CN103614411A - Research method for American ginseng hairy root induction and plant regeneration - Google Patents
Research method for American ginseng hairy root induction and plant regeneration Download PDFInfo
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Abstract
The invention relates to a research method for American ginseng hairy root induction and plant regeneration, belonging to the field of crop biotechnologies. The method comprises the steps of (1) treating an explant; (2) preserving, activating and culturing a strain; (3) optimizing conditions for American ginseng hairy root induction; (4) obtaining and identifying American ginseng hairy roots; (5) determining the liquid culture growth and total saponin content of the American ginseng hairy roots; (6) establishing a regeneration system of the American ginseng hairy roots and performing condition optimization. According to the method, the American ginseng hairy roots are used as a material to induce a regenerated plant in an embryogenesis way, so that a new direction for breeding of American ginseng is opened up. By adopting Ri plasmids, an exogenous gene can be guided in more simply and conveniently; besides, the regenerated plant obtained from hairy roods is a transgenic plant to ensure that the transgenic plant has a characteristic of rooting very easily, so that the difficult problems of difficult root differentiation, or weak growing ability and small amount of formed roots in plant regeneration can be well solved.
Description
Technical field
The present invention relates to Radix Panacis Quinquefolii and send out shape root induction and plant regeneration research method, belong to crop biological technical field.
Background technology
Radix Panacis Quinquefolii (Panax quinque folium L.) American Ginseng, it is China's tradition rare medicinal herbs, it belongs to Araliaceae Panax per nnial herb, be called again the names such as U.S.'s ginseng, Radix Panacis Quinquefolii, American ginseng, Guangdong ginseng, it is the virgin forest that originates in North America the earliest, main product is in France, Canada and the U.S. now, the optimum environment of growth is the hilly country in 2000 meters of left and right of height above sea level, and belong to oceanic climate, be probably distributed in the fully stocked wood of 30 °～47 ° of north latitude, 67 °～125 °, west longitude.China's big area Introducing American Ginseng successfully just from the eighties in 20th century, through the effort of decades, China has developed into the national and consumer nation the biggest in the world that the Atlantic Ocean, the third place in the world ginseng after the U.S., Canada is produced.
Though Radix Panacis Quinquefolii be underground or and over-ground part all contain multiple physiologically active ingredient, comprising Radix Panacis Quinquefolii Saponin, it is mainly triterpene compound, substantially similar to ginsenoside structure.Other composition also comprise flavonoid, volatile oil, protein,, peptide class, amino acid, black alcohols, VITAMIN, polysaccharide, trace element, nucleic acid etc.In Radix Panacis Quinquefolii, topmost effective constituent is Radix Panacis Quinquefolii Saponin, and it is also the most significant material of physiologically active in Radix Panacis Quinquefolii, and people have carried out a large amount of research to Radix Panacis Quinquefolii saponin for this reason, successively therefrom isolates 20 various of monomer saponin(es.Many scholars start to pay close attention to the immunologic function of Radix Panacis Quinquefolii polysaccharide in recent ten years.
Radix Panacis Quinquefolii is the precious medicinal plant of China's tradition, be applied in widely human health care and medical treatment above, and market increases gradually to its demand.Because Radix Panacis Quinquefolii requires the impact of very strict and easy climate and disease and pest on edaphic condition, so production cost is high, the destruction Of resources is serious, and its growth cycle is long especially, to such an extent as to Radix Panacis Quinquefolii output is on the low side, and economic benefit is bad.Therefore to find output and the quality that an effective approach improves Radix Panacis Quinquefolii, improve its productivity effect.
Summary of the invention
The object of the present invention is to provide a kind of Radix Panacis Quinquefolii to send out shape root induction and plant regeneration research method, so that it is difficult to solve better the differentiation of the root that plant regeneration faces, or the few difficult problem of energy for growth radical amount weak and that form.
To achieve these goals, technical scheme of the present invention is as follows.
Radix Panacis Quinquefolii is sent out shape root induction and a plant regeneration research method, comprises the following steps:
(1) explant is processed: (I) seeds of American ginseng is sprouted and processed: through the husky lamination of hiding, process, the seed of breach, is seeded in dish for cultivating, the cultivation of shading, after sowing 1～2 week seedling and growing, by 0.1% mercuric chloride sterilization 6min, sterile water wash 4～5 times.By its blade, the segment of being cut into 1.0cm with leaf children stem and stem section, be connected on MS solid medium preculture as the parent material transforming.(II) processing of Radix Panacis Quinquefolii 1 year, biennial root: after Radix Panacis Quinquefolii 1 year, biennial root surface are cleaned up with clear water, flowing water rinses 3～4h, with after 0.1% mercuric chloride sterilization 10min～20min, aseptic washing 5 times, is cut into the thin slice that 0.2cm is thick and is placed on MS solid medium stand-by.
(2) bacterial classification is preserved, is activated and cultivates: the long-term preservation method of Agrobacterium rhizogenes is that Agrobacterium bacterial classification is dissolved in glycerine, under-20 ℃ of conditions, preserves, and this method can be preserved bacterial classification several years.The method of short-term preservation is that Agrobacterium rhizogenes is inoculated on YEB solid medium and is cultivated, and after growing bacterial plaque, in 4 ℃ of refrigerators, preserves, and this kind of method can be preserved the bacterial classification several months.
Under aseptic condition, the bacterium liquid of preserving with inoculating needle picking is rule on YEB solid plate, and 28 ℃ are placed under dark condition and cultivate, until grow obvious single bacterium colony.Then single colony inoculation of picking is in 50mL liquid YEB substratum, under 120r/min, 28 ℃, dark condition, cultivates 24h, and now YEB liquid nutrient medium is become muddy faint yellowly by the transparent reddish-brown before inoculating, and proves that bacterium normally grows.Get the bacterium liquid that 1mL activated and add in the YEB liquid nutrient medium of 50mL, 120r/min, cultivates under 28 ℃ of dark condition, and when activation bacterium liquid reaches growth logarithmic phase, bacterium liquid now just can be used as to infect and uses.
(3) Radix Panacis Quinquefolii is sent out the optimization of shape root induction condition:
(3a) different strain is sent out the impact of shape root induction rate on Radix Panacis Quinquefolii: select Agrobacterium rhizogenes R1601,8196, A4, tetra-bacterial classifications of rolA to access in YEB liquid nutrient medium after dull and stereotyped activation culture, treat that its bacterium liquid growth concentration reaches OD
600be used for infecting use at=0.6 o'clock.The Radix Panacis Quinquefolii seedling of 2～3 weeks seedling ages, selects well-grown children tender, by its blade, the segment of being cut into 1.0cm with leaf children's stem and stem section, infecting 10～30min without 25 ± 1 ℃ of shaking table 100～120r/min, under aseptic condition, takes out.Sterilizing filter paper blots material surface bacterium liquid, is connected on MS substratum, investigates different strain and Radix Panacis Quinquefolii is sent out to the impact of shape root induction rate.
(3b) bacterial concentration is sent out the impact of shape root induction rate on Radix Panacis Quinquefolii: the R1601 bacterium liquid preparing is diluted to OD with YEB liquid
600value is 0.2,0.4,0.6,0.8 and 1.0, to in the bacterium liquid of Radix Panacis Quinquefolii seedling explant input different concns, contaminate 15～30min, 25 ± 1 ℃ of shaking table 100～120r/min, then take out with aseptic filter paper and blot surperficial bacterium liquid, on common substratum, cultivate after 2d, be transferred to again MS and be total in culture medium, every 7d, change a subculture.
(3c) AS and time of infection are sent out the impact of shape root induction rate on Radix Panacis Quinquefolii: single colony inoculation of picking activates in the 50mL YEB liquid nutrient medium that adds AS, AS concentration is elected 0,100 μ mol/L shaking culture as, until its optical density value, be that 0.6 o'clock tissue segments by Radix Panacis Quinquefolii seedling immerses, time of infection is elected 10min, 15min, 20min, 25min and 30min as.
(3d) different explants is sent out the impact of shape root induction rate on Radix Panacis Quinquefolii: selecting explant is blade, stem section, petiole and the Radix Panacis Quinquefolii callus of 2～3 weeks seedling age Radix Panacis Quinquefolii seedlings, 1 year, biennial American ginseng root, selecting bacterial classification is Agrobacterium rhizogenes R1601, investigates the selection of Radix Panacis Quinquefolii explant to sending out the impact of shape root induction rate.Its Leaf, stem section, petiole are Ye Panfa, and immerged time is 15～30min, and Radix Panacis Quinquefolii callus, Radix Panacis Quinquefolii 1 year, biennial root are dripping method.
