Embryogenesis of photoautotrophic cell cultures of Daucus carota L.

In this paper photoautotrophic carrot (Daucus carota L.) suspension cultures are described which are able to produce somatic embryos. The development of somatic embryos, however, requires a sucrose supplement. Although an elevation of the CO₂ concentration up to 2.3% results in the same level of dry weight production as with sucrose in the medium, somatic embryos could not be observed.Results on the influence of sucrose on some aspects of the photosynthetic apparatus of cultured cells are discussed.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - DW dry weight - ELISA enzyme-linked-immunosorbent-assay - FW fresh weight - IAA indole-3-acetic acid - NAA naphthaleneacetic acid - PEPCase phosphoenol-pyruvate carboxylase - Rubisco Ribulose- 1,5-bisphosphate carboxylase/oxygenase - se somatic embryogenesis     

Plant regeneration from protoplasts of Felicia and Brachycome

Protoplasts were isolated from leaves of axenic shoot cultures of Felicia bergeriana (Kingfisher Daisy) and Brachycome iberidifolia (Swan River Daisy) and from callus cultures of Felicia. Plants were regenerated from all three sources and since both species are of ornamental value (blue flowered) the establishment of plant regeneration provides a basis for their incorporation in somatic hybridisation programmes involving important ornamentals such as Chrysanthemum.Abbreviations BAP 6-benzylaminopurine - IAA indole-3-acetic acid - NAA naphthaleneacetic acid - KIN 6-furfurylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - GA₃ gibberellic acid - MS Murashige and Skoog (1962) - FDA fluorescein diacetate - f. wt. fresh weight     

Cell cultures of the wild sunflower Helianthus maximiliani schrader: growth and secondary metabolite synthesis

Summary Callus cultures derived from leaves, stem, sepals and ray flowers and cell suspension cultures of Helianthus maximiliani Schrader were established and analysed for their phytochemicals. Dark-grown cultures were found to synthesize small amounts of non-toxic, polycyclic diterpenoids when grown on modified MS-medium, while -sitosterol and palmitic acid were found in dark- and light-grown cultures. Red light irradiation did not enhance terpenoid production compared to dark- and light-grown cells.Abbreviations 2,4-D 2,4-Dichlorophenoxyacetic acid - NAA naphthaleneacetic acid - BA N-6-benzyladenine - GA gibberellic acid - TLC thin layer chromatography - GC/MS combined gas chromatography-mass specitroscopy - f wt fresh weight     

Initiation and characterization of a cotton (Gossypium hirsutum L.) photoautotrophic cell suspension culture

A heterotrophic cotton (Gossypium hirsutum L. cv. Stoneville 825) cell suspension culture was adapted to grow photoautotrophically. After two years in continuous photoautotrophic culture at 5% CO₂ (balance air), the maximum growth rate of the photoautotrophic cell line was a 400% fresh weight increase in eight days. The Chl concentration was approximately 500 g per g fresh weight.Elevated CO₂ (1%–5%) was required for culture growth, while the ambient air of the culture room (600 to 700 ul CO₂ 1^–1) or darkness were lethal. The cell line had no net photosynthesis at 350 ul 1^–1 CO₂, 2% O₂, and dark respiration ranged from 29 to 44 mol CO₂ mg^–1 Chl h^–1. Photosynthesis was inhibited by O₂. The approximate 1:1 ratio of ribulose 1,5-bisphosphate carboxylase (RuBPcase) to phosphoenolpyruvate carboxylase (PEPcase) (normally about 6:1 in mature leaves of C₃ plants) was due to low RuBPcase activity relative to that of C₃ leaves, not to high PEPcase activity. The PEPcase activity per unit Chl in the cell line was identical to that of spinach leaves, while the RuBPcase activity was only 15% of the spinach leaf RuBPcase activity. RuBPcase activity in the photoautotrophic cells was not limited by a lack of activation in vivo, since the enzyme in a rapidly prepared cell extract was 73% activated. No evidence of enzyme inactivation by secondary compounds in the cells was found as can be found with cotton leaves. Low RuBPcase activity and high respiration rates are most likely important factors in the low photosynthetic efficiency of the cells at ambient CO₂.Abbreviations Chl chlorophyll - COT heterotrophic cotton cell line - COT-P photoautotrophic cotton cell line - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - Rubisco ribulose 1,5-bisphosphate carboxylase/oxygenase - RuBP ribulose 1,5-bisphosphate - RuBPcase RuBP carboxylase - PEP phosphoenolpyruvate - PEPcase phosphoenolpyruvate carboxylase - MX Murashige and Skoog medium with 0.4 mg 1^–1 2,4-D - KT photomixotrophic medium with 1% sucrose - KT^o KT medium with no carbohydrate - KTP^o KT^o medium supplemented with 0.3 M Picloram - CER CO₂ exchange rate - PCER CO₂ exchange rate in the light     

