CN106718914A - A kind of Chinese yew culture medium - Google Patents
A kind of Chinese yew culture medium Download PDFInfo
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- CN106718914A CN106718914A CN201611209707.9A CN201611209707A CN106718914A CN 106718914 A CN106718914 A CN 106718914A CN 201611209707 A CN201611209707 A CN 201611209707A CN 106718914 A CN106718914 A CN 106718914A
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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 belongs to plant culture technical field, and in particular to a kind of Chinese yew culture medium.Chinese yew culture medium of the present invention, including following component and its concentration：The 50g/L of basal medium B5 40, the 40g/L of sucrose 25, the 1.5mg/L of methyl α-naphthyl acetate 0.5,3 acetic acid of indoles 1 3mg/L, the 1mg/L of 6 benzyl aminoadenine 0.1, the 2.5mg/L of gibberellin 1.5, the 8g/L of casamino acid 5, the 10g/L of wheat-germ oil 1, the 15g/L of Winter jujube juice 5, the 1.5mg/L of polyvinylpyrrolidone 0.5, the 1g/L of cinnamic acid 0.1, the 5mg/L of arachidonic acid 1, the 0.15mg/L of methyl jasmonate 0.01, the 5mg/L of chitosan 1.Culture medium of the present invention can accelerate calli induction, effectively prevent Tissue Browning, at the same time can improve the content of taxol in callus.
Description
Technical field
The invention belongs to plant culture technical field, and in particular to a kind of Chinese yew culture medium.
Background technology
Chinese yew is also known as Japanese yew, and tree-like grace, material is excellent, with gardening ornamental value higher, or preciousness use
Wood species.Anticancer active constituent taxol in Chinese yew can effectively suppress hyperplasia and the breeding of tumour cell, to various cancers
The preventing and treating of disease has significant curative effect, is described as " plant gold " by common people.
The exclusive source of current taxol is extracted from bark of Ramulus et folium taxi cuspidatae, is separated, and the content of taxol is very low, greatly
About a ten thousandth of dry weight or so, Chinese yew genus plants growth is extremely slow in addition, thus in order to solve medicine source of Taxol
Problem, people chemically synthesize, artificial cultivation, fungi fermentation production, and the aspect such as culture plant cell is explored.Due to mesh
Preceding chemical synthesis can only synthesize from taxane substances mostly, and taxane substances content in Chinese yew is equally very low, Gu
Its is costly.If artificial cultivation, more than the 30 years Chinese yew of the age of tree are at least needed to extract taxol, the cycle is long,
Cost is also higher.And cell culture can continuous uniform production, external condition influence is little, and can be in bioreactor
Middle large-scale culture, it is easy to improve yield and carry out the separation and Extraction purifying of product, be the prefered method for carrying out industrialized production.
Chinese patent application (CN105400841A) discloses a kind of Chinese yew culture medium, solves plant in the prior art thin
Born of the same parents' culture medium technical problem relatively low to Taxus chinensis cell cultures density.The culture medium is made respectively with sodium nitrate and dipotassium hydrogen phosphate
It is nitrogen source and phosphorus source, and with the addition of certain calcium chloride, is found surprisingly that on this basis by adding particular types and composition
Chinese medicine contribute to lifted cell culture density.The beneficial discovery based on more than, the present invention is by the preferred Huang of laboratory facilities
Hollyhock, edelweiss, clover, scissors grass, Flos Mume, quassia, flaccid knotweed herb, selaginella tamariscina, Japanese polygala etc. are originally used for mankind's disease
The Chinese medicine composition of disease treatment, gained culture medium surprisingly obtains prominent culture effect so that the culture of yew cell is close
Degree is significantly improved, while its speed of growth is faster.
At present, the subject matter present in Taxus chinensis cell cultures process：One is slow Callus formation；Two is to produce
Callus can not utilize；Three is the easy browning of the callus to be formed；Four is that taxol contains in the callus to be formed
Amount is low.
The content of the invention
To solve the above problems, the invention provides a kind of Chinese yew culture medium.Culture medium of the present invention can accelerate callus
The formation of tissue, effectively prevents Tissue Browning, at the same time can improve the content of taxol in callus.