(3e) NAA concentration is sent out the impact of shape root induction rate on Radix Panacis Quinquefolii: in pre-incubated MS minimum medium, adding a certain amount of NAA is of value to the induction of sending out shape root.The petiole of 2～3 weeks Radix Panacis Quinquefolii seedlings of take is material, adds the concentration of NAA and select to be divided into 0mg/L, 2mg/L, 4mg/L, 6mg/L in MS minimum medium, relatively sends out shape root induction rate to investigate NAA optimal concentration.
(3f) the preculture time is sent out the impact of shape root induction rate on Radix Panacis Quinquefolii: selecting MS+NAA2.0mg/L solid medium is preculture matrix, selecting the stem section of 2～3 weeks Radix Panacis Quinquefolii seedlings is material, the preculture time is elected 0h, 12h, 24h, 48h, 72h, 96h as, to investigate the preculture time, Radix Panacis Quinquefolii is sent out to shape root induction rate.
(3g) a certain amount of NAA is added in the impact that NAA concentration is sent out shape root induction rate on Radix Panacis Quinquefolii in pre-incubated MS minimum medium is of value to the induction of sending out shape root.The petiole of 2～3 weeks Radix Panacis Quinquefolii seedlings of take is material, adds the concentration of NAA and select to be divided into 0mg/L, 2mg/L, 4mg/L, 6mg/L in MS minimum medium, relatively sends out shape root induction rate to investigate NAA optimal concentration.
(3h) incubation time is sent out the impact of shape root induction rate on Radix Panacis Quinquefolii altogether: selecting incubation time is altogether 12h, 24h, 36h, 48h, and culture medium is elected MS as altogether.The stem section of 2～3 weeks Radix Panacis Quinquefolii seedlings of take is material, and preculture substratum is to be 48h the MS+NAA2.0mg/L time, and bacterial classification is R1601, with investigate matrix together incubation time Radix Panacis Quinquefolii is sent out to the impact of shape root induction rate.
(3i) concentration that affects cephamycin (Cef) that cephamycin (Cef) concentration is sent out shape root induction rate to antibacterial and Radix Panacis Quinquefolii is chosen as 200mg/L, 300mg/L, 400mg/L, 500mg/L, 600mg/L, and degerming matrix is chosen as the basic solid medium of MS.Comprehensive relatively fungistatic effect, pollution rate, three indexs of acquisition root of hair number, the optimal concentration of investigation cephamycin (Cef).
(4) Radix Panacis Quinquefolii is sent out acquisition and the evaluation of shape root:
(4a) acquisition of Radix Panacis Quinquefolii root of hair:
1. explant preculture: after the sterilization of Radix Panacis Quinquefolii seedling, its blade is cut into the fritter of 1.0cm * 1.0cm, petiole and stem section are cut into the long section of 1.0cm, Radix Panacis Quinquefolii 1 year, biennial root flowing water rinse and remove surperficial surface dust, after mercuric chloride sterilization, distilled water is rinsed well for 3～5 times, depending on material thickness, be cut into 0.2～0.5cm thin slice, above all material is connect in MS+NAA2mg/L substratum and under dark condition, cultivates 48h for infecting.
2. infect together and cultivate: on ultra-clean aseptic working platform, by pre-incubated explant material, transfer to and be equipped with in the triangular flask that is ready to bacterium liquid, after sealing, be placed on 120r/min, on shaking table under dark condition, infect 10～30min, metainfective explant blots material surface bacterium liquid with aseptic filter paper, is inoculated in MS solid medium, under dark, 25 ℃ of conditions, cultivates altogether 48h.
3. degerming is cultivated: by aseptic water washing 3 times of the infection explant material after common cultivation, aseptic filter paper blots the sterilized water on explant material surface, is transferred on the MS solid medium that contains cephamycin (Cef) 500mg/L and cultivates.Under dark, 25 ℃ of conditions, degerming is cultivated.
(4b) Radix Panacis Quinquefolii callus and 1 year, biennial root are sent out the induction of shape root: with the agrobacterium rhizogene strain 8 μ L that micropipette rifle is drawn activation, drip on the Radix Panacis Quinquefolii callus tangent plane of receiving MS solid medium cuts into slices with American ginseng root.In operating process, avoid bacterium drop to MS solid medium, the material that drips bacterium liquid is placed on to 25 ℃, in the incubator under dark condition, cultivate.After 15 days, remove the dead material of brownization, remaining material transfer is continued to cultivate to fresh MS solid medium.
(4c) PCR Molecular Detection: the Radix Panacis Quinquefolii that infects acquisition is sent out shape root and compared and embody obvious difference with normal American ginseng root in form, such as Radix Panacis Quinquefolii send out shape root can ramp on the MS substratum without hormone, the advantage such as many hairs, multi-branched, apogetropism.And in a shape root liquid medium within, the speed of growth is very fast, is greater than Radix Panacis Quinquefolii callus suspension culture or unconverted root culturd, these phenotypes can be sent out shape root for judgement Radix Panacis Quinquefolii morphologic foundation is provided.By PCR method to Ri plasmid T
lthe rolC gene test of-DNA will further provide molecular biological foundation.
1. design of primers and synthetic:
Primer 1:5 '-GATATATGCCAAATTTACACTAG-3 ';
Primer 2: 5 '-GTTAACAAAGTAGGAAACAGG-3 ';
Above two primers of utilization are by pcr amplification to the upper rolC gene fragment of T-DNA, and expection length is 564bp, and primer is to be synthesized by the biological company limited of Beijing ancient cooking vessel state.
2. CTAB method is extracted Radix Panacis Quinquefolii and is sent out the total DNA of shape root;
3. extract the Ri plasmid of Agrobacterium rhizogenes;
4. PCR loop parameter:
5. PGR reaction system:
Ultrapure water is settled to 25 μ l:
6. agarose gel electrophoresis, takes pictures;
(5) Radix Panacis Quinquefolii is sent out the mensuration of shape root liquid culture increment and total saponin content:
Sending out shape root liquid culture employing MS liquid nutrient medium is minimum medium (3% sucrose, pH value 5.8～6.0), the shape root tip of a root access of sending out of 12 eugonic about 2cm is equipped with in 30mL MS liquid nutrient medium, temperature is 24 ± 1 ℃, shaking speed 100r/min, every 3d sampling, measure the content of sending out shape root dry weight and total saponins, measure one month.All data are the mean value of 3 bottles, repeat twice.
Calibration curve method is to sending out the mensuration of total saponins in shape root: the plot step of typical curve is: 1. get Rg10.020g and be dissolved in methyl alcohol, constant volume is 2mg/mL; 2. get Rg1 reference substance solution 30,60,90,120,150 μ L; 3. be placed in respectively 5 test tubes (10mL, tool plug), methanol constant volume is 150 μ L, with corresponding reagent, compares; 4. test tube is placed in to ice-water bath adds 5% Vanillin alcoholic solution and 72% sulfuric acid shakes up, 60 ℃ of water bath with thermostatic control 20min; 5. after ice-water bath 15min, at 755B spectrophotometer 544nm place, detect.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 (%), as shown in Figure 1.
Send out the mensuration of total saponins in shape root: get fresh the shape root that certain quantity of fluid is cultivated, after distilled water flushing, with filter paper, blot the moisture of sending out shape root surface, weigh and send out shape root fresh weight.Then in vacuum drying oven, at 80 ℃, be dried to constant weight, weigh.Dried shape root pulverized, put into triangular flask, adding a certain amount of concentration is 40% alcohol solvent, and triangle bottleneck is sealed with sealed membrane.Be placed in ultra-sonic generator and carry out separation and Extraction, extraction time 15min, ultrasonic frequency is 28KHz, extracts twice.In ice-water bath, add 5% Vanillin alcoholic solution and 72%H
2sO
4dyeing, 60 ℃ of water bath with thermostatic control 10min, ice-water bath 15nin, surveys absorbancy in 544nm place.
(6) Radix Panacis Quinquefolii is sent out foundation and the condition optimizing of shape root regeneration system:
(6a) Radix Panacis Quinquefolii is sent out the induction of shape root-derived callus: the Radix Panacis Quinquefolii of take is sent out shape root as explant, receive interpolation different concns, 2 of various combination, on the MS substratum of the growth regulators such as 4-D, BA, KT, wherein sucrose concentration is 30g/L, agar content is 6.5g/L, and pH value is 6.0 (before sterilizings), and the sterilizing pressure of substratum is that 0.1MP continues 15～20min.Be put under different illumination conditions, periodicity of illumination is 12h/d, temperature is 24 ± 1 ℃, statistics callus incidence after two weeks (explant number/explant sum of callus occurs) is analyzed different growth regulators and the impact of illumination condition on callus induction, screening best setting material and concentration and illumination condition.
