CN102296083A - Method for inducing and propagating transgenic hairy roots of tetrastigma hemsleyanum dielset gilg - Google Patents
Method for inducing and propagating transgenic hairy roots of tetrastigma hemsleyanum dielset gilg Download PDFInfo
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Abstract
The invention provides a method for inducing and propagating transgenic hairy roots of tetrastigma hemsleyanum dielset gilg. In the invention, specific explants of tetrastigma hemsleyanum dielset gilg are infected with agrobacterium rhizogene K599 and hairy roots are successfully induced by improving the activity of the agrobacterium rhizogene K599, screening infected proper explants and optimizing hairy root induction culture medium; and a root line of hairy roots, which can grow quickly and synthesize medicinal active ingredients efficiently, can be further grown by improving hairy root sterilization conditions and optimizing a culture medium and a condition for propagating hairy roots. The method can provide technical foundation and support for industrial production of tetrastigma hemsleyanum dielset gilg active ingredients for medicinal purpose by using hairy roots, provides a new resolution to the resource replacement problem of rare tetrastigma hemsleyanum dielset gilg medicinal material, promotes the protective utilization and sustained development of a tetrastigma hemsleyanum dielset gilg resource and has a bright development and application prospect.
Description
(1) technical field
The present invention relates to inducing and propagation method of Radix Apioris Fortunei (Radix Lespedezae Buergeri) transgenosis hairly root.
(2) background technology
Radix Apioris Fortunei (Radix Lespedezae Buergeri), have another name called the gold thread hoist, formal name used at school tetratigma hemsleyanum (Tetrastigma hemsleyanum Diels et Gilg), for climbing the Calamus plant in the Vitaceae precipice, be the distinctive medicinal plant of China, have important pharmaceutical use, its herb all can be used as medicine, dry or using fresh herb, best with the medicinal effect of underground root and fruit.
Modern medicine study confirms that extract β-Gu Zaichun Radix Apioris Fortunei (Radix Lespedezae Buergeri) flavones and the polysaccharide of Radix Apioris Fortunei (Radix Lespedezae Buergeri) can effectively suppress tumor cell proliferation, promotes apoptosis of tumor cells, is a kind of extremely rare herbal medicine.But Radix Apioris Fortunei (Radix Lespedezae Buergeri) requires very peculiar to growing environment, it must be grown in the remote, thickly forested mountains of 700 meters of height above sea level and in the shade of crag, above scattered light to be arranged, the next door will have thin water to ooze out, to there be leaf to cover below, throughout the year temperature remains on and just helps its growth about 18 ℃, only in Zhejiang, there is a spot of NATURAL DISTRIBUTION in mountain areas such as Guangxi, Jiangxi, wherein the Radix Apioris Fortunei (Radix Lespedezae Buergeri) in Zhejiang is top grade; And the speed of growth is slow under field conditions (factors).Because it is harsh that Radix Apioris Fortunei (Radix Lespedezae Buergeri) requires the bad border of growing, and adds immoderate people for excavating, the sixth of the twelve Earthly Branches is endangered under field conditions (factors).Though, its tissue culture quick breeding and artificial cultivation technique are achieved success, but it is still very long to form the ripe plant cycle that has the spherical pieces root from plantlet, do not satisfy the market requirement (Wu Zhaolong far away, Lv Jiangming, the present Research of Chinese medicine Radix Apioris Fortunei (Radix Lespedezae Buergeri), Chinese national folk medical magazine, 2006, (78): 15-18; Chen Qiaoli, Gong Jiang, Cao Mengye relies ZCOM, Ni Shifeng, Radix Apioris Fortunei (Radix Lespedezae Buergeri) study of pharmacy new development, Liaoning University of TCM, 2011,13 (1): 72-74).
On the other hand, hairly root (hairy root) is that the T-DNA fragment of the contained Ri plasmid of Agrobacterium rhizogenes is inserted in the vegetable cell genome, integrated and express, the plastidogenetic adventive root of inducing plant.Hairly root has kept the feature of plant primitive root system, the metabolic pathway that has the primitive root system complete on physiology on form; Hairly root originates from unicellular and does not have mosaic, has the stability and the consistence of height in heredity.On using, since hairly root have natural structure, can be on the substratum that does not contain plant hormone growth and genetic stability are high and can synthesize and accumulate secondary metabolites fast, therefore, hairly root can be used as active pharmaceutical ingredients (the Hu ZB that bio-reactor is produced plant, Du M.Hairy root and its application in plant genetic engineering.Journal of Integrative Plant Biology, 2006,48 (2): 121-127).
