CN102630566A - Method for cultivation and production of paclitaxel from induction of taxus chinensis var. mairei axillary bud in vitro - Google Patents

Method for cultivation and production of paclitaxel from induction of taxus chinensis var. mairei axillary bud in vitro Download PDF

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CN102630566A
CN102630566A CN2012101167277A CN201210116727A CN102630566A CN 102630566 A CN102630566 A CN 102630566A CN 2012101167277 A CN2012101167277 A CN 2012101167277A CN 201210116727 A CN201210116727 A CN 201210116727A CN 102630566 A CN102630566 A CN 102630566A
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bud
mairei
wpm
taxol
buds
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CN102630566B (en
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杜亚填
张翔宇
龚雪原
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Jishou University
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Abstract

The invention provides a method for extracting paclitaxel from induction of taxus chinensis var. mairei axillary bud germination in vitro and then cultivation and production. On the basis of a WPM (woody plant medium) minimal medium, the method uses (800-1000) mg/L of (NH4)2SO4 to replace 990 mg/L of K2SO4 to obtain the improved WPM minimal medium, and adding 0.01-4.5 mg/L of TIBA; pruning annual young shoots from a taxus chinensis var. mairei tree, cutting into small stem sections in 2.5cm, sterilizing, inoculating to the improved WPM minimal medium, and performing alternative culture, namely light culture and dark culture, in an artificial climate box for culturing temperature, wherein the temperature is 25+/-1 DEG C in the daytime and is 20+/-1 DEG C in the night, the illumination intensity is 1500lx, and relative humidity is 80%, thus rapidly and successfully inducing axillary buds based on the WPM plus 0.01-4.5 mg/LTIBA. By using the method, the industrialization of the taxus chinensis var. mairei axillary buds can be realized with large scale, short period, high reproduction rate and low cost. Buds can be induced around 15 days with 100% induction rate; and the taxus chinensis var. mairei axillary buds induced by the method is thick and short and has freshly green color. The content of paclitaxel in buds obtained by the method can reach 0.0042% in fifty days, which is higher than that of paclitaxel in natural buds.

