CN106171978B - A kind of tissue culture and rapid propagation method of P. kingianum - Google Patents
A kind of tissue culture and rapid propagation method of P. kingianum Download PDFInfo
- Publication number
- CN106171978B CN106171978B CN201610534811.9A CN201610534811A CN106171978B CN 106171978 B CN106171978 B CN 106171978B CN 201610534811 A CN201610534811 A CN 201610534811A CN 106171978 B CN106171978 B CN 106171978B
- Authority
- CN
- China
- Prior art keywords
- culture
- illumination
- bud
- explant
- days
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
Classifications
-
- 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
Landscapes
- 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 invention discloses a kind of tissue culture and rapid propagation method of P. kingianum, selects explant, sterilizing；It is inoculated into inductive differentiation medium, is in intensity of illumination：1800 2000Lx, under conditions of periodicity of illumination is daily 8 10, cultivation temperature is cultivated 40 50 days under conditions of being 25 27 DEG C；It will be produced in inductive differentiation medium with adventitious bud and the tissue of projection cutting fritter, be inoculated into proliferated culture medium and carry out Multiplying culture, realize the propagation of adventitious bud；By the Multiple Buds obtained in Multiplying culture diameter >=0.5cm, the bud of height >=3cm, cutting is segmented into monomer and cuts off blade, is inoculated into root media, is 1800 2000Lx in intensity of illumination；Periodicity of illumination for first 20 days 10 it is small when, later it is daily 12 it is small when, cultivation temperature be 25 27 DEG C under conditions of cultivate 30 35 days, both can obtain rooted seedling；Finally tame hardening, survival rate more than 95%.
Description
Technical field
The invention belongs to medicinal material planting technology field, is related to a kind of tissue culture and rapid propagation method of P. kingianum.
Background technology
P. kingianum (polygonatum kingianum.coll.ethems) is Liliaceae Polygonatum herbaceous plant, mainly
The ground such as Chinese yunnan, Sichuan, Guizhou are originated in, are grown on hayashishita, bushes or Schattenseite wetland.Its rhizome is used as medicine with bowl spares benefit
Gas, moistening lung enriching yin, enriching kidney essence, curing rheumatism, five viscera settling, suppresses tumour cell and other effects.With deepening continuously for research, people
Medical value and health-care efficacy to sealwort are also gived certainly and approved, are had extensively in terms of research new drug and exploitation health products
Wealthy prospect.Market is increasing to raw materials requirement, and price rises steadily, and wild resource has been unable to meet production requirement, serious shadow
The sustainable exploitation and use to P. kingianum is rung, in order to ensure high quality P. kingianum raw material sources, popularizes in an all-round way and implements Yunnan Huang
Smart industrialization, scale, artificial growth are imperative.
For many years, the artificial growth modes of reproduction of traditional P. kingianum mainly has two kinds：One kind be carried out by seed it is sexual
Breeding.Another kind is to carry out vegetative propagation by rhizome, is collected since P. kingianum seed is difficult, germination rate is low, nursery growth cycle
It is long, production planting cost is added, is unfavorable for being widely applied plantation.This method breeding coefficient of Propagation of Rhizomes is low, and rhizome is used
Amount is big both uneconomical, again limits production planting scale, and seedling problem has become limitation P. kingianum and is manually widely applied kind
The bottleneck of plant.
The content of the invention
The object of the present invention is to provide a kind of tissue culture and rapid propagation method of P. kingianum, the breeding cycle is short, and emergence rate is high, plants into
This is low, is adapted to large area plantation, solves problems of the prior art.
