CN104855288A - Establishment method of Tibetan drug przewalskia tangutica maxim autonomous rooting system - Google Patents
Establishment method of Tibetan drug przewalskia tangutica maxim autonomous rooting system Download PDFInfo
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Abstract
The invention discloses an establishment method of a Tibetan drug przewalskia tangutica maxim autonomous rooting system. The establishment method comprises the following steps: firstly, activating rooting agrobacterium; transferring hypocotyledonary axes of przewalskia tangutica maxim seed aseptic seedlings into an MS solid culture medium, and putting the MS solid culture medium into a biochemical incubator to be subjected to dark culture at 22 DEG C for 48 hours to finish a pre-culture phase of the hypocotyledonary axes; immersing and carry out co-culture by using the rooting agrobacterium; transferring to an MS culture medium containing 500mg/L carbenicillin to be subjected to dark culture at 22 DEG C; and generating white adventitious roots of wound calluses of the hypocotyledonary axes. The establishment method has the beneficial effect that a przewalskia tangutica maxim transgene bioreactor of high-yield scopolamine is researched.
Description
Technical field
The invention belongs to plant biotechnology field, relate to the method for building up of the autonomous root of hair system of Tibetan medicine RADIX PRZEWALSKIAE TANGUTICAE.
Background technology
RADIX PRZEWALSKIAE TANGUTICAE (Przewalskia tangutica Maxim.) has another name called short henbane, tangut przewalskia seed or root, is Solanaceae (Solanaceae) RADIX PRZEWALSKIAE TANGUTICAE platymiscium, is China's Qinghai-Tibet Platean endemic plant, is also the plant that special single becomes to belong to; Be grown on high mountain gravel ground and the aridity grass land of height above sea level 3200-5000 rice.Record according to " Jingzhubencao " and " Tibetan medicine will ", it is the common medicine in Tibetan medicine, and its root and seed and herb all can be used for medicinal purpose, and have the drug effects such as analgesia, spasmolysis, desinsection, anti-inflammatory, for gastrointestinal convulsion pain, diphtheria, anthrax; External application sore, pruitus.Research finds, in its body, the tropane alkaloid of high-load has significant curative effect in treatment neurogenic disease, and hyoscine has good effect as the important intermediate raw material of some pharmaceutical synthesis simultaneously, the feature that side effect is little, also making the demand growth in the international market of this compound very fast, is 10 times of hyoscyamine.Therefore, the hyoscine how obtaining high yield is the focus of research both at home and abroad always.Research and development one of effective way is beyond doubt carried out to high yield alkali plant.1984, Xiao Peigen etc., to the scopolia acutangula in Solanaceae, Anisodus tanguticus, in RADIX PRZEWALSKIAE TANGUTICAEs etc. 54, the tropane alkaloid active ingredient of plant is analyzed, and found that in Tibetan medicine RADIX PRZEWALSKIAE TANGUTICAE, tropane alkaloid content is high, can directly as the raw material of such medicine of industrial production.
Along with the discovery of RADIX PRZEWALSKIAE TANGUTICAE medical value, a large amount of wild resource is excavated, and causes RADIX PRZEWALSKIAE TANGUTICAE wild resource on the one hand in imminent danger, also causes the ecological disruption of highlands fragility on the other hand.Therefore, understand the molecular mechanism of the high yield alkali of RADIX PRZEWALSKIAE TANGUTICAE, the approach exploring a sustainable High-efficient Production tropane alkaloid becomes current problem demanding prompt solution.
