CN109258472A - A kind of tissue culture technique of powder tetrandra root - Google Patents
A kind of tissue culture technique of powder tetrandra root Download PDFInfo
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- CN109258472A CN109258472A CN201811402019.3A CN201811402019A CN109258472A CN 109258472 A CN109258472 A CN 109258472A CN 201811402019 A CN201811402019 A CN 201811402019A CN 109258472 A CN109258472 A CN 109258472A
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/008—Methods for regeneration to complete plants
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Abstract
The invention discloses a kind of tissue culture techniques of powder tetrandra root, are related to the method for culturing seedlings that powder tetrandra root obtains high quality seedling by rapid propagation in vitro technology.The present invention is using powder tetrandra root stem with bud as explant, by processes such as explant disinfection, inducing clumping bud, culture of rootage, acclimatization and transplants to establish powder tetrandra root tissue-culturing quick-propagation system, the popularization of powder tetrandra root breeding and resources development and utilization is accelerated to have important practical significance.
Description
Technical field
The present invention relates to the methods of Plant Tissue Breeding in Plant Biotechnology, specifically, being related to a kind of powder tetrandra root
Tissue culture technique.
Background technique
Powder tetrandra root is menispermaceae plant, and autumn harvesting is cleaned after digging up roots and scrapes off cork, and diameter is at double half of 2cm or more
It rives, dries.Smell is bitter, has dispelling wind and eliminating dampness, inducing diuresis for removing edema.For treating oedema, difficult urination, rheumatic arthralgia, sore.It is long
Supply falls short of demand since phase, in addition artificial predation formula felling causes powder tetrandra root wild resource exhausted, it is therefore necessary to its resource
It is protected.In recent years, the development of resources of powder tetrandra root has certain scale, establishes GAP in Jiangxi, Hubei, Guangdong, Fujian etc.
Planting base plays certain facilitation to the protection work of powder tetrandra root wild resource.Powder tetrandra root mainly passes through seed at present
Sapling multiplication is carried out, but since seminal propagation characters of progenies separates, is unfavorable for high-quality plasm resource protection, constrains high-quality
The popularizing planting of powder tetrandra root.Therefore, it is necessary to establish the tissue culture technique of powder tetrandra root, is quickly bred for its breeding and technology is provided
Support and guarantee accelerate the expansion of its resource to have important practical significance also to meet the needs of China is to powder tetrandra root seedling.It is right
Than file 1, application number: CN201410401162, denomination of invention: the seed tissue culture propagation method of powder tetrandra root, including seed storage,
Seed disinfection, Primary culture, Fiber differentiation, Multiplying culture, the big step of culture of rootage six, which is characterized in that Fiber differentiation step
In, it is cultivated first using induced medium a pair seed expanded of sprouting, induces aseptic seedling, then use Fiber differentiation
Base two carries out continuing to cultivate to aseptic seedling, to reduce the melting brown rate of Multiple Buds and the partial vitrification for eliminating Multiple Buds is existing
As.Two step Multiplying cultures are used in Multiplying culture step, the first step can go out a large amount of Multiple Buds with fast culture, and second step can
With the Multiple Buds for turning out healthy and strong crowd shoots for taking root and part is newly proliferated, the numerous of this tissue culture method is effectively improved
Grow efficiency.The difference of documents and this case.
Summary of the invention
The purpose of the present invention is to provide a kind of tissue culture techniques of powder tetrandra root out, and the present invention is with powder tetrandra root stem with bud
For explant, powder tetrandra root group is established by processes such as explant disinfection, inducing clumping bud, culture of rootage, acclimatization and transplants
Culture rapid propagation system is knitted, to realize the purpose of the present invention.
