CN105063086A - Molecular breeding method fast obtaining large number of transgenosis sedum lineare new species - Google Patents
Molecular breeding method fast obtaining large number of transgenosis sedum lineare new species Download PDFInfo
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Abstract
The invention discloses a molecular breeding method fast obtaining a large number of transgenosis sedum lineare new species. According to the molecular breeding method, blades, stipes and stems of aseptic seedlings are used as transformation accepters, a plant expression vector of target genes is constructed according to the requirement of the breeding purpose, the target genes are guided into a sedum lineare genome through an agrobacterium tumefaciens mediating method, and a regenerated plant is formed through shoot organogenesis way differentiation; a corresponding antibiotic screening transformant of screening marker genes is added during transformation shoot induction, transformation shoot elongation and rooting, and it is verified that an obtained resistance plant is a transformation plant through Southern hybridization and GUS dyeing verification. According to the method, a large number of target gene transformation sedum lineare new species can be obtained by nine weeks, and the urgent demands for breeding sedum lineare fine strains and innovating and researching genetic resources at present can be met. The molecular breeding method has obvious ecological, economic and social benefit significance in sustainable development of sedum lineare.
Description
Technical field
The invention belongs to field of plant genetic, be specifically related to the molecular breeding method of a kind of quick acquisition a large amount of transgenosis Linear Stonecrop Herb new variety.
Background technology
Roof greening increases green space, heat-insulation and heat-preservation, cooling dedusting because it has, purifies air, beautifies the function such as view, energy-saving and water-saving and become the important development research direction that society builds solid space greening.The important element of roof greening engineering one excavates, cultivates the green material application with good characteristic.Current plant breeding method includes traditional hybridization, introduction and acclimatization, bud mutation, physics and chemistry mutagenesis, molecular breeding etc. based on transgenic technology.Various breeding method has its relative merits: traditional cross-breeding is common method, achieved brilliant achievement, but the Cross Combinations breeding technique cycle of routine is long, efficiency is low, poor to the phenotype foresight of offspring, though conventional physicochemical factors induced breeding means also play very important effect cultivating plants in fine quality, but because the method belongs to disposable mutagenesis, process loaded down with trivial details and breeding cycle is long, useful mutation is lower, and physics and chemistry mutagenesis is general only for certain one-phase of Material growth, and vegetable material is different at the Mutagenic Effect of Different growth phases, the mutant character that this point result also in physics and chemistry mutagenesis generation is more single, the quantity of cultivating new variety in recent years reduces gradually, and the method is also impossible operate certain gene exactly and select, also poor to the performance foresight of offspring, and to be based upon with molecular genetics be theoretical basis, the genetically engineered being means with molecular biology and microbiological technique method is exactly by the gene of different sources, by the blueprint designed in advance, build recombination in vitro, then transfered cell, to change the original hereditary property of organism, select to be bred as the transgenosis new variety or germ plasm resource with ideal character through field experiment and land for growing field crops.Genetic engineering breeding can carry out directional transformation to objective trait, short compared with the conventional breeding cycle, the gene (as antiweed, pest-resistant etc.) with dominant character (just regulating and controlling) is adopted to the mode of overexpression; And the plant Inner source gene of negative regulation is generally adopted to the method for antisense or RNAi.Far and away, plant transgenic technology is the focus of current agriculture hi-tech international competition, more existing receive the protection of patent by the cultivate plants method of new variety of molecular breeding technology.
Linear Stonecrop Herb (SedumlineareThunb.) is cauline leaf succulent, and the four seasons are evergreen, and drought-enduring, cold, lean ability is strong, is the essential novel green plants of current each big city roof greening.Therefore, develop breeding technique, to cultivate the Linear Stonecrop Herb new variety with high-quality proterties, there is important Economic development and environment protection meaning.The technology etc. that the domestic and international molecular breeding about Linear Stonecrop Herb, regeneration plant are cultivated at present there is not yet the report of patent documentation and other document.
Summary of the invention
The present invention has filled up the genetic transformation breeding of domestic and international Linear Stonecrop Herb, the blank of regeneration plant Cultivating techniques aspect, provide the molecular breeding method of a kind of quick acquisition a large amount of transgenosis Linear Stonecrop Herb new variety, a large amount of Linear Stonecrop Herb new variety with good character can be obtained in 9 weeks, meet at present to Linear Stonecrop Herb elite plant strain Cultivating techniques in the urgent need to, there is significant ecology, economic and social benefit meaning in Linear Stonecrop Herb Sustainable development.
