CN108913716A - A kind of rapid induction quinoa hairy method - Google Patents
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Abstract
The invention discloses a kind of rapid induction quinoa hairy methods, include the following steps：1) Agrobacterium rhizogenesA4 containing gus gene is constructed；2) quinoa explant infects；Sterile quinoa explant is infected with dip dyeing liquid for shell A or infected liquid B, and dip dyeing liquid for shell A is prepared by wild type Agrobacterium rhizogenesA4, and infected liquid B is prepared by step 1) containing the Agrobacterium rhizogenesA4 of gus gene；3) the quinoa explant after infecting is co-cultured；4) the quinoa explant after co-culturing, which moves in induced medium, to be cultivated, and obtains quinoa hairy；5) hairy progress subculture of quinoa and amplification cultivation；6) to hairy progress GUS dyeing of quinoa, quinoa hairy for by the quinoa explant after infected liquid B dip dyeing through step 3), 4), 5) culture obtain.The present invention can rapidly and efficiently induce more quinoa hairy growths, while can transformation efficiency that is easy, intuitive, quickly detecting hairy.
Description
Technical field
The present invention relates to agricultural biological technical field, specially a kind of rapid induction quinoa hairy method.
Background technique
Quinoa is the annual dicotyledonous herbaceous plant of Amaranthaceae Chenopodiaceae subfamily Chenopodium, originates from South America Andes, is originated in
Ground is Indian due to its nutritive value abundant and unique functional characteristic mainly in Peru, Bolivia and Ecuador
Person is " mother of grain ".Quinoa contains a large amount of good protein, and its amino acid composition is balanced, FAO (Food and Agriculture Organization of the United Nation)
(FAO) formal recommendation its be the optimum mankind perfection " wholefood ", mankind's basic food requirements can be met by being called
Only monomer plant.In the method for gene function verifying for carrying out quinoa, generally again using arabidopsis or callus induction
Raw, growth cycle is long and transformation efficiency is relatively low.And hairy has growth cycle short, convenient for culture, be easy to convert,
The advantages that high conversion rate, but there are no the methods for constructing rapid induction quinoa hairy in currently available technology.And it is existing
For the detection of hairy root induction, the detection that high voltage paper electrophoresis carries out crown gall alkali is commonly used, is tried with nitric acid argentum reagent or phosphomolybdic acid
Agent dyeing, but above-mentioned detection method has that operation is complicated, time-consuming and laborious.
Summary of the invention
The present invention provides a kind of rapid induction quinoa hairy method, can be rapidly and efficiently induction quinoa hairy
Growth, while can transformation efficiency that is easy, intuitive, quickly detecting hairy.
To solve the above problems, the present invention provides a kind of rapid induction quinoa hairy method, include the following steps：
1) Agrobacterium rhizogenesA4 containing gus gene is constructed；
2) quinoa explant infects；
Sterile quinoa explant is infected with dip dyeing liquid for shell A or infected liquid B, and dip dyeing liquid for shell A is prepared by wild type Agrobacterium rhizogenesA4
It obtains, infected liquid B is prepared by step 1) containing the Agrobacterium rhizogenesA4 of gus gene；
3) the quinoa explant after infecting is co-cultured；
4) the quinoa explant after co-culturing, which moves in induced medium, to be cultivated, and obtains quinoa hairy；
5) hairy progress subculture of quinoa and amplification cultivation；
6) to hairy progress GUS dyeing of quinoa, the quinoa hairy is by the quinoa explant after infected liquid B dip dyeing
Through step 3), 4), 5) culture obtain.
Preferably, step 1) is specially：PCR3301 plasmid containing gus gene is added in Agrobacterium rhizogenesA4 competence,
Successively be incubated on ice, liquid nitrogen flash freezer, be transferred to the first YEB fluid nutrient medium culture after metal bath heat shock, the culture of acquisition from
The heart removes supernatant, and residue precipitating moves into the culture of YEB solid medium, obtains monoclonal bacterium, and the monoclonal bacterium moves into second
It is shaked in YEB fluid nutrient medium, obtains bacterium solution, bacterium solution is detected through PCR and screened, and obtains the Agrobacterium rhizogenesA4 containing gus gene.