According to bibliographical information, by infecting of Agrobacterium rhizogenes, in some medicinal plants, realize inducing of hairly root, and from hairly root, separated the acquisition active pharmaceutical ingredients, as: genseng, Ramulus et folium taxi cuspidatae, Vinca etc.And the inducing and breed always and do not capture of Radix Apioris Fortunei (Radix Lespedezae Buergeri) hairly root.
(3) summary of the invention
The object of the invention provides inducing and propagation method of Radix Apioris Fortunei (Radix Lespedezae Buergeri) transgenosis hairly root.
The technical solution used in the present invention is:
Inducing and propagation method of Radix Apioris Fortunei (Radix Lespedezae Buergeri) transgenosis hairly root, described method comprises:
(1) the pre-cultivation of explant: the plant of getting the self-sow of Radix Apioris Fortunei (Radix Lespedezae Buergeri) leans on top 30cm with interior stem section, is cut into the segment of 0.5～1.0cm after cleaning, sterilizing; The stem explants of gained Radix Apioris Fortunei (Radix Lespedezae Buergeri) inserts the MS solid medium that contains naphthylacetic acid 0.5mg/L+ vitamins C 0.1g/L+ halfcystine 0.1g/L or contains in the MS solid medium of naphthylacetic acid 0.5mg/L+ vitamins C 0.1g/L+ activated carbon 1g/L under aseptic condition, at 24 ℃, hide that dark condition is pre-down to be cultivated 20～25 days, explant through pre-the cultivation after callusization slightly; Clean, sterilization carries out according to a conventional method, concrete steps can be as follows: the 3～5min that sterilizes in tap water flushing → 5% liquid detergent solution rinsing 5min → tap water flushing 10min → 75% ethanolic soln scrub surfaces → 0.1% mercuric chloride solution → aseptic water washing 4～6 times;
(2) infect and be total to cultivation: infect stem explants with Agrobacterium rhizogenes K599 through pre-incubated callusization slightly, the stem explants after infecting change in the MS solid medium that contains Syringylethanone 50mg/L+ vitamins C 0.1g/L in 24 ℃, hide under the dark condition and cultivated altogether 1～2 day; It is as follows specifically to infect step: will will put into Erlenmeyer flask through pre-incubated stem explants through the stem section of pre-incubated callusization slightly, adding Agrobacterium rhizogenes K599 infects bacterium liquid makes bacterium liquid submergence explant, and the rotating speed with 50r/min on shaking table infects 5～10min; Infect the end back and take out explant, and blot the bacterium liquid on explant surface with aseptic filter paper with aseptic tweezers;
Infecting bacterium liquid can prepare as follows: the Agrobacterium rhizogenes K599 bacterium liquid of getting incubated overnight, change in the YEB liquid nutrient medium that contains Syringylethanone 10mg/L As+ cucumopine 10mg/L, 28 ℃, 200r/min activation treatment 4 hours, bacterium liquid after the activation is centrifugal, abandon supernatant, stays to be deposited in to add the MS liquid nutrient medium that contains Syringylethanone 20mg/L in the centrifuge tube thalline is suspended; Centrifugal, abandon supernatant, stay to be deposited in the centrifuge tube to add the MS liquid nutrient medium that contains Syringylethanone 20mg/L thalline is suspended; Centrifugal, abandon supernatant, get sedimentary thalline with the MS liquid nutrient medium dilution that contains Syringylethanone 20mg/L, make and infect bacterium liquid.