The present invention is achieved through the following technical solutions：
A kind of Chinese yew culture medium, including following component and its concentration：Basal medium B540-50g/L, sucrose 25-
40g/L, methyl α-naphthyl acetate 0.5-1.5mg/L, indole-3-acetic acid 1-3mg/L, 6- benzyl aminoadenine 0.1-1mg/L, gibberellin 1.5-
2.5mg/L, casamino acid 5-8g/L, wheat-germ oil 1-10g/L, Winter jujube juice 5-15g/L, polyvinylpyrrolidone 0.5-
1.5mg/L, cinnamic acid 0.1-1g/L, arachidonic acid 1-5mg/L, methyl jasmonate 0.01-0.15mg/L, chitosan 1-
5mg/L。
Preferably, the Chinese yew culture medium is made up of following component and its concentration：Basal medium B545g/L, sucrose
30g/L, methyl α-naphthyl acetate 1.0mg/L, indole-3-acetic acid 1.5mg/L, 6- benzyl aminoadenine 0.5mg/L, gibberellin 1.75mg/L,
Casamino acid 7.5g/L, wheat-germ oil 5g/L, Winter jujube juice 10.35g/L, polyvinylpyrrolidone 1.2mg/L, cinnamic acid
0.89g/L, arachidonic acid 1.75mg/L, methyl jasmonate 0.03mg/L, chitosan 3.6mg/L.
Sucrose is main carbon source in Taxus chinensis cell cultures, while the concentration of sucrose grows and yew to yew cell
The generation of alcohol has a major impact.Invention technician has found that sucrose concentration is in the range of 25-40g/L in Chinese yew culture medium
The accumulation of growth and taxol to yew cell has obvious facilitation.
Casamino acid (No. CAS in culture medium of the present invention：9000-71-9) with the addition of gibberellin, purple can be promoted
The growth of China fir alcohol cell.
Hormone is to adjust the main matter that plant cell growth development and metabolite are formed, in culture medium of the present invention,
Methyl α-naphthyl acetate, indole-3-acetic acid, 6- benzyl aminoadenine concentrations ratio is 2:3:When 1, the growth to yew cell promotes
Effect and the summation best results of taxol.
Wheat-germ oil is a kind of grain germ oil with wheat malt as waste, and it has concentrated the nutritious extract of wheat
China, rich in vitamin E, linoleic acid, leukotrienes, sweet eight carbon alcohol and various physiologically active components, with nutritive value very high.
Winter jujube (scientific name：Ziziphus jujuba cv.Dongzao), also known as northern Shandong winter jujube, to freeze jujube, wild goose excessively red etc..Winter jujube
In containing abundant carbohydrate, vitamin C, and CAMP, also containing more VitAVitE, potassium, sodium, iron,
The various trace elements such as copper.After invention technician squeezes the juice the winter jujube after maturation, it is added in Chinese yew culture medium, not only
Can play a part of to prevent callus browning, and yew cell growth can be effectively facilitated.
Arachidonic acid, methyl jasmonate and chitosan are the elicitors for producing taxol, and three's collective effect has
Effect promotes the generation of taxol.
The Chinese yew culture medium preparation method, is divided into following steps：
(1) basal medium B5, sucrose, casamino acid, polyvinylpyrrolidone, cinnamic acid, poly- ammonia accurately are weighed
Base glucose.It is dissolved in 800mL ultra-pure waters, stirs, 116 DEG C of sterilizing 30min obtains solution I；
(2) solution I after sterilizing is cooled to 40 DEG C, the Winter jujube juice for obtaining, wheat-germ oil, methyl α-naphthyl acetate, Yin will be squeezed
Diindyl -3- acetic acid, 6- benzyl aminoadenines, gibberellin, arachidonic acid, methyl jasmonate is aseptically added in solution I
Stir, through 0.22 μm of membrane filtration after, obtain solution II；
(3) pH5-6 of above-mentioned solution II is adjusted, is obtained final product.
Preferably, the pH of the culture medium is 5.5-5.8.
Chinese yew culture medium of the present invention can be used for Taxus chinensis cell suspension cultures, taxus callus induction and subculture training
Support.
Chinese yew culture medium each component of the present invention collocation is reasonable, and each component is each other in corresponding concentration range
Synergy can be played.Chinese yew culture medium of the present invention compared with prior art, with following remarkable result：
(1) culture medium of the present invention be used for taxus callus Fiber differentiation when can improve callus growth rate and
Shortening averages out the more time；
(2) present invention culture can effectively suppress the browning situation of callus；
(3) medium culture base of the present invention can promote the dynamic accumulation of taxol.
Specific embodiment
Below by way of the description of specific embodiment, the invention will be further described, but this is not to limit of the invention
System, those skilled in the art's basic thought of the invention, various modifications may be made or improves, but without departing from this
The basic thought of invention, within the scope of the present invention.
