CN107691225A - The rapid propagation method of buta-buta callus seedling - Google Patents
The rapid propagation method of buta-buta callus seedling Download PDFInfo
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- CN107691225A CN107691225A CN201711123770.5A CN201711123770A CN107691225A CN 107691225 A CN107691225 A CN 107691225A CN 201711123770 A CN201711123770 A CN 201711123770A CN 107691225 A CN107691225 A CN 107691225A
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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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- 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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- Developmental Biology & Embryology (AREA)
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Abstract
The invention discloses a kind of rapid propagation method of buta-buta callus seedling, including：Buta-buta aseptic seedling Inter-node stem-segment is cut into segment as explant；Obtained explant is inoculated in callus inducing medium, callus is cultivated to obtain, obtained callus is forwarded in inducing clumping bud culture medium, and then cultivates to obtain Multiple Buds, obtained Multiple Buds are forwarded in root media, cultivate complete band takes root in strain；Take root in after a plant surface horny is formed and be transplanted in matrix after culture a couple of days after complete band, transplanted to crop field.The present invention solve thes problems, such as buta-buta industrial seedling rearing, has incubation time short, and breeding coefficient is high, and survival rate is high, the advantages that robust plant, can meet the needs of market is to buta-buta seedling.
Description
Technical field
The present invention relates to technical field of plant propagation.It is more particularly related to a kind of buta-buta callus into
The rapid propagation method of seedling.
Background technology
Buta-buta (Aquilaria sinensis (Lour.) Gilg) is Thymelaeceae agalloch eaglewood category perennial evergreen arbor, is
The peculiar precious Medicinal species in China, its resiniferous timber are 2005 editions《Pharmacopoeia of People's Republic of China》Specified in
Medicine agalloch eaglewood.Agalloch eaglewood is rare qi-regulating Chinese medicine.《Compendium of Materia Medica》Be recited as " property is pungent, and tepor is nontoxic, have sending down abnormally ascending, receive kidney, adjust in,
The effect of soothing the liver, impulsion medicine is altered for perfume (or spice) ".And there is the ecological value such as higher economic value and gardens, greening.
Buta-buta only can just produce agalloch eaglewood when by external injury or stimulation, and Edgeworthia chrysantha rate is low under nature.
Because predation formula felling artificial for a long time causes buta-buta wild resource exhausted, buta-buta is in 1987 have been listed in
State Precious, Rare, Endangered three-level protective plant, national two level Top-rated protected wild plants were included in by State Council in 1999.Therefore, people is passed through
The means such as work cultivation, good variety selection and tissue cultures are protected extremely urgent to buta-buta wild resource.
The content of the invention
It is an object of the present invention to provide a kind of rapid propagation method of buta-buta callus seedling, by heavy to soil
Fragrant Inter-node stem-segment carries out callus induction, cultivates a large amount of buta-buta seedlings for being adapted to transplanting in a short time, significantly improves soil
The growth coefficient and seedling quality of agalloch eaglewood seedling, large-scale production can be achieved, meet the needs of in the market is to buta-buta seedling.
In order to realize according to object of the present invention and further advantage, there is provided a kind of buta-buta callus seedling
Rapid propagation method, comprise the following steps：
Step 1: buta-buta aseptic seedling Inter-node stem-segment is cut into segment, explant is obtained；
Step 2: obtained explant is inoculated in into callus inducing medium, callus is cultivated to obtain, by what is obtained
Callus is forwarded in inducing clumping bud culture medium, and then cultivates to obtain Multiple Buds, and obtained Multiple Buds are forwarded into training of taking root
Support in base, cultivate complete band takes root in strain；
It is transplanted in matrix after culture a couple of days, is transplanted to crop field Step 3: being taken root in after complete band after a plant surface horny is formed
Culture.