(3) inducing of hairly root: the stem explants after cultivating is altogether removed surface-moisture and bacterium liquid through thoroughly cleaning, change the MS solid medium that contains indolylacetic acid 0.5mg/L+ vitamins C 0.1g/L+ halfcystine 0.1g/L+ Ticarcillin/Clavulanate Acid 500mg/L over to or contain in the MS solid medium of indolylacetic acid 0.5mg/L+ vitamins C 0.1g/L+ Polyvinylpyrolidone (PVP) 0.1g/L+ Ticarcillin/Clavulanate Acid 500mg/L, in 24 ℃, the following 30～45d that cultivates of dark condition, obtain hairly root;
(4) breeding of hairly root: when hairly root length reaches 2～3cm, cut the Radix Apioris Fortunei (Radix Lespedezae Buergeri) hairly root, after carrying out sterilization, after identifying, forward on the MS solid or the liquid nutrient medium that contain indolylacetic acid 0.1mg/L or MS solid that does not add any plant hormone or the liquid nutrient medium and breed.
Preferably, degerming method is as follows in the described step (4): the Radix Apioris Fortunei (Radix Lespedezae Buergeri) hairly root that cuts is forwarded on the MS solid medium of the MS solid medium that contains indolylacetic acid 0.1mg/L+ vitamins C 0.1g/L+ halfcystine 0.1g/L+ Ticarcillin/Clavulanate Acid 500mg/L or indolylacetic acid 0.1mg/L+ vitamins C 1g/L+ Polyvinylpyrolidone (PVP) 0.1g/L+500mg/L Ticarcillin/Clavulanate Acid, cultivate down at 35 ℃ earlier and carried out high-temperature sterilization treatment in 3 days, move on to 24 ℃ of cultivations down, the switching in 20 days of every interval 1 time again, transfer altogether 2～3 times.
In the step (4), when breeding with solid medium, switching in per 20～30 days once; When breeding with liquid nutrient medium, switching in per 7～15 days once.
The present invention utilizes Agrobacterium rhizogenes K599 to infect the specific explant of Radix Apioris Fortunei (Radix Lespedezae Buergeri), and by improving the vigor of Agrobacterium rhizogenes K599, the suitable explant that screening is infected is optimized the hairly root inducing culture, successfully induces hairly root; On this basis, by improvement hairly root degerming condition, substratum and condition that the optimization hairly root is bred, screening obtain to grow fast, efficiently synthesize the root system of the hairly root of active pharmaceutical ingredients, set up and utilize hairly root to produce the technical system of Radix Apioris Fortunei (Radix Lespedezae Buergeri) active pharmaceutical ingredients fast.
Beneficial effect of the present invention is mainly reflected in: the present invention will provide technical foundation and support for utilizing the hairly root batch production to produce the Radix Apioris Fortunei (Radix Lespedezae Buergeri) active pharmaceutical ingredients; for the substitution of resources problem that solves rare medicinal material Radix Apioris Fortunei (Radix Lespedezae Buergeri) provides a new approach; help the protectiveness utilization and the sustainable development of Radix Apioris Fortunei (Radix Lespedezae Buergeri) resource, have good development prospect.
(4) description of drawings
Fig. 1 for the Radix Apioris Fortunei (Radix Lespedezae Buergeri) explant through pre-the cultivation after callusization slightly;
Fig. 2 infects the white translucent hairly root that induces for the Radix Apioris Fortunei (Radix Lespedezae Buergeri) explant through Agrobacterium rhizogenes;
Fig. 3 infects the white translucent hairly root that induces for the Radix Apioris Fortunei (Radix Lespedezae Buergeri) explant through Agrobacterium rhizogenes;
Fig. 4 is the breeding in a large number on solid medium of Radix Apioris Fortunei (Radix Lespedezae Buergeri) hairly root;
Fig. 5 is the breeding in a large number on liquid substratum of Radix Apioris Fortunei (Radix Lespedezae Buergeri) hairly root.
(5) embodiment
The present invention is described further below in conjunction with specific embodiment, but protection scope of the present invention is not limited in this:
Embodiment 1:
One, the breeding of Agrobacterium rhizogenes K599
In the 50mL Erlenmeyer flask, add 20mL YEB liquid nutrient medium (Beef extract (beef extract) 5g/L, Yeast extract (yeast extract paste) 1g/L, Peptone (peptone) 5g/L, Sucrose (sucrose) 5g/L, MgSO
47H
2O 4g/L, pH 7.4)+50mg/L Str (Streptomycin sulphate), and add the Agrobacterium rhizogenes K599 100 μ L of the wild-type of-80 ℃ of preservations, incubated overnight under 28 ℃, 200r/min condition.Wild-type Agrobacterium rhizogenes K599 preserves (theNational Collection of Plant Pathogenic Bacteria in U K buys and obtains) by the laboratory.
