CN1884490A - Agrobacterium rhizogenes mediated aconitum coreanum and its obtaining method - Google Patents
Agrobacterium rhizogenes mediated aconitum coreanum and its obtaining method Download PDFInfo
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- CN1884490A CN1884490A CNA2006100895233A CN200610089523A CN1884490A CN 1884490 A CN1884490 A CN 1884490A CN A2006100895233 A CNA2006100895233 A CN A2006100895233A CN 200610089523 A CN200610089523 A CN 200610089523A CN 1884490 A CN1884490 A CN 1884490A
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Abstract
The invention discloses a yellow aconite hair root and its acquisition method. The yellow aconite hair root provided by this invention is obtained by selecting yellow aconitic organ as explant materials, performing transfection by hair root agricillin and culturing the hair root. The yellow aconite hair root obtained by this invention can reproduce by fluid culture method, the reproductive speed is quick, the period of production is short (about 40 days or so), and it can not be influenced by the natural condition, which can carry out the intensifited industrial production and can overcome the defects that the speed of reprothropology is slow, the growing cycle is long, the output is low and the hereditary stability is weak in artificial planting of yellow aconite hair root; the medicinal components in hair root-the guanfu base A is of high content, which has wide application foreground and utilization value.
Description
Technical field
The present invention relates to agrobacterium rhizogenes mediated aconitum coreanum and preparation method thereof.
Background technology
Guan-fubase A is the anginal special effect medicine of a kind of treatment, and consumption is atomic just better curative effect.At present, domestic mainly is to extract the Guan-fubase A hyoscine in Radix Aconiti Coreani (Aconitum Coreanum (Levl.) Rapaics) root (Typhoniun Tuber).But natural Radix Aconiti Coreani often is subjected to climatic influences, and seasonal strong, disease and pest etc. also can have a strong impact on the output and the quality of Radix Aconiti Coreani; The Radix Aconiti Coreani growth cycle is long, needs just can gather in 5 years 1 time, is subjected to weather and disease and pest to influence greatly the quality instability; And its root head is little, and per unit area yield is low, seriously restricts the application of Radix Aconiti Coreani on producing.Enlarge the plantation of Radix Aconiti Coreani, must strive ground, do not meet the present situation of China's land resources with the grain plantation.
Agrobacterium rhizogenes (Agrobacterium rhizogenes, Ar.) be Rhizobiaceae, Gram-negative bacteria, different according to the source, Agrobacterium rhizogenes is divided into 8196 and A4 two big classes, and the former separates from apple tree and obtains, intoxicating power a little less than, the latter obtains from the separation of rose plant, and intoxicating power is stronger.The transforming tissue that A4 type bacterial strain causes synthesizes various agropines (agropine), therefore, is referred to as agropine type bacterial strain again usually.Produce many adventive root behind the Agrobacterium rhizogenes infection plant cell, this adventive root growth is rapid, and constantly branch Cheng Maozhuan so be referred to as hairly root, be also referred to as and send out a shape root (hairy root), abbreviates hair root or root of hair as.The Agrobacterium rhizogenes cell contains Ri plasmid (root inducing plasmid), and its size is 200-800kb.Contain T-DNA on the Ri plasmid, its energy transformed plant cells, performance root of hair proterties.The Ri plasmid has the characteristics of common plasmid: be the outer genetic material of bacterial chromosome, can be independently duplicated, and be the closed hoop double-stranded DNA, but self-replacation and have the stability of heredity, the existence to bacterium in particular surroundings plays an important role.But but it has characteristics such as transferability regroup conformability, erasability.
Summary of the invention
The purpose of this invention is to provide a kind of agrobacterium rhizogenes mediated aconitum coreanum and preparation method thereof.
Agrobacterium rhizogenes mediated aconitum coreanum provided by the present invention is to be explant material with the Radix Aconiti Coreani organ, carries out transfection with Agrobacterium rhizogenes, obtains after root of hair is cultivated.
The preparation method of this agrobacterium rhizogenes mediated aconitum coreanum comprises the steps:
1) acquisition of explant: the Radix Aconiti Coreani organ is transferred on the 1/2MS solid medium after sterilization, in the pre-down cultivation of dark condition 2-7 days;
2) transform root of hair: will immerse in the Agrobacterium rhizogenes bacterium liquid through pre-incubated Radix Aconiti Coreani organ and infect 5-20 minute, be inoculated in behind the exhaustion bacterium liquid and contain on the antibiotic 1/2MS solid medium, cultivated through 30-50 days down at 22 ℃-25 ℃, obtain agrobacterium rhizogenes mediated aconitum coreanum.
