CN101629193B - Method for producing anti-cancer alkaloid by culturing cephalotaxus hainanensis li cells - Google Patents
Method for producing anti-cancer alkaloid by culturing cephalotaxus hainanensis li cells Download PDFInfo
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Abstract
The invention relates to a method for producing anti-cancer alkaloid by culturing cephalotaxus hainanensis li cells, in particular to a method for producing anti-cancer alkaloid by culturing plant cells. The method is carried out according to the following steps: (1) screening high-yield active alkaloid cephalotaxus hainanensis li plants; (2) inducing external plants; (3) establishing a high-yield alkaloid suspension cell culturing system; (4) culturing suspension cells by adopting a two-stage method; and (5) separating and purifying alkaloid. The method can obtain active alkaloid with relatively high yield; after culturing in a stirred reactor with an active volume of 2L under proper conditions by adopting the two-stage method, the cell biomass is increased by about 4 times, and the active alkaloid content in the cell culture reaches 0.08% of the dry weight of the cell, and at best, 0.12% of the dry weight of the cell, which is about one order of magnitude higher than the content in the natural plants, therefore, the method provides a technical support for solving the issues, such as rare or endangered specie cephalotaxus hainanensis li protection and resource development and utilization, and also provides a basis for the genetic engineering for further exploring cephalotaxus hainanensis li, and the like.
Description
Technical field
The present invention relates to a kind of culture plant cell and produce the anti-cancer alkaloid method; Specifically be to be means with the plant cell culture technology, through seed of Hainan Plumyew mass cell cultivating and producing anti-cancer alkaloid such as NSC 142196 (deoxyharringtonine), Isoharringtonine (isoharringtonine), harringtonine (harringtonine) and the percephalotaxine multiple alkaloidal methods such as (homoharringtonine) of in the cell biological reactor drum, carrying out.
Background technology
Seed of Hainan Plumyew (Cephalotaxus hainanensis Li) is the cephalotaxaceae plant, and another name cephalotaxus hainanensis, slender lobule bamboo comb China fir are perennial tall and big aiphyllium.Be the distinctive rare tree species of China, its distribution range is very narrow, is detected in partial area such as Hainan and Guangdong, Yunnan.Research shows; Seed of Hainan Plumyew is not only anticancer, and effective alkaloid (like NSC 142196 (deoxyharringtonine), Isoharringtonine (isoharringtonine), harringtonine (harringtonine) and percephalotaxine multiple vegeto-alkalis such as (homoharringtonine)) content is arranged is the highest in the cephalotaxaceae; And curative effect is the most remarkable, is one of best anticancer seeds of generally acknowledging at present.But; At present can't be through vegeto-alkalis such as the synthetic NSC 142196 of manual method, Isoharringtonine, harringtonine and percephalotaxines; Only reliable from seed of Hainan Plumyew vegetable material natural extract, so the seed of Hainan Plumyew raw material guarantee just becomes the key point in this medicine market.But; Because these seeds are that dioecy, seed production and germination rate are very low, these seeds are cut down because of material is good repeatly in addition, and population quantity is few in addition; Be one of most important rare or endangered species of China; Natural resources is very limited, receives factor such as ecological and artificial destruction and causes endangeredly, is difficult to satisfy growing demand.
In addition, seed of Hainan Plumyew over-ground part, particularly bark are for extracting alkaloidal main raw materials such as NSC 142196, Isoharringtonine, harringtonine and percephalotaxine.Satisfy patient of treatment, need vegeto-alkalis such as 2g left and right sides NSC 142196, Isoharringtonine, harringtonine and percephalotaxine approximately; Need 100-1000kg trunk or 10-100kg bark approximately and extract the above vegeto-alkali of 1g, promptly about 10-50 about 50 age seed of Hainan Plumyew.The world market is 1000kg to vegeto-alkali annual requirements such as NSC 142196, Isoharringtonine, harringtonine and percephalotaxines now, and this demand increases just day by day.Rely on the limited resources of natural seed of Hainan Plumyew can not satisfy the demands.But up to this point, though people have effective alkaloid more than can't total man worker synthetic; But people have effective alkaloid more than can synthesizing through the method for half synthetic with the intermediate that extracts from seed of Hainan Plumyew, but its combined coefficient is low, and because the chirality characteristic, its biological activity is difficult to assurance, and with high costs.Therefore, people have to abandon using this type of effective alkaloids for treating relative disease, then utilize the other medicines that curative effect is relatively poor relatively or have certain toxic side effect.So; The invention provides processes such as the separating of a kind of system, purifying, realized that a culture cycle can gather in the crops NSC 142196, Isoharringtonine, harringtonine and percephalotaxine etc. the about 50mgL of effective alkaloid is arranged from the foundation of the selection of high yield vegeto-alkali material, explant induction, high yield vegeto-alkali suspension cell culture system and suspension cell culture to vegeto-alkali
-1The result, for next step through cell cultures produce NSC 142196, Isoharringtonine, harringtonine and percephalotaxine etc. have the effective alkaloid industrialization provide maybe with guarantee; Thereby can for the starting material extraction separation effective alkaloid not arranged in order to natural seed of Hainan Plumyew.These protection and development and use to the seed of Hainan Plumyew natural resources have important theoretical and practice significance.