Preferably, step 1) YEB solid medium is Kan containing 100mg/L (kanamycins) and 100mg/L Rif (Li Fu
It is flat) culture medium.
Preferably, the 2nd YEB fluid nutrient medium of step 1) is Kan containing 100mg/L (kanamycins) and 100mg/L Rif
The culture medium of (rifampin).
Preferably, sterile quinoa explant is 3 days sterile quinoa explants to 2 week old in step 2).
Preferably, time of infection is 8-15 minutes in step 2).
Preferably, the preparation method of step 2) infected liquid A is：Choose OD value (600) be 0.6-1.0 Agrobacterium rhizogenesA4 into
Row centrifugation, the solid being centrifuged are resuspended with 0.5-2.0 times of volume MS fluid nutrient medium and are obtained.
Preferably, the preparation method of step 2) dip dyeing liquid for shell B is：Choosing OD value (600) is that 0.6-1.0 contains gus gene
Agrobacterium rhizogenesA4 is centrifuged, and the solid being centrifuged is resuspended with 0.5-2.0 times of volume MS fluid nutrient medium and is obtained.
Preferably, step 3) co-cultures the time as -72h for 24 hours.
Preferably, step 4) incubation time is 1 week -3 week.
Reagent used in the present invention is existing commodity in the prior art, can be carried out by publicly available approach
Purchase.
Gus gene, that is, beta-glucosiduronatase gene (β-glucuronidase).The gene product is beta-glucosidase acid
Enzyme belongs to hydrolase, stablizes without degradable.Histochemical method is detected with the chloro- 3- indoles of the bromo- 4- of 5--beta-glucosidase acid (X-
Gluc it) is used as reaction substrate, buffer of the tested material containing substrate is impregnated.If gus gene has occurred in histocyte
Conversion, and GUS is given expression to, under appropriate conditions, which can hydrolyze X-Gluc and generate blue product.This is initial by it
Product acts on the bipseudoindoxyl dye to be formed through oxidative dimerization, it, which makes to have the active position GUS or site, is presented blue, with the naked eye or
It can be seen under microscope, and GUS activity can be reflected according to the dyeing depth under to a certain degree.Therefore this method observable is utilized
To expression of the foreign gene in certain organs, tissue or even individual cells.
PCR3301 plasmid map is as shown in Figure 1, there are gus gene and with multiple restriction enzyme sites, starting in plasmid map
Son is CAMV35S, and kanamycin resistance screening can be carried out when carrier construction.The sequence of this plasmid can passage path NCBI>
DNA&RNA>Nucleotide Database inquiry obtains.
Increasing of the YEB culture medium for agrobacterium rhizogenes is cultivated, and every liter contains beef extract 5g, yeast extract 1g, egg
White peptone 5g, sucrose 5g, MgSO4·H2O 0.5g, pH 7.0.
MS fluid nutrient medium is to use most common culture medium at present.Its inorganic salt concentration with higher, can guarantee
Mineral nutrition needed for tissue growth can also accelerate the growth of callus.Since the ion concentration in formula is high, is preparing, storing
During depositing and sterilize etc., even if some ingredients are slightly different, interionic balance will not be influenced.MS fluid nutrient medium is used for
Apparent success can be obtained when cell suspension cultures.
OD value (optical density) is defined as absorbing the optical density (OD) of light substance, it is close with the physics of the substance
Degree should be linearly related.Relationship between OD value and the light intensity for penetrating it meets Lambert-Beer's law, is exponential relationship.
When OD value is equal to 0, to light without absorption, transmissivity 100%.When OD value is equal to infinity, whole can be absorbed
Light, transmissivity 0.OD value (600) is light absorption value of certain solution at 600nm wavelength.
