CN109122323A - The cultural method of Catharanthus Roseus Cell high yield vincaleukoblastinum - Google Patents
The cultural method of Catharanthus Roseus Cell high yield vincaleukoblastinum Download PDFInfo
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- CN109122323A CN109122323A CN201811230765.9A CN201811230765A CN109122323A CN 109122323 A CN109122323 A CN 109122323A CN 201811230765 A CN201811230765 A CN 201811230765A CN 109122323 A CN109122323 A CN 109122323A
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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
- A01H4/005—Methods for micropropagation; Vegetative plant propagation using cell or tissue culture techniques
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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
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Abstract
The present invention provides a kind of cultural method of Catharanthus Roseus Cell high yield vincaleukoblastinum comprising following steps: 1) cultivating Catharanthus roseus calli；2) the Catharanthus Roseus Cell system for stablizing heredity is cultivated；3) the Catharanthus Roseus Cell system that step 2) obtains is inoculated into liquid suspension culture base and carries out Fiber differentiation, to stimulate the accumulation of alkaloid in catharanthus roseus, the inductive condition are as follows: Catharanthus Roseus Cell ties up to liquid suspension culture base culture and starts for 10-15 days, cultivation temperature is reduced to 25~28 DEG C from 32-35 DEG C, it is passed through CO gas and adds niacin into liquid suspension culture base, moulting hormone and silver thiosulfate, after maintaining the temperature to continue culture 2-3 days, renewal cultivation temperature is to 32-35 DEG C, culture 4-5 days, alternately heated up and the culture that cools down, until the culture terminates, obtain the Catharanthus Roseus Cell of high yield vincaleukoblastinum.The present invention has the characteristics that high cell growth speed, vincaleukoblastinum cumulative amount are high.
Description
Technical field
The invention belongs to technical field of cell culture.It is more particularly related to which a kind of Catharanthus Roseus Cell high yield is long
The cultural method of spring alkali.
Background technique
Catharanthus roseus, spring when alias marigold, four, everyday it is new, wild goose is red, 30,000 flowers, be that Apocynaceae Vinca is a kind of
Plant.Currently, more than 100 kinds of terpene indole alkaloids have been isolated from catharanthus roseus, such as vincaleukoblastinum, vincristine, serpentaria
Alkali, vindoline alkali etc., many alkaloids separated from catharanthus roseus all have anti-tumor activity, wherein vincaleukoblastinum is mesh
Medicinal Maximum Value, most widely used alkaloid in preceding catharanthus roseus.However, the intracorporal vinblastine content of catharanthus roseus plant and
Its pettiness, so that the vincaleukoblastinum extracted from catharanthus roseus plant is far from satisfying the market demand.
Summary of the invention
As various extensive and careful research and experiment as a result, it has been found by the inventor that outstanding in liquid
Containing being passed through after carbon monoxide in the case where niacin, moulting hormone and silver thiosulfate, to improve catharanthus roseus thin in floating culture medium
The content of vincaleukoblastinum in born of the same parents.Based on this discovery, the present invention is completed.
It is excellent it is an object of the invention to solve at least the above problems and/or defect, and provide at least to will be described later
Point.
It is a still further object of the present invention to provide a kind of cultural methods of Catharanthus Roseus Cell high yield vincaleukoblastinum, can promote
The growth of Catharanthus Roseus Cell improves cell content in unit time unit volume culture solution, and then improves the content of vincaleukoblastinum.
In order to realize these purposes and other advantages according to the present invention, a kind of Catharanthus Roseus Cell high yield vincaleukoblastinum is provided
Cultural method comprising following steps:
1) full Changchun flower seed is selected, is impregnated 10-20 seconds using weak acid electrolysis water, is washed using aseptic deionized water
Only, it is trained using on the B5 basal medium containing 10-20mg/L vitamin C, 7-9g/L agar and 0.1-0.3g/L oligosaccharide
It supports and obtains aseptic seedling, using the plumular axis of the aseptic seedling as explant, access in callus inducing medium and carry out Fiber differentiation,
Cultivation temperature is 32-35 DEG C, and alternately illumination and dark culturing daily induces callus, then turns callus
Enter in subculture medium, every 20-25 days subcultures are primary, squamous subculture 3-5 times, to obtain stable Catharanthus roseus calli；
2) selection growth is vigorous, quality is loose, the in stable condition uniform Catharanthus roseus calli, and it is outstanding to be inoculated into liquid
In floating culture medium, bead is then added into liquid suspension culture base, and high speed oscillation 8-10min is carried out in earthquake shaking table,
180~200rpm of shaking speed is subsequently placed in illumination shaking table and is cultivated, shaking speed 100-120rpm, intensity of illumination
1500~2000lx, 10~15h/day of light application time, every 20-22 days squamous subcultures 1 time squamous subculture 5~8 times, are stablized
The Catharanthus Roseus Cell system of heredity；
3) the Catharanthus Roseus Cell system that step 2) obtains is inoculated into liquid suspension culture base and carries out Fiber differentiation, with stimulation
The accumulation of alkaloid in catharanthus roseus, the inductive condition are as follows: Catharanthus Roseus Cell ties up to liquid suspension culture base culture the 10-15 days
Start, cultivation temperature is reduced to 25~28 DEG C from 32-35 DEG C, is passed through CO gas and into liquid suspension culture base
Niacin, moulting hormone and silver thiosulfate are added, after maintaining the temperature to continue culture 2-3 days, renewal cultivation temperature to 32-35
DEG C, it cultivates 4-5 days, is alternately heated up and the culture that cools down, up to the culture terminates, the catharanthus roseus of acquisition high yield vincaleukoblastinum is thin
Born of the same parents.
