Rise in chlorotetracycline fluorescence accompanies tracheary element differentiation in suspension cultures ofZinnia

Summary Developing tracheary elements in suspension cultures ofZinnia elegans fluoresce intensely relative to non-differentiating cells when stained with chlorotetracycline (CTC), a fluorescent chelate probe for membrane associated calcium. This suggests that a change in calcium uptake or subcellular distribution accompanies the onset of tracheary element differentiation. A few cells in early differentiating cultures were brightly fluorescent, but did not have visible cell wall thickenings, suggesting that a rise in sequestered calcium may precede visible differentiation. Diffuse CTC fluorescence in early differentiation most likely results from sequestration of calcium in the endoplasmic reticulum. Late in differentiation, CTC fluorescence becomes punctate in appearance, probably due to loss of plasma membrane integrity occurring at the onset of autolysis.Zinnia suspension culture cells were found to be very sensitive to CTC and low concentrations (10 M) were used to assure accurate localization of membrane-associated calcium in healthy cells.Abbreviations CTC chlorotetracycline - DIC differential interference contrast - DiOC₆ 3,3-dihexyloxacarbocyanine iodide - ER endoplasmic reticulum - EGTA ethylene glycol bis-(amino-ethyl ether) N,N,N¹N¹-tetraacetic acid - NPN n-phenylnaphthylamine - OsFeCN osmium tetroxide and potassium ferricyanide - TE tracheary element - TEM transmission electron microscopy     

The release of α-amylase through gibberellin-treated barley aleurone cell walls

Localisation of -amylase (EC 3.2.1.1.) in low-temperature-embedded isolated barley (Hordeum vulgare L.) aleurone has been achieved using rhodamine-labelled secondary antibodies and the protein A-gold technique. Treatment with gibberellic acid (GA₃) resulted in an increase of immunofluorescence in the cytoplasm of aleurone cells and also its appearance in specific regions of the cell walls. Cytoplasmic label was neither perinuclear nor associated specifically with aleurone grains as had been found in earlier work, but was present throughout the cytoplasm of all cells. A relatively high level of labelling occurred in hydrolysed wall regions. Label was also associated with plasmodesmata in both hydrolysed and unhydrolysed wall regions. The pattern of labelling indicates that -amylase is released from aleurone via digested wall channels and that, except for the inner wall layer, unhydrolysed regions are impermeable to the enzyme. It is suggested that the resistant wall tubes around plasmodesmata may facilitate enzyme release by providing a pathway for transfer, especially of wall hydrolases, into the more impermeable parts of the wall.Abbreviations ER endoplasmic reticulum - GA₃ gibberellic acid - RER rough endoplasmic reticulum     

Isolation and culture of cotton ovule epidermal protoplasts (prefiber cells) and analysis of the regenerated wall

Culture protocols were developed and characterization of the regenerated cell walls was performed for protoplasts of cotton (Gossypium hirsutum L., L., var. Acala SJ-2) ovule epidermal cells. This work was undertaken in order to extend studies concerning nutritional effects and regulation of nucleotide sugar incorporation into -1,3- and -1,4-glucan components of cotton fiber cell walls. Protein and carbohydrate polymers and recovered from the culture medium. Analysis of a cellular fraction indicated that the majority of ¹⁴C incorporated from [¹⁴C] glucose was present in the hot-water-soluble fraction of the cells. The majority of label incorporated into cell wall material could be solubilized with acetic-nitric reagent, indicative of noncellulosic material, and characterized as -1,3-linked glucans. Only 5 to 15% of the regenerated cell wall could be characterized as -1,4-linked glucose indicative of cellulose.     

Phytochrome-regulated transfer of fructosidase from cytoplasm to cell wall in Raphanus sativus L. hypocotyls

The far-red absorbing form of phytochrome, P_fr, rapidly increases the rate of transfer of -fructosidase (E.C.3.2.1.26) from the cytoplasm to the cell wall in radish hypocotyls. Far-red light increases the level of enzyme in a particulate fraction: after two hours of light treatment, the particulate enzyme is associated almost exclusively with the endoplasmic reticulum. Transfer from the endoplasmic reticulum to the cell wall involves an incorporation into Golgi bodies and the plasmalemma: these membrane fractions were separated by centrifugation on a discontinuous sucrose density gradient and their degree of purity was determined by the use of known biochemical markers. With respect to -fructosidase, light controls, via P_fr: (1) the total amount, (2) the incorporation into the endoplasmic reticulum and (3) the transfer to the cell-wall. These three processes have different sensitivities to cycloheximide.Abbreviations -FFase -fructosidase - IDPase inosine diphosphatase - SGTase UDPG-sterol glucosyltransferase - NCRase NADPH-cytochrome c-oxydoreductase - NPA N-naphtylphtalamic acid - BSA bovine serum albumine     

