Ethylene precursors and antagonists increase embryogenesis of Hordeum vulgare L. anther culture

The role of ethylene in microspore embryogenesis and regeneration was analyzed by studying the effects of the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) and the ethylene antagonists silver nitrate and silver thiosulphate on the androgenic response of in vitro cultured anthers of seven genotypes of barley. Incorporation of either ACC or silver salts in the culture medium lead to a significant increase in callus induction for five of the seven genotypes tested. The treatment that increased callus induction depended upon genotype. Only anthers cultured on 1 mg l^–1 silver thiosulphate gave rise to fertile plants in all seven genotypes tested.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indole acetic acid - PAA phenyl acetic acid - STS silver thiosulphate - ACC 1-aminocyclopropane-1-carboxylic acid     

Promotion ofin vitro shoot formation from excised roots of silktree (Albizzia julibrissin) by an oxime ether derivative and other ethylene inhibitors

Summary This report describes the regeneration response of excised seedling roots of silktree (Albizzia julibrissin) to added ethylene precursors/generators (1-amino-cyclopropane-1-carboxylic acid [ACC], 2-chloroethylphosphonic acid [CEPA]), biosynthesis inhibitors (aminoethoxyvinylglycine [AVG], an oxime ether derivative [OED={[(ispropylidene)-amino]oxy}-acetic acid-2-(methoxy)-2-oxoethyl ester], CoCl² [Co⁺⁺]), and an ethylene action inhibitor (AgNO₃ [Ag⁺]). When placed on B5 medium, about 50% of the control explants formed shoot buds within 15 days. Addition of ACC or CEPA (1–10 µM) to the culture medium decreased both the percentage of cultures forming shoots and the number of shoots formed per culture. In contrast, AVG and OED (1–10 µM) increased shoot formation to almost 100% and increased the number of shoots formed per culture. Likewise, both Co⁺⁺ and Ag⁺ (1–10 µM) increased shoot regeneration, but the number of shoots produced after 30 days was less than with AVG or OED. The inhibitors of ethylene biosynthesis were partially effective in counteracting the inhibitory effect of ACC on shoot formation. These results suggest that modulation of ethylene biosynthesis and/or action can strongly influence the formation of adventitious shoots from excised roots of silktree.Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid - AVG aminoethoxyvinylglycine - CEPA 2-chloroethylphosphonic acid - OED oxime ether derivative     

Organic nitrogen stimulates caulogenesis from hypocotyl callus of Phyllanthus fraternus

Factors involved in promoting caulogenesis from hypocotyl explants of Phyllanthus fraternus were studied. Hypocotyl explants were cultured on B₅ medium supplemented with 2,4-D or NAA in the presence and absence of BAP (at concentrations 0, 10^–7, 10^–6 and 10^–5M). Adventitious shoots differentiated from callus developed from the cut ends of 12.5% of the hypocotyl segments cultured on medium supplemented with 10^–6M BAP in combination with 10^–6M 2,4-D or 10^–6M NAA. Profuse rooting occurred from the hypocotyl explants on medium supplemented with 10^–6M BAP + 10^–6M NAA. Incorporation of casein hydrolysate in B₅ medium along with 10^–6M BAP + 10^–7M 2,4-D enhanced the frequency of cultures with adventitious shoots upto 68.0%. Glutamine, glutamic acid or proline could partially substitute for the effect of casein hydrolysate. Amongst the hypocotyls from 3–14 d old seedlings, the best caulogenesis was obtained with hypocotyls from 7 d old seedlings both in presence or absence of casein hydrolysate. Best rooting of shoots was achieved on half-strength B₅ medium supplemented with 10^–6M IBA. After hardening, plantlets were successfully transferred to the soil.Abbreviations BAP 6-benzylaminopurine - 2,4-D 2, 4-dichlorophenoxyacetic acid - IBA indole-3-butyric acid - NAA 1-naphthaleneacetic acid - CH casein hydrolysate - Arg L-arginine - Glu L-glutamic acid - Gln L-glutamine - Leu L-leucine - Lys L-lysine - Pro L-proline     

Effect of exogenous amino acids on the intracellular content of proline and other amino acids in Daucus carota cells

