CN1726766A - Method for transforming perennial ryegrass brought in through agrobacterium tumefaciens - Google Patents
Method for transforming perennial ryegrass brought in through agrobacterium tumefaciens Download PDFInfo
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Abstract
A method using Agrobacterium tumefaciens for mediated conversion of perennial ryegrass includes such steps as using the plantlet as explant to induce calli, immersing the calli in the Agrobacterium tumefaciens liquid with plant expression carrier, culturing in culture medium, putting the receptor tissue in the inducing and screening culture medium for inducing resistant colli, culturing in differential culture medium to obtain regenerated plant.
Description
Technical field
The present invention relates to the production method of transgenic turf grass, particularly relate to the method for transforming perennial ryegrass brought in through agrobacterium tumefaciens.
Background technology
Turfgrass is being improved the ecological environment, and aspect such as improve the quality of living plays an important role, and has significant ecological benefits.But because turfgrass exists water consumption big, can not tolerate cold or overheated weather, the green phase has limited it and has used widely than weak points such as weak points.The fast development of modern molecular biology is for the cultivation of turfgrass new varieties provides new technology platform.
At present, known transgenic technology has the foreign gene direct guiding method, as the silicon carbide fibre mediated method, and the protoplast transformation method of polyethylene glycol (PEG) mediation, particle bombardment, electric shock conversion method etc.; And root agrobacterium-mediated transformation.The foreign gene direct guiding method is not subjected to species and genotypic restriction, but this method cost is higher, simultaneously, foreign DNA in most cases is incorporated into genomic site at random with the form that multicopy, front and back polyphone repeats, and easily causes transgene silencing and serial problem such as lose takes place to reset in transgenic progeny.By contrast, the foreign gene conversion method of root Agrobacterium (Agrobacterium tumefaciens) mediation, foreign gene can be inserted genome transcriptionally active district with low copy form, thereby greatly reduce the probability of gene rearrangement and gene silencing, make external source transformed gene energy genetic stability in recipient plant, and this method cost is lower.
The root agrobacterium-mediated transformation is applied to the transgenic research of dicotyledon the earliest, and Horsch in 1984 etc. have set up the agriculture bacillus mediated leaf dish method Plant Transformation technical system of root.Along with people's deepening continuously to the mechanism research of root Agrobacterium-mediated Transformation, last decade utilizes the report of root Agrobacterium success transforming monocots to increase gradually, as paddy rice (Hiei Y, Ohta S, Komari T, Kumashiro T.Efficient transformation of rice (Oryza sativaL.) mediated by Agrobacterium and sequence analysis of the boundaries of theT-DNA.Plant J 6:271-282,1994), corn (Ishida Y, Saito H, Ohta S, Hiei Y, Komari T, Kumashiro T.High efficiency transformation of maize (Zeamays) mediated by Agrobacterium tumefaciens.Nat Biotechnol 4:745-750,1996), wheat (Cheng M, Fry J E, Pang S, Zhou H, Hironaka C M, Duncan D R, Conner T W, Wan Y.Genetic transformation of wheat mediated by Agrobacterium tumefaciens.Plant Physiol 115:971-980,1997), barley (Tingay S, McElroy D, Kalla R, FiegS, Wang M B, Thornton S, Brettell R.Agrobacterium tumefaciens-mediated barleytransformation.Plant J 11:1369-1376,1997; Trifonova A, Madsen S, OlesenA.Agrobacterium-mediated transgene delivery and integration into barleyunder a range of in vitro culture conditiohs.Plant Sci 162:871-880,2001), sugarcane (Arencibia A D, Carmona E R, Tellez P, Chan M T, Yu S M, Trujillo L E, Oramas P.An efficient protocol for sugarcane (Saccharum spp.L.) transformationmediated by Agrobacterium tumefaciens.Transgenic Res 7:213-222,1998) and Chinese sorghum (Zhao Z Y, Cai T, Tagliani L, Miller M, Wang N, Pang H, Rudert M, SchroederS, Hondred D, Seltzer J, Pierce D.Agrobacterium-mediated sorghumtransformation.Plant Mol Biol 44:789-798,2000) etc.These studies show that the agriculture bacillus mediated conversion of root mostly is single site and inserts, and the copy number of about 2/3rds genetically modified plants foreign gene is less than 3.
