Histology and chimeral segregation reveal cell-specific differences in the competence for shoot regeneration and Agrobacterium-mediated transformation in Kohleria internode explants

Summary Internode explants of Kohleria sp. (Gesneriaceae) are capable of regenerating large numbers of adventitious shoots. Regeneration of green shoots from explants of an albino periclinal chimera with genetically green L1, as well as microsurgical removal of the epidermis revealed that shoots originate only from the epidermis. Histological studies further showed that shoots arise from a particular epidermal cell type, viz the basal cell of young glandular trichomes. On the other hand, cells competent for Agrobacterium-mediated transformation are mainly located in vascular tissues, as could be shown by histochemical localization of ß-glucuronidase (GUS) expression in explants that had been inoculated with A. tumefaciens strains carrying binary plasmids with GUS and kanamycin resistance (NPTII) genes. Only 3% of GUS expression events took place in the epidermis. Consequently, shoot regeneration in the presence of kanamycin was very poor. Moreover, most of those shoots proved GUS-negative and did not survive subcultivation on kanamycin-containing medium. Six regenerants, however, were most probably transgenic, as suggested by the ability to produce adventitious shoots in the presence of kanamycin and by polymerase chain reaction (PCR) analysis. To our knowledge, this is the first positive result towards genetic transformation in a taxon of the Gesneriaceae.Abbreviations BA N⁶-benzyladenine - ct cefotaxime - GUS ß-glucuronidase - IAA indole-3-acetic acid - km kanamycin - NPTII neomycin phosphotransferase II - PCR polymerase chain reaction     

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).     

Kanamycin selection in temporary immersion bioreactors allows visual selection of transgenic citrus shoots

In mature citrus transformation, the nptII gene is most commonly used for selection and it is confounded by the high number of non-transformed, escaped shoots that develop on semi-solid kanamycin selection medium, even at high concentrations. Selection in liquid medium with kanamycin in temporary immersion bioreactors might provide a better means of distinguishing between transformed and non-transformed shoots. A dose-response curve was constructed for wild-type Carrizo rootstock in liquid medium to evaluate the effects of kanamycin concentration on the number and the length of microshoots. Kanamycin at 200 mg/l was chosen as the optimal concentration for selection of transgenic mature citrus shoots in bioreactors. At this dose, most non-transgenic microshoots turned yellow and their lengths and numbers were significantly reduced in comparison to the no kanamycin controls. Selection of transgenic shoots in bioreactors was tested after Agrobacterium transformations of mature Carrizo and Valencia using three different binary vectors containing nptII as the selectable marker. Shoots developed on semi-solid medium and were transferred to temporary immersion bioreactors containing liquid MS medium with 200 mg/l kanamycin. After two weeks of culture in bioreactors, 21 dark green shoots were visually selected on the basis of color from a total of 6882 microshoots, and 17 of them (81%) were confirmed as transgenic with either the GUS histochemical assay, GFP fluorescence or PCR. Yellow shoots (5675) to be discarded from pTLAB21 and pCAMBIA2301 transformations were also tested for GUS or GFP expression and only one (0.01%) was positive. Kanamycin selection of mature transgenic shoots in temporary immersion bioreactors permitted transgenics to be visually distinguished on the basis of color, and reduced labor and consumable costs for PCR screening, particularly when reporter genes were not used.     

Factors affecting Agrobacterium-mediated transformation in Citrus and production of sour orange (Citrus aurantium L.) plants expressing the coat protein gene of citrus tristeza virus

