CN103609439B

CN103609439B - Different strain is on the impact of ginseng Hairy root and application method

Info

Publication number: CN103609439B
Application number: CN201310545021.7A
Authority: CN
Inventors: 王�义; 张美萍; 李维; 蒋世翠; 孙春玉; 王康宇; 杨广顺
Original assignee: Jilin Agricultural University
Current assignee: Jilin Agricultural University
Priority date: 2013-11-02
Filing date: 2013-11-02
Publication date: 2015-12-09
Anticipated expiration: 2033-11-02
Also published as: CN103609439A

Abstract

The present invention relates to different strain to the impact of ginseng Hairy root and application method, belong to field of crop biotechnology.Its process comprises following aspect: (1) explant process; (2) preservation of bacterial classification and activation; (3) optimization of ginseng Hairy root inductive condition; (4) acquisition of ginseng Hairy root and qualification; (6) foundation of ginseng Hairy root regeneration system and condition optimizing; (7) research of physio-biochemical characteristics in Hairy root regeneration plant approach.(8) histological observation of ginseng Hairy root and culture thereof and karyotyping.The present invention with Agrobacterium rhizogenes infect ginseng induction Hairy root, propose Hairy root induction some influence factors and Hairy root regeneration plant process, for ginseng Germ-plasma resources protection, innovation preservation provide a new way, for the breeding of ginseng lays the foundation.

Description

Different strain is on the impact of ginseng Hairy root and application method

Technical field

The present invention relates to different strain to the impact of ginseng Hairy root and application method, belong to field of crop biotechnology.

Background technology

Ginseng (PanaxginsengC.A.Mey.) is Araliaceae (Araliaceaae) panax species, from ancient times to the present, and infinite glamour of always glimmering and sacred brilliance.China is the source region of ginseng, and before more than 2,000 years, our ancestors just find and utilize ginseng disease preventing and treating.China's pharmacy ancient books and records Shennong's Herbal the earliest claims, and " main tonifying the five internal organs, peace spirit, determines soul to ginseng, only palpitation with fear, and except perverse trend, improving eyesight, happy intelligence development, clothes are made light of one's life by commiting suicide and prolonged life for a long time." afterwards, in the medical book such as " bright doctor does not record ", treatise on Febrile Diseases, Tang Materia Medica and Compendium of Material Medica, there is detailed description.

Medical science and pharmacological research prove, ginsenoside is one of principle active component of ginseng; It is the primary bioactivity material of ginseng.Ginsenoside can be divided into by its chemical property; Ginsenoside diol type (A type), comprises Ra, Rb1, Rb2, Rc, Rd, Rh2 etc.; Ginsenoside three alcohol type (Type B), comprises Re, Rf, Rg1, Rg2, Rh1 etc.; Oleanolic acid type (C type).

The carbohydrate contained in ginseng mainly contains monose, oligose polysaccharide and mixed polysaccharide, and total sugar content is 4% ~ 6%.Monose mainly contains glucose, fructose, pectinose, rhamnosyl and wood sugar; Disaccharides in oligose is sucrose, maltose and lactose mainly; Trisaccharide mainly contains ginsengtrisaccharide A, B, C, D etc. four kinds; Polysaccharide major part is water miscible, and the water-soluble polysaccharide in ginseng, stem, leaf and fruit is all mixed polysaccharide.Panaxan is made up of ginseng starch and ginseng pectin's two portions, and wherein ginseng pectin is primary pharmacological activity composition.

The root of ginseng, stem, Ye Zhongjun contain total free aminoacids, and the amino acid contained quantity of its each several part and kind are all not quite similar.Zheng Yinan etc. are separated first and obtain arginine derivative final dedust (AFG) from red ginseng.Containing multiple vitamin B group in ginseng, as VB1, VB2, VB12 and nicotinic acid, niacinamide, pantothenic acid.Also containing multiple inorganic elements, Lee to high application Naa Determination in ginseng, pseudo-ginseng all containing the inorganic elements of more than 18 kinds.

The chemical composition of ginseng essential oil mainly contains three classes: the first kind is sesquiterpenoids, and Equations of The Second Kind is long-chain saturated carboxylic acid class, also has a small amount of arene in addition.Sesquiterpenoids is the main component of ginseng essential oil.

Hairy root has the incomparable advantage of many cells, tissue and organ culture because of its characteristic.Compare with organ culture with culture plant cell, Hairy root culture thing has many outstanding advantages: 1. growth rapidly.The Hairy root speed of growth that the leaf mustard that Huang Juhui etc. cultivate transforms is more than 100 times of non-transformed.2. the high and stability of synthesis capability is strong, does not need to add hormone in culturing process.3. bio-transformation function.Utilize the bio-transformation function of Hairy root can produce many new compounds and 4. in substratum, discharge metabolite, this characteristic of root of hair is conducive to separation and Extraction secondary metabolite.5. by gene engineering method, increase the output of secondary substance, Agrobacterium rhizogenes plasmid is a good transgene carrier, goal gene can be imported in vegetable cell.6. fecundity is strong.Root of hair has the reproductive potential of height, can make synthetic seed and preserve for a long time.7. Hairy root is easy to be separated and easily obtain regeneration plant, and this can be significant by the plant of root regeneration for obtaining transgenic plant or foreign gene being forwarded to.8. Hairy root can help the xylophyta being difficult to take root to take root.

In recent years, natural drug particularly plant medicine be just day by day subject to people's attention and welcome.But because the mankind are to the sharply increase of plant amedica demand, wild resource reduces increasingly, and artificial growth medicinal material faces again the problems such as pesticide residue, heavy metal contamination, deterioration of strains and quality control.

Ginseng, as rare medicinal herbs, is keeping healthy and is medically being widely used.Groundwork concentrates on the researchs such as cultivation, medicine, pharmacology.Less about ginseng Study on Genetic Transformation, especially ginseng Hairy root form generation research is less.The present invention with Agrobacterium rhizogenes infect ginseng induction Hairy root, inquire into affect Hairy root induction some questions and Hairy root regeneration plant process, for ginseng Germ-plasma resources protection, innovate preservation provide a new way, for the breeding of ginseng lays the foundation.

Summary of the invention

The object of the invention is to infect ginseng induction Hairy root with Agrobacterium rhizogenes, some influence factors and the Hairy root regeneration plant process of Hairy root induction are proposed, for ginseng Germ-plasma resources protection, innovation preservation provide a new way, for the breeding of ginseng lays the foundation.

To achieve these goals, technical scheme of the present invention is as follows.

This beneficial effect of the invention is:

Different strain is on the impact of ginseng Hairy root and application method, and its process comprises following aspect:

(1) explant process: (I) ginseng seed is sprouted and process: hide Stratificated treatment through husky, the seed ftractureed, is seeded in dish for cultivating, to shade cultivation, after sowing 1 ~ 2 week seedling grows, with 0.1%L mercury sterilization 6min, sterile water wash 5 times.Its blade, band leaf children's stem and stem section are cut into the segment of 1.0cm, are connected to the parent material that preculture on MS solid medium is used as to transform.(II) ginseng 1 year, 2 years, the process of taking root for 3 years: after surface clean water of being taken root for 3 years by ginseng is clean, running water 3 ~ 4h, after 0.1% mercuric chloride sterilization 10 ~ 20min, sterile water wash 5 times, is cut into the thick thin slice of 0.2cm and is placed on MS solid medium stand-by.

(2) preservation of bacterial classification and activation: the long-term preservation method of Agrobacterium rhizogenes is dissolved in glycerine by Agrobacterium, and preserve under-20 DEG C of conditions, this method can preserve bacterial classification several years.The method of short-term preservation is inoculated in by Agrobacterium rhizogenes on YEB solid medium to cultivate, and after growing bacterial plaque, preserve in 4 DEG C of refrigerators, this kind of method can preserve the bacterial classification several months.

Under aseptic condition, preserve bacterium liquid with inoculating needle picking, rule on YEB solid plate, 28 DEG C be placed in dark condition under cultivate, until grow single bacterium colony.Picking single colony inoculation in 50mL liquid YEB substratum, 120r/min, 28 DEG C, cultivate 24h under dark condition, now YEB liquid nutrient medium becomes muddy faint yellow by the transparent reddish-brown before inoculating, and proves that bacterium normally grows.Getting the bacterium liquid that 1mL activated adds in the YEB liquid nutrient medium of 50mL, and 120r/min, cultivates under 28 DEG C of dark condition, and activation bacterium liquid reaches growth logarithmic phase, and now bacterium liquid just can as infection use.

(3) optimization of ginseng Hairy root inductive condition:

(3a) different strain is on the impact of ginseng Hairy root inductivity: select Agrobacterium rhizogenes R1601,8196, A4, rolA, rolB, rolC six bacterial classifications access YEB liquid nutrient medium after flat board activation, treat that its bacterium liquid growth concentration reaches OD ₆₀₀be used for when=0.6 infecting.The ginseng seedling blade of 2 ~ 3 weeks seedling ages, good children is tender for growth selection, and the fritter being aseptically cut into 0.5cm × 0.5cm drops in bacterium liquid, and 25 ± 1 DEG C of shaking table 100 ~ 120r/min infect 15 ~ 30min, take out under aseptic condition.Sterilizing filter paper blots surperficial bacterium liquid, is connected on MS substratum, investigates different strain to the impact of ginseng Hairy root inductivity.

(3b) different explants is on the impact of ginseng Hairy root inductivity: select explant to be the blade of 2 ~ 3 weeks seedling age ginseng seedlings, stem section, petiole and ginseng brood cell, ginseng callus, 1,2,3 year raw ginseng, selection bacterial classification is Agrobacterium rhizogenesA4, investigates the selection of ginseng explant to the impact of Hairy root inductivity.Its Leaf, stem section, petiole, ginseng brood cell are leaf disk method, and immerged time is 15 ~ 30min, and ginseng callus, ginseng are taken root for dripping method for 1,2,3 years.

