Stable transformation of Eustoma grandiflorum by particle bombardment

Explants (7.5±2.5 mm) cut from stems and roots of 3-week-old Eustoma grandiflorum Grise, (lisianthus) cv. Glory White seedlings were bombarded with plasmid pBI221, which harbors the uidA gene encoding β-glucuronidase (GUS) driven by the cauliflower mosaic virus (CaMV) 35S promoter. More than 800 blue spots of GUS-expressing cells were observed per 90 explants. Explants bombarded with pARK22 harboring the bar gene encoding phosphinothricin acetyltransferase driven by the CaMV 35S promoter were selected for bialaphos resistance. Putative transgenic plants were obtained about 3 months after bombardment. Southern blot analysis of putative transgenic plants revealed the presence of the bar gene in their genome. Received: 10 April 1996 / Revision received: 7 November 1997 / Accepted: 22 November 1997     

Green fluorescent protein as an all-purpose reporter in Petunia

Two critical attributes of a reporter gene are ease of scoring for activity and capacity for expression in all cell types. We have examined a variant of the gene encoding green fluorescent protein,mgfp5, for its ability to meet these criteria in petunia. Under regulation of the Cauliflower Mosaic Virus (CaMV) 35S promoter, GFP was detectable in all vegetative and most floral cell types. Promoters from petuniaadhl andadh2 allowed for production of GFP in those few cell types lacking GFP production from the CaMV 35S promoter, verifying its capacity for expression in all cell types. With the appropriate promoter, GFP fluorescence was thus readily detectable throughout the plant. A potential complication is the green autofluorescence exhibited by some plant tissues. This auto-fluorescence is for the most part distinguishable from that contributed by GFP, but under-scores the need for appropriate controls in GFP-reporter-based experiments. An erratum to this article is available at .     

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     

Transgenic <Emphasis Type="Italic">Cucumis sativus</Emphasis> Expressing the Hepatitis B Surface Antigen

A DNA fragment encoding the hepatitis B virus surface antigen was amplified from a positive blood (hepatitis B) sample and introduced into the pET 32c prokaryotic expression vector. The gene encoding the HBV surface protein antigen was introduced into pCAMBIA 3300, and immobilized into Agrobacterium tumefaciens strain LBA4404. Cotyledonary leaf sections of Cucumis sativus (cucumber) cv ‘Swarnamukhi’ were cocultivated with Agrobacterium harboring the binary vector pCAMBIA 3300 carrying the HBV surface antigen gene driven by the CaMV35S promoter and the herbicide resistance gene phosphinothricin. Putative transformed shoots were induced on a Murashige and Skoog (MS) medium containing phosphinothricin, and these were then rooted on MS basal medium supplemented with 1 mg/L Indole 3-butyric acid (IBA). Integration of the T-DNA into in putative transgenic plants was confirmed by PCR and Southern blot analyses. RT-PCR and Northern blot analyses were conducted to determine RNA expression. Levels of expression in transgenic plants were confirmed by Western blot analysis, and quantification of the protein was determined by enzyme linked immuno assay (ELISA). Molecular mass of the recombinant protein was measured by Matrix-Assisted Laser Desorption Ionization-Time-of-Flight (MALDI-TOF) Mass Spectrometry.     

Transgenic fertile Scoparia dulcis L., a folk medicinal plant, conferred with a herbicide-resistant trait using an Ri binary vector

Summary Transgenic herbicide-resistant Scoparia dulcis plants were obtained by using an Ri binary vector system. The chimeric bar gene encoding phosphinothricin acetyltransferase flanked by the promoter for cauliflower mosaic virus 35S RNA and the terminal sequence for nopaline synthase was introduced in the plant genome by Agrobacterium-mediated transformation by means of scratching young plants. Hairy roots resistant to bialaphos were selected and plantlets (R0) were regenerated. Progenies (S1) were obtained by self-fertilization. The transgenic state was confirmed by DNA-blot hybridization and assaying of neomycin phosphotransferase II. Expression of the bar gene in the transgenic R0 and S1 progenies was indicated by the activity of phosphinothricin acetyltransferase. Transgenic plants accumulated scopadulcic acid B, a specific secondary metabolite of S. dulcis, in amounts of 15–60% compared with that in normal plants. The transgenic plants and progenies showed resistant trait towards bialaphos and phosphinothricin. These results suggest that an Ri binary system is one of the useful tools for the transformation of medicinal plants for which a regeneration protocol has not been established.Abbreviations CaMV cauliflower mosaic virus - NPT-II neomycin phosphotransferase - PAT phosphinothricin acetyltransferase - PPT phosphinothricin     

Use of green fluorescent protein as A non-destructive marker for peanut genetic transformation