The effect of tryptophan on the biosynthesis of proline has been investigated. Cells of Daucus carota grown in B5 medium supplemented with 5×10^–4M tryptophan acquired the ability to grow in the presence of inhibitory concentrations of azetidine-2-carboxylic acid, an analog of proline. When trp was added to carrot cell cultures at sub-growth inhibiting concentrations, overproduction of intracellular free proline was observed. An increase was also observed for lys, his, ala, leu and phe. Likewise, the addition of asparagine, glutamic acid and phenylalanine to the medium stimulated the intracellular increase of free proline and other amino acids.Abbreviations A2CA azetidine-2-carboxylic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - 5MT 5-methyltryptophan - P5C pyrroline-5-carboxylic acid - f.wt. fresh weight - d.wt. dry weight     

Ethylene release from green spruce needles involves a combination of ACC-dependent and independent pathways

Aminoethoxyvinylglycine (AVG) and cobalt ions strongly inhibit the conversion of added methionine or aminocyclopropane-1-carboxylic acid (ACC) into ethylene by green-coloured, non-stressed Norway spruce (Picea abies L.) needles but only 30%–40% of basal ethylene formation is affected by such inhibitors. In addition, free radical-mediated ACC-independent ethylene formation (AIEF) of the type released by brown-coloured spruce needles also occurs in extracts from healthy green-coloured needles. Treatment with CdCl₂ (10 mM), Na₂S₂O₅ (5 mM) or FeSO₄ (10 mM) induces 3–7 fold increases in the rates of ethylene evolution from green-coloured needles. However, only Cd²⁺-induced ethylene formation is inhibited by AVG while ethylene induced by S₂O₅ ^2- or Fe²⁺ is insensitive to added AVG although increased levels of ACC have also been detected in these treatments. Nevertheless, ethylene-forming decomposition of the precursors of AIEF is accelerated by S₂O₅ ^- or Fe²⁺ which indicates that the ethylene released from green-coloured spruce needles is formed by a combination of both the ACC-dependent and AIEF pathways.Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid - AVG aminoethoxyvinylglycine - AIEF ACC-independent ethylene formation - EFE ethylene-forming enzyme - MACC N-malonyl(amino)cyclopropane-1-carboxylic acid - DTBN di-tert-butylnitroxide - MNP 2-methyl-2-nitrosopropane - SAM S-adenosylmethionine - TEMPO 2,2,6,6-tetramethyl-1-piperidine-N-oxyl     

Responsiveness of Amaranthus retroflexus seeds to ethephon, 1-aminocyclopropane 1-carboxylic acid and gibberellic acid in relation to temperature and dormancy

Dormant Amaranthus retroflexus seeds do not germinate in the dark at temperatures below 35°C. Fully dormant seeds germinate only at 35–40°C whereas non-dormant ones germinate within a wider range of temperatures (15 to 40°C). Germination of non-dormant seeds requires at least 10% oxygen, but the sensitivity of seeds to oxygen deprivation increases with increasing depth of dormancy. 10^–6 to 10^–4 M ethephon, 10^–3 M 1-aminocyclopropane 1-carboxylic acid (ACC) and 10^–3 M gibberellic acid (GA₃) break this dormancy. In the presence of 10^–3 M GA₃ dormant seeds are able to germinate in the same range of temperatures as non-dormant seeds. The stimulatory effect of GA₃ is less dependent on temperature than that of ethephon, while ACC stimulates germination only at relatively high temperatures (25–30°C). The results obtained are discussed in relation to the possible involvement of endogenous ethylene in the regulation of germination of A. retroflexus seeds.Abbreviations ACC 1-aminocyclopropane 1-carboxylic acid - GA₃ gibberellic acid - SD standard deviation     

Hormonal regulation of iron-stress response in sunflower roots: a morphological and cytological investigation

