing medium is cultivated, obtain callus；Take
Callus, cultivates on adventitious bud induction culture base (adventitious bud induction culture base B), obtains adventitious bud.
The preparation method of the organogenesis of described Ligularia rumicifolia (Drumm.) S.W. Liu. is as follows: take the seed of Ligularia rumicifolia (Drumm.) S.W. Liu., on seed germination medium
Cultivate, obtain aseptic seedling, take the cotyledon of aseptic seedling, prepare organogenesis.
The cultivation cycle of described step (a1) is 40 days.
The cultivation cycle of described step (a2) is 30 days.
The cultivation cycle of described step (b1) is 40 days.
In described step (b2), the cultivation cycle of " cultivating on callus inducing medium " is 30 days.
The cultivation " cultivated on adventitious bud induction culture base (adventitious bud induction culture base B) " in described step (b2)
Cycle is 30-40 days.
The size of described organogenesis is 0.5 × 0.5cm.
The condition of culture cultivated described in any of the above is cultivation temperature 25 ± 2 DEG C, and light application time is 12h/ days, intensity of illumination
For 30-40mmol m-2·s-1。
The present invention also protects a kind of test kit for Ligularia rumicifolia (Drumm.) S.W. Liu. tissue culture, for including following (e1) or (e2) or (e3)
Test kit:
(e1) adventitious bud strengthens bud culture medium and root media；
(e2) adventitious bud strengthens bud culture medium, root media and adventitious bud induction culture base (adventitious bud induction culture base A)；
(e3) adventitious bud strengthens bud culture medium, root media, callus inducing medium and adventitious bud induction culture base
(adventitious bud induction culture base B).
Seed germination medium described in any of the above contains 0.01-1.0mg/L 6-BA.
Seed germination medium described in any of the above specifically can contain 1.0mg/L 6-BA.
Adventitious bud induction culture base described in any of the above (adventitious bud induction culture base A) containing 1.0-3.0mg/L 6-BA and
0.2-4.0mg/L NAA。
Adventitious bud induction culture base described in any of the above (adventitious bud induction culture base A) also can contain 1.0-4.0mg/L6-
BA and 0.5-4.0mg/L 2,4-D.
Adventitious bud induction culture base described in any of the above (adventitious bud induction culture base A) specifically can contain 3.0mg/L 6-BA
With 0.8mg/L NAA.
Adventitious bud induction culture base described in any of the above (adventitious bud induction culture base A) specifically can contain 2.0mg/L 6-BA
With 4.0mg/L 2,4-D.
Callus inducing medium described in any of the above contains 1.0-4.0mg/L NAA.
Callus inducing medium described in any of the above specifically can contain 4.0mg/L NAA.
Adventitious bud induction culture base described in any of the above (adventitious bud induction culture base B) containing 1.0-3.0mg/L TDZ and
0.5-2.0mg/L NAA。
Adventitious bud induction culture base described in any of the above (adventitious bud induction culture base B) also can contain 1.0-4.0mg/L6-
BA and 0.1-2.0mg/L NAA.
Adventitious bud induction culture base described in any of the above (adventitious bud induction culture base B) specifically can contain 3.0mg/L TDZ
With 0.5mg/L NAA.
Adventitious bud induction culture base described in any of the above (adventitious bud induction culture base B) specifically can contain 2.0mg/L 6-BA
With 0.5mg/L NAA.
Bud culture medium of strengthening adventitious bud described in any of the above contains 1.5mg/L 6-BA and 0.1mg/L NAA.
Root media described in any of the above contains 0.5-2.0mg/L IAA.
Root media described in any of the above specifically can contain 0.5mg/L IAA.
Seed germination medium described in any of the above is the MS solid medium containing 0.01-1.0mg/L 6-BA.
Seed germination medium described in any of the above concretely contains the MS solid medium of 1.0mg/L 6-BA.
Adventitious bud induction culture base described in any of the above (adventitious bud induction culture base A) is containing 1.0-3.0mg/L 6-BA
MS solid medium with 0.2-4.0mg/L NAA.
Adventitious bud induction culture base described in any of the above (adventitious bud induction culture base A) can be also containing 1.0-4.0mg/L
The MS solid medium of 6-BA and 0.5-4.0mg/L 2,4-D.
Adventitious bud induction culture base described in any of the above (adventitious bud induction culture base A) concretely contains 3.0mg/L 6-
The MS solid medium of BA and 0.8mg/L NAA.
