Carrot Protoplasts Research Articles

Potassium Transport in Suspension Culture Cells and Protoplasts of Carrot

The properties of potassium transport in carrot (Daucus carota L.) suspension culture cells and their isolated protoplasts were examined. Cells cultured in Murashige and Skoog (MS) medium (Plant Physiol 15: 473-497) were potassium saturated and, consequently, they exhibited little net potassium accumulation. Cells that transport and accumulate potassium were derived from the MS-grown cells by culturing them in a potassium-free modified medium. The transport properties of the modified medium cells included: (a) smooth nonsaturating kinetics with 80% of the maximum rates occurring at 0.1 millimolar KCl, (b) linear transport for at least 75 min, (c) alkaline pH optimum, (d) little accompanying anion uptake with increased malate concentrations balancing net increases in positive charge, and (3) little effect on transport by plasmolysis. Potassium transport activity appeared to be 50% lower in protoplasts isolated from the modified medium cells. Nevertheless, the protoplasts exhibited essentially the same kinetics, time course, pH response, and malate adjustment as the intact cells. We concluded from these results that the low potassium cells and their isolated protoplasts are ideally suited to investigating potassium transport at the cell level without the complications associated with multilayered and highly differentiated tissues.

The effect of fluorescent labeling on calcium-induced fusion of fusogenic carrot protoplasts

Various fluorescent compounds - carboxyfluorescein, scopoletin, fluorescein isothiocyanate (FITC), rhodamine B isothiocyanate (RITC), rhodamine 123, and rhodamine B ethyl ester - were used to study their effects on calcium-induced fusion of fusogenic carrot (Daucus carota L.) protoplasts. These protoplasts normally fused at a high percentage (50-60%) in response to 10 mM calcium, pH 6.0; however, if cells had been labeled with scopoletin, FITC, or RITC, fusion was greatly reduced. In contrast, labeling with carboxyfluorescein, rhodamine 123, or rhodamine B ethyl ester had no detectable effect on calcium-induced fusion. The two rhodamine dyes are shown to be localized in mitochondria.

Oxidation of exogenous NAD(P)H by isolated carrot protoplasts is catalysed by isoperoxidase

A plasma membrane-associated system for the oxidation of exogenous NAD(P)H has been identified in maize root protoplasts (Lin, 1982a, b, 1984) and in protoplasts derived from cell suspensions of sugar cane (Thom & Manetski, 1985). Lin (19826, 1984) reported that this system could be released from intact protoplasts by treatment with trypsin and from his investigations proposed that NADH was oxidized by molecular oxygen via a plasma membrane redox system, which consisted of a quinone pool, cytochrome b and an oxidase. We have investigated the oxidation of exogenous NAD(P)H by protoplasts obtained from carrot cells grown in suspension culture and on the basis of our studies we propose that NAD(P)H is oxidized by isoperoxidase (isoenzymes of peroxidase) extrinsically associated with the plasma membrane of intact protoplasts. Protoplasts were prepared from cultured carrot cells as described by Slabas et a f . (1980). Estimates of viability and intactness were made as described by Widholm (1972). Protoplasts suspensions with greater than 95% viability were routinely used. Addition of 1 .O mM-NAD(P)H to protoplasts markedly increased oxygen consumption as measured with an oxygen electrode. The reaction medium consisted of 0.4 M-sorbitol, 0.5m~-CaCI, and I.OmM-Mes buffer, pH 6.5 at 25°C. The NAD(P)H oxidation was absent or negligible in freshly prepared protoplasts suspensions but became apparent and increased to a maximal value (approximately 75 nmol/l O6 protoplasts per min, i.e. 2.5-3 times basal respiration) 1.5-2 h after preparation. During this period the viability decreased by only 5 7 % . The oxidation of NAD(P)H monitored either by an oxygen electrode or spectrophotometrically (A34-A3,4) had the following characteristics:

Osmoregulation of fusogenic protoplast fusion

Fusion permissive (fusogenic) plant protoplasts have been isolated from wild carrot suspension cultures. Fluorescence, video, and electron microscopy were used to monitor the sequence of fusion events in response to calcium and osmotic stress. Fusion of these protoplasts was enhanced by calcium and inhibited by EGTA. The plasma membranes of these protoplasts were so fusion permissive that osmotic stress, in the absence of external calcium, would stimulate protoplast fusion. Protoplast aggregation but not membrane fusion occurred in a hypertonic solution in the presence of 10 mM calcium. Membrane fusion was not observed until the external osmoticum was lowered. The fusogenic carrot protoplasts provide a model system for studying membrane fusion.