Summary Sunflowers are known to respond to Fe deficiency (-Fe) with a typical root tip swelling and the formation of root hairs and transfer cells in the rhizodermis. The possible regulation of this process was examined by a comparative study of root morphology and cytology of intact seedlings (Helianthus annuus L. cv. Giganteus) under -Fe and hormonal treatment in nutrient solution. Longitudinal sections of -Fe roots showed root tip swelling is due to cessation of cell elongation and isodiarnetric volume increase of the cortical cells. Enhanced cell division in the pericycle leads to the formation of lateral root primordia in the swollen zone. Xylem vessel differentiation is markedly accelerated and accompanied by early differentiation of the casparian band in the endodermis. Exogenous application of IAA (10^–8-10^–7 M) via the nutrient solution to Fe sufficient plants causes symptoms which closely mimick the characteristics of Fe deficiency including root hair development. Moreover, rhizodermal cells produce peripheral protuberances reminiscent of -Fe transfer cells. Ethylene-releasing ethephon (10^–4M) also causes subapical swelling and root hair formation. However, wall protuberance development is less pronounced. ABA (10^–5 M) leads to similar root thickening and root hair formation but without any comparable transfer cell differentiation. From the striking similarities between -Fe and IAA treatment it is concluded that this hormone (possibly in cooperation with ethylene) is involved in the Fe stress response of sunflower roots. The importance of a continuous polar IAA transport for this process is discussed.Abbreviations ABA abscisic acid - ACC 1-aminocyclopropane-1-carboxylic acid - Ethephone 2-chloro-ethylphosphonic acid - Fe(III)-EDTA ethylenediaminetetraacetic ferric-sodium salt - IAA indole-acetic acid - TIBA triiodobenzoic acid     

Regeneration of plants from callus cultures of Origanum vulgare L.

Investigations were undertaken to achieve rapid multiplication and improvement of Origanum vulgare (a herbaceous, ornamental plant well known for its aromatic and medicinal value) through plant regeneration from callus. The explants (cotyledons, hypocotyl and root segments) excised from 15 d old aseptic seedlings were cultured on Gamborg's B₅ medium supplemented with 2,4-D, NAA and BAP individually and in various combinations (at concentrations of 0,10^–7,10^–6 and 10^–5 M). Best callus induction was noted on medium with 10^–7 M 2,4-D alone. The cotyledonary expiants proved to be the best source for compact and nodulated callus. The subcultured cotyledonary calli showed shoot induction when transferred onto media supplemented with BAP alone orin combination with 10^–7M or 10^–6MNAA. However, 10^–5M NAA completely suppressed the shoot inducing ability of BAP. In general, NAA promoted root induction from all explants used including cotyledonary callus. Best shoot induction was obtained on medium supplemented with 10^–6M BAP+10^–6MNAA. Both IBA and NAA at 10^–6 M proved to be equally effective in induction of roots from the cut ends of 15–20 mm long shoots (excised from callus) in half-strength B₅ liquid medium. Rooted shoots were successfully re-established in soil under controlled conditions.Abbreviations 2,4-D 2,4 dichlorophenoxyacetic acid - IBA indole-3-butyric acid - NAA -naphthaleneacetic acid - BAP 6-benzylaminopurine     

Changes in the phenolic metabolism of suspension cultures of Acer pseudoplatanus L. Caused by the addition of 2-(chloroethyl)phosphonic acid (CEPA)

Summary 2-(Chloroethyl)phosphonic acid (CEPA) inhibited the rise in tannin production and phenylalanine ammonia-lyase EC 4.1.1.5 (PAL) activity shown by Acer cells in media containing 9.0×10^–7 M 2,4-D for a period of 2–3 days after its addtion.Abbreviation 2,4-D 2,4-dichlorophenoxyacetic acid     

Plant regeneration from cell suspension-derived protoplasts of Saintpaulia ionantha Wendl

Summary Friable calli were induced on leaf segments of Saintpaulia ionantha Wendl. on B5 medium containing 1 mg l^–1 2,4-D and 2 g l^–1 casein hydrolysate. Cell suspension cultures were readily established from these friable calli and protoplasts could be isolated from the cells with yields of 1–3×10⁷/g f. wt.. By culturing in 0.1 % gellan gum-solidified B5 medium supplemented with 1 mg l^–1 2,4-D and 0.1 M each of sucrose and mannitol at a density of 1×10⁵/ml, the protoplasts divided within 6 days and formed macro-colonies after 2 months of culture. Shoot regeneration from protoplast-derived calli was obtained by sequential treatment of the calli with plant growth regulators: initially with 1 mg l^–1 each of NAA and BA for 2 months followed by 0.01 mg l^–1 NAA and 5 mg l^–1 BA for 4 months. Regenerated plants were established after rooting of the shoots on half-strength MS medium, and successfully transferred to the greenhouse. The regenerated plants grew into flowering stage and showed the same phenotype as the parent plant.Abbreviations BA benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - FDA fluorescein diacetate - f. wt fresh weight - MES 2-(N-morpholino)-ethanesulfonic acid - MS Murashige and Skoog (1962) - NAA -naphthaleneacetic acid - PE plating efficiency     

