Ontogenic Changes in Epicuticular Wax and Chloroplast Integrity of a Cotton (Gossypium hirsutum L.) Leaf

The progressive decline in cotton leaf photosynthesis with season could be accounted for by gaining an insight into ontogenic changes in chloroplast integrity and epicuticular wax ultrastructure. Therefore, the sequence of ultrastructural changes in chloroplast and epicuticular wax morphology were probed in 10-, 20-, 40-, and 60-d-old cotton (Gossypium hirsutum L.) leaves using electron microscopy. Scanning electron microscopy illustrated that the epicuticular wax on the periclinal walls of the convex epidermal cells occurred as striations and persisted as such during the course of leaf aging. The degree of wax spread, however, increased as the leaf progressed towards senescence. Transmission electron microscopy revealed that a 20-d-old photosynthetically active leaf possessed healthy chloroplasts (6.8 m long and an area of 9.7 m²) with absolute membrane integrity depicted by large appressed grana stacks of thylakoids interconnected by non-appressed stroma lamellae. The thylakoid membrane network was oriented parallel to the long axis of the chloroplast and a few small plastoglobuli (1.85 m²) scattered in the stroma. Conversely, membrane integrity was lost with leaf age after 20 d as evidenced by disruption of the grana and stroma lamellae. Concurrent with the membrane damage, extensive occlusion of chloroplast by several large spherical plastoglobuli (5.68 m²) occurred, the rate of occlusion increased with leafage distending the chloroplast as evidenced by proliferation of its cross-sectional area (12.8 m²). Of particular interest was the finding that the plastoglobuli ensued through the chloroplast envelope into the cytoplasm. The progressive loss of chloroplast membrane integrity coupled with increased leaf waxiness may have limited photosynthetic activities of cotton leaves during senescence.     

Photosynthesis of isolated chloroplasts and protoplasts under osmotic stress

1. Isolated intact spinach chloroplasts respond to changes of the sorbitol concentration of the suspending medium as near-perfect osmometers within a large range of osmotic potentials. Under isotonic conditions (=9–10 bar), their average osmotic volume is 24 m³ and the total volume 36 m³. The osmotic volume can be increased to 63 m³ by lowering the sorbitol concentration until a critical osmotic potential of =4 bar is reached. Below that value chloroplasts rupture. Between 10 bar and 4 bar, volume changes are reversible. 2. Increasing the chloroplast volume above 24 m³ causes inhibition of photosynthesis, with 50% inhibition occurring at an osmotic potential of =5–6 bar. This corresponds to an osmotic volume of 45–55 m³. Depending on the duration of hypotonic treatment, inhibition of photosynthesis is more or less reversible. 3. Between 4 and 10 bar, the chloroplast envelope exhibits a very low permeability for ferricyanide, many metabolites, and soluble stroma proteins. 4. Electron transport is not inhibited by swelling of chloroplasts. Also, the ATP/ADP-ratio remains unchanged. 5. The solute concentration in the chloroplasts appears to be optimal for photosynthesis at 10 bar. Increasing the chloroplast volume causes inhibition of photosynthesis by dilution effects.     

Photosynthesis and apparent affinity for dissolved inorganic carbon by cells and chloroplasts of Chlamydomonas reinhardtii grown at high and low CO2 concentrations

Chloroplasts with high rates of photosynthetic O₂ evolution (up to 120 mol O₂· (mg Chl)^-1·h^-1 compared with 130 mol O₂· (mg Chl)^-1·h^-1 of whole cells) were isolated from Chlamydomonas reinhardtii cells grown in high and low CO₂ concentrations using autolysine-digitonin treatment. At 25° C and pH=7.8, no O₂ uptake could be observed in the dark by high- and low-CO₂ adapted chloroplasts. Light saturation of photosynthetic net oxygen evolution was reached at 800 mol photons·m^-2·s^-1 for high- and low-CO₂ adapted chloroplasts, a value which was almost identical to that observed for whole cells. Dissolved inorganic carbon (DIC) saturation of photosynthesis was reached between 200–300 M for low-CO₂ adapted chloroplasts, whereas high-CO₂ adapted chloroplasts were not saturated even at 700 M DIC. The concentrations of DIC required to reach half-saturated rates of net O₂ evolution (K_m(DIC)) was 31.1 and 156 M DIC for low- and high-CO₂ adapted chloroplasts, respectively. These results demonstrate that the CO₂ concentration provided during growth influenced the photosynthetic characteristics at the whole cell as well as at the chloroplast level.Abbreviations Chl chlorophyll - DIC dissolved inorganic carbon - K_m(DIC) coneentration of dissolved inorganic carbon required for the rate of half maximal net O₂ evolution - PFR photon fluence rate - SPGM silicasol-PVP-gradient medium     