Photoautotrophic growth of soybean cells in suspension culture. II. Optimization of culture medium and conditions

We have attempted to optimize the conditions under which a photoautotrophic soybean suspension culture line (SB-P; Horn et al. 1983) is grown. Magnesium, phosphate, and calcium concentrations were varied individually from one-tenth to five times the normal level found in the Murashige and Skoog (1962) recipe. After two subcultures, only phosphate at one-tenth the normal level caused the cells to show a substantial reduction in fresh and dry weight increase and chlorophyll level. Nitrate and ammonium levels were inversely varied in 20 millimolar increments of potassium nitrate and ammonium chloride. Neither N-source alone could support growth through two subcultures. A ratio of 40 millimolar potassium nitrate to 20 millimolar ammonium gave significantly better fresh and dry weight increases than did a ratio of 20:40 or 30:30 but the chlorophyll level was unchanged. The minor salts as a group resulted in a small improvement in growth when provided at twice the normal level.Indole-3-acetic acid at five milligrams per liter resulted in significantly better fresh and dry weight increases than did -naphthaleneacetic acid at any level but the final chlorophyll level was not changed. There was no correlation between growth and kinetin level and this resulted in the discovery that SB-P cells are cytokinin-autotrophic, as are heterotrophic SB cells, with regard to both growth and greening ability. Growing SB-P cells under a 14 h:10 h day:night photoperiod resulted in a slow but inevitable death. Increasing the carbon dioxide level to 10% for four weeks gave no increase in SB-P cell growth or chlorophyll level, but SB-P cells would not grow with carbon dioxide levels below 0.4%. The results clearly show that SB-P cells, despite their tenuous existence, are capable of adapting to a wide range of culture conditions. A simplified and improved culture medium for photoautotrophic cultures is given.Abbreviations SB-P photoautotrophic soybean cells - SB-M photomixotrophic soybean cells - SB-H heterotrophic soybean cells     

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     

Uptake of phosphate and its effect on phenylalanine ammonia-lyase activity and cinnamoyl putrescine accumulation in cell suspension cultures of Nicotiana tabacum

The influence of phosphate on the medium-induced formation of cinnamoyl putrescines in cell cultures of Nicotiana tabcum was investigated. Phosphate added to a phosphate-free production medium was completely accumulated in the cells within 24h after inoculation at initial concentrations up to 2 mM. At higher concentrations phosphate was partly accumulated with an intracellular saturation at approx. 0.65 mmol/g dr. wt. equivalent to approx. 45 mM intracellular concentration. Enhanced activities of phenylalanine ammonialyase and increased product levels of cinnamoyl putrescines, induced by cell transfer into phosphatefree medium were suppressed similarly at initial phosphate concentrations of 0.02–0.5 mM. At the same time growth was stimulated.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - fr. wt. fresh weight - dr. wt. dry weight - MS-medium Murashige-Skoog-medium (Murashige and Skoog 1962)     

Comparison of secondary product accumulation in photoautotrophic,photomixotrophic and heterotrophic Nicotiana tabacum cell suspension cultures

