CN106244625A - A kind of agriculture bacillus mediated tobacco seed genetic transforming method rapidly and efficiently - Google Patents
A kind of agriculture bacillus mediated tobacco seed genetic transforming method rapidly and efficiently Download PDFInfo
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Abstract
The present invention discloses a kind of agriculture bacillus mediated tobacco seed genetic transforming method rapidly and efficiently.The key step of the method includes: ripe tobacco seed is used as genetic transformation material after appropriateness is ground, adjust Agrobacterium bacterial concentration to appropriate value for infecting acceptor material, successively through co-culturing, sowing, GUS histochemical stain and PCR amplification after, identify transgenic tobacco plant.The inventive method without carrying out disinfection, the step such as sterilizing and tissue culture, and major part operating process is without aseptically carrying out, avoid the shortcoming such as loaded down with trivial details, time-consuming brought due to aseptic and group training operation, it is a kind of simple, quick, low cost, repeatability preferably genetic transforming method, quickly initiative transgene tobacco new germ plasm is had great importance and is worth.
Description
Technical field
The invention belongs to agricultural biological technical field and plant genetic engineering field, a kind of agriculture bacillus mediated cigarette
Grass seed method for transformation.
Background technology
Nicotiana tabacum L. is the crop that a kind of economic worth is higher, occupies huge economic market in China.Nicotiana tabacum L. is except being mainly used in
Suck, albumen edible and medicinal outside, also there is relatively broad purposes.Along with the development of plant subject, grind based on Nicotiana tabacum L.
Model plant in studying carefully and applying has played important at aspects such as plant genetic, growth, Physiology and biochemistry and secondary metabolism researchs
Effect.Although China's tobacco planting area and yield all rank first in the world, but Nicotiana tabacum L. is the economy that a kind of yield and quality is laid equal stress on
Crop, the factor such as Different climate condition, pest and disease damage all can have a strong impact on the quality of the growth of cigarette strain, Nicotiana tabacum L., in turn results in greatly
Economic loss.So the requirement that cigarette quality is improved by people is more and more urgent, conventional smoke grass crossing breeding cycle length, take effect
Slowly, there is the phenomenon that unfavorable character gene is chain simultaneously, have a strong impact on new product of tobacco cultivation.At present, genetic engineering is used
It is one of the most efficient breeding mode that technology obtains new product of tobacco.
Since 1984 obtain transgene tobacco first, including particle bombardment, electric shocking method, PEG method, pollen tube channel
Method and the multiple genetic transforming method such as agriculture bacillus mediated have been successfully applied to the initiative of Nicotiana tabacum L. new germ plasm.But PEG method and electric shocking method
Needing by means of plant protoplast culture technique, operation easier is relatively big and plant protoplast may be spontaneous by hormonal action
Merge and produce somatic variation；Although particle bombardment is simple to operate, but economic drain is big, there is exogenous gene and easily loses and sink
Silent phenomenon, the shortcoming such as transgenic progeny easily sudden change；It addition, pollen tube passage method is big by the influence of change of environmental condition, repeatability
Bad, empirical too strong, conversion ratio is on the low side and obtains the shortcomings such as plant late detection difficulty.At present, Agrobacterium is utilized to infect cigarette
It is to study the most deep, the most ripe genetic transforming method that blade of grass sheet obtains transgene tobacco, and it has conversion ratio height, low cost etc.
Advantage, but the method must require strict by tissue culture and Regeneration Ways in operating process, time and effort consuming, work
Amount is big, vitrification, brownization easily occurs in group training material successive transfer culture.So setting up one to be independent of plant tissue culture technique
Nicotiana tabacum L. genetic transforming method significant.
