Regeneration of transgenic plants from the commercial apple cultivar Royal Gala

A transformation system was developed for the commercial apple (Malus X domestica Borkh.) cultivar Royal Gala. Leaf pieces from in vitro-grown shoots were cocultivated for 2 days with Agrobacterium tumefaciens strain LBA4404 containing the binary vectors pKIWI105 or pKIWI110. Shoots were produced on a shooting medium containing kanamycin (50 mg·L^–1). A 2-day incubation period on kanamycin-free medium prior to antibiotic selection enhanced the regeneration of kanamycin-resistant shoots. The majority of the kanamycin-resistant shoots also expressed GUS (-glucuronidase) activity. The putatively transformed shoots were rooted on a medium containing kanamycin (50 mg·L^–1). Rooted plants were established in a greenhouse, and plants transformed with pKIWI110, which contains a mutant Arabidopsis acetolactate synthase gene, were shown to be resistant to the herbicide Glean. Integration of T-DNA into the apple genome was confirmed by PCR and Southern hybridization analyses.Abbreviations NAA -naphthaleneacetic acid - IBA indole-3-butyric acid - BAP 6-benzylaminopurine     

Regeneration of transgenic tamarillo plants

Media were developed to regenerate shoots from leaf pieces of tamarillo (Cyphomandra betacea (Cav.) Sendtner). Shoots were derived via organogenesis and could be easily rooted and transferred to the growth chamber. Transgenic tamarillo plants were produced using the binary vector pKIWI110 in the avirulent Agrobacterium strain LBA4404. All transgenic plants were kanamycin resistant and some plants expressed the D-glucuronidase (gusA) reporter gene and were chlorsulfuron resistant. Molecular evidence for transformation was obtained using PCR (polymerase chain reaction) and Southern hybridization. Inheritance of the transgenic phenotypes was demonstrated in seedling progeny.     

Agrobacterium-mediated transformation of pepino and regeneration of transgenic plants

Regeneration of pepino (Solanum muricatum Ait.) shoots was achieved both by organogenesis and by embryogenesis. Shoots derived via organogenesis were easily rooted and most regenerated plants appeared phenotypically normal. Transgenic plants were obtained using the binary vector pKIWI110 in the avirulent Agrobacterium tumefaciens strain LBA4404. Optimization of transformation protocols was rapidly achieved by monitoring early expression of the GUS (-D-glucuronidase) reporter gene carried on pKIWI110. Transgenic plants expressed GUS and selectable marker genes for kanamycin resistance and chlorsulfuron resistance. PCR (polymerase chain reaction) and Southern analysis provided molecular evidence for transformation.     

Stable transformation of eggplant (Solanum melongena L.) by cocultivation of tissues withAgrobacterium tumefaciens carrying a binary plasmid vector

Stable transformation of eggplant to kanamycin resistance was obtained by cocultivation of cotyledonary and young leaves with the hypervirulent, fully oncogenicAgrobacterium tumefaciens strain A281 carrying plasmid pGA472. No transformation was observed when using the disarmedA. tumefaciens LBA4404 strain carrying pGA472 or when using either strain for cocultivation with eggplant suspension cells.The NPTII enzyme and DNA dot blot assays performed on callus cells growing in the presence of kanamycin indicated both the presence and expression of the foreign gene. The highest proportion of transformed explants was obtained from intact cotyledonary leaf pieces while the highest NPTII enzyme specific activity was detected in callus cells originating from superficially wounded cotyledonary leaf pieces. Kanamycin-resistant plantlets were regenerated after six months in culture.Abbreviations CTAB Cetyltrimethylammonium Bromide - DTT Dithiothreitol - EDTA Ethylenediaminetetraacetic Acid - NAA Naphtaleneacetic Acid - 2,4-D 2,4-Dichlorophenoxyacetic Acid - NPT II Neomycin Phosphotransferase     

Agrobacterium-mediated transformation and plant regeneration of buckwheat (Fagopyrum esculentum Moench.)

Genetic transformation of buckwheat (Fagopyrum esculentum Moench.) and regeneration of transgenic plants were obtained by using Agrobacterium tumefaciens strains as vectors. Buckwheat cotyledons were excised from imbibed seeds, co-cultivated with A. tumefaciens and subjected to previously reported protocols for callus and shoot regeneration. The transformation with oncogenic strains was confirmed by opine and DNA analyses of tumour tissue extracts. Plants were regenerated on cotyledon fragments incubated with strain A281, harboring pGA472, which carries the neomycin phosphotransferase II gene for kanamycin resistance. The transformation of resistant shoot clones was confirmed by NPTII enzyme assay and DNA hybridization. A large number of transformed shoots were rooted and fertile plantlets were raised in the greenhouse. Transgenic plants comprised pin and thrum clones, which were allowed to cross-pollinate. In about 180 R₂ seeds tested for kanamycin resistance, the ratio of resistant to sensitive seedlings was roughly 3:1.Abbreviations BAP 6-benzylaminopurine - 2,4-D dichloro-phenoxyacetic acid - 2iP 6-(, ,-dimethylallyl-amino)-purine - IBA indole-3-butyric acid - IAA indole-3-acetic acid - Km kanamycin - NPTII neomycin phosphotransferase II     

Expression of a chimaeric kanamycin resistance gene introduced into the wild soybeanGlycine canescens using a cointegrate Ri plasmid vector

Seedling hypocotyl explants ofGlycine canescens were inoculated withAgrobacterium rhizogenes carrying a chimaeric NPTII gene cointegrated into the TL-DNA of pRiA4. Transformed roots produced shoots on B5 based medium with 10.0 mgl^–1 BAP, 0.05 mgl^–1 IBA and 50 gml^–1 kanamycin. Cultured roots and regenerated plants expressed NPTII enzyme activity which was correlated with the presence of Ri TL-DNA and the structural sequence of the NPTII gene.Abbreviations BAP 6-benzylaminopurine - BSA bovine serum albumin - DTT dithiothreitol - EDTA ethylenediaminetetraacetic acid - IBA indole-butyric acid - PAGE polyacrylamide gel electrophoresis - NPTII neomycin phosphotransferase II - PMSF phenylmethylsulphonyl fluoride - SDS sodium dodecylsulphate     

Agrobacterium-mediated transformation of commercial melon (Cucumis melo L., cv. Amarillo Oro)

Cotyledon explants of muskmelon (Cucumis melo L., cv. Amarillo Oro) seedlings were co-cultivated with disarmed Agrobacterium tumefaciens strain LBA4404 that contained the binary vector plasmid pBI121.1. The T-DNA region of this binary vector contains the Nopaline synthase/neomycin phosphotransferase II (NPTII) chimeric gene for kanamycin resistance and the Cauliflower Mosaic Virus 35S/-glucuronidase (GUS) chimeric gene. After infection, the cotyledon pieces were placed in induction medium containing 100 mg/l kanamycin. Putative transformed shoots were obtained, followed by the development of morphologically normal plantlets. The transgenic nature of regenerants was demonstrated by polymerase chain reaction, Southern blot analysis, plant growth on medium selective for the transgene (NPTII) and expression of the co-transformed GUS gene. Factors affecting the transformation procedure are discussed.Abbreviations CaMV Cauliflower Mosaic Virus - Cf Cefotaxime - GUS -glucuronidase - Km Kanamycin - MS Murashige and Skoog - NOS nopaline synthase - NPTII neomycin phosphotransferase II - PCR polymerase chain reaction     

Transgenic papaya plants from Agrobacterium-mediated transformation of somatic embryos

Summary Transgenic papaya (Carica papaya L.) plants were regenerated from embryogenic cultures that were cocultivated with a disarmed C58 strain of Agrobacterium tumefaciens containing one of the following binary cosmid vectors: pGA482GG or pGA482GG/cpPRV-4. The T-DNA region of both binary vectors includes the chimeric genes for neomycin phosphotransferase II (NPTII) and ß-glucuronidase (GUS). In addition, the plant expressible coat protein (cp) gene of papaya ringspot virus (PRV) is flanked by the NPTII and GUS genes in pGA482GG/cpPRV-4. Putative transformed embryogenic papaya tissues were obtained by selection on 150 g·ml^–1 kanamycin. Four putative transgenic plant lines were obtained from the cp gene^– vector and two from the cp gene⁺ vector. GUS and NPTII expression were detected in leaves of all putative transformed plants tested, while PRV coat protein expression was detected in leaves of the PRV cp gene⁺ plant. The transformed status of these papaya plants was analyzed using both polymerase chain reaction amplification and genomic blot hybridization of the NPTII and PRV cp genes. Integration of these genes into the papaya genome was demonstrated by genomic blot hybridizations. Thus, like numerous other dicotyledonous plant species, papayas can be transformed with A. tumefaciens and regenerated into phenotypically normal-appearing plants that express foreign genes.Journal Series no. 3757 of the Hawaii Institute of Tropical Agriculture and Human Resources     

Agrobacterium-mediated transformation of Solanum tuberosum L. cv. ‘Russet Burbank’

Stem sections from shoot cultures maintained in vitro were used to produce transgenic plants of the potato, Solanum tuberosum L. cv. Russet Burbank. Stem internode pieces inoculated with Agrobacterium tumefaciens containing coat protein genes from potato virus X and potato virus Y, produced shoots with a frequency of 60% in the absence of selection and 10% on medium containing 100 mg/l kanamycin monosulfate. Regenerated shoots were assayed for kanamycin resistance by placing stem segments on callus induction medium containing an increased level of kanamycin. Of a total 255 regenerated shoots, 47 (18%) were kanamycin resistant. Of the kanamycin resistant shoots, 25 (53%) expressed the PVX or PVY coat protein genes as assayed by enzyme-linked immunosorbent assay or Western immunoblot analysis.     

Transformation of Stylosanthes spp. using Agrobacterium tumefaciens

Tumours were incited on leaf sections of Stylosanthes humilis, S. hamata, S. guianensis and S. scalra following infection by Agrobacterium tumefaciens. The suitability of 2 binary vectors (pGA472, BIN6) for gene transfer in S. humilis was tested and kanamycin-resistant tumour tissue was obtained from infected leaf pieces. The presence and expression of the neomycin phosphotransferase (NPT II) gene in the plant cells was demonstrated by hybridization of the coding region of the NPT II gene of the transposon Tn5 to DNA and RNA of kanamycin resistant tumours and by detection of significant NPT II activity in tissue extracts. Tumours also produced teratomatous shoots expressing the NPT II gene, but these could not be rooted.     

Transformation of lisianthus (Eustoma grandiflorum)

Transformed plants from three cultivars of Eustoma grandiflorum (lisianthus) were produced by cocultivating young leaf pieces with Agrobacterium tumefaciens strain A722 containing the binary vectors pKIWI110 and pLN26. Both vectors contain the selectable marker gene for neomycin phosphotransferase II. pKIWI110 also contains the reporter gene for β-d-glucuronidase, and pLN26, the chalcone synthase antisense gene. Southern DNA analysis revealed that all the kanamycin-resistant transformants tested contained copies of the transgenes integrated in their genome. The two plants transformed with pKIWI110 show β-d-glucuronidase expression in their mature leaves and selected transformants passed on the kanamycin-resistant phenotype to the F1 generation. Received: 8 January 1997 / Revision received: 12 May 1997 / Accepted: 3 June 1997     

Factors affecting Agrobacterium tumefaciens-mediated transformation in several black poplar clones

Transient expression of the uidA reporter gene was used in preliminary experiments with two oncogenic and two disarmed Agrobacterium tumefaciens strains in order to test the efficiency of T-DNA transfer to N084 x Populus nigra and N107 x P. nigra clones. The oncogenic strain A281 pKIWI105 produced the highest average number of GUS spots per leaf disc. In order to optimize the production of transgenic plantlets from different P. nigra clones (San Giorgio, Jean Pourtet, N084 x P. nigra and N107 x P. nigra, respectively), two A. tumefaciens strains (GV2260 p35S GUS, A281pKIWI105) and bacterial concentrations (7×10⁸; 1.2×0⁹ bacteria ml^-1) were used. Following co-cultivation with A281 pKIWI105, the frequency of leaf discs producing kanamycin-resistant calli was not significantly different between the clones and bacteria concentrations used. Transformed shoots were regenerated from all clones, except for Jean Pourtet. Co-cultivation of leaf discs with GV2260 p35S GUS produced very few calli which died when transferred to selective regeneration medium. In addition, the effects of acetosyringone and leaf wounding were evaluated for the San Giorgio and Jean Pourtet clones, using the same strains. Factors which significantly affected the transformation efficiency of leaf explants were the P. nigra clone, the A. tumefaciens strain, and the presence of acetosyringone. Genetic transformation of calli and regenerated plantlets was confirmed by their ability to grow and root on Woody Plant Medium containing kanamycin, by histochemical -glucuronidase assays, and Southern blot hybridization analyses.Abbreviations BA benzyladenine - GUS -glucuronidase - IBA indolebutyric acid - MS Murashige and Skoog - NAA -naphthaleneacetic acid - nptII neomycin phosphotransferase II gene - uidA -glucuronidase gene - WPM Woody Plant Medium     

Kanamycin resistance as a selectable marker for plastid transformation in tobacco

We report on a novel chimeric gene that confers kanamycin resistance on tobacco plastids. The kan gene from the bacterial transposon Tn5, encoding neomycin phosphotransferase (NPTII), was placed under control of plastid expression signals and cloned between rbcL and ORF512 plastid gene sequences to target the insertion of the chimeric gene into the plastid genome. Transforming plasmid pTNH32 DNA was introduced into tobacco leaves by the biolistic procedure, and plastid transformants were selected by their resistance to 50 g/ml of kanamycin monosulfate. The regenerated plants uniformly transmitted the transplastome to the maternal progeny. Resistant clones resulting from incorporation of the chimeric gene into the nuclear genome were also obtained. However, most of these could be eliminated by screening for resistance to high levels of kanamycin (500 g/ml). Incorporation of kan into the plastid genome led to its amplification to a high copy number, about 10000 per leaf cell, and accumulation of NPTII to about 1% of total cellular protein.     

Genetic transformation of strawberry by Agrobacterium tumefaciens using a leaf disk regeneration system

An efficient genetic transformation protocol has been developed for strawberry cv. Redcoat using Agrobacterium tumefadens. The protocol relies on a high frequency (84%) shoot regeneration system from leaf disks. The leaf disks were inoculated with a non-oncogenic Agrobacterium tumefadens strain MP90 carrying a binary vector plasmid pBI121 which contains a chimeric nopaline synthase (NOS) promoter driven neomycin phosphotransferase (NPT II) gene and a cauliflower mosaic virus 35S (CaMV35S) promoter driven, ß-glucuronidase (GUS) marker gene. The inoculated leaf disks, pre-cultured for 10 days on non-selective shoot regeneration medium, formed light green meristematic regions on selection medium containing 50 g/ml kanamycin. These meristematic regions developed into transformed shoots at a frequency of 6.5% on a second selection medium containing 25 g/ml kanamycin. The selected shoots were multiplied on shoot proliferation medium in the presence of kanamycin. All such shoots were resistant to kanamycin and expressed varying levels of NPT II and GUS enzyme activity. Histochemical assays for GUS activity indicated that the 35S promoter was highly active in meristematic cells of shoot and root apices. Molecular analysis of each transgenic clone confirmed the integration of both marker genes into the strawberry genome. Leaf disks prepared from transformed plants, when put through the second selection cycle on kanamycin, formed callus and exhibited GUS activity. The rooted transformed plants were grown in a greenhouse for further characterization. The protocol may be useful for improvement of strawberry through gene manipulations.NRCC No. 31491During the editorial process, a report has appeared on transformation of strawberry (James et al. 1990 Plant Sci 69:79–94).     

Effect of culture conditions on Agrobacterium-mediated transformation in datura

A two step selection procedure is described for high frequency transformation and regeneration of transgenic plants by coculture of leaf discs of Datura innoxia with Agrobacterium tumefaciens carrying binary vectors. Leaf discs were cocultured with disarmed A. tumefaciens vectors pGS Glucl, pGSTRN943, pGV2260 and pBI121, and subcultured on regeneration media containing kanamycin. Kanamycinresistant, putatively transformed callus and vegetative buds were isolated, and subcultured on media containing reduced amounts of growth regulators and kanamycin to induce shooting. Rooted shoots produced normal fertile plants. Transformation frequency was related to duration of preculture, co-culture, and the bacterial strain used. With pGS Glue 1, a 3 day co-culture resulted in 70% of leaf discs being transformed. Transformation was confirmed by histochemical test for GUS activity, by the ability of leaf discs to initiate callus and from NPTII test, and Southern blot analysis. Progeny of the transgenic plants showed Mendelian segregation for kanamycin resistance.     

Introduction and expression of an insect proteinase inhibitor in alfalfa Medicago sativa L.

