CN101185397A - Method for producing taxol by using taxus lateral root proliferation - Google Patents
Method for producing taxol by using taxus lateral root proliferation Download PDFInfo
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Abstract
The invention discloses a method of utilizing taxus lateral root proliferation to produce paclitaxel, the steps are that: (1) annual or biennial taxus branches are selected as cutting materials; (2) after the cuttings are sequentially injected into KMnO4 solution, sucrose solution, and plant growth hormone solution for soaking, the cuttings are conveyed to cutting substrate; (3) the cuttings are pulled out after the roots grow up, and are continually conveyed to the cutting substrate after being processed with hormone; (4) a certain quantity of the lateral roots are cut for producing the paclitaxel; (5) being dried under temperature control at the temperature that is less than or equal to 55 DEG C, pure products of the paclitaxel are obtained after processes of grinding, concentrated organic solvent extraction, column chromatography, solvent recovery, crystallizing and drying; or the cut taxus lateral roots are cultured in a improved Murashige and amp;Skoog fluid nutrient medium, bacteriostat is added in the nutrient medium, 2 to 9 days after fresh taxus lateral roots are induced and processed by elicitors, the pure products of the paclitaxel are extracted. The invention provides a new approach for medicine sources of the paclitaxel, and the shortcomings in artificial cultivation of the taxus are solved of low rooting rate, low survival rate after the roots are cut, slow roots proliferation speed, etc.
Description
Technical field
The invention belongs to agricultural biotechnologies, bio-pharmaceuticals and chemistry of forest product and industrial circle, be specifically related to a kind of method of utilizing taxus lateral root proliferation to produce taxol.
Background technology
Taxol is the important antineoplastic of generally acknowledging in the world at present, and is unique native compound that can promote tubulin polymerization and suppress its depolymerization of finding at present.Taxol occupies the first place of the antineoplastic market share owing to its unique antitumor action mechanism and notable antitumor activity.
At present, being used for studying taxol with clinical testing is that bark from natural Chinese yew plant extracts and obtains mostly.Taxol in bark content is very little, only is the 0.007-0.01% of bark dry weight, the pure product taxol that common every extraction the is 1 kilogram bark 15-30 ton of will using up.It is calculated that treating 1 ovarian cancer patients needs 6 Chinese yews that the age of tree is 60-100, the year number of the infected of whole world cancer is about 1,000 ten thousand people.The Chinese yew growth rate is quite slow, and the tree that diameter is 20 centimetres need grow 100 years.At present, taxol costs an arm and a leg on the market, and per kilogram needs more than 20 ten thousand dollars, under order about of market to the active demand of taxol and juice, has caused wild taxus resource to suffer catastrophic collapse.Therefore, the contradiction between the increasing sharply of the shortage of taxus resource and taxol demand is becoming increasingly acute, and many countries have all dropped into the research and development that a large amount of funds is carried out taxol medicine source, in the hope of solving the world-famous puzzle of taxol medicine source anxiety.The process of chemical synthesis is very complicated, not only needs expensive chemical reagent, and the extremely difficult control of reaction condition, and the per step yield and the total recovery of chemosynthesis process are all very low, are difficult to really be used for suitability for industrialized production.Culture plant cell is produced taxol, and to remain, productive rate less in production scale at present be not very high industrialization problem, and its industrialization is still among research and development.Therefore, Comparatively speaking, the artificial cultivation Chinese yew remains and obtains taxol in a short time, particularly obtains the main path of the semi-synthetic precursor of taxol chemistry.But the tame Chinese yew plant strain growth cycle is still longer, if will put in order the precursor that strain is used for extracting taxol and semi-synthetic usefulness thereof, is difficult to form industrialization.Therefore, at present common way be termly, selectively clip Chinese yew plant branches and leaves and do not injure the normal growth of Chinese yew plant.But content of taxol seldom in the branches and leaves of yew, and in existing 11 kinds of Chinese yew, content of taxol also only is the 0.01-0.03% of dry weight in the branches and leaves of the Taxus x media that content of taxol is the highest.The method mainly is to pass through further chemical semi-synthetic acquisition taxol after extracting the taxol precursor, so it is very high to produce the required cost of taxol with the method, can only satisfy the demand of market to taxol to a certain extent.
