CN108575750A - Gynostemma pentaphylla tissue-culturing rapid propagation culture medium and apply its gynostemma pentaphylla tissue-culturing rapid propagation technique - Google Patents
Gynostemma pentaphylla tissue-culturing rapid propagation culture medium and apply its gynostemma pentaphylla tissue-culturing rapid propagation technique Download PDFInfo
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- CN108575750A CN108575750A CN201810370509.3A CN201810370509A CN108575750A CN 108575750 A CN108575750 A CN 108575750A CN 201810370509 A CN201810370509 A CN 201810370509A CN 108575750 A CN108575750 A CN 108575750A
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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 kind of glue stock indigo plant tissue-culturing rapid propagation culture medium and apply its gynostemma pentaphylla tissue-culturing rapid propagation technique, glue stock indigo plant tissue-culturing rapid propagation culture medium is comprising MS minimal mediums and adds 6 benayl aminopurines and gibberellin in MS minimal mediums respectively, wherein, 6 1.3~1.7mg of benayl aminopurine, 0.4~0.6mg of gibberellin are added in every liter of MS minimal medium.The gynostemma pentaphylla tissue-culturing rapid propagation culture medium of the present invention improves the bud appreciation rate of explant, and gynostemma pentaphylla axillary bud growth is vigorous, and the subculture for being conducive to the later stage is taken root, and economic benefit is improved.
Description
Technical field
The present invention relates to a kind of gynostemma pentaphylla tissue-culturing rapid propagation culture medium and apply its gynostemma pentaphylla tissue-culturing rapid propagation technique.
Background technology
Gynostemma pentaphylla also known as seven leaf courages, gynostemma pentaphyllum makino pellet tea are climing, are Curcurbitaceae, gynostemma pentaphyllum genus herbaceous stem Climbing Plant, in China master
It is distributed in Hunan, Hubei, the provinces such as Yunnan, Guangxi, gynostemma pentaphylla has lowering blood pressure and blood fat, hypoglycemic, strengthening the spleen and stomach, gives protection against cancer, disappears
The functions such as inflammation, be development prospect it is wide shake food dual purpose plant.1986, State Scientific and Technological Commission was in Spark Program, gynostemma pentaphylla
It is classified as the first place of " rare traditional Chinese medicine " leaved for development, on March 5th, 2002, health ministry was included in health products list.
The modes of reproduction of current gynostemma pentaphylla is based on seminal propagation and cutting propagation, but percentage of seedgermination is relatively low, cuttage
Breeding takes work consuming, and two kinds of modes of reproduction breeding coefficients are not high, are subject to seasonal restrictions, seriously limit Fast Propagation in Vitro of Cynostemma
And the research and development of the work such as breeding of new variety.
Invention content
The technical problem to be solved by the present invention is to overcome the deficiencies of existing technologies, a kind of gynostemma pentaphylla tissue-culturing rapid propagation training is provided
Base is supported, it improves the bud appreciation rate of explant, and gynostemma pentaphylla axillary bud growth is vigorous, and the subculture for being conducive to the later stage is taken root, and improves
Economic benefit.
In order to solve the above-mentioned technical problem, the technical scheme is that：A kind of glue stock indigo plant tissue-culturing rapid propagation culture medium, it is wrapped
Minimal medium containing MS also includes to add 6-benzyl aminopurine and gibberellin in MS minimal mediums respectively, wherein every liter
1.3~1.7mg of 6-benzyl aminopurine, 0.4~0.6mg of gibberellin are added in MS minimal mediums.
Further, MS minimal mediums add comprising MS culture mediums and respectively sucrose and agar in MS culture mediums,
In, 25~35g of sucrose is added in every liter of MS culture medium, the PH of agar 4.26~6.26g, MS minimal medium is 5.6~6.
In order to shorten the tissue culture period of gynostemma pentaphylla, the present invention also provides a kind of gynostemma pentaphylla tissue-culturing rapid propagation techniques, it is answered
With glue stock indigo plant tissue-culturing rapid propagation culture medium.
