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PubMed2001

Efficient protoplast regeneration for some homofermentative lactobacilli and pediococci.

Conditions for protoplast regeneration were examined for several strains of homofermentative lactobacilli and pediococci isolated from silage. Attempts to regenerate protoplasts using previously published agar regeneration media for lactobacilli were unsuccessful for most of the strains. Replacing or increasing colloidal substances in a medium containing raffinose and MgCl(2) as osmotic stabilizers enabled efficient regeneration of the protoplasts at a frequency of 10-99%. A medium containing gelatin, polyvinylpyrrolidone (PVP) and no agar was effective for Lactobacillus plantarum, Lactobacillus pentosus and Lactobacillus rhamnosus protoplasts. An agar medium containing PVP (PVP medium) was effective for Pediococcus sp. protoplasts, and addition of agarose to the PVP medium enabled regeneration of Lactobacillus casei protoplasts. A medium containing calcium alginate gel and no agar was effective for Lactobacillus curvatus protoplasts. The type of colloidal substance required for protoplast regeneration varied from species to species. This result suggested that several kinds of media may be necessary to regenerate protoplasts for all the genera of lactobacilli and pediococci.

Bacteriological Techniques↗
PubMed2005

Analysis of amino acids in individual wheat embryonic protoplast.

Amino acids analysis in single wheat embryonic protoplast was performed using capillary electrophoresis equipped with laser-induced fluorescence (CE-LIF), combination with tissue culture technique. Reagent fluorescein isothiocyanate (FITC) was introduced into living protoplasts by electroporation for intracellular derivatization. A special osmotic buffer (0.6 mol/L mannitol, 5 mmol/L CaCl2) was used to keep the osmotic balance of embryonic protoplasts during the protoplasts derivatization. After completion of the derivatization reaction in the protoplasts, a single protoplast was drawn into the capillary tip by electroosmotic flow. Then a 0.1 M NaOH lysing solution was injected by diffusion. The derivatized amino acids were separated by capillary electrophoresis and detected by laser-induced fluorescence detection after the protoplast was lysed Nine amino acids were quantitatively and qualitatively determined and compared in lysate and single protoplast of wheat embryonic cells respectively, with mean concentrations of amino acids ranging from 2.68 x 10(-5) mol/L to 18.18 x 10(-5) mol/L in single protoplast.

Amino Acids↗
PubMed1988

Binding of cowpea chlorotic mottle virus to cowpea protoplasts and relation of binding to virus entry and infection.

Cowpea chlorotic mottle virus (CCMV) and cowpea protoplasts were used to study initial interactions between virus and protoplast. Protoplasts and virus were incubated under varying conditions of temperature, pH, ionic strength, and the presence of added compounds. Both the amount of 35S-labeled virus bound to protoplasts and the percentage of infected cells were determined. At 0 and 25 degrees the amount of virus associated with protoplasts increased with the amount of virus added. With inoculum of 25 x 10(6) virus particles per protoplast, 4 x 10(3) and 14 x 10(3) particles per protoplast were bound at 0 and 25 degrees, respectively. In the presence of polyethylene glycol, 85 x 10(3) associated particles per protoplast were bound at both temperatures and ca. 50% of the protoplasts became infected. No infection occurred in the absence of PEG. Variation of pH or ionic strength in the absence of PEG caused little to no change in binding and no infection. In the presence of PEG, increase of pH resulted in lower binding, but infectivity was not affected. Increasing ionic strength, however, increased both binding and infectivity. The presence of unlabeled CCMV, tobacco mosaic virus coat protein, bovine serum albumin, and polycations during inoculation in the absence of PEG decreased the amount of bound CCMV. In contrast, CCMV coat protein, which has a positively charged N-terminal arm, increased binding. In the presence of PEG the effects were similar, although larger amounts of virus were bound. The percentage of infection was reduced by all additives to 5-25%. Addition of ammonium chloride, which inhibits endocytotic virus uptake in animal cells, during inoculation as well as in culture media, did not reduce infectivity. These data do not support a specific receptor-mediated endocytotic uptake of virus but favor a nonspecific mechanism of entry, possibly through membrane lesions. Observations in the electron microscope support the latter mechanism.

Endocytosis↗
PubMed2006

Sugar effects on membrane damage during desiccation of pea embryo protoplasts.

