CN105063086B - A kind of molecular breeding method for quickly obtaining a large amount of transgenosis sedum lineare new varieties - Google Patents
A kind of molecular breeding method for quickly obtaining a large amount of transgenosis sedum lineare new varieties Download PDFInfo
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Abstract
The invention discloses a kind of molecular breeding method for quickly obtaining a large amount of transgenosis sedum lineare new varieties.The present invention is using the blade, petiole, stem of aseptic seedling as transformation receptor, according to the plant expression vector for needing to build target gene of breeding objective, target gene is imported by sedum lineare genome by Agrobacterium tumefaciens mediated method, regeneration plant is differentiated to form by bud adventitious organogenesis；The corresponding antibiotic-screening transformant of riddled basins is added when transformation bud induction, transformation bud extend and taken root, is hybridized by Southern, GUS dyeing proves that obtained resistant strain is transformant.The inventive method can obtain the substantial amounts of sedum lineare new varieties for turning target gene for 9 weeks, can meet current sedum lineare elite plant strain cultivate, Germplasm enhancement research there is an urgent need to.The present invention has significant ecology, economic and social benefit meaning in sedum lineare sustainable development.
Description
Technical field
The invention belongs to field of plant genetic, and in particular to it is new that one kind quickly obtains a large amount of transgenosis sedum lineares
The molecular breeding method of kind.
Background technology
Roof greening has increase green space, heat-insulation and heat-preservation, cooling dedusting, purification air, beautification landscape, energy-conservation because of it
Water saving etc. function and as today's society structure solid space greening an important development research direction.Roof greening engineering one
Individual important element is excavation, cultivates the green material application with good characteristic.Plant breeding method includes traditional miscellaneous at present
Friendship, introduction and acclimatization, bud mutation, physics and chemistry mutagenesis, molecular breeding based on transgenic technology etc..Various breeding methods have its advantage and disadvantage：
Traditional crossbreeding is common method, has achieved the achievement of splendidness, but conventional Cross Combinations breeding technique cycle
Length, efficiency are low, poor to the phenotype foresight of offspring；Though conventional physicochemical factors induced breeding means are also cultivating plant fine quality
In served critically important, but because this method belongs to disposable mutagenesis, processing is cumbersome and breeding cycle is grown, beneficial mutation
Frequency is relatively low, and physics and chemistry mutagenesis is typically just for a certain stage of Material growth, and vegetable material is in different growth phases
Mutagenic Effect is different, and this point also results in that mutant character caused by physics and chemistry mutagenesis is relatively simple, cultivates new varieties in recent years
Quantity gradually decreases, and this method is also that some gene can not possibly be operated and selected exactly, the performance to offspring
Foresight is also poor；And establish using molecular genetics as theoretical foundation, using molecular biology and microbiological technique method as hand
The genetic engineering of section is exactly by the gene of separate sources, by the blueprint being pre-designed, builds recombination in vitro, is then introduced into
Cell, to change the original hereditary capacity of organism, it is bred as turning base with ideal character by field experiment and crop field selection
Because of new varieties or germ plasm resource.Genetic engineering breeding can be oriented transformation to objective trait, and more conventional breeding cycle is short, for
By the way of gene (such as antiweed, pest-resistant etc.) with dominant character (positive regulation and control) is using overexpressing；And for negative regulation
Plant Inner sources gene is typically using the method for antisense or RNAi.Far and away, plant transgenic technology is current agriculture high-tech state
The focus of border competition, have some protections that patent is received by the method for molecular breeding technology cultivation new variety of plant.
Sedum lineare (Sedum lineare Thunb.) is cauline leaf succulent, and the four seasons are evergreen, and drought-enduring, cold, lean ability is strong,
It is the essential novel green plants of major Urban Roof greening at present.Therefore, breeding technique is developed to cultivate with high-quality character
Sedum lineare new varieties there is important economic development and environmental protection meaning.The molecule both at home and abroad on sedum lineare is educated at present
Technology that kind, regeneration plant are cultivated etc. is there is not yet the report of patent document and other documents.
The content of the invention
The present invention has filled up the blank in terms of the genetic transformation breeding of domestic and international sedum lineare, regeneration plant Cultivating techniques, carries
A kind of molecular breeding method for quickly obtaining a large amount of transgenosis sedum lineare new varieties has been supplied, substantial amounts of tool can be obtained within 9 weeks
Have a sedum lineare new varieties of merit, meet at present to sedum lineare elite plant strain Cultivating techniques there is an urgent need in sedum lineare
There is significant ecology, economic and social benefit meaning in sustainable development.
