A ferrocene-based chemo-dosimeter for colorimetric and electrochemical detection of cyanide and its estimation in cassava flour.

A simple chemo-dosimeter VDP2 bearing a ferrocene moiety was designed, synthesized, and characterized, and exhibited both chromogenic and electrochemical responses selectively for CN- in H2O-DMSO (9 : 1, v/v) medium. The probe VDP2 showed an instantaneous color change from colorless to yellow with CN- that can readily be observed visually. The deprotonation of the benzimidazole -NH, followed by nucleophilic addition of CN- to the olefinic C-atom, as evidenced by 1H and 13C NMR titration experiments, caused the colorimetric and electrochemical responses. The mass spectral study, CV, FTIR and Mulliken charges computed well supported the proposed mechanism. The electrochemical limit of detection was calculated to be 72 nM. The results of DFT and TD-DFT calculations suggested that the colorless nature of the probe VDP2 is due to weak intramolecular charge transfer (ICT) transition and the yellow color of the VDP2+CN adduct is due to through-space ICT transition. Above all, the probe could be an ideal candidate for monitoring cyanide in water samples and cassava flour with practical significance. A simple and convenient colorimetric method was developed to determine cyanide content in cassava flour.

Spectroscopic and Theoretical Studies on the Selective Detection of Cyanide Ions by a Turn-On Fluorescent Chemo-Dosimeter and its Application in Living Cell Imaging.

A new chemo-dosimeter AK4 containing quinoline fluorophore has rationally been designed, synthesised and characterized using 1H and 13C NMR and mass spectral techniques. The probe senses explicitly CN- ion through a dramatic enhancement in fluorescence over other commonly coexistent anions in H2O:DMSO (9:1 v/v) medium over a broad pH range (4-10). 1H NMR titration revealed the deprotonation followed by nucleophilic addition reaction of CN-, which was supported by 13C NMR and mass spectral examinations. The Job's continuous variation method indicated the formation of a 1:1 adduct between AK4 and CN- with a binding constant of 1.62 × 104 M-1. A limit of detection (LOD) towards CN- of 0.69 µM has been determined, which is much lower than the World Health Organization (WHO) recommended limit of CN- in drinking water (1.9 µM). The changes in the optical properties of AK4 upon reaction with CN- were delineated using Density Functional Theory (DFT) and Time-Dependent Density Functional Theory (TD-DFT) calculations. Moreover, fluorescence microscopic studies established that AK4 could be an effective probe for imaging intracellular CN- in HeLa cells.

Multi-Spectroscopic and TD-DFT Studies on Chromogenic and Fluorogenic Detection of Cyanide in an Aqueous Solution.

Different spectroscopic techniques and Density Functional Theory (DFT)/Time-Dependent Density Functional Theory (TDDFT) calculations have been employed to investigate the dual channel CN- detection behaviour of the developed chemo-dosimeter (AK3). The CN- with AK3 reaction triggered a colour change from pale yellow to colourless and enhanced fluorescence. UV-Vis, fluorescence, 1H & 13C NMR and mass techniques coupled with theoretical calculations (Mulliken charges, dihedral angles) revealed that the CN- sensing process mechanism involves deprotonation of the N-H group followed by nucleophilic addition reaction. Detailed TD-DFT calculations showed that the relaxation of excited electrons from LUMO and to two different ground states is responsible for the weak/moderate fluorescence of AK3. Nucleophilic addition of CN- to the C-atom of the CH = CH bridge terminated the π-conjugation between donor and acceptor regions, reduced the coplanarity, decreased the ICT transition and consequently enhanced the fluorescence of the probe. The practical utility of the probe was demonstrated by detecting cyanide in food materials and determining CN- in environmental water samples.

Spectroscopic and TD-DFT studies on the chromo-fluorogenic detection of cyanide ions in organic and aquo-organic media.

A new naked-eye chromogenic and fluorogenic probe KS5 has been developed for the detection of CN- ions in neat DMSO and H2O:DMSO (1:1 v/v) media. The probe KS5 exhibited selectivity towards CN- and F- ions in organic and high selectivity towards CN- ions in aquo-organic media resulting in a colour change from brown to colourless and a turn-on fluorescence response. The probe could able to detect CN- ions via a deprotonation process, which was conceived by consecutive addition of hydroxide and hydrogen ions and confirmed using 1H NMR studies. The limit of detection (LOD) of KS5 towards CN- ions were in the range of 0.07-0.62 µM in both these solvent systems. Suppression of intra-molecular charge transfer (ICT) transition and photoinduced electron transfer (PET) process of KS5 by the added CN- ions are responsible for the chromogenic and fluorogenic changes observed, respectively. Density Functional Theory (DFT) and Time-Dependent Density Functional Theory (TD-DFT) calculations strongly supported the proposed mechanism along with the optical properties of the probe before and after the addition of CN- ions. To prove the practical applicability, KS5 was successfully utilized to detect CN- ions in cassava powder and bitter almonds as well as to determine CN- ions in various real water samples.

