CN110192527A - The fragrant green method for tissue culture of fragrant plant bell bell - Google Patents
The fragrant green method for tissue culture of fragrant plant bell bell Download PDFInfo
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- CN110192527A CN110192527A CN201910584522.3A CN201910584522A CN110192527A CN 110192527 A CN110192527 A CN 110192527A CN 201910584522 A CN201910584522 A CN 201910584522A CN 110192527 A CN110192527 A CN 110192527A
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- 238000000034 method Methods 0.000 title claims abstract description 22
- 239000001963 growth medium Substances 0.000 claims abstract description 40
- 230000006698 induction Effects 0.000 claims description 43
- 206010020649 Hyperkeratosis Diseases 0.000 claims description 41
- 239000002609 medium Substances 0.000 claims description 31
- 239000005631 2,4-Dichlorophenoxyacetic acid Substances 0.000 claims description 26
- 230000001939 inductive effect Effects 0.000 claims description 25
- 239000007787 solid Substances 0.000 claims description 10
- 239000012869 germination medium Substances 0.000 claims description 4
- 230000007226 seed germination Effects 0.000 claims description 4
- 239000003153 chemical reaction reagent Substances 0.000 claims description 2
- 238000002360 preparation method Methods 0.000 claims description 2
- 238000009395 breeding Methods 0.000 abstract description 2
- 230000001488 breeding effect Effects 0.000 abstract description 2
- 239000005648 plant growth regulator Substances 0.000 abstract description 2
- 238000011031 large-scale manufacturing process Methods 0.000 abstract 1
- 241000196324 Embryophyta Species 0.000 description 7
- 238000012549 training Methods 0.000 description 6
- 241000226665 Anaphalis Species 0.000 description 4
- 230000000694 effects Effects 0.000 description 4
- 239000000463 material Substances 0.000 description 4
- 239000002304 perfume Substances 0.000 description 3
- 230000035755 proliferation Effects 0.000 description 3
- 235000013599 spices Nutrition 0.000 description 3
- 238000004659 sterilization and disinfection Methods 0.000 description 3
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 125000003118 aryl group Chemical group 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 230000001965 increasing effect Effects 0.000 description 2
- 238000011081 inoculation Methods 0.000 description 2
- 238000012545 processing Methods 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 230000004083 survival effect Effects 0.000 description 2
- 241001000010 Anaphalis hancockii Species 0.000 description 1
- 241000722824 Ardisia crenata Species 0.000 description 1
- 241000208838 Asteraceae Species 0.000 description 1
- 241000579895 Chlorostilbon Species 0.000 description 1
- 241001677188 Coccus viridis Species 0.000 description 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- 206010054949 Metaplasia Diseases 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 239000010692 aromatic oil Substances 0.000 description 1
- 239000002131 composite material Substances 0.000 description 1
- 239000012141 concentrate Substances 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 239000000645 desinfectant Substances 0.000 description 1
- 230000000249 desinfective effect Effects 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- 229910052876 emerald Inorganic materials 0.000 description 1
- 239000010976 emerald Substances 0.000 description 1
- 239000000835 fiber Substances 0.000 description 1
- 230000035784 germination Effects 0.000 description 1
- 210000004907 gland Anatomy 0.000 description 1
- 241000411851 herbal medicine Species 0.000 description 1
- 238000005286 illumination Methods 0.000 description 1
- 239000007943 implant Substances 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 230000015689 metaplastic ossification Effects 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 239000003415 peat Substances 0.000 description 1
- 235000019362 perlite Nutrition 0.000 description 1
- 239000010451 perlite Substances 0.000 description 1
- 230000008929 regeneration Effects 0.000 description 1
- 238000011069 regeneration method Methods 0.000 description 1
- 239000004576 sand Substances 0.000 description 1
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- 239000008399 tap water Substances 0.000 description 1
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Classifications
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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
-
- 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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- Life Sciences & Earth Sciences (AREA)
- Developmental Biology & Embryology (AREA)
- Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Cell Biology (AREA)
- Botany (AREA)
- Environmental Sciences (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
The present invention provides a kind of fragrant fragrant green method for tissue culture of plant bell bell, and include the following steps (a1): (a1) takes the fragrant green adventitious bud of bell bell, is cultivated on root media, obtains regrowth；The root media contains 0.1-2.0mg/L IAA or 0.1-2.0mg/L IBA or 0.1-2.0mg/L NAA.The plant growth regulator that the present invention passes through addition particular types and concentration in the culture medium used in bell bell fragrant green tissue cultures each stage; so that the fragrant green breeding coefficient of bell bell is high; speed is fast; it can get a large amount of regrowths in a relatively short period of time; it is numerous to be conducive to the fragrant green expansion of bell bell, promote the fragrant green large-scale production of bell bell.
