CN106577291A - Method for callus efficient induction seedling development of stemona sessilifolia - Google Patents
Method for callus efficient induction seedling development of stemona sessilifolia Download PDFInfo
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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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Abstract
The invention discloses a method for callus efficient induction seedling development of stemona sessilifolia. The method comprises the following steps: taking a stemona sessilifolia tender shoot as an explant, and putting the explant into an MS primary induction culture medium to perform induction so as to obtain a sterile test-tube plantlet; putting the test-tube plantlet into an MS callus induction culture medium to perform callus culture; putting an obtained callus into an MS differential culture medium to obtain a large amount of cluster buds; putting the cluster buds into an MS sound seedling culture medium to perform sound seedling culture; and putting obtained robust plants into an MS rooting culture medium to perform rooting culture. A test shows that by adopting the propagation method provided by the invention, the multiplication coefficient of the obtained cluster buds is 40-80 times, the rooting rate of the obtained tissue culture seedlings is 95% or above, and the survival rate of the seedlings transplanted to a seedbed is 90% or above. Therefore, the method disclosed by the invention can make effective use of existing resources to quickly propagate the stemona sessilifolia, solves the problem of industrialized rooting and seedling raising of the stemona sessilifolia, and can fully guarantee a raw material source for medical application development of the stemona sessilifolia.
Description
Technical field
The invention belongs to Radix Stemonae field of tissue culture, more particularly to a kind of Radix Stemonae calluss are efficiently induced into
The method of Seedling.
Background technology
Radix Stemonae (Stemona sessilifolia (Miq.) Miq.) is Stemonaceae Radix Stemonae platymiscium, produces Zhejiang, river
Soviet Union, Anhui, Jiangxi, Shandong, Henan etc. save.Radix Stemonae tuber is《Chinese Pharmacopoeia》(version in 2015) records the three of chinese medicine stemona tuber
One of legal source is planted, has nourishing the lung to keep the adverse QI downward cough-relieving, effect of desinsection and delousion is coughed for new chronic cough, pulmonary tuberculosis cough, pertussis；External
In head louse, body louse, enterobiasiss, pudendal pruritus.Research shows that the principle active component of Radix Stemonae is the exclusive class of Stemonaceae plant
Stemona alkaloids with pyrroles or pyrido Azaazulene mother nucleus structure, such as root are containing (-)-Stemoninine, protostermonine, Stemonidine, different
Stemonidine, sessilistemonine, hordorine, tuberostemonine etc..Modern pharmacological research shows that Stemona alkaloids have drive
Worm, parasite killing, antitussive and antiasthmatic, Neuromuscular Conduction, antitumor and antibacterial etc. are acted on.Jing is investigated,《Jing is herbal for figure》Contained Chuzhou hundred
Portion is Radix Stemonae.
All the time, Radix Stemonae depends on and takes wild product and make medicinal, does not plant due to only digging, and it is relatively slow to add growth, its
Wild resource is increasingly exhausted.Simultaneously as Radix Stemonae has a very limited distribution, Radix Stemonae commodity are almost difficult to find that on market
Medical material, therefore Radix Stemonae artificial growth and foster imperative.At present, though being related to the (patent application of Radix Stemonae tissue culture
Number 201410540445.9 publication date on December 24th, 2014) and equal congener Radix stemonae japonicae (Li Kelie, Radix stemonae japonicae group
Knit culture fast breeding technique, Guangxi Agricultural science, 2005,36：505-506) and radix stemonae tuberosae (Yang Zhende, radix stemonae tuberosae tissue training
Foster fast breeding technique research, modern agriculture science and technology, 2016,2：105-107, the 115) relevant report of tissue culture, but not yet have
The document report of Radix Stemonae callus induction seedling.
The content of the invention
It is efficient that the technical problem to be solved in the present invention is to provide a kind of practical, simple, efficient Radix Stemonae calluss
The method of induction seedling, with effectively utilizes existing resource Radix Stemonae is quickly bred.
To solve above-mentioned technical problem, the present invention is employed the following technical solutions：
The method that Radix Stemonae calluss efficiently induce seedling, with Radix Stemonae tender shoots explant is made, and is placed in MS first generations
Induction is carried out in inducing culture and obtains in vitro cuttings, test tube seedling is placed in MS calli induction medias carries out calluss
Culture, gained calluss are placed in MS division culture mediums and obtain a large amount of clump buds, and clump bud is placed in MS strong seedling culture bases to be carried out
Strong seedling culture, the healthy and strong plant for obtaining is placed in MS root medias and carries out root culture.