(6b) Radix Panacis Quinquefolii is sent out the generation of shape root embryoid: embryoid is occurred for different hormone combinations, concentration and illumination and 2 of different concns and combination got respectively in the impact of growth, 4-D, KT, BA process culture, analyze the impact that each hormone occurs and grows embryoid, filter out the best medium formula that embryoid occurs and grows.Get under dark, illumination condition and cultivate, observe the impact of illumination condition on culture.Illumination provides (about 1200Lx) by fluorescent lamp, and the photoperiod is 12h/d.
Further, the CTAB method in step (4c) is extracted Radix Panacis Quinquefolii to send out the detailed process of the total DNA of shape root as follows:
(1) getting 280mg sends out shape root through the Radix Panacis Quinquefolii of succeeding transfer culture and adds 10%PVP in liquid nitrogen, to be fully ground to Powdered (about 30s～1min);
(2) with spoon, be carefully transferred in the centrifuge tube of 1.5ml, add the Extraction buffer of 660 μ 12 * CTAB of 65 ℃ of preheatings, 65 ℃ of water-bath 45min, vibrate once gently every 5～10min;
(3) the Solution I that adds 200 μ L ice baths to cross, vortex vibration is uniformly distributed cell, places 10min under room temperature:
(4) add the fresh Solution II configuring of 400 μ L, put upside down and mix gently, ice bath 5min;
(5) add 300 μ L Solution III, repeatedly put upside down for several times, solution is uniformly dispersed in the cellular lysate thing of thickness, then put 5min on ice;
(6) the centrifugal 10min of 12000r/min, carefully draws supernatant liquor and adds in 1.5mL centrifuge tube.
(7) add isopyknic saturated phenol, chloroform, by thermal agitation, mix organic phase and water, the centrifugal 5min of 12000r/min.
(8) take out centrifuge tube and be cooled to room temperature, add isopyknic chloroform: primary isoamyl alcohol (24:1), mixes 12000rpm, centrifugal 10min;
(9), with a rifle head that cuts off front, carefully supernatant liquor is transferred in a new 1.5ml centrifuge tube, and added the dehydrated alcohol of-20 ℃ of precoolings of two volumes, see thread precipitation, place 30min, 12000rpm at-20 ℃, centrifugal 5min, obtains faint yellow precipitation;
(10) supernatant liquor that inclines, and with the thieving paper residual solution that exhausts, add 300 μ l ultrapure waters again to dissolve, then add the dehydrated alcohol 600 μ l of-20 ℃ of precoolings, precipitation is spent the night;
(11) 12000rpm, centrifugal 10min, obtains white gelatinous precipitate.The supernatant liquor that inclines, and with the thieving paper residual solution that exhausts, add 70% ethanol 800 μ l washing precipitation 2～3 times;
(12) exhaustion residual solution, puts Bechtop and dries up (approximately needing 10～15min);
(13) add 40～60 μ l ultrapure waters, dissolution precipitation, adds 3 great lRnase, and room temperature is placed after 30min, preserves at-20 ℃.
Further, the detailed process of the Ri plasmid of the extraction Agrobacterium rhizogenes in step (4c) is as follows:
(1) the mono-bacterium colony of picking R1601 is in 10ml YEB substratum, and 28 ℃, 120rpm, incubated overnight;
(2) draw the cultured bacterium liquid of 8ml, add in 10ml centrifuge tube, 4000rpm, centrifugal 5min, the nutrient solution that inclines, adds the STE of 2ml precooling, Eddy diffusion cell.Draw respectively 1ml bacteria suspension, add in 1.5ml centrifuge tube, the centrifugal 2min of 12000rpm, the nutrient solution that inclines, obtains appropriate somatic cells;
(3) the Solution I that adds 200 μ l ice baths to cross, vortex vibration is uniformly distributed cell, under room temperature, places 10min;
(4) add the fresh Solution II configuring of 400 μ l, put upside down and mix gently, ice bath 5min;
(5) add 300 μ l Solution III, repeatedly put upside down for several times, solution is uniformly dispersed in the cellular lysate thing of thickness, then put 5min on ice;
(6) the centrifugal 10min of 12000rpm, carefully draws supernatant liquor and adds in 1.5ml centrifuge tube.
(7) add isopyknic saturated phenol, chloroform, by thermal agitation, mix organic phase and water, the centrifugal 5min of 12000rpm.
(8) carefully drawing supernatant liquor adds in new 1.5ml centrifuge tube, with isopyknic chloroform again extracting once;
(9) carefully draw supernatant liquor and add in new 1.5ml centrifuge tube, add the dehydrated alcohol of 2 times of volumes, the 3mol/LNaAc of 1/10 volume, ice bath 30min, 12000rpm, centrifugal 10min, the supernatant liquor that inclines, is upside down on thieving paper, and solution is flow to end:
(10) add 1ml70% washing with alcohol twice, the centrifugal 5min of 12000rpm, collecting precipitation;
(11) washings that inclines, is inverted on thieving paper and blots remaining solution, on Bechtop, dries up;
(12) add 50 μ l TE resuspended, standby.
Further, the agarose gel electrophoresis detailed process in step (4c) is as follows:
(1) with adhesive tape, seal electrophoresis chamber rubber moulding edge and form a mould;
(2) prepare enough electrophoretic buffers (1 * TBE) in order to fill electrophoresis chamber and preparation gel;
(3) accurately take a certain amount of agar powder and be added to and in the triangular flask that fills a certain amount of electrophoretic buffer, in microwave oven, be heated to melt, then carefully add ethidium bromide, concentration is 0.5 μ g/ml, and rotation is to mixing gelating soln gently;
(4) on mould, add a suitable comb and form well, then pour appropriate warm agarose solution into, after room temperature 30min, remove edge sealing adhesive tape.Gel is put into electrophoresis chamber, make electrophoretic buffer not have the about 1mm of gel;
(5) mix the sample-loading buffer of pcr amplification product sample and 0.2 times of volume;
(6) with liquid-transfering gun and disposable rifle head, sample mix liquid is slowly added in the well of submergence gel, molecular mass standard substance are added in the left side of sample;
(7) cover electrophoresis chamber lid, connect electrode plug, DNA has the negative electrode extreme swimming that starts to face south, and gives the voltage of 1～5V/cm, can observe soon tetrabromophenol sulfonphthalein and enter in colloid from well;
(8) when DNA sample or dimethylbenzene cyanogen FF and tetrabromophenol sulfonphthalein have been moved enough distances in gel, close power source, extract electrode plug and open electrophoresis chamber lid, gel is placed under gel imaging system and is taken pictures.
This beneficial effect of the invention is: the present invention infects Radix Panacis Quinquefolii induction with Agrobacterium rhizogenes and sends out a shape root, has studied the impact that some factors of infecting infect efficiency to sending out shape root.Send out shape root and have very high reproductive potential, can make synthetic seed prolonged preservation, the germplasm of the rare traditional Chinese medicines such as this behaviour Radix Panacis Quinquefolii is preserved provides a new way.The shape root of sending out of Radix Panacis Quinquefolii of simultaneously take passes through embryoid way regeneration induction plant as material, for a new direction has been opened up in the breeding of Radix Panacis Quinquefolii.There is not mosaic problem in the conversion of Agrobacterium rhizogenes, adopts Ri plasmid more simply, easily foreign gene to be imported; The regeneration plant being obtained by root of hair in addition itself is transfer-gen plant, and render transgenic plant has the advantages that to be easy to take root, and the differentiation that can solve well the root that plant regeneration faces is difficult, or the few difficult problem of energy for growth radical amount weak and that form.
Accompanying drawing explanation
Fig. 1 is the mensuration figure of embodiment of the present invention Plays curve.
Fig. 2 is that in the embodiment of the present invention, Agrobacterium rhizogenes R1601 bacterial concentration is sent out the figure that affects of shape root induction rate on Radix Panacis Quinquefolii.
Fig. 3 is AS and the affect figure of infection time on Radix Panacis Quinquefolii inductivity in the embodiment of the present invention.
Fig. 4 is that in the embodiment of the present invention, different explants is sent out the figure that affects of shape root induction rate on Radix Panacis Quinquefolii.
Fig. 5 is that in the embodiment of the present invention, NAA concentration is sent out the figure that affects of shape root induction rate on Radix Panacis Quinquefolii.
Fig. 6 be in the embodiment of the present invention preculture time Radix Panacis Quinquefolii is sent out to the figure that affects of shape root induction rate.