The technical solution adopted in the present invention is that a kind of tissue culture and rapid propagation method of P. kingianum, follows the steps below：
Step 1, selection explant；
Step 2, explant sterilizing；
Step 3, induction differentiation culture：Sterilizing explant is inoculated into inductive differentiation medium, is in intensity of illumination：
1800-2000Lx, under conditions of periodicity of illumination is daily 8-10, cultivation temperature is cultivated 40-50 days under conditions of being 25-27 DEG C,
Generation adventitious bud is expanded in bastem portion, and bastem portion forms projection and differentiation adventitious bud；
Step 4, proliferation and subculture culture：The tissue with adventitious bud and projection produced in inductive differentiation medium is cut into
0.5cm2Fritter, be inoculated into proliferated culture medium and carry out Multiplying culture, realize the propagation of adventitious bud；Intensity of illumination is 1800-
2000Lx, when periodicity of illumination is daily 10-12 small, cultivation temperature is under 25-27 DEG C of condition of culture, is cultivated 25-30 days, is formed
Highly reach the Multiple Buds of 4-5cm；
Step 5, Rooting and hardening-off culture：By the Multiple Buds obtained in Multiplying culture diameter >=0.5cm, the bud of height >=3cm,
Cutting is segmented into monomer and cuts off blade, is inoculated into root media, is 1800-2000Lx in intensity of illumination；Periodicity of illumination is
First 20 days 10 it is small when, later it is daily 12 it is small when, cultivation temperature be 25-27 DEG C under conditions of cultivate 30-35 days, can both be taken root
Seedling；The seedling of rhizome bud diameter ＜ 0.5cm is transferred in the proliferated culture medium of step 4 breeds again；
Step 6, domestication hardening：Culture of rootage 30-35 days, rooted seedling is taken out out of bottle, and training is rinsed well with tap water
Stem is supported, and with 2000 times of carbendazim solution immersion 3-5min are diluted to water, plants in matrix, shading 70%, keeps 65-70%
Air humidity, survival rate more than 95% after 30d.
The present invention is further characterized in that, further, the detailed process of the step 1 selection explant for：Annual 3-4
Month, growth selection are healthy and strong, and yield is high, and character is stablized, no disease and pests harm, and band is sprouted the rhizome of bud.
Further, the detailed process of the step 2 explant sterilizing is：
Step a, the sprout rhizome of bud of band is dug out from soil, washes away soil, and rinsed well with water, cutting length is
Root-like stocks of the 4-5cm with terminal bud, is put into immersion 30min in the agricultural streptomycin solution that concentration is 200ppm and carries out pretreatment and go out
Bacterium, then rinsed well with tap water；
Step b, the explant of pretreatment is put into the mercuric chloride solution that concentration is 0.1% in aseptic working platform, and is added
Enter tween 1-2 drops, continuously rock, taken out after soaking 18-20min, blotted outside with aseptic water washing 4-5 time, then with aseptic filter paper
Implant surface moisture, cutting length is：2-3cm terminal buds, are inoculated into induction differentiation base.
Further, in the step 3, inductive differentiation medium is：
MS+6-BA4mg/L+2.4-D0.3mg/L+IBA0.2mg/L+ sugar 30g/L+ agar 4.5g/L, pH value 5.8-6.
Further, in the step 4, proliferated culture medium is：MS+6-BA3.5mg/L+NAA0.2mg/L+ sugar 30g/L+
Agar 4.5g/L, pH value 5.8-6.
Further, in the step 5, root media is：MS+6-BA0.4mg/L+NAA0.8mg/L+ sugar 30g/L+
Agar 4.5g/L, pH value 5.8-6.
Further, in the step 6, it according to quality parts ratio is turfy soil that matrix, which is,：Perlite=4:1 mixing
Thing.
The beneficial effects of the invention are as follows using plant tissue culture technique, P. kingianum vegetative propagation system is established, is obtained big
High quality seedling is measured, realizes scale, is standardized, industrialization plantation.Solves P. kingianum seedling by plant tissue culture fast breeding technique
The problems such as breeding cycle is grown, and emergence rate is low, and production cost is high, the present invention sterilize through explant, the induction differentiation of rhizome bud, propagation training
Support, the process collective effect such as Rooting and hardening-off culture, explant disinfection inoculation success rate induces bud ratio 90%, propagation up to 80%
Rate is up to 6.8 times, and gained seedling plant height 6-7cm, lamina 4-5 pieces, blade development degree is good, roomy thick and solid, and height of seedling degree is moderate, average root
Stem eye diameter reaches 1.2cm, and individual is larger, uniform in size, domestication hardening survival rate more than 95%.
Embodiment
The technical solution in the embodiment of the present invention is clearly and completely described below, it is clear that described embodiment
Only part of the embodiment of the present invention, instead of all the embodiments.Based on the embodiments of the present invention, the common skill in this area
Art personnel all other embodiments obtained without making creative work, belong to the model that the present invention protects
Enclose.
A kind of tissue culture and rapid propagation method of P. kingianum, follows the steps below.
1st, the selection of explant：In annual 3-4 months, growth selection is healthy and strong, and yield is high, and character is stablized, no disease and pests harm, band
Sprout the rhizome of bud.