Protoplast isolation, culture, and fusion protocols for kenaf (Hibiscus cannabinus)

Protoplast isolation and culture protocols were developed for ten cultivars of Hibiscus cannabinus L. (kenaf). Leaves from seedling lines maintained in vitro were used as donor tissues. Optimal cell wall digestions were achieved with a combination of cellulysin (1.0%) and macerase (0.5%). Average yields ranged from 0.9×10⁵ to 5.9×10⁶ protoplasts g fw^-1 leaf tissue with viability estimates ranging from 53% to 87%. This protocol was ineffective for leaf tissue taken from plants grown in vivo. Protoplasts harvested from plantlets maintained in vitro produced rapidly growing calluses when plated in semi-solid medium after an initial culture in liquid medium. First cell divisions were observed within four to six days after initial culture in medium containing plant growth regulators 2,4-dichlorophenoxyacetic acid (1.4 M) and kinetin (13.8 M). An electrofusion protocol which did not significantly reduce protoplast viabilities was developed for kenaf protoplasts. The maximum fusion frequency (4.6%) was obtained with an electrofusion voltage of 2.0 kV cm^-1.Abbreviations 2,4-d 2,4-dichlorophenoxyacetic acid - BA 6-benzylaminopurine - FDA fluorescein diacetate - MS Murashige and Skoog - NAA 1-naphthaleneacetic acid - PGRs plant growth regulators - SCL seed clonal line     

Ultrastructural cytochemistry of the diaminobenzidine reaction in the aquatic fungus Entophlyctis variabilis

Ultrastructural localization of peroxidatic activity was investigated in the chytrid Entophlyctis variabilis with the 3,3-diaminobenzidine (DAB) cytochemical prodedure. The subcellular distribution of reaction product varied with changes in pH of the DAB medium and with the developmental stage of the fungus. Incubations in the DAB reaction medium at pH 9.2 produced an electron dense reaction product within single membrane bounded organelles which resembled microbodies but which varied in shapes from elongate to oval. At this pH the cell wall also stained darkly. When the pH of the DAB medium was lowered to pH 8.2 or 7.0, DAB oxidation product was localized within mitochondrial cristae as well as in microbodies and zoosporangial walls. As soon as zoospores were completely cleaved out of the zoosporangial cytoplasm, endoplasmic reticulum (ER) also stained. When the wall appeared around the encysted zoospore, ER staining was no longer found. The influence of the catalase inhibitor, aminotriazole, and the inhibitors of heme enzymes, sodium azide and sodium cyanide, on the staining patterns within cells incubated in the DAB media indicates that microbody staining is due to both catalase and peroxidase, mitochondrial staining is due to cytochrome c, and ER staining is due to peroxidase.Abbreviations DAB 3,3-diaminobenzidine-HCl - ER endoplasmic reticulum     

Ultrastructure of the cell wall regeneration of isolated protoplasts of Skimmia japonica thunb

Isolated protoplasts obtained from leaves and from stem callus cultures of Skimmia japonica were cultivated for 72 h to regenerate a new cell wall. During this process the structural changes in the protoplasts and at the surface of the plasmalemma were studied in ultrathin sections and after freeze-fracturing and deep-etching.The cultured protoplasts show an apparent increase in cell organelles compared to the freshly isolated protoplasts. In particular, mitochondria, endoplasmic reticulum, and ribosomes, many of them appear as polysomes, become numerous. Moreover, special connections between the ER and the plasmalemma are visible. Most important are the fracture faces of the plasmalemma with two different arrangements of membrane-bound particles: (1) particles in hexagonal arrays and (2) rows of ca. 14 particles. Their orientation usually conforms with that of the regenerated microfibrils of the cell wall. According to these results the following model for microfibril synthesis and orientation in higher plants is proposed: While the cytoplasmic activity is involved in the production of cellulose precursors and enzymes, the hexagonal arrays may respresent specialized regions for the outward passage of these cellulose precursors. The rows of membrane-associated particles may function as a linear enzyme complex (matrix) for microfibril biosynthesis and orientation.Abbreviations ER endoplasmic reticulum - IAA -indolylacetic acid - 2,4-D 2,4-dichlorophenoxy acetic acid     