Enzymatic ethylene formation from 1-aminocyclopropane-1-carboxylic acid by manganese,a protein fraction and a cofactor of etiolated pea shoots

The cofactor of enzymatic, 1-aminocyclopropane-1-carboxylic acid dependent ethylene formation was concentrated on cation exchange columns. When chelators of cations were added to the homogenates, cofactor activity was lost. Cofactor fractions were partly resistant to oxidation at 600° C. Mn²⁺ substituted for the cofactor in ethylene formation from 1-aminocyclopropane-1-carboxylic acid by a protein fraction isolated from etiolated pea shoots. In addition, Mn²⁺ enhanced the stimulatory effect of the concentrated cofactor. The elution volume for the cofactor on a Sephadex G-25 column was lower than that of MnCl₂. In paper electrophoresis the cofactor migrated to the cathode at pH 10.8 and 2.2. The R_F of cofactor on cellulose plates developed in butanol: acetic acid: H₂O was 0.4. After cellulose chromatography, cofactor activity had to be reconstituted by the addition of MnCl₂. Chelators, anti-oxidants, and catalase were inhibitors of Mn²⁺-cofactor-dependent ethylene formation. The protein necessary for 1-aminocyclopropane-1-carboxylic acid dependent ethylene formation in vitro was seperated from 95–98% of the total protein in homogenates by DE-52 cellulose chromatography and (NH₄)₂SO₄-fractionation.Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid - EDTA ethylenediaminetetraacetic acid - DDTC diethyldithiocarbamate     

In vitro culture of Cucumis sativus L. V. Stabilizing effect of glycine on leaf protoplasts

A method for leaf mesophyll protoplast isolation and plant regeneration of cucumber (Cucumis sativus L.) is described. Using an isolation solution complemented with 0.1 M glycine, 8.2·10⁶ viable protoplasts were isolated from 1 g of fresh leaves. The effect of the growth substances indole-3-acetic acid, naphthalene acetic acid, 2,4,-dichlorophenoxy acetic acid, 6-benzylaminopurine, 2-isopentenyladenine and kinetin at concentrations from 0.5 to 5 mg·1^–1 was studied using the multi-hanging drop technique. The optimal growth substance combination, namely 5 mg·1^–1 naphthalene acetic acid and 3 mg·1^–1 2-isopentenyladenine, together with agarose medium in a so-called bead culture resulted in a plating efficiency of 21%. Some of the colonies obtained regenerated to plantlets which developed to plants.Abbreviations BAP 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxy acetic acid - IAA indole-3-acetic acid - 2iP 2-isopentenyladenine - MS Murashige and Skoog - NAA naphthalene acetic acid     

High-frequency plant regeneration through callus initiation from mature embryos of maize (<Emphasis Type="Italic">Zea Mays</Emphasis> L.)

An efficient maize regeneration system was developed using mature embryos. Embryos were removed from surface-sterilized mature seeds and sliced into halves. They were used as explants to initiate callus on induction medium supplemented with 4.0 mg l^–1 2,4-dichlorophenoxyacetic acid (2,4-D). The induction frequency of primary calli was over 90% for all inbred lines tested. The primary calli were then transferred onto subculture medium supplemented with 2.0 mg l^–1 2,4-D. Following two biweekly subcultures, embryogenic calli were formed. Inclusion of a low concentration (0.2 mg l^–1) of 6-benzylaminopurine (BA) in the subculture medium significantly promoted the formation of embryogenic callus. The addition of silver nitrate (10 mg l^–1) also supported an increased frequency of embryogenesis. The embryogenic callus readily formed plantlets on regeneration medium supplemented with 0.5 mg l^–1 BA. The regenerated plantlets were transferred to half-strength Murashige and Skoog medium supplemented with 0.6 mg l^–1 indole-3-butyric acid to develop healthy roots. The regenerated plantlets were successful on transfer to soil and set seed. Using this system, plantlets were regenerated from seven elite maize inbred lines. The frequency of forming green shoots ranged from 19.8% to 32.4%. This efficient regeneration system provides a solid basis for genetic transformation of maize.Abbreviations BA 6-Benzylaminopurine - 2,4-D 2,4-Dichlorophenoxyacetic acid - IBA Indole-3-butyric acid - KT KinetinCommunicated by M.C. Jordan     