Raw material wheat-germ oil is purchased from Puyang Zhong He Trade Co., Ltd.s；Chitosan is purchased from Henan Jin Run food additives
Plus agent Co., Ltd；Winter jujube juice is obtained by the fresh squeezing of winter jujube that market is bought.
A kind of Chinese yew culture medium of embodiment 1
A kind of Chinese yew culture medium, is made up of following component and its concentration：Basal medium B545g/L, sucrose 30g/L,
Methyl α-naphthyl acetate 1.0mg/L, indole-3-acetic acid 1.5mg/L, 6- benzyl aminoadenine 0.5mg/L, gibberellin 1.75mg/L, casein
Amino acid 7.5g/L, wheat-germ oil 5g/L, Winter jujube juice 10.35g/L, polyvinylpyrrolidone 1.2mg/L, cinnamic acid 0.89g/
L, arachidonic acid 1.75mg/L, methyl jasmonate 0.03mg/L, chitosan 3.6mg/L.
Chinese yew culture medium preparation method, is divided into following steps：
(1) basal medium B5, sucrose, casamino acid, polyvinylpyrrolidone, cinnamic acid, poly- ammonia accurately are weighed
Base glucose.It is dissolved in 800mL ultra-pure waters, stirs, 116 DEG C of sterilizing 30min obtains solution I；
(2) solution I after sterilizing is cooled to 40 DEG C, the Winter jujube juice for obtaining, wheat-germ oil, methyl α-naphthyl acetate, Yin will be squeezed
Diindyl -3- acetic acid, 6- benzyl aminoadenines, gibberellin, arachidonic acid, methyl jasmonate is aseptically added in solution I
Stir, through 0.22 μm of membrane filtration after, obtain solution II；
(3) pH for adjusting above-mentioned solution II is 5.8, is obtained final product.
A kind of Chinese yew culture medium of embodiment 2
A kind of Chinese yew culture medium, by following component and its concentration：Basal medium B540g/L, sucrose 25g/L, naphthalene second
Sour 0.5mg/L, indole-3-acetic acid 1mg/L, 6- benzyl aminoadenine 0.1mg/L, gibberellin 1.5mg/L, casamino acid
5g/L, wheat-germ oil 1g/L, Winter jujube juice 5g/L, polyvinylpyrrolidone 0.5mg/L, cinnamic acid 0.1g/L, arachidonic acid
1mg/L, methyl jasmonate 0.01mg/L, chitosan 1mg/L.
The Chinese yew culture medium preparation method is similar to Example 1.
A kind of Chinese yew culture medium of embodiment 3
A kind of Chinese yew culture medium, by following component and its concentration：Basal medium B550g/L, sucrose 40g/L, naphthalene second
Sour 1.5mg/L, indole-3-acetic acid 3mg/L, 6- benzyl aminoadenine 1mg/L, gibberellin 2.5mg/L, casamino acid 8g/
L, wheat-germ oil 10g/L, Winter jujube juice 15g/L, polyvinylpyrrolidone 1.5mg/L, cinnamic acid 1g/L, arachidonic acid 5mg/
L, methyl jasmonate 0.15mg/L, chitosan 5mg/L.
The Chinese yew culture medium preparation method is similar to Example 1.
A kind of Chinese yew culture medium of comparative example 1
A kind of Chinese yew culture medium, is made up of following component and its concentration：Basal medium B545g/L, sucrose 30g/L,
Methyl α-naphthyl acetate 1.0mg/L, indole-3-acetic acid 1.5mg/L, 6- benzyl aminoadenine 1.5mg/L, gibberellin 1.75mg/L, casein
Amino acid 7.5g/L, wheat-germ oil 5g/L, Winter jujube juice 10.35g/L, polyvinylpyrrolidone 1.2mg/L, cinnamic acid 0.89g/
L, arachidonic acid 1.75mg/L, methyl jasmonate 0.03mg/L, chitosan 3.6mg/L.
The Chinese yew culture medium preparation method is similar to Example 1.
Difference with embodiment 1 is the concentration that increased 6- benzyl aminoadenines.