In order to improve the output and the quality of the agrobacterium rhizogenes mediated aconitum coreanum that is obtained, agrobacterium rhizogenes mediated aconitum coreanum is also through past bacterium cultivation and enlarged culturing; Described to go bacterium to cultivate be that root of hair is cut from explant, cultivates containing on the antibiotic 1/2MS solid medium, and every 3-5 days subculture 1 time carries out 3-5 time; Described enlarged culturing be with through the past root of hair cultivated of bacterium at 1/2MS or 1/2B
5In the liquid nutrient medium, under dark condition, 22 ℃-25 ℃, cultivate.
In cultivating transfection process, bacterial concentration is higher than 1 * 10 in the step 1) Agrobacterium rhizogenes bacterium liquid
6Individual/mL; Used microbiotic has Cephradine, Cefalexin Ampicillin, Pyocianil etc. usually; Concentration is 200mg/L-500mg/L.
In the present invention, the various organs of Radix Aconiti Coreani as blade, root, stem, petiole, filigree or petal etc., all can be used as the material of explant.
The yellow rhizome of Chinese monkshood root of hair that the present invention obtained can carry out growth and breeding in common tissue culture medium (TCM), can cultivate by the mode of liquid culture, reproduction speed is fast, (about about 40 days) with short production cycle, be not subjected to the influence of natural condition, can realize that industry intensification produces, defectives such as the reproduction speed that has been had when having overcome the artificial growth Radix Aconiti Coreani is slow, growth cycle is grown, yield poorly, genetic stability difference; Medicinal composition in the root of hair---Guan-fubase A content height has broad application prospects and utility value.
Description of drawings
Fig. 1 is the photo of agrobacterium rhizogenes mediated aconitum coreanum of the present invention.
Embodiment
With organs such as Radix Aconiti Coreani blade, root, stem, petiole, filigree or petals is explant, transform with the Agrobacterium rhizogenes that has activated, T-DNA on the Agrobacterium rhizogenes Ri plasmid is incorporated among the Radix Aconiti Coreani explant somatocyte nuclear DNA, express forming hairly root, be referred to as root of hair (hairy root) again; Each root of hair is a mono-clonal; Through repeatedly succeeding transfer culture and repeated screening, progressively set up agrobacterium rhizogenes mediated aconitum coreanum suspension culture clone.
The cultivation of embodiment 1, agrobacterium rhizogenes mediated aconitum coreanum and evaluation
One, the cultivation of agrobacterium rhizogenes mediated aconitum coreanum
1, material
The blade that potted plant Radix Aconiti Coreani is fresh, healthy, Agrobacterium rhizogenes A
4
2, transform
1) acquisition of explant: get fresh, the healthy blade of potted plant Radix Aconiti Coreani, clean; With 0.1% mercuric chloride sterilization 10min, rinsed with sterile water 4 times is no less than 1min at every turn, blots surface-moisture then; On super clean bench, blade is cut into 0.5cm * 1.0cm size again, transfers to (5 every bottle) on the sterilized 1/2MS solid medium; Cultivated 3 days in dark condition is pre-down.
2) cultivation of Agrobacterium rhizogenes: the single bacterium colony of picking Agrobacterium rhizogenes, be inoculated in the 5mL liquid YEB, shaking culture is spent the night under 28 ℃ of dark conditions, and it is standby after cultivating 4-6h to be inoculated into 50ml liquid YEB relaying next day.
3) Radix Aconiti Coreani blade and Agrobacterium rhizogenes are cultivated conversion altogether: will immerse cultured Agrobacterium rhizogenes bacterium liquid (〉=1 * 10 through pre-incubated Radix Aconiti Coreani blade
6Individual/mL) in, infect 10min, then, with the exhaustion of bacterium liquid, be inoculated in and contain antibiotic 1/2MS solid medium (used microbiotic is a Cephradine with aseptic filter paper; Concentration is 200mg/L) on, cultivate through 30d approximately and can bear root of hair.
3, cultivate
1) go bacterium to cultivate: cutting is down by the root of hair that grows on the explant, places to contain antibiotic 1/2MS solid medium (used microbiotic is a Cefalexin Ampicillin; Concentration is 300mg/L) on, go bacterium to cultivate, every 3-5d changes subculture 1 time, carries out 34 times;
2) enlarged culturing: aseptic root of hair 1.0g immigration is filled in the 100mL triangular flask of 50mL 1/2MS liquid nutrient medium, under dark condition, 22 ℃-25 ℃, 110rpm rotating speed low suspension is cultivated, and goes down to posterity once in per 30 days, and going down to posterity obtains agrobacterium rhizogenes mediated aconitum coreanum 800g 4 times.
Two, the evaluation of agrobacterium rhizogenes mediated aconitum coreanum
1, root of hair growthhabit
The agrobacterium rhizogenes mediated aconitum coreanum photo that is obtained shows as shown in Figure 1: the agrobacterium rhizogenes mediated aconitum coreanum apogetropism of conversion, and intersection ' unordered ' growth at random, many ' root hairs ', and multi-branched, growth is fast, has typical conversion root of hair feature.