Preferably, callus tissue culture process is carried out under dark condition in step 2, is cultivated 35 days；
Adventitious shoots culture process is carried out under illumination condition, daily illumination 12h, and it is 22~26 DEG C to control temperature, culture 30
My god；
Complete band is taken root in plant incubation and carried out under illumination condition, daily illumination 12h, and it is 22~26 DEG C to control temperature,
Culture 30 days.
Preferably, every liter of callus inducing medium includes 0.1~1.0mg brassin lactones, 0.1~1.0mg
Kinetin, 0.01~0.05mg methyl α-naphthyl acetate, 30mg sucrose and 5mg agar, remaining is MS culture mediums.
Preferably, every liter of inducing clumping bud culture medium include 0.1~0.5mg Thidiazuron, 0.5~2.0mg it is red mould
The agar of element, 0.1~0.5mg kinetin, 30mg sucrose and 5mg, remaining is MS culture mediums.
Preferably, every liter of root media includes 0.1~0.5mg NAA, 10mg sucrose and 5mg agar, remaining
For MS culture mediums.
Preferably, the detailed process of step 3 is：
A small amount of running water is added into the root media, opening is placed 2~4 days, treats that the complete band takes root in a plant table
After face angle matter is formed, the root media for cleaning its root obtains seedling, after the seedling replanting is cultivated 30 days into matrix again
Cultivate in transplanting to crop field.
Preferably, the length of the explant is 1cm.
The present invention comprises at least following beneficial effect：
By taking explant selection, the differentiation culture and hardening and transplanting of the Fiber differentiation of callus, callus
Etc. step, the present invention solve thes problems, such as buta-buta industrial seedling rearing, has incubation time short, breeding coefficient is high, survives
Rate is high, the advantages that robust plant, can meet the needs of market is to buta-buta seedling.
Further advantage, target and the feature of the present invention embodies part by following explanation, and part will also be by this
The research and practice of invention and be understood by the person skilled in the art.
Embodiment
With reference to embodiment, the present invention is described in further detail, to make those skilled in the art with reference to specification
Word can be implemented according to this.
It should be noted that experimental method described in following embodiments, is conventional method unless otherwise specified, institute
Reagent and material are stated, unless otherwise specified, is commercially obtained.
<Embodiment 1>
A kind of rapid propagation method of buta-buta callus seedling, comprises the following steps：
Step 1: buta-buta aseptic seedling Inter-node stem-segment to be cut into the segment of 1cm length, explant is obtained；
Step 2: the explant obtained in step 1 is inoculated into callus inducing medium, under dark condition
Culture obtains callus in 35 days, wherein, the callus inducing medium includes：In MS culture mediums, 0.1mg/L brassins
Ester, 0.1mg/L kinetins, 0.01mg/L methyl α-naphthyl acetates, 30mg/L sucrose and 5mg/L agar；
Step 3: the callus obtained in step 2 is forwarded in inducing clumping bud culture medium, in 22 DEG C, per daylight
Multiple Buds are obtained according to cultivating 30 days under 12h illumination condition, wherein, the inducing clumping bud culture medium includes：MS culture mediums,
0.1mg/L Thidiazurons, 0.5mg/L gibberellin, 0.1mg/L kinetins, 30mg/L sucrose and 5mg/L agar；
Step 4: the Multiple Buds obtained in step 3 are forwarded in root media, in 22~26 DEG C, daily illumination
Cultivated 30 days under 12h illumination condition, obtain completely band root stalwartness plant, wherein, the root media includes：MS culture mediums,
0.1mg/L NAA, 10mg/L sucrose and 5mg/L agar；
Step 5: adding a small amount of running water into the root media, opening is placed 2 days, is treated that complete band root is healthy and strong and is planted
After the surface horny of strain is formed, the complete band root stalwartness plant is taken out, its root culture medium is cleaned and obtains seedling, by the children
After transplantation of seedlings cultivates 30 days into matrix, then it is transplanted to crop field culture.