Fig. 7 is total to incubation time Radix Panacis Quinquefolii to be sent out to the figure that affects of shape root induction rate in the embodiment of the present invention.
Fig. 8 is the affect figure of various ce f concentration on fungistatic effect and induction root of hair number in the embodiment of the present invention.
Fig. 9 is that in the embodiment of the present invention, Radix Panacis Quinquefolii is sent out shape root PCR detected result figure.
Figure 10 is the performance graph that in the embodiment of the present invention, Radix Panacis Quinquefolii is sent out under shape root liquid culture condition growth and saponin content.
Figure 11 is the affect figure of illumination on culture in the embodiment of the present invention.
Embodiment
Below in conjunction with embodiment, further set forth the embodiment of this invention.
Embodiment 1: Radix Panacis Quinquefolii is sent out the optimization of shape root induction condition
(1) different strain is sent out the impact of shape root induction rate on Radix Panacis Quinquefolii: in embodiment, select R1601,8196, A4, tetra-bacterial classifications of rolA to infect the Radix Panacis Quinquefolii seedling tissue segments of 2～3 weeks seedling ages, investigate different strain and Radix Panacis Quinquefolii is sent out to the impact of shape root induction rate, in Table 1.
Table 1 different strain is sent out the impact of shape root induction rate on Radix Panacis Quinquefolii
Pathogenecity between different Agrobacterium rhizogenes bacterial classifications exists very big-difference, the 20d that the Radix Panacis Quinquefolii seedling tissue segments of 2～3 weeks seedling ages that infected by Agrobacterium rhizogenes R1601 is infecting, the granulation tissue of incision adularescent occurs, have some granulation tissue to appear at the place of brownization of wound, it not is clearly that other granulation tissue comes across on the callus expanding.Along with the prolongation of time, the random elongation of granulation tissue, its tip is rendered as ivory buff hat cap, and some is transparent in trunk summary, occurs countless tiny white root hairs after certain length on trunk.After its contact culture medium, do not go deep into substratum, but be attached at media surface continued growth; When contact bottle wall, along the continued growth that makes progress of a bottle wall.
(2) bacterial concentration is sent out the impact of shape root induction rate on Radix Panacis Quinquefolii: bacterial concentration is to affect the important factor that Radix Panacis Quinquefolii is sent out shape root induction rate.Bacterial concentration excessive or too small all cannot, if bacterial concentration is excessive, Agrobacterium rhizogenes growth fraction is rapider, explant occur damage or browning.Otherwise bacterial concentration is too small, explant transforms complete not.Therefore the type that bacterial concentration should be selected with different floristics, explant and different infection times and have difference.Different optical density(OD) R1601 bacteria suspensions to preculture 48h the tissue segments of Radix Panacis Quinquefolii seedling infect, inductivity is as shown in table 2 and Fig. 2.Result shows: when optical density value is OD
600be that 0.6 o'clock inductivity reaches as high as 11.1%, inductivity specific optical density value be 0.4 and 0.8 exceed respectively 5.3 and 3.8 percentage points.
Table 2 Agrobacterium rhizogenes R1601 bacterial concentration is sent out the impact of shape root induction rate on Radix Panacis Quinquefolii
Condition：preconditioning?time=48h.
(3) AS (Syringylethanone) sends out the impact of shape root induction rate on Radix Panacis Quinquefolii with time of infection: relevant research shows to have 9 kind of plant cell releasor molecules to participate in activating the genetic expression of Vir district, they are all water-soluble phenolic compounds, what wherein effect was the strongest is glycoloyl syringone and Syringylethanone, when shape root is sent out in induction, can add this appropriate compounds to promote plasmid T-DNA shift, process and integrate in plant thing cellular genome.By single colony inoculation of picking in adding the 50ml YEB liquid nutrient medium of 100 μ mol/L AS, 120r/min, shaking culture under 28 ℃ of dark condition.Dilution bacterium liquid makes optical density value reach 0.6.By Radix Panacis Quinquefolii explant and the common shaking culture of bacterium liquid, incubation time is respectively 10min, 15min, 20min, 25min and 30min.Not add the bacterium liquid of AS, cultivate same time as a control group, to investigate immerged time and the AS impact on induction of hairy roots rate simultaneously.Table 3 and Fig. 3 show: AS has promoter action to induction of hairy roots rate, and peak rate of conversion reaches 9.8%, and occur when immerged time is 20min, illustrate that the selection of infection time is also very great to the meaning of induction of hairy roots rate.
Table 3AS and infection time are sent out the impact of shape root induction rate on Radix Panacis Quinquefolii
(4) explant selects Radix Panacis Quinquefolii to send out the impact of shape root induction rate: the explant of the plant generally adopting in genetic transformation is all younger tender part, this may be more vigorous because of the histocyte division of young tender part, so the probability that they are successfully integrated by foreign DNA is very large.It is blade, stem section, petiole and the ginseng callus of 2～3 weeks seedling age Radix Panacis Quinquefolii seedlings, 1 year, 2 year year life ginseng that the present embodiment is selected explant.Investigate the selection of explant to sending out the impact of shape root induction rate.Result in table 4 and Fig. 4 shows: petiole is optimum explant, and inductivity is 9.4%, and annual American ginseng root takes second place, and inductivity is 8.3%, and callus does not obtain a shape root.The conversion condition of experimental selection is to be 48h the explant preculture time, and explant and Agrobacterium rhizogenes are cultivated 48h altogether, and culture medium is that MS adds AS100 μ mol/L, OD altogether
600=0.6, the blade of Radix Panacis Quinquefolii, stem section and petiole are Ye Panfa, and immerged time is 30min, and Radix Panacis Quinquefolii callus, Radix Panacis Quinquefolii are taken root for dripping method for 1,2 years.
Table 4 different explants is sent out the impact of shape root induction rate on Radix Panacis Quinquefolii
Condition：OD
600=0.6，preconditioning?time=48h
(5) NAA concentration is sent out the impact of shape root induction rate on Radix Panacis Quinquefolii: as shown in table 5 and Fig. 5, in preculture substratum, add certain density NAA and be of value to the induction that Radix Panacis Quinquefolii is sent out shape root, do not add NAA group inductivity all lower than adding NAA group, it is not add 2.19 times of NAA that NAA2.0mg/L inductivity reaches 6.8%.
Table 5NAA concentration is sent out the impact of shape root induction rate on Radix Panacis Quinquefolii
Condition：OD
600=0.6.
(6) impact of preculture time on Panax quinquefolium Hairy Root inductivity: in the present embodiment, select containing preculture on the MS solid medium of 2mg/LNAA, the preculture time is 0～48h.By table 6 and Fig. 6, can find out that different preculture time conditions are sent out the inductivity impact of shape root to Radix Panacis Quinquefolii under same culture conditions very large, increase in time of preculture 0～48h inductivity and improving gradually, starts to decline and surpass 48h inductivity.Draw thus and adding on the MS solid medium of 2mg/LNAA preculture 48h for best, preculture overlong time or do not have preculture all can reduce inductivity.
The table 6 preculture time is sent out the impact of shape root induction rate on Radix Panacis Quinquefolii
Condition：OD
600=0.6，
(7) incubation time is sent out the impact of shape root induction rate on Radix Panacis Quinquefolii altogether: explant and Agrobacterium altogether incubation time are another important factors that shape root transformation efficiency is sent out in impact.Inductivity that explant is cultivated 48h is altogether the highest (in Table 7, Fig. 7), and finds that preculture 48h cultivates altogether 48h through metainfective explant and obtains better result.Although lengthen incubation time altogether, can increase transforming machine meeting, while cultivating altogether, ask and surpass two days later, can make the excessive breeding of bacterium cause the death of explant.
Table 7 altogether incubation time is sent out the impact of shape root induction rate on Radix Panacis Quinquefolii
Condition：OD
600=0.6，preconditioning?time=48h.
(8) cephamycin (Cef) concentration is on antibacterial and impact inductivity: while utilizing Agrobacterium rhizogenes leaf dish method to carry out Genetic Transformation in Higher Plants, cephamycin (Cef) can effectively suppress the growth of Agrobacterium rhizogenes on explant and substratum.From table 8 and Fig. 8, can find out: cephamycin concentration is within the scope of 200～600mg/L, and along with the increase fungistatic effect of its concentration is just better, the root of hair number of acquisition is between 2～24.Comprehensive root of hair number, fungistatic effect, three indexs of inductivity of obtaining, think that obtain more root of hair number keeps again the optimum cephamycin concentration of better fungistatic effect to should be 500mg/L simultaneously.