Factors influencing transformation frequencies using the Agrobacterium-mediated protocol developed for Citrus seedling internodal stem segments in this laboratory were evaluated, with particular emphasis on decreasing the numbers of ``escape' shoots produced. Although the use of a wild-type ``shooty' Agrobacterium strain allowed relatively high frequencies of β-glucuronidase positive (GUS+) shoots to be produced, none of the shoots were free of wild-type T-DNA and would not root. Both use of a liquid medium/kanamycin overlay and horizontal placement of stem segments increased the efficiency of kanamycin selection. Wounding via particle bombardment prior to Agrobacterium inoculation did not increase transformation frequencies. The concentration of benzyladenine (BA) in the regeneration/selection medium inversely influenced the numbers of shoots that regenerated and the subsequent ability of the shoots to root. Regeneration in the presence of kanamycin also influenced the ability of shoots to root. Many of the shoots that regenerated on selection medium were chimeric for GUS expression, and plants established from such shoots ranged from non-staining to solidly staining for GUS. However, solidly transformed plants with integrated T-DNA were obtained, and these plants have maintained the expression of transgenes over several years. The transgenic plants include ones of sour orange (C. aurantium L.) and Key lime (C. aurantifolia (Christm.) Swing.), two species not previously transformed, and have integrated and express the coat protein gene of citrus tristeza virus. This is the first report of a potentially agriculturally important transgene being expressed in Citrus. Received: 8 October 1996 / Revision received: 1 April 1997 / Accepted: 18 April 1997     

Agrobacterium-mediated genetic transformation of groundnut (arachis hypogaea L.): An assessment of factors affecting regeneration of transgenic plants

Transgenic groundnut (Arachis hypogaea L.) plants were produced efficiently by inoculating different explants withAgrobacterium tumefaciens strain LBA4404 harbouring a binary vector pBM21 containinguidA (GUS) andnptll (neomycin phosphotransferase) genes. Genetic transformation frequency was found to be high with cotyledonary node explants followed by 4 d cocultivation. This method required 3 days of precultivation period before cocultivation withAgrobacterium. A concentration of 75 mg/l kanamycin sulfate was added to regeneration medium in order to select transformed shoots. Shoot regeneration occurred within 4 weeks; excised shoots were rooted on MS medium containing 50 mg/I kanamycin sulfate before transferring to soil. The expression of GUS gene (uidA gene) in the regenerated plants was verified by histochemical and fluorimetric assays. The presence ofuidA andnptll genes in the putative transgenic lines was confirmed by PCR analysis. Insertion of thenptll gene in the nuclear genome of transgenic plants was verified by genomic Southern hybridization analysis. Factors affecting transformation efficiency are discussed.     

Transformation of chicory and expression of the bacterial uidA and nptll genes in the transgenic regenerants

Transgenic chicory plants were obtained from different explantsco-cultured with Agrobacterium tumefaciens. Among tap-root,leaf and cotyledonary tissues, etiolated cotyledons showed thegreatest competence for transformation. The Agrobacterium strainsused contained either pGSGLUC1 or pTDE4 as a vector which carryboth the neomycin phosphotransferase II gene (nptll) for kanamycinresistance and ß-glucuronidase gene (uidA) under thecontrol of different promoters. Transformation was confirmedby NPTII enzymatic assay, histochemical analysis of GUS activityand DNA hybridization. Transgenic plants expressed both markergenes in root and shoot tissues. In leaves, GUS activity wasexpressed in all tissue types, whatever the nature of the promoter.Nevertheless, variable heterogeneous patterns of expressionwere observed in the different root tissues. Differential expression of the GUS fusions controlled by thedual TR or the CaMV 35S promoters are discussed. Key words: Chicory, genetic transformation, GUS activity, kanamycin resistance     

Green fluorescent protein as a screenable marker to increase the efficiency of generating transgenic woody fruit plants

 The green fluorescent protein (GFP) from Aequorea victoria has been introduced into three different citrus genotypes [Citrus aurantium L., C. aurantifolia (Christm.) Swing. and C. sinensis L. Osbeck×Poncirus trifoliata (L.) Raf.] which are considered recalcitrant to transformation, mainly due to low transformation frequencies and to the regeneration of escape shoots at high frequencies from the Agrobacterium-inoculated explants. High-level GFP expression was detected in transgenic cells, tissues and plants. Using GFP as a vital marker has allowed us to localize the sites of transgene expression in specific cells, always occurring in callus tissue formed from the cambium of the cut ends of explants. Whereas green fluorescent shoots regenerated in all cases from this callus, most escapes regenerated directly from explants with almost no callus formation. Thus, development of callus from cambium is a prerequisite for citrus transformation. Furthermore, in vivo monitoring of GFP expression permitted a rapid and easy discrimination of transgenic and escape shoots. The selection of transgenic shoots could be easily favored by eliminating the escapes and/or by performing shoot-tip grafting of the transgenic buds soon after their origin. GFP-expressing shoots have also been observed in citrus explants co-cultivated with Agrobacterium but cultured in a medium without the selective agent kanamycin. This opens the possibility to rescue the transgenic sectors and to regenerate transgenic plants without using selectable marker genes conferring antibiotic or herbicide resistance, which is currently a topic of much discussion for the commercialization of transgenic plants. Received: 28 October 1998 / Accepted: 28 November 1998     

