CN106171981A - The preparation method of a kind of cross-pollinatd plant callus high frequency regeneration system and the application in genetic transformation thereof - Google Patents
The preparation method of a kind of cross-pollinatd plant callus high frequency regeneration system and the application in genetic transformation thereof Download PDFInfo
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- CN106171981A CN106171981A CN201610543125.8A CN201610543125A CN106171981A CN 106171981 A CN106171981 A CN 106171981A CN 201610543125 A CN201610543125 A CN 201610543125A CN 106171981 A CN106171981 A CN 106171981A
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/008—Methods for regeneration to complete plants
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/001—Culture apparatus for tissue culture
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- 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
Abstract
The invention discloses the preparation method of cross-pollinatd plant callus high frequency regeneration system and the application in genetic transformation thereof.The method that present invention also offers cross-pollinatd plant Regeneration in Vitro, the method be the young fringe with cross-pollinatd plant individual plant be outer implant, complete Regeneration in Vitro.It is demonstrated experimentally that utilize in-vitro regeneration method provided by the present invention can screen the cross-pollinatd plant callus high frequency regeneration system obtaining same gene type；By this high frequency regeneration system genetic transformation cross-pollinatd plant, advantage of both having: one is that each independent transformant belongs to homologous genes type, not only improve exogenous gene expression analysis in transgenic line, the most beneficially between transgenic line and parent control strain, carry out the comparative analysis of the aspects such as phenotype；Two is higher for obtaining the efficiency of transgenic line, and can preserve, and can directly utilize later.Therefore, method provided by the present invention has important using value.
Description
Technical field
The present invention relates to biological technical field, be specifically related to obtaining of a kind of cross-pollinatd plant callus high frequency regeneration system
Obtain method and the application in genetic transformation thereof.
Background technology
Wild barley (Hordeum brevisubulatum (Trin.) Link) is that grass family Hordeum is a kind of perennial facultative
The raw good forage of salt, is again short covered barley grass, is a kind of typical cross-pollinatd plant.It is mainly distributed on West Siberia, illiteracy
The provinces and regions such as Gu and China northeast, North China, Xinjiang, Tibet and Inner Mongol, are salinization and the constructive species on Alkalization Meadow grassland, can be at saliferous
Well growing on the soil of amount 0.6～1.0% (mass percent), its salt tolerance can match in excellence or beauty with Puccinellia tenuiflora etc., has weight
The application wanted and scientific research value.At present, wild barley salt tolerant molecule mechanism and resistant gene of salt biological function explore aspect research are less.
The Regeneration in Vitro of wild barley rarely has report, occasionally has been reported that the research having carried out genetic transformation, but transformation efficiency is relatively low.Cut-off mesh
Before, still do not set up stable wild barley genetic conversion system, significantly limit wild barley and turn base based on Regeneration in Vitro
Because of carrying out in a deep going way of breeding and gene function analysis.
Summary of the invention
The technical problem to be solved is to provide the cross-pollinatd plant callus height obtaining same gene type
Frequency regeneration is.
For solving above-mentioned technical problem, a kind of method that present invention firstly provides cross-pollinatd plant Regeneration in Vitro.
The method of cross-pollinatd plant Regeneration in Vitro provided by the present invention, is with the children of described cross-pollinatd plant individual plant
Fringe or the sections being cut into described children's fringe are outer implant, complete Regeneration in Vitro.
The young fringe of described cross-pollinatd plant is the young fringe being developed to 1.5～2.5cm (such as 1.5cm, 2cm or 2.5cm).
A length of the 0.2 of described sections～0.4cm (such as 0.2cm, 0.3cm or 0.4cm).
The genotype of the regrowth carrying out Regeneration in Vitro acquisition according to the method described above is same gene type.And cross-pollination is planted
Thing, the general resource material obtained or kind are mixing genotype.
The method of described cross-pollinatd plant Regeneration in Vitro can comprise the steps:
(1) described outer implant is inoculated in inducing culture to cultivate, it is thus achieved that callus；In described inducing culture
Containing 2～4mg/L 2,4-D, 30～40g/L sucrose and 0.4～0.6g/L caseinhydrolysate；
(2) callus that step (1) obtains is placed in subculture medium to cultivate, it is thus achieved that embryo callus；Institute
State containing 0.8～1.2mg/L 2 in subculture medium, 4-D, 30～40g/L sucrose and 0.4～0.6mg/L 6-BA；
(3) embryo callus that step (2) obtains is placed in division culture medium first to cultivate, it is thus achieved that differentiation Seedling；Point
Change in culture medium first containing 0.8～1.2mg/L 6-BA, 0.15～0.25mg/L zeatin and 30～40g/L sucrose；
(4) the differentiation Seedling that step (3) obtains is placed in root media first to cultivate, it is thus achieved that regrowth；Described take root
Containing 20～30g/L sucrose in culture medium first.
Described inducing culture concretely contains the 2 of 3mg/L, the sucrose of 4-D, 35g/L and the hydrolysis cheese egg of 0.5g/L
White MS solid medium.
Described subculture medium concretely contains the 2 of 1.0mg/L, the sucrose of 4-D, 35g/L and the 6-benzyl ammonia of 0.5mg/L
The MS solid medium of base adenine (6-Benzylaminopurine, 6-BA).
Described division culture medium first concretely contains the zeatin (Zeatin, ZT) of 6-BA, 0.2mg/L of 1.0mg/L
MS solid medium with the sucrose of 35g/L.
Described root media first concretely contains the 1/2MS solid medium of the sucrose of 25g/L.
In described step (1), the condition of described cultivation can be: 25 DEG C～28 DEG C (such as 25 DEG C, 26 DEG C, 27 DEG C or 28 DEG C),
Light culture 4～5 weeks (such as 4 weeks or 5 weeks).
In described step (2), the condition of described cultivation can be: 25 DEG C～28 DEG C (such as 25 DEG C, 26 DEG C, 27 DEG C or 28 DEG C),
Light culture 9～12 weeks (such as 9 weeks, 10 weeks, 11 weeks or 12 weeks)." callus step (1) obtained " can be Callus induction rate
The callus of the individual plant higher than more than 75%.
In described step (3), the condition of described cultivation can be: 25 DEG C～28 DEG C (such as 25 DEG C, 26 DEG C, 27 DEG C or 28 DEG C),
Alternation of light and darkness cultivates 5～8 weeks (such as 5 weeks, 6 weeks, 7 weeks or 8 weeks)." embryo callus step (2) obtained " can be embryo
The embryo callus of the wound healing rate individual plant higher than more than 70%.
In described step (4), the condition of described cultivation can be: 25 DEG C～28 DEG C (such as 25 DEG C, 26 DEG C, 27 DEG C or 28 DEG C),
Alternation of light and darkness cultivates 4～7 weeks (such as 4 weeks, 5 weeks, 6 weeks or 7 weeks)." differentiation Seedling step (3) obtained " can be that differentiation rate is higher than
The differentiation Seedling of the individual plant of more than 80%.