The impact of the different cephamycin concentration of table 8 on fungistatic effect and acquisition root of hair number
Note: ++ for there being a large amount of Agrobacteriums, +++ for there being a small amount of Agrobacterium, ++++for there being minute quantity Agrobacterium
Embodiment 2: Radix Panacis Quinquefolii is sent out acquisition and the evaluation of shape root
(1) Radix Panacis Quinquefolii is sent out the acquisition of shape root: the material after cultivating altogether rinses 3～5 times with 500mg/L cephamycin (Cef) aqueous solution, aseptic filter paper blots material surface moisture content, then material is received on the MS solid medium containing 500mg/L cephamycin (Cef) and is carried out degerming cultivation.Change weekly a subculture, after 5～6 weeks, material is not transferred to and is cultivated containing continuing on antibiotic MS solid medium.In the present embodiment, it is at metainfective about the 30th day (Condition:OD600=0.6, seedlings with stem explants, preconditioning time=48h, AS100 μ mol/L) the earliest that Radix Panacis Quinquefolii is sent out shape root.Send out shape root and appear at wound, can produce several even tens shape roots on an explant material, some explant material is that first dedifferentiation produces loose callus, then on callus surface, grows gradually and sends out a shape root.The shape root of sending out of all generations all shows fast growth, has that tiny white root hair, branch are many, hormone autotrophic.The contrast explant material dying without bacterium liquid inductance is not sent out shape root and is produced, though some explant materials two ends slightly expand, further dead gradually in culturing process on without hormone solid medium.When a shape root grows to 1cm left and right, cut to be connected on MS solid medium and cultivate.
(2) Radix Panacis Quinquefolii is sent out the evaluation of shape root: get blade and the callus 200mg of Radix Panacis Quinquefolii root of hair 100mg and unconverted Radix Panacis Quinquefolii, extract the DNA that sends out shape root according to 2.2.4.3, as detecting sample.Extract the DNA in Agrobacterium rhizogenes and non-transformed Radix Panacis Quinquefolii blade and callus simultaneously, be prepared into positive control and negative control sample.The good various DNA of extraction are carried out to amplified reaction under PCR reaction conditions noted earlier.PCR product after electrophoresis (5V/cm), is observed DNA band and takes pictures on 1.2% sepharose under gel imaging system.Its result is as Fig. 9.As seen from Figure 9, according to the pair of primers that provided, Radix Panacis Quinquefolii is sent out to shape root DNA above and carry out PCR detection, there is the 564bp specific band of expection in result, this band does not occur, illustrates that Ri plasmid T-DNA has been incorporated in the genome of Radix Panacis Quinquefolii in unconverted Radix Panacis Quinquefolii blade and callus.
(3) Radix Panacis Quinquefolii is sent out the performance graph of shape root growth and saponin content: under liquid culture condition, shape root growth sent out by Radix Panacis Quinquefolii and total saponin content curve meets " S " type (seeing Figure 10) substantially.Radix Panacis Quinquefolii is sent out being grown in of shape root after of short duration lag period, just starts to enter logarithmic phase during by the 9th day, and during by the 27th day, growth reaches climax, after the 27th day, almost stops growing, and dry weight no longer increases substantially.The content of total saponins is along with sending out its also raising thereupon of increase of the increment of shape root, and maximum value also appears in total saponin content in the time of the 27th day, and follow-up production just starts to decline gradually.Embodiment 3: Radix Panacis Quinquefolii is sent out the impact of the different growth regulator confrontation of induction (1) the Radix Panacis Quinquefolii induction of hairy roots callus of shape root-derived callus: inductivity analysis in table 9 and the table 10 of Radix Panacis Quinquefolii root of hair callus.The factor that must affect Radix Panacis Quinquefolii root of hair callus induction rate according to the size order of extreme difference is 2,4-D>KT>BA, and the optimum level of each factor is A3B2C3,2,4-D (2.0mg/L), BA (1.0mg/L), KT (0.5mg/L).From variance analysis, the induction to Radix Panacis Quinquefolii root of hair callus, 2,4-D has the greatest impact, and reaches utmost point conspicuous level; KT takes second place, and has reached conspicuous level; The impact of BA is less, acts on not remarkable.The optimal medium of the Radix Panacis Quinquefolii induction of hairy roots callus of then selecting in the present embodiment is: MS+2,4-D2.0mg/L+KT0.5mg/L.
Table 9 orthogonal test and range analysis
Table 10 variance analysis
Note: (P<0.01) represent utmost point conspicuous level; (P<0.05) represent conspicuous level
(2) impact of illumination on Radix Panacis Quinquefolii induction of hairy roots callus: can dedifferentiation be callus (seeing Figure 11 and table 11) at illumination or the dark condition ginseng root of hair of travelling to the West no matter be.But illumination has obvious restraining effect to its growth.Compare with cultivating the Radix Panacis Quinquefolii root of hair callus that induction produces under illumination condition, the callus color of inducing under dark culture condition is more shallow, yellowish or light yellowish brown, and 25d left and right is callus all almost, and callus forms in a large number, and growth is rapidly.But under illumination condition, its growth is suppressed, during 40d, inductivity is still very low, and poor growth.Radix Panacis Quinquefolii root of hair dedifferentiation speed under dark condition is fast and inductivity is the highest as can be seen here.
The impact of table 11 illumination on culture
The callus of Radix Panacis Quinquefolii induction of hairy roots carries out initial 3 middle of the month of subculture, dedifferentiation not exclusively still has the shape root of sending out to produce, this is very unfavorable to the growth of callus, and lower shape radical amount of cytokinin concentration is just more, along with increasing of the prolongation of Subculture Time, the algebraically of transferring, not exclusively dedifferentiation quantity is fewer and feweri.
Embodiment 4: Radix Panacis Quinquefolii is sent out the induction of shape root embryo callus
(1) impact of the subculture number of the callus of initial induction on root of hair embryonic callus induction: the callus subculture number of initial induction and the embryo sexuality of callus have much relations.The embryo callus subculture incidence of initial callus is higher than the callus through subculture, and generally through subculture, the ability of the generation of the callus about year embryo callus subculture obviously goes down.The callus quality of initial induction is hard, fine and close, and soft, loose through the callus of subculture repeatedly, surface water materialization is serious.The embryoid number that the callus induction of initial induction goes out is more than subculture, illustrates that callus lost gradually embryo's generating ability in subculture process.
(2) different plant hormones and the concentration impact on root of hair embryonic callus induction: as shown in table 12, the pistac that induction is produced, the tight shape of quality, the Radix Panacis Quinquefolii root of hair callus of fast growth is received containing in the substratum of different plant-growth regulator, be incubated at containing BA0.5mg/L and 1mg/L2, the minicell bulk that the Radix Panacis Quinquefolii root of hair Transformation of Callus on the substratum of 4-D is yellow-white, loose frangible, and fast growth, in the generation of its visible embryoid in surface; Be incubated at containing 2 of KT0.5mg/L and 1mg/L, the callus propagation on the substratum of 4-D is very fast, and quality is closeer, has the generation of embryoid; Containing 0.5mg/L2, it is green that the callus on the substratum of 4-D is all after succeeding transfer culture; In the concentration of 2,4-D, reach the callus of cultivating on the substratum of 2.0mg/L and also can produce and a kind ofly containing aquosity, white, approach transparence, callus that quality is loose, the further succeeding transfer culture of this class callus, is difficult to break up again.Through succeeding transfer culture, only have the callus of yellow-white cell bulk that embryoid can occur in a large number, therefore think that this callus is embryo callus, so suitably reduce the formation that the concentration of 2,4-D can be induced Radix Panacis Quinquefolii root of hair embryo callus preferably.
The induction of table 12 plant-growth regulator to embryo callus
(3) impact of illumination on root of hair embryonic callus induction: no matter under dark condition or illumination condition, Radix Panacis Quinquefolii root of hair callus has embryo callus subculture to occur, but the dark restraining effect certain to having of embryo callus subculture.As shown in table 13, to compare with the Radix Panacis Quinquefolii root of hair embryo callus of cultivating induction generation under illumination condition, the embryo callus color of inducing under dark culture condition is more shallow, yellowish or light yellowish brown.For embryo callus subculture genetic coefficient, when illumination, it is high when dark that Radix Panacis Quinquefolii is sent out shape root embryo callus incidence, shows that illumination condition is of value to the generation of sending out shape root embryo callus.