A kind of Chinese yew culture medium of comparative example 2
A kind of Chinese yew culture medium, is made up of following component and its concentration：Basal medium B545g/L, sucrose 30g/L,
Methyl α-naphthyl acetate 1.0mg/L, indole-3-acetic acid 1.5mg/L, 6- benzyl aminoadenine 0.5mg/L, gibberellin 1.75mg/L, casein
Amino acid 7.5g/L, wheat-germ oil 5g/L, vitamin C 3.6mgg/L, polyvinylpyrrolidone 1.2mg/L, cinnamic acid
0.89g/L, arachidonic acid 1.75mg/L, methyl jasmonate 0.03mg/L, chitosan 3.6mg/L.
The Chinese yew culture medium preparation method is similar to Example 1.
Difference with embodiment 1 is that Winter jujube juice is replaced with into vitamin C.
A kind of Chinese yew culture medium of comparative example 3
A kind of Chinese yew culture medium, is made up of following component and its concentration：Basal medium B545g/L, sucrose 30g/L,
Methyl α-naphthyl acetate 1.0mg/L, indole-3-acetic acid 1.5mg/L, 6- benzyl aminoadenine 0.5mg/L, gibberellin 1.75mg/L, casein
Amino acid 7.5g/L, wheat-germ oil 5g/L, Winter jujube juice 10.35g/L, polyvinylpyrrolidone 1.2mg/L, cinnamic acid 0.89g/
L, arachidonic acid 5.35mg/L, methyl jasmonate 0.03mg/L.
The Chinese yew culture medium preparation method is similar to Example 1.
Difference with embodiment 1 is to be not added with chitosan, increased arachidonic acid concentration.
The induction of callus of test example 1
(1) explant sterilization
Branches and leaves surface dirt first is washed away with liquid detergent, clear water is rinsed 3-4 hours, is soaked 1-2 minutes with 75% ethanol,
Mercuric chloride routine disinfection 10-15min, sterile distilled water is rinsed 4-6 times and blotted with aseptic paper, standby.
(2) it is inoculated with
The needle for removing stem section (point old stem section and tender stem segmentses) with scalpel is switched to the length of 1cm or so, or with tender
The segment that needle cuts into 0.5-1cm is inoculated on different culture media.The tender sprig of 5-6cm childrens or last year raw sprig cut 2 knives, into
3 sections of upper, middle and lower, are inserted perpendicularly into or are tiltedly put in (culture medium in the culture medium that embodiment 1-3 and comparative example 1-3 are prepared
In agar also added with 2%).Each bottle is inoculated with 4 to 5 pieces of explants.
(3) cultivate
Above-mentioned postvaccinal explant is carried out into dark Fiber differentiation：Under the conditions of 25 DEG C ± 1 DEG C of temperature, humidity 85% ± 5%
Light culture 12 hours.Then dim light Fiber differentiation is carried out：After through light culture 12 hours, switch to 25 DEG C ± 1 DEG C of daily day temperature,
Dim light culture under illumination 1000-1200 μ x, every 12 hours switch conditions are once.
(4) callus growth index
Inductivity (%)：The explant block number of inductivity (%)=grow callus/(access explant block number-dirty
Dye explant block number) × 100；
Average out the more time：Average out more time=always go out more time/explant number
Callus induction rate and average out more that the time is as shown in the following Table 1 in each culture medium.
Callus induction rate and the more time is averaged out in each culture medium of table 1
| Culture medium | Inductivity (%) | Average out more the time (my god) |
| Experimental example 1 | 86 | 7 |
| Experimental example 2 | 79 | 12 |
| Experimental example 3 | 82 | 15 |
| Comparative example 1 | 63 | 18 |
| Comparative example 2 | 67 | 24 |
| Comparative example 3 | 68 | 16 |
As shown in Table 1, embodiment medium culture taxus callus inductivity is apparently higher than comparative example, wherein implementing
The culture medium callus induction rate highest that example 1 is prepared, is 1.3 times of comparative example 3；And red bean in embodiment culture medium
China fir callus averages out more Time transfer receiver ratio culture medium and heals that the time short.It can thus be appreciated that culture medium of the present invention is conducive to callus
The formation of tissue.
Brown stain situation of the callus in squamous subculture in the different culture media of test example 2
The squamous subculture of callus is carried out using embodiment 1-3 and comparative example 1-3 culture mediums, and is observed in incubation
The brown stain situation of callus in each culture medium, as a result as shown in table 2.
Brown stain situation of the callus in squamous subculture in the different culture media of table 2
| Culture medium | Browning degree |
| Embodiment 1 | - |
| Embodiment 2 | - |
| Embodiment 3 | - |
| Comparative example 1 | + |
| Comparative example 2 | ++ |
| Comparative example 3 | + |
Note："+" represents slight browning；" ++ " represents serious browning；" +++ " represents that browning is very serious；"-" represents basic
Without browning.
