CN109234309A - A kind of tobacco Hongda tobacco method for transformation that agrobacterium rhizogenes mediates - Google Patents
A kind of tobacco Hongda tobacco method for transformation that agrobacterium rhizogenes mediates Download PDFInfo
- Publication number
- CN109234309A CN109234309A CN201811334235.9A CN201811334235A CN109234309A CN 109234309 A CN109234309 A CN 109234309A CN 201811334235 A CN201811334235 A CN 201811334235A CN 109234309 A CN109234309 A CN 109234309A
- Authority
- CN
- China
- Prior art keywords
- tobacco
- culture
- agrobacterium rhizogenes
- transformation
- hongda
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
- C12N15/8202—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation by biological means, e.g. cell mediated or natural vector
- C12N15/8205—Agrobacterium mediated transformation
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
- C12N15/8206—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation by physical or chemical, i.e. non-biological, means, e.g. electroporation, PEG mediated
- C12N15/8207—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation by physical or chemical, i.e. non-biological, means, e.g. electroporation, PEG mediated by mechanical means, e.g. microinjection, particle bombardment, silicon whiskers
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Genetics & Genomics (AREA)
- Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Molecular Biology (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Microbiology (AREA)
- Plant Pathology (AREA)
- Biophysics (AREA)
- Cell Biology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Physics & Mathematics (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
The present invention relates to the tobacco Hongda tobacco method for transformation that a kind of agrobacterium rhizogenes mediates, and belong to plant genetic engineering field.This method comprises the following steps: the disinfection of tobacco Hongda tobacco seed the culture of tobacco seedling plant, the preparation of tobacco leaf disc preculture, the bacterium solution of agrobacterium rhizogenes, is infected and is co-cultured, Hairy root culture, Hairy root callus tissue culture, Bud Differentiation culture and regeneration plant culture of rootage.The Transformation of tobacco technology that the present invention is mediated by agrobacterium rhizogenes, establishes stabilization regenerating system of the agrobacterium rhizogenes in the conversion of tobacco Hongda tobacco, and this method can make transformation tissue culture plant have higher rooting rate and higher transformation efficiency simultaneously.
Description
Technical field
The present invention relates to a kind of tobacco Hongda tobacco method for transformation, and in particular to a kind of tobacco that agrobacterium rhizogenes mediates
Hongda tobacco method for transformation, belongs to plant genetic engineering field.
Background technique
Agrobacterium rhizogenes (Agrobacterium rihzogenes) some Plantlet formations can be induced under given conditions
The phenomenon that Hairy root.Agrobacterium rhizogenes can convert infects plant generate Hairy root, be to agrobacterium rhizogenes truly
Further investigation, on infecting position or surrounding can induce and generate many by-products --- Hairy root.Agrobacterium rhizogenes can invade
Contaminate most of dicotyledon, a small amount of monocotyledon and individual special gymnosperms.Currently, nearly 20 in dicotyledon
Most of plant, which can use agrobacterium rhizogenes and convert, in 40 kinds of a section infects generation Hairy root, and passes through plant regeneration
Method typical variation features are embodied in progeny plants.Ri plasmid is a kind of Large plasmid with infectivity, is located at hair
Outside root Agrobacterium chromosome.In laboratory conditions, agrobacterium rhizogenes can infect the explant wound artificially scratched, so that Ri
The rolB gene contained in T-DNA on plasmid is inserted into the specific gene of plant nucleolus, is borrowed in host cell
It helps the DNA replication dna of plant itself to complete itself duplication and expression, synthesizes specific albumen, so that plant be induced to generate hair-like
Root.
Tobacco Hongda tobacco, original name " Lu Mei city cigarette " are Lu Mei city of people's commune of Lu Mei city of south of road county group tobacco growers from " great Jin
It selects good strains in the field for seed and cultivates in member ", choose qualification test by Yunnan Province's flue-cured tobacco local varieties and save flue-cured tobacco cultivars and identify session discussing,
Approved by the higher authorities report, is named as " Hongda tobacco ".Since the kind has resistance strong, wide adaptability, the spies such as stable high yield
Property, therefore after popularization, planted area expands rapidly, and is the Main Cultivars of tobacco now.However, tobacco safflower is big at present
The rooting rate and transformation efficiency of gold dollar are not ideal enough.
Summary of the invention
In order to solve the above-mentioned technical problem, the purpose of the present invention is to provide the tobacco safflowers that a kind of agrobacterium rhizogenes mediates
Big gold dollar method for transformation.The scheme that the present invention solves technical problem is as follows:
A kind of tobacco Hongda tobacco method for transformation that agrobacterium rhizogenes mediates, characterized by the following steps:
Step (1), the disinfection of tobacco Hongda tobacco seed；
Step (2), the culture of tobacco seedling plant: the seed that step (1) obtains is inoculated into MS culture medium, so that the seed is sent out
Bud grows up to seedling；
Step (3) tobacco leaf disc preculture: obtains tobacco leaf disc from seedling obtained in step (2), tobacco leaf disc is placed in
Preculture is carried out in MS culture medium；
Step (4), the bacterium solution preparation of agrobacterium rhizogenes: being transferred to agrobacterium rhizogenes for the plasmid with editor's PDS genetic fragment, will
Agrobacterium rhizogenes after conversion is placed on the YEB solid medium containing corresponding antibiotic and cultivates, dark culturing 2-3d；Picking will
Containing there is the agrobacterium rhizogenes of purpose plasmid to carry out 28 DEG C of dark culturings in the YEB fluid nutrient medium containing corresponding antibiotic, so
Afterwards according to 1:50 ratio carry out liquid oscilaltion expand culture, wait cultivate to bacterial concentration be OD600=0.5-0.8 carries out thallus receipts
Collection finally utilizes MS fluid nutrient medium suspension thalline to OD600=0.6-0.8, to used in follow-up test；
Step (5), infects and co-cultures: the tobacco leaf disc of preculture 3d or so in step (3) being immersed in the bacterium solution of step (4)
10min or so takes out leaf dish and blots bacterium solution in sterilizing filter paper, and puts to co-cultivation 3d or so on MS culture medium；
Step (6) Hairy root culture: the leaf dish after co-culturing in step (5) is taken out, disinfection is placed on containing corresponding antibiotic
MS culture medium on carry out hairy root culture；
Step (7), Hairy root callus tissue culture: will cut in step (6) from the Hairy root that leaf dish edge is grown, be placed in containing
Differentiation callus group is carried out in the MS differential medium of 6-benzyladenine 6-BA, methyl α-naphthyl acetate NAA plant hormone and corresponding antibiotic
Knit culture；
Step (8), Bud Differentiation culture: cutting having the callus that Bud Differentiation is formed in step (7), is placed in containing corresponding anti-
It is cultivated on raw element MS culture medium, it is long to 2-4cm high to Bud Differentiation culture on callus；
Step (9), regeneration plant culture of rootage: Bud Differentiation in step (8) is cut, and is inserted into the MS culture containing corresponding antibiotic
Culture of rootage is carried out on base to get the tobacco Hongda tobacco regeneration plant of agrobacterium rhizogenes mediated transformation PDS gene is arrived.