Desiccation tolerance of protoplasts isolated from germinating pea (Pisum sativum L. cv. 'Alaska') embryonic axes depends, in part, on the osmotic strength and composition of the suspending medium. To determine the reason for this dependence and whether treatment with different solutions results in different types of damage, protoplast recovery and survival were assessed after dehydration to a range of water contents. Protoplasts were derived from germinating axes that had intermediate desiccation tolerance. Protoplasts were isolated and resuspended in buffers containing sucrose/raffinose (85:15, w/w) or sorbitol, which were isotonic or hypertonic to the cells of the embryonic axis, then were flash-dried to a range of water contents. Protoplasts were rehydrated and stained with fluorescein diacetate (FDA) to assess survival and to estimate two types of membrane injury: lysis and the loss of semipermeability. In all treatments, protoplast survival dropped sharply during the initial phase of dehydration due to lysis. Protoplast survival was greater in hypertonic sucrose/raffinose buffer than in isotonic sucrose/raffinose buffer, or in the latter made hypertonic by the addition of sorbitol. When sorbitol was substituted for sucrose/raffinose in either the isolation or desiccation buffer, or both, protoplast survival at intermediate and low hydrations decreased due to a loss of membrane semipermeability. The results indicate that additional sucrose/raffinose is beneficial for the desiccation tolerance of protoplasts, the benefit is not due to a simple osmotic effect, and the benefit is greatest at water contents less than 0.5 g g(-1) DW, where the presence of the sugars appears to protect membrane semipermeability.

Cell Membrane↗
PubMed2001

Blue-light-dependent osmoregulation in protoplasts of Phaseolus vulgaris Pulvini.

Blue light was found to induce shrinkage of the protoplasts isolated from first-leaf lamina pulvini of 18-day-old Phaseolus vulgaris. The response was transient following pulse stimulation, while it was sustainable during continuous stimulation. No apparent difference was found between flexor and extensor protoplasts. Protoplasts of the petiolar segment located close to the pulvinus showed no detectable response. In the plants used, the pulvinus was fully matured and the petiole was ceasing its elongation growth. When younger, 12-day-old, plants were used, however, the petiolar protoplasts did respond to blue light. The pulse-induced response was similar to that in pulvinar protoplasts, although the response to continuous stimulation was transient and differed from that in pulvinar protoplasts. No shrinkage was induced in pulvinar protoplasts when the far-red-light-absorbing form of phytochrome was absent for a period before blue-light stimulation, indicating that the blue-light responsiveness is strictly controlled by phytochrome. Inhibitors of anion channels and H(+)-ATPase abolished the shrinking response, supporting the view that protoplasts shrink by extruding ions. The response of pulvinar protoplasts is probably involved in the blue-light-induced, turgor-based movement of pulvini. The blue-light responding system in pulvini is suggested to have evolved from that functioning in other growing organs.

Adaptation, Physiological↗
PubMed1989

Protoplast isolation and regeneration in Streptomyces clavuligerus.

The regeneration of streptomycete protoplasts is a major step following genetic manipulations such as fusion and DNA-mediated transformation. Reports of studies on the regeneration of protoplasts from Streptomyces clavuligerus are limited and for this reason the experiments described in this paper were carried out. An investigation of protoplast formation and cytology was made to gain further insight into the loss of protoplast viability in osmotically stabilized support media. Protoplasts with the highest regeneration frequency were isolated from mycelium, grown in a two-stage culture system (without glycine), using lysozyme dissolved in a sucrose osmoticum containing 1% bovine serum albumin. The latter promoted improved protoplast viability. A systematic survey was made of the components of regeneration medium R5, previously used for S. clavuligerus, and other potentially advantageous components and conditions, in an attempt to raise the regeneration frequency of the protoplasts. An improved regeneration medium (R6) and protocol which supported higher and more consistent levels of regeneration of S. clavuligerus protoplasts resulted from these experiments. These improved procedures for protoplast isolation and regeneration proved to be suitable for other streptomycete species.

Bacteriological Techniques↗
PubMed1979

Isolation and characterization of protoplasts from Saccharomyces rouxii.