Preferably, the callus inducing medium is to contain 1~2mg/L basic element of cell division, 0.1~0.5mg/L
6-benzyl aminopurine, 20~30g/L sucrose, 3-5mg/L licoflavone and 5~8g/L agar B5 basal medium.
Preferably, the subculture medium is formed by adding phenylalanine in callus inducing medium, phenylpropyl alcohol ammonia
Concentration of the acid in subculture medium is 0.5~1.0mmol/L.
Preferably, the liquid suspension culture base is containing fast added with licoflavone, resveratrol, 6- benzyl amino
Purine, methyl α-naphthyl acetate, 2,4- dichlorphenoxyacetic acid, sucrose B5 basal medium, the concentration of the licoflavone is 0.005-
0.008mg/L, the concentration of resveratrol are 0.001-0.003mg/L, and the concentration of 6-benzyl aminopurine is 0.08-0.1mg/L, naphthalene
The concentration of acetic acid is 0.2-0.3mg/L, and the concentration of 2,4- dichlorphenoxyacetic acids is 0.06-0.09mg/L, the concentration of sucrose is 20-
The B5 basal medium of 30g/L.
It preferably, further include tryptophan and caseinhydrolysate in the fluid nutrient medium, the concentration of the tryptophan is
20-30mg/L, the content of the caseinhydrolysate are 50-70mg/L.
Preferably, the intake of the carbon monoxide is 0.01-0.03m3/h。
Preferably, the concentration of the niacin is 0.05-0.1mg/L, and the concentration of the moulting hormone is 0.06-
0.08mg/L, the concentration of the silver thiosulfate are 0.02-.0.05mg/L.
The present invention is include at least the following beneficial effects: by using weak acid electrolysis water soaking disinfection Changchun flower seed, can be mentioned
8% or more the germination percentage of high Changchun flower seed；Using callus inducing medium of the invention, effectively cell can be avoided to go out
Existing browning, and can be shortened induction duration；It is passed through in the fluid nutrient medium containing niacin, moulting hormone and silver thiosulfate
Carbon monoxide promotes to synthesize vincaleukoblastinum key gene d4h in Catharanthus Roseus Cell and dat gene expression amount increases 1 times, improves
The biosynthesis amount of vincaleukoblastinum；By alternately changing cultivation temperature, vinblastine content in Catharanthus Roseus Cell can be promoted to improve
15% or more.The present invention has the characteristics that high cell growth speed, vincaleukoblastinum cumulative amount are high.
Further advantage, target and feature of the invention will be partially reflected by the following instructions, and part will also be by this
The research and practice of invention and be understood by the person skilled in the art.
Specific embodiment
The present invention is described in further detail below, to enable those skilled in the art's refer to the instruction text being capable of evidence
To implement.
It should be appreciated that such as " having ", "comprising" and " comprising " term used herein do not allot one or more
The presence or addition of a other elements or combinations thereof.
