Electrical patterns of tobacco cells in media containing indole-3-acetic acid or 2,4-dichlorophenoxyacetic acid

A simple, inexpensive, and stable drive-unit for a vibrating probe is described. It was used to measure transcellular electrical currents and their stability in cells from suspension cultures of Nicotiana tabacum L. var. virginica. The cells were highly variable in size, morphology and current-pattern. The magnitude and pattern of the currents depended on the age of the culture, the morphology of the cells and the auxin in the culture medium. Currents in small cell clusters were weakest during the lag-phase of growth and strongest when the cultures were actively growing. The shape of the cells was related to the electrical pattern surrounding them, electrically polar cells tending to be elongated. The proportion of polar cells depended on the auxin composition of the culture medium. About 75% of the cells from suspensions grown in the presence of indole-3-acetic acid (IAA) were electrically polar. These cells normally divided at right angles to their electrical axes to form filaments. Only around 20% of the cells grown in medium containing 2,4-dichlorophenoxyacetic acid (2,4-D) were electrically polar, the remainder had randomly oriented currents and divided in random directions to form irregular clusters rather than filaments. The electrical patterns of cells in 2,4-D were much less stable than those of cells in IAA. When currents were measured repeatedly at fixed locations on cells, those in 2,4-D were about twice as likely to disappear, arise de novo, or change direction as those in IAA. When cells were transferred from 2,4-D to IAA media, the percentage of polar cells increased from 25 to 40 within 1 d, but when they were transferred from IAA to 2,4-D, this percentage decreased from 48 to 26. It is suggested that one of the reasons that 2,4-D suppresses organogenesis in tobacco cultures (and possibly why it also functions as a herbicide) is that it reduces the stability of transcellular currents and disrupts the electrical patterns of cells so that they become less capable of organized polar growth.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid The authors are indebted to the Agricultural and Food Research Council of the UK for their financial support and to the Royal Society for the provision of the vibrating probe. We would also like to thank Dr. A. Lagoa for his help in culturing the cells.     

Effect of exogenous cytokinins on growth and somatic embryogenesis in anise cells (Pimpinella anisum L.)

A cell culture of anise was grown in the presence or absence of 2,4-dichlorophenoxyacetic acid (2,4-D). Application of isopentenyladenine or isopentenyladenosine (4·10^-8 to 4·10^-7 M) to the proembryonic culture (+2,4-D) yielded an increase of the cell density, in contrast to a proembryonic culture grown without exogenous application of cytokinins. Embryogenesis was induced by transferring the cells to a hormone-free medium. Embryo development was promoted by isopentenyladenine and isopentenyladenosine (5·10^-8 to 5·10^-7 M), higher concentrations (5·10^-6 M) inhibited embryogenesis. The effect of cytokinins on embryogenesis was only promotive until the third day of culture, i.e. coincident with cell growth rather than differentiation.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - i6Ade isopentenyladenine - i6Ado isopentenyladenosine     

Plant regeneration from long-term suspension cultures of white clover

A genotype of Trifolium repens L. capable of sustaining high-frequency plant regeneration from long-term (24-month old) cell cultures has been selected. Numerous densely cytoplasmic meristemoids were formed in suspension cultures following the coordinate removal of 2,4-dichlorophenoxyacetic acid (2,4-D) and trichloropicolinic acid (picloram) from the medium and an increase in the NH ₄ ⁺ concentration. Some meristemoids arose from single cells in culture. Increasing the NH ₄ ⁺ concentration in the medium resulted in increased meristemoid formation and decreased the growth rate. Ammonium stimulated meristemoid formation when it was the sole source of nitrogen only if a lethal shift in the pH of the medium was prevented. Meristemoids plated on hormone-free agar medium developed directly into shoots which spontaneously formed roots.Abbreviations 2,4-D dichlorophenoxyacetic acid - MS Murashige-Skoog (1962) medium - NAA -naphthaleneacetic acid - SH Schenk-Hildebrandt (1972) medium     

Establishment of synchrony by starvation and readdition of auxin in suspension cultures of Catharanthus roseus cells

