CN109329052A - Aristolochia contorta tissue-cultured seedling expanding propagation method - Google Patents
Aristolochia contorta tissue-cultured seedling expanding propagation method Download PDFInfo
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- CN109329052A CN109329052A CN201811174107.2A CN201811174107A CN109329052A CN 109329052 A CN109329052 A CN 109329052A CN 201811174107 A CN201811174107 A CN 201811174107A CN 109329052 A CN109329052 A CN 109329052A
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- aristolochia contorta
- culture medium
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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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- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
The invention discloses Aristolochia contorta tissue-cultured seedling expanding propagation methods, and using tender explant, by adjusting hormone ratio, the optimal medium formula of optimization Aristolochia contorta tissue-cultured seedling growth establishes the method system of a set of Aristolochia contorta tissue culture seedling and propagating.It using Aristolochia contorta tissue-cultured seedling expanding propagation method of the invention, can breed to obtain the consistent seedling of a large amount of characters in a short time, meet production needs, and provide reference to provide the Aristolochia contorta industrial seedling rearing of merit.
Description
Technical field
The present invention relates to Plant Tissue Breeding reproduction technique fields, specifically, being related to Aristolochia contorta tissue culture seedling and propagating side
Method.
Background technique
Birthwort [Aristolochia debilis Sieb.et Zucc.] is aristolochiaceae plant Aristolochia contorta or horse
There is disappear cough, the gut purge of clearing lung-heat sending down abnormally ascending, Dingchuan to disappear mole and other effects, be used for lung heat cough and asthma, phlegm for the dry mature fruit of pocket bell, bitter
The illnesss such as middle band blood (Pharmacopoeia of the People's Republic of China, version in 2015).Herba aristolochiae [Fibraurea recisa Pierre] is
There is the branches and leaves of aristolochiaceae plant Aristolochia contorta or birthwort anti-malarial and antineoplastic action (to help religion dragon, Liu Jia, Wu Chen
Surprise waits herba aristolochiae chemical component to isolate and purify research Shanghai Agricultural journal, 2014,30 (6): 116-119.).But birthwort
Main component aristolochic acid can lead to birthwort nephrosis, kidney region fibrosis, upper bladder transitional cell carcinoma etc. (Zhang Ying, Xiao Rong, Huang Jie,
Equal different sources birthwort honey toast front and back HPLC fingerprint map analyzing Chinese Pharmaceutical Journal, 2017,52 (16): 1397-
1402).For Aristolochic acid nephropathy caused by aristolochic acid substance, processing means is mostly used to reach " detoxification deposits effect " at present
Purpose, main includes the methods of sweet toast, vinegar toast, salt toast, ginger toast, alkali system, stir-bake to brown.The content of birthwort aristolochic acid after honey toast
51%~55% is reduced compared with health product, reducing toxicity, (Zhang Jinlian, the phoenix opened, Luo Wenhua wait the preferred freeze-day with constant temperature of orthogonal experiment
Treasure's traditional Chinese medical science traditional Chinese medicines when method honey processs birthwort toxicity reduction processing procedure, 2007,18 (11): 2710-2711).Equal chemically angle above
The research that birthwort processes attenuation is inquired into, fails to thoroughly eliminate birthwort toxicity.
In recent years due to excessively excavating, wild Aristolochia contorta resource is made to face threat.For the wild money for protecting this kind of plant
Source reinforces the development and utilization to Aristolochia contorta, need to widely popularize the cultivating and growing of Aristolochia contorta, to realize its artificial cultivation, first
Solve the problems, such as be exactly seedling breeding, and utilize tissue culture technique, its reproduction speed can be accelerated, meet seedling market
Needs.
Summary of the invention
The object of the present invention is to provide a kind of Aristolochia contorta tissue-cultured seedling expanding propagation methods.
Present inventive concept is as follows: aseptically, using tender explant, by adjusting hormone ratio, optimization north
The optimal medium formula of birthwort tissue-cultured seedling growth, establishes the method system of a set of Aristolochia contorta tissue culture seedling and propagating.
In order to achieve the object of the present invention, in a first aspect, the present invention provides a kind of proliferation for Aristolochia contorta tissue cultures
Culture medium, the proliferated culture medium are to add 0.4mg/L IBA, 1mg/ using 4.3-4.5g/L MS culture medium as minimal medium
L KT, 1mg/L 6-BA, 28-30g/L sucrose and 7.0-8.0g/L agar and formed, adjust pH 5.8-6.2.