The molecular breeding method of quick acquisition of the present invention a large amount of transgenosis Linear Stonecrop Herb new variety, is characterized in that, comprise the following steps:
The acquisition of a, aseptic seedling and explant: get Linear Stonecrop Herb edible tender branch, after sterilization, branch is cut into one joint segment, after be inoculated into 1/2MS substratum one by one, be placed in temperature 25 ± 1 DEG C, illuminance 50 μm of olm
– 2s
– 1cultivate under illumination 12h/ days condition, axil Fiber elongation is branch, the otch at contact substratum place grows adventive root, shoot proliferation is carried out under bud branch being cut out 1/2MS substratum the same terms of transferring, breed the bud branch obtained and be aseptic seedling, cut blade, petiole, stipes respectively as explant from aseptic seedling;
B, the explant prepared by step a is soaked in the agrobacterium tumefaciens bacterium liquid containing the plant expression vector carrying target gene, this plant expression vector contains riddled basins, then explant is taken out, explant is moved in Dual culture base after blotting unnecessary bacterium liquid, put 25 ± 1 DEG C, dark lower cultivation, then the explant after Dual culture is taken out, residual water-content on explant is blotted after rinsing, explant is moved in resistant buds induction screening culture medium, put 25 ± 1 DEG C, cultivate under light, after growing resistant buds, continue to cultivate under being forwarded to resistant buds induction screening culture medium the same terms, and then be forwarded in resistant buds elongation medium, put 25 ± 1 DEG C, cultivate under light, resistant buds extends and produces adventive root, obtain resistant plant,
Described Dual culture base is: MS substratum adds 1mgl
-1bA, 0.1mgl
-1nAA and 20mgl
-1syringylethanone, pH is 5.5;
The Molecular Detection of c, transfer-gen plant: utilize the sequence of the riddled basins on plant expression vector, reporter gene or target gene to carry out PCR Molecular Detection method, Southern hybridizing method or RT-PCR detection method, detects resistant buds or whether resistant plant has been transgenic line;
The transplanting of d, transfer-gen plant: shifted out from culturing bottle by the transfer-gen plant of taking root detected containing target gene, wash away root substratum, field planting, in the basin that cultivation matrix is housed, obtains transplanting successful transfer-gen plant.
Described step b's is pCAMBIA1301 carrier containing target gene containing the plant expression vector carrying target gene, its riddled basins is hygromix phosphotransferase screening-gene, and the resistant buds induction screening culture medium of described step b is add 1mgl in MS substratum
-1bA, 0.1mgl
-1nAA, 10mgl
-1totomycin and 500mgl
-1cephamycin, pH is 5.8; The resistant buds elongation medium of described step b is that 1/2MS substratum adds 10mgl
-1totomycin and 250mgl
-1cephamycin, pH is 5.8.
Described step b's is pCAMBIA3301 carrier containing target gene containing the plant expression vector carrying target gene, and its riddled basins is Glufosinate ammonium resistant gene, and the resistant buds induction screening culture medium of described step b is that MS substratum adds 1mgl
-1bA, 0.1mgl
-1nAA, 5mgl
-1glufosinate ammonium and 500mgl
-1cephamycin, pH is 5.8; The resistant buds elongation medium of described step b is that 1/2MS substratum adds 5mgl
-1glufosinate ammonium and 250mgl
-1cephamycin, pH is 5.8.
Described step b's is pBI121 carrier containing target gene containing the plant expression vector carrying target gene, and its riddled basins is kalamycin resistance gene; The resistant buds induction screening culture medium of described step b is that MS substratum adds 1mgl
-1bA, 0.1mgl
-1nAA, 100mgl
-1kantlex and 500mgl
-1cephamycin, pH is 5.8; The resistant buds elongation medium of described step b is that 1/2MS substratum adds 100mgl
-1kantlex and 250mgl
-1cephamycin, pH is 5.8.
The described agrobacterium tumefaciens containing the plant expression vector carrying target gene builds by the following method: after target gene being inserted expression vector promotor, by freeze-thaw method, the plant expression vector carrying target gene built is imported agrobacterium tumefaciens, through resistance screening, obtain the agrobacterium tumefaciens containing the plant expression vector carrying target gene.
The sterilization of described step a, be Linear Stonecrop Herb edible tender branch tap water is clean after, the carbendazim solution putting into massfraction 0.1% soaks 5-10 minute, then taking-up tap water is clean, use volume fraction 75% more alcohol-pickled 30 seconds, after aseptic water washing 3 times, branch is moved into and sterilizes 10 minutes containing massfraction 2.5% chlorine bleach liquor of massfraction 0.05% tween 80, with aseptic water washing 6 times.
The rinsing of described step b is the aseptic water washing of use containing massfraction 0.05% tween 80 5 ~ 6 times, then with containing 500mgl
-1the aseptic water washing of cephamycin once.
The cultivation matrix of described steps d is vermiculite.
The step be soaked in by explant in described b step in the agrobacterium tumefaciens containing the plant expression vector carrying target gene is first suspended in by the agrobacterium tumefaciens containing the plant expression vector carrying target gene in the MS substratum containing 20mg/l Syringylethanone, its pH is 5.2, and cellar culture is to OD
600=0.4 ~ 0.5, be then soaked in by explant in this agrobacterium tumefaciens bacterium liquid, the time is 10min.
Described MS substratum is international substratum, its composition and collocation method are shown in MurashigeT, SkoogF (1962) (Arevisedmediumforrapidgrowthandbioassaywithtobaccotissue cultures.PhysiolPlant15:473 – 497).Described 1/2MS substratum refers to and is reduced by half by all elements in MS substratum, and sucrose content is 30mgl
-1substratum.