Plant regeneration via somatic embryogenesis in Styrian pumpkin: cytological and biochemical investigations

Somatic embryo formation was induced from cotyledon explants of Styrian pumpkin (Cucurbita pepo L. subsp. pepo var. styriaca Greb.) by using a solid MS medium supplemented with 16.11M NAA and 4.44M BA or 26.85M NAA and 13.32M BA. The callus proliferation was more efficient on medium supplemented with 26.85M NAA and 13.32M BA. In contrast, the embryogenic response was higher on medium with lower concentrations of growth regulators (16.11M NAA and 4.44M BA). The time needed for embryo induction did not depend on medium composition. Embryos in globular stage were transferred to three different maturation media, containing 2.89M GA₃ in combination with 0.54M NAA, 11.42M IAA and growth regulator-free medium. The germination rate was the highest when embryos were cultured on medium with 11.42M IAA. Plantlets grown on this medium achieved maturity suitable for transplantation into soil within 9 to 10weeks. The regenerated plants were successfully transferred into field and developed fertile flowers and set fruits. Biochemical analysis showed significant lower total glutathione levels among in vitro grown plantlets compared to seedlings grown in soil. When the plantlets were transferred into soil, they reached a normal size within a month and the glutathione concentration was comparable to seed-derived plants at the same developmental stage. Transmission electron microscopy was used to investigate possible differences in the ultrastructure of cells from callus cultures, and leaf cells of regenerated and seed-derived plants. Differences in the ultrastructure were found within chloroplasts which contained only single thylakoids, large starch grains and small plastoglobuli in callus cells in comparison to leaf cells, which possessed a well developed thylakoid system, small starch grains and large plastoglobuli.     

Photosynthetic inhibition after long-term exposure to elevated levels of atmospheric carbon dioxide

The effect of long-term exposure to elevated levels of CO₂ on biomass partitioning, net photosynthesis and starch metabolism was examined in cotton. Plants were grown under controlled conditions at 350, 675 and 1000 l l^-1 CO₂. Plants grown at 675 and 1000 l l^-1 had 72% and 115% more dry weight respectively than plants grown at 350 l l^-1. Increases in weight were partially due to corresponding increases in leaf starch. CO₂ enrichment also caused a decrease in chlorophyll concentration and a change in the chlorophyll a/b ratio. High CO₂ grown plants had lower photosynthetic capacity than 350 l l^-1 grown plants when measured at each CO₂ concentration. Reduced photosynthetic rates were correlated with high internal (non-stomatal) resistances and higher starch levels. It is suggested that carbohydrate accumulation causes a decline in photosynthesis by feedback inhibition and/or physical damage at the chloroplast level.Abbreviations Ci internal CO₂ concentration - Chl chlorophyll - DMSO dimethylsulfoxide - HSD honestly significant difference (procedure) - MCW methanolchloroform-water - Pi inorganic phosphate - S.E.M. standard error of mean     

Measurements of manganese in thylakoids of Sinapis alba grown under high-light and low-light conditions

The manganese content of thylakoids and tissues was measured in leaves grown under high- and low-light conditions. Especially when grown in a nutrient medium enriched in manganese (20 M), the thylakoids contained large amounts of manganese, which could be removed by EDTA washing without impairment of the Hill reaction. The unremovable content of manganese was almost the same in thylakoids from plants grown in nutrient media of normal (2 M) and reduced (0.2 M) manganese content. Up to this limit of manganese content, Hill activity did not seem to be impaired. 1.2 atoms Mn per 100 molecules chlorophyll were found in low-light thylakoids and 1.6 atoms Mn in high-light thylakoids. This is similar to the behaviour of other electron transport components, the number of which is also decreased under low-light conditions. However, the decrease in the manganese content is not as striking as the decrease in, for example, the cytochrome f and ferredoxin content. This may be attributed to an invariable pool of manganese which is not involved in the oxygen evolving system. Alternatively, if all of our measured manganese is involved in electron transport to PS II, this could indicate that in low-light chloroplasts the ratio of PS II/PS I components may be somewhat increased.