Embodiment 1
A kind of cultural method of Catharanthus Roseus Cell high yield vincaleukoblastinum comprising following steps:
1) full Changchun flower seed is selected, is impregnated 10 seconds using weak acid electrolysis water, is cleaned, is made using aseptic deionized water
Aseptic seedling is obtained with cultivating on the B5 basal medium containing 10mg/L vitamin C, 7g/L agar and 0.1g/L oligosaccharide, with
The plumular axis of the aseptic seedling is explant, accesses in callus inducing medium and carries out Fiber differentiation, and cultivation temperature is 32 DEG C,
Daily alternately illumination and dark culturing, induces callus, then callus is transferred in subculture medium, and every 25
Its subculture is primary, and squamous subculture 3 times, to obtain stable Catharanthus roseus calli；
2) selection growth is vigorous, quality is loose, the in stable condition uniform Catharanthus roseus calli, and it is outstanding to be inoculated into liquid
In floating culture medium, bead is then added into liquid suspension culture base, and high speed oscillation 8min, shaking table are carried out in earthquake shaking table
Revolving speed 200rpm is subsequently placed in illumination shaking table and is cultivated, shaking speed 100rpm, intensity of illumination 2000lx, when illumination
Between 15h/day, every 22 days squamous subcultures 1 time, squamous subculture 5 times, obtain stablize heredity Catharanthus Roseus Cell system；
3) the Catharanthus Roseus Cell system that step 2) obtains is inoculated into liquid suspension culture base and carries out Fiber differentiation, with stimulation
The accumulation of alkaloid in catharanthus roseus, the inductive condition are as follows: Catharanthus Roseus Cell is tied up to the culture of liquid suspension culture base the 10th day and opened
Begin, cultivation temperature is reduced to 28 DEG C from 32 DEG C, be passed through CO gas and add Buddhist nun gram into liquid suspension culture base
Acid, moulting hormone and silver thiosulfate, after maintaining the temperature to continue culture 2 days, renewal cultivation temperature is cultivated 4 days to 32 DEG C, is handed over
That replaces carries out the culture that heats up and cool down, until the culture terminates, obtains the Catharanthus Roseus Cell of high yield vincaleukoblastinum.
Compared with using alcohol or potassium permanganate disinfection solution, after weak acid electrolysis water soaking disinfection, the seed of catharanthus roseus
Germination percentage improves 8.9%.Be not passed through carbon monoxide and be not added with the test of niacin, moulting hormone and silver thiosulfate
It compares, using the expression quantity of fluorescent quantitative PCR technique measurement d4h and dat gene, the results showed that d4h and dat in the present embodiment
92% and 95% has been respectively increased in the expression quantity of gene.D4h and dat gene is the key enzyme for synthesizing vindoline, and vindoline is
The important prerequisite for synthesizing vincaleukoblastinum and vincristine, and is not passed through carbon monoxide and is not added with niacin, moulting hormone and sulphur
The test of silver thiosulfate is compared, and after the present embodiment Catharanthus Roseus Cell is freeze-dried, detects the content of vindoline and vincaleukoblastinum, hair respectively
Existing vindoline content improves 86%, and vinblastine content improves 95%, illustrates to be passed through carbon monoxide and addition niacin, slough off
Skin hormone and silver thiosulfate can promote cell to synthesize vincaleukoblastinum.
Embodiment 2
A kind of cultural method of Catharanthus Roseus Cell high yield vincaleukoblastinum comprising following steps:
1) full Changchun flower seed is selected, is impregnated 10 seconds using weak acid electrolysis water, is cleaned, is made using aseptic deionized water
Aseptic seedling is obtained with cultivating on the B5 basal medium containing 10mg/L vitamin C, 7g/L agar and 0.1g/L oligosaccharide, with
The plumular axis of the aseptic seedling is explant, accesses in callus inducing medium and carries out Fiber differentiation, and cultivation temperature is 32 DEG C,
Daily alternately illumination and dark culturing, induces callus, then callus is transferred in subculture medium, and every 25
Its subculture is primary, and squamous subculture 3 times, to obtain stable Catharanthus roseus calli；Wherein, the callus inducing medium
To contain the 1mg/L basic element of cell division, 00.5mg/L 6-benzyl aminopurine, 30g/L sucrose, 3mg/L licoflavone and 8g/L agar
B5 basal medium；
2) selection growth is vigorous, quality is loose, the in stable condition uniform Catharanthus roseus calli, and it is outstanding to be inoculated into liquid
In floating culture medium, bead is then added into liquid suspension culture base, and high speed oscillation 8min, shaking table are carried out in earthquake shaking table
Revolving speed 200rpm is subsequently placed in illumination shaking table and is cultivated, shaking speed 100rpm, intensity of illumination 2000lx, when illumination
Between 15h/day, every 22 days squamous subcultures 1 time, squamous subculture 5 times, obtain stablize heredity Catharanthus Roseus Cell system；
3) the Catharanthus Roseus Cell system that step 2) obtains is inoculated into liquid suspension culture base and carries out Fiber differentiation, with stimulation
The accumulation of alkaloid in catharanthus roseus, the inductive condition are as follows: Catharanthus Roseus Cell is tied up to the culture of liquid suspension culture base the 10th day and opened
Begin, cultivation temperature is reduced to 28 DEG C from 32 DEG C, be passed through CO gas and add Buddhist nun gram into liquid suspension culture base
Acid, moulting hormone and silver thiosulfate, after maintaining the temperature to continue culture 2 days, renewal cultivation temperature is cultivated 4 days to 32 DEG C, is handed over
That replaces carries out the culture that heats up and cool down, until the culture terminates, obtains the Catharanthus Roseus Cell of high yield vincaleukoblastinum.