Further, in step (2), the condition of culture of seed are as follows: cultivation temperature be 25 ± 1 DEG C, intensity of illumination 30-50
μmol/(m2S), light application time cultivates 60d under conditions of being 16h/d.
Further, in step (4), the PDS genetic fragment, nucleotide sequence is as shown in SEQ ID NO .1；
The plasmid, nucleotide sequence is as shown in SEQ ID NO.2.
Further, in step (4), agrobacterium rhizogenes MSU440, Ar.Qual, K599 or ATCC15834, plasmid turn
The method for entering agrobacterium rhizogenes is electroporated or chemical conversion.
Further, electroporated to be specifically in step (4):
Agrobacterium rhizogenes competence is placed on ice, is melted to competence, the plasmid of 2-3 μ L mesh is added in competence, slightly
Mix, be immediately inserted into ice, by competence-plasmid mixture be moved quickly to electric shock cup in carry out it is electroporated, after the completion of electric shock
In quick insertion ice, 1mL antibiotic-free YEB liquid is added, is transferred in sterile EP tube, 28 DEG C of shaken cultivation 2-3h；6000rpm
It is centrifuged 1min and collects thallus, leave and take the mixing of 100 μ L or so supernatant.
Further, in step (4), chemical conversion is specifically:
K599 agrobacterium rhizogenes competence is placed on ice, is melted to competence；The plasmid of 2-3 μ L mesh is added in competence,
Quickly, acutely dial tube bottom mixing, after be placed in ice and stand 5min, 5min in liquid nitrogen, 37 DEG C of water-bath 5min, ice bath 5min；
Then it is taken out in ice bath and puts room temperature, the YEB liquid of 700 μ L antibiotic-frees is added, in 28 DEG C of shaken cultivation 2-3h；6000rpm from
Heart 1min collects thallus, leaves and takes the mixing of 100 μ L or so supernatant.
Further, in step (6), after leaf dish is taken out, with the sterile water wash containing 250mg/L carbenicillin,
Suck dry moisture in sterilizing filter paper is placed on the MS culture medium containing corresponding antibiotic and carries out hairy root culture, condition of culture are as follows: 28 DEG C
Illumination cultivation 16h/d, 30-50 μm of ol/ (m of intensity of illumination2S), 25 DEG C of dark culturings 8h/d, 14d.
Further, antibiotic is one in 500mg/L carbenicillin, 50mg/L kanamycins and 50mg/L streptomysin
Kind is a variety of, and the additive amount of carbenicillin, kanamycins and streptomysin is respectively 500mg/L, 50mg/L and 50mg/L.
Further, the condition of culture of step (7) are as follows: 28 DEG C of illumination cultivation 16h/d, 30-50 μm of ol/ of intensity of illumination
(m2S), 25 DEG C of dark culturings 8h/d, 14-21d, every 10d replacement culture medium；
The condition of culture of step (8) are as follows: 28 DEG C of illumination cultivation 16h/d, 30-50 μm of ol/ (m of intensity of illumination2S), 25 DEG C of dark
Cultivate 8h/d, 10d；
The condition of culture of step (9) are as follows: 28 DEG C of illumination cultivation 16h/d, 30-50 μm of ol/ (m of intensity of illumination2S), 25 DEG C of dark
Cultivate 8h/d, 7-10d.
Further, in step (7), the additive amount of 6-benzyladenine 6-BA and methyl α-naphthyl acetate NAA be respectively 2mg/L and
0.2mg/L。
Compared with prior art, the invention has the following beneficial effects:
1, the present invention is directed to tobacco bred Hongda tobacco, using agrobacterium rhizogenes, is turned by the tobacco that agrobacterium rhizogenes mediates
Change technology establishes stabilization regenerating system of the agrobacterium rhizogenes in the conversion of tobacco Hongda tobacco.
2, tradition mediates tobacco using Agrobacterium tumefaciems method, and regeneration plant rooting rate is below 85%.This method simultaneously can
So that transformation tissue culture plant has higher rooting rate and higher transformation efficiency, wherein leaf dish Rooting percent 14d and 21d distinguishes
For 20.37-46.15% and 22.22-76.92%, shoot regeneration frequency 44.38-64.33%, regeneration plant rooting rate is reachable
91.02-92.34%。
Specific embodiment
Below with reference to embodiment, the present invention is described in further detail.
It will be understood to those of skill in the art that the following example is merely to illustrate the present invention, and it should not be regarded as limiting this hair
Bright range.In the examples where no specific technique or condition is specified, described technology or conditions according to the literature in the art
Or it is carried out according to product description.Reagents or instruments used without specified manufacturer is that can be obtained by purchase
Conventional products.
Embodiment 1
The tobacco Hongda tobacco method for transformation that the agrobacterium rhizogenes of the present embodiment mediates, includes the following steps:
The disinfection of step (1) vegetable seeds: taking full tobacco seed to move on superclean bench, with 75% alcohol by plant species
Sub- immersion 30s, aseptic water washing 2 times, then use 1%AgNO310min is sterilized, sterile water soaking and washing 5 times, is inhaled with aseptic filter paper
It is inoculated with after dry vegetable seeds surface moisture, wherein sterile water is through autoclaved ultrapure water.
Step (2) vegetable seeds culture: the vegetable seeds of step (1) is inoculated into MS culture medium, is in cultivation temperature
25 ± 1 DEG C, 30-50 μm of ol/ (m of intensity of illumination2S), light application time is cultivates 60 days under conditions of 16 hours/day, seed hair
Bud grows up to explant seedling；The MS culture medium are as follows: the agar that MS minimal medium adds 30g/L sucrose to add 4g/L, culture medium
PH value be 5.7.
Step (3) tobacco leaf disc preculture: seedling obtained in step (2) is carried out using punch or scalpel
Acquisition tobacco leaf disc is cut, tobacco leaf disc is placed in MS culture medium and carries out preculture, preculture condition are as follows: 28 DEG C of dark culturings
3d, MS culture medium are MS minimal medium.