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Zusammenfassung Der Mangangehalt von Thylakoiden und Gewebe aus Starklicht- und Schwachlichtblättern wurde untersucht. Besonders bei Pflanzen, welche unter erhöhtem Manganangebot (20 M) angezogen wurden, besaßen die Thylakoide sehr viel Mangan, welches durch Waschen mit EDTA entfernt werden konnte, ohne die Hill-Aktivität zu beeinträchtigen. In Thylakoiden aus Pflanzen, welche unter normalem (2 M) und reduziertem (0,2 M) Manganangebot gewachsen waren, unterschied sich der nicht entfernbare Mangangehalt nicht sehr. Dies scheint die untere Grenze des Mangangehalts zu sein, bis zu welchem die Hill-Aktivität noch nicht beeinträchtigt wird. Schwachlicht-Thylakoide besitzen 1,2 Atome Mn pro 100 Chlorophyllmoleküle, während Starklicht-Thylakoide 1,6 Atome Mn pro 100 Chlorophyllmoleküle enthalten. Dies gleicht dem Verhalten anderer Komponenten des Elektronentransports, welche ebenfalls im Starklicht vermehrt vorkommen. Die Unterschiede im Mangangehalt sind jedoch geringer als die Unterschiede im Gehalt von z.B. Cytochrom f und Ferredoxin. Dies könnte auf einen konstanten Anteil von Mangan zurückzuführen sein, welcher nicht am wasserspaltenden System beteiligt ist. Wenn jedoch das gesamte gemessene Mangan am Elektronentransport zum PS II beteiligt ist, könnte dies ein Hinweis sein, daß in Schwachlicht-Chloroplasten sich das Verhältnis der PS II-/PS I-Komponenten etwas vergrößert.

     

Changes of photosynthetic parameters in cucumber leaves under Cu, Cd, and Pb stress

The effects of Cu, Cd, and Pb toxicity on photosynthesis in cucumber leaves (Cucumis sativus L.) were studied by the measurements of gas exchange characteristics, chlorophyll (Chl) fluorescence parameters, and Chl content. Concentrations of metals in sequence of 20 M Cu, 20 and 50 M Cd, and 1 000 M Pb decreased the plant dry mass to 50–60 % after 10 d of treatment whereas 50 M of Cu decreased it to 30 %. The content of Cd in leaves of plants treated with 50 M Cd was three times higher than the contents of Cu and Pb after plant treatment with 50 M Cu or 1 000 M Pb. Hence Cd was transported to leaves much better than Cu and Pb. Nevertheless, the net photosynthetic rate and stomatal conductance in leaves treated with 50 M Cu or Cd were similarly reduced. Thus, Cu was more toxic than Cd and Pb for photosynthesis in cucumber leaves. None of the investigated metals decreased internal CO₂ concentrations. Also the effect of metals on potential efficiency of photosystem 2, PS2 (F_v/F_m) was negligible. The metal dependent reduction of PS2 quantum efficiency (_PS2) after plant adaptation in actinic irradiation was more noticeable. This could imply that reduced demand for ATP and NADPH in a dark phase of photosynthesis caused a down-regulation of PS2 photochemistry. Furthermore, in leaves of metal-treated plants the decrease in water percentage as well as lower contents of Chl and Fe were observed. Thus photosynthesis is not the main limiting factor for cucumber growth under Cu, Cd, or Pb stress.This revised version was published online in March 2005 with corrections to the page numbers.     

Fine structure of abscission zones

Summary Electron micrographs of the zone of separation in flower pedicels of Lycopersicon esculentum and Nicotiana tabacum are presented with particular reference to the indentation of epidermal tissue in the abscission zone, subcellular organelles, and the cell wall. The indentation or groove which delineates the abscission zone extends some distance into the pedicel with branchings off the main groove. These branches are approximately 20 m in width while the main groove averages approximately 200 m in width. Invaginations of the plasmalemma are observed with considerable frequency. within these invaginations one observes a material of about the same density as the cell wall except that it is more fibrillar. Plasmodesmata are also observed, with considerable branching into middle lamellae of cells comprising the abscission zones. Microbodies with crystalloid cores appear with considerable frequency in cells of the abscission zone. The crystalloids appear to be cubical in shape and are composed of parallel sheets of osmiophilic material. The sheets average about 6 m in thickness and are spaced at 4 m intervals. The microbodies with crystalloid cores are observed to be characteristically of two size groupings. In tobacco the microbodies average 900 m and 1,500 m in profile. In tomato they average 300 m and 500 m. Chloroplasts contain a granular component which is membrane-enclosed. The component is large in comparison with the plastid in which it occurs, averaging 1.2–1.4 in diameter in chloroplasts ranging from 1.6 to 2.2 in diameter. The inner membrane of the chloroplast is highly invaginated, and DNA- and phytoferritin-like materials are observed within the plastids. Microtubules with an average diameter of 20 m are observed adjacent and parallel to the plasmalemma, primarily in the corners of the cells. Micrographs of other normally occurring cytoplasmic inclusions are also presented.     

