Improved cell growth and total flavonoids of Saussurea medusa on solid culture medium supplemented with rare earth elements

Callus cultures of Saussurea medusa were cultivated on solid culture medium supplemented with either Ce³⁺, La³⁺, Nd³⁺ or a mixture of rare earth elements. Ce³⁺, 0.05 mM, gave the highest biomass (0.53 g dry wt per flask) and total flavonoids (27.5 mg per flask), which were, 70% and 100% higher than those without Ce³⁺ addition, respectively. Ce³⁺, 0.01–0.1 mM, or La³⁺, 0.05 mM, or the mixture of rare earth elements, 0.025–0.1 mM, can substitute for 6-benzyladenine, and 0.025 mM Ce³⁺ can partly substitute for naphthaleneacetic acid in promoting cell growth and biosynthesis of total flavonoids in S. medusa.     

The effects of CA2+ during the elicitation of shikonin derivatives inOnosma paniculatum cells

Summary A fungal elicitor extracted fromAspergillus oryzae (Ahlb.) Cobn mycelia promoted the production of shikonin derivatives inOnosma paniculatum Bur et Franch cell suspension cultures. Elicitor treatment also increased Ca²⁺ concentration in RM₉ medium, which could be measured earlier than the elicited increase of shikonin formation. Several reagents known to induce Ca²⁺-influx and increase the intracellular-free Ca²⁺ level, such as the addition of Ca (NO₃)₂·4H₂O, the Ca²⁺ ionophore A₂₃₁₈₇, and abscisic acid (ABA), appreciably suppressed the elicitor-promoted shikonin formation inOnosma cells. In contrast, the decrease of intracellular-free Ca²⁺ level by the specific Ca²⁺-chelator ethylene glycol bis (β-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA) or the Ca²⁺—channel blocker, verapamil, enhanced the biosynthesis of shikonin even in the absence of elicitor. Treatment of cells with trifluoperazine (TFP) also stimulated shikonin formation inOnosma cell cultures. A rapid and transient drop of free Ca²⁺ level in one protoplast was directly determined after the addition of elicitor toOnosma cell cultures. The inhibitory effect on shikonin formation by ABA was largely on account of its ability to restore the intracellular Ca²⁺ level lowered by the elicitor. These results suggest that Ca²⁺ play a significant role in an early stage of the elicitation process ofOnosma cells. The rapid drop of cytoplasmic Ca²⁺ carries the elicitor signal and in turn regulates the biosynthesis of shikonin derivatives.     

Reduction of hyperhydricity in the culture of <Emphasis Type="Italic">Lepidium meyenii</Emphasis> shoots by the addition of rare earth elements

Adventitious shoots induced from maca calli on induction media without rare earth elements (REE) had higher water content and lower soluble protein concentration when compared with shoots sprouted from maca seeds. Due to lower activities of antioxidative enzymes, there were higher concentrations of H₂O₂ and malonyldialdehyde (MDA) in adventitious shoots than those in seed shoots. When La³⁺, Ce³⁺ and Nd³⁺ (0.04 mM to 0.1 mM) were added to induction media, induction rates of the adventitious shoots were only affected slightly, but hyperhydricity rates were significantly reduced. La³⁺, Ce³⁺ or Nd³⁺ enhanced the activities of antioxidative enzymes in adventitious shoots, e.g. peroxidase (POD), catalase (CAT), ascorbate peroxidase (APX), superoxide dismutase (SOD), monodehydroascorbate reductase (MDHAR) and glutathione reductase (GR). When the concentrations of La³⁺, Ce³⁺ and Nd³⁺ were 0.1 mM, the oxygen stress in adventitious shoots was decreased to levels similar to seed shoots, where most adventitious shoots grew normally.     

The effect of several factors on somatic embryogenesis and plant regeneration in protoplast cultures of <Emphasis Type="Italic">Gentiana kurroo</Emphasis> (Royle)