Prepared by the bacterium solution of step (4) agrobacterium rhizogenes MSU440: will be with editor's PDS gene using electroporated method
The plasmid of segment is transferred in MSU440 agrobacterium rhizogenes competence.Wherein, the nucleotide sequence of PDS genetic fragment such as SEQ ID
Shown in NO.1.Obtained plasmid pORE-Cas9 is transformed acquisition, nucleotide sequence such as SEQ ID NO.2 according to PBI121
It is shown.
Electroporated concrete operations are first to place MSU440 agrobacterium rhizogenes competence, electric revolving cup on ice, wait feel
Melted by state, electric revolving cup pre-cooling；Secondly the plasmid of 2-3 μ L mesh is added in competence, it is slight to mix, it is immediately inserted into ice, uses
Competence-plasmid mixture is moved quickly in electric shock cup by 200 μ L pipette tips, covers cup lid；Then start electroporation, setting ginseng
Number: the μ of C=25 F, PC=200 ohm, V=2400V take out electric shock cup from ice, and blotting paper dries electric shock cup outer bottom, quickly
It is put into electric turn trough, starting electric shock, after the completion of electric shock in quick insertion ice, 1mL antibiotic-free YEB liquid is added, is transferred to nothing
In bacterium EP pipe, 28 DEG C of shaken cultivation 2h.Finally, 6000rpm centrifugation 1min collects thallus, the mixing of 100 μ L or so supernatant is left and taken, after
Bacterium solution is coated on the YEB solid medium containing 50mg/L streptomysin and 50mg/L kanamycins, it is black under the conditions of 28 DEG C
Dark culture 2-3d.
Picking, which will contain, has the agrobacterium rhizogenes of purpose plasmid in the YEB containing 50mg/L streptomysin Yu 50mg/L kanamycins
In fluid nutrient medium, 28 DEG C of dark culturings are carried out, then liquid oscilaltion is carried out according to 1:50 ratio and expands culture, wait cultivate to bacterium
Liquid concentration is OD600=0.5, microorganism collection is carried out, MS fluid nutrient medium suspension thalline to OD is finally utilized600=0.6, to subsequent
Used in test.
The YEB culture medium is beef extract powder 5g/L, peptone 5g/L, sucrose 5g/L, yeast extract 1g/L, seven hydrations
Magnesium sulfate 0.5g/L, agar 15g/L, 121 DEG C of sterilizing 15min pour into 30mL culture medium in 100mm culture dish after sterilizing, cold
But spare after；MS fluid nutrient medium is that sucrose 30g/L is added in MS minimal medium, 5.7,121 DEG C of pH sterilizing 15min, cold
But spare after.
Step (5), infects and co-cultures, and the tobacco leaf disc of preculture 3d in step (3) is immersed step (4) containing purposeful
10min in the MSU440 agrobacterium rhizogenes suspension bacteria liquid of plasmid takes out leaf dish and blots bacterium solution in sterilizing filter paper, and puts to MS and train
It supports and is co-cultured on base, condition of culture are as follows: 28 DEG C, dark culturing 3d.
Step (6), Hairy root culture take out the leaf dish that 3d is co-cultured in step (5), green with 250mg/L carboxylic benzyl is contained
The sterile water wash of mycin, the suck dry moisture in sterilizing filter paper are placed in containing 500mg/L carbenicillin, that is mould for 50mg/L card
Hairy root culture, condition of culture are carried out on the MS culture medium of element and 50mg/L streptomysin are as follows: 28 DEG C of illumination cultivation 16h/d, illumination
30-50 μm of ol/ (m of intensity2S), 25 DEG C of dark culturings 8h/d, 14d.
Step (7), Hairy root callus tissue culture will be cut in step (6) from the Hairy root that leaf dish edge is grown, about
1-1.5cm long is placed in MS differential medium and carries out differentiation callus tissue culture, wherein MS differential medium is trained substantially in MS
It supports and adds 2.0mg/L 6-benzyladenine 6-BA, 0.2 mg/L methyl α-naphthyl acetate NAA plant hormone and 500mg/L carboxylic benzyl mould in base
Element, 50mg/L kanamycins and 50mg/L streptomysin.
Condition of culture are as follows: 28 DEG C of illumination cultivation 16h/d, 30-50 μm of ol/ (m of intensity of illumination2S), 25 DEG C of dark culturings
8h/d, 14-21d, every 10d replace culture medium.
Step (8), Bud Differentiation culture, by step (7) have Bud Differentiation formed callus cut, be placed in containing
It is cultivated on the MS culture medium of 500mg/L carbenicillin, 50mg/L kanamycins and 50mg/L streptomysin, to callus group
It is long to 2-4cm high, condition of culture to knit Bud Differentiation culture are as follows: 28 DEG C of illumination cultivation 16h/d, 30-50 μm of ol/ of intensity of illumination
(m2S), 25 DEG C of dark culturings 8h/d, 10d.
Step (9), regeneration plant culture of rootage cut Bud Differentiation in step (8), and insertion is green containing 500mg/L carboxylic benzyl
Culture of rootage, condition of culture are carried out on the MS culture medium of mycin, 50mg/L kanamycins and 50mg/L streptomysin are as follows: 28 DEG C of light
According to culture 16h/d, 30-50 μm of ol/ (m of intensity of illumination2S), 25 DEG C of dark culturings 8h/d, 7-10d are obtained through MSU440 root of hair
The tobacco Hongda tobacco regeneration plant of agrobacterium mediation converted PDS gene.
In the present embodiment, MSU440 agrobacterium rhizogenes infect leaf dish Rooting percent 14d and 21d be respectively 46.15% with
76.92%, shoot regeneration frequency 64.33%, regeneration plant rooting rate is up to 92.34%.
Embodiment 2
The tobacco Hongda tobacco method for transformation that the agrobacterium rhizogenes of the present embodiment mediates, includes the following steps:
Step (1)-(3) are same as Example 1；
Step (4): the bacterium solution preparation of agrobacterium rhizogenes Ar.Qual: will be with editor's PDS gene piece using electroporated method
The plasmid of section is transferred in Ar.Qual agrobacterium rhizogenes competence.