A highly selective probe for fluorometric sensing of cyanide in an aqueous solution and its application in quantitative determination and living cell imaging.

A simple fluorescent probe (KS4) containing multiple reaction sites (phenolic -OH, imine and C = C bonds) is successfully synthesized and characterized using 1H NMR, 13C NMR, mass and single crystal XRD techniques. KS4 exhibits high selectivity towards CN- over a wide range of common anions in H2O:DMSO (1:1 v/v) leading to an amazing turn-on fluorescence at 505 nm via deprotonation of the phenolic -OH group. The limit of detection (1.3 µM) for CN- was much below the standard (1.9 µM) set by the World Health Organization (WHO). Stoichiometry of the interaction between KS4 and CN- was ascertained as 1:1 by the Job's plot method and the binding constant was determined to be 1.5x104 M-1. Density Functional Theory (DFT) and Time-Dependent Density Functional Theory (TD-DFT) based theoretical insight has been appealed to understand the optical properties of KS4 before and after the addition of CN- ion. The probe shows respectable real-time applicability for qualitative detection of CN- in almond and cassava powder as well as quantification in real water samples with excellent recoveries (98.8 - 99.8%). In addition, KS4 is found to safe towards living HeLa cells and successfully applied to the detection of endogenous cyanide ions in HeLa cells.

A highly selective and sensitive ratiometric fluorescent probe for quantitative detection of Al(III) in different natural matrices.

A highly selective and sensitive assay of Al(III) using ratiometric fluorescence enhancement is reported in an aqueous solution. The probe (named RS5) exhibits a red-shift of 54 nm upon binding with Al(III) ion. The significant enhancement response of RS5 at 481 nm is attributed to the formation of a 1:1 complex between the probe and Al(III), wherein RS5 acts as a tridentate NNN-donor ligand. The complexation process is ascertained by1H,13C, and27Al NMR and HR-MS spectral techniques. The binding constant of the complex is determined to be 1.3 × 105M-1. The ratiometric change in fluorescence upon complexation with Al(III) is ascribed to an increase in intramolecular charge transfer (ICT) transition along with chelation enhanced fluorescence (CHEF) processes. The probe can be applied for monitoring Al(III) in a pH range of 6-8. The limit of detection (LOD) of RS5 for the examination of Al(III) is found to be 0.3µM. With an aim to understand the sensing behavior of RS5, the optical properties of the probe and its Al(III) complex are investigated using density functional theory (DFT) and time-dependent density functional theory (TD-DFT) methods. The probe is successfully employed for the determination of Al(III), with very high recovery percentages, in natural matrices like deep well water, tap water, drinking water, pond water, river water, bovine serum albumin (BSA) solution and blood serum.

Cell line selection combined with jasmonic acid elicitation enhance camptothecin production in cell suspension cultures of Ophiorrhiza mungos L.

Ophiorrhiza mungos is a herbaceous medicinal plant which contains a quinoline alkaloid, camptothecin (CPT), an anticancer compound. A high-yielding cell line, O. mungos cell line-3 (OMC3) was selected from cell suspension cultures of O. mungos using cell aggregate cloning method and established cell suspension culture. OMC3 cell suspension produced significantly high biomass (9.25 ± 1.3 g/flask fresh weight (FW)) and CPT yield (0.095 ± 0.002 mg g-1 dry weight (DW)) compared with the original cell suspension. Inoculum size of OMC3 cell suspension culture was optimised as 14 g L-1. Media optimisation has shown that 5 % (w/v) sucrose and an increased ammonium/nitrate concentration of 40/20 mM favoured CPT production, whereas 3 % (w/v) sucrose, an ammonium/nitrate concentration of 20/40 mM and 1.25 mM of phosphate favoured biomass accumulation. Jasmonic acid, chitin and salicylic acid was used to elicit CPT production in the original cell suspension culture and achieved significantly high CPT production with jasmonic acid (JA) elicitation. Further, OMC3 cell suspension culture was elicited with JA (50 µM) and obtained 1.12 ± 0.08 mg g-1 DW CPT and 9.52 ± 1.4 g/flask FW (190.4 g L-1 FW). The combination of cell line selection and elicitation has produced 18.66-fold increases in CPT production together with significantly high biomass yield. The study is helpful in the scale-up studies of O. mungos cell suspension culture in suitable bioreactor systems for the production of CPT.