Quinoa is the annual dicotyledonous herbaceous plant of Amaranthaceae Chenopodiaceae subfamily Chenopodium.Originating from South America Andes, originate in
Ground is Indian due to its nutritive value abundant and unique functional characteristic mainly in Peru, Bolivia and Ecuador
Person is " mother of grain ".Quinoa contains a large amount of good protein, and its amino acid composition is balanced.FAO (Food and Agriculture Organization of the United Nation)
(FAO) formal recommendation its be the optimum mankind perfection " wholefood ", mankind's basic food requirements can be met by being called
Only monomer plant.In the method for gene function verifying for carrying out quinoa, generally regenerated using arabidopsis or callus induction,
Growth cycle is long and transformation efficiency is relatively low.
The Ri plasmid of Agrobacterium rhizogenesA4 can induce plant and generate hairy system, and plasmid has the area T-DNA and Vir.It is first
First, agrobacterium rhizogenes is attached to plant cell, stays in space between cells later.Later, Agrobacterium can promote injured plant group
It knits and generates the soluble micromolecule phenolic such as acetosyringone and hydroxyl acetosyringone through shikimic acid route of synthesis, these
Micromolecule phenolic will be used as signaling molecule, promote transfer of the Agrobacterium to plant tissue.Meanwhile these signaling molecules exist
Under the mediation of VirA gene product (a kind of combination receptor protein) on film, so that the Foundation acidification structure of VirG albumen changes
Become, and then activate other areas Vir gene, promotes the formation of T-DNA duplication and T- catenin compound.Then, T- catenin is multiple
Zoarium, by cell membrane transporter, eventually enters into plant nucleolus, T-DNA is integrated into the base of plant under the assistance of transport protein
Among group, and the transcript and expression in plant, so that plant is grown hairy near wound.Hairy with its maternal plant phase
Than, have growth rapidly, inheritance stability and to show the biosynthesis ability identical or bigger with secondary metabolite etc. special
Point.
The present invention uses a kind of rapid induction quinoa hairy method, by 3 days to 2 week old sterile quinoa explants,
Infected 8-15 minutes with infected liquid A and infected liquid B respectively, then carry out co-culture 24-72 hours after, be transferred to screening and culturing into
Row culture, hairy for cutting fast growing after one week from explant are transferred on subculture medium, transfer after two weeks
It expands culture, is obtained after one month hairy a large amount of on to fluid nutrient medium.Infected liquid A is wild type agrobacterium rhizogenes
A4, staining reagent B are the A4 agrobacterium rhizogenes for being transferred to gus reporter gene.
The present invention has filtered out optimal strain and its OD value during quinoa hairy root induction, when optimal co-cultivation
Between and optimal inductive site.And the Agrobacterium rhizogenesA4 with gus gene is constructed, and to the hair for having gus gene
Shape root has carried out GUS dyeing, for detecting transformation efficiency.Present invention chemical dyeing method carries out GUS dyeing, so as to it is easy,
Visual inspection transformation efficiency.Also, method provided by the invention have it is easy to operate, analysis speed it is fast, it is low using reagent price
Honest and clean advantage.
Detailed description of the invention
Fig. 1, pCR3301 plasmid map.
Specific embodiment
It is right With reference to embodiment in order to make those skilled in the art more fully understand the technical solution of invention
The present invention is described in further detail.
At present hairy to be gradually used for improvement of crop cultivar, the production of secondary metabolites and environmental pollution reparation etc. all
It is many-sided.Meanwhile the regeneration plant turned out by hairy root induction is often with the variation having on some special phenotypic characteristics, this
The variation of a little phenotypes is mostly due to the result of T-DNA conversion in Agrobacterium rhizogenes.Using this characteristic, send out grain farmer bacillus oneself
It is used for the Upgrading of crop economical character.And on production secondary metabolites, not only the production cycle is long for traditional agriculture method,
Yield is unstable, but also to be influenced by many factors such as production environment, pest and disease damages, is difficult to obtain expected yield.Together
When, there is also certain deficiencies for traditional culture plant cell method, can often encounter such as cell slow growth, need to add sharp
The problems such as element maintains, while the limits throughput of secondary metabolites, production capacity are also unstable.And the growth of hairy system is fast
Speed, secondary metabolite synthesizes vigorous advantage is that industrialized production is dreamed of.So far, pass through hairy Gen Kepei
The secondaryization metabolite of health output has covered many kinds of substance such as polysaccharide, alkaloids, flavonoids, phenols.Meanwhile having packet
It includes tens kinds of important pharmaceutical ingredients including camptothecine, ginkgol, Puerarin etc. and realizes by hairy and produce.It is purple
The plants such as grass, catharanthus roseus, beet are even more the industrialization for realizing the production of texturing root.