Electroporated concrete operations are, first by Ar.Qual agrobacterium rhizogenes competence, electric revolving cup place on ice, to
Competence is melted, electric revolving cup pre-cooling；Secondly the plasmid of 2-3 μ L mesh is added in competence, it is slight to mix, it is immediately inserted into ice,
Competence-plasmid mixture is moved quickly in electric shock cup with 200 μ L pipette tips, covers cup lid；Then start electroporation, setting ginseng
Number: the μ of C=25 F, PC=200 ohm, V=2400V take out electric shock cup from ice, and blotting paper dries electric shock cup outer bottom, quickly
It is put into electric turn trough, starting electric shock, after the completion of electric shock in quick insertion ice, 1mL antibiotic-free YEB liquid is added, is transferred to nothing
In bacterium EP pipe, 28 DEG C of shaken cultivation 2-3h.Finally, 6000rpm centrifugation 1min collects thallus, the mixing of 100 μ L or so supernatant is left and taken,
Bacterium solution is coated on the YEB solid medium containing 50mg/L streptomysin and 50mg/L kanamycins afterwards, under the conditions of 28 DEG C
Dark culturing 2-3d.Picking, which will contain, has the agrobacterium rhizogenes of purpose plasmid containing 50mg/L streptomysin and 50mg/L kanamycins
YEB fluid nutrient medium in carry out 28 DEG C of dark culturings, then according to 1:50 ratio carry out liquid oscilaltion expand culture, wait cultivate
It is OD to bacterial concentration600=0.5-0.8 carries out microorganism collection, finally utilizes MS fluid nutrient medium suspension thalline to OD600 =
0.6-0.8, to used in follow-up test.
Step (5), infects and co-cultures, and the tobacco leaf disc of preculture 3d in step (3) is immersed step (4) containing purposeful
10min in the Ar.Qual agrobacterium rhizogenes suspension bacteria liquid of plasmid takes out leaf dish and blots bacterium solution in sterilizing filter paper, and puts to MS
It is co-cultured on culture medium, condition of culture are as follows: 28 DEG C, dark culturing 3d.
Step (6)-(8) are same as Example 1；
Step (9), regeneration plant culture of rootage cut Bud Differentiation in step (8), and insertion contains 500mg/L carbenicillin,
Culture of rootage, condition of culture are carried out on the MS culture medium of 50mg/L kanamycins and 50mg/L streptomysin are as follows: 28 DEG C of illumination trainings
Support 16h/d, 30-50 μm of ol/ (m of intensity of illumination2S), 25 DEG C of dark culturings 8h/d, 7-10d are obtained through Ar.Qual root of hair agriculture
The tobacco Hongda tobacco regeneration plant of bacillus mediated transformation PDS gene.Remaining is same as Example 1.
In the present embodiment, Ar.Qual agrobacterium rhizogenes infect leaf dish Rooting percent 14d and 21d be respectively 22.97% with
37.84%, shoot regeneration frequency 53.42%, regeneration plant rooting rate is up to 91.67%.
Embodiment 3
The tobacco Hongda tobacco method for transformation that the agrobacterium rhizogenes of the present embodiment mediates, includes the following steps:
Step (1)-(3) are same as Example 1；
Step (4): the bacterium solution preparation of Agrobacterium rhyzogenesK599: will be with editor's PDS genetic fragment using the method for chemical conversion
Plasmid be transferred in K599 agrobacterium rhizogenes competence.
The concrete operations of chemical conversion are first to place on ice K599 agrobacterium rhizogenes competence, melt to competence；
Secondly the plasmid of 2-3 μ L mesh is added in competence, with quick-moving speed, acutely dials tube bottom and mixes, after be placed in ice and stand
5min, 5min, 37 DEG C of water-bath 5min, ice bath 5min in liquid nitrogen；Then it is taken out in ice bath and puts room temperature, 700 μ L antibiotic-frees are added
YEB liquid, in 28 DEG C of shaken cultivation 2-3h.Finally, 6000rpm centrifugation 1min collects thallus, it is mixed to leave and take 100 μ L or so supernatant
It is even, after bacterium solution is coated on the YEB solid medium containing 50mg/L streptomysin and 50mg/L kanamycins, in 28 DEG C of conditions
Lower dark culturing 2-3d.Picking is by containing there is the agrobacterium rhizogenes of purpose plasmid, containing 50mg/L streptomysin and 50mg/L card, that is mould
28 DEG C of dark culturings are carried out in the YEB fluid nutrient medium of element, are then carried out liquid oscilaltion according to 1:50 ratio and are expanded culture, wait train
Supporting to bacterial concentration is OD600=0.5-0.8 carries out microorganism collection, finally utilizes MS fluid nutrient medium suspension thalline to OD600 =
0.6-0.8, to used in follow-up test.
Step (5), infects and co-cultures, and the tobacco leaf disc of preculture 3d in step (3) is immersed step (4) containing purposeful
10min in the K599 agrobacterium rhizogenes suspension bacteria liquid of plasmid takes out leaf dish and blots bacterium solution in sterilizing filter paper, and puts to MS and cultivate
It is co-cultured on base, condition of culture are as follows: 28 DEG C, dark culturing 3d.
Step (6)-(8) are same as Example 1；
Step (9), regeneration plant culture of rootage cut Bud Differentiation in step (8), and insertion contains 500mg/L carbenicillin,
Culture of rootage, condition of culture are carried out on the MS culture medium of 50mg/L kanamycins and 50mg/L streptomysin are as follows: 28 DEG C of illumination trainings
Support 16h/d, 30-50 μm of ol/ (m of intensity of illumination2S), 25 DEG C of dark culturings 8h/d, 7-10d are obtained through K599 agrobacterium rhizogenes
The tobacco Hongda tobacco regeneration plant of mediated transformation PDS gene.Remaining is same as Example 1.
In the present embodiment, K599 agrobacterium rhizogenes infects leaf dish Rooting percent 7d and 14d is respectively 20.37% and 22.22%, plants
Strain regeneration rate is 49.85%, and regeneration plant rooting rate is up to 91.26%.
