Effects of Chromium(III) Picolinate on Cortisol and DHEAs Secretion in H295R Human Adrenocortical Cells

Dietary chromium(III) picolinate (CrPic) effects on circulating steroid hormones have been reported in various experimental animals. However, direct effects of CrPic on adrenocortical steroidogenesis are uncertain. Therefore, the objective was to determine the effects of CrPic on cortisol and dehydroepiandrosterone sulfate (DHEAs) secretion from H295R cells. In experiment 1, a 24-h exposure to CrPic (0 to 200 μM) had both linear (p < 0.001) and quadratic (p < 0.001) effects on cortisol secretion from forskolin-stimulated cells with the highest cortisol secretion at 0.1 μM of CrPic and the lowest at 200 μM of CrPic. In experiment 2, a 48-h exposure to CrPic (200 μM) decreased cortisol (p < 0.07) release from forskolin-stimulated cells during a 24-h collection period. In experiment 3, a 48-h exposure to CrPic (100 μM) decreased cortisol (p < 0.05) and DHEAs (p < 0.01) from forskolin-stimulated cells during a 24-h sampling period. In experiment 4, a 24-h exposure to forskolin followed by a 24-h exposure to both forskolin and CrPic (100 and 200 μM) decreased both cortisol and DHEAs secretion (p < 0.01). This study suggests that at high concentrations, CrPic inhibits aspects of steroidogenesis in agonist-stimulated adrenocortical cells.     

Chromosome doubling in a <Emphasis Type="Italic">Rosa rugosa</Emphasis> Thunb. hybrid by exposure of in vitro nodes to oryzalin: the effects of node length,oryzalin concentration and exposure time

Chromosome doubling was induced in vitro in a diploid hybrid of Rosa rugosa Thunb. using oryzalin as the spindle inhibitor. Nodal sections, 2 mm long, were exposed to 2.5 or 5 μM oryzalin and 10 mm nodal sections were exposed to 5 μM oryzalin for 0 (controls), 6, 12, 24 and 48 h. The ploidy of the emergent shoots was determined by flow cytometry. The frequency of tetraploid and mixoploid leaves that developed from 2 mm nodal sections exposed to 5 μM oryzalin peaked at 12 h exposure, when 35% of the leaves were tetraploid, but fell after longer exposures. Fewer tetraploid and mixoploid leaves were found when 2 mm nodes were exposed to 2.5 μM oryzalin for 6 and 12 h, indicating that it took longer for a spindle inhibiting concentration of oryzalin to build up in the meristem. However, the frequencies of tetraploid and mixoploid leaves continued to rise after 12 h and were highest at 48 h, when 44% were tetraploid. In treatments with 5 μM oryzalin, the frequencies of tetraploid and mixoploid leaves were lower, at equivalent exposure times, in 10 mm nodes than 2 mm nodes. This suggests that oryzalin diffused to the meristem mainly via the cut surfaces and that access via the epidermis and cuticle was impeded.     

Capsaicin formation in p-fluorophenylalanine resistant and normal cell cultures ofCapsicum frutescens and activity of phenylalanine ammonia lyase

Callus cultures ofCapsicum frutescens capable of producing a maximum of 53 μg capsaicin/g FW were exposed to various levels of p-fluorophenyialanine (PFP) at 100, 400, 1000 and 2000 μM to develop a resistant cell line that over produces capsaicin. After 15 days of culturing on media lacking PFP, cell lines resistant to 100, 400 and 1000 μM registered 18%, 34.5% and 45% increase in capsaicin content over normal cell line (cells not exposed to PFP). Capsaicin accumulation was inhibited in 2000 μM PFP resistant cell line. The profile of phenylalanine ammonia lyase (PAL), the key enzyme in pheny1propanoid pathway in resistant cell cultures was studied and compared with normal cell cultures to understand its role in capsaicin formation. Importantly increased production of capsaicin was obtained using PFP resistant cell lines. The activity profile of PAL had no correlation with capsaicin content in both control and PFP resistant cells.     