The present invention first adopts plant tissue culture technique to obtain a large amount of Linear Stonecrop Herb aseptic seedling used for gene transformation, sets up with the method for bud adventitious organogenesis regeneration plant; Needs structure according to practical study or breeding objective contains by the target gene of CaMV35S promoters driven of tobacco cauliflower mosaic virus and the plant expression vector of riddled basins, expression vector is imported agrobacterium tumefaciens, make agrobacterium tumefaciens and blade, petiole, stipes explant Dual culture, use resistant buds, resistant plant that the corresponding antibiotic-screening of riddled basins obtains, prove it is all genetically modified materials through GUS dyeing, Southern hybridisationdetection technology.Cut blade from aseptic seedling, petiole, stipes explant carry out agrobacterium tumefaciens and contaminate the transfer-gen plant obtaining and can transplant, whole operating process is 9 weeks.Single job 100 leaf explants, the transgenosis bud of about 25 to 50 strains can be obtained, each transgenosis bud cultivated the Multiple Buds that can produce more than 10 through 3 weeks, and the rooting rate of regeneration plant is 100%, and transfer-gen plant is transplanted to the survival rate 100% of soil.
Utilize the inventive method in 9 weeks, just can obtain a large amount of transgenosis Linear Stonecrop Herb new variety, meet at present to Linear Stonecrop Herb elite plant strain Cultivating techniques in the urgent need to, simultaneously also for the innovation, research and development, Sustainable development, cultivation improved Varieties, biological research etc. of Linear Stonecrop Herb germ plasm resource provide a kind of effective research method, there is in Linear Stonecrop Herb Sustainable development significant ecology, economic and social benefit meaning.
Advantage of the present invention:
(1) quickly breeding Linear Stonecrop Herb genetic improvement new variety
Utilize the inventive method can need purposively to improve according to breeding, view or ecology the proterties of Linear Stonecrop Herb, simple to operate, a large amount of Linear Stonecrop Herb new variety with good character can be obtained in 9 weeks, meet at present to Linear Stonecrop Herb elite plant strain Cultivating techniques in the urgent need to, there is significant ecology, economic and social benefit meaning in Linear Stonecrop Herb Sustainable development.Simultaneously also for the innovation, research and development, Sustainable development, cultivation improved Varieties, biological research etc. of Linear Stonecrop Herb germ plasm resource provide a kind of effective research method.
(2) efficient Linear Stonecrop Herb tissue culture plants regeneration system
The Linear Stonecrop Herb tissue culture plants regeneration system that the present invention sets up is very efficient: be 1 shoot proliferation cycle with 21 days, and every 1 shoot proliferation cycle can be bred by 1 bud branch and be formed 10-15 bud branch; Cut blade from aseptic seedling, petiole, stipes explant survive to Transplantation of Regenerated Plantlets soil, whole operating process is 7 weeks, has the explant of more than 95% all can produce regeneration bud, rooting rate 100%, and the regeneration plant of 100% all successfully can transplant subsistence; Derive from the blade of aseptic seedling, petiole, stipes and all can become effective transformation receptor, aseptic seedling can Preservation in sterile condition in laboratory conditions for a long time, and the source of explant is unrestricted, can carry out genetically modified operation at any time.
(3) method of strict effective screening transformant
Respectively through 10mgl
-1totomycin, 5mgl
-1glufosinate ammonium, 100mgl
-1the resistant strain obtained is pressed in kantlex screening, proves to be all genetically modified materials through GUS dyeing, Southern hybrid experiment.Which ensure that the resistant plant obtained by this technological method is transfer-gen plant, decreases the workload that the later stage adopts Molecular Detection.
(4) transformation efficiency is high, repeatability is high
Technological method provided by the invention, through repeatedly repeating experiment, proves that transformation efficiency is 25-50%.A transformation experiment operation easily obtains in a large number because transforming explant used, so just can obtain required transgenic line.
(5) breeding efficiency is high
By above operation, each transformation explant all can produce Multiple Buds, Multiple Buds is again by shoot proliferation, every 1 shoot proliferation cycle can be bred by 1 bud branch and be formed more than 10 bud branches, and plant can be become by rooting development, therefore apply the inventive method and can obtain a large amount of transgenosis strains in 9 weeks, 100% transgenosis strain can successfully be transplanted in soil, ensure that the variety culture success that molecular breeding method is cultivated.
Accompanying drawing explanation
Fig. 1 is that Linear Stonecrop Herb is by bud adventitious organogenesis regeneration plant.Wherein, A be leaf explant when being inoculated in bud inducement culture medium culturing 16 days the otch of explant have the generation of regeneration bud; B is the regeneration bud of leaf explant when cultivating 35 days on bud inducement substratum; The Transplantation of Regenerated Plantlets having 2cm high is equipped with in vermiculite basin growing state when growing 35 days by C.
Fig. 2 is the T-DNA area schematic of plant expression vector.Wherein, 35Spro is the promotor of the CaMV35S of tobacco cauliflower mosaic virus.
