CN104839022B - The abductive approach of tuniclike psammosilene root feather shaped root system - Google Patents
The abductive approach of tuniclike psammosilene root feather shaped root system Download PDFInfo
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Abstract
The abductive approach of tuniclike psammosilene root feather shaped root system is the method for generating hairy root with agrobacterium rhizogenes induction tuniclike psammosilene root explant, and the method includes the CuCl of 10~15mmol/L is used before induction2Aqueous solution handles the step of explant.Invention is pioneering to utilize CuCl2Tuniclike psammosilene root blade is handled, blade is infected by agrobacterium rhizogenes, hairy root can occur within 2-4 days at petiole and leaf margin vein, the root of hair time shortens, and can generate within 3~5 days root of hair, Induction Transformation rate greatly improves, and reaches 60% or more.Then by screening, hairy root fast-growth in no hormone culture-medium.
Description
Technical field
The biotechnology efficiently method of induction and culture feather shaped root system is the present invention relates to the use of, tuniclike psammosilene root is especially efficiently induced
The method of feather shaped root system.
Background technology
Tuniclike psammosilene root (Psammosilene tunicoides W.C.Wu et C.Y.Wu) is that Caryophyllaceae tuniclike psammosilene root belongs to for many years
Raw herbaceous plant also known as RADIX PSAMMOSILENE, Little Caesar, Kunming the root of straight ladybell, Pachysandra axillaris Franch., Talinum crassifolium etc. are that Southwestern China portion is distinctive
It is single to belong to kind of a medicinal plant.Tuniclike psammosilene root is mainly used as medicine with root, and primary medicinal component is triterpene, triterpenoid saponin, cyclic peptide and lactams etc.,
Pharmacological research find Radix Psammosilenes extract mainly have antalgic and inflammation relieving, dissipate stasis of blood hemostasis, antitumor, immunological regulation, it is antibacterial the effects that, use
It is one of the main ingredient of a variety of well-known Chinese patent drugs, such as cloud in the treatment of traumatic injury, rheumatalgia, peratodynia, traumatic bleeding etc.
Southern baiyao series, cure-all series, Yunnan hongyao capsule, Guizhou Jin Gulian capsules, Fujian pain Urapidil etc. have prodigious
Economic value and social benefit.
The medicinal demand of tuniclike psammosilene root is continuously increased in recent years, and wild resource is adopted excessively year after year, private is adopted seriously.Due to forest
Reasons, the tuniclike psammosilene root ecological environments such as felling, vegetation stony desertification, soil layer loss are also seriously destroyed.Along with natural climate shadow
The factors such as sound and tuniclike psammosilene root natural propagation force difference itself, cause tuniclike psammosilene root wild resource quantity drastically to decline, have been used as dilute
There is endangered species to be included in《Chinese Plants Red Data Book》In, belong to Chinese Second Class Key Protected Plant.Since tuniclike psammosilene root medicinal material is mainly by harvesting
Wild resource, harvest cycle is longer, is influenced by external factor such as pest and disease damage, weathers, causes supply is not easy to control to wait reality
Problem.Therefore seek new substitute or new technology to meet the market demand and solve the problems, such as that species conservation is extremely urgent.
Consider, is 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 economic cost of Producing medicinal herbs.Although such as
This, realizes that the floristics commercially produced is limited by means such as cell culture, 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 agrobacterium rhizogenes infects special adventitious root caused by plant cell, have inheritance stability, hormone autotrophic,
The advantages that speed of growth is fast and cometabolism is strong, hairy root is as a kind of transgenic organ, a kind of new medicinal raw material, increasingly by
To the concern of domestic and international researcher.Some researches show that the callus induced as explant using hairy root also keeps hairy
The advantages of root growth speed is fast, inheritance stability.Tuniclike psammosilene root hairy root is prepared and existing outstanding problem during Liquid Culture：
(1) hairy root induction rate is low, and 20% or so, this is because plant cell is determined with characteristic intrinsic in agrobacterium rhizogenes Interaction
Fixed；(2) conglobation property during Liquid Culture makes interior tissue anoxic and nutrient, and gradual aging is even dead, leads to growth speed
Degree declines and then influences crop.
Based on the above issues, the present invention wishes the induction and the culture technique that are further improved tuniclike psammosilene root hairy root, with effective
Solve the problems, such as that tuniclike psammosilene root natural resources lacks and market product is in short supply.