The molecular breeding method of a large amount of transgenosis sedum lineare new varieties of quick acquisition of the present invention, it is characterised in that including
Following steps：
A, the acquisition of aseptic seedling and explant：Take sedum lineare edible tender branch, after sterilization, by branch cut into one section
Segment, after be inoculated into 1/2MS culture mediums one by one, be placed in 25 ± 1 DEG C of temperature, 50 μm of ol m of illuminance–2s–1, illumination 12h/ Heaven's commandments
Cultivated under part, axil Fiber elongation is branch, contacts the otch at culture medium and grows adventitious root, bud branch is cut out and transferred into 1/2MS
Shoot proliferation is carried out under culture medium the same terms, the bud branch for breeding to obtain is aseptic seedling, cuts blade, leaf respectively from aseptic seedling
Handle, stipes are as explant；
B, the step a explants prepared are soaked in the Agrobacterium tumefaciems containing the plant expression vector for carrying target gene
In bacterium solution, the plant expression vector contains riddled basins, then takes out explant, blots explant after unnecessary bacterium solution
Move to and co-culture in base, put 25 ± 1 DEG C, dark is lower to cultivate, and then takes out the explant after co-culturing, explant is blotted after rinsing
Upper residual moisture, explant is moved in resistant budses induction screening and culturing medium, 25 ± 1 DEG C is put, is cultivated under light, resistant budses to be grown
Afterwards, it is forwarded under resistant budses induction screening and culturing medium the same terms and continues to cultivate, is then forwarded to resistant budses elongation training again
Support in base, put 25 ± 1 DEG C, cultivated under light, resistant budses extend and produce adventitious root, obtain resistant plant；
Described co-cultivation base is：MS culture mediums addition 1mg l-1BA、0.1mg l-1NAA and 20mg l-1Acetyl cloves
Ketone, pH 5.5；
C, the Molecular Detection of transfer-gen plant：Using the riddled basins on plant expression vector, reporter gene or
The sequence of target gene enters performing PCR Molecular Detection method, Southern hybridizing methods or RT-PCR detection methods, detect resistant budses or
Whether person's resistant plant has been transgenic line；
D, the transplanting of transfer-gen plant：The transfer-gen plant taken root of the detection containing target gene is moved from blake bottle
Go out, wash away root culture medium, be colonized in the basin equipped with cultivation matrix, obtain transplanting successful transfer-gen plant.
The described step b plant expression vector containing carrying target gene is to contain target gene
PCAMBIA1301 carriers, its riddled basins are hygromix phosphotransferase screening-gene, described step b resistant budses
It is that 1mg l are added in MS culture mediums to induce screening and culturing medium-1BA、0.1mg l-1NAA、10mg l-1Hygromycin and 500mg l-1Head
P0-357, pH 5.8；Described step b resistant budses elongation medium is that 1/2MS culture mediums add 10mg l-1Hygromycin and
250mg l-1Cephalosporin, pH 5.8.
The described step b plant expression vector containing carrying target gene is to contain target gene
PCAMBIA3301 carriers, its riddled basins are cremart resistant gene, and described step b resistant budses induction screening is trained
It is that MS culture mediums add 1mg l to support base-1BA、0.1mg l-1NAA、5mg l-1Cremart and 500mg l-1Cephalosporin, pH are
5.8；Described step b resistant budses elongation medium is that 1/2MS culture mediums add 5mg l-1Cremart and 250mg l-1Cephalo
Mycin, pH 5.8.
Described step b's carries containing the plant expression vector for carrying target gene for the pBI121 containing target gene
Body, its riddled basins are kalamycin resistance gene；Described step b resistant budses induction screening and culturing medium is cultivated for MS
Base addition 1mg l-1BA、0.1mg l-1NAA、100mg l-1Kanamycins and 500mg l-1Cephalosporin, pH 5.8；Described
Step b resistant budses elongation medium is that 1/2MS culture mediums add 100mg l-1Kanamycins and 250mg l-1Cephalosporin,
PH is 5.8.
The described Agrobacterium tumefaciems containing the plant expression vector for carrying target gene builds by the following method：
After target gene is inserted into expression vector promoter, the plant of the carrying target gene built is expressed by freeze-thaw method and carried
Body imports Agrobacterium tumefaciems, through resistance screening, obtains the Agrobacterium tumefaciems containing the plant expression vector for carrying target gene.
Described step a sterilization, it is after sedum lineare edible tender branch is rinsed well with running water, to be put into mass fraction
5-10 minutes are soaked in 0.1% carbendazim solution, then takes out and is rinsed well with running water, then with the alcohol of volume fraction 75%
Immersion 30 seconds, after aseptic water washing 3 times, branch is moved into 2.5% chlorine of mass fraction of the Tween 80 containing mass fraction 0.05%
Acid sodium solution sterilizes 10 minutes, with aseptic water washing 6 times.