2, agrobacterium rhizogenes mediated aconitum coreanum growth curve
The growth curve of this agrobacterium rhizogenes mediated aconitum coreanum is a typical sigmoid curve, cultivates and can breed 20 times in 30 days.
3, the secondary metabolites of agrobacterium rhizogenes mediated aconitum coreanum
After testing, Guan-fubase A content is 0.38% in the agrobacterium rhizogenes mediated aconitum coreanum of the present invention, and is suitable with Guan-fubase A content the Radix Aconiti Coreani root of being gathered from plant.
4, the substratum of agrobacterium rhizogenes mediated aconitum coreanum
Can 1/2MS or 1/2B
5Liquid nutrient medium is as the growth medium of agrobacterium rhizogenes mediated aconitum coreanum, pH5.0-6.0.
In root of hair was cultivated, the effect of sucrose had two: one, as the carbon source and the energy, and the 2nd, regulate nutrient solution osmotic pressure.In substratum, sucrose concentration is advisable with 1.0-3.0% (weight), be lower than 1.0% or be higher than 3.0% all unfavorable to its growth.
This agrobacterium rhizogenes mediated aconitum coreanum can be grown in no hormone nutrient solution, but behind growth hormone such as additional NAA, it is more quick to grow, and concentration is advisable with 0.2mg/L.
5, culture condition is to the influence of agrobacterium rhizogenes mediated aconitum coreanum growth
Under indoor natural light, light is to the stronger restraining effect of agrobacterium rhizogenes mediated aconitum coreanum growth performance, and its biomass only is 1/3 under the dark culturing condition.
Culture temperature is to be advisable at 22-25 ℃.
The cultivation of embodiment 2, agrobacterium rhizogenes mediated aconitum coreanum
Get fresh, the healthy stem of potted plant Radix Aconiti Coreani, clean; With 0.1% mercuric chloride sterilization 10min, rinsed with sterile water 4 times is no less than 1min at every turn, blots surface-moisture then; On super clean bench, blade is cut into 0.5cm * 1.0cm size again, transfers to (5 every bottle) on the sterilized 1/2MS solid medium; In the pre-down 6d that cultivates of dark condition.
Picking Agrobacterium rhizogenes 15834 single bacterium colonies are inoculated in the 5mL liquid YEB, and shaking culture is spent the night under 28 ℃ of dark conditions, and it is standby after cultivating 4-6h to be inoculated into 50ml liquid YEB relaying next day.
To immerse in the cultured Agrobacterium rhizogenes bacterium liquid through pre-incubated Radix Aconiti Coreani stem, infect 20min, and then, with the exhaustion of bacterium liquid, be inoculated in and contain microbiotic 1/2MS solid medium (used microbiotic is a Pyocianil with aseptic filter paper; Concentration is 500mg/L) on, cultivate through 50d approximately and bear root of hair.
Cutting is down by the root of hair that grows on the explant, places to contain antibiotic 1/2MS solid medium (used microbiotic is a Cephradine; Concentration is 400mg/L) on, go bacterium to cultivate, every 4d changes subculture 1 time, carries out 4 times;
Aseptic root of hair 1.0g immigration is filled 50mL 1/2B
5In the 100mL triangular flask of liquid nutrient medium, under dark condition, 22 ℃-25 ℃ of room temperatures, 110rpm rotating speed low suspension is cultivated, and goes down to posterity once in per 30 days, and going down to posterity obtains agrobacterium rhizogenes mediated aconitum coreanum 800g 4 times.
Other organs of Radix Aconiti Coreani can adopt identical with it method to obtain agrobacterium rhizogenes mediated aconitum coreanum through transfection, cultivation.
Claims (10)
1, a kind of agrobacterium rhizogenes mediated aconitum coreanum is to be explant material with the Radix Aconiti Coreani organ, carries out transfection with Agrobacterium rhizogenes, obtains after root of hair is cultivated.
2, agrobacterium rhizogenes mediated aconitum coreanum according to claim 1 is characterized in that: described Radix Aconiti Coreani blade was also being cultivated 2-7 days under dark condition on the 1/2MS solid medium before transfection in advance.
3, agrobacterium rhizogenes mediated aconitum coreanum according to claim 2 is characterized in that: described microbiotic is selected from Cephradine, Cefalexin Ampicillin or Pyocianil; Concentration is 200mg/L-500mg/L.
4, agrobacterium rhizogenes mediated aconitum coreanum according to claim 1 is characterized in that: it is to contain on the antibiotic 1/2MS solid medium to descend through cultivation in 30-50 days to bear root of hair in 22 ℃-25 ℃ that described root of hair is cultivated.
