Factors influencing transient gene expression in electroporated tall fescue protoplasts

For the rapid establishment of optimal conditions for a genetic transformation system for tall fescue, several factors influencing transient gene expression were studied in protoplasts, after the reporter β-glucuronidase gene was introduced by electroporation. In a time-course study of transient gene expression, GUS activity peaked at 24 h after electroporation. Among the different field strength conditions tested, maximum GUS activity was observed at 750 V/cm. Increases in the amount of plasmid DNA to 80 μg/ml led to increased GUS activity. GUS activities increased in linear fashion with increasing protoplast densities up to 2 × 10⁶/ml. Age of suspension cells from which protoplasts were derived influenced transient expression with maximum GUS activity obtained in 3- and 5-day-old suspensions. These results show that monocot and dicot protoplasts respond similarly in electroporation.     

Reporter gene introduction and transient expression in protoplasts of Porphyra yezoensis

The transient expression of foreign genes in the protoplasts of Porphyrayezoensis was examined using three recombinant vectors, pYez-Rub-GUS, pYez-Rub-GFP and pYez-Rub-LUC, which were constructed with the promoter sequence of the ribulose-bisphosphate-carboxylase / oxygenase (Rubisco) gene as a promoter and the bacterial β-glucuronidase (GUS), mutant of green fluorescent protein (S65T-GFP) and firefly luciferase (LUC) genes, respectively, as reporter genes. When the pYez-Rub-GUS was introduced into protoplasts by electroporation, cells stained dark blue by indigotin were observed after the histochemical GUS assay. GUS activity was also detected by quantitative enzyme assays with a chemiluminescent substrate. When the pYez-Rub-GFP was electroporated into protoplasts, the expression of GFP could be detected in vivo observations with fluorescence microscopy. However, the rates of gene expression cells to the total number of cells were different between the GUS and GFP genes. LUC activity was also detected by assay with a chemiluminescent substrate after the introduction of pYez-Rub-LUC into protoplasts, although the activity levels were considerably lower. Relatively high expression rates of introduced GUS genes were observed 3 to 5 days after electroporation. These results show that the promoter sequence of the chloroplast Rubisco gene functions as a promoter of foreign gene expression and that transient expression occurred in protoplasts of P. yezoensis after the introduction of foreign genes. This revised version was published online in August 2006 with corrections to the Cover Date.     

Factors affecting transient gene expression in electroporated Glycine max protoplasts

Electroporation was used to evaluate parameters affecting transient gene expression in Glycine max protoplasts. Protoplast viability and reporter enzyme activity for chloramphenicol acetyl transferase (CAT) and ß-glucuronidase (GUS) depended on the field strength employed. Maximum CAT and GUS activity was obtained when a field strength of 500 V/cm at 1000 F and a protoplast concentration of 1–3 × 10⁶/ml was used. Transformation efficiencies up to approximately 1.6% GUS positive protoplasts were obtained. Transient gene expression increased with increasing plasmid DNA concentration and with the time after electroporation, reaching a maximum after 48 hr. Addition of polyethylene glycol at 5.6% and heat shock (5 rain at 45 °C) given to the protoplasts before adding DNA further enhanced the transformation efficiency. Under the optimized experimental conditions, CAT and GUS activity increased simultaneously, thereby indicating that the increased expression is caused by DNA uptake by more cells rather than greater DNA uptake by the same cells. Our results demonstrate that both GUS and CAT can be used as efficient screenable markers for transformation studies in soybean.Abbreviations CAT chloramphenicol acetyl transferase - GUS ß-glucuronidase - PEG polyethylene glycol     

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     

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).     