Studies show that in a large number, except bark of Ramulus et folium taxi cuspidatae, japanese yew root also is the main enrichment positions of taxol, content of taxol is the 0.05-0.1% of dry weight in the japanese yew root, far above branches and leaves of yew, the precursor substance baccatin III and the 10-that also are rich in taxol biosynthesis simultaneously remove the acetyl baccatin III.At present, the research of the aspects such as technology and artificial cultivation method of taking root about Yew cuttage both at home and abroad is many, still yet there are no a large amount of propagation of utilizing taxus lateral root and the controlled regeneration paclitaxel produced report in next life of lateral root.Even the artificial cultivation of Chinese yew seedling also ubiquity that rooting rate is low, the root growth rate waits problem slowly.Obtain the suitability for industrialized production that a large amount of lateral roots are used for taxol in order to realize utilizing the controlled quantitative shearing of taxus lateral root and breeding repeatedly, just be necessary to take root on the basis of condition optimizing Yew cuttage, further research and development promote taxus lateral root proliferation, improve lateral root and shear back survival rate and multiplication rate.
Summary of the invention
The object of the present invention is to provide a kind of method of utilizing taxus lateral root proliferation to produce taxol, this method provides new approach for the taxol drug source, alleviates the imbalance between supply and demand of taxol growing tension on the medical market; The rooting rate that has also solved present Chinese yew artificial cultivation existence simultaneously is low, root shearing back survival rate is low, the root growth rate waits shortcoming slowly.
The method of utilizing taxus lateral root proliferation to produce taxol provided by the invention, its step comprises:
(1) in spring and summer, middle and upper part, the Chinese yew branch of growing that faces south of selecting 1-2 to give birth to are done the plugged ear material;
(2) the plugged ear base portion being inserted successively concentration is to soak 2-30h respectively in 0.01-0.2% liquor potassic permanganate, 0.5-10% sucrose solution and the 10-500mg/L auxin, and cuttage is intermittently sprayed Gram nutrient solution cultivation suddenly in the matrix of having sterilized again;
Described auxin is the combination of any mass ratio of the sodium salt of a kind of or α-Nai Yisuan and indolebutyric acid in the sodium salt of α-Nai Yisuan, 2,4 dichlorophenoxyacetic acid butyl ester, indolebutyric acid or the sylvite or sylvite.
(3) treat that plugged ear is taken root after, extract plugged ear, being soaked in concentration is 20-120min in the 10-1000mg/L hormone solution, cuttage is intermittently sprayed suddenly the Gram nutrient solution and is cultivated in original cutting medium then;
Described hormone is a kind of or indolebutyric acid in α-Nai Yisuan, 2,4 dichlorophenoxyacetic acid butyl ester, the indolebutyric acid, the combination that α-Nai Yisuan is 0.5: 1～1: 2 by mass ratio;
When (4) treating root vigorous growth, results Chinese yew plantlet is not injuring the condition down cut lateral root of main root, and first order lateral root keeps at least 2/3; The root concentration of shearing the back plant is the parahormone solution soaking 20-120min above-mentioned of the same race of 10-1000mg/L, and cuttage is in original cutting medium then, and intermittently atomizing is cultivated;
(5) to adopting conventional method dry under≤55 ℃ of conditions through shearing the taxus lateral root that obtains behind the aforementioned cuttage cultivation, then through pulverizing, organic solvent extraction concentrates, obtain pure product of paclitaxel behind the column chromatography, crystallization, the dry processing.
Technique scheme can have following preferred version:
In the step (1), the selection leaf bud is full, branch is sturdy, internode is short, the strong Chinese yew branch of growth potential, is cut into 8-10cm, and upper cut is level and smooth, incision a kind of horse raised north of the Grean Wall ear shape, and plugged ear 2/3 following defoliation and side shoot only stays 1-2 side shoot in upper end and blade.
In the step (2), liquor potassic permanganate concentration is 0.04-0.08%, and soak time is 8-24h; Sucrose solution concentration is 1-4%, and soak time is 12-30h; The concentration of auxin is 50-100mg/L, and soak time is 4-6h; Cutting medium is that sand is or/and humus soil.
In step (3), (4), described hormone is a kind of or indolebutyric acid in α-Nai Yisuan, 2,4 dichlorophenoxyacetic acid butyl ester, the indolebutyric acid, the combination that α-Nai Yisuan is 1: 1 by mass ratio; The hormone total concentration is 300-700mg/L, hormone soak time 30-60min.
In the step (5), before carrying out the drying processing, the taxus lateral root with clip is incubated at aseptic liquid improvement Murashige﹠amp earlier; In the Skoog medium, promptly only utilize Murashige﹠amp; The mineral salt composition of Skoog medium, and its integral level dropped to original 1/10-2/3, add medicament in the medium and suppress living contaminants, adopt fungal elicitor or chemical induction to induce simultaneously and handled fresh taxus lateral root 2-9 days.The medicament that suppresses living contaminants be any quality of a kind of in potassium chromate and the potassium permanganate or two kinds than mixed liquor, the medicament total concentration is 30-80mg/L; Any quality that perhaps is a kind of in kanamycin and the streptomycin or two kinds is than mixed liquor, and the medicament total concentration is 20-100mg/L.Fungal elicitor is to separate a kind of in the cell wall polysaccharides extract obtain black-koji mould, Penicillium notatum, streptomycete, yeast or gibberella in the bark of Ramulus et folium taxi cuspidatae, and its concentration is 30-80mg/L; Chemical inducer is any or the wantonly two kinds mixed liquor in the following solution: concentration is the 10-100mg/L methyl jasmonate, and concentration is the 10-100mg/L MDJ, and concentration is the 50-100mg/L salicylic acid, and concentration is the 1-5mg/L ethrel.
