CN106613999B - The tissue culture and rapid propagation method of one seedling medicine Heiguteng exract - Google Patents
The tissue culture and rapid propagation method of one seedling medicine Heiguteng exract Download PDFInfo
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- CN106613999B CN106613999B CN201710147117.6A CN201710147117A CN106613999B CN 106613999 B CN106613999 B CN 106613999B CN 201710147117 A CN201710147117 A CN 201710147117A CN 106613999 B CN106613999 B CN 106613999B
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- seedling
- adventitious bud
- heiguteng exract
- explant
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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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- 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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- Developmental Biology & Embryology (AREA)
- Engineering & Computer Science (AREA)
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- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
The present invention relates to the methods of Plant Tissue Breeding in agricultural biotechnologies, specifically, it is related to the tissue culture and rapid propagation method of a seedling medicine Heiguteng exract, including the following steps carried out successively：The explant of acquisition Heiguteng exract, explant induction, the Multiplying culture of adventitious bud, the culture of rootage of adventitious bud, test tube transplantation of seedlings；The inducing culture of the described explant induction includes：MS culture mediums, 3.0~5.0mg/L KT, 1.5~2.0mg/L, 6 BA, 20~30g/L sucrose, 3.5~4.0g/L agar, 0.5~1.0g/L activated carbons, pH is 5.4~5.8, and the seedling medicine Heiguteng exract test tube seedling transplanting survival rate of method of the invention reaches 95% or more.
Description
Technical field
The present invention relates to the methods of Plant Tissue Breeding in agricultural biotechnologies, specifically, being related to seedling medicine Heiguteng exract
Tissue culture and rapid propagation method.
Background technology
Seedling medicine Heiguteng exract (Periploca forrestil Schltr) also known as black keel, Yunnan periploca spium etc. are Asclepiadaceae thick stick
Salix rattan shape shrub, for the civil people for being widely used in treating the diseases such as closed soft tissue injury, rheumatism and rheumatoid of Miao ethnic group
Race's medicine, rich in substances such as C21 steroids, cardiac glycosides, terpenes.Heiguteng exract is with a long history as a seedling medicine, early in the Western Han Dynastry
Liu to《Shi Shuo Xin Yu says that object is distinguished in garden》In just have：" that I hears Gu is doctor, says seedling father, the skill of a doctor is consummate, and prescription is unique, is generation
The record in the roads Ren Cheng ", the Qing Dynasty《Maguan county annals custom piece》In just have the description of " the Miaos' good medicine synthetism wind-dispelling, effect as god ".By
In the regular buccal Heiguteng exract of Miao ethnic group people, therefore people from Miao ethnic group is not susceptible to suffer from rheumatism.Heiguteng exract has been developed at present chases after wind work
Network capsule, tendon-extending bone-penetrating liquid etc. treat rheumatism Chinese patent drug.
The tissue cultures correlative study of Heiguteng exract is had no in view of the current country and is had been reported that, the present invention selects Heiguteng exract belt segment
Stem section is explant, through induction, is proliferated, takes root, hardening and transplanting and other steps, establishes the tissue cultures skill of seedling medicine Heiguteng exract
Art system.The present invention have the characteristics that it is technical it is strong, of low cost, seedling is fast, can be directly used for the batch production of Heiguteng exract seedling
Production, for promoting the resources development and utilization of seedling medicine Heiguteng exract to have important practical significance.
Currently, at home and abroad there is no the reports of Heiguteng exract tissue culture technique, there is not Heiguteng exract tissue-culturing rapid propagation patent yet
Application.
Invention content
The object of the present invention is to provide the tissue culture and rapid propagation method of a seedling medicine Heiguteng exract, the present invention selects Heiguteng exract belt segment stem
Section is explant, through induction, is proliferated, takes root, hardening and transplanting and other steps, establishes the tissue culture technique of seedling medicine Heiguteng exract
System, to realize the purpose of the present invention.