Described step b rinsing, it is with the aseptic water washing 5~6 times containing the Tween 80 of mass fraction 0.05%, then
With containing 500mg l-1The aseptic water washing of cephalosporin is once.
Described step d cultivation matrix is vermiculite.
Explant is soaked in the Agrobacterium tumefaciems containing the plant expression vector for carrying target gene in described b step
In step be that will be first suspended in containing the Agrobacterium tumefaciems of plant expression vector for carrying target gene containing 20mg/l acetyl
In the MS culture mediums of syringone, its pH is 5.2, cellar culture to OD600=0.4~0.5, explant is then soaked in the root
In cancer Agrobacterium bacterium solution, time 10min.
Described MS culture mediums are international culture medium, and its composition and collocation method are shown in Murashige T, Skoog
F(1962)(A revised medium for rapid growth and bioassay with tobacco tissue
cultures.Physiol Plant15:473–497).Described 1/2MS culture mediums refer to all member members in MS culture mediums
Element halves, and cane sugar content is 30mg l-1Culture medium.
The present invention is first obtained largely for the sedum lineare aseptic seedling used in genetic transformation using plant tissue culture technique, is established
In the method for bud adventitious organogenesis regeneration plant；Contained according to the needs of practical study or breeding objective structure by tobacco cauliflower
The target gene of CaMV35S promoters driving and the plant expression vector of riddled basins of mosaic virus, by expression vector
Agrobacterium tumefaciems is imported, Agrobacterium tumefaciems is co-cultured with blade, petiole, stipes explant, it is corresponding using riddled basins
Antibiotic-screening obtained resistant budses, resistant plant, dyed through GUS, Southern hybridisationdetection technologies prove it is to turn base
The material of cause.From aseptic seedling cut blade, petiole, stipes explant carry out Agrobacterium tumefaciems contaminate to obtain can transplant turn
Gene plant, whole operation process are 9 weeks.100 blade explants of once-through operation, about 25 to 50 strains can be obtained
Transgenosis bud, each transgenosis bud can produce the Multiple Buds of more than 10 through culture in 3 weeks, and the rooting rate of regeneration plant is
100%, transfer-gen plant is transplanted to the survival rate of soil up to 100%.
It is obtained with a large amount of transgenosis sedum lineare new varieties within 9 weeks using the inventive method, meets at present to Fo Jia
Careless elite plant strain Cultivating techniques there is an urgent need to, while also be sedum lineare germ plasm resource innovation, research and development, sustainable development, training
Educate research of improved Varieties, biology etc. and a kind of effective research method is provided, have in sedum lineare sustainable development aobvious
Ecology, the economic and social benefit meaning of work.
Advantages of the present invention：
(1) quickly breeding sedum lineare genetic improvement new varieties
It can be needed purposefully to improve the character of sedum lineare according to breeding, landscape or ecology using the inventive method,
It is simple to operate, the substantial amounts of sedum lineare new varieties with merit can be obtained within 9 weeks, met excellent to sedum lineare at present
Strain Cultivating techniques there is an urgent need to have significant ecology, economic and social benefit meaning in sedum lineare sustainable development.
It is also simultaneously the innovation of sedum lineare germ plasm resource, research and development, sustainable development, the research etc. of cultivating improved Varieties, biology carries
For a kind of effective research method.
(2) efficient sedum lineare tissue culture plants regenerating system
The sedum lineare tissue culture plants regenerating system that the present invention establishes is very efficient：With 21 days for 1 shoot proliferation week
Phase, every 1 shoot proliferation cycle can be bred by 1 bud branch forms 10-15 bud branch；Blade, petiole, stipes are cut from aseptic seedling
Explant survives to Transplantation of Regenerated Plantlets soil, and whole operation process is 7 weeks, and the explant for having more than 95% can produce regeneration
Bud, rooting rate 100%, 100% regeneration plant can successfully transplant subsistence；From the blade, petiole, stem of aseptic seedling
Section can turn into effective transformation receptor, aseptic seedling can Preservation in sterile condition in laboratory conditions for a long time, the source of explant not by
Limitation, can carry out the operation of transgenosis at any time.
(3) method of strict effective screening transformed cells
Respectively through 10mg l-1Hygromycin, 5mg l-1Cremart, 100mg l-1The resistant strain that kanamycins screening pressure obtains,
Dyed through GUS, Southern hybrid experiments prove be transgenosis material.Which ensures that obtained by the technical method
Resistant plant be transfer-gen plant, reduce the later stage use Molecular Detection workload.
(4) transformation efficiency is high, repeatability is high
Technical method provided by the invention is tested through being repeated several times, it was demonstrated that conversion ratio 25-50%.Because outer used in conversion
Implant easily largely obtains, so a transformation experiment operation is obtained with required transgenic line.
