CN106472318B - A kind of method of medicinal dioscorea zingiberensis wright mass production - Google Patents
A kind of method of medicinal dioscorea zingiberensis wright mass production Download PDFInfo
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- CN106472318B CN106472318B CN201610915163.1A CN201610915163A CN106472318B CN 106472318 B CN106472318 B CN 106472318B CN 201610915163 A CN201610915163 A CN 201610915163A CN 106472318 B CN106472318 B CN 106472318B
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- dioscorea zingiberensis
- seedling
- medicinal
- zingiberensis wright
- mass production
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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)
- Engineering & Computer Science (AREA)
- Biotechnology (AREA)
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- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a kind of dioscorea zingiberensis wright rapid propagation methods of efficient low-consume amount, include the following steps：(1) it is explant to select the stem section of excellent medicinal dioscorea zingiberensis wright plant, it is constant in other component contents after explant disinfection, it is cultivated on MS+6 BA0.01~0.05mg/L+ paclobutrazol 3.0~10.0mg/L+ cycocel 3.0~10.0mg/L Solid agar cultures that only inositol content doubles, induces a large amount of budlet clumps；(2) Multiple Buds that step (1) obtains are scaled off, it is divided into 5 plants one clump, it is connected on that other component contents are constant, is proliferated and is taken root on the Solid agar culture of MS+NAA0.02~0.6mg/L+IAA0.02 that only inositol content doubles~0.5~5.0mg/L of 0.6mg/L+ paclobutrazol 0.5~5.0mg/L+ cycocels.This method culture effect is good, and growth coefficient is high, and stability is good, production cost is low, is a kind of rapid propagation method obtaining a large amount of test tube seedlings within the shortest time.
Description
Technical field
The invention belongs to the tissue culture technology fields of medicinal dioscorea zingiberensis wright, and in particular to a kind of efficient medicinal dioscorea zingiberensis wright batch
Quantify the method for production.
Background technology
It is synthesis contraceptive and steroid that dioscorea (Dioscorea), which has important medical value, rhizome sapogenin,
The important starting material of body hormone medicine, occupies an important position in medical industry.《Chinese rare endangered plants name
Record》In be listed in national two level and gradually endanger species and be protected.Shield leaf potato is carried out using plant cell engineering technology and methods
The extensive tissue-culturing rapid propagation of Chinese yam (salvia hispanica L), standardized planting are for promoting the development of pharmacy industry to have important work
With.There is provided a kind of batch production method (grant number of medicinal dioscorea zingiberensis wright forming seedling through one step culture within 2014
ZL201410288573.9), evoked callus, bud, adventitious root can be synchronized, be proliferated and take root be reduced to a step completion, still
The inventive method test tube seedling need to pass through the callus tissue culture stage, improve aberration rate.There is provided a kind of medicinal shield leaf potato within 2015
The method (application number 201510319287.9) that the sterile brachyplast of Chinese yam tissue-cultured seedling takes root, this method solve because Chinese yam stem structure it is special
And lead to difficult problem of taking root, while clonal offspring keeps highly consistent with maternal good characteristic, but the invented party
Effective seedling quantity is few (most 18 plants/bottle) in method single bottle test tube seedling, and test tube seedling is single plant, and bunching effect is poor, and then causes
Test tube seedling transplanting survival rate is low, and single plant is of high cost.The present invention establishes to create the best approach of the efficient seedling of dioscorea zingiberensis wright
The cultivating system of a set of efficient low-consume amount converts achievement in research to actual productivity with most fast speed.
Invention content
The object of the present invention is to provide a kind of dioscorea zingiberensis wright rapid propagation method of efficient low-consume amount, this method culture effects
Good, growth coefficient is high, and stability is good, production cost is low, is a kind of to obtain the quick numerous of a large amount of test tube seedlings within the shortest time
Grow method.