Fig. 3 is the resistant buds turning pCAMBIA1301 plasmid when being inoculated in resistant buds induction screening culture medium 21 days.
Fig. 4 be with agrobacterium tumefaciens EHA105/pCAMBIA1301 Dual culture 3 days after Linear Stonecrop Herb leaf explant gus gene transient expression detected result.
Fig. 5 is that GUS dyeing presents the blue resistant buds turning pCAMBIA1301 plasmid.
Fig. 6 is without the GUS unconverted bud branch dyeed and the resistant buds branch turning pCAMBIA1301 plasmid having GUS to dye.Wherein, figure A does not present blue unconverted bud through GUS dyeing when being inoculated in bud elongation medium 2 weeks, turns dyeing through GUS of pCAMBIA1301 plasmid gained and present the resistant buds of blueness when figure B is and is inoculated into resistant buds elongation medium 2 weeks.
Fig. 7 is the result of Southern hybridization check pCAMBIA1301 plasmid transformant.Wherein, WT is unconverted strain, and display does not have hybridization signal; T1, T2, T3, T4, T5 are pCAMBIA1301 plasmid transformant, all have hybridization signal and are all different transformation plants.
Fig. 8 is unconverted plant when transplanting soil 28 days and the plant turning pCAMBIA1301 plasmid.Figure A and figure B is unconverted plant and the regeneration plant that turns pCAMBIA1301 plasmid plant growing state when transplanting soil incubation 28 days when being about 2cm respectively.
Embodiment
Following examples further illustrate of the present invention, instead of limitation of the present invention.
In following Examples, the concrete experimental technique indicated, all can conventionally carry out, or according to the operation instruction of products production manufacturer used.Material used in following embodiment, reagent etc., if no special instructions, all obtain by commercial sources.
Embodiment 1: the acquisition of aseptic seedling
Buy Linear Stonecrop Herb from market, get well-grown sprout, after clean with tap water, the carbendazim solution putting into massfraction 0.1% soaks 5-10 minute, clean with tap water after taking-up, branch is cut into 5 cm length.30 seconds are soaked with volume fraction 75% ethanol aqueous solution, after using aseptic water washing 3 times again, branch is moved into massfraction 2.5% chlorine bleach liquor (adding massfraction 0.05% tween 80) to sterilize 10 minutes, with aseptic water washing 6 times, then branch is placed in suck dry moisture on aseptic filter paper.Branch is cut into one joint segment, after be inoculated into 1/2MS substratum one by one.Be placed in temperature 25 ± 1 DEG C, illuminance 50 μm of olm
– 2s
– 1, cultivate under illumination 12h/ days condition.Cultivate about 20 days, axil Fiber elongation is bud branch, and the otch at contact substratum place grows adventive root.Shoot proliferation is carried out under cutting out identical fresh 1/2MS substratum the same terms of transferring during the high about 5cm of bud branch.Within general 21 days, be 1 shoot proliferation cycle, every 1 shoot proliferation cycle can be bred by 1 bud branch and be formed 10-15 bud branch, and the bud branch of acquisition is the aseptic seedling that will obtain.
Embodiment 2: the acquisition of regeneration plant
Cut blade respectively from aseptic seedling, petiole, stipes be explant, (MS substratum adds 1mgl to be inoculated in bud inducement substratum
-1benzyladenine (BA) and 0.1mgl
-1a-Naphthaleneaceticacid (NAA), pH5.8), put temperature 25 ± 2 DEG C, illuminance 50 μm of olm
– 2s
– 1, cultivate under illumination 12h/ days condition.When cultivating 16 days, the incision of explant of more than 95% can be observed the generation (Figure 1A) of regeneration bud, transfer to continue to cultivate under identical fresh bud inducement substratum the same terms have a large amount of regeneration bud to grow (Figure 1B) as seen by cultivating 16 days leaf explants with regeneration bud under light, bud elongation medium that regeneration bud is transferred (1/2MS substratum, pH is 5.8) cultivate under the same terms, bud branch Fiber elongation, contact substratum base portion has the generation of a large amount of adventive root, rooting rate 100%, obtains regeneration plant thus.
Shifted out from culturing bottle by regeneration plant high for 2cm, wash away root substratum, field planting, in the basin that vermiculite is housed, covers with preservative film, and preservative film is beaten a little aperture, opens after 5 days, survival rate 100% (Fig. 1 C).Cut blade from aseptic seedling, petiole, stipes explant survive to Transplantation of Regenerated Plantlets soil, whole operating process is 7 weeks.
Embodiment 3: for the cultivation of Linear Stonecrop Herb explant transformed
Be 1 subculture with 21 days, cultivate under constantly 2cm height bud branch being cut switching 1/2MS substratum the same terms, expand numerous aseptic seedling; Cut blade respectively from aseptic seedling, petiole, stipes be explant, contaminate used for agrobacterium tumefaciens.
