CN1324562A - Method of producing green red sage root in biological reactor - Google Patents
Method of producing green red sage root in biological reactor Download PDFInfo
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- CN1324562A CN1324562A CN 00107475 CN00107475A CN1324562A CN 1324562 A CN1324562 A CN 1324562A CN 00107475 CN00107475 CN 00107475 CN 00107475 A CN00107475 A CN 00107475A CN 1324562 A CN1324562 A CN 1324562A
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Abstract
The method relates to the production of salvia root using bio-reactor which is characterized by that respectively inoculating the salvia hair root obtained by rooting agrobacterium induction and salvia drifting root obtained by means of induction of in-vitro tissue culture method into bio-reactor to make culture, through two-step culture, finally can produce pollution-less salvia root. Its advantages are: the multiplication power of salvia hair root and drifting root is above 200 times, as compared with Erlenmeyer flask culture it can implement industrialized productino, said invented product can be made into various medicinal products and health-care products with biological activity, such as oral liquor and capsule, etc.
Description
The invention relates to a kind of method with producing green red sage root in biological reactor.It belongs on the basis of Plant Tissue Breeding method, the gene engineering that grows up the eighties, the technical field of cell engineering.Root of hair is cultivated and adventive root is cultivated as a kind of new technology, the useful medicine that can be mass-produced out, and its prospect is considerable.
The red sage root is China's traditional Chinese medicine, has to activate blood circulation and disperse blood clots inducing meastruation to relieve menalgia function.Contain two active components in the red sage root: fat-soluble diterpene quinone and water-soluble phenolic acid compounds.Diterpene quinone, as salvia miltiorrhiza bge I, tanshinone IIA, Cryptotanshinones etc. have and suppress platelet aggregation, anoxia enduring, improve pharmacological action such as coronary artery blood supply, are the important drugs of treatment disease of cardiovascular system.Phenolic acid compound, as salviandic acid A, tanshin polyphenolic acid B, alkannic acid, Rosmarinic acid, protocatechualdehyde, danshensus etc. have very strong lipotropism matter oxidation, anti-hepatic fibrosis, improve uremia, improve effects such as renal function.But content is low in former plant roots, growth cycle is long owing to effective component in red sage, and home environment pollutes and for reasons such as diseases prevention worm spraying pesticides, makes the supply of bulk drug all can not satisfy the needs of clinical practice on quality and quantity in recent years in addition.Breed the active constituent content height and the pollution-free green red sage root at short notice in a large number and just become the task of top priority that natural resources of Chinese medicinal materials is develop and useedd.It is the new technology that the genetic and cell engineering that grows up the eighties combines that root of hair is cultivated, it is that the T-DNA that contains in the Ri plasmid with agrobacterium rhizogenes is transformed on the DNA in the plant cell, plant cell is induced produce a shape root, this root of hair that forms by agrobacterium rhizogenes infection medicinal plant, fast growth, genetic stability becomes the culture systems that has much potentiality.Obtain root of hair by hairy root culture method as belladonna, henbane, catharanthus roseus, genseng etc.It also is a new technology that grows up the eighties that adventive root is cultivated.Compare adventive root fast growth, genetic stability with cell suspension cultures.Therefore, this organ culture of adventive root system becomes the medicinal plant culture systems that has much potentiality equally.So far, the adventive root culture systems of having set up genseng, radix bupleuri is carried out suitability for industrialized production.By literature search, yellow refining etc., Plant Physiology Communications, 1997,33 (4): 259-261, United States Patent (USP): U.S.Pat.No5908771, Japan Patent: JP02215397 utilizes the cultivation of red sage root root of hair to rest on triangular flask and shakes on bottle level, does not all adopt bioreactor culture, in the screening of bioreactor culture, condition of culture and the method for red sage root root of hair, never seeing has report; The in-vitro inducing of red sage root adventive root, cultivate and utilize the red sage root adventive root aspect that the bioreactor technology culture of ex vivo is induced, at home and abroad not seeing has report.
The purpose of the invention provides a kind of method with producing green red sage root in biological reactor, be agrobacterium rhizogenes is induced the red sage root root of hair that is obtained and to induce the red sage root adventive root that is obtained to be linked into respectively through the in vitro tissue cultivation and cultivate in the bio-reactor, obtain the pollution-free green red sage root through growth medium and the two-steps tissue culture method of producing medium again.