The purpose of the present invention is what is be accomplished by the following way：
A kind of method of medicinal dioscorea zingiberensis wright mass production, includes the following steps：
(1) basic seedling obtains：It is explant to select the stem section of excellent medicinal dioscorea zingiberensis wright plant, at it after explant disinfection
Its component content is constant, and 3.0~10.0mg/L+ of MS+6-BA0.01~0.05mg/L+ paclobutrazols that only inositol content doubles is short strong
It is cultivated on element 3.0~10.0mg/L Solid agar cultures, induces a large amount of budlet clumps；
(2) mass production：The Multiple Buds that step (1) obtains are scaled off, is divided into 5 plants one clump, is connected on other ingredients and contains
Measure constant, MS+NAA0.02~0.6mg/L+IAA0.02 that only inositol content doubles~0.5~5.0mg/ of 0.6mg/L+ paclobutrazols
It is proliferated and is taken root on the Solid agar culture of 0.5~5.0mg/L of L+ cycocels；
(3) test tube transplantation of seedlings：Before the transplanting of test tube seedling bottle outlet, bottle cap hardening 3-5d is opened, the seedling in bottle is first with 1000 times
Carbendazim or Mancozeb spray 3~5 times, take out cleaning root culture medium, 5 plants/Cong Yizhi of seedling is then had into top glass
Transplanting container in, be 20~30 DEG C in temperature, grow under conditions of humidity 80~90%, when growing young leaves, already survive.
Explant is cut into the specification containing 11 bud of section in step (1), is cleaned up first with washing powder, in sterile working
With sterile washing 5~7 times on platform, infiltrated -1 minute 30 seconds with 70% alcohol, place into 0.1% mercuric chloride solution sterilizing 12~
18 minutes, aseptic water washing 5~6 times was spare.
When the clump bud squamous subculture that step (1) induces, goes old stem to stay sprouting clump, be transferred to the identical culture medium of step (1)
In, it is primary every 28-30d subcultures.
Step (2) is that the Multiple Buds for obtaining step (1) scale off, and is divided into 5 plants one clump, 10 clumps every bottle, is transferred to step (2)
In the culture medium, culture 20d is taken root, and is transplanted.
Step (1) and the cultivation temperature of (2) they are 24 ± 2 DEG C, in 1000~2000lx low light environments, daily 10~12 hours
Illumination cultivation.
Step (1) it is preferable to use culture medium：Other component contents are constant, the MS+6-BA that only inositol content doubles
0.05mg/L+ paclobutrazol 6.0mg/L+ cycocels 3.0mg/L；Or it is preferable to use culture mediums：Other component contents are constant, only flesh
The MS+6-BA 0.02mg/L+ paclobutrazol 4.0mg/L+ cycocels 6.0mg/L that alcohol content doubles.
Step (2) it is preferable to use culture medium：Other component contents are constant, the MS+NAA that only inositol content doubles
0.1mg/L+IAA0.2mg/L+ paclobutrazol 1.0mg/L+ cycocels 2.0mg/L；Or it is preferable to use culture mediums：Other ingredients contain
Measure the MS+NAA 0.2mg/L+IAA 0.2mg/L+ paclobutrazol 1.5mg/L+ cycocels 1.0mg/ constant, only inositol content doubles
L。
Compared with prior art, the present invention the beneficial effects of the present invention are：
1, place, the inventive method improve the sterile brachyplast culture efficiency of Chinese yam in view of the shortcomings of the prior art, directly
It connects from sterile brachyplast axil, forms bud clump, clonal offspring can be kept and maternal excellent spy without callus phase
The high consistency of property.
2, the present invention needs to design cultural method according to production, first with a large amount of budlet clumps of one-step inducing, inhibits the formation of root,
Improve proliferation times, accelerate the breeding of basic seedling, proliferation times reach 15 or more, the general proliferation times of the prior art 4-6 it
Between；Every bottle obtains effective 50 plants of seedling or more, and general every bottle of the effective bud of the prior art between 10 and 20, substantially increases culture
Efficiency has saved production cost.
3, when efficient mass production, synchronous induced bud clump extends and takes root, and forming intact plant only needs 20d or so；5 plants
One Cong Jinhang is transplanted, and seedling-slowing stage is short, and transplanting survival rate is up to 100%.
