Analysis of conditions forAgrobacterium-mediated transformation of tobacco cells in suspension

We have developed anAgrobacterium-mediated transformation system, using tobacco cell suspensions, that permits evaluation of factors affecting transformation within seven days of co-cultivation. Tobacco cell transformation was determined by monitoring -glucuronidase (GUS) activity detected in plant cell extracts. The use of a chimeric gene construct, 35S-GUS/INT, containing a portable intron in theuidA reading frame, assured only plant-specific GUS expression. During the co-cultivation period, induction of the bacterialvir-region was monitored using a heterologous gene construct composed of avirB promoter fragment from pTiC58 fused to the chloramphenicol acetyltranferase (CAT) gene ofTn9. Tobacco cell transformants were confirmed by antibiotic selection of transformed plant cells and by X-Gluc staining. Maximum transformation was obtained when plant suspension cultures were growing rapidly which also was coincidental with elevated levels of bacterialvir-region expression. One week after co-cultivation, the transformed cultures exhibited a stable pattern of GUS activity which remained constant without antibiotic selection. The system was used to compare the virulence of a number ofAgrobacterium strains. GUS activity of plant cells co-cultivated with a strain containing a cointegrate plasmid was 3-fold higher than that of one with a binary configuration of the T-DNA. When the co-cultivatingAgrobacterium strain also carried the plasmid used to monitorvir induction, the frequency of transformation was reduced by as much, as 97%.     

Centrifugation Assisted Agrobacterium tumefaciens-mediated Transformation (CAAT) of embryogenic cell suspensions of banana (Musa spp. Cavendish AAA and Lady finger AAB)

Centrifugation-assisted Agrobacterium-mediated transformation (CAAT) protocol, developed using banana cultivars from two economically important genomic groups (AAA and AAB) of cultivated Musa, is described. This protocol resulted in 25-65 plants/50mg of settled cell volume of embryogenic suspension cells, depending upon the Agrobacterium strain used, and gave rise to hundreds of morphologically normal, transgenic plants in two banana cultivars from the two genomic groups. Development of a highly efficient Agrobacterium-mediated transformation protocol for a recalcitrant species like banana, especially the Cavendish group (AAA) cultivars, required the identification and optimisation of the factors affecting T-DNA delivery and subsequent plant regeneration. We used male-flower-derived embryogenic cell suspensions of two banana cultivars (Cavendish and Lady Finger) and Agrobacterium strains AGL1 and LBA4404, harbouring binary vectors carrying hpt (hygromycin phosphotransferase) and gusA (-glucuronidase) or nptII (neomycin phosphotransferase) and a modified gfp (green fluorescent protein) gene in the T-DNA, to investigate and optimise T-DNA delivery and tissue culture variables. Factors evaluated included pre-induction of Agrobacterium, conditions and media used for inoculation and co-cultivation, and the presence of acetosyringone and Pluronic F68 in the co-cultivation media. One factor that led to a significant enhancement in transformation frequency was the introduction of a centrifugation step during co-cultivation. Post co-cultivation liquid-media wash and recovery step helped avoid Agrobacterium overgrowth on filters supporting suspension culture cells. Marker-gene expression and molecular analysis demonstrated that transgenes integrated stably into the banana genome. T-DNA:banana DNA boundary sequences were amplified and sequenced in order to study the integration profile.     