At present, large quantity research shows, contaminate plant explants through agrobacterium rhizogenes and can produce the autonomous hair shape root of hormone, hair shape root have compared with plant normal root hormone autotrophic, growth conditions simple, grow rapid, its secondary metabolites content is high and the stable and advantage such as differentiation degree is high, not easily make a variation, and effectively can solve the problem that people utilize plant production secondary metabolite.The plant utilizing agrobacterium rhizogenes to contaminate plant acquisition Hairy root at present comprises 20 sections 40 and belongs to 160 various plants, mostly is the dicotyledon plant that wherein transformation frequency is higher and mainly concentrates on Solanaceae, composite family, Cruciferae and Convolvulaceae etc.The secondary metabolite utilizing hair shape root successfully to obtain mainly contains saponin(e, green onion quinone (Ko et al, 1998), tropane alkaloid (mano et al, 1989), trichosanthin (Qiu Deyou etc., 1996), taxol (Huang Zunxi etc., 1997), qinghaosu (Liu Chunchao etc., 1999), alkaloid (Trypsteen et al, 1991), Diterpenes (Hu et al, 1993), volatile oil (Kennedy et al) etc.Also be widely used in producing tropane alkaloid, as the plants such as the henbane seed in plant of Solanaceae, Tangut Anisodus Radix, datura all establish induction of hairy roots and cultivating system, but because a variety of causes is at present still not used for industrial production.Utilize agrobacterium rhizogenes to contaminate RADIX PRZEWALSKIAE TANGUTICAE plant explants, producing at the Hairy root without autonomous growth on hormone culture-medium, to be the effective way of sustainable use RADIX PRZEWALSKIAE TANGUTICAE plant production tropane alkaloid, is also the basis of research RADIX PRZEWALSKIAE TANGUTICAE high yield base molecule mechanism.
Summary of the invention
The object of the present invention is to provide the method for building up of the autonomous root of hair system of Tibetan medicine RADIX PRZEWALSKIAE TANGUTICAE, solve the problem that current RADIX PRZEWALSKIAE TANGUTICAE plant root growth is slow.
The technical solution adopted in the present invention is carried out according to following steps:
Step 1: the activation of agrobacterium rhizogenes; The agrobacterium rhizogenes of Cord blood is taken out, agrobacterium rhizogenes culture medium flat plate is rule, then the constant incubator being placed on 25 DEG C cultivates 48h, and in time growing bacterial plaque, picking list bacterium colony is rule again on agrobacterium rhizogenes culture medium flat plate, the constant incubator being placed on 25 DEG C cultivates 48h, in time growing bacterial plaque, picking list colony inoculation in the triangular flask containing 30mL agrobacterium rhizogenes liquid nutrient medium, 28 DEG C, 12-15h is cultivated in 180rpm concussion, and being cultured to bacterium liquid light absorption value concentration is OD
600=0.6-0.8, then proceed in 50mL centrifuge tube by bacterium liquid, 6000r/min, centrifugal 10min, resuspended to OD=0.6 with MS liquid nutrient medium, bacteria suspension is for subsequent use;
Step 2: the preculture of explant; The RADIX PRZEWALSKIAE TANGUTICAE seed asepsis seedling of growth about 15-20d is taken out, cut seed asepsis shoot root portion and hypocotyl with upper part, the hypocotyl cut is forwarded in MS solid culture medium, is positioned over light culture 48h in 22 DEG C of biochemical cultivation cases, complete the hypocotylar preculture stage;
Step 3: agrobacterium rhizogenes is contaminated and Dual culture; Take out with preculture after hypocotyl, be positioned in the clean culture dish of sterilizing, bacteria suspension is poured in culture dish, period does not stop to shake culture dish, contaminate 9-10min, then the hypocotyl after dip-dye is taken out, removing excess surface bacterium liquid, then be seeded in MS solid culture medium, be positioned over 28 DEG C of biochemical cultivation cases and cultivate 48h;
Step 4: degerming cultivation; Aseptically, be forwarded to by the hypocotyl completing Dual culture in the MS medium containing 500mg/L carbenicillin and carry out light culture under 22 DEG C of conditions, hypocotyl wound callus place produces white adventive root.