Summary Photoautotrophic, photomixotrophic and heterotrophic Nicotiana tabacum cell suspension cultures were compared for the constitutive accumulation of secondary metabolites and the elicitor-induced formation of the phytoalexin capsidiol. Nicotine and chlorogenic acid were found in high amounts in the heterotrophic cultures and in moderate concentrations in photomixotrophic but not in photoautotrophic cells. Nicotinic acid-N-glucoside occured in all culture types; in photoautotrophic and photomixotrophic cells the formation of N-methylnicotinic acid (trigonelline) was also observed. Treatment with a fungal elicitor led to substantial accumulation of capsidiol in heterotrophic and photomixotrophic cells and in only low levels in photoautotrophic cultures. Elicitor-treated photomixotrophic cells showed a pronounced increase in cell wall-bound phenolics. The levels of nicotine, nicotinic acid-N-glucoside and trigonelline were not affected by elicitation.Abbreviations hcc heterotrophic cell culture - mcc photomixotrophic cell culture - pcc photoautotrophic cell culture - fr.wt. freshweight - nic-N-glc nicotinic acid-N-glucoside - PMG Phytophthora megasperma f. sp. glycínea - HPLC high performance liquid chromatography - GC gas chromatography - TLC thin layer chromatography - 2,4D 2,4-dichlorophenoxyacetic acid - Kin kinetin - BAP 6-benzylaminopurine - NAA -naphthylacetic acid     

Plant regeneration from protoplasts of Dimorphotheca and Rudbeckia

Protoplasts were isolated from leaves, shoots, cotyledons, ray florets and callus cultures of Dimorphotheca aurantiaca (syn. D. sinuata) (Cape Marigold, Star of the Veldt) and Rudbeckia hirta, R. laciniata and R. purpurea; species of ornamental value. For Dimorphotheca, plants were regenerated from protoplasts of all sources apart from the ray floret, whilst for the Rudbeckia species, although protoplast division was induced in most cases, only leaf mesophyll protoplasts of R. hirta c.v. Marmalade gave plants. The establishment of plant regeneration for these ornamental species, from protoplasts, now provides a basis for their somatic hybridisation.Abbreviations BAP 6-benzylaminopurine - IAA indole-3-acetic acid - NAA naphthaleneacetic acid - K kinetin - GA₃ gibberellic acid - MS Murashige and Skoog (1962) - f.wt. fresh weight     

A microtest system for the serial assay of phytotoxic compounds using photoautotrophic cell suspension cultures of Chenopodium rubrum

Chenopodium rubrum photoautotrophic cell suspensions were grown in plastic tissue culture dishes under photoautotrophic conditions. Growth was monitored by measuring cell number, packed cell volume, chlorophyll content and oxygen production. Such microtiter dishes are suitable systems for the serial assay of growth inhibition and various physiological effects (i.e. chlorophyll fluorescence, cell viability, oxygen production) of photoautotrophic cells as caused by herbicides and fungal phytotoxins. The applicability of the test system is discussed.Abbreviations pcv packed cell volume - fr.w. fresh weight - rpm revol. per minute - DMSO dimethyl sulfoxide - PMS phenazine methosulfate - NBT nitro-blue tetrazolium chloride     

Establishment and characterization of photoautotrophic protoplast-derived cultures ofNicotiana plumbaginifolia

A procedure is described for the rapid establishment of photoautotrophic protoplast-derived cultures ofNicotiana plumbaginifolia. Photoautotrophic growth was induced by lowering the glucose concentration to 2.5 g.l^–1 in the protoplast culture medium and by omitting glucose from the subsequent dilution medium. Four week-old highly viable suspensions were plated on an agar-medium without glucose in unsealed Petri dishes and kept in illuminated chambers flushed with 0.05 % or 2 % CO₂. Air-grown calli had net photosynthesis rates of 1.8 and 17 moles CO₂.g^–1 fresh wt.h^–1 in air at 0.034 % CO₂ and in air enriched with 1 % CO₂, respectively. Calli grown in 2 % CO₂ exhibited lower rates of net photosynthesis at the two CO₂ concentrations tested (0 and 7.5 moles CO₂.g^–1 fresh wt.h^–1, respectively). The contribution of photosynthesis to growth was estimated to be 80 % in air-grown calli and more than 90 % in calli grown in 2 % CO₂. The suitability of this photoautotrophic culture procedure is discussed with regard to the screening of photosynthetic mutants or transformants from protoplasts.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - BAP 6-benzylaminopurine - IAA indoleacetic acid - Rubisco ribulose 1,5-bisphosphate carboxylase/oxygenase     