PEG-mediated expression of GUS and CAT genes in protoplasts from embryogenic suspension cultures of Picea glauca

ß-Glucuronidase (GUS) and chloramphenicol acetyl transferase (CAT) were used as reporter proteins in protoplasts from embryogenic suspension cultures of Picea glauca (Moench) Voss (white spruce). Plasmid DNA enclosing chimeric GUS and CAT constructs, using the cauliflower mosaic virus 35S promoter, was introduced into Picea glauca protoplasts using polyethylene glycol (PEG). Transient expression was detected 12 to 40 h after PEG-mediated DNA delivery. Dose-response curves using covalently closed circular plasmid DNA, in the absence of carrier DNA, have been obtained for each of these reporter genes. Linearized plasmid DNA gave lower levels of expression than covalently closed circular plasmid DNA when assayed 40 h after PEG-mediated DNA transfer. The use of carrier DNA (herring sperm DNA), in combination with covalently closed circular plasmid DNA, increased the level of expression of GUS by about 50%. CAT expression was enhanced if PEG-mediated delivery was performed on ice rather than at room temperature. The highest level of expression for CAT, and the lowest signal-to-noise ratio, was found 24 h after PEG-mediated DNA transfer. Both GUS and CAT provided results that were quantifiable and can therefore be used as reporter genes in Picea glauca.Abbreviations CAT chloramphenicol acetyl transferase - GUS ß-glucuronidase - CaMV cauliflower mosaic virus - NOS nopaline synthase - CCC covalently closed circular DNA - L linear DNA - PEG polyethylene glycol - HS herring sperm DNA - P protoplasts - PCM protoplast culture medium - MES morpholinoethane-sulfonic acid - Cm chloramphenicol - Ac acetylated - MUG 4-methyl umbelliferyl ß-D-glucuronide - TLC thin layer chromatography     

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     

Physical,chemical and physiological parameters for electroporation-mediated gene delivery into rice protoplasts

Transformation of cereal protoplasts has been reported using several methods; however, the efficiencies of transformations are still very low. We have evaluated a number of parameters that influence electroporation-mediated DNA uptake and have also compared the efficiency of transient GUS activity and stable transformation obtained using an optimized electroporation method with that of the PEG method. The electroporation conditions tested were ionic composition of buffer, ionic strength, resistivity of buffer, type of anions, voltage, and capacitance.Protoplasts isolated from suspension cultures derived from immature embryos of rice (cvs Radon and IR-54) were used for this study. Stable transformation or transient GUS expression experiments were carried out using a plasmid construct containing the CaMV 35S promoter driving thebar gene and a rice actin promoter driving thegus A (uid A) gene (pAG35bar). Electroporation under optimized conditions resulted in about 13-fold higher GUS activities compared to the PEG method. Protoplast survival following optimized electroporation conditions was 55–60%, compared to 35–40% with the PEG treatment. Protoplasts isolated from a suspension culture at different ages gave substantially different levels of transient GUS expression following electroporation-mediated DNA uptake. In contrast, the age of the suspension culture did not influence PEG-mediated DNA uptake and transient GUS activities, which remained low throughout the culture period examined (21 months). Putatively transformed calluses were selected after three to four weeks on medium containing phosphinothricin as the selection agent. The transformation frequencies ranged from 6.2×10^–5 to 5.4×10^–4 with the electroporation method compared to 1.3×10^–5 to 5.3×10^–5 with the PEG method. Southern blot analysis of PPT-resistant calluses obtained by the electroporation-mediated transformation showed simple intergration patterns of integrated DNA in most of the transformants.     

Competence for gene transfer by electroporation in a sub-population of protoplasts from uniform carrot cell suspension cultures

We have investigated the basis for increased transient reporter gene expression following electroporation of protoplasts from uniform carrot cell suspension cultures at increasing DNA concentrations. Use of a combination of histochemical and fluorometric GUS gene assays allowed differentiation between increases due to a higher proportion of expressing protoplasts and increases due to higher expression by each expressing protoplast. A plateau of 20–25% expressing protoplasts was reached by 50 g ml^–1 DNA but total expression continued to increase in direct proportion to applied DNA concentration up to at least 100 g ml^–1. This indicates the existence of a subpopulation of protoplasts competent for the uptake and expression of genes by electroporation.     