Embodiment 4
The tobacco Hongda tobacco method for transformation that the agrobacterium rhizogenes of the present embodiment mediates, includes the following steps:
Step (1)-(3) are same as Example 1；
Step (4): the bacterium solution preparation of agrobacterium rhizogenes ATCC15834: will be with editor's PDS gene using electroporated method
The plasmid of segment is transferred in ATCC15834 agrobacterium rhizogenes competence.Electroporated concrete operations are first will
ATCC15834 agrobacterium rhizogenes competence, electric revolving cup are placed on ice, are melted to competence, electric revolving cup pre-cooling；Secondly in competence
The middle plasmid that 2-3 μ L mesh is added, it is slight to mix, it is immediately inserted into ice, it is with 200 μ L pipette tips that competence-plasmid mixture is quick
It moves on in electric shock cup, covers cup lid；Then start electroporation, parameter: the μ of C=25 F, PC=200 ohm, V=2400V is set, it will be electric
It hits cup to take out from ice, blotting paper dries electric shock cup outer bottom, is quickly put into electric turn trough, starting electric shock, fast after the completion of electric shock
In speed insertion ice, 1mL antibiotic-free YEB liquid is added, is transferred in sterile EP tube, 28 DEG C of shaken cultivation 2-3h.Finally,
6000rpm is centrifuged 1min and collects thallus, leaves and takes the mixing of 100 μ L or so supernatant, after bacterium solution is coated on to that is mould containing 50mg/L card
On the YEB solid medium of element, dark culturing 2-3d under the conditions of 28 DEG C.Picking, which will contain, has the agrobacterium rhizogenes of purpose plasmid to exist
28 DEG C of dark culturings are carried out in YEB fluid nutrient medium containing 50mg/L kanamycins, then carry out liquid according to 1:50 ratio
Oscillation expand culture, wait cultivate to bacterial concentration be OD600=0.5-0.8 carries out microorganism collection, finally utilizes MS Liquid Culture
Base suspension thalline is to OD600=0.6-0.8, to used in follow-up test.
Step (5), infects and co-cultures: the tobacco leaf disc of preculture 3d in step (3) is immersed step (4) containing purposeful
10min in the ATCC15834 agrobacterium rhizogenes suspension bacteria liquid of plasmid, take out leaf dish bacterium solution is blotted in sterilizing filter paper, and put to
It is co-cultured on MS culture medium, condition of culture are as follows: 28 DEG C, dark culturing 3d.
Step (6), Hairy root culture: the leaf dish that 3d is co-cultured in step (5) is taken out, green with 250mg/L carboxylic benzyl is contained
The sterile water wash of mycin, the suck dry moisture in sterilizing filter paper are placed in containing 500mg/L carbenicillin, that is mould for 50mg/L card
Hairy root culture, condition of culture are carried out on the MS culture medium of element are as follows: 28 DEG C of illumination cultivation 16h/d, 30-50 μm of ol/ of intensity of illumination
(m2S), 25 DEG C of dark culturings 8h/d, 14d.
Step (7) Hairy root callus tissue culture: will be cut in step (6) from the Hairy root that leaf dish edge is grown, about 1-
1.5cm long is placed in containing 2.0mg/L 6-benzyladenine 6-BA, 0.2 mg/L methyl α-naphthyl acetate NAA plant hormone and 500mg/L carboxylic
Differentiation callus tissue culture, condition of culture are carried out in the MS differential medium of parasiticin and 50mg/L kanamycins are as follows: 28 DEG C
Illumination cultivation 16h/d, 30-50 μm of ol/ (m of intensity of illumination2S), 25 DEG C of dark culturings 8h/d, 14-21d, every 10d replacement culture
Base.
Step (8), Bud Differentiation culture: by step (7) have Bud Differentiation formed callus cut, be placed in containing
It is cultivated on 500mg/L carbenicillin and the MS culture medium of 50mg/L kanamycins, to Bud Differentiation culture on callus
It grows to 2-4cm high, condition of culture are as follows: 28 DEG C of illumination cultivation 16h/d, 30-50 μm of ol/ (m of intensity of illumination2S), 25 DEG C of dark trainings
Support 8h/d, 10d.
Step (9), regeneration plant culture of rootage: Bud Differentiation in step (8) is cut, and insertion is green containing 500mg/L carboxylic benzyl
Culture of rootage, condition of culture are carried out on the MS culture medium of mycin and 50mg/L kanamycins are as follows: 28 DEG C of illumination cultivation 16h/d, light
According to 30-50 μm of ol/ (m of intensity2S), 25 DEG C of dark culturings 8h/d, 7-10d, acquisition are mediated through ATCC15834 agrobacterium rhizogenes
Convert the tobacco Hongda tobacco regeneration plant of PDS gene.Remaining is same as Example 1.
In the present embodiment, ATCC15834 agrobacterium rhizogenes infect leaf dish Rooting percent 7d and 14d be respectively 56.72% with
64.18%, shoot regeneration frequency 44.38%, regeneration plant rooting rate is up to 91.02%.
As it can be seen that MSU440, Ar.Qual, K599 and ATCC15834 agrobacterium rhizogenes with editor's PDS gene convert cigarette
Regeneration plant rooting rate all with higher is obtained after the big gold dollar of safflower.With editor PDS gene MSU440 with
The agrobacterium rhizogenes of ATCC15834, leaf dish Rooting percent with higher.And MSU440 and Ar.Qual with editor's PDS gene
Agrobacterium rhizogenes, shoot regeneration frequency with higher, therefore, in test containing editor PDS gene plasmid four kinds of root of hair agricultures
In bacillus, the MSU440 agrobacterium rhizogenes containing editor's PDS gene plasmid is in editor's PDS genetic transformation tobacco Hongda tobacco
When, leaf dish Rooting percent, shoot regeneration frequency and rooting rate are all higher.
The above shows and describes the basic principles and main features of the present invention and the advantages of the present invention.The technology of the industry
Personnel are it should be appreciated that the present invention is not limited to the above embodiments, and the above embodiments and description only describe this
The principle of invention, without departing from the spirit and scope of the present invention, various changes and improvements may be made to the invention, these changes
Change and improvement all fall within the protetion scope of the claimed invention.The claimed scope of the invention by appended claims and its
Equivalent thereof.