Invention content
Aiming firstly at for the present invention provides a kind of abductive approach of tuniclike psammosilene root feather shaped root system, is to induce gold with agrobacterium rhizogenes
Iron locks the method that explant generates hairy root, including the CuCl of 10~15mmol/L is used before induction2Aqueous solution handles explant
The step of.
The present invention is pioneering to utilize CuCl2Tuniclike psammosilene root blade is handled, blade is infected by agrobacterium rhizogenes, in petiole and leaf margin
Hairy root can occur within 2-4 days at vein, the root of hair time shortens, and can generate within 3~5 days root of hair, and Induction Transformation rate greatly improves,
Reach 60% or more.Then by screening, hairy root fast-growth in no hormone culture-medium.
It is outer for handling as a preferred technical solution, in the abductive approach of tuniclike psammosilene root feather shaped root system of the present invention
The CuCl of implant2Concentration of aqueous solution is 10~12mmol/L.
" processing " described in the above method should be understood as the contact of general significance, preferably use CuCl2Water
Solution impregnates 10~15min of explant.
Explant described in the method for the present invention is the tuniclike psammosilene root aseptic seedling for removing leaf margin surrounding tissue but retaining petiole
Blade.
Specifically, the present invention provides one of the preferred embodiment of abductive approach of above-mentioned tuniclike psammosilene root feather shaped root system, including
Following steps：
(1) tuniclike psammosilene root tests for sterility is taken, leaf margin surrounding tissue is removed, retains petiole, with the CuCl of 10~15mmol/L2
After 10~15min of aqueous solution soaking explant, explant surface moisture is blotted；
(2) with agrobacterium rhizogenes dip dyeing step (1) treated explant, access co-cultures in base, under the conditions of 26 ± 1 DEG C
Light culture is for 24 hours；
The co-cultivation base is the MS solid mediums of the acetosyringone containing 100umol/L；
(3) culture of step (2) is transferred to antibacterial subculture medium, and 3~6d squamous subcultures are primary, until bacterium is cleared；
The antibacterial subculture medium is containing 500mg/L Cefotaxime Sodiums without hormone 1/2MS solid mediums；
The antibacterial squamous subculture is：Under the conditions of 26 ± 1 DEG C, light culture is stood；
(4) the culture access of step (3) carries out black dull suspension shaken cultivation without hormone 1/2MS liquid minimal mediums,
Oscillation shaking speed is 110~120rmin-1。
It is outer with agrobacterium rhizogenes dip dyeing described in the step (2) in the abductive approach of above-mentioned tuniclike psammosilene root feather shaped root system
The method of implant includes the following steps：
(a) agrobacterium rhizogenes ACCC10060 bacterium solutions are inoculated in YEB solid mediums, 25 DEG C of ± 1 time 2~3d of light culture
It obtains agrobacterium rhizogenes ACCC10060 and clones thalline；
(b) picking agrobacterium rhizogenes ACCC10060 clones thalline, is inoculated in YEB culture mediums, 25 DEG C of ± 1 shaken cultivations；
It is 0.6 to take the bacterium solution that 1 parts by volume OD values are 0.6~0.8 to be placed in shaken cultivation to OD values in 100 parts by volume YEB fluid nutrient mediums
When collect bacterium solution, 3500r.m-1Lower centrifugation collects thalline in 10 minutes；
(c) gained thalline is uniformly blended into the MS solid mediums of 100 parts by volume acetosyringones containing 100umol/L；
(d) 10~15 minutes in the culture medium by the dip dyeing of tuniclike psammosilene root explant prepared by step (c), table is cleaned after taking-up
Face obtains the explant of agrobacterium rhizogenes dip dyeing.
Specific implementation mode
Without specified otherwise, using oleanolic acid standard items as reference substance in the present invention, surveyed using vanilla root rot colorimetric method
Determine total saponin content in tuniclike psammosilene root tissue or culture (Li Jingbin etc.,《CHINA JOURNAL OF CHINESE MATERIA MEDICA》2011,36 (5)：547).Its
In, the culture of tuniclike psammosilene root includes but not limited to hairy root, callus, suspended cell culture etc., total saposins in culture
It extracts and is described as follows with the specific method measured：
The extraction of total saposins：Quality is weighed after the culture of acquisition is dried and is crushed, and three are extracted with 70% alcohol reflux
It is secondary；Merge extracting solution and ethyl alcohol is recovered under reduced pressure to dry cream；It will get dry extract with 2 times of amount water dissolutions, filtering, filtrate equimultiple oil
Ether is except lipid materials such as depigmentatons；Aqueous after grease removal is extracted five times with equimultiple water-saturated n-butanol, obtained n-butanol extraction
Liquid is taken to be evaporated to dryness under reduced pressure cream.It gets dry extract and is dried under reduced pressure to constant weight to get to the thick total saposins of tuniclike psammosilene root.