Further, described MS liquid culture based formulas is:
NH
4nO
341.25g, KNO
347.50g, KH
2pO
44.25g, is dissolved in 300mL distilled water, is settled to 500mL and draws 20mL/L;
CaCl
22H
2o 22g, is settled to 500mL, draws 10mL/L;
MgSO
47H
2o 18.50g, is settled to 500mL, draws 10mL/L;
MnSO
44H
2o 2230mg or MnSO
4h
2o 1690mg, ZnSO
47H
2o 860mg, H
3bO
3620mg, KI 83mg, NaMoO
42H
2o 25mg, CuSO
45H
2o 2.5mg, CoCl
26H
2o2.5mg, is settled to 500ml, draws 5mL/L;
Glycine 0.1g, nicotinic acid thiamine 0.005g, nicotinic acid pyridoxine 0.025g, nicotinic acid 0.025g, is settled to 500mL, draws 10mL/L;
Inositol 5g, is settled to 500mL, draws 10mL/L;
Na
2-EDTA 1.865g, FeSO
47H
2o 1.390g, is settled to 500mL, draws 10mL/L.
Further, described MS solid culture based formulas is: NH
4nO
341.25g, KNO
347.50g, KH
2pO
44.25g, is dissolved in 300mL distilled water, is settled to 500mL and draws 20mL/L;
CaCl
22H
2o 22g, is settled to 500mL, draws 10mL/L;
MgSO
47H
2o 18.50g, is settled to 500mL, draws 10mL/L;
MnSO
44H
2o 2230mg or MnSO
4h
2o 1690mg, ZnSO
47H
2o 860mg, H
3bO
3620mg, KI 83mg, NaMoO
42H
2o 25mg, CuSO
45H
2o 2.5mg, CoCl
26H
2o2.5mg, is settled to 500ml, draws 5mL/L;
Glycine 0.1g, nicotinic acid thiamine 0.005g, nicotinic acid pyridoxine 0.025g, nicotinic acid 0.025g, is settled to 500mL, draws 10mL/L;
Inositol 5g, is settled to 500mL, draws 10mL/L;
Na
2-EDTA 1.865g, FeSO
47H
2o 1.390g, is settled to 500mL, draws 10mL/L; Sucrose 30g/L; Agar 7g/L.
The invention has the beneficial effects as follows the RADIX PRZEWALSKIAE TANGUTICAE transgenosis root of hair bio-reactor that have developed high yield hyoscine.
Accompanying drawing explanation
Fig. 1 is that different monoclonal root of hair ties up to without the screening schematic diagram on hormone MS medium;
Fig. 2 is RADIX PRZEWALSKIAE TANGUTICAE transgenic hairy root RPt01 growth curve schematic diagram.
Embodiment
Below in conjunction with embodiment, the present invention is described in detail.
The method for building up step of Tibetan medicine RADIX PRZEWALSKIAE TANGUTICAE of the present invention autonomous root of hair system is as follows:
The activation of the 1st step agrobacterium rhizogenes; Aseptically, the agrobacterium rhizogenes preserved by low temperature (-80 DEG C) is taken out, YEB culture medium flat plate is rule, then the constant incubator being placed on 25 DEG C cultivates 48h (hour), in time growing obvious bacterial plaque, picking list bacterium colony repeats at YEB medium (agrobacterium rhizogenes medium) flat lining out, the constant incubator being placed on 25 DEG C equally cultivates 48h, in time growing obvious bacterial plaque, picking list colony inoculation is in the triangular flask containing 30mL YEB liquid nutrient medium, 28 DEG C, 12-15h is cultivated in 180rpm concussion, and being cultured to concentration is OD
600(bacterium liquid light absorption value)=0.6-0.8 is advisable.Then proceed in 50mL centrifuge tube by bacterium liquid, 6000r/min, centrifugal 10min, resuspended to OD=0.6 with MS liquid nutrient medium (formula sees appendix, and does not add agar), bacteria suspension is for subsequent use.
The preculture of the 2nd step explant; Aseptically, by grow 15-20d (my god) left and right RADIX PRZEWALSKIAE TANGUTICAE seed asepsis seedling (cotyledon just stretched and children tender, do not grow true leaf) take out, seed asepsis shoot root portion and hypocotyl is carefully cut with upper part with scalpel, the hypocotyl cut is forwarded in MS solid culture medium and (sees attached list), be positioned over light culture 48h in 22 DEG C of biochemical cultivation cases, complete explant and hypocotylar preculture stage.