Summary of the invention
The key technical problem that the present invention solves is through screening seed of Hainan Plumyew high yield NSC 142196, Isoharringtonine, harringtonine and percephalotaxine etc. the effective alkaloid plant to be arranged; Explant induction; The foundation of high yield vegeto-alkali suspension cell culture system and suspension cell culture to vegeto-alkali separate, process such as purifying, for having the effective alkaloid industrialization that technical guarantee is provided through cell cultures production NSC 142196, Isoharringtonine, harringtonine and percephalotaxine etc.These protection and development and use to the seed of Hainan Plumyew natural resources have important theoretical and practice significance.
The technical scheme that the present invention addresses the above problem comprises the steps:
(1) high yield has the screening of effective alkaloid seed of Hainan Plumyew plant: gather the adult plant of seed of Hainan Plumyew of different sources, carry out effective alkaloid analysis, select the highest plant of content to originate as explant;
(2) explant induction: get the highest plant bark of effective alkaloid as explant, clean up with zero(ppm) water, put into the about 3-8min of about 0.5-2.0% chlorine bleach liquor vacuum filtration sterilization after filter paper blots, sterilized water washes 3-6 time repeatedly then; Explant is cut into about 0.5cm * 0.5cm square, is inoculated on the following callus inducing medium: 0.5-1MS (Murashige and Skoog Stock substratum)+0.01-0.1mgL
-16-BA (6-benzyl purine)+0.5-1mgL
-1NAA (naphthylacetic acid)+0.05-0.5mgL
-1KT (kinetin)+0-100mgL
-1Inositol+0.2-0.6% agar+1-5% sucrose+1-5% coconut milk, pH value 5.0-6.0; Place periodicity of illumination 16hd again behind the dark cultivation 2d
-1, intensity of illumination 2000-3000Lux cold light lamp under, continue in 25 ± 2 ℃ of the envrionment temperatures to cultivate that (every month subculture once in 3 months; The explant of choosing, cut the vigorous callus of band growth with aseptic scalper and aseptic nipper during subculture is inoculated into new substratum), just obtain required callus.
(3) foundation of high yield vegeto-alkali suspension cell culture system: get callus and be inoculated into and shake cultivation: 0.5-1MS+0.01-0.1mgL in the following substratum
-16-BA+0.5-1mgL
-1NAA+0.05-0.5mgL
-1KT+0-100mgL
-1Inositol+1-5% sucrose+1-5% coconut milk, pH value 5.0-6.0; Place periodicity of illumination 16hd again behind the dark cultivation 2d
-1, intensity of illumination 2000-3000Lux cold light lamp under, 25 ± 2 ℃ of envrionment temperatures, shaking speed is 60-200rpm; Continue to cultivate 25-35d follow-up generation 1 time; Measuring suspension cell line behind subculture 5-10 time respectively produces effective alkaloid efficient is arranged; Selecting to produce has the high person of effective alkaloid efficient to be high yield vegeto-alkali suspension cell culture system.