Physiological behavior of Scenedesmus sp. during exposure to elevated levels of Cu and Zn and after withdrawal of metal stress

Summary. A 48 h exposure of Scenedesmus sp. to sublethal concentrations of Cu (2.5 and 10 μM) and Zn (5 and 25 μM) caused a concentration-dependent inhibition of growth, photosynthesis, respiration, NO₃ ⁻ uptake, and nitrate reductase (EC 1.6.6.1) activity, and a reduction in protein, carbohydrate, and photosynthetic-pigment levels with a concomitant increase in intracellular levels of the test metals. After exposure, algal cells were transferred to the basal medium without the excess level of test metals, to study the recovery of various processes. The growth of the test algae had not recovered up to 12 h after transfer to the basal medium, but some physiological parameters such as photosynthesis and respiration recovered within 6 h. The quicker recovery of photosynthesis and respiration might be used as acclimatory responses as they prepare a background for the recovery of other parameters, including growth, of the test alga by generating energy, forming photosynthate, and establishing the usual catabolism to attain normal conditions. Most of the processes recovered completely or almost completely after being stressed with 2.5 μM Cu or 5 μM Zn. However, the maintenance of a relatively high level of Cu and Zn in the cells previously exposed to 10 μM Cu and 25 μM Zn slowed down the recovery of different processes, which did not fully recover even at the end of the experiment after 96 h. The present study demonstrates that a chain of metabolic events, beginning with respiration and photosynthesis and continuing with assimilation and uptake of nutrients and subsequent restoration of other metabolic processes, is involved in the recovery of the algae from Cu and Zn stress. Each studied parameter seems to play an important role in balancing the cellular homeostasis during recovery from metal stress. Correspondence and reprints: Department of Bioscience and Biotechnology, Banasthali Vidyapith, Banasthali 304 022, Rajasthan, India.     

The use of human epidermal keratinocytes in culture as a model for studying the biochemical mechanisms of sulfur mustard toxicity

Human epidermal keratinocytes in culture were studied to evaluate their usefulness in demonstrating toxic events following exposure to sulfur mustard. Exposure of keratinocytes to sulfur mustard over a concentration range of 1–1000 μM HD, reduced NAD+ levels from 96% to 32% of control levels. When keratinocytes were exposed to a concentration of 300 μM HD, NAD+ levels began to fall at 1 hour and reached a plateau of 47% of control levels at 4 hours. Niacinamide, an inhibitor of the enzyme poly(ADP-ribose) polymerase, partially protected mustard-exposed cells against NAD+ depletion. It also protected cellular viability as assessed by vital staining 24 hours after exposure. This protection was not seen in long-term (72 hr) cultures. These studies suggest that human epidermal keratinocytes in culture can serve as a usefulin vitro model for research into the biochemical mechanisms of sulfur mustard-induced cutaneous injury.     

Pathognomonic genetic expression profile within peripheral blood mononuclear cells of rheumatic heart disease patients

In this study we have tested the effects of d-propranolol (D-Pro) on the iron uptake, iron release and oxidative response of iron-loaded cells in a cellular model of iron-overload using isolated rat peritoneal macrophages incubated with iron-dextran (Fe-D). Pretreatment of macrophages with D-Pro (5–200 μ M) prior to Fe-D exposure decreased the cellular iron content and partially prevented iron release from latex-activated macrophages. Release of reactive oxygen species from activated cells was detected by dichlorodihydrofluorescein (DCDHF, 5 μ M) oxidation. We found that loading cells with Fe-D increased their response to latex, which was prevented by the lysosomotropic antioxidant agent D-Pro (10 μ M).     

Effect of Strontium Ranelate on Hydrogen Peroxide-Induced Apoptosis of CRL-11372 Cells

In vitro and in vivo studies have proven strontium to be an osteoinductive trace element. The effect of strontium ranelate (SR) on H₂O₂-induced apoptosis of CRL-11372 cells and optimization of its anti-apoptotic dose were the aims of this study. After 1 h of pretreatment with SR 1 μM, 50 μM, 100 μM, 500 μM, and 1,000 μM concentrations, CRL-11372 osteoblasts were exposed to 100 μM H₂O₂ for periods of 6–12 h. The same experiments were repeated without H₂O₂. The apoptotic index and viability of cells were assessed quantitatively with a fluorescent dye and qualitatively with agarose gel electrophoresis. Concentrations of 1–100 μM of SR with a 6-h treatment and only 1 μM concentration with a 12-h treatment inhibited the apoptotic effect of H₂O₂ on cultured osteoblasts significantly (P < 0.05). SR was shown to inhibit H₂O₂-induced apoptosis of CRL-11372 cells in a dose-dependent manner.     