3rd step agrobacterium rhizogenes dip-dye and Dual culture are aseptically, with tweezers carefully take out with preculture after hypocotyl, be positioned in the clean culture dish of sterilizing, poured into by bacteria suspension in culture dish, period does not stop to shake culture dish, contaminate 9-10min, then with tweezers, the hypocotyl after dip-dye is taken out, with sterilizing filter paper removing excess surface bacterium liquid, be then seeded in MS solid culture medium, be positioned over 28 DEG C of biochemical cultivation cases, Dual culture 48h.
The degerming cultivation of 4th step aseptically, the hypocotyl completing Dual culture is forwarded in the MS medium containing 500mg/L carbenicillin and carries out light culture under 22 DEG C of conditions, until about 27d, hypocotyl wound callus place produces white adventive root successively, if there is pollution to continue to be seeded in except on bacterium culture medium, until completely degerming, condition of culture is 22 DEG C of light culture.
The Molecular Identification of the 5th step Hairy root extracts the genomic DNA of root of hair with reference to CTAB method.Genomic DNA is used as the amplification template of genes of interest RolB gene.RolB gene primer sequence: F primer sequence: 5 '-TCTCGCGAGAAGATGCAGAA-3 ' R primer sequence: 5 '-TAAAGGTTGCCTGCAGGGG-3 '.The reaction system of PCR sees attached list 3, and amplification condition is 94 DEG C of denaturation 8min, 94 DEG C, 45sec; 53 DEG C, 30sec; 72 DEG C, 1min; 35 circulations, 72 DEG C extend 10min.Amplified production carries out agarose electrophoresis.
The expansion of the 6th step root of hair system is numerous to be seeded in without on hormone MS medium with judgement that is hormone autonomous growth type by the RADIX PRZEWALSKIAE TANGUTICAE root of hair system clip tip of a root 3-4cm through Molecular Identification, the root of hair that can grow continuously and healthily ties up to squamous subculture under 22 DEG C of conditions, and the expansion completing root of hair system is numerous.
The liquid suspension culture of the 7th step root of hair system, step by step amplification are cultivated and the results clip root of hair tip of a root 3-4cm of root of hair is seeded in 30mL without in hormone MS liquid nutrient medium, 22 DEG C, 100rpm dark condition low suspension cultivates 35-40d, clip root of hair tip of a root 3-4cm is seeded in 100mL without in hormone MS liquid nutrient medium, amplifies cultivation step by step under similarity condition.Taking out completing the root of hair amplifying cultivation from triangular flask, blotting surface moisture with filter paper, drying in 45 DEG C of baking ovens, as the alkaloidal material of mensuration.
The alkaloidal mensuration of the alkaloidal mensuration of 8th step, with reference to (Wang et al.2002) method, does not elaborate here.
YEB culture medium prescription (taking distilled water as solvent) as shown in table 1, table 2 is PCR reaction system (20ul).
Table 1
Table 2
This experiment utilizes agrobacterium rhizogenes to contaminate the different explants of RADIX PRZEWALSKIAE TANGUTICAE seed asepsis seedling first, induction produces can at the hormone without autonomous growth on hormone MS medium from principal mode root of hair system, and different root of hair system is screened, by round pcr Molecular Identification, amplify the characterizing gene rolB gene of agrobacterium rhizogenes, show to transform successfully.On this basis, we determine it without the growth curve in hormone MS liquid nutrient medium, have understood the growth characteristics of root of hair in 30ml liquid nutrient medium and best harvest time.And cultivate the amplification step by step that it has carried out 30-100-1000ml, experimental result shows, the root of hair of RADIX PRZEWALSKIAE TANGUTICAE all can grow, along with the harvest yield of the amplification root of hair of culture systems also increases step by step under different liquids condition of culture.Utilize HPLC to measure the content of four kinds of tropane alkaloids in different root tissue, result shows, in RADIX PRZEWALSKIAE TANGUTICAE root of hair, the content of hyoscine is a little more than the content of perennial wild middle hyoscine.Therefore, RADIX PRZEWALSKIAE TANGUTICAE hairy root culture system to replace wild to produce the high-efficient biological reactor of hyoscine, and this is that the sustainable exploitation utilization of this resource is laid a good foundation.