Method as a kind of new expansion taxol medicine source approach, the present invention is by optimizing cutting medium and increasing the cuttage root-taking rate that HORMONE TREATMENT promotes Chinese yew, and improve the part lateral root by suitable cut mode and HORMONE TREATMENT and shear the survival rate of plant afterwards, promote the regeneration and the propagation of root.The lateral root of results is directly used in taxol and extracts, or stripped the extraction after elicitor is induced processing obtains more taxol.Particularly, the present invention has following characteristics:
1. the method with harvesting Chinese yew branches and leaves is the same, can't hurt the growth of Chinese yew plant behind a part of lateral root of clip Chinese yew, can recover and increase the quantity and the length of lateral root by the propagation that promotes Chinese yew plant root.Therefore, this method can obtain taxol constantly under the situation of not destroying existing natural resources;
2. content of taxol is significantly higher than its branches and leaves in the lateral root of Chinese yew, promote the propagation of Chinese yew tree root by artificial cultivation, its part lateral root of clip is directly used in the extraction taxol termly, can avoid also will could obtaining taxol behind the picture branches and leaves clip, so this method can significantly reduce the production cost of taxol through extracting with complicated chemical semi-synthetic process;
3. by the lateral root in-vitro inducing, the precursor substance in the taxus lateral root can be converted into taxol, further improve the content of taxol in the lateral root, significantly improve the recovery rate of taxol, further reduce production costs.
Embodiment
Embodiment 1
In March then, select life in 1 year, middle and upper part, the leaf bud of growth is full, branch is sturdy, internode is short, the strong Taxus x media branch of growth potential is done the plugged ear material on the sunny side.The Chinese yew branch is cut into the long stem section of 8cm as plugged ear, and upper cut is level and smooth, incision a kind of horse raised north of the Grean Wall ear shape, and plugged ear 2/3 following defoliation and side shoot only stays 1 side shoot in upper end and blade.The stem section 30 strain plugged ears of Chinese yew after the cutting are tied into 1 bundle, its base portion 1/4 (about 2cm) is inserted 0.04%KMnO
4Soak 8h in the solution, clean with flushing with clean water; Soak 12h in the sucrose solution with plugged ear base portion insertion 1% again, clean with flushing with clean water again; 2%FeSO is used in cuttage after soaking 2h with the 10mg/L α-Nai Yisuanna at last in advance
4In the sand that spray solution was sterilized, place the shady and cool ventilation place, insert the back and spray 1 Hoagland (Gram suddenly) nutrient solution every 3d.The statistics rooting rate is 74% after 30 days.
Embodiment 2
In March then, select life in 1 year, middle and upper part, the leaf bud of growth is full, branch is sturdy, internode is short, the strong southern enqlish yew branch of growth potential is done the plugged ear material on the sunny side.The Chinese yew branch is cut into the long stem section of 8cm as plugged ear, and upper cut is level and smooth, incision a kind of horse raised north of the Grean Wall ear shape, and plugged ear 2/3 following defoliation and side shoot only stays 1 side shoot in upper end and blade.The stem section 30 strain plugged ears of Chinese yew after the cutting are tied into 1 bundle, its base portion 1/4 (about 2cm) is inserted 0.04%KMnO
4Soak 8h in the solution, clean with flushing with clean water; Soak 12h in the sucrose solution with plugged ear base portion insertion 1% again, clean with flushing with clean water again; Use 500mg/L2 at last, 2%FeSO was used in cuttage after the 4-dichlorobenzene oxygen butyl acetate soaked 2h in advance
4In the sand that spray solution was sterilized, place the shady and cool ventilation place, insert the back and spray 1 Hoagland (Gram suddenly) nutrient solution every 3d.The statistics rooting rate is 65% after 30 days.