A system of synchronous cell division was established by starvation of auxin and its readdition to suspension cultures of cells of Catharanthus roseus L. cv. Little-Pinky. When cells in the stationary phase were transferred to fresh medium free of 2,4-dichlorophenoxyacetic acid (2,4-D), cells were arrested preferentially at the G₁ phase. After cells had been cultured for 2 days in medium without 2,4-D, readdition of 2,4-D induced the synchronous division of cells. In this system, 70–80% of cells divided synchronously within 3 to 4h, and the mitotic index increased sharply in parallel with the increase in cell number. Active synthesis of DNA was demonstrated by measurements of incorporation of [³H]-thymidine into the DNA fraction. The induction of cell division by the addition of 2,4-D was inhibited by treating cells with analogues of auxin, such as 2,4,6-trichlorophenoxyacetic acid and p-chlorophenoxyisobutyric acid.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - DAPI 4,6-diamidino-2-phenylindole - IAA indole-3-acetic acid - MS Murashige & Skoog - NAA -naphthalenacetic acid - PCIB p-chlorophenoxyisobutyric acid - 2,4,6-T 2,4,6-trichlorophenoxyacetic acid     

Molecular weight distribution of cellulose in primary cell walls

The distribution pattern of the degree of polymerization (DP) of cellulose present in the cell walls of mesophyll- and suspension-cultured cells of tobacco was compared to that of newly synthesized ¹⁴C-labeled cellulose from regenerating tobacco protoplasts and suspension-cultured cells. The cellulose was nitrated, and, after fractionation according to differences in solubility in acetone/water, the DP pattern of labeled or unlabeled cellulose nitrate was determined by viscosity measurements. A low (DP<500) and high DP-fraction (DP>2500) of cellulose were predominant in the cell walls of protoplasts, suspension — cultured cells, and mesophyll cells. The average DP of the high molecular weight fraction of cellulose in the cell walls of mesophyll was higher (DP4,000) than in protoplasts or suspension — cultured cells (DP 2,500-3,000). In all cell walls tested, minor amounts of cellulose molecules with a broad spectrum of a medium DP were present. Pulse — chase experiments with either protoplasts or suspension —cultured cells showed that a large proportion of the low and medium DP-cellulose are a separate class of structural components of the cellulose network. The results are discussed in relation to the organization of cellulose in the primary cell wall.Abbreviations DP degree of polymerisation - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid     

Ionic basis of currents in somatic embryos of Daucus carota

A vibrating probe was used to measure extracellular electrical currents around developing somatic embryos in two lines (RCC27, RCC48) of cultured cells of Daucus carota L. at the heart and torpedo stages. At pH 5.5, an inward current of 1.2±0.1 A·cm^-2 (n=23) was detected at the cotyledon, and an outward current of 1.0±0.1 A·cm^-2 (n=22) was found at the radicle in torpedostage embryos from the RCC27 line. At a pH of 5.75 the currents increased by 0.2–0.3 A·cm^-2 (n=60–62). In a few cases an additional small inward current was detected at the tip of the radicle in toepedo-stage embryos from RCC27 line. Such an inward current at the radicle seemed to appear earlier, some time after the heart stage, in embryos from the RCC48 line.Both extracellular pH measurements (using microelectrodes filled with ion-sensitive resin) and ion-substitution studies were carried out in order to ascertain the ionic composition of the currents in torpedo-stage embryos from the RCC27 line. Regions adjacent to the cotyledon and radicle, at the points of current entry and exit, were found to be more acidic by 0.02±0.01 (n=14) and 0.07±0.01 (n=12) pH units, respectively, than the bulk medium. Removal of K⁺ from the medium reversibly reduced the currents to about 25% of their original value at both cotyledon and radicle. Deletion of Cl^- decreased the currents slightly. Removal of Ca²⁺ resulted in a rapid doubling of currents. Addition of either N,N-dicyclohexylcarbodiimide or tetraethyl ammonium chloride substantially reduced overall currents, and their removal resulted in partial recovery of the currents. It is suggested that the inward current at the cotyledon is comprised largely of K⁺ influx and the outward current at the radicle is mainly the result of active H⁺ efflux.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - MS Murashige and Skoog     

Direct differentiation of tracheary elements in cultured explants of gamma-irradiated tubers of Helianthus tuberosus

