CN108220325A - A kind of preparation method of agrobacterium rhizogenes conversion Radix Notoginseng cell - Google Patents
A kind of preparation method of agrobacterium rhizogenes conversion Radix Notoginseng cell Download PDFInfo
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- CN108220325A CN108220325A CN201611125478.2A CN201611125478A CN108220325A CN 108220325 A CN108220325 A CN 108220325A CN 201611125478 A CN201611125478 A CN 201611125478A CN 108220325 A CN108220325 A CN 108220325A
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Abstract
The present invention relates to a kind of preparation methods of agrobacterium rhizogenes conversion Radix Notoginseng cell.The technical solution that the present invention takes is as follows：For the Radix Notoginseng hairy root of the existing well-grown solid culture in choice experiment room or Liquid Culture as explant evoked callus, concrete operations are as follows：Hairy root is cut into segment, callus can be induced in 34 weeks or so by being inoculated in be placed in 25 ± 1 DEG C of darkroom on the MS solid mediums added with 1.0+0.5 mg/L of 2,4 D of mg/L, 6 BA and cultivate；Then the embryo callus of yellow particle shape is accessed, 40 d are cultivated under 25 ± 1 DEG C, 12 h/d illumination conditions.
Description
Technical field
The invention belongs to field of plant cell engineering technology, specifically say it is a kind of agrobacterium rhizogenes conversion Radix Notoginseng cell
Preparation method, and the method for producing effective secondary metabolite.
Background technology
Radix Notoginseng（(Panaxnotoginseng (Burk) F.H.Chen）For Araliaceae Panax, perennial herb is dicotyledonous
Plant, and have the title of invaluable, pseudo-ginseng, radix notoginseng etc., it is the distinctive rare medicinal plant in China.It is Radix Notoginseng bitter but slightly sweet taste, mild-natured,
Mainly it is used as medicine with root tuber and rhizome, main medicinal ingredient has arasaponin（PNS）, dencichine, brass, volatile oil, ammonia
Base acid, carbohydrate and various trace elements etc..It is found through pharmacological research, there is promoting blood circulation and removing blood stasis, detumescence ding-tong, anti-swollen in the effect of Radix Notoginseng
Knurl, strengthening by means of tonics, hypoglycemic, antithrombotic etc. are mainly used to traumatic injury, hemoptysis, spit blood, have blood in stool.Radix Notoginseng is numerous Chinese patent drugs
One of main component, such as Yunnan Baiyao, ' Xintongning ' piece, FUFANG SANQI JIAONANG etc..Therefore, Radix Notoginseng has very big economy
Value and social benefit.
The medical value of Radix Notoginseng is constantly known in recent years, therefore its medicinal demand is continuously increased, wild money
Excessive, private adopt seriously is exploited year after year in source.Due to deforestation, vegetation stony desertification, soil layer loss etc., the ecological environment of Radix Notoginseng
Also it is seriously destroyed.Along with factors such as natural climate influence and Radix Notoginseng natural propagation force differences itself, cause Radix Notoginseng wild
Raw resource quantity drastically declines.
Since pseudo-ginseng is mainly by harvesting wild resource, harvest cycle is longer, by the external factor shadow such as pest and disease damage, weather
It rings big, supply is caused the practical problems such as to be difficult to control.Although the GAP cultivating and growings area of Radix Notoginseng is also constantly expanding,
Soil environment in the course of cultivation deteriorates and continuous cropping obstacle harm also becomes increasingly conspicuous.At present, notoginseng area area is up to total
The 15% of cultivated area, the loss thereby resulted in every year is in up to ten million members.Continuous cropping obstacle, which has become, influences Genuine producing area Wenshan Prefecture
The important restriction factor of Radix Notoginseng industry sustainable development.Therefore, seek new substitute or new technology with meet the market demand and
Solve the problems, such as that species conservation is extremely urgent.
Consider, be to solve the problems, such as this maximally efficient approach using biological technique method.On the one hand can alleviate
The scarcity of plant resources protects precious wild plant resource, avoids predatory exploitation；On the other hand pass through the cell of scale
Culture technique can make the effective substance of plant cell controlled syntheses with the secondary metabolite of orientation adjustment cell, to improve, adjust
Active ingredients from traditional Chinese medicinal content is controlled, its medical value and curative effect is improved, reduces the financial cost of Producing medicinal herbs.Although such as
This, the floristics commercially produced by the realization of the means such as cell culture is limited, and main cause is heredity and medicinal ingredient life
The stability difference of object synthesis and biomass is small etc. in the unit interval, therefore, screening high-quality high yielding cell sarain is to solve above-mentioned bottle
The key technique of neck problem.
