Stably transformed herbicide resistant callus of sugarcane via microprojectile bombardment of cell suspension cultures and electroporation of protoplasts

Stably transformed callus of a hybrid sugarcane cultivar (Saccharum species hybrid, CP72-1210) was achieved following high velocity microprojectile bombardment of suspension culture cells, and electroporation of protoplasts. A three-day old cell suspension culture (SC88) was bombarded with gold particles coated with pBARGUS plasmid DNA containing the ß-glucuronidase (GUS) reporter gene and the bar selectable gene that confers resistance to the herbicide basta. The pBARGUS plasmid was also electroporated into the protoplasts of another cell line (SCPP). Colonies resistant to basta were recovered from both sources. Stable integration of the bar gene in the resistant cell lines was confirmed by Southern analysis. In addition, phosphinothricin acetyltransf erase (PAT) activity was also demonstrated in the transformed cell lines.Abbreviations GUS ß-glucuronidase - 2,4-D 2,4-dichlorophenoxyacetic acid - BAP benzylaminopurine - PMSF phenylmethylsulfonyl fluoride - MES 2[N-Morpholino]ethanesulfonic acid - HEPES [N-2-hydroxyethyl] piperazine-N-[2-ethane sulfonic acid] - PAT Phosphinothricin acetyltransferase - CTAB cetyltrimethylammonium bromide     

Bialaphos selection of stable transformants from maize cell culture

Summary Stable transformed Black Mexican Sweet (BMS) maize callus was recovered from suspension culture cells bombarded with plasmid DNA that conferred resistance to the herbicide bialaphos. Suspension culture cells were bombarded with a mixture of two plasmids. One plasmid contained a selectable marker gene, bar, which encoded phosphinothricin acetyl transferase (PAT), and the other plasmid encoded a screenable marker for -glucuronidase (GUS). Bombarded cells were selected on medium containing the herbicide bialaphos, which is cleaved in plant cells to yield phosphinothricin (PPT), an inhibitor of glutamine synthetase. The bialaphos-resistant callus contained the bar gene and expressed PAT as assayed by PPT inactivation. Transformants that expressed high levels of PAT grew more rapidly on increasing concentrations of bialaphos than transformants expressing low levels of PAT. Fifty percent of the bialaphos-resistant transformants tested (8 of 16) expressed the nonselected gene encoding GUS.     

Efficient Agrobacterium-mediated transformation of Arabidopsis thaliana using the bar gene as selectable marker

Summary We have established an efficient Agrobacterium-mediated transformation procedure for Arabidopsis thaliana genotype C24 using the chimeric bialaphos resistance gene (bar) coding for phosphinothricin acetyltransferase (PAT). Hypocotyl explants from young seedlings cocultivated with agrobacteria carrying a bar gene were selected on shoot-inducing media containing different concentrations of phosphinothricin (PPT) which is an active component of bialaphos. We found that 20 mg/l of PPT completely inhibited the control explants from growing whereas the explants transformed with the bar gene gave rise to multiple shoots resistant to PPT after 3 weeks under the same selection conditions. The transformation system could also be applied to root explants. Resulting plantlets could produce viable seeds in vitro within 3 months after preparation of the explants. The stable inheritance of the resistance trait, the integration and expression of the bar gene in the progeny were confirmed by genetic tests, Southern analysis and PAT enzyme assay, respectively. In addition, the mature plants in soil showed tolerance to the herbicide Basta.Abbreviations bar bialaphos resistance gene - CIM callus-inducing medium - DTNB 5,5-dithiobis(2-nitrobenzoic acid) - GM germination medium - HPT hygromycin phosphotransferase - MS Murashige and Skoog salts - NPTII neomycin phosphotransferase II - PAT phosphinothricin acetyltransferase - PPT phosphinothricin - SIM shoot-inducing medium     

Regeneration of herbicide resistant transgenic rice plants following microprojectile-mediated transformation of suspension culture cells

Summary Suspension cells of Oryza sativa L. (rice) were transformed, by microprojectile bombardment, with plasmids carrying the coding region of the Streptomyces hygroscopicus phosphinothricin acetyl transferase (PAT) gene (bar) under the control of either the 5 region of the rice actin 1 gene (Act1) or the cauliflower mosaic virus (CaMV) 35S promoter. Subsequently regenerated plants display detectable PAT activity and are resistant to BASTA^TM, a phosphinothricin (PPT)-based herbicide. DNA gel blot analyses showed that PPT resistant rice plants contain a bar-hybridizing restriction fragment of the expected size. This report shows that expression of the bar gene in transgenic rice plants confers resistance to PPT-based herbicide by suppressing an increase of ammonia in plants after spraying with the herbicide.     