Description
Technical field
The present invention relates to a kind of fragrant green method for tissue culture of fragrant plant bell bell.
Background technique
Bell bell fragrant green (Anaphalis hancockii Maxim) is composite family (Compositae) anaphalis
(Anaphalis) plant, complete stool have aromatic odor, are important one of spice berry.The fragrant green application value of bell bell is very high,
Complete stool can extract aromatic oil；Also it can be used as ornamental plant, be a kind of natural dried flower material, with the aroma that the several years is without cease
Taste；Pharmaceutically acceptable, all herbal medicine has clearing heat and detoxicating effect.In addition, there are also the effects of desinsection.
People concentrate on to vegetalizations such as the fragrant blueness of yellow gland, Anaphalis marguuritacea, milkywhite pearleverlasting herb, fragrant blueness the research of anaphalis plant
The extraction and analysis studied point, and have no the research report in terms of tissue cultures.
Summary of the invention
The object of the present invention is to provide a kind of fragrant green method for tissue culture of fragrant plant bell bell.
The fragrant green method for tissue culture of fragrant plant bell bell provided by the present invention, includes the following steps (a1):
(a1) the fragrant green adventitious bud of bell bell is taken, is cultivated on root media, obtains regrowth；
In above method step (a1), the root media contains 0.1-2.0mg/L IAA or 0.1-2.0mg/L IBA
Or 0.1-2.0mg/LNAA, the root media can specifically contain 0.1mg/L IAA or 0.1-0.5mg/L IBA or 0.1mg/
L NAA；
(a1) in, the time of the culture can be 45d；
The root media also contains MS solid medium.
In above-mentioned method for tissue culture, the adventitious bud can be the adventitious bud being prepared by following (b1) or (b2),
Or the clump bud being prepared by following (b3) or (b4):
(b1) the fragrant green petiole explant of bell bell is taken, it is enterprising in adventitious bud induction culture base (adventitious bud induction culture base A)
Row culture, obtains adventitious bud；
(b2) the fragrant green blade explant of bell bell is taken, it is enterprising in adventitious bud induction culture base (adventitious bud induction culture base B)
Row culture, obtains adventitious bud；
(b3) the fragrant green petiole explant of bell bell is taken, is cultivated in petiole clump bud inducement cultivation base, obtains clump bud；
(b4) the fragrant green blade explant of bell bell is taken, is cultivated in blade clump bud inducement cultivation base, obtains clump bud；
In (b1), adventitious bud induction culture base A contains 1.0-4.0mg/L TDZ, the adventitious bud induction culture base A
2.0mg/L TDZ can specifically be contained；
Or, adventitious bud induction culture base A contains 1.0-4.0mg/L KT, the adventitious bud induction culture base A can specifically contain
There is 3.0mg/L KT；
The adventitious bud induction culture base A also contains MS solid medium.
In (b1), the time of the culture can be 45d.
In (b2), adventitious bud induction culture base B contains 1.0-4.0mg/L TDZ, the adventitious bud induction culture base B
4.0mg/L TDZ can specifically be contained；
Or, adventitious bud induction culture base B contains 1.0-4.0mg/L KT, the adventitious bud induction culture base B can specifically contain
There is 3.0mg/L KT；
The adventitious bud induction culture base B also contains MS solid medium.
In (b2), the time of the culture can be 45d.
In above-mentioned (b3), petiole clump bud inducement cultivation base contains the 6-BA of 1.0-4.0mg/L, the petiole clump bud induction training
Feeding base is specifically containing the 6-BA of 2.0mg/L；
Or, petiole clump bud inducement cultivation base contains the KT of 2, the 4-D and 1.0-4.0mg/L of 1.0-4.0mg/L, the petiole
The KT of the specific 2,4-D containing 1.0mg/L and 4.0mg/L of clump bud inducement cultivation base；
The petiole clump bud inducement cultivation base also contains MS solid medium.