Description

A kind of method of southern enqlish yew axillalry bud in-vitro inducing cultivating and producing taxol
Technical field
The present invention relates to biological field, specifically, a kind of method of southern enqlish yew axillalry bud in-vitro inducing cultivating and producing taxol.
Background technology
Chinese yew is the endangered natural rare plants for anticancer of generally acknowledging in the world, is the ancient seeds that quaternary glacier carries over, the history in existing 2,500,000 years.Chinese yew genus plants is distributed in the Northern Hemisphere, is high megaphanerophyte, and population distributes rare, and growth rate is slow, and regeneration capacity is poor, so for a long time, also do not had to form the Chinese yew raw material forest base that big rule are touched in the world wide.China classifies it as one-level rare and endangered protective plant, and the United Nations also prohibites felling.Chinese yew genus plants has 11 kinds in the whole world, southern enqlish yew ( Taxus chinensisVar. Mairei) be the mutation of Chinese yew in China.
Amos etc. (1981) find in the WPM medium, to add low concentration 6-BA and help the bud generation; High concentration 6-BA then suppresses the bud elongation growth.Barnes (1983) finds containing on the WPM medium of 1mg/LBA, and the stem apex axillary bud growth of T. canadensis is better.Chee (1995) is seeded in it on 1/2B5+10 μ M6-BA medium in the research of the stripped embryonal induction indefinite bud of yewtree, has induced the bud original hase on the embryo that exsomatizes behind the 14d.Yang Zhenguo etc. (1997) utilize taxus chinensis in northeast sprout terminal bud to make explant, are minimal medium with MS, have induced the bud of growing thickly.Cheng Guangyou etc. (1997) discovery 6-BA/IBA ratio and taxus chinensis in northeast stem apex induce the amount of growth of lateral bud and callus growth amount that much relations are arranged, and can produce indefinite bud when ratio is big.Great this grade of benevolence tower (2004) discovers that the bud differentiation of taxus chinensis in northeast stem apex is not too responsive to the hormone of different quality concentration with growth, and 6-BA relatively helps the differentiation of bud, and KT helps the generation of callus.Horse impartial (2007) is an explant with the Taxus x media stem with bud, and the starting rate in MS+0.05 mg/L6-BA+0.5 mg/LNAA medium is the highest, has reached 89.55%, and finds that the 6-BA of low concentration more helps the startup of bud.It is explant that Tang Dao sides etc. (2008) adopt the southern enqlish yew stem with bud; The condition of research southern enqlish yew tissue culture bud propagation and plant regeneration; The result shows: the most suitable shoot proliferation inducing culture of spray of the animated period of growing spring, its optimal medium is DCR+0.1mg/L IBA+0.05mg/L6-BA.Zhang Shengxi etc. (2010) discover that DCR+ 0.5 mg/L6-BA+0.8 mg/LNAA also relatively is fit to the southern enqlish yew explant and directly induces and sprout.But using TIBA carries out the Chinese yew genus plants shoot proliferation and induces the aspect also not see report is arranged.
Retrieval through to the prior art document is found; Number of patent application: CN200610000193.6; Publication number: CN100999719; Denomination of invention: the inducing and plant regeneration method of sequoia sempervirens blade indefinite bud and somatic embryo, this method are to be explant with the sequoia sempervirens stem apex, on the SH medium of BA 0.5mg/L+KT 0.2mg/L+IBA 0.2mg/L and BA0.5mg/L+IBA 0.5mg/L, have induced indefinite bud and somatic embryo.It is different fully with the used culture medium prescription of present technique that this method is used medium; This technology can realize extensive, short period, high reproductive rate, the batch production production cheaply of sequoia sempervirens nursery stock, and for disclose cell differentiation, growth, that form takes place and zygotic embryo produces is machine-processed significant.Number of patent application: CN200610136929.2; Publication number: CN101209028; Denomination of invention: Chinese yew electromagnetic pulse fast breeding and industrialization kind method for planting; This method adopts computer environment control technology and electromagnetic pulse technology to combine with open fast breeding technology, utilizes " totipotency " and " holography property " of plant, and utilization is to plant growth environment factor analysis expert systemic-function; Breeding plant conditions such as the temperature in self adaptation adjusting seedbed, humidity, oxygen, density of carbon dioxide gas, nutrient; Create the environmental condition of suitable strong plantlets and rootage for the stripped material of different plants, realize the fast breeding of plant, obtain the Chinese yew plantation seedling of batch production high-quality in enormous quantities within a short period of time with Miao Fanmiao.Though these methods all to the fast numerous effect preferably that has of Chinese yew, all are not mentioned to employing TIBA and induce the southern enqlish yew shoot proliferation.And above technology is all fast numerous in a large number with Chinese yew to be culturing purposes, and the present invention also is to produce anti-cancer medicine paclitaxel through test tube gemmule inducing culture.
Summary of the invention
The purpose of this invention is to provide a kind of method of under isolated condition, producing taxol, for being that raw material are produced taxol a kind of new path is provided with the southern enqlish yew through southern enqlish yew axillalry bud inducing culture.
For realizing the object of the invention, the technical scheme that the present invention adopted is: a kind of southern enqlish yew axillary bud sprouting of under isolated condition, inducing carries out the method that cultivating and producing is extracted taxol then, may further comprise the steps:
(1) clip is given birth to the tender tip then from the southern enqlish yew trees, and tripping cuts into the long little stem section of 2～3 cm;
(2) add water in the beaker, add 0.5g washing powder in every 500mL water, stir; To shear good little stem section then and put into beaker stirring and washing 30min; Take out the back and wash 2h, change aseptic superclean bench then over to, alcohol-pickled sterilization 30s with 70～75% with running water; Sterile water wash 4 times is used 0.1～0.25% HgCl again 2Solution soaking sterilization 8min, sterile water wash 6 times is dried surface water reserve inoculation and is used in superclean bench;
(3) the morphology lower end of having got step (2) ready explant is inserted in the improvement WPM medium of additional 0.01～4.5mg/L TIBA and is carried out the axillalry bud inducing culture;
(4) place climatic cabinate to cultivate step (3) inoculated and cultured bottle, cultivation temperature: 25 ± 1 ℃ of daytimes, 20 ± 1 ℃ of evenings; Carry out 12h and alternately cultivate intensity of illumination: 1500lx, relative moisture 80 ± 5%;
(5) cultivation was gathered in the crops bud after 50 days, detected the content of taxol in the bud, produced and extracted taxol.
Improvement WPM minimal medium described in the step (2) is on the basis of WPM minimal medium, with the K of 990mg/L 2SO 4(NH4) with (800～1000) mg/L 2SO 4Replacement.
The concentration of TIBA is 0.1～4.0mg/L. described in the step (2)
The optium concentration of said TIBA is 3.0mg/L.
Beneficial effect of the present invention:
(1) the present invention adopts TIBA that southern enqlish yew stem section is carried out axillalry bud to induce first, has that induction time is short, the inductivity advantages of higher, can about 15 days, induce and sprout, and inductivity is 100%.
(2) adopt this method to induce the southern enqlish yew axillalry bud sprout of generation short and sturdy, color is bud green.
(3) content of taxol can reach 0.0042% in 50 days in the bud of employing this method acquisition, and content is high in the more natural bud.
Below in conjunction with specific embodiment the present invention is described in further detail.
Embodiment
Embodiment 1
(1) clip is given birth to the tender tip then from the southern enqlish yew trees, and tripping cuts into the little stem section of 2.5cm.
(2) in beaker, add running water, in water, add a small amount of washing powder, add 0.5g washing powder in every 500mL water, stir; To shear good little stem section then and put into beaker stirring and washing 30min, take out, with changing aseptic superclean bench over to behind the running water flushing 2h, the alcohol-pickled sterilization 30s with 70%, sterile water wash 4 times is used 0.1% HgCl again 2Solution soaking sterilization 8min, sterile water wash 6 times is dried surface water reserve inoculation and is used in superclean bench.
(3) 2,3 of additional 0.1mg/L is inserted in the morphology lower end of having got step (2) ready explant, in the improvement WPM medium of 5-Triiodobenzoic acid (2,3,5-Triiodobenzoic acid is called for short TIBA), carries out the axillalry bud inducing culture.The WPM medium of said improvement is on the basis of WPM minimal medium, with the K of 990mg/L 2SO 4(NH with 800mg/L 4) 2SO 4Replacement.
(4) cultivation temperature is cultivated in inoculation back in climatic cabinate: daytime (25 ± 1) ℃, evening (20 ± 1) ℃, carry out 12h and alternately cultivate intensity of illumination: 1500lx, relative moisture 80 ± 5%.
(5) cultivated 25 days, the germination rate of axillalry bud reaches 16.67%, and average bud reaches 1.21 cm.
(6) cultivate after 50 days, the content of taxol fails to detect in the bud.
Embodiment 2
(1) clip is given birth to the tender tip then from the southern enqlish yew trees, and tripping cuts into the little stem section about 2cm.
(2) in beaker, add tap water, in water, add a small amount of washing powder, add 0.5g washing powder in every 500mL water, stir; To shear good little stem section then and put into beaker and stir 30min, and take out, with the alcohol-pickled sterilization 30s that changes over to behind the running water flushing 2h in the aseptic superclean bench with 72%, sterile water wash 4 times is used 0.2% HgCl again 2Solution soaking sterilization 8min, sterile water wash 6 times is dried surface water reserve inoculation and is used in superclean bench.
(3) additional 3.0mg/L 2,3 is inserted in the morphology lower end of having got step (2) ready explant, carries out the axillalry bud inducing culture in the improvement WPM medium of 5-Triiodobenzoic acid (2,3,5-Triiodobenzoic acid is called for short TIBA).The WPM medium of said improvement is on the basis of WPM minimal medium, with the K of 990mg/L 2SO 4(NH with 900mg/L 4) 2SO 4Replacement.
(4) cultivation temperature is cultivated in inoculation back in climatic cabinate: daytime (25 ± 1) ℃, evening (20 ± 1) ℃, carry out 12h and alternately cultivate intensity of illumination: 1500lx, relative moisture 80 ± 5%.
(5) cultivated 15 days, the germination rate of axillalry bud reaches 100%, and average bud reaches 1.82 ㎝.
(6) cultivate after 50 days, the content of taxol is 0.0042% (shoot dry matter) in the bud.
 