Genetic transformation of cocoa leaf cells using Agrobacterium tumefaciens

Leaf strips from cocoa tree (Theobroma cacao L.) clones ICS-16 and SIC-5 were cocultivated with the supervirulent Agrobacterium tumefaciens strain A281-Kan. A281-Kan contains a wild-type Ti plasmid and an additional plasmid, pGPTV-Kan, which confers kanamycin resistance to transformed plant cells after integration and expression of the neomycin phosphotransferase II (nptII) gene. Transformed cells were selected on callusing medium containing 100 g ml^-1 kanamycin. NptII assays confirmed that kanamycin-resistant cultures of ICS-16 and SIC-5 expressed the nptII gene, whereas control cultures did not. Genomic Southern blot analyses demonstrated single T-DNA insertions into ICS-16 and SIC-5. T-DNA/cocoa DNA border regions from transformed cultures were cloned and sequenced, revealing that in both transformed cell lines, the right T-DNA border was at the 5 end of the 25 bp right border repeat. Cocoa DNA probes from the T-DNA/cocoa DNA insertion sites were used in Southern blot analyses and showed that T-DNA from pGPTV-Kan had inserted into a unique region in ICS-16 and into a repetitive region in SIC-5. This study establishes that foreign genes can be inserted and expressed in cocoa using A. tumefaciens-mediated gene transfer.     

Agrobacterium tumefaciens-mediated transformation of severalRubus genotypes and recovery of transformed plants

Experiments in shoot regeneration and virulentAgrobacterium tumefaciens-mediated transformation were used to develop a protocol forRubus transformation. This protocol was then used to produce transformedRubus plants fromin vitro internodes inoculated with anAgrobacterium tumefaciens encoding neomycin phosphotransferase on its disarmed T-DNA. Two transformed plants were selected from 800 inoculations on a medium containing 10 µg ml^–1 kanamycin. Results indicated that this level of kanamycin successfully selected against non-transformed cells but did not reduce the number of transformed, kanamycin-resistant, shoots formed. Enzyme assays and Southern blot analysis verified the presence of the -glucuronidase gene in the plant genome.     

Inactivation of the nopaline synthase gene by double transformation: reactivation by segregation of the induced DNA

Tobacco plants were transformed with derivatives of a binary vector pMON505 and two kanamycin resistant lines that were nopaline positive were selected for second transformation. The plasmids used for the second transformation were derivatives of pMON850 which carries the nopaline synthase gene in addition to a gene for gentamicin resistance. Insertion of each transgene was confirmed by Southern hybridization. Surprisingly, we found that more than 50% of the doubly transformed tobacco plants were nopaline negative. Tobacco plants that were transformed only by the second vector exhibited nopaline accumulation. DNA methylation patterns at the HpaII site in the promoter region of the nopaline synthase gene did not correlate with the nopaline phenotype. In some plant lines, seedlings of the R1 generation which segregated out the second T-DNA insertion recovered the nop⁺ phenotype. These results indicate that nopaline accumulation was inhibited by the presence of the second T-DNA.Abbreviations T-DNA transferred DNA - NPTII neomycin phosphotransferase II - uidA -glucuronidase - Km kanamycin - Gm gentamicin - nop⁺ nopaline positive - nop^– nopaline negative - MS medium, Murashige-Skoog medium     

The use of selectable markers for the isolation of plant-DNA/T-DNA junction fragments in a cosmid vector

Summary DNA of the crown gall tumor line W38T37:: Tn7-1 was partially digested with Sau3A to an average molecular weight of 25 Md and ligated either directly or after size fractionation to BamHI cut cosmid pJC81 DNA. After in vitro packaging in phage particles and transduction to E. coli HB101, recombinants that expressed the Tn7 coded resistances to spectinomycin and trimethoprim were selected. The recombinant plasmids thus isolated contained part or the whole of Tn7 together with adjacent T-DNA. Four independent, large clones are described, three containing the left border of the T-DNA, one containing the right border and an intact copy of the Tn7 transposon. In this case all the Tn7 encoded genes were shown to have remained fully functional since the reisolated Tn7 was found to be capable of normal transposition in E. coli. The T-DNA in the W38T37::Tn7 tumor line is flanked both to the left and to the right by highly AT rich repetitive plant sequences. These results further demonstrate that foreign genes can be transferred, integrated and stably maintained in chromosomes of plant cells without undergoing any observable rearrangements. This method of cosmid cloning combined with direct selection for the desired recombinant colonies is of general application for the genomic cloning of transformed eukaryotic cells.     