(5) breeding efficiency is high
Being operated more than, each explant that converts can produce Multiple Buds, and Multiple Buds and can is by shoot proliferation, and every 1
The individual shoot proliferation cycle can be bred by 1 bud branch forms more than 10 bud branches, and can rooting development into plant, therefore application this
Inventive method can obtain substantial amounts of transgenic line within 9 weeks, and 100% transgenic line successfully can be transplanted in soil, ensure
The variety culture success that molecular breeding method is cultivated.
Brief description of the drawings
Fig. 1 is that sedum lineare passes through bud adventitious organogenesis regeneration plant.Wherein, A is that blade explant is inoculated in bud induction
The otch of explant has the generation of regeneration bud during medium culture 16 days；B is that blade explant is cultivated on bud inducement cultivation base
Regeneration bud at 35 days；C is will have 2cm high Transplantation of Regenerated Plantlets that growing state when being grown 35 days in vermiculite basin is housed.
Fig. 2 is the T-DNA area schematics of plant expression vector.Wherein, 35S pro are tobacco cauliflower mosaic virus
CaMV35S promoter.
Fig. 3 is the resistant budses for turning pCAMBIA1301 plasmids when being inoculated in resistant budses induction screening and culturing medium 21 days.
Fig. 4 is the sedum lineare blade explant GUS after being co-cultured 3 days with Agrobacterium tumefaciems EHA105/pCAMBIA1301
Gene transient expression testing result.
Fig. 5 is the resistant budses for turning pCAMBIA1301 plasmids that blueness is presented in GUS dyeing.
Fig. 6 is without the GUS unconverted bud branches dyed and the resistant budses branch for turning pCAMBIA1301 plasmids for having GUS to dye.Its
In, it when being inoculated in bud elongation medium 2 weeks is inoculation through unconverted bud of the GUS dyeing without blueness is presented, figure B that figure A, which is,
Turn to dye the resistant budses that blueness is presented through GUS obtained by pCAMBIA1301 plasmids during to resistant budses elongation medium 2 weeks.
Fig. 7 is the result of Southern hybridization check pCAMBIA1301 plasmid transformants.Wherein, WT is unconverted strain, is shown
Show no hybridization signal；T1, T2, T3, T4, T5 are pCAMBIA1301 plasmid transformants, have hybridization signal and are different turn
Change strain.
Unconverted plant when Fig. 8 is transplanting soil 28 days and the plant for turning pCAMBIA1301 plasmids.Scheme A and figure B difference
Growth when being unconverted plant and soil incubation 28 days is transplanted when turning the regeneration plant about 2cm of pCAMBIA1301 plasmid plant
Situation.
Embodiment
Following examples are to further explanation of the invention, rather than limitation of the present invention.
The experimental method not indicated specifically in following Examples, can conventionally be carried out, or be given birth to according to product used
Produce the operation instruction of manufacturer.Material used, reagent etc., unless otherwise specified, can pass through commercial sources in following embodiments
Obtain.
Embodiment 1：The acquisition of aseptic seedling
Sedum lineare is bought from market, takes well-grown sprout, after being rinsed well with running water, be put into mass fraction
5-10 minutes are soaked in 0.1% carbendazim solution, are rinsed well after taking-up with running water, branch is cut into 5 cms length
Degree.Soaked 30 seconds with the alcohol water blend of volume fraction 75%, then with after aseptic water washing 3 times, branch is moved into mass fraction
2.5% liquor natrii hypochloritis (addition mass fraction 0.05% Tween 80) sterilization 10 minutes, with aseptic water washing 6 times, then will
Branch is placed in suck dry moisture on aseptic filter paper.By branch cut into one section segment, after be inoculated into one by one 1/2MS culture
Base.It is placed in 25 ± 1 DEG C of temperature, 50 μm of ol m of illuminance–2s–1, illumination cultivates under the conditions of 12h/ days.Culture 20 days or so, axil
Fiber elongation is bud branch, contacts the otch at culture medium and grows adventitious root.Cut out during the high about 5cm of bud branch transfer it is fresh into identical
Shoot proliferation is carried out under 1/2MS culture medium the same terms.It is within general 21 days 1 shoot proliferation cycle, every 1 shoot proliferation cycle
It can be bred by 1 bud branch and form 10-15 bud branch, the bud branch of acquisition is obtained aseptic seedling.