The application in cross-pollinatd plant genetic transformation of the method for any of the above-described described cross-pollinatd plant Regeneration in Vitro
Fall within protection scope of the present invention.
For solving above-mentioned technical problem, present invention also offers the acquisition of cross-pollinatd plant callus high frequency regeneration system
Method.
The preparation method of cross-pollinatd plant provided by the present invention callus high frequency regeneration system, it may include walk as follows
Rapid:
(1) it is outer implant with the young fringe of cross-pollinatd plant individual plant or the sections that is cut into described children's fringe, by described outer planting
Body is inoculated in described inducing culture and cultivates, it is thus achieved that callus；
(2), after completing step (1), take the Callus induction rate callus of individual plant higher than more than 75%, be placed in described in continue
Culture base is cultivated, it is thus achieved that embryo callus；
(3), after completing step (2), take the embryo callus of the embryo callus subculture rate individual plant higher than more than 70%, be placed in institute
State division culture medium first to cultivate, it is thus achieved that differentiation Seedling；
(4), after completing step (3), take the differentiation Seedling of the differentiation rate individual plant higher than more than 80%, be placed in described root culture
Ji Jia cultivates, it is thus achieved that regrowth.
In the preparation method of above-mentioned cross-pollinatd plant callus high frequency regeneration system, the young fringe of described cross-pollinatd plant
For being developed to the young fringe of 1.5～2.5cm (such as 1.5cm, 2cm or 2.5cm).A length of the 0.2 of described sections～0.4cm (as
0.2cm, 0.3cm or 0.4cm).Described high frequency regeneration system can be Callus induction rate higher than more than 75%, embryo callus subculture rate is higher than
The callus general name that the young fringe of more than the 70% cross-pollinatd plant individual plant being higher than more than 80% with differentiation rate is induced.
Preparation method according to above-mentioned cross-pollinatd plant callus high frequency regeneration system, it is thus achieved that cross-pollinatd plant more
Injured tissue high frequency regeneration system is same gene type.
Above, the computing formula of described Callus induction rate is: the outer implant number of Callus induction rate=generation callus/
Outer implant sum × 100% of inoculation.
Above, the computing formula of described embryo callus subculture rate is: embryo callus subculture rate=generation embryo callus number/inoculation
Outer implant sum × 100%.
Above, the computing formula of described differentiation rate is: the callus number of differentiation rate=differentiation and seedling emergence/be inoculated in differentiation
Callus number × 100% in culture medium.
For solving above-mentioned technical problem, present invention also offers the genetic transforming method of cross-pollinatd plant.
The genetic transforming method of cross-pollinatd plant provided by the present invention, comprises the steps:
(1) it is outer implant with the young fringe of cross-pollinatd plant individual plant or the sections that is cut into described children's fringe, by described outer planting
Body is inoculated in inducing culture and cultivates, it is thus achieved that callus；Containing 2～4mg/L 2 in described inducing culture, 4-D, 30
～40g/L sucrose and 0.4～0.6g/L caseinhydrolysate；
(2) after completing step (1), described callus is placed in subculture medium and cultivates, it is thus achieved that embryo callus subculture group
Knit；Containing 0.8～1.2mg/L 2 in described subculture medium, 4-D, 30～40g/L sucrose and 0.4～0.6mg/L6-BA；
(3), after completing step (2), described embryo callus is carried out genetic transformation.
Above-mentioned genetic transforming method, in described step (3), described " described embryo callus is carried out genetic transformation " depends on
Secondary comprise the steps:
(A) take recombinational agrobacterium, described embryo callus is infected；Described recombinational agrobacterium is by containing purpose
The recombinant expression carrier of gene imports and sets out what Agrobacterium obtained；
(4) after completing step (A), take described embryo callus, carry out drying and other treatment；
(5), after completing step (4), take described embryo callus, be placed in and co-culture base, co-culture；Described co-culture base
In containing 0.8～1.2mg/L 2,4-D, 0.4～0.6mg 6-BA, 30～40g/L sucrose and 40～60mg/L acetosyringones；
(6) after completing step (5), take described embryo callus, be sequentially placed into primary screening culture medium, postsearch screening training
Support and cultivate in base and three screening culture medium, it is thus achieved that resistant calli；Containing 2,4-D in described primary screening culture medium
0.8～1.2mg/L, sucrose 30～40g/L, Ticarcillin/Clavulanate Acid 120～180mg/L and hygromycin 15～25mg/L；Described postsearch screening
Containing 2 in culture medium, 4-D 0.8～1.2mg/L, sucrose 30～40g/L, Ticarcillin/Clavulanate Acid 120～180mg/L and hygromycin 20～
30mg/L；Containing 2 in described three screening culture medium, 4-D 0.8～1.2mg/L, sucrose 30～40g/L, Ticarcillin/Clavulanate Acid 120～
180mg/L and hygromycin 25～35mg/L；
(7) after completing step (6), take described resistant calli, be placed in division culture medium second and cultivate, it is thus achieved that differentiation
Seedling；In described division culture medium second containing 6-BA 0.8～1.2mg/L, zeatin 0.15～0.25mg/L and sucrose 30～40g,
Ticarcillin/Clavulanate Acid 120～180mg/L and hygromycin 15～25mg/L；
(8) after completing step (7), take described differentiation Seedling, be sequentially placed into root media second and root media third is cultivated,
Obtain transgenic seedling；Containing sucrose 20～30g/L, Ticarcillin/Clavulanate Acid 120～180mg/L and hygromycin 25 in described root media second
～35mg/L；In described root media third containing sucrose 20～30g/L, Ticarcillin/Clavulanate Acid 120～180mg/L and hygromycin 45～
55mg/L。
Described inducing culture concretely contains the 2 of 3mg/L, the sucrose of 4-D, 35g/L and the hydrolysis cheese egg of 0.5g/L
White MS solid medium.
Described subculture medium concretely contains the 2 of 1.0mg/L, the sucrose of 4-D, 35g/L and the 6-benzyl ammonia of 0.5mg/L
The MS solid medium of base adenine (6-Benzylaminopurine, 6-BA).
The described base that co-cultures can be to infecting addition agar in culture medium, is 6～8g/L to its concentration, the cultivation obtained
Base.
The described base that co-cultures can be to infecting addition agar in culture medium, is 7g/L to its concentration, the culture medium obtained.
The solute of culture medium is infected concretely: NH described in every 1L4NO3 1650mg、KNO3 1900mg、KH2PO4
170mg、MgSO4·7H2O 370mg、CaCl2·2H2O 440mg、FeSO4·7H2O 27.80mg、Na2EDTA 37.30mg、
MnSO4·4H2O 22.30mg、ZnSO4·7H2O 8.60mg、H3BO3 6.20mg、KI 0.83mg、Na2MOO4·
2H2O0.25mg、CuSO4·5H2O 0.025mg、COCl2·6H2O 0.025mg, inositol 100.00mg, VB10.1mg、
VB60.5mg, nicotinic acid 0.5mg, glycine 2.0mg, 2,4-D 1.0mg, 6-BA 0.5mg, sucrose 35g and acetosyringone
50mg, described in infect the solvent of culture medium be water, described in infect the pH value of culture medium be 5.1～5.3 (such as 5.1,5.2 or 5.3).