Adventitious bud induction culture base described in any of the above (adventitious bud induction culture base A) concretely contains 2.0mg/L 6-
The MS solid medium of RA and 4.0mg/L 2,4-D.
Callus inducing medium described in any of the above is the MS solid medium containing 1.0-4.0mg/L NAA.
Callus inducing medium described in any of the above concretely contains the MS solid medium of 4.0mg/L NAA.
Adventitious bud induction culture base described in any of the above (adventitious bud induction culture base B) is containing 1.0-3.0mg/L TDZ
MS solid medium with 0.5-2.0mg/L NAA.
Adventitious bud induction culture base described in any of the above (adventitious bud induction culture base B) can be also containing 1.0-4.0mg/L
The MS solid medium of 6-BA and 0.1-2.0mg/L NAA.
Adventitious bud induction culture base described in any of the above (adventitious bud induction culture base B) concretely contains 3.0mg/L
The MS solid medium of TDZ and 0.5mg/L NAA.
Adventitious bud induction culture base described in any of the above (adventitious bud induction culture base B) concretely contains 2.0mg/L 6-
The MS solid medium of BA and 0.5mg/L NAA.
It is the MS solid training containing 1.5mg/L 6-BA and 0.1mg/L NAA that adventitious bud described in any of the above strengthens bud culture medium
Support base.
Root media described in any of the above is the 1/2MS culture medium containing 0.5-2.0mg/L IAA.
Root media described in any of the above concretely contains the 1/2MS culture medium of 0.5mg/L IAA.
The present invention also protects described in-vitro culture method or the application in Ligularia rumicifolia (Drumm.) S.W. Liu. expanding propagation of the described test kit.
MS solid medium described in any of the above is the MS solid medium shown in detailed description of the invention.
1/2MS culture medium described in any of the above is the 1/2MS culture medium shown in detailed description of the invention.
The present invention carries out Study on tissue culture using the cotyledon of Ligularia rumicifolia (Drumm.) S.W. Liu. aseptic seedling as outer implant, establish Ligularia rumicifolia (Drumm.) S.W. Liu. from
Body cultivates regenerating system.Plant it is demonstrated experimentally that utilize Ligularia rumicifolia (Drumm.) S.W. Liu. in-vitro culture method of the present invention can be successfully obtained Ligularia rumicifolia (Drumm.) S.W. Liu. regeneration
Strain, transplants regeneration plant, survival rate 90%.The present invention has for effectively protecting and utilize wild Ligularia rumicifolia (Drumm.) S.W. Liu. resource
Significance, may advantageously facilitate the large-scale production of Ligularia rumicifolia (Drumm.) S.W. Liu. simultaneously.
Detailed description of the invention
Below example facilitates a better understanding of the present invention, but does not limit the present invention.Experiment in following embodiment
Method, if no special instructions, is conventional method.Test material used in following embodiment, if no special instructions, is certainly
Routine biochemistry reagent shop is commercially available.Quantitative test in following example, is respectively provided with three times and repeats experiment, and result is made even
Average.Condition of culture in embodiment is cultivation temperature 25 ± 2 DEG C, and light application time is 12h/ days, and intensity of illumination is 30-
40mmol·m-2·s-1。
MS solid medium: CaCl2·2H2O 440mg, KH2PO4170mg, MgSO4·7H2O 370mg, NH4NO3
1650mg, KNO31900mg, KI 0.83mg, CoCl2·6H2O 0.025mg, H3BO36.2mg, Na2MoO4·2H2O
0.25mg, MnSO4·4H2O 22.3mg, CuSO4·5H2O 0.025mg, ZnSO4·7H2O 8.6mg, Na2-EDTA·2H2O
37.3mg, FeSO4·7H2O 27.8mg, VB1 0.1mg, VB6 0.5mg, nicotinic acid 0.5mg, inositol 100mg, glycine 2mg, sugarcane
Sugar 30g, agar 7g, it is 5.8 that deionized water adds to 1L, pH.121 DEG C of autoclaving 20min.