Neuroprotective Effects of Chalcones from <Emphasis Type="Italic">Myracrodruon urundeuva</Emphasis> on 6-Hydroxydopamine-Induced Cytotoxicity in Rat Mesencephalic Cells

In the present work, we showed that a chalcone-enriched fraction (CEF) isolated from the stem bark of a Brazilian medicinal plant, Myracrodruon urundeuva, presents neuroprotective actions on 6-hydroxydopamine (6-OHDA)-induced neuronal cell death, in rat mesencephalic cells. In the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium] assay, which is an index of cell viability, CEF (1–100 μg/ml) reversed in a concentration-dependent manner the 6-OHDA-induced cell death. While cells exposed to 6-OHDA (40 μM) showed an increased concentration of thiobarbituric acid reactive substances (TBARS), the pretreatment with CEF (10–100 μg/ml) significantly decreased the 6-OHDA-induced TBARS formation, indicative of a neuroprotection against lipoperoxidation. Furthermore, the drastic increase of nitrite levels induced by 6-OHDA, indicative of nitric oxide formation and free radicals production, was prevented by CEF. Double staining with acridine orange/ethidium bromide showed that cultures exposed to 6-OHDA (40 and 200 μM) presented an increase of apoptotic and necrotic cell numbers in a concentration-dependent manner. CEF (100 μg/ml) protected cells from apoptosis and necrosis and increased number of cells presenting a normal morphology. The immunohistochemical analysis for tyrosine hydroxylase (TH) positive neurons indicated that 6-OHDA (40 and 200 μM) caused a concentration-dependent loss of TH+ and TH− neurons. CEF protected both cells types from 6-OHDA-induced cell death. All together, our results demonstrated neuroprotective effects of chalcones, which are able to reduce oxidative stress and apoptotic injury caused by 6-OHDA. Our findings suggest that chalcones could provide benefits, along with other therapies, in neurodegenerative injuries, such as Parkinson’s disease.     

Real-time in vivo visualization of oxidative stress in duckweed (Lemna minor L.)

An experimental set-up which enabled non-invasive, real-time reactive oxygen species (ROS) visualization on a whole plant level was constructed. In the test organism, Lemna minor L. (common duckweed), apoplastic and symplastic oxidative stress was evaluated by exposure to menadione (50 μM), menadione (50 μM) + ascorbate (100 μM) or neither for control. Menadione (50 μM) caused a statistically significant increase in H₂DCFDA fluorescence in the apoplast after 60 minutes of exposure. The addition of ascorbate (100 μM) in the test medium significantly decreased apoplastic oxidative stress. 50 μM menadione caused an increase in symplastic H₂DCFDA fluorescence in 57% of fronds. The exposure of L. minor plants to both menadione and ascorbate decreased the rate of fluorescence intensity accumulation in the symplast to control levels. The method has proven to be quick and straightforward and could be applied to a range of chemicals in various physiological and toxicological plant studies. The advantages of the set-up and different possible artefacts are discussed.     

Zinc alleviates mercury-induced oxidative stress in <Emphasis Type="Italic">Pfaffia glomerata</Emphasis> (Spreng.) Pedersen

The possible role of zinc (Zn) to reverse the oxidative stress caused by mercury (Hg) was investigated in Pfaffia glomerata plantlets. Thirty-day-old acclimatized plantlets of P. glomerata were exposed to four treatments: control, 50 μM Zn, 50 μM Hg and 50 μM Zn + 50 μM Hg for 9 days. In Zn + Hg treatment, shoot and root Hg concentrations were 59 and 24% smaller than that plants exposed to 50 μM Hg added alone. An increase in the Zn concentration in the shoot of plants exposed to Zn + Hg occurred, although in the roots Zn concentration was not altered, when compared to the control. Fresh and dry weights, as well as the activity of δ-aminolevulinic acid dehydratase (δ-ALA-D) in Hg-treated plants were significantly reduced. Percentage survival, fresh and dry weights and δ-ALA-D activity of plants treated by 50 μM Zn + 50 μM Hg were greater than of that treated by Hg alone. Moreover, Zn treatment reduced the lipid peroxidation caused by Hg, being this effect related to increased root superoxide dismutase activity, and shoot catalase and ascorbate peroxidase activities. In conclusion, the presence of Zn in the substrate caused a significant reduction in the oxidative stress induced by Hg.     