Unusually short chalcogen bonds involving organoselenium: insights into the Se-N bond cleavage mechanism of the antioxidant ebselen and analogues.

Structural studies on the polymorphs of the organoselenium antioxidant ebselen and its derivative show the potential of organic selenium to form unusually short Se⋅⋅⋅O chalcogen bonds that lead to conserved supramolecular recognition units. Se⋅⋅⋅O interactions observed in these polymorphs are the shortest such chalcogen bonds known for organoselenium compounds. The FTIR spectral evolution characteristics of this interaction from solution state to solid crystalline state further validates the robustness of this class of supramolecular recognition units. The strength and electronic nature of the Se⋅⋅⋅O chalcogen bonds were explored using high-resolution X-ray charge density analysis and atons-in-molecules (AIM) theoretical analysis. A charge density study unravels the strong electrostatic nature of Se⋅⋅⋅O chalcogen bonding and soft-metal-like behavior of organoselenium. An analysis of the charge density around Se-N and Se-C covalent bonds in conjunction with the Se⋅⋅⋅O chalcogen bonding modes in ebselen and its analogues provides insights into the mechanism of drug action in this class of organoselenium antioxidants. The potential role of the intermolecular Se⋅⋅⋅O chalcogen bonding in forming the intermediate supramolecular assembly that leads to the bond cleavage mechanism has been proposed in terms of electron density topological parameters in a series of molecular complexes of ebselen with reactive oxygen species (ROS).

Vivipary in Ophiorrhiza mungos L. - a rare phenomenon in angiosperms.

Vivipary, the precocious germination of seeds within the parent plant, is a specialised feature of evolutionary and biological importance that ensures survival of a plant. Reports on vivipary in angiosperms are rare, accounting for <0.1% of flowering plants. Here, we report a remarkable case of occurrence of vivipary in Ophiorrhiza mungos. A study was conducted to collect information on the morphology of the capsules that support vivipary, environmental factors that induce vivipary, survival mode and the survival of viviparous seedlings. The hydroscopic movement of the cup-shaped capsules of O. mungos was found to help in viviparous germination during the rainy season. Of the total seeds in a capsule, 70% showed viviparous germination. The seedlings remaining inside the capsule attain a height of 0.98 ± 0.4 cm and reach the ground when the capsule falls. On the ground, seedlings obtain easy anchorage to the substratum since they have already germinated. Vivipary appears to be an adaptation of O. mungos to the rainy season for ensuring viable offspring. This suggests that vivipary in this species might be artificially induced by continuous spraying with water to rescue seeds in all seasons for use in large-scale propagation to meet increasing market demand and conservation of this valuable anticancer medicinal herb.

Solvent microenvironments and copper binding alters the conformation and toxicity of a prion fragment.

The secondary structures of amyloidogenic proteins are largely influenced by various intra and extra cellular microenvironments and metal ions that govern cytotoxicity. The secondary structure of a prion fragment, PrP(111-126), was determined using circular dichroism (CD) spectroscopy in various microenvironments. The conformational preferences of the prion peptide fragment were examined by changing solvent conditions and pH, and by introducing external stress (sonication). These physical and chemical environments simulate various cellular components at the water-membrane interface, namely differing aqueous environments and metal chelating ions. The results show that PrP(111-126) adopts different conformations in assembled and non-assembled forms. Aging studies on the PrP(111-126) peptide fragment in aqueous buffer demonstrated a structural transition from random coil to a stable ß-sheet structure. A similar, but significantly accelerated structural transition was observed upon sonication in aqueous environment. With increasing TFE concentrations, the helical content of PrP(111-126) increased persistently during the structural transition process from random coil. In aqueous SDS solution, PrP(111-126) exhibited ß-sheet conformation with greater α-helical content. No significant conformational changes were observed under various pH conditions. Addition of Cu(2+) ions inhibited the structural transition and fibril formation of the peptide in a cell free in vitro system. The fact that Cu(2+) supplementation attenuates the fibrillar assemblies and cytotoxicity of PrP(111-126) was witnessed through structural morphology studies using AFM as well as cytotoxicity using MTT measurements. We observed negligible effects during both physical and chemical stimulation on conformation of the prion fragment in the presence of Cu(2+) ions. The toxicity of PrP(111-126) to cultured astrocytes was reduced following the addition of Cu(2+) ions, owing to binding affinity of copper towards histidine moiety present in the peptide.