Embodiment 2：The acquisition of regeneration plant
It is explant to cut blade, petiole, stipes respectively from aseptic seedling, and being inoculated in bud inducement cultivation base, (MS culture mediums add
Add 1mg l-1Benzyladenine (BA) and 0.1mg l-1A-Naphthalene acetic acid (NAA), pH 5.8), put
In 25 ± 2 DEG C of temperature, 50 μm of ol m of illuminance–2s–1, illumination cultivates under the conditions of 12h/ days.During culture 16 days more than 95% it is outer
The generation (Figure 1A) for having regeneration bud can be observed in the incision of implant, and 16 days blade explants for having regeneration bud will be cultivated under light
Continue to cultivate under switching fresh bud inducement cultivation base the same terms of identical it is visible have a large amount of regeneration buds developments (Figure 1B), will regenerate
Cultivated under bud switching bud elongation medium (1/2MS culture mediums, pH 5.8) the same terms, bud branch Fiber elongation, contact culture medium
Base portion has the generation of a large amount of adventitious roots, rooting rate 100%, thus obtains regeneration plant.
Regeneration plant high 2cm is removed from blake bottle, root culture medium is washed away, is colonized in the basin equipped with vermiculite,
Covered with preservative film, a little apertures are beaten on preservative film, are opened after 5 days, survival rate is up to 100% (Fig. 1 C).Leaf is cut from aseptic seedling
Piece, petiole, stipes explant survive to Transplantation of Regenerated Plantlets soil, and whole operation process is 7 weeks.
Embodiment 3：Culture for the sedum lineare explant of conversion
With 21 days for 1 subculture, constantly the high bud branches of 2cm are cut and cultivated under switching 1/2MS culture medium the same terms, are expanded numerous
Aseptic seedling；It is explant to cut blade, petiole, stipes respectively from aseptic seedling, for used in Agrobacterium tumefaciems dip-dye.
Embodiment 4：The preparation of engineering strain containing target gene plant expression vector
1st, the preparation of Agrobacterium tumefaciems EHA105/pCAMBIA1301 bacterium solutions
By freeze-thaw method by pCAMBIA1301 carriers (pCAMBIA1301 carriers contain by CaMV 35S promoters drive β-
Glucose aldoside enzyme reporter gene gus and hygromix phosphotransferase screening-gene hpt can as screening-gene, target gene
Need to be placed in behind CaMV 35S promoters (Fig. 2) according to breeding objective, the present embodiment is with β-glucose aldoside enzyme report base
Because gus is as experiment test gene) Agrobacterium tumefaciems EHA105 is imported, then it is seeded in containing 50mg l–1Kanamycins and
50mg l–1In the YEP culture mediums of rifampin, cultivate 24 hours under the conditions of 28 DEG C, by the way that bacterium solution is collected by centrifugation, bacterium cell is suspended
Containing 20mg l-1In the MS fluid nutrient mediums (pH5.2) of acetosyringone, OD600=0.4~0.5, thus contained
The Agrobacterium tumefaciems EHA105 bacterium solutions of pCAMBIA1301 carriers, are named as Agrobacterium tumefaciems EHA105/pCAMBIA1301.
2nd, the preparation of Agrobacterium tumefaciems EHA105/pCAMBIA3301 bacterium solutions
By freeze-thaw method by pCAMBIA3301 carriers (pCAMBIA3301 carriers contain by CaMV 35S promoters drive β-
Glucose aldoside enzyme reporter gene gus and cremart resistant gene bar can be according to breeding mesh as screening-gene, target gene
Mark is needed to be placed in behind CaMV 35S promoters, and the present embodiment is surveyed using β-glucose aldoside enzyme reporter gene gus as experiment
Try gene) Agrobacterium tumefaciems EHA105 is imported, then it is seeded in containing 50mg l–1Kanamycins and 50mg l–1Rifampin
In YEP culture mediums, cultivate 24 hours under the conditions of 28 DEG C, by the way that bacterium solution is collected by centrifugation, bacterium cell is suspended in containing 20mg l-1Second
In the MS fluid nutrient mediums (pH5.2) of acyl syringone, OD600=0.4~0.5, thus obtain containing pCAMBIA3301 carriers
Agrobacterium tumefaciems EHA105 bacterium solutions, are named as Agrobacterium tumefaciems EHA105/pCAMBIA3301.
3rd, the preparation of Agrobacterium tumefaciems EHA105/pBI121 bacterium solutions
By freeze-thaw method, by pBI121 carriers, (pBI121 carriers contain drives β-glucose aldose by CaMV 35S promoters
Glycosides enzyme reporter gene gus and kalamycin resistance gene NPT II can be according to breeding objective need as screening-gene, target gene
It is placed in behind CaMV 35S promoters, the present embodiment is used as experiment test base using β-glucose aldoside enzyme reporter gene gus
Cause) Agrobacterium tumefaciems EHA105 is imported, then it is seeded in containing 50mg l–1Kanamycins and 50mg l–1The YEP trainings of rifampin
Support in base, cultivate 24 hours under the conditions of 28 DEG C, by the way that bacterium solution is collected by centrifugation, bacterium cell is suspended in containing 20mg l-1Acetyl fourth
In the MS fluid nutrient mediums (pH5.2) of ketone musk, OD600=0.4~0.5, the crown gall agriculture bar containing pBI121 carriers is thus obtained
Bacterium EHA105 bacterium solutions, are named as Agrobacterium tumefaciems EHA105/pBI121.