(3c) pre-incubation time is on the impact of ginseng Hairy root inductivity: select MS solid medium to be preculture matrix, the stem section selecting 2 ~ 3 weeks ginseng seedlings is material, pre-incubation time elects 12h, 24h, 48h, 72h, 96h as, to investigate the impact of pre-incubation time on ginseng Hairy root inductivity.

(3d) NAA concentration is on the impact of ginseng Hairy root inductivity: in pre-incubated MS minimum medium, add the induction that a certain amount of NAA is of value to Hairy root.With the stem section of 2 ~ 3 weeks ginseng seedlings for material, the concentration of adding NAA in MS minimum medium is chosen as 0mg/L, 2mg/L, 4mg/L, 6mg/L, compares Hairy root inductivity and investigates NAA optimal concentration.

(3e) matrix together incubation time on the impact of ginseng Hairy root inductivity: generally speaking, high salt culture medium such as MS, LS etc. contribute to the formation of Hairy root, low salt culture medium such as B5 etc. then contributes to bacterial multiplication, so in sterilizing process after Hairy root is formed, degerming more difficult.This experimental selection Dual culture time be 12h, 24h, 36,48h, Dual culture substratum elects MS, 1/2MS as.Explant is the blade of seedling, and precultivation medium is the MS+NAA2.0mg/L time is 48h, and bacterial classification is A4, with investigate matrix together incubation time on the impact of ginseng Hairy root inductivity.

(3f) bacterial concentration is on the impact of ginseng Hairy root inductivity: the bacterium liquid YEB liquid prepared is diluted to OD ₆₀₀value is 0.2,0.4,0.6,0.8 and 1.0, ginseng seedling petiole is dropped in the bacterium liquid of different concns and contaminate 15 ~ 30min, 25 ± 1 DEG C of shaking table 100 ~ 120r/min, then taking-up aseptic filter paper blots the bacterium liquid on surface, after 2d cultivated by Dual culture base, be transferred to again on MS Dual culture substratum, change a subculture every 7d.

(3g) AS and time of infection are on the impact of ginseng Hairy root inductivity: single colony inoculation of picking activates in the 30mLYEB liquid nutrient medium adding AS, AS concentration elects 0,100,200 μm of ol/L shaking culture as, the stem section of ginseng seedling immersed when its optical density value is 0.6, time of infection elects 10min, 15min, 20min, 25min and 30min as.

(3h) Cef concentration 200 be chosen as on antibacterial and concentration that is impact that is that infect: Cef, 300,400,500,600mg/L, degerming matrix is chosen as the basic solid substrate of MS.The comprehensive fungistatic effect of Integrated comparative, pollution rate, acquisition root of hair number three indexs, investigate the optimal concentration of Cef.

(4) acquisition of ginseng Hairy root and qualification:

(4a) acquisition of ginseng poultry: 1. explant preculture: after the sterilization of ginseng seedling, its blade is cut into the fritter of 1.0cm × 1.0cm, petiole and stem are cut into the long section of 1.0cm, ginseng 1 year, 2 years, 3 years running water of taking root remove surperficial surface dust, after mercuric chloride sterilization, distilled water washes down for 3 ~ 5 times, be cut into 0.2 ~ 0.5cm thin slice depending on material thickness, above material be connected in MS+NAA2mg/L substratum and cultivate 48h under dark condition and be used for infecting.2. infect and Dual culture: on Bechtop, by pre-incubated explant, transfer in the triangular flask being equipped with and getting bacterium liquid ready, 120r/min is placed on after sealing, shaking table under dark condition infects 10 ~ 30min, metainfective explant aseptic filter paper blots surperficial bacterium liquid, is inoculated in MS solid medium, Dual culture 48h under dark, 25 DEG C of conditions.3. degerming cultivation: by the infected material aseptic water washing after Dual culture three times, aseptic filter paper blots the sterilized water on explant surface, is transferred on the MS solid medium containing Cef500mg/L and cultivates.Degerming cultivation under dark, 25 DEG C of conditions.

(4b) induction of ginseng callus and 1 year, 2 years, 3 years Hairy root of taking root: draw with micropipet the bacterial strain 8 μ L activated and drip on the ginseng callus tangent plane of receiving MS solid medium and ginseng cut into slices.Avoid bacterium drop on MS solid medium, 25 DEG C, cultivate under dark condition.The death of 2 weeks rear removing brownization, transfer on fresh MS solid medium and continue to cultivate.

(4c) PCR Molecular Detection: the Hairy root infecting acquisition morphologically embodies obvious difference compared with normal ginseng, as Hairy root can without ramp on the substratum of hormone, the features such as many hairs, multi-branched, apogetropisms.In Hairy root liquid medium within, growth rapidly, is greater than ginseng callus suspension culture or unconverted root culturd, and these phenotypes can for judging that Hairy root provides morphologic foundation.By PCR method to plasmid T _lthe rolB gene test of-DNA will provide molecular biological foundation further.

1. design of primers and synthesis: plasmid T _l-DNA sequence analysis result, devises special primer prolc1 and prolc2 of rolB gene.

Primer 1:5 '-GCTCTTGCAGTGCTAGATTT-3 ';

Primer 2: 5 '-GAAGGTGCAAGCTACCTCTC-3 ';

Utilize above primer by the fragment of rolB gene on pcr amplification to T-DNA, expection length is 423bp, and by Shanghai, the biological company limited of raw work synthesizes.

2. CTAB method extracts ginseng Hairy root STb gene;

3. the Ri plasmid of Agrobacterium rhizogenes is extracted;

4. PCR loop parameter

5. PCR reaction system

Ultrapure water is settled to 20 μ L

6. agargel electrophoresis, takes a picture under imaging system.

(5) mensuration of ginseng Hairy root liquid and solid culture increment and total saponin content:

Hairy root liquid culture adopts 1/2MS liquid nutrient medium to be minimum medium (3% sucrose, pH value 5.8 ~ 6.0), the access of the Hairy root tip of a root of 10 eugonic about 2cm is equipped with in 30mL1/2MS liquid nutrient medium, temperature is 24 ± 1 DEG C, shaking speed 100r/min, every 5d sampling, measure the content of Hairy root dry weight and Radix Ginseng total saponins, measure one and a half months.All data are the mean value of 3 bottles, repeat 3 times.

Total saponin content in drawing standard curve determination Hairy root: 1. get Rg10.020g and be dissolved in methyl alcohol, constant volume is 2mg/mL; 2. Rg1 reference substance solution 30,60,90,120,150 μ L is got; 3. be placed in 5 test tubes (10mL, tool plug) respectively, methanol constant volume is 150 μ L, compares with corresponding reagent; 4. test tube is placed in ice-water bath and adds 5% Vanillin alcoholic solution and 72% sulfuric acid shakes up, 60 DEG C of water bath with thermostatic control 20min; 5. detect at 755 spectrophotometer 544nm places after ice-water bath 15min.Take ginseng as standard substance colorimetric estimation, according to typical curve Y=503.29X+4.456, R ²=0.9924 tries to achieve sample saponin content (%), and typical curve is see accompanying drawing 1.

The mensuration of total saponins in Hairy root: get the fresh Hairy root that certain quantity of fluid is cultivated, after distilled water flushing, blot the moisture on Hairy root surface with filter paper, weighs Hairy root fresh weight.Then in vacuum drying oven, be dried to constant weight at 80 DEG C, weigh.Dried Hairy root is pulverized, puts into triangular flask, add the alcohol solvent that a certain amount of concentration is 40%, and triangle bottleneck sealed membrane is sealed.Be placed in ultra-sonic generator and carry out separation and Extraction, extraction time 15min, ultrasonic frequency is 28KHz, extracts twice.5% Vanillin alcoholic solution and 72%H is added in ice-water bath ₂sO ₄dyeing, 60 DEG C of water bath with thermostatic control 10min, ice-water bath 15min, survey absorbancy in 544nm place.

(6) foundation of ginseng Hairy root regeneration system and condition optimizing:

(6a) induction of ginseng Hairy root callus: with ginseng Hairy root for explant, be placed in interpolation different concns, 2 of various combination, on the MS substratum of the growth regulatory substances such as 4-D, BA, KT, sucrose concentration is 30g/L, agar 6.5g/L, pH value is adjusted to 6.0 (before sterilizings), and sterilization pressure is that 0.1MP continues 15 ~ 20min.Under being positioned over different illumination intensity, inducing temperature is 24 ± 1 DEG C, periodicity of illumination is 12h, the low light level is cultivated, add up callus induction rate (producing the explant number/explant sum of callus) after 12d and analyze all kinds of growth regulatory substance and intensity of illumination to the size of callus induction effect, screen best kind and concentration combination and intensity of illumination.

(6b) organ of ginseng Hairy root occurs:

1. hormon concentration, combination and illumination are on the impact of Multiple Buds growth and differ entiation: the different concns getting KT, IBAGA35.0mg/L growth regulatory substance of different concns and combination respectively combines and processes culture, analyze the impact of each material on culture growth, differentiation, filter out the best medium formula that growing way and biomass all reach desirable level.Get dark, the low light level, high light three illumination conditions, observe light to the impact of culture.Wherein the low light level is normal daylighting degree (about 1000Lx) in culturing room, and the photoperiod is with normal daily variation.High light is provided (about 2000Lx) by fluorescent lamp, and the photoperiod is 10h/d.

2. hormon proportioning is on the impact of taking root: NAA, IBA, GA of getting different concns and combination respectively ₃the different concns combination of 5.0mg/L growth regulatory substance processes culture, analyzes each material to root growing way and the impact of situation of taking root, and filters out the best medium formula that growing way and biomass all reach desirable level.