Preferably, the formula of the proliferated culture medium are as follows: MS 4.43g/L+IBA 0.4mg/L+KT 1.0mg/L+6-BA
1..0mg/L+ sucrose 30.0g/L+ agar 7.0g/L, pH 6.0.
Second aspect, the present invention provide application of the proliferated culture medium in Aristolochia contorta tissue cultures.
The third aspect, the present invention provide the preparation method of the proliferated culture medium.The following steps are included:
1) MS minimal medium is prepared；
2) each hormone mother liquor is prepared:
IBA mother liquor: weighing IBA (indolebutyric acid) 10mg, is first dissolved in a small amount of (≤1mL) ethyl alcohol, adds 10mL water constant volume, be made into
1mg/mL solution is saved in -20 DEG C；
KT mother liquor: weighing KT (6- glycosyl adenine phosphate) 10mg, and first plus on a small quantity (≤1mL) 1mol/L HCl solution dissolves
Afterwards, add 10mL water constant volume, be made into 1mg/mL solution, saved in -20 DEG C；
6-BA mother liquor: weighing 6-BA (6-benzyladenine) 10mg, and first plus on a small quantity (≤1mL) 1mol/L HCl solution dissolves
Afterwards, add 10mL water constant volume, be made into 1mg/mL solution, saved in -20 DEG C；
3) it is proportionally added into above-mentioned each hormone mother liquor into minimal medium, is eventually adding agar, adjusts pH5.8-6.2, it is high
After temperature sterilizing (120-125 DEG C of sterilizing 20-30min), cooling, inverted plate to obtain the final product.
Fourth aspect, the present invention provide a kind of Aristolochia contorta tissue-cultured seedling expanding propagation method, are with the bud of Aristolochia contorta, stem
Point, blade or stem with bud are explant, are inoculated into the proliferated culture medium after cleaning and sterilizing, in 22-28 DEG C of alternation of light and darkness
Culture 28-35 days.
Further, condition of culture are as follows: 25 DEG C, daily illumination, dark each 12h.
In the present invention, intensity of illumination 1500-2000lux.
Cleaning and sterilizing method particularly includes: explant is rinsed with clear water, the moisture on explant surface is blotted after cleaning；With
75% alcohol impregnates 30-40s, is subsequently placed in 0.1%HgCl210-12min is impregnated, is rinsed 4~5 times with aqua sterilisa, blots explant
The moisture in body surface face can be used to be inoculated with.
Further, materials object is Aristolochia contorta bud, cleaning and sterilizing method particularly includes: take bud, rinsed with clear water, washed
The moisture on sprout surface is blotted after net；30-40s is impregnated with 75% alcohol, is subsequently placed in 0.1%HgCl210-12min is impregnated, is used
Aqua sterilisa rinses 4~5 times, blots the moisture on sprout surface, can be used to be inoculated with.
In the present invention, MS minimal medium is referring to Liao Weiwei, and Xie Junfeng, pay is green, and is waited to do taking root for hilllock birthwort tissue-cultured seedling
Cultivate tropical agricultural science, 2014,34 (7), 65-68.
By above-mentioned technical proposal, the present invention at least have following advantages and the utility model has the advantages that
(1) Aristolochia contorta tissue-cultured seedling expanding propagation method of the invention is used, can breed to obtain a large amount of characters one in a short time
The seedling of cause meets production needs, and provides reference to provide the Aristolochia contorta industrial seedling rearing of merit.
(2) the culture medium prescription system of optimization Aristolochia contorta tissue-cultured seedling growth provided by the invention are as follows: 4.3-4.5g/L
MS+IBA 0.4mg/L+KT 1mg/L+6-BA 1mg/L+28-30g/L sucrose+7.0-8.0g/L agar.Under the formula system,
Aristolochia contorta tissue-cultured seedling healthy appearance, the speed of growth is fast, and growth cycle (about 30 days) Tissue vaccine biomass is up to 11.63g
Left and right.
Detailed description of the invention
Fig. 1 is Aristolochia contorta tissue-cultured seedling growing state in the embodiment of the present invention 1.Wherein, A is 6 days pictures of growth；B makes a living
Long 12 days pictures；C is 18 days pictures of growth；D is 24 days pictures of growth；E is 30 days pictures of growth.
Specific embodiment
The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention..Unless otherwise specified, embodiment
Used in the conventional means that are well known to those skilled in the art of technological means, raw materials used is commercial goods.
IBA (CAS:133-32-4) used in the following embodiment, KT (CAS:K3378), 6-BA (CAS:B5478) are purchased
From Sigma company.