China's quinoa Introduced Cultivars and breeding work are started late at present, and the biotechnology applications of quinoa and exploitation are more slow
Slowly, it therefore improves to the hairy root induction speed of quinoa and genetic transformation efficiency, and simplifies testing process, it is aobvious to improve detection speed
It obtains very necessary.
The present invention provides a kind of rapid induction quinoa hairy method, includes the following steps：
1) Agrobacterium rhizogenesA4 containing gus gene is constructed, specially：Hair is added in pCR3301 plasmid containing gus gene
It in root Agrobacterium A4 competence, is successively incubated on ice, liquid nitrogen flash freezer, is transferred to the first YEB fluid nutrient medium after metal bath heat shock
Supernatant is removed in culture, the culture centrifugation of acquisition, and residue precipitating moves into the culture of YEB solid medium, obtains monoclonal bacterium, institute
It states and is shaked in monoclonal bacterium the 2nd YEB fluid nutrient medium of immigration, obtain bacterium solution, bacterium solution is detected through PCR and screened, and acquisition contains GUS
The Agrobacterium rhizogenesA4 of gene.YEB solid medium is Kan containing 100mg/L (kanamycins) and 100mg/L Rif (Li Fu
It is flat) culture medium.2nd YEB fluid nutrient medium is Kan containing 100mg/L (kanamycins) and 100mg/L Rif (rifampin)
Culture medium.The sequence of pCR3301 plasmid can be inquired by following network address.
https://www.ncbi.nlm.nih.gov/nucleotide/KF206152.1？Report=genbank&
log$
2) quinoa explant infects；
Sterile quinoa explant is infected with dip dyeing liquid for shell A or infected liquid B, and sterile quinoa explant is 3 days to the sterile of 2 week old
Quinoa explant, time of infection are 8-15 minutes, and dip dyeing liquid for shell A is prepared by wild type Agrobacterium rhizogenesA4, and infected liquid B is by step
Rapid 1) the Agrobacterium rhizogenesA4 containing gus gene prepares；The preparation method of infected liquid A is：Choosing OD value (600) is 0.6-
1.0 Agrobacterium rhizogenesA4s are centrifuged, and the solid being centrifuged is resuspended with 0.5-2.0 times of volume MS fluid nutrient medium and is obtained.Leaching
The preparation method of dye liquor B is：Choosing OD value (600) is that the Agrobacterium rhizogenesA4 that 0.6-1.0 contains gus gene is centrifuged, from
The solid that gains in depth of comprehension arrive is resuspended with 0.5-2.0 times of volume MS fluid nutrient medium and is obtained.
3) the quinoa explant after infecting is co-cultured；The time is co-cultured as -72h for 24 hours
4) the quinoa explant after co-culturing, which moves in induced medium, to be cultivated, and obtains quinoa hairy；Incubation time is 1
- 3 week of week.
5) hairy progress subculture of quinoa and amplification cultivation；
6) to hairy progress GUS dyeing of quinoa, the quinoa hairy is by the quinoa explant after infected liquid B dip dyeing
Through step 3), 4), 5) culture obtain.
The present invention induces quinoa hairy by Agrobacterium rhizogenesA4, has filtered out optimal induction time, concentration and training
Time etc. is supported, and constructs the Agrobacterium rhizogenesA4 with gus gene, and GUS has been carried out to hairy with gus gene
Dyeing is for detecting transformation efficiency.Present invention firstly provides a kind of rapid induction quinoa hairy methods, secondly with chemistry dye
Color method carries out GUS dyeing, can easy, visual inspection transformation efficiency.
