Transient expression of the GUS gene in a unicellular marine green alga,<Emphasis Type="Italic">Chlorella</Emphasis> sp.<Emphasis Type="Italic">MACC/C95</Emphasis>, via electroporation

A transient expression system for a unicellular marine green alga,Chlorella sp.MACC/C95, was developed using a reporter GUS gene coded for by plasmid pBI121. The results demonstrated a high transformation efficiency could be achieved by using electroporation to deliver DNA into intact cells and the CaMV35S promoter to drive the foreign gene expression inChlorella sp.MACC/C95. The use of a carrier DNA coupled with osmosis treatment improved the transformation efficiency, while linearization of the plasmid had minor effects. Investigation of the effects of DNA concentration and growth phases ofChlorella sp.MACC/C95 on transformation efficiency indicated that the highest level of transient expression was observed when 6 μg mL⁻¹ of plasmid DNA and cells 2–6 days old were used.     

Efficient transient expression and transformation of PEG-mediated gene uptake into mesophyll protoplasts of pepper (<Emphasis Type="Italic">Capsicum annuum</Emphasis> L.)

PEG-mediated transformation was used for gene delivery and evaluation of various parameters affecting the transient expression of a gene for ß-glucuronidase (gus) in mesophyll protoplasts of Capsicum annuum. Transient expression was found to be dependent on PEG concentration and exposure time of plasmid DNA to protoplasts as well as the amount of plasmid DNA. Maximum GUS activity was obtained when protoplasts were applied to 40% concentration and molecular weight was 6,000 of PEG solution with 30 min of exposure time. Protoplasts of pepper were transformed with a vector, pCAMBIA::Ac, which contained a pCAMBIA1302 T-DNA vector carrying a maize transposable element, Ac (activator), a selection marker HPT (hygromycin phosphotransferase), and a GFP-coding region driven by the 35S promoter in the presence of PEG. Approximately 30% of the protoplasts expressed GFP. Visibly transformed colonies were obtained from protoplasts after 2 months of culture and GFP was expressed. Southern hybridization confirmed the presence of Ac in the pepper genome.     

Genotype- and promoter-induced variability in transient β-glucuronidase expression in pea protoplasts

Summary Leaf mesophyll protoplasts isolated from pea (Pisum sativum L.) genotypes Century and PI244253 showed transient expression of -glucuronidase (GUS) when electroporated with plasmid DNA containing various promoter-leader sequence constructs driving the GUS gene. The optimum conditions for transient expression were: using protoplasts isolated from leaf material that had been kept in the dark for 90 h; electroporating at 250 V and 960 F; and using 125 g of calf thymus carrier DNA and 75 of plasmid DNA. PI244253 had 5 to 20 times the GUS activity levels of Century. Similar levels of transient expression were obtained using either the nopaline synthase or cauliflower mosaic virus 35S (35S) promoters. These levels were lower than that obtained using a duplicated 35S promoter derivative. The presence of an untranslated coat protein mRNA leader sequence from alfalfa mosaic virus between each promoter and the GUS gene resulted in increased GUS activity. Leaf mesophyll protoplasts and root protoplasts of PI244253 did not differ in levels of transient expression.NRCC No. 30910     

Bifunctional Reporter Genes: Construction and Expression in Prokaryotic and Eukaryotic Cells

Bifunctional reporter proteins were constructed to combine Clostridium thermocellum lichenase (LicBM2) with Aequorea victoria green fluorescent protein (GFP) or with Escherichia coli -glucuronidase (GUS). The major properties of the initial proteins were preserved in the hybrid ones: LicBM2 was active at 65°C, GFP fluoresced, and GUS hydrolyzed its substrates. LicBM2 remained active after extension of its C or N end. Bifunctional reporter systems were shown to provide a convenient tool for studying the gene expression regulation in prokaryotic (E. coli) and eukaryotic (Saccharomyces cerevisiae, mammalian) cells, advantages of one reporter compensating for drawbacks of the other.     

