Cadmium Accumulation in Relation to Organic Acids and Nonprotein Thiols in Leaves of the Recently Found Cd Hyperaccumulator <Emphasis Type="Italic">Rorippa globosa</Emphasis> and the Cd-accumulating Plant <Emphasis Type="Italic">Rorippa islandica</Emphasis>

It has been proposed that organic acid and nonprotein thiol are involved in the hyperaccumulation of metals. In this study, Cd accumulation, organic acid, and nonprotein thiol production and their relationships in the leaves of Cd-hyperaccumulator Rorippa globosa were examined and compared with a closely related species, Rorippa islandica. The results showed that there was no reduction in biomass of R. globosa when treated with 25 μg Cd g⁻¹ (T₂), despite Cd accumulation in the leaves was up to 158.2 μg g⁻¹ DW. On the other hand, the growth of Cd-treated R. islandica was obviously inhibited as it accumulated more than 100 μg g⁻¹ DW in the leaves. Therefore, R. islandica behaved as a Cd-accumulating plant. The Cd treatments could significantly induce the synthesis of acetic acid in both species, suggesting that acetic acid, as the most abundant organic acid, might be related to the Cd accumulation. Significant positive correlations between Cd concentrations and both tartaric and malic acid concentrations in the leaves of R. globosa were observed. There was a significant positive correlation between Cd concentrations and acetic acid concentrations in the leaves of R. islandica. This trend of tartaric and malic acids in the leaves of R. globosa and acetic acid in the leaves of R. islandica might be related to Cd accumulation. In addition, a quadratic relationship was obviously observed for NP-SH contents and total Cd concentrations in the leaves of R. globosa, indicating that NP-SH was significantly related to Cd accumulation and tolerance.     

Sulphur protects mustard (<Emphasis Type="Italic">Brassica campestris</Emphasis> L.) from cadmium toxicity by improving leaf ascorbate and glutathione

In a pot-soil culture ameliorative effect of sulphur (S) (0 or 40 mg S kg⁻¹ soil) on cadmium (Cd) (0, 25, 50 and 100 mg Cd kg⁻¹ soil)-induced growth inhibition and oxidative stress in mustard (Brassica campestris L.) cultivar Pusa Gold was studied. Cadmium at 100 mg kg⁻¹ soil caused maximum increase in the contents of Cd and thiobarbituric acid reactive substances (TBARS) in leaves. Maximum reductions in growth (plant dry mass, leaf area), chlorophyll content, net photosynthetic rate (P_N) and the contents of ascorbate (AsA), glutathione (GSH) were observed with 100 mg Cd kg⁻¹ soil compared to control. The application of S helped in reducing Cd toxicity, which was greater for 25 and 50 mg Cd kg⁻¹ soil) compared to 100 mg Cd kg⁻¹ soil. Addition of S to Cd-treated plants showed decrease in Cd and TBARS content in leaves and restoration of growth and photosynthesis through increase in the contents of AsA and GSH. Net photosynthetic rate and plant dry mass were strongly and positively correlated with the contents of AsA and GSH. It is suggested that S may ameliorate Cd toxicity and protects growth and photosynthesis of mustard involving AsA and GSH.     

The influence of cadmium on life-history parameters and gut microflora of <Emphasis Type="Italic">Archegozetes longisetosus</Emphasis> (Acari: Oribatida) under laboratory conditions

We tested the effect of cadmium (25, 130 μg Cd g⁻¹), administered via Chinese cabbage (Brassica chinensis L.) as food on life-history parameters and gut microflora of tritonymphs and adults of the oribatid mite, Archegozetes longisetosus Aoki. Both concentrations of Cd had an adverse effect on offspring mortality, and the higher concentration also reduced female fecundity, as well as the number of bacteria, fungi and actinomycetes, and it changed the community structure of bacteria; the proportion of gram-negative bacteria increased while that of gram-positive bacteria declined. Interestingly, at the lower Cd concentration microflora was more abundant and diverse than in the control group, especially in the tritonymphs, although the mean activity of gut microflora was reduced. The higher Cd concentration reduced microflora activity both in the tritonymphs and adults.     