(7) research of physio-biochemical characteristics in Hairy root regeneration plant approach:

(7a) Coomassie brilliant G-250 Determination Staining protein content:

Bovine serum albumin is made into the standard protein solution containing protein 100 μ g/mL.Being X-coordinate with protein concn, take absorbancy as ordinate zou drawing standard curve: Y=0.0052X-0.0253R ²=0.9905;

Take each 1g of fresh sample and put into mortar, add 2mL distilled water and grind to form homogenate, transfer in centrifuge tube, use 6mL distilled water wash mortar again, washings is collected in same centrifuge tube, under room temperature (20 ~ 25 DEG C), place 1h fully to extract, then the centrifugal 20min of 4000r/min, discard precipitation, supernatant liquor proceeds to 10mL volumetric flask, and with distilled water constant volume.

Separately get two 10mL tool plug test tubes, pipette samples extracting solution of protein 0.1mL respectively, put into tool plug scale test tube, adding 5mL Coomassie brilliant G-250 solution, fully mix, is contrast with blank after placing 2min, colorimetric under 595nm, record light absorption value, and check in protein content by typical curve, calculate according to the following formula:

The typical curve value (μ g) that in formula, X-absorbancy per sample checks in;

V _t-extracting solution cumulative volume (mL);

W-sample Fresh Yuxincao (g);

V _sapplication of sample amount (mL) during-mensuration;

N-extension rate;

(7b) anthrone method measures soluble sugar content:

Analytical pure glucose is dried to constant weight at 80 DEG C, accurately takes 0.100g, be configured to the Standard glucose solution of 100 μ g/mL.Being X-coordinate with glucose concn, take absorbancy as ordinate zou drawing standard curve: Y=0.0064X+0.0035R ²=0.9991;

Take material 0.20g, shred and put into Zhi Kedu test tube, add 8mL distilled water, plastic film sealing, in boiling water, extract 30min (extracting 2 times), extracting liquid filtering enters in 25mL volumetric flask, repeatedly rinse test tube and residue, be settled to scale, draw 0.5mL sample liquid in test tube, adding distil water 1.5mL, add 5mL anthrone sulphate reagent, densitometric under 630nm wavelength, calculate according to the following formula:

The sugar amount (μ g) that in formula, C-checks in from typical curve; V _t-extracting solution cumulative volume (mL); V _sthe sample volume (mL) of taking during-mensuration; W-tissue weight (g); N-extension rate;

(7c) mensuration of starch content: accurately take the pure starch of 100mg, be configured to the reference liquid of the starch-containing 100 μ g of every milli L, being X-coordinate with starch concentration, take absorbancy as ordinate zou drawing standard curve: Y=0.0021X+0.0359R ²=0.9658;

By extracting the later residue of soluble sugar, moving in 50mL volumetric flask, adding 20mL hot distilled water, put into boiling water bath and boil 15min, then add 9.2mol/LHCLO ₄2mL extracts 15min, after cooling, and mixing, the centrifugal 10min of 2500r/min, and use distilled water constant volume, add 5mL anthrone sulphate reagent, densitometric under 630nm wavelength, calculate according to the following formula:

The starch quality (μ g) that in formula, C-is obtained by typical curve; V-extracts liquid measure (mL); W-tissue weight (g);

Liquid draw volume (mL) during a-colour developing;

(7d) Peroxidase Activity Determination: the preparation of reaction mixture: 100mol/LpH6.0 phosphoric acid buffer 50mL is in beaker, add methyl catechol 28 μ l, heated and stirred on magnetic stirring apparatus, until methyl catechol dissolves, after solution cooling, add 30%H ₂o ₂19 μ L, mix, and are stored in refrigerator for subsequent use.

The 2g that draws materials puts into mortar, adds 5mLpH6.0 phosphoric acid buffer and grinds to form homogenate, with the centrifugal 15min of 4000r/min, supernatant liquor proceeds in 20mL volumetric flask, and residue extracts once with 5mL phosphoric acid buffer again, and supernatant liquor is incorporated in volumetric flask, be settled to scale, for subsequent use under storing in low temperature.

Get mixed solution 3mL and phosphoric acid buffer 1mL for contrast, mixed solution 3mL adds enzyme liquid 1mL and starts timing immediately, surveys absorbance under 470nm wavelength, every the 1min number of degrees 1 time, calculates according to the following formula:

Δ A in formula ₄₇₀the internal absorbance change of-reaction times; W-material weight (g); The T-reaction times (min);

V _t-extract enzyme liquid cumulative volume (mL); V _senzyme liquid long-pending (mL) is taken during-mensuration;

(7e) catalase activity measures:

Get the phosphoric acid buffer that each 1g of different times material adds the precooled pH7.8 of 3mL, put into mortar and grind to form homogenate, be transferred in 25mL volumetric flask, rinse mortar several merging damping fluid and be settled to scale, be placed in 5 DEG C of refrigerators to leave standstill 10min and get liquid, the centrifugal 15min of 4000r/min, gets supernatant liquor recentrifuge, obtains supernatant liquor 5 DEG C and saves backup.

Get 3,10mL test tube, wherein two is mensuration one is blank, specifically in table 1.

Table 1 activity of catalase measures

Peroxide unit: mL

S0 pipe boils 1min in boiling water, cooling.The 25 DEG C of preheatings of all test tubes, add the H of 0.3mL0.1mol/L by pipe ₂o ₂, timing immediately, 240nm surveys light absorption value, at interval of 1min reading once:

{ΔA}_{240} = A_{S 0} - \frac{A_{S 1} + A_{S 2}}{2}

A in formula _s0-reaction times internal reference pipe absorbancy; A _s1, A _s2-sample determination pipe absorbancy;

W-sample Fresh Yuxincao (g); The t-reaction times (min); V _t-extract enzyme liquid cumulative volume (mL);

V _senzyme liquid long-pending (mL) is taken during-mensuration; 0.1 represents A ₂₄₀decline 0.1 time a unit of enzyme activity (U);

(7f) polyphenol oxidase activity measures:

Get 2g somatic embryo occur in each period material be placed in precooling mortar, add 0.2gPVP, 5mL0.05mol/LpH8.0 phosphoric acid buffer and fully grind to form homogenate.Centrifugal 15min under 3000r/min4 DEG C of condition, gets supernatant r/min and enters in 25mL volumetric flask, again extract residue with damping fluid, and merge supernatant, constant volume, cryopreservation is for subsequent use, in table 2.

Table 2 polyphenol oxidase enzyme activity determination

Oxidase unit: mL

30 DEG C of constant temperature water bath 10min, measure light absorption value under 398nm wavelength.

A ₃₉₈the internal absorbance change of-reaction times; W-material weight (g); The t-reaction times (min);

(8) histological observation of ginseng Hairy root and culture thereof and karyotyping:

(8a) paraffin section and displaing microstructure observing: 1. fix: choose Hairy root, the callus of induction of hairy roots and embryo callus, be divided into the fritter being less than about 1cm, 24h fixed by material FAA stationary liquid, and r/min enters 70% alcohol and is stored in 4 DEG C of refrigerators; 2. dehydration, transparent, saturating wax and embedding: through 70% under room temperature, 85%, 95%, 100%, in the ethanol of 100%, dewater each 2h, ethanol: dimethylbenzene=1:1 spends the night, the each 1.5h of pure dimethylbenzene secondary, dimethylbenzene: paraffin=1:1, transparent, the saturating wax process of 50 DEG C of 12h, paraffin refined wax secondary 60 DEG C of each 12h, finally be embedded in melt paraffin, to be cooledly solidify; 3. section, paster: wax stone, after finishing, is cut into the continuous wax band of 10 μm, then carries out paster with rotary microtome, 35 DEG C of exhibition sheets dry 12h; 4. dyeing, mounting: siderotil-hematoxylin staining dyeing, balsam mounting, observes under TS100 type inverted microscope and take a picture.

(8b) karyotyping: 1. draw materials and pre-treatment: get the Hairy root tip of a root 0.5cm when morning 9 ~ 10, callus 0.5cm × 0.5cm fritter, Multiple Buds 0.5cm blade tip with santochlor solution at room temperature treatment 6 ~ 12h.2. fix: pretreated is stated material distilled water flushing 3 ~ 5 times, then move in Ka Nuoshi stationary liquid and fix 24h under 4 ~ 15 DEG C of conditions, distilled water flushing, put into 0.075mol/L liquid hypotonic, under 25 ~ 30 DEG C of conditions, Hairy root process 6min, callus and bud 30min, proceed in 70% ethanol after 70% alcohol flushing 2 times and save backup.3. acidolysis: material is taken out from 70% ethanol, use distilled water rinsing. then put at the 1mol/LHCI of 60 DEG C of water-bath preheatings, dissociate 10 ~ 15min under 60 DEG C of constant temperatures, takes out with distilled water flushing 2 ~ 3 times (about 10min).4. film-making: the material after rinsing that dissociates is placed in 30 ~ 40 DEG C, the siderotil aqueous solution of 4% dyes 1h, distilled water wash 4 ~ 5 times. each about 5min, is placed in the 5% phenodin aqueous solution and dyes 2h, distilled water flushing 5 ~ 10min, the acetic acid 20min of 45%, compressing tablet.During compressing tablet, get the tip of a root with tweezers and be placed on slide glass, cut off root tips meristematic tissue.Be cut into thin slice, drip the acetic acid of 45%, smash material to pieces rapidly, add cover glass compressing tablet.The slice, thin piece selecting Chromosome spread good is freezing takes off cover plate, puts into baking oven, 40 DEG C of dry 1h.More than seasoning 24h under room temperature, dimethylbenzene is transparent, neutral gum mounting.

