Enhanced wedelolactone content in in vitro-raised genetically uniform Wedelia chinensis under the influence of CuSO4.

In the present study, we addressed the imperative for potent anticancer agents through Wedelia chinensis, a medicinal plant abundant in the robust antihepatotoxic and antitumor compound wedelolactone. Hindrances in conventional propagation methods due to cross-pollination and habitat degradation prompted us to pioneer in vitro rapid multiplication using plant tissue culture. Optimal outcomes were attained employing Murashige and Skoog (MS) medium supplemented with Indole-3-butyric acid (IBA) (0.5 mg/L) and Kinetin (KN) (5.0 mg/L), yielding 97.67% shoot regeneration and 81.67% rooting from nodal explants. Transplanted plantlets exhibited a 92% survival rate. We established a wedelolactone extraction protocol using toluene:ethyl acetate:formic acid (5:4:1) for High-performance thin-layer chromatography (HPTLC) analysis, trailblazing wedelolactone quantification and 2C DNA analysis in W. chinensis via flow cytometry. Experiments under heavy metal stress with CuSO4 unveiled physiological responses, with peak wedelolactone content [193.90 µg/g dry weight (dw)] in vitro at 75 µM CuSO4, surpassing in vivo levels (89.95 µg/g dw) by 116%. By pioneering successful in vitro rapid multiplication and enhanced wedelolactone content, we bridge a critical gap in the conservation and production of this medicinal plant. Our findings not only offer a sustainable means of propagation but also present a viable strategy for elevating the yield of potent bioactive molecules like wedelolactone, holding immense promise for the development of novel therapeutic interventions and addressing the pressing healthcare challenges of our time.

Multi-Fold Enhancement of Tocopherol Yields Employing High CO2 Supplementation and Nitrate Limitation in Native Isolate Monoraphidium sp.

Tocopherols are the highly active form of the antioxidant molecules involved in scavenging of free radicals and protect the cell membranes from reactive oxygen species (ROS). In the present study, we focused on employing carbon supplementation with varying nitrate concentrations to enhance the total tocopherol yields in the native isolate Monoraphidium sp. CABeR41. The total tocopherol productivity of NRHC (Nitrate replete + 3% CO2) supplemented was (306.14 µg·L-1 d-1) which was nearly 2.5-fold higher compared to NRVLC (Nitrate replete + 0.03% CO2) (60.35 µg·L-1 d-1). The best tocopherol productivities were obtained in the NLHC (Nitrate limited + 3% CO2) supplemented cells (734.38 µg·L-1 d-1) accompanied by a significant increase in cell biomass (2.65-fold) and total lipids (6.25-fold). Further, global metabolomics using gas chromatography-mass spectrometry (GC-MS) was done in the defined conditions to elucidate the molecular mechanism during tocopherol accumulation. In the present study, the Monoraphidium sp. responded to nitrogen limitation by increase in nitrogen assimilation, with significant upregulation in gamma-Aminobutyric acid (GABA). Moreover, the tricarboxylic acid (TCA) cycle upregulation depicted increased availability of carbon skeletons and reducing power, which is leading to increased biomass yields along with the other biocommodities. In conclusion, our study depicts valorization of carbon dioxide as a cost-effective alternative for the enhancement of biomass along with tocopherols and other concomitant products like lipids and carotenoids in the indigenous strain Monoraphidium sp., as an industrial potential strain with relevance in nutraceuticals and pharmaceuticals.

Harnessing the Potential of Roots of Traditional Power Plant: Ocimum.

