CN109234308A - A kind of Agrobacterium tumefaciens mediated transgenosis woaded blue regeneration plant method for transformation - Google Patents
A kind of Agrobacterium tumefaciens mediated transgenosis woaded blue regeneration plant method for transformation Download PDFInfo
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- CN109234308A CN109234308A CN201811250112.7A CN201811250112A CN109234308A CN 109234308 A CN109234308 A CN 109234308A CN 201811250112 A CN201811250112 A CN 201811250112A CN 109234308 A CN109234308 A CN 109234308A
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- C12N15/8205—Agrobacterium mediated transformation
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Abstract
The present invention provides a kind of Agrobacterium tumefaciens mediated transgenosis woaded blue regeneration plant method for transformation, it the described method comprises the following steps: the woaded blue explant after preculture is immersed in the activated Agrobacterium tumefaciems bacterium solution with target gene, shaking makes to take out after coming into full contact with, aseptic paper is blotted to be inoculated in co-culture medium after extra bacterium solution and be carried out dark culture 1-4 days, explant is transferred to resistance screening induced medium and carries out illumination cultivation, experience proliferation, it takes root, the drop of strong sprout anti-screening and culturing is after 30-120 days, positive transgenic seedling is transferred to earth culture progress hardening culture through water planting transition and grows up to woaded blue regeneration plant；The woaded blue explant is woaded blue seedling leaf.The present invention directly uses the sterile miaoye of woaded blue as explant, and materials are convenient, and woaded blue regeneration plant can be turned out in compared with short cycle, and required equipment is simple, and operating technology is easily mastered.
Description
[technical field]
The invention belongs to the field of plant genetic project technology in Plant Biotechnology, in particular to a kind of crown gall
The transgenosis woaded blue regeneration plant method for transformation of mediated by agriculture bacillus.
[background technique]
The genetic transformation of plant cell is a key link of plant genetic engineering.From Abel etc. (1986) by tobacco
Mosaic virus (TMV) capsid protein imports tobacco cell, after the transgenic tobacco plant for obtaining anti-TMV virus, successively development
Many plant genetic transformation methods and technology.
Cruciferae isatis Isatis indigotica Fort. is China's successive dynasties conventional Chinese medicine, and root claims plate blue
Root, leaf claim folium isatidis, there is the effect of clearing heat and detoxicating, cool blood relieving sore-throat, can prevent and treat viral infection, popularity encephalitis B, stream
Row sexuality emits, acute, chronic hepatitis, and mumps etc. is effectively.
Agrobacterium tumefaciems Agrobacterium tumefaciens is a kind of natural bio-carrier, can pass through its T-DNA
Transfer, outer source DNA molecule is integrated into recipient plant Matrix attachment region and realizes genetic transformation.Agrobacterium tumefaciens mediated base
Because conversion system is a more perfect plant conversion system, there is very extensive host range, can be used for most of Shuangzis
Leaf plant and some monocotyledonous conversions.
At present report relevant to woaded blue genetic conversion system have woaded blue quick breeding method for tissue culture, device outside woaded blue root
Official's method for generation, agrobacterium rhizogenes mediate hairy transgenic method of woaded blue, using cotyledon and hypocotyl as the woaded blue of explant
Transform insect-resistant gene method, there is not yet using blade as the report of the woaded blue regeneration plant transgenic method of explant.
It is successfully many because being known as to influence Agrobacterium tumefaciens transformation, type, position, physiological status including transformed plant；
Type, cell suspending liquid density, culture medium composition and condition of culture of Agrobacterium tumefaciems etc..
Different plants will be by Agrobacterium tumefaciems induced synthesis regeneration plant, and obtains high-quality based on this
Transgenic plant still will pay laborious task.
[summary of the invention]
The technical problem to be solved in the present invention is to provide a kind of Agrobacterium tumefaciens mediated transgenosis woaded blue regeneration plant
Method for transformation directly uses the sterile miaoye of woaded blue as explant, and materials are convenient, and woaded blue regeneration can be turned out in compared with short cycle
Plant, required equipment is simple, and operating technology is easily mastered.