4, newest tissue culture technology according to previous studies produces 1,000,000 plants of dioscorea zingiberensis wright tissue-cultured seedling, i.e. " forming seedling through one step culture method "
18 plant/bottle of test tube seedling for taking root at most is formed, 5.5 ten thousand bottles of tissue-cultured seedling need to be produced.The technology of the present invention produces 1,000,000 plants of shield leaves
Chinese yam tissue-cultured seedling, every bottle of effective rooted seedling are 50 plants or more, it is only necessary to produce 10,000 bottles of basic seedlings, 20,000 bottles of rooted seedlings add up to 30,000
Bottle.The present invention not only reduces sterile working step, and has saved culturing room space, simplifies health seedling production routine,
Production cost is reduced, production efficiency is improved.
Description of the drawings
Fig. 1 is the growing state of induced bud clump at the sterile brachyplast axil of dioscorea zingiberensis wright；
Fig. 2 is the growing state of dioscorea zingiberensis wright basis seedling high efficiently multiplying；
Fig. 3 is the growing state that dioscorea zingiberensis wright bud clump synchronizes elongation and takes root；
Fig. 4 is the root growth situation that dioscorea zingiberensis wright bud clump synchronizes elongation and takes root；
Fig. 5 is the growing state for the dioscorea zingiberensis wright test tube seedling for transplanting 7d；
Fig. 6 is the growing state for the dioscorea zingiberensis wright test tube seedling for transplanting 40d.
Specific implementation mode
It is intended to further illustrate the present invention with reference to embodiments, is not intended to limit the present invention.Embodiment 1, tissue culture rapid
Speed breeding dioscorea zingiberensis wright
One, culture medium is prepared and sterilizes
Primary subculture medium is used for basic seedling and breeds：(the other constituent contents of MS are constant, and only inositol content adds for MS improvement
Times)+6-BA 0.05mg/L+ paclobutrazol 6.0mg/L+ cycocels 3.0mg/L；
Elongation and root media are used for mass production：(the other constituent contents of MS are constant, and only inositol content adds for MS improvement
Times)+NAA0.1mg/L+IAA0.2mg/L+ paclobutrazol 1.0mg/L+ cycocels 2.0mg/L；
At 121 DEG C of the above culture medium, sterilize 20 minutes.
Two, efficiently basic seedling breeding
Dioscorea zingiberensis wright (salvia hispanica L) stem section is taken, is cleaned up first with washing powder, in aseptic operating platform
Upper sterile washing 5~7 times, is infiltrated 1 minute with 70% alcohol, places into 0.1% mercuric chloride solution and sterilize 12~18 minutes,
Stem section is transferred to equipped in plastic culture bottle in Initial culture base (80 milliliters of capacity), each cultivates by aseptic water washing 5~6 times
Bottle explant number is 10 or so, is cultivated under following condition of culture：24 DEG C or so, daily illumination in 10 hours, illumination
Intensity is 1000~2000lx or so；Stem section is inoculated with 50.Culture 2 weeks or so, initially forms bud point from stem section axil, continues
It cultivates 4 weeks, forms 15 or more bud at 100% stem section axil, up to 22, induce Multiple Buds growing state such as Fig. 1 institutes
Show.When squamous subculture, clump bud is divided into fritter, primary every 30d subcultures, in each subculture, each bud clump is proliferated 15 times or more,
Multiple Buds growing state is as shown in Figure 2.This step needs to inhibit the formation of root, to prevent influencing the breeding of basic seedling.According to production
Scale breeds sufficient basic seedling, then carries out mass production.
Three, efficient mass production
Above-mentioned 50 strains are taken to carry out successive propagation, the specific method is as follows：The bud clump that step (1) is obtained presses 5 plants one clump
Specification separate, inoculation is cultivated in the medium, and culture medium is the elongation prepared of front and root media, condition of culture
It is bred with aforementioned base seedling；After cultivating 20d or so, grow up to the seedling of 4-6cm high per bud Cong Jun, while 5-10 item roots are grown from base portion
Intact plant.The seedling growing state that Multiple Buds synchronize elongation growth and take root is as shown in Figure 3；Multiple Buds synchronize elongation growth and
The root growth situation taken root is as shown in Figure 4.