The callus induction success rate of the present embodiment is 95.6%, than the induction success rate for using common induced medium
Improve 15% or more.Be not passed through carbon monoxide and be not added with the test phase of niacin, moulting hormone and silver thiosulfate
Than using the expression quantity of fluorescent quantitative PCR technique measurement d4h and dat gene, the results showed that d4h the and dat base in the present embodiment
93% and 96% has been respectively increased in the expression quantity of cause.D4h and dat gene is the key enzyme for synthesizing vindoline, and vindoline is to close
At the important prerequisite of vincaleukoblastinum and vincristine, and it is not passed through carbon monoxide and is not added with niacin, moulting hormone and thio
The test of silver sulfate is compared, and the vindoline content in the present embodiment Catharanthus Roseus Cell improves 96%, and vinblastine content improves
103%, illustrate that being passed through carbon monoxide and addition niacin, moulting hormone and silver thiosulfate can promote cell to synthesize Changchun
Alkali.
Embodiment 3
A kind of cultural method of Catharanthus Roseus Cell high yield vincaleukoblastinum comprising following steps:
1) full Changchun flower seed is selected, is impregnated 10 seconds using weak acid electrolysis water, is cleaned, is made using aseptic deionized water
Aseptic seedling is obtained with cultivating on the B5 basal medium containing 10mg/L vitamin C, 7g/L agar and 0.1g/L oligosaccharide, with
The plumular axis of the aseptic seedling is explant, accesses in callus inducing medium and carries out Fiber differentiation, and cultivation temperature is 32 DEG C,
Daily alternately illumination and dark culturing, induces callus, then callus is transferred in subculture medium, and every 25
Its subculture is primary, and squamous subculture 3 times, to obtain stable Catharanthus roseus calli；Wherein, the callus inducing medium
To contain the 1mg/L basic element of cell division, 00.5mg/L 6-benzyl aminopurine, 30g/L sucrose, 3mg/L licoflavone and 8g/L agar
B5 basal medium；The subculture medium is formed by adding phenylalanine in callus inducing medium, phenylalanine
Concentration in subculture medium is 1.0mmol/L；
2) selection growth is vigorous, quality is loose, the in stable condition uniform Catharanthus roseus calli, and it is outstanding to be inoculated into liquid
In floating culture medium, bead is then added into liquid suspension culture base, and high speed oscillation 8min, shaking table are carried out in earthquake shaking table
Revolving speed 200rpm is subsequently placed in illumination shaking table and is cultivated, shaking speed 100rpm, intensity of illumination 2000lx, when illumination
Between 15h/day, every 22 days squamous subcultures 1 time, squamous subculture 5 times, obtain stablize heredity Catharanthus Roseus Cell system；
3) the Catharanthus Roseus Cell system that step 2) obtains is inoculated into liquid suspension culture base and carries out Fiber differentiation, with stimulation
The accumulation of alkaloid in catharanthus roseus, the inductive condition are as follows: Catharanthus Roseus Cell is tied up to the culture of liquid suspension culture base the 10th day and opened
Begin, cultivation temperature is reduced to 28 DEG C from 32 DEG C, be passed through CO gas and add Buddhist nun gram into liquid suspension culture base
Acid, moulting hormone and silver thiosulfate, after maintaining the temperature to continue culture 2 days, renewal cultivation temperature is cultivated 4 days to 32 DEG C, is handed over
That replaces carries out the culture that heats up and cool down, until the culture terminates, obtains the Catharanthus Roseus Cell of high yield vincaleukoblastinum.
Catharanthus Roseus Cell can be promoted to keep growth activity by adding phenylalanine in subculture medium, keep catharanthus roseus thin
Born of the same parents keep vigorous splitting ability, and vitro growth rates improve 3% or more.Be not passed through carbon monoxide and be not added with Ni Ke
Acid, moulting hormone are compared with the test of silver thiosulfate, using the expression of fluorescent quantitative PCR technique measurement d4h and dat gene
Amount, the results showed that 110% and 121% has been respectively increased in the expression quantity of the d4h and dat gene in the present embodiment.D4h and dat base
Because being to synthesize the key enzyme of vindoline, and vindoline is the important prerequisite for synthesizing vincaleukoblastinum and vincristine, and is not passed through an oxygen
Change carbon and the test for being not added with niacin, moulting hormone and silver thiosulfate are compared, the text in the present embodiment Catharanthus Roseus Cell
More spirit contents improve 133%, and vinblastine content improves 128%, illustrate to be passed through carbon monoxide and addition niacin, husking
Hormone and silver thiosulfate can promote cell to synthesize vincaleukoblastinum.