Recovery of transgenic trees after electroporation of poplar protoplasts

Protoplasts from leaflets ofin vitro cuttings were electroporated in osmotically adjusted and buffered solutions containing plasmid DNA: pABD1, carrying thenptII gene for resistance to neomycin; pGH1, carrying a mutant acetolactate synthase gene,als, for resistance to sulfonylurea; and pGSFR781A, carrying a synthetic phosphinothricin acetyltransferase (pat) for resistance to phosphinothricin (Basta). Gene transfer was repeatedly efficient, without use of carrier DNA, in the range of one transformant for 10⁵ to 10⁴ protoplast-derived cell colonies. This was probably due to the high plating efficiency (30%) of protoplasts in our culture process. Selection for expression of foreign genes was applied in liquid medium and repeatedly achieved with 30 M paromomycin for NPTII, 200 nM chlorsulfuron for the mutant ALS ofArabidopsis and 25 M phosphinothricin for PAT expression. Integration of foreign genes into genomic DNA of resistant poplar trees was demonstrated by Southern blot hybridizations, which revealed that for some transformants practically no other part of the vector plasmid than the selected gene was integrated.Effective processes for protoplast culture, efficient selection at the cell colony stage and gene transfer will provide new possibilities in poplar breeding.     

An efficient particle bombardment system for the genetic transformation of asparagus (Asparagus officinalis L.)

The microprojectile bombardment method was used to transfer DNA into embryogenic callus of asparagus (Asparagus officcinalis L.) and to produce stably transformed asparagus plants. Embryogenic callus, derived from UC 157 and UC72 asparagus cultivars, was bombarded with tungsten particles coated with plasmid DNA that contained genes encoding hygromycin phosphotransferase, phosphinothricin acetyl transferase and -glucuronidase. Putatively transformed calli were identified from the bombarded tissue after 4 months selection on 25 mg/L hygromycin B plus 4 mg/L phosphinothricin (PPT). By selecting embryogenic callus on hygromycin plus PPT the overall transformation and selection efficiencies were substantially improved over selection with hygromycin or PPT alone, where no transgenic clones were recovered. The transgenic nature of the selected material was demonstrated by GUS histochemical assays and Southern blot hybridization analysis. Transgenic asparagus plants were found to withstand the prescribed levels of the PPT-based herbicide BASTATM for weed control.Abbreviations GUS -glucuronidase - HPT hygromycin phosphotransferase - bar phosphinothricin acetyl transferase gene - PPT phosphophinothricin - NAA naphthalene acetic acid - 2iP 2-isopentenyl adenine     

Agrobacterium-mediated transformation of apple (Malus x domestica Borkh.): an assessment of factors affecting regeneration of transgenic plants