Exposure of Jerusalem artichoke (Helianthus tuberosus) tubers to 20 krad doses of -irradiation inhibits mitosis and DNA synthesis in cultures subsequently inititated from such material. When cultures were initiated from immature, developing tubers, tracheary elements differentiated from parenchyma cells in response to auxin in the culture medium. The capacity for direct differentiation in irradiated tissues declined with tuber maturity, and in fully mature tubers xylem differentiation only occurred in non-irradiated controls, following a period of cell division. An hypothesis concerning changes in developmental plasticity of cells in relation to the cell cycle is discussed.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - [³H]TdR tritiated thymidine     

Quantitation of gibberellins and the metabolism of [3H]gibberellin A1 during somatic embryogenesis in carrot and anise cell cultures

In a carrot (Daucus carota L.) cell line lacking the ability to undergo somatic embryogenasis, and in carrot and anise (Pimpinella anisum L.) cell lines in which embryogenesis could be regulated by presence or absence of 2,4-dichlorophen-oxyacetic acid (2,4-D), in the medium (+2,4-D=no embryogenesis,-2,4-D=embryo differentiation and development), the levels of endogenous gibberellin(s) (GA) were determined by the dwarfrice bioassay, and the metabolism of [³H]GA₁ was followed. Embryos harvested after 14 d of subculture in-2,4-D had low levels (0.2–0.3 g g^-1 dry weight) of polar GA (e.g. GA₁-like), but much (3–22 times) higher levels of less-polar GA (GA_4/7-like); GA₁, GA₄ and GA₇ are native to these cultures. Conversely, the undifferentiated cells in a non-embryogenic strain, and proembryos of an embryogenic strain (+2,4-D) showed very high levels of polar GA (2.9–4.4 g g^-1), and somewhat reduced levels of less-polar GA. Cultures of anise undergoing somatic embryo development (-2,4-D) metabolized [³H]GA₁ very quickly, whereas proembryo cultures of anise (+2,4-D) metabolized [³H]GA₁ slowly. The major metabolites of [³H]GA₁ in anise were tentatively identified as GA₈-glucoside (24%), GA₈ (15%), GA₁-glucoside (8%) and the ¹⁽¹⁰⁾GA₁-counterpart (2%). Thus, high levels of a GA₁-like substance and a reduced ability to metabolize GA₁ are correlated with the absence of embryo development, while lowered levels of GA₁-like substance and a rapid metabolism of GA₁ into GA₈ and GA-conjugates are correlated with continued embryo development. Exogenous application of GA₃ is known to reduce somatic embryogenesis in carrot cultures; GA₄ was found to have the same effect in anise cultures. Thus, a role (albeit negative) in somatic embryogenesis for a polar, biologically active GA is implied.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - GA gibberellin(s) or gibberellin-like substances - GC-RC gas chromatography-radiochromatogram counting - HPLC high-presare liquid chromatography - Rt retention time - TLC thinlaver chromatography     

Metabolism of 2,4-dichlorophenoxyacetic acid in cell suspension cultures of soybean (Glycine max L.) and wheat (Triticum aestivum L.)

Cell suspension cultures of soybean (Glycine max L.) and wheat (Triticum aestivum L.) incorporated 2,4-dichlorophenoxyacetic acid (2,4-D) into a metabolite fraction which was insoluble in ethanol, water, and hot sodium dodecylsulphate. Further treatment with hot dimethylformamide solubilized a material which by the following criteria appeared to consist of 2,4-D derivatives covalently bound to lignin: i) co-chromatography of radioactivity and of UV-absorbing material upon gel permeation chromatography; ii) spectral similarity with authentic lignins (IR- and UV-spectra, phloroglucinol reaction), 2,4-D appeared to be incorporated as the intact molecule, as shown by comparison of ring- and sidechain-labeled 2,4-D and by detection of monohydroxylated and intact 2,4-D as the major radioactive products of acid hydrolysis. The same compounds were released from the metabolite material which could not be solubilized in dimethylformamide. The incorporation of xenobiotics or their metabolites into lignin, followed by deposition in the cell wall, is suggested as a general pathway for local excretion and detoxification by plant cells.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - 4-OH-2,5-D 4-hydroxy-2,5-dichlorophenoxyacetic acid - SDS sodium dodecylsulphate - DMF dimethylformamide     

Ionic currents flow throughMicrasterias andClosterium cells during expansion of the primary cell wall