Protoplasts were isolated from cell suspensions derived from cotyledon and hypocotyl Gentiana kurroo (Royle). Cell walls were digested with an enzyme cocktail containing cellulase, macerozyme, driselase, hemicellulase and pectolyase in CPW solution. Protoplast viability ranged from 88 to 96%. Three techniques of culture and six media were evaluated in terms of their efficiency in producing viable cultures and regenerating whole plants. With liquid culture, cell division occurred in only a low number of the protoplasts isolated, and no plant regeneration was successful. Cell division occurred within 2 or 3 days in case of agarose solidified media. After 10 days of culture, the number of dividing cells was the highest with modified MS medium in which NH₄NO₃ was replaced with 3.0 g l⁻¹ glutamine. The best results were obtained with agarose bead cultures: plating efficiency was 68.7% and 58.1% for protoplasts isolated from cotyledon and hypocotyl derived suspensions, respectively. The results were achieved with using medium containing 0.5 mg l⁻¹ 2,4-D + 1.0 mg l⁻¹ kinetin or 2.0 mg l⁻¹ BAP + 1.0 mg l⁻¹ dicamba + 0.1 mg l⁻¹ NAA + 80 mg l⁻¹ adenine sulfate. Protocalluses transferred on the following composition of plant growth regulators: 0.5 mg l⁻¹ 2,4-D + 1.0 mg l⁻¹ kinetin or 1.0 mg l⁻¹ kinetin + 0.5 mg l⁻¹ GA₃ + 80.0 mg l⁻¹ adenine sulfate developed in embryogenic cultures. However, the best embryo production occurred with the first one. Later embryos were transferred to half-strength MS mineral salts to promote plants formation. Flow cytometry studies revealed increased amounts of DNA in about one third of the regenerants.     

Research of the entry of rare earth elements Eu3+ and La3+ into plant cell

Whether rare earth elements can enter into plant cells remains controversial. This article discusses the ultracellular structural localization of lanthanum (La³⁺) and europium (Eu³⁺) in the intact plant cells fed by rare earth elements Eu³⁺ and La³⁺. Eu-TTA fluorescence analysis of the plasmalemma, cytoplast, and mitochondria showed that Eu³⁺ fluorescence intensities in such structures significantly increased. Eu³⁺ can directly enter or be carried by the artificial ion carrier A23187 into plant cells through the calcium ion (Ca²⁺) channel and then partially resume the synthesis of amaranthin in the Amaranthus caudatus growing in the dark. Locations of rare earth elements La³⁺ and Eu³⁺ in all kinds of components of cytoplasmatic organelles were determined with transmission electron microscope, scanning electron microscope, and energy-dispersive X-ray microanalysis. The results of energy-dispersive X-ray microanalysis indicated that Eu³⁺ and La³⁺ can be absorbed into plant cells and bind to the membranes of protoplasm, chloroplast, mitochondrion, cytoplast, and karyon. These results provide experimental evidence that rare earth elements can be absorbed into plant cells, which would be the basis for interpreting physiological and biochemical effects of rare earth elements on plant cells.     

Effects of divalent metal cations on lovastatin biosynthesis from <Emphasis Type="Italic">Aspergillus terreus</Emphasis> in chemically defined medium

Effects of six divalent metal cations: Fe²⁺, Ca²⁺, Zn²⁺, Mg²⁺, Cu²⁺and Mn²⁺ on fungal cell growth and lovastatin biosynthesis were investigated by submerged cultivation of Aspergillus terreus in a modified chemically defined medium. The influences of different initial concentrations of the above six metal cations were also examined at 1, 2, and 5 mM, respectively. Cu²⁺ apparently inhibited the cell growth, but had no influence on biosynthesis of lovastatin. All of Fe²⁺, Ca²⁺, Zn²⁺, Mg²⁺ and Mn²⁺ promoted the cell growth and lovastatin biosynthesis in different extents. The highest biomass of 13.8 ± 0.5 g l⁻¹ and specific lovastatin titres of 49.2 ± 1.4 mg gDCW⁻¹ were obtained at the level of 2 and 5 mM in the presence of Zn²⁺, respectively. The values were improved double and 14.4-fold. Excess Zn²⁺ inhibited the cell growth, but enhanced lovastatin biosynthesis with an increment of 17.6 mg l⁻¹ per mM. The interactions of all metal cations slightly inhibited the lovastatin production comparing with the existence of Zn²⁺, Fe²⁺ and Mg²⁺ solely, yet remarkably improved the cell growth. These results suggest that the divalent metal ions Zn²⁺ or Fe²⁺ influence the production by regulating the action of key enzymes such as LovD or LovF in lovastatin biosynthesis.     