Cells of the osmotolerant yeast Saccharomyces rouxii were transformed to protoplasts in good yield (85%) by digesting cell walls with snail-gut enzyme in the presence of 10 mM dithioerythritol, 0.1 M sodium phosphate buffer (pH 6.8), and 2.0 M KCl. The requirement for 2.0 M KCl compares with that for S. bisporus var. mellis (another osmotolerant species) and contrasts with the 0.3 to 0.8 M KCl concentrations used in the preparation of most yeast protoplasts. Short digestions (60 min or less) produced mostly spheroplasts; longer incubations (90 min or more) yielded mostly protoplasts as judged by electron micrographs. These protoplasts could be transferred to 1.0 M KCl or 2.0 M sorbitol without lysing, but lysis was pronounced in 0.5 M KCl or 1.0 M mannitol and complete in 0.02 M KCl. Protoplasts were separated from isolated cell wall remnants and debris by centrifugation on a linear gradient of Ficoll 400 (35 to 17.5%, wt/vol) containing 2.0 M KCl. Both crude and fractionated protoplast preparations contained vesicles which were identified with the periplasmic bodies of whole cells. Some of the periplasmic bodies were connected to protoplasts by fine pedicels; others appeared free. Independent degeneracy of periplasmic bodies was occasionally observed. beta-Fructofuranosidase (EC 3.2.1.26) activity is cryptic (physically) in cells of S. rouxii in contrast to the expressed enzyme (periplasmic space) of other Saccharomyces species. This enzyme remains cryptic in protoplast preparations of S. rouxii but is expressed upon lysis. The same specific activities were found per unit cell or protoplast. The possible association of the cryptic enzyme with periplasmic bodies is discussed.

Organoids↗
PubMed1969

Analytical studies on regeneration of protoplasts of Geotrichum candidum by quantitative thin-layer-agar plating.

A preparation of pure protoplasts of Geotrichum candidum became osmotically stable and colonies developed when the protoplasts were embedded in stabilizing thin-layer-agar and incubated with stabilizing basal medium. When growing protoplasts were exposed to distilled water and then reincubated with basal medium, the process of regeneration of protoplasts could be quantitatively demonstrated by counting colonies. The process was divided into three phases, lag, logarithmic, and stationary. Furthermore, the state of regeneration of protoplasts at each phase could be seen in detail by microscopic studies of protoplasts under similar growth conditions. In the lag phase, which lasted for 2 hr after inoculation, protoplasts were completely destroyed when placed in distilled water. During the logarithmic phase, from 2 to 5 hr after inoculation, protoplasts rapidly became osmotically stable and about 18% of them were growing. In the stationary phase, most protoplasts developed germ tubes within 2 hr. These results suggested that there are two main phases, although individual cells passed through three different conditions, osmotically labile, osmotically stable, and growing. No apparent structure of cell wall material could be detected by electron microscopy on the surface of the membrane of these osmotically stable cells.

Agar↗
PubMed1996

The alignment of the axis of asymmetry in regenerating protoplasts of the moss, Ceratodon purpureus, is determined independently of axis polarity.

Ceratodon protoplasts regenerate by polar outgrowth to form cell filaments. The kinetics of regeneration show that some cellular event has to be completed before regeneration can be initiated. The development of the regeneration axis is strongly influenced by light, with axis alignment and axis polarity being fixed independently. We define axis alignment as the relationship of the regeneration axis to the incident light, independent of polarity. Thus protoplasts regenerating directly towards, or directly away from the light source are defined as being similarly aligned but with opposite polarity. Protoplasts that regenerate in unidirectional red light form axes that are aligned parallel to the light direction, with about 70% being polarised towards the light and about 30% away. In unidirectional blue or white light, almost all protoplasts regenerate towards the light but axis alignment is determined less stringently. Re-orientation of protoplasts regenerating in unidirectional light shows that axis alignment is fixed between 8 and 9 hours before protoplasts regenerate and that axis polarity is fixed later. When protoplasts are removed from directional light to either non-directional light or to darkness, regeneration axes continue to be aligned by the earlier directional stimulus for at least 24 hours. Thus although axis alignment is fixed only about 8 hours before regeneration, in the absence of contradictory information about directionality in the light environment, protoplasts retain a memory of light direction for much longer. However, both reorientation and removal from a directional light field have profound effects on axis polarity; the pattern observed in undisturbed protoplasts being lost. To account for these observations, we propose that separate gradients are established independently to determine the alignment and polarity of the regeneration axis respectively. The alignment gradient is established rapidly and is steeper in red than in blue or white light, the polarity gradient is established slowly and is steeper in white or blue light than in red. These studies will now allow a genetic dissection of these processes in moss.