Described primary screening culture medium concretely contains the 2 of 1.0mg/L, the sucrose of 4-D, 35g/L, the spy of 150mg/L
The MS solid medium of the hygromycin of Mei Ting and 20mg/L.
Described postsearch screening culture medium concretely contains the 2 of 1.0mg/L, the sucrose of 4-D, 35g/L, the spy of 150mg/L
The MS solid medium of the hygromycin of Mei Ting and 25mg/L.
Described three screening culture medium concretely contain the 2 of 1.0mg/L, the sucrose of 4-D, 35g/L, the spy of 150mg/L
The MS solid medium of the hygromycin of Mei Ting and 30mg/L.
Described division culture medium second concretely contain ZT and 35g/L of 6-BA, 0.2mg/L of 1.0mg/L sucrose,
The MS solid medium of the Ticarcillin/Clavulanate Acid of 150mg/L and the hygromycin of 20mg/L.
The tide that described root media second concretely contains the sucrose of 25g/L, the Ticarcillin/Clavulanate Acid of 150mg/L and 30mg/L is mould
The 1/2MS solid medium of element.
Described root media third concretely contains the 1/2MS solid training of the sucrose of 25g/L and the hygromycin of 50mg/L
Support base.
In described step (1), the condition of described cultivation is: 25 DEG C～28 DEG C (such as 25 DEG C, 26 DEG C, 27 DEG C or 28 DEG C), secretly
Cultivate 4～5 weeks (such as 4 weeks or 5 weeks).The young fringe of described cross-pollinatd plant for being developed to 1.5～2.5cm (such as 1.5cm, 2cm or
Young fringe 2.5cm).A length of the 0.2 of described sections～0.4cm (such as 0.2cm, 0.3cm or 0.4cm).
In described step (2), the condition of described cultivation is: 25 DEG C～28 DEG C (such as 25 DEG C, 26 DEG C, 27 DEG C or 28 DEG C), secretly
Cultivate 9～12 weeks (such as 9 weeks, 10 weeks, 11 weeks or 12 weeks).
In described step (A), the method for described " infecting " can be as follows: drops in described embryo callus by infecting bacterium solution
Surface, then application of vacuum 5～15min (such as 5min, 10min or 15min)；The described bacterium solution that infects is that recombinational agrobacterium bacterium is hanged
Liquid.Described vacuum can be 0.4～0.6 atmospheric pressure.Described infect bacterium solution preparation method be: described recombinational agrobacterium is suspended
Culture medium is infected in described.The described OD infecting bacterium solution600nmValue is 0.4～0.6.
In described step (4), the method for described " drying and other treatment " can be as follows: described embryo callus is placed in aseptic bar
Desiccation culture under part.Described embryo callus concretely is moved to be covered with aseptic filter paper by the method for described " drying and other treatment "
In sterile petri dish, process one day at superclean bench.
In described step (5), the condition of described cultivation is: 25 DEG C～27 DEG C (such as 25 DEG C, 26 DEG C or 27 DEG C), light culture 3
～4 days (such as 3 days or 4 days).
In described step (6), the condition of described cultivation is: 25 DEG C～28 DEG C (such as 25 DEG C, 26 DEG C, 27 DEG C or 28 DEG C), light
Dark alternate culture 8～10 weeks (such as 8 weeks, 9 weeks or 10 weeks).
In described step (7), the condition of described cultivation is: 25 DEG C～28 DEG C (such as 25 DEG C, 26 DEG C, 27 DEG C or 28 DEG C), light
Dark alternate culture 6～8 weeks (such as 6 weeks, 7 weeks or 8 weeks).
In described step (8), the condition of described cultivation is: 25 DEG C～28 DEG C (such as 25 DEG C, 26 DEG C, 27 DEG C or 28 DEG C), light
Dark alternate culture 4～7 weeks (such as 4 weeks, 5 weeks, 6 weeks or 7 weeks).The transgenic seedling obtained by above-mentioned genetic transforming method, all with
The genetic transformation that same gene shaped material is carried out.
For solve above-mentioned technical problem, present invention also offers for cross-pollinatd plant Regeneration in Vitro test kit first or
Test kit second for cross-pollinatd plant genetic transformation.
Described test kit first contains described inducing culture and/or described subculture medium and/or described division culture medium
First and/or described root media first.
Described test kit second contain described inducing culture and/or described subculture medium and/or described in co-culture base and/
Or described primary screening culture medium and/or described postsearch screening culture medium and/or described three screening culture medium and/or described point
Change culture medium second and/or described root media second and/or described root media third.
Any of the above-described described cross-pollinatd plant can be unifacial leaf cross-pollinatd plant.Described unifacial leaf cross-pollinatd plant
Concretely wild barley.
For solving above-mentioned technical problem, present invention also offers the genetic transforming method of a kind of plant.
The genetic transforming method of plant provided by the present invention, including step (a1) or (a2):
(a1) bacterium solution will be infected and drop in plant callus surface；
(a2) the described bacterium solution that infects is dropped in plant callus surface, drying and other treatment.
The described bacterium solution that infects is recombinational agrobacterium bacteria suspension；Described recombinational agrobacterium is by by the restructuring containing genes of interest
Expression vector imports the Agrobacterium that sets out and obtains.
In the genetic transforming method of above-mentioned plant, described callus can be embryo callus.
In described step (a1), described " bacterium solution will be infected and drop in plant callus surface " also include application of vacuum 5～
The step (such as 5min, 10min or 15min) of 15min.Described vacuum can be 0.4～0.6 atmospheric pressure.
In described step (a2), the method for described " drying and other treatment " can be as follows: is placed in aseptic by described plant callus
Under the conditions of desiccation culture.Described plant callus concretely is moved to be covered with aseptic filter paper by the method for described " drying and other treatment "
Sterile petri dish in, superclean bench process one day.
In the genetic transforming method of above-mentioned plant, described plant can be cross-pollinatd plant.Described cross-pollinatd plant can
For unifacial leaf cross-pollinatd plant.Described unifacial leaf cross-pollinatd plant concretely wild barley.
Above, the preparation method of described cross-pollinatd plant children's fringe is: by cross-pollinatd plant planting seed in nutrition
Earth culture is supported, and then transplants land for growing field crops, and Second Year takes children's fringe from sheath.
Above, the described i.e. illumination cultivation of alternation of light and darkness cultivation and light culture are alternately.Intensity of illumination during illumination cultivation is
2000Lx.The cycle that alternation of light and darkness is cultivated is particularly as follows: 16 hours illumination cultivation/8 hour dark culturing.