Two, infect the preparation of liquid
1, gets the bacterium liquid of 1mL incubated overnight, change in 20mL YEB+10mg/L As (Syringylethanone)+10mg/L Cus (cucumopine) liquid nutrient medium, 28 ℃, 200r/min activation treatment 4 hours.
2, the bacterium liquid 1.5mL that gets activation treatment is in the centrifuge tube of 2mL, the centrifugal 3min of 5000r/min.
3, abandon supernatant, stay the MS+20mg/L As liquid nutrient medium that is deposited in adding 2.0mL in the centrifuge tube thalline to be suspended the centrifugal 3min of 5000r/min.
4, repeating step 3 once that is: stays the MS+20mg/LAs liquid nutrient medium that is deposited in adding 2.0mL in the centrifuge tube thalline to be suspended the centrifugal 3min of 5000r/min.
5, use the bacterium liquid of MS+20mg/LAs to dilute 10 times the thalline of centrifugation in the step 4 with MS liquid, make and infect liquid.
Three, the Radix Apioris Fortunei (Radix Lespedezae Buergeri) hairly root induces and degerming
1, the sterilization of Radix Apioris Fortunei (Radix Lespedezae Buergeri) explant
Gather the plant of self-sow from mountain, county, Lishui City Jinyun, Zhejiang Province, the plant of self-sow of getting Radix Apioris Fortunei (Radix Lespedezae Buergeri) by top 30cm with the 5min → aseptic water washing 5 times of sterilizing in interior stem section → tap water flushing → 5% (v/v) liquid detergent solution rinsing 5min → tap water flushing 10min → 75% (v/v) ethanolic soln scrub surfaces → 0.1% (w/w) mercuric chloride solution → on the aseptic technique platform, explant the is cut into segment of 0.5～1.0cm.
2, the pre-cultivation of explant
The stem explants of Radix Apioris Fortunei (Radix Lespedezae Buergeri) is inserted under aseptic condition in MS+NAA (naphthylacetic acid) 0.5mg/L+0.1g/L Vc (vitamins C)+0.1g/L Cys (halfcystine) solid medium,, hide dark condition pre-the cultivation 25 days down at 24 ℃.
3, infect
Explant through pre-the cultivation after callusization (Fig. 1) slightly, the Erlenmeyer flask of putting into 50mL through the stem section of pre-incubated callusization slightly respectively, what add 20mL in every bottle infects bacterium liquid, make bacterium liquid submergence explant, rotating speed with 50r/min on shaking table infects 5min, take out explant with aseptic tweezers then, and blot the bacterium liquid on explant surface with aseptic filter paper.
4, cultivate altogether
The explant that has infected is inoculated in the solid medium of MS+As (Syringylethanone) 50mg/L+0.1g/L Vc and cultivated altogether 1 day, 24 ℃, hide dark.
5, wash bacterium
The explant of cultivating altogether is transferred in the liquid nutrient medium of MS+Timentin (Ticarcillin/Clavulanate Acid) 500mg/L, on shaking table, washed bacterium 1 hour, use aseptic water washing then 4～5 times, and blot the moisture or the bacterium liquid on surface with filter paper with the rotating speed of 100r/min.
6, the Radix Apioris Fortunei (Radix Lespedezae Buergeri) hairly root induces
The explant of washing bacterium is changed in MS+0.5mg/L IAA (indolylacetic acid)+0.1g/L Vc+0.1g/L Cys+500mg/L Timentin solid medium, and 24 ℃, dark condition is cultivated down.Behind 35d, can induce white translucent hairly root (Fig. 2).