We have previously developed a protocol for efficient gene transfer and regeneration of transgenic calli following cocultivation of apple (cv. Jonagold) explants with Agrobacterium tumefaciens (De Bondt et al. 1994, Plant Cell Reports 13: 587–593). Now we report on the optimization of postcultivation conditions for efficient and reproducible regeneration of transgenic shoots from the apple cultivar Jonagold. Factors which were found to be essential for efficient shoot regeneration were the use of gelrite as a gelling agent and the use of the cytokinin-mimicing thidiazuron in the selective postcultivation medium. Improved transformation efficiencies were obtained by combining the hormones thidiazuron and zeatin and by using leaf explants from in vitro grown shoots not older than 4 weeks after multiplication. Attempts to use phosphinothricin acetyl transferase as a selectable marker were not successful. Using selection on kanamycin under optimal postcultivation conditions, about 2% of the leaf explants developed transgenic shoots or shoot clusters. The presence and expression of the transferred genes was verified by -glucuronidase assays and Southern analysis. The transformation procedure has also been succesfully applied to several other apple cultivars.Abbreviations BAP benzylaminopurine - CTAB hexadecyltrimethylammoniumbromide - Na₂EDTA ethylenediamine-tetra-acetate ferric-sodium salt - FeNaEDTA ethylenediamine-tetra-acetate ferric-sodium salt - GA₃ gibberellic acid 3 - GusA -glucuronidase - gusA -glucuronidase gene of Escherichia coli - IAA indole acetic acid - IBA indole butyric acid - 2iP N6-2-isopentenyl adenine - NAA naphthalene acetic acid - nptII neomycinphosphotransferase II gene - bar phosphinothricin acetyl transferase gene - PCR polymerase chain reaction - PPT phosphinothricin - STS silver thiosulphate - T-DNA transferred DNA - TDZ thidiazuron - X-Gluc 5-bromo-4-chloro-3-indolyl -D-glucuronide - Zea trans-Zeatin     

Herbicide resistant transgenic papaya plants produced by an efficient particle bombardment transformation method

A system for the production of transgenic papaya (Carica papaya L.) plants using zygotic embryos and embryogenic callus as target cells for particle bombardment is described. Phosphinothricin (bar ) and kanamycin (npt II) resistance genes were used as selectable markers, and the gus gene (uidA) as a reporter gene. Selection with 100 mg/l kanamycin and 4 mg/l phosphinothricin (PPT) yielded a total of over 90 resistant embryogenic colonies from three independent experiments using embryogenic callus as a target tissue. This represents an efficiency of 60 transgenic clones per gram of fresh weight callus bombarded. The efficiency of genetic transformation using zygotic embryos was lower, as only 8 independent resistant clones were recovered out of 645 bombarded zygotic embryos, giving a efficiency of 1.24%. Subsequent subculture of transgenic somatic embryos both from zygotic embryos and embryogenic callus led to the development of plants with apparently normal morphology. Histological, fluorimetric assay for GUS, NPT II assay and DNA analysis (Southern hybridization) showed that kanamycin /PPT resistant plants carried and expressed the transgenes.Abbreviations Gus -glucuronidase - NPTII neomycin phophotransferase II - bar phophinothricin acetyl transferase gene - Pat phosphinothricin acetyl transferase - PPT phosphinothricin - Km kanamycin - 2,4-D 2,4-dichlorophenoxyacetic acid - K kinetin - BAP benzylaminopurine - IBA indolbutyric acid     

Efficient regeneration of Brassica oleracea hypocotyl protoplasts and high frequency genetic transformation by direct DNA uptake

Summary Efficient regeneration (80%) and high frequency genetic transformation (10–33%) were achieved by culturing protoplasts isolated from hypocotyl tissues of six day old Brassica oleracea seedlings and by subjecting these protoplasts to PEG mediated direct plasmid uptake. Three different plasmid vectors carrying marker genes for resistance to methotrexate (dhfr), hygromycin (hpt) and phosphinotricin (bar) were constructed and used for transformation. Large number of normal, fertile transformants were obtained with vectors carrying hpt and bar genes. No transformants could be regenerated for resistance to methotrexate as it severely suppressed shoot differentiation.Abbreviations bar/PAT bialaphos resistance gene/phosphinotricin acetyltransferase - 2,4-D 2,4-di-chlorophenoxyacetic acid - dhfr/DHPR dihydrofolate reductase gene/enzyme - gus/GUS -glucuronidase gene/enzyme - hpt/HPT hygromycin phosphotransferase gene/enzyme - Kn kinetin - PEG polyethylene glycol - RH relative humidity     

Stable transformation via electroporation into maize Type II callus and regeneration of fertile transgenic plants