Green fluorescent protein: an in vivo reporter of plant gene expression

Summary Protoplasts were isolated from H89, an embryogenic sweet orange (Citrus sinensis (L.) Osbeck cv. Hamlin) suspension culture, and electroporated with p35S-GFP, a plasmid carrying the gene for the green fluorescent protein (GFP) from the bioluminescent jellyfish Aequorea victoria. p35S-GFP was constructed by replacing the GUS coding sequence of pBI221 with a functional GFP gene, thereby placing the GFP gene under the control of the CaMV 35S promoter. Protoplasts were viewed by incident-light fluorescence microscopy twentyfour h after electroporation. 20–60% of the protoplasts emitted an intense green light when illuminated with blue (450–490 nm) light.Abbreviations GUS -glucuronidase - LUC luciferase - NPTII neomycinphosphotransferase - CaMV cauliflower mosaic virus - MUG 4-methylumbelliferyl -D-glucuronide     

Transgenic peach plants (<Emphasis Type="Italic">Prunus persica</Emphasis> L.) produced by genetic transformation of embryo sections using the green fluorescent protein (GFP) as an <Emphasis Type="Italic">in vivo</Emphasis> marker

The main obstacle to genetic engineering of fruit tree species is the regeneration of transformed plantlets. Transformation events in peach (Prunus persica L.) have been reported using particle bombardment or Agrobacteriummediated transformation of immature embryos. However, the regeneration of plants from transgenic tissues is still difficult and the recovery of non-chimeric plants has not been reported to date. In this paper we describe an efficient, reliable transformation and regeneration system to produce transgenic peach plants using embryo sections of mature seeds as starting material. This represents an important advantage due to the availability of such material throughout the year. A. tumefaciens strain C58 (pMP90) containing the binary plasmid pBin19 was used as vector system for transformation. We used the Nospro-nptII-Noster cassette as a selectable marker and the CaMV35Spro-sgfp-CaMV35Ster cassette as a vital reporter gene coding for an improved version of the green fluorescent protein (sGFP). In vitro cultured embryo sections were Agrobacterium-cocultivated and, after selection, transgenic shoots were regenerated. Shoots that survived exhibited high-level of sGFP expression mainly visible in the young leaves of the apex. In vivo monitoring of GFP expression permitted an early, rapid and easy discrimination of both transgenic and escape buds. After elimination of escapes, transgenic shoots were rooted in vitro and the recovered plantlets were screened using PCR amplification. Southern analysis confirmed stable genomic integration of the sgfp transgene. The high levels of GFP expression were also maintained in the second generation of transgenic peach plants.     

Transient gene expression in cassava using high-velocity microprojectiles

The bacterial gene encoding -glucuronidase (GUS) was transiently expressed in cassava leaves following the introduction of the gene by microparticle bombardment. The DNA expression vector used to introduce the reporter gene is a pUC 19 derivative and consisted of a CaMV 35S promoter (P35S), the GUS coding region and 7S polyadenylation region. Several other promoters and regulating sequences were tested for efficiency in cassava leaves. Two derivatives of the P35S, one including a partial duplication of the upstream region of the P35S and the other containing a tetramer of the octopine synthase enhancer, were found to be expressed at three times the level of the P35S in cassava leaves. The ubiquitin 1 promoter fromArabidopsis thaliana was expressed at the same level as the P35S. No influence on the level of expression was observed when different 3 ends were used. The biolistic transient gene expression system in cassava leaves allows rapid analysis of gene constructs and can serve as a preliminary screen for chimeric gene function in the construction of transgenic cassava plants.     