Further, the CTAB method in above-mentioned steps (4c) extracts ginseng Hairy root STb gene step:

(1) get 280mg to add 10%PVP through the ginseng Hairy root of succeeding transfer culture in liquid nitrogen, be fully ground to powdery (about 30s ~ 1min);

(2) be carefully transferred in the centrifuge tube of 1.5mL with spoon, add the Extraction buffer of 660 μ L2 × CTAB of 65 DEG C of preheatings, 65 DEG C of water-bath 45min, vibrate once gently every 5 ~ 10min;

(3) take out Eppendorff pipe and be cooled to room temperature, add isopyknic chloroform: primary isoamyl alcohol (24:1), mixing, 12000r/min, centrifugal 10min;

(4) cut off the rifle head of front with one, carefully supernatant liquor is transferred in a new 1.5mL centrifuge tube, and add the dehydrated alcohol of-20 DEG C of precoolings of two volumes, namely see thread precipitation, place 30min at-20 DEG C, 12000r/min, centrifugal 5min, obtains pale yellow precipitate;

(5) incline supernatant liquor, and to exhaust residual solution with thieving paper, adds 300 μ L ultrapure waters and again dissolve, then add the dehydrated alcohol 600 μ L of-20 DEG C of precoolings, precipitates overnight;

(6) 12000r/min, centrifugal 10min, obtain the gelatinous precipitate of white.Incline supernatant liquor, and to exhaust residual solution with thieving paper, adds 70% ethanol 800 μ L washing precipitation 2 ~ 3 times;

(7) exhaust residual solution, puts Bechtop and dry up (about needing 10 ~ 15min);

(8) add 40 ~ 60 μ L ultrapure waters, dissolution precipitation, add 3 μ LRnase, after room temperature places 30min, preserve at-20 DEG C.

Further, the Ri plasmid procedure extracting Agrobacterium rhizogenes in above-mentioned steps (4c) is:

(1) the mono-bacterium colony of picking R1601 is in 10mLYEB substratum, 28 DEG C, 120r/min, incubated overnight;

(2) draw the cultured bacterium liquid of 8mL, add in 10mL centrifuge tube, 4000r/min, centrifugal 5min, incline nutrient solution, adds the STE of 2mL precooling, Eddy diffusion cell.Draw 1mL bacteria suspension respectively, add in 1.5mL centrifuge tube, the centrifugal 2min of 12000r/min, incline nutrient solution, obtains appropriate somatic cells;

(3) enter the SolutionI that 200 μ L ice baths are crossed, vortex oscillation makes cell be uniformly distributed, and ambient temperatare puts 10min;

(4) add the fresh SolutionII configured of 400 μ L, put upside down mixing gently, ice bath 5min;

(5) add 300 μ LSolutionIII, repeatedly put upside down for several times, solution is uniformly dispersed in the cellular lysate thing of thickness, then puts 5min on ice;

(6) the centrifugal 10min of 12000r/min, careful Aspirate supernatant adds in 1.5mL centrifuge tube;

(7) isopyknic saturated phenol, chloroform is added, by thermal agitation mixing organic phase and aqueous phase, the centrifugal 5min of 12000r/min;

(8) careful Aspirate supernatant adds in new 1.5mL centrifuge tube, with isopyknic chloroform again extracting once;

(9) careful Aspirate supernatant adds in new 1.5mL centrifuge tube, adds the dehydrated alcohol of 2 times of volumes, the 3mol/LNaAc of 1/10 volume, ice bath 30min, 12000r/min, centrifugal 10min, incline supernatant liquor, is upside down on thieving paper, and solution is flow to end;

(10) the centrifugal 5min of 1mL70% washing with alcohol twice, 12000r/min is added, collecting precipitation;

(11) incline washings, is inverted on thieving paper and blots remaining solution, Bechtop dries up;

(12) 50 μ LTE are added resuspended, for subsequent use.

Further, in above-mentioned steps (4c), agargel electrophoresis step is:

(1) seal electrophoresis chamber rubber moulding edge with article tape and form a mould;

(2) enough electrophoretic buffers (1 × TAE) are prepared in order to fill electrophoresis chamber and preparation gel;

(3) precise agar powder is added in the triangular flask filling a certain amount of electrophoretic buffer and is heated to melt in microwave oven, and carefully add ethidium bromide, final concentration is 0.5 μ g/mL, gently revolves to mix gelating soln.

(4) on mould, put suitable comb and form well, then pour agarose solution warm in right amount into, after room temperature 30min, remove edge sealing adhesive tape.Gel is put into electrophoresis chamber, makes electrophoretic buffer not have gel to be about 1mm;

(5) sample-loading buffer of pcr amplification product sample and 0.2 times of volume is mixed;

(6) slowly add in the well of submergence gel with the disposable rifle head of micropipet by sample mix liquid, molecular mass standard is added in the left side of sample;

(7) shut electrophoresis chamber lid, connect electrode plug, DNA extreme swimming on the sunny side, gives the voltage of 1 ~ 5V/cm, observes tetrabromophenol sulfonphthalein very soon and enter in colloid from well;

(8) when DNA sample or tetrabromophenol sulfonphthalein and dimethylbenzene cyanogen FF have moved enough distances in gel, close power source, extract electrode plug and open electrophoresis chamber lid, taking a picture under gel is placed in gel imaging system.

Beneficial effect of the present invention is: the present invention infects ginseng induction Hairy root with Agrobacterium rhizogenes, propose some influence factors and the Hairy root regeneration plant process of Hairy root induction, for ginseng Germ-plasma resources protection, innovation preservation provide a new way, for the breeding of ginseng lays the foundation.

Accompanying drawing explanation

Fig. 1 is the mensuration figure of embodiment of the present invention standard curve.

Fig. 2 be in the embodiment of the present invention Agrobacterium rhizogenesA4 bacterial concentration to the effect diagram of ginseng inductivity.

Fig. 3 be in the embodiment of the present invention AS and infection time to the effect diagram of ginseng inductivity.

Fig. 4 be in the embodiment of the present invention different explants to the effect diagram of ginseng Hairy root inductivity.

Fig. 5 be in the embodiment of the present invention pre-incubation time to the effect diagram of ginseng inductivity.

Fig. 6 be in the embodiment of the present invention NAA concentration to the effect diagram of ginseng inductivity.

Fig. 7 be in the embodiment of the present invention Dual culture time to the effect diagram of ginseng inductivity.

Fig. 8 be in the embodiment of the present invention various ce f concentration to the effect diagram of fungistatic effect and Bud polarization.

Fig. 9 is ginseng Hairy root PCR detected result figure in the embodiment of the present invention.

Figure 10 is the performance graph of growth and saponin content under ginseng Hairy root liquid culture condi in the embodiment of the present invention.

Figure 11 be in the embodiment of the present invention 5 kinds of different substratum to the effect diagram of ginseng Hairy root growth multiple.

Figure 12 be in the embodiment of the present invention illumination to the effect diagram of culture.

Figure 13 be in the embodiment of the present invention illumination to the effect diagram of Differentiation ration of adventitious buds.

Figure 14 be in the embodiment of the present invention illumination to the effect diagram of indefinite bud growth rate.

Figure 15 is different development stage soluble protein content figure (1: ginseng Hairy root in the embodiment of the present invention; 2: ginseng Hairy root callus; 3: ginseng Hairy root forms the callus of bud).

Figure 16 is different development stage soluble sugar content figure (1: ginseng Hairy root in the embodiment of the present invention; 2: ginseng Hairy root callus; 3: ginseng Hairy root forms the callus of bud).

Figure 17 is different development stage starch content comparison diagram (1: ginseng Hairy root in the embodiment of the present invention; 2: ginseng Hairy root callus; 3: ginseng Hairy root forms the callus of bud).

Figure 18 is developmental stage peroxidase activity figure (1: ginseng Hairy root different in the embodiment of the present invention; 2: ginseng Hairy root callus; 3: ginseng Hairy root forms the callus of bud).

Figure 19 is developmental stage catalase activity figure (1: ginseng Hairy root different in the embodiment of the present invention; 2: ginseng Hairy root callus; 3: ginseng Hairy root forms the callus of bud).

Figure 20 is different times polyphenol oxidase activity figure (1: ginseng Hairy root in the embodiment of the present invention; 2: ginseng Hairy root callus; 3: ginseng Hairy root forms the callus of bud).

Embodiment

The embodiment of this invention is set forth further below in conjunction with embodiment.

Embodiment 1: the optimization of ginseng Hairy root inductive condition

(1) different strain is on the impact of ginseng Hairy root inductivity: select in the present embodiment R1601,8196, A4, rolA, rolB, rolC six bacterial classifications infect the ginseng seedling blade of 2 ~ 3 weeks seedling ages, investigate different strain to the impact of ginseng Hairy root inductivity, it the results are shown in Table 3.

Table 3 different strain is on the impact of ginseng Hairy root inductivity

Pathogenecity between different strain also exists very big-difference, the 16d that the ginseng seedling blade of 2 ~ 3 weeks seedling ages infected by Agrobacterium rhizogenesA4 is infecting, the granulation tissue of the incision adularescent of blade occurs, have some to appear at blade wound brownization place, other to come across on callus that blade expands and not obvious.As time goes on, white particle organizes random elongation growth, and it is most advanced and sophisticated is ivory buff hat cap, and trunk is slightly transparent, after certain length, trunk occurs tiny white root hair.Do not go deep into matrix after its contact culture medium, but be attached at stromal surface continued growth; When contacting bottle wall, then along the upwards continued growth of bottle wall.

(2) bacterial concentration is on the impact of ginseng Hairy root inductivity: bacterial concentration is the important factor affecting ginseng Hairy root inductivity.Because of the difference of floristics, explant type and time of infection, require that Agrobacterium rhizogenes bacterial concentration is also variant, bacterial classification concentration is excessive, and bacterial growth is rapid, and brownization or damage easily appear in T suppression cell metabolic process explant, and not easily degerming; Concentration is too rare, and the bacterium that can be attached to cell very little, is not enough to form enough Hairy root, thus affects inductivity.The petiole of A4 Agrobacterium rhizogenes suspension to the ginseng seedling of preculture 48h of different optical density(OD) infects, and the impact of inductivity is as shown in table 4.Result shows: optical density value is OD ₆₀₀=0.6 is the most suitable, and inductivity can reach 10.8%, specific optical density value be 0.4 and 0.8 inductivity exceed 4.8% and 3.7% respectively, the effect diagram of Agrobacterium rhizogenesA4 bacterial concentration to ginseng inductivity is shown in accompanying drawing 2.