5, according to the arbitrary described agrobacterium rhizogenes mediated aconitum coreanum of claim 1-4, it is characterized in that: described Radix Aconiti Coreani organ is blade, root, stem, petiole, filigree or petal.
6, a kind of method that obtains agrobacterium rhizogenes mediated aconitum coreanum comprises the steps:
1) acquisition of explant: the Radix Aconiti Coreani organ is transferred on the 1/2MS solid medium after sterilization, in the pre-down cultivation of dark condition 2-7 days;
2) transform root of hair: will immerse in the Agrobacterium rhizogenes bacterium liquid through pre-incubated Radix Aconiti Coreani organ and infect 5-20 minute, be inoculated in behind the exhaustion bacterium liquid and contain on the antibiotic 1/2MS solid medium, cultivated through 30-50 days down at 22 ℃-25 ℃, obtain agrobacterium rhizogenes mediated aconitum coreanum.
7, method according to claim 6 is characterized in that: described agrobacterium rhizogenes mediated aconitum coreanum is also through past bacterium cultivation and enlarged culturing; Described to go bacterium to cultivate be that root of hair is cut from explant, cultivates containing on the antibiotic 1/2MS solid medium, and every 3-5 days subculture 1 time carries out 3-5 time; Described enlarged culturing be with through the past root of hair cultivated of bacterium in 1/2MS or 1/2B5 liquid nutrient medium, under dark condition, 22 ℃-25 ℃, cultivate.
8, according to claim 6 or 7 described preparation methods, it is characterized in that: described microbiotic is selected from Cephradine, Cefalexin Ampicillin or Pyocianil; Concentration is 200mg/L-500mg/L.
8, according to claim 6 or 7 described methods, it is characterized in that: described Radix Aconiti Coreani organ is blade, root, stem, petiole, filigree or petal.
9, according to claim 6 or 7 described methods, it is characterized in that: step 2) bacterial concentration is higher than 1 * 10 in the described Agrobacterium rhizogenes bacterium liquid
6Individual/mL.
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| CNB2006100895233A CN100420741C (en) | 2006-06-30 | 2006-06-30 | Agrobacterium rhizogenes mediated aconitum coreanum and its obtaining method |
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| CN100420741C CN100420741C (en) | 2008-09-24 |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102499078A (en) * | 2011-10-18 | 2012-06-20 | 李昌禹 | Transgenic aconitum coreanum seedling and production method thereof |
| CN101715665B (en) * | 2009-12-30 | 2012-08-22 | 雅安三九中药材科技产业化有限公司 | Method for improving germination rate of monkshood seed |
| CN103798040A (en) * | 2014-02-19 | 2014-05-21 | 云南和创药业有限公司 | Efficient cultivation method of plateau section aconitum vilmorimianum kom |
| CN106212275A (en) * | 2016-07-21 | 2016-12-14 | 西南科技大学 | A kind of Aconitum earmichaeli seedling tissue culture quick breeding renovation process |
| CN107372125A (en) * | 2017-09-14 | 2017-11-24 | 河南科技学院 | A kind of indefinite bud method of rhizome of Chinese monkshood holder callus induction and differentiation |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1285138A (en) * | 1999-08-23 | 2001-02-28 | 赵寿经 | Method for producing ginsent hairy root and ginsent seponin by hairy root agricillin A4 bacterial strain inducing explant of ginsent root |
-
2006
- 2006-06-30 CN CNB2006100895233A patent/CN100420741C/en not_active Expired - Fee Related
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101715665B (en) * | 2009-12-30 | 2012-08-22 | 雅安三九中药材科技产业化有限公司 | Method for improving germination rate of monkshood seed |
| CN102499078A (en) * | 2011-10-18 | 2012-06-20 | 李昌禹 | Transgenic aconitum coreanum seedling and production method thereof |
| CN103798040A (en) * | 2014-02-19 | 2014-05-21 | 云南和创药业有限公司 | Efficient cultivation method of plateau section aconitum vilmorimianum kom |
| CN106212275A (en) * | 2016-07-21 | 2016-12-14 | 西南科技大学 | A kind of Aconitum earmichaeli seedling tissue culture quick breeding renovation process |
| CN106212275B (en) * | 2016-07-21 | 2019-02-12 | 西南科技大学 | A kind of Aconitum earmichaeli seedling tissue culture quick breeding regeneration method |
| CN107372125A (en) * | 2017-09-14 | 2017-11-24 | 河南科技学院 | A kind of indefinite bud method of rhizome of Chinese monkshood holder callus induction and differentiation |
| CN107372125B (en) * | 2017-09-14 | 2019-11-05 | 河南科技学院 | A kind of indefinite bud method of rhizome of Chinese monkshood holder callus induction and differentiation |
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| CN100420741C (en) | 2008-09-24 |
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