Agrobacterium is the gram negative bacteria being widely present in a kind of soil.Research finds, after plant sustains damage, and agriculture bar
Bacterium can infect most of dicotyledon and part monocotyledon, and plant injury will produce a species specific biology
Alkali crown gall alkali (opines), enters in agrobatcerium cell, causes the processing of T-DNA, enters plant cell and is incorporated into cell
On Matrix attachment region.If inserted in the suitable agrobatcerium T-DNA built by genes of interest, exogenous gene will be along with T-DNA
Insert in Plant Genome, and along with the Differentiation of plant cell, produce transfer-gen plant.The Nicotiana tabacum L. of agrobacterium-mediated transformation is lost
The acceptor material that biography Study on Transformation is used generally includes blade, embryo callus etc..The difference of these material source plants
Growth and development stage, has spatio-temporal difference, need to take different conversion and cultural method for different materials, operate
Journey is relatively complicated, in order to improve transformation efficiency, it is thus achieved that new tobacco bred, it should use ideal genetic transformation material,
It should have the advantage that first, and material processing mode is relatively uniform, has high duplication and universality；Second, material breaks up
Degree is low, and cellular omnipotency is strong, has the probability developing into whole plant；3rd, material easily obtains, and reduces and draws materials to reality
The impact tested, it is simple to test；4th, material quantity is relatively large, beneficially genetic transformation success.Due to tobacco seed subnumber
Amount is relatively big, and material is homogeneous, and repeatability is high, and easily obtains, and easily preserves, and cellular omnipotency is strong, is by Nicotiana tabacum L. Study on Genetic Transformation
Ideal material.Tradition Nicotiana tabacum L. Agrobacterium genetic transformation generally requires by plant tissue culture technique, and outer implant
Sterilization is one of key factor, and disinfecting time is too short, the easy microbiological contamination of tissue culture procedures；Overlong time, disinfecting substance can kill
Plant cell, affects group training effect.Therefore, a Nicotiana tabacum L. genetic transforming method being independent of tissue culture is set up for initiative cigarette
Grass new germ plasm is particularly important.
Summary of the invention
For the demand in above-mentioned field, the invention provides one and grow tobacco genetic transforming method, thus avoid tradition
Shortcoming in Nicotiana tabacum L. genetic transforming method is loaded down with trivial details including plant tissue culture technique, it is desirable to stringent asepsis requirements, in incubation
Growth cycle is long, more high to the conditions dictate of material, establishes the something lost of a kind of efficient quick agriculture bacillus mediated tobacco seed
Pass method for transformation.
The most agriculture bacillus mediated a kind of tobacco seed genetic transforming method, employing following steps:
1) choosing full seed is experiment material, destroys seed coat thus its embryo exposed with grinding pestle grinding seed；
2) adjustment engineering Agrobacterium bacterium solution containing destination carrier is to debita spissitudo, in conjunction with vacuum infiltration method, by Agrobacterium
Import the blastocyte of Nicotiana tabacum L.；
3) after aseptic filter paper draws unnecessary bacterium solution, the seed after infecting is sowed at and co-cultures in ware, and 28 DEG C of dark condition are cultivated
2d。
4) tobacco seed is moved into the soil of suitable growth, it is carried out Routine Management, to its grow to the 3-4 leaf phase carry out turn
Genetic tobacco plant is identified, screening.
Described vacuum infiltration method is to be soaked by aseptic ultra-pure water resuspended Agrobacterium bacterium solution by the tobacco seed exposing embryo,
Evacuation processes.
The condition that described evacuation processes is: soak 8-10min under 15Kpa pressure.
Its OD of Agrobacterium bacterium solution in described re-suspension liquid600For 0.3-0.5.
Described Agrobacterium bacterium solution adds acetosyringone (acetosyringone, AS) and surfactant (Silwet
L-77)。
Described Agrobacterium is Agrobacterium tumefaciems (Agrobacterium tumefaciens) bacterial strain LBA4404, destination carrier
For carrying quinolate phosphoribosyl transferase (Quinolinate phosphoribosyltransferases
Transferase) gene QPT and the plant expression vector pQPT of screening report fusion gene (GUS ∷ NPT II).
Described step 3) in culture medium be desaccharide MS fluid medium.
The method that described transfer-gen plant identifies, screen is GUS histochemical stain and pcr analysis, screening GUS dyeing and
The tobacco plant that PCR amplification is positive.
Before described tobacco seed carries out genetic transformation, all without disinfecting.
Described tobacco bred is K326 (Nicotiana tabacum L.var.K326).
Described step 1) in seed be collect ripe tobacco seed, in 40 DEG C of dry for standby.
The present invention, with Nicotiana tabacum L. mature seed as material, processes, with exposed embryo as receptor through grinding seed coat；Need not nothing
Collarium border, under vacuum, utilizes agriculture bacillus mediated, exogenous gene imports tobacco gene group, thus obtains transgenic cigarette
Grass.
