Linear forms of plasmid DNA are superior to supercoiled structures as active templates for gene expression in plant protoplasts

Introduction of the plasmids pUC8CaMVCAT and pNOSCAT into plant protoplasts is known to result in transient expression of the chloramphenicol acetyl transferase (CAT) gene. Also, transfection with the plasmid pDO432 results in transient appearance of the luciferase enzyme. In the present work we have used these systems to study the effect of DNA topology on the expression of the above recombinant genes. Linear forms of the above plasmids exhibited much higher activity in supporting gene expression than their corresponding super-coiled structures. CAT activity in protoplasts transfected with the linear forms of pUC8CaMVCAT and pNOSCAT was up to ten-fold higher than that observed in protoplasts transfected by the supercoiled template of these plasmids. This effect was observed in protoplasts derived from two different lines of Petunia hybrida and from a Nicotiana tabacum cell line. Transfection with the relaxed form of pUC8CaMVCAT resulted in very low expression of the CAT gene.Northern blot analysis revealed that the amount of poly(A)⁺ RNA extracted from protoplasts transformed with the linear forms of the DNA was about 10-fold higher than that found in protoplasts transformed with supercoiled DNA.Southern blot analysis revealed that about the same amounts of supercoiled and linear DNA molecules were present in nuclei of transfected protoplasts. No significant quantitative differences have been observed between the degradation rates of the various DNA templates used.     

Transient expression of electroporated DNA in monocotyledonous and dicotyledonous species

Transient expression of electroporated DNA was monitored in protoplasts of several monocot and dicot species by assaying for expression of chimeric chloramphenicol acetyltransferase (CAT) gene constructions. Expression was obtained in the dicot species of Daucus carota, Glycine max and Petunia hybrida and the monocot species of Triticum monococcum, Pennisetum purpureum, Panicum maximum, Saccharum officinarum, and a double cross, trispecific hybrid between Pennisetum purpureum, P. americanum, and P. squamulatum. Recovery and viability of protoplasts after electroporation decreased with increasing voltages and capacitance while CAT activity increased up to a critical combination of voltage and capacitance beyond which the activity dramatically decreased. The optimal compromise between DNA uptake and expression versus cell survival was determined for D. carota and applied successfully to the other species. Maximum transient expression occurred 36 hours after electroporation of D. carota. The potential for using this procedure to rapidly assay gene function in dicot and monocot cells and application of this technique to obtain transformed cereals is discussed.     

Flow cytometric quantification of electroporation-mediated uptake of macromolecules into plant protoplasts

Summary Flow cytometry was used to provide a rapid and accurate assessment of electroporation-induced uptake of macromolecules into plant protoplasts. Rice protoplasts were electroporated in the presence of fluorescein isothiocyanate-conjugated dextran (FITC-dextran). After washing, the protoplasts were resuspended in a solution containing propidium iodide which intercalates with DNA, but which is excluded by an intact plasma membrane. Electroporation in the presence of FITC-dextran gave rise to populations of protoplasts that fluoresced green or yellow due to the presence of non-conjugated FITC. Non-viable protoplasts fluoresced red because of their inability to exclude propidium iodide molecules. Flow cytometry was used to resolve and quantify these protoplast populations and thus identify optimal conditions for macromolecule uptake. A direct relationship was observed between FITC-dextran uptake and transient gene expression following plasmid uptake. Thus, simultaneous electroporation of protoplasts with foreign DNA and FITC-dextran followed by fluorescence activated cell sorting may permit partial selection of transformed cells and so reduce the need for a selectable marker.Abbreviations ADC analogue to digital converter - CAT chloramphenicol acetyl transferase (enzyme) - cat chloramphenicol acetyl transferase (gene) - CPW solution cell and protoplast wash solution - DC direct current - EF electrofusion - FALS forward angle light scatter - FITC fluorescein isothiocyanate - FITC-dextran fluorescein isothiocyanate conjugated dextran - PI propidium iodide - PMT photomultipliertube - TLC thin layer chromatography     