Embodiment 4
A kind of cultural method of Catharanthus Roseus Cell high yield vincaleukoblastinum comprising following steps:
1) full Changchun flower seed is selected, is impregnated 10 seconds using weak acid electrolysis water, is cleaned, is made using aseptic deionized water
Aseptic seedling is obtained with cultivating on the B5 basal medium containing 10mg/L vitamin C, 7g/L agar and 0.1g/L oligosaccharide, with
The plumular axis of the aseptic seedling is explant, accesses in callus inducing medium and carries out Fiber differentiation, and cultivation temperature is 32 DEG C,
Daily alternately illumination and dark culturing, induces callus, then callus is transferred in subculture medium, and every 25
Its subculture is primary, and squamous subculture 3 times, to obtain stable Catharanthus roseus calli；Wherein, the callus inducing medium
To contain the 1mg/L basic element of cell division, 00.5mg/L 6-benzyl aminopurine, 30g/L sucrose, 3mg/L licoflavone and 8g/L agar
B5 basal medium；The subculture medium is formed by adding phenylalanine in callus inducing medium, phenylalanine
Concentration in subculture medium is 1.0mmol/L；
2) selection growth is vigorous, quality is loose, the in stable condition uniform Catharanthus roseus calli, and it is outstanding to be inoculated into liquid
In floating culture medium, bead is then added into liquid suspension culture base, and high speed oscillation 8min, shaking table are carried out in earthquake shaking table
Revolving speed 200rpm is subsequently placed in illumination shaking table and is cultivated, shaking speed 100rpm, intensity of illumination 2000lx, when illumination
Between 15h/day, every 22 days squamous subcultures 1 time, squamous subculture 5 times, obtain stablize heredity Catharanthus Roseus Cell system；Wherein, described
Liquid suspension culture base is containing added with licoflavone, resveratrol, 6-benzyl aminopurine, methyl α-naphthyl acetate, 2,4- Dichlorophenoxy
The concentration of the B5 basal medium of acetic acid, sucrose, the licoflavone is 0.005mg/L, and the concentration of resveratrol is
0.001mg/L, the concentration of 6-benzyl aminopurine are 0.08mg/L, and the concentration of methyl α-naphthyl acetate is 0.2mg/L, 2,4- dichlorphenoxyacetic acids
Concentration be 0.06mg/L, the B5 basal medium that the concentration of sucrose is 30g/L；
3) the Catharanthus Roseus Cell system that step 2) obtains is inoculated into liquid suspension culture base and carries out Fiber differentiation, with stimulation
The accumulation of alkaloid in catharanthus roseus, the inductive condition are as follows: Catharanthus Roseus Cell is tied up to the culture of liquid suspension culture base the 10th day and opened
Begin, cultivation temperature is reduced to 28 DEG C from 32 DEG C, be passed through CO gas and add Buddhist nun gram into liquid suspension culture base
Acid, moulting hormone and silver thiosulfate, after maintaining the temperature to continue culture 2 days, renewal cultivation temperature is cultivated 4 days to 32 DEG C, is handed over
That replaces carries out the culture that heats up and cool down, until the culture terminates, obtains the Catharanthus Roseus Cell of high yield vincaleukoblastinum.
Compared with using common liq culture medium, in liquid suspension culture, the speed of growth improves the cell of the present embodiment
8.5%.Compared with not being passed through carbon monoxide and being not added with the test of niacin, moulting hormone and silver thiosulfate, use
The expression quantity of fluorescent quantitative PCR technique measurement d4h and dat gene, the results showed that the table of the d4h and dat gene in the present embodiment
108% and 105% has been respectively increased up to amount.D4h and dat gene is the key enzyme for synthesizing vindoline, and vindoline is synthesis length
The important prerequisite of spring alkali and vincristine, and is not passed through carbon monoxide and is not added with niacin, moulting hormone and thiosulfuric acid
The test of silver is compared, and the vindoline content in the present embodiment Catharanthus Roseus Cell improves 115%, and vinblastine content improves
111%, illustrate that being passed through carbon monoxide and addition niacin, moulting hormone and silver thiosulfate can promote cell to synthesize Changchun
Alkali.