Electric field mediated stable transformation of carrot protoplasts with naked DNA

We have developed an electroporation procedure for the transformation of carrot protoplasts with Ti-plasmid DNA from Agrobacterium tumefaciens. The uptake of pTiC58 into carrot protoplasts was mediated by high voltage electrical pulses at field strengths from 0.5 to 3.8 kV/cm. Protoplast regeneration, somatic embryogenesis and plantlet regeneration were unaffected by the electroporation conditions selected for DNA uptake. Uptake of plasmid pTiC58 resulted in hormone independent regeneration of carrot protoplasts. Transformed somatic embryos were detected in carrot cultures 45 days after electroporation. The transformed somatic embryos developed into teratomas which synthesized nopaline. Hybridization was obtained between a labeled T-DNA fragment from pTiC58 and DNA fragments from 4 month old teratomas regenerated from electro-transformed protoplasts. Based on the number of somatic embryos regenerated after electro-transformation, a frequency of 1.6×10(2) transformants/10(4) somatic embryos/μg pTiC58 DNA was obtained.

Expression of genes transferred into monocot and dicot plant cells by electroporation.

We have developed a general method for electrically introducing DNA into plant cells. Gene transfer occurs when a high-voltage electric pulse is applied to a solution containing protoplasts and DNA. Carrot protoplasts were used as a model system to optimize gene-transfer efficiency, which was measured 24-48 hr after electroporation by the amount of chloramphenicol acetyltransferase activity resulting from the expression of the introduced chimeric plasmids. Gene-transfer efficiency increased with the DNA concentration and was affected by the amplitude and duration of the electric pulse as well as by the composition of the electroporation medium. Our optimized gene-transfer conditions were effective when applied to tobacco and maize protoplasts, demonstrating that the method is applicable to both monocot and dicot protoplasts.

Monoclonal antibody to intermediate filament antigen cross-reacts with higher plant cells.

The monoclonal antibody (anti-IFA) raised (Pruss et al., 1981, Cell 27:419-428) against an intermediate filament antigen, which is widespread throughout phylogeny, has been shown here to cross-react with higher plants. On immunoblotting, anti-IFA cross-reacted with proteins in homogenates of carrot suspension cells and of meristematic cells from onion root tips. A 50-kD cross-reactive protein was enriched in a fraction that consisted of detergent-insoluble bundles of 7-nm fibrils from carrot protoplasts (Powell et al., 1982, J. Cell Sci. 56:319-335). By use of indirect immunofluorescence, anti-IFA stained formaldehyde-fixed onion meristematic cells and carrot protoplasts in patterns approximating those obtained with monoclonal anti-tubulins. That anti-IFA was not recognizing plant tubulins was established by use of immunoblots of two-dimensional gels on which the proteins that comprised isolated fibrillar bundles and taxol-purified carrot tubulins had been separated. The two groups of proteins had different positional coordinates: anti-IFA recognized the fibrillar bundle proteins, and anti-tubulins recognized plant microtubule proteins with no cross-reaction to the heterologous proteins. Likewise, formaldehyde-fixed taxol microtubules from carrot cells could be stained with anti-tubulin but not with anti-IFA. It is concluded that an epitope common to intermediate filaments from animals co-distributes with microtubules in higher plant cells.

Carrot protoplast fusion

Carrot protoplasts have been used successfully in many protoplast fusion studies. These studies have demonstrated 1) whether or not the expression of certain resistances is dominant or recessive, 2) the development of a “universla hybridzer,” 3) the use of chemicals to inactivate one parent, 50 the complementation of albino mutants, 5) interspecific, intergeneric, and interkingdom fusions, and 6) the dominant expression of plant regeneration ability, and 7) the occurrence of somatic segregation in fusion hybrids.

Fusion of Spinach Mesophyll Protoplasts with Carrot Root Parenchyma Protoplasts and the Effect on Spinach Chloroplasts

Mesophyll protoplasts of spinach (Spinacia oleracea L.) have been fused, using polyethylene glycol, with root parenchyma protoplasts of carrot (Daucus carota L.) to enable studies of spinach chloroplasts in a foreign cytoplasm. After 24 h culture, electron microscopy of the fused protoplasts showed many intact chloroplasts, but some chloroplasts were completely disrupted, showing loss of chloroplast envelope and stroma. Spinach protoplasts showed normal chloroplast structure 24 h after PEG treatment. These observations are consistent with a type of incompatibility between some spinach chloroplasts and carrot cytoplasm.

Calcium-induced fusion of fusogenic wild carrot protoplasts

Rather than selecting for a chemical fusogen one can select for a fusogenic plant membrane (i.e., one that will fuse readily). Wild carrot suspension culture cells can be grown under conditions which cause the released protoplasts to have a high potential to fuse. Protoplast fusion is enhanced by calcium and inhibited by EGTA. When 10mM calcium (pH6.0) is added, fusion percentages of 60% are common. The mild fusion treatment appears to have no effect on callus regeneration and differentiation.

8-azaguanine resistant carrot cell mutants and their use as universal hybridizers

We isolated a carrot cell mutant resistant to 8-azaguanine. Into this line a second mutation for resistance to α-amanitin was introduced. The double mutant was shown to be selectable in α-amanitin and counter-selectable in HAT medium. Resistance to Azg and sensitivity to HAT were correlated with an alteration of the enzyme HPRT. The properties of the double mutant as universal hybridizer were verified in a fusion with wild-type carrot protoplasts.

Characterization of Intraspecific Somatic Hybrids of Carrot Obtained by Fusion of Iodoacetate-inactivated A2CA-Resistant and Sensitive Protoplasts

Appearance of A2CA-resistant colonies from a mixed population of iodoacetate-inactivated A2CA-resistant and sensitive carrot protoplasts was dependent upon fusion. No colonies arose from controls in which mixed unfused (or homologously fused) population of parental protoplasts were plated in order to rule out cross-feeding. Suspension cultures obtained from resistant colonies were tested for level of resistance to A2CA, uptake of the analogue and endocellular content of free proline. All these parameters were found to be intermediate between those of parental lines. Unexpectedly the chromosome number of hybrid lines was found to be very close to that of the sensitive parental line suggesting that extensive chromosome loss had occurred in fusion products. These experiments show that resistance to A2CA due to proline overproduction behaves as a semidominant trait.

Orientation and Integrity of Plasma Membrane Vesicles Obtained from Carrot Protoplasts

Two fractions enriched in plasma membrane derived from suspension-cultured carrot (Daucus carota L.) cells were examined to determine if they differed from each other either in physical nature or in orientation. Parameters studied included the protein composition of purified membranes derived from trypsinized and nontrypsinized protoplasts as well as from trypsinized purified plasma membranes, the effect of inhibitors and membrane perturbants on ATPase activity, the binding of [acetyl-(14)C]concanavalin A to purified membrane fractions, and the competitive removal of [acetyl-(14)C]concanavalin A from purified membranes derived from [acetyl-(14)C]concanavalin A-labeled protoplasts. One fraction (at density of 1.102 grams per cubic centimeter on Renografin gradients) appears to be a mixed population of ;tightly' sealed vesicles with the majority being rightside-out vesicles of plasma membrane, and the other fraction (density 1.128 grams per cubic centimeter) apparently is a population of predominantly ;leaky' vesicles and/or nonvesicular fragments of plasma membrane, a large portion of which appear to be ;leaky' inside-out vesicles. In addition, it is shown that plasma membrane-enriched fractions can be distinguished from cellular endomembranes on the basis of protein and glycoprotein composition.

Poly(ethylene glycol)-induced fusion of plant protoplasts. A spin-label study

Carrot protoplasts were used to study the mechanism of poly(ethylene glycol)-induced fusion in vivo. In contrast to human erythrocytes, plant protoplasts can be induced to undergo massive fusions by adding recrystallized PEG 6000. ESR spectra of 5-doxylstearic acid (5DS)-labeled protoplasts indicate that PEG 6000 ( > 20% w/v ) causes a decrease in membrane fluidity and the appearance of a fluid signal indicating isotropic motion of the fatty acid spin label. The same ESR spectra are obtained if spin-labeled protoplasts are pretreated with glutaraldehyde. The 3-carboxyl-2,2,5,5-tetramethylpyrroline-1-oxyl derivative of poly(ethylene glycol) does not appear to intercalate into the hydrophobic region of the lipid bilayer, however, the apparent extraction of the 5DS-label from the protoplast membrane indicates that poly(ethylene glycol) can interact with membrane lipids.

Effects of Conditions of Incubation on the Binding of DNA-Loaded Liposomes to Plant Protoplasts

Summary The binding of DNA-loaded, positively charged, multilamellar vesicles and negatively charged, reverse evaporation vesicles to tobacco and carrot Protoplasts has been investigated. It was found that polyethylene glycol (PEG) 6000 increased binding of both types of liposomes to protoplasts. In addition, this binding was cooperative and competition between DNAloaded and empty liposomes could not be detected. A differential effect of pH on positively charged and negatively charged liposome binding to protoplasts was observed. The first ones bound best at pH 5.6 whereas the latter bound slightly better at pH 9.0. High salt medium considerably unpaired binding of positively charged liposomes while it affected negatively charged ones to a much lesser extent. It was also observed that the amount of DNA bound to protoplasts after liposome-mediated transfer decreased with time after resuspension of protoplasts in fresh medium. Less donor DNA degradation was observed when it was originally sequestered in negatively charged liposomes as compared to positively charged liposomes. Incorporation of soybean agglutinin into liposomes strongly increased their binding to protoplasts.