At the beginning of MS for inducing culture based on MS minimal mediums, addition 1.0～2.0mg/L 6-benzyl aminopurines,
0.2～1.0mg/L heteroauxings, 0.1～0.5mg/L kinetins, 30mg/L sucrose, 5mg/L agar, the pH of culture medium is 5.8；
MS calli induction medias add 0.5～1.5mg/L 2,4- Dichlorophenoxy second based on MS minimal mediums
Acid, 0.1～0.5mg/L kinetins, 30mg/L sucrose, 5mg/L agar, the pH of culture medium is 5.8；
MS division culture mediums add 1.0～3.0mg/L zeatin, 0.1～0.5mg/L based on MS minimal mediums
Indolebutyric acid, 0.10～0.50mg/L kinetins, 30mg/L sucrose, 5mg/L agar, the pH of culture medium is 5.8；
MS strong seedling cultures base based on MS minimal mediums, addition 1.0～2.0mg/L 6-benzyl aminopurines, 0.2～
0.5mg/L naphthalene acetic acids, 30mg/L sucrose, 5mg/L agar, the pH of culture medium is 5.8；
MS root medias based on 1/2MS minimal mediums, addition 0.2～1.0mg/L naphthalene acetic acids, 10g/L sucrose,
5g/L agar, 1.5g/L activated carbons, the pH of culture medium is 5.8.
The method that above-mentioned Radix Stemonae calluss efficiently induce seedling, according to the following steps operation is carried out：
(1) selection of explant and sterilization
The tender shoots for taking Radix Stemonae makees explant, successively with 2% liquid detergent aqueous solution soaking of volumetric concentration 5 minutes, wire
Tap water is rinsed 15～20 minutes, and the 100mg/L0.1% mercuric chloride of the drop tween 20 of addition 2～3 is sterilized 8～10 minutes, sterilized water
Rinse 3～5 times, finally remove surface moisture content with sterilized filter paper, obtain explant；
(2) just for inducing culture acquisition in vitro cuttings
By step (1) explant be inoculated into MS just for inducing culture in, be 22～26 degree in temperature, intensity of illumination
1500lux, light application time is to cultivate 30 days under conditions of 8～10 hours/day, obtains in vitro cuttings；
(3) induction of callus
The in vitro cuttings that step (2) is obtained are cut into into 1cm or so stem sections, in being placed in MS calli induction medias,
Temperature is 22～26 degree, is cultivated 30 days under dark condition, obtains calluss；
(4) calluss differentiation culture
The calluss that step (3) is obtained are placed in MS division culture mediums, are 22～26 degree in temperature, intensity of illumination
1500lux, light application time is to cultivate 30 days under conditions of 8～10 hours/day, obtains a large amount of Multiple Buds；
(5) strong seedling culture
The Multiple Buds that step (4) is obtained are cut into into single bud, is 22～26 in temperature in being placed in MS strong seedling culture bases
Degree, intensity of illumination 1500lux, light application time is to cultivate 30 days under conditions of 8～10 hours/day, obtains healthy and strong plant；
(6) root culture
The healthy and strong plant that step (5) is obtained is placed in into MS root media cultures, is 22～26 degree in temperature, intensity of illumination
1500lux, light application time is to cultivate 30 days under conditions of 8～10 hours/day, obtains band root whole plant；
(7) seedling exercising and transplanting
Tap water is added in tissue culture bottle, bottle cap is unclamped, seedling exercising one week after surface horny is formed, seedling is taken out, cleaned
Root culture medium, is transplanted to immediately in sand bed, and after growing one month in sand bed land for growing field crops is transplanted to.
For the problem that Radix Stemonae field source shortage, artificial growth are difficult, inventor establishes a kind of Radix Stemonae
The method that calluss efficiently induce seedling, with Radix Stemonae tender shoots explant is made, and be placed in MS is just carried out in inducing culture
Induction obtains in vitro cuttings, and test tube seedling is placed in MS calli induction medias carries out callus culture, gained wound healing group
Knit to be placed in MS division culture mediums and obtain a large amount of clump buds, clump bud is placed in MS strong seedling culture bases carries out strong seedling culture, obtains
Healthy and strong plant is placed in MS root medias and carries out root culture.Test shows, using growing thickly that the propagation method of the present invention is obtained
Bud growth coefficient is 40～80 times, obtains tissue cultured seedling rooting rate more than 95%, and transplanting seedbed survival rate is more than 90%.Cause
This, the present invention can effectively utilizes existing resource quickly breed Radix Stemonae, solve Radix Stemonae batch production and take root the asking of nursery
Topic, and can fully ensure its medical usage exploitation needed raw material source.