Bryopsida↗
PubMed1989

Actin is associated with the formation of the cell wall in reverting protoplasts of the fission yeast Schizosaccharomyces pombe.

To clarify the involvement of actin in the formation of the yeast cell wall, reverting protoplasts of Schizosaccharomyces pombe were used as a simple model system. Actin of reverting protoplasts was labeled with rhodamine-conjugated phalloidin and observed by conventional fluorescence microscopy and laser scanning confocal microscopy. A close spatial as well as temporal relationship between actin and cell wall formation was observed in protoplast reversion. That is, the site of actin 'dots' in the reverting protoplasts coincided with the site of new wall formation and the timing of rearrangement of actin coincided with the initiation of cell wall formation and with the timing of cell wall expansion. Treatment of reverting protoplasts with cytochalasin D (CD) further clarified the close relationship between actin and cell wall organization. The effect of CD was dose dependent. A high dose of CD caused the absence of actin as well as the complete inhibition of cell wall formation. A low dose of CD caused weakly stained unlocalized actin, which induced grossly aberrant cell wall deposition as well as substantial changes in the morphology of the reverting protoplasts. These results demonstrated that actin is associated with initiation of cell wall formation, the proper deposition of cell wall materials, and maintaining the normal morphology of reverting protoplasts. Scanning electron microscopy revealed the presence of a fibrillar net structure on the surface of non-treated control reverting protoplasts. However, the absence of a fibrillar network on the surface of reverting protoplasts was observed in the presence of a high concentration of CD. Lack of localization of microfibrils as well as poor development of the fibrillar network were also observed in the presence of a low concentration of CD. Recovery experiments confirmed the close relationship between actin and cell wall formation.

Actins↗
PubMed1986

Immunofluorescence of the microtubular skeleton in growing and drug-treated yeast protoplasts.

The microtubular system in growing protoplasts of Saccharomyces uvarum was visualized by immunofluorescence using the monoclonal antitubulin antibody TU 01. We confirmed the coexistence of regular spindle configuration and extensive cytoplasmic networks in growing protoplasts and also observed a distinct distortion of cytoplasmic microtubules in association with wall removal. After a short period for recovery of protoplasts in nutrient medium a restitution of cytoplasmic microtubules and their resumed contact with the protoplast surface was observed. Treatment of growing protoplasts with nocodazole resulted in the disappearance of spindle and cytoplasmic microtubules in the relevant fraction of the protoplast population. In carbendazime (MBC)-arrested protoplasts spindle microtubules were absent but cytoplasmic microtubules associated with spindle pole bodies were clearly visible. Microtubule reassembly on spindle pole bodies occurred within 30 min after washing out nocodazole as well as carbendazime. The approach using protoplasts suggests a simple way in which the differential effect of antimicrotubule agents can be experimentally tested and the microtubule organizing activity of yeast protoplasts visualized at the population level.

Benzimidazoles↗
PubMed1985

Protoplast formation from submerged mycelium and from spore germinants of Streptomyces coelicolor.

Stages in the formation of protoplasts from S. coelicolor strain A3(2) have been studied by transmission electron microscopy. Protoplasts liberated from submerged mycelial growth were variable in size and were released when digestion of the cell wall by lysozyme had completely or almost completely taken place. Protoplasts did not fully adopt the typical rounded shape until after release. A single region of cytoplasm gave rise to more than one protoplast unit. Protoplasts released from spore germinants escaped from the tip of the germ tube, which was the region of the cell wall most susceptible to digestion. Protoplasts derived from spore germinants were more consistent in size and rounded up more rapidly. If a cross-wall had formed in a germinant then it gave rise to separate protoplasts from each cellular compartment. Protoplasts of either type contained a single DNA region. These studies give an indication of the cellular organization of a streptomycete colony, which can be visualized as a multinucleated assemblage of cellular units in a common cytoplasm. The assembly of units separates into a number of protoplasts on digestion of the cell wall.

Microscopy, Electron↗
PubMed1984

[Optical density changes in bacterial protoplast suspensions exposed to membrane-active antibiotics].