Headspace ethylene accumulation on Stizolobium hassjoo hairy root culture producing L-3,4-dihydroxyphenylalanine

The addition of 1-aminocyclopropane-1-carboxylic acid (ethylene precursor), or 2-chloroethylphosphonic acid (ethephon, an ethylene-releasing compound) decreased root dry weight and l-DOPA (l-3,4-dihydroxyphenylalanine) accumulation in hairy root cultures of Stizolobium hassjoo. The inhibition caused by ethephon-mediated ethylene release was alleviated by 0.5 mg CoCl₂ l^–1 as an inhibitor of ethylene biosynthesis. The action of ethylene was inhibited by 1.5 mg AgNO₃ l^–1. Ethylene thus lowers hairy root formation and l-DOPA production; CoCl₂ decreases ethylene formation leading to a considerably improved root dry weight and l-DOPA production.     

Stimulation ofDaucus carota somatic embryogenesis by inhibitors of ethylene synthesis: cobalt and nickel

The effects of Co²⁺ and Ni²⁺ on ethylene production and somatic embryogenesis by carrot (Daucus carota L.) cell cultures were studied. At concentrations of 10 M to 50 M, CoCl₂ effectively inhibited ethylene production by embryogenic cultures and significantly stimulated somatic embryogenesis. The observed increase of embryo number was proportional to the inhibition level of ethylene production. However, CoCl₂ had no effect when Ethephon was supplied. Nickel also reduced ethylene production, but to a slightly lesser extent than CoCl₂, bringing about a lower increase in the number of somatic embryos. The role of ethylene on somatic embryogenesis is discussed.Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - SAM S-adenosyl-methionine     

Formation of plantlets through somatic embryogeny in the Himalayan blue poppy,Meconopsis simplicifolia (Papaveraceae)

Summary Exuberant and subculturable calli could be induced from only hypocotyl and leaf segments of ca 4-month-old seedlings of Meconopsis simplicifolia cultured on Murashige & Skoog's medium supplemented with 10^–6M kinetin + 10^–5M -naphthalene acetic acid. Suspension cultures were initiated from the calli in a similar medium but with 10^–5M 2,4-dichlorophenoxy acetic acid in place of -naphthalene acetic acid. In ca 80% of the suspension cultures somatic embryos differentiated freely (80–85%) as well as on the surface of small clumps of tissue (15–20%). Somatic embryos that developed beyond heart-shaped stage were transferred to agar-solidified Murashige & Skoog's medium free of growth substances. When maintained in 10 h light and 14 h dark the somatic embryos developed into plantlets bearing cauline leaves. From seed sowing to raising normal plantlets via callus required 28 weeks; on average 80 plantlets were obtained from one explant in three passages.Abbreviations 2,4-D 2,4-dichlorophenoxy acetic acid - FAA formalin-acetic acid-alcohol - GA₃ gibberellic acid - Kn kinetin - MS Murashige & Skoog's medium (Murashige and Skoog 1962) - NAA -naphthalene acetic acid     

Somatic embryogenesis and regeneration of plants in the bamboo Dendrocalamus strictus

Somatic embryogenesis leading to plant regeneration has been achieved in the bamboo, Dendrocalamus strictus, by culturing seeds (caryopses) on B₅ basal medium supplemented with 2,4-dichlorophenoxyacetic acid. Callus cultures obtained from the embryonal end of the seeds differentiated chlorophyllous embryoids. On transfer to a germination medium (B₅ liquid, sucrose, indolebutyric acid, and -naphthaleneacetic acid) 40% of the embryoids developed into plantlets. Further development of the plantlets occured on B₅ liquid medium (half strength) + sucrose (1%) + IBA (5 × 10^–7M) + NAA (10^–7M).Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - IBA Indolebutyric acid - NAA -naphthaleneacetic acid     