Genus Ocimum of Labiatae is well known in all traditional medicinal systems like Ayurveda, Unani, Siddha, and Homeopathy. The pharmaceutical activities of different species of Ocimum attributed to all plant parts. Roots are the most significant vital organ of the plant, as they absorb water and nutrients from soil and transport to aerial parts of the plants. Roots of Ocimum were found helpful with free-radical scavenging activity to improve physical and mental strength as well as to treat diabetes, malaria, and liver problems. Antibacterial activity of Ocimum roots and its main component, rosmarinic acid, is very beneficial to protect against several human pathogens, including bacteria and viruses. Being so important in every way, roots of Ocimum need healthy rhizosphere. Bacteria, fungi, nematodes, types of soil, fungicide, pesticides, salt, radioactive elements, as well as heavy metal contaminations, affect roots and overall growth of Ocimum in positive or negative ways. Each component of rhizosphere (natural, treatment or contamination) affects the roots, which highlights current ecological scenario to discover biosafe and more productive approaches. For such prestigious organ of Ocimum, development of in vitro root cultures and hairy root cultures assists to reduce the efforts and timing of the traditional cultivation process along with elimination of negative factors in rhizosphere. Different strains of Agrobacterium rhizogenes, various media compositions, as well as discrete treatments, like elicitors, on nonidentical species or cultivars of Ocimum boost the root induction, biomass, and accumulation of phytoceuticals differently. Hairy roots and in vitro roots of Ocimum accumulate higher quantity of therapeutic metabolites. These metabolites include several phenolics (like rosmarinic acid, 3-hydroxybenzoic acid, m-coumaric acid, p-coumaric acid, caffeic acid, ferulic acid, vanillic acid, chicoric acid, and lithospermic acid), triterpenes (such as betulinic acid, 3-epimaslinic acid, alphitolic acid, euscaphic acids, oleanolic acid, and ursolic acid) as well as flavonoids (flavones, flavonols, and dihydroflavonols). This review highlights pharmaceutical applications of Ocimum roots, a great deal of rhizosphere components and in vitro culturing techniques to enhance biomass as well as chief phytoceuticals.

Zingiber officinale: Its antibacterial activity on Pseudomonas aeruginosa and mode of action evaluated by flow cytometry.

Biofilm formation, low membrane permeability and efflux activity developed by Pseudomonas aeruginosa, play an important role in the mechanism of infection and antimicrobial resistance. In the present study we evaluate the antibacterial effect of Zingiber officinale against multi-drug resistant strain of P. aeruginosa. The study explores antibacterial efficacy and time-kill study concomitantly the effect of herbal extract on bacterial cell physiology with the use of flow cytometry and inhibition of biofilm formation. Z. officinale was found to inhibit the growth of P. aeruginosa, significantly. A major decline in the Colony Forming Units was observed with 3 log10 at 12 h of treatment. Also it is found to affect the membrane integrity of the pathogen, as 70.06 ± 2.009% cells were found to stain with Propidium iodide. In case of efflux activity 86.9 ± 2.08% cells were found in Ethidium bromide positive region. Biofilm formation inhibition ability was found in the range of 68.13 ± 4.11% to 84.86 ± 2.02%. Z.officinale is effective for killing Multi-Drug Resistant P. aeruginosa clinical isolate by affecting the cellular physiology and inhibiting the biofilm formation.

Protective effect of Berberis aristata against peritonitis induced by carbapenem-resistant Escherichia coli in a mammalian model.

OBJECTIVES: Berberis aristata is known to contain a variety of phenolic compounds contributing to its holistic capability of mitigating bacterial multidrug resistance. METHODS: B. aristata stem bark extract was prepared and was characterised using liquid chromatography-mass spectrometry (LC-MS). The antimicrobial efficacy of the extract against carbapenem-resistant Escherichia coli was assessed in vivo in an animal model using Sprague Dawley rats. Microbial counts in blood and urine, physical health status, haematological and biochemical analysis of blood, and histopathology of the kidney were assessed as the study endpoints. RESULTS: An aquo-alcoholic extract of B. aristata (PTRC-2111-A) was found to effectively manage peritonitis induced by carbapenem-resistant E. coli in a rat model at a single post-exposure prophylactic dose of 0.5mg/kg body weight (BW). The extract was also found to show a no observed adverse effect level (NOAEL) up to a dose of 2000mg/kg BW. Physical, immunological, haematological, biochemical and histopathological aberrations were found to be restored to normal in the herbal-treated group at a dose of 0.5mg/kg BW. CONCLUSIONS: The antimicrobial and hepatorenal protective ability of PTRC-2111-A could be attributed to the presence of isoquinoline alkaloids.

