CN1556201A - Solid liquid two step culturing method for producing useful metabolism product in plant cell culturing - Google Patents
Solid liquid two step culturing method for producing useful metabolism product in plant cell culturing Download PDFInfo
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Abstract
A two-step solid-liquid culture method of plant cells for preparing the useful metabolic product includes preparing basic culture medium, preparing solid culture medium, inoculating the fastly growing plant cells to it, culturing to obtain calli of plant, preparing liquid culture medium, inoculating the calli to it, catalytically synthesizing the target metabolic product, collecting aggregate and culture liquid, and separating and puring the target metabolic product.
Description
Technical field
This patent is the novel method that a kind of culture plant cell is produced secondary metabolite, is applicable to various culture plant cell, and is more effective for the production with the meta-bolites that suppresses the cell fission function especially.
Background technology
In the technical field that culture plant cell is produced secondary metabolite, perplex for a long time or the biggest obstacle that hinders its industrialization be because of the vegetable cell rate of propagation slowly, the meta-bolites generated time is long and active constituent content is low etc., and production cost that factor causes is too high.Sum up previous studies and can find that the proliferative amount of vegetable cell in solid medium can reach 5～20 times/30 days, and the proliferative amount in the liquid medium within was generally 2～4 times/30 days.Though the rate of propagation of cell is very fast in solid culture, because its substratum mass transfer is bad and don't be beneficial to the synthetic of its secondary metabolite.In a lot of culture plant cell, because its secondary metabolite can suppress synthesizing or cell fission of DNA after accumulating in cell, so its cell proliferation and meta-bolites are synthetic conflicting.Culture plant cell method in the past is main the same with the microorganism cells culture method, take cell cultured continuously or suspension culture, just in earlier stage promote cell proliferation in cultivation, changing culture condition in the later stage promotes product synthetic, do like this because the proliferative amount of cell is less, so also limited for the product resultant quantity in later stage.
Summary of the invention
The objective of the invention is to solve existing culture plant cell and produce in the secondary metabolite technology, plant cell growth and its meta-bolites be synthetic to have mutual inhibiting contradiction, promotes the cell fast breeding, realization target meta-bolites high yield.
Technical scheme of the present invention is as follows: the solid-liquid two-steps tissue culture method of culture plant cell production desirable metabolites is characterized in that this method comprises the steps:
1) in plant tissue and cell culture medium, add 0.1～4mg growth hormone, 0.1～4mg phytokinin by every liter of nutrient solution, and 20～60g sucrose or glucose, adjust pH is 5.5～7, as minimum medium;
2) get described minimum medium, to wherein making solid medium by every liter of nutrient solution adding 7～10g agar or the efficient peptizer of 2g; The cell strain system of the quick growth that filters out is seeded on the described solid medium, under 20～25 ℃ of lucifuges or irradiation condition, cultivates, obtain plant callus;
3) get described minimum medium, promote target product synthetic precursor substance or elicitor, medium pH value is adjusted to 5.5～7, behind sterilization, be made into liquid nutrient medium to wherein adding;
4) with step 2) in the plant callus that obtains sieve (the callus of kindred plant is not to the requirement difference in aperture) with the stainless (steel) wire of certain pore size, the plant callus particle of a certain size after will sieving then is seeded on the described liquid nutrient medium, under 20～25 ℃ of lucifuges or irradiation condition, the target meta-bolites is synthesized in catalysis;
5) collect cell mass and nutrient solution, and obtain the target meta-bolites by separation and purification.
Plant tissue of the present invention and cell culture medium are Murashige ﹠amp; Skoog substratum, Miller substratum, Gamborg substratum, White substratum, Heller substratum, Linsmaier ﹠amp; Skoog substratum or sss substratum.
Growth hormone of the present invention is preferably indolylacetic acid, α-Nai Yisuan or 2,4 dichlorphenoxyacetic acids.
Phytokinin of the present invention is preferably 6-benzyl aminopurine or kinetin.