Embodiment 3
In April then, select life in 1 year, middle and upper part, the leaf bud of growth is full, branch is sturdy, internode is short, the strong southern enqlish yew branch of growth potential is done the plugged ear material on the sunny side.The Chinese yew branch is cut into the long stem section of 8cm as plugged ear, and upper cut is level and smooth, incision a kind of horse raised north of the Grean Wall ear shape, and plugged ear 2/3 following defoliation and side shoot only stays 1 side shoot in upper end and blade.The stem section 30 strain plugged ears of Chinese yew after the cutting are tied into 1 bundle, its base portion 1/4 (about 2cm) is inserted 0.04%KMnO
4Soak 8h in the solution, clean with flushing with clean water; Soak 12h in the sucrose solution with plugged ear base portion insertion 1% again, clean with flushing with clean water again; 2%FeSO is used in cuttage after soaking 2h with the 500mg/L α-Nai Yisuan at last in advance
4In the sand that spray solution was sterilized, place the shady and cool ventilation place, insert the back and spray 1 Hoagland (Gram suddenly) nutrient solution every 3d.The statistics rooting rate is 68% after 30 days.
Embodiment 4
In July then, select life in 1 year, middle and upper part, the leaf bud of growth is full, the thick ` of branch strong, internode is short, the strong southern enqlish yew branch of growth potential is done the plugged ear material on the sunny side.The Chinese yew branch is cut into the long stem section of 8cm as plugged ear, and upper cut is level and smooth, incision a kind of horse raised north of the Grean Wall ear shape, and plugged ear 2/3 following defoliation and side shoot only stays 1 side shoot in upper end and blade.The stem section 30 strain plugged ears of Chinese yew after the cutting are tied into 1 bundle, its base portion 1/4 (about 2cm) is inserted 0.04%KMn0
4Soak 8h in the solution, clean with flushing with clean water; Soak 12h in the sucrose solution with plugged ear base portion insertion 1% again, clean with flushing with clean water again; 2%FeSO is used in cuttage after using 10mg/L NAA+IBA (1: 100) to soak 2h at last in advance
4In the sand that spray solution was sterilized, place the shady and cool ventilation place, insert the back and spray 1 Hoagland (Gram suddenly) nutrient solution every 3d.The statistics rooting rate is 68% after 30 days.
Embodiment 5
In July then, select life in 1 year, middle and upper part, the leaf bud of growth is full, branch is sturdy, internode is short, the strong southern enqlish yew branch of growth potential is done the plugged ear material on the sunny side.The Chinese yew branch is cut into the long stem section of 8cm as plugged ear, and upper cut is level and smooth, incision a kind of horse raised north of the Grean Wall ear shape, and plugged ear 2/3 following defoliation and side shoot only stays 1 side shoot in upper end and blade.The stem section 30 strain plugged ears of Chinese yew after the cutting are tied into 1 bundle, its base portion 1/4 (about 2cm) is inserted 0.04%KMnO
4Soak 8h in the solution, clean with flushing with clean water; Soak 12h in the sucrose solution with plugged ear base portion insertion 1% again, clean with flushing with clean water again; 2%FeSO is used in cuttage after using 10mg/L NAA+IBA (100: 1) to soak 2h at last in advance
4In the sand that spray solution was sterilized, place the shady and cool ventilation place, insert the back and spray 1 Hoagland (Gram suddenly) nutrient solution every 3d.The statistics rooting rate is 61% after 30 days.
Embodiment 6
In July then, select life in 1 year, middle and upper part, the leaf bud of growth is full, branch is sturdy, internode is short, the strong southern enqlish yew branch of growth potential is done the plugged ear material on the sunny side.The Chinese yew branch is cut into the long stem section of 8cm as plugged ear, and upper cut is level and smooth, incision a kind of horse raised north of the Grean Wall ear shape, and plugged ear 2/3 following defoliation and side shoot only stays 1 side shoot in upper end and blade.The stem section 30 strain plugged ears of Chinese yew after the cutting are tied into 1 bundle, its base portion 1/4 (about 2cm) is inserted 0.04%KMnO
4Soak 8h in the solution, clean with flushing with clean water; Soak 12h in the sucrose solution with plugged ear base portion insertion 1% again, clean with flushing with clean water again; 2%FeSO is used in cuttage after using 100mg/L NAA+IBA (1: 1) to soak 2h at last in advance
4In the sand that spray solution was sterilized, place the shady and cool ventilation place, insert the back and spray 1 Hoagland (Gram suddenly) nutrient solution every 3d.The statistics rooting rate is 88% after 30 days.