At present, perennial ryegrass transgenic technology majority still with suspended culture cell or protoplast as the acceptor material that transforms, adopting direct guiding method to carry out foreign gene transforms, the defective of this method is that the cultivation difficulty of acceptor material is high and cost is big, and the cycle that obtains transfer-gen plant is longer, and frequency is lower.For example, Heleen M.van der Mass etc. have obtained non-embryonal suspension cultured cell (the Heleen M.van der Mass of system of English ryegrass of stable conversion gusA gene with particle bombardment, Eliza R.de Jong, Saskia Rueb, Lambert A.M.Hensgensand Frans A.Krens, Stable transformation and long-term expression of the gusAreporter gene in callus lines of perennial ryegrass (Lolium perenne L.) .PlantMolecular Breeding, 24:401-405,1994), the frequency of its resistant calli is 3.57 * 10 of total transformant
-6, because of this cell-line does not have embryo, so fail to obtain transfer-gen plant.Nineteen ninety-five, Spangenberg G etc. has reported method (the Spangenberg G that transforms English ryegrass embryonal suspension cultured cell with particle bombardment, Wang ZY, Wu XL, Nagel J, Potrykus I, Transgenic perennial ryegrass (Loliumperenne) plants from microprojectil bombardment of embryogenic suspensioncells.Plant Sci.108:209-217,1995), used this method cotransformation 96 ware suspended culture cells (every ware has the 200-250mg suspended culture cell), obtain 36 kanamycin-resistant callus tissues, 23% differentiation of kanamycin-resistant callus tissue obtains plant.1997, Ye X. etc. also utilizes particle bombardment successfully to transform Annual Ryegrass embryonal suspension cultured cell (Ye X., Wang Z Y, Wu X, Potrykus I., Spangerberg G, Transgenic Italinryegrass (Lolium multiflorum) plants from microprojectil bombardment ofembryogenic suspension cells.Plant Cell Reports.16:379-384.1997), used this method cotransformation 156 ware suspended culture cells (every ware has the 200-250mg suspended culture cell), obtain 93 kanamycin-resistant callus tissues, 33% of kanamycin-resistant callus tissue is divided into plant.
The superiority that the root agrobacterium-mediated transformation has it to replace in the genetic transformation technology of plant, but it is subjected to the restriction of the genotype of recipient plant and the state of receptor tissue's material or the like factor, so foundation and the enforcement of this technology on a new recipient plant is very difficult.
Summary of the invention
The purpose of this invention is to provide a kind of inducing, conversion and differentiation frequency are higher, and the cycle is the method for short transforming perennial ryegrass brought in through agrobacterium tumefaciens.
A kind of method of transforming perennial ryegrass brought in through agrobacterium tumefaciens may further comprise the steps:
1) with the embryo is the explant induction callus; Callus inducing medium is to have added 2,4 dichloro benzene ethoxyacetic acid and 6-benzyl aminopurine on the basis of MS minimal medium;
2) callus is soaked in the root Agrobacterium bacterium liquid that has plant expression vector;
3) callus that bacterium liquid was soaked places on the common culture medium and cultivates altogether; Culture medium is to have added 2,4 dichloro benzene ethoxyacetic acid and 6-benzyl aminopurine on the basis of MS minimal medium altogether;
4) receptor tissue is placed induce and carry out inducing of resistant calli on the screening culture medium; It is to have added the 2,4 dichloro benzene ethoxyacetic acid on the basis of MS minimal medium, 6-benzyl aminopurine and selectivity antibiotic that resistant calli is induced screening culture medium;
5) resistant calli with growth places and makes it differentiate regeneration plant on the differential medium; Differential medium is to have added 6-benzyl aminopurine and kinetin on the basis of MS minimal medium.