Sequence table
<120>the tobacco Hongda tobacco method for transformation that a kind of agrobacterium rhizogenes mediates
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 23
<212> DNA
<213>artificial sequence
<400> 1
gctgcatgga aagatgatga tgg 23
<210> 2
<211> 12133
<212> DNA
<213>artificial sequence (pORE-Cas9)
<400> 2
gatcgttcaa acatttggca ataaagtttc ttaagattga atcctgttgc cggtcttgcg 60
atgattatca tataatttct gttgaattac gttaagcatg taataattaa catgtaatgc 120
atgacgttat ttatgagatg ggtttttatg attagagtcc cgcaattata catttaatac 180
gcgatagaaa acaaaatata gcgcgcaaac taggataaat tatcgcgcgc ggtgtcatct 240
atgttactag atccctaggg aagttcctat tccgaagttc ctattctctg aaaagtatag 300
gaacttcttt gcgtattggg cgctcttggc ctttttggcc accggtcgta cggttaaaac 360
caccccagta cattaaaaac gtccgcaatg tgttattaag ttgtctaagc gtcaatttgt 420
ttacaccaca atatatcctg ccaccagcca gccaacagct ccccgaccgg cagctcggca 480
caaaatcacc actcgataca ggcagcccat cagtccacta gacgctcacc gggctggttg 540
ccctcgccgc tgggctggcg gccgtctatg gccctgcaaa cgcgccagaa acgccgtcga 600
agccgtgtgc gagacaccgc agccgccggc gttgtggata cctcgcggaa aacttggccc 660
tcactgacag atgaggggcg gacgttgaca cttgaggggc cgactcaccc ggcgcggcgt 720
tgacagatga ggggcaggct cgatttcggc cggcgacgtg gagctggcca gcctcgcaaa 780
tcggcgaaaa cgcctgattt tacgcgagtt tcccacagat gatgtggaca agcctgggga 840
taagtgccct gcggtattga cacttgaggg gcgcgactac tgacagatga ggggcgcgat 900
ccttgacact tgaggggcag agtgctgaca gatgaggggc gcacctattg acatttgagg 960
ggctgtccac aggcagaaaa tccagcattt gcaagggttt ccgcccgttt ttcggccacc 1020
gctaacctgt cttttaacct gcttttaaac caatatttat aaaccttgtt tttaaccagg 1080
gctgcgccct gtgcgcgtga ccgcgcacgc cgaagggggg tgccccccct tctcgaaccc 1140
tcccggcccg ctctcgcgtt ggcagcatca cccataattg tggtttcaaa atcggctccg 1200
tcgatactat gttatacgcc aactttgaaa acaactttga aaaagctgtt ttctggtatt 1260
taaggtttta gaatgcaagg aacagtgaat tggagttcgt cttgttataa ttagcttctt 1320
ggggtatctt taaatactgt agaaaagagg aaggaaataa taaatggcta aaatgagaat 1380
atcaccggaa ttgaaaaaac tgatcgaaaa ataccgctgc gtaaaagata cggaaggaat 1440
gtctcctgct aaggtatata agctggtggg agaaaatgaa aacctatatt taaaaatgac 1500
ggacagccgg tataaaggga ccacctatga tgtggaacgg gaaaaggaca tgatgctatg 1560
gctggaagga aagctgcctg ttccaaaggt cctgcacttt gaacggcatg atggctggag 1620
caatctgctc atgagtgagg ccgatggcgt cctttgctcg gaagagtatg aagatgaaca 1680
aagccctgaa aagattatcg agctgtatgc ggagtgcatc aggctctttc actccatcga 1740
catatcggat tgtccctata cgaatagctt agacagccgc ttagccgaat tggattactt 1800
actgaataac gatctggccg atgtggattg cgaaaactgg gaagaagaca ctccatttaa 1860
agatccgcgc gagctgtatg attttttaaa gacggaaaag cccgaagagg aacttgtctt 1920
ttcccacggc gacctgggag acagcaacat ctttgtgaaa gatggcaaag taagtggctt 1980
tattgatctt gggagaagcg gcagggcgga caagtggtat gacattgcct tctgcgtccg 2040
gtcgatca 2048
Sequence table
<120>the tobacco Hongda tobacco method for transformation that a kind of agrobacterium rhizogenes mediates
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 23
<212> DNA
<213>artificial sequence ()
<400> 1
gctgcatgga aagatgatga tgg 23
<210> 2
<211> 12133
<212> DNA
<213>artificial sequence (pORE-Cas9)
<400> 2
gatcgttcaa acatttggca ataaagtttc ttaagattga atcctgttgc cggtcttgcg 60
atgattatca tataatttct gttgaattac gttaagcatg taataattaa catgtaatgc 120
atgacgttat ttatgagatg ggtttttatg attagagtcc cgcaattata catttaatac 180
gcgatagaaa acaaaatata gcgcgcaaac taggataaat tatcgcgcgc ggtgtcatct 240
atgttactag atccctaggg aagttcctat tccgaagttc ctattctctg aaaagtatag 300
gaacttcttt gcgtattggg cgctcttggc ctttttggcc accggtcgta cggttaaaac 360
caccccagta cattaaaaac gtccgcaatg tgttattaag ttgtctaagc gtcaatttgt 420
ttacaccaca atatatcctg ccaccagcca gccaacagct ccccgaccgg cagctcggca 480
caaaatcacc actcgataca ggcagcccat cagtccacta gacgctcacc gggctggttg 540
ccctcgccgc tgggctggcg gccgtctatg gccctgcaaa cgcgccagaa acgccgtcga 600
agccgtgtgc gagacaccgc agccgccggc gttgtggata cctcgcggaa aacttggccc 660
tcactgacag atgaggggcg gacgttgaca cttgaggggc cgactcaccc ggcgcggcgt 720
tgacagatga ggggcaggct cgatttcggc cggcgacgtg gagctggcca gcctcgcaaa 780
tcggcgaaaa cgcctgattt tacgcgagtt tcccacagat gatgtggaca agcctgggga 840
taagtgccct gcggtattga cacttgaggg gcgcgactac tgacagatga ggggcgcgat 900
ccttgacact tgaggggcag agtgctgaca gatgaggggc gcacctattg acatttgagg 960
ggctgtccac aggcagaaaa tccagcattt gcaagggttt ccgcccgttt ttcggccacc 1020
gctaacctgt cttttaacct gcttttaaac caatatttat aaaccttgtt tttaaccagg 1080
gctgcgccct gtgcgcgtga ccgcgcacgc cgaagggggg tgccccccct tctcgaaccc 1140
tcccggcccg ctctcgcgtt ggcagcatca cccataattg tggtttcaaa atcggctccg 1200
tcgatactat gttatacgcc aactttgaaa acaactttga aaaagctgtt ttctggtatt 1260
taaggtttta gaatgcaagg aacagtgaat tggagttcgt cttgttataa ttagcttctt 1320
ggggtatctt taaatactgt agaaaagagg aaggaaataa taaatggcta aaatgagaat 1380
atcaccggaa ttgaaaaaac tgatcgaaaa ataccgctgc gtaaaagata cggaaggaat 1440
gtctcctgct aaggtatata agctggtggg agaaaatgaa aacctatatt taaaaatgac 1500
ggacagccgg tataaaggga ccacctatga tgtggaacgg gaaaaggaca tgatgctatg 1560
gctggaagga aagctgcctg ttccaaaggt cctgcacttt gaacggcatg atggctggag 1620
caatctgctc atgagtgagg ccgatggcgt cctttgctcg gaagagtatg aagatgaaca 1680
aagccctgaa aagattatcg agctgtatgc ggagtgcatc aggctctttc actccatcga 1740
catatcggat tgtccctata cgaatagctt agacagccgc ttagccgaat tggattactt 1800
actgaataac gatctggccg atgtggattg cgaaaactgg gaagaagaca ctccatttaa 1860
agatccgcgc gagctgtatg attttttaaa gacggaaaag cccgaagagg aacttgtctt 1920
ttcccacggc gacctgggag acagcaacat ctttgtgaaa gatggcaaag taagtggctt 1980
tattgatctt gggagaagcg gcagggcgga caagtggtat gacattgcct tctgcgtccg 2040
gtcgatca 2048
Claims (10)
1. the tobacco Hongda tobacco method for transformation that a kind of agrobacterium rhizogenes mediates, characterized by the following steps:
Step (1), the disinfection of tobacco Hongda tobacco seed；