Embodiment 4: the preparation of the engineering strain containing target gene plant expression vector
1, the preparation of agrobacterium tumefaciens EHA105/pCAMBIA1301 bacterium liquid
By freeze-thaw method, using pCAMBIA1301 carrier, (pCAMBIA1301 carrier contains by CaMV35S promoters driven β-glucal Glycosylase reporter gene gus and hygromix phosphotransferase screening-gene hpt as screening-gene, target gene can need to be placed in (Fig. 2) after CaMV35S promotor according to breeding objective, the present embodiment is using β-glucal Glycosylase reporter gene gus as experiment test gene) import agrobacterium tumefaciens EHA105, be then seeded in containing 50mgl
– 1kantlex and 50mgl
– 1in the YEP substratum of Rifampin, cultivate 24 hours under 28 DEG C of conditions, by collected by centrifugation bacterium liquid, mycetocyte is suspended in containing 20mgl
-1in the MS liquid nutrient medium (pH5.2) of Syringylethanone, OD600=0.4 ~ 0.5, obtains the agrobacterium tumefaciens EHA105 bacterium liquid containing pCAMBIA1301 carrier, called after agrobacterium tumefaciens EHA105/pCAMBIA1301 thus.
2, the preparation of agrobacterium tumefaciens EHA105/pCAMBIA3301 bacterium liquid
By freeze-thaw method, using pCAMBIA3301 carrier, (pCAMBIA3301 carrier contains by CaMV35S promoters driven β-glucal Glycosylase reporter gene gus and Glufosinate ammonium resistant gene bar as screening-gene, target gene can need to be placed in after CaMV35S promotor according to breeding objective, the present embodiment is using β-glucal Glycosylase reporter gene gus as experiment test gene) import agrobacterium tumefaciens EHA105, be then seeded in containing 50mgl
– 1kantlex and 50mgl
– 1in the YEP substratum of Rifampin, cultivate 24 hours under 28 DEG C of conditions, by collected by centrifugation bacterium liquid, mycetocyte is suspended in containing 20mgl
-1in the MS liquid nutrient medium (pH5.2) of Syringylethanone, OD600=0.4 ~ 0.5, obtains the agrobacterium tumefaciens EHA105 bacterium liquid containing pCAMBIA3301 carrier, called after agrobacterium tumefaciens EHA105/pCAMBIA3301 thus.
3, the preparation of agrobacterium tumefaciens EHA105/pBI121 bacterium liquid
By freeze-thaw method, using pBI121 carrier, (pBI121 carrier contains by CaMV35S promoters driven β-glucal Glycosylase reporter gene gus and kalamycin resistance gene NPTII as screening-gene, target gene can need to be placed in after CaMV35S promotor according to breeding objective, the present embodiment is using β-glucal Glycosylase reporter gene gus as experiment test gene) import agrobacterium tumefaciens EHA105, be then seeded in containing 50mgl
– 1kantlex and 50mgl
– 1in the YEP substratum of Rifampin, cultivate 24 hours under 28 DEG C of conditions, by collected by centrifugation bacterium liquid, mycetocyte is suspended in containing 20mgl
-1in the MS liquid nutrient medium (pH5.2) of Syringylethanone, OD600=0.4 ~ 0.5, obtains the agrobacterium tumefaciens EHA105 bacterium liquid containing pBI121 carrier, called after agrobacterium tumefaciens EHA105/pBI121 thus.
Embodiment 5: the acquisition turning the hygromycin regeneration plant of pCAMBIA1301
Explant prepared by embodiment 3 is soaked in the agrobacterium tumefaciens EHA105/pCAMBIA1301 bacterium liquid prepared by embodiment 4, time is 10 minutes, take out explant, be placed on aseptic paper and blot unnecessary bacterium liquid, after explant moved to Dual culture base (MS substratum adds 1mgl
-1bA, 0.1mgl
-1nAA and 20mgl
-1syringylethanone, pH is 5.5), put 25 ± 1 DEG C, dark lower cultivation 3 days.Take out the explant after Dual culture, with the aseptic water washing containing massfraction 0.05% tween 80 5 ~ 6 times, finally with containing 500mgl
-1the aseptic water washing of cephamycin once, is put explant in aseptic paper, is blotted water, and (MS substratum adds 1mgl explant to be moved to resistant buds induction screening culture medium
-1bA, 0.1mgl
-1nAA, 10mgl
-1totomycin and 500mgl
-1cephamycin, pH is 5.8).Put 25 ± 1 DEG C, cultivate 18 days under light.
Continue cultivation under explant (Fig. 3) with resistant buds being transferred to identical fresh resistant buds induction screening culture medium the same terms and after 2 weeks, be forwarded to resistant buds elongation medium (1/2MS substratum interpolation 10mgl
-1totomycin and 250mgl
-1cephamycin, pH is 5.8), put 25 ± 1 DEG C, cultivate under light, can be observed the elongation of resistant buds and a large amount of generations of adventive root.Obtain the hygromycin regeneration plant (resistant plant) turning pCAMBIA1301 thus.
