CN106922539A - A kind of tissue culture and rapid propagation method of Chinese medicine cissus assamica - Google Patents
A kind of tissue culture and rapid propagation method of Chinese medicine cissus assamica Download PDFInfo
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- CN106922539A CN106922539A CN201710294063.6A CN201710294063A CN106922539A CN 106922539 A CN106922539 A CN 106922539A CN 201710294063 A CN201710294063 A CN 201710294063A CN 106922539 A CN106922539 A CN 106922539A
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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 present invention relates to the method for Plant Tissue Breeding in agricultural biotechnologies, specifically, it is related to a kind of tissue culture and rapid propagation method of Chinese medicine cissus assamica, including the following steps for carrying out successively：Obtain explant, explant Fiber differentiation, squamous subculture, culture of rootage, the transplanting of cissus assamica；The explant inducing culture of described explant Fiber differentiation operation includes：MS culture mediums, the BA of 2.0~4.0mg/L 6,0.5~1.0mg/L NAA, 20~30g/L sucrose, 3.5~4.0g/L agar, 60~100ml/L coconut milk, 0.1~0.3g/L activated carbons, explant inducing culture pH is 5.4~5.8, present invention selection cissus assamica stem-segment with node is explant, through Fiber differentiation, squamous subculture, culture of rootage and transplanting and other steps, its inductivity has reached more than 88%, survival rate is to more than 94%, the tissue-culturing rapid propagation of Chinese medicine cissus assamica is realized, it is achieved thereby that the purpose of the present invention.
Description
Technical field
The present invention relates to the method for Plant Tissue Breeding in agricultural biotechnologies, specifically, it is related to a kind of red back of the body of Chinese medicine
The tissue culture and rapid propagation method of silk.
Background technology
Chinese medicine cissus assamica, i.e. Vitaceae Cissus plant Ku Lang rattans (Cissus assamica (Laws.) Craib.)
Also known as Radix Cissi assamicae, bryony, left side rattan etc., its is mild-natured, with draw out the pus detumescence, blood stasis removing analgesic the effects such as, it is among the people to be usually used in
The treatment of the diseases such as traumatic injury, rheumatic arthritis, ulcerative carbuncle, pyogenic infections, snake bite, particularly venomous snake bite, research find the red back of the body
Silk has anti-Endothelin short of money to act on, and its ethanol extract or acetic acid ethyl ester extract or resveratrol isolated therefrom exist
Overall and in vitro level is respectively provided with obvious antagonism to ET-1, the rat-isolated aorta bar that notable antagonism is caused by ET-1
Shrink and in dose dependent, and can rat blood pressure rises caused by antagonism ET-1 amplitude, therefore cissus assamica is wide
There is the good reputation of " panacea " of antidoting against snake bite in west.Cissus assamica is distributed mainly on Guangxi, Guangdong, Jiangxi, Fujian, Guizhou, Yunnan, Hunan etc.
Ground, in being common in mountain valley small stream side woods, in border or hillside.Due to cissus assamica grow condition particularity, it is slow-growing and
Excessively excavated, its wild resource falls sharply.
In view of the domestic artificial breeding to Chinese medicine cissus assamica does not make a breakthrough at present, the red back of the body silk of present invention selection
Silk ribbon stem segment is explant, through Fiber differentiation, squamous subculture, culture of rootage and transplanting and other steps, realizes the red back of the body of Chinese medicine
The tissue-culturing rapid propagation of silk.The features such as present invention has strong operability, low cost, process is simple, can be directly used for cissus assamica kind
The factorial praluction of seedling, for promoting cissus assamica industrialization development to have important practical significance.
At present, the report of cissus assamica tissue culture technique is at home and abroad there is no, does not also have cissus assamica tissue-culturing rapid propagation special
The application of profit.
The content of the invention
It is an object of the invention to provide a kind of tissue culture and rapid propagation method of Chinese medicine cissus assamica, present invention selection cissus assamica band
Stem segment is explant, and through Fiber differentiation, squamous subculture, culture of rootage and transplanting and other steps, its inductivity has reached 88%
More than, survival rate to more than 94% realizes the tissue-culturing rapid propagation of Chinese medicine cissus assamica, it is achieved thereby that mesh of the invention
's.