The impact of table 13 illumination on the induction of embryo callus
Embodiment 5: embryoid induction obtains regeneration plant
(1) hormone and somatic embryo occur: when embryo callus being transferred to hormone concentration reduction or removing the substratum of 2,4-D, will start somatic embryo and occur.About one month, can produce the early stage embryoid of macroscopic oyster white, quantity is a lot, also easily from callus, separates.The globular embryo in this period is comprised of the basically identical cell of size, is early stage globular embryo.During 40d, just can be observed a large amount of globular embryos, there is certain differentiation in their cell now, is globular embryo in late period.Minority globular embryo further breaks up, and through heart-shaped embryonic development, becomes cotyledon shape embryo.The impact that hormone concentration reduces the results are shown in following table (table 14).
Table 14 different hormone combinations sends out on Radix Panacis Quinquefolii the impact that shape root somatic embryo occurs
By table 14, can be obtained, the optimal medium that Radix Panacis Quinquefolii is sent out the generation of shape root somatic embryo is: MS+BA0.5mg/L
(2) impact of illumination on somatic embryo propagation: the propagation impact that the illumination condition of cultivation is sent out shape root somatic embryo to Radix Panacis Quinquefolii is very remarkable.Illumination condition is the essential condition that Radix Panacis Quinquefolii is sent out shape root somatic cell proliferation, the somatic embryo forming under illumination not only quantity is many, and regular shape, majority is all the solid bead of compact structure, and such somatic embryo can pass through quickly globular embryo, heart-shape embryo, torpedo-shape embryo and cotyledonary embryos stage and then develop into plant; And under dark condition, almost can not form new somatic embryo, and the somatic embryo development having formed is slow.As shown in Table 15.
The impact of table 15 illumination on somatic embryo propagation
(3) impact that substratum is sprouted somatic embryo: because somatic embryo is nonsynchronous, on somatic embryo proliferated culture medium, will form the somatic embryo of various different times, select the outer material in torpedo stage or cotyledon period of most somatic embryo to proceed to germination medium.If continue cultivated on former substratum, somatic embryo great majority rest on torpedo embryo or cotyledonary embryos period, seldom can sprout into plantlet, and somatic embryo is transferred to somatic embryo on germination medium, could sprout into plantlet.Concrete outcome is in Table 16.
Table 16 substratum sends out on Radix Panacis Quinquefolii the impact that shape root somatic embryo is sprouted
By table 16, can be obtained, the optimal medium that Radix Panacis Quinquefolii is sent out the sprouting of shape root somatic embryo is: MS.
Claims (4)
1. Radix Panacis Quinquefolii is sent out shape root induction and a plant regeneration research method, it is characterized in that: comprise the following steps:
(1) explant is processed: (I) seeds of American ginseng is sprouted and processed: through the husky lamination of hiding, process, the seed of breach, is seeded in dish for cultivating, the cultivation of shading, after sowing 1～2 week seedling and growing, by 0.1% mercuric chloride sterilization 6min, sterile water wash 4～5 times; By its blade, the segment of being cut into 1.0cm with leaf children stem and stem section, be connected on MS solid medium preculture as the parent material transforming; (II) processing of Radix Panacis Quinquefolii 1 year, biennial root: after Radix Panacis Quinquefolii 1 year, biennial root surface are cleaned up with clear water, flowing water rinses 3～4h, with after 0.1% mercuric chloride sterilization 10min～20min, aseptic washing 5 times, is cut into the thin slice that 0.2cm is thick and is placed on MS solid medium stand-by;
(2) bacterial classification is preserved, is activated and cultivates: the long-term preservation method of Agrobacterium rhizogenes is that Agrobacterium bacterial classification is dissolved in glycerine, under-20 ℃ of conditions, preserves; The method of short-term preservation is that Agrobacterium rhizogenes is inoculated on YEB solid medium and is cultivated, and after growing bacterial plaque, in 4 ℃ of refrigerators, preserves; Under aseptic condition, the bacterium liquid of preserving with inoculating needle picking is rule on YEB solid plate, and 28 ℃ are placed under dark condition and cultivate, until grow obvious single bacterium colony; Then single colony inoculation of picking is in 50mL liquid YEB substratum, under 120r/min, 28 ℃, dark condition, cultivates 24h, and now YEB liquid nutrient medium is become muddy faint yellowly by the transparent reddish-brown before inoculating, and proves that bacterium normally grows; Get the bacterium liquid that 1mL activated and add in the YEB liquid nutrient medium of 50mL, 120r/min, cultivates under 28 ℃ of dark condition, and when activation bacterium liquid reaches growth logarithmic phase, bacterium liquid now just can be used as to infect and uses;
(3) Radix Panacis Quinquefolii is sent out the optimization of shape root induction condition:
(3a) different strain is sent out the impact of shape root induction rate on Radix Panacis Quinquefolii: select Agrobacterium rhizogenes R1601,8196, A4, tetra-bacterial classifications of rolA to access in YEB liquid nutrient medium after dull and stereotyped activation culture, treat that its bacterium liquid growth concentration reaches OD
600be used for infecting use at=0.6 o'clock; The Radix Panacis Quinquefolii seedling of 2～3 weeks seedling ages, selects well-grown children tender, by its blade, the segment of being cut into 1.0cm with leaf children's stem and stem section, infecting 10～30min without 25 ± 1 ℃ of shaking table 100～120r/min, under aseptic condition, takes out; Sterilizing filter paper blots material surface bacterium liquid, is connected on MS substratum, investigates different strain and Radix Panacis Quinquefolii is sent out to the impact of shape root induction rate;
(3b) bacterial concentration is sent out the impact of shape root induction rate on Radix Panacis Quinquefolii: the R1601 bacterium liquid preparing is diluted to OD with YEB liquid
600value is 0.2,0.4,0.6,0.8 and 1.0, to in the bacterium liquid of Radix Panacis Quinquefolii seedling explant input different concns, contaminate 15～30min, 25 ± 1 ℃ of shaking table 100～120r/min, then take out with aseptic filter paper and blot surperficial bacterium liquid, on common substratum, cultivate after 2d, be transferred to again MS and be total in culture medium, every 7d, change a subculture;
(3c) AS and time of infection are sent out the impact of shape root induction rate on Radix Panacis Quinquefolii: single colony inoculation of picking activates in the 50mL YEB liquid nutrient medium that adds AS, AS concentration is elected 0,100 μ mol/L shaking culture as, until its optical density value, be that 0.6 o'clock tissue segments by Radix Panacis Quinquefolii seedling immerses, time of infection is elected 10min, 15min, 20min, 25min and 30min as:
(3d) different explants is sent out the impact of shape root induction rate on Radix Panacis Quinquefolii: selecting explant is blade, stem section, petiole and the Radix Panacis Quinquefolii callus of 2～3 weeks seedling age Radix Panacis Quinquefolii seedlings, 1 year, biennial American ginseng root, selecting bacterial classification is Agrobacterium rhizogenes R1601, investigates the selection of Radix Panacis Quinquefolii explant to sending out the impact of shape root induction rate; Its Leaf, stem section, petiole are Ye Panfa, and immerged time is 15～30min, and Radix Panacis Quinquefolii callus, Radix Panacis Quinquefolii 1 year, biennial root are dripping method;
(3e) NAA concentration is sent out the impact of shape root induction rate on Radix Panacis Quinquefolii: in pre-incubated MS minimum medium, adding a certain amount of NAA is of value to the induction of sending out shape root; The petiole of 2～3 weeks Radix Panacis Quinquefolii seedlings of take is material, adds the concentration of NAA and select to be divided into 0mg/L, 2mg/L, 4mg/L, 6mg/L in MS minimum medium, relatively sends out shape root induction rate to investigate NAA optimal concentration;
(3f) the preculture time is sent out the impact of shape root induction rate on Radix Panacis Quinquefolii: selecting MS+NAA2.0mg/L solid medium is preculture matrix, selecting the stem section of 2～3 weeks Radix Panacis Quinquefolii seedlings is material, the preculture time is elected 0h, 12h, 24h, 48h, 72h, 96h as, to investigate the preculture time, Radix Panacis Quinquefolii is sent out to shape root induction rate;
(3g) NAA concentration is sent out the impact of shape root induction rate on Radix Panacis Quinquefolii: the petiole of 2～3 weeks Radix Panacis Quinquefolii seedlings of take is material, in MS minimum medium, add the concentration of NAA and select to be divided into 0mg/L, 2mg/L, 4mg/L, 6mg/L, relatively send out shape root induction rate to investigate NAA optimal concentration;
(3h) incubation time is sent out the impact of shape root induction rate on Radix Panacis Quinquefolii altogether: selecting incubation time is altogether 12h, 24h, 36h, 48h, and culture medium is elected MS as altogether; The stem section of 2～3 weeks Radix Panacis Quinquefolii seedlings of take is material, and preculture substratum is to be 48h the MS+NAA2.0mg/L time, and bacterial classification is R1601, with investigate matrix together incubation time Radix Panacis Quinquefolii is sent out to the impact of shape root induction rate;