The effect of membrane active antibiotics, such as gramicidin S, its derivatives and carbonyl-conjugated pentaens on suspended bacterial protoplasts resulted in lysis of the protoplasts accompanied by a marked decrease in the optical density (OD) of the suspensions. However, when the drug concentrations were lower than those inducing the lysis, an increase in the suspension OD by 10-30 per cent as compared to the control values of the OD was often observed. The increase in the protoplast suspension OD was most pronounced with the use of the drugs with a relatively low lytic activity or under the conditions lowering the lytic activity of the antibiotics. Under such conditions no agglutination of the protoplasts was observed. The comparative estimation of the protoplast diameters by the method of Klenin et al. in the control suspensions of M. lysodeikticus protoplasts and in the suspensions with a stable increase in the OD showed that the OD increase was associated with swelling of the protoplasts: an increase in the suspension OD by approximately 30 per cent corresponded to an increase in the protoplast diameter by approximately 15 per cent. The observed increasing of the suspension OD must be due to the fact that the membrane active antibiotics induced a decrease in the osmotic stability of the protoplasts not sufficient for their lysis.

Anti-Bacterial Agents↗
PubMed1973

Purification of enzymatically isolated mesophyll protoplasts from c(3), c(4), and crassulacean Acid metabolism plants using an aqueous dextran-polyethylene glycol two-phase system.

Enzymatic digestion of leaf segments with 2% cellulase, in combination with a pectinase in some species, yields intact protoplasts mixed with epidermal tissue, vascular tissue, broken protoplasts, and chloroplasts. Epidermal and vascular tissue are removed with sieves of various porosity. Intact protoplasts in the filtrate are separated from other components by an aqueous two-phase system which consists of dextran-polyethylene glycol, with sorbitol and sodium phosphate. Intact protoplasts partition at the interphase, while chloroplasts and broken protoplasts partition in the lower phase when the separation is facilitated by low speed centrifugation. The optimum conditions for purification of maize mesophyll protoplasts with high yields are centrifugation of the two-phase system at 300g for 6 minutes at 2 C with a mixture including 0.46 m sorbitol, 10 mm sodium phosphate, 5.5% polyethylene glycol 6000, and 10% dextran of average molecular weight of 20,000 to 40,000. The collection of protoplasts at the inter-phase was proportional to the amount of chlorophyll added over a wide range of concentrations regardless of the initial contamination of the preparation by other cellular debris. The two-phase system is applicable for protoplast purification from a wide variety of species, including C(3), C(4), and Crassulacean acid metabolism plants, regardless of protoplast size.

Journal Article↗
PubMed1977

Quantitative analysis of the fate of exogenous DNA in Nicotiana protoplasts.

After a 5-hour incubation of protoplasts of Nicotiana tabacum L. ;Xanthi' with (3)H-DNA (7.26 mug/ml) from N. tabacum L. ;Xanthi nc' 3.5% of the initial radioactivity was found in acid-insoluble substances of the protoplasts. The addition of DEAE-dextran and poly-l-lysine to the incubation medium nearly doubled radioactivity adsorption. The absorption was inhibited by 2,4-dinitrophenol, KCN, and low temperature (0 C); this inhibition could not be reversed by exogenous ATP. About 500 tobacco plants established from protoplasts of a normally tobacco-mosaic virus-susceptible cultivar that had been allowed to absorb DNA prepared from a resistant cultivar did not show transfer of the virus-resistant gene.A detailed analysis was performed of the disposition of exogenous DNA in plant protoplasts, by employing Escherichia coli(3)H-DNA and Nicotiana glutinosa protoplasts. In 5 to 20 hours, about 10% of the (3)H-DNA entered the protoplasts. Competition experiments between the (3)H-DNA and unlabeled DNA or thymidine showed that the entry occurred as undegraded (3)H-DNA. Examination of intraprotoplast fractions revealed that 60 to 80% of the absorbed radioactivity resided in the "soluble" fraction of the cytoplasm and 20% in the nuclear fraction. The mitochondrion fraction also contained measurable radioactivity. Sizing on sucrose density gradients showed that the bulk of the absorbed E. coli DNA had been depolymerized. Of the incorporated radioactivity, 15% was accountable as DNA, exogenous as well as resynthesized, and 15% as RNA, protein, and other cell constituents. DNA/DNA hybridization test indicated that 17.6% of the re-extractable (3)H-DNA retained homology with the E. coli DNA; this was equivalent to 2.6% of the absorbed radioactivity. Resynthesized receptor protoplast DNA was represented by a fraction at least 1.7% of the total absorbed radioactivity. The amount of bacterial DNA remaining in protoplasts suggests that each protoplast retained 2.3 x 10(-15)g donor DNA, or approximately half of the E. coli genome.

