CN102994544A - Method for cultivating transgenetic gloxinia plant through agrobacterium-mediated transformation - Google Patents
Method for cultivating transgenetic gloxinia plant through agrobacterium-mediated transformation Download PDFInfo
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Abstract
The invention relates to a regeneration method for gloxinia plant, in particular to a method for cultivating transgenetic gloxinia plate through agrobacterium-mediated transformation, which comprises the following steps of taking young leaves of gloxinia as explant, removing epibiotic antibiotic in the culture after precultivation of the explant so as to be cultivated with bacterium liquid of agrobacterium strain EHA105 of bacterium metabolin, eliminating bacterium, thus obtaining transgenetic plant through direct differentiation after resistance screening. Due to the adoption of the technical scheme, gloxinia can be transformed efficiently and rapidly through agrobacterium-mediated transformation, therefore, the transformed plant is obtained and transgenetic gloxinia plant is successfully obtained for the first time, and better result is achieved.
Description
Technical field
The present invention relates to the gloxinia plant regeneration method, relate in particular to the method that agriculture bacillus mediated transgenosis is cultivated the gloxinia plant.
Background technology
Flowers are as the fresh and alive commodity of viewing and admiring, at Christmas, demand is large before the major holiday such as New Year's Day, the Spring Festival, its price differs up to 7,8 times at different times such as demand peak, general, stagnations, usually forms and supplies with climax and the dislocation of demand peak phase, causes very large production and liquidity risk.Therefore, use the Target Flowering flower variety that the modern genetic engineering technology is cultivated independent intellectual property right, not only be conducive to protect flower-grower's economic interests, more be conducive to promote the high-tech content of China's industry of flowers and plants, rank among the ranks of world flower production power.
Gloxinia (Sinningia speciosa) originates in Brazil, is the perennial flower bulbs of Gesneriaceae, is a kind of fabulous spring and summer indoor potted flower in season, all over the world extensively cultivation.The gloxinia explant very easily breaks up, and can directly break up seedling at the explant edge without callus on the MS substratum of additional 2.0 mg/L 6-BA and 0.2 mg/L NAA, and growth is also than faster.
As Chinese invention patent (application number: 200410061427.9 applyings date: 2004-12-24), a kind of culture method of Sinningia speciosa is disclosed, take tissue culture without offspring as material, its step is, at first the tender tip of gloxinia downcut; Next is that young sprout is inserted in the matrix; The 3rd is the pouring nutritive medium; The 4th is to take root the seedling field planting in identical matrix.But the transgenic research of gloxinia had no report always both at home and abroad.
Summary of the invention
In order to solve above-mentioned technical problem, the purpose of this invention is to provide the method that a kind of agriculture bacillus mediated transgenosis is cultivated the gloxinia plant, the method can rapidly and efficiently transform gloxinia, obtains transformed plant.
In order to realize above-mentioned purpose, the present invention has adopted following technical scheme:
Agriculture bacillus mediated transgenosis is cultivated the method for gloxinia plant, the method adopts the gloxinia young leaflet tablet as explant, explant is trained microbiotic remaining in rear and the removal culture in advance and the agrobacterium strains EHA105 bacterium liquid of bacterium metabolite is trained altogether, take off bacterium, after passing through resistance screening again, directly differentiation obtains transfer-gen plant.
As preferably, the OD of above-mentioned agrobacterium strains EHA105 bacterium liquid
600Be 0.2 ~ 0.4, the time of training is 2 ~ 4 days altogether.
As preferably, the method for above-mentioned common training is as follows: take out pre-incubated blade in aseptic small beaker, add in the bacterium liquid for preparing and soak 3 ~ 8min, shaken several times; Take out blade, blot unnecessary bacterium liquid at aseptic filter paper, blade back places on the basic MS solid medium up, 25 ℃, secretly cultivates 2 ~ 4d.
As preferably, above-mentioned agrobacterium strains EHA105 bacterium liquid is cultivated to adopt and is contained kantlex and Rifampin LB liquid nutrient medium is cultivated.