(3i) cephamycin (Cef) concentration is sent out the impact of shape root induction rate on antibacterial and Radix Panacis Quinquefolii: the concentration of cephamycin (Cef) is chosen as 200mg/L, 300mg/L, 400mg/L, 500mg/L, 600mg/L, and degerming matrix is chosen as the basic solid medium of MS; Comprehensive relatively fungistatic effect, pollution rate, three indexs of acquisition root of hair number, the optimal concentration of investigation cephamycin (Cef);
(4) Radix Panacis Quinquefolii is sent out acquisition and the evaluation of shape root:
(4a) acquisition of Radix Panacis Quinquefolii root of hair:
1. explant preculture: after the sterilization of Radix Panacis Quinquefolii seedling, its blade is cut into the fritter of 1.0cm * 1.0cm, petiole and stem section are cut into the long section of 1.0cm, Radix Panacis Quinquefolii 1 year, biennial root flowing water rinse and remove surperficial surface dust, after mercuric chloride sterilization, distilled water is rinsed well for 3～5 times, depending on material thickness, be cut into 0.2～0.5cm thin slice, above all material is connect in MS+NAA2mg/L substratum and under dark condition, cultivates 48h for infecting;
2. infect together and cultivate: on ultra-clean aseptic working platform, by pre-incubated explant material, transfer to and be equipped with in the triangular flask that is ready to bacterium liquid, after sealing, be placed on 120r/min, on shaking table under dark condition, infect 10～30min, metainfective explant blots material surface bacterium liquid with aseptic filter paper, is inoculated in MS solid medium, under dark, 25 ℃ of conditions, cultivates altogether 48h;
3. degerming is cultivated: by aseptic water washing three times of the infection explant material after common cultivation, aseptic filter paper blots the sterilized water on explant material surface, be transferred on the MS solid medium that contains cephamycin (Cef) 500mg/L and cultivate, under dark, 25 ℃ of conditions, degerming is cultivated;
(4b) Radix Panacis Quinquefolii callus and 1 year, biennial root are sent out the induction of shape root: with the agrobacterium rhizogene strain 8 μ L that micropipette rifle is drawn activation, drip on the Radix Panacis Quinquefolii callus tangent plane of receiving MS solid medium cuts into slices with American ginseng root; In operating process, avoid bacterium drop to MS solid medium, the material that drips bacterium liquid is placed on to 25 ℃, in the incubator under dark condition, cultivate; After 15d, remove the dead material of brownization, remaining material transfer is continued to cultivate to fresh MS solid medium;
(4c) PCR Molecular Detection: by PCR method to Ri plasmid T
lthe rolC gene test of-DNA will further provide molecular biological foundation, and detailed process is as follows:
1. design of primers and synthetic: design primer 1 and primer 2:
Primer 1:5 '-GATATATGCCAAATTTACACTAG-3 ';
Primer 2: 5 '-GTTAACAAAGTAGGAAACAGG-3 ';
Above two primers of utilization are by pcr amplification to the upper rolC gene fragment of T-DNA, and expection length is 564bp;
2. CTAB method is extracted Radix Panacis Quinquefolii and is sent out the total DNA of shape root; 3. extract the Ri plasmid of Agrobacterium rhizogenes;
4. PCR loop parameter:
5. PCR reaction system:
Ultrapure water is settled to 25 μ l:
6. agarose gel electrophoresis, takes pictures;
(5) Radix Panacis Quinquefolii is sent out the mensuration of shape root liquid culture increment and total saponin content: sending out shape root liquid culture employing MS liquid nutrient medium is minimum medium (3% sucrose, pH value 5.8～6.0), the shape root tip of a root access of sending out of 12 eugonic about 2cm is equipped with in 30mL MS liquid nutrient medium, temperature is 24 ± 1 ℃, shaking speed 100r/min, every 3d sampling, measure the content of sending out shape root dry weight and total saponins, measure one month; All data are the mean value of 3 bottles, repeat twice;
Adopt calibration curve method to sending out the mensuration of total saponins in shape root: the plot step of typical curve is: 1. get Rg10.020g and be dissolved in methyl alcohol, constant volume is 2mg/mL; 2. get Rg1 reference substance solution 30,60,90,120,150 μ L; 3. be placed in respectively 5 test tubes (10mL, tool plug), methanol constant volume is 150 μ L, with corresponding reagent, compares; 4. test tube is placed in to ice-water bath adds 5% Vanillin alcoholic solution and 72% sulfuric acid shakes up, 60 ℃ of water bath with thermostatic control 20min; 5. after ice-water bath 15min, at 755B spectrophotometer 544nm place, detect, 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 (%);
Send out the mensuration of total saponins in shape root and get fresh shape root of liquid culture, after distilled water flushing, with filter paper, blot the moisture of sending out shape root surface, weigh and send out shape root fresh weight; Then in vacuum drying oven, at 80 ℃, be dried to constant weight, weigh, dried shape root pulverized, put into triangular flask, add the alcohol solvent that a certain amount of concentration is 40%, and triangle bottleneck is sealed with sealed membrane; Be placed in ultra-sonic generator and carry out separation and Extraction, extraction time 15min, ultrasonic frequency is 28KHz, extracts twice; In ice-water bath, add 5% Vanillin alcoholic solution and 72%H
2sO
4dyeing, 60 ℃ of water bath with thermostatic control 10min, ice-water bath 15min, surveys absorbancy in 544nm place;
(6) Radix Panacis Quinquefolii is sent out foundation and the condition optimizing of shape root regeneration system:
(6a) Radix Panacis Quinquefolii is sent out the induction of shape root-derived callus: the Radix Panacis Quinquefolii of take is sent out shape root as explant, receive interpolation different concns, 2 of various combination, 4-D, BA, on the MS substratum of the growth regulators such as KT, wherein sucrose concentration is 30g/L, agar content is 6.5g/L, pH value is 6.0 (before sterilizings), the sterilizing pressure of substratum is that 0.1MP continues 15～20min, be put under different illumination conditions, periodicity of illumination is 12h/d, temperature is 24 ± 1 ℃, statistics callus incidence after two weeks (explant number/explant sum of callus occurs) is analyzed different growth regulators and the impact of illumination condition on callus induction, screening best setting material and concentration and illumination condition,
(6b) Radix Panacis Quinquefolii is sent out the generation of shape root embryoid: embryoid is occurred for different hormone combinations, concentration and illumination and 2 of different concns and combination got respectively in the impact of growth, 4-D, KT, BA process culture, analyze the impact that each hormone occurs and grows embryoid, filter out the best medium formula that embryoid occurs and grows; Get under dark, illumination condition and cultivate, observe the impact of illumination condition on culture; Illumination provides (about 1200Lx) by fluorescent lamp, and the photoperiod is 12h/d.
2. Radix Panacis Quinquefolii according to claim 1 is sent out shape root induction and plant regeneration research method, it is characterized in that: the detailed process that the CTAB method extraction Radix Panacis Quinquefolii in described step (4c) is sent out the total DNA of shape root is as follows:
(1) getting 280mg sends out shape root through the Radix Panacis Quinquefolii of succeeding transfer culture and adds 10%PVP in liquid nitrogen, to be fully ground to Powdered (about 30s～1min);
(2) with spoon, be carefully transferred in the centrifuge tube of 1.5ml, add the Extraction buffer of 660 μ 12 * CTAB of 65 ℃ of preheatings, 65 ℃ of water-bath 45min, vibrate once gently every 5～10min;
(3) the Solution I that adds 200 μ L ice baths to cross, vortex vibration is uniformly distributed cell, under room temperature, places 10min;
(4) add the fresh Solution II configuring of 400 μ L, put upside down and mix gently, ice bath 5min;
(5) add 300 μ L Solution III, repeatedly put upside down for several times, solution is uniformly dispersed in the cellular lysate thing of thickness, then put 5min on ice;
(6) the centrifugal 10min of 12000r/min, carefully draws supernatant liquor and adds in 1.5mL centrifuge tube;
(7) add isopyknic saturated phenol, chloroform, by thermal agitation, mix organic phase and water, the centrifugal 5min of 12000r/min;
(8) take out centrifuge tube and be cooled to room temperature, add isopyknic chloroform: primary isoamyl alcohol (24:1), mixes 12000rpm, centrifugal 10min;
(9), with a rifle head that cuts off front, carefully supernatant liquor is transferred in a new 1.5ml centrifuge tube, and added the dehydrated alcohol of-20 ℃ of precoolings of two volumes, see thread precipitation, place 30min, 12000rpm at-20 ℃, centrifugal 5min, obtains faint yellow precipitation;
(10) supernatant liquor that inclines, and with the thieving paper residual solution that exhausts, add 300 μ l ultrapure waters again to dissolve, then add the dehydrated alcohol 600 μ l of-20 ℃ of precoolings, precipitation is spent the night;
(11) 12000rpm, centrifugal 10min, obtains white gelatinous precipitate, the supernatant liquor that inclines, and with the thieving paper residual solution that exhausts, add 70% ethanol 800 μ l washing precipitation 2～3 times;
(12) exhaustion residual solution, puts Bechtop and dries up (approximately needing 10～15min);
(13) add 40～60 μ l ultrapure waters, dissolution precipitation, adds 3 μ l Rnase, and room temperature is placed after 30min, preserves at-20 ℃.