Relative to prior art, the present invention's has the prominent advantages that：
(1) callus culture is carried out to Radix Stemonae, quickly obtains Multiple Buds, a large amount of being adapted to is cultivated at short notice
The Radix Stemonae seedling of transplanting, significantly improves the growth coefficient and seedling quality of Radix Stemonae seedling, accomplishes scale production,
Meet the demand on market.
(2) zeatin and kinetins added in MS division culture mediums belongs to frequently seen plants growth regulator, cheap easy
, the cost consumption in Radix Stemonae tissue culture can be substantially reduced, reduce the purpose of cost.
(3) adding 6-benzyl aminopurine and naphthalene acetic acid in MS strong seedling culture bases can promote bud to grow tall lamina.
(4) the repeatable operation of the present invention, method is simple.
(5) present invention lays the foundation for the transgenic research of follow-up Radix Stemonae.
Specific embodiment
Embodiment 1
(1) selection of explant and sterilization
The tender shoots for taking Radix Stemonae makees explant, successively with 2% liquid detergent aqueous solution soaking of volumetric concentration 5 minutes, wire
Tap water is rinsed 15～20 minutes, and the 100mg/L0.1% mercuric chloride of the drop tween 20 of addition 2～3 is sterilized 8～10 minutes, sterilized water
(the autoclaved distilled water of Jing) is rinsed 3～5 times, finally removes surface moisture content with sterilized filter paper, obtains explant；
(2) just for inducing culture acquisition in vitro cuttings
By step (1) explant be inoculated into MS just for inducing culture in, be 22～26 degree in temperature, intensity of illumination
1500lux, light application time is to cultivate 30 days under conditions of 8～10 hours/day, obtains in vitro cuttings；
Wherein, MS is just for inducing culture based on MS minimal mediums, addition 1.0mg/L6- benayl aminopurines,
0.5mg/L heteroauxings, 0.2mg/L kinetins, 30mg/L sucrose, 5mg/L agar, the pH of culture medium is 5.8；
(3) induction of callus
The in vitro cuttings that step (2) is obtained are cut into into 1cm or so stem sections, in being placed in MS calli induction medias,
Temperature is 22～26 degree, is cultivated 30 days under dark condition, obtains calluss；
Wherein, MS calli induction medias add 0.5mg/L 2,4- Dichlorophenoxy second based on MS minimal mediums
Acid, 0.2mg/L kinetins, 30mg/L sucrose, 5mg/L agar, the pH of culture medium is 5.8；
(4) calluss differentiation culture
The calluss that step (3) is obtained are placed in MS division culture mediums, are 22～26 degree in temperature, intensity of illumination
1500lux, light application time is to cultivate 30 days under conditions of 8～10 hours/day, obtains a large amount of Multiple Buds；
Wherein, MS division culture mediums add 1.0mg/L zeatin, 0.3mg/L indole fourths based on MS minimal mediums
Acid, 0.2mg/L kinetins, 30mg/L sucrose, 5mg/L agar, the pH of culture medium is 5.8；
(5) strong seedling culture
The Multiple Buds that step (4) is obtained are cut into into single bud, is 22～26 in temperature in being placed in MS strong seedling culture bases
Degree, intensity of illumination 1500lux, light application time is to cultivate 30 days under conditions of 8～10 hours/day, obtains healthy and strong plant；
Wherein, MS strong seedling cultures base adds 1.0mg/L 6-benzyl aminopurines, 0.5mg/ based on MS minimal mediums
L naphthalene acetic acids, 30mg/L sucrose, 5mg/L agar, the pH of culture medium is 5.8；
(6) root culture
The healthy and strong plant that step (5) is obtained is placed in into MS root media cultures, is 22～26 degree in temperature, intensity of illumination
1500lux, light application time is to cultivate 30 days under conditions of 8～10 hours/day, obtains band root whole plant；
Wherein, MS root medias are based on 1/2MS minimal mediums, addition 0.5mg/L naphthalene acetic acids, 10g/L sucrose,
5g/L agar, 1.5g/L activated carbons, the pH of culture medium is 5.8.