A kind of tissue culture technique of powder tetrandra root of the invention, includes the following steps:
Step 1, inducing clumping bud: choosing robust growth without the treelet current year of disease pest raw stem with bud as explant, with washing
Clean essence aqueous solution soaking 14min, then rinses 35min with tap water, dry it is spare after surface moisture, in superclean bench with
70%~80% ethyl alcohol impregnates 14s, and 0.1% mercuric chloride solution sterilizes 25min, then with sterile water wash 8 times, being cut into length after drying is
The stem section of 1.5cm is inoculated into induced medium, first dark culture 25 days full under the conditions of 30 DEG C, and it is small to be subsequently placed in daily illumination 15
When, intensity of illumination 900lx, cultivation temperature is cultivated under the conditions of being 30 DEG C, until induced synthesis Multiple Buds；Induced medium are as follows:
MS+6mg/L6-BA+2mg/L NAA+0.9mmol/L La(NO3)2+ 0.15% active carbon of+0.9% agar of+3.9% sucrose, pH value are
5.9;
Step 2, Multiplying culture: the resulting Multiple Buds of step 1 are transferred on proliferated culture medium and carry out Multiplying culture, are first existed after inoculation
Full dark culture germinates a lateral bud at axil under the conditions of 30 DEG C, is subsequently placed in daily illumination 16 hours, intensity of illumination is
900lx, culture under conditions of cultivation temperature is 30 DEG C is until each Multiple Buds are elongated to 5cm；Proliferated culture medium are as follows: MS+3mg/L6-
+ 0.2% active carbon of+0.8% agar of BA+0.9mg/L NAA+3.9% sucrose, pH value 5.9;
Step 3, culture of rootage: the Multiple Buds bud that step 1 or step 2 height obtained are about 5cm is cut and is inoculated into life
Culture of rootage is carried out on root culture medium, it is first dark culture 10 days full under the conditions of 30 DEG C after inoculation, it is small to be subsequently placed in daily illumination 15
When, intensity of illumination 1900lx, cultivation temperature is cultivated under conditions of being 30 DEG C to taking root；Root media are as follows: 1/2MS+4mg/L
+ 0.2% active carbon of+0.9% agar of NAA+3.9% sucrose, pH value 5.9;
Step 4, acclimatization and transplants: after taking root 33 days in powder tetrandra root bottle, after being placed in natural lighting lower refining seedling 8 days, root culture is cleaned
Base is transplanted by yellow soil: in the matrix that river sand=3:2 is mixed into.
Compared with prior art the invention has the advantages that the present invention obtains the nursery of high quality seedling by rapid propagation in vitro technology
Method.Using powder tetrandra root stem with bud as explant, waited by explant disinfection, inducing clumping bud, culture of rootage, acclimatization and transplants
Journey accelerates the popularization of powder tetrandra root breeding and resources development and utilization to have to establish powder tetrandra root tissue-culturing quick-propagation system
Important realistic meaning.
Specific embodiment
It the following examples are further illustrations of the invention, is not limitation of the present invention.
Embodiment 1:
(1) inducing clumping bud: the stem with bud of treelet current year life of the robust growth without disease pest is chosen as explant, uses dish washing liquid
Then aqueous solution soaking 6min rinses 11min with tap water, dry spare after surface moisture.In superclean bench with 75% second
Alcohol impregnates 9s, and 0.1% mercuric chloride solution sterilizes 12min, then with sterile water wash 5 times, and the stem section that length is 2.0cm is cut into after drying
It is inoculated into induced medium, it is first dark culture 18 days full under the conditions of 26 DEG C, it is subsequently placed in daily illumination 15 hours, intensity of illumination
For 900x, cultivation temperature cultivated under the conditions of being 26 DEG C 41 days can induced synthesis Multiple Buds, inductivity 68.8%.The induction
Culture medium is MS+3mg/L6-BA+0.7mg/L NAA+0.2mmol/L La (NO3)2+ 0.40% agar+0.06% of+3.0% sucrose is living
Property charcoal, pH value 5.9.