Table 4 Agrobacterium rhizogenesA4 bacterial concentration is on the impact of ginseng inductivity

(3) AS and the impact of time of infection on ginseng Hairy root inductivity: there are some researches show that the vegetable cell releasor molecule activating the genetic expression of Vir district relevant has 9 kinds, be soluble phenolic compounds, wherein, Syringylethanone and the effect of glycoloyl syringone the strongest, when inducing Hairy root, these compound treatment can be used to promote T-DNA to vegetable cell transfer, processing and to integrate.In general trend, the interpolation of AS has promoter action to the stem section rate that A4 infects ginseng seedling as shown in Table 5, improve its transformation efficiency, peak rate of conversion reaches 10.7%, time of infection 20min in general trend is optimum, illustrate that the selection of immerged time also has a significant impact for infection rate, AS and the effect diagram of infection time to ginseng inductivity are shown in accompanying drawing 3.

Table 5AS and infection time are on the impact of ginseng inductivity

(4) different explants is on the impact of ginseng Hairy root inductivity: select explant to be the blade of 2 ~ 3 weeks seedling age ginseng seedlings, stem section, petiole and ginseng gemma, ginseng callus, 1,2,3 year raw ginseng.Investigate A4 and infect ginseng, the selection of explant is on the impact of Hairy root inductivity.Result in table 6 shows: petiole is optimum explant, and inductivity is that 9.0%, 1 year raw ginseng takes second place, and inductivity is 8.5%, and gemma and callus do not obtain Hairy root.Conversion condition is pre-incubation time is 48h, Dual culture 48h, and Dual culture substratum is that MS adds AS100 μm of ol/L, OD ₆₀₀=0.6, blade, stem section, petiole, ginseng gemma are leaf disk method, and immerged time is 30min, and ginseng callus, ginseng are taken root for dripping method for 1,2,3 years.The effect diagram of different explants to ginseng Hairy root inductivity is shown in Fig. 4.

Table 6 different explants is on the impact of ginseng Hairy root inductivity

(5) pre-incubation time is on the impact of ginseng Hairy root inductivity: under same culture conditions, the inductivity impact of different pre-incubation time condition on ginseng Hairy root is very large as can be seen from Table 7, preculture 12 ~ 48h inductivity increase in time and increasing gradually, this may be because preculture can make the cell at edge, explant wound enter vigorous division stage fast, thus improves inductivity.Start to decline more than 48h inductivity.Find out thus, preculture 48h is advisable for induction ginseng Hairy root.The effect diagram of pre-incubation time to ginseng inductivity is shown in accompanying drawing 5.

Table 7 pre-incubation time is on the impact of ginseng Hairy root inductivity

(6) NAA concentration is on the impact of ginseng Hairy root inductivity: as shown in table 8 and Fig. 6, the induction that NAA is of value to ginseng Hairy root is added in precultivation medium, add NAA group inductivity all higher than not adding group, it is 2.33 times that do not add NAA that NAA2.0mg/L inductivity reaches 7.0%.

Table 8NAA concentration is on the impact of ginseng Hairy root inductivity

(7) matrix together incubation time on the impact of ginseng Hairy root inductivity: explant and Agrobacterium Dual culture time and matrix are the important factors affecting Hairy root transformation efficiency.Explant is the blade of seedling, and precultivation medium is the MS+NAA2.0mg/L time is 48h, and bacterial classification is A4, select the Dual culture time be 12h, 24h, 36,48h, Dual culture substratum elects MS, 1/2MS as.As shown in table 9 and Fig. 7, result is that Dual culture matrix MS is better than 1/2MS, and the Dual culture time is the most suitable with 48h.The prolongation Dual culture time can increase conversion probability, but Dual culture overlong time, bacterium amount reproduction will be made to increase follow-up degerming difficulty and easily cause explant death.

Table 9 Dual culture time and matrix are on the impact of ginseng Hairy root inductivity

(8) cephamycin (Cef) concentration is on antibacterial and impact that is that infect: when adopting leaf disk method to carry out Genetic Transformation in Higher Plants, Cef effectively can suppress the growth of Agrobacterium on plant materials and substratum.As can be seen from table 10 and Fig. 8: its concentration is within the scope of 200 ~ 600mg/L, and concentration is larger, fungistatic effect is better, and the root of hair number of acquisition is between 3 ~ 25.Comprehensive fungistatic effect, pollution rate, acquisition root of hair number three indexs, think and keep good fungistatic effect to should be 500mg/L with the optimal concentration obtaining more root of hair number.

Table 10 various ce f concentration is on fungistatic effect and the impact obtaining root of hair number

Note: ++ for there being a large amount of Agrobacterium, +++ for there being a small amount of Agrobacterium, ++++for there being minute quantity Agrobacterium

Embodiment 2: the qualification of ginseng Hairy root

The material 500mg/LCef aqueous solution after Dual culture rinses 3 times, and aseptic filter paper blotting material surface moisture content, is inoculated on the MS solid medium containing 500mg/L head Cef and carries out degerming cultivation.Every 7d changes once except bacterium culture medium, is transferred to and does not cultivate containing on any antibiotic MS solid medium after 4 ~ 5 weeks.

Ginseng Hairy root in the present embodiment the earliest 27d after infection produces (Condition:OD ₆₀₀=0.6, seedlingswithstemexplants, preconditioningtime=48h, AS100 μm of ol/L).Hairy root appears at wound place, and one piece of explant material can produce one to several Hairy root, some materials then first produce loose callus, on callus, then have Hairy root to grow gradually.The Hairy root of all generations all show growth rapidly, adularescent root hair, branch be many, without the need to adding outer rim hormone, being attached at media surface or prolonging a bottle wall and upwards grow.Control material without bacterium liquid inductance dye does not then have Hairy root to produce, though some explant two ends are slightly expanded, and death gradually in without culturing process further on hormone solid medium.Cut to be connected on MS solid medium when hair-like more than root growth 1cm and cultivate.

(1) qualification of ginseng Hairy root: get ginseng Hairy root 100mg and without the ginseng seedling blade infected and 1 year each 200mg of raw ginseng, according to preceding method, extract the DNA in Hairy root and 1 year raw ginseng, be prepared into detection sample.Extract the DNA in Ri plasmid and the non-transformed blade of ginseng simultaneously, prepare positive control and negative control sample.Various for preparation DNA is carried out amplified reaction under aforementioned PCR reaction conditions.Product after electrophoresis (5V/cm), is taken a picture under gel imaging system and is observed DNA band and take pictures on the sepharose of 1.2%.Its result as shown in Figure 9.As can be seen from Figure 9, pair of primers according to design carries out PCR detection to ginseng Hairy root, there is the specific band (423bp) of expecting in result, unconverted leaves of panax ginseng and 1 year raw ginseng then do not have amplified band to occur, prove that tDNA has been integrated in the genome of ginseng Hairy root.

(2) growth and the performance graph of saponin content under ginseng Hairy root liquid culture condi: under liquid culture condi, the increment of ginseng Hairy root and saponin content change and substantially meet " S " type curve (Figure 10 is shown).Ginseng Hairy root to be transferred in liquid nutrient medium and can a few days ago to enter of short duration lag period in switching from solidified MS media, after the lag period of short period of time, namely logarithmic phase is entered at 15d, until 35d reaches the climax that growth rate increases, grow after 35d slow down gradually, saponin content declines to some extent after 45d.

(3) different solid medium is on the impact of ginseng Hairy root increment: solid medium MS, 1/2MS, 1/3MS, 1/4MS and 1/10MS are on the impact (shown in Figure 11) of the increment of ginseng Hairy root and total saponin content.As can be seen from Figure 11 in solidified MS media, 30d increased times reaches as high as 46.5 times, and most I reaches 35 times, average out to 35 times.The poorest average out to of 1/10MS 2.29 times.

Embodiment 3: the induction of ginseng Hairy root callus

(1) impact of different growth regulator confrontation callus induction: adopt L ₉(3 ⁴) orthogonal test research BA, KT and 2,4-D be to the influence power of the inducing action of ginseng Hairy root callus.Level of factor is in table 11, and the sample number of each experiment is greater than 30, and test repetition 3 times, adopts point system log, and the average result SPSSV13.0 of 3 tests analyzes, in table 12, table 13.

Table 11 evoked callus experimental factor level design

Table 12 orthogonal test scheme and test-results

Table 13 variance analysis

DependentVariable: ginseng Hairy root Callus induction rate

aRSquared＝.980(AdjustedRSquared=.919)

Note: (P<0.01) represents pole conspicuous level; (P<0.05) conspicuous level is represented

From variance analysis (table 13), for the induction of root of hair callus, 2,4-D has the greatest impact, and KT takes second place, and all reaches conspicuous level.The impact of BA is less, acts on not obvious.According to the above best inducing culture choosing ginseng Hairy root callus of analyzing be: MS+2,4-D2.0mg/L+KT0.2mg/L.

(2) illumination is on the impact of callus: ginseng ginseng Hairy root callus, no matter can occur under the condition of illumination or dark, but illumination all has obvious restraining effect to both growths.With cultivate compared with the callus of inducing and producing under illumination condition, the callus color under light culture condition is more shallow, yellowish or sundown.Dedifferentiation speed is fast, and about 25d is whole callus almost, and callus is formed in a large number, and growth rapidly.And under high light, its growth is suppressed, during 40d, inductivity is still very low.From inductivity, the highest under dark condition, minimum under high light, falling between under the low light level, is about 80%; From increment, fast growth when growing comparatively illumination under dark condition.In table 14 and Figure 12.Callus carries out initial 2 middle of the month of subculture, dedifferentiation not exclusively still has Hairy root to produce, unfavorable to the growth of callus, and cytokinin concentration lower Hairy root quantity is more, along with growth, transfer the increasing of algebraically of Subculture Time, incomplete dedifferentiation quantity is fewer.

