共查询到20条相似文献,搜索用时 15 毫秒
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T. Sretenović-Rajičić S. Ninković B. Uzelać B. Vinterhalter D. Vinterhalter 《Russian Journal of Plant Physiology》2007,54(5):653-658
Two inbred lines of Brassica oleracea L. var. capitata were transformed with two Agrobacterium tumefaciens strains harboring resistance to herbicide Basta: AGL1/pDM805 and LBA4404/pGKB5 (LB5-1). Inoculated cotyledons and hypocotyls
provided equally good explants and manifested a high percentage of shoot regeneration on MS medium supplemented with 1 mg/l
benzyladenine and 0.5 mg/l indole-3-butyric acid. The P34I5 genotype was superior to P22I5 in shoot regeneration (48.1 vs. 26.9%), multiplication, and acclimation in the greenhouse (76 vs. 40%). A. tumefaciens AGL1/pDM805 provided more regenerated shoots per explant, especially in the case of cotyledon explants, and the higher transformation
rate (up to 35% vs. up to 12%) as compared to LB5-1. Putative transformants survived spraying with 10–30 mg/l phosphinothricin.
Transformation was confirmed by GUS assay and PCR analysis in T0 and T1 generations.
Published in Russian in Fiziologiya Rastenii, 2007, vol. 54, No. 5, pp. 738–743.
The text was submitted by the authors in English. 相似文献
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<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated genetic transformation of <Emphasis Type="Italic">Perilla frutescens</Emphasis> 总被引:3,自引:0,他引:3
A reproducible plant regeneration and an Agrobacterium tumefaciens-mediated genetic transformation protocol were developed for Perilla frutescens (perilla). The largest number of adventitious shoots were induced directly without an intervening callus phase from hypocotyl explants on MS medium supplemented with 3.0 mg/l 6-benzylaminopurine (BA). The effects of preculture and extent of cocultivation were examined by assaying -glucuronidase (GUS) activity in explants infected with A. tumefaciens strain EHA105 harboring the plasmid pIG121-Hm. The highest number of GUS-positive explants were obtained from hypocotyl explants cocultured for 3 days with Agrobacterium without precultivation. Transgenic perilla plants were regenerated and selected on MS basal medium supplemented with 3.0 mg/l BA, 125 mg/l kanamycin, and 500 mg/l carbenicillin. The transformants were confirmed by PCR of the neomycin phosphotransferase II gene and genomic Southern hybridization analysis of the hygromycin phosphotransferase gene. The frequency of transformation from hypocotyls was about 1.4%, and the transformants showed normal growth and sexual compatibility by producing progenies. 相似文献
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Notocactus scopa cv. Soonjung was subjected to in planta Agrobacterium tumefaciens-mediated transformation with vacuum infiltration, pin-pricking, and a combination of the two methods. The pin-pricking combined with vacuum infiltration (20-30 cmHg for 15 min) resulted in a transformation efficiency of 67-100%, and the expression of the uidA and nptII genes was detected in transformed cactus. The established in planta transformation technique generated a transgenic cactus with higher transformation efficiency, shortened selection process, and stable gene expression via asexual reproduction. All of the results showed that the in planta transformation method utilized in the current study provided an efficient and time-saving procedure for the delivery of genes into the cactus genome, and that this technique can be applied to other asexually reproducing succulent plant species. 相似文献
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A high-frequency and simple procedure for Agrobacterium tumefaciens-mediated genetic transformation of the medicinal plant Salvia miltiorrhiza was developed. Leaf discs were pre-cultured on MS medium supplemented with 6.6 μmol l−1 BAP and 0.5 μmol l−1 NAA for one day, then co-cultured with A. tumefaciens strain EHA105 harboring the plasmid pCAMBIA 2301 for three days on the same medium. Regenerated buds were obtained on selection
medium (co-culture medium supplemented with 60 mg l−1 kanamycin and 200 mg l−1 cefotaxime) after two cycles’ culture of 10 days each and then transferred to fresh MS medium with 60 mg l−1 kanamycin for rooting. Fifteen days later, the rooted plantlets were obtained and then successfully transplanted to soil.