<Embodiment 2>
A kind of rapid propagation method of buta-buta callus seedling, comprises the following steps：
Step 1: buta-buta aseptic seedling Inter-node stem-segment to be cut into the segment of 1cm length, explant is obtained；
Step 2: the explant obtained in step 1 is inoculated into callus inducing medium, under dark condition
Culture obtains callus in 35 days, wherein, the callus inducing medium includes：In MS culture mediums, 1.0mg/L brassins
Ester, 1.0mg/L kinetins, 0.05mg/L methyl α-naphthyl acetates, 30mg/L sucrose and 5mg/L agar；
Step 3: the callus obtained in step 2 is forwarded in inducing clumping bud culture medium, in 26 DEG C, per daylight
Multiple Buds are obtained according to cultivating 30 days under 12h illumination condition, wherein, the inducing clumping bud culture medium includes：MS culture mediums,
0.5mg/L Thidiazurons, 2.0mg/L gibberellin, 0.5mg/L kinetins, 30mg/L sucrose and 5mg/L agar；
Step 4: the Multiple Buds obtained in step 3 are forwarded in root media, in 26 DEG C, daily illumination 12h
Cultivated 30 days under illumination condition, obtain completely band root stalwartness plant, wherein, the root media includes：MS culture mediums, 0.5mg/
L NAA, 10mg/L sucrose and 5mg/L agar；
Step 5: adding a small amount of running water into the root media, opening is placed 4 days, is treated that complete band root is healthy and strong and is planted
After the surface horny of strain is formed, the complete band root stalwartness plant is taken out, its root culture medium is cleaned and obtains seedling, by the children
After transplantation of seedlings cultivates 30 days into matrix, then it is transplanted to crop field culture.
<Embodiment 3>
A kind of rapid propagation method of buta-buta callus seedling, comprises the following steps：
Step 1: buta-buta aseptic seedling Inter-node stem-segment to be cut into the segment of 1cm length, explant is obtained；
Step 2: the explant obtained in step 1 is inoculated into callus inducing medium, under dark condition
Culture obtains callus in 35 days, wherein, the callus inducing medium includes：In MS culture mediums, 0.5mg/L brassins
Ester, 0.5mg/L kinetins, 0.03mg/L methyl α-naphthyl acetates, 30mg/L sucrose and 5mg/L agar；
Step 3: the callus obtained in step 2 is forwarded in inducing clumping bud culture medium, in 24 DEG C, per daylight
Multiple Buds are obtained according to cultivating 30 days under 12h illumination condition, wherein, the inducing clumping bud culture medium includes：MS culture mediums,
0.3mg/L Thidiazurons, 1.2mg/L gibberellin, 0.3mg/L kinetins, 30mg/L sucrose and 5mg/L agar；
Step 4: the Multiple Buds obtained in step 3 are forwarded in root media, in 24 DEG C, daily illumination 12h
Cultivated 30 days under illumination condition, obtain completely band root stalwartness plant, wherein, the root media includes：MS culture mediums, 0.3mg/
L NAA, 10mg/L sucrose and 5mg/L agar；
Step 5: adding a small amount of running water into the root media, opening is placed 3 days, is treated that complete band root is healthy and strong and is planted
After the surface horny of strain is formed, the complete band root stalwartness plant is taken out, its root culture medium is cleaned and obtains seedling, by the children
After transplantation of seedlings cultivates 30 days into matrix, then it is transplanted to crop field culture.
<Comparative example 1>
A kind of rapid propagation method of buta-buta callus seedling, its process is substantially the same manner as Example 3, difference
It is：The segment of 1cm length is cut into step 1 from buta-buta aseptic seedling stem with bud, every section includes a bud, obtains explant
Body.
<Comparative example 2>
A kind of rapid propagation method of buta-buta callus seedling, its process is substantially the same manner as Example 3, difference
It is：1 × 1cm fritter is cut into step 1 from buta-buta tests for sterility, obtains explant.