2nd, the sterilizing of explant：
(1) the sprout rhizome of bud of band is dug out from soil, washes away soil, and scrubbed clean under tap water.Cut length
The root-like stock with terminal bud for 4-5cm is spent, immersion 30min in the agricultural streptomycin solution that concentration is 200ppm is put into and is pre-processed
Sterilizing, then rinsed well with tap water.
Explant is using subterraneous root stem eye, and grow nonparasitically upon another plant many miscellaneous bacterias on its surface, particularly bacterium, comes one to sterilization zone
Determine difficulty.It is seriously polluted according to conventional sterilization procedures.Extend sterilization time, sterilization effect is good, but explant is damaged tight
Weight, is unfavorable for the induction differentiation of bud.This step can kill a large amount of bacteriums in explant surface, play preferable sterilization effect, and reduce
Damage to explant, sterilization effect can improve 40%.
(2) explant of pretreatment is put into the mercuric chloride solution that concentration is 0.1% in aseptic working platform, and adds and spit
Warm 1-2 drops, continuously rock, and are taken out after soaking 18-20min, and explant is blotted with aseptic water washing 4-5 times, then with aseptic filter paper
Surface moisture, cutting length is：2-3cm terminal buds, are inoculated into induction differentiation base.
3rd, induction differentiation culture：Inductive differentiation medium：MS+6-BA4mg/L+2.4-D0.3mg/L+IBA0.2mg/L+ sugar
30g/L+ agar 4.5g/L, pH value 5.8-6, sterilizing explant is inoculated into inductive differentiation medium, is in intensity of illumination：
1800-2000Lx, under conditions of periodicity of illumination is daily 8-10, cultivation temperature is cultivated 40-50 days under conditions of being 25-27 DEG C,
Generation adventitious bud is expanded in bastem portion, and bastem portion forms projection and differentiation adventitious bud.Average mark rate is 85%, average each rhizome
Bud breaks up 2.8 adventitious buds.
4th, proliferation and subculture culture：The tissue with adventitious bud and projection produced in inductive differentiation medium is cut into
0.5cm2Fritter, be inoculated into：MS+6-BA3.5mg/L+NAA0.2mg/L+ sugar 30g/L+ agar 4.5g/L, pH value 5.8-6's
Multiplying culture is carried out in proliferated culture medium, realizes the propagation of adventitious bud；Intensity of illumination is 1800-2000Lx, and periodicity of illumination is every
When its 10-12 is small, cultivation temperature is under 25-27 DEG C of condition of culture, is cultivated 25-30 days, can both form the clump that height reaches 4-5cm
Sprout.Differentiation and proliferation rate is up to 6.8 times.
5th, Rooting and hardening-off culture：
By the Multiple Buds obtained in Multiplying culture diameter >=0.5cm, the bud of height >=3cm, cutting is segmented into monomer and cuts off
Blade, is inoculated into MS+6-BA0.4mg/L+NAA0.8mg/L+ sugar 30g/L+ agar 4.5g/L, the root media of pH value 5.8-6
In, it is 1800-2000Lx in intensity of illumination；Periodicity of illumination for first 20 days 10 it is small when, later it is daily 12 it is small when, cultivation temperature is
Cultivated 30-35 days under conditions of 25-27 DEG C, both can obtain rooted seedling；Up to more than 95%, rhizome bud diameter exists its rooting rate
More than 1.2cm, height of seedling degree reach 6-7cm, lamina 4-5 pieces, and each rhizome bud is averaged root system up to 8-10 bars.Rhizome bud diameter ＜
The seedling of 0.5cm is transferred in the proliferated culture medium of step 4 breeds again.
6th, hardening is tamed
Culture of rootage 30-35 days, rooted seedling is taken out out of bottle, and culture stem is rinsed well with tap water, and with 2000 times
Carbendazim solution soaks 3-5min, and (matrix is according to quality parts ratio is turfy soil for plantation to matrix：Perlite=4:1) in,
Shading 70%, keeps the air humidity of 65-70%, survival rate more than 95% after 30d.
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the scope of the present invention.It is all
Any modification, equivalent replacement, improvement and so within the spirit and principles in the present invention, are all contained in protection scope of the present invention
It is interior.