Hairy root is that the special adventitious root generated after plant cell is infected using agrobacterium rhizogenes, have inheritance stability,
Hormone autotrophic, the speed of growth are fast and the advantages that cometabolism is strong.Hairy root is a kind of new medicinal as a kind of transgenic organ
Raw material is increasingly subject to the concern of domestic and international researcher.Experiments have shown that the callus group induced by the use of hairy root as explant
It knits, it may have the advantages that speed of growth of hairy root is fast, inheritance stability, compared with common Radix Notoginseng callus, solid culture item
Higher cell yield can be obtained in the part lower short period, while saponin content is also higher.Hairy root is in suspension culture system
There is conglobation property down, with the extension of incubation time, internal hairy root tissue due to not enough oxygen supplies and
Nutrient Cycling is supplied and gradually aging even browning death, and the speed of growth is caused to decline so as to influence crop.Therefore, it utilizes
The callus of hairy root establishes plant cell suspension cultures and optimum culture condition, can solve the root amplification culture of puzzlement hairy
Agglomerating sex chromosome mosaicism in the process.
Invention content
The purpose of the present invention is disclose a kind of preparation method of agrobacterium rhizogenes conversion Radix Notoginseng cell.
The technical solution that the present invention takes is as follows：
1. the Radix Notoginseng hairy root of the existing well-grown solid culture in choice experiment room or Liquid Culture is induced as explant
Callus, concrete operations are as follows：Hairy root is cut into segment, is inoculated in added with 1.0 mg/L 2 ,+0.5 mg/ of 4-D
On the MS solid mediums of L 6-BA, callus can be induced in 3-4 weeks or so by being placed in 25 ± 1 DEG C of darkroom culture；Then
The embryo callus of yellow particle shape is accessed, 40 d are cultivated under 25 ± 1 DEG C, 12 h/d illumination conditions.
2. the callus induced is detached with explant, the callus isolated is inoculated in added with 1.0 mg/L
Squamous subculture is carried out on the MS solid mediums of+0.2 mg/L 6-BA+0.3mg/L KT of 2,4-D（25 ± 1 DEG C of light cultures）,
25d or so subcultures on this culture medium are primary.The callus color and luster that newly induces is dim, soft, moisture is larger and growth speed
Degree is slower, needs the squamous subculture by 5 times or more, callus gradually show loose quality, easy dispersion, do not secrete it is glutinous
The graininess of liquid, white approximately passes through the squamous subculture of 10 times or so, callus growth speed at this time and very steady
It is fixed, while the material suitable for doing cell suspension cultures.On aseptic operating platform, with tweezers by the Radix Notoginseng callus after preculture
It is transferred in the bacterium solution of the agrobacterium rhizogenes prepared with blade, stem section and infects 30 min, 130 r/min, 28 DEG C make pre- training
It can adequately be contacted between foster explant and agrobacterium rhizogenes.After infecting, with aseptic filter paper by the callus group of taking-up
It knits and is blotted with the bacterium solution of blade surface, be then transferred to the acetosyringone containing 0,20,40,60,80,100 mg/L again
In MS solid mediums, 25 ± 1 DEG C, carry out co-culturing 2 d under the conditions of light culture.
3. on aseptic operating platform, by formed during co-culturing bacterial plaque Radix Notoginseng callus and blade, stem section with sterile
Water clean 2-3 times, then again with aseptic filter paper blot Radix Notoginseng callus and blade, stem section surface excessive moisture after will
It is transferred on the MS solid mediums containing 500 mg/L Cefotaxime Sodiums progress degerming culture, week about will be into
Row squamous subculture is primary, and gradually reduces the concentration of degerming antibiotic until around Radix Notoginseng callus and blade, stem section
Bacterial plaque completely disappears.
4. when hairy root long derived to 2-3 cm or so, hairy root is cut, and is transferred to containing 200 mg/
On the MS solid mediums of L kanamycins, 25 ± 1 DEG C, cultivated under 12 h/d illumination conditions, to be sieved in incubation
Select the as Radix Notoginseng hairy root that the speed of growth with kalamycin resistance is very fast, branch is more.
The foundation of plant cell suspension cultures：
The present invention first selection 150ml triangular flasks do shake flask test, per bottled liquid measure be 50ml, be placed in 25 ± 1 DEG C,
Light culture on 110 ± 5r/min shaking tables, cultivation cycle 20d.