After obtaining regeneration plant, in order to improve the survival rate of transplanting, the regeneration plant that differentiates is transplanted to soil after preferably placing and taking root on the root media again; Described root media is the MS minimal medium.
The concentration of used culture medium additive is in said process: the 2,4 dichloro benzene ethoxyacetic acid concentration of described callus inducing medium is 1-5mg/l, and 6-benzyl aminopurine concentration is 0.1-0.5mg/l; The described 2,4 dichloro benzene ethoxyacetic acid concentration of culture medium altogether is 1-5mg/l, and 6-benzyl aminopurine concentration is 0.1-0.5mg/l; It is 1-5mg/l that described resistant calli is induced the 2,4 dichloro benzene ethoxyacetic acid concentration of screening culture medium, and 6-benzyl aminopurine concentration is 0.1-0.5mg/l, and the selectivity antibiotic is that concentration is hygromycin or the G418 of 30-100mg/l; The 6-benzyl aminopurine concentration of described differential medium is 1-3mg/l, and kinetin concentration is 0.1-0.5mg/l.
Induce in order to make, the effect of conversion and differentiation is better, also contains the caseinhydrolysate that concentration is 0-2g/l in the described callus inducing medium; Described inducing also contained the caseinhydrolysate that concentration is 0-2g/l in the screening culture medium; Press as screening, also containing concentration in described differentiation screening culture medium and the root media is the selectivity antibiotic of 10-50mg/l.Antibiotic is preferably hygromycin or G418.
For the growth that makes resistant calli is not subjected to the influence of environment, also containing concentration in the described resistant calli inducing culture is the antibiotic of the inhibition root Agrobacterium growth of 250mg/l.Described antibiotic is preferably carbenicillin or cephalosporin.
For the effect that makes conversion reaches best, the described time that callus is soaked in the root Agrobacterium bacterium liquid that has plant expression vector is 5-10 minute; It is 60-80 hour that the described callus that bacterium liquid was soaked places the time of cultivating altogether on the common culture medium.
The present invention has set up the ripe perennial ryegrass mature seed embryo callus induction differentiated system of a cover, and set up the agriculture bacillus mediated transformation technology of root on this basis, utilize method of the present invention, the kanamycin-resistant callus tissue induction frequency can reach 30-60%, wherein about 30% can finally be divided into transfer-gen plant, need 4-5 month from callus of induce to obtaining the whole cycle of transfer-gen plant, no matter all be significantly improved than prior art from conversion ratio or from obtaining the transfer-gen plant cycle, for the cultivation of transgenosis perennial ryegrass provides simple and reliable new method, solved the problem that people for a long time expect the transgenosis perennial ryegrass breeding difficulty that solves always, build a reliable and practical platform for the transgenic breeding of perennial ryegrass, had important theory and practice significance.
Description of drawings
Fig. 1 shows the callus after 2 weeks of inoculation.
Fig. 2 shows the callus after 7 weeks.
Fig. 3 shows that the root Agrobacterium infects the back 3 days callus glucuronidase activity testing result of common cultivation.
Fig. 4 shows that the root Agrobacterium infects the fresh flaxen resistant calli that 3-4 grew after week.
Fig. 5 shows that resistant calli goes out budlet through induction.
Fig. 6 is the blade glucuronidase activity testing result of transgenosis seedling.
Fig. 7 shows the transgenic turf grass of transplant survival.
Embodiment
Embodiment 1, root agrobacterium-mediated transformation transform English ryegrass
One, the inducing culture of English ryegrass (Lolium perenne L.) callus
1, callus induction
Choose English ryegrass (the Lolium perenne L.) seed of mature and plump, in distilled water, soak after 4 hours under the room temperature and peel off kind of a skin, put into distilled water again and continue soaked overnight.Soaked peeling seed crosses twice with 70% alcohol earlier, again with 30% clorox sterilization 30 minutes, aseptic water washing three times.Under aseptic condition,, callus inducing medium will be inserted again after the embryo crosscut into two respectively with embryo complete peeling from the seed that disinfects.Inoculum density: 50/ware, plate diameter 55mm.