Arabidopsis ammonium transporters,AtAMT1;1 and AtAMT1;2, have different biochemical properties and functional roles

We have compared the biochemical properties of two different Arabidopsis ammonium transporters, AtAMT1;1 and AtAMT1;2, expressed in yeast, with the biophysical properties of ammonium transport in planta. Expression of the AtAMT1;1 gene in Arabidopsis roots increased approximately four-fold in response to nitrogen deprivation. This coincided with a similar increase in high-affinity ammonium uptake by these plants. The biophysical characteristics of this high-affinity system (K_m for ammonium and methylammonium of 8 M and 31 M, respectively) matched those of AtAMT1;1 expressed in yeast (K_m for methylammonium of 32 M and K_i for ammonium of 1–10 M). The same transport system was present, although less active, in nitrate-fed roots. Ammonium-fed plants exhibited the lowest rates of ammonium uptake and appeared to deploy a different transporter (K_m for ammonium of 46 M). Expression of AtAMT1;2 in roots was insensitive to changes in nitrogen nutrition. In contrast to AtAMT1;1, AtAMT1;2 expressed in yeast exhibited biphasic kinetics for methylammonium uptake: in addition to a high-affinity phase with a K_m of 36 M, a low-affinity phase with a K_m for methylammonium of 3.0 mM was measured. Despite the presence of a putative chloroplast transit peptide in AtAMT1;2, the protein was not imported into chloroplasts in vitro. The electrophysiological data for roots, together with the biochemical properties of AtAMT1;1 and Northern blot analysis indicate a pre-eminent role for AtAMT1;1 in ammonium uptake across the plasma membrane of nitrate-fed and nitrogen-deprived root cells.     

Immunolocalization of abscisic acid by monoclonal antibodies in Lavandula stoechas L. leaves

A monoclonal antibody was used to localize abscisic acid in Lavandula stoechas L. plants treated with 1 M abscisic acid. ABA localization was performed using EDC as the only fixative, which preserves antigenicity and improves the specificity of monoclonal antibodies. A relationship was observed between the effect of abscisic acid on chloroplast ultrastructure and ABA accumulation. Gold particles accumulated on swollen chloroplasts. Our findings provide cytochemical evidence that the chloroplast is a trapping compartment, and yield valuable information on the relation between chloroplast ultrastructure and ABA accumulation.Abbreviations ABA abscisic acid - BSA bovine serum albumin - EDC 1-(3-dimethyl-aminopropyl)-3 ethyl carbodiimide - IgG immunoglobulin G - mAB monoclonal antibodies - pAB polyclonal antibodies     

Effect of CO2 enrichment and nitrogen availability on resource acquisition and resource allocation in a grass,Bromus mollis

Summary The effects of CO₂ enrichment on the growth, biomass partitioning, photosynthetic rates, and leaf nitrogen concentration of a grass, Bromus mollis (C₃), were investigated at a favorable and a low level of nitrogen availability. Despite increases in root: shoot ratios, leaf nitrogen concentrations were decreased under CO₂ enrichment at both nitrogen levels. For the low-nitrogen treatment, this resulted in lower photosynthetic rates measured at 650 l/l for the CO₂-enriched plants, compared to photosynthetic rates measured at 350 l/l for the non-enriched plants. At higher nitrogen availability, photosynthetic rates of plants grown and measured at 650 l/l were greater than photosynthetic rates of the non-enriched plants measured at 350 l/l. Water use efficiency, however, was increased in enriched plants at both nitrogen levels. CO₂ enrichment stimulated vegetative growth at both high and low nitrogen during most of the vegetative growth phase but, at the end of the experiment, total biomass of the high and low CO₂ treatments did not differ for plants grown at low nitrogen availability. While not statistically significant, CO₂ tended to stimulate seed production at high nitrogen and to decrease it at low nitrogen.     