Gene transfer by electroporation in betulaceae protoplasts: Alnus incana

The Escherichia coli -glucuronidase gene (GUS) was introduced into Alnus incana (L.) Moench protoplasts by electroporation. Level of GUS transient gene expression was increased by increasing DNA concentrations of pBI 221 plasmid and was affected by the amplitude and duration of the applied electric pulse as well as by the presence of polyethylene glycol (PEG) in the electroporation medium. An optimal level of GUS activity was obtained after electroporation with a capacitive discharge of 500 V/cm and 71 ms-duration. This transformation procedure is simple and efficient. These results motivated us to investigate this method as a possible way of achieving the stable transformation of actinorhizal alder.Abbreviations CaMV cauliflower mosaic virus - CPW salts, Cocking-Power-White salts - kb kilobase(s) - MU 4-methyl umbelliferyl - MUG 4-methyl umbelliferyl glucuronide - F microfarad(s) - NOS nopaline synthase     

Potato granule-bound starch synthase promoter-controlled GUS expression: regulation of expression after transient and stable transformation

Chimaeric genes of promoter sequences from the potato gene encoding granule-bound starch synthase (GBSS) and the -glucuronidase (GUS) reporter gene were used to study GBSS expression and regulation. Analysis of stable transformants revealed that a GBSS promoter sequence of 0.4 kb was sufficient to result in tissue-dependent GUS expression: levels in stably transformed microtubers exceeded levels in corresponding leaves by orders of magnitude. GBSS-GUS constructs could be transiently expressed in leaf protoplasts from wild-type and amylose-free potato lines, etuberosumSolanum brevidens, Nicotiana tabacum andArabidopsis thaliana. Transient expression levels in potato leaf protoplasts were clearly lower than in corresponding suspension cell protoplasts. This lower expression in leaf protoplasts could not be elevated by increasing DNA concentrations during transfection. Light incubation of electroporated suspension cell protoplasts reduced transient GBSS-GUS expression, whereas incubation of transfected protoplasts in media with different sucrose concentrations did not affect transient expression levels. However, electroporated protoplasts, isolated from suspensions, which had been grown on media with increasing amounts of sucrose showed a sucrose concentration-dependent transient expression profile. This indicates that studying GBSS regulation by transient expression experiments needs pre-treatment of the protoplast source. Sequence data of the GBSS promoter were compared to those of two other potato alleles.     

Direct gene transfer to protoplasts of two genotypes of Asparagus officinalis L. by electroporation

Callus-derived protoplasts of two genotypes of asparagus (Asparagus officinalis L.) were electroporated to introduce the -glucuronidase gene (GUS). The level of GUS transient gene expression and the viability of the protoplasts were influenced by the voltage and duration of the electric pulse. The transient expression level was enhanced by increasing the plasmid DNA concentration and by the presence of polyethylene glycol (PEG) in the electroporation medium. A considerable increase in GUS activity was observed in presence of both PEG and upon heat-shock treatments compared to PEG treatment alone. An optimal level of GUS activity was obtained after electroporation with a capacitive discharge of 500 V/cm and 94 ms duration in both genotypes. The two genotypes differed in their responses to in vitro culture and also showed variable levels of transient expression. The present technique was found to be suitable to obtain transgenic plants as the histochemical GUS assay revealed GUS activity in the protoplast-derived micro-colonies as well as in callus tissues.Abbreviations CaMV cauliflower mosaic virus - CAT chloramphenicol acetyltransferase - CPW salts Cocking-Power-White salts - MES 2-[N-Morpholino]ethanesulfonic acid - MU 4-methyl umbelliferone - MUG 4-methyl umbelliferyl glucuronide - F microfarad - NAA naphthaleneacetic acid - NOS nopaline synthase - NPT II neomycin phosphotransferase - X-Gluc 5-bromo-4-chloro-3-indolyl--D-glucuronide     

High level of GUS gene expression driven by pollen-specific promoters in electroporated lily pollen protoplasts