Every liter of callus inducing medium composed as follows: on the basis of MS minimal medium, add the auxin substance 2,4 dichlorophenoxyacetic acid (2,4-D) 3mg, cytokinin-like substance 6-benzyl aminopurine 0.1mg, caseinhydrolysate 1000mg, pH 5.8.
Induction frequency: the 3rd day embryo in inoculation back begins to sprout, and adds up the frequency of sprouting during two weeks and can reach more than 70%, and the result as shown in Figure 1.
2, callus subculture
Use above-mentioned callus of induce medium, per two to three weeks are changeed ware once with callus.In primary stage of inoculation, callus be ivory buff, dry, growth rate is very fast, 2-3 after week callus be ivory buff particle or bulk; some tool ramentum shape projection of callus surface; about 6-7 enters quick vegetative stage after week, as shown in Figure 2, can carry out the root Agrobacterium and infect this moment.Callus is as shown in table 1 at different phase growth rate comparative result.
Table 1 different phase callus Growth velocity ratio
| The callus incubation time | Callus average weight (mg) | Callus average weight (mg) after 2 weeks | Growth rate (%) | Per day growth rate (%) |
| 6 all 7 weeks | 30.5±5.24 27.7±8.23 | 67.6±21.12 87.6±48.43 | 125.47 215.77 | 8.96 15.41 |
Two, the root agrobacterium-mediated transformation transforms
Used root agrobacterium strains is for having selectable marker gene hygromycin phosphotransferase gene (hpt) and reporter gene beta-glucosiduronatase gene (β-glucuronidase, uidA) LBA4404 (Hoekem, A.1983, Abinary plant vector strategy based on separation of vir and T region of theAgrobacterium tumefaciens Ti-plasmid.Nature 303,179-180), beta-glucosiduronatase gene has first intron, can only express in eukaryotic.
1, the cultivation of root Agrobacterium
From the root Agrobacterium bacterial classification of-70 ℃ of preservations bacterium liquid that takes a morsel, cultivate in containing on the corresponding antibiotic YEB solid culture medium line.28 ℃ of dark cultivations after 3-4 days are got single bacterium colony and are transferred once again on same solid culture medium, are cultured to the growth animated period.Scrape and get bacterial plaque and be resuspended in liquid nutrient medium, leave standstill 2h in 28 ℃, be diluted to naked eyes with the same liquid medium again and see muddiness (OD slightly
600=0.1), prepares against the usefulness of conversion.Liquid nutrient medium is to add acetosyringone 50-100 μ M, 30g/L sucrose in the MS minimal medium.
YEB solid culture medium composition (1L): beef extract, 5g; Peptone, 5g; Yeast extract, 10g; Sucrose, 5g; MgSO
47H
20,0.5g; Agar, 1.5%; PH 7.2.
2, the root Agrobacterium is infected
Enter the callus of quick vegetative stage in the optional step one.
Placing callus through the resuspended bacterial concentration that leaves standstill is OD
600Infected 10 minutes in=0.1 the LBA4404 root Agrobacterium bacterium liquid, remove bacterium liquid, on aseptic filter paper, callus is dried (about 5 minutes), place the common culture medium that is covered with one deck aseptic filter paper, 25 ℃ of dark cultivations 3 days.Used altogether culture medium be step-callus inducing medium.
Carry out glucuronidase activity dyeing after cultivating altogether immediately, the result as shown in Figure 3, instantaneous coloration result shows more locus coeruleus number, the instantaneous conversion frequency can reach about 80%.
Three, the acquisition of transformed plant
1, the screening of resistant calli
Cultivate the back altogether with inducing screening culture medium that callus is screened.To be connected to through the callus of cultivating altogether and induce screening culture medium at first to screen for 2 weeks, the resistant calli that as seen is fresh ivory buff grows out, as shown in Figure 4.