Removal of fluoride ions from aqueous solution by conducting polypyrrole.

Conducting polypyrrole was found to possess potential efficiency to remove fluoride ions from aqueous solutions. The amount of fluoride ions removed per unit mass of the adsorbent, at 30 degrees C from 10 mg/L fluoride ion solution, was estimated to be 6.37 mg/g. Batch sorption studies have been carried out to determine the effect of initial concentration of the sorbate, agitation time, adsorbent dose, pH, co-ions and temperature on the removal of fluoride by polypyrrole. The sorptive removal was found to be strongly pH and temperature dependent. The experimental data fitted well to the Freundlich and Dubinin-Radushkevick (D-R) isotherms. Thermodynamic parameters such as DeltaH(0), DeltaS(0) and DeltaG(0) were calculated indicating that the adsorption process was spontaneous and exothermic. FT-IR spectral studies suggested that the mechanism of fluoride ions removal follow chemisorption.

Defluoridation of water via doping of polyanilines.

The potentiality of polyaniline and poly (m-methyl aniline) to remove fluoride from water via doping was investigated. The influence of pH, dosage of polyanilines, initial fluoride concentration and temperature on the amount of fluoride removed by the polyanilines were studied. The amount of fluoride removed at pH 7.0 by 50mg/50 ml dose was found to be 0.78 mg/g. The data of fluoride removal fitted well with Langmuir and Freundlich isotherms. Thermodynamic parameters computed show that the adsorption process is endothermic in nature. FT-IR, X-ray and EDAX patterns of the polyanilines before and after exposure to fluoride ions suggest that the defluoridation occurs via doping of fluoride ions onto these polymers.

A protocol for high frequency regeneration through nodal explant cultures and ex vitro rooting of Plumbago rosea L.

A rapid clonal multiplication scheme comprising direct multiple shoot initiation and downsizing of the node with buds proliferated upon during subculture was developed for Plumbago rosea. Sixty five per cent of the nodes (approximately 2.0 cm) dissected out of young shoots from field grown plants and cultured in MS agar medium containing 3% sucrose and 15.4 microM BAP remained contamination free and responded at 95% rate with callusing at basal cut end and axillary bud break in 5 days followed by the formation of 2.41 +/- 0.14 shoots of 0.87 +/- 0.14 cm length in 3 weeks. Though differences in frequency and number of buds formed between nodes of 1-5 positions from the young shoots was negligible, the shoots emanated from the youngest node were shorter (0.92 +/- 0.19 cm) than those (2.3 +/- 0.50 cm) of the mature 5th node. Synergistic influence of BAP and auxins on caulogenesis was absent. Bud emergence in shorter (approximately 0.5 cm) nodes was delayed up to 3 weeks and extensive callus proliferation from the cut basal end overlapped the 8.2 +/- 0.37 axillary shoots/buds formed after 7 weeks. Reduction in the size (downsized) of the 2.0 cm node with buds to 1.0 cm by dissecting out the basal internodal segment having the callus and subculture of them (approximately 1.0 cm) with buds in contact with the medium for 3 weeks contributed to maximum multiplication of 42.1 +/- 5.40 shoot buds. Division of the shoot cluster and transfer of 2-3 shoots (0.5-1.5 cm) in a clump to MS basal liquid medium induced elongation of the shoots to 4.1 +/- 0.18 cm in 2 weeks. Shoots of 3.0-4.2 cm length were rooted within 3 weeks at 100% efficiency in vitro or ex vitro without hardening. In vitro rhizogenesis in presence of 0.49 microM IBA is recommended for enhanced rooting and high yield of commercially important tuberous roots during cultivation in the field.

Assemblages of prion fragments: novel model systems for understanding amyloid toxicity.