Maize Type II callus tissue was used as the plant material for genetic transformation via electroporation. Plasmid DNA containing a selectable marker gene (either neomycin phosphotransferase (npt-II) or phosphinothricin acetyl transferase (bar)), and a screenable marker gene (gus A) was incubated with the tissue prior to electroporation. Electroporated callus tissue was placed on selection medium containing kanamycin sulfate or Bast. No kanamycin resistant colonies were recovered whereas four independent Basta resistant callus isolates were recovered from a total of 544 cuvettes electroporated. After 8 to 16 weeks on the Basta containing medium, selected calli were isolated and maintained in individual selection plates for 4 to 6 weeks until sufficient tissue accumulated. Enzyme assays and DNA analyses were performed to verify the transformation events. Several plants were regenerated from individual callus isolates. The plants derived from one callus isolate were male sterile while those derived from the other isolates were both male and female fertile. Most plants showed Basta resistance. DNA analyses confirmed the presence of the introduced bar gene(s) in the primary regenerants and their progeny. The integration patterns of the inserted DNA appeared to be complex.     

Transgenic herbicide-resistant Atropa belladonna using an Ri binary vector and inheritance of the transgenic trait

Summary Transgenic Atropa belladonna conferred with a herbicide-resistant trait was obtained by transformation with an Ri plasmid binary vector and plant regeneration from hairy roots. We made a chimeric construct, pARK5, containing the bar gene encoding phosphinothricin acetyltransferase flanked with the promoter for cauliflower mosaic virus 35S RNA and the 3 end of the nos gene. Leaf discs of A. belladonna were infected with Agrobacterium rhizogenes harboring an Ri plasmid, pRi15834, and pARK5. Transformed hairy roots resistant to bialaphos (5 mg/l) were selected and plantlets were regenerated. The integration of T-DNAs from pRi15834 and pARK5 were confirmed by DNA-blot hybridization. Expression of the bar gene in transformed R₀ tissues and in backcrossed F1 progeny with a nontransformant and self-fertilized progeny was indicated by enzymatic activity of the acetyltransferase. The transgenic plants showed resistance towards bialaphos and phosphinothricin. Tropane alkaloids of normal amounts were produced in the transformed regenerants. These results present a successful application of transformation with an Ri plasmid binary vector for conferring an agronomically useful trait to medicinal plants.Abbreviations CaMV cauliflower mosaic virus - NPT-II neomycin phosphotransferase II - PAT phosphinothricin acetyltransferase - PPT phosphinothricin     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated genetic transformation and development of herbicide-resistant sugarcane (<Emphasis Type="Italic">Saccharum</Emphasis> species hybrids) using axillary buds

Direct regeneration from explants without an intervening callus phase has several advantages, including production of true type progenies. Axillary bud explants from 6-month-old sugarcane cultivars Co92061 and Co671 were co-cultivated with Agrobacterium strains LBA4404 and EHA105 that harboured a binary vector pGA492 carrying neomycin phosphotransferase II, phosphinothricin acetyltransferase (bar) and an intron containing -glucuronidase (gus-intron) genes in the T-DNA region. A comparison of kanamycin, geneticin and phosphinothricin (PPT) selection showed that PPT (5.0 mg l^–1) was the most effective selection agent for axillary bud transformation. Repeated proliferation of shoots in the selection medium eliminated chimeric transformants. Transgenic plants were generated in three different steps: (1) production of putative primary transgenic shoots in Murashige-Skoog (MS) liquid medium with 3.0 mg l^–1 6-benzyladenine (BA) and 5.0 mg l^–1 PPT, (2) production of secondary transgenic shoots from the primary transgenic shoots by growing them in MS liquid medium with 2.0 mg l^–1 BA, 1.0 mg l^–1 kinetin (Kin), 0.5 mg l^–1 -napthaleneacetic acid (NAA) and 5.0 mg l^–1 PPT for 3 weeks, followed by five more cycles of shoot proliferation and selection under same conditions, and (3) rooting of transgenic shoots on half-strength MS liquid medium with 0.5 mg l^–1 NAA and 5.0 mg l^–1 PPT. About 90% of the regenerated shoots rooted and 80% of them survived during acclimatisation in greenhouse. Transformation was confirmed by a histochemical -glucuronidase (GUS) assay and PCR amplification of the bar gene. Southern blot analysis indicated integration of the bar gene in two genomic locations in the majority of transformants. Transformation efficiency was influenced by the co-cultivation period, addition of the phenolic compound acetosyringone and the Agrobacterium strain. A 3-day co-cultivation with 50 M acetosyringone considerably increased the transformation efficiency. Agrobacterium strain EHA105 was more effective, producing twice the number of transgenic shoots than strain LBA4404 in both Co92061 and Co671 cultivars. Depending on the variety, 50–60% of the transgenic plants sprayed with BASTA (60 g l^–1 glufosinate) grew without any herbicide damage under greenhouse conditions. These results show that, with this protocol, generation and multiplication of transgenic shoots can be achieved in about 5 months with transformation efficiencies as high as 50%.Abbreviations BA 6-Benzyladenine - CaMV Cauliflower mosaic virus - GUS -Glucuronidase - Kin Kinetin - NAA -Naphthaleneacetic acid - Nos Nopaline synthase - nptII Neomycin phosphotransferase II - PCR Polymerase chain reaction - PPT Phosphinothricin - YEP Yeast extract and peptone     