Semi-continuous production of sanguinarine and dihydrosanguinarine by Papaver somniferum L. cell suspension cultures treated with fungal homogenate

Papaver somniferum L. (opium poppy) cells were elicited with a Botrytis sp. homogenate and cultured by a semi-continuous process. Elicitation induced synthesis of sanguinarine and dihydrosanguinarine. Significant release of both alkaloids into the culture medium occurred. Medium exchange at 2-day intervals enabled product recovery from spent medium and maintained culture viability. Culture growth was not inhibited by elicitor treatment necessitating sub-culture prior to re-elicitation. Re-elicited cultures displayed an increasing sensitivity (reduced growth rate, higher alkaloid yield) to the elicitor with each successive treatment and did not survive a fourth elicitation.Abbreviations DW dry weight - FW fresh weight - 2,4-D 2,4-dichlorophenoxyacetic acid - SGE sanguinarine - DSGE dihydrosanguinarine Publication 29143 from the National Research Council of Canada     

Photoautotrophic potato cells: Transition from heterotrophic to autotrophic growth

Cells of potato (Solanum tuberosum L.) were obtained which were capable of photoautotrophic growth in liquid suspension culture under a photon flux density of 90–110 μmol m^?2 s^?1 PAR and in an atmosphere enriched with 2% CO₂. These photoautotrophic cells contained between 100 to 200 μg Chl (g fresh weight)^?1 and fixed CO₂ at a maximum rate of 16 μmol CO₂ (g fresh weight)^?1h^?1. In order to obtain cells capable of photoautotrophic growth it was necessary to adapt highly chlorophyllous heterotrophic cells (>50 μg Chl (g fresh weight)^?1) for growth in medium with 2.5 g sucrose 1^?1 (photomixotrophic cells). The photomixotropic cells had a Chl content of ca 100 μg Chl (g fresh weight)^?1 and were capable of photosynthetic activity which allowed them to survive after sugars had been depleted from the medium. It was from the photomixotrophic cells that cells capable of photoautotrophic growth were obtained. Heterotrophic cells initially established in liquid medium with 25 g sucrose I^?1 from chlorophyllous callus contained about 50 to 150 μg Chl (g fresh weight)^?1. However, after 5 to 10 passages the Chl content decreased to a maximum of 15 μg Chl (g fresh weight)^?1. These cells could not be adapted to photomixotrophic or photoautotrophic growth. These cells also were not able to regain Chl or initiate high rates of CO₂ fixation during the stationary phase of growth as did photomixotrophic cells or chlorophyllous heterotrophic cells. The loss of Chl exhibited by the cells during adaption to heterotrophic growth could be attributed at least in part to unbalanced growth (when cell division and growth exceeds Chl accumulation). Sucrose appeared to have an inhibitory effect directly on photosynthesis independent of Chl accumulation.     

Photosynthetic characteristics of photomixotrophic and photoautotrophic cell suspension cultures ofChenopodium rubrum