Agrobacterium-mediated Transformation of Sunflower (Helianthus annuusL.): A Simple Protocol

A simple and reproducibleAgrobacterium tumefaciens-mediatedtransformation system was developed for sunflower (HelianthusannuusL.) ‘KBSH-1’. The objective was to substantiallyeliminate thein vitroregeneration component from the transformationprotocol. Two-d-old seedlings with one cotyledon detached wereinfected withAgrobacterium tumefaciensstrain LBA 4404/pKIWI105harbouring genes for ß-glucuronidase (GUS) and neomycinphosphotransferase (NPT II). Following co-cultivation, seedlingswere grown aseptically for 5 d on a growth regulator-free basalmedium supplemented with 250 µg ml^-1cefotaxime (a bacteriostatused to control gram negative bacteria). Seedlings were screenedfor transient GUS expression and the shoot portions of the putativetransformants were utilized for propagation as transgenic plants.The excised shoots that initiated roots following selectionwere subsequently transferred to a glasshouse. Molecular analysisof transgenic plants confirmed concordance between the presenceof foreign genes and enzyme activity. The transformation regimefacilitated rapid generation of up to 2% phenotypically normalfertile plants containing functional transgenes. The transmissionand integration of the marker genes in the progeny is demonstrated.Copyright1999 Annals of Botany Company Sunflower (Helianthus annuusL.), transformation,Agrobacterium,simple protocol.     

Study of different antibiotic combinations for use in the elimination of Agrobacterium with kanamycin selection in carnation

The effect of several β-lactam antibiotics on shoot regeneration, growth and rooting of carnation (Dianthus caryophyllus L.), and their use in combination with kanamycin in Agrobacterium-mediated genetic transformation studies, was determined. Carbenicillin, cefotaxime and ticarcillin increased the regeneration rate when added alone in non-inoculated explants; whereas, with inoculated explants, this effect was only observed in ticarcillin-containing medium. Cefotaxime inhibited root growth in both transgenic and non-transgenic shoots. Rooting of non-transgenic shoots was completely inhibited in all culture media containing kanamycin. The different antibiotics used, alone or in combination, did not prevent the occurrence of false positive shoots, but it was possible to select transgenic shoots when rooting was induced in a kanamycin + ticarcillin-containing medium. Regenerated transformed shoots were free of Agrobacterium after culturing in rooting medium, as was proven by the PCR analysis for the nptI gene, the antibiogram and the culture of tissue pieces of transgenic shoots on LB broth. The use of kanamycin and timentin with or without carbenicillin, was very useful in the transformation procedure, for the elimination of Agrobacterium in regenerated shoots before their transfer to greenhouse conditions and also in the selection of transgenic versus false-positive shoots. This revised version was published online in June 2006 with corrections to the Cover Date.     

Agrobacterium-mediated transformation of Citrus stem segments and regeneration of transgenic plants

Summary A method for Agrobacterium-mediated transformation of Citrus and organogenic regeneration of transgenic plants is reported. Internodal stem segments were co-cultured with Agrobacterium harboring binary vectors that contained the genes for the scorable marker ß-glucuronidase (GUS) and the selectable marker NPT-II. A low but significant percentage ( 5%) of the shoots regenerated in the presence of 100 g/ml kanamycin were GUS⁺. Polymerase chain reaction (PCR) analysis confirmed that GUS⁺ shoots contained T-DNA. Two plants established in soil were shown to be transgenic by Southern analysis.     