Plant regeneration of the mining ecotype <Emphasis Type="Italic">Sedum alfredii</Emphasis> and cadmium hyperaccumulation in regenerated plants

An efficient micropropagation system for mining ecotype Sedum alfredii Hance, a newly identified Zn/Cd hyperaccumulator, was developed. Frequency of callus induction reached up to 70% from leaves incubated on Murashige and Skoog (MS) medium supplemented with 1.0 mg l⁻¹ 2,4-dichlorophenoxy acetic acid (2,4-D) and 0.5 mg l⁻¹ 6-benzyladenine (BA), and 83% from internodal stem segments grown on MS medium with 0.1 mg l⁻¹ 2,4-D and 0.1 mg l⁻¹ BA. Callus proliferated rapidly on MS medium containing 0.2 mg l⁻¹ 2,4-D and 0.05 mg l⁻¹ thidiazuron. The highest number of adventitious buds per callus (17.3) and frequency of shoot regeneration (93%) were obtained when calli were grown on MS medium supplemented with 2.0 mg l⁻¹ BA and 0.3 mg l⁻¹ α-naphthalene acetic acid (NAA). Elongation of shoots was achieved when these were incubated on MS medium containing 3.0 mg l⁻¹ gibberellic acid. Induction of roots was highest (21.4 roots per shoot) when shoots were transferred to MS medium containing 2.0 mg l⁻¹ indole 3-butyric acid rather than either indole 3-acetic acid or NAA. When these in vitro plants were acclimatized and transferred to the greenhouse, and grown in hydroponic solutions containing 200 μM cadmium (Cd), they exhibited high efficiency of Cd transport, from roots to shoots, and hyperaccumulation of Cd.     

Fungal autolysate as a nutrient supplement for ethanol and chitosan production by <Emphasis Type="Italic">Mucor indicus</Emphasis>

Mucor indicus can be used to produce ethanol from a variety of sugars, including pentose’s. An extract of it, produced by autolysis, could replace yeast extract in culture medium with improved production of ethanol. At 10 g l⁻¹, the extract gave a higher ethanol yield (0.47 g g⁻¹) and productivity (0.71 g l⁻¹ h⁻¹) compared to medium containing yeast extract (yield 0.45 g g⁻¹; productivity 0.67 g l⁻¹ h⁻¹).     

Cadmium accumulation in relation to organic acids in leaves of Solanum nigrum L. as a newly found cadmium hyperaccumulator

The influence of various cadmium concentrations on organic acid levels in leaves of the Cd hyperaccumulator, Solanum nigrum L. and a closely related species, Solanum melongena L., were investigated. In particular, the relationship of organic acids with Cd accumulation in the two plants was investigated. The results showed that Cd accumulation in the shoots of S. nigrum was significantly higher than that of S. melongena. The accumulation of Cd in the leaves of S. nigrum ranged from 2.0 to 167.8 μg g⁻¹ dry weight (DW), but only from 1.2 to 64.0 μg g⁻¹ DW in S. melongena. Solanum melongena was considerably less tolerant to Cd than S. nigrum. Approximately 20% of the total Cd in S. nigrum leaves was water-soluble, suggesting that some accumulated Cd was associated with water-soluble compounds such as organic acids. Malic acid in the leaves of S. nigrum was the most abundant organic acid [up to 115.6–145.7 μmol g⁻¹ fresh weight (FW)], but this acid was not significantly affected by the Cd concentration in soil. However, the level of malic acid in S. melongena plants was much lower, only 16.3–75.4 μmol g⁻¹ FW. The significant positive correlations between total Cd and water-soluble Cd concentrations and both acetic and citric acid concentrations in the leaves of S. nigrum were observed. In contrast, there was no correlation between concentrations of the two acids and Cd concentrations in the leaves of S. melongena. These results indicated that acetic and citric acids in the leaves of S. nigrum might be related to its Cd hyperaccumulation.     

Cost-benefit relationships in sclerophyllous leaves of the ‘Bana’ vegetation in the Amazon region