Embodiment 7
In May then, select life in 2 years, middle and upper part, the leaf bud of growth is full, branch is sturdy, internode is short, the strong taxus chinensis in northeast branch of growth potential is done the plugged ear material on the sunny side.The Chinese yew branch is cut into the long stem section of 10cm as plugged ear, and upper cut is level and smooth, incision a kind of horse raised north of the Grean Wall ear shape, and plugged ear 2/3 following defoliation and side shoot only stays 2 side shoots in upper end and blade.The stem section 100 strain plugged ears of Chinese yew after the cutting are tied into 1 bundle, its base portion 1/2 (about 4cm) is inserted 0.08%KMnO
4Soak 24h in the solution, clean with flushing with clean water; Soak 30h in the sucrose solution with plugged ear base portion insertion 4% again, clean with flushing with clean water again; 5%FeSO is used in cuttage after soaking 8h with 50mg/L indolebutyric acid potassium at last in advance
4In the humus soil that spray solution was sterilized, place the shady and cool ventilation place, insert the back and spray 1 Hoagland nutrient solution every 3d.The statistics rooting rate is 82% after 30 days.
Embodiment 8
In March then, select life in 1 year, middle and upper part, the leaf bud of growth is full, branch is sturdy, internode is short, the strong Chinese Chinese yew branch of growth potential is done the plugged ear material on the sunny side.The Chinese yew branch is cut into the long stem section of 9cm as plugged ear, and upper cut is level and smooth, incision a kind of horse raised north of the Grean Wall ear shape, and plugged ear 2/3 following defoliation and side shoot only stays 2 side shoots in upper end and blade.The stem section 50 strain plugged ears of Chinese yew after the cutting are tied into 1 bundle, its base portion 1/3 (about 3-4cm) is inserted 0.06%KMnO
4Soak 12h in the solution, clean with flushing with clean water; Soak 24h in the sucrose solution with plugged ear base portion insertion 2% again, clean with flushing with clean water again; 3%FeSO is used in cuttage after soaking 6h with the 100mg/L α-Nai Yisuanna at last in advance
4In sand+humus soil that spray solution was sterilized (1: 1), place the shady and cool ventilation place, insert the back and spray 1 Hoagland nutrient solution every 3d.The statistics rooting rate is 93% after 30 days.
Embodiment 9
Cuttage placed the shady and cool ventilation place after the plugged ear that will take root in cutting medium sand, sand+humus soil (1: 1) and humus soil used the IBA of 100mg/L to soak 10min in original cutting medium, inserted the back and sprayed 1 Hoagland nutrient solution every 2d.The amount of taking root after half a year is respectively 21, and 30,28, average root length is respectively 8.6cm, 12.3cm, 9.0cm.
Embodiment 10
Cuttage placed the shady and cool ventilation place after the plugged ear that will take root in cutting medium sand, sand+humus soil (1: 1) and humus soil used the IBA of 1000mg/L to soak 120min in original cutting medium, inserted the back and sprayed 1 Hoagland nutrient solution every 4d.The amount of taking root after half a year is respectively 18, and 27,24, average root length is respectively 6.5cm, 10.6cm, 8.0cm.
Embodiment 11
Cuttage was in original cutting medium after the plugged ear that will take root in cutting medium sand, sand+humus soil (1: 1) and humus soil used the IBA+NAA (1: 1) of 1000mg/L to soak 30min, place the shady and cool ventilation place, insert the back and spray 1 Hoagland nutrient solution every 3d.The amount of taking root after half a year is respectively 9, and 34,22, average root length is respectively 8.4cm, 12.8cm, 9.4cm.
Embodiment 12
Cuttage was in original cutting medium after the plugged ear that will take root in cutting medium sand, sand+humus soil (1: 1) and humus soil used the IBA+NAA (1: 1) of 500mg/L to soak 30min, place the shady and cool ventilation place, insert the back and spray 1 Hoagland nutrient solution every 3d.The amount of taking root after half a year is respectively 24, and 38,32, average root length is respectively 9.9cm, 14.8cm, 11.2cm.
Embodiment 13
Cuttage was in original cutting medium after the plugged ear that will take root in cutting medium sand, sand+humus soil (1: 1) and humus soil used the IBA+NAA (1: 1) of 50mg/L to soak 10min, place the shady and cool ventilation place, insert the back and spray 1 Hoagland nutrient solution every 3d.The amount of taking root after half a year is respectively 10, and 14,12, average root length is respectively 5.3cm, 4.4cm, 6.5cm.
Embodiment 14
The growth of Taxus x media plantlet that HORMONE TREATMENT is crossed was gathered in the crops after half a year.Do not injuring the condition down cut lateral root of main root, first order lateral root keeps 2/3.Cuttage was in original cutting medium after former plant was soaked 10min with 100mg/L IBA, and intermittently atomizing is cultivated.After 30 days, former plant survival rate is 83%; 10 of newborn first order lateral root quantity average out to; The average root of new root is long to be 1.4cm.
Embodiment 15
The growth of Taxus x media plantlet that HORMONE TREATMENT is crossed was gathered in the crops after half a year.Do not injuring the condition down cut lateral root of main root, first order lateral root keeps 2/3.Cuttage was in original cutting medium after former plant used the IBA of 1000mg/L to soak 120min, and intermittently atomizing is cultivated.After 30 days, former plant survival rate is 87%; 12 of newborn first order lateral root quantity average out to; The average root length of new root is respectively 1.7cm.