In (b3), the time of the culture can be 45d.
In above-mentioned (b4), blade clump bud inducement cultivation base contains the 6-BA of 1.0-4.0mg/L, the blade clump bud induction training
Feeding base is specifically containing the 6-BA of 2.0mg/L；Or
Blade clump bud inducement cultivation base contains the KT of the 2,4-D and 1.0-4.0mg/L of 1.0-4.0mg/L, the blade clump
The KT of the specific 2,4-D containing 1.0mg/L and 4.0mg/L of bud inducement cultivation base；
The blade clump bud inducement cultivation base also contains MS solid medium.
In (b4), the time of the culture can be 45d.
In above-mentioned tissue cultures, before carrying out culture of rootage to adventitious bud, it can also be prepared to above by (b1), (b2)
Obtained adventitious bud or the operation by (b3), (b4) the clump bud progress Multiplying culture being prepared；
The Multiplying culture carries out on proliferated culture medium；
The proliferated culture medium contains 2.0-4.0mg/L 6-BA and 1.0-4.0mg/L NAA, the proliferated culture medium tool
Body can contain 3.0mg/L 6-BA and 1.0mg/L NAA；
The proliferated culture medium also contains MS solid medium.；
The time of the Multiplying culture can be 45d.
When the clump bud that (b3), (b4) is prepared carries out Multiplying culture, need first to be cut into the clump bud individually
Bud carries out Multiplying culture again.
Above-mentioned (b1), (b2), (b3), in (b4), the petiole, blade explant the preparation method is as follows:
The fragrant green seed of bell bell is taken, is cultivated on seed germination medium, obtains seedling, grown directly from seeds with bell bell perfume (or spice) blueness
The blade of seedling, petiole are as explant；
The seed germination medium is MS solid medium.
The time of the culture can be 20-30d, concretely 30d.
The fragrant green method for tissue culture of above-mentioned bell bell can also further comprise carrying out hardening to gained regrowth then to transplant
Operation.
The present invention can also further comprise that the operation of callus, institute are prepared by the fragrant green petiole of bell bell, blade explant
Adventitious bud, or the extraction for aromatic substance etc. can be further trained by stating callus.
It is described to operate concretely:
(c1) the fragrant green petiole explant of bell bell is taken, is cultivated on callus inducing medium A, obtains callus group
It knits；
(c2) the fragrant green blade explant of bell bell is taken, is cultivated on callus inducing medium B, obtains callus group
It knits；
In (c1), callus inducing medium A contains 1.0-4.0mg/L 2,4-D, the callus induction
Culture medium A can specifically contain 1.0mg/L 2,4-D；
Or callus inducing medium A contains 1.0-4.0mg/L2,4-D and 1.0-4.0mg/LKT, the callus
Induced medium A can specifically contain 4.0mg/L 2,4-D and 2.0mg/L KT；
The callus inducing medium A further includes MS solid medium.
The time cultivated on callus inducing medium A can be 45d.
In (c2), callus inducing medium B contains 1.0-4.0mg/L2,4-D, the callus induction training
1.0mg/L2,4-D can specifically be contained by supporting base B；Or
Callus inducing medium B contains 1.0-4.0mg/LKT and 1.0-4.0mg/L2,4-D, and the callus lures
4.0mg/L2,4-D and 2.0mg/L KT can specifically be contained by leading culture medium B；
The callus inducing medium B further includes MS solid medium.
The time cultivated on callus inducing medium B can be 45d.
The condition of the culture of any description above is 25-26 DEG C of cultivation temperature, light application time 12h/d, and intensity of illumination is
2000-3000lx。
The present invention also provides a kind of kits for the fragrant green tissue cultures of bell bell, are to include in following (e1)-(e9)
The kit of any one:
(e1) root media；
(e2) root media and the adventitious bud induction culture base A；
(e3) root media and the adventitious bud induction culture base B；
(e4) root media and the petiole clump bud inducement cultivation base；
(e5) root media and the blade clump bud inducement cultivation base；
(e6) root media, the adventitious bud induction culture base A and the proliferated culture medium；
(e7) root media, the adventitious bud induction culture base B and the proliferated culture medium；
(e8) root media, the petiole clump bud inducement cultivation base and the proliferated culture medium；
(e9) root media, the blade clump bud inducement cultivation base and the proliferated culture medium；
Above-mentioned method for tissue culture or kit also belong to of the invention in the fragrant green application expanded in numerous of fragrant plant bell bell
Protection scope.