Studies of cotyledon protoplast cultures from Brassica napus,B. campestris and B. oleracea. I: Cell wall regeneration and cell division

Protoplasts isolated from cotyledons of a number of cultivars of Brassica napus, B. campestris and B. oleracea were cultured in different media to study the characteristics of cell wall regeneration and cell division at early stages of culture. Time course analysis using Calcolfluor White staining indicated that cell wall regeneration began in some protoplasts 2–4 h following isolation in all cultivars. 30–70% of cultured cotyledon protoplasts exhibited cell wall regeneration at 24 h and about 60–90% at 72 h after the initiation of culture. Results also indicated that a low percentage (0.4–5.4%) of cultured cotyledon protoplasts entered their first cell division one day after initial culture in all twelve cultivars. The percentage of dividing cells increased linearly up to 40% from 1 to 7 day, indicating that cotyledon protoplasts of Brassica had a high capacity for cell division. Factors that influence the level of cell wall regeneration and cell division during cotyledon protoplast culture have been investigated in this study. Cotyledons from seedlings germinated in a dark/dim light regime provided a satisfactory tissue source for protoplast isolation and culture for all Brassica cultivars used. The percentages of protoplasts exhibiting cell wall regeneration and division were significantly influenced by cultivar and species examined, with protoplasts from all five cultivars of B. campestris showing much lower rates of cell wall regeneration than those of B. napus and B. oleracea over 24–120 h, and with the levels of cell division in B. napus cultivars being much higher than those in B. campestris and B. oleracea over 1–9 days. The capacity of cell wall regeneration and cell division in cotyledon protoplast culture of the Brassica species appears under strong genetic control. Cell wall regeneration in protoplast culture was not affected by the culture medium used. In contrast, the composition of the culture medium played an important role in determining the level of cell division, and the interaction between medium type and cultivars was very significant.Abbreviations BA benzylaminopurine - CPW Composition of Protoplast Washing-solution - CW Calcolfluor White - EDTA ethylenediamine-tetraacetic acid - KT Kinetin - Md MS modified Murashige and Skoog medium - 2,4-d 2,4-dichlorophenoxyacetic acid - NAA -naphthaleneacetic acid - IAA indole-3-acetic acid - PAR photosynthetically active radiation - SDS sodium dodecyl sulfate     

Lignin synthesis and its related enzymes as markers of tracheary-element differentiation in single cells isolated from the mesophyll of Zinnia elegans

Mesophyll cells isolated from Zinnia elegans L. cv. Canary Bird were cultured for 96 h in a liquid medium containing 0.1 mg l^-1 -naphthaleneacetic acid and 1 mg l^-1 benzyladenine in which both differentiation of tracheary elements (TE) and cell division were induced, or in a medium containing 0.1 mg l^-1 -naphthaleneacetic acid and 0.001 mg l^-1 benzyladenine, in which cell division was induced but TE differentiation was not. Lignification was found to occur only in the former medium, fairly synchronously after 76 h of culture, 5 h later than the onset of visible secondary wall thickening. Changes in the soluble phenolics were not correlated with TE differentiation. Of three important enzymes which have been reported to play a role in TE differentiation, the activity of phenylalanine ammonia-lyase (EC 4.3.1.5) in the TE-inductive culture was higher than that in the control culture between 72 and 96 h of culture, when TE differentiation progressed and lignin was synthesized actively. O-Methyltransferase (EC 2.1.1.6) activity was higher in the control culture than in the TE-inductive culture, indicating that this enzyme was not a marker enzyme of TE differentiation. The activities of peroxidases (EC 1.11.1.7), one extractable and the other nonextractable, with CaCl₂ from the cell walls, reached peaks at 72 h (just before lignification) and 84 h of culture (active lignin synthesis), respectively, in the TE-inductive culture only, whereas the activity of soluble peroxidase showed a similar pattern of increase in the TE-inductive to the control culture. These results indicate that phenylalanine ammonia-lyase and peroxidase bound to the cell walls can be marker proteins for the differentiation of TE.Abbreviations OMT O-methyltransferase - PO peroxidase - PAL phenylalanine ammonia-lyase - TE tracheary element(s)     

