Publications (12)18.15 Total impact
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Article: Isopentenyl transferase gene expression offers the positive selection of marker-free transgenic plant of Kalanchoe blossfeldiana
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ABSTRACT: The technologies allowing the production of transgenic plants without selectable marker genes, is of great interest in public and environmental safety. For generating such marker-free transgenic plants, possibility has been offered by Multi-Auto-Transformation [MAT] vector system, which combines positive selection, using the isopentenyl transferase (ipt) gene, with a site-specific recombination that generates marker-free plants. In this study Agrobacterium tumefaciens strain EHA105 harboring an ipt-type MAT vector, pMAT21, containing lacZ, gus genes and the removable cassette in the T-DNA region was used to produce marker-free transgenic Kalanchoe blossfeldiana Poelln., employing ipt gene as the selectable marker gene. Co-cultivated explants were cultured on hormone- and selective agent-free MS medium, and 85% of the regenerated shoots showed ipt-shooty phenotype with GUS expression. Forty-one morphologically normal shoots were produced during the subculture. More than ninety percent of the normal shoots were ipt −, gus − but lacZ + as determined by PCR analyses. These results indicate that the ipt phenotype was clearly distinguishable from non-transgenic as well as transgenic marker-free shoots. This study opens interesting perspective for the generation of marker-free transgenic K. blossfeldiana with objective useful transgene.Plant Cell Tissue and Organ Culture 04/2012; 97(3):237-242. · 3.09 Impact Factor -
Article: Agrobacterium-mediated transformation of protocorm-like bodies in Cattleya
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ABSTRACT: Protocorm-like bodies (PLBs) of Cattleya orchid CM2450 were cocultivated with Agrobacterium tumefaciens strain EHA101 carrying either plasmid pIG121-Hm harboring genes coding for neomycin phosphotransferase II, hygromycin phosphotransferase, and β-glucuronidase (GUS) or plasmid pBBRacdS harboring these same genes along with a gene coding for 1-aminocyclopropane-1-carboxylate (ACC) deaminase. PLBs were maintained in a liquid New Dogashima (ND) medium and then added to a bacterial suspension culture (OD600≈0.6) yielding dilution ratios of either 1:10 or 1:40, and these were incubated for either 30min, 3h, or 6h. Hygromycin-resistant secondary PLBs were induced after 4weeks of culture on an ND medium containing 1.0mgL−1 naphthaleneacetic acid (NAA), 0.1mgL−1 benzyladenine (BA), 10mgL−1 hygromycin, 20mgL−1 meropenem, 10gL−1 sucrose, and solidified with 2.5gL−1 gellan gum. The highest frequency of transformation was obtained when PLBs were inoculated with 1:10 Agrobacterium liquid culture for 3h. The transformation of hygromycin-resistant plantlets regenerated from different sites of inoculated PLBs was confirmed by histochemical GUS assay, polymerase chain reaction (PCR) analysis, and Southern blot hybridization. Keywords Agrobacterium tumefaciens - Cattleya -Orchid-Protocorm-like body-TransformationPlant Cell Tissue and Organ Culture 04/2012; 103(1):41-47. · 3.09 Impact Factor -
Article: Metabolic engineering of Lilium × formolongi using multiple genes of the carotenoid biosynthesis pathway
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ABSTRACT: Lilium×formolongi was genetically engineered by Agrobacterium-mediated transformation with the plasmid pCrtZW-N8idi-crtEBIY, which contains seven enzyme genes under the regulation of the CaMV 35S promoter. In the transformants, ketocarotenoids were detected in both calli and leaves, which showed a strong orange color. In transgenic calli, the total amount of carotenoids [133.3μg/g fresh weight (FW)] was 26.1-fold higher than in wild-type calli. The chlorophyll content and photosynthetic efficiency in transgenic orange plantlets were significantly lowered; however, after several months of subculture, they had turned into plantlets with green leaves that showed significant increases in chlorophyll and photosynthetic efficiency. The total carotenoid contents in leaves of transgenic orange and green plantlets were quantified at 102.9 and 135.2μg/g FW, respectively, corresponding to 5.6- and 7.4-fold increases over the levels in the wild-type. Ketocarotenoids such as echinenone, canthaxanthin, 3′-hydroxyechinenone, 3-hydroxyechinenone, and astaxanthin were detected in both transgenic calli and orange leaves. A significant change in the type and composition of ketocarotenoids was observed during the transition from orange transgenic plantlets to green plantlets. Although 3′-hydroxyechinenone, 3-hydroxyechinenone, astaxanthin, and adonirubin were absent, and echinenone and canthaxanthin were present at lower levels, interestingly, the upregulation of carotenoid biosynthesis led to an increase in the total carotenoid concentration (+31.4%) in leaves of the transgenic green plantlets. KeywordsCarotenoid-Ketocarotenoid- Agrobacterium-mediated transformation- Lilium×formolongi -Metabolic engineering-Multi-gene constructPlant Biotechnology Reports 04/2012; 4(4):269-280. · 1.19 Impact Factor -
Article: Increased resistance to cucumber mosaic virus (CMV) in Lilium transformed with a defective CMV replicase gene.