Genetic transformation of prickly-pear cactus (Opuntia ficus-indica) by Agrobacterium tumefaciens

A system for genetic transformation of an elite prickly pear cactus (Opuntia ficus-indica L., cultivar Villa Nueva) by Agrobacterium tumefaciens was developed. Beginning with direct bacterial infection by using a hypodermic syringe to the meristematic tissue termed areoles, transgenic plants were obtained by selection with 100 mg l⁻¹ kanamycin. Transient and stable GUS activities were monitored on kanamycin-resistant shoots and regenerated plants, respectively. Genetic transformation of regenerated plants growing under selection was demonstrated by PCR and Southern blot analysis; transgene copy number in the genome of transgenic plants ranged from two to six, while the transformation frequency obtained by the system reported here was of 3.2%. This method may be useful for routine transformation and introduction of several important genes in prickly pear cactus.     

Transformation of Sugarbeet (Beta vulgaris) by Agrobacterium tumefaciens

A method has been developed for the regeneration of transformedplants of the commercially important crop sugarbeet (Beta vulgarisL.), using Agrobacterium tumefaciens. Binary vectors were used,carrying both screenable and selectable genes. Plant regenerationfrom shoot-base tissues was found to be relatively rapid andfrequent compared with petioles or leaf tissue. Inoculationof cultured shoot-base tissues resulted in the production oftransformed plants, as determined by (1) introduced resistanceto kanamycin, (2) introduced CAT or GUS activity, and (3) Southernblot analysis to show the integration of foreign DNA. The transformationfrequency was found to be dependent upon explant source, plantgenotype and selection conditions used. Key words: Agrobacterium tumefaciens, sugarbeet (Beta vulgaris L.), transformation.     

Agrobacterium-mediated transformation of strawberry calli and recovery of transgenic plants

Transformed calli and shoots of strawberry (Fragaria × ananassa Duch.) cv. Redcoat were obtained using Agrobacterium tumefaciens carrying plasmid pB1121. Inoculated leaf explants produced transgenic calli at a frequency of 3% on selection medium containing 50 g/ml kanamycin. Twenty per cent of selected caili regenerated, giving rise to transgenic shoots. All transgenic calli and shoots expressed substantial amounts of GUS and NPT-II activity. The Southern blot analysis confirmed the insertion of both marker genes into the strawberry genome as single and multiple copy inserts. The transgenic shoots elongated on rooting medium in the presence of 25 g/ml kanamycin, but exhibited reduced rooting ability.Abbreviations BA benzyladenine - NAA 1-naphthaleneacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - IBA indole-3-butyric acid - NPT-II neomycin phosphotransferase(EC 2.7.1.95) - GUS -glucuronidase(EC 3.2.1.31) - X-GLUC 5-bromo-4-chloro-3-indolyl-glucuronide - 4-MU 4-methylumbelliferone NRCC No. 31227     

Selection for kanamycin resistance in transformed petunia cells leads to the co-amplification of a linked gene

A cell suspension culture was established from a transgenic petunia (Petunia hybrida L.) plant which carried genes encoding neomycin phosphotransferase II (nptII) and -glucuronidase (uidA, GUS). Two selection experiments were performed to obtain cell lines with increased resistance to kanamycin. In the first, two independently selected cell lines grown in the presence of 350 g/ml kanamycin were eight to ten-fold more resistant to kanamycin than unselected cells. Increased resistance was correlated with amplification of the nptII gene and an increase in nptII mRNA levels. Selection for kanamycin resistance also produced amplification of the linked GUS gene, resulting in increased GUS mRNA levels and enzyme activity. Selected cells grown in the absence of kanamycin for twelve growth cycles maintained increased copy numbers of both genes, and GUS enzyme activity was also stably overexpressed. In a second selection experiment, a cell line grown continuously in medium containing 100 g/ml kanamycin exhibited higher nptII and GUS gene copy numbers and an increase in GUS enzyme activity after eleven growth cycles. In this cell line, amplification of the two genes was accompanied by DNA rearrangement.     