The present invention is implemented as follows:
A kind of Agrobacterium tumefaciens mediated transgenosis woaded blue regeneration plant method for transformation, it is characterised in that: the method packet
Include following steps:
Woaded blue explant after preculture is immersed in the activated Agrobacterium tumefaciems bacterium solution with target gene, shaking
Make to take out after coming into full contact with, aseptic paper is blotted to be inoculated in co-culture medium after extra bacterium solution and be carried out dark culture 1-4 days, explant
Body be transferred to resistance screening induced medium carry out illumination cultivation, experience be proliferated, take root, the drop of strong sprout anti-screening and culturing 30-120 days
Afterwards, positive transgenic seedling is transferred to earth culture progress hardening culture through water planting transition and grows up to woaded blue regeneration plant；
The woaded blue explant is woaded blue seedling leaf.
Further, the woaded blue explant is the 0.5-3cm that woaded blue tests for sterility is made into2Leaf dish.
Further, the target gene is the PHB-Flag label gene of shikimate kinase gene or zero load.
Further, the Agrobacterium tumefaciems bacterium solution is EHA105 or GV3101.
Further, the scheme of the preculture is MS or 1/2MS solid medium, and dark culture 1-48 hours, temperature was
24-30℃。
Further, the shaking is placed in the measure come into full contact in shaken cultivation case, and temperature is 25-37 DEG C, turns
Speed is 0-200rpm.
Further, the co-culture medium is MS or 1/2MS solid medium, wherein the aminoadenine of benzyl containing 6-
(6-BA) 0.1-2.0mg/L, methyl α-naphthyl acetate (NAA) 0.1-2.0mg/L, PH 5.5-6.0.
Further, the illumination cultivation condition be light intensity 1000-4000Lux, 18-30 DEG C of temperature, irradiation time 6-
24h。
Further, the resistance screening induced medium is MS or 1/2MS solid medium, wherein the amino gland of benzyl containing 6-
Purine (6-BA) 0.1-2.0mg/L, methyl α-naphthyl acetate (NAA) 0.1-2.0mg/L, cefalexin (Cef) 0-500mg/L, hygromycin
(Hyg) 0-20mg/L, PH 5.5-6.0.
Further, described take root, the culture medium in strong sprout step be respectively as follows: root media be MS or 1/2MS solid
Culture medium, wherein containing methyl α-naphthyl acetate (NAA) 0.1-2.0mg/L, cefalexin (Cef) 0-500mg/L, hygromycin (Hyg) 0-20mg/
L, PH 5.5-6.0；Strong seedling culture base is MS or 1/2MS solid medium, wherein contain cefalexin (Cef) 0-500mg/L, tide
Mycin (Hyg) 0-20mg/L, PH 5.5-6.0.
Further, the qualification program of the positive transgenic seedling is that the woaded blue of strong seedling culture in step is taken to regenerate miaoye
Piece, using CTAB method extract total DNA, using gained total DNA be template identify whether the resistance containing over-express vector PHB-Flag
Genetic fragment hpt and target gene fragment.
Further, the qualification program of the positive transgenic seedling is specially the woaded blue regrowth for taking strong seedling culture in step
Whether blade extracts the total DNA of positive transgenic seedling leaf by CTAB method, contain using gained total DNA as template through PCR identification
The resistance gene fragment hpt and target gene fragment of over-express vector PHB-Flag.
Further, the hardening condition of culture is as follows: the volume ratio of each component is Nutrition Soil: pumice: sandy soil=2:1:
1, light intensity 1000-4000Lux, 18-30 DEG C of temperature, irradiation time 6-24h.
The present invention has the advantage that
Inventive method materials are convenient, directly use the sterile miaoye of woaded blue as explant, established on this basis with blade
For the method that the Agrobacterium tumefaciens mediated woaded blue of explant forms regeneration plant genetic system, used instrument is not only reduced
Expense makes experimental implementation technology be easy to carry out in common lab, and to be carried out from now on using transgenic technology to woaded blue
Genetic improvement provides technical support.
[Detailed description of the invention]
The present invention is further illustrated in conjunction with the embodiments with reference to the accompanying drawings.
Fig. 1 is the woaded blue aseptic seedling of the embodiment of the present invention 1.
Fig. 2 is that the woaded blue regeneration plant of the embodiment of the present invention 1 is taken root.