Cell wall-lytic activity in Chlorella fusca

The soluble fraction of homogenates of synchronous Chlorella fusca was tested for carbohydrate-lyzing activities. With isolated cell walls and -1,4-mannan or carboxymethyl cellulose as substrates, a sharp increase in activity occurred shortly before release of the daughter cells followed by a decline during release. The lytic activities were partially purified by ammonium sulphate precipitation and analyzed by gel filtration on a calibrated column. Apparent molecular weights were 27,000 for cell wall autolysin(s) and -1,4-mannanase, 36,000 for carboxymethyl cellulase and 70,000 for another -1,4-mannanase. Incubation of isolated cell walls with an enzyme preparation purified by ammonium sulphate precipitation resulted in release of up to 70% of the cell wall carbohydrate as monosaccharide, predominantly mannose and glucose. The carbohydrate released in vivo into the culture medium shortly before and during liberation of the daughter cells consisted largely of polymeric material with rhamnose, fucose and mannose as main constitutents. Upon poisoning the cells with NaN₃ or carbonyl cyanide p-trifluoromethoxy-phenylhydrazone, however, a monosaccharide fraction consisting of mannose and glucose was predominant in the medium. It is suggested that the major products of cell wall lysis in vivo are monosaccharides which are rapidly taken up and metabolized by the developing daughter cells in an energy-dependent manner.     

Induction of berberine biosynthesis by cytokinins in Thalictrum minus cell suspension cultures

Production of berberine could be induced by adding 6-benzylaminopurine (BAP) to Thalictrum minus cells, cultured in suspension in a medium containing 2,4-dichlorophenoxyacetic acid (2,4-D), early in the growth cycle. In the presence of BAP, the precursor, L-tyrosine, was rapidly converted into berberine which was then released into the medium, whereas substantial amounts of the intermediates, tyramine and dopamine, accumulated in non-berberine-producing cells grown in the same 2,4-D-containing medium without BAP. These results suggest that BAP activated enzymatic reactions subsequent to the formation of the amines in the biosynthesis of berberine.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - BAP 6-benzylaminopurine - NAA 1-naphthaleneacetic acid - IAP 6-isopentenylaminopurine - LS medium Linsmaier-Skoog medium - Growth medium LS medium containing 10^-6 M 2,4-D     

Gibberellic-acid-stimulated Ca2+ accumulation in endoplasmic reticulum of barley aleurone: Ca2+ transport and steady-state levels

The steady-state levels of Ca²⁺ within the endoplasmic reticulum (ER) and the transport of ⁴⁵Ca²⁺ into isolated ER of barley (Hordeum vulgare L. cv. Himalaya) aleurone layers were studied. The Ca²⁺-sensitive dye indo-1. Endoplasmic reticulum was isolated and purified from indo-1-loaded protoplasts, and the Ca²⁺ level in the ER was measured using the Ca²⁺-sensitive dye indo-1. Endoplasmic reticulum was isolated and purified from indo-1-loaded protoplasts, and the Ca²⁺ level in the lumen of the ER was determined by the fluorescence-ratio method to be at least 3 M. Transport of ⁴⁵Ca²⁺ into the ER was studied in microsomal fractions isolated from aleurone layers incubated in the presence and absence of gibberellic acid (GA₃) and Ca²⁺. Isopycinic sucrose density gradient centrifugation of microsomal fractions isolated from aleurone layers or protoplasts separates ER from tonoplast and plasma membranes but not from the Golgi apparatus. Transport of ⁴⁵Ca²⁺ occurs primarily in the microsomal fraction enriched in ER and Golgi. Using monensin and heat-shock treatments to discriminate between uptake into the ER and Golgi, we established that ⁴⁵Ca²⁺ transport was into the ER. The sensitivity of ⁴⁵Ca²⁺ transport to inhibitors and the K_m of ⁴⁵Ca²⁺ uptake for ATP and Ca²⁺ transport in the microsomal fraction of barley aleurone cells. The rate of ⁴⁵Ca²⁺ transport is stimulated several-fold by treatment with GA₃. This effect of GA₃ is mediated principally by an effect on the activity of the Ca²⁺ transporter rather than on the amount of ER.Abbreviations CCR cytochrome-c reductase - DCCD dicyclohexylcarbodiimide - EGTA ethylene glycol bis(-aminoethyl ether)-N,N,N,N-tetraacetic acid - ER endoplasmic reticulum - FCCP carbonylcyanide p-trifluoromethoxyphenyl hydrazone - GA₃ gibberellic acid - IDPase inosine diphosphatase - Mon monensin     

Ultrastructure of the salt glands of the mangrove,Avicennia marina (Forssk.) Vierh., as indicated by the use of selective membrane staining