Visualization of site-specific recombination catalyzed by a recombinase from Zygosaccharomyces rouxii in Arabidopsis thaliana

Excision of a DNA segment can occur in Arabidopsis thaliana by reciprocal recombination between two specific recombination sites (RSs) when the recombinase gene (R) from Zygosaccharomyces rouxii is expressed in the plant. To monitor recombination events, we generated several lines of transgenic Arabidopsis plants that carried a cryptic -glucuronidase (GUS) reporter gene which was designed in such a way that expression of the reporter gene could be induced by R gene-mediated recombination. We also made several transgenic lines with an R gene linked to the 35S promoter of cauliflower mosaic virus. Each transgenic line carrying the cryptic reporter gene was crossed with each line carrying the R gene. Activity of GUS in F₁ and F₂ progeny was examined histochemically and recombination between two RSs was analyzed by Southern blotting and the polymerase chain reaction. In seedlings and plantlets of F₁ progeny and most of the F₂ progeny, a variety of patterns of activity of GUS, including sectorial chimerism in leaves, was observed. A small percentage of F₂ individuals exhibited GUS activity in the entire plant. This pattern of expression was ascribed to germinal recombination in the F₁ generation on the basis of an analysis of DNA structure by Southern blotting. These results indicate that R gene-mediated recombination can be induced in both somatic and germ cells of A. thaliana by cross-pollination of parental transgenic lines.     

Promoters for pregenomic RNA of banana streak badnavirus are active for transgene expression in monocot and dicot plants

Two putative promoters from Australian banana streak badnavirus (BSV) isolates were analysed for activity in different plant species. In transient expression systems the My (2105 bp) and Cv (1322 bp) fragments were both shown to have promoter activity in a wide range of plant species including monocots (maize, barley, banana, millet, wheat, sorghum), dicots (tobacco, canola, sunflower, Nicotiana benthamiana, tipu tree), gymnosperm (Pinus radiata) and fern (Nephrolepis cordifolia). Evaluation of the My and Cv promoters in transgenic sugarcane, banana and tobacco plants demonstrated that these promoters could drive high-level expression of either the green fluorescent protein (GFP) or the -glucuronidase (GUS) reporter gene (uidA) in vegetative plant cells. In transgenic sugarcane plants harbouring the Cv promoter, GFP expression levels were comparable or higher (up to 1.06% of total soluble leaf protein as GFP) than those of plants containing the maize ubiquitin promoter (up to 0.34% of total soluble leaf protein). GUS activities in transgenic in vitro-grown banana plants containing the My promoter were up to seven-fold stronger in leaf tissue and up to four-fold stronger in root and corm tissue than in plants harbouring the maize ubiquitin promoter. The Cv promoter showed activities that were similar to the maize ubiquitin promoter in in vitro-grown banana plants, but was significantly reduced in larger glasshouse-grown plants. In transgenic in vitro-grown tobacco plants, the My promoter reached activities close to those of the 35S promoter of cauliflower mosaic virus (CaMV), while the Cv promoter was about half as active as the CaMV 35S promoter. The BSV promoters for pregenomic RNA represent useful tools for the high-level expression of foreign genes in transgenic monocots.     

Delivery of plasmid DNA into intact plant cells by electroporation of plasmolyzed cells

This report describes the delivery of plasmid DNA containing either the β-glucuronidase (GUS) or the green fluorescent protein (GFP) reporter gene into intact plant cells of bamboo callus, lilium scales, and Nicotiana benthamiana suspension culture cells. By first plasmolyzing the tissues or cells with 0.4 m sucrose in the presence of plasmid DNA, electroporation effectively delivers plasmid DNA into the intact plant cells. Transient expression of the GUS gene, as revealed by histochemical assays, showed the presence of blue-staining areas in the electroporated tissues. A short exposure of cells to 2% DMSO (dimethyl sulfoxide) prior to plasmolysis elevated the level of transient GUS activity. When plasmid DNA containing a synthetic GFP gene was used, a strong green fluorescence was observed in N. benthamiana suspension culture cells that were subjected to plasmolysis and electroporation. These results suggest that plasmolysis brings the plasmid DNA into the void space that is in close vicinity to the plasmalemma, allowing electroporation to efficiently deliver the plasmid DNA into intact plant cells. Received: 15 June 1998 / Revision received: 18 August 1998 / Accepted: 28 August 1998     