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ABSTRACT: Lilium cv Acapulco was transformed with a defective cucumber mosaic virus (CMV) replicase gene (CMV2-GDD) construct using Agrobacterium tumefaciens. Four lines were analyzed for gene expression and resistance to CMV-O strain. Expression of the CMV2-GDD gene in the transgenic plants was confirmed by reverse transcription PCR (RT-PCR). When these four lines were mechanically inoculated with CMV-O, no signal of coat protein (CP) messages using RT-PCR was detected in newly produced leaves of two transgenic lines. Dot-immunobinding assay (DIBA) of CP was performed to examine the presence of the CMV in the newly produced leaves of challenged plants. Results, similar to those obtained with RT-PCR of the CP messages, were observed in DIBA. Therefore, our results imply that the two lines show increased levels of resistance to CMV, and CMV-GDD replicase gene is an effective construct that has protection against CMV in Lilium.Biotechnology Letters 02/2011; 33(6):1249-55. · 1.68 Impact Factor -
Article: Production of marker-free disease-resistant potato using isopentenyl transferase gene as a positive selection marker.
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ABSTRACT: The use of antibiotic or herbicide resistant genes as selection markers for production of transgenic plants and their continuous presence in the final transgenics has been a serious problem for their public acceptance and commercialization. MAT (multi-auto-transformation) vector system has been one of the different strategies to excise the selection marker gene and produce marker-free transgenic plants. In the present study, ipt (isopentenyl transferase) gene was used as a selection marker gene. A chitinase gene, ChiC (isolated from Streptomyces griseus strain HUT 6037) was used as a gene of interest. ChiC gene was cloned from the binary vector, pEKH1 to an ipt-type MAT vector, pMAT21 by gateway cloning and transferred to Agrobacterium tumefaciens strain EHA105. The infected tuber discs of potato were cultured on hormone- and antibiotic-free MS medium. Seven of the 35 explants infected with the pMAT21/ChiC produced shoots. The same antibiotic- and hormones-free MS medium was used in subcultures of the shoots (ipt like and normal shoots). Molecular analyses of genomic DNA from transgenic plants confirmed the integration of gene of interest and excision of the selection marker in 3 of the 7 clones. Expression of ChiC gene was confirmed by Northern blot and western blot analyses. Disease-resistant assay of the marker-free transgenic, in vitro and greenhouse-grown plants exhibited enhanced resistance against Alternaria solani (early blight), Botrytis cinerea (gray mold) and Fusarium oxysporum (Fusarium wilt). From these results it could be concluded that ipt gene can be used as a selection marker to produce marker-free disease-resistant transgenic potato plants on PGR- and antibiotic-free MS medium.Plant Cell Reports 12/2010; 30(4):587-97. · 2.27 Impact Factor -
Article: An efficient Agrobacterium tumefaciens-mediated genetic transformation of ‘‘Egusi’’ melon (Colocynthis citrullus L.)
01/2010; -
Article: Agrobacterium-mediated transformation of protocorm-like bodies in Cymbidium.
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ABSTRACT: Genetically transformed plants of Cymbidium were regenerated after cocultivating protocorm-like bodies (PLB) with Agrobacterium tumefaciens strain EHA101 (pIG121Hm) that harbored genes for beta-glucuronidase (gus), hygromycin phosphotransferase (hpt) and neomycin phosphotransferase II (nptII). PLB of three genotypes maintained in liquid new Dogashima medium (NDM), were subjected to transformation experiments. The PLB inoculated with Agrobacterium produced secondary PLB, 4 weeks after transfer onto 2.5 g L(-1) gellan gum-solidified NDM containing 10 g L(-1) sucrose, 20 mg L(-1) hygromycin and 40 mg L(-1) meropenem. Transformation efficiency was affected by genotype and the presence of acetosyringone during cocultivation. The highest transformation efficiency was obtained when PLB from the genotype L4 were infected and cocultivated with Agrobacterium on medium containing 100 muM acetosyringone. Transformation of the hygromycin-resistant plantlets regenerated from different sites of inoculated PLB was confirmed by histochemical GUS assay, PCR analysis and Southern blot hybridization.Plant Cell Reports 07/2007; 26(6):735-43. · 2.27 Impact Factor -
Article: Production of marker-free transgenic Nierembergia caerulea using MAT vector system.