Metallothionein response to cadmium and zinc exposures compared in two freshwater bivalves, Dreissena polymorpha and Corbicula fluminea

Metallothionein (MT) response to cadmium (Cd) and zinc (Zn) bioaccumulation after single or combined direct exposure was compared in two freshwater bivalves, Dreissena polymorpha (zebra mussel) and Corbicula fluminea (Asiatic clam). Bivalves were exposed to 0.133 μM Cd and/or 15.3 μM Zn, with metal and MT concentrations analysed in the whole soft body after 1, 3, 10 and 24 days of exposure and compared with controls. Results showed significant increase in MT concentrations in both species exposed to Cd and Cd+Zn with a higher accumulation of the protein compared to the control in D. polymorpha for nevertheless similar Cd levels accumulated with time. Exposure to Zn alone led to a significant increase in MT concentrations only in C. fluminea, whereas there was a lack of MT gene induction in the zebra mussels which was confirmed by MT mRNA quantification in gills (RT-PCR). Mussel mortality after 10 days of exposure to Zn and Cd + Zn is discussed with regard to detoxification mechanisms, which include metallothioneins.     

Response of Sulfide:Quinone Oxidoreductase to Sulfide Exposure in the Echiuran Worm Urechis unicinctus

Sulfide is a natural, widely distributed, poisonous substance, and sulfide:quinone oxidoreductase (SQR) is responsible for the initial oxidation of sulfide in mitochondria. In this study, we examined the response of SQR to sulfide exposure (25, 50, and 150 μM) at mRNA, protein, and enzyme activity levels in the body wall and hindgut of the echiuran worm Urechis unicinctus, a benthic organism living in marine sediments. The results revealed SQR mRNA expression during sulfide exposure in the body wall and hindgut increased in a time- and concentration-dependent manner that increased significantly at 12 h and continuously increased with time. At the protein level, SQR expression in the two tissues showed a time-dependent relationship that increased significantly at 12 h in 50 μM sulfide and 6 h in 150 μM, and then continued to increase with time while no significant increase appeared after 25 μM sulfide exposure. SQR enzyme activity in both tissues increased significantly in a time-dependent manner after 50 μM sulfide exposure. We concluded that SQR expression could be induced by sulfide exposure and that the two tissues studied have dissimilar sulfide metabolic patterns. A U. unicinctus sulfide-induced detoxification mechanism was also discussed.     

The effects of anhydrous ammonia on membrane stability ofP.hymatotrichum omnivorum

Sclerotia and mycelium ofPhymatotrichum omnivorum were exposed to anhydrous ammonia (NH₃) and then observed with an electron microscope in order to determine the effects of the NH₃ treatment on the fungal membranes. Sclerotia were exposed to four rates of NH₃: 28, 56, 84, and 112μg NH₃/ml of air for 24 hours. At 28μg/ml, the plasmalemma became wavy and the mitochondrial cristae began to swell and disperse. At 56μg NH₃/ml the plasmalemma showed breakage and formation of vesicles, and all other membrane systems within the cell were broken and distorted. All membranes were totally disrupted and no organelles were recognizable at 84μg NH₃/ml. Mycelium was exposed to 2, 4, 8, 20, and 40μg NH₃/ml for one minute. Damage to cell membranes was not observed at NH₃ conc. up to 4μg/ ml. At 8μg NH₃/ml the plasmalemma was broken and the mitochondria were disrupted. At 20μg/ ml and above, all internal organization was destroyed.     

Alleviation of Salt Stress in Common Bean (Phaseolus vulgaris) by Exogenous Abscisic Acid Supply

In this work the effect of abscisic acid (ABA) and 100 mM NaCl on common bean (Phaseolus vulgaris var. Coco) growth, nitrogenase activity, and nodule metabolism was studied. Experiments were carried out in a controlled environmental chamber and plants, at the vegetative growth stage (16 days old), were treated with ABA (1 μM and 10 μM) and 48 h later were exposed to saline treatment. Results revealed that plant dry weight, nodule dry weight, nitrogen fixation (acetylene reduction activity and ureides content), and most enzymes of ammonium and ureides metabolism were affected by both ABA and NaCl. The addition of 1 μM ABA to the nutrient solution before the exposure to salt stress reduced the negative effect of NaCl. Based on our results, we suggest that ABA application improves the response of Phaseolus vulgaris symbiosis under saline stress conditions, including the nitrogen fixation process and enzymes of ammonium assimilation and purine catabolism.     