The results of studies of Micrasterias rotata (Grev.) Ralfs, M. thomasiana Archer (biradiate and uniradiate forms) and Closterium sp. using one- and two-dimensional vibrating probes show that transcellular ionic currents are detectable only around cells undergoing expansion of the primary cell wall (half-cell); current enters local regions of expansion and exits over both the rigid surface of the secondary wall and regions of the primary wall where hardening of the wall prevents further expansion. Current densities remain at steady levels until expansion stops with maturation of the primary wall, whereupon currents are no longer detectable. The temporal and spatial correlation between the currents and regions of wall expansion is particularly evident because morphogenesis of the half-cell is a determinate process. Measurements of inward currents ranged from 0.1 to 5.4 A · cm^–2, and outward currents ranged from-0.05 to -1.5 A · cm^–2 measured at 18 from the cell surface. The results of ion substitution and channel-blocker studies indicate that the currents may be carried at least in part by Ca²⁺, Cl^–, H⁺ and K⁺ ions. The possible role of a Ca²⁺ influx during tip growth in desmids is discussed.This work was conducted at the National Vibrating Probe Facility, Marine Biological Laboratory, Woods Hole, Mass., USA. Dr. Lionel F. Jaffe, Director of the Facility, and Dr. Jeremy D. PickettHeaps, University of Colorado, Boulder, USA, provided valuable guidance and support, and gave unstinting encouragement during these studies. Dr. Franklin M. Harold provided support for the writing of this paper during C.L.T.'s postdoctoral year at the National Jewish Center for Immunology and Respiratory Research, Denver. Mr. Alan Shipley and Mr. Steve Dixon provided talented technical assistance. C.L.T. is grateful for support received from a National Institutes of Health Pre-doctoral Training Grant in the Department of Molecular, Cellular and Developmental Biology, University of Colorado. The work was supported by N.I.H. grants 5 P41 RR01395 and 3 P41 RR01395-02S1 (to L.F.J.), National Science Foundation grants No. BSR 82 14199 and PCM 83 09331 (to J.P.-H.), and No. DCB 86 18694 (to F.M.H.).     

Auxin-induced rapid changes in translatable mRNAs in tobacco cell suspension

When 2,4-dichlorophenoxyacetic acid (2,4-D)-dependent tobacco cell suspensions, one normal and one transformed by Agrobacterium tumefaciens, were subcultured on hormone-lacking medium the stationary phase of the cell cycle was reached earlier than on medium containing 2,4-D. Addition of the auxin 2,4-D could restore cell division activity within 10–12 h for the most rapidly reacting cell line. The cell-division response was characterized as being auxin-specific and optimal with 2,4-D at 2.2 10^-6 M. Although the cell lines used showed different characteristics, both reacted with a rapid increase in at least three mRNA species within 1 or 2 h after 2,4-D application. Two, 2,4-D-induced protein spots, seen after in-vitro translation, had the same characteristics (MWs 35 kilodaltons (kDa) and 25 kDa with isoelectric points of 7.1 and 6.3, respectively) in both cell lines. Water-treated controls did not show alterations in the translatable mRNA populations. This indicates that the accumulation of the corresponding mRNAs is an early hormone-induced event. Since cell division is the only measurable reaction found after auxin application, cell systems as described here offer excellent possibilities for studying early auxin-induced changes at the molecular level preceding mitosis.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - kDa kilodalton     

Correlation between the presence of membrane-bound auxin binding and root regeneration in cultured tobacco cells

When cell-suspension cultures and callus tissue from Nicotiana tabacum are grown on medium containing -naphthaleneacetic acid (NAA) and kinetin, three classes of auxin-binding proteins can be detected. When the herbicide 2,4-dichlorophenoxyacetic acid is used instead of both NAA and kinetin, one of these sites, which is membranebound, disappears. After retransferring cells to medium containing NAA and kinetin, this membrane-bound site reappears after four to eight weeks. This reappearance is correlated with the ability of the cells to regenerate roots.Abbreviations IAA indole-3-acetic acid - NAA -naphthaleneacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid     