Step (2), the culture of tobacco seedling plant: the seed that step (1) obtains is inoculated into MS culture medium, so that the seed is sent out
Bud grows up to seedling；
Step (3) tobacco leaf disc preculture: obtains tobacco leaf disc from seedling obtained in step (2), tobacco leaf disc is placed in
Preculture is carried out in MS culture medium；
Step (4), the bacterium solution preparation of agrobacterium rhizogenes: being transferred to agrobacterium rhizogenes for the plasmid with editor's PDS genetic fragment, will
Agrobacterium rhizogenes after conversion is placed on the YEB solid medium containing corresponding antibiotic and cultivates, dark culturing 2-3d；Picking will
Containing there is the agrobacterium rhizogenes of purpose plasmid to carry out 28 DEG C of dark culturings in the YEB fluid nutrient medium containing corresponding antibiotic, so
Afterwards according to 1:50 ratio carry out liquid oscilaltion expand culture, wait cultivate to bacterial concentration be OD600=0.5-0.8 carries out thallus receipts
Collection finally utilizes MS fluid nutrient medium suspension thalline to OD600=0.6-0.8, to used in follow-up test；
Step (5), infects and co-cultures: the tobacco leaf disc of preculture 3d or so in step (3) being immersed in the bacterium solution of step (4)
10min or so takes out leaf dish and blots bacterium solution in sterilizing filter paper, and puts to co-cultivation 3d or so on MS culture medium；
Step (6) Hairy root culture: the leaf dish after co-culturing in step (5) is taken out, disinfection is placed on containing corresponding antibiotic
MS culture medium on carry out hairy root culture；
Step (7), Hairy root callus tissue culture: will cut in step (6) from the Hairy root that leaf dish edge is grown, be placed in containing
Differentiation callus group is carried out in the MS differential medium of 6-benzyladenine 6-BA, methyl α-naphthyl acetate NAA plant hormone and corresponding antibiotic
Knit culture；
Step (8), Bud Differentiation culture: cutting having the callus that Bud Differentiation is formed in step (7), is placed in containing corresponding anti-
It is cultivated on raw element MS culture medium, it is long to 2-4cm high to Bud Differentiation culture on callus；
Step (9), regeneration plant culture of rootage: Bud Differentiation in step (8) is cut, and is inserted into the MS culture containing corresponding antibiotic
Culture of rootage is carried out on base to get the tobacco Hongda tobacco regeneration plant of agrobacterium rhizogenes mediated transformation PDS gene is arrived.
2. the tobacco Hongda tobacco method for transformation that agrobacterium rhizogenes according to claim 1 mediates, it is characterised in that: step
Suddenly in (2), the condition of culture of seed are as follows: cultivation temperature be 25 ± 1 DEG C, 30-50 μm of ol/ (m of intensity of illumination2S), when illumination
Between to cultivate 60d under conditions of 16h/d.
3. the tobacco Hongda tobacco method for transformation that agrobacterium rhizogenes according to claim 1 mediates, it is characterised in that: step
Suddenly in (4), the PDS genetic fragment, nucleotide sequence is as shown in SEQ ID NO.1；The plasmid, nucleotides sequence
Column are as shown in SEQ ID NO.2.
4. the tobacco Hongda tobacco method for transformation that agrobacterium rhizogenes according to claim 1 mediates, it is characterised in that: step
Suddenly in (4), agrobacterium rhizogenes MSU440, Ar.Qual, K599 or ATCC15834, the method that plasmid is transferred to agrobacterium rhizogenes is
Electroporated or chemical conversion.
5. the tobacco Hongda tobacco method for transformation that agrobacterium rhizogenes according to claim 4 mediates, it is characterised in that: step
Suddenly electroporated to be specifically in (4):
Agrobacterium rhizogenes competence is placed on ice, is melted to competence, the plasmid of 2-3 μ L mesh is added in competence, slightly
Mix, be immediately inserted into ice, by competence-plasmid mixture be moved quickly to electric shock cup in carry out it is electroporated, after the completion of electric shock
In quick insertion ice, 1mL antibiotic-free YEB liquid is added, is transferred in sterile EP tube, 28 DEG C of shaken cultivation 2-3h；6000rpm
It is centrifuged 1min and collects thallus, leave and take the mixing of 100 μ L or so supernatant.
6. the tobacco Hongda tobacco method for transformation that agrobacterium rhizogenes according to claim 4 mediates, it is characterised in that: step
Suddenly in (4), chemical conversion is specifically:
K599 agrobacterium rhizogenes competence is placed on ice, is melted to competence；The plasmid of 2-3 μ L mesh is added in competence,
Quickly, acutely dial tube bottom mixing, after be placed in ice and stand 5min, 5min in liquid nitrogen, 37 DEG C of water-bath 5min, ice bath 5min；
Then it is taken out in ice bath and puts room temperature, the YEB liquid of 700 μ L antibiotic-frees is added, in 28 DEG C of shaken cultivation 2-3h；6000rpm from
Heart 1min collects thallus, leaves and takes the mixing of 100 μ L or so supernatant.
7. the tobacco Hongda tobacco method for transformation that agrobacterium rhizogenes according to claim 1 mediates, it is characterised in that: step
Suddenly in (6), after leaf dish is taken out, with the sterile water wash containing 250mg/L carbenicillin, the suck dry moisture in sterilizing filter paper,
It is placed on the MS culture medium containing corresponding antibiotic and carries out hairy root culture, condition of culture are as follows: 28 DEG C of illumination cultivation 16h/d, illumination
30-50 μm of ol/ (m of intensity2S), 25 DEG C of dark culturings 8h/d, 14d.