Promotion target product synthetic precursor substance of the present invention or elicitor are phenylalanine, tyrosine, methyl jasmonate (mj) or coffic acid.
In step 2) in, the cell strain system of the quick growth that filters out is seeded on the described solid medium, under 20～25 ℃ of lucifuges or irradiation condition, cultivate and obtained plant callus in 15～25 days.
Described plant callus is seeded in the rotating and culturing device or bio-reactor that described liquid nutrient medium is housed, and under 20～25 ℃ of lucifuges or irradiation condition, cultivates 2-10 days synthetic target products.
A kind of being improved to of the present invention: utilize two kinds of different plant tissues and cell culture medium, make two kinds of minimum mediums according to method described in the step 1), in step 2) and step 3) in respectively choose a kind of minimum medium, be used for preparing solid medium and liquid nutrient medium.
Take the solid-liquid two-steps tissue culture method biomass of vegetable cell to be risen to 5～20 times (4 weeks) in a culture cycle, in 2～10 days, the content of target meta-bolites is brought up to the several times of seed cell～tens of times in the liquid culture step in the solid culture step.
Embodiment
The present invention will be further described below in conjunction with specific embodiment.
Embodiment 1
(1) at Murashige ﹠amp; In Skoog (MS) improved culture medium, by the α-Nai Yisuan (NAA) of every liter of nutrient solution adding 0.1～4mg, the kinetin of 0.1～4mg (kinetin), and 30～50g sucrose; With acid or alkali the pH value is adjusted to 5.5～7, as minimum medium.
(2) get described minimum medium,,, be divided in and cultivate in the vessel after the dissolving of heating by every liter of nutrient solution adding efficient peptizer of 2g (Gellan gum) to wherein, through 125 ℃ of high temperature, 0.1Mpa pressure is sterilized down after be cooled to solid medium; The callus of cistanche deserticda high yield strain system that filters out is seeded on the described solid medium, under 25 ℃ of lucifuge conditions, cultivated 20～25 days, obtain plant callus.
(3) get described minimum medium, promote target product synthetic precursor substance phenylalanine 50～500mg and tyrosinase 15 0～400mg to wherein adding by every liter of nutrient solution, medium pH value is adjusted to 5.5～7, divide then and install in incubator or the bio-reactor, through 125 ℃ of high temperature, 0.1Mpa pressure carries out cooling process after the sterilization down, is made into liquid nutrient medium.
(4) callus that will in step (2), obtain, with the aperture is that the stainless (steel) wire of 0.5～1mm sieves callus, be seeded in then in the liquid nutrient medium of step (3) preparation, in rotating and culturing device or bio-reactor in 25 ℃, the synthetic target product of catalysis was finished synthetic reaction process after 5～10 days under lucifuge or the irradiation condition;
(5) harvested cell extracts with nutrient solution and separates its effective constituent, obtains the target meta-bolites.
Embodiment 2:
(1) in Gamborg (B5) improved culture medium, by 2,4 dichlorphenoxyacetic acids of every liter of nutrient solution adding 0.1～4mg, the 6-benzyl aminopurine of 0.1～4mg (BA), and 30～50g sucrose; With acid or alkali the pH value is adjusted to 5.5～7, as minimum medium.
(2) get described minimum medium,,, be divided in and cultivate in the vessel after the dissolving of heating by every liter of nutrient solution adding 7～10g agar to wherein, through 125 ℃ of high temperature, 0.1Mpa pressure is sterilized down after be cooled to solid medium; The Taxus chinensis callus high yield strain system that filters out is seeded on the described solid medium, under 25 ℃ of lucifuge conditions, cultivated 20～25 days, obtain plant callus.
(3) get described minimum medium, promote target product synthetic precursor substance phenylalanine 50～500mg and elicitor methyl jasmonate (mj) 100mg to wherein adding by every liter of nutrient solution, medium pH value is adjusted to 5.5～7, divide then and install in incubator or the bio-reactor, through 125 ℃ of high temperature, 0.1Mpa pressure carries out cooling process after the sterilization down, is made into liquid nutrient medium.