Embodiment 5
A kind of cultural method of Catharanthus Roseus Cell high yield vincaleukoblastinum comprising following steps:
1) full Changchun flower seed is selected, is impregnated 10 seconds using weak acid electrolysis water, is cleaned, is made using aseptic deionized water
Aseptic seedling is obtained with cultivating on the B5 basal medium containing 10mg/L vitamin C, 7g/L agar and 0.1g/L oligosaccharide, with
The plumular axis of the aseptic seedling is explant, accesses in callus inducing medium and carries out Fiber differentiation, and cultivation temperature is 32 DEG C,
Daily alternately illumination and dark culturing, induces callus, then callus is transferred in subculture medium, and every 25
Its subculture is primary, and squamous subculture 3 times, to obtain stable Catharanthus roseus calli；Wherein, the callus inducing medium
To contain the 1mg/L basic element of cell division, 00.5mg/L 6-benzyl aminopurine, 30g/L sucrose, 3mg/L licoflavone and 8g/L agar
B5 basal medium；The subculture medium is formed by adding phenylalanine in callus inducing medium, phenylalanine
Concentration in subculture medium is 1.0mmol/L；
2) selection growth is vigorous, quality is loose, the in stable condition uniform Catharanthus roseus calli, and it is outstanding to be inoculated into liquid
In floating culture medium, bead is then added into liquid suspension culture base, and high speed oscillation 8min, shaking table are carried out in earthquake shaking table
Revolving speed 200rpm is subsequently placed in illumination shaking table and is cultivated, shaking speed 100rpm, intensity of illumination 2000lx, when illumination
Between 15h/day, every 22 days squamous subcultures 1 time, squamous subculture 5 times, obtain stablize heredity Catharanthus Roseus Cell system；Wherein, described
Liquid suspension culture base is containing added with licoflavone, resveratrol, 6-benzyl aminopurine, methyl α-naphthyl acetate, 2,4- Dichlorophenoxy
Acetic acid, sucrose, tryptophan and caseinhydrolysate B5 basal medium, the concentration of the licoflavone is 0.005mg/L, white Chenopodiaceae
The concentration of reed alcohol is 0.001mg/L, and the concentration of 6-benzyl aminopurine is 0.08mg/L, and the concentration of methyl α-naphthyl acetate is 0.2mg/L, 2,4-
The concentration of dichlorphenoxyacetic acid is 0.06mg/L, the concentration of sucrose is 30g/L, and the concentration of the tryptophan is 20mg/L, described
The content of caseinhydrolysate is 50mg/L；
3) the Catharanthus Roseus Cell system that step 2) obtains is inoculated into liquid suspension culture base and carries out Fiber differentiation, with stimulation
The accumulation of alkaloid in catharanthus roseus, the inductive condition are as follows: Catharanthus Roseus Cell is tied up to the culture of liquid suspension culture base the 10th day and opened
Begin, cultivation temperature is reduced to 28 DEG C from 32 DEG C, be passed through CO gas and add Buddhist nun gram into liquid suspension culture base
Acid, moulting hormone and silver thiosulfate, after maintaining the temperature to continue culture 2 days, renewal cultivation temperature is cultivated 4 days to 32 DEG C, is handed over
That replaces carries out the culture that heats up and cool down, until the culture terminates, obtains the Catharanthus Roseus Cell of high yield vincaleukoblastinum.
Compared with being not added with the test of tryptophan and caseinhydrolysate, adds tryptophan and caseinhydrolysate can be to vincaleukoblastinum
Synthesis have the function of just regulating and controlling, research shows that addition tryptophan and caseinhydrolysate after can enhance catharanthine synzyme
Activity 52% and enhancing vincaleukoblastinum synthase activity 75%.Be not passed through carbon monoxide and be not added with niacin, moulting hormone
It is compared with the test of silver thiosulfate, using the expression quantity of fluorescent quantitative PCR technique measurement d4h and dat gene, the results showed that this
108% and 115% has been respectively increased in the expression quantity of d4h and dat gene in embodiment.D4h and dat gene is synthesis vindoline
Key enzyme, and vindoline is the important prerequisite for synthesizing vincaleukoblastinum and vincristine, and is not passed through carbon monoxide and is not added with
Niacin, moulting hormone are compared with the test of silver thiosulfate, and the vindoline content in the present embodiment Catharanthus Roseus Cell improves
112%, vinblastine content improves 109%, illustrates to be passed through carbon monoxide and addition niacin, moulting hormone and thiosulfuric acid
Silver can promote cell to synthesize vincaleukoblastinum.