Table 14 illumination is on the impact of culture

Embodiment 4: the organ of ginseng Hairy root occurs

(1) plant hormone kind and concentration are on the impact of indefinite bud: the impact of different growth regulator confrontation bud, in table 15, therefrom shows, through succeeding transfer culture in the KT substratum of different concns, its proliferation rate and differentiation rate are just in time contrary; When KT reaches 2.0mg/L, differentiation rate is higher is about 20%, but growth rate is lower; When KT is 1.0mg/L, its result antithesis.Choose No. 3 by result and be combined as ginseng Hairy root callus adventitious bud inducing and growth matrix.

The impact of table 15 different growth regulator confrontation bud

(2) the illumination impact of breeding differentiation adventitious buds: as shown in table 16 and Figure 13, Figure 14, under dark and illumination condition, ginseng indefinite bud has differentiation.For the coefficient of differentiation of indefinite bud when alternate illumination, ginseng Hairy root is than high when dark and illumination, and when adventitious bud proliferation under illumination condition, its growth coefficient, than high during alternate illumination, shows that illumination condition is of value to the growth of Hairy root callus indefinite bud.

The impact that table 16 illumination is bred differentiation adventitious buds

(3) hormon proportioning is on the impact of taking root: the aseptic seedling of the stalwartness growing into 3 ~ 4cm is divided into individual plant, be inoculated into add hormon not containing ammonium nitrate 1/2MS+GA35.0mg/L substratum in root induction.IBA0.5mg/L is better than IBA1.0mg/L rooting efficiency as can be seen from Table 17, and NAA1.0mg/L is better than NAA2.0mg/L rooting efficiency.

After 2 months, just can grow many fleshy roots at seedling base portion, after whole plant to be formed, open sealed membrane, at room temperature hardening 2 ~ 3d, takes out seedling from bottle, clean root substratum, suck dry moisture on filter paper, then immigration is equipped with in the flowerpot of the matrix (perlite: vermiculite: sand=1:1:1) of sterilizing, front several times with the pouring of 1.0mg/LNAA nutritive medium, regularly water afterwards, cover to keep certain humidity with plastic cloth, remove after several days, can transplant.

Table 17 different substrates is on the impact of taking root

Embodiment 5: the research of physio-biochemical characteristics in Hairy root regeneration plant approach

Plant materials regeneration plant is a complicated process.The physiological acoustic signals of cell is the basis of tissue morphology change.The essence that plant materials regeneration plant occurs is the result that control cytogene is expressed in turn by space-time, and protein, sugar, starch and enzyme are all the products of genetic expression.Therefore, the physiological and biochemical index of cell also presents regular change, directly can reflect the Gene Activity of cell.Enzyme in plant soma and matter and energy metabolism closely related, participate in again division and the differentiation of cell, the change of its activity can affect phytomorph and occur simultaneously.

(1) soluble protein content: the change of Hairy root organ generation regeneration plant albumen in period as seen from Figure 15, by Hairy root to callus again to the callus having bud to produce, protein content reduces gradually.The albumen change of ginseng group is very mild.

(2) soluble polysaccharide content: the change of ginseng candies content and albumen are similar to as seen from Figure 16, are decline of walking unhurriedly substantially.The content of albumen raised gradually in the ginseng Hairy root stage, entered callus for falling after rising, and the phase lasts produced at bud reduces.

(3) Zulkovsky starch content: starch is the most general energy reserve substance of vegetable cell, starch content number can as of a cytodifferentiation mark, its growth and decline and cell fission, tissue differentiation and orga-nogenesis have substantial connection.In plant cell development process, starch is utilized the variation tendency homopolysaccharide of starch content in a large number as growing signal, higher at embryo callus content in period, starch accumulation may be the prerequisite that cell breaks up further.As can be seen from Figure 17, the change of starch content is substantially identical with the change of sugar.

(4) Peroxidase Activity Determination: identical in ginseng group variation tendency as shown in Figure 18, the vigor arriving bud peroxidase again to callus through Hairy root reduces gradually.The enzyme activity at ginseng poultry's initial stage is 3.4 times of callus late period.

(5) catalase activity measures: ginseng group is fallen after rising as shown in Figure 19, and the callus stage that its enzyme activity is the highest is 2.06 times of Hairy root, 2.79 times of bud respectively.

(6) polyphenoloxidase: all present S curve shape in the change of ginseng group different steps polyphenoloxidase as seen from Figure 20, its variation tendency is all the decline of root of hair stage, and the callus stage rises, and obviously declines again during bud.The ratio that its enzyme activity is high with minimum is 3.78 times.

The above is the preferred embodiment of the present invention; it should be pointed out that for those skilled in the art, under the premise without departing from the principles of the invention; can also make some improvements and modifications, these improvements and modifications are also considered as protection scope of the present invention.

Claims

1. for the application method that different strain affects ginseng Hairy root, it is characterized in that: its process comprises following aspect:

(1) explant process: (I) ginseng seed is sprouted and process: hide Stratificated treatment through husky, the seed ftractureed, is seeded in dish for cultivating, to shade cultivation, after sowing 1 ~ 2 week seedling grows, with 0.1% mercuric chloride sterilization 6min, sterile water wash 5 times; Its blade, band leaf children's stem and stem section are cut into the segment of 1.0cm, are connected to the explant material that preculture on MS solid medium is used as to transform; (II) ginseng 1 year, 2 years, the process of taking root for 3 years: after surface clean water of being taken root for 3 years by ginseng is clean, running water 3 ~ 4h, after 0.1% mercuric chloride sterilization 10 ~ 20min, sterile water wash 5 times, is cut into the thick thin slice of 0.2cm and is placed on MS solid medium stand-by;

(2) preservation of bacterial classification and activation: the long-term preservation method of Agrobacterium rhizogenes is dissolved in 20% glycerine by Agrobacterium, preserves under-80 DEG C of conditions; The method of short-term preservation is inoculated in by Agrobacterium rhizogenes on YEB solid medium to cultivate, and after growing bacterial plaque, preserves in 4 DEG C of refrigerators; Under aseptic condition, preserve bacterium liquid with inoculating needle picking, rule on YEB solid plate, 28 DEG C be placed in dark condition under cultivate, until grow single bacterium colony; Picking single colony inoculation in 50mL liquid YEB substratum, 120r/min, 28 DEG C, cultivate 24h under dark condition, now YEB liquid nutrient medium becomes muddy faint yellow by the transparent reddish-brown before inoculating, and proves that bacterium normally grows; Getting the bacterium liquid that 1mL activated adds in the YEB liquid nutrient medium of 50mL, and 120r/min, cultivates under 28 DEG C of dark condition, and activation bacterium liquid reaches growth logarithmic phase, and now bacterium liquid is just as infecting use;

(3) optimization of ginseng Hairy root inductive condition, comprising:

(3a) different strain is on the impact of ginseng Hairy root inductivity: select Agrobacterium rhizogenes R1601,8196, A4, rolA, rolB, rolC six bacterial classifications access YEB liquid nutrient medium after flat board activation, treat that its bacterium liquid growth concentration reaches OD ₆₀₀be used for when=0.6 infecting; The ginseng seedling blade of 2 ~ 3 weeks seedling ages, good children is tender for growth selection, and the fritter being aseptically cut into 0.5cm × 0.5cm drops in bacterium liquid, and 25 ± 1 DEG C of shaking table 100 ~ 120r/min infect 15 ~ 30min, take out under aseptic condition; Sterilizing filter paper blots surperficial bacterium liquid, is connected on MS substratum, investigates different strain to the impact of ginseng Hairy root inductivity;

(3b) different explants is on the impact of ginseng Hairy root inductivity: select explant to be the blade of 2 ~ 3 weeks seedling age ginseng seedlings, stem section, petiole and ginseng brood cell, ginseng callus, 1 year, 2 years, 3 years raw ginsengs, selection bacterial classification is Agrobacterium rhizogenesA4, investigates the selection of ginseng explant to the impact of Hairy root inductivity; Its Leaf, stem section, petiole, ginseng brood cell are leaf disk method, and immerged time is 15 ~ 30min, and ginseng callus, ginseng are taken root for dripping method in 1 year, 2 years, 3 years;

(3c) pre-incubation time is on the impact of ginseng Hairy root inductivity: select MS solid medium to be preculture matrix, the stem section selecting 2 ~ 3 weeks ginseng seedlings is material, pre-incubation time elects 12h, 24h, 48h, 72h, 96h as, to investigate the impact of pre-incubation time on ginseng Hairy root inductivity;

(3d) NAA concentration is on the impact of ginseng Hairy root inductivity: with the stem section of 2 ~ 3 weeks ginseng seedlings for material, the concentration of adding NAA in MS minimum medium is chosen as 0mg/L, 2mg/L, 4mg/L, 6mg/L, compares Hairy root inductivity and investigates NAA optimal concentration;

(3e) matrix together incubation time on the impact of ginseng Hairy root inductivity: select the Dual culture time be 12h, 24h, 36,48h, Dual culture substratum elects MS, 1/2MS as; Explant is the blade of seedling, and precultivation medium is the MS+NAA2.0mg/L time is 48h, and bacterial classification is A4, with investigate matrix together incubation time on the impact of ginseng Hairy root inductivity;