Experiment flow
1, the optimum condition of orthogonal design screening Tibetan medicine RADIX PRZEWALSKIAE TANGUTICAE induction of hairy roots is as shown in table 3.
Table 3 biological factors produces the impact of hair shape root to induction Tibetan medicine RADIX PRZEWALSKIAE TANGUTICAE
On superclean bench, the seed asepsis seedling of growth about 20d is taken out, cut on MS medium that hypocotyl part is seeded in without hormone, 22 DEG C of light culture two days.Hypocotyl is taken out according to the experimental design shown in table, hypocotyl is immersed in different time in the agrobacterium rhizogenes bacterium liquid of variable concentrations, period does not stop to rock, and after having contaminated, hypocotyl taking-up aseptic filter paper is blotted excess surface bacterium liquid and is seeded in 28 DEG C of Dual culture 48h in former medium.Hypocotyl two days one-periods of Dual culture are seeded in degerming cultivation in the MS medium of the carbenicillin containing 500mg/L, condition of culture 22 DEG C of light culture, until about 27d produces adventive root from wound, adventive root clip tip of a root 3-4cm is seeded in without on hormone MS medium, if there is pollution to continue to be seeded in except on bacterium culture medium, until completely degerming, condition of culture is 22 DEG C of light culture.This part of test results shows, bacterial classification, dip-dye concentration, immerged time produce hair shape root to RADIX PRZEWALSKIAE TANGUTICAE seed asepsis seedling hypocotyl and have obvious impact.It is OD=1.0 that MSU44O contaminates concentration, and immerged time is that 7min inductivity is the highest, is 33.33%, but follow-up observation finds, the adventive root that its induction produces can not without autonomous growth on hormone MS medium.ATCC10060 contaminate concentration be OD=0.6, immerged time 9min, inductivity is 23.33%, induction produce root of hair can on MS medium autonomous growth.Therefore, determine that the inductive condition of the most applicable Tibetan medicine RADIX PRZEWALSKIAE TANGUTICAE seed asepsis seedling hypocotyl generation root of hair is bacterial classification ATCC10060, dip-dye concentration is OD=0.6, immerged time 9min.Fig. 1 is that different monoclonal root of hair ties up to without the screening on hormone MS medium.
2, Tibetan medicine RADIX PRZEWALSKIAE TANGUTICAE hormone is from the screening of principal mode root of hair system
By the observation to completely degerming different hair shape roots, mainly contain three types, A, can the root system of autonomous growth, B, poky root system, C, without the root system producing callus autonomous on hormone culture-medium.Tie up to without the growing state on hormone MS medium to understand dissimilar root of hair further, different ABC is labeled as respectively RPt01,02,03 root system screens in without hormone MS medium, condition of culture is 22 DEG C of light culture, (the measurement way of main root is walk from the tip of a root to end with the growth track of fine rule around root, then stretching for fine rule ruler is measured), result shows that RPt01 root system can autonomous growth braning factor average every day be 0.44, and main root grows 0.84cm every day.
3, the Molecular Identification of Tibetan medicine RADIX PRZEWALSKIAE TANGUTICAE monoclonal root of hair system
This part utilizes round pcr from RADIX PRZEWALSKIAE TANGUTICAE adventive root, amplify the characterizing gene RolB gene of agrobacterium rhizogenes, and what show acquisition can be that the hormone of Tibetan medicine RADIX PRZEWALSKIAE TANGUTICAE generation after agrobacterium rhizogenes is contaminated is from principal mode transgenic hairy root without the adventive root of autonomous growth on hormone MS medium.