It is above-mentioned to be elaborated to be of the invention, it is below the embodiment of the present invention.
Embodiment one
The building of Agrobacterium rhizogenesA4 containing gus gene.
(1) 10 μ L pCR3301 plasmids are taken to thaw on ice 10min with liquid-transfering gun, taking-up is stored in -80 DEG C of refrigerators
The Agrobacterium rhizogenesA4 competence prepared, is placed on and melts 10min on ice.
(2) 10 μ L pCR3301 are added in the Agrobacterium rhizogenesA4 competence thawed, under gently shaking three with hand,
It is incubated for 15-30min on ice, transformed Agrobacterium is put into liquid nitrogen rapidly, the quick-frozen time is 8min.
(3) the agrobacterium rhizogenes competence in liquid nitrogen, metal bath heat shock are quickly removed with tweezers, temperature is kept for 37 DEG C continue
Time be 5min.
(4) the first YEB fluid nutrient medium is added in the Agrobacterium competence for taking out heat shock, and volume is 500 μ L and is not added
Any antibiotic, the condition of culture are 28 DEG C of temperature, shaking speed 200rpm, incubation time 4-5h.
(5) the use of low speed centrifuge revolving speed is 5000rpm centrifugation 10min, the transparent supernatant of 300 μ L is sucked out with liquid-transfering gun
Body, remaining precipitating carry out screening and culturing on solid medium in next step.
(6) thallus of previous step precipitating carries out coated plate using sterilized glass bar, cultivates on YEB solid medium,
Solid medium Kan containing 100mg/L and 100mg/L Rif.
(7) solid medium is placed in constant incubator and is incubated overnight, 28 DEG C of temperature, plate first just placement 2h be inverted again
Cultivate 2d.It chooses and grows mellow and full smooth monoclonal bacterium, be put into the 2nd YEB liquid of Kan containing 100mg/L and 100mg/L Rif
Bacterium is shaken in culture medium, bacterium solution is muddy after 6h, carries out bacterium solution PCR.Choosing testing result is that positive Agrobacterium rhizogenesA4 carries out
Subsequent experimental.
Embodiment two
The rapid induction that quinoa is hairy
(1) infected liquid A：It is centrifuged by (600) 0.6 Agrobacterium A4 of 50mlOD value, with 2.0 times of volume MS fluid nutrient mediums
Be resuspended to obtain mix it is spare.
(2) infected liquid B：(600) 0.6 Agrobacterium A4 of 50mlOD value is centrifuged with gus reporter gene, with 2.0 times of bodies
Product MS fluid nutrient medium be resuspended to obtain mix it is spare.
(3) the sterile quinoa explant in two bottles of 3-4 days ages is taken.
(4) one groups are added to 50 milliliters of infected liquid A, are completely soaked hypocotyl and cotyledon in a liquid, infect 8 points
Clock.Another set is added in 50 milliliters of infected liquid B.
(5) 25 DEG C are added in MS fluid nutrient medium, and dark culture is for 24 hours.
(6) after dark culture terminates, under shifting after MS solid medium one week to two weeks containing 100mg/ml cephalo
Plumular axis and cotyledon are differentiated to form hairy.
(7) it cuts off 2-3CM hairy and carries out screening and culturing on 100mg/ml cephalo MS solid medium is added.Two weeks with
The fast quinoa of the speed of growth hairy is filtered out afterwards carries out squamous subculture on the MS solid medium of the cephalo containing 50mg/ml.After
Be commissioned to train it is 2-3 times feeding after, obtain quinoa hairy of detoxification.
(8) 2-3CM hairy is cut off to expand culture on MS fluid nutrient medium is added.It is fast so as to grow,
Hairy of quinoa more than lateral root branch.
(9) hairy progress GUS dyeing of the quinoa for B infected liquid being infected and being cultivated can catch color, and discovery becomes blue.
Embodiment three
The rapid induction that quinoa is hairy
(1) infected liquid A：Centrifugation is carried out by (600) 1.0 Agrobacterium A4 of 50mlOD value to be carried out with 0.5 times of MS fluid nutrient medium
Resuspension obtains mixing spare.