(3f) bacterial concentration is on the impact of ginseng Hairy root inductivity: the bacterium liquid YEB liquid prepared is diluted to OD ₆₀₀value is 0.2,0.4,0.6,0.8 and 1.0, ginseng seedling petiole is dropped in the bacterium liquid of different concns and contaminate 15 ~ 30min, 25 ± 1 DEG C of shaking table 100 ~ 120r/min, then taking-up aseptic filter paper blots the bacterium liquid on surface, after 2d cultivated by Dual culture base, be transferred to again on MS Dual culture substratum, change a subculture every 7d;

(3g) AS and time of infection are on the impact of ginseng Hairy root inductivity: single colony inoculation of picking activates in the 30mLYEB liquid nutrient medium adding AS, AS concentration elects 0,100,200 μm of ol/L shaking culture as, the stem section of ginseng seedling immersed when its optical density value is 0.6, time of infection elects 10min, 15min, 20min, 25min and 30min as;

(3h) Cef concentration 200 be chosen as on antibacterial and concentration that is impact that is that infect: Cef, 300,400,500,600mg/L, degerming matrix is chosen as the basic solid substrate of MS; The comprehensive fungistatic effect of Integrated comparative, pollution rate, acquisition root of hair number three indexs, investigate the optimal concentration of Cef;

(4) acquisition of ginseng Hairy root and qualification:

(4a) acquisition of ginseng poultry: 1. explant preculture: after the sterilization of ginseng seedling, its blade is cut into the fritter of 1.0cm × 1.0cm, petiole and stem are cut into the long section of 1.0cm, ginseng 1 year, 2 years, 3 years running water of taking root remove surperficial surface dust, after mercuric chloride sterilization, distilled water washes down for 3 ~ 5 times, be cut into 0.2 ~ 0.5cm thin slice depending on material thickness, above material be connected in MS+NAA2mg/L substratum and cultivate 48h under dark condition and be used for infecting; 2. infect and Dual culture: on Bechtop, by pre-incubated explant, transfer in the triangular flask being equipped with and getting bacterium liquid ready, 120r/min is placed on after sealing, shaking table under dark condition infects 10 ~ 30min, metainfective explant aseptic filter paper blots surperficial bacterium liquid, is inoculated in MS solid medium, Dual culture 48h under dark, 25 DEG C of conditions; 3. degerming cultivation: by the infected material aseptic water washing after Dual culture three times, aseptic filter paper blots the sterilized water on explant surface, is transferred on the MS solid medium containing Cef500mg/L and cultivates, degerming cultivation under dark, 25 DEG C of conditions;

(4b) induction of ginseng callus and 1 year, 2 years, 3 years Hairy root of taking root: draw with micropipet the bacterial strain 8 μ L activated and drip on the ginseng callus tangent plane of receiving MS solid medium and ginseng cut into slices, avoid bacterium drop on MS solid medium, 25 DEG C, cultivate under dark condition, the death of 2 weeks rear removing brownization, transfer on fresh MS solid medium and continue to cultivate;

(4c) PCR Molecular Detection: adopt PCR method to plasmid T _lthe rolB gene test of-DNA, step is:

1. design of primers and synthesis: plasmid T _l-DNA sequence analysis result, devises special primer prolc1 and prolc2 of rolB gene:

Primer 1:5 '-GCTCTTGCAGTGCTAGATTT-3 ';

Primer 2: 5 '-GAAGGTGCAAGCTACCTCTC-3 ';

Utilize above primer by the fragment of rolB gene on pcr amplification to T-DNA, expection length is 423bp;

2. CTAB method extracts ginseng Hairy root STb gene;

3. the Ri plasmid of Agrobacterium rhizogenes is extracted;

4. PCR loop parameter:

94 DEG C of denaturation 5min;

5min is extended after 72 DEG C;

5. PCR reaction system:

Ultrapure water is settled to 20 μ L;

6. agargel electrophoresis, takes a picture under imaging system;

(5) mensuration of ginseng Hairy root liquid and solid culture increment and total saponin content: Hairy root liquid culture adopts 1/2MS liquid nutrient medium to be minimum medium, the access of the Hairy root tip of a root of 10 eugonic 2cm is equipped with in 30mL1/2MS liquid nutrient medium, temperature is 24 ± 1 DEG C, shaking speed 100r/min, sample every 5d, measure the content of Hairy root dry weight and Radix Ginseng total saponins, measure one and a half months; All data are the mean value of 3 bottles, repeat 3 times; Total saponin content in drawing standard curve determination Hairy root: 1. get Rg10.020g and be dissolved in methyl alcohol, constant volume is 2mg/mL; 2. Rg1 reference substance solution 30,60,90,120,150 μ L is got; 3. be placed in 5 test tubes respectively, methanol constant volume is 150 μ L, compares with corresponding reagent; 4. test tube is placed in ice-water bath and adds 5% Vanillin alcoholic solution and 72% sulfuric acid shakes up, 60 DEG C of water bath with thermostatic control 20min; 5. detect at 755 spectrophotometer 544nm places after ice-water bath 15min; Take ginseng as standard substance colorimetric estimation, according to typical curve Y=503.29X+4.456, R ²=0.9924 tries to achieve sample saponin content; In Hairy root, the measuring method of total saponins is, gets the fresh Hairy root that certain quantity of fluid is cultivated, after distilled water flushing, blots the moisture on Hairy root surface with filter paper, weighs Hairy root fresh weight; Then in vacuum drying oven, be dried to constant weight at 80 DEG C, weigh; Dried Hairy root is pulverized, puts into triangular flask, add the alcohol solvent that a certain amount of concentration is 40%, and triangle bottleneck sealed membrane is sealed; Be placed in ultra-sonic generator and carry out separation and Extraction, extraction time 15min, ultrasonic frequency is 28KHz, extracts twice; 5% Vanillin alcoholic solution and 72%H is added in ice-water bath ₂sO ₄dyeing, 60 DEG C of water bath with thermostatic control 10min, ice-water bath 15min, survey absorbancy in 544nm place;

(6a) induction of ginseng Hairy root callus: with ginseng Hairy root for explant, be placed in interpolation different concns, 2 of various combination, on the MS substratum of 4-D, BA, KT growth regulatory substance, sucrose concentration is 30g/L, agar 6.5g/L, before sterilizing, pH value is adjusted to 6.0, and sterilization pressure is that 0.1MP continues 15 ~ 20min; Under being positioned over different illumination intensity, inducing temperature is 24 ± 1 DEG C, and periodicity of illumination is 12h, and the low light level is cultivated, add up callus induction rate after 12d and analyze all kinds of growth regulatory substance and intensity of illumination to the size of callus induction effect, screen best kind and concentration combination and intensity of illumination;

(6b) organ of ginseng Hairy root occurs: 1. hormon concentration, combination and illumination are on the impact of Multiple Buds growth and differ entiation: the different concns getting KT, IBAGA35.0mg/L growth regulatory substance of different concns and combination respectively combines and processes culture, analyze the impact of each material on culture growth, differentiation, filter out the best medium formula that growing way and biomass all reach desirable level; Get dark, the low light level, high light three illumination conditions, observe light to the impact of culture; Wherein the low light level is normal daylighting degree in culturing room, and the photoperiod is with normal daily variation; High light is provided by fluorescent lamp, and the photoperiod is 10h/d; 2. hormon proportioning is on the impact of taking root: NAA, IBA, GA of getting different concns and combination respectively ₃the different concns combination of 5.0mg/L growth regulatory substance processes culture, analyzes each material to root growing way and the impact of situation of taking root, and filters out the best medium formula that growing way and biomass all reach desirable level;

(7a) Coomassie brilliant G-250 Determination Staining protein content: bovine serum albumin is made into the standard protein solution containing protein 100 μ g/mL; Being X-coordinate with protein concn, take absorbancy as ordinate zou drawing standard curve: Y=0.0052X-0.0253R ²=0.9905; Take each 1g of fresh sample and put into mortar, add 2mL distilled water and grind to form homogenate, transfer in centrifuge tube, use 6mL distilled water wash mortar again, washings is collected in same centrifuge tube, at room temperature places 1h fully to extract, then the centrifugal 20min of 4000r/min, discard precipitation, supernatant liquor proceeds to 10mL volumetric flask, and with distilled water constant volume; Separately get two 10mL tool plug test tubes, pipette samples extracting solution of protein 0.1mL respectively, put into tool plug scale test tube, adding 5mL Coomassie brilliant G-250 solution, fully mix, is contrast with blank after placing 2min, colorimetric under 595nm, record light absorption value, and check in protein content by typical curve, adopt following formula to calculate:

The typical curve value that in formula, X-absorbancy per sample checks in, unit is μ g; V _t-extracting solution cumulative volume, unit is mL; W-sample Fresh Yuxincao, unit is g; V _sapplication of sample amount during-mensuration, unit is mL; N-extension rate;

(7b) anthrone method measures soluble sugar content: dried to constant weight at 80 DEG C by analytical pure glucose, accurately take 0.100g, be configured to the Standard glucose solution of 100 μ g/mL; Being X-coordinate with glucose concn, take absorbancy as ordinate zou drawing standard curve: Y=0.0064X+0.0035R ²=0.9991; Take material 0.20g, shred and put into Zhi Kedu test tube, add 8mL distilled water, plastic film sealing, in boiling water, extract 30min, extract 2 times, extracting liquid filtering enters in 25mL volumetric flask, repeatedly rinses test tube and residue, be settled to scale, draw 0.5mL sample liquid in test tube, adding distil water 1.5mL, adds 5mL anthrone sulphate reagent, densitometric under 630nm wavelength, adopts following formula to calculate:

The sugar amount that in formula, C-checks in from typical curve, unit is μ g; V _t-extracting solution cumulative volume, unit is mL; V _sthe sample volume of taking during-mensuration, unit is mL; W-tissue weight, unit is g; N-extension rate;