This experimental selection oleanolic acid standard items are reference substance, total using vanilla root rot colorimetric method for determining tuniclike psammosilene root
Saponin content.
The preparation of reference substance solution：About 20mg oleanolic acids standard items (120 DEG C are dried under reduced pressure to constant weight) accurately are weighed, are used
After absolute ethyl alcohol dissolving, constant volume is in 50mL volumetric flasks to get oleanolic acid reference substance liquid.
The preparation of sample solution：Accurately about 30mg is weighed to dry to the thick total saposins of tuniclike psammosilene root of constant weight, it is molten with absolute ethyl alcohol
Xie Hou, constant volume is in 50mL volumetric flasks to get sample solution.
The determination of maximum absorption wavelength：It is accurate to measure standard solution 0.6mL and sample solution 5mL, respectively it is placed in 10mL capacity
In bottle, solvent is removed in volatilization in 75 DEG C of water-baths, adds a concentration of 5% vanillic aldehyde-glacial acetic acid solution (50mg newly prepared
Vanillic aldehyde is dissolved in 10mL glacial acetic acid) 0.4mL, perchloric acid 1.6mL shakes up, in 60 DEG C of heating water bath 25min, after taking-up immediately
3min is cooled down with flowing water, is shaken up, ethyl acetate constant volume is added, the Duplicate Samples to be not added with standard solution and sample solution are sky
In vain, UV scanning is carried out at 400~800nm, as a result contrast solution has larger absorption with sample solution at 535nm.
The drafting of standard curve：It is accurate draw oleanolic acid standard solution 0.20,0.40,0.60,0.80,1.00,
1.20mL is respectively put into 1~No. 6 10mL measuring bottle, ethyl alcohol is flung in 70 DEG C of water-baths, and light absorption value (A) is measured at 535nm.
Each concentration parallel determination 3 times, using the average value of A values 3 times as ordinate, oleanolic acid concentration C (mgmL-1) painted for abscissa
Calibration curve processed, calculating its regression equation is：Y=30.871C -0.1503, r=0.9997.
Tuniclike psammosilene root saponin content measures：Correct amount takes sample solution 5mL, is placed in 10mL volumetric flasks, in 75 DEG C of water-baths
Volatilization removes solvent, adds a concentration of 5% vanillic aldehyde-glacial acetic acid solution 0.4mL newly prepared, and perchloric acid 1.6mL shakes up,
In 60 DEG C of heating water bath 25min, 3min is cooled down with flowing water immediately after taking-up, is shaken up, ethyl acetate constant volume is added, to be not added with sample
The Duplicate Samples of solution are blank, and light absorption value is measured at 535nm, and parallel determination 3 times calculates average value, is returned according to standard curve
Return equation calculation total saponin content.
Precision, stability, reproducibility, mean sample recovery rate data are satisfied by pharmacopoeial requirements in methodology validation.
Following specific embodiments of the present invention are to further illustrate the present invention, and are not construed as any to the content of present invention
Restriction in meaning.In the present invention, unless otherwise specified, it is carbon source that culture medium, which uses 30g/L sucrose,.
The efficient induction of 1 tuniclike psammosilene root feather shaped root system of embodiment
(1) tuniclike psammosilene root tests for sterility is taken, leaf margin surrounding tissue is removed, with the CuCl of 10mmol/L2Outside aqueous solution soaking
After implant 10min, aseptic filter paper blots explant excess surface moisture；
(2) with agrobacterium rhizogenes dip dyeing step (1) treated explant：
2a. agrobacterium rhizogenes ACCC10060 bacterium solutions are inoculated in YEB solid mediums, 25 DEG C of ± 1 time 2~3d of light culture
It obtains agrobacterium rhizogenes ACCC10060 and clones thalline；
2b. picking agrobacterium rhizogenes ACCC10060 clones thalline, is inoculated in YEB culture mediums, 25 DEG C of ± 1 shaken cultivations；
It takes the bacterium solution that 1ml OD values are 0.6~0.8 to be placed in 100ml YEB fluid nutrient mediums when shaken cultivation to OD values is 0.6 to collect
Bacterium solution centrifuges under 3500rpm 10 minutes and collects thalline；
Thalline is uniformly blended into the MS solid mediums of 100ml acetosyringones containing 100umol/L obtained by 2c.；
2d. disseminates tuniclike psammosilene root explant in the culture medium prepared by step (c) 10~15 minutes, and table is cleaned after taking-up
Face obtains the explant of agrobacterium rhizogenes dip dyeing.