(7) seedling exercising and transplanting
A small amount of tap water is added in tissue culture bottle, bottle cap is unclamped, seedling exercising one week after surface horny is formed, seedling is taken out,
Clean root culture medium, is transplanted to immediately in sand bed, and after growing one month in sand bed land for growing field crops is transplanted to.
As a result：Adventitious buds proliferation coefficient is 40 times, obtains tissue cultured seedling rooting rate more than 95%, transplants seedbed survival rate and exists
More than 90%.
Embodiment 2
It is substantially the same manner as Example 1, it is only following different：
MS adds 1.5mg/L 6-benzyl aminopurines, 0.3mg/L just for inducing culture based on MS minimal mediums
Heteroauxing, 0.5mg/L kinetins, 30mg/L sucrose, 5mg/L agar, the pH of culture medium is 5.8；
MS calli induction medias based on MS minimal mediums, add 1.0mg/L 2,4- dichlorphenoxyacetic acids,
0.5mg/L kinetins, 30mg/L sucrose, 5mg/L agar, the pH of culture medium is 5.8；
MS division culture mediums based on MS minimal mediums, addition 1.0mg/L zeatin, 0.5mg/L indolebutyric acids,
0.2mg/L kinetins, 30mg/L sucrose, 5mg/L agar, the pH of culture medium is 5.8；
MS strong seedling cultures base adds 1.0mg/L6- benayl aminopurines, 0.5mg/L naphthalene second based on MS minimal mediums
Acid, 30mg/L sucrose, 5mg/L agar, the pH of culture medium is 5.8；
MS root medias add 0.5mg/L naphthalene acetic acids, 10g/L sucrose, 5g/L based on 1/2MS minimal mediums
Agar, 1.5g/L activated carbons, the pH of culture medium is 5.8.
As a result：Adventitious buds proliferation coefficient is 70 times, obtains tissue cultured seedling rooting rate more than 95%, transplants seedbed survival rate and exists
More than 90%.
Embodiment 3
It is substantially the same manner as Example 1, it is only following different：
MS adds 2mg/L6- benayl aminopurines, 0.5mg/L Yin just for inducing culture based on MS minimal mediums
Indolylbutyric acid, 0.5mg/L kinetins, 30mg/L sucrose, 5mg/L agar, the pH of culture medium is 5.8；
MS calli induction medias based on MS minimal mediums, add 1.5mg/L2,4- dichlorphenoxyacetic acids,
0.2mg/L kinetins, 30mg/L sucrose, 5mg/L agar, the pH of culture medium is 5.8；
MS division culture mediums based on MS minimal mediums, addition 3.0mg/L zeatin, 0.5mg/L indolebutyric acids,
0.2mg/L kinetins, 30mg/L sucrose, 5mg/L agar, the pH of culture medium is 5.8；
MS strong seedling cultures base adds 2.0mg/L6- benayl aminopurines, 0.2mg/L naphthalene second based on MS minimal mediums
Acid, 30mg/L sucrose, 5mg/L agar, the pH of culture medium is 5.8；
MS root medias add 1.0mg/L naphthalene acetic acids, 10g/L sucrose, 5g/L based on 1/2MS minimal mediums
Agar, 1.5g/L activated carbons, the pH of culture medium is 5.8.
As a result：Adventitious buds proliferation coefficient is 80 times, obtains tissue cultured seedling rooting rate more than 95%, transplants seedbed survival rate and exists
More than 90%.
Claims (3)
1. a kind of method that Radix Stemonae calluss efficiently induce seedling, it is characterised in that：Explant is made with Radix Stemonae tender shoots
Body, is placed in MS and just obtains in vitro cuttings for carrying out induction in inducing culture, and test tube seedling is placed in into MS calli induction medias
In carry out callus culture, gained calluss are placed in MS division culture mediums and obtain a large amount of clump buds, clump bud are placed in into MS and strengthened
Strong seedling culture is carried out in Seedling culture medium, the healthy and strong plant for obtaining is placed in MS root medias and carries out root culture.