The transgenic nature of the regenerated plants was confirmed by PCR, Southern hybridization analysis and GUS histochemical
assay. Averagely, 1.1 independent verified transgenics per explant plated were obtained through this protocol. Adopting this
procedure, positive transformed plants could be obtained within 2–3 months from mature seeds germination to transplant to
soil, and more than 1,000 transgenic plants with several engineered constructs encoding different genes of interest were produced
in our lab in the past two years. 相似文献
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Ufuk Celikkol Akcay M. Mahmoudian H. Kamci M. Yucel H. A. Oktem 《Plant cell reports》2009,28(3):407-417
A simple and reproducible Agrobacterium-mediated transformation protocol for a recalcitrant legume plant, lentil (Lens culinaris M.) is reported. Application of wounding treatments and efficiencies of three Agrobacterium tumefaciens strains, EHA105, C58C1, and KYRT1 were compared for T-DNA delivery into lentil cotyledonary node tissues. KYRT1 was found
to be on average 2.8-fold more efficient than both EHA105 and C58C1 for producing transient β-glucuronidase (GUS) gene (gus) expression on cotyledonary petioles. Wounding of the explants, use of an optimized transformation protocol with the application
of acetosyringone and vacuum infiltration treatments in addition to the application of a gradually intensifying selection
regime played significant roles in enhancing transformation frequency. Lentil explants were transformed by inoculation with
Agrobacterium tumefaciens strain, KYRT1 harboring a binary vector pTJK136 that carried neomycin phosphotransferase gene (npt-II) and an intron containing gusA gene on its T-DNA region. GUS-positive shoots were micrografted on lentil rootstocks. Transgenic lentil plants were produced
with an overall transformation frequency of 2.3%. The presence of the transgene in the lentil genome was confirmed by GUS
assay, PCR, RT-PCR and Southern hybridization. The transgenic shoots grafted on rootstocks were successfully transferred to
soil and grown to maturity in the greenhouse. GUS activity was detected in vegetative and reproductive organs of T0, T1, T2 and T3 plants. PCR assays of T1, T2 and T3 progenies confirmed the stable transmission of the transgene to the next generations. 相似文献
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We developed an efficient gene transfer method mediated by Agrobacterium tumefaciens for introgression of new rice for Africa (NERICA) cultivars, which are derivatives of interspecific hybrids between Oryza glaberrima Steud. and O. sativa L. Freshly isolated immature embryos were inoculated with A. tumefaciens LBA4404 that harbored binary vector pBIG-ubi::GUS or pIG121Hm, which each carried a hygromycin-resistance gene and a GUS
gene. Growth medium supplemented with 500 mg/l cefotaxime and 20 mg/l hygromycin was suitable for elimination of bacteria
and selection of transformed cells. Shoots regenerated from the selected cells on MS medium containing 20 g/l sucrose, 30 g/l
sorbitol, 2 g/l casamino acids, 0.25 mg/l naphthaleneacetic acid, 2.5 mg/l kinetin, 250 mg/l cefotaxime, and 20 mg/l hygromycin.
The shoots developed roots on hormone-free MS medium containing 30 mg/l hygromycin. Integration and expression of the transgenes
were confirmed by PCR, Southern blot analysis, and histochemical GUS assay. Stable integration, expression, inheritance, and
segregation of the transgenes were demonstrated by molecular and genetic analyses in the T0 and T1 generations. Most plants were normal in morphology and fertile. The transformation protocol produced stable transformants
from 16 NERICA cultivars. We also obtained transformed plants by inoculation of calluses derived from mature seeds, but the
frequency of transformation was lower and sterility was more frequent. 相似文献
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Bo Yu Hong Zhai Yuping Wang Ning Zang Shaozhen He Qingchang Liu 《Plant Cell, Tissue and Organ Culture》2007,90(3):265-273
Efficient Agrobacterium tumefaciens-mediated transformation was achieved using embryogenic suspension cultures of sweetpotato (Ipomoea batatas (L.) Lam.) cv. Lizixiang. Cell aggregates from embryogenic suspension cultures were cocultivated with the A. tumefaciens strain EHA105 harboring a binary vector pCAMBIA1301 with gusA and hygromycin phosphotransferase II gene (hpt II) genes. Selection culture was conducted using 25 mg l−1 hygromycin. A total of 2,218 plants were regenerated from the inoculated 1,776 cell aggregates via somatic embryogenesis.