The present invention provide technical scheme be：A kind of tissue culture and rapid propagation method of Chinese medicine cissus assamica, including carry out successively
Following steps：Obtain explant, explant Fiber differentiation, squamous subculture, culture of rootage, the transplanting of cissus assamica；
The explant inducing culture of described explant Fiber differentiation operation includes：MS culture mediums, 2.0~4.0mg/
L6-BA, 0.5~1.0mg/LNAA, 20~30g/L sucrose, 3.5~4.0g/L agar, 60~100ml/L coconut milk, 0.1~
0.3g/L activated carbons, explant inducing culture pH is 5.4~5.8.
In the tissue culture and rapid propagation method of above-mentioned Chinese medicine cissus assamica, the method that described explant Fiber differentiation is operated
For：The belt segment rattan of 1.5~2.5cm is cut into after the belt segment rattan sterilization of the cissus assamica as explant that will be collected
And be inoculated into inducing culture, being placed in 25~28 DEG C carries out full light culture induced synthesis adventitious bud by 45~50 days.
In the tissue culture and rapid propagation method of above-mentioned Chinese medicine cissus assamica, the used subculture training of described squamous subculture operation
Foster base includes：MS culture mediums, 1.0~2.0mg/L 6-BA, 0.1~0.3mg/LNAA, 20~30g/L sucrose, 3.5~4.0g/L
Agar, 0.1~0.5g/L activated carbons, subculture medium pH are 5.4~5.8.
In the tissue culture and rapid propagation method of above-mentioned Chinese medicine cissus assamica, described squamous subculture operation is specially：By explant
The adventitious bud that the operation of body Fiber differentiation is obtained is cut into the stem section of 1~1.5cm long and is inoculated into culture 25~30 in subculture medium
It is capable of achieving the subculture of adventitious bud.
In the tissue culture and rapid propagation method of above-mentioned Chinese medicine cissus assamica, the used training of taking root of described culture of rootage operation
Foster base includes：MS, 0.5~1.0mg/LABT-6,2.0~4.0mg/LNAA, 15~20g/L sucrose, 3.0~4.0g/L agar,
0.1~0.3g/L activated carbons, root media pH is 5.4~5.8.
In the tissue culture and rapid propagation method of above-mentioned Chinese medicine cissus assamica, the method for described culture of rootage operation is：From base
Portion cuts the adventitious bud of 1.5~2.0cm of length that described squamous subculture operation is obtained and is inoculated into root media cultivates 25
Taking root for test tube seedling can be realized within~30 days.
In the tissue culture and rapid propagation method of above-mentioned Chinese medicine cissus assamica, the specific method of described transplanting operation is：Will be through
The good test tube seedling of the culture of rootage root growth that obtains of operation cleans the training of root in the natural light lower refining seedling 1~3 day in greenhouse
It is 1 that foster base is transplanted in volume ratio:Seedling is cultivated in 1 fertile soil, bark mixed-matrix and obtains final product seedling.
In the tissue culture and rapid propagation method of above-mentioned Chinese medicine cissus assamica, bar is cultivated in squamous subculture operation, culture of rootage behaviour
Part is：Cultivation temperature is 25~28 DEG C, and intensity of illumination is 2500~3000lx, and light application time is 10~12 hours/day.
Beneficial effect：
The present invention realizes the tissue-culturing rapid propagation of Chinese medicine cissus assamica using plant tissue culture technique, and the present invention has operation
Property strong, low cost, process is simple the features such as, can be directly used for the factorial praluction of cissus assamica seedling.
Specific embodiment
With reference to specific embodiment, technical scheme is described in further detail, but do not constitute it is right
Any limitation of the invention.
Embodiment one
(1) explant collection：In the wild choose robust growth, without obvious disease Ku Lang rattan plant middle and upper part, on the sunny side fill
Real belt segment rattan is explant, carries out water conservation moisturizing treatment after collection immediately and takes back laboratory in time.
