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.     

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.     

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     

Gene Transfer to Lentil Protoplasts by Lipofection and Electroporation

Abstract

Cationic liposomes made of dipalmitoylphosphatidylcholine and stearylamine (9:1) were prepared by reverse-phase evaporation and were able to interact spontaneously with plasmid DNA. The loaded vesicles delivered a β-glucuronidase (GUS)-carrying plasmid to lentil Lens culinaris) protoplasts, leading to transient expression of the GUS reporter gene. The transfection efficiency achieved by using stearylamine-containing liposomes (lipofection) was comparable to the one obtained by electroporating the protoplasts at 500 μF and different field strengths. Furthermore, the combination of electroporation and lipofection, though reducing cell survival, increased the activity of the reporter enzyme detected in the cell lysates, yielding transient expression levels higher than those recorded after lipofection or electroporation alone.     

Activity of a maize ubiquitin promoter in transgenic rice

We have used the maize ubiquitin 1 promoter, first exon and first intron (UBI) for rice (Oryza sativa L. cv. Taipei 309) transformation experiments and studied its expression in transgenic calli and plants. UBI directed significantly higher levels of transient gene expression than other promoter/intron combinations used for rice transformation. We exploited these high levels of expression to identify stable transformants obtained from callus-derived protoplasts co-transfected with two chimeric genes. The genes consisted of UBI fused to the coding regions of the uidA and bar marker genes (UBI:GUS and UBI:BAR). UBI:GUS expression increased in response to thermal stress in both transfected protoplasts and transgenic rice calli. Histochemical localization of GUS activity revealed that UBI was most active in rapidly dividing cells. This promoter is expressed in many, but not all, rice tissues and undergoes important changes in activity during the development of transgenic rice plants.     

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     

Transient gene expression in electroporated bean cotyledon protoplasts

A protocol has been developed for the introduction of foreign DNA into cell protoplasts from immature bean cotyledons. The method yields high amounts of the reporter enzymes β-glucuronidase (GUS) and chloramphenicol acetyl transferase (CAT) when cognate genes are driven by the promoter and upstream sequences of a bean β-phas gene. Comparisons with expression in stable tobacco transformants indicate that transient assays can be used to investigate enhancer function. This techniques should be applicable to other gene systems as well.     

Transient GFP expression in Nicotiana plumbaginifolia suspension cells: the role of gene silencing,cell death and T-DNA loss

The transient nature of T-DNA expression was studied with a gfp reporter gene transferred to Nicotiana plumbaginifolia suspension cells fromAgrobacterium tumefaciens. Individual GFP-expressing protoplasts were isolated after 4 days' co-cultivation. The protoplasts were cultured without selection and 4 weeks later the surviving proto-calluses were again screened for GFP expression. Of the proto-calluses initially expressing GFP, 50% had lost detectable GFP activity during the first 4 weeks of culture. Multiple T-DNA copies of the gfp gene were detected in 10 of 17 proto-calluses lacking visible GFP activity. The remaining 7 cell lines contained no gfp sequences. Our results confirm that transiently expressed T-DNAs can be lost during growth of somatic cells and demonstrate that transiently expressing cells frequently integrate multiple T-DNAs that become silenced. In cells competent for DNA uptake, cell death and gene silencing were more important barriers to the recovery of stably expressing transformants than lack of T-DNA integration.     

Introduction and expression of foreign DNA in isolated spinach chloroplasts by electroporation

An electroporation-mediated method for the study of foreign gene expression within chloroplasts has been developed. The chloroplast expression vector pHD203-GUS, which consists of coding regions for β-glucuronidase (GUS) and chloramphenicol acetyltransferase (CAT) separated by a double psbA promoter fragment from pea (in opposite orientation) was electroporated into spinach chloroplasts and the transient gene expression was examined. Conditions for the expression of the reporter genes have been optimized. Both CAT and GUS activities were detected in chloroplasts electroporated with pHD203-GUS, but not with nuclear expression vector pBI221 or negative control pUC18. No GUS activity was detected when pHD203-GUS was electroporated into spinach protoplasts. Dot immunoblot analysis using anti-GUS antibody confirmed the existence of GUS protein in chloroplasts electroporated with chloroplast-specific vector but not the negative controls, excluding the possibilities of endogenous GUS or bacterial contamination. The expression of GUS protein in treated chloroplasts was further confirmed by Western blot analysis.     