1 Aristolochia contorta tissue-cultured seedling expanding propagation method of embodiment
1, the proliferated culture medium for Aristolochia contorta tissue cultures
The proliferation culture medium formula are as follows: MS 4.43g/L+IBA 0.4mg/L+KT 1.0mg/L+6-BA 1..0mg/L+
Sucrose 30.0g/L+ agar 7.0g/L, pH 6.0.
Preparation method is as follows:
1) MS minimal medium is prepared；
2) each hormone mother liquor is prepared:
IBA mother liquor: precision weighs IBA 10mg, is first dissolved in a small amount of (about 500 μ L) ethyl alcohol, adds 10mL water constant volume, be made into
1mg/mL solution is saved in -20 DEG C；
KT mother liquor: precision weighs KT 10mg, first plus on a small quantity after the dissolution of (about 500 μ L) 1mol/L HCl solution, adds 10mL water
Constant volume is made into 1mg/mL solution, saves in -20 DEG C；
6-BA mother liquor: precision weighs 6-BA 10mg, first plus on a small quantity after the dissolution of (about 500 μ L) 1mol/L HCl solution, adds
10mL water constant volume, is made into 1mg/mL solution, saves in -20 DEG C；
3) it is proportionally added into above-mentioned each hormone mother liquor into minimal medium, is eventually adding agar, adjusts pH6.0, high temperature goes out
After bacterium (120-125 DEG C of sterilizing 20-30min), cooling, inverted plate to obtain the final product.
2, Aristolochia contorta tissue-cultured seedling expanding propagation method
It uses the bud of Aristolochia contorta for explant, filters out the optimal tissue-cultured seedling culture formula system of Aristolochia contorta, establish
A kind of method system of Aristolochia contorta tissue culture seedling and propagating.Explant disinfectant program: taking bud, and tap water sufficiently rinses, and clear water is cleaned
The moisture on surface is blotted with blotting paper afterwards；30s is impregnated with 75% alcohol, is subsequently placed in 0.1%HgCl2Middle immersion 10min, with going out
Bacterium water rinses 4~5 times and thoroughly cleans remaining mercury ion；The moisture for blotting surface with blotting paper again, is connected to equipped with above-mentioned proliferation
In the plate of culture medium.It is 25 DEG C that incubator temperature, which is arranged, alternation of light and darkness culture, daily illumination, dark each 12h, intensity of illumination
For 1500-2000lux.Count Aristolochia contorta tissue-cultured seedling increment.
Fig. 1 is Aristolochia contorta tissue-cultured seedling growing state.It can be seen from figure 1 that the tissue-cultured seedling growth of induction is preferably, 6 days whens, grow
Tender seedling, about 1~2cm out；Tissue-cultured seedling grows into 3~4cm at 12 days；Tissue-cultured seedling grows into 4~5cm at 18 days；24 days
When tissue-cultured seedling grow into 6~7cm；Tissue-cultured seedling grows into 8~9cm at 30 days.
Influence of the above-mentioned proliferated culture medium to tissue-cultured seedling increment is as shown in table 1, and biomass repeats to be respectively as follows: three times
11.1g、12.7g、11.1g。
The increment of 1 Aristolochia contorta tissue-cultured seedling of table
The result shows that Aristolochia contorta tissue-cultured seedling healthy appearance, the speed of growth is fast under the formula system, a growth cycle
(about 30 days) Tissue vaccine biomass is up to 11.63g or so.
Take other positions (such as stem apex, blade or stem with bud) equal indifference in tissue culture effect.
The optimization of 2 Aristolochia contorta tissue-cultured seedling of embodiment expansion traditional font system
The present embodiment is by carrying out appropriate adjustment (table 2) to the hormon and proportion that add in proliferated culture medium, in turn
It proves to be MS+IBA 0.4mg/L+KT 1.0mg/L+6-BA 1.0mg/L using formula, sucrose 3%, agar 0.8%, pH6's
Proliferated culture medium carries out tissue culture, and callus goes out faster, vigorous, the no browning of growth, most suitable Aristolochia contorta tissue-cultured seedling
Expand numerous.Specific experiment method is the same as embodiment 1.
2 Aristolochia contorta tissue-cultured seedling formulation optimization of table
Wherein, each hormone mother liquor method method is as follows:
2,4-D mother liquors: weighing 2,4-D (2,4- dichlorphenoxyacetic acid) 10mg, is first dissolved in a small amount of (about 500 μ L) 1mol/L
After NaOH solution dissolution, add 10mL water constant volume, be made into 1mg/mL solution, is saved in -20 DEG C.