(2) Fiber differentiation：When step (1) gathers back laboratory explant first night flushed under running water, it is placed in ultra-clean
Sterilized 5 seconds in 75% ethanol solution in workbench, after aseptic water washing uses aseptic filter paper suck dry moisture 5 times afterwards, be placed in
Sterilized 15 minutes in 0.1% mercuric chloride solution, with being cut into 1.5~2.5cm after aseptic filter paper suck dry moisture after aseptic water washing 5 times
Left and right belt segment rattan simultaneously be inoculated into inducing culture, be placed in 25 DEG C carry out full light culture by 45 days induced synthesis it is indefinite
Bud, inductivity is 90.1%, and dirty rate is less than 25%.Described inducing culture is：MS culture mediums+4.0mg/L6-BA+1.0mg/
LNAA+20g/L sucrose+3.5g/L agar+60ml/L coconut milk+0.1g/L activated carbons, pH is 5.4.
(3) squamous subculture：Step (2) adventitious bud that obtains of induction is cut into the stem section that is about 1~1.5cm and be inoculated into after
For 25 days subcultures of i.e. achievable adventitious bud are cultivated in culture medium, growth coefficient reaches 6 times.Described subculture medium is：MS
Culture medium+2.0mg/L 6-BA+0.3mg/LNAA+20~30g/L sucrose+3.5g/L agar+0.1g/L activated carbons, pH is 5.4.
(4) culture of rootage：The adventitious bud for being about 1.5~2.0cm that step (3) subculture obtains is cut from base portion and be inoculated into
Culture can realize taking root for test tube seedling in 25 days in root media, and rooting rate reaches 100%.Described root media is：
MS+1.0mg/LABT-6 (root-inducing powder)+4.0mg/LNAA+15g/L sucrose+3.0g/L agar+0.1g/L activated carbons, pH is 5.4.
(5) transplant：By the good test tube seedling of step (4) root growth in the natural light lower refining seedling 1 day in greenhouse, root is cleaned
Culture medium transplant in volume ratio be 1:Seedling is cultivated in 1 fertile soil, bark mixed-matrix and obtains final product seedling, after transplanting 30 days into
Motility rate reaches 95.6%.
The condition of culture of above-mentioned steps (3)~(4) is：Cultivation temperature is 25 DEG C, and intensity of illumination is 2000lx, light application time
It is 10 hours/day.
Embodiment two
(1) explant collection：In the wild choose robust growth, without obvious disease Ku Lang rattan plant middle and upper part, on the sunny side fill
Real belt segment rattan is explant, carries out water conservation moisturizing treatment after collection immediately and takes back laboratory in time.
(2) Fiber differentiation：When step (1) gathers back laboratory explant first night flushed under running water, it is placed in ultra-clean
Sterilized 7 seconds in 75% ethanol solution in workbench, after aseptic water washing uses aseptic filter paper suck dry moisture 6 times afterwards, be placed in
Sterilized 17 minutes in 0.1% mercuric chloride solution, with being cut into 1.5~2.5cm after aseptic filter paper suck dry moisture after aseptic water washing 6 times
Left and right belt segment rattan simultaneously be inoculated into inducing culture, be placed in 27 DEG C carry out full light culture by 47 days induced synthesis it is indefinite
Bud, inductivity is 94.7%, and pollution rate is less than 20%.Described inducing culture is：MS culture mediums+3.0mg/L6-BA+
0.8mg/LNAA+25g/L sucrose+3.8g/L agar+80ml/L coconut milk+0.2g/L activated carbons, pH is 5.6.
(3) squamous subculture：Step (2) adventitious bud that obtains of induction is cut into the stem section that is about 1~1.5cm and be inoculated into after
For 27 days subcultures of i.e. achievable adventitious bud are cultivated in culture medium, growth coefficient reaches 5 times.Described subculture medium is：MS
Culture medium+1.5mg/L 6-BA+0.2mg/L NAA+25g/L sucrose+3.8g/L agar+0.3g/L activated carbons, pH is 5.6.
(4) culture of rootage：The adventitious bud for being about 1.5~2.0cm that step (3) subculture obtains is cut from base portion and be inoculated into
Culture can realize taking root for test tube seedling for 28 days in root media, and rooting rate is 96%.Described root media is：MS+
0.7mg/LABT-6 (root-inducing powder)+3.0mg/LNAA+17g/L sucrose+3.5g/L agar+0.2g/L activated carbons, pH is 5.6.