应用稀土调控藏红花胚性愈伤组织的生长与分化

为提高藏红花(Crocus sativus)胚性愈伤组织的繁殖系数与出芽率, 以建立藏红花离体快繁体系, 解决其资源短缺问题, 采用两步法, 用稀土调控其胚性愈伤组织的生长与分化。结果表明, 在添加了0.25 mg·L^–1 NAA、3 mg·L^–1 6-BA和400 mg·L^–1 CH的B₅固体培养基中, 0.04 mmol·L^–1 La³⁺促进胚性愈伤组织生长的效果最佳, 繁殖系数为12, 是不添加稀土处理组的1.48倍; 在添加了0.25 mg·L^–1 NAA、3 mg·L^–1 6-BA和400 mg·L^–1 CH的1/2 B₅固体培养基中, 0.06 mmol·L^–1 Ce³⁺促进胚性愈伤组织分化出芽的效果最佳, 出芽率高达84.5%, 是不添加稀土处理组的1.81倍, 且高于国外报道的出芽率(40%)。初步解决了藏红花胚性愈伤组织生长慢和出芽率低等问题, 为建立高效稳定的藏红花离体快繁体系奠定了基础。     

Ca<Superscript>2+ </Superscript>and Cu<Superscript>2+ </Superscript>supplementation increases mannitol production by <Emphasis Type="Italic">Candida magnoliae</Emphasis>

Supplementation with CaCl₂·2H₂O (50 mg l⁻¹) or CuSO₄·5H₂O (10 mg l⁻¹) improved mannitol production by Candida magnoliae by 14.5 and 18.6% (25 and 32 g/L), respectively. When used in combination, they acted synergistically: Ca²⁺ decreased the intracellular concentration of mannitol 30%, whereas Cu²⁺ increased the intracellular activity of mannitol dehydrogenase 1.6-times more than control. Ca²⁺ probably works by altering the permeability of cells to mannitol, whereas, Cu²⁺ increases the activity of an enzyme responsible for mannitol biosynthesis.     

Enhancement of xylitol productivity and yield using a xylitol dehydrogenase gene-disrupted mutant of Candida tropicalis under fully aerobic conditions

The effects of glycerol and the oxygen transfer rate on the xylitol production rate by a xylitol dehydrogenase gene (XYL2)-disrupted mutant of Candida tropicalis were investigated. The mutant produced xylitol near the almost yield of 100% from d-xylose using glycerol as a co-substrate for cell growth and NADPH regeneration: 50 g d-xylose l⁻¹ was completely converted into xylitol when at least 20 g glycerol l⁻¹ was used as a co-substrate. The xylitol production rate increased with the O₂ transfer rate until saturation and it was not necessary to control the dissolved O₂ tension precisely. Under the optimum conditions, the volumetric productivity and xylitol yield were 3.2 g l⁻¹ h⁻¹ and 97% (w/w), respectively.     

Biodegradation of 2,4-dichlorophenol by a <Emphasis Type="Italic">Bacillus</Emphasis> consortium

As part of our effort at establishing microbial consortia of relevance for the bioremediation of xenobiotics polluted environments in Mexico, we assessed the aerobic biodegradation of 2,4-dichlorophenol (2,4-DCP) by a consortium of four Bacillus species that were isolated from a polluted soil by enrichment using a mixture of chlorophenols. The bacterial consortium effectively biodegraded 2-chlorophenol, 3-chlorophenol and 2,4-dichlorophenol at degradation rates of between 1.7 and 6.7 μmoles l⁻¹ h⁻¹. In the presence of NH₄Cl or KNO₂ as nitrogen sources_, 2,4-DCP was variously degraded. Under both conditions, cell biomass attained highest values of 350 and 450 mg l⁻¹ respectively, while the amounts of 2,4-DCP metabolized in 21 days reached peak values of 2.1 and 2.5 mM representing between 70 and 85% degradation respectively. Chloride releases during the same period were highest at 4.7 mM and 5.3 mM in the presence of the two nitrogen sources. The presence of free-chloride in the culture medium had a significant impact on the catabolism of 2,4-dichlorophenol.     