Technical solution provided by the invention is：The tissue culture and rapid propagation method of one seedling medicine Heiguteng exract, including carry out successively as
Lower step：The explant of acquisition Heiguteng exract, explant induction, the Multiplying culture of adventitious bud, the culture of rootage of adventitious bud, test tube seedling
Transplanting；
The inducing culture of the described explant induction includes：MS culture mediums, 3.0~5.0mg/L KT, 1.5~2.0mg/
L6-BA, 20~30g/L sucrose, 3.5~4.0g/L agar, 0.5~1.0g/L activated carbons, pH are 5.4~5.8.
In the tissue culture and rapid propagation method of above-mentioned seedling medicine Heiguteng exract, the method for explant induction is：By Heiguteng exract
Explant is cleaned disinfect after be cut into the belt segment rattan of 1.5~2.5cm or so and be inoculated into inducing culture, be placed in 25
~28 DEG C of full light cultures of progress 30~35 days can induced synthesis adventitious bud.
In the tissue culture and rapid propagation method of above-mentioned seedling medicine Heiguteng exract, the proliferated culture medium packet used in the Multiplying culture of adventitious bud
It includes：MS culture mediums, 2.0~3.0mg/L KT, 0.5~1.0mg/L NAA, 20~30g/L sucrose, 3.5~4.0g/L agar,
0.1~0.5g/L activated carbons, pH are 5.4~5.8.
In the tissue culture and rapid propagation method of above-mentioned seedling medicine Heiguteng exract, the method for the Multiplying culture of the adventitious bud is specific
For：The adventitious bud induced by explant is cut into be about the stem section of 1~1.5cm and be inoculated into proliferated culture medium and is cultivated
25~30 days proliferation that adventitious bud can be realized.
In the tissue culture and rapid propagation method of above-mentioned seedling medicine Heiguteng exract, the root media packet used in the culture of rootage of adventitious bud
It includes：1/2MS culture mediums, 1.0~2.0mg/L GGR, 0.5~1.0mg/L NAA, 15~20g/L sucrose, 3.0~4.0g/L fine jades
Fat, 0.1~0.3g/L activated carbons, pH are 5.4~5.8.
In the tissue culture and rapid propagation method of above-mentioned seedling medicine Heiguteng exract, the method for the culture of rootage of the adventitious bud is：From
The base portion of the adventitious bud obtained by Multiplying culture cuts the adventitious bud for being about 1.5~2.0cm and is inoculated into root media
Culture can realize taking root for test tube seedling in 25~30 days.
In the tissue culture and rapid propagation method of above-mentioned seedling medicine Heiguteng exract, the method for the test tube transplantation of seedlings is specially：It will be through
Well-grown test tube seedling that culture of rootage obtains is crossed in the natural light lower refining seedling 1~3 day in greenhouse, the culture medium for cleaning root moves
It is 1 to plant in volume ratio:Cultivation seedling is up to seedling in 1 fertile soil, coconut chaff mixed-matrix.
In the tissue culture and rapid propagation method of above-mentioned seedling medicine Heiguteng exract, the explant induction, adventitious bud Multiplying culture
Condition of culture be：Cultivation temperature is 25~28 DEG C, and intensity of illumination is 2000~3000lx, and light application time is 10~12 small
When/day.
In the tissue culture and rapid propagation method of above-mentioned seedling medicine Heiguteng exract, the specific method of the explant of Heiguteng exract is obtained
For：Robust growth, the Heiguteng exract plant middle and upper part without apparent disease are chosen in the wild, the substantial belt segment rattan that faces south is explant
Body carries out water conservation moisturizing processing immediately after acquisition.
Advantageous effect：
The present invention establishes the tissue culture technique system of seedling medicine Heiguteng exract, present invention tool using plant tissue culture technique
There is the features such as technical strong, of low cost, seedling is fast, survival rate reaches 95% or more, can be directly used for Heiguteng exract seedling
It is factory produced, for promoting the resources development and utilization of seedling medicine Heiguteng exract to have important practical significance.
Specific implementation mode
With reference to embodiment, technical scheme of the present invention is described in further detail, but do not constituted pair
Any restrictions of the present invention.