Transformation of the monocot Alstroemeria by Agrobacterium rhizogenes

An efficient procedure is described for transformation of calli of the monocotyledonous plant Alstroemeria by Agrobacterium rhizogenes. Calli were co-cultivated with A. rhizogenes strain A13 that harbored both a wild-type Ri-plasmid and the binary vector plasmid pIG121Hm, which included a gene for neomycin phosphotransferase II (NPTII) under the control of the nopaline synthase (NOS) promoter, a gene for hygromycin phosphotransferase (HPT) under the control of the cauliflower mosaic virus (CaMV) 35S promoter, and a gene for -glucuronidase (GUS) with an intron fused to the CaMV 35S promoter. Inoculated calli were plated on medium that contained cefotaxime to eliminate bacteria. Four weeks later, transformed cells were selected on medium that contained 20 mg L^–1 hygromycin. A histochemical assay for GUS activity revealed that selection by hygromycin was complete after eight weeks. The integration of the T-DNA of the Ri-plasmid and pIG121Hm into the plant genome was confirmed by PCR. Plants derived from transformed calli were produced on half-strength MS medium supplemented with 0.1 mg L^–1 GA₃ after about 5 months of culture. The presence of the gusA, nptII, and rol genes in the genomic DNA of regenerated plants was detected by PCR and Southern hybridization, and the expression of these transgenes was verified by RT-PCR.     

The Agrobacterium rhizogenes pRi TL-DNA segment as a gene vector system for transformation of plants

Summary A plant gene transfer system was developed from the Agrobacterium rhizogenes pRi15834 TL-DNA region. Intermediate integration vectors constructed from ColE1-derived plasmids served as cloning vectors in Escherichia coli and formed cointegrates into the TL-DNA after transfer to A. rhizogenes. An A. rhizogenes strain with pBR322 plasmid sequences replacing part of the TL-DNA was also constructed. Plasmids unable to replicate in Agrobacterium can integrate into this TL-DNA by homologous recombination through pBR322 sequences. No loss of pathogenicity was observed with the strains formed after integration of intermediate vectors or strains carrying pBR322 in the TL-DNA segment. Up to 15 kb of DNA have been transferred to plant cells with these systems. The T-DNA from a binary vector was cotransformed into hairy roots which developed after transfer of the wild-type pRi T-DNA. Tested on Lotus corniculatus the TL-derived vector system transformed 90% of the developed roots and the T-DNA from the binary vector was cotransformed into 60% of the roots. Minimum copy numbers of one to five were found. Both constitutive and organ-specific plant genes were faithfully expressed after transfer to the legume L. corniculatus.     

Agrobacterium tumefaciens-mediated transformation of Allium cepa L.: the production of transgenic onions and shallots

This paper describes the development of a reliable transformation protocol for onion and shallot (Allium cepa L.) which can be used year-round. It is based on Agrobacterium tumefaciens as a vector, with three-week old callus, induced from mature zygotic embryos, as target tissue. For the development of the protocol a large number of parameters were studied. The expression of the uidA gene coding for -glucuronidase was used as an indicator in the optimization of the protocol. Subspecies (onion and shallot) and cultivar were important factors for a successful transformation: shallot was better than onion and for shallot cv. Kuning the best results were obtained. Also, it was found that constantly reducing the size of the calli during subculturing and selection by chopping, thus enhancing exposure to the selective agent hygromycin, improved the selection efficiency significantly. Furthermore, callus induction medium and co-cultivation period showed a significant effect on successful stable transformation. The usage of different Agrobacterium strains, callus ages, callus sources and osmotic treatments during co-cultivation did not influence transformation efficiency. The highest transformation frequency (1.95%), was obtained with shallot cv. Kuning. A total of 11 independent transformed callus lines derived from zygotic embryos were produced: seven lines from shallot and four lines from onion. Large differences in plantlet production were observed among these lines. The best line produced over 90 plantlets. Via PCR the presence of the uidA and hpt (hygromycin phosphotransferase) genes could be demonstrated in these putative transformed plants. Southern hybridization showed that most lines originated from one transformation event. However, in one line plants were obtained indicating the occurrence and rescue of at least three independent transformation events. This suggested that T-DNA integration occurred in different cells within the callus. Most transgenic plants only had one copy of T-DNA integrated into their genomes. FISH performed on 12 plants from two different lines representing two integration events showed that original T-DNA integration had taken place on the distal end of chromosomes 1 or 5. A total of 83 transgenic plants were transferred to the greenhouse and these plants appeared to be diploid and normal in morphology.     