Effect of aquo-alchoholic extract of Glycyrrhiza glabra against Pseudomonas aeruginosa in Mice Lung Infection Model.

The prevalence of lung infection caused by Pseudomonas aeruginosa strains that are classified as multi-drug resistant has increased considerably and is mainly attributed to relative insufficiency of potent chemotherapeutic modalities. The present study was conducted to evaluate the antimicrobial activity of aquo-alcoholic extract of Glycyrrhiza glabra against the P. aeruginosa causing lung infection in Swiss albino mice. The study involves evaluation of lethal dose of P. aeruginosa in Swiss albino mice and analysis of disease manifestation that includes bacteremia, hypothermia, reduction in body weight and other parameters for 48h of infection. Physical manifestations of infected mice showed a significant decline in body temperature that is 29±0.57°C (at 48th h) from 38.81±0.33°C (0h) and 30% weight loss was observed at the end of the study. Further the efficacy of G. glabra extract against lung infection induced with the calculated lethal dose was evaluated by employing bacteremia, histopathology and radiological analysis. Bacterial burden showed that 2.30±0.02 Log10CFU/mL at day 7, a significant decline in the bacterial load as compared to day 1 when the bacterial burden was found to be 3.32±0.1 Log10CFU/mL. Histopathological results showed more diffuse and patchy accumulation of inflammatory cells within the alveolar space also the infiltrates were noted in all the lung section of infected mice. In treated animal group improved lung histology was seen with the exudates were less seen in D1 dose (20mg/kg) and disappeared in D2 dose (80mg/kg). The study clearly declares that the G. glabra extract is effective against lung infection caused by P. aeruginosa at dose of 80mg/kg. The LCMS results revealed that the extract contains Glycyrrhizin, Stigmasterol and Ergosterol, Licochalcone and Glabridin. The current study expected to further exploit the biomedical properties of this extract in the preparation of a potent regimen against such threatening pathogen.

Augmenting the potency of third-line antibiotics with Berberis aristata: In vitro synergistic activity against carbapenem-resistant Escherichia coli.

The aim of this study was to analyse the in vitro synergistic antibacterial potential of an aquoethanolic extract of the stem bark of Berberis aristata (PTRC-2111-A) with third-line antibiotics against carbapenem-resistant Escherichia coli. PTRC-2111-A was prepared and was characterised using phytochemical- and bioactivity-based fingerprinting. Fourier transform infrared spectroscopy (FTIR) and liquid chromatography-mass spectrometry (LC-MS) analyses were performed, and superoxide and hydroxyl scavenging activities were assessed in conjunction with in vitro antimicrobial efficacy testing against the test micro-organism. Analysis of drug combinations of PTRC-2111-A and third-line antibiotics was performed using CompuSyn software. PTRC-2111-A from B. aristata was found to have seven common functional groups in comparison with the pre-identified marker compound quercetin, and phytochemical quantitation analysis revealed the presence of 25.44% alkaloids. Moreover, PTRC-2111-A was found to contain isoquinoline alkaloids, namely berbamine, berberine, reticuline, jatrorrhizine, palmatine and piperazine, as elucidated in the LC-MS analysis. Analysis of combinations of PTRC -2111-A and antibiotics revealed synergistic behaviour [fractional inhibitory concentration index (FICI)<1] with colistin, tigecycline and amoxicillin/clavulanate potassium (Augmentin(®)), whereas antagonism (FICI>1) was seen with ertapenem and meropenem.