3. Radix Panacis Quinquefolii according to claim 1 is sent out shape root induction and plant regeneration research method, it is characterized in that: the detailed process of the Ri plasmid of the extraction Agrobacterium rhizogenes in described step (4c) is as follows:
(1) the mono-bacterium colony of picking R1601 is in 10ml YEB substratum, and 28 ℃, 120rpm, incubated overnight;
(2) draw the cultured bacterium liquid of 8ml, add in 10ml centrifuge tube 4000rpm, centrifugal 5min, nutrient solution inclines, the STE that adds 2ml precooling, Eddy diffusion cell, draws respectively 1ml bacteria suspension, add in 1.5ml centrifuge tube, the centrifugal 2min of 12000rpm, the nutrient solution that inclines, obtains appropriate somatic cells;
(3) the Solution I that adds 200 μ l ice baths to cross, vortex vibration is uniformly distributed cell, under room temperature, places 10min;
(4) add the fresh Solution II configuring of 400 μ l, put upside down and mix gently, ice bath 5min;
(5) add 300 μ l Solution III, repeatedly put upside down for several times, solution is uniformly dispersed in the cellular lysate thing of thickness, then put 5min on ice;
(6) the centrifugal 10min of 12000rpm, carefully draws supernatant liquor and adds in 1.5ml centrifuge tube;
(7) add isopyknic saturated phenol, chloroform, by thermal agitation, mix organic phase and water, the centrifugal 5min of 12000rpm;
(8) carefully drawing supernatant liquor adds in new 1.5ml centrifuge tube, with isopyknic chloroform again extracting once;
(9) carefully draw supernatant liquor and add in new 1.5ml centrifuge tube, add the dehydrated alcohol of 2 times of volumes, the 3mol/LNaAc of 1/10 volume, ice bath 30min, 12000rpm, centrifugal 10min, the supernatant liquor that inclines, is upside down on thieving paper, and solution is flow to end;
(10) add 1ml70% washing with alcohol twice, the centrifugal 5min of 12000rpm, collecting precipitation;
(11) washings that inclines, is inverted on thieving paper and blots remaining solution, on Bechtop, dries up;
(12) add 50 μ l TE resuspended, standby.
4. Radix Panacis Quinquefolii according to claim 1 is sent out shape root induction and plant regeneration research method, it is characterized in that: the agarose gel electrophoresis detailed process in described step (4c) is as follows:
(1) with adhesive tape, seal electrophoresis chamber rubber moulding edge and form a mould;
(2) prepare enough electrophoretic buffers (1 * TBE) in order to fill electrophoresis chamber and preparation gel;
(3) accurately take a certain amount of agar powder and be added to and in the triangular flask that fills a certain amount of electrophoretic buffer, in microwave oven, be heated to melt, then carefully add ethidium bromide, concentration is 0.5 μ g/ml, and rotation is to mixing gelating soln gently;
(4) on mould, add a suitable comb and form well, then pour appropriate warm agarose solution into, after room temperature 30min, remove edge sealing adhesive tape, gel is put into electrophoresis chamber, make electrophoretic buffer not have the about 1mm of gel;
(5) mix the sample-loading buffer of pcr amplification product sample and 0.2 times of volume;
(6) with liquid-transfering gun and disposable rifle head, sample mix liquid is slowly added in the well of submergence gel, molecular mass standard substance are added in the left side of sample;
(7) cover electrophoresis chamber lid, connect electrode plug, DNA has the negative electrode extreme swimming that starts to face south, and gives the voltage of 1～5V/cm, can observe soon tetrabromophenol sulfonphthalein and enter in colloid from well;
(8) when DNA sample or dimethylbenzene cyanogen FF and tetrabromophenol sulfonphthalein have been moved enough distances in gel, close power source, extract electrode plug and open electrophoresis chamber lid, gel is placed under gel imaging system and is taken pictures.
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104904491A (en) * | 2014-03-10 | 2015-09-16 | 廉美兰 | Method for improving saponin content in American ginseng adventive roots cultured in reactor with ginseng pathogenic bacterium elicitor |
| CN105087599A (en) * | 2015-09-07 | 2015-11-25 | 昆明理工大学 | Application of panax japonicus transcription factor gene PjERF1 |
| CN105087601A (en) * | 2015-09-07 | 2015-11-25 | 昆明理工大学 | Application of panax japonicus transcription factor gene PjWRKY1 |
| CN105087600A (en) * | 2015-09-07 | 2015-11-25 | 昆明理工大学 | Application of panax japonicus transcription factor gene PjbHLH1 |
| CN105441461A (en) * | 2016-01-06 | 2016-03-30 | 昆明理工大学 | Application of panaxnotoginseng transcription factor gene PnWRKY1 |
| CN105861546A (en) * | 2016-06-06 | 2016-08-17 | 江苏农林职业技术学院 | Gene transformation method for summer black grapes |
| CN108719052A (en) * | 2018-06-11 | 2018-11-02 | 中国农业科学院特产研究所 | The positive and negative first familiar generation rataria rescue culture method of ginseng, American Ginseng |
-
2013
- 2013-11-02 CN CN201310545022.1A patent/CN103614411A/en active Pending
Non-Patent Citations (4)
| Title |
|---|
| 刘志洋等: "不同年生西洋参的植物形态和有效成分含量比较", 《吉林农业》 * |
| 宫秀杰: "发根农杆菌Ri质粒转化西洋参条件的研究", 《中国优秀硕士学位论文全文数据库农业科技辑》 * |
| 谢海云等: "西洋参发根器官发生及再生植株研究", 《北方园艺》 * |
| 靳浩淼: "诱导子对西洋参发状根的影响_", 《中国优秀硕士学位论文全文数据库》 * |
Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104904491A (en) * | 2014-03-10 | 2015-09-16 | 廉美兰 | Method for improving saponin content in American ginseng adventive roots cultured in reactor with ginseng pathogenic bacterium elicitor |
| CN105087599A (en) * | 2015-09-07 | 2015-11-25 | 昆明理工大学 | Application of panax japonicus transcription factor gene PjERF1 |
| CN105087601A (en) * | 2015-09-07 | 2015-11-25 | 昆明理工大学 | Application of panax japonicus transcription factor gene PjWRKY1 |
| CN105087600A (en) * | 2015-09-07 | 2015-11-25 | 昆明理工大学 | Application of panax japonicus transcription factor gene PjbHLH1 |
| CN105087601B (en) * | 2015-09-07 | 2018-06-15 | 昆明理工大学 | A kind of application of panax japonicus majoris transcription factor gene PjWRKY1 |
| CN105087599B (en) * | 2015-09-07 | 2018-06-15 | 昆明理工大学 | A kind of application of panax japonicus majoris transcription factor gene PjERF1 |
| CN105087600B (en) * | 2015-09-07 | 2018-06-15 | 昆明理工大学 | A kind of application of panax japonicus majoris transcription factor gene PjbHLH1 |
| CN105441461A (en) * | 2016-01-06 | 2016-03-30 | 昆明理工大学 | Application of panaxnotoginseng transcription factor gene PnWRKY1 |
| CN105441461B (en) * | 2016-01-06 | 2019-02-05 | 昆明理工大学 | A kind of application of Radix Notoginseng transcription factor gene PnWRKY1 |
| CN105861546A (en) * | 2016-06-06 | 2016-08-17 | 江苏农林职业技术学院 | Gene transformation method for summer black grapes |
| CN108719052A (en) * | 2018-06-11 | 2018-11-02 | 中国农业科学院特产研究所 | The positive and negative first familiar generation rataria rescue culture method of ginseng, American Ginseng |
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