Gene constructs that contained the -glucuronidase (GUS) gene under the control of a pollen-specific Zm13 promoter from maize and a LAT52 promoter from tomato were introduced by electroporation into pollen protoplasts isolated from bicellular pollen grains of Lilium longiflorum. After 20 h in culture, the pollen protoplasts exhibited the apparent expression of GUS in a fluorometric assay. The GUS activity induced under the control of the Zm13 promoter was over 10 000 times higher than activity in the control (with no DNA or without electroporation). By contrast, the GUS gene was nearly silent in the lily microspore protoplasts and generative cell protoplasts. The GUS activity driven by the Zm13 and LAT52 promoters was also detected by a cytochemical assay. The frequency of blue-staining pollen protoplasts was about 70% in the case of the Zm13 promoter. The efficiency of gene transfer by electroporation was much higher than by particle bombardment. This protoplast-specific electroporation system is suitable for rapid and reliable examination of pollen-specific promoters, being as good as the particle bombardment system.     

An improved method for the generation and transfection of protoplasts from Cucumis sativus Cotyledons

Summary A protocol was developed for the preparation of Cucumis sativus var Straight 8 protoplasts that incorporates a two-step Ficoll^® gradient and results in a high percentage of viable, debris-free protoplasts suitable for the transient expression of foreign genes. Polyethylene glycol and electroporation were compared for their effect on protoplast transfection with commonly used reporter genes. Using a polyethylene glycol method, cucumber protoplasts transfected with a plasmid containing the -glucuronidase gene showed high expression levels, while protoplasts transfected with a plasmid containing the chloramphenicol acetyl transferase gene showed levels of activity that were barely distinguishable from mock-transfected controls. Tomato ringspot virus genomic RNA was also transfected into the protoplasts, and the assembly of viral particles was confirmed.     

The effects of promoter on transient expression in conifer cell lines

Summary Protoplasts from suspension cultures of somatic embryos of white spruce (Picea glauca Moench Voss) were electroporated with plasmids containing the chimeric genes for chloramphenicol acetyl transferase (CAT) or -glucuronidase (GUS), under control of one of three promoters. Transient CAT gene expression of approximately equal magnitude resulted when the CAT gene was fused to either the cauliflower mosaic virus (CaMV) 35S promoter or the nopaline synthase (NOS) promoter. When the CAT gene was fused to a tandem repeat CaMV 35S promoter (pPBI-363), CAT enzyme activity compared to NOS or 35S promoters increased up to eightfold (cell line WS-34), and were up to 100-fold greater than control (electroporated without plasmid). Comparatively, protoplasts of black spruce (Picea mariana Mill) and jack pine (Pinus banksiana Lamb.), electroporated with pPBI-363, produced increases in CAT activity compared to control of 90-fold and 70-fold, respectively. White spruce (WS-34) protoplasts were subsequently electroporated with the GUS gene fused to the tandem repeat CaMV 35S promoter. Comparatively, GUS enzyme activity increased up to tenfold compared to GUS fused to a CaMV 35S promoter. The results indicated that transient expression of the CAT and GUS genes was influenced by the type of promoter and cell line used, as well as by electroporation conditions.NRCC No. 30498     

Antisense regulation of the rice waxy gene expression using a PCR-amplified fragment of the rice genome reduces the amylose content in grain starch

The waxy gene encodes a granule-bound starch synthase. A 1.0-kb portion of the sequence of the rice waxy gene, which includes the region between exon 4 and exon 9, was inserted in an antisense orientation between the 35 S promoter and the GUS gene of pBI221. The resultant plasmid, pWXA23, was introduced into rice protoplasts by electroporation. GUS activity was clearly detected in derived callus lines, suggesting that the antisense component of the fusion gene was also expressed. Transgenic rice plants were regenerated from these callus lines and their GUS activity was confirmed. Some of the rice seeds from these transformants showed a significant reduction in the amylose content of grain starch, even though they had become polyploid. These results suggest that even when intron sequences are included, antisense constructs can bring about a reduced level of expression of a target gene. The utility of GUS, included as a reporter gene, for the simple detection of expression of an antisense gene, was apparent from these results.