Fig. 3 is that the woaded blue regeneration plant PCR amplification of the embodiment of the present invention 1 identifies electrophoretogram.
Fig. 4 is the transgenosis woaded blue regeneration plant in 1 water planting of the embodiment of the present invention and earth culture.
[specific embodiment]
The present invention relates to a kind of Agrobacterium tumefaciens mediated transgenosis woaded blue regeneration plant method for transformation, the method includes
Following steps:
Woaded blue explant after preculture is immersed in the activated Agrobacterium tumefaciems bacterium solution with target gene, shaking
Make to take out after coming into full contact with, aseptic paper is blotted to be inoculated in co-culture medium after extra bacterium solution and be carried out dark culture 1-4 days, explant
Body be transferred to resistance screening induced medium carry out illumination cultivation, experience be proliferated, take root, the drop of strong sprout anti-screening and culturing 30-120 days
Afterwards, positive transgenic seedling is transferred to earth culture progress hardening culture through water planting transition and grows up to woaded blue regeneration plant；
The woaded blue explant is the 0.5-3cm that woaded blue tests for sterility is made into2Leaf dish.
The target gene is the PHB-Flag label gene of shikimate kinase gene or zero load.
The Agrobacterium tumefaciems bacterium solution is EHA105 or GV3101.
The scheme of the preculture is MS or 1/2MS solid medium, and dark culture 1-48 hours, temperature was 24-30 DEG C.
The shaking is placed in the measure come into full contact in shaken cultivation case, and temperature is 25-37 DEG C, revolving speed 0-
200rpm。
The co-culture medium is MS or 1/2MS solid medium, wherein the aminoadenine of benzyl containing 6- (6-BA) 0.1-
2.0mg/L, methyl α-naphthyl acetate (NAA) 0.1-2.0mg/L, PH 5.5-6.0.
The illumination cultivation condition be light intensity 1000-4000Lux, 18-30 DEG C of temperature, irradiation time 6-24h.
The resistance screening induced medium is MS or 1/2MS solid medium, wherein the aminoadenine of benzyl containing 6- (6-
BA) 0.1-2.0mg/L, methyl α-naphthyl acetate (NAA) 0.1-2.0mg/L, cefalexin (Cef) 0-500mg/L, hygromycin (Hyg) 0-
20mg/L, PH 5.5-6.0.
It is described take root, the culture medium in strong sprout step be respectively as follows: root media be MS or 1/2MS solid medium,
In contain methyl α-naphthyl acetate (NAA) 0.1-2.0mg/L, cefalexin (Cef) 0-500mg/L, hygromycin (Hyg) 0-20mg/L, PH 5.5-
6.0；Strong seedling culture base is MS or 1/2MS solid medium, wherein containing cefalexin (Cef) 0-500mg/L, hygromycin (Hyg)
0-20mg/L, PH 5.5-6.0.
The qualification program of the positive transgenic seedling is specially that the woaded blue of strong seedling culture in step is taken to regenerate seedling leaf, is passed through
CTAB method extracts the total DNA of positive transgenic seedling leaf, whether contains overexpression load through PCR identification using gained total DNA as template
The resistance gene fragment hpt and target gene fragment of body PHB-Flag.
The hardening condition of culture is as follows: the volume ratio of each component is Nutrition Soil: pumice: sandy soil=2:1:1, light intensity
1000-4000Lux, 18-30 DEG C of temperature, irradiation time 6-24h.
1/2MS the and MS culture medium that the present invention selects is the common culture medium of plant cell engineering, is widely used in plant
Object tissue cultures, work well.Plant culture used in the present invention is specifically added to different plants according to different culture periods
The hygromycin of object hormone ratio, the antibiotic of various concentration and various concentration screening.
The full name of CTAB method of the present invention is cetyl trimethyl smelling hinge method, which can extract plant gene
Group belongs to general molecular biology routine operation.
PCR of the present invention is " polymerase chain reaction ", is well known to those skilled in the art experimental method, according to
PCR primer and PCR reaction condition, those skilled in the art can amplify purpose base according to common sense and empirically determined reaction system
Cause.
Below in conjunction with specific embodiment, the present invention is further illustrated.