Each salt-excreting gland of the mangrove Avicennia marina (Forsskål) Vierh. consists of two to four collecting cells, one stalk cell, and eight to twelve excretory cells. Differential membrane staining by zinc iodide-osmium tetroxide (as a post-fixative) or phosphotungstic acid (as a section-stain) was used to characterise the ultrastructure of the glands. A large amount of tubular endoplasmic reticulum was found in the stalk and excretory cells of the gland, but not in the collecting cells. The ultrastructural arrangement of the endoplasmic reticulum indicates that salt is loaded from the apoplasm into the endoplasmic reticulum of the symplasm at the base of the stalk cell, traverses both cell types in the endoplasmic reticulum, and is excreted at the outer edge of the gland by an eccrine-type mechanism. Increasing development of the tubular endoplasmic reticulum accompanied differentiation of the gland cells.Abbreviations ER endoplasmic reticulum - PTA phosphotungstic acid - ZIO zinc iodide-osmium tetroxide     

Regeneration from Laminaria japonica Areschoug (Laminariales, Phaeophyceae) protoplasts isolated with bacterial alginase

Summary The conditions for effective isolation of viable protoplasts from Laminaria japonica with an alginase produced by marine bacterium Alteromonas sp. and a commercially available cellulase were investigated. The highest yields of viable protoplasts (7.910.4x10⁶ cells g^–1 FW) were obtained with a hypertonic solution containing 50 % seawater, 25 mM MgCl₂, 5 mM HEPES buffer system, and 0.5 M mannitol. Protoplasts were not obtained from thalli of L. japonica when an abalone alginase (abalone acetone powder; AAP: Sigma) was used instead of the bacterial alginase. The isolated protoplasts were cultured in an PESI medium at 5 °C. Complete cell wall formation was observed within 7 days, and dividing cells were first observed in a 9-day-old culture. Some protoplasts regenerated into sheet-shaped thalli and rhizoid structures were also observed on some thalli after 30 to 40 days in culture. This is the first report of protoplast regeneration into plantlets of L. japonica Areschoug (Laminariales, Phaeophyceae).Abbreviations FW Flesh weight - AAP Abalone acetone powder - HEPES N-2-hydroxy-ethylpiperazine-N-2-ethanesulfonic acid - Tris Tris(hyrdoxymethyl)aminomethane - PESI Provasoli's enriched seawater with iodine     

Incorporation of Mevalonic Acid into a Lipid Intermediate in Cell Wall Synthesis and Nucleic Acid Fraction by Lactobacillus heterohiochii

It was, using the particulate enzyme from Micrococcus lysodeikticus, revealed that most of 2-¹⁴C-mevalonic acid incorporated into the cell of Lactobacillus heterohiochii H-1 was incorporated into the lipid intermediate of cell wall biosynthesis. About 10% of the radioactivities incorporated into the cells was, however, found in nucleic acid fraction which was extracted from lysozyme treated cells with phenol. Most of the radioactivities in the nucleic acid fraction was eluted at the beginning of the elution pattern from Sephadex G-200 or MAK-column. The material is different from tRNA and rRNA.     

Molecular cloning and characterization of isomultiflorenol synthase, a new triterpene synthase from Luffa cylindrica, involved in biosynthesis of bryonolic acid.

An oxidosqualene cyclase cDNA, LcIMS1, was isolated from cultured cells of Luffa cylindrica Roem. by heterologous hybridization with cDNA of Glycyrrhiza glabra beta-amyrin synthase. Expression of LcIMS1 in yeast lacking endogenous oxidosqualene cyclase activity resulted in the accumulation of isomultiflorenol, a triterpene. This is consistent with LcIMS1 encoding isomultiflorenol synthase, an oxidosqualene cyclase involved in bryonolic acid biosynthesis in cultured Luffa cells. The deduced amino-acid sequence of LcIMS1 shows relatively low identity with other triterpene synthases, suggesting that isomultiflorenol synthase should be classified into a new group of triterpene synthases. The levels of isomultiflorenol synthase and cycloartenol synthase mRNAs, which were measured with gene-specific probes, correlated with the accumulation of bryonolic acid and phytosterols over a growth cycle of the Luffa cell cultures. Isomultiflorenol synthase mRNA was low during the early stages of cell growth and accumulated to relatively high levels in the late stages. Induction of this mRNA preceded accumulation of bryonolic acid. In contrast, cycloartenol synthase mRNA accumulated in the early stages of the culture cycle, whereas phytosterols accumulated at the same relative rate throughout the whole growth cycle. These results suggest independent regulation of these two genes and of the accumulation of bryonolic acid and phytosterols.     