Four, test tube transplantation of seedlings：
Before the transplanting of test tube seedling bottle outlet, bottle cap hardening 3-5d is opened, the seedling in bottle is first with 1000 times of carbendazim or Dai Sen
MnZn is sprayed 3~5 minutes, and cleaning root culture medium is taken out, and is then had 5 plants/Cong Yizhi of seedling in the transplanting container for pushing up glass,
It is 20~30 DEG C in temperature, is grown under conditions of humidity 80~90%, 7d or so restoration ecosystems, 20d or so grows young leaves, 40d
Left and right 100% grows young leaves.The growing state for transplanting the dioscorea zingiberensis wright test tube seedling of 7d or so is as shown in Figure 5；Transplant 40d's or so
The growing state of dioscorea zingiberensis wright test tube seedling is as shown in Figure 6.
Dioscorea zingiberensis wright tissue culture propagation provided by the present invention, stem section are explant, and materials are easy, and quantity is big,
Genetic stability is high, the advantages such as well-grown.During Initial culture, without callus phase, directly from stem section leaf
Multiple Buds are formed at armpit, not only the speed of growth is fast, but also breeding coefficient is big.The mass production stage, using synchronous elongation growth
With the method taken root, health seedling production routine is simplified, the newly-invented tissue culture technology of forefathers produces 1,000,000 plants of dioscorea zingiberensis wrights
Tissue-cultured seedling, i.e. " forming seedling through one step culture method " form the tissue-cultured seedling that 18 plants/bottle are used to take root, and need to produce 5.5 ten thousand bottles of tissue-cultured seedling.Skill of the present invention
Art produces 1,000,000 plants of dioscorea zingiberensis wright tissue-cultured seedling, and every bottle of effective seedling is 50 plants or more, it is only necessary to about 30,000 bottles of subculture rooted seedlings of production.This
Invention not only reduces sterile working step, and has saved culturing room space, simplifies health seedling production routine, reduces
Production cost improves production efficiency.The present invention not only high efficiently multiplying, proliferation times reach 15 or more, effective seedling reach 50 plants/
Bottle, and clump bud is transplanted, and bunching effect is strong, and survival rate is up to 100%.
Embodiment 2, tissue-culturing quick-propagation dioscorea zingiberensis wright
One, culture medium is prepared and sterilizes
Primary subculture medium：MS improves (the other constituent contents of MS are constant, and only inositol content doubles)+6-BA0.02mg/L
+ paclobutrazol 4.0mg/L+ cycocels 6.0mg/L；
Elongation and root media：MS improves (the other constituent contents of MS are constant, and only inositol content doubles)+NAA0.2mg/L
+ IAA0.3mg/L+ paclobutrazol 1.5mg/L+ cycocels 1.0mg/L.
At 121 DEG C of the above culture medium, sterilize 20 minutes.
Take dioscorea zingiberensis wright (salvia hispanica L) stem section, basic operation method consistent with embodiment 1；Effect and reality
Example 1 is applied to be not much different.
Claims (5)
1. a kind of method of medicinal dioscorea zingiberensis wright mass production, which is characterized in that include the following steps：
（1）Basic seedling obtains：It is explant to select the stem section of excellent medicinal dioscorea zingiberensis wright plant, after explant disinfection it is other at
Divide content constant, MS+6-BA0.02~0.05mg/L that only inositol content doubles+paclobutrazol 4.0~6.0mg/L+ cycocels
It is cultivated on the Solid agar culture of 3.0~6.0mg/L, induces a large amount of budlet clumps；
（2）Mass production：By step（1）The Multiple Buds of acquisition scale off, and are divided into 5 plants one clump, are connected on other component contents not
Become, 1.0~1.5mg/L of the mg/L of the MS+ NAA0.1~0.2 mg/L+ paclobutrazols of+IAA0.2~0.3 that only inositol content doubles
It is proliferated and is taken root on the Solid agar culture of 1.0~2.0mg/L of+cycocel；
（3）Test tube transplantation of seedlings：Before the transplanting of test tube seedling bottle outlet, bottle cap hardening 3-5 d are opened, the seedling in bottle is first more with 1000 times
Bacterium spirit or Mancozeb are sprayed 3~5 times, take out cleaning root culture medium, 5 plants/Cong Yizhi of seedling is then had to the shifting of top glass
In cultivation receptacle, it is 20~30 DEG C in temperature, grows under conditions of humidity 80~90%, when growing young leaves, that is, survived.