Conditions that affect binding of DNA by cultured plant cells and protoplasts: An autoradiographic analysis

Summary The binding of the¹⁴C-labelledSalmonella typhimurium DNA or³H-labelled soybean SB-1 DNA to cultured soybean cells (Glycine max L. Merr.) (SB-1) could be increased at least 100-fold by choosing the proper incubation conditions. The uptake of DNA by cells could completely be inhibited by the addition of an excess of unlabelled thymidine, indicating that the observed uptake of DNA by cells most probably is simply uptake of DNA degradation products. Autoradiograms, prepared from SB-1 protoplasts that were previously incubated with DNA, showed that the DNA was not associated with the protoplasts, but only with aggregates of cell wall material contaminating the protoplast preparation. When protoplasts and DNA were incubated in the presence of DEAE-dextran, the amount of DNAse resistant radioactivity increased 40 times. Again, the autoradiograms showed that most if not all DNAse-resistant material was associated with cell wall materials. Our observation that it is cell wall contaminants in protoplast preparations which account for most of the DNA binding demonstrates the need for caution in interpreting experiments on the binding and uptake of DNA by plant protoplasts.NRCC No. 16353.     

Transient expression of the plasmid pCaMVCAT in plant protoplasts following transformation with polyethyleneglycol

Petunia and carrot protoplasts have been transformed with the plasmid pCaMVCAT by the use of polyethyleneglycol (PEG) as a facilitator. Transformation was revealed by the appearance of the chloramphenicol-acetyl transferase (CAT) enzyme within the transformed cells. Maximal activity of the CAT enzyme was detected within 15 h following transformation, while after 60 h, its activity was significantly reduced, indicating transient expression of the CAT gene. The efficiency of transformation was highly dependent on the presence of CaCl2 in the transformation system, was stimulated by non-functional carrier DNA and was independent on the molecular weight (MW) of PEG used.     

Nuclear RNA quantification in protoplast cell-cycle phases

Using acridine orange staining and flow cytometry the DNA and RNA levels (arbitrary units) of individual cells may be established. Here, this method has been applied to nuclei isolated from plant protoplasts during culture. The specificity of the technique has been validated for such plant material; ribonuclease markedly reduced nuclear staining without modifying the DNA histogram; ribonuclease inhibitor prevented the action of released cell nucleases; and protoplasts cultivated with actinomycin D did not synthesize RNA. First RNA synthesis was evident 18 h after Petunia hybrida protoplasts had been put into culture. An increase of RNA above a critical level was required for cells to be able to initiate DNA replication from G1, termed G1B. G2 nuclei had an RNA:DNA ratio similar to that of G1 nuclei.     

Analysis of transfer of tumor-inducing plasmids from Agrobacterium tumefaciens to Petunia protoplasts.

Petunia protoplasts were infected with the virulent Agrobacterium tumefaciens strain A348 or the avirulent strain A136 (lacking a Ti plasmid). The infection process was stopped at various time intervals up to 24 h after inoculation, and the DNA from the plant cells was isolated. Southern blot analysis indicated that the DNA isolated from infected Petunia cells was not detectably contaminated by bacterial DNA from lysed Agrobacterium cells. Analysis of the DNA from the virulent infections suggested that the transferred DNA (T-DNA) may be transferred to the plant cell rapidly (within 2 to 6 h) after the bacteria bind to the cell wall and that the T-DNA may exist in a rearranged state which is stable over the time period investigated. Dot blot analysis indicated that regions far outside the T-DNA may be transferred to the plant cell. Most of the DNA transferred to the plant cell during the initial hours of infection is rapidly degraded.     