As most preferably, the preparation method of above-mentioned agrobacterium strains EHA105 bacterium liquid is as follows:
1) take out the agrobacterium strains EHA105 that contains recombinant plasmid from-70 ℃ of refrigerators, containing microbiotic Kan 100 mg/L, line activation on the LB solid medium of Rif 80 mg/L is until grow the single bacterium colony of Agrobacterium on the substratum;
2) get 5～10ml LB liquid nutrient medium, add microbiotic-kantlex and Rifampin, making its final concentration is Kan 100mg/L, Rif 80mg/L; Picking list bacterium colony adds wherein from fresh flat board, shakes up, and in 28 ℃, 180 r/min incubated overnight are to logarithmic phase, OD
600Be about 0.8～1.0;
3) draw above-mentioned bacterium liquid 1ml in the 1.5ml centrifuge tube, 4 ℃, the centrifugal 2min of 4000rpm removes supernatant, uses 30ml 1/2 MS suspension Agrobacterium again, shakes about 1-2h again, shows slightly muddy to bacterium liquid, OD
600Be 0.2 ~ 0.4 to get final product, for subsequent use.
As preferably, the above-mentioned bacterium that takes off is adopted the washing of Ticarcillin/Clavulanate Acid sterile solution.Because after existing Pyocianil and cephamycin used continuously, the resistant calli differentiation frequency was lower, therefore selected in the present invention Ticarcillin/Clavulanate Acid (160 mg/L) degerming, can obtain like this differentiation capability that bacteria-eliminating efficacy does not affect again the resistance tissue.
As preferably, the marker gene that above-mentioned resistance screening adopts is hpt, adopts Totomycin as selective pressure.In the conversion of gloxinia, existing kantlex screening can be disturbed the regeneration of green plant, and most plant is responsive to Totomycin comparison kantlex, and it is more effective than the kantlex that Totomycin is used for Plant Transformation.Still all be to select Totomycin as the resistance screening mark.
As preferably, the concentration of above-mentioned Totomycin is 20mg/L.
As preferably, the resistance of above-mentioned resistance screening selects substratum to be: MS+6-BA 2 mg/L+NAA 0.3 mg/L+
Hgy20mg/L+Tm 160 mg/L; Carry out two-wheeled and select to cultivate, the every wheel continues 12 ~ 15d.
As preferably, the pre-training of above-mentioned explant get the gloxinia aseptic seedling in, the blade on top, cut a circle along leaf margin, receive on the basic MS solid medium that contains 80 ~ 120 uM Syringylethanones, 25 ℃, secretly cultivate 20 ~ 30h.
The present invention by agrobacterium-mediated transformation, can rapidly and efficiently transform gloxinia owing to adopted above-mentioned technical scheme, obtains transformed plant and successfully obtains first the gloxinia transfer-gen plant, has obtained preferably achievement.
Description of drawings
The group training process of Fig. 1 transgenosis gloxinia.
The tissue culture and inducement of Fig. 2 transgenosis gloxinia breaks up and takes root.
The regeneration of Fig. 3 gloxinia transformed plant.
Fig. 4 different concns hygromycin selection is depressed the Leaves of Sinningia speciosa of cultivation.
Embodiment
Embodiment 1: the cultivation of Agrobacterium
(1) take out the agrobacterium strains EHA105 that contains recombinant plasmid from-70 ℃ of refrigerators, line activation on the LB solid medium that contains microbiotic (Kan 100 mg/L, Rif 80 mg/L) is until grow the single bacterium colony of Agrobacterium on the substratum;
(2) get 5～10ml LB liquid nutrient medium, add microbiotic---kantlex and Rifampin, making its final concentration is Kan 100mg/L, Rif 80mg/L.Picking list bacterium colony adds wherein from fresh flat board, shakes up, and in 28 ℃, 180 r/min incubated overnight are to logarithmic phase, OD
600Be about 0.8～1.0;
(3) draw above-mentioned bacterium liquid 1ml (1ml * 2) in the 1.5ml centrifuge tube, 4 ℃, the centrifugal 2min of 4000rpm removes supernatant.Use 30ml 1/2 MS suspension Agrobacterium (add in advance 2ml 1/2 MS, inhale with liquid-transfering gun and beat evenly) again, shake about 1-2h again, show slightly muddy to bacterium liquid, OD600 is about 0.3 and gets final product, and is for subsequent use.
Embodiment 2: the processing of explant
Get the gloxinia aseptic seedling in, the blade on top, cut a circle along leaf margin, receive on the basic MS solid medium that contains 100 uM Syringylethanones (As), 25 ℃, secretly cultivate 1d.
Embodiment 3: transform
(1) takes out pre-incubated blade in aseptic small beaker, add in the bacterium liquid for preparing and soak about 5min shaken several times;
(2) take out blade, blot unnecessary bacterium liquid at aseptic filter paper, blade back places on the basic MS solid medium up, 25 ℃, secretly cultivates 3 d.