This study estimated the construction const (CC) and maintenance cost (MC) of leaf tissue on the basis of dry mass (CC_Mass, MC_Mass) and leaf area (CC_Area, MC_Area), as well as the maximum leaf gas exchange capacity, so as to examine leaf cost:benefit relationship in six dominant species of the ‘Bana’ vegetation. Minimum and maximum CC_Mass averaged 1.71 ± 0.03 and 1.78 ± 0.03 g glucose g⁻¹. The CC_Mass showed a statistically significant positive correlation with crude fibre, and tended to decline as leaves were larger. Thus, smaller leaves tended to be built out of a more expensive material than that found in species bearing larger leaves. The average CC_Area of the ‘Bana’ species was 376 ± 15 g glucose m⁻². A robust correlation was found between CC_Area with leaf dry mass to leaf area ratio, as well as with leaf thickness, but not with leaf density. MC_Mass (g glucose g⁻¹ day⁻¹) and MC_Area (g glucose m⁻² day⁻¹⁾ were positively correlated. Maximum and minimum MC_Mass increased significantly with protein and lipid content, respectively. Maximum carbon assimilation (A _max) was positively correlated with CC_Area. All the species operated at high stomatal conductance (g _s) and C _i/C _a which suggested low short-term water use efficiency. Potential nitrogen use efficiency (PNUE = A _max/N) averaged 35.4 ± 1.8 mmol CO₂ mol⁻¹ N. As the sclerophylly index (g crude fibre g⁻¹ protein) increased, the ratio of CC_Area to A _max increased significantly. This result suggests a trade-off between investments in an expensive resistant sclerophyllous leaf which should maximize carbon gain in the long term.     

Characterization of Cadmium-Binding Ligands from Roots of <Emphasis Type="Italic">Echinochloa crusgalli</Emphasis> var. <Emphasis Type="Italic">frumentacea</Emphasis>

Herbaceous Echinochloa crusgalli var. frumentacea is highly resistant to a wide range of heavy metal concentrations. However, its detoxification mechanism has not been reported yet. We exposed plants to 100 μM CdCl₂ for 7 days then examined cadmium accumulation and its subcellular distribution in binding to ligands. Cd concentration increased over the exposure period to a saturation point at day 5 when its level reached 1,732.41 μg g⁻¹ dry weight, an amount about 820 times greater than that found in the control. In the roots, most Cd was distributed in the insoluble fraction, perhaps because of an absorption mechanism at the root surface. Our profile for distribution revealed two Cd-binding ligand peaks: a high molecular weight of 60 kDa and a 2.5-kDa Cd-binding ligand. The latter increased with time and had a typical phytochelatin (PC) amino acid composition of predominantly cysteine, glutamate, and glycine (16.5%, 16.6%, and 11.9%, respectively). The ratio of glutamate/cysteine/glycine was 1.4:1.4:1.0, which is similar to that for other typical PCs.     

Physiological and antioxidant responses of <Emphasis Type="Italic">Mentha pulegium</Emphasis> (Pennyroyal) to salt stress

Mentha pulegium L. is a medicinal and aromatic plant belonging to the Labiatae family present in the humid to the arid bioclimatic regions of Tunisia. We studied the effect of different salt concentrations on plant growth, mineral composition and antioxidant responses. Physiological and biochemical parameters were assessed in the plant organs after 2 weeks of salt treatment with 25, 50, 75 and 100 mM NaCl. Results showed that, growth was reduced even by 25 mM, and salt effect was more pronounced in shoots (leaves and stems) than in roots. This growth decrease was accompanied by a restriction in tissue hydration and K⁺ uptake, as well as an increase in Na⁺ levels in all organs. Considering the response of antioxidant enzymes to salt, leaves and roots reacted differently to saline conditions. Leaf and root guaiacol peroxidase activity showed an increase by different concentration of NaCl, but superoxide dismutase activity in the same organs showed a slight modification in NaCl-treated leaves and roots. Moreover, polyphenol contents and antioxidant activity were analysed in M. pulegium leaves and roots under salt constraint. The analysis showed an increase of total polyphenol content (2.41–8.17 mg gallic acid equivalent g⁻¹ dry weight) in leaves. However, methanol extract of leaves at 100 mM NaCl displayed the highest DPPH· scavenging ability with the lowest IC₅₀ value (0.27 μg ml⁻¹) in comparison with control which exhibited IC₅₀ equal to 0.79 μg ml⁻¹.     

Carbon source pulsed feeding to attain high yield and high productivity in poly(3-hydroxybutyrate) (PHB) production from soybean oil using <Emphasis Type="Italic">Cupriavidus necator</Emphasis>

Poly(3-hydroxybutyrate) (PHB) biosynthesis from soybean oil by Cupriavidus necator was studied using a bench scale bioreactor. The highest cell concentration (83 g l⁻¹) was achieved using soybean oil at 40 g l⁻¹ and a pulse of the same concentration. The PHB content was 81% (w/w), PHB productivity was 2.5 g l⁻¹ h⁻¹, and the calculated Y_p/s value was 0.85 g g⁻¹. Growth limitation and the onset of PHB biosynthesis took place due to exhaustion of P, and probably also Cu, Ca, and Fe.     