β-glucuronidase gene expression and mRNA stability in oat protoplasts

Summary Protoplasts derived from oat (Avena sativa L.) suspension culture cells (7 days after subculturing) were electroporated with plasmid DNA containing the Escherichia coli uidA gene encoding the ß-glucuronidase reporter enzyme. Consistently high enzyme activity was observed with electroporation conditions of 500 F and 1125 volts/cm. Enzyme activity and mRNA accumulation time courses were determined. The maximum enzyme activity was detected at 24 hours after electroporation, while the maximum mRNA level was detected at 12 hours after electroporation. ß-glucuronidase mRNA was in vitro synthesized with and without a 5 methylated cap and then electroporated into protoplasts. Only capped mRNA produced significant enzyme activity. By electroporating radiolabeled, in vitro synthesized mRNA, the ß-glucuronidase mRNA half-life was estimated to be 35 minutes in oat protoplasts.Abbreviations GUS ß-glucuronidase - mRNA messenger RNA - ICP insecticidal crystal proteins - OCS octopine synthase - CAT chloramphenicol acetyltransferase - nt nucleotide - kb kilobase - MSOD3 Murashige and Skoog media with zero 2,4-dichlorophenoxy acetic acid and 3% sucrose - MU 4-methyl umbelliferone; ATA: aurintricarboxylic acid     

Transient gene expression in electroporated Solanum protoplasts

Electroporation was used to evaluate parameters important in transient gene expression in potato protoplasts. The protoplasts were from leaves of wild potato Solanum brevidens, and from leaves, tubers and suspension cells of cultivated Solanum tuberosum cv. Désirée. Reporter enzyme activity, chloramphenicol acetyl transferase (CAT) under the control of the cauliflower mosaic virus (CaMV) 35S promoter, depended on the field strength and the pulse duration used for electroporation. Using field pulses of 85 ms duration, the optimum field strengths for maximum CAT activity were: S. brevidens mesophyll protoplasts –250 V/cm; Désirée mesophyll protoplasts –225 V/cm; Désirée suspension culture protoplasts –225 V/cm; and Désirée tuber protoplasts –150 V/cm. The optimum field strengths correlated inversely with the size of the protoplasts electroporated; this is consistent with biophysical theory. In time courses, maximum CAT activity (in Désirée mesophyll protoplasts) occurred 36–48 h after electroporation. Examination at optimised conditions of a chimaeric gene consisting of a class II patatin promoter linked to the -glucuronidase (gus) gene, showed expression (at DNA concentrations between 0–10 pmol/ml) comparable to the CaMV 35S promoter in both tuber and mesophyll protoplasts. At higher DNA concentrations (20–30 pmol/ml) the patatin promoter directed 4–5 times higher levels of gus expression. Implications and potential contributions towards studying gene expression, in particular of homologous genes in potato, are discussed.     