Alternative to antibiotics against Pseudomonas aeruginosa: Effects of Glycyrrhiza glabra on membrane permeability and inhibition of efflux activity and biofilm formation in Pseudomonas aeruginosa and its in vitro time-kill activity.

The multi-drug resistance offered by Pseudomonas aeruginosa to antibiotics can be attributed towards its propensity to develop biofilm, modification in cell membrane and to efflux antibacterial drugs. The present study explored the activity of Glycyrrhiza glabra and one of its pure compounds, glycyrrhizic acid against P. aeruginosa and their mechanism of action in terms of the effect on membrane permeability, efflux activity, and biofilm formation were determined. Minimum inhibitory concentrations were determined by using broth dilution technique. The minimum bactericidal concentrations were assessed on agar plate. The MIC of the extract and glycyrrhizic acid was found to be 200 and 100 µg ml(-1), respectively. The MBC was found to be 800 and 400 µg ml(-1) in the case of extract and glycyrrhizic acid, respectively. Time -dependent killing efficacy was also estimated. Flowcytometric analysis with staining methods was used to determine the effect of extract and glycyrrhizic acid at 2 × MIC on different physiological parameters and compared it with the standard (antibiotic). The growth of P. aeruginosa was significantly inhibited by extract and the pure compound. The herbal extract and the glycyrrhic acid were also found to effective in targeting the physiological parameters of the bacteria that involve cell membrane permeabilization, efflux activity, and biofilm formation. This study reports the antipseudomonal action of Glycyrrhiza glabra and one of its compound and provides insight into their mode of action.

In vitro bactericidal activity of promising nutraceuticals for targeting multidrug resistant Pseudomonas aeruginosa.

OBJECTIVE: We evaluated the bactericidal activity of nutraceuticals against multidrug resistant Pseudomonas aeruginosa. The nutritionally valued herbs were screened on the basis of a matrix modeling approach and molecular docking based validation analysis. METHODS: The database of 38 herbs developed earlier using fuzzy logic based scoring analysis was subjected to molecular docking based validation. The molecular docking (Hex 6.12) analyses of predominant phytoligands (∼10 per herb) against exoenzyme S of P. aeruginosa filtered potent herbs were selected. The preauthenticated bacterial inoculum (10(8) CFU/mL) was added to the sterile nutrient broth impregnated with standardized aqueous-alcoholic herbal extracts (1-1600 µg/mL). After overnight incubation at 37°C, antibacterial activity was evaluated in terms of minimum inhibitory and minimum bactericidal concentrations. RESULTS: Five herbs were selected on the basis of fuzzy set scoring, an herbal informatics model, and validation analysis based on energy of docking (i.e., Evalue of 380) phytoligands with maximum scoring obtained by Glycyrrhiza glabra. Among the 5 nutraceuticals, G. glabra showed maximum bactericidal activity significantly (P < 0.05) higher than Amikacin, a standard antibiotic, which was in consonance with in silico bioprospection. Zingiber officinale, despite a low Evalue, showed considerably higher inhibition attributed to its higher flavonoid content as compared to other herbs. CONCLUSION: G. glabra (licorice), a flavoring agent; Z. officinale (ginger), a condiment; and Mentha piperita (mint), a fragrance component, showed significant therapeutic potential against multidrug resistant strains of P. aeruginosa.

In vitro bactericidal activity of Berberis aristata extract against clinical isolates of carbapenem-resistant Escherichia coli.