Embodiment 5
A kind of cultural method of Catharanthus Roseus Cell high yield vincaleukoblastinum comprising following steps:
1) full Changchun flower seed is selected, is impregnated 10 seconds using weak acid electrolysis water, is cleaned, is made using aseptic deionized water
Aseptic seedling is obtained with cultivating on the B5 basal medium containing 10mg/L vitamin C, 7g/L agar and 0.1g/L oligosaccharide, with
The plumular axis of the aseptic seedling is explant, accesses in callus inducing medium and carries out Fiber differentiation, and cultivation temperature is 32 DEG C,
Daily alternately illumination and dark culturing, induces callus, then callus is transferred in subculture medium, and every 25
Its subculture is primary, and squamous subculture 3 times, to obtain stable Catharanthus roseus calli；Wherein, the callus inducing medium
To contain the 1mg/L basic element of cell division, 00.5mg/L 6-benzyl aminopurine, 30g/L sucrose, 3mg/L licoflavone and 8g/L agar
B5 basal medium；The subculture medium is formed by adding phenylalanine in callus inducing medium, phenylalanine
Concentration in subculture medium is 1.0mmol/L；
2) selection growth is vigorous, quality is loose, the in stable condition uniform Catharanthus roseus calli, and it is outstanding to be inoculated into liquid
In floating culture medium, bead is then added into liquid suspension culture base, and high speed oscillation 8min, shaking table are carried out in earthquake shaking table
Revolving speed 200rpm is subsequently placed in illumination shaking table and is cultivated, shaking speed 100rpm, intensity of illumination 2000lx, when illumination
Between 15h/day, every 22 days squamous subcultures 1 time, squamous subculture 5 times, obtain stablize heredity Catharanthus Roseus Cell system；Wherein, described
Liquid suspension culture base is containing added with licoflavone, resveratrol, 6-benzyl aminopurine, methyl α-naphthyl acetate, 2,4- Dichlorophenoxy
Acetic acid, sucrose, tryptophan and caseinhydrolysate B5 basal medium, the concentration of the licoflavone is 0.005mg/L, white Chenopodiaceae
The concentration of reed alcohol is 0.001mg/L, and the concentration of 6-benzyl aminopurine is 0.08mg/L, and the concentration of methyl α-naphthyl acetate is 0.2mg/L, 2,4-
The concentration of dichlorphenoxyacetic acid is 0.06mg/L, the concentration of sucrose is 30g/L, and the concentration of the tryptophan is 20mg/L, described
The content of caseinhydrolysate is 50mg/L；
3) the Catharanthus Roseus Cell system that step 2) obtains is inoculated into liquid suspension culture base and carries out Fiber differentiation, with stimulation
The accumulation of alkaloid in catharanthus roseus, the inductive condition are as follows: Catharanthus Roseus Cell is tied up to the culture of liquid suspension culture base the 10th day and opened
Begin, cultivation temperature is reduced to 28 DEG C from 32 DEG C, is passed through 0.03m3/ h CO gas and into liquid suspension culture base
0.1mg/L niacin, 0.08mg/L moulting hormone and 0.05mg/L silver thiosulfate are added, the temperature is maintained to continue culture 2 days
Afterwards, renewal cultivation temperature is cultivated 4 days to 32 DEG C, is alternately heated up and the culture that cools down obtains high until the culture terminates
Produce the Catharanthus Roseus Cell of vincaleukoblastinum.
Compared with being not added with the test of tryptophan and caseinhydrolysate, adds tryptophan and caseinhydrolysate can be to vincaleukoblastinum
Synthesis have the function of just regulating and controlling, research shows that addition tryptophan and caseinhydrolysate after can enhance catharanthine synzyme
Activity 55% and enhancing vincaleukoblastinum synthase activity 80%.Be not passed through carbon monoxide and be not added with niacin, moulting hormone
It is compared with the test of silver thiosulfate, using the expression quantity of fluorescent quantitative PCR technique measurement d4h and dat gene, the results showed that this
128% and 125% has been respectively increased in the expression quantity of d4h and dat gene in embodiment.D4h and dat gene is synthesis vindoline
Key enzyme, and vindoline is the important prerequisite for synthesizing vincaleukoblastinum and vincristine, and is not passed through carbon monoxide and is not added with
Niacin, moulting hormone are compared with the test of silver thiosulfate, and the vindoline content in the present embodiment Catharanthus Roseus Cell improves
132%, vinblastine content improves 129%, illustrates to be passed through carbon monoxide and addition niacin, moulting hormone and thiosulfuric acid
Silver can promote cell to synthesize vincaleukoblastinum.
Although the embodiments of the present invention have been disclosed as above, but its is not only in the description and the implementation listed
With.It can be applied to various suitable the field of the invention completely.It for those skilled in the art, can be easily
Realize other modification.Therefore without departing from the general concept defined in the claims and the equivalent scope, the present invention is simultaneously unlimited
In specific details and embodiment shown and described herein.