β-glucuronidase (GUS) assay and PCR, dot blot and Southern blot analyses of the regenerated plants randomly sampled showed
that 90.37% of the regenerated plants were transgenic plants. The number of integrated T-DNA copies varied from 1 to 4. Transgenic
plants, when transferred to soil in a greenhouse and a field, showed 100% survival. No morphological variations were observed
in the ex vitro transgenic plants. These results exceed all transformation experiments reported so far in the literature in
quantity of independent events per transformation experiment in sweetpotato. 相似文献
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AsAgrobacterium tumefaciens, which has long been used to transform plants, is known to transfer T-DNA to budding yeast,Saccharomyces cerevisiae, a variety of fungi were subjected to theA. tumefaciens-mediated transformation to improve their transformation frequency and feasibility. TheA. tumefaciens-mediated transformation of chestnut blight fungus,Cryphonectria parasitica, is performed in this study as the first example of transformation of a hardwood fungal pathogen. The transfer of the binary
vector pBIN9-Hg, containing the bacterial hygromycin B phosphotransferase gene under the control of theAspergillus nidulans trpC promoter and terminator, as a selectable marker, led to the selection of more than 1,000 stable, hygromycin B-resistant transformants
per 1×106 conidia ofC. parasitica. The putative transformants appeared to be mitotically stable. The transformation efficiency appears to depend on the bacterial
strain, age of the bacteria cell culture and ratio of fungal spores to bacterial cells. PCR and Southern blot analysis indicated
that the marker gene was inserted at different chromosomal sites. Moreover, three transformants out of ten showed more than
two hybridizing bands, suggesting more than two copies of the inserted marker gene are not uncommon. 相似文献
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Meiru Li Hongqing Li Huawu Jiang Guojiang Wu 《Plant Cell, Tissue and Organ Culture》2008,93(3):249-255
Broussonetia papyrifera is well-known for its bark fibers, which are used for making paper, cloth, rope etc. This is the first report of a successful
genetic transformation protocol for B. papyrifera using Agrobacterium tumefaciens. Callus was initiated at a frequency of about 100% for both leaf and petiole explants. Shoots formed on these calli with
a success rate of almost 100%, with 14.08 and 8.36 shoots regenerating from leave-derived and petiole-derived callus, respectively.
For genetic transformation, leaf explants of B. papyrifera were incubated with A. tumefaciens strain LBA4404 harboring the binary vector pCAMBIA 1301 which contains the hpt gene as a selectable marker for hygromycin resistance and an intron-containing β-glucuronidase gene (gus-int) as a reporter gene. Following co-cultivation, leaf explants were cultured on Murashige and Skoog (Physiol Plant 15:473,
1962) (MS) medium supplemented with 1.5 mg l−1 benzyladenine (BA) and 0.05 mg l−1 indole-3-butyric acid (IBA) (CI medium) containing 5 mg l−1 hygromycin and 500 mg l−1 cefotaxime, in the dark. Hygromycin-resistant calli were induced from leaf explants 3 weeks thereafter. Regenerating shoots
were obtained after transfer of the calli onto MS medium supplemented with 1.5 mg l−1 BA, 0.05 mg l−1 IBA, and 0.5 mg l−1 gibberellic acid (GA3) (SI medium), 5 mg l−1 hygromycin and 250 mg l−1 cefotaxime under fluorescent light. Finally, shoots were rooted on half strength MS medium (1/2 MS) supplemented with 10 mg l−1 hygromycin. Transgene incorporation and expression was confirmed by PCR, Southern hybridisation and histochemical GUS assay.