Factors affecting Agrobacterium tumefaciens-mediated transformation of peppermint

 Substantial improvement in peppermint (Mentha x piperita L. var. Black Mitcham) genetic transformation has been achieved so that the frequency of transgenic plants regenerated (percent of leaf explants that produced transformed plants) was 20-fold greater than with the original protocol. Essential modifications were made to conditions for Agrobacterium tumefaciens co-cultivation that enhanced infection, and for selection of transformed cells and propagules during regeneration. A systematic evaluation of co-cultivation parameters established that deletion of coconut water from the co-cultivation medium resulted in substantially increased transient β-Glucuronidase (GUS) activity, in both the frequency of explants expressing gusA and the number of GUS foci per explant (>700 explants). Co-cultivation on a tobacco cell feeder layer also enhanced A. tumefaciens infection. Enhanced transformation efficiencies were further facilitated by increased selection pressure mediated by higher concentrations of kanamycin in the medium during shoot induction, regeneration, and rooting: from 20 to 50 mg/l in shoot induction/regeneration medium and from 15 to 30 mg/l in rooting medium. Raising the concentration of kanamycin in media substantially lowered the number of "escapes" without significant reduction in plant regeneration. These modifications to the protocol yielded an average transformation frequency of about 20% (>2000 explants) based on expression of GUS activity or the tobacco antifungal protein, osmotin, in transgenic plants. Genetic transformation of peppermint has been enhanced to the extent that biotechnology is a viable alternative to plant breeding and clonal selection for improvement of this crop. Received: 7 December 1998 / Revision received: 27 April 1999 / Accepted: 14 May 1999     

Transgenic peppermint (Mentha×piperita L.) plants obtained by cocultivation with Agrobacterium tumefaciens

The first transgenic peppermint (Mentha×piperita L. cultivar Black Mitcham) plants have been obtained by Agrobacterium-mediated transformation by cocultivation with morphogenically responsive leaf explants. Basal leaf explants with petioles, from leaves closest to the apex of in-vitro-culture-maintained shoots (5 cm), exhibited optimal shoot organogenetic responsiveness on medium supplemented with thidiazuron (8.4 μm). Shoot formation occurred at sites of excision on the leaf blade and petiole either directly from cells of the explant or via a primary callus. Analyses of transient GUS activity data indicated that DNA delivery by microprojectile bombardment was more effective than Agrobacterium infection. However, no transgenic plants were obtained from over 22,000 leaf explants after particle bombardment. Cocultivation of leaf explants with Agrobacterium strain EHA 105 and kanamycin selection produced transgenic plants. Greater transient and stable -glucuronidase (GUS) activities were detected in explants or propagules transformed with the construct where gusA was driven by the pBISN1 promoter rather than a CaMV 35S promoter. Eight plants were subsequently regenerated and verified as transgenic based on detection of the nptII transgene by PCR and Southern blot analyses. The Southern analyses indicated that the plants were derived from eight unique transformation events. All transgenic plants appeared morphologically normal. Analyses of GUS activities in leaves sampled from different portions of these transgenic plants, 10 months after transfer to the greenhouse, indicated that six out of the eight original regenerants were uniformly transformed, i.e., did not exhibit chimeric sectors. Received: 12 December 1997 / Revision received: 3 June 1997 / Accepted: 18 July 1997     

Stable transformation of papaya via microprojectile bombardment

Summary Stable transformation of papaya (Carica papaya L.) has been achieved following DNA delivery via high velocity microprojectiles. Three types of embryogenic tissues, including immature zygotic embryos, freshly explanted hypocotyl sections, and somatic embryos derived from both, were bombarded with tungsten particles carrying chimeric NPTII and GUS genes. All tissue types were cultured prior to and following bombardment on half-strength MS medium supplemented with 10 mg 1^–1 2,4-D, 400 mg 1^–1 glutamine, and 6% sucrose. Upon transfer to 2,4-D-free medium containing 150 mg 1^–1 kanamycin sulfate, ten putative transgenic isolates produced somatic embryos and five regenerated leafy shoots. Leafy shoots were produced six to nine months following bombardment. Tissues from 13 of these isolates were assayed for NPTII activity, and 10 were positive. Six out of 15 isolates assayed for GUS expression were positive. Three isolates were positive for both NPTII and GUS,Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - GUS -glucuronidase - X-gluc 5-Br-4-Cl-3-indolyl--D-glucuronic acid - CaMV cauliflower mosaic virus - NOS nopaline synthase - NPTII neomycin phosphotransferase II Journal Series no. 3448 of the Hawaii Institute of Tropical Agriculture and Human Resources     

Agrobacterium tumefaciens-mediated genetic transformation of mungbean (Vigna radiata L. Wilczek) — a recalcitrant grain legume