Production of bialaphos-resistant Nierembergia repens by electroporation

Transgenic plants with the herbicide-resistance gene (bar gene) were obtained via organogenesis from isolated mesophyll protoplasts of Nierembergia repens after applying electroporation. Transient β-glucuronidase (GUS) activity of electroporated protoplasts assayed 2 days after applying an electric pulse showed that optimum condition (transient GUS activity 319 pmol 4 MU/mg per min and plating efficiency 2.43%) for electroporation was 0.5 kV/cm in field strength and 100 μF in capacitance. The protoplasts electroporated with the bar gene at this condition initiated formation of microcolonies on medium after 2 weeks. After 4 weeks of culture, equal volume of fresh 1/2-strength Murashige and Skoog (MS) medium containing 0.2 mg/l bialaphos was added for selection of transformed colonies. After 6 weeks of culture, growing colonies were transferred onto regeneration medium containing 1.0 mg/l bialaphos, on which they formed adventitious shoots 1–2 months after electroporation. The adventitious shoots rooted easily after transfer onto MS medium with bialaphos lacking plant-growth regulators. Transformation of these regenerants with the bar gene was confirmed by Southern analysis. Some of the transformants showed strong resistance to the application of bialaphos solution at 10.0 mg/l.     

Transient gene expression in electroporated Picea glauca protoplasts

The reporter gene for chloramphenicol acetyltransferase (CAT) was introduced into white spruce (Picea glauca (Moench) Voss.) protoplasts by electroporation. CAT transient gene expression was increased by increasing the concentration of pCaMVCN plasmid and was affected by the level of the applied voltage. Highest CAT activities were obtained after electroporation with a pulse of 350V.cm^–1 having an exponential decay constant of approximately 105ms. Linearized plasmid constructs gave much higher levels of CAT activity than circular plasmid. Attempts to use the Escherichia coli -glucuronidase gene (-GUS) as a marker gene revealed very high levels of -GUS-like activity in electroporated protoplasts. This activity was mainly due to a small molecule and may mask successful transformation since -GUS-like activity increased when plasmid DNA was present during electroporation.Abbreviations CAT chloramphenicol acetyltransferase - -GUS -glucuronidase - MUG 4-methyl umbelliferyl glucuronide - F microfarads NRCC No. 29150     

Factors affecting transient gene expression in protoplasts isolated from very slowly growing embryogenic callus cultures of wheat (Triticum aestivum L.)

Protoplasts isolated from embryogenic (Mustang and Chinese Spring) and non-embryogenic (Mit) calli of wheat (Triticum aestivum L.) genotypes transiently expressed -glucuronidase (GUS) activity when electroporated with a plasmid containing the GUS gene and driven by an enhanced 35S promoter and a TMV leader sequence. Conditions for the maximum expression of GUS activity were: electroporation of the freshly isolated protoplasts at 250 Vcm^-1 and 250 F for 2 s using 50 g/ml of plasmid DNA; incubation of the protoplasts with the plasmid before the pulse for 2 h; and a 15-min recovery period on ice after the pulse. In general, a higher GUS activity was obtained in protoplasts of non-embryogenic (NE) callus origin than in those of embryogenic (E) callus origin. Only GUS constructs containing a duplicate 35S promoter derivative resulted in a significant level of GUS expression. The presence of the TMV viral leader sequence in the pAGUS1-TN2 plasmid construct resulted in a significant increase of GUS activity in the electroporated protoplasts of both callus types. On the other hand, protoplasts electroporated with the Adh1 promoter and intron showed a threefold less GUS activity than those electroporated with pAGUS1-TN2. Optimized conditions for DNA uptake and expression were very similar for protoplasts of both callus types. The importance of these findings for the successful regeneration of transgenic and fertile wheat plants is discussed.     