Efficient production of lactic acid from sucrose and corncob hydrolysate by a newly isolated <Emphasis Type="Italic">Rhizopus oryzae</Emphasis> GY18

The aim of this study is to investigate production of l-lactic acid from sucrose and corncob hydrolysate by the newly isolated R. oryzae GY18. R. oryzae GY18 was capable of utilizing sucrose as a sole source, producing 97.5 g l⁻¹ l-lactic acid from 120 g l⁻¹ sucrose. In addition, the strain was also efficiently able to utilize glucose and/or xylose to produce high yields of l-lactic acid. It was capable of producing up to 115 and 54.2 g l⁻¹ lactic acid with yields of up to 0.81 g g⁻¹ glucose and 0.90 g g⁻¹ xylose, respectively. Corncob hydrolysates obtained by dilute acid hydrolysis and enzymatic hydrolysis of the cellulose-enriched residue were used for lactic acid production by R. oryzae GY18. A yield of 355 g lactic acid per kg corncobs was obtained after 72 h incubation. Therefore, sucrose and corncobs could serve as potential sources of raw materials for efficient production of lactic acid by R. oryzae GY18.     

Salicylic acid-mediated alleviation of cadmium toxicity in hemp plants in relation to cadmium uptake,photosynthesis, and antioxidant enzymes

To assess the role of salicylic acid (SA) in alleviating cadmium (Cd) toxicity in hemp (Cannabis sativa L.) plants, the growth parameters, Cd accumulation, photosynthetic performance and activities of major antioxidant enzymes were investigated in hemp seedlings treated with 500 μM SA, under 0, 25, 50, and 100 mg Cd kg⁻¹ sands (DW) conditions, respectively. Cd exposure resulted in a small reduction in biomass (12.0–26.9% for root, and 8.7–29.4% for shoot, respectively), indicating hemp plants have innate capacity to tolerant Cd stress. This was illustrated by little inhibition in photosynthetic performance, unchanged malondialdehyde content, and enhancement of superoxide dismutase (SOD) and peroxidases (POD) activities in hemp plants. Cd content in root is 25.0–29.5 times’ higher than that in shoot, suggesting the plant can be classified as a Cd excluder. It is concluded that SA pretreatment counteracted the Cd-induced inhibition in plant growth. The beneficial effects of SA in alleviating Cd toxicity can be attributed to the SA-induced reduction of Cd uptake, improvement of photosynthetic capacity, and enhancement of SOD and POD activities.     

Fed-batch production of a bioflocculant from <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis>

The constant-rate fed-batch production of the polygalacturonic acid bioflocculant REA-11 was studied. A controlled sucrose-feeding strategy resulted in a slight improvement in biomass and a 7% reduction in flocculating activity compared with the batch process. When fed with a 3 g l⁻¹ urea solution, the flocculating activity was enhanced to 720 U ml⁻¹ in 36 h. High cell density (2.12 g l⁻¹) and flocculating activity (820 U ml⁻¹) were obtained in a 10-l fermentor by feeding with a sucrose-urea solution, with values of nearly two times and 50% higher than those of the batch process, respectively. Moreover, the residual sucrose declined to 2.4 g l⁻¹, and residual urea decreased to 0.03 g l⁻¹. Even higher flocculating activity of 920 U ml⁻¹ and biomass of 3.26 g l⁻¹ were obtained by feeding with a sucrose-urea solution in a pilot scale fermentation process, indicating the potential industrial utility of this constant-rate feeding strategy in bioflocculant production by Corynebacterium glutamicum.     

Rate of dehydration,state of subcellular organisation and nature of cryoprotection are critical factors contributing to the variable success of cryopreservation: studies on recalcitrant zygotic embryos of <Emphasis Type="Italic">Haemanthus montanus</Emphasis>