Genetically Transformed Plant Roots as a Model for Studying Specific Metabolism and Symbiotic Contacts of the Root System

Genetic transformation of plants mediated by Ri plasmid ofAgrobacterium rhizogenes occupies a special place in plant cell engineering, since this technique based on a natural phenomenon allows cultivation of isolated growing plant roots on hormone-free media. Application of wild-type unmodified agrobacterial strains allows us to obtain root cultures capable of long-term growth in vitro due to an increased sensitivity of the cells to auxins while other biochemical properties remain unaltered. A collection of pRi T-DNA transformed roots of certain dicotyledons was made; some strains in it are used to study synthesis of secondary metabolites in root cells. Thein vitro cultivated roots could synthesize root-specific metabolites, which makes possible their application for large-scale biotechnological production of ecologically pure crude drugs. Cocultivation of pRi T-DNA transformed roots with arbuscular mycorrhizal fungi makes possible vital study of all stages of obligate symbiont development and interaction with plant roots. Dual axenic culture of AM fungi and pRi T-DNA transformed plants can be used to make a collection of the most valuable endomycorrhizal fungal species and to produce considerable quantities of homogeneous fungal inoculums.     

A functional analysis of T-DNA gene 6b: the fine tuning of cytokinin effects on shoot development

Summary The physiological function in planta of T-DNA gene 6b was studied under various experimental conditions. For this purpose the coding region of gene 6b was cloned behind the 1-promoter of the TR-DNA to enhance expression of the gene product in transformed plant cells. Expression of the recombinant gene in leaf discs of Nicotiana tabacum altered the capacity for shoot formation of the discs, induced by exogenous (i.e. BAP in the growth medium or agrobacterial trans-zeatin produced under control of gene tzs) or endogenous cytokinins (i.e. isopentenyladenosine produced under control of T-DNA gene 4). The data obtained indicate a reduction of cytokinin activity within the plant cells by the product of T-DNA gene 6b.Abbreviations AMP adenosine-5-monophosphate - BAP 6-benzylaminopurine     

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     

Optimization of Agrobacterium tumefaciens-Mediated Transformation of Xylaria grammica EL000614, an Endolichenic Fungus Producing Grammicin

An endolichenic fungus Xylaria grammica {"type":"entrez-nucleotide","attrs":{"text":"EL000614","term_id":"124922317"}}EL000614 produces grammicin, a potent nematicidal pyrone derivative that can serve as a new control option for root-knot nematodes. We optimized an Agrobacterium tumefaciens-mediated transformation (ATMT) protocol for X. grammica to support genetic studies. Transformants were successfully generated after co-cultivation of homogenized young mycelia of X. grammica with A. tumefaciens strain AGL-1 carrying a binary vector that contains the bacterial hygromycin B phosphotransferase (hph) gene and the eGFP gene in T-DNA. The resulting transformants were mitotically stable, and PCR analysis showed the integratin of both genes in the genome of transformants. Expression of eGFP was confirmed via fluorescence microscopy. Southern analysis showed that 131 (78.9%) out of 166 transformants contained a single T-DNA insertion. Crucial factors for producing predominantly single T-DNA transformants include 48 h of co-cultivation, pre-treatment of A. tumefaciens cells with acetosyringone before co-cultivation, and using freshly prepared mycelia. The established ATMT protocol offers an efficient tool for random insertional mutagenesis and gene transfer in studying the biology and ecology of X. grammica.     