Embodiment one
(1) explant acquires：Robust growth is chosen in the wild, the Heiguteng exract plant middle and upper part without apparent disease, is filled on the sunny side
Real belt segment rattan is explant, carries out water conservation moisturizing after acquisition immediately and handles and take back laboratory in time.
(2) explant induces：When step (1) acquires back laboratory explant first night flushed under tap water, it is placed in super
Sterilize 15 seconds in 75% ethanol solution in net workbench, after using aseptic filter paper suck dry moisture after aseptic water washing 5 times, sets
Sterilized 15 minutes in 0.1% mercuric chloride solution, after aseptic water washing 5 times be cut into 1.5 after aseptic filter paper suck dry moisture~
The belt segment rattan of 2.5cm or so is simultaneously inoculated into inducing culture, and being placed in 25 DEG C of full light cultures of progress 30 days can induced synthesis
Adventitious bud, inductivity 93.1%, pollution rate are less than 10%.The inducing culture is：MS culture medium+5.0mg/L KT+
2.0mg/L 6-BA+20g/L sucrose+3.5g/L agar+0.5g/L activated carbons, pH 5.4.
(3) adventitious bud proliferation：The adventitious bud that step (2) induction obtains is cut into and is about the stem section of 1~1.5cm and is inoculated into
The 25 days proliferation that adventitious bud can be realized is cultivated in proliferated culture medium, growth coefficient reaches 6 times.The proliferated culture medium is：
MS culture medium+3.0mg/L KT+1.0mg/L NAA+20g/L sucrose+3.5g/L agar+0.1g/L activated carbons, pH 5.4.
(4) culture of rootage：Step (3) the obtained adventitious bud for being about 1.5~2.0cm of proliferation is cut from base portion and is inoculated into
Taking root for test tube seedling can be realized by being cultivated 25 days in root media, and rooting rate reaches 93% or more.The root media
For：1/2MS culture medium+2.0mg/L GGR (root-inducing powder)+1.0mg/L NAA+15g/L sucrose+3.0g/L agar+0.1g/L lives
Property charcoal, pH 5.4.
(5) it transplants：Natural light lower refining seedling 1 day by the good test tube seedling of step (4) root growth in greenhouse cleans root
Culture medium transplanting in volume ratio be 1:Cultivation seedling is up to seedling in 1 fertile soil, coconut chaff mixed-matrix, after transplanting 30 days
Survival rate reaches 95% or more.
The condition of culture of above-mentioned steps (3)~(4) is：Cultivation temperature is 25 DEG C, intensity of illumination 2000lx, light application time
For 10 hours/day.
Embodiment two
(1) explant acquires：Robust growth is chosen in the wild, the Heiguteng exract plant middle and upper part without apparent disease, is filled on the sunny side
Real belt segment rattan is explant, carries out water conservation moisturizing after acquisition immediately and handles and take back laboratory in time.
(2) explant induces：When step (1) acquires back laboratory explant first night flushed under tap water, it is placed in super
Sterilize 17 seconds in 75% ethanol solution in net workbench, after using aseptic filter paper suck dry moisture after aseptic water washing 6 times, sets
Sterilized 18 minutes in 0.1% mercuric chloride solution, after aseptic water washing 6 times be cut into 1.5 after aseptic filter paper suck dry moisture~
The belt segment rattan of 2.5cm or so is simultaneously inoculated into inducing culture, and being placed in 27 DEG C of full light cultures of progress 33 days can induced synthesis
Adventitious bud, inductivity 90%, pollution rate are less than 8%.The inducing culture is：MS culture medium+4.0mg/L KT+
1.7mg/L 6-BA+25g/L sucrose+3.7g/L agar+0.8g/L activated carbons, pH 5.6.
(3) adventitious bud proliferation：The adventitious bud that step (2) induction obtains is cut into and is about the stem section of 1~1.5cm and is inoculated into
The 28 days proliferation that adventitious bud can be realized is cultivated in proliferated culture medium, growth coefficient reaches 5.3 times.The proliferated culture medium
For：MS culture medium+2.5mg/L KT+0.8mg/L NAA+25g/L sucrose+3.7g/L agar+0.3g/L activated carbons, pH 5.6.