The present invention is by being added particular types and concentration in the culture medium used in bell bell fragrant green tissue cultures each stage
Plant growth regulator so that the fragrant green breeding coefficient of bell bell is high, speed is fast, can get a large amount of regeneration in a relatively short period of time
Regrowth is carried out transplanting survival rate up to 100% by seedling, so that the expansion for being conducive to the fragrant blueness of bell bell is numerous, promotes the fragrant green scale of bell bell
Metaplasia produces.
Detailed description of the invention
Fig. 1 is aseptic seed seedling, wherein A is the seed seedling cultivated after 4d, and B is the seed seedling cultivated after 20d；
Fig. 2 is inducing effect of the MS+6-BA2.0mg/L culture medium to adventitious bud, wherein A is outside the blade after cultivating 10d
Implant, the clump bud that B is generated when being blade explant Fiber differentiation 45d.
Specific embodiment
The present invention will be described below by way of specific embodiments, but the present invention is not limited thereto.
Experimental method used in following embodiments is conventional method unless otherwise specified；Institute in following embodiments
Reagent, material etc., are commercially available unless otherwise specified.
The content (mg/L) of the composition of MS culture medium (pH 5.8) and each ingredient employed in following embodiments are as follows:
Embodiment
1, the acquisition of material
This experiment material used is the fragrant green seed of bell bell, pick up from Dongling Mountain (40 ° 1 ' 51 of north latitude ", 115 ° of east longitude
27 ' 19 ") the mesophorbium, coryphile of height above sea level 2100-2330m.
2, the culture of the disinfection treatment of seed and aseptic seedling
Best disinfection scheme are as follows: alcohol disinfecting 30s, 10% disinfectant with hydrogen peroxide 15min, pollution rate 0.
The fragrant green seed of bell bell after disinfection is inoculated in MS culture medium, 4d or so starts to sprout；15d or so germination rate reaches
To 98.0%, every seed grows 2 cotyledons；When culture is to 20d, 1-2 piece true leaf (Fig. 1) is born.
3, the induction of callus
3.1, the inductive effect of 2,4-D
The blade of the fragrant green seedling of bell bell to have cultivated 30d, petiole are as explant.Explant is inoculated in MS respectively
In culture medium, every bottle is inoculated with 5, and each processing is inoculated with 50 explants, is repeated 3 times, the induction of different explants is counted after 45d
Situation calculates inductivity.
The fragrant green petiole explant of bell bell is inoculated in the 2,4-D (1.0,2.0,3.0,4.0mg/L) of addition various concentration
Explant change can be observed in MS culture medium, after 5d to expand, when culture is to 10d, petiole appearance significantly is expanded, and is cultivated to 20d
When, there is the appearance of granular yellow callus at petiole both ends.Continuing culture can find that callus persistently increases, mostly loose
Yellow green is presented in state.Inductivity is 23.33%-75.33% when 45d.Wherein when 2,4-D concentration is 1.0mg/L, petiole
Callus induction rate reaches 75.33%.
Be inoculated in using the fragrant green blade of bell bell as explant add various concentration 2,4-D (1.0,2.0,3.0,
4.0mg/L) in MS culture medium, when culture is to 20d, there is granular yellow callus in blade surrounding.Inductivity is when 45d
32.67%-98.00%.When 2,4-D concentration is 1.0mg/L, the inductivity of callus is up to 98.00%.
3.2, the combination inductive effect of 2,4-D and KT
Bell bell perfume (or spice) leafiness piece explant is inoculated into the MS culture medium of 2, the 4-D and KT of addition various concentration, analysis statistics
As a result learn various concentration 2,4-D and KT can be different degrees of induce callus, and the inductivity of callus compared with
It is high.When the concentration that the concentration of 2,4-D is 4.0mg/L, KT is 2.0mg/L, the inductivity of 45d callus reaches highest, is
94.67% (table 1).Callus is in green, rarefaction.
By the fragrant green petiole explant of bell bell be inoculated into MS+2,4-D (1.0,2.0,3.0,4.0mg/L) and KT (1.0,
2.0,3.0,4.0mg/L) it is cultivated in culture medium, as a result, it has been found that, when the concentration that the concentration of 2,4-D is 4.0mg/L, KT is
When 2.0mg/L, the inductivity of 45d petiole callus reaches up to 85.33% (table 1).