(4) callus that will in step (2), obtain, with the aperture is that the stainless (steel) wire of 0.5～1mm sieves callus, be seeded in then in the liquid nutrient medium of step (3) preparation, in rotating and culturing device or bio-reactor in 25 ℃, the synthetic target product of catalysis was finished synthetic reaction process after 2～7 days under lucifuge or the irradiation condition;
(5) harvested cell extracts with nutrient solution and separates its effective constituent, obtains the target meta-bolites.
Embodiment 3:
(1) at Murashige ﹠amp; In Skoog (MS) improved culture medium, by the indolylacetic acid (IAA) of every liter of nutrient solution adding 0.1～4mg, the 6-benzyl aminopurine of 0.1～4mg (BA), and 30～50g sucrose; With acid or alkali the pH value is adjusted to 5.5～7, as first kind of minimum medium.
In the Miller improved culture medium, by the indolylacetic acid (IAA) of every liter of nutrient solution adding 0.1～4mg, the 6-benzyl aminopurine of 0.1～4mg (BA), and 20～50g sucrose; With acid or alkali the pH value is adjusted to 5.5～7, as second kind of minimum medium.
(2) get described first kind of minimum medium,,, be divided in and cultivate in the vessel after the dissolving of heating by every liter of nutrient solution adding 7～10g agar to wherein, through 125 ℃ of high temperature, 0.1Mpa pressure is sterilized down after be cooled to solid medium; The callus of cistanche deserticda high yield strain system that filters out is seeded on the described solid medium, under 25 ℃ of lucifuge conditions, cultivated 20～25 days, obtain plant callus.
(3) get described second kind of minimum medium, promote target product synthetic precursor substance coffic acid to wherein adding by every liter of nutrient solution, medium pH value is adjusted to 5.5～7, divide then and install in incubator or the bio-reactor, through 125 ℃ of high temperature, 0.1Mpa pressure carries out cooling process after the sterilization down, is made into liquid nutrient medium.
(4) callus that will in step (2), obtain, with the aperture is that the stainless (steel) wire of 0.5～1mm sieves callus, be seeded in then in the liquid nutrient medium of step (3) preparation, in rotating and culturing device or bio-reactor in 25 ℃, the synthetic target product of catalysis was finished synthetic reaction process after 5～10 days under lucifuge or the irradiation condition;
(5) harvested cell extracts with nutrient solution and separates its effective constituent, obtains the target meta-bolites.
Claims (8)
1. the solid-liquid two-steps tissue culture method of culture plant cell production desirable metabolites is characterized in that this method comprises the steps:
1) in plant tissue and cell culture medium, add 0.1～4mg growth hormone, 0.1～4mg phytokinin by every liter of nutrient solution, and 20～60g sucrose or glucose, adjust pH is 5.5～7, as minimum medium;
2) get described minimum medium, to wherein making solid medium by every liter of nutrient solution adding 7～10g agar or the efficient peptizer of 2g; The cell strain system of the quick growth that filters out is seeded on the described solid medium, under 20～25 ℃ of lucifuges or irradiation condition, cultivates, obtain plant callus;
3) get described minimum medium, promote target product synthetic precursor substance or elicitor, medium pH value is adjusted to 5.5～7, behind sterilization, be made into liquid nutrient medium to wherein adding;
4) with step 2) in the plant callus that obtains through the stainless (steel) wire screening after, be seeded on the described liquid nutrient medium, under 20～25 ℃ of lucifuges or irradiation condition, the target meta-bolites is synthesized in catalysis;
5) collect cell mass and nutrient solution, and obtain the target meta-bolites by separation and purification.
2. solid-liquid two-steps tissue culture method according to claim 1 is characterized in that: described plant tissue and cell culture medium are Murashige ﹠amp; Skoog substratum, Miller substratum, Gamborg substratum, White substratum, Heller substratum, Linsmaier ﹠amp; Skoog substratum or sss substratum.