(2) infected liquid B：(600) 1.0 Agrobacterium A4 of 50mlOD value carries out 0.5 times of volume of centrifugation with gus reporter gene
MS fluid nutrient medium be resuspended to obtain mix it is spare.
(3) the sterile quinoa explant of two bottle of 2 week old is taken.
(4) one groups are added to 50 milliliters of infected liquid A, are completely soaked hypocotyl and cotyledon in a liquid, infect 15 points
Clock.Another set is added in 50 milliliters of infected liquid B.
(5) 25 DEG C are added in MS fluid nutrient medium, dark culture 72h.
(6) after dark culture terminates, under shifting after MS solid medium two weeks to three weeks containing 100mg/ml cephalo
Plumular axis and cotyledon are differentiated to form hairy.
(7) it cuts off 2-3CM hairy and carries out screening and culturing on 100mg/ml cephalo MS solid medium is added.Two weeks with
The fast quinoa of the speed of growth hairy is filtered out afterwards carries out squamous subculture on the MS solid medium of the cephalo containing 50mg/ml.After
Be commissioned to train it is 2-3 times feeding after, obtain quinoa hairy of detoxification.
(8) 2-3CM hairy is cut off to expand culture on MS fluid nutrient medium is added.It is fast so as to grow,
Hairy of quinoa more than lateral root branch.
(9) hairy progress GUS dyeing of the quinoa for B infected liquid being infected and being cultivated can catch color, and discovery becomes blue.
Example IV
Quinoa hairy rapid induction method
(1) infected liquid A：Centrifugation is carried out by (600) 0.8 Agrobacterium A4 of 50mlOD value to be carried out with isometric MS fluid nutrient medium
Resuspension obtains mixing spare.
(2) infected liquid B：(600) 0.8 Agrobacterium A4 of 50mlOD value carries out the isometric MS of centrifugation with gus reporter gene
Fluid nutrient medium be resuspended to obtain mix it is spare.
(3) the sterile quinoa explant of two bottle of one week old is taken.
(4) one groups are added to 50 milliliters of infected liquid A, are completely soaked hypocotyl and cotyledon in a liquid, infect 10 points
Clock.Another set is added in 50 milliliters of infected liquid B.
(5) 25 DEG C are added in MS fluid nutrient medium, dark culture 48h.
(6) after dark culture terminates, under shifting after MS solid medium one week to two weeks containing 100mg/ml cephalo
Plumular axis and cotyledon are differentiated to form hairy.
(7) it cuts off 2-3CM hairy and carries out screening and culturing on 100mg/ml cephalo MS solid medium is added.Two weeks with
The fast quinoa of the speed of growth hairy is filtered out afterwards carries out squamous subculture on the MS solid medium of the cephalo containing 50mg/ml.After
Be commissioned to train it is 2-3 times feeding after, obtain quinoa hairy of detoxification.
(8) 2-3CM hairy is cut off to expand culture on MS fluid nutrient medium is added.It is fast so as to grow,
Hairy of quinoa more than lateral root branch.
(9) NILA for B infected liquid being infected and being cultivated, which carries out GUS dyeing, can catch color, and discovery becomes blue.
The result of embodiment two to example IV is characterized as below shown in table.
Table 1, the result of embodiment two to example IV
It is above-mentioned the experimental results showed that, embodiment two to example IV successfully induction discharging quinoa hairy, wherein under
The inductivity embodiment two of plumular axis is optimal, reaches 77.78%, optimal to the inductivity embodiment three of cotyledon, reaches 40%.
The above is only the preferred embodiment of the present invention, it is noted that above-mentioned preferred embodiment is not construed as pair
Limitation of the invention, protection scope of the present invention should be defined by the scope defined by the claims..For the art
For those of ordinary skill, without departing from the spirit and scope of the present invention, several improvements and modifications can also be made, these change
It also should be regarded as protection scope of the present invention into retouching.