8. the tobacco Hongda tobacco method for transformation that agrobacterium rhizogenes according to claim 1 mediates, it is characterised in that: anti-
Raw element is one of 500mg/L carbenicillin, 50mg/L kanamycins and 50mg/L streptomysin or a variety of, carboxylic benzyl mould
The additive amount of element, kanamycins and streptomysin is respectively 500mg/L, 50mg/L and 50mg/L.
9. the tobacco Hongda tobacco method for transformation that agrobacterium rhizogenes according to claim 1 mediates, it is characterised in that:
The condition of culture of step (7) are as follows: 28 DEG C of illumination cultivation 16h/d, 30-50 μm of ol/ (m of intensity of illumination2S), 25 DEG C of dark trainings
8h/d, 14-21d are supported, every 10d replaces culture medium；
The condition of culture of step (8) are as follows: 28 DEG C of illumination cultivation 16h/d, 30-50 μm of ol/ (m of intensity of illumination2S), 25 DEG C of dark trainings
Support 8h/d, 10d；
The condition of culture of step (9) are as follows: 28 DEG C of illumination cultivation 16h/d, 30-50 μm of ol/ (m of intensity of illumination2S), 25 DEG C of dark trainings
Support 8h/d, 7-10d.
10. the tobacco Hongda tobacco method for transformation that agrobacterium rhizogenes according to claim 1 mediates, it is characterised in that:
In step (7), the additive amount of 6-benzyladenine 6-BA and methyl α-naphthyl acetate NAA are respectively 2mg/L and 0.2mg/L.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811334235.9A CN109234309A (en) | 2018-11-09 | 2018-11-09 | A kind of tobacco Hongda tobacco method for transformation that agrobacterium rhizogenes mediates |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811334235.9A CN109234309A (en) | 2018-11-09 | 2018-11-09 | A kind of tobacco Hongda tobacco method for transformation that agrobacterium rhizogenes mediates |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN109234309A true CN109234309A (en) | 2019-01-18 |
Family
ID=65078045
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201811334235.9A Pending CN109234309A (en) | 2018-11-09 | 2018-11-09 | A kind of tobacco Hongda tobacco method for transformation that agrobacterium rhizogenes mediates |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN109234309A (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109371057A (en) * | 2018-11-05 | 2019-02-22 | 云南中烟工业有限责任公司 | A kind of method of tobacco high throughput genetic transformation |
| 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 |
| CN114085867A (en) * | 2021-11-22 | 2022-02-25 | 云南中烟工业有限责任公司 | Method for improving genetic transformation rate and tissue culture efficiency of tobacco gene editing |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103045609A (en) * | 2012-11-16 | 2013-04-17 | 云南省烟草农业科学研究院 | Complementary DNA (cDNA) sequence of tobacco NtFT1 genes and transient expression thereof for inducing tobacco early blossoming |
-
2018
- 2018-11-09 CN CN201811334235.9A patent/CN109234309A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103045609A (en) * | 2012-11-16 | 2013-04-17 | 云南省烟草农业科学研究院 | Complementary DNA (cDNA) sequence of tobacco NtFT1 genes and transient expression thereof for inducing tobacco early blossoming |
Non-Patent Citations (2)
| Title |
|---|
| L. SPANO等: "Regeneration of Plants From Callus Cultures of Roots Induced by Agrobacterium rhizogenes on Tobacco", 《Z. PJLANZENPHYSIOL. BD. 》 * |
| 侯丽丽等: "烟草毛状根多倍体诱导及其植株再生", 《生物工程学报》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109371057A (en) * | 2018-11-05 | 2019-02-22 | 云南中烟工业有限责任公司 | A kind of method of tobacco high throughput genetic transformation |
| 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 |
| CN114085867A (en) * | 2021-11-22 | 2022-02-25 | 云南中烟工业有限责任公司 | Method for improving genetic transformation rate and tissue culture efficiency of tobacco gene editing |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| JP2952041B2 (en) | Improved method for AGROBACTERIUM-mediated transformation of cultured soybean cells | |
| AU611652B2 (en) | Process for the genetic modification of monocotyledonous plants | |
| CN101331227B (en) | Method for improving conversion efficiency | |
| Mysore et al. | Arabidopsis ecotypes and mutants that are recalcitrant to Agrobacterium root transformation are susceptible to germ‐line transformation | |
| CN109234309A (en) | A kind of tobacco Hongda tobacco method for transformation that agrobacterium rhizogenes mediates | |
| CN110295185A (en) | A method of citrus is improved to canker resistance based on CsWAKL08 overexpression | |
| KR19990076718A (en) | Growth and / or Screening Methods of Plant Raw Materials | |
| CN110257421A (en) | The construction method of cabbage type rape gene mutation body PTG8 a kind of and its application | |
| US5567599A (en) | Method for producing transformed chrysanthemum plants | |
| Li et al. | High frequency generation of fertile transgenic rice plants after PEG-mediated protoplast transformation | |
| CN104770294B (en) | A kind of protocorm based on the sprouting of iris seed is the breeding method of acceptor | |
| CN106244625A (en) | A kind of agriculture bacillus mediated tobacco seed genetic transforming method rapidly and efficiently | |
| CN1377420A (en) | Method of improving gene transfer efficiency into plant cells | |
| CN101831459B (en) | Method for acquiring transgene cotton | |
| OA10503A (en) | Genetic modification of plants | |
| CN108901844B (en) | Method for constructing lycoris genus genetic transformation system | |
| CN108588002B (en) | Method for obtaining embryogenic callus of millet for genetic transformation and genetic transformation | |
| CN110358791A (en) | Mung bean genetic transforming method | |
| CN105695504B (en) | A kind of method that mediated by agriculture bacillus infects the Mongolian willow of conversion | |
| JP2008259497A (en) | Method for creating transformant of domestic variety of soybean through agrobacterium and method for acquiring seed of current generation and progeny of transformant in short period of time | |
| CN114164229A (en) | Method for obtaining novel strawberry germplasm with high regeneration efficiency by using CRISPR/Cas9 gene editing vector and application | |
| US6646185B2 (en) | Genetic transformation method for zoysiagrass | |
| CN113025645A (en) | Method for obtaining gypsophila paniculata transgenic plant by taking callus as receptor | |
| CN105886527A (en) | Agrobacterium tumefaciens mediated transformation system for efficiently obtaining transgenic plants of paspalum vaginatum and application thereof | |
| CN100334218C (en) | Effectively producing transgene plant using auxin precursor |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |