A high‐throughput transformation system allows the regeneration of marker‐free plum plants (Prunus domestica)

A high‐throughput transformation system previously developed in our laboratory was used for the regeneration of transgenic plum plants without the use of antibiotic selection. The system was first tested with two experimental constructs, pGA482GGi and pCAMBIAgfp94(35S) that contain selective marker and reporter genes. Transformation was monitored by GUS detection, and estimated transformation efficiencies were 5.7% and 17.7% for pGA482GGi and pCAMBIAgfp94(35S), respectively. Subsequently, an intron‐hairpin‐RNA (ihpRNA) construct, carrying the Plum Pox Virus coat protein (ppv‐cp) gene, without selectable or reporter marker genes was designed. Five transgenic lines were regenerated as confirmed by DNA blot analysis. We believe that this is the first report on the production of marker‐free plants transformed with a potential agronomically important trait in a Prunus species.     

Coat protein mediated resistance to Plum Pox Virus in Nicotiana clevelandii and N. benthamiana

Transgenic Nicotiana benthamiana and N. clevelandii plants expressing the coat protein of Plum Pox Virus under the control of the 35S promoter from Cauliflower Mosaic Virus were engineered by Agrobacterium tumefaciens mediated transformation. The phenomenon of virus resistance was observed at different levels when transgenic plants, expressing the coat protein and control plants were compared after challenge infection with Plum Pox Virus. N. clevelandii coat protein transgenic plants circumvent virus accumulation. After an initial increase in virus titer similar to the control plants, some coat protein expressing plants showed a reduced accumulation of virus and inhibition of the systemic spread, characterized by decrease of the virus titer and formation of new symptomless leaves. In other N. clevelandii coat protein expressing plants virus accumulation was inhibited and disease symptoms never appeared. N. benthamiana coat protein expressing plants were also protected. After a temporary virus accumulation, virus titer decreased without the appearance of symptoms with the exception of a few plants, which showed a delay of thirty days in the development of symptoms post challenge infection.Abbreviations PPV Plum Pox Virus - CP coat protein - CaMV Cauliflower Mosaic Virus - CP+ coat protein expressing plant - CP– control plant = non coat protein expressing plant - TMV Tobacco Mosaic Virus - NPTII neomycin phosphotransferaseII - IBA indole-3-butyric acid - BAP 6-benzylaminopurine; - MS Murashige Skoog - ELISA enzyme linked immunosorbent assay     

Beet necrotic yellow vein virus coat protein-mediated protection in sugarbeet (Beta vulgaris L.) protoplasts

Transformed Beta vulgaris L. suspension cultures were obtained after cocultivation of sugarbeet cells with Agrobacterium tumefaciens harbouring a binary vector containing the coat protein gene of beet necrotic yellow vein virus inserted between the kanamycin resistance gene and a ß-glucuronidase reporter gene. Protoplasts were isolated both from untransformed cells, and from transformed cells expressing the viral coat protein, and both were then infected with beet necrotic yellow vein virus. Comparison of the levels of infectivity shows that the expression of the coat protein gene in sugarbeet protoplasts mediates high levels of protection against infection by beet necrotic yellow vein virus.Abbreviations TMV Tobacco Mosaic Virus - CP Coat Protein - BNYVV Beet Necrotic Yellow Vein Virus - ß-Glu ß-glucuronidase - MS Murashige and Skoog (1962) - PEG Polyethylene glycol - npt neomycin phosphotransferase - nos nopaline synthase - FITC fluoresceine isothiocyanate - IAA indole acetic acid - BAP benzyl amino purine - MES 2-[N-Morpholino]ethane sulfonic acid - IgG Immunoglobulin G - nt nucleotide     

Agrobacterium tumefaciens-mediated transformation of Pisum sativum L. using binary and cointegrate vectors

Epicotyl segments and nodus expiants from etiolated seedlings of Pisum sativum were transformed using Agrobacterium tumefaciens strains GV 2260 (p35S GUS INT) and GV 3850 HPT carrying either a neomycin- or hygromycinphosphotransferase-gene as selectable markers. The transgenic character of hygromycin- or kananamycin-resistant tissue was confirmed by detection of nopaline or neomycinphosphotransferase-II- and ß-glucuronidase activity in crude extracts of resistant tissues. Up to 5 % of developing shoots from shoot proliferating nodi were regenerated via organogenesis to kanamycin-resistant plantlets. Transformation frequency in vitro was found to be influenced by expiant source, A. tumefaciens strain, pea genotype and duration of cocultivation. Acetosyringone did not increase the transformation rate.Abbrevations GUS ß-glucuronidase - NAA 1-naphthyl-acetic-acid - BA 6-benzylaminopurine - NPT-II neomycinphosphotransferase-II - HPT hygromycinphosphotransferase     

Transformation of vitis tissue by different strains of Agrobacterium tumefaciens containing the T-6b gene

Summary Stem pieces and leaf disks of Vitis spp. were cocultured with Agrobacterium tumefaciens strains carrying the UidA (ß-glucuronidase = GUS) gene. The transformation efficiency was highly increased by using a modified T-6b gene (a gene from pTiTm4) which interferes with normal growth and allows regeneration of normal Nicotiana rustica plants (Tinland 1990). The strains first tested on stem segments were subsequently tested in a leaf explant system. On leaves the transformation efficiency of the strains was much lower than with stems. Both the T-6b gene and the hsp 70-T-6b gene (a modified T-6b gene under the control of a heat shock promoter) allowed the initiation of GUS-positive buds.Abbreviations GUS ß-glucuronidase - BAP benzylaminopurine - X-gluc 5-bromo-4-chloro-3-indolyl glucuronide     

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).     

Regeneration of fertile transgenic indica (group 1) rice plants following microprojectile transformation of embryogenic suspension culture cells

Regenerable embryogenic suspensions of elite Indica (group 1) rice varieties IR24, IR64, IR72 and an advanced Indica rice breeding line IR57311-95-2-3 were established within 6–8 weeks from 3–4 week old calli derived from mature seeds. Transgenic rice plants were obtained by introducing a plasmid carrying genes encoding hygromycin phosphotransferase (hph, conferring resistance to hygromycin B) and ß-glucuronidase (uidA), both driven by the CaMV 35S promoter, via particle bombardment of embryogenic suspensions. The effect of osmotic conditioning on transformation was evaluated. Regenerated plants were resistant to hygromycin B and expressed the uidA (GUS) gene. The growth of mother plants (R₀) was normal and seeds were produced. Southern blot analysis of R₀ and R₁ plants showed that hygromycin resistant plants contained intact hph genes that were inherited in a Mendelian fashion. A protocol for a simple, efficient, repeatable, genotype- and environment-independent Indica rice transformation system is described.Abbreviations 2,4-D 2,4-dichlorophenoxy acetic acid - NAA -naphthalene acetic acid - kb kilobase - GUS ß-glucuronidase - hph hygromycin B phosphotransferase     

Agrobacterium-mediated transformation of the desiccation-tolerant plant Craterostigma plantagineum

Summary An efficient procedure for Agrobacterium tumefaciens- mediated transformation of the desiccation-tolerant plant Craterostigma plantagineum has been developed. Leaf explants were inoculated with A. tumefaciens strain GV3101 carrying the gene for kanamycin- or hygromycin-resistance and the ßglucuronidase reporter gene. Parameters which affected the transformation efficiency were the age of the explant, the degree of wounding and the presence of an antioxidant in the medium. Under optimal conditions, calli originated in more than 80% of leaf explants. Transformed plants were obtained from more than 50% of the cultured calli during regeneration in the presence of a suitable antibiotic. The stable integration of T-DNA was confirmed by Southern blot analysis and its expression by assays for ß-glucuronidase activity.Abbreviations GUS ß-glucuronidase - MUG 4-methyl-umbelliferyl ß-D-glucuronide - ABA abscisic acid - NPTII neomycin phosphotransferase II - CaMV cauliflower mosaic virus - MSAR modified MS medium - MS Murashige and Skoog     

Effect of an enhanced CaMV 35S promoter and a fruit-specific promoter on uida gene expression in transgenic tomato plants

Summary Two different promoters, a cauliflower mosaic virus (CaMV) 35S promoter with a 5′-untranslated leader sequence from alfalfa mosaic virus RNA4 (designated as CaMV 35S/AMV) and an E-8 fruit-ripening-specific promoter, were compared to evaluate their effects on expression of the uidA reporter gene in transgenic tomato plants. In order to generate sufficient numbers of transgenic tomato plants, both a reliable regeneration system and an efficient Agrobacterium transformation protocol were developed using 8-d-old cotyledons of tomato (Lycopersicon ecsulentum Mill. cv. Swifty Belle). Two sets of constructs, both derivatives of the binary vector pBI121, were used in transformation of tomato whereby the uidA gene was driven either by the CaMV 35S/AMV or the E-8 fruit-ripening-specific promoter. Southern blot hybridization confirmed the stable integration of the chimeric uidA gene into the tomato genome. Fruit and leaf tissues were collected from T₀ and T₁ plants, and assayed for β-glucuronidase (GUS) enzyme activity. As expected, both vegetative and fruit tissues of transgenic plants carrying the uidA gene under the control of CaMV 35S/AMV showed varying levels of GUS activity, while no expression was observed in vegetative tissues of transgenic plants carrying the uidA gene driven by the E-8 promoter. All fruits from transgenic plants produced with both sets of constructs displayed expression of the uidA gene. However, when this reporter gene was driven by the CaMV 35S/AMV, GUS activity levels were significantly higher than when it was driven by the E-8 fruit-specific promoter. The presence/absence of the uidA gene in T₁ plants segregated in a 3∶1 Mendelian ratio.     

Plant transformation by particle bombardment of embryogenic pollen

Summary Direct delivery of DNA into embryogenic pollen was used to produce transgenic plants in tobacco. A plasmid bearing the ß-glucuronidase (GUS) marker gene in fusion with the 35S-promoter was introduced by microprojectile bombardment into mid-binucleate pollen of Nicotiana tabacum that had been induced to form embryos by a starvation treatment. In cytochemical expression assays, 5 out of 10⁴ pollen grains were GUS⁺. Visual selection by staining with a non-lethal substrate for GUS was used to manually isolate transformed embryos. From the initial population of embryogenic GUS⁺ pollen, 1–5% developed into multicellular structures and 0.02% formed regenerable embryos. Two haploid transformants were regenerated. GUS expression was detected in different parts of the plants, and Southern analysis confirmed stable integration of the foreign DNA. Diploidisation was induced by injection of colchicine into the stem near adventitious buds. Offspring from selfings and backcrosses of one transformant were tested for GUS expression and by Southern blots. All F1-plants were transgenic, in accordance with Mendelian inheritance.Abbreviations GUS ß-glucuronidase - CaMV Cauliflower Mosaic Virus - MCS multicellular structure - NPTII neomycin phosphotransferase - PEG polyethylene glycol - X-gluc 5-bromo-4-chloro-3-indolyl glucuronide - DAPI 4,6-diamidino-2-phenylindole - Tris Tris(hydroxymethyl)aminomethane hydrochloride - EDTA ethylenedinitrilo tetraacetic acid, disodium salt dihydrate     

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     

Effects of tissue type and promoter strength on transient GUS expression in sugarcane following particle bombardment

Effects of tissue type and promoter strength on transient GUS expression in the sugarcane (Saccharum spp. hybrids) cultivar NCo 310 were evaluated following microprojectile bombardment of leaf explants. GUS expression was histochemically or fluorometrically measured 48 h after delivery of the uidA gene. High levels of GUS expression were obtained in leaf segments isolated from young, expanding sugarcane leaves cultured for 1, 3, or 6 d prior to bombardment. The promoter derived from the maize ubiquitin 1 gene (Ubi-1) produced significantly more GUS foci and higher GUS activity levels compared to the recombinant Emu, rice actin 1 (Act1), and CaMV 35S promoters. Our transient expression system should facilitate efforts to identify promoters and elements which will regulate desired gene expression patterns in sugarcane and aid in development of an efficient stable transformation system.Abbreviations Act1 rice actin 1 gene - CaMV cauliflower mosaic virus - GUS ß-glucuronidase - Ubi-1 maize ubiquitin 1 gene - uidA GUS gene - X-Glu 5-bromo-4-chloro-3-indoylglucuronide     

Evaluation of microprojectile-mediated DNA delivery and reporter genes for genetic transformation of the Mediterranean cypress (Cupressus sempervirens L.)

Embryonal-suspensor tissue (EST) of Mediterranean cypress (Cupressus sempervirens L.) was tested for microprojectile-DNA delivery (by the PDS-1000/He device) for different subculture periods (9, 15, and 21 days) using the plasmid vectors pRT99GUS [containing the β-glucuronidase (GUS) and neomycin phosphotransferase (NPT II) genes, and the CaMV 35S promoter], pBI426 (with a GUS::NPT II fusion gene under the control of a duplicated 35S RNA promoter), and pCGUδ0 (containing the GUS gene with the ubiquitin intron, under the control of the sunflower ubiquitin promoter). The relative strengths of the promoters as determined by GUS assays were sunflower ubiquitin>35S-35S-AMVE>35S. The highest expression level was observed when 15-day-subcultured EST was bombarded with the pCGUδ0 gene construct, which also showed high activity of the chloramphenicol acetyltransferase and NPT II genes. Green fluorescent areas were observed on EST when bombarded with the p35S-GFP plasmid, carrying the gene for the green fluorescent protein from the bioluminescent jellyfish Aequorea victoria. Received: 18 November 1996/ Revision received: 19 February 1997/ Accepted: 20 November 1997     

Transgenic poplars: expression of chimeric genes using four different constructs

Summary Leaf or stem explants of a hybrid poplar clone (Populus tremula X Populus alba), sensitive to Agrobacterium tumefaciens, were co-cultivated either by an octopine or a nopaline disarmed A. tumefaciens modified strain. Transformed poplar shoots were readily regenerated from explants. The protocol was improved using the nopaline disarmed strain C58/pMP90 with the binary vector pBI121. This protocol was then used to test three other vectors. The first one, possessing a nptII gene fused to the CaMV 19S promoter, permitted regeneration of transformed shoots in presence of 50 to 100 mg/l kanamycin. The two other vectors carried an additional nptII gene under the control of the CaMV 35S or CaMV 35S promoter with a double enhancer sequence (CaMV 70). CaMV 70 promoter provided consistently higher level of gene expression than the other promoters in both callus and leaf tissues.Abbreviations CaMV Cauliflower Mosaïc Virus - 2iP 2-isopentenyladenine - GUS and gus ß-glucuronidase - NAA 1-naphthaleneacetic acid - NPTII and nptII neomycin phosphotransferase II - NOS Nopaline synthase, X-Gluc: 5-bromo-4-chloro-3-indolyl ß-D glucuronide - Ap ampicillin - Gn gentamycin - Km kanamycin - Rf rifampicin - St streptomycin This work is dedicated to the late Marie France Michel who initiated the poplar biotechnology project at INRA.     

Agrobacterium tumefaciens-mediated transformation of Solanum gilo Raddi as influenced by explant type

An efficient system for Agrobacterium tumefaciens-mediated transformation of Solanum gilo was established. The marker genes for kanamycin resistance and ß-glucuronidase expression were introduced. A comparison between cotyledon and hypocotyl explants showed that while regeneration was better from hypocotyl explants, cotyledon explants gave better transformation efficiency (46% vs. 32%). Four levels of kanamycin selection (100, 150, 200 and 250 mg/l) were tested for effect on transformation efficiency with each type of explant. Lower levels of kanamycin worked better using cotyledon explants, while higher levels of kanamycin worked better for hypocotyl explants. All nine t₀ plants tested for expression of the kan ^r gene were positive. The progeny of three of these plants showed a pattern of classical Mendelian inheritance (3 to 1) for both the kan ^r and the ß-glucuronidase genes.Abbreviations MS Murashige and Skoog (1962) medium - 2,4-D 2,4-Dichlorophenoxyacetic acid - NPTII neomycin phosphotransferase - GUS ß-glucuronidase     

Agrobacterium-mediated transformation of quaking aspen (Populus tremuloides) and regeneration of transgenic plants

Summary Agrobacterium-mediated gene transformation of Populus tremuloides Michx was accomplished by co-cultivation of leaf disks excised from greenhouse plants with Agrobacterium tumefaciens containing a binary Ti-plasmid vector harboring chimeric neomycin phosphotransferase (NPT II) and ß-glucuronidase (GUS) genes. Shoot regeneration in the presence of kanamycin was achieved when thidiazuron (TDZ) was used as a plant growth regulator. Transformation was verified by amplification of NPT II and GUS gene fragments from genomic DNA of transgenic plants with polymerase chain reaction (PCR) and integration of these genes into nuclear genome of transgenic plants was confirmed by genomic Southern hybridization analysis. Histochemical assay revealed the expression of GUS gene in leaf, stem and root tissues of transgenic plants, further confirming the integration and expression of T-DNA in these plants. This protocol allows effective transformation and regeneration of quaking aspen using greenhouse-grown materials as an explant source. Whole plant regeneration from cuttings of fieldgrown mature quaking aspen and hybrid poplar (P. alba x P. grandidentata) was also readily achieved by using this protocol, which represents a potential system for producing transgenic quaking aspen and hybrid poplar of valuable genotypes.Abbreviations AMV RNA4 Alfalfa mosaic virus RNA4 - BA 6-benzyladenine - CaMV cauliflower mosaic virus - 2,4-D 2,4-dichlorophenoxyacetic acid - EDTA ethylenediaminetetraacetic acid - FAA formalin-acetic acid-alcohol - GUS ß-glucuronidase - NAA 1-naphthylacetic acid - NPT II neomycin phosphotransferase II - PCR polymerase chain reaction - SDS sodium dodecyl sulphate - TE Tris-Cl/EDTA - TDZ N-phenyl-N-1,2,3-thiadiazol-5-yl-urea (thidiazuron) - WPM woody plant medium (Lloyd and McCown 1980) - X-GLUC 5-bromo-4-chloro-3-indolyl-ß-glucuronic acid     

Screening for stable transformants and stability of β-glucuronidase gene expression in suspension cultured cells of tall fescue (Festuca arundinacea)

Summary By screening cell colonies derived from protoplasts of tall fescue (Festuca arundinacea), transformed with a rice actin-1-promoter-ß-glucuronidase gene construct, several ß-glucuronidase positive callus clones were obtained. Two callus clones with different GUS expression were derived from these. One was light blue after X-gluc staining, and expression of the ß-glucuronidase gene was stable over repeated subculture, while another stained intensely blue, and expression of the ß-glucuronidase gene was unstable. Southern blot analysis showed that only one copy of the ß-glucuronidase gene was integrated into the genome, and that these two clones appeared to have the same integration pattern. Treatment with 5-azacytidine maintained GUS expression in the unstable line but had no effect on reactivating expression of the GUS gene after expression had been lost. Following the screening procedure the callus clones would only regenerate albino plants.Abbreviations X-gluc 5-bromo-4-chloro-3-indolylglucuronide - GUS ß-glucuronidase - CaMV cauliflower mosaic virus - PEG polyethylene glycol - AZC 5-azacytidine - SDS sodium dodecyl sulphate - UV ultraviolet - EDTA ethylenediaminetetra-acetic acid disodium salt - SSPE salt-sodium-phosphate-EDTA - SSC standard saline citrate - hpt hygromycin phosphotransferase     

Transformation of arctic bramble (Rubus arcticus L.) by Agrobacterium tumefaciens

Genetic transformation of arctic bramble (Rubus arcticus L.) was achieved utilizing a Ti-plasmid vector system of Agrobacterium tumefaciens. Internodal stem segments were inoculated with Agrobacterium strain EHA101 carrying a T-DNA with the CaMV 35 S promoter-gus-int marker gene from which β-glucuronidase (GUS) is expressed only in plants. Regenerants were produced on Murashige and Skoog medium. Growth of Agrobacterium was inhibited with cefotaxime. Kanamycin was used as the selective agent for the transformants. Regenerants were assayed by histochemical GUS staining, and by Southern analysis using a gus-int probe. Transgenic arctic bramble plants containing gus-int and expressing GUS were recovered. Expression has been stable for 3 years in micropropagation. Received: 22 October 1997 / Revision received: 7 January 1998 / Accepted: 2 February 1998     

Far upstream activating promoter regions are responsible for expression of the BnC1 cruciferin gene from Brassica napus

Summary Cruciferin is the major seed storage protein in Brassica napus. As much as 1.9 kbp of the BnC1 cruciferin gene promoter have been sequenced and analyzed. Promoter fragments with 5 deletions from –2500 to –v202 were fused with the ß-glucuronidase reporter gene and used for Nicotiana tabacum transformation. ß-glucuronidase could be specifically expressed in transgenic tobacco seeds under the control of the BnC1 promoter and regulatory elements were found to be dispersed over 1903 bp. An almost 5-fold increase in ß-glucuronidase expression was obtained when the promoter length was increased from –379 to –498, and another 10-fold increase was observed when sequences between –1266 and –1903 were added. Histochemical analysis shows that the region between –844 and –1266 directs the expression of the chimeric gene specifically to the root apical meristem.Abbreviations GUS ß-glucuronidase - MU 4-methyl umbelliferone - MUG 4-methyl-umbelliferyl-ß-D-glucuronide - X-gluc 5-bromo-4-chloro-3-indolyl-ß-D-glucuronide     

Genetic transformation of Populus nigra by Agrobacterium tumefaciens

Two clones of Populus nigra L. were tested in vivo and in vitro for their susceptibility to three different Agrobacterium tumefaciens wild-type strains evaluating number and size of resulting calluses. Strain C58 proved to be the most virulent.Various parameters affecting Agrobacterium-mediated transformation of P. nigra clones were further analyzed using ß-glucuronidase gene transient expression. The clone Jean Pourtet proved to be more susceptible than the clone San Giorgio. A. tumefaciens strain A281 pKIWI105 proved to be the most virulent. The optimal procedure involved dipping of leaf discs into a bacterial suspension (7×10⁸ cells/ml) for 20 min, followed by a 48 h co-cultivation period on semi-solid regeneration medium.Leaf explants were co-cultivated with two disarmed A. tumefaciens strains. Plantlets of San Giorgio were regenerated, tested for ß-glucuronidase activity and rooted on selective medium containing kanamycin. Polymerase chain reaction analysis and Southern blot hybridization confirmed the integration of the neomycin phosphotransferase II gene into the poplar genome.Abbreviations BAP 6-benzyl-aminopurine - CaMV Cauliflower Mosaic Virus - 2,4-D 2,4-dichlorophenoxyacetic acid - GUS and gus ß-glucuronidase - hpt hygromycin phosphotransferase - IBA indole-3-butyric acid - KIN kinetin - LB Luria Bertani - MS Murashige and Skoog - NAA ßnaphthaleneacetic acid - NOS Nopaline synthase - NPTII and nptII neomycin phosphotransferase II - PCR Polymerase chain reaction - PVC poly-vinyl-cloride - SDS sodium dodecyl sulfate - SSC sodium cloride-sodium citrate - Tris tris(hydroxymethyl)amino-methane - WPM Woody Plant Medium