(4) suspension cell culture: the employing two-phase method is carried out suspension cell culture, and promptly cell strain system is cultivated for some time earlier with the accumulation living weight in growth medium, changes the production substratum again over to and impels a large amount of synthetic effective alkaloids that have of cell.System changes in the following growth medium through aseptic technique with high yield vegeto-alkali suspension cell culture: pH value 5.0-6.5; Carbonaceous sources concentration is that 2-6% (W/V), inorganic nitrogen-sourced ionic concn are that 40-80mM, phosphate concn are 2-6mM; Hormonal readiness is 2; 4-D (2,4 dichlorophenoxyacetic acid) is 0.5-2mgL
-1, 6-BA is 0.5-2mgL
-1, NAA is 0.5-3mgL
-1, KT is 0.5-2mgL
-1, GA (Plant hormones regulators,gibberellins) is 0.5-2mgL
-1, ZT (anti-zein) is 0.5-2.5mgL
-1And the liquid growth medium of 0.5-1MS nutrition source carries out suspension culture; Under the cold light lamp of intensity of illumination 2000-3000Lux, 25 ± 2 ℃ of envrionment temperatures; Shaking table and stirring reactor rotating speed are 60-250rpm; The air flow 0.1-0.5vvm of reactor drum cultivated after 25-35 days, changed and produced substratum continuation suspension culture; Producing substratum is that pH is 5.0-6.0; Carbonaceous sources concentration is that 2-4% (W/V), inorganic nitrogen-sourced ionic concn are that 30-60mM, phosphate concn are 1-3mM; Hormone kind and horizontal extent are constant; And the liquid nutrient medium of 0.5-1MS nutrition source, under aforesaid culture condition, continue to cultivate 25-35 days;
(5) vegeto-alkali isolation and purification: the above-mentioned cultured cells of harvested cell, grind, ammoniacal liquor is moistening; Add the chloroform submergence, airtight, put the 24h that vibrates on the shaking table; The leaching clear liquid, be evaporated to dried, the dissolve with methanol residue; Further in the pair cell culture have effective alkaloid to adopt half preparation HPLC method to carry out separation and purification (moving phase is methyl alcohol-second eyeball-water (29: 27: 44), and to use ammoniacal liquor to regulate pH be about 8.5, flow velocity 1ml/min; Detect wavelength: 290nm, column temperature: 35 ℃, sample size: 100 μ L.Use method of the present invention; Can obtain output higher effective vegeto-alkali; Adopt the stirring reactor of 2L effective volume to carry out two-step approach cultivation 50-70 days under optimum conditions, cellular biomass increases to about 4 times, and effective alkaloid reaches dried cell weight 0.08% in the cell culture.Best-case can reach 0.12% of dried cell weight, and this result exceeds an about one magnitude than content in the natural phant.
3, beneficial effect
The invention has the beneficial effects as follows: use method of the present invention; Can obtain output higher effective vegeto-alkali; Adopt the stirring reactor of 2L effective volume to carry out two-step approach cultivation 50-70 days under optimum conditions; Cellular biomass increases to about 4 times, and effective alkaloid reaches dried cell weight 0.08% in the cell culture.Best-case can reach 0.12% of dried cell weight, and this result exceeds an about one magnitude than content in the natural phant.Thereby with resources development and utilization technical guarantee is provided for solving the protection of rare or endangered species seed of Hainan Plumyew; Do not decided the basis for the genetically engineered of further exploring seed of Hainan Plumyew etc. yet.
Four, embodiment
The specific embodiment of the invention is illustrated below in conjunction with instance: required various medicine and reagents all can commercially availablely obtain in the substratum, press the adapted of ordinary method shown in the catalogue, and institute is general for industry.
Embodiment 1:
(1) high yield has the screening of effective alkaloid seed of Hainan Plumyew plant: gather adult about 60 strains of plant of seed of Hainan Plumyew in different population source, carry out effective alkaloid analysis, select the highest plant of content to originate as explant;
(2) explant induction: get the highest plant bark of effective alkaloid as explant, clean up with zero(ppm) water, put into 0.5% chlorine bleach liquor's vacuum filtration sterilization 8min after filter paper blots, sterilized water washes 3 times repeatedly then; Explant is cut into about 0.5cm * 0.5cm square, is inoculated on the following callus inducing medium: 0.5MS (Murashige andSkoog Stock substratum)+0.01mgL
