CN106613953A - Method for tissue culture of stevia rebaudiana - Google Patents
Method for tissue culture of stevia rebaudiana Download PDFInfo
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- CN106613953A CN106613953A CN201610948120.3A CN201610948120A CN106613953A CN 106613953 A CN106613953 A CN 106613953A CN 201610948120 A CN201610948120 A CN 201610948120A CN 106613953 A CN106613953 A CN 106613953A
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- China
- Prior art keywords
- stevia rebaudiana
- explant
- culture
- culture medium
- antibacterial liquid
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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
Abstract
The invention belongs to the technical field of tissue culture production and specifically relates to a method for tissue culture of stevia rebaudiana. The method comprises a step 1) of preparing disinfection liquid, a step 2) of obtaining explants, a step 3) of disinfecting a medium square vase and the explants, a step 4) of differentiating explant buds, and a step 5) of causing the buds to root and become seedlings. The method for tissue culture of stevia rebaudiana has easiness in material taking, plenty of seed sources can be subjected to expanding propagation quickly, the propagation cost can be reduced, the growth cycle is short, the reproductive rate is high, the method facilitates management and is favorable for industrial production and automatic monitoring, and manpower, material resources and field crop land are greatly saved.
Description
Technical field
The invention belongs to tissue culture production technical field, and in particular to a kind of STEVIA REBAUDIANA tissue culture method.
Background technology
STEVIA REBAUDIANA, is the composite family sweetleaf Chrysanthemum herbaceos perennial for originating in South America Paraguay, due to STEVIA REBAUDIANA institute
The steviol glycoside for containing has blood sugar processed, reduces blood pressure, enhances metabolism and treat diabetes, obesity, adjust hydrochloric acid in gastric juice, recover
Effect of neural fatigue, and physics, stable chemical nature, without Fermented feature, its product has the long shelf-life, especially
Be suitable as sweetener, products and health products additive agent of patient's food such as diabetes, hypertension etc., with wide market prospects and
Range of application.
Since STEVIA REBAUDIANA and successfully expansion plantation are introduced a fine variety in 20 century 70 years from China, there is going through for more than 30 years
History, but due to the Breeding trait of stevia rebaudianum, traditional modes of reproduction includes seed, cuttage mode.But with the method for seed growing,
Survival rate is low；And cutting propagation causes quality deterioration, and both mode of reproduction time length, needs in a large number with stem apex, band armpit
The materials such as stem section, the seed of bud.
The content of the invention
It is an object of the invention to provide a kind of STEVIA REBAUDIANA tissue culture method, can reduce the cost bred, growth cycle is short, breeding
Rate is high, facilitate and manage, be conducive to industrialized production and automatically-monitored, has greatly saved human and material resources and field crops
Soil.
The invention provides following technical scheme：
A kind of STEVIA REBAUDIANA tissue culture method, comprises the following steps,
1）The preparation of antibacterial liquid, the vitamin C aqueous solution of the 400-600 times of liquid of ziram with 10% is mixed, the ziram solution
The volume ratio of 400-600 times of liquid and the 10% vitamin C aqueous solution is 2：1, obtain antibacterial liquid A；Prepare 0.2% HgCl2Solution, obtains
To antibacterial liquid B；
2）The acquisition of explant, takes the not aging blade in STEVIA REBAUDIANA top, using solution planing tool along STEVIA REBAUDIANA blade stem position
Put it is crosscutting go out long 15-20 mm, the blade bar of wide 10-15 mm；
3）The sterilization of culture medium square vase and explant, by culture medium square vase immersion 20-30 minutes in the antibacterial liquid B are put into, and are used
Aseptic water washing 2-4 time, each 3-5 minutes, by the blade bar immersion 10-20 minutes in antibacterial liquid A is put into, and uses aseptic washing
Only, it is then placed in being soaked 20-30 minutes in antibacterial liquid B, is cleaned with sterilized water；
4）Explant Bud Differentiation, explant is inoculated in the culture medium square vase equipped with bud inducement cultivation base, at 28-30 DEG C,
Light light culture 15-20 days, explant differentiation is sprouted, and the bud inducement cultivation base is MS+ methyl α-naphthyl acetate 0.1-0.3 mg/L+6- benzyls
Purine 0.4-1.0 mg/L+ agar 8-10 g/L；
5）Bud is taken root seedling, when step 4）In bud length to 3-5 cm, be separated from each other into individual plant, and be forwarded to equipped with taking root
In the culture medium square vase of culture medium, at 28-30 DEG C, light light culture 20-25 days grows a plurality of and white root hair, institute occurs
Root media is stated for 1/2MS+ heteroauxin 0.1-0.2 mg/L+ methyl α-naphthyl acetate 0.1-0.2 mg/L+ agar 5-7 g/L+ sulfuric acid
Ferrous 0.5-1.5 mg/L；
Preferably, the step 3）The specification of middle culture medium square vase is 500 ml.