Embodiment 1:
(1) the recombination PHB-Flag plasmid for carrying target gene is extracted
By the Escherichia coli Trans1-T1 containing recombinant plasmid after bacterial examination, sequence verification, it is inoculated into the liquid of kanamycins
In body culture medium (reagent preparation is purchased from Sheng Gong biotech firm), it is incubated overnight under conditions of 37 DEG C, 200rpm；It is mentioned with plasmid is small
Kit (being purchased from Quan Shijin biotechnology company) extracts recombinant plasmid.This method belongs to general molecular biology routine operation.
(2) prepare the woaded blue explant that genetic transformation uses
Diploid Seeds of Isatis indigotica picks up from pharmaceutical college of The 2nd Army Medical College botanical garden, is carried out disinfection place using routine disinfection method
Reason, sterilisation step are as follows: flowing water rinses 4h, and 75% alcohol embathes 1min, and aseptic water washing 3 times, 0.1% mercuric chloride vibrates
8min is washed, aseptic water washing 5 times or more, is inoculated on MS culture medium, in 25 DEG C of illumination (2000Lux) 16h, 18 DEG C of dark situations
Grow up to aseptic seedling in the temperature-controlling chamber of 8h, as shown in Figure 1, wherein a is Seeds of Isatis indigotica sprouting, b is woaded blue aseptic seedling.
It from the leaf dish of clip 0.5-3cm2 size in woaded blue aseptic seedling, is inoculated on precultivation medium, turns as heredity
The receptor of change；
The culture medium and leaf dish precultivation medium of woaded blue aseptic seedling are equal are as follows: and MS solid medium+30g/L glucose+
7.5g agar, PH 5.6-5.8；
(3) acquisition of the Agrobacterium tumefaciems engineering bacteria containing the PHB-Flag recombinant plasmid for carrying target gene SK.
By the recombinant plasmid of extraction by freeze-thaw method import EHA105 Agrobacterium tumefaciems in, then through antibiotic resistance screening and
The identification of PCR bacterial examination, obtains the Agrobacterium tumefaciems containing recombinant plasmid.
(4) genetic transformation of woaded blue blade explant
With rifampin containing 100mg/L (Rif, Agrobacterium tumefaciems resistance) and 75mg/L kanamycins, (Kan, PHB-Flag are carried
Body resistance) YEB culture medium activation culture to OD600 be 0.6 ± 0.1 Agrobacterium tumefaciems engineering bacteria, at room temperature 5000rpm from
Heart 10min, aseptic condition go down supernatant, with isometric fresh 1/2MS fluid nutrient medium resuspension containing acetosyringone (As)
Thallus.28 DEG C, after 100rpm oscillation incubation 30min, the good leaf dish of preculture is disseminated in bacterium solution in superclean bench
10min, during which constantly jiggling keeps its dip dyeing abundant.Explant is taken out, residual bacterium solution is absorbed with aseptic filter paper, is inoculated in altogether
Culture medium, 25 DEG C dark culture 2 days.
The explant of co-cultivation is transferred to except the progress degerming culture under 25 DEG C of illumination conditions on bacterium culture medium.
Co-culture medium are as follows: MS solid medium+30g/L glucose+1.0mg/L 6-BA+0.1mg/LNAA, PH
5.6-5.8；
Except bacterium culture medium are as follows: MS solid medium+30g/L glucose+1.0mg/L 6-BA+0.1mg/L NAA+500mg/
L Cef, PH 5.6-5.8；
(5) screening and culturing transgenic regenerated plant
Leaf dish is transferred on the anti-screening and culturing medium of drop, and illumination cultivation is to Multiple Buds are grown at 25 DEG C, replacement culture every 2 weeks
Base is once until the withered and yellow no power of regeneration of leaf dish is stopped.If bacterium phenomenon of overflowing occurs in leaf dish, has depending on Multiple Buds upgrowth situation and do not exist together
Reason, can carefully remove free of contamination Multiple Buds and be put into the anti-screening and culturing medium of new drop, otherwise can lose when Multiple Buds are up to 0.5cm
It abandons；
It is gently removed when the long seedling to 1-2cm high of the Multiple Buds that are grown in leaf dish and is transferred to proliferation screening and culturing medium
On, the illumination cultivation at 25 DEG C；
Regrowth after Multiplying culture it is long to 3cm or so when, move it into screening and culturing medium of taking root and continue to cultivate, the phase
Between, in case of bacterium of overflowing, then the culture medium of the antibiotic containing high concentration is moved into after cleaning out the long bacterium position of regrowth again, closely
Observe its condition of rooting；
Gained regrowth root long is then transferred into strong sprout screening and culturing medium one to 1cm or so and continues to cultivate, removing
After all plant hormones, regrowth can normally take root (fibrous root) as shown in Fig. 2, wherein a be take root incubation time grow it is thick
Root, b are the fibrous root that grows of woaded blue strong seedling culture period, when its root long is to about 2cm, then transfer them to strong sprout screening and culturing medium
It is cultivated in two.