Direct Organogenesis from Mature Leaf and Petiole Explants of Eryngium Foetidum L.

Eryngium foetidum L. plants were regenerated from mature leaf and petiole explants through direct organogenesis without intervening callus phase. From leaf explants, adventitious multiple shoots raised on Murashige and Skoog (MS) medium supplemented with 4.43 M benzylaminopurine (BAP) and 0.57 M indole-3-acetic acid (IAA), whereas in petiole explants shoot regeneration occurred at 8.86 M BAP and 0.57 M IAAA. 80% of the leaf explants and 44% of petiole explants produced shoots after four weeks of culture. The regenerated plants were rooted on MS medium supplemented with 2.46 M indole-3-butyric acid and 2.88 M gibberellic acid. The plants were successfully established in the soil and showed 70.9% survival in the field.     

Axillary shoot induction and plant regeneration in Plantago ovata Forssk

Axillary shoot induction and plant regeneration were obtained in Plantago ovata. The optimum medium for inducing axillary shoots was Murashige & Skoog (MS) medium [5] supplemented with 4.6 M kinetin and 0.05 M NAA. Rooting of shoots was best on half-strength MS medium containing 5.0 M IBA and 0.05 M kinetin. The regenerated plants were similar to the control plants in karyotypic and phenotypic details.     

IAA and ZR content in leek (Allium porrum L.), as influenced by P nutrition and arbuscular mycorrhizae,in relation to plant development

Leek plants (Allium porrum L.), infected or not with the arbuscular mycorrhizal (AM) fungus, Glomus mosseae, were grown in a sand-hydroponic system, fed with a nutrient solution containing 3.2 or 96 M P and analyzed for root IAA and ZR content, to assess the role played by the fungus and P nutrition on host hormonal balance. IAA was analyzed by HPLC-fluorimetry, ZR by HPLC-UV coupled with a bioassay based on the expression of a phytohormone-regulated GUS reporter gene. Shoot and root weights and shoot FW-DW ratio enhancements, as well as root-to-shoot DW ratio decrement in mycorrhizal plants, were related to P nutrition. Shoot P concentration was increased by mycorrhizae at both P levels, but was comparable in AM plants grown at 3.2 M P and non mycorrhizal (NM) plants at 96 M P. Mycorrhizae and P increased IAA at substantially similar values, while P increased ZR much more than mycorrhizae did. These results are discussed in relation to root architecture modifications induced by the AM fungus.     

Regeneration of peach plants from callus derived from immature embryos

Summary Peach plants were repeatedly regenerated from immature embryos but not from callus derived from mature embryos. A white, nodular, highly regenerative callus was obtained when friable, primary callus from immature embryos was transferred from medium containing 4.5 M 2,4-dichlorophenoxyacetic acid and 0.44 M benzyladenine (BA) to media containing 0.27 M -naphthaleneacetic acid (NAA) and 2.2 M BA. This callus retained its morphogenetic potential for a minimum of three subcultures. Green nodular callus, that lacked regenerative capacity, was produced from primary callus derived from mature embryos. Maximum regeneration of shoots occurred when highly regenerative callus was transferred to a medium in which the NAA concentration was reduced five times and the BA concentration was increased two times. Regenerated shoots were rooted in the dark on a medium containing 28.5 M indoleacetic acid. Cytogenetic analysis of regenerated plants indicated that all plants were diploid, 2n = 2x = 16. Phenotypic evaluation of regenerated plants, grown under field conditions, is now in progress.     

Investigations of the polarity of the photo-induced electrical signal of chloroplast suspensions

This paper reports data and considerations relevant to the question of what determines the polarity of the voltages induced between electrodes in a suspension of chloroplasts when irradiated with a flash of light from a laser or flash-lamp. We found positive polarity (electrode nearest the light source positive) with excitation by ns pulses at 694, 539 and 530 nm wavelength. This and the earlier finding (Meszéna et al. (1988) Studia Biophysica 126:77–86), confirmed in this work, of negative polarity at 420 nm confirm, in part, the action spectrum reported by Gräber and Trissl (1981 FEBS Let 123:95–99) using 50 s flashes. Gräber and Trissl also showed that swelling the chloroplasts can reverse the polarity.Negative polarity is expected on the basis of a simple light-gradient in the sample together with what is known about photosynthetic charge movements. The cause of positive polarities has eluded explanation. Duration of flash was suspected. We tried a random series of short flashes averaging about 10 s apart and found that all simply duplicated the first flash. If there is any effect of light following the first flash it must occur in less than about 10 s.We suggest that the polarity is determined by a complicated interference pattern of the light in the chloroplast which can focus it onto different parts, front or back, depending upon the wavelength of the light and the structure of the chloroplast.Abbreviations DCMU 3-(3, 4-dichlorophenyl)-1, 1 dimethylurea - PSII photosystem II, the oxygen emitting system in green plant photosynthesis - EDTA ethylenediaminetetraacetic acid - Hepes N-2-Hydroxyethylpiperazine-N'-2-ethanesulfonic acid     