Agrobacterium-mediated transformation of Vigna radiata L. Wilczek has been achieved. Hypocotyl and primary leaves excised from 2-day-old in-vitro grown seedlings produced transgenic calli on B₅ basal medium supplemented with 5×10⁻⁶ M BAP, 2.5×10⁻⁶ M each of 2,4-D and NAA and 50 mg l⁻¹ kanamycin after co-cultivation with Agrobacterium tumefaciens strains, LBA4404 (pTOK233), EHA105 (pBin9GusInt) and C58C1 (pIG121Hm) all containing β-glucuronidase (gusA) and neomycin phosphotransferase II (nptII) marker genes. Transformed calli were found resistant to kanamycin up to 1000 mg^.l⁻¹. Gene expression of kanamycin resistance (nptII) and gusA in transformed calli was demonstrated by nptII assay and GUS histochemical analysis, respectively. Stable integration of T-DNA into the genome of transformed calli of mungbean was confirmed by Southern blot analysis. Transgenic calli could not regenerate shoots on B₅ or B₅ containing different cytokinins or auxins alone or in combination. However, for the first time, transformed green shoots showing strong GUS activity were regenerated directly from cotyledonary node explants cultured after co-cultivation with LBA4404 (pTOK233) on B₅ medium containing 6-benzylaminopurine (5×10⁻⁷ M) and 75 mg l⁻¹ kanamycin. The putative transformed shoots were rooted on B₅+indole-3-butyric acid (5×10⁻⁶ M) within 10–14 days and resulted plantlets subsequently developed flowers and pods with viable seeds in vitro after 20 days of root induction. The stamens, pollen grains and T₀ seeds showed GUS activity. Molecular analysis of putative transformed plants revealed the integration and expression of transgenes in T₀ plants and their seeds.     

Meristem transformation of sunflower via Agrobacterium

Summary For transformation of sunflower (Helianthus annuus L. cv. Zebulon), shoot apical meristems were dissected from seeds and cocultivated with a disarmed Agrobacterium tumefaciens strain harboring a binary vector carrying genes encoding GUS- and NPTII-activity. The influence of the media conditions, the time of cocultivation and the stage of the developing seed on shoot development and meristem transformation was analysed. Transformants were selected by their ability to grow on kanamycin. Transformation was confirmed by assays for GUS and NPTII. GUS-positive shoots were rooted on rockwool and transferred to soil. Transformation of shoot meristem cells occurred at low frequencies. Chimaeric expression of the two genes was observed in transformed plants. Integration of the foreign DNA in the sunflower genome was confirmed with the polymerase chain reaction.Abbreviations GUS ß-Glucuronidase - NPTII Neomycin phosphotransferase II     

Transformation and Characterization of Transgenic Plants of Solanum dulcamara L.--Incidence of Transgene Silencing

A transformation system is described for Solanum dulcamara usingthe supervirulentAgrobacterium tumefaciens strain 1065, carryingboth the ß-glucuronidase (gus) and neomycin phosphotransferaseII (npt II) genes adjacent to the right and left T-DNA borders,respectively. Leaf explants were more efficient for the productionof transformed plants compared to stem explants on medium containing50 mg l^-1of kanamycin sulphate. A 1:10 (v:v) dilution of anovernight culture ofAgrobacterium gave optimal transformationin terms of transgenic plant regeneration. From a total of 174kanamycin-resistant plants selected by their antibiotic resistance,16 failed to exhibit GUS activity. Southern analysis revealedthat these GUS-negative transformants originated from threeindependently transformed cell lines. Restriction enzyme analysesshowed that the GUS-negative plants had both the gus and nptII genes integrated into their genome (one plant had a singlecopy of each gene; the other two plants had multiple copies),with major rearrangement of the gus gene occurring in plantswith several copies of the transgene. GUS-negative plants showedleaf malformations, delayed flowering and a reduction in flower,fruit and seed production compared to GUS-positive and non-transformed(control) plants. Although gene silencing of the gus gene occurred,albeit at a low frequency (9.2%), the transformation systemdescribed generates large numbers of phenotypically normal,stably transformed plants. Copyright 2000 Annals of Botany Company Agrobacterium -mediated transformation, gene silencing, Solanum dulcamara L. (Bittersweet, Woody Nightshade), T-DNA truncation, transgene expression