Anti- screening and culturing medium drops are as follows: MS solid medium+30g/L glucose+1.0mg/L 6-BA+0.1mg/L NAA+
300mg/L Cef+10mg/L Hyg, PH 5.6-5.8；
It is proliferated screening and culturing medium are as follows: MS solid medium+30g/L glucose+1.0mg/L 6-BA+0.3mg/L NAA+
300mg/L Cef+15mg/L Hyg, PH 5.6-5.8；
It takes root screening and culturing medium are as follows: MS+30g/L glucose+0.5mg/L NAA+100mg/L Cef+15mg/L Hyg, PH
5.6-5.8；
Strong sprout screening and culturing medium one are as follows: MS+30g/L glucose+Cef 100mg/L+15mg/L Hyg, PH 5.6-5.8；
Strong sprout screening and culturing medium two are as follows: MS+30g/L glucose+15mg/L Hyg, PH 5.6-5.8；
(6) PCR amplification identifies regeneration plant
It takes the woaded blue in strong seedling culture to regenerate seedling leaf 1, total DNA is extracted using CTAB method, using gained total DNA as template
Identification whether resistance gene fragment hpt and target gene fragment containing over-express vector PHB-Flag.
Hpt fragment amplification primer are as follows:
Hpt-F:CGATTTGTGTACGCCCGACAGTC,
Hpt-R:CGATGTAGGAGGGCGTGGATATG；
Target gene fragment amplimer are as follows:
PhbSK-F:aaaGGATCCatggaggccagggtttctca；
Rbcs-R:attaacttcggtcattagaggc.
The nucleotide of the target gene SK (biological name in its source is Escherichia coli (Escherichia coli))
Sequence as shown in SEQ ID NO.1, the nucleotide sequence of the hpt fragment amplification primer hpt-F as shown in SEQ ID NO.2,
The nucleotide sequence of the hpt fragment amplification primer hpt-R as shown in SEQ ID NO.3, draw by the target gene fragment amplification
The nucleotide sequence of object phbSK-F is as shown in SEQ ID NO.4；The nucleotide of the target gene fragment amplimer Rbcs-R
Sequence is as shown in SEQ ID NO.5, and said gene sequence is referring in particular to attachment nucleotides sequence list.
PCR reaction system is (20 μ L): Template DNA 1 μ L, Forward Primer (10 μM) 1 μ L,
1 μ L, 2rTaq PCR SuperMix of Reverse Primer (10 μM), 10 7 μ L of μ L, ddH2O.
PCR reaction condition are as follows: 94 DEG C of 5min, 30 circulations (94 DEG C of 30s, 55 DEG C of 30s, 72 DEG C of 60s), 72 DEG C of 7min.
PCR product is through agarose gel electrophoresis and ultraviolet detection.The amplified band size of hpt is 812bp, target gene SK
Show for 1385bp, such as Fig. 3, each swimming lane is expressed as follows in Fig. 3: M:DL-2000DNA molecule Marker, N:EHA105 empty bacterium are negative
Control, the number of each positive regeneration plant of number swimming lane expression, the BcSK-OVX-1, BcSK-OVX-2 identified as shown in Figure 3,
It is to have PHB-flag resistance base in BcSK-OVX-5, BcSK-OVX-6, BcSK-OVX-8, BcSK-OVX-9, BcSK-OVX-14
Because of the electrophoretic band of hpt and target fragment rbcsr, the rbcsr detected in the regeneration plant of target gene is transferred to due to inserting
The segment of 400-500bp, so the more corresponding fragment lengths of purpose band, illustrate that these regeneration plants are transgenic positive
Plant.