Claims (1)
1. a kind of tissue culture and rapid propagation method of P. kingianum, it is characterised in that follow the steps below：
Step 1, selection explant；
Step 2, explant sterilizing；
Step 3, induction differentiation culture：Sterilizing explant is inoculated into inductive differentiation medium, is in intensity of illumination：1800-
2000Lx, when periodicity of illumination is daily 8-10 small, cultivation temperature is cultivated 40-50 days under conditions of being 25-27 DEG C, and bastem portion is expanded
Adventitious bud is produced, bastem portion forms projection and differentiation adventitious bud；
Step 4, proliferation and subculture culture：The tissue with adventitious bud and projection produced in inductive differentiation medium is cut into
0.5cm2Fritter, be inoculated into proliferated culture medium and carry out Multiplying culture, realize the propagation of adventitious bud；Intensity of illumination is 1800-
2000Lx, when periodicity of illumination is daily 10-12 small, cultivation temperature is under 25-27 DEG C of condition of culture, is cultivated 25-30 days, is formed
Highly reach the Multiple Buds of 4-5cm；
Step 5, Rooting and hardening-off culture：By the Multiple Buds diameter >=0.5cm obtained in proliferation and subculture culture, the bud of height >=3cm,
Cutting is segmented into monomer and cuts off blade, is inoculated into root media, is 1800-2000Lx in intensity of illumination；Periodicity of illumination is
First 20 days 10 it is small when, later it is daily 12 it is small when, cultivation temperature be 25-27 DEG C under conditions of cultivate 30-35 days, you can taken root
Seedling；The seedling of rhizome bud diameter ＜ 0.5cm is transferred in the proliferated culture medium of step 4 breeds again；
Step 6, domestication hardening：Culture of rootage 30-35 days, rooted seedling is taken out out of bottle, and culture stem is rinsed well with tap water,
And with 2000 times of carbendazim solution immersion 3-5min of dilution are watered, plant in matrix, shading 70%, keep the sky of 65-70%
Air humidity degree, survival rate more than 95% after 30d；
The detailed process of the step 1 selection explant for：In annual 3-4 months, growth selection is healthy and strong, and yield is high, and character is steady
Fixed, no disease and pests harm, band is sprouted the rhizome of bud；
The detailed process of step 2 explant sterilizing is：
Step a, the sprout rhizome of bud of band is dug out from soil, washes away soil, and rinsed well with water, it is 4- to cut length
Root-like stocks of the 5cm with terminal bud, is put into immersion 30min in the agricultural streptomycin solution that concentration is 200ppm and carries out pretreatment sterilizing,
Rinsed well again with tap water；
Step b, the explant of pretreatment is put into the mercuric chloride solution that concentration is 0.1% in aseptic working platform, and adds and spits
Warm 1-2 drops, continuously rock, and are taken out after soaking 18-20min, and explant is blotted with aseptic water washing 4-5 times, then with aseptic filter paper
Surface moisture, cutting length is：2-3cm terminal buds, are inoculated into inductive differentiation medium；
In the step 3, inductive differentiation medium is：MS+6-BA4mg/L+2,4-D0.3mg/L+IBA0.2mg/L+ sugar 30g/
L+ agar 4.5g/L, pH value 5.8-6；
In the step 4, proliferated culture medium is：MS+6-BA3.5mg/L+NAA0.2mg/L+ sugar 30g/L+ agar 4.5g/L, pH
Value 5.8-6；
In the step 5, root media is：MS+6-BA0.4mg/L+NAA0.8mg/L+ sugar 30g/L+ agar 4.5g/L, pH
Value 5.8-6；
In the step 6, it according to quality parts ratio is turfy soil that matrix, which is,：Perlite=4:1 mixture.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610534811.9A CN106171978B (en) | 2016-07-08 | 2016-07-08 | A kind of tissue culture and rapid propagation method of P. kingianum |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610534811.9A CN106171978B (en) | 2016-07-08 | 2016-07-08 | A kind of tissue culture and rapid propagation method of P. kingianum |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN106171978A CN106171978A (en) | 2016-12-07 |
| CN106171978B true CN106171978B (en) | 2018-05-15 |
Family
ID=57472594
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610534811.9A Expired - Fee Related CN106171978B (en) | 2016-07-08 | 2016-07-08 | A kind of tissue culture and rapid propagation method of P. kingianum |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106171978B (en) |