1/2MS culture medium: CaCl2·2H2O 220mg, KH2PO485mg, MgSO4·7H2O 185mg, NH4NO3
825mg, KNO3950mg, KI 0.83mg, CoCl2·6H2O 0.025mg, H3BO36.2mg, Na2MoO4·2H2O 0.25mg,
MnSO4·4H2O 22.3mg, CuSO4·5H2O 0.025mg, ZnSO4·7H2O 8.6mg, Na2-EDTA·2H2O 37.3mg,
FeSO4·7H2O 27.8mg, VB1 0.1mg, VB6 0.5mg, nicotinic acid 0.5mg, inositol 100mg, glycine 2mg, sucrose 30g,
Agar 7g, it is 5.8 that deionized water adds to 1L, pH.121 DEG C of autoclaving 20min.
Embodiment 1, the isolated culture of Ligularia rumicifolia (Drumm.) S.W. Liu.
One, the collection of Ligularia rumicifolia (Drumm.) S.W. Liu. and disinfecting
Ligularia rumicifolia (Drumm.) S.W. Liu. in July, 2013 pick up from Milin County, Tibet south she ditch height above sea level 3104m the open place of sylvan life, and according to " in
State's flora " the morphological characteristic standard of described Ligularia rumicifolia (Drumm.) S.W. Liu. confirmed, in kind it is consistent with title.By the fruit of Ligularia rumicifolia (Drumm.) S.W. Liu. in room
Temperature is lower chooses the fruit of mature and plump with distilled water immersion 24h (distilled water that every 8h more renews) after 24h, after peelling off peel, and will
Ligularia rumicifolia (Drumm.) S.W. Liu. seed is first with the ethanol surface sterilization 20s of 75% (volume fraction) on superclean bench, then (quality is divided with 0.1%
Number) mercuric chloride sterilization 2min, with aseptic water washing 6 times.
Two, the acquisition of aseptic seedling
Take the seed after step one processes, be inoculated in respectively containing 0mg/L, 0.01mg/L, 0.1mg/L or 1.0mg/L 6-BA
MS solid medium in cultivate 30 days, obtain aseptic seedling.
Statistics seed germination rate, is inoculated in the seed germination of MS solid medium containing 0.01mg/L-0.1mg/L 6-BA
Rate reaches 73.33%-87.33%, wherein, is inoculated in the seed germination rate of MS solid medium containing 0.1mg/L 6-BA
Height, reaches 87.33%.When in MS solid medium without 6-RA, seed germination rate is 76.67%, when MS solid culture
When in base, the concentration of 6-BA is 1.0mg/L, seed germination rate is 68.00%.
Three, the induction of callus
For step 2 is cultivated in the MS solid medium containing 0.1mg/L 6-BA the aseptic seedling obtained for 30 days, take son
Leaf, is cut into the stripping and slicing of about 0.5 × 0.5cm, be inoculated in containing variable concentrations NAA (0,0.5,1.0,2.0,3.0,4.0,
MS solid medium 5.0mg/L) is cultivated 30 days, callus induction.
The inductivity of record callus, result shows: the NAA of higher concentration is conducive to induction cotyledon to produce wound healing group
Knit.When the NAA concentration added is 3.0-4.0mg/L, the inductivity of callus is 51.33%-54.67%；NAA concentration is
Cotyledon can also be induced during 1.0-2.0mg/L to produce callus, and inductivity is 36.00%-38.67%；When NAA concentration raises
During to 5.0mg/L, callus induction rate is only 2.00%；Can not induce more when being 0.5mg/L without NAA or NAA concentration
Injured tissue.According to statistical result, the Cotyledon Callus inductivity being inoculated in the MS solid medium containing 4.0mg/L NAA reaches
The highest, it is 54.67%.
Four, the induction of adventitious bud
1, step 3 will be cultivated in the MS solid medium containing 4.0mg/L NAA the callus inoculation obtained for 30 days
In containing variable concentrations TDZ (1.0,2.0,3.0,4.0mg/L) and NAA (0.5,1.0,2.0,4.0,6.0mg/L) MS solid
Culture medium is cultivated.Bottle-green bud point occurred after 20 days, after 30 days, generates adventitious bud.Statistics adventitious bud induction frequency, knot
Fruit display: each concentration group all can success evoking adventive bud.When the TDZ concentration in MS solid medium is 1.0mg/L-3.0mg/L,
When NAA concentration is 0.5mg/L-2.0mg/L, adventitious bud induction frequency is higher, for 29.33%-49.42%；Wherein, TDZ concentration is
When 2.0mg/L, NAA concentration is 0.5mg/L, adventitious bud induction frequency reaches 45.60%；TDZ concentration is that 3.0mg/L, NAA concentration is
During 0.5mg/L and 1.0mg/L, adventitious bud induction frequency respectively reaches 49.42% and 48.00%.When TDZ concentration is increased to 4.0mg/
During L, adventitious bud induction frequency is relatively low, is only inoculated in the callus adventitious bud induction frequency of MS solid medium containing 0.5mg/LNAA
Can reach 37.33%, the inductivity of remaining concentration group is below 20%.When the concentration of NAA is increased to 4.0mg/L, each concentration
The adventitious bud induction frequency of group is below 25%.According to statistical result, it is inoculated in the MS containing 3.0mg/L TDZ, 0.5mg/L NAA solid
The callus adventitious bud induction frequency of body culture medium is the highest.