Embodiment 3
(1) clip is given birth to the tender tip then from the southern enqlish yew trees, and tripping cuts into the stem section about 3cm.
(2) in beaker, add tap water, in water, add a small amount of washing powder, add 0.5g washing powder in every 500mL water, stir; To shear good stem section then and put into beaker and stir 30min, and take out, with the alcohol-pickled sterilization 30s that changes over to behind the running water flushing 2h in the aseptic superclean bench with 75%, sterile water wash 4 times is used 0.25% HgCl again 2Solution soaking sterilization 8min, sterile water wash 6 times is dried surface water reserve inoculation and is used in superclean bench.
(3) 2,3 of additional 4.0mg/L is inserted in the morphology lower end of having got step (2) ready explant, carries out the axillalry bud inducing culture in the improvement WPM medium of 5-Triiodobenzoic acid (2,3,5-Triiodobenzoic acid is called for short TIBA).The WPM medium of said improvement is on the basis of WPM minimal medium, with the K of 990mg/L 2SO 4(NH with 1000mg/L 4) 2SO 4Replacement.
(4) cultivation temperature is cultivated in inoculation back in climatic cabinate: daytime (25 ± 1) ℃, evening (20 ± 1) ℃, carry out 12h and alternately cultivate intensity of illumination: 1500lx, relative moisture 80 ± 5%.
(5) cultivated 23 days, the germination rate of axillalry bud reaches 22.22%, and average bud length is 1.12 cm.
(6) cultivate after 50 days, the content of taxol fails to detect in the bud.
Except that above-mentioned three embodiment, to the TIBA of variable concentrations, the applicant has done repeatedly experiment, and the result is as shown in the table:
Tested number TIBA concentration (mg/L) Sprout time (d) Germination rate (%) Average bud long (cm) Content of taxol (%) ?
1 0.01 － 0 － － ?
2 0.05 － 0 － － ?
3 0.1 25 16.67 1.21 － ?
4 0.5 25 22.22 1.35 － ?
5 1.0 25 22.22 1.42 － ?
6 1.5 20 33.33 1.58 － ?
7 2.0 18 55.56 1.74 0.0024 ?
8 2.5 16 61.11 1.73 0.0038 ?
9 3.0 15 100 1.82 0.0042 ?
10 3.5 15 83.33 1.67 0.0040 ?
11 4.0 23 22.22 1.12 － ?
12 4.5 － 0 － － ?
Experimental result shows: TIBA concentration is during less than 0.1 mg/L or more than or equal to 4.5mg/L, and the germination rate of axillalry bud is zero, promptly can not induce the generation bud; TIBA concentration is between 0.1 mg/L～2.0 mg/L the time, and the germination rate of axillalry bud is relatively low; When TIBA concentration was 3.0mg/L, the germination rate of axillalry bud reached the highest, was 100%, and wherein the content of taxol also reaches and is up to 0.0042% (dry weight).