Claims (10)
1. a kind of rapid induction quinoa hairy method, which is characterized in that include the following steps：
1) Agrobacterium rhizogenesA4 containing gus gene is constructed；
2) quinoa explant infects；
Sterile quinoa explant is infected with dip dyeing liquid for shell A or infected liquid B, and the dip dyeing liquid for shell A is prepared by wild type Agrobacterium rhizogenesA4
It obtains, the infected liquid B is prepared by the Agrobacterium rhizogenesA4 described in step 1) containing gus gene；
3) the quinoa explant after infecting is co-cultured；
4) the quinoa explant after co-culturing, which moves in induced medium, to be cultivated, and obtains quinoa hairy；
5) hairy progress subculture of quinoa and amplification cultivation；
6) to hairy progress GUS dyeing of quinoa, the quinoa hairy is by the quinoa explant after infected liquid B dip dyeing through step
It is rapid 3), 4), 5) culture obtain.
2. rapid induction quinoa hairy method according to claim 1, which is characterized in that the step 1) is specific
For：PCR3301 plasmid containing gus gene is added in Agrobacterium rhizogenesA4 competence, be successively incubated on ice, liquid nitrogen flash freezer,
The first YEB fluid nutrient medium culture is transferred to after metal bath heat shock, supernatant is removed in the culture centrifugation of acquisition, and residue precipitating moves into
YEB solid medium culture obtains monoclonal bacterium, and the monoclonal bacterium moves into the 2nd YEB fluid nutrient medium and shakes, and obtains bacterium
Liquid, the bacterium solution are detected through PCR and are screened, and obtain the Agrobacterium rhizogenesA4 containing gus gene.
3. rapid induction quinoa hairy method according to claim 2, which is characterized in that the YEB solid culture
Base is the culture medium of Kan containing 100mg/L (kanamycins) and 100mg/L Rif (rifampin).
4. rapid induction quinoa hairy method according to claim 3, which is characterized in that the 2nd YEB liquid
Culture medium is the culture medium of Kan containing 100mg/L (kanamycins) and 100mg/L Rif (rifampin).
5. rapid induction quinoa hairy method according to claim 4, which is characterized in that sterile in the step 2)
Quinoa explant is 3 days sterile quinoa explants to 2 week old.
6. rapid induction quinoa hairy method according to claim 5, which is characterized in that infected in the step 2)
Time is 8-15 minutes.
7. rapid induction quinoa hairy method according to claim 6, which is characterized in that the step 2) infected liquid
The preparation method of A is：Choosing OD value (600) is that 0.6-1.0 Agrobacterium rhizogenesA4 is centrifuged, the solid 0.5- being centrifuged
2.0 times of volume MS fluid nutrient mediums, which are resuspended, to be obtained.
8. rapid induction quinoa hairy method according to claim 7, which is characterized in that the step 2) dip dyeing liquid for shell
The preparation method of B is：Choosing OD value (600) is that the Agrobacterium rhizogenesA4 that 0.6-1.0 contains gus gene is centrifuged, and is centrifuged
The solid arrived is resuspended with 0.5-2.0 times of volume MS fluid nutrient medium and is obtained.
9. rapid induction quinoa hairy method according to claim 8, which is characterized in that the step 3) co-cultures
Time is -72h for 24 hours.
10. rapid induction quinoa hairy method according to claim 9, which is characterized in that the step 4) culture
Time is 1 week -3 week.
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| CN110172474A (en) * | 2019-05-22 | 2019-08-27 | 中国药科大学 | Salvia chinensis hairy induction and quick propagation method |
| US11299700B1 (en) | 2021-02-19 | 2022-04-12 | Acequia Biotechnology, Llc | Bioreactor containers and methods of growing hairy roots using the same |
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| CN110172474A (en) * | 2019-05-22 | 2019-08-27 | 中国药科大学 | Salvia chinensis hairy induction and quick propagation method |
| CN110172474B (en) * | 2019-05-22 | 2023-03-21 | 中国药科大学 | Induction and rapid propagation method for hairy roots of purple ginseng |
| US11299700B1 (en) | 2021-02-19 | 2022-04-12 | Acequia Biotechnology, Llc | Bioreactor containers and methods of growing hairy roots using the same |
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