Cryostorage of Cloned Amino Acid Analog-Resistant Carrot and Tobacco Suspension Cultures

Five clones were isolated from five different amino acid analog-resistant Daucus carota L. var. Sativa and Nicotiana tabacum L. cv. Xanthi cell lines. The individual clones were similar in their resistance to dl-5-methyltryptophan, S-(2-aminoethyl)-l-cysteine, or azetidine-2-carboxylic acid, and in their corresponding free amino acid levels.The cell suspensions were stored using a controlled freezing rate at -196 degrees C with concentrations up to 40% of the four cryoprotectants: mannitol, proline, dimethylsulfoxide, glycerol, and combinations of dimethyl-sulfoxide and glycerol. No less than 55% post-thaw viability, determined by phenosafranin dye exclusion, was obtained after storage using a cryoprotectant mixture of 10% glycerol and 10% dimethylsulfoxide. Growth of the cryostored cells could be obtained consistently only by using feeder plate methodology with this combination of cryoprotectants. Post-thaw viability and percentage of cells demonstrating growth, as estimated by growth kinetics, were found to be similar. This indicates that little selection occurred during the freezing and recovery process. In addition, the amino acid analog-resistant traits were unaltered following cryostorage.Suspension cultures of Datura innoxia Mill. were frozen similarly with maximum post-thaw viability of 38%, but subsequent growth was not obtained.Protoplasts of D. innoxia, tobacco and carrot were also cryostored using a mixture of 10% dimethylsulfoxide and 10% glycerol as cryoprotectants. Viabilities of no less than 40% were obtained, however, only the carrot protoplasts regenerated cell walls and underwent cell division.

Transcription in nuclei isolated from carrot protoplasts: effects of exogenous DNA

Transcription in nuclei isolated from carrot protoplasts: effects of exogenous DNA

Novel features of the plant cytoskeleton

Carrot protoplasts, prepared by avoiding plasmolysis and by extraction with Triton X-100 under isotonic conditions, reveal features not seen when incomplete cytoskeletons are prepared from protoplasts by hypotonic lysis. These include: the retention on the polylysine-coated substratum of the nucleus which is invariably associated with a bundle(s) of 5–7 nm fibrils, and longer microtubules than are otherwise seen.

Liposome-mediated delivery of DNA to carrot protoplasts

The encapsulation of DNA within liposomes and subsequent fusion of the liposomes with carrot (Daucus carota L.) protoplasts were examined to determine optimum conditions for effective liposome-mediated delivery of DNA to protoplasts. Escherichia coli [(3)H]DNA could be encapsulated with 50% efficiency using encapsulation volumes as low as 0.5 ml. Incorporation of liposome-encapsulated [(3)H]DNA by carrot protoplasts increased linearly for 2.5 h, and increasing the ratio of protoplasts to liposomes increased the total amount of radioactive label incorporated within the protoplasts. Liposome-mediated incorporation of [(3)H]DNA by protoplasts increased over a range of polyethylene glycol concentrations up to 20%, but Ca(2+) did not increase liposome-mediated incorporation when present in the liposome-protoplast incubation mixture. Optimum incorporation was observed when the pH of the liposome-protoplast incubation medium was decreased to 4.8. Encapsulation experiments using DNA of the plasmid pBR322 indicated that an average of 200-1,000 intact copies of pBR322 were sequestered within each nucleus after liposome delivery.

β-Glucan synthetase activities in regenerating protoplasts from carrot suspension cultures

The activities of β-glucan synthetases I and II from particulate fractions of protoplast homogenates were compared to the phases of cell wall regeneration in cultivated carrot protoplasts. β-1,4-Glucan synthetase (I), assayed at low UDP-glucose (UDPG) and high Mg 2+ levels showed a lag phase of 15–20 h. The specific activity increased 2-fold during the first 3 days of cultivation. Cultivated carrot cells from the exponential growth period did not exhibit any increase in the activity of the particulate fraction. Inhibitors of RNA and protein synthesis prevented an increase suggesting that enzyme protein synthesis is a prerequisite for this process. β-Glucan synthetase II, presumably producing β-1,3-glucans at high substrate concentrations in the absence of Mg 2+, showed an immediate rise for 20 h, but then declined. The phase of cell wall regeneration was determined by the incorporation of [ 14C]glucose into cellulose.