Selection 5 kinds of culture mediums of MS, 1/2MS, B5,1/2B5 and N6 do minimal medium experiment, are determined by experiment 1/
2MS fluid nutrient mediums are best minimal medium.
On the basis of 1/2MS is best minimal medium, 2 are selected, tetra- kinds of hormones of 4-D, 6-BA, NAA, KT carry out orthogonal
Experiment, the experimental results showed that, the best hormone combinations of hairy root-derived callus suspension culture are 1.0mg/L 2,4-D+0.2mg/L
6-BA+0.6mg/L NAA+0.2mg/L KT。
Using 1/2MS+1.0mg/L 2,4-D+0.2mg/L 6-BA+0.6mg/L NAA+0.2mg/L KT as culture medium into
Row inoculum concentration is tested, and finally determining optimum inoculation amount is 4-6%（W/v, g（FW）/ml）.
In conclusion the best medium of Radix Notoginseng suspension cell culture is 1/2MS+1.0mg/L 2,4-D+0.2mg/L
6-BA+0.8mg/L NAA+0.2mg/L KT, inoculum concentration 4-6%（W/v, g（FW）/ml）.Radix Notoginseng hairy root suspension cell culture
The foundation of system includes 4 steps：The induction of hairy root and solid culture, the Liquid Culture of hairy root, the callus group of hairy root
It is numerous with expanding to knit induction, the culture of suspension cell shaking flask and amplification are cultivated.The initial several generations cell granulations cultivated that suspend are usually larger, can
The methods of with by being sieved under aseptic condition, removes bulky grain, by the squamous subculture of 5-8 times or so, suspension culture system by
Gradually tend towards stability.
The detection method of the thick total saposins of Radix Notoginseng:
Cell culture harvest object grind into powder and is sieved for the measure of saponin content after drying to constant weight, selection is neat
Pier tartaric acid standard items are reference substance, and using vanilla root rot colorimetric method for determining content of the total saponins in radix notoginseng, specific method is referring to Lee
Refined wait of scape writes " foundation of tuniclike psammosilene root hairy root induction and cultivating system "《CHINA JOURNAL OF CHINESE MATERIA MEDICA》2011,36 (5)：547.
The expansion culture of hairy root-derived callus cell：After hairy root-derived callus is obtained, solid culture can be passed through
Or the method for Liquid Culture carries out continuous expand and cultivates, to obtain a large amount of cell products.Wherein solid culture can lead to
It crosses continuous expansion culture and obtains final cell products, complete the production of a large amount of cells；One can also be obtained in solid culture
After quantitative cell, it is transferred to the production that bioreactor completes a large amount of cells.
Specific embodiment
Embodiment 1：
(1) under sterile conditions, the graininess Radix Notoginseng callus turned out is marked wound to be placed on without any hormone
MS solid mediums in.Likewise, the blade of the regeneration plant of Radix Notoginseng and stem section are cut and leave wound, it is put into and is free of
In the MS solid mediums of any hormone.Then in 25 ± 3 DEG C, light culture condition, 2 d of preculture.After preculture
The blade and stem section of callus and Radix Notoginseng will all be used for bacterium solution and infect, and obtain Radix Notoginseng callus.
(2) on aseptic operating platform, the Radix Notoginseng callus after preculture and blade, stem section are transferred to and prepared with tweezers
Agrobacterium rhizogenes bacterium solution in infect 30 min, 130 r/min, 28 DEG C make explant and the agrobacterium rhizogenes of preculture
Between can adequately contact.After infecting, the bacterium solution of the callus of taking-up and blade surface is blotted with aseptic filter paper,
Then it is transferred to again in the MS solid mediums of the acetosyringone containing 0,20,40,60,80,100 mg/L, 25 ± 1
DEG C, it carries out co-culturing 2 d under the conditions of light culture.
(3) 1 collarium single bacterium colony of picking is inoculated in 50mL liquid YEB culture mediums, and 25 DEG C ± 3 shaken cultivation 12h, OD values are 0.3
~1.2. takes 1ml bacterium solutions to be placed in shaken cultivation in 100mlYEB fluid nutrient mediums, cultivates 6h.OD values are 0.3~0.9.3500
5~15min is centrifuged under rpm and collects thalline.
(4) thalline that (3) are collected is placed in MS fluid nutrient mediums simultaneously mixing.