(4) culture of rootage：Step (3) the obtained adventitious bud for being about 1.5~2.0cm of proliferation is cut from base portion and is inoculated into
Taking root for test tube seedling, rooting rate 91.5% can be realized by being cultivated 27 days in root media.The root media is：1/
2MS culture medium+1.5mg/L GGR (root-inducing powder)+0.7mg/L NAA+18g/L sucrose+3.5g/L agar+0.2g/L activated carbons,
PH is 5.6.
(5) it transplants：Natural light lower refining seedling 2 days by the good test tube seedling of step (4) root growth in greenhouse cleans root
Culture medium transplanting in volume ratio be 1:Cultivation seedling is up to seedling in 1 fertile soil, coconut chaff mixed-matrix, after transplanting 30 days
Survival rate is 96.7%.
The condition of culture of above-mentioned steps (3)~(4) is：Cultivation temperature is 26 DEG C, intensity of illumination 2300lx, light application time
For 11 hours/day.
Embodiment three
(1) explant acquires：Robust growth is chosen in the wild, the Heiguteng exract plant middle and upper part without apparent disease, is filled on the sunny side
Real belt segment rattan is explant, carries out water conservation moisturizing after acquisition immediately and handles and take back laboratory in time.
(2) explant induces：When step (1) acquires back laboratory explant first night flushed under tap water, it is placed in super
Sterilize 20 seconds in 75% ethanol solution in net workbench, after using aseptic filter paper suck dry moisture after aseptic water washing 7 times, sets
Sterilized 20 minutes in 0.1% mercuric chloride solution, after aseptic water washing 7 times be cut into 1.5 after aseptic filter paper suck dry moisture~
The belt segment rattan of 2.5cm or so is simultaneously inoculated into inducing culture, and being placed in 28 DEG C of full light cultures of progress 35 days can induced synthesis
Adventitious bud, inductivity 87.3%, pollution rate are less than 3%.The inducing culture is：MS culture medium+3.0mg/L KT+
1.5mg/L 6-BA+30g/L sucrose+4.0g/L agar+0.5~1.0g/L activated carbons, pH 5.8.
(3) adventitious bud proliferation：The adventitious bud that step (2) induction obtains is cut into and is about the stem section of 1~1.5cm and is inoculated into
The 30 days proliferation that adventitious bud can be realized is cultivated in proliferated culture medium, growth coefficient reaches 4 times.The proliferated culture medium is：
MS culture medium+2.0mg/L KT+0.5mg/L NAA+30g/L sucrose+4.0g/L agar+0.5g/L activated carbons, pH 5.8.
(4) culture of rootage：Step (3) the obtained adventitious bud for being about 1.5~2.0cm of proliferation is cut from base portion and is inoculated into
Taking root for test tube seedling can be realized by being cultivated 30 days in root media, and rooting rate reaches 90% or more.The root media
For：1/2MS culture medium+1.5mg/L GGR (root-inducing powder)+0.5mg/L NAA+20g/L sucrose+4.0g/L agar+0.3g/L lives
Property charcoal, pH 5.8.
(5) it transplants：Natural light lower refining seedling 3 days by the good test tube seedling of step (4) root growth in greenhouse cleans root
Culture medium transplanting in volume ratio be 1:Cultivation seedling is up to seedling in 1 fertile soil, coconut chaff mixed-matrix, after transplanting 30 days
Survival rate reaches 95% or more.
The condition of culture of above-mentioned steps (3)~(4) is：Cultivation temperature is 28 DEG C, intensity of illumination 2500lx, light application time
For 12 hours/day.
The above embodiment is a preferred embodiment of the present invention, but embodiments of the present invention are not by above-described embodiment
Limitation, it is other it is any without departing from the spirit and principles of the present invention made by changes, modifications, substitutions, combinations, simplifications,
Equivalent substitute mode is should be, is included within the scope of the present invention.