7, the degerming of Radix Apioris Fortunei (Radix Lespedezae Buergeri) hairly root
When hairly root length reaches 2～3cm, cut the Radix Apioris Fortunei (Radix Lespedezae Buergeri) hairly root, forward on the MS+IAA0.1mg/L+0.1g/L Vc+0.1g/L Cys+500mg/L Timentin solid medium, pyroprocessing sterilization 3 days under 35 ℃ of temperature condition earlier moves on to 24 ℃ again and cultivates down.Every interval switching in 20 days 1 time is carried out 2 times altogether, promptly reaches the purpose of degerming.
Four, the PCR of hairly root identifies
Extract the hairly root genomic dna according to ordinary method, sequence according to rolB among the Ri T-DNA and rolC gene, the design amplification designs rolB and rolC primer respectively, rolB-P1:5 '-ggcctctaatccaaacgtga-3 ', rolB-P2:5 '-ccaacctcacatcacaatgc-3 ' and rolC-P1:5 '-tgatgcgatgcttttatgga-3 ', rolC-P2:5 '-tggcataaaggtcgaaggtc-3 '.The same ordinary method of PCR reaction system and response procedures.Be that reaction system is: contain ddH in per 35 μ L volumes
2O22.5 μ L, 10 * PCR Buffer (contains 15mmol/L Mg
2+) 3.5 μ L, dNTP (2mmol/L) 2 μ L, 2 each 2 μ L of primer, dna profiling 2 μ L, Taq DNA polymerase (2u/ μ L) 1 μ L.Response procedures is: after 94 ℃ of 5min sex change, carry out 30 circulations by the setting of 94 ℃ of 45s, 55 ℃ of 45s, 72 ℃ of 90s, reaction finishes the back and extends 10min down at 72 ℃, and is standby 4 ℃ of preservations at last.Amplified production is electrophoresis 1.5h (5V/cm) on 1.2% sepharose, and ethidium bromide (EtBr) dyeing is observed and Taking Pictures recording with U.S. Bio/Rad gel imaging system.Infect by Agrobacterium rhizogenes K599 that to induce the hairly root that forms can amplify corresponding to rolB and rolC mrna length respectively be the band of 388bp and 267bp.
Five, the breeding of Radix Apioris Fortunei (Radix Lespedezae Buergeri) hairly root
Aseptic hairly root after identifying is forwarded on the MS+IAA 0.1mg/L solid medium to switching in per 20～30 days 1 time.
Can increase volume (Fig. 4) about 1 times in per 20～30 days at hairly root on the solid medium.
Six, the analysis of hairly root active pharmaceutical ingredients
Adopting ultraviolet-visible spectrophotometry, is reference substance with the spherical pieces root of self-sow, at wavelength 500nm place the total flavones in the sample is carried out assay.In the hairly root, its general flavone content is more than 15mg/g, and is suitable with general flavone content in the Pear-Shaped piece root of self-sow.
Embodiment 2:
One, the breeding of Agrobacterium rhizogenes K599
With embodiment 1.
Two, infect the preparation of liquid
With embodiment 1.
Three, the Radix Apioris Fortunei (Radix Lespedezae Buergeri) hairly root induces and degerming
1, the sterilization of Radix Apioris Fortunei (Radix Lespedezae Buergeri) explant
With embodiment 1.
2, the pre-cultivation of explant
The stem explants of Radix Apioris Fortunei (Radix Lespedezae Buergeri) is inserted under aseptic condition in the solid medium of MS+NAA 0.5mg/L+0.1g/L Vc+1g/L activated carbon,, hide dark condition pre-the cultivation 20～25 days down at 24 ℃.
3, infect
Explant through pre-the cultivation after callusization slightly, the Erlenmeyer flask of putting into 50mL through the stem section of pre-incubated callusization slightly respectively, what add 25mL in every bottle infects bacterium liquid, make bacterium liquid submergence explant, rotating speed with 50r/min on shaking table infects 10min, take out explant with aseptic tweezers then, and blot the bacterium liquid on explant surface with aseptic filter paper.
4, cultivate altogether
The explant that has infected is inoculated in the solid medium of MS+As (Syringylethanone) 50mg/L+0.1g/L Vc and cultivated altogether 2 days, 24 ℃, hide dark.
5, wash bacterium
The explant of cultivating altogether is transferred in the liquid nutrient medium of MS+Timentin (Ticarcillin/Clavulanate Acid) 500mg/L, on shaking table, washed bacterium 1 hour, use aseptic water washing then 4～5 times, and blot the moisture or the bacterium liquid on surface with filter paper with the rotating speed of 100r/min.