Embodiment 5：Turn the acquisition of pCAMBIA1301 hygromycin regeneration plant
Explant prepared by embodiment 3 is soaked in the Agrobacterium tumefaciems EHA105/ prepared by embodiment 4
In pCAMBIA1301 bacterium solutions, the time is 10 minutes, takes out explant, is placed on aseptic paper and blots unnecessary bacterium solution, after by explant
Body, which moves to, co-cultures base (MS culture mediums addition 1mg l-1BA、0.1mg l-1NAA and 20mg l-1Acetosyringone, pH 5.5),
25 ± 1 DEG C are put, the lower culture of dark 3 days.The explant after co-culturing is taken out, with containing the sterile of the Tween 80 of mass fraction 0.05%
Water rinses 5~6 times, finally uses and contains 500mg l-1The aseptic water washing of cephalosporin once, is put explant in aseptic paper, blotted
Water, explant is moved into resistant budses induction screening and culturing medium, and (MS culture mediums add 1mg l-1BA、0.1mg l-1NAA、10mg l-1
Hygromycin and 500mg l-1Cephalosporin, pH 5.8).25 ± 1 DEG C are put, is cultivated 18 days under light.
Explant (Fig. 3) with resistant budses is transferred to the fresh resistant budses induction screening and culturing medium the same terms of identical
Under continue culture 2 weeks after be forwarded to resistant budses elongation medium (1/2MS culture mediums addition 10mg l-1Hygromycin and 250mg l-1
Cephalosporin, pH 5.8), 25 ± 1 DEG C are put, is cultivated under light, the elongation of resistant budses and a large amount of generations of adventitious root can be observed.
It is derived from turning pCAMBIA1301 hygromycin regeneration plant (resistant plant).
Embodiment 6：Turn the acquisition of pCAMBIA3301 anti-cremart regeneration plant
Explant prepared by embodiment 3 is soaked in the Agrobacterium tumefaciems EHA105/ prepared by embodiment 4
In pCAMBIA3301 bacterium solutions, experimental implementation is same as Example 5, unlike, resistant budses used induce screening and culturing medium into
It is divided into：MS culture mediums addition 1mg l-1BA、0.1mg l-1NAA、5mg l-1Cremart and 500mg l-1Cephalosporin, pH are
5.8；Resistant budses elongation medium composition used is：1/2MS culture mediums addition 5mg l-1Cremart and 250mg l-1Cephalo is mould
Element, pH 5.8.It is derived from turning pCAMBIA3301 anti-cremart regeneration plant (resistant plant).
Embodiment 7：Turn the acquisition of pBI121 anti-kanamycins regeneration plant
Explant prepared by embodiment 3 is soaked in the Agrobacterium tumefaciems EHA105/pBI121 bacterium solutions prepared by embodiment 4
In, experimental implementation is same as Example 5, unlike, resistant budses used induction screening and culturing based component is：MS culture mediums add
Add 1mg l-1BA、0.1mg l-1NAA、100mg l-1Kanamycins and 500mg l-1Cephalosporin, pH 5.8；Resistance used
Bud elongation medium composition is：1/2MS culture mediums addition 100mg l-1Kanamycins and 250mg l-1Cephalosporin, pH are
5.8.It is derived from turning pBI121 anti-kanamycins regeneration plant (resistant plant).
Embodiment 8：The Molecular Detection of transfer-gen plant
Because pCAMBIA1301 contains gus reporter genes, can be carried out by Jefferson etc. (1987) GUS colouring methods
The expression of dyeing detection gus genes.Take the explant after being co-cultured 3 days with Agrobacterium tumefaciems EHA105/pCAMBIA1301
Body, resistant budses, unconverted bud branch and transformation bud branch are dipped in X-Gluc dyeing liquors respectively, are incubated 3~5 hours in 37 DEG C, are used
70% ethanol decolorization, observe blue-colored situation.It can be seen that after being co-cultured 3 days with Agrobacterium tumefaciems EHA105/pCAMBIA1301
Explant (Fig. 4) surface have obvious locus coeruleus, illustrate Agrobacterium tumefaciems EHA105/pCAMBIA1301 can adsorb contaminate Fo Jia
Blueness is also presented in careless explant, resistant budses (Fig. 5) and transformation bud branch (Fig. 6), and unconverted bud (Fig. 6) is then blue without presenting
Color, illustrate to operate more than, gus genes have successfully imported sedum lineare and the stable expression in transformant.Using screening
Gene hpt specific primer：HFw (5 '-CGATCTTAGCCAGACGAGCGGGTTC-3 ') and HRe (5 '-GCTGGGGCG
TCGGTTTCCACTATCGG-3 ') synthesising probing needle, the method hybridized by Southern, in resistant plant T1, T2, T3, T4, T5
In it is observed that hybridization signal, and banding pattern is different (Fig. 7), and it is different transgenic lines to illustrate T1, T2, T3, T4, T5
System, and unconverted strain WT (wild type) does not have signal, explanation passes through above this method again, and hpt genes successfully import
The genome of sedum lineare.