Claims (7)
1. the tissue culture and rapid propagation method of a seedling medicine Heiguteng exract, it is characterised in that：Including the following steps carried out successively：Obtain black bone
Rattan plant middle and upper part, belt segment rattan substantial on the sunny side are explant, the proliferation of explant induced synthesis adventitious bud, adventitious bud is trained
The foster, culture of rootage of adventitious bud, test tube transplantation of seedlings；
The inducing culture of the described explant induction includes：MS culture mediums, 3.0~5.0mg/L KT, 1.5~2.0mg/L 6-
BA, 20~30g/L sucrose, 3.5~4.0g/L agar, 0.5~1.0g/L activated carbons, pH are 5.4~5.8；The adventitious bud
Proliferated culture medium used in Multiplying culture includes：MS culture mediums, 2.0~3.0mg/L KT, 0.5~1.0mg/L NAA, 20~
30g/L sucrose, 3.5~4.0g/L agar, 0.1~0.5g/L activated carbons, pH are 5.4~5.8；The culture of rootage of the adventitious bud
Root media used includes：1/2MS culture mediums, 1.0~2.0mg/L GGR, 0.5~1.0mg/L NAA, 15~20g/L
Sucrose, 3.0~4.0g/L agar, 0.1~0.3g/L activated carbons, pH are 5.4~5.8.
2. the tissue culture and rapid propagation method of seedling medicine Heiguteng exract according to claim 1, it is characterised in that：The explant induction
Method be：By Heiguteng exract explant is cleaned disinfect after be cut into the belt segment rattan of 1.5~2.5cm and be inoculated into induction training
It supports in base, being placed in 25~28 DEG C of full light cultures of progress 30~35 days can induced synthesis adventitious bud.
3. the tissue culture and rapid propagation method of seedling medicine Heiguteng exract according to claim 1, it is characterised in that：The increasing of the adventitious bud
The method for growing culture is specially：The adventitious bud induced by explant is cut into the stem section of a length of 1~1.5cm and is inoculated into
25~30 days proliferation that adventitious bud can be realized is cultivated in proliferated culture medium.
4. the tissue culture and rapid propagation method of seedling medicine Heiguteng exract according to claim 1, it is characterised in that：The life of the adventitious bud
The method of root culture is：The adventitious bud of a length of 1.5~2.0cm is cut from the base portion of the adventitious bud obtained by Multiplying culture and is connect
Taking root for test tube seedling can be realized by being cultivated 25~30 days in kind to root media.
5. the tissue culture and rapid propagation method of seedling medicine Heiguteng exract according to claim 1, it is characterised in that：The test tube transplantation of seedlings
Method be specially：Natural light lower refining seedling 1~3 day by the well-grown test tube seedling obtained by culture of rootage in greenhouse,
The culture medium transplanting of clean root is 1 in volume ratio:Cultivation seedling is up to seedling in 1 fertile soil, coconut chaff mixed-matrix.
6. the tissue culture and rapid propagation method of seedling medicine Heiguteng exract according to any one of claims 1 to 5, it is characterised in that：Described is outer
Implant induces, the condition of culture of the Multiplying culture of adventitious bud is：Cultivation temperature be 25~28 DEG C, intensity of illumination be 2000~
3000lx, light application time are 10~12 hours/day.
7. the tissue culture and rapid propagation method of seedling medicine Heiguteng exract according to any one of claims 1 to 5, it is characterised in that：It obtains
The specific method of the explant of Heiguteng exract is：In the wild choose robust growth, the Heiguteng exract plant middle and upper part without apparent disease, to
The substantial belt segment rattan of sun is explant, carries out water conservation moisturizing processing after acquisition immediately.
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| CN109302984B (en) * | 2018-09-20 | 2022-03-01 | 河北农业大学 | Periploca sepium rapid propagation method |
| CN115005097A (en) * | 2022-05-26 | 2022-09-06 | 桂林医学院 | Rapid propagation method of Yao medicine Mallotus japonicus |
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