It is inoculated in step 3 is cultivated in the MS solid medium containing 4.0mg/L NAA the callus obtained for 30 days
Containing variable concentrations 6-BA (1.0,2.0,3.0,4.0mg/L) and NAA (0.1,0.5,1.0,2.0mg/L) MS solid medium
After middle cultivation 20 days, bottle-green bud point occurs, after cultivating 30 days, carry out successive transfer culture, after 10 days, start to generate adventitious bud.
Statistics adventitious bud induction frequency, result show: each concentration group all can successful evoking adventive bud, and inductivity is all higher than 20%.Work as training
Supporting 6-BA concentration in base is 1.0mg/L-3.0mg/L, NAA concentration when being 0.5mg/L, and adventitious bud induction frequency is 55.50-
69.56%.When in culture medium 6-BA concentration be 2.0-4.0mg/L, NAA concentration be 1.0mg/L time, adventitious bud induction frequency also up to
Arrive as 51.54%-63.25%.According to statistical result, it is inoculated in the MS solid training containing 2.0mg/L 6-RA and 0.5mg/L NAA
The callus adventitious bud induction frequency supporting base reaches the highest, is 69.56%, and every piece of callus can differentiate about 3 not
Normal bud, adventitious bud is green, and form is normal.
Reaching a conclusion based on the above results, the optimal medium of callus induction differentiation adventitious bud is containing 2.0mg/L
The MS solid medium of 6-BA and 0.5mg/L NAA.
3, for step 2 is cultivated in the MS solid medium containing 0.1mg/L 6-BA the aseptic seedling obtained for 30 days, take
Cotyledon, is cut into the stripping and slicing of about 0.5 × 0.5cm, be seeded in containing variable concentrations 6-BA (1.0,2.0,3.0,4.0mg/L) and 2,
4-D (0.5,1.0,2.0,4.0mg/L) MS solid medium in, after cultivating 20 days, cotyledon incision forms projection, cultivates 40
After it, projection is divided into adventitious bud.Statistics adventitious bud induction frequency, result shows: each concentration group all can success evoking adventive bud.When
When in culture medium, the concentration of 6-BA is 1.0mg/L, adventitious bud induction frequency is on the low side；When in culture medium, the concentration of 6-BA is 2.0mg/L-
4.0mg/L, when the concentration of 2,4-D is 4.0mg/L, adventitious bud induction frequency reaches 44.95%-55.18%.According to statistical result,
The adventitious bud induction frequency being inoculated in the MS solid medium containing 2.0mg/L 6-BA and 4.0mg/L 2,4-D reaches the highest, for
55.18%.
For step 2 is cultivated in the MS solid medium containing 0.1mg/L 6-BA the aseptic seedling obtained for 30 days, take son
Leaf, is cut into the stripping and slicing of about 0.5 × 0.5cm, be seeded in containing variable concentrations 6-BA (1.0,2.0,3.0,4.0mg/L) and NAA
(0.2,0.4,0.8,1.0,2.0,4.0mg/L) MS solid medium in, cultivate the wound healing generating a small amount of white for about 15 days
Tissue, differentiates the bud point of green on callus, cultivates and be divided into adventitious bud in about 40 days.Statistics adventitious bud induction frequency,
Result shows: when in culture medium the concentration of 6-BA be 1.0mg/L-3.0mg/L, NAA concentration be 0.2mg/L-4.0mg/L time, all
Can success evoking adventive bud.When the concentration of 6-BA is increased to 4.0mg/L, it is impossible to induce adventitious bud.As 6-in culture medium
The concentration of BA is 3.0mg/L, NAA concentration when being 0.2mg/L-0.8mg/L, and adventitious bud induction frequency can reach 60.00%-
72.33%.When in culture medium 6-BA concentration be 1.0mg/L, NAA concentration be 4.0mg/L time, adventitious bud induction frequency reaches
62.17%.When in culture medium 6-BA concentration be 2.0mg/L, NAA concentration be 1.0mg/L time, adventitious bud induction frequency reaches
70.36%.According to statistical result, it is inoculated in the adventitious bud of MS solid medium containing 3.0mg/L 6-BA and 0.8mg/L NAA
Inductivity reaches the highest, is 72.33%.