By adventitious buds proliferation coefficient, rooting rate and the shifting of embodiment 1, embodiment 2, embodiment 3 and comparative example 1, comparative example 2
Plant after survival rate is counted and obtain result, be shown in Table 1.
Table 1, adventitious buds proliferation coefficient, rooting rate and transplanting survival rate contrast table
Adventitious buds proliferation coefficient refers to bud number caused by single stem section propagation growth, and rooting rate refers to caused by single stem section
Multiple Buds bud number of taking root accounts for the ratio of total bud number, is not difficult to find out from table 1, explant is done compared to using stem with bud or blade fragment
Body carries out callus seedling culture, and doing explant using Inter-node stem-segment carries out the culture of callus seedling in adventitious buds proliferation system
It is greatly increased on number, rooting rate and transplanting survival rate.
<Comparative example 3>
A kind of rapid propagation method of buta-buta callus seedling, its process is substantially the same manner as Example 3, difference
It is：Brassin lactones concentration is 0.05mg/L in the callus inducing medium, kinetin concentration is 0.05mg/L, naphthalene
Acetic acid concentration is 0.005mg/L.
<Comparative example 4>
A kind of rapid propagation method of buta-buta callus seedling, its process is substantially the same manner as Example 3, difference
It is：Brassin lactones concentration is 1.5mg/L in the callus inducing medium, kinetin concentration is 1.5mg/L, naphthalene second
Acid concentration is 0.1mg/L.
Each example in embodiment 1, embodiment 2, embodiment 3 and comparative example 3, comparative example 4 is sampled 100, by more
After injured tissue culture, the inductivity and the speed of growth of callus are counted, the results are shown in Table 2.
Table 2, the inductivity of callus and speed of growth contrast table
| Embodiment 1 | Embodiment 2 | Embodiment 3 | Comparative example 3 | Comparative example 4 | |
| Inductivity | 100% | 100% | 100% | 90% | 83% |
| The speed of growth | Quickly | Quickly | Quickly | Typically | Slowly |
The inductivity of callus refers to that the number that the complete dedifferentiation of explant is callus accounts for the ratio of sampling sum,
The speed of growth refers to the time that the complete dedifferentiation of explant is callus needs, is not difficult to find out from table 2, callus induction training
Support brassin lactones in base, three kinds of compositions of kinetin and methyl α-naphthyl acetate too high levels or it is too low to the inductivity of callus with
And the speed of growth has negative effect.
<Comparative example 5>
A kind of rapid propagation method of buta-buta callus seedling, its process is substantially the same manner as Example 3, difference
Be in：Thidiazuron concentration is 0.05mg/L in the inducing clumping bud culture medium, gibberellin concentration is 0.25mg/L, kinetin
Concentration is 0.05mg/L.
<Comparative example 6>
A kind of rapid propagation method of buta-buta callus seedling, its process is substantially the same manner as Example 3, difference
Be in：Thidiazuron concentration is 1.0mg/L in the inducing clumping bud culture medium, gibberellin concentration is 3.0mg/L, kinetin is dense
Spend for 1.0mg/L.
Each example in embodiment 1, embodiment 2, embodiment 3 and comparative example 5, comparative example 6 is sampled 100, by clump
Sprout after Fiber differentiation, adventitious buds proliferation coefficient is counted and calculates the average value of each, the results are shown in Table 3.
Table 3, adventitious buds proliferation index contrast table
It is not difficult to find out from table 3, the concentration of Thidiazuron, gibberellin and kinetin is too low to induction in inducing clumping bud culture medium
The effect that callus produces Multiple Buds is not strong, and the excessive concentration of Thidiazuron, gibberellin and kinetin meeting strong inhibition is cured
Injured tissue produces Multiple Buds.
<Comparative example 7>
A kind of rapid propagation method of buta-buta callus seedling, its process is substantially the same manner as Example 3, difference
Be in：NAA concentration is 0.05mg/L in the root media.
<Comparative example 8>
A kind of rapid propagation method of buta-buta callus seedling, its process is substantially the same manner as Example 3, difference
Be in：NAA concentration is 1.0mg/L in the root media.