The obtained explant access through agrobacterium rhizogenes dip dyeing is co-cultured in base, light culture under the conditions of 26 ± 1 DEG C
24h；The co-cultivation base is the MS solid mediums of the acetosyringone containing 100umol/L；
(3) culture of step (2) is transferred to antibacterial subculture medium, and 3~6d squamous subcultures are primary, until bacterium is cleared；
The antibacterial subculture medium is containing 500mg/L Cefotaxime Sodiums without hormone 1/2MS solid mediums；
The antibacterial squamous subculture is：Under the conditions of 26 ± 1 DEG C, light culture is stood；
(4) the culture access of step (3) carries out black dull suspension shaken cultivation without hormone 1/2MS liquid minimal mediums,
Oscillation shaking speed is 110~120rmin-1。
In the present embodiment incubation, 2-4 days after inoculation, hairy root can be occurred at petiole and leaf margin vein, 3~5
It can generate root of hair, and the root of hair time is shorter；Induction Transformation rate is more than 60%；Then by screening, hairy root is trained in no hormone
Support fast-growth in base.
Embodiment 2：The comparison of tuniclike psammosilene root feather shaped root system induces
According to the method for embodiment 1, but omit in step (1) with the CuCl of 10mmol/L2Aqueous solution soaking explant
The step of 10min, carries out subsequent step with clean tuniclike psammosilene root explant, and induction tuniclike psammosilene root hairy root generates.
In the present embodiment incubation, 4-6 days after inoculation, hairy root occurs at petiole and leaf margin vein, produces within 5~8 days
Hair tonic root, root of hair time are longer relative to embodiment 1；Induction Transformation rate is less than 30%..
Embodiment 3：The induction of tuniclike psammosilene root callus
Respectively with the good hairy root of growth conditions in Examples 1 and 2, and removal leaf margin surrounding tissue tuniclike psammosilene root without
Vaccine blade is explant, is labeled as explant I, explant II and explant III, respectively evoked callus.
(1) material that will act as explant cuts into segment, is inoculated in addition 1.0mg/L 2,4-D+1.0mg/L 6-BA+
It on the MS solid mediums of 2.0mg/L NAA+30g/L sucrose, is placed in 25 ± 1 DEG C of light culture rooms and cultivates, induce within 3~4 weeks
Callus；
(2) it after detaching the callus induced with explant, is inoculated in added with 0.5mg/L 2,4-D+1.0mg/L
Squamous subculture is carried out on the MS culture mediums of 6-BA+0.5mg/L NAA+0.2mg/L KT+30g/L sucrose, condition of culture is 25 ± 1
DEG C, light culture.25d or so subcultures on this culture medium are primary.
The equal color and luster of callus that explant I is newly induced is dim, soft, moisture is larger and the speed of growth is slower, by 5
Secondary squamous subculture, callus gradually show quality it is loose, it is easy dispersion, do not secrete mucus, white graininess, approximately pass through
10 times or so squamous subcultures, callus growth speed at this time and highly stable suspend training suitable for establishing cell
The system of supporting.
Under similarity condition, the callus that explant II is induced passes through 15 squamous subcultures, the growth of callus
Speed and situation are similar to the explant I callus of 10 subcultures, can be used for establishing cell suspension cultures system.
Under similarity condition, the callus that explant III is induced passes through 18 squamous subcultures, the growth of callus
Speed and situation are similar to the explant I callus of 10 subcultures, can be used for establishing cell suspension cultures system.
Embodiment 4：Establish tuniclike psammosilene root plant cell suspension cultures
With in embodiment 3, respectively with explant I inductions and the callus through 10 subcultures and explant III inductions
Gained and callus through 18 subcultures are material, establish golden cable plant cell suspension cultures i and iii respectively.