Using this protocol, transgenic B. papyrifera plants containing desirable new genes can be obtained in approximately 3 months with a transformation frequency as high as
44%. 相似文献
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Summary To achieve reliable stable transformation of sweet potato, we first developed efficient shoot regeneration for stem explants,
leaf disks, and petioles of sweet potato (Ipomoea batatas (L.) Lam.) cultivar Beniazuma. The shoot regeneration protocol enabled reproducible stable transformation mediated by Agrobacterium tumefaciens strain EHA105. The binary vector pIG121Hm contains the npt II (pnos) gene for kanamycin (Km) resistance, the hpt (p35S) gene for hygromycin (Hyg) resistance, and the gusA (p35S) reporter gene for β-glucuronidase (GUS). After 3 d co-cultivation, selection of calluses from the three explant types
began first with culture on 50 mg l−1 of Km for 6 wk and then transfer to 30 mg l−1 of Hyg for 6–16 wk in Linsmaier and Skoog (1965) medium (LS) also containing 6.49 μM 4-fluorophenoxyacetic acid and 250 mgl−1 cefotaxime in the dark. The selected friable calluses regenerated shoots in 4 wk on LS containing 15.13 μM abscisic acid and 2.89 μM gibberellic acid under a 16h photoperiod of 30 μmol m−2s−1. The two-step selection method led to successful recovery of transgenic shoots from stem explants at 30.8%, leaf dises 11.2%,
and petioles 10.7% stable transformation efficiencies. PCR analyses of 122 GUS-positive lines revealed the expected fragment
for hpt. Southern hybridization of genomic DNA from 18 independent transgenic lines detected the presence of the gusA gene. The number of integrated T-DNA copies varied from one to four. 相似文献
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To establish a procedure for Agrobacterium tumefaciens-mediated transformation of golden pothos (Epipremnum aureum) plants, the effects of selection antibiotics and the preculture period of stem explants before A. tumefaciens infection were examined. Explants were co-cultivated with A. tumefaciens EHA105, harboring the plasmid pGWB2/cGUS, on a somatic embryo-inducing medium supplemented with acetosyringone. Resulting
transgenic somatic embryos were screened on an antibiotic selection medium, and the transgenic pothos plants were regenerated
on a germination medium. Hygromycin was the optimum selection antibiotic tested. The preculture period significantly affected
the transformation efficiency, with explants precultured for one-day showing the best efficiency (5–30%). Both transformed
hygromycin-resistant embryos and regenerated plants showed β-glucuronidase activity. Southern blot analysis confirmed transgene
integration into the pothos genome. This reproducible transformation system for golden pothos may enable the molecular breeding
of this very common indoor plant. 相似文献
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The dwarf pomegranate (Punica granatum L. var. nana) is a dwarf ornamental plant that has the potential to be the model plant of perennial fruit trees because it bears fruits
within 1 year of seedling. We established an Agrobacterium-mediated transformation system for the dwarf pomegranate. Adventitious shoots regenerated from leaf segments were inoculated
with A. tumefaciens strain EHA105 harboring the binary vector pBin19-sgfp, which contains neomycin phosphotransferase (npt II) and green fluorescent protein (gfp) gene as a selectable and visual marker, respectively. After co-cultivation, the inoculated adventitious shoots were cut
into small pieces to induce regeneration, and then selected on MS medium supplemented with 0.5 μM α-naphthaleneacetic acid
(NAA), 5 μM N6-benzyladenine (BA), 0.3% gellan gum, 50 mg/l kanamycin, and 10 mg/l meropenem. Putative transformed shoots were regenerated
after 6–8 months of selection. PCR and PCR-Southern blot analysis revealed the integration of the transgene into the plant
genome. Transformants bloomed and bore fruits within 3 months of being potted, and the inheritance of the transgene was confirmed
in T1 generations. The advantage of the transformation of dwarf pomegranate was shown to be the high transformation rate. The establishment
of this transformation system is invaluable for investigating fruit-tree-specific phenomena. 相似文献
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Clarke JL Spetz C Haugslien S Xing S Dees MW Moe R Blystad DR 《Plant cell reports》2008,27(6):1027-1038