1 various concentration 2,4-D of table combines the inductive effect to callus with KT
Note: data withIt indicates, same letter does not indicate significant difference.P < 0.05.
4, the induction of adventitious bud
4.1, inductive effect of the TDZ to adventitious bud
Petiole explant is inoculated in TDZ (1.0,2.0,3.0,4.0mg/L) MS culture medium of addition various concentration, training
When supporting to 20d, emerald granular callus can persistently increase, and then green bud point occur, and bud point gradually forms not
Normal bud.Inductivity is 28.20%-46.67% when 45d.When the concentration of TDZ reaches 2.0mg/L, the inductivity of adventitious bud reaches
It is 46.67% to highest.
The fragrant green blade explant of bell bell is inoculated in TDZ (1.0,2.0,3.0,4.0mg/L) MS of addition various concentration
It is cultivated in culture medium, the inductivity of adventitious bud is increased with the raising of TDZ concentration, and inductivity is 66.02%- when 45d
92.65%.When TDZ concentration is 4.0mg/L, adventitious bud induction frequency is up to 92.65%.
4.2, inductive effect of the KT to adventitious bud
The fragrant green petiole explant of bell bell is inoculated into the MS culture medium of (1.0,2.0,3.0,4.0mg/L) MS+KT,
Inductivity when 45d is 32.33%-52.34%.When KT reaches 3.0mg/L, petiole adventitious bud induction frequency is up to
52.34%.
The fragrant green blade explant of bell bell is inoculated into the MS culture medium of (1.0,2.0,3.0,4.0mg/L) MS+KT, with
The raising of KT concentration, the inductivity of adventitious bud have the tendency that first increasing and reduce afterwards, inductivity when 45d is 34.00%-
91.96%.When the concentration of KT is 3.0mg/L, the inductivity highest of adventitious bud is 91.96%.
4.3, inductive effect of the 6-BA to adventitious bud
The fragrant green petiole explant of bell bell is inoculated in 6-BA (1.0,2.0,3.0,4.0mg/L) MS of addition various concentration
In culture medium, when culture is to 10d, the appearance of petiole explant significantly is expanded, and when cultivating to 20d, is had at explant edge
Granular yellow callus and green bud point occur, and then form green clump bud.Clump bud inductivity when 45d is 13.33%-
58.00%.When the concentration of 6-BA is 2.0mg/L, the inductivity of petiole clump bud is respectively 58.00%
The fragrant green blade explant of bell bell is inoculated in 6-BA (1.0,2.0,3.0,4.0mg/L) MS of addition various concentration
In culture medium, discovery 6-BA can induce bell bell perfume (or spice) leafiness piece and generate clump bud, higher to the inductivity of clump bud.Clump bud induces when 45d
Rate is 28.00%-78.00%.When the concentration of 6-BA is 2.0mg/L, the inductivity of blade explant clump bud is 78.00%
(Fig. 2).
4.4, the inductive effect of 2,4-D and KT combination
By the fragrant green blade inoculation of bell bell to MS+2,4-D (1.0,2.0,3.0,4.0mg/L)+KT (1.0,2.0,3.0,
4.0mg/L) in MS culture medium, as a result, it has been found that, low concentration 2,4-D whens ratio is more easier to induce clump bud in high concentration, when 2,4-D
Concentration is 1.0mg/L, KT concentration when being 4.0mg/L, cultivates 45d, and the inductivity of clump bud is up to 87.25% (table 2).
After 2, the 4-D and KT that add various concentration in MS culture medium, the effect that induction petiole generates clump bud is different, when 2,4-
When the concentration of D is 1.0mg/L, the inductivity of clump bud is significantly higher than the inductivity of other concentration.2,4-D1.0mg/L,KT
When 4.0mg/L, 45d is cultivated, the inductivity of clump bud is up to 59.33%.
Inductive effect of the 2,4-D and KT of 2 various concentration of table to clump bud
Note: data withIt indicates, same letter does not indicate significant difference.P < 0.05.