-16-BA (6-benzyl purine)+0.5mgL
-1NAA (naphthylacetic acid)+0.05mgL
-1KT (kinetin)+0.2% agar+1% sucrose+1% coconut milk, pH value 5.0; Place periodicity of illumination 16hd again behind the dark cultivation 2d
-1, intensity of illumination 2000Lux cold light lamp under, continue in 25 ± 2 ℃ of the envrionment temperatures to cultivate 3 months (every month subculture once), just obtain required callus;
(3) foundation of high yield vegeto-alkali suspension cell culture system: get callus and be inoculated into and shake cultivation: 0.5MS+0.01mgL in the following substratum
-16-BA+0.5mgL
-1NAA+0.05mgL
-1KT+1% sucrose+1% coconut milk, pH value 5.0; Place periodicity of illumination 16hd again behind the dark cultivation 2d
-1, intensity of illumination 2000Lux cold light lamp under, 25 ± 2 ℃ of envrionment temperatures, shaking speed is 60rpm; Continue to cultivate about 25d follow-up generation 1 time; Measuring suspension cell line behind the subculture 5 times respectively produces effective alkaloid efficient is arranged; Selecting to produce has the high person of effective alkaloid efficient to be high yield vegeto-alkali suspension cell culture system;
(4) suspension cell culture: system changes in the following substratum through aseptic technique with high yield vegeto-alkali suspension cell culture: pH value 5.0; Carbonaceous sources concentration is that 2% (W/V), inorganic nitrogen-sourced ionic concn are that 40mM, phosphate concn are 2mM; Hormonal readiness is 2; 4-D (2,4 dichlorophenoxyacetic acid) is 0.5mgL
-1, 6-BA is 0.5mgL
-1, NAA is 0.5mgL
-1, KT is 0.5mgL
-1, GA (Plant hormones regulators,gibberellins) is 0.5mgL
-1, ZT (anti-zein) is 0.5mgL
-1And the liquid growth medium of 0.5MS nutrition source carries out suspension culture; Under the cold light lamp of intensity of illumination 2000Lux, 25 ± 2 ℃ of envrionment temperatures, shaking table and stirring reactor rotating speed are 60rpm, the air flow 0.1vvm of reactor drum; After cultivating 25d, change and produce substratum continuation suspension culture; Producing substratum is that pH is 5.0; Carbonaceous sources concentration is that 2% (W/V), inorganic nitrogen-sourced ionic concn are that 30mM, phosphate concn are 1mM; Hormone kind and horizontal extent are constant, also contain the liquid nutrient medium of 0.5MS nutrition source simultaneously, under aforesaid culture condition, continue to cultivate 25d;
(5) vegeto-alkali isolation and purification: the above-mentioned cultured cells of harvested cell, grind, ammoniacal liquor is moistening, adds the chloroform submergence; Airtight, put the 24h that vibrates on the shaking table, the leaching clear liquid; Be evaporated to dried, the dissolve with methanol residue, further in the pair cell culture have effective alkaloid to adopt half preparation HPLC method to carry out separation and purification (moving phase is methyl alcohol-second eyeball-water (29: 27: 44); And to use ammoniacal liquor to regulate pH be about 8.5, and flow velocity 1ml/min detects wavelength: 290nm; Column temperature: 35 ℃, sample size: 100 μ L, effective alkaloid reaches dried cell weight 0.07% in the cell culture.
Embodiment 2:
(1) high yield has the screening of effective alkaloid seed of Hainan Plumyew plant: gather adult about 60 strains of plant of seed of Hainan Plumyew in different population source, carry out effective alkaloid analysis, select the highest plant of content to originate as explant;
(2) explant induction: get the highest plant bark of effective alkaloid as explant, clean up with zero(ppm) water, put into 2.0% chlorine bleach liquor's vacuum filtration sterilization 3min after filter paper blots, sterilized water washes 6 times repeatedly then; Explant is cut into about 0.5cm * 0.5cm square, is inoculated on the following callus inducing medium: 1MS (Murashige and SkoogStock substratum)+0.1mgL
-16-BA (6-benzyl purine)+1mgL
-1NAA (naphthylacetic acid)+0.5mgL
-1KT (kinetin)+100mgL
-1Inositol+0.6% agar+5% sucrose+5% coconut milk, pH value 6.0; Place periodicity of illumination 16hd again behind the dark cultivation 2d
-1, intensity of illumination 3000Lux cold light lamp under, continue in 25 ± 2 ℃ of the envrionment temperatures to cultivate 3 months (every month subculture once), just obtain required callus;