(5) transplant：By the good test tube seedling of step (4) root growth in the natural light lower refining seedling 2 days in greenhouse, root is cleaned
Culture medium transplant in volume ratio be 1:Seedling is cultivated in 1 fertile soil, bark mixed-matrix and obtains final product seedling, after transplanting 30 days into
Motility rate is more than 95%.
The condition of culture of above-mentioned steps (3)~(4) is：Cultivation temperature is 27 DEG C, and intensity of illumination is 2300lx, light application time
It is 11 hours/day.
Embodiment three
(1) explant collection：In the wild choose robust growth, without obvious disease Ku Lang rattan plant middle and upper part, on the sunny side fill
Real belt segment rattan is explant, carries out water conservation moisturizing treatment after collection immediately and takes back laboratory in time.
(2) Fiber differentiation：When step (1) gathers back laboratory explant first night flushed under running water, it is placed in ultra-clean
Sterilized 10 seconds in 75% ethanol solution in workbench, after aseptic water washing uses aseptic filter paper suck dry moisture 7 times afterwards, be placed in
Sterilized 20 minutes in 0.1% mercuric chloride solution, with being cut into 1.5~2.5cm after aseptic filter paper suck dry moisture after aseptic water washing 7 times
Left and right belt segment rattan simultaneously be inoculated into inducing culture, be placed in 28 DEG C carry out full light culture by 50 days induced synthesis it is indefinite
Bud, inductivity is 88%, and pollution rate is less than 15%.Described inducing culture is：MS culture mediums+2.0mg/L6-BA+0.5mg/
LNAA+30g/L sucrose+4.0g/L agar+100ml/L coconut milk+0.3g/L activated carbons, pH is 5.8.
(3) squamous subculture：Step (2) adventitious bud that obtains of induction is cut into the stem section that is about 1~1.5cm and be inoculated into after
For 30 days subcultures of i.e. achievable adventitious bud are cultivated in culture medium, growth coefficient reaches 4.1 times.Described subculture medium is：
MS culture medium+1.0mg/L 6-BA+0.1mg/LNAA+30g/L sucrose+4.0g/L agar+0.5g/L activated carbons, pH is 5.8.
(4) culture of rootage：The adventitious bud for being about 1.5~2.0cm that step (3) subculture obtains is cut from base portion and be inoculated into
Culture can realize taking root for test tube seedling for 30 days in root media, and rooting rate is 89%.Described root media is：MS+
0.5mg/LABT-6 (root-inducing powder)+2.0mg/LNAA+20g/L sucrose+4.0g/L agar+0.3g/L activated carbons, pH is 5.8.
(5) transplant：By the good test tube seedling of step (4) root growth in the natural light lower refining seedling 3 days in greenhouse, root is cleaned
Culture medium transplant in volume ratio be 1:Seedling is cultivated in 1 fertile soil, bark mixed-matrix and obtains final product seedling, after transplanting 30 days into
Motility rate is 94%.
The condition of culture of above-mentioned steps (3)~(4) is：Cultivation temperature is 28 DEG C, and intensity of illumination is 2500lx, light application time
It is 12 hours/day.
Above-described embodiment is the present invention preferably implementation method, but embodiments of the present invention are not by above-described embodiment
Limitation, it is other it is any without departing from Spirit Essence of the invention and the change, modification, replacement made under principle, combine, simplification,
Equivalent substitute mode is should be, is included within protection scope of the present invention.
Claims (8)
1. a kind of tissue culture and rapid propagation method of Chinese medicine cissus assamica, it is characterised in that：Including the following steps for carrying out successively：Obtain red
Carry on the back explant, explant Fiber differentiation, squamous subculture, culture of rootage, the transplanting of silk；
The explant inducing culture of described explant Fiber differentiation operation includes：MS culture mediums, 2.0~4.0mg/L 6-
BA, 0.5~1.0mg/L NAA, 20~30g/L sucrose, 3.5~4.0g/L agar, 60~100ml/L coconut milk, 0.1~
0.3g/L activated carbons, explant inducing culture pH is 5.4~5.8.