Embodiment 16
The growth of Taxus x media plantlet that HORMONE TREATMENT is crossed was gathered in the crops after half a year.Do not injuring the condition down cut lateral root of main root, first order lateral root keeps 2/3.Cuttage was in original cutting medium after former plant used the NAA of 300mg/L to soak 60min, and intermittently atomizing is cultivated.After 30 days, former plant survival rate is 92%; 17 of newborn first order lateral root quantity average out to; The average root of new root is long to be 2.4cm.
Embodiment 17
The growth of Taxus x media plantlet that HORMONE TREATMENT is crossed was gathered in the crops after half a year.Do not injuring the condition down cut lateral root of main root, first order lateral root keeps 2/3.Cuttage was in original cutting medium after former plant used the NAA of 700mg/L to soak 30min, and intermittently atomizing is cultivated.After 30 days, former plant survival rate is 95%; 22 of newborn first order lateral root quantity average out to; The average root of new root is long to be 2.1cm.
Embodiment 18
The growth of Taxus x media plantlet that HORMONE TREATMENT is crossed was gathered in the crops after half a year.Do not injuring the condition down cut lateral root of main root, first order lateral root keeps 2/3.Cuttage was in original cutting medium after former plant used the IBA+NAA (1: 1) of 500mg/L to soak 30min, and intermittently atomizing is cultivated.After 30 days, former plant survival rate is 98%; 25 of newborn first order lateral root quantity average out to; The average root of new root is long to be 2.8cm.
Embodiment 19
The growth of Taxus x media plantlet that HORMONE TREATMENT is crossed was gathered in the crops after half a year.Do not injuring the condition down cut lateral root of main root, first order lateral root keeps 1/2.Cuttage was in original cutting medium after former plant used the IBA of 50mg/L to soak 30min, and intermittently atomizing is cultivated.After 30 days, former plant survival rate is 34%; 5 of newborn first order lateral root quantity average out to; The average root of new root is long to be 1.1cm.
Embodiment 20
The growth of Taxus x media plantlet that HORMONE TREATMENT is crossed was gathered in the crops after half a year.Do not injuring the condition down cut lateral root of main root, first order lateral root keeps 1/2.Cuttage was in original cutting medium after former plant used the IBA+NAA (1: 1) of 500mg/L to soak 30min, and intermittently atomizing is cultivated.After 30 days, former plant survival rate is 55%; 8 of newborn first order lateral root quantity average out to; The average root of new root is long to be 1.5cm.
Embodiment 21
The growth of Taxus x media plantlet that HORMONE TREATMENT is crossed was gathered in the crops after half a year.Do not injuring the condition down cut lateral root of main root, first order lateral root keeps 2/3.50 plantlets are gathered in the crops fresh 153.2g altogether.Adopt conventional method to pulverize, organic solvent extraction obtains taxol.Taxol recovery rate 63%, content of taxol 0.045%.
Embodiment 22
The Taxus x media lateral root of results is incubated in the sterile liquid MS medium that contains 1/10 mineral salt composition, adds the 30mg/L potassium bichromate in the medium, adds the sub-methyl jasmonate of 10mg/L chemical induction, 30 ± 1 ℃ of dark cultivations 4 days.Adopt conventional method to pulverize, organic solvent extraction obtains taxol.Taxol recovery rate 60%, content of taxol 0.061%.
The MS medium is the naming with two inventors, and full name is Murashige﹠amp; The Skoog medium.
Embodiment 23
The Taxus x media lateral root of results is incubated in the aseptic liquid nutrient medium that contains 1/4MS mineral salt composition, add the 50mg/L potassium bichromate in the medium, add sub-methyl jasmonate of 100mg/L chemical induction and the sub-Penicillium notatum cell wall polysaccharides of 80mg/L biotic induce extract simultaneously, 30 ± 1 ℃ of dark cultivations 3 days.Adopt conventional method to pulverize, organic solvent extraction obtains taxol.Taxol recovery rate 60%, content of taxol 0.088%.
Embodiment 24
The Taxus x media lateral root of results is incubated in the aseptic liquid nutrient medium that contains 2/3 MS mineral salt composition, add 50mg/L potassium permanganate in the medium, add sub-methyl jasmonate of 100mg/L chemical induction and the sub-Penicillium notatum cell wall polysaccharides of 80mg/L biotic induce extract simultaneously, 30 ± 1 ℃ of dark cultivations 3 days.Adopt conventional method to pulverize, organic solvent extraction obtains taxol.Taxol recovery rate 60%, content of taxol 0.088%.