The present invention needs not move through tissue culture, directly operates with seed, has the advantage that
The first, simple to operate.In addition to the cultivation of Agrobacterium, other steps are without operating in an aseptic environment.Avoid
Plant tissue culture course's necessary culture medium preparation, the sterilization of outer implant, callus induction, subculture, train altogether, screen, regenerate
Bud inducement, a series of complicated processes such as take root.
The second, growth cycle is short.Obtain transfer-gen plant and only need one growth cycle of Nicotiana tabacum L., relative to traditional tissue training
The method required time supported is shorter.
3rd, repeatability is high with universality.Unified to the processing mode of material, easy, easily repeat.
4th, high financial profit.Directly use Agrobacterium bacterium solution to infect tobacco seed genetic transformation receptor, utilize soil to train
Support Nicotiana tabacum L., decrease during Conventional reformat a series of manpower and materials needed for plant tissue culture and put into, can be greatly reduced
Genetic transformation cost.
The term of the present invention and culture medium:
In the present invention, Agrobacterium cultivates general employing YEP culture medium (yeast extract 10g/L+ peptone 10g/L+NaCl
5g/L, pH 7.2, adds 15g/L agar in solid medium) cultivate.
Accompanying drawing explanation
Fig. 1 plant expression vector structure chart,
Wherein A is plant expression vector pSH737, and B is plant expression vector pQPT,
Seed conversion process agriculture bacillus mediated for Fig. 2,
A: the tobacco seed that after grinding, seed coat is impaired；B: the tobacco seed co-cultured after infecting；C: application of vacuum seed with
Agrobacterium infects the mixture of liquid；D: training terminates rear planting seed in seedlings nursing plate altogether；E: infect seed germination.
Fig. 3 GUS histochemical stain and PCR identify
Wt: represent wild-type tobacco plants (Wild-type plants)；Tp: represent transgenic tobacco plant
(Transgenic plants)
Fig. 4 QPT gene PCR testing result
M:5000bp DNA Marker；P: positive plasmid compares；1-3: transfer-gen plant；N: negative control
Detailed description of the invention
Below in conjunction with embodiment, the present invention is described in further detail.
Embodiment: use this method to obtain Nicotiana tabacum L. K326 transfer-gen plant.
1, plant expression vector construction and the activation of engineering Agrobacterium
Studying the plant expression vector pSH737 (A in Fig. 1) to preserve is that initial carrier is transformed.In pSH737 carrier
Containing a multiple clone site (multiple cloning site, MCS), containing Xba I, BamH I, Xma I, Sma I,
The restriction enzyme sites such as Asp718, Kpn I and EcoR I.Respectively with BamH I and Kpn I double digestion plant expression vector pSH737 and
The two ends of synthetic are with BamH I and the QPT genetic fragment of Kpn I restriction enzyme site, afterwards at T4Under DNA ligase effect
It is attached, is built into plant expression vector pQPT (in Fig. 1 B, the public can be provided by this carrier).QPT gene code quinoline
Acid phosphoric acid phosphoribosynltransferase, this enzyme catalysis quinolinic acid forms NAMN (NAMN), enters pyridine nucleotide circulation with this,
And then formation nicotinic acid.Research shows, QPT is the rate-limiting step of nicotine biosynthetic branch approach pyridine nucleotide circulation.For
Cause the change of tobacco plant alkaloid and cause physiological shaped in transfer-gen plant after research Nicotiana tabacum L. overexpression QPT
The change of state, utilizes Agrobacterium freeze-thaw method that the plant expression vector pQPT built is imported competence Agrobacterium tumefaciems
(A.tumefaciens) in bacterial strain LBA4404, and tobacco seed is carried out quick genetic transformation.Screening in Vector map
Reporter gene is fusion gene, i.e. GUS ∷ NPT II, wherein NPT II gene code neomycin phosphotransferase (neomycin
Phosphotransferase), neomycin and kanamycin all there is resistance.Agrobacterium tumefaciems (A.tumefaciens) bacterial strain
LBA4404, carries the pQPT carrier of quinolate phosphoribosyl transferase gene, and streak inoculation (contains in YEP solid medium
On 20mg/L rifampicin (Rif)+50mg/L kanamycin (Kan).It is placed in incubator, after cultivating 2d under 28 DEG C of dark conditions, takes
Go out culture medium picking list bacterium colony in YEP solid medium (containing 20mg/L rifampicin (Rif)+50mg/L kanamycin (Kan)
On, it is placed in incubator, after cultivating 2d under 28 DEG C of dark conditions, resuspended with aseptic ultra-pure water, observe its bacterial concentration, and survey it
OD600Value, until bacterium solution OD600=0.3-0.5.