Every liter of resistant calli is induced the composed as follows of screening culture medium: on the basis of MS minimal medium, add 2,4 dichlorophenoxyacetic acid (2,4-D) 3mg, 6-benzyl aminopurine 0.1mg, caseinhydrolysate 1000mg, hygromycin 100mg, carbenicillin 250mg, pH 5.8.
The antagonism callus is carried out programmed screening after carrying out the screening first time again, and screening 1-2 week breaks up again on the postsearch screening medium.
2, the differentiation of resistant calli
To reach the above resistant calli of 0.5cm through the diameter of screening and be put on the differentiation screening culture medium and break up, and in 25 ℃ of following illumination cultivation (16h light/8h is dark).There is green budlet to occur after 10 days, can be grown to seedling after the about week, as shown in Figure 5.The average differentiation frequency of resistant calli is 62%.
Every liter of differential medium composed as follows: on the basis of MS minimal medium, add 6-benzyl aminopurine 1.0mg, kinetin 0.2mg, hygromycin 50mg, pH 5.8.
3, taking root of transformed plant:
The seedling that will break up before taking root is removed the unnecessary callus of leaf and bottom on top, be placed on the differential medium in the triangular flask slow seedling and carry out culture of rootage about 10 days again, after being inoculated in root media, visible white young root grows after about 5 days, can form flourishing ripe root system about afterwards three weeks, the frequency of taking root of transformed plant is 65.4%.
Root media is the MS minimal medium, hygromycin 30mg.
4, the glucuronidase activity qualitative detection of transgenic regenerated plant
Take off blade from the transformed plant of taking root, be soaked in the dyeing liquor, and place 37 ℃ of dark overnight incubation.Clean the dyeing blade with the 50mM sodium phosphate buffer, then blade is soaked in fixer (absolute ethyl alcohol: glacial acetic acid) 1 hour, and with 25%, 50%, 70%, 90%, 100% ethanol respectively decoloured 1 hour, direct visual observation, and the blade of transfer-gen plant is dyed blueness, the result as shown in Figure 6, it is transfer-gen plant that 78.6% plant is arranged.
Dyeing liquor is composed as follows: the Na of 0.1M
2HPO
4/ KH
2PO
4(Ph7.0, Sigma, America), 10mMEDTA (pH8.0, Promega, America), the 5mM potassium ferricyanide (Beijing Chemical Plant), 5mM potassium ferrocyanide (Beijing Chemical Plant), 1m M X-Gluc (5-bromo-4-chloro-3indolyl-β D-glucuronide, Sigma is America) with 0.1% triton x-100 (worker is given birth in Shanghai).
5, the transplanting of transformed plant
The seedling that will take root is completely taken out from triangular flask, cleans medium, plants in the vermiculite of the bacterium of going out and cultivates.Begin several days surface coverage plastic films in order to keep moistening, begin suitably to open the film venting after three days, deflation time is wilted with not dehydration of blade and is as the criterion.Film can be removed fully after one week.Transplanting survival rate is 98%, as shown in Figure 7.
Embodiment 2, root agrobacterium-mediated transformation transform English ryegrass
One, the inducing culture of English ryegrass (Lolium perenne L.) callus
L, callus induction
Choose English ryegrass (the Lolium perenne L) seed of mature and plump, in distilled water, soak after 4 hours under the room temperature and peel off kind of a skin, put into distilled water again and continue soaked overnight.Soaked peeling seed crosses twice with 70% alcohol earlier, again with 30% clorox sterilization 30 minutes, aseptic water washing three times.With embryo complete peeling from the seed that disinfects, callus inducing medium will be inserted again after the embryo crosscut into two respectively.Inoculum density: 100/ware, plate diameter 75mm.
Every liter of callus inducing medium composed as follows: on the basis of MS minimal medium, add the auxin substance 2,4 dichlorophenoxyacetic acid (2,4-D) 4.0mg/l, 6-benzyl aminopurine concentration is 0.2mg/l caseinhydrolysate 500mg/l, pH 5.8.
Induction frequency: the 3rd day embryo in inoculation back begins to sprout, and adding up the frequency of sprouting during two weeks can reach more than 70%.