Evaluation and Comparison of the GUS, LUC and GFP Reporter System for Gene Expression Studies in Plants

Abstract: The detailed analysis of the expression pattern of a plant gene can give important clues about its function in plant development, cell differentiation and defence reactions. Gene expression studies have been greatly facilitated by the employment of proteins like β-glucuronidase (GUS), green fluorescent protein (GFP), and firefly luciferase (LUC) as reporters of gene activity. The application of reporter genes in plants, specifically in the field of gene expression studies, has expanded over the years from a mere tool to quantify (trans) gene expression in tissue samples, to real-time imaging of in planta promoter dynamics. To correctly interpret the activity that is given by each reporter, it is important to have a good understanding of the intrinsic properties of the different reporter proteins. Here we discuss those properties of GUS, LUC and GFP that are of interest in gene expression studies.     

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.     

Expression of green-fluorescent protein gene in sweet potato tissues

Green-fluorescent protein (GFP) gene expression, transient and stable after electroporation and particle bombardment, was analyzed in tissues of sweet potato cv.Beauregard. Leaf and petiole tissues were used for protoplast isolation and electroporation. After 48 h, approximately 25–30% of electroporated mesophyll cell protoplasts regenerated cell walls, and of these, 3% expressed GFP. Stable expression of GFP after four weeks of culture was observed in 1.0% of the initial GFP positive cells. In a separate experiment, we observed 600–700 loci expressing GFP 48 h after bombarding leaf tissue or embryogenic calli, and stable GFP-expressing sectors were seen in leaf-derived embryogenic calli after four weeks of protoplast culture without selection. These results demonstrate GFP gene expression in sweet potato tissues. Screening for GFP gene expression may prove useful to improve transformation efficiency and to facilitate detection of transformed sweet potato plants.     

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.     

β-Glucuronidase gene and green fluorescent protein gene expression in de-exined pollen of Nicotiana tabacum by microprojectile bombardment

 Gene constructs containing the β-glucuronidase (GUS) gene or green fluorescent protein (GFP) gene under the control of pollen-specific promoter Zm13-260 from maize were introduced by particle bombardment into de-exined pollen of Nicotiana tabacum. The de-exined pollen exhibited transient expression of the GUS or GFP gene as indicated by histochemical and fluorescent assay, respectively. The frequency of de-exined pollen transformation with the GUS or GFP gene was approximately 6 and 3 times higher, respectively, than that of pollen with intact walls, indicating that pollen deprived of the exine barrier responded better to foreign gene transfer than did the original. Cytological observation of GUS-expressing pollen grains showed that introduced gold particles were visible in the cytoplasm and vegetative nucleus as well as in the generative nucleus. GFP-expressing pollen tubes were observed in the style even after pollination. Received: 28 October 1997 / Revision accepted: 13 April 1998     

Exine-Detached Pollen of Nicotiana tabacum as an Electroporation Target for Gene Transfer

In developing alternative systems for plant transformation the authors investigated the use of male gametophyte as the foreign gene receptor. However, delivery of foreign DNA into pollen is difficult because of the existence of a thick exine, therefore a new experimental system was developed using exine-detached pollen (EDP) of Nicotiana tabacum as an electroporation target which was also compared with germinating pollen (GP) and pollen grains (P). A transient GUS expression assay was conducted to analyze the effects of different electroporation conditions and promoter activity. The pollen-specific promoter Zml3 from Zea mays mediated high level of GUS gene expression but CaMV 35S only had very low activity in both EDP and GP. The optimal field strength for gene transfer was obtained at 750 V/cm for EDP and 1250 V/cm for GP when the time constant of pulse was 13 ms. The GUS activity in EDP had a 5-fold increase as compared with GP and P respectively. The level of GUS gene expression was slightly increased when adding 10 % PEG into the electroporation buffer. This result indicates that pollen deprived of exine responds much better to foreign gene transfer than the previously used intact pollen grains and may be a better vector to introduce, via pollen tube, genes into the egg cell and offsprings.     

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.     

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).