Families Citing this family (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107182780A (en) * | 2017-04-28 | 2017-09-22 | 玉溪市祥馨农业技术开发有限公司 | The method for culturing seedlings of P. kingianum |
| CN107821169A (en) * | 2017-12-11 | 2018-03-23 | 桂林亦元生现代生物技术有限公司 | A kind of tissue culture method of P. kingianum seedling |
| CN107926712A (en) * | 2017-12-28 | 2018-04-20 | 临沧市云瑞堂生物科技有限公司 | A kind of P. kingianum stem tuber and stem apex quickly breed the tissue culture method of seedling |
| CN108812321B (en) * | 2018-07-09 | 2021-09-14 | 重庆市药物种植研究所 | Tissue culture rapid propagation method of polygonatum kingianum |
| CN111434218A (en) * | 2019-01-15 | 2020-07-21 | 湖北民族学院 | Tissue culture rapid propagation method for rejuvenation of polygonatum sibiricum varieties |
| CN111837953A (en) * | 2020-07-22 | 2020-10-30 | 国药种业有限公司 | Rapid breeding method of sealwort seedlings |
| CN113678737B (en) * | 2021-10-11 | 2022-05-17 | 浙江省亚热带作物研究所 | Open tissue culture method for polygonatum cyrtonema |
| CN115644067B (en) * | 2022-12-09 | 2023-03-14 | 云南省农业科学院药用植物研究所 | Method for producing polygonatum kingianum dihaploid |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA2466653C (en) * | 2001-11-15 | 2012-02-21 | Council Of Scientific And Industrial Research | Media compositions for faster growth of polygonatum cirrhifolium royle |
| CN102550413B (en) * | 2011-12-26 | 2013-04-10 | 福建省三明市农业科学研究所 | Rapid propagation method for tube seedlings of high-grade polygonatum cyrtonema hua |
| CN105532448B (en) * | 2015-12-03 | 2017-12-22 | 普洱市玉林林业开发有限公司 | A kind of method of P. kingianum tissue cultures |
| CN105580734B (en) * | 2016-01-14 | 2017-09-15 | 遵义市龙驰生物科技有限公司 | A kind of highly effective revulsion induction method of the in vitro rhizome of sealwort |
-
2016
- 2016-07-08 CN CN201610534811.9A patent/CN106171978B/en not_active Expired - Fee Related
Also Published As
| Publication number | Publication date |
|---|---|
| CN106171978A (en) | 2016-12-07 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN106171978B (en) | A kind of tissue culture and rapid propagation method of P. kingianum | |
| CN104429965B (en) | The method that high eyebrow Herba Anoectochili roxburghii seed is bred without hormone quickly tissue culture | |
| CN102301951B (en) | Method for rapidly propagating roots of subprostrate sophora by tissue culture | |
| CN107182780A (en) | The method for culturing seedlings of P. kingianum | |
| CN103250645B (en) | Rapid propagation and transplantation method of blumea balsamifera | |
| CN102227976A (en) | Litsea cubeba pers asexual quick propagation and seedling breeding method | |
| CN103348920B (en) | Rapid propagation method for high quality seedlings of Kyara | |
| CN103270952B (en) | Sinkiang drug mulberry tissue culture and rapid propagation culture medium | |
| CN104041412A (en) | Rapid propagation method for tissue culture of Guizhou hemiboea cavaleriei | |
| CN106577281A (en) | High-seedling-rate culture method for tissue culture of Polygala fallax stems | |
| CN105325296A (en) | Jackfruit tissue culture rapid propagation method | |
| CN107864865A (en) | A kind of efficient hardening technology and domesticating cultivation method of haw ginseng tissue-cultured seedling | |
| CN111165352A (en) | Novel space breeding tissue culture seedling raising method for gentiana rigescens | |
| CN107211891B (en) | Konjak callus direct seeding and rapid propagation technology | |
| CN106172016A (en) | A kind of Cortex Acanthopancis tissue culture and rapid propagation method | |
| CN109819892B (en) | Tissue culture method of good single plant of tsaoko | |
| CN102939903A (en) | Ginger detoxification, sterilization and rapid propagation method | |
| CN105432466B (en) | Method with plant regeneration occurs for a kind of pittosporum tobira somatic embryo | |
| CN103621399A (en) | Peony tissue culture method | |
| CN108719067A (en) | A kind of tissue culture and rapid propagation method of paris polyphylla | |
| CN104686342A (en) | Asexual and rapid propagation technology for styrax tonkinensis | |
| CN115024221A (en) | Method for rapidly propagating tissue culture seedlings of large-leaf morinda officinalis and application of method | |
| CN107873524A (en) | A kind of serpentgrass stem segment tissue culture fast breeding method | |
| CN107821169A (en) | A kind of tissue culture method of P. kingianum seedling | |
| JP6530584B2 (en) | Method of producing seedlings of licorice genus plant |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20180515 Termination date: 20200708 |