As one approach to alleviating the need for insecticide spraying, our objective is to express protein insecticides in transgenic alfalfa. To initiate these studies, a cDNA encoding the protease inhibitor (PI) anti-elastase from Manduca sexta was placed under the control of the CaMV 35S promoter, inserted into pAN 70, and transferred into leaf and petiole sections of alfalfa (Medicago sativa L.) using Agrobacterium tumefaciens mediated gene transfer. Transformation rates were 10% of all explants exposed to Agrobacterium. More than 1000 transgenic plants containing the PI have been recovered. Transgenic plants were initially identified when leaf explants from the regenerated plants formed callus in the presence of 50 g/ml kanamycin, and subsequently the presence of the PI gene was confirmed by southern analysis. The 35S promoter-PI fusion produced up to 0.125% of total protein as PI protein in leaves, roots, and flowers. Progeny analysis demonstrated Mendelian segregation of the NPTII gene (observed as kanamycin resistance) and the PI (confirmed by southern analysis). Accumulation of the anti-elastase PI insecticide in transgenic alfalfa reduced the onset of thrip predation, suggesting that this methodology can establish insect resistance within this agronomically important legume.Abbreviations Km kanamycin - PI protease inhibitor - SDS Sodium dodecyl sulfate - NPTII neomycin phosphotransferase II - PCR polymerase chain reaction - SH Shenk and Hildebrandt (1972) medium     

A simple and efficient gene transfer system of trifoliate orange (Poncirus trifoliata Raf.)

Summary A simple and efficient gene transfer system of trifoliate orange (Poncirus trifoliata Raf.) was developed using epicotyl segments. The segments were infected with Agrobacterium harboring the binary vector pBI121 or pBI101-O12-p1. Both vectors contained the neomycin phosphotransferase II (NPTII) and the -glucuronidase (GUS) genes. In the plasmid pBI101-O12-p1, the GUS gene was directed to the promoter region of ORF12 (rolC) of the Ri plasmid. On a selection medium containing 100 or 200 g/ml kanamycin, adventitious shoots were formed from 21.7–44.6% of the segments. Histochemical GUS assay showed that 55.4–87.7% of the shoots expressed the GUS gene. The stable integration of this gene was also confirmed by polymerase chain reaction (PCR) analysis and by Southern blot analysis. When the pBI101-O12-p1 plasmid was used, the GUS activity was found to be located in phloem cells of leaf, stem and root. More than 100 transformed plants were obtained using this method within 2–3 months.     

Genetic transformation of commercial breeding lines of alfalfa (Medicago sativa)

Bio-engineering technologies are now routinely used for the genetic improvement of many agricultural crops. However, breeding lines of Medicago sativa are not easily amenable to genetic transformation and therefore cannot benefit from the molecular tools that have been developed for genetic manipulations. This paper describes a strategy that has been developed to transfer DNA into commercially important breeding lines of winter-hardy alfalfa via Agrobacterium infection. Three highly regenerative genotypes have been selected from ca 1000 genotypes within 11 breeding lines. They have been used as basic material for an extensive genetic transformation trial. Combinations of genotypes (11.9, 8.8, 1.5) expression vectors (pGA482, pGA643, pBibKan) and bacterial strains (C58, A281, LBA4404) were tested for their ability to produce stable transgenic material. Putative transgenic plantlets were further screened by nptII-specific PCR amplification, Southern hybridization and recallusing assays. One genotype (1.5) gave only one transformant out of 432 individual trials. With the two other genotypes, efficiency of transformation (kanamycin-resistant calluses obtained/explant tested) ranged from 0 to 0.92 depending on the strain/vector combination used. Statistical interactions underline the possibility of obtaining good genotype-strain-vector combinations for alfalfa transformation. Predicted transformation probability indicates that with strain LBA4404 containing the vector pGA482 and genotype 11.9, transformation efficiency is above 60% and 10% or more of the calluses retain embryogenic potential. PCR amplification and Southern hybridization of randomly chosen regenerated plantlets demonstrated that all embryos developing on 50 g ml^-1 kanamycin had a stable genomic insertion of nptII. Sexual crosses with untransformed genotypes showed that segregation of the transgenic trait followed Mendelian heredity.     

Genetic transformation of Medicago species by Agrobacterium tumefaciens and electroporation of protoplasts

Shoot and leaf segments of a non-regenerable Medicago sativa L. genotype were cocultivated with the shooty mutant of Agrobacterium tumefaciens carrying the pGV 2206 plasmid. Transformed callus lines were selected and regenerated on the hormone free B5 medium. Southern blot analysis demonstrated integration of T-DNA in to the genome of the regenerated plants.Transgenic plants resistant to kanamycin were obtained by electroporation of Medicago borealis protoplasts with the pGA 472 plasmid DNA.Abbreviations 2.4 D 2.4 dichlorophenoxyacetic acid - BAP 6-benzyladenine - T-DNA transferred DNA into plants from Ti-plasmid of A. tumefaciens