Neuroprotective Effect of Crocin on Acrylamide-induced Cytotoxicity in PC12 cells

Acrylamide (ACR) is a potent neurotoxic in human and animal models. In this study, the effect of crocin, main constituent of Crocus sativus L. (Saffron) on ACR-induced cytotoxicity was evaluated using PC12 cells as a suitable in vitro model. The exposure of PC12 cells to ACR reduced cell viability, increased DNA fragmented cells and phosphatidylserine exposure, and elevated Bax/Bcl-2 ratio. Results showed that ACR increased intracellular reactive oxygen species (ROS) in cells and ROS played an important role in ACR cytotoxicity. The pretreatment of cells with 10–50 μM crocin before ACR treatment significantly attenuated ACR cytotoxicity in a dose-dependent manner. Crocin inhibited the downregulation of Bcl-2 and the upregulation of Bax and decreased apoptosis in treated cells. Also, crocin inhibited ROS generation in cells exposed to ACR. In conclusion, our results indicated that pretreatment with crocin protected cells from ACR-induced apoptosis partly by inhibition of intracellular ROS production.     

Arabidopsis thaliana and Thlaspi caerulescens respond comparably to low zinc supply

The main objective of this research was to study the response of Arabidopsis thaliana L. and Thlaspi caerulescens J. & C. Presl to different Zn supplies. The A. thaliana plants were exposed to Zn-deficiency (0 and 0.05 μM Zn) and compared to the plants grown on media containing standard Zn (2 μM). T. caerulescens plants were also exposed to Zn-deficiency (0.05 μM Zn), but as this is a Zn hyperaccumulator species, also to high Zn (1,000 μM Zn). Plants were compared to plants grown on standard Zn media (100 μM Zn). Both A. thaliana and T. caerulescens were found to be heavily affected by Zn deficiency, showing similar retarded growth and reduced reproduction phenotypes, and even less reduction in biomass production in T. caerulescens than in A. thaliana. T. caerulescens plants were similarly affected when grown on high Zn concentrations, with comparable effects on reproductive tissues as seen on low Zn supply.     

Effect of nitrate concentration and UVR on photosynthesis,respiration, nitrate reductase activity,and phenolic compounds in <Emphasis Type="Italic">Ulva rigida</Emphasis> (Chlorophyta)

Seaweeds growing in the intertidal zone are exposed to fluctuating nitrate and ultraviolet radiation (UVR) levels. While it has been shown that elevated UVR levels and the decrease of nitrate concentration can reduce photosynthetic levels in seaweeds, less is known about the combined effect of nitrate levels and UVR on metabolism and photoprotection mechanisms of intertidal species. Consequently, the objective of this study was to evaluate the effect of nitrate concentration and UVR treatments on photosynthesis, respiration, nitrate reductase activity and phenolic compound levels of Ulva rigida (Chlorophyta). There was a two- to threefold increase in maximal gross photosynthesis (GP_max) and respiration rates, as nitrate increased from 0 to 50 μM NO₃⁻. Similarly, nitrate reductase activity increased linearly from low values in algae incubated at 0 μM NO₃ to high values in tissue incubated at 50 μM NO₃⁻. Phenolic compounds in the tissue of U. rigida increased approximately 60% under 50 μM NO₃⁻ relative to those incubated at 0 μM NO₃⁻. Algae exposed to UVR (8 h) showed a significant decrease in the effective quantum yield and respiration, however, no effect was observed in the phenolic compounds levels. Full recovery of effective quantum yield was observed after U. rigida was transferred for 48 h to low PAR. Nitrate reductase also decreased after an 8-h UVR exposure, but no differences were observed among the nitrate treatments. This study shows that high nitrate levels reduced the negative effect of UVR on the effective quantum yield and increased the recovery of key metabolic enzymes. It is possible that the increase of phenolic compounds in the thallus of U. rigida under high nitrate levels provide a photoprotective mechanism when exposed to high UV levels during low tides.     