Ti Plasmid Homologous Sequences Present in Tissues from Agrobacterium Plasmid-transformed Petunia Protoplasts

Suspension cell protoplasts of albino Petunia hybrida have beentransformed by isolated Agrobacterium tumefaciens Ti plasmid.Uptake of octopine Ti plasmid (pTiACH5) into protoplasts wasstimulated by poly-L-ornithine and polyethylene glycol (PEG).The frequency and efficiency of transformation of protoplaststo phytohormone autotrophy was compared using the two uptakeagents with various concentrations of plasmid. Transformationwas most efficient with PEG-mediated uptake, 5 µg of Tiplasmid per 10⁶ protoplasts giving a frequency of 6?10^–5.Octopine was not synthesised in any of the transformants afterthe second subculture on hormone-free medium. DNA-DNA hybridisationshowed the presence of DNA homologous to the T-DNA region ofpTiACH5 in all clones analysed. (Received November 9, 1981; Accepted January 29, 1982)     

Lipid synthesis in callus and protoplasts during culture: Analysis of dividing protoplasts of Petunia hybrida and non-dividing protoplasts of Parthenocissus tricuspidata

The purpose of the investigation was to study the process, by which protoplasts derived from callus tissue overcome the hydrolysis of membrane phospholipids, which is induced by the isolation. Dividing protoplasts from Petunia hybrida L. (line SFla) and non-dividing protoplasts from Parthenocissus tricurpidara L. crown gall (Morel strain) were fed with sodium [1-¹⁴C]-acetate for 1 h. The incorporation was carried out at protoplast isolation and after 1, 2, 4 and 7 days of culture. The rates of fatty acid and lipid synthesis were then followed. Isolation stress induced diversions in the utilization of the precursor via triacylglycerol ( Petuunia ) or diacylglycerol ( Partheno-cissus ) and a decrease in the synthesis of oleic acid ( Parrhenocissus ). During the first 2 days of culture, the rate of phospholipid synthesis was similar in both systems. Later on, an increase in the rate and in the efficiency of phospholipid synthesis was observed in dividing Petunia protoplasts. The accelerated rate of lipid synthesis allowed them to recover the same partitioning and content in membrane phospholipids as the reference cells in the callus. This process seemed to bc related to cell division, since it was not observed in the non-dividing protoplasts of Parthenocissus .     

An ultrastructural investigation on the polyethylene glycol-induced adhesion of tobacco nuclei and uptake of micronuclei by soybean protoplasts

Nuclei isolated from tobacco protoplasts were induced to be taken up by soybean protoplasts using a protocol involving polyethylene glycol (PEG), osmotic shock and pH shift. Transmission electron microscopy revealed that PEG treatment condensed the chromatin of the isolated nuclei. Close adhesion of isolated nuclei to the plasma membrane of protoplasts following PEG treatment, was observed by both scanning and transmission electron microscopic methods. Ultrastructural observations were also made on the formation of micronuclei in tobacco cells following the treatment with amiprophosmethyl (APM). Nuclei and micronuclei isolated from APM-treated cells were induced to be taken up by soybean protoplasts. A single case of uptake of an isolated micronucleus was observed by transmission electron microscopy. The observations on the effects of PEG on the isolated nuclei, micronuclei and protoplasts are discussed in relation to the possible mechanism of uptake of nuclei by protoplasts using PEG.     

Study on the Multidrug Resistance 1 Gene Transfection Efficiency Using Adenovirus Vector Enhanced by Ultrasonic Microbubbles In Vitro

As gene delivery reagents, microbubbles have been successfully used in combination with ultrasound. Shock wave exposure has been shown to transfect cells with naked DNA in vitro, but it has not been tested whether the addition of microbubbles would enhance DNA uptake with adenovirus vector. Therefore, the aim of this study was to study the efficacy and safety of multidrug resistance 1 (MDR1) gene transfer into the bone marrow mononuclear cells of rabbits using adenovirus vector enhanced by ultrasound with microbubbles in vitro. The transfection rate of the MDR1 gene was significantly increased by ultrasound microbubbles with adenovirus. After ultrasonic irradiation, there were transient holes in the cell membrane, which disappeared after irradiation by ultrasound for 24 h. The temporary swelling of the organelles was reversible. Our in vitro findings conclusively demonstrate that the exogenous MDR1 gene transfer into the mononuclear cells of rabbits with adenovirus vector was enhanced by the ultrasonic microbubbles and this transfection technique is safe.     