(7) the hardening culture of woaded blue regrowth
When at the top of woaded blue regrowth length to culture bottle, well-grown woaded blue regrowth is taken out from culture tank, root is used
Tap water is rinsed well, and appropriate tap water (amount is limited to submerge root) is added in former culture tank, and woaded blue regrowth is put into culture
Bottle, mouth cover preservative film (preservative film pricks hole with toothpick and is conducive to breathe freely), the illumination cultivation at 25 DEG C；
After culture about 1 week, the regrowth of (having new fibrous root to grow) in good condition is transplanted in hardening culture medium, in 25
DEG C, humidity 65% is cultivated under the conditions of light intensity 2000Lux, as shown in figure 3, wherein a is the transgenosis woaded blue regeneration in water planting
Plant, b are the transgenosis woaded blue regeneration plant in earth culture.
Hardening culture medium are as follows: Nutrition Soil: pumice: sandy soil=2:1:1.
Inventive method directly uses the sterile miaoye of woaded blue as explant, obtains Agrobacterium tumefaciens mediated transgenosis woaded blue again
Raw plant, materials are convenient, not only reduce the expense of used instrument, make experimental implementation technology be easy common lab into
Row, and technical support is provided to carry out genetic improvement to woaded blue using transgenic technology from now on.
Although specific embodiments of the present invention have been described above, those familiar with the art should be managed
Solution, we are merely exemplary described specific embodiment, rather than for the restriction to the scope of the present invention, it is familiar with this
The technical staff in field should be covered of the invention according to modification and variation equivalent made by spirit of the invention
In scope of the claimed protection.
Sequence table
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<120>a kind of Agrobacterium tumefaciens mediated transgenosis woaded blue regeneration plant method for transformation
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tctcacttca gcgttggcgt acgcaccacc cctctcttca aaaaggtcag ttaacaactt 180
catggtcttt gaataaggat caccagaatc atgatgcaac aaaggccgag aattggttcc 240
gactgccgta agcctgtgtg ccaatgcttc caagggtaca tctagccaaa cagttatacc 300
atttcccatg tgtctccaat tgattggtcg aaccacagca ccaccacccg tggatacaat 360
caattgatgc cttgaagaca acttgtgcaa aacctcagtc tcattatctc tgaagaaatt 420
ttcgccatgt agtttgaaga tttcggcaac agtgtatcca ccagctgcct cctctattag 480
tgcatcacag tcacaaaatg aatatccaag ggccttggcc acaatcttgc ccactgttgt 540
ttttccagat cccatcattc caacaagata tatgcatcgt ccattcaggt atggtagaat 600
ctcttctgat ttattcttca tgatctttga ctcatcacct aatgctggaa aactgccaca 660
ctgcattaac gaagctgaat tgttttcaga agtacatgag acctctaaag caagaggttc 720
atgaaatctt ccctttttgc tgagccgttg gcaattcagt tttcgataca actgttgctc 780
cactttgttt cgcccaatgg gcaagtaacc gctgggtttt cttacaaatt ttcccagatc 840
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attaacttcg gtcattagag gc 22
Claims (13)
1. a kind of Agrobacterium tumefaciens mediated transgenosis woaded blue regeneration plant method for transformation, it is characterised in that: the method includes
Following steps:
Woaded blue explant after preculture is immersed in the activated Agrobacterium tumefaciems bacterium solution with target gene, shaking makes to fill
Tap is taken out after touch, and aseptic paper is blotted to be inoculated in co-culture medium after extra bacterium solution and be carried out dark culture 1-4 days, explant turn
Entering resistance screening induced medium and carries out illumination cultivation, experience is proliferated, takes root, the drop of strong sprout anti-screening and culturing is after 30-120 days,
Positive transgenic seedling is transferred to earth culture progress hardening culture through water planting transition and grows up to woaded blue regeneration plant；
The woaded blue explant is woaded blue seedling leaf.
2. the Agrobacterium tumefaciens mediated transgenosis woaded blue regeneration plant method for transformation of one kind according to claim 1, special
Sign is: the woaded blue explant is the 0.5-3cm that woaded blue tests for sterility is made into2Leaf dish.