BACKGROUND: Berberis aristata is known to contain a variety of phenolic compounds, flavonoids such as quercetin attributing towards its holistic capability of mitigating multidrug resistance. METHODS: B. aristata stem bark extract was prepared and characterized using phytochemical and bioactivity-based fingerprinting. Anti-oxidant and anti-lipid peroxidation profiling was also done in conjunction with in vitro anti-microbial efficacy testing against the test microorganism i. e., New Delhi Metallo-ß-lactamase-1 (NDM-1) Escherichia coli. RESULTS: Aquo-alcoholic (1:1) extract of B. aristata (PTRC-2111-A), containing 3.0±0.02 µg of QUERCETIN/mg of dried extract, exhibited [flavonoid/polyphenol: F/P (quercetin %) ~ 0.16(0.06 %)]. The bioactivity fingerprint profile of PTRC-2111-A included IC50 ratio [DPPH/NOS]=0.064 as functional standardized value having IC50 (DPPH Scavenging)=16±0.5 µg/mL and IC50 (Nitric Oxide Scavenging)=250±0.5 µg/mL respectively. The reducing ability and anti-lipid peroxidation equivalent (extract: standard) of PTRC-2111-A with respect to standard was estimated to be 3.44 (ascorbic acid) and 0.78 (quercetin) respectively. In vitro anti-microbial activity evaluated against sts-09 multidrug-resistant strain of carbapenem-resistant E. coli was found to be 25 µg/mL. CONCLUSIONS: B. aristata was found to contain a number of phytoconstituents, which acts in a synergistic manner to provide significant bactericidal potential against carbapenem-resistant E. coli.

Assessment of aquo-ethanolic extract of Camellia sinensis against Carbapenem Resistant Escherichia coli: In Vivo Trials in a Murine Model.

The prevalence of Carbapenem Resistant Escherichia coli (CRE) has increased considerably during the last decade, which can be ascribed to relative scarcity of effective non toxic antimicrobial agents. The present study was conducted to evaluate the antimicrobial activity of aquo-ethanolic (1:1) extract of leaves of Camellia sinensis (PTRC-31911-A) against Carbapenem Resistant Escherichia coli at preclinical level using peritonitis infection model in Sprague Dawley rats. Efficacy analysis of PTRC-31911-A involved enumeration of CRE colonies in blood and urine samples of test animals for a period of 5 days from infection. A reduction in microbial count of biological fluids was considered as the primary endpoint of the selected murine model. Physical, biochemical, hematological and histological indices of toxicity were employed as secondary relative indicators of the induced disease. Physical manifestations of infected rats included significantly high body temperature (TempInfected=103.18°F, ∼5% increase) and noteworthy reduction in weight (WeightInfected=126.83g, ∼15% decrease) as compared to control. Significant (P<0.05) increase in total white blood cells, eosinophil and monocyte counts as well as a significant decrease (P<0.05) in erythrocytes count, hematocrit volume, red blood cell distribution width and hemoglobin concentration were observed in the infected group as compared to the control group. Furthermore, noteworthy increase in liver and kidney function test parameters were observed in case of infected groups. All the hematological and biochemical parameters were found to be within optimum range in case of treatment group, indicating restoration of homeostasis. Histopathological studies also presented symptoms of hemorrhage and glomerular damage with structural distortion in glomerular capillary loops of infected groups, which were later recovered in treated groups, indicating the nephro-protective potential of PTRC-31911-A. The study clearly points out that Camellia sinensis extract (PTRC-31911-A; single dose of 5mg/Kg bwt; oral,+24h) is highly effective against Carbapenem Resistant Escherichia coli owing mainly to the presence of flavonoids and polyphenolic compounds, identified by LCMS. Ongoing studies are expected to further unravel the mechanism of action and bioactivity determinants of this broad spectrum plant extract.

Anti-hemolytic, hemagglutination inhibition and bacterial membrane disruptive properties of selected herbal extracts attenuate virulence of Carbapenem Resistant Escherichia coli.