2, callus subculture
With embodiment 1.
Two, the root agrobacterium-mediated transformation transforms
Used root agrobacterium strains is for having selectable marker gene neomycin phosphotransferase gene (npt-II) and reporter gene beta-glucosiduronatase gene (β-glucuronidase, uidA) AGLl (Lazo, G.R., 1991A DNA transformation competent Arabidopsis genomic library in Agrobacterium.Biotechnology 9,963-967), the beta-glucosiduronatase gene gene has first intron, can only express in eukaryotic.
1, the cultivation of root Agrobacterium
With embodiment 1.
2, the root Agrobacterium is infected
Enter the callus of quick vegetative stage in the optional step one.Placing callus through the resuspended bacterial concentration that leaves standstill is O.D
600Infected 7 minutes in=0.1 the AGL1 root Agrobacterium bacterium liquid, remove bacterium liquid, on aseptic filter paper, callus is dried (about 5 minutes), place the common culture medium that is covered with one deck aseptic filter paper, 25 ℃ of dark cultivations 3 days.Used altogether culture medium be step-callus inducing medium.
Carry out glucuronidase activity dyeing after cultivating altogether immediately, show that the instantaneous conversion frequency can reach about 90%.
Three, the acquisition of transformed plant
1, the screening of resistant calli
Cultivate the back altogether with inducing screening culture medium that callus is screened.To be connected to through the callus of cultivating altogether induces screening culture medium at first to screen for 2 weeks, as seen the resistant calli that is fresh ivory buff grows out, and the test of glucuronidase activity qualitative detection shows foreign gene existing expression in resistant calli.
Every liter of resistant calli is induced the composed as follows of screening culture medium: on the basis of MS minimal medium, add 2,4 dichlorophenoxyacetic acid (2,4-D) 3mg, 6-benzyl aminopurine concentration is 0.2mg/l, caseinhydrolysate 500mg, G418 100mg, the 250mg cephalosporin, pH5.8.
The antagonism callus is carried out programmed screening, and screening 1-2 week breaks up again on the postsearch screening medium.
2, the differentiation of resistant calli
To reach the above resistant calli of 0.5cm through the diameter of screening and be put on the differentiation screening culture medium and break up, and in 25 ℃ of following illumination cultivation (16h light/8h is dark).There is green budlet to occur after 10 days, can be grown to seedling after the about week.The differentiation frequency of resistant calli is 45%.
Every liter of differential medium composed as follows: on the basis of MS minimal medium, add 6-benzyl aminopurine 2.5mg, kinetin 0.2mg, G418 50mg, pH 5.8.
3, taking root of transformed plant:
The seedling that will break up before taking root is removed the unnecessary callus of leaf and bottom on top, be placed on the differential medium in the triangular flask slow seedling and carry out culture of rootage about 10 days again, after being inoculated in root media, visible white young root grows after about 5 days, can form flourishing ripe root system about afterwards three weeks, the frequency of taking root of transformed plant is 65%.
Every liter of root media composed as follows: on the basis of MS minimal medium, add G418 30mg.
4, the glucuronidase activity qualitative detection of transgenic regenerated plant
Detection method is with embodiment 1, and the result shows that it is transfer-gen plant that 80% plant is arranged.
5, the transplanting of transformed plant
The process of transplanting is with embodiment 1.Transplanting survival rate is 99%.
Claims (10)
1, a kind of method of transforming perennial ryegrass brought in through agrobacterium tumefaciens may further comprise the steps:
1) with the embryo is the explant induction callus; Callus inducing medium is to have added 2,4 dichloro benzene ethoxyacetic acid and 6-benzyl aminopurine on the basis of MS minimal medium;
2) callus is soaked in the root Agrobacterium bacterium liquid that has plant expression vector;
3) callus that bacterium liquid was soaked places on the common culture medium and cultivates altogether; Culture medium is to have added 2,4 dichloro benzene ethoxyacetic acid and 6-benzyl aminopurine on the basis of MS minimal medium altogether;
4) receptor tissue is placed induce and carry out inducing of resistant calli on the screening culture medium; It is to have added 2,4 dichloro benzene ethoxyacetic acid, 6-benzyl aminopurine and selectivity antibiotic on the basis of MS minimal medium that resistant calli is induced screening culture medium;
5) resistant calli with growth places and makes it differentiate regeneration plant on the differential medium; Differential medium is to have added 6-benzyl aminopurine, kinetin and selectivity antibiotic on the basis of MS minimal medium.