(5) Radix Notoginseng 10~30min of callus is disseminated with the bacterium solution obtained after (4) mixing, it is extra with aseptic paper absorption after taking-up
Bacterium solution, the Radix Notoginseng callus of dip dyeing is linked into the MS solid cultures containing 100umol/L acetosyringones, 0.9% agar
1~3d is cultivated in base.
(6) MS solid medium of the callus access containing 500mg/L Cefotaxime Sodiums after (5) are co-cultured is enterprising
Row degerming culture, will carry out that squamous subculture is primary week about, and gradually reduce the concentration of degerming antibiotic until Radix Notoginseng
Bacterial plaque around callus and blade, stem section completely disappears.
(7) by the use of no hormone 1/2MS fluid nutrient mediums as amplification culture medium, by the hair-like derived of 1g after antibacterial processing
Root is put into the amplification culture medium of 30mL, dark suspension shaken cultivation, and oscillation shaking speed is 110~120rmin-1。
(8) when hairy root long derived to 2-3 cm or so, hairy root is cut, and is transferred to containing 200
On the MS solid mediums of mg/L kanamycins, 25 ± 1 DEG C, cultivated under 12 h/d illumination conditions, in incubation
Filter out the as Radix Notoginseng hairy root that the speed of growth with kalamycin resistance is very fast, branch is more.
Embodiment 2：
(1) under sterile conditions, the graininess Radix Notoginseng callus turned out is marked wound to be placed on without any hormone
MS solid mediums in.Likewise, the blade of the regeneration plant of Radix Notoginseng and stem section are cut and leave wound, it is put into and is free of
In the MS solid mediums of any hormone.Then in 25 ± 1 DEG C, light culture condition, preculture 2d.Being cured after preculture
The blade and stem section of injured tissue and Radix Notoginseng will all be used for bacterium solution and infect, and obtain Radix Notoginseng callus.
(2) on aseptic operating platform, the Radix Notoginseng callus after preculture and blade, stem section are transferred to and prepared with tweezers
Agrobacterium rhizogenes bacterium solution in infect 30 min, 130 r/min, 28 DEG C make explant and the agrobacterium rhizogenes of preculture
Between can adequately contact.After infecting, the bacterium solution of the callus of taking-up and blade surface is blotted with aseptic filter paper,
Then it is transferred to again in the MS solid mediums of the acetosyringone containing 0,20,40,60,80,100 mg/L, 25 ± 1
DEG C, it carries out co-culturing 2 d under the conditions of light culture.
(3) 1 collarium single bacterium colony of picking is inoculated in 50mL liquid YEB culture mediums, and 25 DEG C ± 3 shaken cultivation 12h, OD values are 0.3
~1.2. takes 1ml bacterium solutions to be placed in shaken cultivation in 100mlYEB fluid nutrient mediums, cultivates 6h.OD values are 0.3~0.9.3500
5~15min is centrifuged under rpm and collects thalline.
(4) thalline that (3) are collected is placed in MS fluid nutrient mediums simultaneously mixing.
(5) Radix Notoginseng 10~30min of callus is disseminated with the bacterium solution obtained after (4) mixing, it is extra with aseptic paper absorption after taking-up
Bacterium solution, the Radix Notoginseng callus of dip dyeing is linked into the MS solid cultures containing 100umol/L acetosyringones, 0.9% agar
1~3d is cultivated in base.
(6) MS solid medium of the callus access containing 500mg/L Cefotaxime Sodiums after (5) are co-cultured is enterprising
Row degerming culture, will carry out that squamous subculture is primary week about, and gradually reduce the concentration of degerming antibiotic until Radix Notoginseng
Bacterial plaque around callus and blade, stem section completely disappears.
(7) by the use of no hormone 1/2MS fluid nutrient mediums as amplification culture medium, by the Hairy root derived of 1g after antibacterial processing
It is put into the amplification culture medium of 30mL, dark suspension shaken cultivation, oscillation shaking speed is 110~120rmin-1。
(8) when hairy root long derived to 2-3 cm or so, hairy root is cut, and is transferred to containing 200
On the MS solid mediums of mg/L kanamycins, 24 ± 1 DEG C, cultivated under 10 h/d illumination conditions, in incubation
Filter out the as Radix Notoginseng hairy root that the speed of growth with kalamycin resistance is very fast, branch is more.