Further, include in processing step：
(1) explant sterilizes；
(2) explant is handled：The both ends for removing explant, to reduce browning probability；
(3) explant is inoculated with：The morphology lower end of explant is inserted into glue stock indigo plant tissue-culturing rapid propagation culture medium；
(4) culture of explant：Explant after inoculation in step (3) is positioned in incubator and carries out bud increment training
It supports.
Further, explant is the stem section with growing point of gynostemma pentaphylla.
Further, in step (1), it is to be impregnated in 75% alcoholic solution that the explant rinsed well, which is positioned over mass concentration,
8~12s, after be transferred in sterile water and rinse 3~4 times, then the mercuric chloride solution 4~5min of soaking disinfection for being 0.1% with mass concentration,
Last be transferred to again in sterile water is rinsed 3~5 times, that is, completes the disinfection of explant.
Further for increasing alcoholic solution and the contact area and mercuric chloride solution of explant and the contact area of explant,
In step (1) during being impregnated explant with alcoholic solution and being impregnated explant with mercuric chloride solution, it is molten constantly to shake alcohol
Liquid and mercuric chloride solution.
Further, the condition of culture in step (4) in incubator is：
Temperature：26.5~27.5 DEG C；
Light application time：12h；
Intensity of illumination is：3000Lx.
Further, fluorescent tube is cultivated by plant in incubator to complete the condition of culture of light application time and intensity of illumination.
After using above-mentioned technical proposal, trained substantially with the MS of the 6-benzyl aminopurine and gibberellin that are added to appropriate weight
Base is supported as glue stock indigo plant tissue-culturing rapid propagation culture medium, improves bud appreciation rate, and gynostemma pentaphylla axillary bud growth is vigorous, is conducive to the later stage
Subculture is taken root, and economic benefit is improved；Currently, gynostemma pentaphylla tissue-culturing rapid propagation is mostly from callus, the present invention by after technologic improvement,
Using the stem section with growing point as explant, directly axillary bud is proliferated, shortens the period of entire tissue culture.
Specific implementation mode
In order that the present invention can be more clearly and readily understood, the present invention is made into one below according to specific embodiment
Step detailed description.
Embodiment one
A kind of glue stock indigo plant tissue-culturing rapid propagation culture medium, it includes MS minimal mediums, also includes to add to train substantially in MS respectively
Support the 6-benzyl aminopurine and gibberellin in base, wherein addition 6-benzyl aminopurine 1.3mg in every liter of MS minimal medium, it is red
Mycin 0.4mg.
MS minimal mediums are comprising MS culture mediums and add sucrose and agar in MS culture mediums respectively, wherein every liter
Sucrose 25g is added in MS culture mediums, the PH of agar 4.26g, MS minimal medium is 5.6.MS minimal mediums go out by high temperature
Bacterium.
A kind of gynostemma pentaphylla tissue-culturing rapid propagation technique, it applies glue stock indigo plant tissue-culturing rapid propagation culture medium.
Include in processing step：
(1) explant sterilizes；
(2) explant is handled：The both ends for removing explant, to reduce browning probability；
(3) explant is inoculated with：The morphology lower end of explant is inserted into glue stock indigo plant tissue-culturing rapid propagation culture medium；
(4) culture of explant：Explant after inoculation in step (3) is positioned in incubator and carries out bud increment training
It supports.
Explant is the stem section with growing point of gynostemma pentaphylla.
In step (1), it is to impregnate 8s in 75% alcoholic solution that the explant rinsed well, which is positioned over mass concentration, rear to turn
Enter in sterile water and rinse 3 times, then the mercuric chloride solution soaking disinfection 4min for being 0.1% with mass concentration, is finally transferred to sterile water again
It is middle to rinse 3 times, that is, complete the disinfection of explant.
In order to increase the contact area of alcoholic solution and explant and the contact area of mercuric chloride solution and explant, step
(1) in alcoholic solution impregnate explant and with mercuric chloride solution impregnate explant during, constantly shake alcoholic solution and
Mercuric chloride solution.
Condition of culture in step (4) in incubator is：
Temperature：26.5℃；
Light application time：12h；
Intensity of illumination is：3000Lx.