2, method according to claim 1 is characterized in that: the described regeneration plant that differentiates is transplanted to soil after placing and taking root on the root media again; Described root media is the MS minimal medium.
3, method according to claim 1 and 2 is characterized in that: the 2,4 dichloro benzene ethoxyacetic acid concentration of described callus inducing medium is 1-5mg/l, and 6-benzyl aminopurine concentration is 0.1-0.5mg/l; The described 2,4 dichloro benzene ethoxyacetic acid concentration of culture medium altogether is 1-5mg/l, and 6-benzyl aminopurine concentration is 0.1-0.5mg/l; It is 1-5mg/l that described resistant calli is induced the 2,4 dichloro benzene ethoxyacetic acid concentration of screening culture medium, and 6-benzyl aminopurine concentration is 0.1-0.5mg/l, and the selectivity antibiotic is that concentration is hygromycin or the G418 of 50-100mg/l; The 6-benzyl aminopurine concentration of described differential medium is 1-4mg/l, and kinetin concentration is 0.1-1.0mg/l.
4, method according to claim 1 and 2 is characterized in that: also contain the caseinhydrolysate that concentration is 0-2g/l in the described callus inducing medium; Described inducing also contained the 6-benzyl aminopurine that concentration is 0.1-0.5mg/l in the screening culture medium, concentration is the caseinhydrolysate of 0-2g/l; Also containing concentration in described differentiation screening culture medium and the root media is the selectivity antibiotic of 20-50mg/l.
5, method according to claim 3 is characterized in that: also contain the caseinhydrolysate that concentration is 0-2g/l in the described callus inducing medium; Described inducing also contained the 6-benzyl aminopurine that concentration is 0.1-0.5mg/l in the screening culture medium, concentration is the caseinhydrolysate of 0-2g/l; Also containing concentration in described differentiation screening culture medium and the root media is the selectivity antibiotic of 20-50mg/l.
6, method according to claim 4 is characterized in that: described antibiotic is hygromycin or G418.
7, method according to claim 1 and 2 is characterized in that: also containing concentration in the described resistant calli inducing culture is the antibiotic of the inhibition root Agrobacterium growth of 250mg/l.
8, method according to claim 7 is characterized in that: described antibiotic is carbenicillin or cephalosporin.
9, method according to claim 1 and 2 is characterized in that: described explant becomes the crosscut of turfgrass mature seed embryo two parts to obtain.
10, method according to claim 1 and 2 is characterized in that: the described time that callus is soaked in the root Agrobacterium bacterium liquid that has plant expression vector is 5-10 minute; It is 60-80 hour that the described callus that bacterium liquid was soaked places the time of cultivating altogether on the common culture medium.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102268450A (en) * | 2010-06-07 | 2011-12-07 | 中国科学院成都生物研究所 | Genetic transformation method of Lolium perenne L. |
| CN101451150B (en) * | 2007-12-03 | 2012-01-25 | 韩国农村振兴厅 | Method for enhancing conversion efficiency of garlic by agrobacterium tumefaciens and the garlic prepared thereby |
-
2004
- 2004-07-30 CN CNB2004100701065A patent/CN100371448C/en not_active Expired - Lifetime
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101451150B (en) * | 2007-12-03 | 2012-01-25 | 韩国农村振兴厅 | Method for enhancing conversion efficiency of garlic by agrobacterium tumefaciens and the garlic prepared thereby |
| CN102268450A (en) * | 2010-06-07 | 2011-12-07 | 中国科学院成都生物研究所 | Genetic transformation method of Lolium perenne L. |
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