The purpose of the invention is achieved in that the present invention is a technical method of producing green red sage root with bio-reactor, it is the new technology content in the genetic and cell engineering field that further grows up on Plant Tissue Breeding method basis, under aseptic situation, with agrobacterium rhizogenes R1600, a large amount of red sage root root of hair that a kind of bacterial strain inducing among R1000 and the A4 obtains and the red sage root adventive root culture materials that obtains by method for culturing in-vitro tissues, be linked in the bio-reactor respectively and cultivate, it is to adopt growth medium through the first step, be 67-V, SH, B5, the N6 medium and second step adopt and produce medium, be MS, the White medium, and add w/v the glossy ganoderma below 5%, sesame rues, dusty yeast, biological Mycophyta elicitor of Asparagus and glucose, sucrose, fructose, the jasmine acids, the two-steps tissue culture method of the abiotic class elicitor of dimethyl sulfoxide (DMSO) is cultivated, and final production goes out a large amount of pollution-free green red sage roots.
Further specify below in conjunction with embodiment.
Embodiment 1:, adopt two-steps tissue culture method to cultivate and produce green red sage root by bio-reactor with red sage root root of hair of the present invention.
Concrete grammar is: utilize red sage root aseptic seedling that the Plant Tissue Breeding method obtained as material and agrobacterium rhizogenes directly altogether best cultivation carry out inducing of root of hair.The red sage root root of hair that is obtained is carried out earlier successive transfer culture make it propagation on solid growth culture media.Adopt 10 liters spherical airlift reactor and 75 liters cylindricality airlift bioreactor during red sage root root of hair bioreactor culture.Dress 5000-7500ml liquid growth medium in 10 liters the spherical airlift reactor.The entire reaction apparatus is linked good back 0.12Mpa, 121 ℃ of autoclavings 20 minutes, the red sage root root of hair that 3-3.68 restrains is inserted in the cooling back on superclean bench, and throughput is 0.05vvm.Adorn 40 in 75 liters the cylindricality airlift bioreactor, 000-50,000ml liquid growth medium, steam sterilizing, the red sage root root of hair of 15.00-19.00 gram is inserted in the cooling back, and throughput is 0.05vvm.All trainings are all carried out under 25 ℃ and the low light level.Change the production medium in the time of 35 days into, continue to cultivate results root of hair and culture fluid after 5 days again, measure its fresh weight, when drying to weight for 70 ℃, measure its dry weight, promptly obtain the pollution-free green red sage root.
Embodiment 2:, adopt two-steps tissue culture method to cultivate and produce green red sage root by bio-reactor with red sage root adventive root of the present invention.
Concrete grammar is: after the root of red sage root field plant is rinsed well with running water, with the mercuric chloride sterilization of 75% alcohol and 0.2% 1 minute and 10 minutes, promptly obtained aseptic red sage root 5 times through aseptic water washing respectively.Aseptic red sage root is cut into the fritter of 0.1 centimetre of size, be linked on the MS solid culture medium that only contains 0.01-10mg/L6-furfuryl group purine and cultivate, afterwards, be transferred to and just can induce a large amount of red sage root adventive root on the MS solid culture medium that contains the 0.01-2.0g/L active carbon but do not have hormone.The red sage root adventive root that is obtained is carried out earlier successive transfer culture make it propagation on solid growth culture media.Adopt 10 liters spherical airlift reactor during red sage root adventive root bioreactor culture, interior dress 5000-7500ml liquid growth medium.The entire reaction apparatus is linked good back 0.12Mpa, 121 ℃ of autoclavings 20 minutes, the red sage root adventive root that 3-3.5 restrains is inserted in the cooling back on superclean bench, and throughput is 0.05vvm.All trainings are all carried out under 25 ℃ and the low light level.Change the production medium in the time of 35 days into, results adventive root and culture fluid after continuing to cultivate 5 days are measured its fresh weight, measure its dry weight when drying to weight for 70 ℃, promptly obtain the pollution-free green red sage root.
The invention has the following advantages: 1. adopt bioreactor to carry out two-steps tissue culture method and cultivate pellet The propagation multiple of ginseng root of hair and adventitious root is very suitable for red sage root root of hair and adventitious root more than 200 times Growth and the accumulation of tanshinone and water soluble ingredient. 2. cultivate with triangular flask and compare, utilize biological Reactor carries out red sage root root of hair and adventitious root is cultivated the industrialization life that can carry out red sage root root of hair and adventitious root Produce, be lasting development and utilization and the green material medicine of the rare Chinese herbal medicine resource resemble the red sage root Production open up a new way. Red sage root bioreactor culture thing can be developed into oral liquid, The biologically active medicine and health care products such as capsule. 4. the present invention utilizes the training of bioreactor culture technology The red sage root adventitious root that the red sage root root of hair that hair care root Agrobacterium is induced and the group training method that exsomatizes are induced, extensive Produce green red sage root, limited by natural conditions so method can fundamentally solve the Salvia miltiorrhiza source Problem, thereby bring huge economic and social benefit.