Fluorescent tube is cultivated to complete the condition of culture of light application time and intensity of illumination by plant in incubator.In the present embodiment
In, it is that T5 plants cultivate fluorescent tube that plant, which cultivates fluorescent tube, but not limited to this.
Embodiment two
A kind of glue stock indigo plant tissue-culturing rapid propagation culture medium, it includes MS minimal mediums, also includes to add to train substantially in MS respectively
Support the 6-benzyl aminopurine and gibberellin in base, wherein addition 6-benzyl aminopurine 1.5mg in every liter of MS minimal medium, it is red
Mycin 0.5mg.
MS minimal mediums are comprising MS culture mediums and add sucrose and agar in MS culture mediums respectively, wherein every liter
Sucrose 30g is added in MS culture mediums, the PH of agar 5.26g, MS minimal medium is 5.8.MS minimal mediums go out by high temperature
Bacterium.
A kind of gynostemma pentaphylla tissue-culturing rapid propagation technique, it applies glue stock indigo plant tissue-culturing rapid propagation culture medium.
Include in processing step：
4, gynostemma pentaphylla tissue-culturing rapid propagation technique according to claim 3, which is characterized in that include in processing step：
(1) explant sterilizes；
(2) explant is handled：The both ends for removing explant, to reduce browning probability；
(3) explant is inoculated with：The morphology lower end of explant is inserted into glue stock indigo plant tissue-culturing rapid propagation culture medium；
(4) culture of explant：Explant after inoculation in step (3) is positioned in incubator and carries out bud increment training
It supports.
Explant is the stem section with growing point of gynostemma pentaphylla.
In step (1), it is to impregnate 10s in 75% alcoholic solution that the explant rinsed well, which is positioned over mass concentration, after
It is transferred in sterile water and rinses 4 times, then the mercuric chloride solution soaking disinfection 5min for being 0.1% with mass concentration, be finally transferred to again sterile
It is rinsed in water 5 times, that is, completes the disinfection of explant.
In order to increase the contact area of alcoholic solution and explant and the contact area of mercuric chloride solution and explant, step
(1) in alcoholic solution impregnate explant and with mercuric chloride solution impregnate explant during, constantly shake alcoholic solution and
Mercuric chloride solution.
Condition of culture in step (4) in incubator is：
Temperature：27℃；
Light application time：12h；
Intensity of illumination is：3000Lx.
Fluorescent tube is cultivated to complete the condition of culture of light application time and intensity of illumination by plant in incubator.In the present embodiment
In, it is that T5 plants cultivate fluorescent tube that plant, which cultivates fluorescent tube, but not limited to this.
Embodiment three
A kind of glue stock indigo plant tissue-culturing rapid propagation culture medium, it includes MS minimal mediums, also includes to add to train substantially in MS respectively
Support the 6-benzyl aminopurine and gibberellin in base, wherein addition 6-benzyl aminopurine 1.7mg in every liter of MS minimal medium, it is red
Mycin 0.6mg.
MS minimal mediums are comprising MS culture mediums and add sucrose and agar in MS culture mediums respectively, wherein every liter
Sucrose 35g is added in MS culture mediums, the PH of agar 6.26g, MS minimal medium is 6.MS minimal mediums pass through high-temperature sterilization.
A kind of gynostemma pentaphylla tissue-culturing rapid propagation technique, it applies glue stock indigo plant tissue-culturing rapid propagation culture medium.
Include in processing step：
(1) explant sterilizes；
(2) explant is handled：The both ends for removing explant, to reduce browning probability；
(3) explant is inoculated with：The morphology lower end of explant is inserted into glue stock indigo plant tissue-culturing rapid propagation culture medium；
(4) culture of explant：Explant after inoculation in step (3) is positioned in incubator and carries out bud increment training
It supports.
Explant is the stem section with growing point of gynostemma pentaphylla.
In step (1), it is to impregnate 12s in 75% alcoholic solution that the explant rinsed well, which is positioned over mass concentration, after
It is transferred in sterile water and rinses 4 times, then the mercuric chloride solution soaking disinfection 5min for being 0.1% with mass concentration, be finally transferred to again sterile
It is rinsed in water 5 times, that is, completes the disinfection of explant.