PCAMBIA1301, pCAMBIA3301 and pBI121 contain gus reporter genes, and embodiment 5-7 is turned
PCAMBIA1301 hygromycin regeneration plant, the anti-cremart regeneration plant for turning pCAMBIA3301, the anti-card for turning pBI121
That mycin regeneration plant, the expression of its gus gene is detected by GUS colouring methods, found respectively through 10mg l-1Tide is mould
Element, 5mg l-1Cremart, 100mg l-1The resistant plant that kanamycins screening pressure obtains has GUS dyeing, therefore, sedum lineare
Screening pressure 10mg l are respectively adopted during conversion-1Hygromycin, 5mg l-1Cremart, 100mg l-1Kanamycins may insure to be obtained
The resistant plant obtained is transfer-gen plant, reduces workload of the later stage using Molecular tools detection transfer-gen plant.
By above method, substantial amounts of transfer-gen plant can be obtained in 9 weeks, with 10mg l-1Hygromycin is that screening is pressed
To the hygromycin plant for turning pCAMBIA1301 transformation frequency be 50% or so, with 100mg l-1Kanamycins is screening
The transformation frequency for pressing the obtained anti-kanamycins plant for turning pBI121 is 50% or so, and with 5mg l-1Cremart is pressed for screening
The transformation frequency of what is obtained turn pCAMBIA3301 anti-cremart plant is then 25% or so.
Embodiment 9：The transplanting of transgenic regenerated plant
From aseptic seedling cut blade, petiole, stipes explant carry out Agrobacterium tumefaciems contaminate to obtain can transplant turn
Gene plant, whole operation process are 9 weeks.Will be above 2cm transfer-gen plant (resistant plant prepared by embodiment 5-7) from
Removed in blake bottle, wash away root culture medium, be colonized in the basin equipped with vermiculite, covered with preservative film, beaten on preservative film
Aperture, opened after 5 days, survival rate is up to 100%.
The hygromycin regeneration plant for turning pCAMBIA1301 for being about 2cm by height prepared by embodiment 5 and high about 2cm
Unconverted strain regeneration plant (be prepared according to embodiment 1 and the methods described of embodiment 2, as control) respectively from culture
Removed in bottle, wash away root culture medium, be colonized in the basin equipped with vermiculite, covered with preservative film, a little apertures are beaten on preservative film,
Opened after 5 days, after being colonized 28 days, the growing state of the two is compared in observation, as shown in Figure 8.It can be seen that turn
The regeneration plant upgrowth situation of pCAMBIA1301 hygromycin regeneration and unconverted strain is essentially identical, shows sedum lineare heredity
The success of transformation tissue culture system construction.
Claims (6)
1. a kind of molecular breeding method for quickly obtaining a large amount of transgenosis sedum lineare new varieties, it is characterised in that including following step
Suddenly：
A, the acquisition of aseptic seedling and explant：Take sedum lineare edible tender branch, after sterilization, by branch cut into one section cut
Section, after be inoculated into 1/2MS culture mediums one by one, be placed in 25 ± 1 DEG C of temperature, 50 μm of ol m of illuminance–2s–1, 12h/ days conditions of illumination
Lower culture, axil Fiber elongation are branch, contact the otch at culture medium and grow adventitious root, and bud branch is cut out to transfer and trained into 1/2MS
Support and carry out shoot proliferation under base the same terms, the bud branch for breeding to obtain is aseptic seedling, cuts blade, leaf respectively from aseptic seedling
Handle, stipes are as explant；
B, the step a explants prepared are soaked in the Agrobacterium tumefaciems bacterium solution containing the plant expression vector for carrying target gene
In, the plant expression vector contains riddled basins, then takes out explant, moves to explant after blotting unnecessary bacterium solution
Co-culture in base, put 25 ± 1 DEG C, dark is lower to cultivate, and then takes out the explant after co-culturing, is blotted after rinsing residual on explant
Remaining moisture, explant is moved in resistant budses induction screening and culturing medium, 25 ± 1 DEG C is put, is cultivated under light, after resistant budses are grown,
It is forwarded under resistant budses induction screening and culturing medium the same terms and continues to cultivate, is then forwarded to resistant budses elongation medium again
In, 25 ± 1 DEG C are put, is cultivated under light, resistant budses extend and produce adventitious root, obtain resistant plant；Described co-cultivation base is：MS
Culture medium addition 1mg l-1BA、0.1mg l-1NAA and 20mg l-1Acetosyringone, pH 5.5；