(2) Multiplying culture: step (1) resulting Multiple Buds are transferred on proliferated culture medium and carry out Multiplying culture, after inoculation
First dark culture full under the conditions of 26 DEG C germinates a lateral bud at axil, is subsequently placed in daily illumination 15 hours, intensity of illumination
For 900lx, cultivation temperature, which cultivates 34 days each Multiple Buds under conditions of being 26 DEG C, can be elongated to 5cm, proliferation rate 5.0.It is described
Proliferated culture medium is+0.07% active carbon of+0.40% agar of MS+2mg/L6-BA+0.2mg/L NAA+2.5% sucrose, and pH value is
5.9。
(3) culture of rootage: the Multiple Buds bud that step (1) or step (2) height obtained are about 5cm is cut and is inoculated with
Culture of rootage is carried out on to root media, it is first dark culture 6 days full under the conditions of 26 DEG C after inoculation, it is subsequently placed in daily illumination 8
Hour, intensity of illumination 2300lx, cultivation temperature is cultivated 42 days and can be taken root under conditions of being 26 DEG C, rooting rate 74.4%.
(4) acclimatization and transplants: after taking root 22 days in powder tetrandra root bottle, after being placed in natural lighting lower refining seedling 7 days, root culture is cleaned
Base is transplanted by yellow soil: in the matrix that river sand=3:2 is mixed into.Transplanting survival rate is 88%.
Embodiment 2:
(1) inducing clumping bud: the stem with bud of treelet current year life of the robust growth without disease pest is chosen as explant, uses dish washing liquid
Then aqueous solution soaking 10min rinses 20min with tap water, dry spare after surface moisture.In superclean bench with 75%
Ethyl alcohol impregnates 9s, and 0.1% mercuric chloride solution sterilizes 15min, then with sterile water wash 6 times, and the stem that length is 2.0cm is cut into after drying
Section is inoculated into induced medium, first dark culture 20 days full under the conditions of 26 DEG C, is subsequently placed in daily illumination 16 hours, illumination is strong
Degree be 1400x, cultivation temperature be 26 DEG C under the conditions of cultivate 35 days can induced synthesis Multiple Buds, inductivity 75.9%.It is described to lure
Leading culture medium is MS+7mg/L6-BA+1.0mg/L NAA+0.5mmol/L La (NO3)2+ 0.45% agar+0.15% of+2.5% sucrose
Active carbon, pH value 5.9.
(2) Multiplying culture: step (1) resulting Multiple Buds are transferred on proliferated culture medium and carry out Multiplying culture, after inoculation
First dark culture full under the conditions of 26 DEG C germinates a lateral bud at axil, is subsequently placed in daily illumination 17 hours, intensity of illumination
For 1800lx, cultivation temperature, which cultivates 33 days each Multiple Buds under conditions of being 26 DEG C, can be elongated to 5cm, proliferation rate 6.29.Institute
Stating proliferated culture medium is+0.2% active carbon of+0.36% agar of MS+1.8mg/L6-BA+0.9mg/L NAA+2.6% sucrose, and pH value is
5.9。
(3) culture of rootage: the Multiple Buds bud that step (1) or (2) height obtained are about 5cm is cut and is inoculated into life
Culture of rootage is carried out on root culture medium, it is first dark culture 5 days full under the conditions of 26 DEG C after inoculation, it is small to be subsequently placed in daily illumination 16
When, intensity of illumination 1900lx, cultivation temperature is cultivated 32 days and can be taken root under conditions of being 26 DEG C, rooting rate 90.9%.
(4) acclimatization and transplants: after taking root 25 days in powder tetrandra root bottle, after being placed in natural lighting lower refining seedling 6 days, root culture is cleaned
Base is transplanted by yellow soil: in the matrix that river sand=3:2 is mixed into.Transplanting survival rate is 91%.