In order to increase the contact area of alcoholic solution and explant and the contact area of mercuric chloride solution and explant, step
(1) in alcoholic solution impregnate explant and with mercuric chloride solution impregnate explant during, constantly shake alcoholic solution and
Mercuric chloride solution.
Condition of culture in step (4) in incubator is：
Temperature：27.5℃；
Light application time：12h；
Intensity of illumination is：3000Lx.
Fluorescent tube is cultivated to complete the condition of culture of light application time and intensity of illumination by plant in incubator.In the present embodiment
In, it is that T5 plants cultivate fluorescent tube that plant, which cultivates fluorescent tube, but not limited to this.
In general, it is exogenous hormone that traditional bud proliferation, which need to add 6-benzyl aminopurine, by constantly testing and optimizing
Improvement, and a small amount of gibberellin added, to improve the speed of growth of bud and play the role of strong sprout.In specific experiment process
In, using the tender stem segments with growing point as explant, is sterilized by alcoholic solution, mercuric chloride solution, be then seeded into addition
On the MS minimal mediums of exogenous hormone.
Such as table 1, when individually using 6-benzyl aminopurine as exogenous hormone, in low content, the armpit of most explants
Bud can be proliferated, but the negligible amounts of single explant bud proliferation, with 6-benzyl aminopurine in every liter of MS minimal medium
The quantity of the bud proliferation of the gradual increase of weight, bud appreciation rate and single explant gradually increases, and proliferation bud growth is vigorous, when
The weight of 6-benzyl aminopurine reaches 1.5mg when being in every liter of MS minimal medium, and proliferation bud reaches optimum growh state, but still
There is the case where bud appearance of individual explants is not rised in value, when the weight of 6-benzyl aminopurine in every liter of MS minimal medium continues to increase
Gao Shi, although the quantity of bud proliferation is also more, bud growing way starts to die down, or even vitrification phenomenon occurs.
Table 1：The increment of 6-benzyl aminopurine Gynostemma pentaphyllum Makino axillary bud and life of addition different weight in every liter of MS minimal medium
The influence of long situation
Note：+ poor, ++ preferably, +++ it is vigorous
Such as table two, to reach better effect, every liter of MS minimal medium adds the basis of the 6-benzyl aminopurine of 1.5mg
On, and it is added to suitable gibberellin, the gibberellin of low content can improve the growing way of proliferation bud, in every liter of MS minimal medium
Effect is best when the weight of the gibberellin of addition reaches 0.5mg, and bud appreciation rate is 100%, and growth is vigorous, is conducive to the later stage
Subculture is taken root, and the content of gibberellin continues to increase, and to proliferation bud growing way there is no gain effect, bud growing way is weaker.
Table 2：The 6-benzyl aminopurine of addition 1.5mg and the gibberellin of different weight are to strand in every liter of MS minimal medium
Blue axillary bud increment and the influence of growing state
Note：+ poor, ++ preferably, +++ it is vigorous
Working principle of the present invention is as follows：
The present invention using be added to appropriate weight 6-benzyl aminopurine and gibberellin MS minimal mediums it is blue as glue stock
Tissue-culturing rapid propagation culture medium improves bud appreciation rate, and gynostemma pentaphylla axillary bud growth is vigorous, and the subculture for being conducive to the later stage is taken root, and improves
Economic benefit；Currently, gynostemma pentaphylla tissue-culturing rapid propagation is mostly from callus, the present invention is by after technologic improvement, with growing point
Stem section is explant, is directly proliferated axillary bud, shortens the period of entire tissue culture.
Particular embodiments described above, pair present invention solves the technical problem that, technical solution and advantageous effect carry out
It is further described, it should be understood that the above is only a specific embodiment of the present invention, is not limited to this
Invention, all within the spirits and principles of the present invention, any modification, equivalent substitution, improvement and etc. done should be included in this hair
Within bright protection domain.