5, the Multiplying culture of adventitious bud
By single adventitious bud (clump bud made from step 4.3 is cut into single adventitious bud) be inoculated in MS+6-BA (2.0,
3.0,4.0mg/L) in+NAA (1.0,2.0,3.0,4.0mg/L) culture medium, observation counts the amplification coefficient of single adventitious bud.Training
Support 45d, the results showed that, generation clump bud can be induced in every kind of culture medium to some extent.MS+6-BA3.0mg/L+NAA
1.0mg/L culture medium cultivation effect is best, proliferation rate 98.67%, and each simple bud can averagely expand 6 buds.Therefore, MS+6-
BA 3.0mg/L+NAA 1.0mg/L is the optimum medium of adventitious bud proliferation.
The 6-BA and NAA of 3 various concentration of table combine the influence to adventitious bud proliferation
Note: data withIt indicates, same letter does not indicate significant difference.P < 0.05.
5, culture of rootage
It chooses in 3cm or so morphologically normal adventitious bud (clump bud prepared by step 4.3) access MS culture medium, every group of processing
Inoculation 50, is repeated 3 times, and cultivates 45d, and observation, which is taken root, situation and counts rooting rate, several and root long of taking root.
Influence of IAA, the IBA of 4 various concentration of table to root induction
Note: data withIt indicates, same letter does not indicate significant difference.P < 0.05.
When medium supplemented IAA and IBA, there is higher rooting rate.It is raw in the culture medium of MS+IBA0.1mg/L
Root rate is up to 98.67%, and item number of averagely taking root is 22.33, and adventitious root length is about 3.23cm (table 4).Medium supplemented
Also it can get 82.00% rooting rate when 0.1mg/L NAA, but with the raising of its concentration, rooting rate is decreased obviously.
5, hardening and transplanting
Triangular flask sealed membrane is opened, regrowth is first subjected to hardening 7d or so, the training of root is carefully washed out with tap water
Support base, transplanting is to there is peat soil: perlite: in sand=4:3:2 mixed-matrix, being placed in relative humidity is 80%-90%, temperature
To cultivate in 25 DEG C or so of environment, 30d rear blade is grown up, and petiole elongation, survival rate is up to 100%.
Claims (10)
1. a kind of fragrant green method for tissue culture of fragrant plant bell bell, includes the following steps (a1):
(a1) the fragrant green adventitious bud of bell bell is taken, is cultivated on root media, obtains regrowth.
2. according to the method described in claim 1, it is characterized by: the root media contains 0.1- in step (a1)
2.0mg/L IAA or 0.1-2.0mg/L IBA or 0.1-2.0mg/LNAA.
3. method according to claim 1 or 2, it is characterised in that: the adventitious bud is by following (b1) or (b2) system
Standby obtained adventitious bud, or the clump bud being prepared by following (b3) or (b4):
(b1) the fragrant green petiole explant of bell bell is taken, is cultivated on normal bud induced medium A, obtains adventitious bud；
(b2) the fragrant green blade explant of bell bell is taken, is cultivated on adventitious bud induction culture base B, obtains adventitious bud；
(b3) the fragrant green petiole explant of bell bell is taken, is cultivated in petiole clump bud inducement cultivation base, obtains clump bud；
(b4) the fragrant green blade explant of bell bell is taken, is cultivated in blade clump bud inducement cultivation base, obtains clump bud.
4. according to the method described in claim 3, it is characterized by: adventitious bud induction culture base A contains 1.0- in (b1)
4.0mg/L TDZ；Or, adventitious bud induction culture base A contains 1.0-4.0mg/L KT；
(b2) in, adventitious bud induction culture base B contains 1.0-4.0mg/L TDZ；Or, adventitious bud induction culture base B contains 1.0-
4.0mg/L KT；
(b3) in, petiole clump bud inducement cultivation base contains the 6-BA of 1.0-4.0mg/L；Or, petiole clump bud inducement cultivation base contains
The KT of the 2,4-D and 1.0-4.0mg/L of 1.0-4.0mg/L；
(b4) in, blade clump bud inducement cultivation base contains the 6-BA of 1.0-4.0mg/L；Or, blade clump bud inducement cultivation base contains
The KT of the 2,4-D and 1.0-4.0mg/L of 1.0-4.0mg/L.
5. method according to any of claims 1-4, it is characterised in that: the method for tissue culture is at (a1) to not
It further include the operation that Multiplying culture is carried out to adventitious bud or clump bud before normal bud carries out culture of rootage；
The Multiplying culture carries out on proliferated culture medium；
The proliferated culture medium contains 2.0-4.0mg/L6-BA and 1.0-4.0mg/L NAA.