3. the Agrobacterium tumefaciens mediated transgenosis woaded blue regeneration plant method for transformation of one kind according to claim 1, special
Sign is: the target gene is the PHB-Flag label gene of shikimate kinase gene or zero load.
4. the Agrobacterium tumefaciens mediated transgenosis woaded blue regeneration plant method for transformation of one kind according to claim 1, special
Sign is: the Agrobacterium tumefaciems bacterium solution is EHA105 or GV3101.
5. the Agrobacterium tumefaciens mediated transgenosis woaded blue regeneration plant method for transformation of one kind according to claim 1, special
Sign is: the scheme of the preculture is MS or 1/2MS solid medium, and dark culture 1-48 hours, temperature was 24-30 DEG C.
6. the Agrobacterium tumefaciens mediated transgenosis woaded blue regeneration plant method for transformation of one kind according to claim 1, special
Sign is: the shaking is placed in the measure come into full contact in shaken cultivation case, and temperature is 25-37 DEG C, revolving speed 0-
200rpm。
7. the Agrobacterium tumefaciens mediated transgenosis woaded blue regeneration plant method for transformation of one kind according to claim 1, special
Sign is: the co-culture medium is MS or 1/2MS solid medium, wherein the aminoadenine of benzyl containing 6- (6-BA) 0.1-
2.0mg/L, methyl α-naphthyl acetate (NAA) 0.1-2.0mg/L, PH 5.5-6.0.
8. the Agrobacterium tumefaciens mediated transgenosis woaded blue regeneration plant method for transformation of one kind according to claim 1, special
Sign is: the illumination cultivation condition be light intensity 1000-4000Lux, 18-30 DEG C of temperature, irradiation time 6-24h.
9. the Agrobacterium tumefaciens mediated transgenosis woaded blue regeneration plant method for transformation of one kind according to claim 1, special
Sign is: the resistance screening induced medium is MS or 1/2MS solid medium, wherein the aminoadenine of benzyl containing 6- (6-BA)
0.1-2.0mg/L, methyl α-naphthyl acetate (NAA) 0.1-2.0mg/L, cefalexin (Cef) 0-500mg/L, hygromycin (Hyg) 0-20mg/
L, PH 5.5-6.0.
10. the Agrobacterium tumefaciens mediated transgenosis woaded blue regeneration plant method for transformation of one kind according to claim 1, special
Sign is: it is described take root, the culture medium in strong sprout step be respectively as follows: root media be MS or 1/2MS solid medium, wherein
Containing methyl α-naphthyl acetate (NAA) 0.1-2.0mg/L, cefalexin (Cef) 0-500mg/L, hygromycin (Hyg) 0-20mg/L, PH 5.5-
6.0；Strong seedling culture base is MS or 1/2MS solid medium, wherein containing cefalexin (Cef) 0-500mg/L, hygromycin (Hyg)
0-20mg/L, PH 5.5-6.0.
11. the Agrobacterium tumefaciens mediated transgenosis woaded blue regeneration plant method for transformation of one kind according to claim 1, special
Sign is: the qualification program of the positive transgenic seedling is that the woaded blue of strong seedling culture in step is taken to regenerate seedling leaf, using CTAB
Method extract total DNA, using gained total DNA be template identify whether the resistance gene fragment hpt containing over-express vector PHB-Flag
And target gene fragment.
12. the Agrobacterium tumefaciens mediated transgenosis woaded blue regeneration plant method for transformation of one kind according to claim 11,
Be characterized in that: the qualification program of the positive transgenic seedling is specially that the woaded blue of strong seedling culture in step is taken to regenerate seedling leaf, is led to
The total DNA that CTAB method extracts positive transgenic seedling leaf is crossed, whether contains overexpression through PCR identification using gained total DNA as template
The resistance gene fragment hpt and target gene fragment of carrier PHB-Flag.
13. the Agrobacterium tumefaciens mediated transgenosis woaded blue regeneration plant method for transformation of one kind according to claim 1, special
Sign is: the hardening condition of culture is as follows: the volume ratio of each component is Nutrition Soil: pumice: sandy soil=2:1:1, light intensity 1000-
4000Lux, 18-30 DEG C of temperature, irradiation time 6-24h.
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