Effects of sequential procedures required for cryopreservation of embryos excised from the recalcitrant seeds of Haemanthus montanus were assessed ultrastructurally and in conjunction with respiratory activity and the rate of protein synthesis. Fresh material (water content, 5.05 ± 0.92 g g⁻¹ dry mass) afforded ultrastructural evidence of considerable metabolic activity, borne out by respiratory rates. Neither exposure to glycerol nor sucrose as penetrating and non-penetrating cryoprotectants, respectively, brought about degradative changes, although increased vacuolation and autophagy accompanied both, while respiratory and protein synthetic activity were not adversely affected. Glycerol-cryoprotected embryos flash dried to water contents >0.4 g g⁻¹ showed organised ultrastructural features and considerable autophagy consistent with metabolic activity, and although respiratory activity was lower, protein synthesis rate was enhanced relative to fresh material. However, at water contents <0.4 g g⁻¹, embryo tissue presented a mosaic of cells of variable density and ultrastructural status, but trends in rates of respiration and protein synthesis remained similar. Flash drying after sucrose exposure was accompanied by considerable ultrastructural abnormality particularly at water contents <0.4 g g⁻¹, lysis of individual and groups of cells and considerable depression of respiration, but not of protein synthesis. Success, assessed as ≥50% axes forming seedlings after cryogen exposure, was obtained only when glycerol-cryoprotected embryos at water contents >0.4 g g⁻¹—in which the degree of vacuolation remained moderate—were rapidly cooled. The outcomes of this study are considered particularly in terms of the stresses imposed by prolonged, relatively slow dehydration and ultimate water contents, on embryos showing considerable metabolic activity.     

Glycerol/Glucose Co-Fermentation: One More Proficient Process to Produce Propionic Acid by <Emphasis Type="Italic">Propionibacterium acidipropionici</Emphasis>

Cosubstrates fermentation is such an effective strategy for increasing subject metabolic products that it could be available and studied in propionic acid production, using glycerol and glucose as carbon resources. The effects of glycerol, glucose, and their mixtures on the propionic acid production by Propionibacterium acidipropionici CGMCC1.2225 (ATCC4965) were studied, with the aim of improving the efficiency of propionic acid production. The propionic acid yield from substrate was improved from 0.475 and 0.303 g g⁻¹ with glycerol and glucose alone, respectively, to 0.572 g g⁻¹ with co-fermentation of a glycerol/glucose mixture of 4/1 (mol/mol). The maximal propionic acid and substrate conversion rate were 21.9 g l⁻¹ and 57.2% (w/w), respectively, both significantly higher than for a sole carbon source. Under optimized conditions of fed-batch fermentation, the maximal propionic acid yield and substrate conversion efficiency were 29.2 g l⁻¹ and 54.4% (w/w), respectively. These results showed that glycerol/glucose co-fermentation could serve as an excellent alternative to conventional propionic acid fermentation.     

Hydrophobic response of the fungus <Emphasis Type="Italic">Rhinocladiella similis</Emphasis> in the biofiltration with volatile organic compounds with different polarity

Rhinocladiella similis biodegraded volatile organic compounds (VOCs) of different polarity in gas-phase biofilters. Elimination capacities, (EC) of 74 g_hexane m⁻³ h⁻¹, 230 g_ethanol m⁻³ h⁻¹, 85 g_toluene m⁻³ h⁻¹ and 30 g_phenol m⁻³ h⁻¹ were obtained. EC values correlated with the solubility of the VOCs. R. similis grown with n-hexane or ethanol in biofilters packed with Perlite showed that the surface hydrophobicity was higher with n-hexane than ethanol. The hydrophobin-like proteins extracted from the mycelium produced with n-hexane (15 kDa) were different from those in the ethanol biofilter (8.5 kDa and 7 kDa).     

Metallothionein as Potential Biomarker of Cadmium Exposure in Persian Sturgeon (<Emphasis Type="Italic">Acipenser persicus</Emphasis>)

Metallothionein (MT) concentration in gills, liver, and kidney tissues of Persian sturgeon (Acipenser persicus) were determined following exposure to sublethal levels of waterborne cadmium (Cd) (50, 400, and 1,000 μg l⁻¹) after 1, 2, 4, and 14 days. The increases of MT from background levels were 4.6-, 3-, and 2.8-fold for kidney, liver, and gills, respectively. The results showed that MT level change in the kidney is time and concentration dependent. Also, cortisol measurement revealed elevation at the day 1 of exposure and followed by MT increase in the liver. Cd concentrations in the cytosol of experimental tissues were measured, and the results indicated that Cd levels in the cytosol of liver, kidney, and gills increased 240.71-, 32.05-, and 40.16-fold, respectively, 14 days after exposure to 1,000 μg l⁻¹ Cd. The accumulation of Cd in cytosol of tissues is in the order of liver > gills > kidney. Pearson correlation coefficients showed that the MT content in kidney is correlated with Cd concentration, the value of which is more than in liver and gills. Thus, kidney can be considered as a tissue indicator in A. persicus for waterborne Cd contamination.     