We report the conformational and toxic properties of two novel fibril-forming prion amyloid sequences, GAVVGGLG (PrP(119-126)) and VVGGLGG (PrP(121-127)). The conformational preferences of these fragments were studied in differing microenvironments of TFE/water mixtures and SDS solution. Interestingly, with an increase in TFE concentration, PrP(119-126) showed a helical conformational propensity, whereas PrP(121-127) adopted a more random coil structure. In 5% SDS, PrP(119-126) showed more alpha-helical content than in TFE solution, and PrP(121-127) exhibited a predominantly random coil conformation. However, both peptides took a random coil conformation in water, and over time the random coil transformed into a beta-sheet structure with a significant percentage of helical conformation and beta-turn structure in PrP(119-126) and PrP(121-127), respectively, as observed with CD spectroscopy. The aged fibrils of PrP(119-126) were insoluble in SDS, and PrP(121-127) was extractable with SDS solution. These fibrils were characterized by transmission electron microscopy. Both PrP(119-126) and PrP(121-127) formed stable monolayer's consisting of multimeric assemblages at the air-water interface. Monomeric PrP(119-126) was more toxic to astrocytes than the control Abeta peptide; however, the fibrillar form of PrP(119-126) was less toxic to astrocytes. PrP(121-127) elicited moderate toxicity in both soluble and fibrillar forms on astrocytes. Furthermore, quenching experiments using acroyl-labeled PrP(119-126) and PrP(121-127) with eosin-labeled synaptosomal membrane revealed that these prion fragments bind to anion-exchange protein. The binding of PrP(119-126) and PrP(121-127) with a membrane microdomain (lipid raft) was also analyzed using pyrenated derivatives. We conclude that the formation of PrP(119-126) and PrP(121-127) fibrils is a concentration-dependent process that involves coil to sheet conversion with aging. PrP(119-126), the sequence with intrinsic helical propensity, is more toxic in monomer form, and the fibril formation in this case seems to be protective to cells. For PrP(121-127), the SDS-soluble fibrils are more cytotoxic, indicating that a higher order assemblage structure is required for cytotoxic activity of this peptide.

In vitro mass multiplication of Ophiorrhiza mungo Linn.

A protocol for in vitro mass multiplication of plants through seedling (shoot) cultures was established for Ophiorrhiza mungo. Maximum number of adventitious shoots per shoot culture (10.4 +/- 1.72) was initiated on MS solid medium supplemented with BAP (2.22 microM) after 3 weeks. Shoots were further multiplied (12.8 +/- 2.8) through subculture of intact shoots and reculture of nodal segments of aseptic shoots (6.5 +/- 0.94) in MS solid medium containing BAP (0.89 microM). Shoot elongation (1.27 +/- 0.12 cm) was achieved in the medium containing GA3 (1.44 microM) in two weeks. Rooting was favoured in basal agar medium supplemented with IBA (12.3 microM) plus NAA (1.07 microM). The plants were successfully established (100%) in the pots containing sand and top soil (1:1) mixture in a period of two weeks.

Calix[8]arene-mediated self-assembly of tetrapeptide H-Leu-Leu-Ile-Leu-OMe.

Conformational analysis of peptide 1, H-Leu-Leu-Ile-Leu-OMe on complexing with macro cycle calix[8]arene has been carried out using (1)H-NMR and FTIR spectroscopic techniques. Stoichiometry of the complex formed in the 1:8 ratio was evidenced by a Job plot. NMR studies of the above peptide show a marked downfield shift and an increase in (3)J values for NH resonances on complexing with calix[8]arene. The characteristic NOE connectivity between N(i+1)H and C(ialpha)H confirm beta-sheet conformation in the complexed state. Both (1)H-NMR and FTIR results indicate that the alpha-amino group of Leu I is proximal to the macrocycle and is involved in hydrogen bond formation with phenolic hydrogen atom of the calix[8]arene. This suggests that calix[8]arene provides a suitable platform for peptide 1 to self-assemble in a parallel beta-sheet conformation. The nature of calix[8]arene interaction with peptide 1 has been studied using dynamic NMR studies, which concludes that a bifurcated hydrogen bonding interaction exists in the molecular interfaces of the assembly.

Conformational polymorphism of the amyloidogenic peptide homologous to residues 113-127 of the prion protein.