Factors affecting transformation efficiency of embryogenic callus of Upland cotton (<Emphasis Type="Italic">Gossypium hirsutum</Emphasis>) with <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>

A reliable and high-efficiency system of transforming embryogenic callus (EC) mediated by Agrobacterium tumefaciens was developed in cotton. Various aspects of transformation were examined in efforts to improve the efficiency of producing transformants. LBA4404 and C58C3, harboring the pgusBin19 plasmid containing neomycin phosphortransferase II (npt-II) gene as a selection marker, were used for transformation. The effects of Agrobacterium strains, acetosyringone (AS), co-cultivation temperature, co-cultivation duration, Agrobacterium concentration and physiological status of EC on transformation efficiency were evaluated. Strain LBA4404 proved significantly better than C58C3. Agrobacterium at a concentration of 0.5 × 10⁸ cells ml^–1 (OD₆₀₀=0.5) improved the efficiency of transformation. Relatively low co-cultivation temperature (19 °C) and short co-cultivation duration (48 h) were optimal for developing a highly efficient method of transforming EC. Concentration of AS at 50 mg l^–1 during co-cultivation significantly increased transformation efficiency. EC growing 15 days after subculture was the best physiological status for transformation. An overall scheme for producing transgenic cotton is presented, through which an average transformation rate of 15% was obtained.     

Agrobacterium-mediated genetic transformation of a Dendrobium orchid

A protocol was developed to obtain stable transgenic orchids (Dendrobium nobile) via Agrobacterium-mediated transformation of protocorm-like bodies (PLBs). Agrobacterium tumefaciens strains AGL1 and EHA105 were used, with each containing a binary vector pCAMBIA1301 with the hpt gene as a selectable marker for hygromycin resistance and an intron-containing -glucuronidase gene (gus-int) as a reporter gene. PLBs were co-cultivated with A. tumefaciens, which had been activated with 100 M acetosyringone (AS), for 2–3 days until the growth of A. tumefaciens was observed on co-cultivation medium containing 100 M AS. Following co-cultivation, PLBs were cultured on selective medium containing 30 mg l^–1 hygromycin and 250 mg l^–1 cefotaxime. Proliferating PLBs were repeatedly selected for hygromycin resistance. A high efficiency of transformation (18%) was obtained with a total of 73 stably transformed lines produced. Incorporation and expression of the transgenes were confirmed by Southern blot analysis and GUS histochemical assay.     

Assessment of the efficiency of cotransformation of the T-DNA of disarmed binary vectors derived from Agrobacterium tumefaciens and the T-DNA of A. rhizogenes

Co-transfer of Agrobacterium rhizogenes T-DNA and T-DNA from the A. tumefaciens binary vector pBin19 (Bevan, 1984) was studied in detail using Nicotiana rustica. High frequencies of co-transfer of T-DNA's were observed, even when no selection pressure was exerted. Increased levels of pBin19 T-DNA were found in hairy root cultures with selection at higher levels of kanamycin sulphate (50–200 g ml^–1). Several other species were also transformed by A. rhizogenes carrying pBin19 and A. rhizogenes harbouring a different binary factor, pAGS125 (Van den Elzen et al., 1985), was used to transform N. rustica hairy roots to confer hygromycin B resistance.     

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     

Production of transgenic lily plants by<Emphasis Type="Italic"> Agrobacterium</Emphasis>-mediated transformation

A system for the production of transgenic plants was developed for the Oriental hybrid lily, Lilium cv. Acapulco, by Agrobacterium-mediated genetic transformation. Filament-derived calli were co-cultivated with A. tumefaciens strain EHA101/pIG121Hm, which harbored a binary vector carrying the neomycin phosphotransferase II, hygromycin phosphotransferase, and intron-containing -glucuronidase genes in the T-DNA region. Six hygromycin-resistant (Hyg^r) culture lines were obtained from 200 calli by scratching them with sandpaper prior to inoculation and using NH₄NO₃-free medium for co-cultivation and a hygromycin-containing regeneration medium for selection. Hyg^r culture lines regenerated shoots, which developed into plantlets following transfer to a plant growth regulator-free medium. All of these plantlets were verified to be transgenic by GUS histochemical assay and inverse PCR analysis.Abbreviations AS Acetosyringone (3,5-dimethoxy-4-hydroxy-acetophenone) - BA Benzyladenine - CaMV Cauliflower mosaic virus - GUS -Glucuronidase - HPT Hygromycin phosphotransferase - Hyg^r Hygromycin-resistant - NOS Nopaline synthase - NPTII Neomycin phosphotransferase II - PGR Plant growth regulator - PIC Picloram (4-amino-3,5,6-trichloropicolinic acid)Communicated by H. Ebinuma     