Embodiment 25
The Taxus x media lateral root of results is incubated in the aseptic liquid nutrient medium that contains 2/3 MS mineral salt composition, add the 20mg/L kanamycin in the medium, add sub-methyl jasmonate of 25mg/L chemical induction and the sub-Penicillium notatum cell wall polysaccharides of 50mg/L biotic induce extract simultaneously, 30 ± 1 ℃ of dark cultivations 3 days.Adopt conventional method to pulverize, organic solvent extraction obtains taxol.Taxol recovery rate 60%, content of taxol 0.093%.
Embodiment 26
The Taxus x media lateral root of results is incubated in the aseptic liquid nutrient medium that contains 1/2 MS mineral salt composition, add the 100mg/L kanamycin in the medium, add sub-MDJ of 25mg/L chemical induction and 5mg/L ethrel simultaneously, 30 ± 1 ℃ of dark cultivations 3 days.Adopt conventional method to pulverize, organic solvent extraction obtains taxol.Taxol recovery rate 60%, content of taxol 0.075%.
Embodiment 27
The Taxus x media lateral root of results is incubated in the aseptic liquid nutrient medium that contains 1/2 MS mineral salt composition, add the 50mg/L kanamycin in the medium, add sub-MDJ of 25mg/L chemical induction and 50mg/L salicylic acid simultaneously, 30 ± 1 ℃ of dark cultivations 3 days.Adopt conventional method to pulverize, organic solvent extraction obtains taxol.Taxol recovery rate 60%, content of taxol 0.075%.
Embodiment 28
The Taxus x media lateral root of results is incubated in the aseptic liquid nutrient medium that contains 1/4 MS mineral salt composition, add the 50mg/L potassium bichromate in the medium, add sub-methyl jasmonate of 25mg/L chemical induction and the sub-Penicillium notatum cell wall polysaccharides of 50mg/L biotic induce extract simultaneously, 30 ± 1 ℃ of dark cultivations 6 days.Adopt conventional method to pulverize, organic solvent extraction obtains taxol.Taxol recovery rate 59%, content of taxol 0.121%.
Claims (7)
1. method of utilizing taxus lateral root proliferation to produce taxol, its step comprises:
(1) in spring and summer, middle and upper part, the Chinese yew branch of growing that faces south of selecting 1-2 to give birth to are done the plugged ear material;
(2) the plugged ear base portion being inserted successively concentration is to soak 2-30h respectively in 0.01-0.2% liquor potassic permanganate, 0.5-10% sucrose solution and the 10-500mg/L auxin, and cuttage is intermittently sprayed Gram nutrient solution cultivation suddenly in the matrix of having sterilized again;
Described auxin is the combination of the arbitrary proportion of the sodium salt of a kind of or α-Nai Yisuan and indolebutyric acid in the sodium salt of α-Nai Yisuan, 2,4 dichlorophenoxyacetic acid butyl ester, indolebutyric acid or the sylvite or sylvite;
(3) treat that plugged ear is taken root after, extract plugged ear, being soaked in concentration is 20-120min in the 10-1000mg/L hormone solution, cuttage is intermittently sprayed suddenly the Gram nutrient solution and is cultivated in original cutting medium then;
Described hormone is a kind of or indolebutyric acid in α-Nai Yisuan, 2,4 dichlorophenoxyacetic acid butyl ester, the indolebutyric acid, the combination that α-Nai Yisuan is 0.5: 1～1: 2 by mass ratio;
When (4) treating root vigorous growth, results Chinese yew plantlet is not injuring the condition down cut lateral root of main root, and first order lateral root keeps at least 2/3; The root concentration of shearing the back plant is the parahormone solution soaking 20-120min above-mentioned of the same race of 50-1000mg/L, and cuttage is in original cutting medium then, and intermittently atomizing is cultivated;
(5) to adopting conventional method dry under≤55 ℃ of conditions through shearing the taxus lateral root that obtains behind the aforementioned cuttage cultivation, then through pulverizing, organic solvent extraction concentrates, obtain pure product of paclitaxel behind the column chromatography, crystallization, the dry processing.
2. method according to claim 1, it is characterized in that: in the step (1), the selection leaf bud is full, branch is sturdy, internode is short, the strong Chinese yew branch of growth potential, be cut into 8-10cm, upper cut is level and smooth, incision a kind of horse raised north of the Grean Wall ear shape, plugged ear 2/3 following defoliation and side shoot only stays 1-2 side shoot in upper end and blade.
3. method according to claim 1 is characterized in that: in the step (2), liquor potassic permanganate concentration is 0.04-0.08%, and soak time is 8-24h; Sucrose solution concentration is 1-4%, and soak time is 12-30h; The concentration of auxin is 50-100mg/L, and soak time is 4-6h; Cutting medium is that sand is or/and humus soil.