Prospects of using marine actinobacteria as probiotics in aquaculture

In the present study, optimum culture conditions for the production of extracellular polysaccharides (EPS) in submerged culture of an edible mushroom, Laetiporus sulphureus var. miniatus and their stimulatory effects on insulinoma cell (RINm5F) proliferation and insulin secretion were investigated. The maximum mycelial growth (4.1 g l⁻¹) and EPS production (0.6 g l⁻¹) in submerged flask culture were achieved in a medium containing 30 g l⁻¹ maltose, 2 g l⁻¹ soy peptone, and 2 mM MnSO₄·5H₂O at an initial pH 2.0 and temperature 25°C. In the stirred-tank fermenter under optimized medium, the concentrations of mycelial biomass and EPS reached a maximum level of 8.1 and 3.9 g l⁻¹, respectively. Interestingly, supplementation of deep sea water (DSW) into the culture medium significantly increased both mycelial biomass and EPS production by 4- and 6.7-fold at 70% (v/v) DSW medium, respectively. The EPS were proved to be glucose-rich polysaccharides and were able to increase proliferation and insulin secretary function of rat insulinoma RINm5F cells, in a dose-dependent manner. In addition, EPS also strikingly reduced the streptozotocin-induced apoptosis in RINm5F cells indicating the mode of the cytoprotective role of EPS on RINm5F cells.     

Enhancement of phenylethanoid glycosides biosynthesis in cell cultures of <Emphasis Type="Italic">Cistanche deserticola</Emphasis> by osmotic stress

The effect of osmotic stress on cell growth and phenylethanoid glycosides (PeGs) biosynthesis was investigated in cell suspension cultures of Cistanche deserticola Y. C. Ma, a desert medicinal plant grown in west region of China. Various initial sucrose concentrations significantly affected cell growth and PeGs biosynthesis in the suspension cultures, and the highest dry weight and PeGs accumulation reached 15.9 g l⁻¹-DW and 20.7 mg g⁻¹-DW respectively at the initial osmotic stress of 300 mOsm kg⁻¹ where the sucrose concentration was 175.3 mM. Stoichiometric analysis with different combinations of sucrose and non-metabolic sugar (mannitol) or non-sugar osmotic agents (PEG and NaCl) revealed that osmotic stress itself was an important factor for enhancing PeGs biosynthesis in cell suspension cultures of C. deserticola. The maximum PeGs contents of 26.9 and 23.8 mg g⁻¹-DW were obtained after 21 days at the combinations of 87.6 mM sucrose with 164.7 mM mannitol (303 mOsm kg⁻¹) or 20 mM PEG respectively, which was higher than that of C. deserticola cell cultures grown under an initial sucrose concentration of 175.3 mM after 30 days. The stimulated PeGs accumulation in the cell suspension cultures was correlated to the increase of phenylalanine ammonium lyase (PAL) activity induced by osmotic stress.     

Effect of nutritional factors on shikonin derivative formation in Lithospermum callus cultures

Some nutritional factors affecting the biosynthesis of shikonin derivatives in callus cultures of Lithospermum erythrorhizon were examined. High sucrose concentrations increased the content of shikonin derivatives, but neither glucose nor fructose was effective for shikonin derivative formation. High concentrations of nitrogen sources inhibited or retarded shikonin derivative formation and streptomycin sulphate stimulated their biosynthesis. Addition of ascorbic acid increased the content of shikonin derivatives. Among some precursors tested only l-phenylalanine had a positive effect. At high concentrations, Ca²⁺ and Fe²⁺ inhibited the biosynthesis of shikonin derivatives.     

Oxygen uptake, heart rate and blood lactate concentration during a normal training session of an aerobic dance class

The aim of this research was to investigate the physiological responses and, in particular, the participation of lactic acid anaerobic metabolism in aerobic dance, which is claimed to be pure aerobic exercise. In contrast to previous studies, that have put subjects in very unfamiliar situations, the parameters were monitored in the familiar context of gymnasium, practice routine and habitual instructor. A group of 30 skilled fairly well-trained women performed their usual routine,␣a combination of the two styles: low (LI) and high impact (HI), and were continuously monitored for heart rate (HR) and every 8 min for blood lactate concentration ([La⁻]_b). Of the group, 15 were tested to determine their maximal aerobic power (V˙O_2max) using a cycleergometer. They were also monitored during the routine for oxygen uptake (V˙O₂) by a light telemetric apparatus. The oxygen pulses of the routine and of the corresponding exercise intensity in the incremental test were not statistically different. The mean values in the exercise session were: peak HR 92.8 (SD 7.8)% of the subject's maximal theoretical value, peak V˙O₂ 99.5 (SD 12.4)% of V˙O_2max, maximal [La⁻]_b 6.1 (SD 1.7) mmol · l^−l, and mean 4.8 (SD 1.3) mmol · l^−l. Repeated measures ANOVA found statistically significant differences between the increasing [La⁻]_b values (P < 0.001). In particular, the difference between the [La⁻]_b values at the end of the mainly LI phase and those of the LI-HI combination phase, and the difference between the samples during the combination LI-HI phase were both statistically significant (both P= 0.002 and P= 0.002). The similar oxygen pulses confirmed the validity of the present experiment design and the reliability of HR monitoring in this activity. The HR, V˙O₂ and, above all, the increase of [La⁻]_b to quite high values, showing a non steady state, demonstrated the high metabolic demand made by this activity that involved lactic acid metabolism at a much higher level than expected. Accepted: 23 September 1997     