Embodiment 6: the acquisition turning the anti-Glufosinate ammonium regeneration plant of pCAMBIA3301
Be soaked in by explant prepared by embodiment 3 in the agrobacterium tumefaciens EHA105/pCAMBIA3301 bacterium liquid prepared by embodiment 4, experimental implementation is identical with embodiment 5, unlike, resistant buds induction screening and culturing based component used is: MS substratum adds 1mgl
-1bA, 0.1mgl
-1nAA, 5mgl
-1glufosinate ammonium and 500mgl
-1cephamycin, pH is 5.8; Resistant buds elongation medium composition used is: 1/2MS substratum adds 5mgl
-1glufosinate ammonium and 250mgl
-1cephamycin, pH is 5.8.Obtain the anti-Glufosinate ammonium regeneration plant (resistant plant) turning pCAMBIA3301 thus.
Embodiment 7: the acquisition turning the anti-kantlex regeneration plant of pBI121
Be soaked in by explant prepared by embodiment 3 in the agrobacterium tumefaciens EHA105/pBI121 bacterium liquid prepared by embodiment 4, experimental implementation is identical with embodiment 5, unlike, resistant buds induction screening and culturing based component used is: MS substratum adds 1mgl
-1bA, 0.1mgl
-1nAA, 100mgl
-1kantlex and 500mgl
-1cephamycin, pH is 5.8; Resistant buds elongation medium composition used is: 1/2MS substratum adds 100mgl
-1kantlex and 250mgl
-1cephamycin, pH is 5.8.Obtain the anti-kantlex regeneration plant (resistant plant) turning pBI121 thus.
Embodiment 8: the Molecular Detection of transfer-gen plant
Because pCAMBIA1301 contains gus reporter gene, the expression of staining examine gus gene can be carried out by the GUS dyeing process of (1987) such as Jefferson.Get with agrobacterium tumefaciens EHA105/pCAMBIA1301 Dual culture 3 days after explant, resistant buds, unconverted bud branch and transform bud branch and be dipped in X-Gluc staining fluid respectively, in 37 DEG C of insulations 3 ~ 5 hours, use 70% ethanol decolorization, observe blue-colored situation.There is obvious locus coeruleus on visible explant (Fig. 4) surface with agrobacterium tumefaciens EHA105/pCAMBIA1301 Dual culture after 3 days, illustrate that agrobacterium tumefaciens EHA105/pCAMBIA1301 can adsorb and contaminate Linear Stonecrop Herb explant, resistant buds (Fig. 5) and conversion bud branch (Fig. 6) also all present blueness, unconverted bud (Fig. 6) does not then present blueness, illustrate by above operation, gus gene successfully imported Linear Stonecrop Herb and in transformant stably express.Adopt the Auele Specific Primer of screening-gene hpt: HFw (5 '-CGATCTTAGCCAGACGAGCGGGTTC-3 ') and HRe (5 '-GCTGGGGCGTCGGTTTCCACTATCGG-3 ') synthesising probing needle, by the method that Southern is hybridized, at resistant plant T1, T2, T3, T4, all hybridization signal can be observed in T5, and banding pattern different (Fig. 7), T1 is described, T2, T3, T4, T5 is different transgenic lines, and unconverted strain WT (wild-type) does not have signal, again illustrate by above the method, hpt gene has successfully imported the genome of Linear Stonecrop Herb.
PCAMBIA1301, pCAMBIA3301 and pBI121 are all containing gus reporter gene, the hygromycin regeneration plant turning pCAMBIA1301 that embodiment 5-7 is obtained, turn the anti-Glufosinate ammonium regeneration plant of pCAMBIA3301, turn the anti-kantlex regeneration plant of pBI121, detected the expression of its gus gene by GUS dyeing process, find respectively through 10mgl
-1totomycin, 5mgl
-1glufosinate ammonium, 100mgl
-1kantlex screening presses the resistant plant obtained all to have GUS to dye, and therefore, adopts screening pressure 10mgl when Linear Stonecrop Herb transforms respectively
-1totomycin, 5mgl
-1glufosinate ammonium, 100mgl
-1kantlex can guarantee that obtained resistant plant is transfer-gen plant, reduces the workload that the later stage adopts Molecular tools detection transfer-gen plant.
By above method, in 9 weeks, a large amount of transfer-gen plants can be obtained, with 10mgl
-1totomycin is that screening presses the transformation frequency turning the hygromycin plant of pCAMBIA1301 obtained to be about 50%, with 100mgl
-1kantlex is that screening presses the transformation frequency turning the anti-kantlex plant of pBI121 obtained to be about 50%, and with 5mgl
-1glufosinate ammonium is that screening presses the transformation frequency turning the anti-Glufosinate ammonium plant of pCAMBIA3301 obtained to be then about 25%.
Embodiment 9: the transplanting of transgenic regenerated plant
Cut blade from aseptic seedling, petiole, stipes explant carry out agrobacterium tumefaciens and contaminate the transfer-gen plant obtaining and can transplant, whole operating process is 9 weeks.Shifted out from culturing bottle by transfer-gen plant (resistant plant prepared by embodiment 5-7) higher than 2cm, wash away root substratum, field planting is in the basin that vermiculite is housed, cover with preservative film, preservative film is beaten a little aperture, opens after 5 days, survival rate 100%.