Neoplastic progression in crown gall in tobacco without elevated auxin levels

We have isolated two stable variants from a crown-gall teratoma tissue of tobacco (Nicotiana tabacum L.) transformed by Agrobacterium tumefaciens strain A66, a mutant of the virulent A6 strain containing an insertion sequence in the tumor-inducing (Ti) plasmid at the locus coding for auxin biosynthesis. Normally tobacco cells transformed by strain A66 spontaneously form shoots in culture and will not grow on hormone-free medium unless shoots develop. The variant tissue lines, isolated from the teratoma tissue after prolonged culture in the dark, grew as friable and unorganized tissues on hormone-free growth medium. Growth of the variants was more sensitive to auxin feeding than growth of the parental teratoma line, and the auxin dose-response curves of the variant lines were similar to those obtained with A6-transformed tobacco cells. Southern blot analysis of DNA from the parental teratoma line and one of the variants showed no differences in copy number or organization of the oncogenic DNA sequence (T-DNA) transferred from the bacterium, indicating that the variant phenotype did not result from reversion of the A66 mutation. Radio-immunoassay analysis showed similar levels of indole-3-acetic acid (IAA) in the variants and parental teratoma line (3–50 and 38–42 pmol·(gFW)^-1, respectively), whereas an A6-transformed cell line contained much higher IAA levels (150–1200 pmol·(g FW)^-1). Low levels of the ethylene precursor 1-aminocyclopropane-1-carboxylic acid in the variants and the parental teratoma line (<5 nmol·(g FW)^-1) as compared with that found in the A6-transformed line (>100 nmol· (g FW)^-1) provided additional, indirect evidence for low auxin levels in the variant lines. These results indicate that crown-gall teratoma tissues of tobacco may switch to the unorganized, auxin-sensitive phenotype without an increase in auxin content.Abbreviations IAA indole-3-acetic acid - kb kilobase - NAA -naphthalene acetic acid - NAM -naphthaleneacetamide - T-DNA DNA transferred from the Ti plasmid to the plant - T_L-DNA the left transferred region of pTiA6 containing the T-DNA oncogenes     

Transgenic grasspea (Lathyrus sativus L.): Factors influencing Agrobacterium-mediated transformation and regeneration

A reproducible procedure was developed for genetic transformation of grasspea using epicotyl segment co-cultivation with Agrobacterium. Two disarmed Agrobacterium tumefaciens strains, EHA 105 and LBA 4404, both carrying the binary plasmid p35SGUSINT with the neomycin phosphotransferase II (nptII) gene and the -glucuronidase (gus)-intron, were studied as vector systems. The latter was found to have a higher transforming ability. Several key factors modifying the transformation rate were optimized. The highest transformation rate was achieved using hand-pricked explants for infection with an Agrobacterium culture corresponding to OD₆₀₀0.6 and diluted to a cell density of 10⁹ cells ml^–1 for 10 min, followed by co-cultivation for 4 days in a medium maintained at pH 5.6. Putative transformed explants capable of forming shoots were selected on regeneration medium containing kanamycin (100 g ml^–1). We achieved up to 36% transient expression based on the GUS histochemical assay. Southern hybridization of genomic DNA of the kanamycin-resistant GUS-expressive shoots to a gus-intron probe substantiated the integration of the transgene. Transformed shoots were rooted on half-strength MS containing 0.5 mg l^–1 indole-3-acetic acid, acclimated in vermi-compost and established in the experimental field. Germ-line transformation was evident through progeny analysis. Among T₁ seedlings of most transgenic plant lines, kanamycin-resistant and -sensitive plants segregated in a ratio close to 3:1.