6. method according to any one of claims 1-5, it is characterised in that: (b1), (b2), (b3), in (b4), it is described
Petiole, blade explant the preparation method is as follows:
The fragrant green seed of bell bell is taken, is cultivated on seed germination medium, obtains seedling, with the fragrant green seedling of bell bell
Blade, petiole are as explant；
The seed germination medium is MS solid medium.
7. method according to claim 1-6, it is characterised in that: the method for tissue culture further includes by bell bell
Fragrant green petiole, blade explant prepare the operation of callus,
The operation are as follows:
(c1) the fragrant green petiole explant of bell bell is taken, is cultivated on callus inducing medium A, obtains callus；
(c2) the fragrant green blade explant of bell bell is taken, is cultivated on callus inducing medium B, obtains callus.
8. the method stated according to claim 7, it is characterised in that: in (c1), callus inducing medium A contains 1.0-
4.0mg/L2,4-D；Or, callus inducing medium A contains 1.0-4.0mg/L2,4-D and 1.0-4.0mg/LKT；
(c2) in, callus inducing medium B contains 1.0-4.0mg/L2,4-D；Or, callus inducing medium B contains
1.0-4.0mg/LKT and 1.0-4.0mg/L2,4-D.
9. a kind of kit for the fragrant green tissue cultures of bell bell, being includes the reagent of any one in following (e1)-(e9)
Box:
(e1) root media；
(e2) root media and adventitious bud induction culture base A；
(e3) root media and adventitious bud induction culture base B；
(e4) root media and petiole clump bud inducement cultivation base；
(e5) root media and blade clump bud inducement cultivation base；
(e6) root media, adventitious bud induction culture base A and proliferated culture medium；
(e7) root media, adventitious bud induction culture base B and proliferated culture medium；
(e8) root media, petiole clump bud inducement cultivation base and proliferated culture medium；
(e9) root media, blade clump bud inducement cultivation base and proliferated culture medium；
The root media contains 0.1-2.0mg/L IAA or 0.1-2.0mg/L IBA or 0.1-2.0mg/LNAA；
Adventitious bud induction culture base A contains 1.0-4.0mg/L TDZ, or, adventitious bud induction culture base A contains 1.0-4.0mg/L
KT；
Adventitious bud induction culture base B contains 1.0-4.0mg/L TDZ, or, adventitious bud induction culture base B contains 1.0-4.0mg/L
KT；
Petiole clump bud inducement cultivation base contains the 6-BA of 1.0-4.0mg/L, or, petiole clump bud inducement cultivation base contains 1.0-
The KT of the 2,4-D and 1.0-4.0mg/L of 4.0mg/L；
Blade clump bud inducement cultivation base contains the 6-BA of 1.0-4.0mg/L, or, blade clump bud inducement cultivation base contains 1.0-
The KT of the 2,4-D and 1.0-4.0mg/L of 4.0mg/L；
The proliferated culture medium contains 2.0-4.0mg/L6-BA and 1.0-4.0mg/L NAA.
10. the described in any item method for tissue culture of claim 1-8 or kit as claimed in claim 9 are in fragrant plant bell
The fragrant green application expanded in numerous of bell.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106234226A (en) * | 2016-08-23 | 2016-12-21 | 中央民族大学 | A kind of in-vitro culture method of Ligularia rumicifolia (Drumm.) S.W. Liu. |
| CN106386484A (en) * | 2016-08-30 | 2017-02-15 | 云南金碧制药有限公司 | Anaphalis bulleyana cultivation and breeding method |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106234226A (en) * | 2016-08-23 | 2016-12-21 | 中央民族大学 | A kind of in-vitro culture method of Ligularia rumicifolia (Drumm.) S.W. Liu. |
| CN106386484A (en) * | 2016-08-30 | 2017-02-15 | 云南金碧制药有限公司 | Anaphalis bulleyana cultivation and breeding method |
Non-Patent Citations (1)
| Title |
|---|
| P.SENTHILKUMAR等: "Conservation of an endemic medicinal plant, Anaphalis eliptica DC. by employing plant tissue culture technique", 《JOURNAL OF APPLIED AND NATURAL SCIENCE》 * |
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