Preferably, the step 4）The intensity of illumination of middle optical culture be 1300 luxs, step 5）The illumination of middle optical culture
Intensity is 1800 luxs.
The STEVIA REBAUDIANA method for tissue culture that the present invention is provided, draws materials easily, can expand rapidly it is numerous go out a large amount of introduces a collections, can reduce
The cost of breeding, growth cycle is short, breeding potential is high, conveniently manage, be conducive to industrialized production and automatically-monitored, greatly saves
The about soil of human and material resources and field crops.
Specific embodiment
Embodiment 1
A kind of STEVIA REBAUDIANA tissue culture method, comprises the following steps,
1）The preparation of antibacterial liquid, the vitamin C aqueous solution of the 400 times of liquid of ziram with 10% is mixed, the ziram solution 400
The volume ratio of times liquid and the 10% vitamin C aqueous solution is 2：1, obtain antibacterial liquid A；Prepare 0.2% HgCl2Solution, obtains antibacterial
Liquid B；
2）The acquisition of explant, takes the not aging blade in STEVIA REBAUDIANA top, using solution planing tool along STEVIA REBAUDIANA blade stem position
Put it is crosscutting go out long 15 mm, the blade bar of wide 10 mm；
3）The sterilization of culture medium square vase and explant, culture medium square vase is put in the antibacterial liquid B and is soaked 20 minutes, with aseptic
Water is rinsed 2 times, 3 minutes every time, the blade bar is put into into immersion 10- minutes in antibacterial liquid A, is cleaned with sterilized water, Ran Houfang
Enter in antibacterial liquid B and soak 20 minutes, cleaned with sterilized water；
4）Explant Bud Differentiation, explant is inoculated in the culture medium square vase equipped with bud inducement cultivation base, at 28 DEG C, brightness
Culture 15 days, explant differentiation is sprouted, and the bud inducement cultivation base is the mg/ of 0.1 mg/L+6- benzyl purines of MS+ methyl α-naphthyl acetates 0.4
The g/L of L+ agar 8；
5）Bud is taken root seedling, when step 4）In bud length to 3 cm, be separated from each other into individual plant, and be forwarded to equipped with training of taking root
In the culture medium square vase of foster base, at 28 DEG C, light light culture 20 days grows a plurality of and white root hair occurs, the training of taking root
Foster base is the mg/L of 5 g/L+ ferrous sulfate of 1/2MS+ heteroauxins 0.1 mg/L+ methyl α-naphthyl acetates, 0.1 mg/L+ agar 0.5；
The step 3）The specification of middle culture medium square vase is 500 ml.
The step 4）The intensity of illumination of middle optical culture be 1300 luxs, step 5）The intensity of illumination of middle optical culture is
1800 luxs.
Embodiment 2
A kind of STEVIA REBAUDIANA tissue culture method, comprises the following steps,
1）The preparation of antibacterial liquid, the vitamin C aqueous solution of the 500 times of liquid of ziram with 10% is mixed, the ziram solution 500
The volume ratio of times liquid and the 10% vitamin C aqueous solution is 2：1, obtain antibacterial liquid A；Prepare 0.2% HgCl2Solution, obtains antibacterial
Liquid B；
2）The acquisition of explant, takes the not aging blade in STEVIA REBAUDIANA top, using solution planing tool along STEVIA REBAUDIANA blade stem position
Put it is crosscutting go out long 18 mm, the blade bar of wide 13 mm；
3）The sterilization of culture medium square vase and explant, culture medium square vase is put in the antibacterial liquid B and is soaked 25 minutes, with aseptic
Water is rinsed 3 times, 4 minutes every time, the blade bar is put in antibacterial liquid A and is soaked 15 minutes, is cleaned with sterilized water, is then placed in
Soak 25 minutes in antibacterial liquid B, cleaned with sterilized water；
4）Explant Bud Differentiation, explant is inoculated in the culture medium square vase equipped with bud inducement cultivation base, at 29 DEG C, brightness
Culture 18 days, explant differentiation is sprouted, and the bud inducement cultivation base is the mg/ of 0.2 mg/L+6- benzyl purines of MS+ methyl α-naphthyl acetates 0.7
The g/L of L+ agar 9；
5）Bud is taken root seedling, when step 4）In bud length to 4 cm, be separated from each other into individual plant, and be forwarded to equipped with training of taking root
In the culture medium square vase of foster base, at 29 DEG C, light light culture 23 days grows a plurality of and white root hair occurs, the training of taking root
Foster base is the mg/L of 6 g/L+ ferrous sulfate of 1/2MS+ heteroauxins 0.15 mg/L+ methyl α-naphthyl acetates, 0.15 mg/L+ agar 1.0；
The step 3）The specification of middle culture medium square vase is 500 ml.