When the described plant expression vector containing carrying target gene is the pCAMBIA1301 carriers containing target gene,
Its riddled basins is hygromix phosphotransferase screening-gene, and described resistant budses induction screening and culturing medium is MS culture mediums
Middle addition 1mg l-1BA、0.1mg l-1NAA、10mg l-1Hygromycin and 500mg l-1Cephalosporin, pH 5.8；Described is anti-
Property bud elongation medium be that 1/2MS culture mediums add 10mg l-1Hygromycin and 250mg l-1Cephalosporin, pH 5.8；
Or carried when described containing the plant expression vector for carrying target gene for the pCAMBIA3301 containing target gene
During body, its riddled basins is cremart resistant gene, and described resistant budses induce screening and culturing medium to be added for MS culture mediums
1mg l-1BA、0.1mg l-1NAA、5mg l-1Cremart and 500mg l-1Cephalosporin, pH 5.8；Described resistant budses elongation
Culture medium is that 1/2MS culture mediums add 5mg l-1Cremart and 250mg l-1Cephalosporin, pH 5.8；
Or when the described plant expression vector containing carrying target gene is the pBI121 carriers containing target gene,
Its riddled basins is kalamycin resistance gene；Described resistant budses induction screening and culturing medium is that MS culture mediums add 1mg
l-1BA、0.1mg l-1NAA、100mg l-1Kanamycins and 500mg l-1Cephalosporin, pH 5.8；Described resistant budses elongation
Culture medium is that 1/2MS culture mediums add 100mg l-1Kanamycins and 250mg l-1Cephalosporin, pH 5.8；
C, the Molecular Detection of transfer-gen plant：Utilize the riddled basins on plant expression vector, reporter gene or target
The sequence of gene enters performing PCR Molecular Detection method, Southern hybridizing methods or RT-PCR detection methods, detects resistant budses or anti-
Whether property plant has been transgenic line；
D, the transplanting of transfer-gen plant：The transfer-gen plant taken root of the detection containing target gene is removed from blake bottle, washed
Root culture medium is removed, is colonized in the basin equipped with cultivation matrix, obtains transplanting successful transfer-gen plant.
2. the molecular breeding method according to claim 1 for quickly obtaining a large amount of transgenosis sedum lineare new varieties, its feature
It is, the described Agrobacterium tumefaciems containing the plant expression vector for carrying target gene builds by the following method：Will
Target gene is inserted after expression vector promoter, the plant expression vector for the carrying target gene that will be built by freeze-thaw method
Agrobacterium tumefaciems is imported, through resistance screening, obtains the Agrobacterium tumefaciems containing the plant expression vector for carrying target gene.
3. the molecular breeding method according to claim 1 for quickly obtaining a large amount of transgenosis sedum lineare new varieties, its feature
It is, described step a sterilization, is after sedum lineare edible tender branch is rinsed well with running water, to be put into mass fraction 0.1%
Carbendazim solution in soak 5-10 minutes, then take out and rinsed well with running water, then alcohol-pickled with volume fraction 75%
30 seconds, after aseptic water washing 3 times, branch is moved into the sodium hypochlorite of mass fraction 2.5% of the Tween 80 containing mass fraction 0.05%
Solution disinfection 10 minutes, with aseptic water washing 6 times.
4. the molecular breeding method according to claim 1 for quickly obtaining a large amount of transgenosis sedum lineare new varieties, its feature
It is, described step b rinsing, is with the aseptic water washing 5~6 times containing the Tween 80 of mass fraction 0.05%, Ran Houyong
Contain 500mg l-1The aseptic water washing of cephalosporin is once.
5. the molecular breeding method according to claim 1 for quickly obtaining a large amount of transgenosis sedum lineare new varieties, its feature
It is, described step d cultivation matrix is vermiculite.
6. the molecular breeding method according to claim 1 for quickly obtaining a large amount of transgenosis sedum lineare new varieties, its feature
It is, explant is soaked in the Agrobacterium tumefaciems bacterium containing the plant expression vector for carrying target gene in described b step
Step in liquid is that first the Agrobacterium tumefaciems containing the plant expression vector for carrying target gene is suspended in containing 20mg/l second
In the MS culture mediums of acyl syringone, its pH is 5.2, cellar culture to OD600=0.4~0.5, explant is then soaked in this
In Agrobacterium tumefaciems bacterium solution, time 10min.
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