2. the tissue culture and rapid propagation method of Chinese medicine cissus assamica according to claim 1, it is characterised in that：Described explant is lured
Lead culture operation method be：1.5 are cut into after the belt segment rattan sterilization of the cissus assamica as explant that will be collected
The belt segment rattan of~2.5cm is simultaneously inoculated into inducing culture, be placed in 25~28 DEG C carry out full light culture 45~50 days by lure
Lead to form adventitious bud.
3. the tissue culture and rapid propagation method of Chinese medicine cissus assamica according to claim 1, it is characterised in that：Described squamous subculture
The used subculture medium of operation includes：MS culture mediums, 1.0~2.0mg/L 6-BA, 0.1~0.3mg/L NAA, 20~
30g/L sucrose, 3.5~4.0g/L agar, 0.1~0.5g/L activated carbons, subculture medium pH are 5.4~5.8.
4. the tissue culture and rapid propagation method of Chinese medicine cissus assamica according to claim 3, it is characterised in that：Described squamous subculture
Operation is specially：The adventitious bud that the operation of explant Fiber differentiation is obtained is cut into the stem section of 1~1.5cm long and subculture training is inoculated into
Support 25~30 days subcultures of i.e. achievable adventitious bud of culture in base.
5. the tissue culture and rapid propagation method of Chinese medicine cissus assamica according to claim 1, it is characterised in that：Described culture of rootage
The used root media of operation includes：MS, 0.5~1.0mg/L ABT-6,2.0~4.0mg/L NAA, 15~20g/L sugarcanes
Sugar, 3.0~4.0g/L agar, 0.1~0.3g/L activated carbons, root media pH are 5.4~5.8.
6. the tissue culture and rapid propagation method of Chinese medicine cissus assamica according to claim 5, it is characterised in that：Described culture of rootage
The method of operation is：The adventitious bud of 1.5~2.0cm of length that described squamous subculture operation is obtained is cut from base portion and be inoculated into life
Culture can realize taking root for test tube seedling in 25~30 days in root culture medium.
7. the tissue culture and rapid propagation method of Chinese medicine cissus assamica according to claim 1, it is characterised in that：Described transplanting operation
Specific method be：The good test tube seedling of the root growth for obtaining will be operated in the natural light lower refining seedling 1 in greenhouse through culture of rootage
~3 days, it was 1 that the culture medium of clean root is transplanted in volume ratio:Seedling is cultivated in 1 fertile soil, bark mixed-matrix and obtains final product kind
Seedling.
8. according to the tissue culture and rapid propagation method of any described Chinese medicine cissus assamicas of claim 1-7, it is characterised in that：Squamous subculture
Condition of culture is in operation, culture of rootage behaviour：Cultivation temperature is 25~28 DEG C, and intensity of illumination is 2500~3000lx, illumination
Time is 10~12 hours/day.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107182793A (en) * | 2017-07-25 | 2017-09-22 | 广西壮族自治区中国科学院广西植物研究所 | A kind of tissue culture and rapid propagation method of precious jade anther-wings stem Cissus repens |
| CN115005098A (en) * | 2022-05-26 | 2022-09-06 | 桂林理工大学 | Rapid propagation method of folk Chinese herbal medicine Shujincao |
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| CN102144547A (en) * | 2011-01-14 | 2011-08-10 | 四川农业大学 | Method for quickly breeding and transplanting grape stock unit |
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| CN102144547A (en) * | 2011-01-14 | 2011-08-10 | 四川农业大学 | Method for quickly breeding and transplanting grape stock unit |
Non-Patent Citations (2)
| Title |
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| 刘秀芳等: "珍奇园艺植物珠帘藤的组织培养", 《西南师范大学学报(自然科学版)》 * |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107182793A (en) * | 2017-07-25 | 2017-09-22 | 广西壮族自治区中国科学院广西植物研究所 | A kind of tissue culture and rapid propagation method of precious jade anther-wings stem Cissus repens |
| CN115005098A (en) * | 2022-05-26 | 2022-09-06 | 桂林理工大学 | Rapid propagation method of folk Chinese herbal medicine Shujincao |
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