Expression of a multitude of virulence factors by multi-drug resistant microbial strains, e.g., Carbapenem Resistant Escherichia coli (Family: Enterobacteriaceae; Class: Gammaproteobacteria), is responsible for resistance against beta-lactam antibiotics. Hemolysin production and induction of hemagglutination by bacterial surface receptors inflicts direct cytotoxicity by destroying host phagocytic and epithelial cells. We have previously reported that Berberis aristata, Camellia sinensis, Cyperus rotundus Holarrhena antidysenterica and Andrographis paniculata are promising herbal leads for targeting Carbapenem resistant Escherichia coli. These herbal leads were analyzed for their anti-hemolytic potential by employing spectrophotometric assay of hemoglobin liberation. Anti-hemagglutination potential of the extracts was assessed by employing qualitative assay of visible RBC aggregate formation. Camellia sinensis (PTRC-31911-A) exhibited anti-hemolytic potential of 73.97 ± 0.03%, followed by Holarrhena antidysenterica (PTRC-8111-A) i.e., 68.32 ± 0.05%, Berberis aristata (PTRC-2111-A) i.e., 60.26 ± 0.05% and Cyperus rotundus (PTRC-31811-A) i.e., 53.76 ± 0.03%. Comprehensive, visual analysis of hemagglutination inhibition revealed that only Berberis aristata (PTRC-2111-A) and Camellia sinensis (PTRC-31911-A) exhibited anti-hemagglutination activity. However, Andrographis paniculata (PTRC-11611-A) exhibited none of the inhibitory activities. Furthermore, the pair wise correlation analysis of the tested activities with quantitative phytochemical descriptors revealed that an increased content of alkaloid; flavonoids; polyphenols, and decreased content of saponins supported both the activities. Additionally, flow cytometry revealed that cell membrane structures of CRE were damaged by extracts of Berberis aristata (PTRC-2111-A) and Camellia sinensis (PTRC-31911-A) at their respective Minimum Inhibitory Concentrations, thereby confirming noteworthy antibacterial potential of both these extracts targeting bacterial membrane; hemolysin and bacterial hemagglutination.

Attenuation of adhesion, quorum sensing and biofilm mediated virulence of carbapenem resistant Escherichia coli by selected natural plant products.

The multi-drug resistance offered by Carbapenem Resistant Escherichia coli (Family: Enterobacteriaceae; Class: Gammaproteobacteria) against third line antibiotics can be attributed towards its ability to develop biofilm. Such process involves adhesion and quorum-sensing induced colonization leading to biomass development. The present study explored the anti-adhesion, anti-quorum sensing and anti-biofilm potential of 05 pre-standardized potent herbals. Berberis aristata (PTRC-2111-A) exhibited maximum potential in all these activities i.e. 91.3% ± 0.05% (Anti-adhesion), 96.06% ± 0.05% (Anti-Quorum sensing) and 51.3% ± 0.07% (Anti-Biofilm formation) respectively. Camellia sinensis (PTRC-31911-A) showed both anti-adhesion (84.1% ± 0.03%) and anti-quorum sensing (90.0%) potential while Holarrhena antidysenterica (PTRC-8111-A) showed only anti-quorum sensing potential as compared to standards/antibiotics. These findings were in line with the molecular docking analysis of phytoligands against Lux S and Pilin receptors. Furthermore, the pairwise correlation analysis of the tested activities with qualitative, quantitative and bioactivity functional descriptors revealed that an increased content of alkaloid, moderate content of flavonoids and decreased content of tannins supported all the three activities. In addition, nitric oxide and superoxide scavenging activity were found to be correlated with anti-quorum sensing activity. The findings indicated clearly that B. aristata (Family: Berberidaceae) and C. sinensis (Family: Theaceae) were potent herbal leads with significant therapeutic potential which further needs to be explored at pre-clinical level in the future.

Camellia sinensis Ameliorates the Efficacy of Last Line Antibiotics Against Carbapenem Resistant Escherichia coli.