Each example in embodiment 1, embodiment 2, embodiment 3 and comparative example 7, comparative example 8 is sampled 100, by life
After root culture, rooting rate is counted and calculates the average value of each, the results are shown in Table 4.
Table 4, rooting rate contrast table
| Embodiment 1 | Embodiment 2 | Embodiment 3 | Comparative example 7 | Comparative example 8 | |
| Rooting rate | 100% | 100% | 100% | 70% | 40% |
It is not difficult to find out from table 4, the too low Multiple Buds that can not effectively facilitate of NAA concentration are taken root in root media, and are taken root
NAA excessive concentration can suppress Multiple Buds and takes root again in culture medium.
Although embodiment of the present invention is disclosed as above, it is not restricted in specification and embodiment listed
With it can be applied to various suitable the field of the invention completely, can be easily for those skilled in the art
Other modification is realized, therefore under the universal limited without departing substantially from claim and equivalency range, it is of the invention and unlimited
In specific details and shown here as the embodiment with description.
Claims (7)
1. a kind of rapid propagation method of buta-buta callus seedling, it is characterised in that comprise the following steps：
Step 1: buta-buta aseptic seedling Inter-node stem-segment is cut into segment, explant is obtained；
Step 2: obtained explant is inoculated in into callus inducing medium, callus is cultivated to obtain, the callus that will be obtained
Tissue is forwarded in inducing clumping bud culture medium, and then cultivates to obtain Multiple Buds, and obtained Multiple Buds are forwarded into root media
In, cultivate complete band takes root in strain；
It is transplanted to Step 3: being taken root in after complete band after a plant surface horny is formed in matrix after culture a couple of days, transplants to crop field and cultivate.
2. the rapid propagation method of buta-buta callus seedling as claimed in claim 1, it is characterised in that in step 2 more
Injured tissue incubation is carried out under dark condition, is cultivated 35 days；
Adventitious shoots culture process is carried out under illumination condition, daily illumination 12h, and it is 22~26 DEG C to control temperature, is cultivated 30 days；
Complete band is taken root in plant incubation and carried out under illumination condition, daily illumination 12h, and it is 22~26 DEG C to control temperature, culture
30 days.
3. the rapid propagation method of buta-buta callus seedling as claimed in claim 2, it is characterised in that every liter of callus group
Knit inducing culture and include 0.1~1.0mg brassin lactones, 0.1~1.0mg kinetin, 0.01~0.05mg naphthalene second
The agar of acid, 30mg sucrose and 5mg, remaining is MS culture mediums.
4. the rapid propagation method of buta-buta callus seedling as claimed in claim 2, it is characterised in that every liter of Multiple Buds
Inducing culture includes 0.1~0.5mg Thidiazuron, 0.5~2.0mg gibberellin, 0.1~0.5mg kinetin, 30mg
The agar of sucrose and 5mg, remaining is MS culture mediums.
5. the rapid propagation method of buta-buta callus seedling as claimed in claim 2, it is characterised in that every liter of training of taking root
NAA, 10mg sucrose and 5mg agar that base includes 0.1~0.5mg are supported, remaining is MS culture mediums.
6. the rapid propagation method of buta-buta callus seedling as claimed in claim 2, it is characterised in that the tool of step 3
Body process is：
A small amount of running water is added into the root media, opening is placed 2~4 days, treats that the complete band takes root in the surface of strain
After cutin is formed, the root media for cleaning its root obtains seedling, is moved again after the seedling replanting is cultivated 30 days into matrix
Plant to crop field and cultivate.
7. the rapid propagation method of buta-buta callus seedling as claimed in claim 1, it is characterised in that the explant
Length be 1cm.
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| CN104542308A (en) * | 2015-02-03 | 2015-04-29 | 广西壮族自治区药用植物园 | Rapid callus reproduction method for dioscorea bulbifera |
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