As can be seen from Table 2, the Chinese yew culture medium that embodiment 1-3 is prepared can effectively suppress the brown of callus
Change.Wherein, there is serious browning in the culture medium of comparative example 2, and do not occur browning in embodiment 1, illustrate the addition of Winter jujube juice
It is obvious to suppressing the effect of callus browning phenomenon.
The measure of content of taxol in the callus of test example 3
Callus after being cultivated 30 days in each culture medium of above-mentioned test example 1 is dried to constant weight at 60 DEG C, is ground extremely
0.25mm.With chloroform: methyl alcohol=6: 4 refluxing extractions 3 times, concentration, the de- ester of petroleum ether, then reduced vacuum low temperature drying is to perseverance
Weight.The accurate testing sample that weighs is appropriate, plus flowing phased soln, is made into the solution to be measured that concentration is 0.3mg/mL, accurate sample introduction point
Analysis.Result is as shown in table 3.
Yew alcohol content in the different culture media callus of table 3
As shown in Table 3, content of taxol is above comparative example in the callus that embodiment 1-3 medium cultures are obtained,
Content of taxol improves 85.4% compared with comparative example 1 in the culture medium callus of embodiment 1.
Claims (4)
1. a kind of Chinese yew culture medium, it is characterised in that including following component and its concentration：Basal medium B540-50g/L,
Sucrose 25-40g/L, methyl α-naphthyl acetate 0.5-1.5mg/L, indole-3-acetic acid 1-3mg/L, 6- benzyl aminoadenine 0.1-1mg/L are red
Mycin 1.5-2.5mg/L, casamino acid 5-8g/L, wheat-germ oil 1-10g/L, Winter jujube juice 5-15g/L, polyvinyl pyrrole
Alkanone 0.5-1.5mg/L, cinnamic acid 0.1-1g/L, arachidonic acid 1-5mg/L, methyl jasmonate 0.01-0.15mg/L, poly- ammonia
Base glucose 1-5mg/L.
2. Chinese yew culture medium according to claim 1, it is characterised in that be made up of following component and its concentration：Basis training
Foster base B545g/L, sucrose 30g/L, methyl α-naphthyl acetate 1.0mg/L, indole-3-acetic acid 1.5mg/L, 6- benzyl aminoadenine 0.5mg/L,
Gibberellin 1.75mg/L, casamino acid 7.5g/L, wheat-germ oil 5g/L, Winter jujube juice 10.35g/L, polyvinylpyrrolidine
Ketone 1.2mg/L, cinnamic acid 0.89g/L, arachidonic acid 1.75mg/L, methyl jasmonate 0.03mg/L, chitosan
3.6mg/L。
3. the preparation method of Chinese yew culture medium according to claim 1 or claim 2, it is characterised in that be divided into following steps：
(1) basal medium B5, sucrose, casamino acid, polyvinylpyrrolidone, cinnamic acid, poly- amino Portugal accurately are weighed
Sugar.It is dissolved in 800mL ultra-pure waters, stirs, 116 DEG C of sterilizing 30min obtains solution I；
(2) solution I after sterilizing is cooled to 40 DEG C, the Winter jujube juice for obtaining, wheat-germ oil, methyl α-naphthyl acetate, indoles -3- will be squeezed
Acetic acid, 6- benzyl aminoadenines, gibberellin, arachidonic acid, stirring is equal during methyl jasmonate aseptically adds solution I
It is even, through 0.22 μm of membrane filtration after, obtain solution II；
(3) pH to 5-6 of above-mentioned solution II is adjusted, is obtained final product.
4. the preparation method of Chinese yew culture medium according to claim 3, it is characterised in that the pH of the culture medium is adjusted to
5.5-5.8。
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| CN108841778A (en) * | 2018-04-28 | 2018-11-20 | 大连普瑞康生物技术有限公司 | A kind of yew cell tissue culture |
| CN110972952A (en) * | 2019-12-26 | 2020-04-10 | 上海帝巨智能科技有限公司 | Tissue rapid propagation culture method of Chinese yew |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108841778A (en) * | 2018-04-28 | 2018-11-20 | 大连普瑞康生物技术有限公司 | A kind of yew cell tissue culture |
| CN110972952A (en) * | 2019-12-26 | 2020-04-10 | 上海帝巨智能科技有限公司 | Tissue rapid propagation culture method of Chinese yew |
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