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ABSTRACT: Agrobacterium tumefaciens strain EHA105 harboring an ipt-type MAT vector, pNPI132, was used to produce morphologically normal transgenic Nierembergia caerulea cv. Mont Blanc employing ipt gene as the selectable marker gene. beta-glucuronidase (GUS) gene was used as model gene of interest. The MAT vector system is a positive selection system that gives the advantage of regeneration to the transgenic cells without killing the non-transgenic cells. Infected explants were cultured on hormone- and antibiotic-free MS medium, and 65% of the regenerated shoots developed ipt shooty phenotype-morphologically abnormal shoots, within approximately 3 months after co-cultivation. Twenty morphologically normal shoots were produced from 12 transgenic ipt shoots 7 months after co-cultivation. The normal shoots rooted well on hormone-free MS medium. Ninety percent of the normal shoots were ipt (-), GUS(+) and excision(+) as determined by PCR and Southern blot analyses. These results indicate that ipt-type MAT vector system can be used successfully in Nierembergia to produce marker-free transgenic plants without using phytohormones and selective chemical agents.Plant Cell Reports 10/2006; 25(9):914-9. · 2.27 Impact Factor -
Article: Transgenic Phalaenopsis plants with resistance to Erwinia carotovora produced by introducing wasabi defensin gene usingAgrobacterium method
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ABSTRACT: Transgenic plants over-expressing wasabi defensin gene were successfully produced in Phalaenopsis orchid by Agrobacterium-mediated transformation method. Embryogenic cell suspension culture of Phalaenopsis Wataboushi ‘#6.13’ was infected with A. tumefaciens strain EHA101 carrying a plasmid containing wasabi defensin gene and selectable marker nptII, hpt genes. Plantlets were regenerated through somatic embryogenesis from the calli selected on hygromycincontaining medium. Transformation of plantlets with wasabi defensin gene was confirmed by PCR analysis. Southern blot analysis confirmed successful integration of 1–4 copies of the gene. Production of the 5 kDa wasabi defensin protein with varying levels was confirmed in the leaf extracts of different transgenic clones using Western blot analysis. Most of the transgenic plants showed strong resistance to Erwinia carotovora, which causes soft rot disease in the control plant. These results suggest the usefulness of this gene for conferring the resistance to various diseases of Phalaenopsis and possibly other orchids.Plant Biotechnology 01/2006; -
Article: Transgenic Phalaenopsis plants with resistance to Erwinia carotovora produced by introducing wasabi defensin gene using Agrobacterium method
Plant Biotechnology. 01/2006; -
Article: ESTABLISHMENT OF CALLUS CULTURE WITH HIGH PLANT REGENERATION ABILITY FROM LEAF SEGMENTS OF LYSIONOTUS PAUCIfLORUS MAxIM.
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ABSTRACT: REFERENCES Chen C. H., Goeden-Kallemeyn Y. (1979). In vitro induction of tetraploid plants from colchicines-treated diploid daylily callus. Euphytica, 28: 705-709. Geier T., Sangwan R. S. (1996). Histology and chimeral segregation reveal cell-specific differences in the competence for shoot regeneration and Agrobacterium-mediated transformation in Kohleria internode explants.. In vitro culture for preservation of triploid Senno (Lychnis senno Siebold et Zucc., 2n=36), a valuable and rare ornamental plant.. Detection of protopast-derived DNA tetraploid Lisianthus (Eustoma grandiflorum) plants by leaf and flower characteristics and by flow cytometry. Plant Cell, Tissue and Organ Culture, 38: 53-55. Lo K. H., Giles K. L., Sawhney V. K. (1997a). Acquisition of competence for shoot regeneration in leaf discs of Saintpaulia ionantha × confusa hybrid (African violet) cultured in vitro. Plant Cell Reports, 6:416-420.Plant Cell Reports Plant Cell Reports Plant Science Larkin P. J., Scowcroft W. R. Theoretical and Applied Genetics. 01/2006; 6(60):180-186. -
Article: Agrobacterium-mediated transformation of Phalaenopsis by targeting protocorms at an early stage after germination.
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ABSTRACT: A transformation procedure for phalaenopsis orchid established by using immature protocorms for Agrobacterium infection was aimed at the introduction of target genes into individuals with divergent genetic backgrounds. Protocorms obtained after 21 days of culture on liquid New Dogashima medium were inoculated with Agrobacterium strain EHA101(pIG121Hm) harboring both beta-glucuronidase (GUS) and hygromycin resistance genes. Subculture of the protocorms on acetosyringone-containing medium 2 days before Agrobacterium inoculation gave the highest transformation efficiencies (1.3-1.9%) based on the frequency of hygromycin-resistant plants produced. Surviving protocorms obtained 2 months after Agrobacterium infection on selection medium containing 20 mg l(-1) hygromycin were cut transversely into two pieces before transferring to recovery medium without hygromycin. Protocorm-like bodies (PLBs) proliferated from pieces of protocorms during a 1-month culture on recovery medium followed by transfer to selection medium. Hygromycin-resistant phalaenopsis plants that regenerated after the re-selection culture of PLBs showed histochemical blue staining due to GUS. Transgene integration of the hygromycin-resistant plants was confirmed by Southern blot analysis. A total of 88 transgenic plants, each derived from an independent protocorm, was obtained from ca. 12,500 mature seeds 6 months after infection with Agrobacterium. Due to the convenient protocol for Agrobacterium infection and rapid production of transgenic plants, the present procedure could be utilized to assess expression of transgenes under different genetic backgrounds, and for the molecular breeding of phalaenopsis.Plant Cell Reports 08/2005; 24(5):297-303. · 2.27 Impact Factor
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Institutions
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2005–2012
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Chiba University
- • Graduate School of Horticulture
- • Faculty of Horticulture
Chiba-shi, Chiba-ken, Japan
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