Embodiment 4: take off bacterium and screening and culturing
(1) secretly cultivate 3d after, from substratum, collect blade in the small beaker of sterilization, through following process washing: the 2 times → aseptic filter paper of aseptic washing that rinsed with sterile water 3 times → add 50ml contains 160mg/L Ticarcillin/Clavulanate Acid (Tm) blots;
(2) then change resistance over to and select substratum 1. to go up, carry out two-wheeled and select to cultivate, the every wheel continued for 2 weeks;
1. Totomycin (hgy) selects substratum: MS+6-BA 2 mg/L+NAA 0.3 mg/L+hgy 20mg/L+Tm 160 mg/L;
(3) with substratum 1. the hygromycin resistance callus transposition of the upper regeneration substratum of sprouting 2. go up;
Sprout substratum 2.: MS+KT 2.5 mg/L+NAA 1.9 mg/L+ GA3 1.0 mg/L;
(4) will be transplanted to proliferated culture medium the plant that 2. substratum of sprouting grows to 2～3cm 3. continue to cultivate;
Proliferated culture medium is 3.: MS+KT 2.0 mg/L+NAA 0.2 mg/L+ GA3 1.0 mg/L;
(5) plant that proliferated culture medium is grown to 2～3cm on 3. moves into root media and 4. goes up, and does root culture;
Root media is 4.: 1/2 MS+NAA, 0.2 mg/L+ sucrose 2%.
Embodiment 5: the PCR of transformed plant identifies
1, choose transformed plant, extract genomic dna with CTAB-free method trace, do pcr template, identify:
(1) spire that takes a morsel, in liquid nitrogen bath, clay into power with mortar, powder is moved into the 1.5ml centrifuge tube, add 0.6ml CTAB-free damping fluid (200mmol/L Tris-HCl, 50mmol/L EDTA, 250mmol/L NaCl, 1% mercaptoethanol), the concussion mixing is placed on places 10min on ice.
(2) 4 ℃, the centrifugal 5min of 8000r/min, careful supernatant discarded adds 0.3ml and is preheated to 3 * CTAB damping fluid (3% CTAB of 65 ℃, 100mmol/L Tris-HCl, 25mmol/L EDTA, 1.5mol/L NaCl, 1% mercaptoethanol), resuspended precipitation, 65 ℃ of water-bath 30min.Add equal-volume chloroform/primary isoamyl alcohol, put upside down gently mixing, static to layering.
(3) the centrifugal 5min of 5000rpm moves on to upper water in the new 1.5ml centrifuge tube mutually, adds the dehydrated alcohol of two volumes, leaves standstill 5min after putting upside down mixing, and DNA that will be cotton-shaped with suction pipe precipitates and forwards new 1.5ml centrifuge tube to.
(4) precipitate 2 times with 1ml 70% washing with alcohol, dry air 20min is dissolved in 20 μ l TE(RNase) damping fluid, 4 ℃ save backup.
2, the Trizol method is extracted gloxinia RNA
(1) 75% alcohol wipe pilot region, whole experimentation need be worn gloves and mouth mask, and in time changes.
(2) get 0.6mg gloxinia spire, the liquid nitrogen grinding sample treats that sample grinds fully, gets aseptic spoon (placing in advance the liquid nitrogen precooling), will organize powder to be transferred to the 1.5ml centrifuge tube of precooling, and every pipe packing 0.1 restrains sample.
(3) add rapidly 1ml Trizol reagent (sample volume be no more than Trizol reagent volume 10%), shake rapidly mixing (can first mixed sample be placed on ice if sample is more); Only put 5～10min under the room temperature and be beneficial to dissociating of nucleic acid protein complex body.
(4) 4 ℃, the centrifugal 10min of 12000 rpm draws supernatant, moves to new 1.5ml centrifuge tube.
(5) add the chloroform of 200 μ l, acutely sway 15～30sec, leave standstill about 5min under the room temperature.
(6) 4 ℃, the centrifugal 15min of 12000 rpm changes supernatant over to new centrifuge tube (400 μ l) (do not touch precipitation, the general absorption is no more than 800 μ l as far as possible).
(7) the volume Virahol that doubles, mixing precipitates 10min under the room temperature gently.
(8) 4 ℃, the centrifugal 15min of 12000rpm abandons supernatant.
(9) add 1ml 75% ethanol (the water configuration that DEPC processes) and clean RNA, vibration moments later (must make precipitation suspend, to guarantee washes clean), and the centrifugal 5min of 10000 rpm carefully abandons supernatant.