Journal Article↗
PubMed1977

Stabilization of Oat Leaf Protoplasts through Polyamine-mediated Inhibition of Senescence.

Protoplasts isolated from Avena sativa L. leaves undergo progressive senescence when incubated aseptically in 0.6 m mannitol with or without added nutrients. This senescence is manifested by morphological deterioration and ultimate lysis of protoplasts, by a decrease in incorporation of [(3)H]uridine and [(3)H]leucine into macromolecules, and by a sharp increase in ribonuclease activity.The presence in the incubation medium of l-arginine, l-lysine, certain polyamines related to these amino acids (cadaverine, putrescine, spermidine), Ca(2+), or streptomycin stabilizes the protoplasts. Protoplasts incubated with 10 mml-arginine or l-lysine show an initial inhibition of [(3)H]uridine incorporation, but with time, incorporation is restored to levels greater than in control protoplasts. The rise in ribonuclease activity of protoplasts is completely inhibited if the protoplasts are incubated with 10 mml-arginine. Greater incorporation of [(3)H]uridine into RNA of aging protoplasts is also maintained by appropriate concentration of cadaverine, putrescine, spermidine, Ca(2+), or streptomycin in the incubation medium; the same concentrations of these substances stabilize the protoplasts against additional lysis.

Journal Article↗
PubMed1991

Relationship between Respiration and Photosynthesis in Guard Cell and Mesophyll Cell Protoplasts of Commelina communis L.

A mass spectrometric method combining (16)O/(18)O and (12)C/(13)C isotopes was used to quantify the unidirectional fluxes of O(2) and CO(2) during a dark to light transition for guard cell protoplasts and mesophyll cell protoplasts of Commelina communis L. In darkness, O(2) uptake and CO(2) evolution were similar on a protein basis. Under light, guard cell protoplasts evolved O(2) (61 micromoles of O(2) per milligram of chlorophyll per hour) almost at the same rate as mesophyll cell protoplasts (73 micromoles of O(2) per milligram of chlorophyll per hour). However, carbon assimilation was totally different. In contrast with mesophyll cell protoplasts, guard cell protoplasts were able to fix CO(2) in darkness at a rate of 27 micromoles of CO(2) per milligram of chlorophyll per hour, which was increased by 50% in light. At the onset of light, a delay observed for guard cell protoplasts between O(2) evolution and CO(2) fixation and a time lag before the rate of saturation suggested a carbon metabolism based on phosphoenolpyruvate carboxylase activity. Under light, CO(2) evolution by guard cell protoplasts was sharply decreased (37%), while O(2) uptake was slowly inhibited (14%). A control of mitochondrial activity by guard cell chloroplasts under light via redox equivalents and ATP transfer in the cytosol is discussed. From this study on protoplasts, we conclude that the energy produced at the chloroplast level under light is not totally used for CO(2) assimilation and may be dissipated for other purposes such as ion uptake.

Journal Article↗
PubMed1988

Flow cytometric characterization of the chlorophyll contents and size distributions of plant protoplasts.

We have employed flow cytometry for the characterization of populations of protoplasts prepared from tobacco (Nicotiana tabacum) leaf tissues. We first investigated the possibility of using flow cytometric analysis of the emission of chlorophyll autofluorescence for measurement of the chlorophyll contents of leaf protoplasts. Defined numbers of leaf protoplasts were sorted according to different, nonoverlapping windows placed on the one-dimensional histograms of chlorophyll autofluorescence emission. The amounts of cellular chlorophyll were measured in cell-free extracts of these sorted protoplasts using fluorometry. A high degree of correlation (r2 = 0.983) was observed between these two parameters. We then examined the distribution of protoplast diameters in these protoplast populations through the use of pulse-width time-of-flight (TOF) analysis. Through sorting of protoplasts using a series of narrow, nonoverlapping TOF windows, we were able to demonstrate that the TOF parameter was linearly correlated with protoplast diameter, over the range of 15-55 micron (r2 greater than 0.99). We also compared the use of fluorescein diacetate (FDA) fluorochromasia and chlorophyll autofluorescence as the source of fluorescent signals for TOF analysis. We found that the presence of chloroplasts introduced distortions into the measurement of apparent size afforded by TOF analysis of FDA fluorochromasia. These results are discussed in terms of the application of techniques of flow analysis and sorting for the measurement of gene expression within the various different cell types found in plant tissues and organs.

Cell Separation↗