Direct gene transfer to protoplasts: Fate of the transferred genes

Direct gene transfer to protoplasts is one of several methods developed for the production of transgenic plants. This method utilizes the efficient uptake of DNA from the surrounding medium by protoplasts (cell wall-less plant cells). Where a suitable protoplast system exists large numbers of transformant clones can be efficiently produced and often regenerated to normal fertile plants. This review concentrates on the fate of the DNA which is taken up into the protoplasts. Particular emphasis is given to the factors which can influence the integration and form of the transferred DNA, the expression of transferred genes, and the inheritance in further generations of those genes. The information available suggests (1) that DNA is taken up by a large proportion of the cells in a transformation mixture, (2) that this DNA forms complexes sometimes involving carrier DNA, (3) that fewer cells actually take up DNA into the nucleus, and (4) that the complex may be rearranged and/or amplified and then integrated into the genome. If the DNA is arranged in such a way that a gene can be expressed it does so in a normal manner and is stably inherited both mitotically and meiotically.     

Protoplast transformation of glutamate-producing bacteria with plasmid DNA

A method for polyethylene glycol-induced protoplast transformation of glutamate-producing bacteria with plasmid DNA was established. Protoplasts were prepared from cells grown in the presence of penicillin by treatment with lysozyme in a hypertonic medium. The concentration of penicillin during growth affected the efficiency of formation, regeneration, and polyethylene glycol-induced DNA uptake of protoplasts. Regeneration of protoplasts was accomplished on a hypertonic agar medium containing sodium succinate and yeast extract. The spectinomycin and streptomycin resistance plasmid pCG4, originally from Corynebacterium glutamicum T250, could transform various glutamate-producing bacteria such as C. glutamicum, Corynebacterium herculis, Brevibacterium flavum, and Microbacterium ammoniaphilum. The plasmid was structurally unchanged and stably maintained in new hosts. The transformation frequency of most competent protoplasts with pCG4 DNA isolated from primary transformants was high (ca. 10(6) transformants per microgram of covalently closed circular DNA) but was still two orders of magnitude below the frequency of transfection with modified DNA of the bacteriophage phi CGI. The difference was ascribed to the involvement of regeneration in transformation.     

Stable transformation of moth bean Vigna aconitifolia via direct gene transfer

Direct gene transfer proved to be an efficient transformation method for Vigna aconitifolia, a member of the legume family. Kanamycin resistant calli and plants were regenerated from heat shocked protoplasts treated with PEG and plasmid DNA containing the coding region for aminoglycoside phosphotransferase gene (NPT II). The plant cultivar used was an important factor in attaining higher transformation frequencies. Transformation was confirmed by Southern blot analysis using a non-radioactive detection system. Attempts to transform mesophyll and suspension cultured cells by this method were unsuccessful. Protoplasts electroporated with the plasmid pCAP212, which codes for chloramphenicol acetyltransferase, exhibited transient expression of this gene two days after treatment while electroporated cells did not show this enzyme activity. It is therefore assumed that the DNA uptake is prevented by the cell wall.     

Heat-inducible expression of FLP gene in maize cells

The soybean heat-shock gene promoter ( Gmhsp 17.5-E ) has been used to direct expression of gusA and FLP genes in maize cells. At inducible temperatures, in transient expression assays, gusA gene expression controlled by the heat-shock promoter is about 10-fold higher than the expression directed by the CaMV 35S promoter. The Gmhsp 17.5-E promoter preserves its regulatory functions in heterologous maize cells after random integration into genomic DNA.
Heat-shock inducible expression of the FLP gene was investigated by co-transformation of the FLP expression vector (pHsFLP) and a recombination test vector (pUFNeo-FmG) into maize protoplasts. Co-transformed protoplasts were incubated at 42°C for 2 h. This treatment induced recombination of 20–25% of the available FRT sites in transient assays. As a result of heat-shock treatment of stably co-transformed maize cells, activation of gusA gene expression and an associated decrease or elimination of NPT-II activity in transgenic maize lines was observed. Molecular evidence was obtained of the expected DNA excision process catalyzed by the FLP protein in maize transgenic cells. Thus, the experiments presented in this paper indicate that the FLP protein can recognize and subsequently recombine the FRT target sites that had integrated into plant genomic DNA, and that regulated expression of the FLP gene is possible in maize cells using the soybean heat-shock promoter.     