4, the mensuration of Tibetan medicine RADIX PRZEWALSKIAE TANGUTICAE transgenic hairy root RPt01 growth curve as shown in Figure 2.
By RPt01 clip 4-5cm, be seeded in 30ml liquid nutrient medium, every bottle graft kind 3 roots.This part of test results shows, hormone can vigorous growth in without hormone MS liquid nutrient medium from principal mode transgenic hairy root RPt01, and during 35d, dry weight, fresh weight reach maximum and are respectively, 0.14g/30ml and 2.05g/30ml.
HPLC method is utilized to measure the content of four kinds of tropane alkaloids in the different root tissue of RADIX PRZEWALSKIAE TANGUTICAE, the size order showing anisodamine, anisodine, atropine and total alkaloids in different root tissue is perennial wild > transgenosis Hairy root > seed asepsis shoot root, and the content size order of hyoscine be transgenosis root of hair a little more than perennial wild, 3.36 times higher than seed asepsis shoot root.Although the content of perennial wild middle total alkaloids turns root 4.76 times higher than transgenosis hair, but wild RADIX PRZEWALSKIAE TANGUTICAE root growth is slow, it is high that transgenic hairy root has reproduction coefficient, is easy to the advantages such as suitability for industrialized production, and the content of hyoscine is a little more than perennial wild.Therefore, it is the effective way replacing its wild High-efficient Production tropane alkaloid that Tibetan medicine RADIX PRZEWALSKIAE TANGUTICAE transgenosis hair turns root, is worth of widely use.
This experiment utilizes agrobacterium rhizogenes to contaminate the hypocotyl of Tibetan medicine RADIX PRZEWALSKIAE TANGUTICAE seed asepsis seedling, obtaining can at the transgenic hairy root system RPt01 without autonomous growth on hormone MS medium, and to its inductive condition, growth characteristics, produce alkali ability to study, result shows, the RPt01 root system utilizing transgenic technology to obtain is the high-efficient biological reactor that can replace perennial wild production tropane alkaloid, and it is as high in reproduction coefficient more to have advantage than perennial wild, condition, be easy to pilot scale culture etc., explore the effective way of this plant resources of sustainable exploitation utilization, molecular mechanism simultaneously for understanding RADIX PRZEWALSKIAE TANGUTICAE high yield alkali is laid a good foundation.
The above is only to better embodiment of the present invention, not any pro forma restriction is done to the present invention, every any simple modification done above embodiment according to technical spirit of the present invention, equivalent variations and modification, all belong in the scope of technical solution of the present invention.
Claims (3)
1. the method for building up of the autonomous root of hair system of Tibetan medicine RADIX PRZEWALSKIAE TANGUTICAE, is characterized in that carrying out according to following steps:
Step 1: the activation of agrobacterium rhizogenes; The agrobacterium rhizogenes of Cord blood is taken out, agrobacterium rhizogenes culture medium flat plate is rule, then the constant incubator being placed on 25 DEG C cultivates 48h, and in time growing bacterial plaque, picking list bacterium colony is rule again on agrobacterium rhizogenes culture medium flat plate, the constant incubator being placed on 25 DEG C cultivates 48h, in time growing bacterial plaque, picking list colony inoculation in the triangular flask containing 30mL agrobacterium rhizogenes liquid nutrient medium, 28 DEG C, 12-15h is cultivated in 180rpm concussion, and being cultured to bacterium liquid light absorption value concentration is OD
600=0.6-0.8, then proceed in 50mL centrifuge tube by bacterium liquid, 6000r/min, centrifugal 10min, resuspended to OD=0.6 with MS liquid nutrient medium, bacteria suspension is for subsequent use;
Step 2: the preculture of explant; The RADIX PRZEWALSKIAE TANGUTICAE seed asepsis seedling of growth about 15-20d is taken out, cut seed asepsis shoot root portion and hypocotyl with upper part, the hypocotyl cut is forwarded in MS solid culture medium, is positioned over light culture 48h in 22 DEG C of biochemical cultivation cases, complete the hypocotylar preculture stage;
Step 3: agrobacterium rhizogenes is contaminated and Dual culture; Take out with preculture after hypocotyl, be positioned in the clean culture dish of sterilizing, bacteria suspension is poured in culture dish, period does not stop to shake culture dish, contaminate 9-10min, then the hypocotyl after dip-dye is taken out, removing excess surface bacterium liquid, then be seeded in MS solid culture medium, be positioned over 28 DEG C of biochemical cultivation cases and cultivate 48h;
Step 4: degerming cultivation; Aseptically, be forwarded to by the hypocotyl completing Dual culture in the MS medium containing 500mg/L carbenicillin and carry out light culture under 22 DEG C of conditions, hypocotyl wound callus place produces white adventive root.