2, Nicotiana tabacum L. genetic transformation receptor prepares
Take K326 mature seed, seed is put into and 1.5mL centrifuge tube coordinates glass grinding rod appropriateness grind seed coat, thus
Expose its embryo (A in Fig. 2).
3, the genetic transformation of Nicotiana tabacum L.
Ready tobacco seed material is put into resuspended Agrobacterium bacterium solution, adds surface activity by the concentration of 200 μ L/L
The concentration of agent (SILWET 1-77) and 1mL/L adds acetosyringone (As), application of vacuum 8-10min under 15Kpa pressure
(C in Fig. 2), outwells bacterium solution, is placed in clean culture dish, adds aseptic ultra-pure water and cleans 2-3 time, is inhaled by surface liquid filter paper
Dry.Draw 2mL desaccharide MS fluid medium to be laid in culture dish (diameter 9cm), the subset after Agrobacterium is infected is put
It is placed in culture dish center, under 28 DEG C of dark conditions, cultivates 2d (B in Fig. 2).Planting seed after co-culturing afterwards is in soil
Germination and growth is to the 2-3 leaf phase.(D in Fig. 2, E), co-culturing base is sterilizing MS fluid medium (removing sucrose).
4, transgenic tobacco plant is identified
4.1 transformation of tobacco GUS histochemical stains are identified
Tobacco seed after cultivating is seeded in soil, waits that it grows, and its Nicotiana tabacum L. tender leaf is carried out GUS systematism
Learning dyeing detection, concrete grammar is as follows: clip Nicotiana tabacum L. tender leaf, puts in PCR pipe, and (the bromo-4-of 5-is chloro-to add the X-Gluc of 40 μ L
3-indole-β-D-Glucose glycosides) dyeing liquor, place 12h, sucking-off X-Gluc for 37 DEG C, add ethanol (75%) solution of 200ul,
Its chlorophyll is taken off, compares with matched group, filter out the positive plant (Fig. 3) of blueness.
4.2 transformation of tobacco PCR detections
Take the plant of GUS dyeing tests positive, use DNAsecure Plant Kit (TIANGEN company) test kit
Extract the DNA of tobacco leaf.Concrete grammar is as follows:
(1) take the Nicotiana tabacum L. fresh blade 100mg that GUS detection is positive, shred to mortar, add liquid nitrogen, be fully ground.
(2) blade after grinding, be equipped with the 2mL of 400 μ L buffer LP1 and 6 μ L RNase A (10mg/mL) from
Heart pipe, vortex vibration 1min, room temperature places 10min.
(3) 130 μ L buffer LP2, liquid-transfering gun piping and druming mixing, vortex vibration 1min are added.
(4) 12,000rpm is centrifuged 5min, is moved to by supernatant in new 2mL centrifuge tube.
(5) the buffer LP3 of 1.5 times of volumes, immediately fully vibration mixing 15sec are added, until flocculent deposit occurs.
(6) previous step gained solution and flocculent deposit are all added in an adsorption column CB3 that (adsorption column puts into collecting pipe
In), 12,000rpm are centrifuged 30sec, outwell waste liquid, and adsorption column CB3 puts in collecting pipe.
(7) adding 600 μ L rinsing liquid PW in adsorption column CB3,12,000rpm are centrifuged 30sec, outwell waste liquid, will absorption
Post CB3 puts in collecting pipe.
(8) previous action is repeated.
(9) putting back in collecting pipe by adsorption column CB3,12,000rpm are centrifuged 2min, outwell waste liquid.Adsorption column CB3 is placed in
Room temperature places several minutes, thoroughly dries rinsing liquid remaining in adsorbing material.
(10) adsorption column CB3 is proceeded in a clean centrifuge tube, the unsettled dropping 50-to the middle part of adsorbed film
200 μ L elution buffer TE, room temperature placement 2-5min, 12,000rpm are centrifuged 2min, are collected in centrifuge tube by solution.