Claims (7)
1. a kind of cultural method of Catharanthus Roseus Cell high yield vincaleukoblastinum, which comprises the following steps:
1) full Changchun flower seed is selected, is impregnated 10-20 seconds using weak acid electrolysis water, is cleaned, is made using aseptic deionized water
It is obtained with being cultivated on the B5 basal medium containing 10-20mg/L vitamin C, 7-9g/L agar and 0.1-0.3g/L oligosaccharide
Aseptic seedling accesses in callus inducing medium using the plumular axis of the aseptic seedling as explant and carries out Fiber differentiation, culture temperature
Degree is 32-35 DEG C, and alternately illumination and dark culturing daily induces callus, callus is then transferred to subculture
In culture medium, every 20-25 days subcultures are primary, squamous subculture 3-5 times, to obtain stable Catharanthus roseus calli；
2) selection growth is vigorous, quality is loose, the in stable condition uniform Catharanthus roseus calli, is inoculated into liquid suspension training
It supports in base, bead is then added into liquid suspension culture base, high speed oscillation 8-10min, shaking table are carried out in earthquake shaking table
180~200rpm of revolving speed is subsequently placed in illumination shaking table and is cultivated, shaking speed 100-120rpm, intensity of illumination 1500
~2000lx, 10~15h/day of light application time, every 20-22 days squamous subcultures 1 time squamous subculture 5~8 times, obtain and stablize heredity
Catharanthus Roseus Cell system；
3) the Catharanthus Roseus Cell system that step 2) obtains is inoculated into liquid suspension culture base and carries out Fiber differentiation, to stimulate Changchun
Spend the accumulation of middle alkaloid, the inductive condition are as follows: Catharanthus Roseus Cell ties up to liquid suspension culture base culture and opens for 10-15 days
Begin, cultivation temperature is reduced to 25~28 DEG C from 32-35 DEG C, CO gas is passed through and adds into liquid suspension culture base
Add niacin, moulting hormone and silver thiosulfate, after maintaining the temperature to continue culture 2-3 days, renewal cultivation temperature to 32-35
DEG C, it cultivates 4-5 days, is alternately heated up and the culture that cools down, up to the culture terminates, the catharanthus roseus of acquisition high yield vincaleukoblastinum is thin
Born of the same parents.
2. the cultural method of Catharanthus Roseus Cell high yield vincaleukoblastinum according to claim 1, which is characterized in that the callus group
Knit induced medium be containing 1~2mg/L basic element of cell division, 0.1~0.5mg/L 6-benzyl aminopurine, 20~30g/L sucrose,
The B5 basal medium of 3-5mg/L licoflavone and 5~8g/L agar.
3. the cultural method of Catharanthus Roseus Cell high yield vincaleukoblastinum according to claim 1, which is characterized in that described after being commissioned to train
It supports base to be formed by adding phenylalanine in callus inducing medium, concentration of the phenylalanine in subculture medium is 0.5
~1.0mmol/L.
4. the cultural method of Catharanthus Roseus Cell high yield vincaleukoblastinum according to claim 1, which is characterized in that the liquid is outstanding
Floating culture medium is containing added with licoflavone, resveratrol, 6-benzyl aminopurine, methyl α-naphthyl acetate, 2,4- dichlorphenoxyacetic acid, sugarcane
The B5 basal medium of sugar, the concentration of the licoflavone are 0.005-0.008mg/L, and the concentration of resveratrol is 0.001-
0.003mg/L, the concentration of 6-benzyl aminopurine are 0.08-0.1mg/L, and the concentration of methyl α-naphthyl acetate is 0.2-0.3mg/L, 2,4- dichloros
The B5 basal medium that the concentration of phenoxy acetic acid is 0.06-0.09mg/L, the concentration of sucrose is 20-30g/L.
5. the cultural method of Catharanthus Roseus Cell high yield vincaleukoblastinum according to claim 4, which is characterized in that the liquid training
Supporting further includes tryptophan and caseinhydrolysate in base, and the concentration of the tryptophan is 20-30mg/L, and the caseinhydrolysate contains
Amount is 50-70mg/L.
6. the cultural method of Catharanthus Roseus Cell high yield vincaleukoblastinum according to claim 1, which is characterized in that an oxidation
The intake of carbon is 0.01-0.03m3/h。
7. the cultural method of Catharanthus Roseus Cell high yield vincaleukoblastinum according to claim 1, which is characterized in that the niacin
Concentration be 0.05-0.1mg/L, the concentration of the moulting hormone is 0.06-0.08mg/L, and the concentration of the silver thiosulfate is
0.02-.0.05mg/L。
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