2. according to the method for building up of the autonomous root of hair system of Tibetan medicine RADIX PRZEWALSKIAE TANGUTICAE described in claim 1, it is characterized in that: described MS liquid culture based formulas is:
NH
4nO
341.25g, KNO
347.50g, KH
2pO
44.25g, is dissolved in 300mL distilled water, is settled to 500mL and draws 20mL/L;
CaCl
22H
2o 22g, is settled to 500mL, draws 10mL/L;
MgSO
47H
2o 18.50g, is settled to 500mL, draws 10mL/L;
MnSO
44H
2o 2230mg or MnSO
4h
2o 1690mg, ZnSO
47H
2o 860mg, H
3bO
3620mg, KI 83mg, NaMoO
42H
2o 25mg, CuSO
45H
2o 2.5mg, CoCl
26H
2o2.5mg, is settled to 500ml, draws 5mL/L;
Glycine 0.1g, nicotinic acid thiamine 0.005g, nicotinic acid pyridoxine 0.025g, nicotinic acid 0.025g, is settled to 500mL, draws 10mL/L;
Inositol 5g, is settled to 500mL, draws 10mL/L;
Na
2-EDTA 1.865g, FeSO
47H
2o 1.390g, is settled to 500mL, draws 10mL/L.
3. according to the method for building up of the autonomous root of hair system of Tibetan medicine RADIX PRZEWALSKIAE TANGUTICAE described in claim 1, it is characterized in that: described MS solid culture based formulas is: NH
4nO
341.25g, KNO
347.50g, KH
2pO
44.25g, is dissolved in 300mL distilled water, is settled to 500mL and draws 20mL/L;
CaCl
22H
2o 22g, is settled to 500mL, draws 10mL/L;
MgSO
47H
2o 18.50g, is settled to 500mL, draws 10mL/L;
MnSO
44H
2o 2230mg or MnSO
4h
2o 1690mg, ZnSO
47H
2o 860mg, H
3bO
3620mg, KI 83mg, NaMoO
42H
2o 25mg, CuSO
45H
2o 2.5mg, CoCl
26H
2o2.5mg, is settled to 500ml, draws 5mL/L;
Glycine 0.1g, nicotinic acid thiamine 0.005g, nicotinic acid pyridoxine 0.025g, nicotinic acid 0.025g, is settled to 500mL, draws 10mL/L;
Inositol 5g, is settled to 500mL, draws 10mL/L;
Na
2-EDTA 1.865g, FeSO
47H
2o 1.390g, is settled to 500mL, draws 10mL/L;
Sucrose 30g/L; Agar 7g/L.
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| CN106718884A (en) * | 2016-11-29 | 2017-05-31 | 青岛松良基因科技有限公司 | RADIX PRZEWALSKIAE TANGUTICAE seedling and its method for culturing seedlings |
| 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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| CN106718884B (en) * | 2016-11-29 | 2019-01-15 | 青岛松良基因科技有限公司 | RADIX PRZEWALSKIAE TANGUTICAE seedling and its method for culturing seedlings |
| 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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