(1) loose quality, easy dispersion, white granular callus not slimy are chosen for establishing suspension cell
Cultivating system；
(2) callus of selection is immersed in the sterile oxalic acid calcium solution of a concentration of 10mmol/L, static processing 30min
Callus is taken out afterwards and blots surface moisture with aseptic filter paper；
(3) by step (2) treated callus according to 4~6% (gFW/ml, callus fresh weight g/ml culture mediums)
Inoculum concentration be seeded in suspension medium (150ml triangular flasks, liquid amount 50ml), 25 ± 1 DEG C, on 110 ± 5r/min shaking tables
Light culture；The suspension medium is mMS+1.5mg/L 2,4-D+0.5mg/L 6-BA+0.25mg/L NAA+0.1mg/L
KT+30g sucrose/L；MMS culture mediums therein are the improvement basal medium of the present invention, are added on the basis of MS culture mediums
KCl makes potassium concentration reach 50mmol/L and obtain.
The period of above-mentioned steps (3) suspension culture is 20d.
The initial several generations cell granulations of plant cell suspension cultures i and iii are larger, can be by being sieved under aseptic condition
The methods of bulky grain is removed, plant cell suspension cultures i passes through 5-8 squamous subculture, and suspension culture system gradually tends to
Stablize.Plant cell suspension cultures iii passes through 12 squamous subcultures, and suspension culture system gradually tends towards stability, but growth conditions
Not as good as plant cell suspension cultures i.
5. tuniclike psammosilene root plant cell suspension cultures condition optimizing of embodiment
(1) MS, 1/2MS, mMS, B5,1/2B are selected5And N66 kinds of culture mediums do minimal medium experiment, speed of growth difference
For：0.43,0.72,0.85,0.38,0.40 and 0.35gL-1·d-1(DW), it is most to be determined by experiment mMS fluid nutrient mediums
Good minimal medium.
Wherein, mMS culture mediums are the improvement basal medium of the present invention, are to add KCl on the basis of MS culture mediums, make
Potassium concentration reaches 50mmol/L and obtains.
(2) on the basis of mMS is best minimal medium, 2 are selected, tetra- kinds of hormones of 4-D, 6-BA, NAA, KT carry out just
Experiment is handed over, the experimental results showed that, the best hormone prescription of hairy root-derived callus suspension culture is 1.5mg/L2,4-D+0.5mg/
L 6-BA+0.25mg/L NAA+0.1mg/L KT+30g/L sucrose；
(3) with mMS+1.5mg/L 2,4-D+0.5mg/L 6-BA+0.25mg/L NAA+0.1mg/L KT+30g/L sucrose
Inoculum concentration experiment is carried out for culture medium, it is final to determine that optimum inoculation amount is 4~6% (w/v, g, fw/ml)；
In conclusion the best medium condition of tuniclike psammosilene root suspension cell culture is：
MMS+1.5mg/L 2,4-D+0.5mg/L 6-BA+0.25mg/L NAA+0.1mg/L KT+30g sucrose/L, wherein
MMS culture mediums are to add KCl on the basis of MS culture mediums, and potassium concentration is made to reach 50mmol/L.
Optimal inoculum concentration is 4~6% (w/v, g (FW)/ml).
6. tuniclike psammosilene root suspension cell of embodiment amplifies cultivating system
According to the fluid nutrient medium formula composition that embodiment 5 determines, culture medium is prepared, sucrose concentration 30g/L utilizes 2.5L
Bubbling style reactor is amplified culture, liquid amount 1.5L, 121 DEG C of moist heat sterilization 20min.In view of the oxygen-supply quantity of reactor
Higher than shaking flask culture, selection is inoculated with the hairy root-derived callus of solid culture with 4% inoculum concentration, and inoculation is placed on 25 ± 1
It is cultivated under the conditions of DEG C.1.5L culture medium inoculated hairy root-derived callus 60g, 28~30d of culture or so, harvest culture fresh weight can
Up to 200g, culture is placed in 60 DEG C of baking ovens and dries by fresh weight growth rate 220% or so, harvest dry weight 12g or so.