Conformational transitions are thought to be the prime mechanism of amyloid formation in prion diseases. The prion proteins are known to exhibit polymorphic behavior that explains their ability of "conformation switching" facilitated by structured "seeds" consisting of transformed proteins. Oligopeptides containing prion sequences showing the polymorphism are not known even though amyloid formation is observed in these fragments. In this work, we have observed polymorphism in a 15-residue peptide PrP (113-127) that is known to form amyloid fibrils on aging. To see the polymorphic behavior of this peptide in different solvent environments, circular dichroism (CD) spectroscopic studies on an aqueous solution of PrP (113-127) in different trifluoroethanol (TFE) concentrations were carried out. The results show that PrP (113-127) have sheet preference in lower TFE concentration whereas it has more helical conformation in higher TFE content (>40%). The structural transitions involved in TFE solvent were studied using interval-scan CD and FT-IR studies. It is interesting to note that the alpha-helical structure persists throughout the structural transition process involved in amyloid fibril formation implicating the involvement of both N- and C-terminal sequences. To unravel the role of the N-terminal region in the polymorphism of the PrP (113-127), CD studies on another synthetic peptide, PrP (113-120) were carried out. PrP(113-120) exhibits random coil conformation in 100% water and helical conformation in 100% TFE, indicating the importance of full-length sequence for beta-sheet formation. Besides, the influence of different chemico-physical conditions such as concentration, pH, ionic strength, and membrane like environment on the secondary structure of the peptide PrP (113-127) has been investigated. At higher concentration, PrP (113-127) shows features of sheet conformation even in 100% TFE suggesting aggregation. In the presence of 5% solution of sodium dodecyl sulfate, PrP (113-127) takes high alpha-helical propensity. The environment-dependent conformational polymorphism of PrP (113-127) and its marked tendency to form stable beta-sheet structure at acidic pH could account for its conformation switching behavior from alpha-helix to beta-sheet. This work emphasizes the coordinative involvement of N-terminal and C-terminal sequences in the self-assembly of PrP (113-127).

In vitro mass multiplication and production of roots in Plumbago rosea.

Mass multiplication of Plumbago rosea was achieved by indirect organogenesis in young stem, leaf and root explant cultures of 6-9-month-old plants. All the explants responded similarly in a hormonal regime of 2.5 mg/L BA and 1.5 mg/L NAA with the formation of nodular callus in 4 weeks; the callus was divided and subcultured at 4-week intervals in the presence of 3.0 mg/L BA to produce up to 23.5 +/- 1.6 shoots in 18 weeks and then at 2.0 mg/L BA to produce up to 79.6 +/- 1.5 shoots in 23 weeks. The shoots of 2.0-3.5 cm length were rooted easily in half-strength MS agar medium supplemented with 0.1 mg/L IBA and rooted plants established within 4 weeks at a 95-98 % rate without hardening. Eight weeks after establishment, the micropropagated plants were transferred to experimental plots and cultivated for 10 months to obtain a significantly higher number (18.0 +/- 0.5) of larger tuberous roots (137.4 +/- 3.4 g fw/plant) compared to conventional rooted cuttings (14.0 +/- 1.7, 47.9 +/- 1.6 g fw/plant). During this period, the concentration of the root-specific compound, plumbagin recorded per g dw (1.5 %), was higher than that of conventionally propagated plants (0.9-1.0 %). The early formation of plumbagin-rich tuberous roots holds significant potential for the commercial cultivation of the micropropagated plants.

Sonication induced sheet formation at the air-water interface.

A hydrophobic pentadecapeptide, AGAAAA-GAVVGGLGG (1), part of the prion sequence PrP (106-127), on fresh aqueous dissolution takes a mixture of random and sheet conformations which forms a stable monolayer with a high beta-sheet content when compressed at the air-water interface. This also develops into a kinetically stabilized beta-sheet structure on sonication.

In vitro multiplication of Nothapodites foetida (Wight.) Sleumer through seedling explant cultures.

In vitro multiplication of Nothapodites foetida (Wight.) Sleumer was achieved using axenic seedling explant cultures. Isolated nodes (1.0-1.2 cm) and shoot tips (1.0-1.5 cm) cultured in Murashige and Skoog's agar medium containing varying concentrations of TDZ, BA and combinations of 2iP and GA3. Single shoot (0.8-1.2 cm) was regenerated in each culture after 6 weeks. Axillary shoots were then excised and recultured for 8 weeks in medium containing TDZ (0.05 mgL-1) which formed shoots (about 4 in no.; 2 cm) from the basal node. Axillary branches (2) which formed on 60% of these shoots after 10-12 weeks of culture were separated and recultured in the same medium for 8 weeks. Three shoots (0.8-1.0 cm) per culture were regenerated. Shoots of 0.8-1.8 cm length were subcultured on a low cytokinin (0.01 mgL-1 TDZ) regime to induce shoot elongation (2.0-3.5 cm) in 4 weeks. Shoot cuttings were rooted (60%) in the medium containing IBA (1.5 mgL-1). Rooted plantlets established in pots (90%) after hardening resumed normal growth in 3 months.