Agrobacterium plasmid extracts, and uses the TIANGEN company little extraction reagent kit of plasmid to extract, and concrete grammar is as follows:
(1) column equilibration step: (adsorption column is put in collecting pipe) adds the balance liquid BL of 500 μ L in adsorption column CP3,
12,000rpm are centrifuged 1min, outwell the waste liquid in collecting pipe, and adsorption column is put back in collecting pipe.
(2) taking bacterium solution 1-5ml of incubated overnight, add in centrifuge tube, 12,000rpm are centrifuged 1min, draw supernatant.
(3) in the centrifuge tube leave bacterial sediment, add 250 μ L solution P1, use pipettor or vortex oscillator thorough
Suspended bacterial precipitates.
(4) in centrifuge tube, add 250 μ L solution P2, leniently spin upside down 6-8 time and make thalline fully crack.
(5) in centrifuge tube, add 350 μ L solution P3, the most leniently spin upside down 6-8 time, fully mix, now will
White flock precipitate occurs.12,000rpm are centrifuged 10min, now form precipitation bottom centrifuge tube.
(6) the supernatant pipettor that previous step is collected is transferred in adsorption column CP 3 that (adsorption column puts into collecting pipe
In), note sucking-off precipitation of trying not.12,000rpm are centrifuged 30-60s, outwell the waste liquid in collecting pipe, by adsorption column CP 3
Put in collecting pipe.
(7) adding 600 μ L rinsing liquid PW in adsorption column CP 3,12,000rpm are centrifuged 30-60s, outwell in collecting pipe
Waste liquid, puts into adsorption column CP 3 in collecting pipe.
(8) step (7) is repeated.
(9) putting in collecting pipe by adsorption column CP 3,12,000rpm are centrifuged 2min, remove rinsing remaining in adsorption column
Liquid.
(10) adsorption column CP 3 is placed in a clean centrifuge tube, drips 50-100 μ l to the middle part of adsorbed film
Elution buffer EB, room temperature placement 2min, 12,000rpm are centrifuged 1min, are collected in centrifuge tube by plasmid solution.
QPT gene is carried out PCR detection, respectively by transgene tobacco DNA, Agrobacterium plasmid product, carry out as template
PCR expands；Its gene primer is by the synthesis of the handsome biological company limited in Shanghai.Primer sequence is: Forward primer:5 '-
Cgcacaatcccactatcctt-3 ': Reverse primer:5 '-ttagagctttgccgacacct-3 ', PCR response procedures
For: preheat 94 DEG C of 3min；(94℃30s；58℃1min；68 DEG C of 2min 30s) 35 circular response；68℃5min；16 DEG C of guarantors
Deposit.
PCR after completion of the reaction, takes 5 μ L amplified productions, electrophoresis in the agarose gel electrophoresis of 1%, Bio-Rad gel imaging
Observe under system instrument and take a picture.PCR amplification shows, 3 strain positive plants all can amplify target stripe (Fig. 4).
4.3 seed genetic transformation rate statistics
Often approving and forwardingization tobacco seed about 100, co-culture after planting statistics sprouting and obtain tobacco seedlings about 14-20 strain, through GUS group
The plant about 3-5 strain that weave chemistry dyeing and PCR are positive after expanding, seed germination rate is 14%-20%, and conversion ratio is 15%-
36%.
Claims (10)
1. a most agriculture bacillus mediated tobacco seed genetic transforming method, employing following steps:
1) choosing full seed is experiment material, destroys seed coat thus its embryo exposed with grinding pestle grinding seed；
2) adjustment engineering Agrobacterium bacterium solution containing destination carrier is to debita spissitudo, in conjunction with vacuum infiltration method, is imported by Agrobacterium
The blastocyte of Nicotiana tabacum L.；
3), after aseptic filter paper draws unnecessary bacterium solution, the seed after infecting is sowed in culture dish cultivates 2d in 28 DEG C of dark condition.
4) tobacco seed is moved into the soil of suitable growth, it is carried out Routine Management, grow to the 3-4 leaf phase to it and carry out transgenic
Tobacco plant is identified, screening.