Summary Cell suspension cultures ofChenopodium rubrum have been grown for more than 2 years photoautotrophically with CO₂ as sole carbon source. Average increase in fresh weight is appr. 600% within 14 days. The chlorophyll content of photoautotrophic cells (200 g/g fresh weight) is much higher than of photomixotrophic cells (50 g/g fresh weight). The photosynthetic activity of the cells (190 moles CO₂×mg^–1 chlorophyllXh^–1) is comparable to the values found with intact leaves. As shown by short-term¹⁴CO₂ photosynthesis, both, the photomixotrophic and the photoautotrophic cell suspension cultures assimilate CO₂ predominantly via the Calvin pathway.Major differences were found with cells from either exponential or stationary phase of growth with regard to differential labelling of 3-phosphoglyceric acid, malate, sucrose and glucose/fructose.In vitro measurements of carboxylation reactions only partially corroborate our findings with¹⁴CO₂ incorporation. The ratio of ribulosebisphosphate to phosphoenolpyruvate carboxylase activity is 4.7 for leaves of C.rubrum, 1.2 for photoautotrophic cells during stationary growth and 0.5 for cells during exponential growth phase, however, 0.18 was found for photomixotrophic cells. Though the¹⁴CO₂ incorporation into 3-phosphoglyceric acid is clearly higher than into malate, thein vitro activity of phosphoenolpyruvatecarboxylase is 2–6 fold higher than that of ribulosebisphosphate carboxylase. We postulate that anaplerotic reactions of the tricarboxylic acid cycle are involved in the regulation of phosphoenolpyruvate carboxylase.Abbreviations 2,4-D didilorophenoxyacetic acid - EDTA ethylene-diamine-tetraacetic acid - fr. w. fresh weight - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - PGA 3-phosphoglyceric acid - PPO 2,5-diphenyloxazole - PEP phosphoenolpyruvate - RuBP nbulosebisphosphate     

Plant regeneration from in vitro cultures of anthers and mature seeds of rice (Oryza sativa L.) cv. Basmati-370

Pollen plants were obtained from anther-derived calli of the indica rice variety Basmati-370. Anther-response (anthers producing pollen derived calli) and plant regeneration frequency from the pollen derived calli. was very low. Donor plants which flowered at the average max/min. temperature of 34.2°/23.3°C gave a significantly higher anther-response to in vitro techniques, than did those which flowered at 29.1°/16.4°C. Somatic callus induction and subsequent plant regeneration was readily obtained from mature seed embryos. While 2,4-D or 2,4,5-T (1 or 2 mg/l) proved highly efficient for callus induction, tryptophan (50 or 100 mg/l) induced a high frequency of green plants from the calli.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - 2,4,5-T 2,4,5-trichlorophenoxyacetic acid - NAA naphthaleneacetic acid - IAA indoleacetic acid - K kinetin - BA benzyladenine - Trp tryptophan - CW coconut water     

Plantlets from somatic callus tissue of the East Indian Rosewood (Dalbergia latifolia Roxb.)

Callus-mediated shoot bud formation was demonstrated in Dalbergia latifolia Roxb. (East Indian Rosewood). Cultures were raised from shoot explants of six year-old plants on Murashige and Skoog (MS) medium supplemented with naphthaleneacetic acid (NAA) and benzyladenine (BA). A sequential treatment of callus with increasing BA levels and decreasing NAA ensured shoot bud induction. Rooting of shoots was achieved by a three-step culture procedure involving 1) White's(W) liquid medium containing indoleacetic acid (IAA), naphthaleneacetic acid and indolebutyric acid (IBA), 2) half-strength MS agar-solidified medium with charcoal (0.25%) and 3) half-strength MS liquid medium.Abbreviations BA Benzyladenine - IAA Indoleacetic acid - IBA Indolebutyric acid - MS Murashige and Skoog - NAA a-naphthaleneacetic acid - PVP Polyvinylpyrrolidone - W White's medium - 2,4-D 2,4-dichlorophenoxyacetic acid     

Photoautotrophic Growth and Photosynthesis in Cell Suspension Cultures of Chenopodium rubrum

A method is described for growing cell suspension cultures of Chenopodium rubrum photoautotrophically for prolonged periods of time. By using a two-tier culture vessel the growth medium with the cells was separated from the CO₂ reservoir. Definite CO₂ concentrations were established by a K₂CO₃/KHCO₃ buffer. Photoautotrophic growth in C. rubrum cell suspension cultures was correlated with the CO₂ level. At 0.5% CO₂ the cell cultures contained 68 μg chlorophyll/g fresh weight and showed an increase in fresh weight of about 80% in 18 days. At 1% CO₂ an increase in fresh weight of 165% in 18 days was observed. The chlorophyll content rose up to 84 μg/g fresh weight. The photoautotrophic growth was also greatly influenced by the 2,4-D content of the medium. Cell growth was enhanced by lowering the auxin concentration. Best growth was attained (210% increase in fresh weight) at 10^?8M 2,4-D. The photosynthetic activity of the cells was measured by the light dependent ¹⁴CO₂ incorporation. At 0.5% CO₂ the cell suspensions assimilated about 100 μmol CO₂/mg chlorophyll × h. In the presence of 1% CO₂ the light driven assimilation was raised up to 185 μmol CO₂/mg chlorophyll × h. In both cases, the dark incorporation of CO₂ was merely 1.8% of the values obtained in light.     