(10) room temperature leaves standstill 5～15min, and dry RNA precipitation adds 20～50 μ l DEPC water dissolution (65 ℃ of water-bath 10min).
(11) DNaseI processes, and system is as follows:
(12) add 200 μ l DEPC-H2O and 200 μ l chloroforms, static to layering behind the concussion mixing, 4 ℃, the centrifugal 5min of 12000rpm.
(13) get 200 μ l supernatants to new centrifuge tube, add the 3M sodium-acetate (pH5.2) of 1/10 volume, the dehydrated alcohol of 2.5 times of volumes is placed more than the 4h for-20 ℃.
(14) the centrifugal 15min of 12000rpm abandons supernatant, 75% alcohol washing precipitation, and drying adds an amount of DEPC-H2O dissolving, measures concentration and purity, is stored in-70 ℃ of refrigerators, and is for subsequent use.
3, sxemiquantitative RT-PCR
Common reverse transcription
(1) preparation following template ribonucleic acid/primer mixed solution in the Microtube pipe, full dose 12 μ l.
(2) 70 ℃ the insulation 10min after rapidly on ice more than the chilling 2min.
(3) the centrifugal several seconds makes the denaturing soln of template ribonucleic acid/primer be gathered in Microtube pipe bottom.
(4) the following inverse transcription reaction liquid of preparation in above-mentioned Microtube pipe.
| Reagent name | Usage quantity |
| Above-mentioned template ribonucleic acid/primer denaturing soln | 12 μl |
| 5×M- |
4 μl |
| Each 10 mM of dNTP Mixture() | 1 μl |
| RNase Inhibitor（40 U/μl） | 0.5 μl |
| RTase M-MLV（RNase H-）（200 U/μl） | 0.5 μl |
| RNase free dH2O | up to 20 μl |
(5) 42 ℃ of insulation 1h.
Cooled on ice behind (6) 70 ℃ of insulation 15min adds isopyknic ddH2O dilution, and the cDNA solution that obtains can be directly used in the synthetic or pcr amplification of 2nd-Strand cDNA etc., and 1 μ l～5 μ l are used in the suggestion of the usage quantity of cDNA solution during pcr amplification.
Sxemiquantitative PCR
At first draw 1 μ l RT product and do template, the gene expression abundance of first round PCR(actin that carries out house-keeping gene actin is higher, generally sets 26～28 circulations and gets final product), 20 μ l PCR systems, draw 10 μ l product electrophoresis, use the brightness of Image J software analysis actin band.Suitably adjust the template amount of each sample according to analytical results, and supply residual quantity with ddH2O, carry out second of actin and take turns PCR, with this repeatedly until the brightness of actin electrophoretic band is basically identical, the addition of a record sample template, carrying out successively the cycle number of each specific gene of PCR(of specific gene decides according to its gene expression abundance), and electrophoresis takes pictures, and records the expression pattern of each specific gene.
Embodiment 6: go out transplantation of seedlings
The positive seedling of identifying is taken out from bottle, and flush away agar plants in the vermiculite of 6:4/perlite mixed-matrix, and the potassium permanganate with 0.1% irrigates, the maintenance of shading.
Claims (10)
1. agriculture bacillus mediated transgenosis is cultivated the method for gloxinia plant, it is characterized in that: the method adopts the gloxinia young leaflet tablet as explant, explant is trained microbiotic remaining in rear and the removal culture in advance and the agrobacterium strains EHA105 bacterium liquid of bacterium metabolite is trained altogether, take off bacterium, after passing through resistance screening again, directly differentiation obtains transfer-gen plant.
2. the method for agriculture bacillus mediated transgenosis cultivation gloxinia plant according to claim 1 is characterized in that: the OD of agrobacterium strains EHA105 bacterium liquid
600Be 0.2 ~ 0.4, the time of training is 2 ~ 4 days altogether.
3. agriculture bacillus mediated transgenosis according to claim 2 is cultivated the method for gloxinia plant, the method that it is characterized in that common training is as follows: take out pre-incubated blade in aseptic small beaker, add in the agrobacterium strains EHA105 bacterium liquid for preparing and soak 3 ~ 8min, shaken several times; Take out blade, blot unnecessary bacterium liquid at aseptic filter paper, blade back places on the basic MS solid medium up, 25 ℃, secretly cultivates 2 ~ 4d.
4. it is characterized in that according to claim 1 and 2 or the 3 described agriculture bacillus mediated transgenosiss method of cultivating the gloxinia plant: agrobacterium strains EHA105 bacterium liquid is cultivated to adopt and is contained kantlex and Rifampin LB liquid nutrient medium is cultivated.