Method the most according to claim 1, described vacuum infiltration method is to be surpassed with aseptic by the tobacco seed exposing embryo
Pure water resuspended Agrobacterium bacterium solution is soaked, and evacuation processes.
Method the most according to claim 2, the condition that described evacuation processes is: soak 8-10min under 15Kpa pressure.
Method the most according to claim 3, its OD of Agrobacterium bacterium solution in described re-suspension liquid600For 0.3-0.5.
Method the most according to claim 4, adds acetosyringone (AS) and surfactant in described Agrobacterium bacterium solution
(Silwet L-77)。
Method the most according to claim 1, described Agrobacterium is agrobacterium tumefaciens bacterial strain LBA4404, and destination carrier is for taking
Band quinolate phosphoribosyl transferase (Quinolinate phosphoribosyltransferases transferase) base
Because of QPT and the pQPT carrier of kanamycin (kanamycin) resistant gene.
Method the most according to claim 6, transfer-gen plant identifies, the method screened is GUS histochemical stain and PCR
Analyzing, screening GUS dyeing and PCR expand positive tobacco plant.
Method the most according to claim 1, described step 3) in culture medium be desaccharide MS fluid medium.
Method the most according to claim 1, described step 1) in seed be the ripe tobacco seed collected, in 40 DEG C of bakings
Dry standby；Before described tobacco seed carries out genetic transformation, all without disinfecting.
Method the most according to claim 1, described tobacco bred is K326 (Nicotiana tabacum
L.var.K326)。
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109315290A (en) * | 2018-11-14 | 2019-02-12 | 云南中烟工业有限责任公司 | A method of induction Hongda tobacco Hairy root and plant regeneration |
| CN109825524A (en) * | 2019-02-26 | 2019-05-31 | 上海迈其生物科技有限公司 | A kind of herbicide basta resistance of mediated by agriculture bacillus imports the method for transformation of tobacco |
| CN110423744A (en) * | 2019-07-29 | 2019-11-08 | 甘肃农业大学 | A kind of extracting method of fructus lycii chloroplast genomic dna |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103205459A (en) * | 2013-03-20 | 2013-07-17 | 广州甘蔗糖业研究所 | Agrobacterium-mediated sugarcane genetic transformation method with vacuum infiltration assistance |
| CN104232683A (en) * | 2014-09-28 | 2014-12-24 | 江苏农林职业技术学院 | Agrobacterium tumefaciens-mediated tobacco transgenic method |
| CN104450779A (en) * | 2014-12-15 | 2015-03-25 | 贵州大学 | Fast and efficient agrobacterium-mediated rice stem tip genetic transformation method |
-
2016
- 2016-10-09 CN CN201610877696.5A patent/CN106244625B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103205459A (en) * | 2013-03-20 | 2013-07-17 | 广州甘蔗糖业研究所 | Agrobacterium-mediated sugarcane genetic transformation method with vacuum infiltration assistance |
| CN104232683A (en) * | 2014-09-28 | 2014-12-24 | 江苏农林职业技术学院 | Agrobacterium tumefaciens-mediated tobacco transgenic method |
| CN104450779A (en) * | 2014-12-15 | 2015-03-25 | 贵州大学 | Fast and efficient agrobacterium-mediated rice stem tip genetic transformation method |
Non-Patent Citations (2)
| Title |
|---|
| DANE K.等: "Rapid， EfficientProduction of Homozygous Transgenic Tobacco Plants with Agrobacterium tumefaciens: A Seed-to-Seed Protocol", 《PLANT MOLECULAR BIOLOGY REPORTER》 * |
| 郭丽红等: "根癌农杆菌法转化烟草的条件探索", 《云南大学学报(自然科学版)》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109315290A (en) * | 2018-11-14 | 2019-02-12 | 云南中烟工业有限责任公司 | A method of induction Hongda tobacco Hairy root and plant regeneration |
| CN109825524A (en) * | 2019-02-26 | 2019-05-31 | 上海迈其生物科技有限公司 | A kind of herbicide basta resistance of mediated by agriculture bacillus imports the method for transformation of tobacco |
| CN110423744A (en) * | 2019-07-29 | 2019-11-08 | 甘肃农业大学 | A kind of extracting method of fructus lycii chloroplast genomic dna |
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| CN106244625B (en) | 2019-07-12 |
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