Epithelial Barrier Modulation by a Channel Forming Peptide

NC-1059 is a synthetic channel-forming peptide that provides for ion transport across, and transiently reduces the barrier integrity of, cultured epithelial monolayers derived from canine kidney (MDCK cells). Experiments were conducted to determine whether epithelial cells derived from other sources were similarly affected. Epithelial cells derived from human intestine (T-84), airway (Calu-3), porcine intestine (IPEC-J2) and reproductive duct (PVD9902) were grown on permeable supports. Basal short circuit current (I _sc) was <3 μA cm⁻² for T-84, IPEC-J2 and PVD9902 cell monolayers and <8 μA cm⁻² for Calu-3 cells. Apical NC-1059 exposure caused, in all cell types, an increase in I _sc to >15 μA cm⁻², indicative of net anion secretion or cation absorption, which was followed by an increase in transepithelial conductance (in mS cm⁻²: T-84, 1.6 to 62; PVD9902, 0.2 to 51; IPEC-J2, 0.3 to 26; Calu-3, 2.3 to 13). These results are consistent with the peptide affecting transcellular ion movement, with a likely effect also on the paracellular route. NC-1059 exposure increased dextran permeation when compared to basal permeation, which documents an effect on the paracellular pathway. In order to evaluate membrane ion channels, experiments were conducted to study the dose dependence and stability of the NC-1059-induced membrane conductance in Xenopus laevis oocytes. NC-1059 induced a dose-dependent increase in oocyte membrane conductance that remained stable for greater than 2 h. The results demonstrate that NC-1059 increases transcellular conductance and paracellular permeation in a wide range of epithelia. These effects might be exploited to promote drug delivery across barrier epithelia.     

The influence of bisnaphthalimidopropyl polyamines on DNA instability and repair in Caco-2 colon epithelial cells

Bisnaphthalimido compounds bis-intercalate to DNA via the major groove and are potentially potent cancer therapeutics. Previously, we incorporated natural polyamines as linkers connecting the two naphthalimido ring moieties to create a series of soluble bisnaphthalimidopropyl polyamines (BNIPPs). Here, extending earlier work on bisnaphthalimidopropylspermidine (BNIPSpd)-induced apoptosis in colon adenocarcinoma Caco-2 cells, we compare the cytotoxicity and genotoxicity of BNIPSpd relative to the spermine and oxaspermine derivatives, bisnaphthalimidopropylspermine (BNIPSpm) and bisnaphthalimidopropyloxaspermine (BNIPOSpm). The order of cytotoxicity after 24 h was BNIPSpd (IC₅₀ = 0.47 μM) > BNIPSpm (IC₅₀ = 10.04 μM) > BNIPOSpm (IC₅₀ >50 μM). After a 72-h BNIPOSpm exposure, an IC₅₀ = 10.25 μM was achieved. With 4-h exposure to BNIPSpd or BNIPSpm or 12-h exposure to BNIPOSpm, concentrations ≥1 μM induced a significant dose-dependent increase in DNA damage as measured by alkaline single-cell gel electrophoresis. The longer incubation times required for BNIPOSpm to induce DNA strand breaks reflect a slower rate of BNIPOSpm cellular distribution as monitored via BNIPP fluorescence within the cells. Moreover, exposure to a non-genotoxic concentration of BNIPSpd, BNIPSpm (0.1 μM for 4 h) or BNIPOSpm (0.1 μM for 12 h) induced a significant decrease in repair of oxidative DNA damage induced by hydrogen peroxide. In conclusion, BNIPP exposure in Caco-2 cells is associated with significant induction of DNA damage and inhibition of DNA repair at non-genotoxic concentrations. The latter is a novel consequence of BNIPP–cell interactions which adds to the spectrum of therapeutically relevant activities that may be exploited for the design and development of naphthalimide-based therapeutics.     

Histological observations of morphogenesis in petiole derived callus of Amorphophallus rivieri Durieu in vitro

The initiation and development of somatic embryos and organogenic shoots and corm-like structures (CLSs) from petiole-derived calli of Amorphophallus rivieri Durieu were observed histologically. The petioles were cultured on Murashige and Skoog (MS) medium supplemented with 5.37 μM α-naphthaleneacetic acid (NAA) and 4.44 μM N⁶-benzylaminopurine (6-BA) for callus induction. The shoot and corm organogenesis occurred from the compact calli when they were transferred to a medium containing 0.54 μM NAA and 4.44 μM 6-BA. A combination of 13.57 μM 2,4-dichlorophenoxyacetic acid (2,4-D) and 8.88 μM 6-BA or 24.18 μM NAA and 6.66 μM 6-BA was optimum for induction of somatic embryos, which failed to produce plantlets because of their structural abnormalities. Shoot regeneration predominantly happened through organogenesis although somatic embryogenesis infrequently occurred. The subepidermal cells of the compact callus converted to competent cells and started divisions, which resulted in formation of the meristemoids. The meristemoid cells continued division to develop into bud primordia. Subepidermal cells could also form the globular structures. Subsequently, these globoids developed into CLSs from which plantlets regenerated during subculture. Meanwhile, the CLSs were capable to form cormels, which could be a promising way for the propagation of A. rivieri.