Construction of a GFP-BAR plasmid and its use for switchgrass transformation

 A dual marker plasmid comprising the reporter gene sgfp (green fluorescent protein) and the selectable bar gene (Basta tolerance) was constructed by replacing the uidA (β-glucuronidase, GUS) gene in a uidA-bar construct with sgfp. A particle inflow gun was used to propel tungsten particles coated with this plasmid into immature inflorescence-derived embryogenic callus of switchgrass (Panicum virgatum L.). GFP was observed in leaf tissue and pollen of transgenic plants. Nearly 100 plants tolerant to Basta were obtained from the experiments, and Southern blot hybridization confirmed the presence of both the bar and sgfp genes. Plants regenerated from in vitro cultures of transgenic plants grew on medium with 10 mg l^–1 bialaphos. When the pH indicator chlorophenol red was in the medium, the transgenic plantlets changed the medium from red to yellow. Basta tolerance was observed in T₁ plants resulting from crosses between transgenic and nontransgenic control plants, indicating inheritance of the bar transgene. Received: 11 May 2000 / Revision received: 21 August 2000 / Accepted: 22 August 2000     

Tissue-specific expression of the rolC promoter of the Ri plasmid in transgenic rice plants

Summary Transgenic rice plants were obtained from protoplasts treated with two plasmids by electroporation. Primary transformants were selected on the basis of resistance to hygromycin, conferred by one of the co-transferred plasmids. Out of 26 hygromycin-resistant plants 2 showed the reporter gene activity due to another plasmid possessing a chimeric gene consisting of the promoter (about 900 by upstream non-coding region) of the ORF12 gene (roIC of the Ri plasmid and the coding region for -glucuronidase (GUS). Using a colorimetric reaction, the GUS enzyme was found to be localized in vascular tissues, demonstrating the similar expression of the roIC gene promoter in monocots and dicots (Sugaya et al. 1989; Schmülling et al. 1989).     

Transfection of insect cell lines using polyethylenimine

Insect cell lines have been widely used in recombinant baculovirus expression systems and transient gene expression studies. Critical to these applications have been the transfection of foreign DNA. This has been frequently done using labor intensive and cytotoxic liposome-based transfection reagents. In the current study we have optimized a new kind of polyethylenimine-based DNA transfection reagent on the Spodoptera frugiperda Sf9 insect cell line. A plasmid vector that transiently expresses green fluorescent protein (GFP) was effectively delivered into Sf9 cells. A transfection efficiency of 54% and cell viability of 85–90% were obtained for Sf9 cells. The developed transfection protocol has now been successfully used to transfect eight insect cell lines derived from Bombyx mori, Trichoplusia ni, Helicoverpa zea, Heliothis virescens and S. frugiperda with GFP and GUS with transfection efficiencies of at least 45%. This method provides high heterologous protein expression levels, transfection efficacy and cell viability, and could be used for transient gene expression in other lepidopteran cell lines.

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.     

Optimised DNA delivery into Coffea arabica suspension culture cells by particle bombardment

An optimised bombardment protocol to introduce DNA into Coffea arabica suspension culture cells was developed. Osmotic preconditioning of cells and physical bombardment parameters including Helium pressure, gap and target distances affecting DNA delivery were evaluated by monitoring transient expression of the uidA gene driven by the CaMV35S promoter. The highest transient GUS expression was obtained when cells were subjected to a 0.5 M mannitol–sorbitol pre-treatment 4 h prior to bombardment and a Helium pressure of 1550 psi, a 9-mm gap distance and 12 cm target distance as physical bombardment parameters. The optimised protocol was tested with two coffee promoters: -tubulin and arabicin, which presented similar activity to the CaMV35S promoter in suspension culture cells by fluorometric GUS assays. GUS expression was reduced in bombarded tissue culture leaves, and only the CaMV35S and arabicin promoters showed histochemical activity in coffee endosperms. This is the first report of optimization of particle bombardment on coffee suspension culture cells, equivalent CaMV35S activity for a coffee promoter and transient -glucoronidase expression in coffee endo-sperms.     

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.     