The step 4）The intensity of illumination of middle optical culture be 1300 luxs, step 5）The intensity of illumination of middle optical culture is
1800 luxs.
Embodiment 3
A kind of STEVIA REBAUDIANA tissue culture method, comprises the following steps,
1）The preparation of antibacterial liquid, the vitamin C aqueous solution of the 600 times of liquid of ziram with 10% is mixed, the ziram solution 600
The volume ratio of times liquid and the 10% vitamin C aqueous solution is 2：1, obtain antibacterial liquid A；Prepare 0.2% HgCl2Solution, obtains antibacterial
Liquid B；
2）The acquisition of explant, takes the not aging blade in STEVIA REBAUDIANA top, using solution planing tool along STEVIA REBAUDIANA blade stem position
Put it is crosscutting go out long 20 mm, the blade bar of wide 15 mm；
3）The sterilization of culture medium square vase and explant, culture medium square vase is put in the antibacterial liquid B and is soaked 30 minutes, with aseptic
Water is rinsed 4 times, 5 minutes every time, the blade bar is put in antibacterial liquid A and is soaked 20 minutes, is cleaned with sterilized water, is then placed in
Soak 30 minutes in antibacterial liquid B, cleaned with sterilized water；
4）Explant Bud Differentiation, explant is inoculated in the culture medium square vase equipped with bud inducement cultivation base, at 30 DEG C, brightness
Culture 20 days, explant differentiation is sprouted, and the bud inducement cultivation base is the mg/ of 0.3 mg/L+6- benzyl purines of MS+ methyl α-naphthyl acetates 1.0
The g/L of L+ agar 10；
5）Bud is taken root seedling, when step 4）In bud length to 5 cm, be separated from each other into individual plant, and be forwarded to equipped with training of taking root
In the culture medium square vase of foster base, at 30 DEG C, light light culture 25 days grows a plurality of and white root hair occurs, the training of taking root
Foster base is the mg/L of 7 g/L+ ferrous sulfate of 1/2MS+ heteroauxins 0.2 mg/L+ methyl α-naphthyl acetates, 0.2 mg/L+ agar 1.5；
The step 3）The specification of middle culture medium square vase is 500 ml.
The step 4）The intensity of illumination of middle optical culture be 1300 luxs, step 5）The intensity of illumination of middle optical culture is
1800 luxs.
The preferred embodiments of the present invention are the foregoing is only, the present invention is not limited to, although with reference to aforementioned reality
Apply example to be described in detail the present invention, for a person skilled in the art, it still can be to aforementioned each enforcement
Technical scheme described in example is modified, or carries out equivalent to which part technical characteristic.All essences in the present invention
Within god and principle, any modification, equivalent substitution and improvements made etc. should be included within the scope of the present invention.