Activity of yeast FLP recombinase in maize and rice protoplasts.

We have demonstrated that a yeast FLP/FRT site-specific recombination system functions in maize and rice protoplasts. FLP recombinase activity was monitored by reactivation of beta-glucuronidase (GUS) expression from vectors containing the gusA gene inactivated by insertion of two FRTs (FLP recombination targets) and a 1.31 kb DNA fragment. The stimulation of GUS activity in protoplasts cotransformed with vectors containing FRT inactivated gusA gene and a chimeric FLP gene depended on both the expression of the FLP recombinase and the presence and structure of the FRT sites. The FLP enzyme could mediate inter- and intramolecular recombination in plant protoplasts. These results provide evidence that a yeast recombination system can function efficiently in plant cells, and that its performance can be manipulated by structural modification of the FRT sites.     

Transient and stable expression of marker genes in cotransformedPetunia protoplasts in relation to X-ray and UV-irradiation

Irradiation of protoplasts with X-rays or ultraviolet light does not seem to influence the level of transient expression of foreign DNA inPetunia protoplasts, whereas the number of stably transformed colonies is significantly raised. This may indicate that irradiation influences integration and/or the expression of marker genes and does not result in enhanced uptake rates of plasmids into protoplasts and cell nuclei. Co-transformation with plasmids carrying a gene for kanamycin resistance (neomycin phosphotransferase II) and a gene for hygromycin resistance (hygromycin phosphotransferase) revealed that the cotransformation rates were not stimulated by irradiation when measuring expression. Twenty-five kanamycin resistant but hygromycin sensitive colonies were examined with Southern or slot blotting and all were found to contain the coding sequence for the hygromycinphosphotransferase gene in their genomes. No obvious differences regarding copy number of integrated genes were observed when comparing transformed colonies derived from irradiated and non-irradiated protoplasts.     

Genetic Recombination of Transforming Deoxyribonucleic Acid Molecules with the Recipient Genome and Among Themselves in Protoplasts of Bacillus subtilis

Hirokawa, Hideo (Southwest Center for Advanced Studies, Dallas, Tex.), and Yonosuke Ikeda. Genetic recombination of transforming deoxyribonucleic acid molecules with the recipient genome and among themselves in protoplasts of Bacillus subtilis. J. Bacteriol. 92:455-463. 1966.-Re-extraction of transforming deoxyribonucleic acid (DNA) from protoplasts of Bacillus subtilis is much more efficient than from intact competent cells. This facilitated the detection of physical recombination between donor and recipient DNA molecules, as indicated by a high cotransfer index of ind(+) and his(+) markers which were originally located in exogenous and endogenous DNA molecules, respectively. This recombinant DNA was extracted after 30 min of incubation of ind his(+) protoplasts with ind(+)his DNA, previously extracted from a corresponding mutant strain of B. subtilis. The intracellular formation of recombinant molecules (ind(+)his(+)) bearing markers from two different exogenous DNA species was also detected 15 min after exposure of ind his recipient protoplasts to a mixture of ind(+)his and ind his(+) donor DNA molecules. The unity of the recombinant molecule was ascertained by dilution experiments and by its being resistant to ribonuclease and trypsin treatment (but being sensitive to deoxyribonuclease). The formation of recombinant molecules showed an inverse kinetics to that of the intracellularly induced loss of linkage between the corresponding markers in the wild-type DNA, thus suggesting a breakage and reunion process which is also favored by the absence of DNA synthesis in the protoplasts and the effect of some specific inhibitors.