6, the Radix Apioris Fortunei (Radix Lespedezae Buergeri) hairly root induces
The explant of washing bacterium is changed in the solid medium of MS+0.5mg/L IAA+0.1g/L Vc+0.1g/LPVP (Polyvinylpyrolidone (PVP))+500mg/L Timentin, 24 ℃, dark condition is cultivated down.Behind 40d, can induce white translucent hairly root (Fig. 3).
7, the degerming of Radix Apioris Fortunei (Radix Lespedezae Buergeri) hairly root
When hairly root length reaches 2～3cm, cut the Radix Apioris Fortunei (Radix Lespedezae Buergeri) hairly root, forward on the MS+IAA0.1mg/L+1g/L Vc+0.1g/L PVP+500mg/L Timentin solid medium, pyroprocessing sterilization 3 days under 35 ℃ of temperature condition earlier moves on to 24 ℃ again and cultivates down.Every interval switching in 20 days 1 time is carried out 3 times altogether, promptly reaches the purpose of degerming.
Four, the PCR of hairly root identifies
With embodiment 1.
Five, the breeding of Radix Apioris Fortunei (Radix Lespedezae Buergeri) hairly root
Aseptic hairly root after identifying forwarded to change in the MS+IAA 0.1mg/L MS liquid nutrient medium, 60～90r/min, 24 ℃ of suspension culture, switching in per 7～15 days is once.Can increase the volume (Fig. 5) about 1 times in per 7～15 days.
Six, the analysis of hairly root active pharmaceutical ingredients
Adopting ultraviolet-visible spectrophotometry, is reference substance with the spherical pieces root of self-sow, at wavelength 500nm place the total flavones in the sample is carried out assay.In the hairly root, general flavone content is suitable in the Pear-Shaped piece root of its general flavone content and self-sow.
SEQUENCE?LISTING
＜110〉Hangzhou Pedagogic University
＜120〉Radix Apioris Fortunei (Radix Lespedezae Buergeri) transgenosis hairly root induces and propagation method
<130>
<160> 4
<170> PatentIn?version?3.4
<210> 1
<211> 20
<212> DNA
<213> Unknown
<220>
＜223〉artificial sequence
<400> 1
ggcctctaat?ccaaacgtga 20
<210> 2
<211> 20
<212> DNA
<213> Unknown
<220>
＜223〉artificial sequence
<400> 2
ccaacctcac?atcacaatgc 20
<210> 3
<211> 20
<212> DNA
<213> Unknown
<220>
＜223〉artificial sequence
<400> 3
tgatgcgatg?cttttatgga 20
<210> 4
<211> 20
<212> DNA
<213> Unknown
<220>
＜223〉artificial sequence
<400> 4
tggcataaag?gtcgaaggtc 20
Claims (5)
1. inducing and propagation method of Radix Apioris Fortunei (Radix Lespedezae Buergeri) transgenosis hairly root, described method comprises:
(1) the pre-cultivation of explant: the plant of getting the Radix Apioris Fortunei (Radix Lespedezae Buergeri) self-sow leans on top 30cm with interior stem section, is cut into the segment of 0.5～1.0cm after cleaning, sterilizing; The stem explants of gained Radix Apioris Fortunei (Radix Lespedezae Buergeri) inserts the MS solid medium that contains naphthylacetic acid 0.5mg/L+ vitamins C 0.1g/L+ halfcystine 0.1g/L or contains in the MS solid medium of naphthylacetic acid 0.5mg/L+ vitamins C 0.1g/L+ activated carbon 1g/L under aseptic condition, at 24 ℃, hide dark condition pre-the cultivation 20～25 days down;
(2) infect and be total to cultivation: infect through pre-incubated stem explants with Agrobacterium rhizogenes K599, the stem explants after infecting change in the MS solid medium that contains Syringylethanone 50mg/L+ vitamins C 0.1g/L in 24 ℃, hide under the dark condition and cultivated altogether 1～2 day;
(3) inducing of hairly root: the stem explants after cultivating is altogether removed surface-moisture and bacterium liquid through thoroughly cleaning, change the MS solid medium that contains indolylacetic acid 0.5mg/L+ vitamins C 0.1g/L+ halfcystine 0.1g/L+ Ticarcillin/Clavulanate Acid 500mg/L over to or contain in the MS solid medium of indolylacetic acid 0.5mg/L+ vitamins C 0.1g/L+ Polyvinylpyrolidone (PVP) 0.1g/L+ Ticarcillin/Clavulanate Acid 500mg/L, in 24 ℃, the following 30～45d that cultivates of dark condition, obtain hairly root;
(4) breeding of hairly root: when hairly root length reaches 2～3cm, cut the Radix Apioris Fortunei (Radix Lespedezae Buergeri) hairly root, after carrying out sterilization, after identifying, forward on the MS solid or the liquid nutrient medium that contain indolylacetic acid 0.1mg/L or MS solid that does not add any plant hormone or the liquid nutrient medium and breed.