3. solid-liquid two-steps tissue culture method according to claim 1 and 2, it is characterized in that: utilize two kinds of different plant tissues and cell culture medium, make two kinds of minimum mediums according to method described in the step 1), in step 2) and step 3) in respectively choose a kind of minimum medium, be used for preparing solid medium and liquid nutrient medium.
4. solid-liquid two-steps tissue culture method according to claim 1 is characterized in that: described growth hormone is preferably indolylacetic acid, α-Nai Yisuan or 2,4 dichlorphenoxyacetic acids.
5. solid-liquid two-steps tissue culture method according to claim 1 is characterized in that: described phytokinin is preferably 6-benzyl aminopurine or kinetin.
6. the method for acquisition Herba Cistanches secondary metabolite according to claim 1, it is characterized in that: described promotion target product synthetic precursor substance or elicitor are phenylalanine, tyrosine, methyl jasmonate (mj) or coffic acid.
7. solid-liquid two-steps tissue culture method according to claim 1, it is characterized in that: in step 2) in, the cell strain system of the quick growth that filters out is seeded on the described solid medium, under 20～25 ℃ of lucifuges or irradiation condition, cultivates and obtained plant callus in 15～25 days.
8. solid-liquid two-steps tissue culture method according to claim 1, it is characterized in that: described plant callus is seeded in the rotating and culturing device or bio-reactor that described liquid nutrient medium is housed, under 20～25 ℃ of lucifuges or irradiation condition, cultivate 2-10 days synthetic target products.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104983638A (en) * | 2015-07-20 | 2015-10-21 | 珀莱雅化妆品股份有限公司 | Method for preparing high-gamma-aminobutyric-acid-content tea extract |
| CN107960171A (en) * | 2017-07-26 | 2018-04-27 | 甘肃汇勤生物科技有限公司 | A kind of cultural method for improving Cistanche tubulosa yield and quality |
| CN108220348A (en) * | 2017-10-25 | 2018-06-29 | 浙江工业大学 | The method of Rice Callus asymmetric reduction p- phenylpropyl alcohol ketone class compounds |
| CN109644869A (en) * | 2018-12-20 | 2019-04-19 | 长沙学院 | The method for obtaining Gardenoside by tissue cultures |
| CN113265368A (en) * | 2021-04-27 | 2021-08-17 | 陶氏益农生物工程（青岛）有限公司 | Plant cell culture method for producing high-content mineral elements |
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| US11299700B1 (en) | 2021-02-19 | 2022-04-12 | Acequia Biotechnology, Llc | Bioreactor containers and methods of growing hairy roots using the same |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1185926C (en) * | 2002-03-22 | 2005-01-26 | 清华大学 | Method for obtaining secondary metabolism product of broomrape by using biotechnology |
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2004
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104983638A (en) * | 2015-07-20 | 2015-10-21 | 珀莱雅化妆品股份有限公司 | Method for preparing high-gamma-aminobutyric-acid-content tea extract |
| CN104983638B (en) * | 2015-07-20 | 2017-12-22 | 珀莱雅化妆品股份有限公司 | A kind of preparation method of high content gamma aminobutyric acid tea extract |
| CN107960171A (en) * | 2017-07-26 | 2018-04-27 | 甘肃汇勤生物科技有限公司 | A kind of cultural method for improving Cistanche tubulosa yield and quality |
| CN108220348A (en) * | 2017-10-25 | 2018-06-29 | 浙江工业大学 | The method of Rice Callus asymmetric reduction p- phenylpropyl alcohol ketone class compounds |
| CN109644869A (en) * | 2018-12-20 | 2019-04-19 | 长沙学院 | The method for obtaining Gardenoside by tissue cultures |
| CN109644869B (en) * | 2018-12-20 | 2021-08-10 | 长沙学院 | Method for obtaining geniposide by tissue culture |
| CN113265368A (en) * | 2021-04-27 | 2021-08-17 | 陶氏益农生物工程（青岛）有限公司 | Plant cell culture method for producing high-content mineral elements |
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