2. the method that Radix Stemonae calluss according to claim 1 efficiently induce seedling, it is characterised in that：
At the beginning of the MS for inducing culture based on MS minimal mediums, addition 1.0～2.0mg/L 6-benzyl aminopurines,
0.2～1.0mg/L heteroauxings, 0.1～0.5mg/L kinetins, 30mg/L sucrose, 5mg/L agar, the pH of culture medium is 5.8；
The MS calli induction medias add 0.5～1.5mg/L 2,4- Dichlorophenoxy second based on MS minimal mediums
Acid, 0.1～0.5mg/L kinetins, 30mg/L sucrose, 5mg/L agar, the pH of culture medium is 5.8；
The MS division culture mediums add 1.0～3.0mg/L zeatin, 0.1～0.5mg/L based on MS minimal mediums
Indolebutyric acid, 0.10～0.50mg/L kinetins, 30mg/L sucrose, 5mg/L agar, the pH of culture medium is 5.8；
The MS strong seedling cultures base based on MS minimal mediums, addition 1.0～2.0mg/L 6-benzyl aminopurines, 0.2～
0.5mg/L naphthalene acetic acids, 30mg/L sucrose, 5mg/L agar, the pH of culture medium is 5.8；
The MS root medias based on 1/2MS minimal mediums, addition 0.2～1.0mg/L naphthalene acetic acids, 10g/L sucrose,
5g/L agar, 1.5g/L activated carbons, the pH of culture medium is 5.8.
3. the method that Radix Stemonae calluss according to claim 2 efficiently induce seedling, it is characterised in that by following
Step operation is carried out：
(1) selection of explant and sterilization
The tender shoots for taking Radix Stemonae makees explant, and successively with 2% liquid detergent aqueous solution soaking of volumetric concentration 5 minutes, wire is originally
Water is rinsed 15～20 minutes, and the mercuric chloride of 100mg/L 0.1% of the drop tween 20 of addition 2～3 is sterilized 8～10 minutes, aseptic water washing
3～5 times, finally surface moisture content is removed with sterilized filter paper, obtain explant；
(2) just for inducing culture acquisition in vitro cuttings
By step (1) explant be inoculated into MS just for inducing culture in, be 22～26 degree in temperature, intensity of illumination
1500lux, light application time is to cultivate 30 days under conditions of 8～10 hours/day, obtains in vitro cuttings；
(3) induction of callus
The in vitro cuttings that step (2) is obtained are cut into into 1cm stem sections, is 22 in temperature in being placed in MS calli induction medias
～26 degree, cultivate 30 days under dark condition, obtain calluss；
(4) calluss differentiation culture
The calluss that step (3) is obtained are placed in MS division culture mediums, are 22～26 degree in temperature, intensity of illumination
1500lux, light application time is to cultivate 30 days under conditions of 8～10 hours/day, obtains a large amount of Multiple Buds；
(5) strong seedling culture
The Multiple Buds that step (4) is obtained are cut into into single bud, is 22～26 degree in temperature in being placed in MS strong seedling culture bases, light
According to intensity 1500lux, light application time is to cultivate 30 days under conditions of 8～10 hours/day, obtains healthy and strong plant；
(6) root culture
The healthy and strong plant that step (5) is obtained is placed in into MS root media cultures, is 22～26 degree in temperature, intensity of illumination
1500lux, light application time is to cultivate 30 days under conditions of 8～10 hours/day, obtains band root whole plant；
(7) seedling exercising and transplanting
Tap water is added in tissue culture bottle, bottle cap is unclamped, seedling exercising one week after surface horny is formed, seedling is taken out, clean root
Culture medium, is transplanted to immediately in sand bed, and after growing one month in sand bed land for growing field crops is transplanted to.
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| CN109042325A (en) * | 2018-08-20 | 2018-12-21 | 王远能 | The tissue cultivating method of rag wood |
| CN109924089A (en) * | 2019-03-28 | 2019-06-25 | 四川大学 | A kind of tuber of stemona Industrialization of seeds and seedlings mating system |
| CN114467749A (en) * | 2022-01-21 | 2022-05-13 | 贵州民族大学 | Culture medium and method for rapid breeding of stemona tuberosa seedlings |
| CN115735771A (en) * | 2022-11-30 | 2023-03-07 | 上海世泽生物科技有限公司 | High-efficiency in-vitro propagation method of radix stemonae |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN115735771B (en) * | 2022-11-30 | 2023-08-04 | 上海世泽生物科技有限公司 | Efficient in-vitro propagation method for radix stemonae |
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