Claims (5)
1. method with producing green red sage root in biological reactor, it is characterized in that: the technology that adopts Plant Tissue Breeding, agrobacterium rhizogenes induced the red sage root root of hair that obtained and induce the red sage root adventive root that is obtained through the in vitro tissue cultivation as propagating materials, be linked in the bio-reactor respectively and cultivate, produce a large amount of pollution-free green red sage roots through growth medium and production medium two-steps tissue culture method.
2. the method with producing green red sage root in biological reactor according to claim 1 is characterized in that: red sage root root of hair be adopt agrobacterium rhizogenes R1600, R1000 and A4 induce obtain.
3. the method with producing green red sage root in biological reactor according to claim 1, it is characterized in that: red sage root adventive root is to obtain by method for culturing in-vitro tissues.
4. the method with producing green red sage root in biological reactor according to claim 1, it is characterized in that: growth medium is 67-V, SH, B5, N6 medium.
5. the method with producing green red sage root in biological reactor according to claim 1, it is characterized in that: the production medium is MS, White medium, and adds w/v at the glossy ganoderma below 5%, the sesame that rues, dusty yeast, the biological Mycophyta elicitor of Asparagus and glucose, sucrose, fructose, jasmine acids, the abiotic class elicitor of dimethyl sulfoxide (DMSO).
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| CN 00107475 CN1324562A (en) | 2000-05-18 | 2000-05-18 | Method of producing green red sage root in biological reactor |
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| CN 00107475 CN1324562A (en) | 2000-05-18 | 2000-05-18 | Method of producing green red sage root in biological reactor |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100397980C (en) * | 2006-03-07 | 2008-07-02 | 天津大学 | Airlift biological reactor for culturing non-fixed roots of Danshen |
| CN102408991A (en) * | 2011-07-28 | 2012-04-11 | 东北林业大学 | Bioreactor system for large scale culture of medicinal plants |
| CN101491571B (en) * | 2008-01-23 | 2012-06-13 | 上海中医药大学 | Hair root of salvia miltiorrhiza containing various compounds of high content and culturing technique thereof |
| CN103555758A (en) * | 2013-10-22 | 2014-02-05 | 中国农业科学院特产研究所 | Mountain PanaxGinseng C.A.Meyer hairy roots and cultivation method thereof |
| CN103798137A (en) * | 2014-01-25 | 2014-05-21 | 潍坊职业学院 | Tissue culture rapid propagation method of salvia miltiorrhiza bge |
| US11299700B1 (en) | 2021-02-19 | 2022-04-12 | Acequia Biotechnology, Llc | Bioreactor containers and methods of growing hairy roots using the same |
-
2000
- 2000-05-18 CN CN 00107475 patent/CN1324562A/en active Pending
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100397980C (en) * | 2006-03-07 | 2008-07-02 | 天津大学 | Airlift biological reactor for culturing non-fixed roots of Danshen |
| CN101491571B (en) * | 2008-01-23 | 2012-06-13 | 上海中医药大学 | Hair root of salvia miltiorrhiza containing various compounds of high content and culturing technique thereof |
| CN102408991A (en) * | 2011-07-28 | 2012-04-11 | 东北林业大学 | Bioreactor system for large scale culture of medicinal plants |
| CN102408991B (en) * | 2011-07-28 | 2014-11-05 | 东北林业大学 | Bioreactor system for large scale culture of medicinal plants |
| CN103555758A (en) * | 2013-10-22 | 2014-02-05 | 中国农业科学院特产研究所 | Mountain PanaxGinseng C.A.Meyer hairy roots and cultivation method thereof |
| CN103798137A (en) * | 2014-01-25 | 2014-05-21 | 潍坊职业学院 | Tissue culture rapid propagation method of salvia miltiorrhiza bge |
| US11299700B1 (en) | 2021-02-19 | 2022-04-12 | Acequia Biotechnology, Llc | Bioreactor containers and methods of growing hairy roots using the same |
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