Claims (9)
1. a kind of glue stock indigo plant tissue-culturing rapid propagation culture medium, it includes MS minimal mediums, it is characterised in that：Also include to add respectively
6-benzyl aminopurine in MS minimal mediums and gibberellin, wherein 6-benzyl aminopurine is added in every liter of MS minimal medium
1.3~1.7mg, 0.4~0.6mg of gibberellin.
2. glue stock indigo plant tissue-culturing rapid propagation culture medium according to claim 1, it is characterised in that：MS minimal mediums are trained comprising MS
It supports base and adds sucrose and agar in MS culture mediums respectively, wherein add 25~35g of sucrose, fine jade in every liter of MS culture medium
The PH of fat 4.26~6.26g, MS minimal medium is 5.6~6.
3. a kind of gynostemma pentaphylla tissue-culturing rapid propagation technique, it is characterised in that：Its application such as claim 1~2 any one of them glue stock
Blue tissue-culturing rapid propagation culture medium.
4. gynostemma pentaphylla tissue-culturing rapid propagation technique according to claim 3, which is characterized in that include in processing step：
(1) explant sterilizes；
(2) explant is handled：The both ends for removing explant, to reduce browning probability；
(3) explant is inoculated with：The morphology lower end of explant is inserted into glue stock indigo plant tissue-culturing rapid propagation culture medium；
(4) culture of explant：Explant after inoculation in step (3) is positioned in incubator and carries out bud increment culture.
5. gynostemma pentaphylla tissue-culturing rapid propagation technique according to claim 4, it is characterised in that：Explant is that the band of gynostemma pentaphylla is grown
The stem section of point.
6. gynostemma pentaphylla tissue-culturing rapid propagation technique according to claim 4, it is characterised in that：In step (1), by what is rinsed well
It is that 8~12s is impregnated in 75% alcoholic solution that explant, which is positioned over mass concentration, after be transferred in sterile water and rinse 3~4 times, then use
Mercuric chloride solution 4~5min of soaking disinfection that mass concentration is 0.1%, is finally transferred in sterile water and rinses 3~5 times, that is, complete again
The disinfection of explant.
7. gynostemma pentaphylla tissue-culturing rapid propagation technique according to claim 6, it is characterised in that：With alcoholic solution in step (1)
During impregnating explant and impregnating explant with mercuric chloride solution, alcoholic solution and mercuric chloride solution are constantly shaken.
8. gynostemma pentaphylla tissue-culturing rapid propagation technique according to claim 4, it is characterised in that：Training in step (4) in incubator
Foster condition is：
Temperature：26.5~27.5 DEG C；
Light application time：12h；
Intensity of illumination is：3000Lx.
9. gynostemma pentaphylla tissue-culturing rapid propagation technique according to claim 8, it is characterised in that：Pass through plant cultivation light in incubator
Pipe completes the condition of culture of light application time and intensity of illumination.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112075341A (en) * | 2020-09-08 | 2020-12-15 | 桂林丰润莱生物科技股份有限公司 | Method for rapidly culturing gypenosides |
| CN116158350A (en) * | 2023-03-01 | 2023-05-26 | 浙江觅得优生物科技有限公司 | Gynostemma pentaphylla tissue culture medium and cultivation method of adventitious roots of gynostemma pentaphylla |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112075341A (en) * | 2020-09-08 | 2020-12-15 | 桂林丰润莱生物科技股份有限公司 | Method for rapidly culturing gypenosides |
| CN112075341B (en) * | 2020-09-08 | 2023-04-25 | 桂林丰润莱生物科技股份有限公司 | Method for rapidly culturing gynostemma pentaphylla total saponins |
| CN116158350A (en) * | 2023-03-01 | 2023-05-26 | 浙江觅得优生物科技有限公司 | Gynostemma pentaphylla tissue culture medium and cultivation method of adventitious roots of gynostemma pentaphylla |
| CN116158350B (en) * | 2023-03-01 | 2023-09-08 | 浙江觅得优生物科技有限公司 | Gynostemma pentaphylla tissue culture medium and cultivation method of adventitious roots of gynostemma pentaphylla |
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