Agrobacterium-mediated transformation for poinsettia (Euphorbia pulcherrima Willd. Ex Klotzsch) is reported here for the first time. Internode stem explants of poinsettia cv. Millenium were transformed by Agrobacterium tumefaciens, strain LBA 4404, harbouring virus-derived hairpin (hp) RNA gene constructs to induce RNA silencing-mediated resistance to Poinsettia mosaic virus (PnMV). Prior to transformation, an efficient somatic embryogenesis system was developed for poinsettia cv. Millenium in which about 75% of the explants produced somatic embryos. In 5 experiments utilizing 868 explants, 18 independent transgenic lines were generated. An average transformation frequency of 2.1% (range 1.2-3.5%) was revealed. Stable integration of transgenes into the poinsettia nuclear genome was confirmed by PCR and Southern blot analysis. Both single- and multiple-copy transgene integration into the poinsettia genome were found among transformants. Transgenic poinsettia plants showing resistance to mechanical inoculation of PnMV were detected by double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA). Northern blot analysis of low molecular weight RNA revealed that transgene-derived small interfering (si) RNA molecules were detected among the poinsettia transformants prior to inoculation. The Agrobacterium-mediated transformation methodology developed in the current study should facilitate improvement of this ornamental plant with enhanced disease resistance, quality improvement and desirable colour alteration. Because poinsettia is a non-food, non-feed plant and is not propagated through sexual reproduction, this is likely to be more acceptable even in areas where genetically modified crops are currently not cultivated. 相似文献
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Six plasmids carrying a snowdrop lectin (Galanthus nivalis agglutinin, GNA) and one of three selection markers were successfully transferred into two sugarcane cultivars (FN81–745
and Badila) via Agrobacterium-mediated transformation. Agrobacterium strains LBA4404, EHA105 and A281 that harboured a super-binary vector were used for sugarcane transformation. The use of
the hygromycin (Hyg) resistance gene (hpt II), phosphinothrincin (PPT) resistance gene (bar) or G418 resistance gene (npt II) as a screenable marker facilitated the initial selection of GNA transgenic sugarcane callus with different efficiencies
and helped the rapid segregation of individual transformation events. All the three selective marker genes were controlled
by CaMV 35S promoter, while GNA gene was controlled by promoter of RSs-1 (rice sucrose synthase-1) or Ubi (maize ubiquitin).
Factors important to successful transformation mediated by Agrobacterium tumefaciens were optimized, which included concentration of A. tumefaciens, medium composition, co-cultivated methods with plant tissue, strain virulence and different selective marker genes. An efficient
protocol for sugarcane transformation mediated by A. tumefaciens was established. The GNA gene has been integrated into sugarcane genome as demonstrated by PCR and Southern dot blotting
detections. The preliminary results from bioassay demonstrated a significant resistance of the transgenic sugarcane plants
to woolly aphid (Ceratovacuna lanigera Zehnther) indicating thus the possibility for obtaining a transgenic sugarcane cultivar with resistance to woolly aphid. 相似文献
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We have developed a system to produce transgenic plants in tea (Camelia sinensis [L.] O. Kuntze) viaAgrobacterium tumefaciens-mediated transformation of embryogenic calli. Cotyledon-derived embryogenic callus cultures were cocultivated with anA. tumefaciens strain (AGL 1) harboring a binary vector carrying the hygromycin phosphotransferase (hpt II), glucuronidase (uid A), and green fluorescent protein (GFP) genes in the tDNA region. Following cocultivation, embryogenic calli were cultured in medium containing 500 mg/L carbenicillin
for 1 wk and cultured on an antibiotic selection medium containing 75 mg/L hygromycin for 8–10 wk. Hygromycin-resistant somatic
embryos were selected. The highest production efficiency of hygromycin-resistant calli occurred with cocultivation for 6–7