5. agriculture bacillus mediated transgenosis according to claim 4 is cultivated the method for gloxinia plant, and it is characterized in that: the preparation method of agrobacterium strains EHA105 bacterium liquid is as follows:
1) take out the agrobacterium strains EHA105 that contains recombinant plasmid from-70 ℃ of refrigerators, containing microbiotic Kan 100 mg/L, line activation on the LB solid medium of Rif 80 mg/L is until grow the single bacterium colony of Agrobacterium on the substratum;
2) get 5～10ml LB liquid nutrient medium, add microbiotic-kantlex and Rifampin, making its final concentration is Kan 100mg/L, Rif 80mg/L; Picking list bacterium colony adds wherein from fresh flat board, shakes up, and in 28 ℃, 180 r/min incubated overnight are to logarithmic phase, OD
600Be about 0.8～1.0;
3) draw above-mentioned bacterium liquid 1ml in the 1.5ml centrifuge tube, 4 ℃, the centrifugal 2min of 4000rpm removes supernatant, uses 30ml 1/2 MS suspension Agrobacterium again, shakes about 1-2h again, shows slightly muddy to bacterium liquid, OD
600Be 0.2 ~ 0.4 to get final product, for subsequent use.
6. the method for agriculture bacillus mediated transgenosis cultivation gloxinia plant according to claim 1 is characterized in that: take off bacterium and adopt the washing of Ticarcillin/Clavulanate Acid sterile solution.
7. agriculture bacillus mediated transgenosis according to claim 1 is cultivated the method for gloxinia plant, it is characterized in that: the marker gene that resistance screening adopts is hpt, adopts Totomycin as selective pressure.
8. agriculture bacillus mediated transgenosis according to claim 7 is cultivated the method for gloxinia plant, and it is characterized in that: the concentration of Totomycin is 20mg/L.
9. agriculture bacillus mediated transgenosis according to claim 8 is cultivated the method for gloxinia plant, it is characterized in that: the resistance of resistance screening selects substratum to be: MS+6-BA 2 mg/L+NAA 0.3 mg/L+
Hgy20mg/L+Tm 160 mg/L; Carry out two-wheeled and select to cultivate, the every wheel continues 12 ~ 15d.
10. agriculture bacillus mediated transgenosis according to claim 1 is cultivated the method for gloxinia plant, it is characterized in that: the pre-training of explant get the gloxinia aseptic seedling in, the blade on top, cut a circle along leaf margin, receive on the basic MS solid medium that contains 80 ~ 120 uM Syringylethanones, 25 ℃, secretly cultivate 20 ~ 30h.
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| CN103734015A (en) * | 2014-01-17 | 2014-04-23 | 济南市农业科学研究院 | Method for culturing genetically modified gloxinia under mediation of agrobacterium tumefaciens |
| CN103858763A (en) * | 2014-03-19 | 2014-06-18 | 广西壮族自治区林业科学研究院 | Sinningia speciosa tissue culture propagation and tuber storage and re-propagation method |
| CN103875470A (en) * | 2013-12-05 | 2014-06-25 | 济南市农业科学研究院 | Dwarf culture method for gloxinias |
| CN104686328A (en) * | 2015-02-21 | 2015-06-10 | 杨业云 | Method for establishing in-vitro regeneration system of sinningia speciosa |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103875470A (en) * | 2013-12-05 | 2014-06-25 | 济南市农业科学研究院 | Dwarf culture method for gloxinias |
| CN103875470B (en) * | 2013-12-05 | 2016-01-20 | 济南市农业科学研究院 | Gloxinia dwarfing culture method |
| CN103734015A (en) * | 2014-01-17 | 2014-04-23 | 济南市农业科学研究院 | Method for culturing genetically modified gloxinia under mediation of agrobacterium tumefaciens |
| CN103858763A (en) * | 2014-03-19 | 2014-06-18 | 广西壮族自治区林业科学研究院 | Sinningia speciosa tissue culture propagation and tuber storage and re-propagation method |
| CN103858763B (en) * | 2014-03-19 | 2015-11-18 | 广西壮族自治区林业科学研究院 | A kind of method that gloxinia tissue culture propagation and stem tuber are preserved and bred |
| CN104686328A (en) * | 2015-02-21 | 2015-06-10 | 杨业云 | Method for establishing in-vitro regeneration system of sinningia speciosa |
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Application publication date: 20130327 |