Above, the preparation method of described MS solid medium can be: by NH4NO3 1650mg、KNO3 1900mg、
KH2PO4 170mg、MgSO4·7H2O 370mg、CaCl2·2H2O 440mg、FeSO4·7H2O 27.80mg、
Na2EDTA37.30mg、MnSO4·4H2O 22.30mg、ZnSO4·7H2O 8.60mg、H3BO3 6.20mg、KI 0.83mg、
Na2MOO4·2H2O 0.25mg、CuSO4·5H2O 0.025mg、COCl2·6H2O 0.025mg, inositol 100.00mg,
VB10.1mg、VB60.5mg, nicotinic acid 0.5mg, glycine 2.0mg and agar 7g are dissolved in 1L distilled water, regulate pH to 5.8.
Above, the preparation method of described 1/2MS solid medium can be: by NH4NO3 825mg、KNO3 950mg、
KH2PO4 85mg、MgSO4·7H2O 185mg、CaCl2·2H2O 220mg、FeSO4·7H2O 13.90mg、
Na2EDTA18.65mg、MnSO4·4H2O 11.15mg、ZnSO4·7H2O 4.30mg、H3BO3 3.10mg、KI 0.415mg、
Na2MOO4·2H2O 0.125mg、CuSO4·5H2O 0.0125mg、COCl2·6H2O 0.0125mg, inositol 50.00mg,
VB10.05mg、VB60.25mg, nicotinic acid 0.25mg, glycine 1.0mg and agar 7g are dissolved in 1L distilled water, regulate pH to 5.8.
Cross-pollinatd plant, the general resource material obtained or kind are mixing genotype.And it is demonstrated experimentally that the present invention
The genotype of the regrowth that the cross-pollinatd plant in-vitro regeneration method that provided obtains is same gene type, utilizes the method can
Screening obtains the cross-pollinatd plant callus high frequency regeneration system of same gene type；Different by this high frequency regeneration system genetic transformation
Flower pollinated plant, has following both sides advantage: one is that each independent transformant belongs to homologous genes type, outside not only improving
Source gene expression analysis in transgenic line, the most beneficially carries out the sides such as phenotype between transgenic line and parent control strain
The comparative analysis in face is the most necessary for cross-pollinatd plant；Two is higher for obtaining the efficiency of transgenic line, and this high frequency is again
Raw system is not only the guarantee of high conversion, and can preserve, and can directly utilize later, save the loaded down with trivial details journey of screening-gene type
Sequence, saves man power and material.Therefore, method provided by the present invention has important using value.
Accompanying drawing explanation
Fig. 1 is the process of Regeneration in Vitro.
Fig. 2 is Molecular Detection result.
Fig. 3 is GUS staining analysis result.
Detailed description of the invention
Being further described in detail the present invention below in conjunction with detailed description of the invention, the embodiment be given is only for explaining
The bright present invention rather than in order to limit the scope of the present invention.
Experimental technique in following embodiment, if no special instructions, is conventional method.
Material used in following embodiment, reagent etc., if no special instructions, the most commercially obtain.
Culture medium used in following embodiment is as follows:
MS solid medium: by NH4NO3 1650mg、KNO3 1900mg、KH2PO4 170mg、MgSO4·7H2O
370mg、CaCl2·2H2O 440mg、FeSO4·7H2O 27.80mg、Na2EDTA 37.30mg、MnSO4·4H2O
22.30mg、ZnSO4·7H2O 8.60mg、H3BO3 6.20mg、KI 0.83mg、Na2MOO4·2H2O 0.25mg、CuSO4·
5H2O0.025mg、COCl2·6H2O 0.025mg, inositol 100.00mg, VB10.1mg、VB60.5mg, nicotinic acid 0.5mg, glycine
2.0mg and agar 7g is dissolved in 1L distilled water, regulates pH to 5.8.
1/2MS solid medium: by NH4NO3 825mg、KNO3 950mg、KH2PO4 85mg、MgSO4·7H2O
185mg、CaCl2·2H2O 220mg、FeSO4·7H2O 13.90mg、Na2EDTA 18.65mg、MnSO4·4H2O
11.15mg、ZnSO4·7H2O 4.30mg、H3BO3 3.10mg、KI 0.415mg、Na2MOO4·2H2O 0.125mg、CuSO4·
5H2O 0.0125mg、COCl2·6H2O 0.0125mg, inositol 50.00mg, VB10.05mg、VB60.25mg, nicotinic acid 0.25mg,
Glycine 1.0mg and agar 7g is dissolved in 1L distilled water, regulates pH to 5.8.
Inducing culture: containing the 2 of 3mg/L, the MS solid training of the sucrose of 4-D, 35g/L and the caseinhydrolysate of 0.5g/L
Support base.
Subculture medium: containing the 2 of 1.0mg/L, the sucrose of 4-D, 35g/L and the 6-benzyl aminoadenine (6-of 0.5mg/L
Benzylaminopurine, 6-BA) MS solid medium.
Division culture medium first: the zeatin (Zeatin, ZT) of 6-BA, 0.2mg/L containing 1.0mg/L and the sugarcane of 35g/L
The MS solid medium of sugar.
Root media first: the 1/2MS solid medium of the sucrose containing 25g/L.
LB resistance culture base: by 10g tryptone, 5g yeast extract, 5g NaCl, 1g D-Glucose, 30mg rifampicin and
100mg kanamycin is dissolved in 1L distilled water, and regulation pH value is to 7.0.
Infect culture medium: by NH4NO3 1650mg、KNO3 1900mg、KH2PO4 170mg、MgSO4·7H2O 370mg、
CaCl2·2H2O 440mg、FeSO4·7H2O 27.80mg、Na2EDTA 37.30mg、MnSO4·4H2O 22.30mg、
ZnSO4·7H2O 8.60mg、H3BO3 6.20mg、KI 0.83mg、Na2MOO4·2H2O 0.25mg、CuSO4·
5H2O0.025mg、COCl2·6H2O 0.025mg, inositol 100.00mg, VB10.1mg、VB60.5mg, nicotinic acid 0.5mg, glycine
2.0mg, 2,4-D 1.0mg, 6-BA 0.5mg, sucrose 35g and acetosyringone (Acetosyringone, As) 50mg are dissolved in 1L
Distilled water, regulates pH to 5.2.
Co-culture base: to infecting addition agar in culture medium, be 7g/L to its concentration, the culture medium obtained.
Primary screening culture medium: containing the 2 of 1.0mg/L, the sucrose of 4-D, 35g/L, the Ticarcillin/Clavulanate Acid of 150mg/L and 20mg/L
The MS solid medium of hygromycin.
Postsearch screening culture medium: containing the 2 of 1.0mg/L, the sucrose of 4-D, 35g/L, the Ticarcillin/Clavulanate Acid of 150mg/L and 25mg/L
The MS solid medium of hygromycin.
Three screening culture medium: containing the 2 of 1.0mg/L, the sucrose of 4-D, 35g/L, the Ticarcillin/Clavulanate Acid of 150mg/L and 30mg/L
The MS solid medium of hygromycin.