Summary The ability to non-destructively visualize transient and stable gene expression has made green fluorescent protein (GFP) a most efficient reporter gene for routine plant transformation studies. We have assessed two fluorescent protein mutants, enhanced GFP (EGFP) and enhanced yellow fluorescent protein (EYFP), under the control of the CaMV35S promoter, for their transient expression efficiencies after particle bombardment of embryogenic cultures of the peanut cultivar, Georgia Green. A third construct (p524EGFP.1) that expressed EGFP from a double 35S promoter with an AMV enhancer sequence also was compared. The brightest and most dense fluorescent signals observed during transient expression were from p524EGFP. 1 and EYFP. Optimized bombardment conditions consisted of 0.6 μm diameter gold particles, 12410 kPa bombardment pressure, 95 kPa vacuum pressure, and pretreatment with 0.4 M mannitol. Bombardments with p524EGFP.1 produced tissue sectors expressing GFP that could be visually selected under the fluorescence microscope over multiple subcultures. Embryogenic lines selected for GFP expression initially may have been chimeric since quantitative analysis of expression sometimes showed an increase when GFP-expressing lines, that also contained a hygromycin-resistance gene, subsequently were cultured on hygromycin. Transformed peanut plants expressing GFP were obtained from lines selected either visually or on hygromycin. Integration of the gfp gene in the genomic DNA of regenerated plants was confirmed by Southern blot hybridization and transmission to progeny.     

Expression of the B subunit of <Emphasis Type="Italic">E. coli</Emphasis> heat-labile enterotoxin in tobacco using a herbicide resistance gene as a selection marker

The B subunit of Escherichia coli heat-labile enterotoxin (LTB) has been transformed to plants for use as an edible vaccine. We have developed a simple and reliable Agrobacterium-mediated transformation method to express synthetic LTB gene in N. tabacum using a phosphinothricin acetyltransferase (bar) gene as a selectable marker. The synthetic LTB gene adapted to the coding sequence of tobacco plants was cloned to a plant expression vector under the control of the ubiquitin promoter and transformed to tobacco by Agrobacterium-mediated transformation. Transgenic plants were selected in the medium supplemented with 5 mg l^-1 phosphinothricin (PPT). The amount of LTB protein detected in the transgenic tobacco was approximately 3.3% of the total soluble protein, approximately 300-fold higher than in the plants generated using the native LTB gene under the control of the CaMV 35S promoter. The transgenic plants that were transferred to a greenhouse had harvested seeds that proved to be resistant to herbicide. Thus, the described protocol could provide a useful tool for the transformation of tobacco plants.     

Assessment of the Effectiveness of a Nuclear-Launched TMV-Based Replicon as a Tool for Foreign Gene Expression in Plants in Comparison to Direct Gene Expression from a Nuclear Promoter

An environmentally safe Tobacco Mosaic Virus (TMV)-based expression replicon was constructed that lacks movement protein (MP) and coat protein (CP), and which expresses the green fluorescent protein (GFP) gene from a full CP subgenomic promoter. The TMV replicon, whose cDNA was positioned between an enhanced Cauliflower Mosaic Virus 35S promoter (CaMV) and a self-cleaving hammerhead ribozyme with a downstream nopaline synthase gene polyadenylation signal [nos-poly(A)], was assessed for its effectiveness to accumulate GFP upon agroinfiltration into plant leaves compared to a control construct in which GFP was directly expressed from the enhanced CaMV 35S promoter. It was determined that individually expressing cells produced ca. 9-fold more GFP from the TMV-based replicon than from the enhanced 35S promoter. In contrast, GFP measurements from total leaf extracts determined that leaves infiltrated with the TMV-based replicon produced ca. 7-fold less GFP than the control construct. These apparently contradictory results can be explained by the low infectivity of the TMV-based replicon as it was found that the number of foci expressing GFP produced in leaves agroinfiltrated with the TMV-based replicon was ca. 66-fold lower than produced by the control.     

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.     

Stable transformation ofArabidopsis with thebar gene using particle bombardment

A plasmid pARK 22 harbouring thebar gene encoding phosphinothricin acetyltransferase (PAT) under the control of the cauliflower mosaic virus (CaMV) 35S promoter and nopaline synthase (NOS) terminator was constructed and introduced into root sections ofArabidopsis thaliana using the pneumatic particle gun. The root sections that had been bombarded with this plasmid gave four to eight times higher yield of drug-resistant calluses than those sections bombarded with pCaMVNEO or pCH, which respectively contain the neomycin phosphotransferase and hygromycin phosphotransferase genes. Among a number of primary transformant (T₀) plants obtained from independent bialaphos-resistant calluses, three were studied by Southern blot hybridization and PAT enzyme activity analyses, confirming the stable integration of the foreign gene into theArabidopsis genome and its expression in plants. The progeny analysis showed transmission of the foreign gene and its expression in up to the T₂ generation. Some of the T₁ progeny showed morphological abnormalities. Thus, thebar gene can be used effectively to allow selection of transgenicA. thalianna plants.     