Evaluation of putative seed-specific promoters for Linum usitatissimum

The GUS reporter gene was used to test four different putativeseed-specific promoters in developing and mature seeds, leaves and roots fromlinseed flax (Linum usitatissimum). The promoters testedincluded the regulatory regions of the -ketoacyl-CoA synthase gene (KCS)and the napin protein gene from Brassica napus, thepromoter regions of the 'unknown seed protein' (USP), and a legumin proteingene(LeB4) from Vicia faba and the CaMV 35S promoter (positivecontrol). The promoter-GUS constructs were inserted into L.usitatissimum via Agrobacterium mediatedtransformation, and GUS activity evaluated using histochemical andfluorimetrical assays. All the promoters showed some activity, but only CaMV35S, LeB4 and USP exhibited an expression level high enough to be useful inlinseed flax. Plants with USP-GUS showed the earliest GUS activity at 5 to 6days after flowering (daf) and persisting until 40 daf. Expression of GUS underthe control of the LeB4 promoter was measurable 11 daf and was still detectableat 40 daf. The KCS-GUS construct showed a low level of GUS activity between 14daf and 40 daf. Plants transformed with USP-GUS or LeB4-GUS exhibited a lowlevel of GUS activity in leaves and roots of some of the transformants,indicating the need for generating large numbers of primary transformants,followed by careful evaluation and selection for ones with not only the desiredlevel of expression, but also the desired spatial and temporal expression.     

Transgenic and herbicide resistant pearl millet (Pennisetum glaucum L.) R.Br. via microprojectile bombardment of scutellar tissue

Four different pearl millet breeding lines were transformed and led to the regeneration of fertile transgenic plants. Scutellar tissue was bombarded with two plasmids containing the bar selectable marker and the -glucuronidase reporter gene (gus or uidA) under control of the constitutive CaMV 35S promoter or the maize Ubiquitin1 promoter (the CaMV 35S is not a maize promoter). For the delivery of the DNA-coated microprojectiles, either the particle gun PDS 1000/He or the particle inflow gun was used. The calli and regenerants were selected for their resistance to the herbicide Basta (glufosinate ammonium) mediated by the bar gene. Putative transformants were screened for enzyme activity by painting selected leaves or spraying whole plants with an aqueous solution of the herbicide Basta and by the histochemical GUS assay using cut leaf segments. PCR and Southern blot analysis of genomic DNA indicated the presence of introduced foreign genes in the genomic DNA of the transformants. Five regenerated plants represent independent transformation events and have been grown to maturity and set seed. The integration of the bar selectable and the gus reporter gene was confirmed by genomic Southern blot analysis in all five plants. All five plants had multiple integrations of both marker genes. To date, the T₁ progeny of three out of four lines generated by the PDS particle gun shows co-segregating marker genes, indicating an integration of the bar and the gus gene at the same locus in the genome.     

Isolation and characterization of a novel plant promoter that directs strong constitutive expression of transgenes in plants

A novel, constitutively expressed gene, designated MtHP, was isolated from the model legume species Medicago truncatula. Sequence analysis indicates that MtHP most likely belongs to the PR10 multi-gene family. The MtHP promoter was fused to a -glucuronidase gene to characterize its expression in different plant species. Transient assay by microprojectile bombardment and hairy root transformation by Agrobacterium rhizogenes revealed GUS expression in leaf, stem, radicle and root in M. truncatula. Detailed analysis in transgenic Arabidopsis plants demonstrated that the promoter could direct transgene expression in different tissues and organs at various developmental stages; its expression pattern was similar to that of CaMV35S promoter, and the level of expression was higher than the reporter gene driven by CaMV35S promoter. Deletion analysis revealed that even a 107 bp fragment of the promoter could still lead to a moderate level of expression. The promoter was further characterized in white clover (Trifolium repens), a widely grown forage legume species. Strong constitutive expression was observed in transgenic white clover plants. Compared with CaMV35S promoter, the level of GUS activity in transgenic white clover was higher when the transgene was driven by MtHP promoter. Thus, the promoter provides a useful alternative to the CaMV35S promoter in plant transformation for high levels of constitutive expression.