Embodiment 4：
（1）The Radix Notoginseng hairy root of the existing well-grown solid culture in choice experiment room or Liquid Culture is lured as explant
Callus is led, concrete operations are as follows：Hairy root is cut into segment, is inoculated in added with 1.0 mg/L 2,4-D+0.5
On the MS solid mediums of mg/L 6-BA, callus can be induced in 3-4 weeks or so by being placed in 25 ± 1 DEG C of darkroom culture；
Then the embryo callus of yellow particle shape is accessed, 40 d are cultivated under 25 ± 1 DEG C, 12 h/d illumination conditions；
（2）The callus induced with explant is detached, the callus isolated is inoculated in added with 1.0 mg/
Squamous subculture is carried out on the MS solid mediums of+0.2 mg/L 6-BA+0.3mg/L KT of L 2,4-D, in 25 ± 1 DEG C of dark trainings
It supports；25d or so subcultures on this culture medium are primary, and the callus color and luster newly induced is dim, soft, moisture is larger and grows
Speed is slower, needs the squamous subculture by 5 times or more, callus gradually show loose quality, easy dispersion, do not secrete it is glutinous
The graininess of liquid, white approximately passes through the squamous subculture of 10 times or so, callus growth speed at this time and very steady
It is fixed, while the material suitable for doing cell suspension cultures.On aseptic operating platform, with tweezers by the Radix Notoginseng callus after preculture
It is transferred in the bacterium solution of the agrobacterium rhizogenes prepared with blade, stem section and infects 30 min, 130 r/min, 28 DEG C make pre- training
It can adequately be contacted between foster explant and agrobacterium rhizogenes.After infecting, with aseptic filter paper by the callus group of taking-up
It knits and is blotted with the bacterium solution of blade surface, be then transferred to the acetosyringone containing 0,20,40,60,80,100 mg/L again
In MS solid mediums, 25 ± 1 DEG C, carry out co-culturing 2 d under the conditions of light culture；
（3）On aseptic operating platform, the Radix Notoginseng callus of bacterial plaque and blade, stem section nothing will be formed during co-culturing
Bacterium water clean 2-3 times, then again with aseptic filter paper blot Radix Notoginseng callus and blade, stem section surface excessive moisture after
Progress degerming culture on the MS solid mediums containing 500 mg/L Cefotaxime Sodiums is transferred to, week about will
Progress squamous subculture is primary, and gradually reduces the concentration of degerming antibiotic until around Radix Notoginseng callus and blade, stem section
Bacterial plaque completely disappear；
（4）When hairy root long derived to 2-3 cm or so, hairy root is cut, and is transferred to containing 200
On the MS solid mediums of mg/L kanamycins, 25 ± 1 DEG C, cultivated under 12 h/d illumination conditions, in incubation
Filter out the as Radix Notoginseng hairy root that the speed of growth with kalamycin resistance is very fast, branch is more.Suspension cell culture
The foundation of system：
The present invention first selection 150ml triangular flasks do shake flask test, per bottled liquid measure be 50ml, be placed in 25 ± 1 DEG C,
Light culture on 110 ± 5r/min shaking tables, cultivation cycle 20d.
Selection 5 kinds of culture mediums of MS, 1/2MS, B5,1/2B5 and N6 do minimal medium experiment, are determined by experiment 1/
2MS fluid nutrient mediums are best minimal medium.
On the basis of 1/2MS is best minimal medium, 2 are selected, tetra- kinds of hormones of 4-D, 6-BA, NAA, KT carry out orthogonal
Experiment, the experimental results showed that, the best hormone combinations of hairy root-derived callus suspension culture are 1.0mg/L 2,4-D+0.2mg/L
6-BA+0.6mg/L NAA+0.2mg/L KT。
Using 1/2MS+1.0mg/L 2,4-D+0.2mg/L 6-BA+0.6mg/L NAA+0.2mg/L KT as culture medium into
Row inoculum concentration is tested, and finally determining optimum inoculation amount is 4-6%（W/v, g（FW）/ml）.
In conclusion the best medium of Radix Notoginseng suspension cell culture is 1/2MS+1.0mg/L 2,4-D+0.2mg/L
6-BA+0.8mg/L NAA+0.2mg/L KT, inoculum concentration 4-6%（W/v, g（FW）/ml）.Radix Notoginseng hairy root suspension cell culture
The foundation of system includes 4 steps：The induction of hairy root and solid culture, the Liquid Culture of hairy root, the callus group of hairy root
It is numerous with expanding to knit induction, the culture of suspension cell shaking flask and amplification are cultivated.The initial several generations cell granulations cultivated that suspend are usually larger, can
The methods of with by being sieved under aseptic condition, removes bulky grain, by the squamous subculture of 5-8 times or so, suspension culture system by
Gradually tend towards stability.