Claims (1)
1. a kind of tissue culture technique of powder tetrandra root, it is characterised in that the following steps are included:
Step 1, inducing clumping bud: choosing robust growth without the treelet current year of disease pest raw stem with bud as explant, with washing
Clean essence aqueous solution soaking 14min, then rinses 35min with tap water, dry it is spare after surface moisture, in superclean bench with
70%~80% ethyl alcohol impregnates 14s, and 0.1% mercuric chloride solution sterilizes 25min, then with sterile water wash 8 times, being cut into length after drying is
The stem section of 1.5cm is inoculated into induced medium, first dark culture 25 days full under the conditions of 30 DEG C, and it is small to be subsequently placed in daily illumination 15
When, intensity of illumination 900lx, cultivation temperature is cultivated under the conditions of being 30 DEG C, until induced synthesis Multiple Buds；Induced medium are as follows:
MS+6mg/L6-BA+2mg/L NAA+0.9mmol/L La(NO3)2+ 0.15% active carbon of+0.9% agar of+3.9% sucrose, pH value are
5.9;
Step 2, Multiplying culture: the resulting Multiple Buds of step 1 are transferred on proliferated culture medium and carry out Multiplying culture, are first existed after inoculation
Full dark culture germinates a lateral bud at axil under the conditions of 30 DEG C, is subsequently placed in daily illumination 16 hours, intensity of illumination is
900lx, culture under conditions of cultivation temperature is 30 DEG C is until each Multiple Buds are elongated to 5cm；Proliferated culture medium are as follows: MS+3mg/L6-
+ 0.2% active carbon of+0.8% agar of BA+0.9mg/L NAA+3.9% sucrose, pH value 5.9;
Step 3, culture of rootage: the Multiple Buds bud that step 1 or step 2 height obtained are about 5cm is cut and is inoculated into life
Culture of rootage is carried out on root culture medium, it is first dark culture 10 days full under the conditions of 30 DEG C after inoculation, it is small to be subsequently placed in daily illumination 15
When, intensity of illumination 1900lx, cultivation temperature is cultivated under conditions of being 30 DEG C to taking root；Root media are as follows: 1/2MS+4mg/L
+ 0.2% active carbon of+0.9% agar of NAA+3.9% sucrose, pH value 5.9;
Step 4, acclimatization and transplants: after taking root 33 days in powder tetrandra root bottle, after being placed in natural lighting lower refining seedling 8 days, root culture is cleaned
Base is transplanted by yellow soil: in the matrix that river sand=3:2 is mixed into.
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| CN201811402019.3A CN109258472A (en) | 2018-11-22 | 2018-11-22 | A kind of tissue culture technique of powder tetrandra root |
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| CN201811402019.3A CN109258472A (en) | 2018-11-22 | 2018-11-22 | A kind of tissue culture technique of powder tetrandra root |
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| RU2399665C1 (en) * | 2009-03-10 | 2010-09-20 | Общество с ограниченной ответственностью "Скульт" | Method for preparing stephaglabrine sulphate in stephania glabra miers cell culture |
| CN104170732A (en) * | 2014-08-13 | 2014-12-03 | 安徽医科大学 | Seed tissue culture and propagation method of fourstamen stephania roots |
| CN104686341A (en) * | 2015-02-22 | 2015-06-10 | 梁仕华 | Tissue culture technique of aquilaria sinensis |
| CN107494282A (en) * | 2017-10-17 | 2017-12-22 | 李正美 | A kind of Yellowmouth Dutchmanspipe Root method for tissue culture |
| CN107873518A (en) * | 2017-12-11 | 2018-04-06 | 桂林亦元生现代生物技术有限公司 | A kind of tissue culture method of Fourstamen Stephania Root seedling |
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2018
- 2018-11-22 CN CN201811402019.3A patent/CN109258472A/en not_active Withdrawn
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| RU2399665C1 (en) * | 2009-03-10 | 2010-09-20 | Общество с ограниченной ответственностью "Скульт" | Method for preparing stephaglabrine sulphate in stephania glabra miers cell culture |
| CN104170732A (en) * | 2014-08-13 | 2014-12-03 | 安徽医科大学 | Seed tissue culture and propagation method of fourstamen stephania roots |
| CN104686341A (en) * | 2015-02-22 | 2015-06-10 | 梁仕华 | Tissue culture technique of aquilaria sinensis |
| CN107494282A (en) * | 2017-10-17 | 2017-12-22 | 李正美 | A kind of Yellowmouth Dutchmanspipe Root method for tissue culture |
| CN107873518A (en) * | 2017-12-11 | 2018-04-06 | 桂林亦元生现代生物技术有限公司 | A kind of tissue culture method of Fourstamen Stephania Root seedling |
Non-Patent Citations (1)
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| 闫志刚等: "天仙藤的组织培养及植株再生", 《植物生理学通讯》 * |
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Application publication date: 20190125 |