Callus production from photoautotrophic soybean cell culture protoplasts

Summary Protoplasts were prepared from a photoautotrophic (PA) cell line of Glycine max (soybean). A yield of 75 to 90% after two to three hours digestion in a mixture of 1% Cellulase R10, 0.2% Pectolyase Y23 and 2% Driselase was obtained. Cell division and colony formation occurred from approximately 18% of the plated protoplasts. The cultured protoplasts were as sensitive to the herbicide atrazine, a photosynthetic inhibitor, as the original PA cells under the same conditions. Protoplasts and cells of a heterotrophic (HT) soybean culture were not as sensitive to atrazine. The isolated protoplasts retained the PA characteristics of the parental culture in the callus and cell suspension cultures obtained from the protoplasts. The chromosome numbers in the parental cell line and in cells derived from the isolated protoplasts (both PA and HT) were found to be largely (99%) the normal diploid number of 40.Abbreviations BA Benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - HT Heterotrophic - MES 2-(morpholino) ethane sulfonic acid - NAA Naphthaleneacetic acid - PA Photoautotrophic - PCM Protoplast culture medium     

Establishment and physiological analyses of photoautotrophic callus cultures of the fern Platycerium coronarium (Koenig) Desv. under CO2 enrichmen

Gametophyte-derived callus cultures of Platycerium coronariumcould be maintained under photoautotrophic conditions on Murashigeand Skoog medium supplemented with 2µM 2,4-dichlorophenoxyaceticacid (2,4-D) and with CO₂ enrichment. Progressive reductionof sucrose from the medium resulted in a reduction in growth,but an increase in total chlorophyll content. When subculturingwas delayed beyond 2 weeks, callus cells differentiated intogametophytes on the medium with 0.2 sucrose and no CO₂ enrichment.Enriching the photoautotrophic cultures on 2µM 2, 4-Dwith 1% CO₂ resulted in about 1.7-fold increase in fresh weightwithin 42 d. Total chlorophyll content was generally higherwith 1% CO₂ enrichment than with 10%. F_v/F_m ratio was higherfor callus on low levels of sucrose (>0.5%) than that onsucrose 1.0%. An increase in autofluorescence of chloroplasts,but not the size, was observed with decreasing sucrose levelsin the medium. Autofluorescence decreased with increase in CO₂from 0.03%. Our data are in agreement with the view that long-termexposure to high levels of decrease in photosynthetic capacity. Key words: Platycerium coronarium, stag's horn fern, autofluorescence of chloroplasts, confocal laser scanning microscope, F_v/F_m ratio, photoautotrophic callus     

The isolation and culture of protoplasts from an embryogenic cell suspension culture of Picea glauca (moench) voss

Conditions were standardized for the isolation and culture of protoplasts from an embryogenic cell suspension culture of Picea glauca. A combination of 0.5% Cellulase R-10, 0.25% Macerozyme, 0.25% Driselase, 0.25% Rhozyme HP-150 with 0.5M mannitol and 5 mM CaCl₂.2H₂O produced an average of 4.5 × 10⁶ protoplasts per gram fresh weight of cells. Of the several protoplast culture media tested, von Arnold and Eriksson and Kao and Michayluk (KM8P) media best supported mitotic divisions of protoplasts. A density of 10⁵ protoplasts per ml and the addition of 5 mM glutamine to the culture medium was necessary to induce sustained divisions and microcallus formation. Microcalli grew into subculturable callus using a nurse culture technique.Abbreviations BAP benzylaminopurine - 2,4-D 2,4-dichlorophenoxy-acetic acid - FDA fluorescein diacetate NRCC No. 27937