(according to embodiment 1 and embodiment 2, method prepares height prepared by embodiment 5 to be about the hygromycin regeneration plant turning pCAMBIA1301 of 2cm and the high regeneration plant being about the unconverted strain of 2cm, shift out from culturing bottle respectively in contrast), wash away root substratum, field planting, in the basin that vermiculite is housed, covers with preservative film, and preservative film is beaten a little aperture, open after 5 days, field planting is after 28 days, the growing state of both observation and comparisons, as shown in Figure 8.As can be seen from the figure, the hygromycin regeneration turning pCAMBIA1301 is substantially identical with the regeneration plant upgrowth situation of unconverted strain, shows that Linear Stonecrop Herb genetic transformation regeneration system successfully constructs.
Claims (9)
1. obtain a molecular breeding method for a large amount of transgenosis Linear Stonecrop Herb new variety fast, it is characterized in that, comprise the following steps:
The acquisition of a, aseptic seedling and explant: get Linear Stonecrop Herb edible tender branch, after sterilization, branch is cut into one joint segment, after be inoculated into 1/2MS substratum one by one, be placed in temperature 25 ± 1 DEG C, illuminance 50 μm of olm
– 2s
– 1cultivate under illumination 12h/ days condition, axil Fiber elongation is branch, the otch at contact substratum place grows adventive root, shoot proliferation is carried out under bud branch being cut out 1/2MS substratum the same terms of transferring, breed the bud branch obtained and be aseptic seedling, cut blade, petiole, stipes respectively as explant from aseptic seedling;
B, the explant prepared by step a is soaked in the agrobacterium tumefaciens bacterium liquid containing the plant expression vector carrying target gene, this plant expression vector contains riddled basins, then explant is taken out, explant is moved in Dual culture base after blotting unnecessary bacterium liquid, put 25 ± 1 DEG C, dark lower cultivation, then the explant after Dual culture is taken out, residual water-content on explant is blotted after rinsing, explant is moved in resistant buds induction screening culture medium, put 25 ± 1 DEG C, cultivate under light, after growing resistant buds, continue to cultivate under being forwarded to resistant buds induction screening culture medium the same terms, and then be forwarded in resistant buds elongation medium, put 25 ± 1 DEG C, cultivate under light, resistant buds extends and produces adventive root, obtain resistant plant,
Described Dual culture base is: MS substratum adds 1mgl
-1bA, 0.1mgl
-1nAA and 20mgl
-1syringylethanone, pH is 5.5;
The Molecular Detection of c, transfer-gen plant: utilize the sequence of the riddled basins on plant expression vector, reporter gene or target gene to carry out PCR Molecular Detection method, Southern hybridizing method or RT-PCR detection method, detects resistant buds or whether resistant plant has been transgenic line;
The transplanting of d, transfer-gen plant: shifted out from culturing bottle by the transfer-gen plant of taking root detected containing target gene, wash away root substratum, field planting, in the basin that cultivation matrix is housed, obtains transplanting successful transfer-gen plant.
2. the molecular breeding method of quick acquisition according to claim 1 a large amount of transgenosis Linear Stonecrop Herb new variety, it is characterized in that, described step b's is pCAMBIA1301 carrier containing target gene containing the plant expression vector carrying target gene, its riddled basins is hygromix phosphotransferase screening-gene, and the resistant buds induction screening culture medium of described step b is add 1mgl in MS substratum
-1bA, 0.1mgl
-1nAA, 10mgl
-1totomycin and 500mgl
-1cephamycin, pH is 5.8; The resistant buds elongation medium of described step b is that 1/2MS substratum adds 10mgl
-1totomycin and 250mgl
-1cephamycin, pH is 5.8.
3. the molecular breeding method of quick acquisition according to claim 1 a large amount of transgenosis Linear Stonecrop Herb new variety, it is characterized in that, described step b's is pCAMBIA3301 carrier containing target gene containing the plant expression vector carrying target gene, its riddled basins is Glufosinate ammonium resistant gene, and the resistant buds induction screening culture medium of described step b is that MS substratum adds 1mgl
-1bA, 0.1mgl
-1nAA, 5mgl
-1glufosinate ammonium and 500mgl
-1cephamycin, pH is 5.8; The resistant buds elongation medium of described step b is that 1/2MS substratum adds 5mgl
-1glufosinate ammonium and 250mgl
-1cephamycin, pH is 5.8.
4. the molecular breeding method of quick acquisition according to claim 1 a large amount of transgenosis Linear Stonecrop Herb new variety, it is characterized in that, described step b's is pBI121 carrier containing target gene containing the plant expression vector carrying target gene, and its riddled basins is kalamycin resistance gene; The resistant buds induction screening culture medium of described step b is that MS substratum adds 1mgl
-1bA, 0.1mgl
-1nAA, 100mgl
-1kantlex and 500mgl
-1cephamycin, pH is 5.8; The resistant buds elongation medium of described step b is that 1/2MS substratum adds 100mgl
-1kantlex and 250mgl
-1cephamycin, pH is 5.8.