Expression of the JIM8 cell wall epitope in carrot somatic embryogenesis

Certain single cells in carrot (Daucus carota L.) suspension cultures react with the monoclonal antibody JIM8, and it has been proposed that these cells represent a transitional stage in somatic embryo formation. Shortly after isolation of the single cells by sieving, up to 80% of the cells react with JIM8. Within 4 d, JIM8 labelling becomes restricted to 1% of the single cells. To obtain evidence for the proposed correlation between expression of the JIM8 cell wall epitope and somatic embryo formation the developmental fate of carrot single cells labelled with JIM8 was determined by cell tracking. The results, obtained by recording 43 000 cells, show that only few JIM8-labelled cells give rise to embryos, and most somatic embryos develop from cells devoid of the JIM8 cell wall epitope. We therefore conclude that the presence of the JIM8 cell wall epitope does not coincide with the ability of single suspension cells to form embryos.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - AGP arabino galactan protein - B5-0 Gamborg's B5 medium - B5-0.2 Gamborg's B5 medium supplemented with 0.2 M 2,4-D - FITC fluoresceïn isothiocyanate - PBS phosphate-buffered saline     

Developmental patterns during direct somatic embryogenesis in protoplast cultures of european larch (Larix decidua Mill.)

Summary Protoplasts isolated from embryogenic suspension cultures of European larch (Larix decidua Mill.) were cultured in thin alginate layers using a nylon mesh to enable a monitoring of the development of single cells. The patterns of cell division and differentiation are characterized and compared with zygotic embryogenesis to which homologies can only be drawn to some extent when the protoplasts grow in an auxinfree environment. Already at 2.5 M both 2,4-dichlorophenoxyacetic acid or indole-3-acetic acid cause vacuolation and elongation of individual cells, thus disturbing the process of somatic embryogenesis which generally lacks the precise quantitative patterns occurring in vivo. Prior to the formation of an embryo, a proembryonal mass develops. Oligonucleated products of spontaneous protoplast fusions are able to cellularize even without preceding karyokinesis and perform a normal embryogenic program.Abbreviations BAP N⁶-benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - MES 2-(N-morpholino)ethanesulfonic acid - PEM proembryonal mass     

Quantitative studies of embryogenesis in normal and 5-methyltryptophan-resistant cell lines of wild carrot

The frequency of embryo formation was determined in normal and 5-methyltryptophan-resistant (5-MT^r) cell lines of wild carrot (Daucus carota L.) grown in the presence or absence of 2-isopentenyladenine (2-ip) and 2,4-dichlorophenoxyacetic acid (2,4-D). 2-ip stimulated the intitation of embryo formation and also accelerated embryo development. 2.4-D inhibited embryo differentiation at several stages: at 0.1 mg/l, it stopped regeneration at the earliest stage, resulting in callus growth instead of embryo formation; at 0.04 mg/l 2,4-D, some globular embryos were produced, but they did not develop into more advanced embryos. Variant cell lines with higher levels of auxin (indole-3-acetic acid, IAA) were used to study the effect of an elevated endogenous concentration of auxin on embryogenesis. IAA at these concentrations suppressed regeneration in the same manner as the exogenous auxin, 2,4-D, did. This result confirms the hypothesis that high levels of IAA are responsible for the suppression of regeneration in the 5-MT^r cell lines.     

Inhibition of auxin-stimulated growth of pea stem segments by a specific nonasaccharide of xyloglucan

Hemicellulose extracted from cell walls of suspension-cultured rose (Rosa Paul's Scarlet) cells was digested with cellulase from Trichoderma viride. The quantitatively major oligosaccharide products, a nonasaccharide and a heptasaccharide derived from xyloglucan, were purified by gel permeation chromatography. The nonasaccharide was found to inhibit the 2,4-dichlorophenoxy-acetic-acid-induced elongation of etiolated pea (Pisum sativum) stem segments. This confirms an earlier report (York et al., 1984, Plant Physiol. 75, 295–297). The inhibition of elongation by the nonasaccharide showed a maximum at around 10^-9M with higher and lower concentrations being less effective. The heptasaccharide did not significantly inhibit elongation at 10^-7–10^-10M and also did not affect the inhibition caused by the nonasaccharide when co-incubated with the latter.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - XG xyloglucan - XG7 xyloglucan heptasaccharide (Glc₄·Xyl₃) - XG9 xyloglucan nonasaccharide (Glc₄·Xyl₃·Gal·Fuc)     

Regulation of phenylalanine ammonia-lyase genes in carrot suspension cultured cells