Claims (4)

1. under isolated condition, induce the southern enqlish yew axillary bud sprouting to carry out the method that cultivating and producing is extracted taxol then for one kind, may further comprise the steps:
(1) clip is given birth to the tender tip then from the southern enqlish yew trees, and tripping cuts into the long little stem section of 2～3 cm;
(2) add water in the beaker, add 0.5g washing powder in every 500mL water, stir; To shear good little stem section then and put into beaker stirring and washing 30min; Take out the back and wash 2h, change aseptic superclean bench then over to, alcohol-pickled sterilization 30s with 70～75% with running water; Sterile water wash 4 times is used 0.1～0.25% HgCl again 2Solution soaking sterilization 8min, sterile water wash 6 times is dried surface water reserve inoculation and is used in superclean bench;
(3) the morphology lower end of having got step (2) ready explant is inserted in the improvement WPM medium of additional 0.01～4.5mg/L TIBA and is carried out the axillalry bud inducing culture;
(4) place climatic cabinate to cultivate step (3) inoculated and cultured bottle, cultivation temperature: 25 ± 1 ℃ of daytimes, 20 ± 1 ℃ of evenings; Carry out 12h and alternately cultivate intensity of illumination: 1500lx, relative moisture 80 ± 5%;
(5) cultivation was gathered in the crops bud after 50 days, detected the content of taxol in the bud, produced and extracted taxol.
2. the southern enqlish yew axillary bud sprouting of under isolated condition, inducing according to claim 1 carries out the method that cultivating and producing is extracted taxol then; It is characterized in that; Improvement WPM minimal medium described in the step (2) is on the basis of WPM minimal medium, with the K of 990mg/L 2SO 4(NH4) with (800～1000) mg/L 2SO 4Replacement.
3. the southern enqlish yew axillary bud sprouting of under isolated condition, inducing according to claim 1 and 2 carries out the method that cultivating and producing is extracted taxol then, it is characterized in that the concentration of TIBA is 0.1～4.0mg/L described in the step (2).
4. the southern enqlish yew axillary bud sprouting of under isolated condition, inducing according to claim 3 carries out the method that cultivating and producing is extracted taxol then, it is characterized in that the optium concentration of said TIBA is 3.0mg/L.
CN 201210116727 2012-04-20 2012-04-20 Method for cultivation and production of paclitaxel from induction of taxus chinensis var. mairei axillary bud in vitro Expired - Fee Related CN102630566B (en)

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CN110558228A (en) * 2019-09-18 2019-12-13 江苏红豆杉健康科技股份有限公司 Rapid propagation method of taxus mairei
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CN114041423A (en) * 2021-12-22 2022-02-15 上海金和生物制药有限公司 In-vitro culture method of taxus chinensis rich in taxanes

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