Claims (3)
1. a kind of STEVIA REBAUDIANA tissue culture method, it is characterised in that comprise the following steps,
1）The preparation of antibacterial liquid, the vitamin C aqueous solution of the 400-600 times of liquid of ziram with 10% is mixed, the ziram solution
The volume ratio of 400-600 times of liquid and the 10% vitamin C aqueous solution is 2：1, obtain antibacterial liquid A；Prepare 0.2% HgCl2Solution, obtains
To antibacterial liquid B；
2）The acquisition of explant, takes the not aging blade in STEVIA REBAUDIANA top, using solution planing tool along STEVIA REBAUDIANA blade stem position
Put it is crosscutting go out long 15-20 mm, the blade bar of wide 10-15 mm；
3）The sterilization of culture medium square vase and explant, by culture medium square vase immersion 20-30 minutes in the antibacterial liquid B are put into, and are used
Aseptic water washing 2-4 time, each 3-5 minutes, by the blade bar immersion 10-20 minutes in antibacterial liquid A is put into, and uses aseptic washing
Only, it is then placed in being soaked 20-30 minutes in antibacterial liquid B, is cleaned with sterilized water；
4）Explant Bud Differentiation, explant is inoculated in the culture medium square vase equipped with bud inducement cultivation base, at 28-30 DEG C,
Light light culture 15-20 days, explant differentiation is sprouted, and the bud inducement cultivation base is MS+ methyl α-naphthyl acetate 0.1-0.3 mg/L+6- benzyls
Purine 0.4-1.0 mg/L+ agar 8-10 g/L；
5）Bud is taken root seedling, when step 4）In bud length to 3-5 cm, be separated from each other into individual plant, and be forwarded to equipped with taking root
In the culture medium square vase of culture medium, at 28-30 DEG C, light light culture 20-25 days grows a plurality of and white root hair, institute occurs
Root media is stated for 1/2MS+ heteroauxin 0.1-0.2 mg/L+ methyl α-naphthyl acetate 0.1-0.2 mg/L+ agar 5-7 g/L+ sulfuric acid
Ferrous 0.5-1.5 mg/L.
2. a kind of STEVIA REBAUDIANA tissue culture method according to claim 1, it is characterised in that the step 3）Middle culture medium square vase
Specification be 500 ml.
3. a kind of STEVIA REBAUDIANA tissue culture method according to claim 1, it is characterised in that the step 4）The light of middle optical culture
According to intensity be 1300 luxs, step 5）The intensity of illumination of middle optical culture is 1800 luxs.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
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| CN201610948120.3A CN106613953A (en) | 2016-11-03 | 2016-11-03 | Method for tissue culture of stevia rebaudiana |
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| Application Number | Priority Date | Filing Date | Title |
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| CN201610948120.3A CN106613953A (en) | 2016-11-03 | 2016-11-03 | Method for tissue culture of stevia rebaudiana |
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| CN106613953A true CN106613953A (en) | 2017-05-10 |
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| CN201610948120.3A Withdrawn CN106613953A (en) | 2016-11-03 | 2016-11-03 | Method for tissue culture of stevia rebaudiana |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113950246A (en) * | 2019-06-15 | 2022-01-18 | 拜耳公司 | Stable formulations of dithiocarbamates |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103766222A (en) * | 2014-02-12 | 2014-05-07 | 四川农业大学 | Stevia rebaudiana tissue culture method and culture medium thereof |
| CN103858757A (en) * | 2012-12-11 | 2014-06-18 | 丰益(上海)生物技术研发中心有限公司 | Stevia rebaudiana bertoni tissue culture method adopting leaf as explant, and special culture medium thereof |
| CN104642353A (en) * | 2013-11-21 | 2015-05-27 | 苏州田园农业技术开发有限公司 | Low temperature resistant capsicum seed initiator |
| CN105393919A (en) * | 2015-12-01 | 2016-03-16 | 广西壮族自治区药用植物园 | Tissue culture and rapid propagation method for kadsura coccinea |
-
2016
- 2016-11-03 CN CN201610948120.3A patent/CN106613953A/en not_active Withdrawn
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103858757A (en) * | 2012-12-11 | 2014-06-18 | 丰益(上海)生物技术研发中心有限公司 | Stevia rebaudiana bertoni tissue culture method adopting leaf as explant, and special culture medium thereof |
| CN104642353A (en) * | 2013-11-21 | 2015-05-27 | 苏州田园农业技术开发有限公司 | Low temperature resistant capsicum seed initiator |
| CN103766222A (en) * | 2014-02-12 | 2014-05-07 | 四川农业大学 | Stevia rebaudiana tissue culture method and culture medium thereof |
| CN105393919A (en) * | 2015-12-01 | 2016-03-16 | 广西壮族自治区药用植物园 | Tissue culture and rapid propagation method for kadsura coccinea |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113950246A (en) * | 2019-06-15 | 2022-01-18 | 拜耳公司 | Stable formulations of dithiocarbamates |
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Application publication date: 20170510 |