d in the presence of 400 μM acetosyringone (AS). Hygromycin-resistant somatic embryos developed into complete plantlets in
regeneration medium containing half-strength Murashige and Skoog (MS) salts with 1 mg/L benzyl amino purine (BAP) and 9 mg/L
giberellic acid (GA3). Transformants were subjected to GFP expression analysis, β-glucuronidase (GUS) histochemical assay, PCR analysis, and Southern
hybridization to confirm gene integration. 相似文献
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Luo H Hu Q Nelson K Longo C Kausch AP Chandlee JM Wipff JK Fricker CR 《Plant cell reports》2004,22(9):645-652
Genetic transformation of creeping bentgrass mediated by Agrobacterium tumefaciens has been achieved. Embryogenic callus initiated from seeds (cv. Penn-A-4) was infected with an A. tumefaciens strain (LBA4404) harboring a super-binary vector that contained an herbicide-resistant bar gene driven either by the CaMV 35S promoter or a rice ubiquitin promoter. Plants were regenerated from 219 independent transformation events. The overall stable transformation efficiency ranged from 18% to 45%. Southern blot and genetic analysis confirmed transgene integration in the creeping bentgrass genome and normal transmission and stable expression of the transgene in the T1 generation. All independent transformation events carried one to three copies of the transgene, and a majority (60–65%) contained only a single copy of the foreign gene with no apparent rearrangements. We report here the successful use of Agrobacterium for the large-scale production of transgenic creeping bentgrass plants with a high frequency of a single-copy transgene insertion that exhibit stable inheritance patterns.Abbreviations
2,4-D:
2,4-Dichlorophenoxyacetic acid
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bar:
Bialaphos resistance gene
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GUS:
-Glucuronidase
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PPT:
Phosphinothricin
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ubi:
Ubiquitin
Communicated by J.M. Widholm 相似文献
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An innovative and efficient genetic transformation protocol for European chestnut is described in which embryogenic cultures are used as the target material. When somatic embryos at the globular or early-torpedo stages were cocultured for 4 days with Agrobacterium tumefaciens strain EHA105 harbouring the pUbiGUSINT plasmid containing marker genes, a transformation efficiency of 25% was recorded. Murashige and Skoog culture medium containing 150 mg/l of kanamycin was used as the selection medium. The addition of acetosyringone was detrimental to the transformation efficiency. Transformation was confirmed by a histochemical -glucuronidase (GUS ) assay, PCR and Southern blot analyses for the uidA (GUS) and nptII (neomycin phosphotransferase II) genes. At present, 93 GUS-positive chestnut embryogenic lines are being maintained in culture. Low germination rates (6.3%) were recorded for the transformed somatic embryos. The presence of the transferred genes in leaves and shoots derived from the germinated embryos was also verified by the GUS assay and PCR analysis. 相似文献
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An improved protocol for genetic transformation of juvenile explants of Carrizo (Citrus sinensis Osb. × Poncirus trifoliata L. Raf.), Duncan (Citrus paradisi Macf.), Hamlin (Citrus sinensis (L.) Osbeck) and Mexican Lime (Citrus aurantifolia Swingle) cultivars using a vector containing a bifunctional egfp-nptII fusion gene is described. Several parameters were investigated to optimize genetic transformation of these four cultivars.
It was determined that a short preincubation in hormone rich liquid medium and subculture of Agrobacterium for 3 h in YEP medium containing 100 μM acetosyringone were required for improvement of transformation efficiency. Co-cultivation
duration as well as addition of acetosyringone to co-cultivation medium also played an important role in transformation efficiency
as did OD600 value of the Agrobacterium suspension used for transformation. We regenerated numerous EGFP expressing transgenic lines from all four cultivars. Based
on these results, we conclude that genetic transformation of citrus is cultivar specific and optimization of conditions for
maximum transgenic production are required for each individual cultivar. 相似文献