In vitro micropropagation of Arctostaphylos uva-ursi (L.) Sprengel.: Comparison between two methodologies

In vitro micropropagation of Arctostaphylos uva-ursi was performed to increase the number of ground cover species able to serve as substitute for members of the Rosaceae susceptible to fire blight. Explants (node segments) excised from plants growing in the greenhouse were established in vitro on a medium containing 10 M -naphthaleneacetic acid (NAA) and activated charcoal (2 g I^-1). Using in vitro grown shoots, two propagation procedures were used:- Culture of nodal fragments with 50 M NAA resulted in the growth of 6 to 7 nodes every 4 weeks, yielding 1 700 almost rootable shoots after 4 subcultures;- Development of axillary shoots obtained with media containing 25 M benzyladenine (BA) and 20 M indoleacetic acid (IAA) yielded almost 500 rootable shoots after 4 subcultures. The rate of propagation decreased after the 3^rd subculture.Percentage of in vitro rooted shoots reached 98% with diluted micronutrients and 10 M NAA but 31% of the plants died during acclimatization.Abbreviations BA benzyladenine - BM basal medium - HID high intensity discharge - IAA indoleacetic acid - IBA indolebutyric acid - NAA -naphthaleneacetic acid - PAR photosynthetic active radiation - 2iP 2-isopentenyladenine     

Micropropagation and ecorestoration of Vanda spathulata,an exquisite orchid

Node explants collected from flowering plants of Vanda spathulata, an endemic and exquisite orchid of Peninsular India and Sri Lanka, were cultured in Mitra medium with combinations of 4.4–88.8 m 6-benzyl adenine (BA) and 0.0–114.2 m indole-3-acetic acid (IAA). Combinations of 44.4 m BA with 17.1 or 28.5 m IAA and 66.6 mM BA with 28.5 or 40.0 m IAA induced maximum formation of 12.6 and 12.1 shoots / node, respectively, in a 6-month period. Subcultured nodal explants produced maximum of 6.1 shoots at combinations of 22.2–44.4 m 21 BA and 5.7–28.5 m IAA. Rooting of shoots occurred in medium containing 75 g l^–1 banana pulp and 5.7 m IAA within 3–9 weeks. Plantlets of 2–5 cm length possessing two to five roots established easily in community pots at 80–90% rates without hardening. Community potted plants introduced into forest segments at Ponmudi and Palode in Southern Western Ghats of India established at a rate of 50–70%.     

Influence of plant growth regulators,basal media and carbohydrate levels on the in vitro development of Pinus ponderosa (Dougl. ex Law.) cotyledon explants

Applications of in vitro screening techniques for Pinus ponderosa resistance to Peridermium harknessii could be beneficial in a tree breeding program. Plant growth regulators, basal media formula and carbohydrate levels were examined to determine the various effects each would have on excised cotyledon growth and development. Proliferating green callus was initiated from cotyledon explants on SH basal medium containing 4.4 M BA:5.4 M NAA and 1% sucrose. Subsequent subculturing onto LS medium supplemented with 44.0 M BA: 5.4 M NAA and 2% sucrose improved callus maintenance. The highest frequency of caulogenesis from cotyledon explants occurred on a modified GD medium containing 44.0 M BA: 0.054 M NAA and 4% glucose. The influence of nitrogen source, osmoticum and medium salt concentrations are discussed relative to callus initiation and caulogenesis.Abbreviations BA 6-benzyladenine - NAA -naphthaleneacetic acid - IAA indole-3-acetic acid - IBA indole-3-butyric acid - 2,4-D 2,4-dichlorophenoxyacetic acid - CD Campbell & Durzan - GD Gresshoff & Doy - LP Le Poivre - LS Linsmaier & Skoog - MC McCown - SH Schenk & Hildebrandt