(3) foundation of high yield vegeto-alkali suspension cell culture system: get callus and be inoculated into and shake cultivation: 1MS+0.1mgL in the following substratum
-16-BA+1mgL
-1NAA+0.5mgL
-1KT+100mgL
-1Inositol+5% sucrose+5% coconut milk, pH value 6.0; Place periodicity of illumination 16hd again behind the dark cultivation 2d
-1, intensity of illumination 3000Lux cold light lamp under, 25 ± 2 ℃ of envrionment temperatures, shaking speed is 200rpm; Continue to cultivate about 35d follow-up generation 1 time; Measuring suspension cell line behind the subculture 10 times respectively produces effective alkaloid efficient is arranged; Selecting to produce has the high person of effective alkaloid efficient to be high yield vegeto-alkali suspension cell culture system;
(4) suspension cell culture: system changes in the following substratum through aseptic technique with high yield vegeto-alkali suspension cell culture: pH value 6.5; Carbonaceous sources concentration is that 6% (W/V), inorganic nitrogen-sourced ionic concn are that 80mM, phosphate concn are 6mM; Hormonal readiness is 2; 4-D (2,4 dichlorophenoxyacetic acid) is 2mgL
-1, 6-BA is 2mgL
-1, NAA is 3mgL
-1, KT is 2mgL
-1, GA (Plant hormones regulators,gibberellins) is 2mgL
-1, ZT (anti-zein) is 2.5mgL
-1And the liquid growth medium of 1MS nutrition source carries out suspension culture; Under the cold light lamp of intensity of illumination 3000Lux, 25 ± 2 ℃ of envrionment temperatures, shaking table and stirring reactor rotating speed are 250rpm, the air flow 0.5vvm of reactor drum; After cultivating 35d, change and produce substratum continuation suspension culture; Producing substratum is that pH is 6.0; Carbonaceous sources concentration is that 4% (W/V), inorganic nitrogen-sourced ionic concn are that 60mM, phosphate concn are 3mM; Hormone kind and horizontal extent are constant, also contain the liquid nutrient medium of 1MS nutrition source simultaneously, under aforesaid culture condition, continue to cultivate 35d;
(5) vegeto-alkali isolation and purification: vegeto-alkali isolation and purification: the above-mentioned cultured cells of harvested cell, grind, ammoniacal liquor is moistening, adds the chloroform submergence; Airtight, put the 24h that vibrates on the shaking table, the leaching clear liquid; Be evaporated to dried, the dissolve with methanol residue, further in the pair cell culture have effective alkaloid to adopt half preparation HPLC method to carry out separation and purification (moving phase is methyl alcohol-second eyeball-water (29: 27: 44); And to use ammoniacal liquor to regulate pH be about 8.5, and flow velocity 1ml/min detects wavelength: 290nm; Column temperature: 35 ℃, sample size: 100 μ L, effective alkaloid reaches dried cell weight 0.10% in the cell culture.
Implement by technological method of the present invention, composition relative proportion difference can cause having the difference of effective alkaloid production efficiency in the substratum, and in a word, numerical value is more near optimum value, and result of implementation is unreasonable to be thought.
Claims (1)
1. the method for producing anti-cancer alkaloid by culturing cephalotaxus hainanensis li cells is characterized in that carrying out according to following step:
(1) high yield has the screening of effective alkaloid seed of Hainan Plumyew plant: gather the adult plant of seed of Hainan Plumyew of different sources, carry out effective alkaloid analysis, select the highest plant of content to originate as explant;
(2) explant induction: get the highest plant bark of effective alkaloid as explant, clean up with zero(ppm) water, put into 0.5-2.0% chlorine bleach liquor vacuum filtration sterilization 3-8min after filter paper blots, sterilized water washes 3-6 time repeatedly then; Explant is cut into about 0.5cm * 0.5cm square, is inoculated on the following callus inducing medium: 0.5-1Murashige and Skoog Stock substratum+0.01-0.1mgL
-16-benzyl purine+0.5-1mgL
-1Naphthylacetic acid+0.05-0.5mgL
-1Kinetin+0-100mgL
-1Inositol+0.2-0.6% agar+1-5% sucrose+1-5% coconut milk, pH value 5.0-6.0; Place periodicity of illumination 16hd again behind the dark cultivation 2d
-1, intensity of illumination 2000-3000Lux cold light lamp under, continue in 25 ± 2 ℃ of the envrionment temperatures to cultivate 3 months; Every month subculture once; The explant of choosing, cut the vigorous callus of band growth with aseptic scalper and aseptic nipper during subculture is inoculated into new substratum, just obtains required callus;