Reaching a conclusion based on the above results, the optimal medium of induction cotyledon differentiation adventitious bud is containing 3.0mg/L 6-BA
MS solid medium with 0.8mg/L NAA.
Five, the strong bud of adventitious bud is cultivated
Within 40 days, obtain step 4 is cultivated in the MS solid medium containing 3.0mg/L 6-BA and 0.8mg/L NAA
Adventitious bud be inoculated into containing variable concentrations 6-BA (1.0,1.5,3.0mg/L) and NAA (0.1,0.2,0.4,0.8,1.0,2.0,
MS solid medium 4.0mg/L) carries out strong bud cultivate.Research finds, after cultivating 40 days, is inoculated in containing 1.5mg/L 6-BA
Having obvious strong sprout with the adventitious bud of the MS solid medium of 0.1mg/L NAA, increase effect, remaining group is all without the most strong bud
Effect.
Six, root culture
After step 5 being cultivated 40 days in the MS solid medium containing 1.5mg/L 6-BA and 0.1mg/L NAA not
Normal bud is seeded in MS culture medium (without any plant growth regulator) and cultivates, and does not has Adventitious root initiation after 30 days.
After step 5 being cultivated 40 days in the MS solid medium containing 1.5mg/L 6-BA and 0.1mg/L NAA not
Normal bud be inoculated into containing variable concentrations NAA (0.5,1.0,2.0,3.0,4.0mg/L) MS culture medium and containing variable concentrations IAA
(0.5,1.0,2.0,3.0,4.0mg/L) MS culture medium in cultivate 30 days, adventitious bud gradually withered death.Containing 0.5mg/
In the MS solid medium culture medium of L IAA, it is 1-2cm adventitious root that each adventitious bud grows 2-3 bar brown by hair, root length, raw
Root rate is 30%.
After step 5 being cultivated 40 days in the MS solid medium containing 1.5mg/L 6-BA and 0.1mg/L NAA not
Normal bud is seeded in 1/2MS culture medium cultivation, does not has adventitious root to generate after 30 days.
After step 5 being cultivated 40 days in the MS solid medium containing 1.5mg/L 6-BA and 0.1mg/L NAA not
Normal bud is seeded in the 1/2MS culture medium containing variable concentrations IAA (0.5,1.0,2.0mg/L) to be cultivated 30 days, bottom adventitious bud
The tip of a root, subsequently tip of a root elongation occur, grow the faint yellow adventitious root of 2-3 bar, root length 3-5cm, average rooting rate is respectively
70.00%, 32.22% and 23.33%.Therefore, the suitableeest root media of Ligularia rumicifolia (Drumm.) S.W. Liu. adventitious bud is 1/ containing 0.5mg/L IAA
2MS culture medium.
Seven, seedling exercising and transplanting
Will be equipped with step 6 taper of gained regrowth after the 1/2MS culture medium culturing containing 0.5mg/L IAA 30 days
Bottle closure film was opened, the sterilizing room regrowth seedling exercising to taking root 1 week.Choose blade compared with big, petiole is sturdy, grow fine again
Raw Seedling takes out, and carefully washes away the culture medium of root, reduces the injury to root system.Regrowth is transplanted to peat soil: perlite: husky
In the mixed-matrix of=4: 3: 2 compositions, it is placed in the environment of relative humidity 80%-90%, temperature 20-25 DEG C cultivation 30 days, becomes
Motility rate reaches 90%.
Claims (10)
1. an in-vitro culture method for Ligularia rumicifolia (Drumm.) S.W. Liu., comprises the steps (a1) and (a2):
(a1) take the adventitious bud of Ligularia rumicifolia (Drumm.) S.W. Liu., strengthen at adventitious bud and cultivate in bud culture medium；
(a2), after completing step (a1), take adventitious bud, root media is cultivated, obtain regrowth.