Total saponin content measures in 7. tuniclike psammosilene root material of embodiment and culture
The culture of gained in above-described embodiment 1~4 and the saponin content in the tuniclike psammosilene root seedling of material source are carried out
It measures, as a result as shown in table 1 below.It can be seen that：
The saponin content of tuniclike psammosilene root cell culture is above the saponin content of tuniclike psammosilene root original plant, under condition of suspension culture
Saponin content increases than saponin content under the conditions of solid culture, no matter under which kind of condition of culture, hairy root callus group
The saponin content knitted is above common tuniclike psammosilene root callus, and the saponin content of hairy root-derived callus suspended culture cell can
Up to 0.92%, it is not much different with hairy root saponin content 0.97%, illustrates that it maintains the inheritance stability of hairy root, secondary generation
Thank to the strong advantage of ability.
Table 1
Claims (4)
1. the abductive approach of tuniclike psammosilene root feather shaped root system is the side for generating hairy root with agrobacterium rhizogenes induction tuniclike psammosilene root explant
Method, it is characterised in that the CuCl including using 10~15mmol/L before induction2Aqueous solution handles the step of explant；
The explant is the tuniclike psammosilene root tests for sterility for removing leaf margin surrounding tissue but retaining petiole；
The processing is to use CuCl210~15min of aqueous solution soaking explant.
2. according to the method described in claim 1, it is characterized in that, the CuCl2Concentration of aqueous solution is 10~12mmol/L.
3. according to the method described in claim 1, including the following steps：
(1) tuniclike psammosilene root tests for sterility is taken, leaf margin surrounding tissue is removed, retains petiole, with the CuCl of 10~15mmol/L2It is water-soluble
After liquid impregnates 10~15min of explant, explant surface moisture is blotted；
(2) with agrobacterium rhizogenes dip dyeing step (1) treated explant, access is co-cultured in base, is secretly trained under the conditions of 26 ± 1 DEG C
It supports for 24 hours；
The co-cultivation base is the MS solid mediums of the acetosyringone containing 100umol/L；
(3) culture of step (2) is transferred to antibacterial subculture medium, and 3~6d squamous subcultures are primary, until bacterium is cleared；
The antibacterial subculture medium is containing 500mg/L Cefotaxime Sodiums without hormone 1/2MS solid mediums；
The antibacterial squamous subculture is：Under the conditions of 26 ± 1 DEG C, light culture is stood；
(4) the culture access of step (3) carries out black dull suspension shaken cultivation, oscillation without hormone 1/2MS liquid minimal mediums
Shaking speed is 110~120rmin-1。
4. according to the method described in claim 3, it is characterized in that, the side of explant is disseminated in step (2) with agrobacterium rhizogenes
Method includes the following steps：
(a) agrobacterium rhizogenes ACCC10060 bacterium solutions are inoculated in YEB solid mediums, and 2~3d of light culture is obtained at 25 ± 1 DEG C
Agrobacterium rhizogenes ACCC10060 clones thalline；
(b) picking agrobacterium rhizogenes ACCC10060 clones thalline, is inoculated in YEB culture mediums, 25 ± 1 DEG C of shaken cultivations；Take 1
The bacterium solution that parts by volume OD values are 0.6~0.8 is placed in 100 parts by volume YEB fluid nutrient mediums when shaken cultivation to OD values is 0.6 and receives
Collect bacterium solution, 3500rm-1Lower centrifugation collects thalline in 10 minutes；
(c) gained thalline is uniformly blended into the MS solid mediums of 100 parts by volume acetosyringones containing 100umol/L；
(d) 10~15 minutes in the culture medium by the dip dyeing of tuniclike psammosilene root explant prepared by step (c), clean surface after taking-up,
Obtain the explant of agrobacterium rhizogenes dip dyeing.
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Citations (1)
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Patent Citations (1)
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Non-Patent Citations (5)
| Title |
|---|
| Ri质粒诱导药用植物毛状根技术及应用;杨秀淦等;《广东林业科技》;20121231;第28卷(第5期);第67-73页 * |
| Tanshinone production and isoprenoid pathways in Salvia miltiorrhiza hairy roots induced by Ag+ and yeast elicitor;Xiuchun Ge等;《Plant Science》;20051231;第168卷;第487–491页 * |
| 发根农杆菌A4菌株转化红豆草途径及转化组织植株再生研究;徐子勤等;《兰州大学学报（自然科学版）》;19941231;第30卷(第2期);第96-100页 * |
| 毛状根发展现状研究;刘莉莉等;《北方园艺》;20141231(第24期);第178-182页 * |
| 金铁锁毛状根诱导及培养体系的建立;李景滨等;《中国中医杂志》;20110331;第36卷(第5期);第547-551页 * |
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