Expression of phosphinothricin acetyltransferase from the root specific par promoter in transgenic tobacco plants is sufficient for herbicide tolerance

Summary The pat gene, coding for phosphinothricin acetyltransferase (PAT) from Streptomyces viridochromogenes, was cloned behind the par promoter of the hemoglobin gene from Parasponia andersonii, Introduction into tobacco (Nicotiana tabacum) resulted in predominantly root specific PAT expression. Application of 5 l/ha BASTA^® (herbicidal component: phosphinothricin) did not effect growth morphology and vigor of the plants. After application of 20 l/ha BASTA^® the plants showed herbicide damage. Nevertheless, they all recovered by forming new undamaged leaves and resumed full growth despite virtually non-detectable expression of the PAT enzyme in the leaves.Abbreviations BAP 6-benzylaminopurine - CaMV Cauliflower Mosaic Virus - IAA indole-3-acetic acid - kb kilobases - LB Luria-Bertani - MS Murashige and Skoog - par Parasponia andersonii - PAT phosphinothricin acetyltransferase - ppt phosphinothricin - TCA trichloric acid     

Viral and chloroplastic signals essential for initiation and efficiency of translation in Agrobacterium tumefaciens

The construction of high-level protein expression vectors using the CaMV 35S promoter in concert with highly efficient translation initiation signals for Agrobacterium tumefaciens is a relatively less explored field compared to that of Escherichia coli. In the current study, we experimentally investigated the capacity of the CaMV 35S promoter to direct GFP gene expression in A. tumefaciens in the context of different viral and chloroplastic translation initiation signals. GFP expression and concomitant translational efficiency was monitored by confocal microscopy and Western blot analysis. Among all of the constructs, the highest level of translation was observed for the construct containing the phage T7 translation initiation region followed by the chloroplastic Rubisco Large Subunit (rbcL) 58-nucleotide 5′ leader region including its SD-like sequence (GGGAGGG). Replacing the SD-like (GGGAGGG) with non SD-like (TTTATTT) or replacing the remaining 52 nucleotides of rbcL with nonspecific sequence completely abolished translation. In addition, this 58 nucleotide region of rbcL serves as a translational enhancer in plants when located within the 5′ UTR of mRNA corresponding to GFP. Other constructs, including those containing sequences upstream of the coat proteins of Alfalfa Mosaic Virus, or the GAGG sequence of T4 phage or the chloroplastic atpI and/or PsbA 5′ UTR sequence, supported low levels of GFP expression or none at all. From these studies, we propose that we have created high expression vectors in A. tumefaciens and/or plants which contain the CaMV 35S promoter, followed by the translationally strong T7 SD plus RBS translation initiation region or the rbcL 58-nucleotide 5′ leader region upstream of the gene of interest.     

Herbicide-resistant Indica rice plants from IRRI breeding line IR72 after PEG-mediated transformation of protoplasts

The commercially important Indica rice cultivar Oryza sativa cv. IR72 has been transformed using direct gene transfer to protoplasts. PEG-mediated transformation was done with two plasmid constructs containing either a CaMV 35S promoter/HPH chimaeric gene conferring resistance to hygromycin (Hg) or a CaMV 35S promoter/BAR chimaeric gene conferring resistance to a commercial herbicide (Basta) containing phosphinothricin (PPT). We have obtained so far 92 Hg^r and 170 PPT^r IR72 plants from protoplasts through selection. 31 Hg^r and 70 PPT^r plants are being grown in the greenhouse to maturity. Data from Southern analysis and enzyme assays proved that the transgene was stably integrated into the host genome and expressed. Transgenic plants showed complete resistance to high doses of the commercial formulations of PPT.     

The investigation of optimal bombardment parameters for transient and stable transgene expression in Sorghum

Summary This report outlines the development of optimized particle inflow gun (PIG) parameters for producing transgenic sorghum (Sorghum bicolor (L.) Moench). Both transient and stable expression were examined when determining these parameters. The uidA reporter gene (GUS) encoding β-glucuronidase was used in transient experiments and the green fluorescent protein (GFP) used to monitor stable expression. Initially, optimization was conducted using leaf segments, as the generation of sorghum callus in sufficiently large quantities is time-consuming. Following leaf optimization, experiments were conducted using callus, identifying a high similarity between the two tissue types (r _s=0.83). High levels of GUS expression were observed in both leaf and callus material when most distant from the DNA expulsion point, and using a pressure greater than 1800 kPa. A higher level of expression was also observed when the aperture of the helium inlet valve was constricted. Using the optimized conditions (pressure of 2200 kPa, distance to target tissue of 15 cm from the expulsion point, and the aperture of the helium inlet valve at one full turn), three promoters (Ubiquitin, Actinl and CaMV 35S) were evaluated over a 72-h period using GUS as the reporter gene. A significantly higher number of GUS foci were counted with the Ubiquitin construct over this period, compared to the Actinl and CaMV 35S constructs. Stable callus sectors (on 2 mg 1⁻¹ bialaphos) with GFP expression were visualized for as long as 6 wk post-bombardment. Using this optimized protocol, several plants were regenerated after having been bombarded with the pAHC20 construct (containing the bar gene), with molecular evidence confirming integration.     

Introduction and constitutive expression of a rice chitinase gene in bread wheat using biolistic bombardment and the bar gene as a selectable marker

 Our long-term goal is to control wheat diseases through the enhancement of host plant resistance. The constitutive expression of plant defense genes to control fungal diseases can be engineered by genetic transformation. Our experimental strategy was to biolistically transform wheat with a vector DNA containing a rice chitinase gene under the control of the CaMV 35 S promoter and the bar gene under control of the ubiquitin promoter as a selectable marker. Immature embryos of wheat cv ‘Bobwhite’ were bombarded with plasmid pAHG11 containing the rice chitinase gene chi11 and the bar gene. The embryos were subcultured on MS2 medium containing the herbicide bialaphos. Calli were then transferred to a regeneration medium, also containing bialaphos. Seventeen herbicide-resistant putative transformants (T₀) were selected after spraying with 0.2% Liberty, of which 16 showed bar gene expression as determined by the phosphinothricin acetyltransferase (PAT) assay. Of the 17 plants, 12 showed the expected 35-kDa rice chitinase as revealed by Western blot analysis. The majority of transgenic plants were morphologically normal and self-fertile. The integration, inheritance and expression of the chi11 and bar genes were confirmed by Southern hybridization, PAT and Western blot analysis of T₀ and T₁ transgenic plants. Mendelian segregation of herbicide resistance was observed in some T₁ progenies. Interestingly, a majority of the T₁ progeny had very little or no chitinase expression even though the chitinase transgene was intact. Because PAT gene expression under control of the ubiquitin promoter was unaffected, we conclude that the CaMV 35 S promoter is selectively inactivated in T₁ transgenic wheat plants. Received: 12 May 1998 / Accepted: 15 May 1998     

Transformation and expression of a stilbene synthase gene of Vitis vinifera L. in barley and wheat for increased fungal resistance

 Transformation of barley and wheat via particle bombardment with a gene derived from Vitis vinifera L. (Vst1 gene) resulted in the expression of the foreign phytoalexin, resveratrol, in the transformed plants. Transgenic barley plants were regenerated from microspores and transgenic wheat plants from immature embryos were both selected on Basta. Stable integration of the gene in the genomes of transgenic barley and wheat plants, as well as their progeny, was analysed by Southern-blot analysis. The induction of the stilbene synthase promoter and the transient expression of stilbene synthase-specific mRNA after induction by wounding and infection were proofed in T₁ and T₂ progeny plants. An enhanced expression of the Vst1 gene under control of the stilbene synthase promoter was observed with enhancer sequences from the cauliflower mosaic virus 35s (CaMV 35s) promoter. The enzyme activity of the stilbene synthase was analysed in T₁ progeny plants. The first pathological results indicated an increased resistance of transgenic barley plants to Botrytis cinerea used as a model experimental system. Received: 5 November 1997 / Accepted: 11 November 1997     

Effects of pollen-synthesized green fluorescent protein on pollen grain fitness

Genetically engineered pollen with a visible marker gene could be useful to monitor the movement of transgenic pollen provided there are no negative physiological or fitness effects of expressing such a gene. In this study, we measured the fitness of Nicotiana tabacum cv. Xanthi pollen expressing the marker gene green fluorescent protein (GFP). Average pollen tube germination frequencies and pollen tube growth rates in vitro were measured in three different types of plants: (1) plants producing GFP in pollen cells only (LAT59-GFP), (2) plants synthesizing GFP under the control of a constitutive promoter (CaMV 35S) in which no GFP was produced in pollen, and (3) non-transgenic plants. Pollen synthesizing the GFP protein did not differ significantly in average pollen germination frequencies from pollen without GFP (P=0.65). Average pollen tube growth rates over a 5-h period did not differ significantly between transgenic and non-transgenic types (R²=0.89, 0.98, and 0.95, respectively, for GFP-tagged, 35S-GFP, and wild type). Overall, GFP expression in pollen grains of tobacco was not found to have an effect on pollen fitness under the controlled experimental conditions of this study.     

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.