2. the method for claim 1, it is characterised in that: the preparation method of described adventitious bud is following (b1) or (b2):
(b1) take the organogenesis of Ligularia rumicifolia (Drumm.) S.W. Liu., the adventitious bud induction culture base of named adventitious bud induction culture base A is carried out
Cultivate, obtain adventitious bud；
(b2) take the organogenesis of Ligularia rumicifolia (Drumm.) S.W. Liu., callus inducing medium is cultivated, obtain callus；Take wound healing
Tissue, cultivates on the adventitious bud induction culture base of named adventitious bud induction culture base B, obtains adventitious bud.
3. method as claimed in claim 2, it is characterised in that: the preparation method of the organogenesis of described Ligularia rumicifolia (Drumm.) S.W. Liu. is as follows: take
The seed of Ligularia rumicifolia (Drumm.) S.W. Liu., cultivates on seed germination medium, obtains aseptic seedling, takes the cotyledon of aseptic seedling, prepares cotyledon and cuts
Block.
4. method as claimed in claim 3, it is characterised in that: described seed germination medium contains 0.01-1.0mg/L6-
BA。
5. the method as described in claim 2-4 is arbitrary, it is characterised in that: described adventitious bud induction culture base A contains 1.0-
3.0mg/L 6-BA and 0.2-4.0mg/L NAA or described adventitious bud induction culture base A contains 1.0-4.0mg/L6-BA and 0.5-
4.0mg/L 2,4-D；
Described callus inducing medium contains 1.0-4.0mg/L NAA；
Described adventitious bud induction culture base B contains 1.0-3.0mg/L TDZ and 0.5-2.0mg/L NAA or described Induce aerosor
Culture medium B contains 1.0-4.0mg/L 6-BA and 0.1-2.0mg/L NAA.
6. the method as described in claim 2-5 is arbitrary, it is characterised in that: described adventitious bud induction culture base A contains 3.0mg/L
6-BA and 0.8mg/L NAA or described adventitious bud induction culture base A contains 2.0mg/L 6-BA and 4.0mg/L2,4-D；
Described callus inducing medium contains 4.0mg/L NAA；
Described adventitious bud induction culture base B contains 3.0mg/L TDZ and 0.5mg/L NAA or described adventitious bud induction culture base B
Containing 2.0mg/L 6-BA and 0.5mg/L NAA.
7. the method as described in claim 1-6 is arbitrary, it is characterised in that: described adventitious bud is strengthened bud culture medium and is contained 1.5mg/L
6-BA and 0.1mg/L NAA；Described root media contains 0.5-2.0mg/L IAA.
8. the method as described in claim 1-7 is arbitrary, it is characterised in that: described adventitious bud is strengthened bud culture medium and is contained 1.5mg/L
6-BA and 0.1mg/L NAA；Described root media contains 0.5mg/L IAA.
9., for a test kit for Ligularia rumicifolia (Drumm.) S.W. Liu. tissue culture, it is to include following (e1) or (e2) or the test kit of (e3):
(e1) adventitious bud strengthens bud culture medium and root media；Bud culture medium of strengthening described adventitious bud contain 1.5mg/L 6-BA and
0.1mg/L NAA；Described root media contains 0.5-2.0mg/L IAA.
(e2) adventitious bud strengthens bud culture medium, root media and adventitious bud induction culture base A；Described adventitious bud is strengthened bud culture medium and is contained
There is 1.5mg/L 6-BA and 0.1mg/L NAA；Described root media contains 0.5-2.0mg/L IAA；Described Induce aerosor
Culture medium A contains 1.0-3.0mg/L 6-BA and 0.2-4.0mg/L NAA or described adventitious bud induction culture base A and contains 1.0-
4.0mg/L 6-BA and 0.5-4.0mg/L 2,4-D；
(e3) adventitious bud strengthens bud culture medium, root media, callus inducing medium and adventitious bud induction culture base B；Institute
State adventitious bud to strengthen bud culture medium and contain 1.5mg/L 6-BA and 0.1mg/L NAA；Described root media contains 0.5-2.0mg/L
IAA；Described callus inducing medium contains 4.0mg/L NAA；Described adventitious bud induction culture base B contains 3.0mg/L
TDZ and 0.5mg/L NAA or described adventitious bud induction culture base B contains 2.0mg/L 6-BA and 0.5mg/L NAA.
10. the arbitrary described method of claim 1-8 or the application in Ligularia rumicifolia (Drumm.) S.W. Liu. expanding propagation of the test kit described in claim 9.
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