2. the method for medicinal dioscorea zingiberensis wright mass production according to claim 1, which is characterized in that
Step（1）It is middle that explant is cut into the specification containing 11 bud of section, it is cleaned up first with washing powder, on aseptic operating platform
It with sterile washing 5~7 times, is infiltrated -1 minute 30 seconds with 70% alcohol, places into 0.1% mercuric chloride solution and sterilize 12~18 points
Clock, aseptic water washing 5~6 times are spare.
3. the method for medicinal dioscorea zingiberensis wright mass production according to claim 1, which is characterized in that step（1）Induction
When the clump bud squamous subculture gone out, goes old stem to stay sprouting clump, be transferred to step（1）In identical culture medium, every 28-30d subcultures one
It is secondary.
4. the method for medicinal dioscorea zingiberensis wright mass production according to claim 1, which is characterized in that step（2）Being will
Step（1）The Multiple Buds of acquisition scale off, and are divided into 5 plants one clump, 10 clumps every bottle, are transferred to step（2）In the culture medium, culture
20 d are taken root, and are transplanted.
5. the method for medicinal dioscorea zingiberensis wright mass production according to claim 1, which is characterized in that step（1）With（2）
Cultivation temperature be 24 ± 2 DEG C, in 1000~2000lx low light environments, daily 10~12 hours illumination cultivations.
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| CN109392709A (en) * | 2018-11-05 | 2019-03-01 | 宋兆霞 | A kind of yellow ginger breeding method improving Determination of Diosgenin |
| CN109618924B (en) * | 2018-11-07 | 2022-08-09 | 中国科学院亚热带农业生态研究所 | Method suitable for reversing vitrified test tube plantlets of various plants |
| CN109644871B (en) * | 2019-01-21 | 2022-01-11 | 花垣县精虹生物科技发展有限公司 | Batch production method suitable for various orchidaceous medicinal plants |
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| JP2628610B2 (en) * | 1988-07-09 | 1997-07-09 | 住友化学工業株式会社 | Soybean embryo culture method |
| CN104067938B (en) * | 2014-06-24 | 2016-06-08 | 中国科学院亚热带农业生态研究所 | The method of medicinal Rhizome of Peltate Yam group training forming seedling through one step culture mass production |
| CN104221862B (en) * | 2014-09-10 | 2015-10-21 | 长沙县龙泽农业科技有限公司 | The method of medicinal Dioscorea camposita group training forming seedling through one step culture mass production |
| CN104904601B (en) * | 2015-06-11 | 2017-04-05 | 中国科学院亚热带农业生态研究所 | The method that the aseptic brachyplast of medicinal Rhizoma Dioscoreae Zingiberensiss tissue cultured seedling takes root |
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Address after: 410125 No. 644 Yuanda Second Road, Changsha City, Hunan Province Co-patentee after: Kunming Pharmaceutical Group Chongqing Wuling Pharmaceutical Co. Ltd. Patentee after: Institute of Subtropical Agriculture, Chinese Academy of Sciences Address before: 410125 No. 644 Yuanda Second Road, Changsha City, Hunan Province Co-patentee before: Chongqing Huafang Wulingshan Pharmaceutical Co., Ltd. Patentee before: Institute of Subtropical Agriculture, Chinese Academy of Sciences |
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Granted publication date: 20180810 Termination date: 20191020 |