NAA mother liquor: weighing NAA (methyl α-naphthyl acetate) 10mg, is first dissolved in a small amount of (about 500 μ L) ethyl alcohol, adds 10mL water constant volume, be made into
1mg/mL solution is saved in -20 DEG C.
Although above the present invention is described in detail with a general description of the specific embodiments,
On the basis of the present invention, it can be modified or is improved, this will be apparent to those skilled in the art.Cause
This, these modifications or improvements, fall within the scope of the claimed invention without departing from theon the basis of the spirit of the present invention.
Claims (8)
1. being used for the proliferated culture medium of Aristolochia contorta tissue cultures, which is characterized in that the proliferated culture medium is with 4.3-4.5g/
L MS culture medium is minimal medium, addition 0.4mg/L IBA, 1mg/L KT, 1mg/L 6-BA, 28-30g/L sucrose and 7.0-
8.0g/L agar and formed, adjust pH5.8-6.2.
2. proliferated culture medium according to claim 1, which is characterized in that formula are as follows: MS 4.43g/L+IBA 0.4mg/L+
KT 1.0mg/L+6-BA 1..0mg/L+ sucrose 30.0g/L+ agar 7.0g/L, pH 6.0.
3. application of the proliferated culture medium as claimed in claim 1 or 2 in Aristolochia contorta tissue cultures.
4. Aristolochia contorta tissue-cultured seedling expanding propagation method, which is characterized in that be with the bud of Aristolochia contorta, stem apex, blade or stem with bud
Explant is inoculated into proliferated culture medium as claimed in claim 1 or 2 after cleaning and sterilizing, in 22-28 DEG C of alternation of light and darkness culture 28-
35 days.
5. according to the method described in claim 4, it is characterized in that, condition of culture are as follows: 25 DEG C, daily illumination, dark each 12h.
6. according to the method described in claim 4, it is characterized in that, intensity of illumination is 1500-2000lux.
7. according to the described in any item methods of claim 4-6, which is characterized in that cleaning and sterilizing method particularly includes: by explant
Body is rinsed with clear water, and the moisture on explant surface is blotted after cleaning；30-40s is impregnated with 75% alcohol, is subsequently placed in 0.1%
HgCl210-12min is impregnated, is rinsed 4~5 times with aqua sterilisa, is blotted the moisture on explant surface, can be used to be inoculated with.
8. the method according to the description of claim 7 is characterized in that cleaning and sterilizing method particularly includes: take bud, rushed with clear water
It washes, the moisture on sprout surface is blotted after cleaning；30s is impregnated with 75% alcohol, is subsequently placed in 0.1%HgCl210min is impregnated, is used
Aqua sterilisa rinses 4~5 times, blots the moisture on sprout surface, can be used to be inoculated with.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201811174107.2A CN109329052A (en) | 2018-10-09 | 2018-10-09 | Aristolochia contorta tissue-cultured seedling expanding propagation method |
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| Application Number | Priority Date | Filing Date | Title |
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| CN201811174107.2A CN109329052A (en) | 2018-10-09 | 2018-10-09 | Aristolochia contorta tissue-cultured seedling expanding propagation method |
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101288381A (en) * | 2007-04-18 | 2008-10-22 | 天津市园林绿化研究所 | Collective medium for flower tissue culture |
| CN103168693A (en) * | 2013-04-12 | 2013-06-26 | 广西橡胶研究所 | Proliferation culture medium for aristolochia longgonensis tissue culture and application of proliferation culture medium |
| CN105265320A (en) * | 2015-11-18 | 2016-01-27 | 广西壮族自治区药用植物园 | Tissue culture propagation method for herba aristolochia mollissima |
-
2018
- 2018-10-09 CN CN201811174107.2A patent/CN109329052A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101288381A (en) * | 2007-04-18 | 2008-10-22 | 天津市园林绿化研究所 | Collective medium for flower tissue culture |
| CN103168693A (en) * | 2013-04-12 | 2013-06-26 | 广西橡胶研究所 | Proliferation culture medium for aristolochia longgonensis tissue culture and application of proliferation culture medium |
| CN105265320A (en) * | 2015-11-18 | 2016-01-27 | 广西壮族自治区药用植物园 | Tissue culture propagation method for herba aristolochia mollissima |
Non-Patent Citations (2)
| Title |
|---|
| 昝琦等: "北马兜铃再生系建立的研究", 《农业与技术》 * |
| 邵红等: "北马兜铃组培快繁技术", 《北方园艺》 * |
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Application publication date: 20190215 |