The effects of abscisic acid and methyl jasmonate on 1-aminocyclopropane 1-carboxylic acid conversion to ethylene in hypocotyl segments of sunflower seedlings,and their control by calcium and calmodulin

The conversion of 1-aminocyclopropane 1-carboxylic acid (ACC) to ethylene by hypocotyl segments of sunflower (Helianthus annuus L.) seedlings was inhibited by abscisic acid (ABA) and methyl jasmonate (Me-Ja), and this inhibitory effect increased with increasing concentration of both growth regulators. On the contrary, CaCl, enhanced ACC conversion to ethylene at the concentrations of 10^-4 M and 5 x 10^-4 M, however lower and higher concentrations had no significant action. CaCl, (5 x 10^-4M) seemed to magnify the inhibition of the reaction induced by ABA, whereas it reduced (5 x 10^-4M) and even abolished (10^-3M) the inhibitory action of Me-Ja. The results obtained with a Ca²⁺ chelator (EGTA), a Ca²⁺ channel blocker (nifedipine) and calmodulin antagonists (W7 and TFP), given in association with ABA or Me-Ja, suggested that calcium was involved in the inhibition of ACC conversion to ethylene by ABA and Me-Ja through an interaction with calmodulin. However, the mechanism of action of the two growth regulators seemed to be different, since all treatments which resulted in a decrease in cytosolic Ca²⁺ concentration or in calmodulin action induced a decrease in the effect of ABA and an increase in the effect of Me-Ja.Abbreviations ABA abscisic acid - ACC 1-aminocyclopropane 1-carboxylic acid - EFE ethylene for enzyme - EGTA ethylene glycol-bis-2-aminoethyl tetraacetic acid - Me-Ja methyl jasmonate - NIF nifedipine - TFP trifluoperazine dihydrochloride - W7 N-(6-aminohexyl)5-chloro-l-naphthalenesulfonamide hydrochloride     

Effect of Nitrate and Aminoethoxyvinylglycine on Cicer Arietinum L. Nodules

The chickpea (Cicer arietinum L.) cv. HC-1 was raised in earthen pots filled with dune sand in screenhouse. At vegetative stage, i.e. 40 – 45 d after sowing, 10, 20 and 40 mM NO₃ ^– was applied through rooting medium. After 24 h of NO₃ ^– treatments an ethylene biosynthesis inhibitor, aminoethoxyvinylglycine (AVG) in concentration 5 M was given. A conspicuous increase in (5 – 9 fold) ethylene evolution in nodules was noticed after NO₃ ^– treatments. This rise was parallel to the increase in 1-aminocyclopropane-1-carboxylic acid (ACC) content and ACC oxidase activity. On the contrary, a sharp decline in ACC content, ACC oxidase activity and ethylene evolution was observed when AVG was given. A decrease of in acetylene reduction assay (ARA) with NO₃ ^– treatments was associated with decline in cytosolic pH (from 6.12 to 5.45), leghemoglobin (Lb) content, accumulation of H₂O₂ and with the loss of membrane integrity. The lipid peroxidation, followed as MDA production and electrolyte leakage increased with NO₃ ^– treatments, however, the level of MDA was brought down in AVG-treated nodules. Results confirm that ethylene might be involved in mechanism by which the functioning of nodules is adversely affected by NO₃ ^–.     

Calcium increases the yield of somatic embryos in carrot embryogenic suspension cultures

An upward shift in the concentration of calcium present in the medium during somatic embryogenesis increased the number of embryos produced approximately two-fold. This was observed when embryogenic suspension cells grown in 2,4-D medium with the normal calcium concentration of 10^–3 M were transferred to hormone-free medium containing 10^–2 M calcium and when embryogenic suspension cells grown in 2,4-D medium containing 10^–4 M calcium were transferred to hormone-free medium with 10^–3 M calcium. At calcium concentrations between 6·10^–3 and 10^–2 M globular stage somatic embryos were found in cultures supplemented with 2·10^–6 M of 2,4-D indicating that elevated calcium counteracts the inhibitory effect of 2,4-D on somatic embryogenesis. No qualitative changes were found in the pattern of extracellular polypeptides as a result of growth and embryogenesis in media with different calcium concentrations.