Division culture medium second: the sucrose of ZT and 35g/L of 6-BA, 0.2mg/L containing 1.0mg/L, the Te Mei of 150mg/L
The MS solid medium of the hygromycin of spit of fland and 20mg/L.
Root media second: the 1/2MS of the hygromycin of sucrose, the Ticarcillin/Clavulanate Acid of 150mg/L and 30mg/L containing 25g/L is solid
Body culture medium.
Root media third: the sucrose containing 25g/L and the 1/2MS solid medium of the hygromycin of 50mg/L.
Plasmid pTCK303 is recorded in as in Publication about Document: Wang Z., Chen C.B., Xy Y.Y., Jiang R.X., Han
Y., Xu Z.H., Chong K.A Practical Vector for Efficient Knockdown of Gene
Expression in Rice(Oryza sativa L.).2004.Plant Molecular Biology Reporter22
(4):409–417.
GUS reacts mixed liquor: solute and concentration thereof are 10mmol/L EDTA-Na2、0.5mmol/L K3Fe(CN)6、
0.5mmol/L K4Fe(CN)6·3H2O, 1mg/mL X-gluc and 0.1% (percent by volume) TritonX-100, solvent is for steaming
Distilled water；PH value is 7.0.
Embodiment 1, the preparation method of wild barley callus high frequency regeneration system of same gene type
Wild barley is cross-pollinatd plant, and the general resource material obtained or kind are mixing genotype.Due to difference
Tissue culture's regeneration capacity of genotype and genetic transformation efficiency are the most different, therefore provide the wild barley wound healing group of same gene type
Knit high frequency regeneration system to the tissue culture of wild barley or/and gene studies is most important.
The preparation method of the wild barley callus high frequency regeneration system of the same gene type that the present invention provides, is with wild barley
Individual plant children's fringe is outer implant.Wild barley is many tillers type grass, and an individual plant can extract multiple children out at heading stage simultaneously
Fringe, provides necessary material condition for obtaining the wild barley callus high frequency regeneration system of same gene type.
The preparation method of the wild barley callus high frequency regeneration system of same gene type is as follows:
One, the induction of callus line (hereinafter referred to as wound healing system)
1, choose full wild barley seed, be seeded in greenhouse the flowerpot filling Nutrition Soil, treat wild barley seedling length extremely
During two one heart stages of leaf, it is transplanted into scientific research and testing field, Beijing City Agriculture and Forestry Institute.Second Year after transplanting, fills after wild barley is turned green
Foot waters, and is numbered wild barley individual plant (see in Fig. 1 (a)), and it is (the i.e. material of same gene type that an individual plant is one
Material), one be all children's whole calluss of inducing of fringe be a wound healing system.When Young spike development to 1.5～2.5cm,
From wild barley base portion, stem is cut with shears, divest the sheath of outside, obtain children's fringe (see in Fig. 1 (b), for ensureing wild barley children
Fringe growth conditions is preferable, again waters before taking children's fringe).
2, after completing step 1, children's fringe is placed in superclean bench, first wipes with the ethanol water of 70% (percent by volume)
Wash surface, then scratch the sheath of inside with aseptic dissecting needle, rotate gently at children's fringe tuberosity, extract wrapped children's fringe out, use
This outer implant is cut into the sections of 0.2cm～0.4cm by aseptic nipper, be inoculated on inducing culture (during inoculation in units of being,
And number), 25 DEG C of light culture 5 weeks, obtain white and the callus of structure not consolidation.
Two, the subculture of wound healing system
After completing step one, in units of being, add up the Callus induction rate (outer planting of Callus induction rate=generation callus
Outer implant sum × 100% of body number/inoculation), by the callus inoculation of the Callus induction rate wound healing system higher than more than 75%
On subculture medium, 25 DEG C of light culture 12 weeks (changing the newest subculture medium every 3 weeks during subculture), obtain yellow and
The embryo callus (in Fig. 1 (c)) of structure consolidation and non embryogenic callus.
By scanning electron microscopic observation embryo callus, result shows, embryo callus surface has many undifferentiated
Embryogenic cell masses structure (in Fig. 1 (d)), and non embryogenic callus surface is without this structure.
Three, the differentiation of wound healing system
After completing step 2, in units of being add up embryo callus subculture rate (embryo callus subculture rate=generation embryo callus number/
Outer implant sum × 100% of inoculation), the embryo callus of the embryo callus subculture rate wound healing system higher than more than 70% is inoculated in
In division culture medium first, alternation of light and darkness cultivates 6 weeks (changing the newest division culture medium first during differentiation culture every 3 weeks),
To differentiation Seedling (in Fig. 1 (e)).Wherein alternation of light and darkness cultivation (i.e. illumination cultivation and light culture are alternately) condition is: 25 DEG C.Illumination is trained
Intensity of illumination when supporting is 2000Lx, and the cycle that alternation of light and darkness is cultivated is particularly as follows: 16 hours illumination cultivation/8 hour dark culturing.
Four, the taking root and the acquisition of regrowth of wound healing system
After completing step 2, being for unit statistical rate (the callus number of differentiation rate=differentiation and seedling emergence/be inoculated in
Callus number × 100% on division culture medium), proceed to take root by the differentiation Seedling of the differentiation rate wound healing system higher than more than 80%
Culture medium first carries out alternation of light and darkness cultivation, and when root length length is to more than 3cm, (incubation time about 4～7 weeks) completes regrowth
Take root, it is thus achieved that healthy and strong regrowth ((f), (g) and (h) in Fig. 1).Wherein alternation of light and darkness cultivates (i.e. illumination cultivation and light culture
Alternately) condition is: 25 DEG C.Intensity of illumination during illumination cultivation is 2000Lx, and the cycle that alternation of light and darkness is cultivated is particularly as follows: 16 hours
Illumination cultivation/8 hour dark culturing.
Five, transplant
Take step 4 and obtain healthy and strong regrowth, remove sealed membrane seedling exercising, then regrowth is taken out from triangular flask, wash
Go on root the root media first of attachment, be transplanted in the most autoclaved Vermiculitum and turfy soil 11 (volume ratio) substrate in
Cultivating (in Fig. 1 (i)) in greenhouse, early stage notices that moisturizing is sheltered from heat or light, and is gradually increased light transmission capacity after 3～5 days, can conventional manage after one week
Reason, seedling replanting survival rate more than 90%.
Through above-mentioned steps, it is thus achieved that (it is high that high frequency regeneration wound healing system refers to Callus induction rate for 3 high frequency regeneration wound healing systems
In more than 75%, embryo callus subculture rate is higher than more than 70% with the wound healing group that induced of differentiation rate individual plant children's fringe more than 80%
Knit general name), it is respectively designated as wound healing system S1, wound healing system S2 and wound healing system S3, in units of being, adds up 3 high frequency regeneration wound healing systems
Occurrence frequency, result occurrence frequency is 10% (the individual plant number of occurrence frequency=high frequency regeneration wound healing coefficient/inoculation).