(7c) mensuration of starch content: accurately take the pure starch of 100mg, be configured to the reference liquid of the starch-containing 100 μ g of every milli L, being X-coordinate with starch concentration, take absorbancy as ordinate zou drawing standard curve: Y=0.0021X+0.0359R ²=0.9658; By extracting the later residue of soluble sugar, moving in 50mL volumetric flask, adding 20mL hot distilled water, put into boiling water bath and boil 15min, then add 9.2mol/LHCLO ₄2mL extracts 15min, after cooling, and mixing, the centrifugal 10min of 2500r/min, and use distilled water constant volume, add 5mL anthrone sulphate reagent, densitometric under 630nm wavelength, adopt following formula to calculate:

The starch quality that in formula, C-is obtained by typical curve, unit is μ g; V-extracts liquid measure, and unit is mL; W-tissue weight, unit is g; Liquid draw volume during a-colour developing, unit is mL;

(7d) Peroxidase Activity Determination: the preparation of reaction mixture: 100mol/LpH6.0 phosphoric acid buffer 50mL is in beaker, add methyl catechol 28 μ l, heated and stirred on magnetic stirring apparatus, until methyl catechol dissolves, after solution cooling, add 30%H ₂o ₂19 μ L, mix, and are stored in refrigerator for subsequent use; The 2g that draws materials puts into mortar, adds 5mLpH6.0 phosphoric acid buffer and grinds to form homogenate, with the centrifugal 15min of 4000r/min, supernatant liquor proceeds in 20mL volumetric flask, and residue extracts once with 5mL phosphoric acid buffer again, and supernatant liquor is incorporated in volumetric flask, be settled to scale, for subsequent use under storing in low temperature; Get mixed solution 3mL and phosphoric acid buffer 1mL for contrast, mixed solution 3mL adds enzyme liquid 1mL and starts timing immediately, surveys absorbance under 470nm wavelength, every the 1min number of degrees 1 time:

Δ A in formula ₄₇₀the internal absorbance change of-reaction times; W-material weight, unit is g; In the T-reaction times, unit is min; V _t-extracting enzyme liquid cumulative volume, unit is mL; V _stake enzyme liquid during-mensuration to amass, unit is mL;

(7e) catalase activity measures: get the phosphoric acid buffer that each 1g of different times material adds the precooled pH7.8 of 3mL, put into mortar and grind to form homogenate, be transferred in 25mL volumetric flask, rinse mortar several merging damping fluid and be settled to scale, be placed in 5 DEG C of refrigerators to leave standstill 10min and get liquid, the centrifugal 15min of 4000r/min, gets supernatant liquor recentrifuge, obtains supernatant liquor 5 DEG C and saves backup; Get 3,10mL test tube, wherein two is that mensuration one is for blank; SO pipe boils 1min in boiling water, cooling; The 25 DEG C of preheatings of all test tubes, add the H of 0.3mL0.1mol/L by pipe ₂o ₂, timing immediately, 240nm surveys light absorption value, at interval of 1min reading once, adopts following formula to calculate:

Δ A_{240} = A_{S 0} - \frac{A_{S 1} + A_{S 2}}{2}

A in formula _s0-reaction times internal reference pipe absorbancy; A _s1, A _s2-sample determination pipe absorbancy; W-sample Fresh Yuxincao, unit is g; In the t-reaction times, unit is min; V _t-extracting enzyme liquid cumulative volume, unit is mL; V _stake enzyme liquid during-mensuration to amass, unit is mL;

0.1 represents A ₂₄₀decline 0.1 time a unit of enzyme activity U;

(7f) polyphenol oxidase activity measures: get 2g somatic embryo occur in each period material be placed in precooling mortar, add 0.2gPVP, 5mL0.05mol/LpH8.0 phosphoric acid buffer and fully grind to form homogenate; Centrifugal 15min under 3000r/min4 DEG C of condition, gets supernatant and adds in 25mL volumetric flask, again extract residue with damping fluid, and merge supernatant, constant volume, cryopreservation is for subsequent use; 30 DEG C of constant temperature water bath 10min, measure light absorption value under 398nm wavelength, adopt following formula to calculate:

In formula, A ₃₉₈the internal absorbance change of-reaction times; W-material weight, unit is g; In the t-reaction times, unit is min; V _t-extracting enzyme liquid cumulative volume, unit is mL; V _stake enzyme liquid during-mensuration to amass, unit is mL;

(8a) paraffin section and displaing microstructure observing: 1. fix: choose Hairy root, the callus of induction of hairy roots and embryo callus, be divided into the fritter being less than about 1cm, 24h fixed by material FAA stationary liquid, and r/min enters 70% alcohol and is stored in 4 DEG C of refrigerators; 2. dehydration, transparent, saturating wax and embedding: through 70% under room temperature, 85%, 95%, 100%, in the ethanol of 100%, dewater each 2h, ethanol: dimethylbenzene=1: 1 spends the night, the each 1.5h of pure dimethylbenzene secondary, dimethylbenzene: paraffin=1: 1, transparent, the saturating wax process of 50 DEG C of 12h, paraffin refined wax secondary 60 DEG C of each 12h, finally be embedded in melt paraffin, to be cooledly solidify; 3. section, paster: wax stone, after finishing, is cut into the continuous wax band of 10 μm, then carries out paster with rotary microtome, 35 DEG C of exhibition sheets dry 12h; 4. dyeing, mounting: siderotil-hematoxylin staining dyeing, balsam mounting, observes under TS100 type inverted microscope and takes a picture;

(8b) karyotyping: 1. draw materials and pre-treatment: get the Hairy root tip of a root 0.5cm when morning 9 ~ 10, callus 0.5cm × 0.5cm fritter, Multiple Buds 0.5cm blade tip with santochlor solution at room temperature treatment 6 ~ 12h; 2. fix: pretreated is stated material distilled water flushing 3 ~ 5 times, then move in Ka Nuoshi stationary liquid and fix 24h under 4 ~ 15 DEG C of conditions, distilled water flushing, put into 0.075mol/L liquid hypotonic, under 25 ~ 30 DEG C of conditions, Hairy root process 6min, callus and bud 30min, proceed in 70% ethanol after 70% alcohol flushing 2 times and save backup; 3. acidolysis: taken out from 70% ethanol by material, uses distilled water rinsing. and then put at the 1mol/LHCI of 60 DEG C of water-bath preheatings, dissociate 10 ~ 15min under 60 DEG C of constant temperatures, takes out and use distilled water flushing 2 ~ 3 times; 4. film-making: the material after rinsing that dissociates is placed in 30 ~ 40 DEG C, the siderotil aqueous solution of 4% dyes 1h, distilled water wash 4 ~ 5 times. each 5min, is placed in the 5% phenodin aqueous solution and dyes 2h, distilled water flushing 5 ~ 10min, the acetic acid 20min of 45%, compressing tablet; During compressing tablet, get the tip of a root with tweezers and be placed on slide glass, cut off root tips meristematic tissue; Be cut into thin slice, drip the acetic acid of 45%, smash material to pieces rapidly, add cover glass compressing tablet; The slice, thin piece selecting Chromosome spread good is freezing takes off cover plate, puts into baking oven, 40 DEG C of dry 1h; More than seasoning 24h under room temperature, dimethylbenzene is transparent, neutral gum mounting.

2. application method according to claim 1, is characterized in that: the CTAB method in described step (4c) is extracted ginseng Hairy root STb gene step and is:

(1) get 280mg to add 10%PVP through the ginseng Hairy root of succeeding transfer culture be fully ground to powdery in liquid nitrogen;

(3) take out centrifuge tube and be cooled to room temperature, add isopyknic chloroform: primary isoamyl alcohol=24: 1, mixing, 12000r/min, centrifugal 10min;

(5) incline supernatant liquor, and to exhaust residual solution with thieving paper, adds 300 μ L ultrapure waters and again dissolve, then add the dehydrated alcohol 600. μ L of-20 DEG C of precoolings, precipitates overnight;

(6) 12000r/min, centrifugal 10min, obtain the gelatinous precipitate of white; Incline supernatant liquor, and to exhaust residual solution with thieving paper, adds 70% ethanol 800 μ L washing precipitation 2 ~ 3 times;

(7) exhaust residual solution, puts Bechtop and dry up;

3. application method according to claim 1, is characterized in that: the Ri plasmid procedure extracting Agrobacterium rhizogenes in described step (4c) is:

(2) draw the cultured bacterium liquid of 8mL, add in 10mL centrifuge tube, 4000r/min, centrifugal 5min, incline nutrient solution, adds the STE of 2mL precooling, Eddy diffusion cell; Draw ImL bacteria suspension respectively, add in 1.5m centrifuge tube, the centrifugal 2min of 12000r/min, incline nutrient solution, obtains appropriate somatic cells;

(6) the centrifugal 10min of 12000r/min, careful Aspirate supernatant adds in 1.5mLEppendorff pipe;

(9) careful Aspirate supernatant adds in new 1.5mL centrifuge tube, and add the dehydrated alcohol of 2 times of volumes, the 3mol/LNaAc of 1/10 volume, ice bath 30min, 12000r/min, centrifugal 10min, incline supernatant liquor, is upside down on thieving paper, and solution is flow to end;

(12) 50 μ LTE are added resuspended, for subsequent use.

4. application method according to claim 1, is characterized in that: in described step (4c), agargel electrophoresis step is:

(2) enough electrophoretic buffers are prepared in order to fill electrophoresis chamber and preparation gel;

(3) precise agar powder is added in the triangular flask filling a certain amount of electrophoretic buffer and is heated to melt in microwave oven, and carefully add ethidium bromide, final concentration is 0.5 μ g/mL, gently revolves to mix gelating soln;

(4) on mould, put suitable comb and form well, then pour agarose solution warm in right amount into, after room temperature 30min, remove edge sealing adhesive tape; Gel is put into electrophoresis chamber, makes electrophoretic buffer not have gel 1mm;