Induction of anthocyanin synthesis occurs during metabolic differentiation in carrot suspension cultured cells grown in medium lacking 2,4-dichlorophenoxyacetic acid (2,4-D), and is closely correlated with embryogenesis. Anthocyanin synthesis may also be induced by light-irradiation under different culture conditions. The phenylalanine ammonia-lyase (PAL) gene (TRN-PAL), which was transiently induced by the transfer effect, was also rapidly induced after light-irradiation. However, TRN-PAL was not involved in anthocyanin synthesis. A second PAL gene, ANT-PAL, was involved in anthocyanin synthesis. ANT-PAL was induced during metabolic differentiation in medium lacking 2,4-D parallel with the induction of chalcone synthase (CHS). PAL genes in the carrot genome are expressed differentially depending on the nature of the environmental stimulus, e.g. transfer effect and light, and other parameters which also affect anthocyanin synthesis.Abbreviations CHS chalcone synthase - 2,4-D 2,4-dichlorophenoxyacetic acid - GUS -glucuronidase - Luc firefly luciferase - PAL phenylalanine ammonia-lyase - UV ultraviolet     

Variation in endoplasmic-reticulum-associated glycoproteins of carrot cells cultured in vitro

Glycoproteins extracted from microsomes of in-vitro-cultured cells of Daucus carota L. cv. US-Harumakigosun were analysed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and detected by peroxidase-conjugated concanavalin A. The appearance of a glycoprotein with M_r 31 000 (GP 31) was correlated with the ability of cells to form somatic embryos. GP 31 appeared in embryogenic cells cultured in 2,4-dichlorophenoxyacetic acid (2,4-D)-containing medium, but not in somatic embryos and non-embryogenic cells; it disappeared when the cultures were transferred to auxin-free medium. Another glycoprotein with M_r 32 000 (GP 32) was detected only in non-embryogenic cells, regardless of the presence or absence of 2,4-D. Both glycoproteins, GP 31 and GP 32, were associated with the rough endoplasmic reticulum and were extractable with 0.05% deoxycholate.Abbreviations Con A concanavalin A - 2,4-D 2,4-dichlorophenoxyacetic acid - ER endoplasmic reticulum - GP 31, GP 32 a glycoprotein with an apparent molecular mass of 31 or 32 kdalton - kDa kilodalton - MS Murashige and Skoog - M_r apparent molecular mass - SDS-PAGE sodium dodecylsulfate-polyacrylamide gel electrophoresis     

Microcallus formation from maize protoplasts prepared from embryogenic callus

Conditions have been developed that induce maize (Zea mays L.) protoplasts to re-synthesize cell walls and to initiate cell divisions. Two types of embryogenic maize callus were used as a source of protoplasts: a heterogeneous callus (Type I) derived from immature embryos after three weeks in culture, and a friable, rapidly growing callus (Type II) selected from portions of the Type I callus. Many variables in the growth conditions of the donor tissue (type of medium, transfer schedule, age of callus), protoplast isolation solutions (pH, osmolarity, type and concentration of cell wall hydrolyzing enzymes, addition of polyamines) and conditions (amount of time in enzyme, amount of tissue per volume of enzyme incubation medium, agitation, preplasmolysis of source tissue, type of callus), and purification procedures (filtration and-or flotation), were found to affect both yield and viability of protoplasts (based upon fluorescein-diacetate staining). Our isolation procedure yielded high numbers of viable, uninucleated maize callus protoplasts which were densely cytoplasmic and varied in size from 20 to 50 m in diameter. Protoplasts plated in solid medium formed walls and divided several times. Of several gelling agents tested for protoplast propagation, only agarose resulted in protoplasts capable of sustained divisions leading to the formation of microcalli. Plating efficiency was established over a wide range of protoplast densities (10³–10⁷ protoplasts/ml). Highest plating efficiency (25%) was obtained at 1·10⁶ protoplasts/ml). The resulting microcalli grew to be dense clusters of about 0.1–0.5 mm in diameter and then stopped growing. Nurse cultures of maize and carrot (Daucus carota L.), were used to establish that individual protoplasts (not contaminating cells or cell clusters) formed walls and divided. Nurse cultures also increased the efficiency of microcallus formation from protoplasts.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - MS Murashige and Skoog (1962) salts - MS 1D Murashige and Skoog salts with 1 mg/l 2,4-D - MS 2D Murashige and Skoog salts with 2 mg/l 2,4-D - N₆ medium of Chu et al. (1975) - NN67-mod medium of Nitsch and Nitsch (1967) as modified in the present paper - FDA fluorescein diacetate - LMP low melting point