Purification and characterization of dihydroorotase fromPseudomonas putida

Dihydroorotase was purified to homogeneity fromPseudomonas putida. The relative molecular mass of the native enzyme was 82 kDa and the enzyme consisted of two identical subunits with a relative molecular mass of 41 kDa. The enzyme only hydrolyzed dihydro-l-orotate and its methyl ester, and the reactions were reversible. The apparentK _m andV _max values for dihydro-l-orotate hydrolysis (at pH 7.4) were 0.081 mM and 18 μmol min⁻¹ mg⁻¹, respectively; and those forN-carbamoyl-dl-aspartate (at pH 6.0) were 2.2 mM and 68 μmol min⁻¹ mg⁻¹, respectively. The enzyme was inhibited by metal ion chelators and activated by Zn²⁺. However, excessive Zn²⁺ was inhibitory. The enzyme was inhibited by sulfhydryl reagents, and competitively inhibited byN-carbamoylamino acids such asN-carbamoylglycine, with aK _i value of 2.7 mM. The enzyme was also inhibited noncompetitively by pyrimidine-metabolism intermediates such as dihydrouracil and orotate, with aK _i value of 3.4 and 0.75 mM, respectively, suggesting that the enzyme activity is regulated by pyrimidine-metabolism intermediates and that dihydroorotase plays a role in the control of pyrimidine biosynthesis.     

Two distinct cell sources of H2O2 in the lignifying Zinnia elegans cell culture system

Summary. The use of transdifferentiating Zinnia elegans mesophyll cells has proved useful in investigations of the process of xylem differentiation from cambial derivatives. Cultured mesophyll cells can be induced by external stimuli to proceed through temporally controlled developmental programs which conclude in the formation of single-cell-derived dead vascular tracheids and parenchyma-like elements. However, there is a gap in our knowledge concerning the role played by reactive oxygen species (O₂⁻ and H₂O₂) in the development of these vascular elements. In this study, we show by the following four independent and highly selective methods that transdifferentiating Z. elegans mesophyll cells are capable of producing reactive oxygen species: the 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) assay, which monitors O₂⁻ production, and the xylenol orange, 2,7-dichlorofluorescein diacetate, and CeCl₃ assays, which monitor H₂O₂ production and localization. The joint use of these biochemical (XTT and xylenol orange) assays and cytochemical (2,7-dichlorofluorescein diacetate and CeCl₃) probes revealed that transdifferentiating Z. elegans mesophyll cells do not show an oxidative burst but live in a strongly oxidative state during the entire culture period. In this state, H₂O₂ is produced by both tracheary and parenchyma-like elements, the nonlignifying parenchyma-like cells acting quantitatively as the main source. The existence of these two sources of H₂O₂ in this in vitro cell culture system may be especially relevant during the later stages of tracheary cell wall lignification, in which lignifying tracheary elements become hollow. In the case of differentiating tracheary elements, H₂O₂ was located in the same place and at the same time as the onset of tracheary element lignification, i.e., at the primary cell wall during secondary thickening, supporting the view that the H₂O₂ produced by this in vitro culture system is destined for use during lignin biosynthesis. Correspondence and reprints: Departamento de Biologia Vegetal, Facultad de Biologia, Universidad de Murcia, 30100 Murcia, Spain.     