Embodiment 2, use the wild barley callus high frequency regeneration system of the same gene type of embodiment 1 to set up wild barley to lose
Pass method for transformation
One, embryo callus is obtained
1, take the young fringe of the wild barley of wound healing system S1, divest outside sheath and be placed in superclean bench, first with 70% (volume
Percentage ratio) ethanol water clean surface, then scratch the tender sheath of children with aseptic dissecting needle, rotate children's fringe tuberosity gently
Place, extracts wrapped children's fringe out and as outer implant, this outer implant is cut into aseptic nipper the sections of 0.2cm～0.4cm, connects
Kind on inducing culture, 25 DEG C of light culture 4 weeks, obtain white and the callus of structure not consolidation.
2, after completing step 1, the callus of white and structure not consolidation is inoculated on subculture medium, 25 DEG C of dark trainings
Support 9 weeks (during subculture, changing the newest subculture medium every 3 weeks), obtain yellow and the embryo callus of structure consolidation.
Two, Agrobacterium infects the preparation of liquid
1, using electric shocking method that plasmid pTCK303 is converted Agrobacterium tumefaciems GV3101, picking recombinational agrobacterium, by this restructuring
The named GV3101/pTCK303 of Agrobacterium.
2, GV3101/pTCK303 monoclonal step 1 obtained is inoculated into LB resistance culture base, 28 DEG C, 160rpm vibration
Cultivate 12h, obtain cultivating bacterium solution.By this cultivation bacterium solution 5000rpm, centrifugal 10min, the thalline of collection is with infecting cultivation basic weight
Outstanding, obtain Agrobacterium and infect liquid.To infect the OD of culture medium600nmFor reference, adjust Agrobacterium and infect the OD of liquid600nmValue is to 0.4
～0.6.
Three, embryo callus is infected
Embryo callus is transferred to be covered with the sterile petri dish of aseptic filter paper, then infects liquid with Agrobacterium and carry out dripping
Dye converts, and to ensure that every piece of embryo callus is infected immersion dye by Agrobacterium, but is not soaked in Agrobacterium and infects in liquid.Drip
The operating procedure that dye converts particularly as follows: infect drop surface (the unnecessary agriculture at embryo callus with pipettor by Agrobacterium
Bacillus is infected liquid and is i.e. sopped up by the aseptic filter paper being layered in sterile petri dish), then evacuation (0.4～0.6 atmospheric pressure) place
Reason 10min.
Test in triplicate, each superinfection embryo callus at least 30.
Four, drying and other treatment
After completing step 3, move to be covered with in the sterile petri dish of aseptic filter paper by embryo callus, drying and other treatment 1d
(purpose is to make later stage callus occur without water stainization and browning phenomenon).
Five, co-culture
After completing step 4, embryo callus is placed in and is covered with co-culturing of one layer of aseptic filter paper and on base, co-cultures 3 days.
Co-culture condition: 25～27 DEG C, light culture.
Six, screening and culturing
1, after completing step 5, first for embryo callus aquesterilisa is fully washed and (i.e. cleans the sterilizing to washing
Water is limpid), then with the solution washing 1 time of the Ticarcillin/Clavulanate Acid containing 200mg/L and 0.02% (percent by volume) Tween20,
Finally being placed in primary screening culture medium, alternation of light and darkness is cultivated three weeks, obtains resistant calli.
2, after completing step 1, being placed in by resistant calli in postsearch screening culture medium, alternation of light and darkness is cultivated three weeks.
3, after completing step 2, being placed in by resistant calli in three screening culture medium, alternation of light and darkness is cultivated three weeks.
Above-mentioned alternation of light and darkness is cultivated the condition of (i.e. illumination cultivation and light culture are alternately) and is: 25～26 DEG C.Illumination cultivation
Time intensity of illumination be 2000Lx, the cycle that alternation of light and darkness is cultivated is particularly as follows: 16 hours illumination cultivation/8 hour dark culturing.
Seven, differentiation culture
After completing step 6, resistant calli being placed in division culture medium second, alternation of light and darkness cultivates (differentiation training in 6～8 weeks
Support period and changed the newest division culture medium second every 3 weeks), obtain breaking up Seedling.Wherein alternation of light and darkness cultivate (i.e. illumination cultivation and
Light culture is alternately) condition is: 25～26 DEG C.Intensity of illumination during illumination cultivation is 2000Lx, the cycle tool that alternation of light and darkness is cultivated
Body is: 16 hours illumination cultivation/8 hour dark culturing.
Eight, take root and screening and culturing in strong sprout
After completing step 6, differentiation Seedling is placed in root media second and carries out alternation of light and darkness cultivation.Treat that root length length is to 1cm
Time, cut off root, then be placed in root media third and carry out alternation of light and darkness cultivation, when root length length is to more than 3cm, (incubation time is about
4～7 weeks) i.e. complete taking root of regrowth, it is thus achieved that healthy and strong regrowth, i.e. intend transgenic resistance plant.Wherein alternation of light and darkness training
(i.e. illumination cultivation and light culture are alternately) condition of supporting is: 25 DEG C.Intensity of illumination during illumination cultivation is 2000Lx, and alternation of light and darkness is trained
The cycle supported is particularly as follows: 16 hours illumination cultivation/8 hour dark culturing.
Nine, the acquisition of wild type wild barley
Take the young fringe of the wild barley of wound healing system S1, children's fringe is divested outside sheath and is placed in superclean bench, first with 70%
The ethanol water of (percent by volume) cleans surface, then scratches, with aseptic dissecting needle, the sheath that children is tender, rotates children's fringe gently
At tuberosity, extract wrapped children's fringe out and as outer implant, with aseptic nipper, this outer implant is cut into the joint of 0.2cm～0.4cm
Section, is inoculated on inducing culture, 25 DEG C of light culture 4～5 weeks, obtains embryo callus.Then according to step in embodiment 1
The method of two to four, it is thus achieved that healthy and strong regrowth, by named for this regrowth wild type wild barley (i.e. by regular regeneration program
Rather than the regrowth come by genetic transformation).
Ten, the detection of transgenic resistance plant is intended
1, the Molecular Detection of transgenic resistance plant is intended
Extract the blade of plan transgenic resistance plant and the genomic DNA of the blade of wild type wild barley respectively and as mould
Plate, carries out PCR amplification with primer to 1；The genomic DNA intending the blade of transgenic resistance plant is replaced with water, right as feminine gender
According to；The genomic DNA intending the blade of transgenic resistance plant is replaced, as positive control with plasmid pTCK303.Primer to 1 by
P1:5 '-aaggaatcggtcaatacactacatgg-3 ' and P2:5 '-aacagttcctgattaaccacaaacc-3 ' composition.