2. the method for claim 1, it is as follows to it is characterized in that infecting step in described (2): will put into Erlenmeyer flask through pre-incubated stem explants, adding Agrobacterium rhizogenes K599 infects bacterium liquid makes bacterium liquid submergence explant, and the rotating speed with 50r/min on shaking table infects 5～10min.
3. method as claimed in claim 2, it is characterized in that the described bacterium liquid that infects is prepared by following method: the Agrobacterium rhizogenes K599 bacterium liquid of getting incubated overnight, change in the YEB liquid nutrient medium that contains Syringylethanone 10mg/L As+ cucumopine 10mg/L, 28 ℃, 200r/min activation treatment 4 hours, bacterium liquid after the activation is centrifugal, abandon supernatant, stays to be deposited in to add the MS liquid nutrient medium that contains Syringylethanone 20mg/L in the centrifuge tube thalline is suspended; Centrifugal, abandon supernatant, stay to be deposited in the centrifuge tube to add the MS liquid nutrient medium that contains Syringylethanone 20mg/L thalline is suspended; Centrifugal, abandon supernatant, get precipitation with the MS liquid nutrient medium dilution that contains Syringylethanone 20mg/L, make and infect bacterium liquid.
4. the method for claim 1, it is characterized in that degerming method is as follows in the described step (4): the Radix Apioris Fortunei (Radix Lespedezae Buergeri) hairly root that cuts is forwarded on the MS solid medium of the MS solid medium that contains indolylacetic acid 0.1mg/L+ vitamins C 0.1g/L+ halfcystine 0.1g/L+ Ticarcillin/Clavulanate Acid 500mg/L or indolylacetic acid 0.1mg/L+ vitamins C 1g/L+ Polyvinylpyrolidone (PVP) 0.1g/L+500mg/L Ticarcillin/Clavulanate Acid, cultivate down at 35 ℃ earlier and carried out high-temperature sterilization treatment in 3 days, move on to 24 ℃ of cultivations down, the switching in 20 days of every interval 1 time again, transfer altogether 2～3 times.
5. the method for claim 1 is characterized in that in the step (4), and when breeding with solid medium, switching in per 20～30 days once; When breeding with liquid nutrient medium, switching in per 7～15 days once.
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| CN110885852A (en) * | 2019-12-05 | 2020-03-17 | 浙江省农业科学院 | Method for efficiently inducing formation of transgenic hairy roots of bottle gourds |
| CN111621517A (en) * | 2020-06-05 | 2020-09-04 | 杭州师范大学 | Method for inducing hairy roots of common turmeric |
| CN112544440A (en) * | 2020-12-23 | 2021-03-26 | 江苏师范大学 | Method for cultivating new transgenic or gene-edited seedlings of root tuber plants |
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| CN110885852A (en) * | 2019-12-05 | 2020-03-17 | 浙江省农业科学院 | Method for efficiently inducing formation of transgenic hairy roots of bottle gourds |
| CN111621517A (en) * | 2020-06-05 | 2020-09-04 | 杭州师范大学 | Method for inducing hairy roots of common turmeric |
| CN112544440A (en) * | 2020-12-23 | 2021-03-26 | 江苏师范大学 | Method for cultivating new transgenic or gene-edited seedlings of root tuber plants |
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