Activity of a chimeric promoter with the doubled CaMV 35S enhancer element in protoplast-derived cells and transgenic plants in maize

A reproducible and efficient transformation system has been developed for maize that is based on direct DNA uptake into embryogenic protoplasts and regeneration of fertile plants from protoplast-derived transgenic callus tissues. Plasmid DNA, containing the -glucuronidase (GUS) gene, under the control of the doubled enhancer element (the –208 to –46 bp upstream fragment) from CaMV 35S promoter, linked to the truncated (up to –389 bp from ATG) promoter of wheat, -amylase gene was introduced into protoplasts from suspension culture of HE/89 genotype. The constructed transformation vectors carried either the neomycin phosphotransferase (NPTII) or phosphinothricin acetyltransferase (PAT) gene as selective marker. The applied DNA uptake protocol has resulted at least in 10–20 resistant calli, or GUS-expressing colonies after treatment of 10⁶ protoplasts. Vital GUS staining of microcalli has made possible the shoot regeneration from the GUS-stained tissues. 80–90% of kanamycin or PPT resistant calli showed GUS activity, and transgenic plants were regenerated from more than 140 clones. Both Southern hybridization and PCR analysis showed the presence of introduced foreign genes in the genomic DNA of the transformants. The chimeric promoter, composed of a tissue specific monocot promoter, and the viral enhancer element specified similar expression pattern in maize plants, as it was determined by the full CaMV 35S promoter in dicot and other monocot plants. The highest GUS specific activity was found in older leaves with progressively less activity in young leaves, stem and root. Histochemical localization of GUS revealed promoter function in leaf epidermis, mesophyll and vascular bundles, in the cortex and vascular cylinder of the root. In roots, the meristematic tip region and vascular tissues stained intensively. Selected transformants were grown up to maturity, and second-generation seedlings with segregation for GUS activity were obtained after outcrossing. The GUS-expressing segregants carried also the NPTII gene as shown by Southern hybridization.     

Transformation of potato (Solanum tuberosum) cv. Mantiqueira using Agrobacterium tumefaciens and evaluation of herbicide resistance

Summary In search of establishing a system for genetic transformation of Brazilian potato cultivars, Agrobacterium tumefaciens carrying the plasmid pGV1040, was used to transform leaf discs of three cultivars of local importance, i.e., Aracy, Baronesa and Mantiqueira. This plasmid contains marker genes for resistance to kanamycin and phosphinothricin plus the gene for the enzyme -glucuronidase. A two step regeneration/selection procedure produced shoots of potato cultivar Mantiqueira with in vitro resistance to kanamycin and to phosphinothricin. After transfer to the greenhouse, the potentially transgenic plants, sprayed with the herbicide Finale^® (20% a.i.; Hoechst^®) remained green as compared to control clones that died immediately afterwards. Southern blot analysis and histochemical and fluorimetric assay for -glucuronidase indicated that the gene coding for the enzyme was integrated in the potato genome and could be expressed in potato tissues. No success was obtained for transformation of cultivars Aracy and Baronesa using this procedure.Abbreviations NAA Naphthalene Acetic Acid - BAP Benzyl-aminopurine - GA₃ Gibberellic Acid - PPT Phosphinothricin - PAT Phosphinothricin Acetyl Transferase     

Transgenic radish (Raphanus sativus L. longipinnatus Bailey) by floral-dip method – plant development and surfactant are important in optimizing transformation efficiency

Transgenic radish (Raphanus sativus L. longipinnatus Bailey) plants were produced from the progeny of plants which were dipped into a suspension of Agrobacterium carrying both the -glucuronidase (gusA) gene and a gene for resistance to the herbicide Basta (bar) between T-DNA border sequences. The importance of development of the floral-dipped plant and presence of surfactant in the inoculation medium were evaluated in terms of transgenic plant production. Plants dipped at the primary bolt stage of growth, into a suspension of Agrobacterium containing 0.05% (v/v) Silwet L-77 resulted in optimum transformation efficiency, with 1.4% from 1110 seeds. The presence of Pluronic F-68 or Tween 20 in the inoculation medium was beneficial towards transgenic plant output compared to treatments without surfactant. Putative transformed T1 plants were efficiently selected by spraying with 0.03% (v/v) Basta and all herbicide-resistant plants tested positive for GUS activity when analysed both histochemically and fluorometrically. Southern analysis revealed that both the gusA and bar genes integrated into the genome of transformed plants and segregated as dominant Mendelian traits. These results demonstrate that radish can be genetically modified for the improvement of this important vegetable crop.