For the ordinary skill in the art, specific embodiment is only exemplarily described the present invention,
Obviously the present invention specific implementation is not subject to the restrictions described above, as long as employ the inventive concept and technical scheme of the present invention into
The improvement of capable various unsubstantialities or it is not improved by the present invention design and technical solution directly apply to other occasions
, within protection scope of the present invention.
Claims (1)
1. a kind of preparation method of agrobacterium rhizogenes conversion Radix Notoginseng cell, it is characterised in that：Include the following steps：
（1）The Radix Notoginseng hairy root of the existing well-grown solid culture in choice experiment room or Liquid Culture is lured as explant
Callus is led, concrete operations are as follows：Hairy root is cut into segment, is inoculated in added with 1.0 mg/L 2,4-D+0.5
On the MS solid mediums of mg/L 6-BA, callus can be induced in 3-4 weeks or so by being placed in 25 ± 1 DEG C of darkroom culture；
Then the embryo callus of yellow particle shape is accessed, 40 d are cultivated under 25 ± 1 DEG C, 12 h/d illumination conditions；
（2）The callus induced with explant is detached, the callus isolated is inoculated in added with 1.0 mg/L
Squamous subculture is carried out on the MS solid mediums of 2,4-D+0.2 mg/L 6-BA+0.3mg/L KT, in 25 ± 1 DEG C of dark trainings
It supports；25d or so subcultures on this culture medium are primary, and the callus color and luster newly induced is dim, soft, moisture is larger and grows
Speed is slower, needs the squamous subculture by 5 times or more, callus gradually show loose quality, easy dispersion, do not secrete it is glutinous
The graininess of liquid, white approximately passes through the squamous subculture of 10 times or so, callus growth speed at this time and very steady
It is fixed, while the material suitable for doing cell suspension cultures；On aseptic operating platform, with tweezers by the Radix Notoginseng callus after preculture
It is transferred in the bacterium solution of the agrobacterium rhizogenes prepared with blade, stem section and infects 30 min, 130 r/min, 28 DEG C make pre- training
It can adequately be contacted between foster explant and agrobacterium rhizogenes；After infecting, with aseptic filter paper by the callus group of taking-up
It knits and is blotted with the bacterium solution of blade surface, be then transferred to the acetosyringone containing 0,20,40,60,80,100 mg/L again
In MS solid mediums, 25 ± 1 DEG C, carry out co-culturing 2 d under the conditions of light culture；
（3）On aseptic operating platform, the Radix Notoginseng callus of bacterial plaque and blade, stem section nothing will be formed during co-culturing
Bacterium water clean 2-3 times, then again with aseptic filter paper blot Radix Notoginseng callus and blade, stem section surface excessive moisture after
Progress degerming culture on the MS solid mediums containing 500 mg/L Cefotaxime Sodiums is transferred to, week about will
Progress squamous subculture is primary, and gradually reduces the concentration of degerming antibiotic until around Radix Notoginseng callus and blade, stem section
Bacterial plaque completely disappear；
（4）When hairy root long derived to 2-3 cm or so, hairy root is cut, and is transferred to containing 200
On the MS solid mediums of mg/L kanamycins, 25 ± 1 DEG C, cultivated under 12 h/d illumination conditions, in incubation
Filter out the as Radix Notoginseng hairy root that the speed of growth with kalamycin resistance is very fast, branch is more.
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| CN201611125478.2A Pending CN108220325A (en) | 2016-12-09 | 2016-12-09 | A kind of preparation method of agrobacterium rhizogenes conversion Radix Notoginseng cell |
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| CN114517174A (en) * | 2020-11-19 | 2022-05-20 | 北京化工大学 | Engineering bacterium for synthesizing dencichine and application |
| CN117247891A (en) * | 2023-09-22 | 2023-12-19 | 云南农业大学 | Pseudo-ginseng callus tissue culture and hairy root induction method thereof |
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| CN117247891A (en) * | 2023-09-22 | 2023-12-19 | 云南农业大学 | Pseudo-ginseng callus tissue culture and hairy root induction method thereof |
| CN117247891B (en) * | 2023-09-22 | 2024-04-02 | 云南农业大学 | Pseudo-ginseng callus tissue culture and hairy root induction method thereof |
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