A structural glycoprotein,containing hydroxyproline,isolated from the cell wall of Chlamydomonas reinhardii

Summary A soluble extract from purified cell walls of C. reinhardii has been separated by gel filtration into three fractions which together account for 94% of the cell wall. The major fraction (accounting for 70% of the extract) is a glycoprotein, with a molecular wt. in sodium perchlorate of 298,000, which can be split into 4 electrophoretically distinct species. It contains 35% protein with high levels of hydroxyproline, arabinose and galactose, and is capable of self assembly into crystalline structures identical to those found within the cell wall. The second fraction (25% of the extract) is a similar glycoprotein, but contains 24% protein, a higher proportion of mannose, and is incapable of self assembly. The third fraction (3–6% of the extract) is shown to be an adsorbed impurity from the growth medium used.Abbreviations CB or CBB coomassie brilliant blue - PAS periodic acid Schiff's reagent - PPO 2,5 diphenyloxazole - PO-POP 1,4-di[2-(5phenyloxazolyl]benzene - SDS sodium dodecyl sulphate This is the fourth paper in a series entitled Structure, composition and morphogenesis of the cell wall of Chlamydomonas reinhardii. The last paper in this series was Hills et al. (1975)     

On the secretion of α-amylase by barley aleurone layers after incubation in gibberellic acid

Summary Gel filtration and centrifugation studies were used to study the distribution of -amylase activity in homogenates of barley (Hordeum vulgare L.) aleurone layers. The results obtained were consistent with the hypothesis that -amylase is secreted via membrane-bound vesicles. The -amylase activity in an homogenate of barley aleurone layers was derived not only from the enzyme retained in the aleurone cells but also from enzyme previously secreted from the cells but apparently retained by the cell walls. The amount of -amylase retained by the cell wall was influenced by factors such as the buffer in which the layers were incubated or the presence of Actinomycin D in the incubation medium.Abbreviations GA₃ gibberellic acid - RER rough endoplasmic reticulum - Act. D Actinomycin D     

The pig yolk sac II

Summary Since previous morphological studies have revealed abundant rough endoplasmic reticulum in the yolk sac endoderm, pig yolk sac explants from 30 day old embryos were incubated for 3–12 h with [³H]-l-leucine in order to study their protein biosynthesis. They were fractionated into a 12,000×g-pellet, 105,000×g-pellet, and supernatant. Newly synthesized proteins in these tissue fractions, and proteins discharged into the culture medium, were analysed with the aid of scintillation technique and identified by column chromatography, SDS-polyacrylamide gel electrophoresis with urea, isoelectrofocusing, and 2D-electrophoresis. Most of the radioactivity incorporated into the tissue fractions was regained from the coarse pellet and was located in the molecular weight region between 70,000 and 45,000 daltons, indicating that most of the newly synthesized proteins are membrane bound and include albumin. Albumin, an acid protein of a MW around 80,000 daltons, and many neutral and basic peptides were present in the culture medium. The yolk sac endodermal cells of the pig synthesize less proteins than those of the cat, although the pig cells display much larger amounts of endoplasmic reticulum.     

Somatic embryogenesis and plant regeneration from isolated protoplasts of Lavatera thuringiaca

Embryogenic cell suspensions of Lavatera thuringiaca L. were established from leaf petiole and shoot regeneration was achieved when cells were plated on medium without growth regulators. We tested three methods for protoplast culture, isolated from a one-year old embryogenic cell suspension, to determine the best conditions for L. thuringiaca protoplast culture and shoot regeneration. The highest protoplast plating efficiency was obtained with the agaroseembedded method, reaching 30%, while the nursing culture method gave 5% when the protoplasts were plated over Whatman paper No. 2. However, the same nursing culture failed to produce protoplast-derived microcalluses when the protoplasts were plated on a nitrocellulose filter. The liquid thin layer method gave the lowest plating efficiency with only 0.5%. Shoot regeneration from protoplast-derived microcalluses was achieved in two steps; first, globular embryo development was favored in medium low in auxin (2,4-d and BA at 0.01 and 0.05 mg 1^-1, respectively), second, the globular embryos further differentiate into shoots in medium without growth regulators or in medium containing GA₃ (0.5 to 1.0 mg 1^-1).Abbreviations 2,4-d 2,4-dichlorophenoxyacetic acid - BA benzyladenine - GA₃ gibberellic acid - NAA -naphthaleneacetic acid - IBA indole-3-butyric acid