(3) foundation of high yield vegeto-alkali suspension cell culture system: get callus and be inoculated into and shake cultivation in the following substratum: 0.5-1Murashige and Skoog Stock substratum+0.01-0.1mgL
-16-benzyl purine+0.5-1mgL
-1Naphthylacetic acid+0.05-0.5mgL
-1Kinetin+0-100mgL
-1Inositol+1-5% sucrose+1-5% coconut milk, pH value 5.0-6.0; Place periodicity of illumination 16hd again behind the dark cultivation 2d
-1, intensity of illumination 2000-3000Lux cold light lamp under, 2 ℃ of envrionment temperature 25 scholars, shaking speed is 60-200rpm; Continue to cultivate 25-35d follow-up generation 1 time; Measuring suspension cell line behind subculture 5-10 time respectively produces effective alkaloid efficient is arranged; Selecting to produce has the high person of effective alkaloid efficient to be high yield vegeto-alkali suspension cell culture system;
(4) suspension cell culture: adopt two-phase method to carry out suspension cell culture; Be that cell strain system is cultivated for some time earlier with the accumulation living weight in growth medium; Change the production substratum again over to and impel a large amount of synthetic effective alkaloids that have of cell; System changes in the following growth medium through aseptic technique with high yield vegeto-alkali suspension cell culture: pH value 5.0-6.5; Carbonaceous sources concentration with quality and volume count 2-6%, inorganic nitrogen-sourced ionic concn is that 40-80mM, phosphate concn are 2-6mM, hormonal readiness is that 2,4 dichlorophenoxyacetic acid is 0.5-2mgL
-1, the 6-benzyl purine is 0.5-2mgL
-1, naphthylacetic acid is 0.5-3mgL
-1, kinetin is 0.5-2mgL
-1, Plant hormones regulators,gibberellins is 0.5-2mgL
-1, anti-zein is 0.5-2.5mgL
-1And the liquid growth medium of 0.5-1Murashige and Skoog Stock nutrition source carries out suspension culture; Under the cold light lamp of intensity of illumination 2000-3000Lux, 2 ℃ of envrionment temperature 25 scholars; Shaking table and stirring reactor rotating speed are 60-250rpm; The air flow 0.1-0.5vvm of reactor drum cultivated after 25-35 days, changed and produced substratum continuation suspension culture; Producing substratum is that pH is 5.0-6.0; Carbonaceous sources concentration is for being that 30-60mM, phosphate concn are 1-3mM with quality and volumeter 2-4%, inorganic nitrogen-sourced ionic concn; Hormone kind and horizontal extent are constant; And the liquid nutrient medium of 0.5-1Murashige and Skoog Stock nutrition source, under aforesaid culture condition, continue to cultivate 25-35 days;
(5) vegeto-alkali isolation and purification: gather in the crops above-mentioned cultured cells, grind, ammoniacal liquor is moistening, adds the chloroform submergence; Airtight, put the 24h that vibrates on the shaking table, the leaching clear liquid is evaporated to dried; The dissolve with methanol residue, further the effective alkaloid that has in the pair cell culture adopts half preparation HPLC method to carry out separation and purification, and moving phase is methyl alcohol-acetonitrile-water 29: 27: 44, and use ammoniacal liquor adjusting pH is about 8.5; Flow velocity 1ml/min, detect wavelength: 290nm, column temperature: 35 ℃, sample size: 100 μ L.
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| CN103120189A (en) * | 2013-02-06 | 2013-05-29 | 杨凌农科大无公害农药研究服务中心 | Cephalotaxus sinensis Li. plant-virus-resisting agent and preparation method thereof |
| CN103725725B (en) * | 2013-12-30 | 2016-04-20 | 中国热带农业科学院热带作物品种资源研究所 | The non-cells,primordial suspension culture of seed of Hainan Plumyew is utilized to produce the method for taxol and precursor baccatin III thereof |
| CN104293855A (en) * | 2014-10-15 | 2015-01-21 | 中国计量学院 | Application of trichoderma brevicompactum transformant in improvement of trichodermin yield |
| CN104293853B (en) * | 2014-10-15 | 2018-04-13 | 洛阳理工学院 | A kind of industrialized preparing process of cephalotaxus alkaloids |
| CN110628784B (en) * | 2019-10-31 | 2022-10-25 | 中国热带农业科学院热带作物品种资源研究所 | NCS-like PR10 gene and application thereof |
| US11299700B1 (en) | 2021-02-19 | 2022-04-12 | Acequia Biotechnology, Llc | Bioreactor containers and methods of growing hairy roots using the same |
| CN115572748B (en) * | 2022-11-24 | 2023-03-10 | 海南大学三亚南繁研究院 | Method for producing antitumor deoxy harringtonine by using suspension culture cells |
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