Promotion of indole alkaloid production in Catharanthus roseus cell cultures by rare earth elements

Production of the indole alkaloids, ajmalicine or catharanthine, in cell suspension cultures of Catharanthus roseus was enhanced by cerium (CeO₂ and CeCl₃), yttrium (Y₂O₃) and neodymium (NdCl₃). The yield of ajmalicine in these treated-cultures reached 51 mg l^–1 (CeO₂), 40 mg l^–1 (CeCl₃), 41 mg l^–1 (Y₂O₃) and 49 mg l^–1 (NdCl₃) while catharanthine production reached to 36 mg l^–1 (CeO₂) and 31 mg l^–1 (CeCl₃). A major portion of increased alkaloids was released into medium in these treatments. But Sm₂O₃, SmCl₃, La₂O₃, LaCl₃, complex of chromium (III)-titanium (IV) and NaSeO₄ treatments had little effect on alkaloid production of C. roseus cell cultures.     

The effects of La(III) on the peroxidation of membrane lipids in wheat seedling leaves under osmotic stress

The physiological effects of the rare earth ion La³⁺ on the peroxidation of membrane lipids in wheat (Triticum aestivum L.) seedling leaves under osmotic stress were determined. With the passage of time under osmotic stress, the inhibition ability of lanthanum ions to the relative membrane permeability and concentration of malondialdehyde, Superoxide radicals, and hydrogen peroxide caused by osmotic stress increased substantially, but no changes were noted in ferrous and relative water content. It indicated that lanthanum ions could not retain the water content because of osmotic stress. However, La³⁺ appears to decrease the production of OH by reducing the content of O₂ and H₂O₂ of Haber-Weiss and Fenton reactions, which efficiently alleviated peroxidation of membrane lipids under osmotic stress and, to some degree, protected the membrane from injury of free radicals. Thus, La³⁺ increased the tolerance ability of plant to osmotic stress, which could assure the function of membrane normal temporally after stressed.     

Growth-associated biosynthesis of astaxanthin in heterotrophic Chlorella zofingiensis (Chlorophyta)

The green microalga Chlorella zofingiensis can produce the ketocarotenoid astaxanthin under heterotrophic culture conditions. Here we report the growth-associated biosynthesis of astaxanthin in this biotechnologically important alga. With glucose as sole carbon and energy source, C. zofinginesis grew fast in the dark with rapid exhaustion of nitrogen and carbon sources from media, leading to a high specific growth rate (0.034 h⁻¹). Cultures started at a cell concentration of about 3.4 × 10⁹ cells l⁻¹ reached, after 6 days, standing biomass values of 1.6 × 10¹¹ cells or 8.5 g dry weight l⁻¹. Surprisingly, the biosynthesis of astaxanthin was found to start at early exponential phase, independent of cessation of cell division. A general trend was observed that the culture conditions benefiting cell growth also benefited astaxanthin accumulation, indicating that astaxanthin was a growth-associated product in this alga. The maximum cell dry biomass and astaxanthin yield were 11.75 g l⁻¹ and 11.14 mg l⁻¹ (about 1 mg g⁻¹), simultaneously obtained in the fed-batch culture with a combined glucose–nitrate mixture addition, which were the highest ever reported in dark-heterotrophic algal cultures. The possible reasons why dark-heterotrophic C. zofingiensis could produce astaxanthin during the course of cell growth were discussed.     

Factors influencing efficiency of somatic embryogenesis of Gentiana kurroo (Royle) cell suspension

In this paper, we would like to show unexpected morphogenic potential of cell suspensions derived from seedling explants of Gentiana kurroo (Royle). Suspension cultures were established with the use of embryogenic callus derived from seedling explants (root, hypocotyl and cotyledons). Proembryogenic mass proliferated in liquid MS medium supplemented with 0.5 mg l⁻¹ 2,4-D and 1.0 mg l⁻¹ Kin. The highest growth coefficient was achieved for root derived cell suspensions. The microscopic analysis showed differences in aggregate structure depending on their size. To assess the embryogenic capability of the particular culture, 100 mg of cell aggregates was implanted on MS agar medium supplemented with Kin (0.0–2.0 mg l⁻¹), GA₃ (0.0–2.0 mg l⁻¹) and AS (80.0 mg l⁻¹). The highest number of somatic embryos was obtained for cotyledon-derived cell suspension on GA₃-free medium, but the best morphological quality of embryos was observed in the presence of 0.5–1.0 mg l⁻¹ Kin, 0.5 mg l⁻¹ GA₃ and 80.0 mg l⁻¹ AS. The morphogenic competence of cultures also depended on the size of the aggregate fraction and was lower when size of aggregates decreased. Flow cytometry analysis reveled luck of uniformity of regenerants derived from hypocotyl suspension and 100% of uniformity for cotyledon suspension.