Bioethanol production in batch mode by a native strain of <Emphasis Type="Italic">Zymomonas mobilis</Emphasis>

Two wild strains of Zymomonas mobilis were isolated (named as ML1 and ML2) from sugar cane molasses obtained from different farms of Santander, Colombia. Initially, selection of the best ethanol-producer strains was carried out using ethanol production parameters obtained with a commercial strain Z. mobilis DSM 3580. Three isolated strains were cultivated in a culture medium containing yeast extract, peptone, glucose and salts, at pH 6 and 32°C with stirring rate of 65 rpm during 62 h. The best results of ethanol production were obtained with the native strain ML1, reaching a maximum ethanol concentration of 79.78 g l⁻¹. ML1 and ML2 strains were identified as Z. mobilis, according to the morphology, biochemical tests and molecular characterization by PCR of specific DNA sequences from Z. mobilis. Subsequently, the effect of different nitrogen sources on production of ethanol was evaluated. The best results were obtained using urea at a 0.73 g/l. In this case, maximum concentration of ethanol was 83.81 g l⁻¹, with kinetic parameters of yield of ethanol on biomass (Y_P/X) = 69.01(g g⁻¹), maximum volumetric productivity of ethanol (Qp_max) = 2.28 (g l⁻¹ h⁻¹), specific productivity of ethanol (q_P) = 3.54 (h⁻¹) and specific growth rate (μ) = 0.12 h⁻¹. Finally, we studied the effect of different culture conditions (pH, temperature, stirring, C/N ratio) with a Placket-Burman′s experimental design. This optimization indicated that the most significant variables were temperature and stirring. In the best culture conditions a significant increase in all variables of response was achieved, reaching a maximum ethanol concentration of 93.55 g l⁻¹.     

Xylitol production by recombinant Corynebacterium glutamicum under oxygen deprivation

Wild-type Corynebacterium glutamicum produced 0.6 g l⁻¹ xylitol from xylose at a productivity of 0.01 g l⁻¹ h⁻¹ under oxygen deprivation. To increase this productivity, the pentose transporter gene (araE) from C. glutamicum ATCC31831 was integrated into the C. glutamicum R chromosome. Consequent disruption of its lactate dehydrogenase gene (ldhA), and expression of single-site mutant xylose reductase from Candida tenuis (CtXR (K274R)) resulted in recombinant C. glutamicum strain CtXR4 that produced 26.5 g l⁻¹ xylitol at 3.1 g l⁻¹ h⁻¹. To eliminate possible formation of toxic intracellular xylitol phosphate, genes encoding xylulokinase (XylB) and phosphoenolpyruvate-dependent fructose phosphotransferase (PTS^fru) were disrupted to yield strain CtXR7. The productivity of strain CtXR7 increased 1.6-fold over that of strain CtXR4. A fed-batch 21-h CtXR7 culture in mineral salts medium under oxygen deprivation yielded 166 g l⁻¹ xylitol at 7.9 g l⁻¹ h⁻¹, representing the highest bacterial xylitol productivity reported to date.     

Camptothecin accumulation in various organ cultures of <Emphasis Type="Italic">Camptotheca acuminata</Emphasis> Decne grown in different culture systems

Levels of camptothecin (CPT) and 10-hydroxycamptothecin (HCPT) were determined in different cultures of Camptotheca acuminata grown either in a Temporary Immersion System (TIS) or on solid medium. CPT was also detected in liquid culture medium. HPLC analysis showed significant differences in CPT contents in all tissues analysed and the highest CPT contents were found in shoots grown on solid medium and in TIS with a mean of 2.2 and 2.5 mg g⁻¹ DW, respectively. The highest content of CPT detected in seedlings was 1.96 mg g⁻¹ DW; while that of somatic embryos at cotyledonary stage and regenerated plants were 0.87 and 1.23 mg g⁻¹ DW, respectively. It was also shown that shoots cultured in TIS secreted substantial amount of CPT into the liquid medium. After 4 weeks in culture a mean of 6, 05 and 12, 6 μg g⁻¹ FW were determined at 4 and 8 immersion cycles daily (IC d⁻¹), respectively. This aspect opens new possibilities regarding the isolation of CTP using TIS culture systems.