Aquo-ethanolic extract of Camellia sinensis (PTRC-31911-A), standardized using Fourier transform infrared analysis, was found to have seven common functional groups in comparison with pre-identified marker compound 'quercetin'. Phyto-chemical quantitation analysis revealed the presence of 10.65 µg/mg of flavonoids. The bioactivity fingerprint profile of PTRC-31911-A includes IC50 (Hydroxyl radical site specific scavenging) = 11.36 ± 0.5 µg/mL, IC80 (Hydroxyl radical non-site specific scavenging) = 26.44 ± 0.5 µg/mL and IC50 (Superoxide ion scavenging) = 10.141 ± 0.5 µg/mL. The drug combination analysis of PTRC-31911-A with five third-line antibiotics was carried out against carbapenem-resistant Escherichia coli. The analysis of combination of PTRC-31911-A (6.25-1000 µg/mL) and antibiotics (6.25-1000 µg/mL) revealed synergistic behaviour (fractional inhibitory concentration indices < 1) with tigecycline, ertapenem, meropenem, colistin and augmentin. The lead combination of PTRC-31911-A + ertapenem or meropenem showed maximum augmentative potential at 50 and 100 µg/mL, respectively, with nearly five-fold decrease in minimum inhibitory concentrations as compared with respective antibiotics alone. The synergistic effects implied that the antibacterial combinations of PTRC-31911-A and ertapenem, meropenem, colistin, tigecycline or augmentin would be more effective than a single monotherapy with either of the antibacterial agent.

Genetic fidelity of in vitro regenerants, encapsulation of shoot tips and high diosgenin content in Dioscorea bulbifera L., a potential alternative source of diosgenin.

Dioscorea bulbifera L. containing the pharmaceutically important compound, diosgenin, was regenerated in vitro through nodal segments on supplemented Murashige and Skoog medium (MS). Diosgenin was at 12 mg g(-1)dry wt in 12-week-old plantlets raised on MS with various growth hormones. Random amplified polymorphic DNA (RAPD) analysis showed genetic fidelity of regenerants. Encapsulation of shoot tips in 3% (w/v) calcium alginate for storage and germplasm exchange was achieved.

Abiotic metal stress enhances diosgenin yield in Dioscorea bulbifera L. cultures.

Lower concentrations of CuSO(4) (25-75 microM) in the MS medium supplemented with 0.1 mg l(-1) IAA+5.0 mg l(-1) Kn+500 mg l(-1) CH+10 mg l(-1) Cyst hyd enhanced the growth of regenerants of Dioscorea bulbifera L. CuSO(4) (75 microM) induced an appreciable diosgenin yield in the regenerants compared to those obtained on media without Cu. The presence of Cu thus seems to stimulate diosgenin production. The regenerants also differentiated bulbils on lower concentrations of Cu. At CuSO(4) (100 microM), however, cultures showed poor growth as well as a low diosgenin yield. Increased proline and protein contents were recorded in cultures grown on Cu-enriched media.

In vitro micropropagation, differentiation of aerial bulbils and tubers and diosgenin content in Dioscorea bulbifera.

Dioscorea bulbifera could be micropropagated through nodal segments and bulbils. The best medium for regeneration and bulbil differentiation was MS + 0.5 microM IAA (indole-3-acetic acid) + 20.0 microM Kn (kinetin) + 500 mg/L CH (casein hydrolysate) + activated charcoal (20 %). Diosgenin content was maximum in regenerants grown on MS + 5.0 microM IAA + 20.0 microM Kn + 500 mg/L CH. T.s of bulbils could also be used for direct plantlet differentiation as well as bulbil differentiation on MS + 10.0 microM IAA + 20.0 microM Kn + (in mg/L) 30 each of Asp (asparagine) + Arg (arginine) + Gln (glutamine) + 10 Ad (adenine) + 500 CH + 10 Cyst hyd (cysteine hydrochloride). Diosgenin yield in plantlets reached a maximum after 20 weeks. The results indicate that micropropagation, bulbil formation and tuberisation can be achieved in vitro in D. bulbifera, hitherto a less exploited plant, and can further be used for obtaining enhanced levels of diosgenin.