4. method according to claim 1 is characterized in that: in above-mentioned steps (3), (4), hormone is a kind of or indolebutyric acid in α-Nai Yisuan, 2,4 dichlorophenoxyacetic acid butyl ester, the indolebutyric acid, the combination that α-Nai Yisuan is 1: 1 by mass ratio; The hormone total concentration is 300-700mg/L, hormone soak time 30-60min.
5. according to claim 1,2,3 or 4 described methods, it is characterized in that: in the step (5), before carrying out the drying processing, the taxus lateral root with clip is incubated at aseptic liquid improvement Murashige﹠amp earlier; In the Skoog medium, promptly only utilize Murashige﹠amp; The mineral salt composition of Skoog medium, and its integral level dropped to original 1/10-2/3, add medicament in the medium and suppress living contaminants, adopt fungal elicitor or chemical induction to induce simultaneously and handled fresh taxus lateral root 2-9 days.
6. method according to claim 5 is characterized in that: the medicament that suppresses living contaminants be any quality of a kind of in potassium bichromate and the potassium permanganate or two kinds than mixed liquor, the medicament total concentration is 30-80mg/L; Any quality that perhaps is a kind of in kanamycin and the streptomycin or two kinds is than mixed liquor, and the medicament total concentration is 20-100mg/L.
7. method according to claim 5 is characterized in that: fungal elicitor is to separate a kind of in the cell wall polysaccharides extract obtain black-koji mould, Penicillium notatum, streptomycete, yeast or gibberella in the bark of Ramulus et folium taxi cuspidatae, and its concentration is 30-80mg/L;
Chemical inducer is any or the wantonly two kinds mixed liquor in the following solution: concentration is the 10-100mg/L methyl jasmonate, and concentration is the 10-100mg/L MDJ, and concentration is the 50-100mg/L salicylic acid, and concentration is the 1-5mg/L ethrel.
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| CN103314759A (en) * | 2013-07-04 | 2013-09-25 | 丹阳市保得生态农林开发有限公司 | Method for improving osmanthus cutting rooting |
| CN103772028A (en) * | 2014-01-17 | 2014-05-07 | 陕西鼎天济农腐殖酸制品有限公司 | Humic acid root fertilizer and production method thereof |
| CN103858624A (en) * | 2012-12-10 | 2014-06-18 | 杨万培 | Method for improving breeding efficiency of Chinese yew |
| CN104094765A (en) * | 2014-07-28 | 2014-10-15 | 重庆市碚圣农业科技股份有限公司 | Culture and collection method capable of improving Taxus media biomass |
| CN105638723A (en) * | 2016-01-27 | 2016-06-08 | 湖北祥瑞丰生态果业有限公司 | Rooting agent formula for taxus chinensis branch cutting rooting and preparation method thereof |
| CN106613654A (en) * | 2016-12-12 | 2017-05-10 | 浙江大学宁波理工学院 | Regulation method of increasing taxus polysaccharide content in Taxus Chinensis Var.Mairei leaves |
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| US5642587A (en) * | 1993-05-17 | 1997-07-01 | Grow International Corp. | Compressed dormancy process for increased plant growth |
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| CN103858624A (en) * | 2012-12-10 | 2014-06-18 | 杨万培 | Method for improving breeding efficiency of Chinese yew |
| CN103314759A (en) * | 2013-07-04 | 2013-09-25 | 丹阳市保得生态农林开发有限公司 | Method for improving osmanthus cutting rooting |
| CN103772028A (en) * | 2014-01-17 | 2014-05-07 | 陕西鼎天济农腐殖酸制品有限公司 | Humic acid root fertilizer and production method thereof |
| CN104094765A (en) * | 2014-07-28 | 2014-10-15 | 重庆市碚圣农业科技股份有限公司 | Culture and collection method capable of improving Taxus media biomass |
| CN104094765B (en) * | 2014-07-28 | 2016-06-01 | 重庆市碚圣农业科技股份有限公司 | A kind of cultivation and collection method improving Taxus x media biomass |
| CN105638723A (en) * | 2016-01-27 | 2016-06-08 | 湖北祥瑞丰生态果业有限公司 | Rooting agent formula for taxus chinensis branch cutting rooting and preparation method thereof |
| CN106613654A (en) * | 2016-12-12 | 2017-05-10 | 浙江大学宁波理工学院 | Regulation method of increasing taxus polysaccharide content in Taxus Chinensis Var.Mairei leaves |
| CN109463210A (en) * | 2018-10-17 | 2019-03-15 | 广东红树林生态科技有限公司 | A kind of breeding method of the Lao Shu le root of quick harvesting |
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