5. the molecular breeding method of quick acquisition according to claim 1 a large amount of transgenosis Linear Stonecrop Herb new variety, it is characterized in that, the described agrobacterium tumefaciens containing the plant expression vector carrying target gene builds by the following method: after target gene being inserted expression vector promotor, by freeze-thaw method, the plant expression vector carrying target gene built is imported agrobacterium tumefaciens, through resistance screening, obtain the agrobacterium tumefaciens containing the plant expression vector carrying target gene.
6. the molecular breeding method of quick acquisition according to claim 1 a large amount of transgenosis Linear Stonecrop Herb new variety, it is characterized in that, the sterilization of described step a, be Linear Stonecrop Herb edible tender branch tap water is clean after, the carbendazim solution putting into massfraction 0.1% soaks 5-10 minute, then taking-up tap water is clean, use volume fraction 75% more alcohol-pickled 30 seconds, after aseptic water washing 3 times, branch is moved into and sterilizes 10 minutes containing massfraction 2.5% chlorine bleach liquor of massfraction 0.05% tween 80, with aseptic water washing 6 times.
7. the molecular breeding method of quick acquisition according to claim 1 a large amount of transgenosis Linear Stonecrop Herb new variety, is characterized in that, the rinsing of described step b, is the aseptic water washing of use containing massfraction 0.05% tween 80 5 ~ 6 times, then with containing 500mgl
-1the aseptic water washing of cephamycin once.
8. the molecular breeding method of quick acquisition according to claim 1 a large amount of transgenosis Linear Stonecrop Herb new variety, is characterized in that, the cultivation matrix of described steps d is vermiculite.
9. the molecular breeding method of quick acquisition according to claim 1 a large amount of transgenosis Linear Stonecrop Herb new variety, it is characterized in that, the step be soaked in by explant in described b step in the agrobacterium tumefaciens bacterium liquid containing the plant expression vector carrying target gene is first suspended in by the agrobacterium tumefaciens containing the plant expression vector carrying target gene in the MS substratum containing 20mg/l Syringylethanone, its pH is 5.2, and cellar culture is to OD
600=0.4 ~ 0.5, be then soaked in by explant in this agrobacterium tumefaciens bacterium liquid, the time is 10min.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105284626A (en) * | 2015-12-04 | 2016-02-03 | 吉林大学 | Method for directly generating adult seedlings by genetic transformation of septate internodes of mature seed seedlings of maize inbred line |
| CN110042106A (en) * | 2018-01-17 | 2019-07-23 | 天津大学 | Sedum lineare SLJGR gene and its application |
| CN111621519A (en) * | 2020-06-16 | 2020-09-04 | 彩星(淄博)生物科技有限公司 | Genetic transformation method and application of succulent plant |
| CN115386591A (en) * | 2022-09-05 | 2022-11-25 | 中国科学院华南植物园 | Molecular breeding method of monochantia sonchifolia |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103233041A (en) * | 2013-05-06 | 2013-08-07 | 福建农林大学 | Rapid agrobacterium-mediated transgenic method |
| EP2698432A1 (en) * | 2012-08-17 | 2014-02-19 | University of Copenhagen | Agrobacterium rhizogenes transformation and expression of rol genes in Kalanchoë |
-
2015
- 2015-08-21 CN CN201510518685.3A patent/CN105063086B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP2698432A1 (en) * | 2012-08-17 | 2014-02-19 | University of Copenhagen | Agrobacterium rhizogenes transformation and expression of rol genes in Kalanchoë |
| CN103233041A (en) * | 2013-05-06 | 2013-08-07 | 福建农林大学 | Rapid agrobacterium-mediated transgenic method |
Non-Patent Citations (3)
| Title |
|---|
| E.S.YOON ET AL: "Recovery of Basta-resistant Sedum erythrostichum cia Agrobacterium-mediated transformation", 《PLANT CELL REPORTS》 * |
| 汤聪 等: "高温高湿环境佛甲草栽培基质的研制", 《草业科学》 * |
| 齐小晴 等: "药用植物佛甲草愈伤组织的诱导和培养", 《生物技术》 * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105284626A (en) * | 2015-12-04 | 2016-02-03 | 吉林大学 | Method for directly generating adult seedlings by genetic transformation of septate internodes of mature seed seedlings of maize inbred line |
| CN110042106A (en) * | 2018-01-17 | 2019-07-23 | 天津大学 | Sedum lineare SLJGR gene and its application |
| CN110042106B (en) * | 2018-01-17 | 2021-11-02 | 天津大学 | Sedum lineare SLJGR gene and application thereof |
| CN111621519A (en) * | 2020-06-16 | 2020-09-04 | 彩星(淄博)生物科技有限公司 | Genetic transformation method and application of succulent plant |
| CN115386591A (en) * | 2022-09-05 | 2022-11-25 | 中国科学院华南植物园 | Molecular breeding method of monochantia sonchifolia |
| CN115386591B (en) * | 2022-09-05 | 2024-05-28 | 中国科学院华南植物园 | Molecular breeding method of single herba Cichorii |
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