Extract respectively and intend the blade of transgenic resistance plant and the leaves genomic DNA of wild type wild barley and as mould
Plate, carries out PCR amplification with primer to 2；The genomic DNA intending the blade of transgenic resistance plant is replaced with water, right as feminine gender
According to；The genomic DNA intending the blade of transgenic resistance plant is replaced, as positive control with plasmid pTCK303.Primer to 2 by
F1:5 '-AAGCCAGACAGAGTGTGATATCT-3 ' and F2:5 '-AACATTACATTGACGCAGGTGAT-3 ' composition.
Experimental result is shown in that (upper figure is that figure below is for using primer to 1 experimental result carrying out PCR amplification with primer to (a) in Fig. 2
To 2 experimental results carrying out PCR amplification, M is DNA Marker, and swimming lane 10 to 30 is the blade to intend transgenic resistance plant
Genomic DNA is the experimental result of template, and CK is the experimental result with the leaves genomic DNA of wild type wild barley as template,
Plasmid is positive control, and water is negative control).If the genomic DNA primer intending transgenic resistance plant amplifies about 1
The band of 300bp, amplifies the band of about 570bp simultaneously with primer to 2, then this plan transgenic resistance plant is transgenic sun
Property plant；Wild type wild barley and water primer all can not expand acquisition 300bp band to 1, all can not amplify 2 with primer
570bp band.Result shows, intending there are seven strains in transgenic resistance plant is transgenic positive plant, by its most named OE-
1～OE-7.
2, the fluorescence quantitative PCR detection of transgenic positive plant
RNeasy Plant Mini test kit (QIAGEN Products) is used to extract wild type wild barley, OE-1, OE-
2, total serum IgE the reverse transcription of OE-3, OE-4 or OE-5 is cDNA, and with this cDNA as template, passes through fluorescence quantitative PCR detection
The relative expression quantity (with GAPDH gene as reference gene) of gus gene.
The primer identifying gus gene is GUS-RT-F:5 '-CTCCTACCGTACCTCGCATTAC-3 ' and GUS-RT-R:
5’-ACGCGCTATCAGCTCTTTAATC-3’。
The primer identifying GAPDH gene is GAPDH-RT-F:5 '-TTTCGGAAGGATCGGGAG-3 ' and GAPDH-RT-R:
5’-ACAGCCTTGGCAGCACCA-3’。
Wherein quantitative fluorescent PCR reaction system includes 10 μ L SYBR Premix Ex Taq (TaKaRa Products), just
The each 0.8 μ L of reverse primer (10 μMs), template cDNA 3 μ L, ddH2O 5.4μL.PCR reaction condition is: 94 DEG C of denaturations 15min；
94 DEG C of degeneration 10s, 60 DEG C of annealing 30s, 72 DEG C extend 30s, 40 circulations.
Using in wild type wild barley, the relative expression quantity of gus gene is as 1, in OE-1, OE-2, OE-3, OE-4 and OE-5
The relative expression quantity of gus gene is shown in (b) in Fig. 2 (wherein WT is wild type wild barley).OE-1, OE-2, OE-3, OE-4 and OE-5
The relative expression quantity of middle gus gene be respectively 43 times of the relative expression quantity of gus gene in wild type wild barley, 32 times, 52 times,
35 times and 94 times.Result shows, gus gene is the most successfully incorporated in the genome of OE-1, OE-2, OE-3, OE-4 and OE-5 also
Transcriptional level is expressed.
3, the GUS staining analysis of transgenic positive plant
Take wild type wild barley, non-transgenic positive plant, the blade of OE-1, OE-2, OE-3, OE-4 or OE-5, by it
Fully being immersed in GUS reaction mixed liquor, 37 DEG C are incubated overnight, water-soluble with 50% ethanol water, 70% ethanol the most successively
Liquid and the decolouring of 100% ethanol solution, observe the staining conditions of blade.
Experimental result is shown in that (blade that in (a), a left side is OE-4, the right side is the blade of wild type wild barley to Fig. 3；B in (), a left side is OE-
The blade of 5, the right side is the blade of wild type wild barley；C () is the blade of OE-1；D () is the blade of OE-2；E () is the leaf of OE-3
Sheet；F () and (g) is the blade of non-transgenic positive plant；H () is that the blade of transgenic positive plant (is from left to right followed successively by
OE-1, OE-2, OE-3, OE-4 and OE-5)；I () is the blade of non-transgenic positive plant)；K () is transgenic positive plant
Sheath (being from left to right followed successively by OE-1, OE-2, OE-3, OE-4 and OE-5)).Result shows, OE-1, OE-2, OE-3, OE-4 and
The blade of OE-5 and sheath equal pigmentable after GUS dyes, and the relative expression of GUS staining power and the gus gene in step 2
Measure basically identical.
According to the frequency of GUS coloration result statistics transgenic positive plant, result shows, the heredity provided according to the present invention
The frequency of the transgenic positive plant that method for transformation obtains is about 5%.
Claims (10)
1. a method for cross-pollinatd plant Regeneration in Vitro is the young fringe with described cross-pollinatd plant individual plant or uses described children
The sections that fringe is cut into is outer implant, completes Regeneration in Vitro.
2. the method for claim 1, it is characterised in that: the method for described cross-pollinatd plant Regeneration in Vitro includes as follows
Step:
(1) described outer implant is inoculated in inducing culture to cultivate, it is thus achieved that callus；Described inducing culture contains
2～4mg/L 2,4-D, 30～40g/L sucrose and 0.4～0.6g/L caseinhydrolysate；
(2) callus that step (1) obtains is placed in subculture medium to cultivate, it is thus achieved that embryo callus；Described continue
Containing 0.8～1.2mg/L 2 in culture base, 4-D, 30～40g/L sucrose and 0.4～0.6mg/L 6-BA；
(3) embryo callus that step (2) obtains is placed in division culture medium first to cultivate, it is thus achieved that differentiation Seedling；Differentiation training
Support in base first containing 0.8～1.2mg/L 6-BA, 0.15～0.25mg/L zeatin and 30～40g/L sucrose；
(4) the differentiation Seedling that step (3) obtains is placed in root media first to cultivate, it is thus achieved that regrowth；Described root culture
Containing 20～30g/L sucrose in base first.
3. method as claimed in claim 1 or 2, it is characterised in that:
In described step (1), the condition of described cultivation is: 25 DEG C～28 DEG C, light culture 4～5 weeks；
In described step (2), the condition of described cultivation is: 25 DEG C～28 DEG C, light culture 9～12 weeks；
In described step (3), the condition of described cultivation is: 25 DEG C～28 DEG C, and alternation of light and darkness is cultivated 5～8 weeks；
In described step (4), the condition of described cultivation is: 25 DEG C～28 DEG C, and alternation of light and darkness is cultivated 4～7 weeks.
4. claims 1 to 3 arbitrary described method application in cross-pollinatd plant genetic transformation.
5. a preparation method for cross-pollinatd plant callus high frequency regeneration system, comprises the steps:
(1) it is outer implant with the young fringe of cross-pollinatd plant individual plant or the sections that is cut into described children's fringe, described outer implant is connect
Plant and cultivate in inducing culture described in claim 2, it is thus achieved that callus；
(2), after completing step (1), take the callus of the Callus induction rate individual plant higher than more than 75%, be placed in claim 2
Described subculture medium is cultivated, it is thus achieved that embryo callus；
(3) after completing step (2), take the embryo callus of the embryo callus subculture rate individual plant higher than more than 70%, be placed in right and want
Division culture medium first described in 2 is asked to cultivate, it is thus achieved that differentiation Seedling；
(4) after completing step (3), take the differentiation Seedling of the differentiation rate individual plant higher than more than 80%, be placed in described in claim 2 raw
Root culture medium first is cultivated, it is thus achieved that regrowth.
6. a genetic transforming method for cross-pollinatd plant, in turn includes the following steps:
(1) it is outer implant with the young fringe of described cross-pollinatd plant individual plant or the sections that is cut into described children's fringe, by described outer planting
Body is inoculated in inducing culture and cultivates, it is thus achieved that callus；Containing 2～4mg/L2 in described inducing culture, 4-D, 30
～40g/L sucrose and 0.4～0.6g/L caseinhydrolysate；
(2), after completing step (1), described callus is placed in subculture medium and cultivates, it is thus achieved that embryo callus；Institute
State containing 0.8～1.2mg/L 2 in subculture medium, 4-D, 30～40g/L sucrose and 0.4～0.6mg/L6-BA；
(3), after completing step (2), described embryo callus is carried out genetic transformation.
7. method as claimed in claim 6, it is characterised in that: in described step (3), described " to described embryo callus
Carry out genetic transformation " can comprise the steps: successively
(A) take recombinational agrobacterium, described embryo callus is infected；Described recombinational agrobacterium is by containing genes of interest
Recombinant expression carrier import set out what Agrobacterium obtained；
(4) after completing step (A), take described embryo callus, carry out drying and other treatment；
(5), after completing step (4), take described embryo callus, be placed in and co-culture base, co-culture；Described co-culturing in base contains
Have 0.8～1.2mg/L 2,4-D, 0.4～0.6mg 6-BA, 30～40g/L sucrose and 40～60mg/L acetosyringones；
(6) after completing step (5), take described embryo callus, be sequentially placed into primary screening culture medium, postsearch screening culture medium
Cultivate with in three screening culture medium, it is thus achieved that resistant calli；Containing 2 in described primary screening culture medium, 4-D 0.8
～1.2mg/L, sucrose 30～40g/L, Ticarcillin/Clavulanate Acid 120～180mg/L and hygromycin 15～25mg/L；Described postsearch screening is cultivated
Containing 2 in base, 4-D 0.8～1.2mg/L, sucrose 30～40g/L, Ticarcillin/Clavulanate Acid 120～180mg/L and hygromycin 20～30mg/
L；Containing 2 in described three screening culture medium, 4-D 0.8～1.2mg/L, sucrose 30～40g/L, Ticarcillin/Clavulanate Acid 120～180mg/L
With hygromycin 25～35mg/L；
(7) after completing step (6), take described resistant calli, be placed in division culture medium second and cultivate, it is thus achieved that differentiation Seedling；
Containing 6-BA 0.8～1.2mg/L, zeatin 0.15～0.25mg/L and sucrose 30～40g, spy in described division culture medium second
U.S. spit of fland 120～180mg/L and hygromycin 15～25mg/L；
(8) after completing step (7), take described differentiation Seedling, be sequentially placed into root media second and root media third is cultivated, it is thus achieved that
Transgenic seedling；In described root media second containing sucrose 20～30g/L, Ticarcillin/Clavulanate Acid 120～180mg/L and hygromycin 25～
35mg/L；In described root media third containing sucrose 20～30g/L, Ticarcillin/Clavulanate Acid 120～180mg/L and hygromycin 45～
55mg/L。
8. method as claimed in claim 7, it is characterised in that:
In described step (A), the method for described " infecting " is as follows: will infect bacterium solution and drop in the surface of described embryo callus,
Then application of vacuum 5～15min；The described bacterium solution that infects is recombinational agrobacterium bacteria suspension；
In described step (4), the method for described " drying and other treatment " is as follows: be placed under aseptic condition by described embryo callus dry
Dry cultivation.
9. the method as described in claim 6 to 8 is arbitrary, it is characterised in that:
In described step (1), the condition of described cultivation is: 25 DEG C～28 DEG C, light culture 4～5 weeks；
In described step (2), the condition of described cultivation is: 25 DEG C～28 DEG C, light culture 9～12 weeks；
In described step (5), the condition of described cultivation is: 25 DEG C～27 DEG C, light culture 3～4 days；
In described step (6), the condition of described cultivation is: 25 DEG C～28 DEG C, and alternation of light and darkness is cultivated 8～10 weeks；
In described step (7), the condition of described cultivation is: 25 DEG C～28 DEG C, and alternation of light and darkness is cultivated 6～8 weeks；
In described step (8), the condition of described cultivation is: 25 DEG C～28 DEG C, and alternation of light and darkness is cultivated 4～7 weeks.
10. the test kit first for cross-pollinatd plant Regeneration in Vitro or the test kit for cross-pollinatd plant genetic transformation
Second:
Described test kit first contains inducing culture described in claim 2 and/or described subculture medium and/or described differentiation
Culture medium first and/or described root media first；
Described test kit second contains inducing culture described in claim 6 and/or described subculture medium and/or described training altogether
Support base and/or described primary screening culture medium and/or described postsearch screening culture medium and/or described three screening culture medium and/
Or described division culture medium second and/or described root media second and/or described root media third.
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| CN201610543125.8A Pending CN106171981A (en) | 2016-07-11 | 2016-07-11 | The preparation method of a kind of cross-pollinatd plant callus high frequency regeneration system and the application in genetic transformation thereof |
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| CN107027630A (en) * | 2017-05-31 | 2017-08-11 | 甘肃农业大学 | A kind of method that salt sward blade separate living tissue high-efficiency regeneration system is set up |
| CN115710590A (en) * | 2022-11-29 | 2023-02-24 | 中国科学院青岛生物能源与过程研究所 | Wild barley agrobacterium-mediated callus infection method |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107027630A (en) * | 2017-05-31 | 2017-08-11 | 甘肃农业大学 | A kind of method that salt sward blade separate living tissue high-efficiency regeneration system is set up |
| CN107027630B (en) * | 2017-05-31 | 2019-03-01 | 甘肃农业大学 | A kind of method that salt sward blade separate living tissue high-efficiency regeneration system is established |
| CN115710590A (en) * | 2022-11-29 | 2023-02-24 | 中国科学院青岛生物能源与过程研究所 | Wild barley agrobacterium-mediated callus infection method |
| CN115710590B (en) * | 2022-11-29 | 2024-02-02 | 中国科学院青岛生物能源与过程研究所 | Agrobacterium tumefaciens mediated callus infection method |
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Application publication date: 20161207 |