S. L. Kothari

MetaMapp: mapping and visualizing metabolomic data by integrating information from biochemical pathways and chemical and mass spectral similarity

BackgroundExposure to environmental tobacco smoke (ETS) leads to higher rates of pulmonary diseases and infections in children. To study the biochemical changes that may precede lung diseases, metabolomic effects on fetal and maternal lungs and plasma from rats exposed to ETS were compared to filtered air control animals. Genome- reconstructed metabolic pathways may be used to map and interpret dysregulation in metabolic networks. However, mass spectrometry-based non-targeted metabolomics datasets often comprise many metabolites for which links to enzymatic reactions have not yet been reported. Hence, network visualizations that rely on current biochemical databases are incomplete and also fail to visualize novel, structurally unidentified metabolites.ResultsWe present a novel approach to integrate biochemical pathway and chemical relationships to map all detected metabolites in network graphs (MetaMapp) using KEGG reactant pair database, Tanimoto chemical and NIST mass spectral similarity scores. In fetal and maternal lungs, and in maternal blood plasma from pregnant rats exposed to environmental tobacco smoke (ETS), 459 unique metabolites comprising 179 structurally identified compounds were detected by gas chromatography time of flight mass spectrometry (GC-TOF MS) and BinBase data processing. MetaMapp graphs in Cytoscape showed much clearer metabolic modularity and complete content visualization compared to conventional biochemical mapping approaches. Cytoscape visualization of differential statistics results using these graphs showed that overall, fetal lung metabolism was more impaired than lungs and blood metabolism in dams. Fetuses from ETS-exposed dams expressed lower lipid and nucleotide levels and higher amounts of energy metabolism intermediates than control animals, indicating lower biosynthetic rates of metabolites for cell division, structural proteins and lipids that are critical for in lung development.ConclusionsMetaMapp graphs efficiently visualizes mass spectrometry based metabolomics datasets as network graphs in Cytoscape, and highlights metabolic alterations that can be associated with higher rate of pulmonary diseases and infections in children prenatally exposed to ETS. The MetaMapp scripts can be accessed at http://metamapp.fiehnlab.ucdavis.edu.

Micronutrient optimization results into highly improved in vitro plant regeneration in kodo (Paspalum scrobiculatum L.) and finger (Eleusine coracana (L.) Gaertn.) millets

Basal medium constituents and their concentration play an important role in growth and morphogenesis of plant tissues cultured in vitro. In this study effect of different inorganic nutrients (CoCl2, MnSO4, ZnSO4, CuSO4 and AgNO3) on callus induction and plant regeneration in Paspalum scrobiculatum and Eleusine coracana was examined. A 5× and 3× increase in regeneration response at enhanced levels of CuSO4 was noted for kodo and finger millets, respectively. Significant improvement in plant regeneration was also observed with the increase in levels of Co and Mn. Addition of AgNO3 to the basal medium also had a stimulatory effect on callus induction and plant regeneration. Optimization of nutrient level in the basal medium has highly significant role in obtaining maximum regeneration response from explants and callus culture.

Factors influencing Agrobacterium tumefaciens-mediated genetic transformation of Eleusine coracana (L.) Gaertn

Agrobacterium-mediated transformation protocol has been developed for Eleusine coracana (var. PR-202) by varying several factors which influence T-DNA delivery. Green nodular regenerative calli with meristematic nodules of seed origin were used as the target tissue for Agrobacteriumtumefaciens-mediated gene transfer. The highest frequency of transformation (44.4%) was observed when callus was infected, co-cultivated and incubated at 22°C. Incorporation of higher level of CuSO4 in the regeneration medium had significantly positive effect on the recovery of transformed plants. PCR analysis of T0 and T1 generation plants with nptII-specific primers revealed the amplification of nptII gene. Southern blot analysis of six regenerated plants confirmed selectable marker gene integration in three plants. This is a first report on Agrobacterium-mediated genetic transformation of finger millet and will pave the way for further studies in this and other millet crops.

Adventitious shoot regeneration and in vitro biosynthesis of steroidal lactones in Withania coagulans (Stocks) Dunal

A micropropagation system through leaf explant culture has been developed for Withania coagulans. Shoot bud proliferation occurred through both adventitious and de novo routes depending on the hormonal regime of the culture medium. Green compact nodular organogenic callus developed on Murashige and Skoog (MS) medium supplemented with 2.3xa0μM kinetin (Kn) and lower levels of 6–benzyladenine (BA) (13.3xa0μM) while multiple adventitious shoot bud differentiation occurred on medium fortified with 2.3xa0μM kinetin (Kn) and higher levels of BA (22.2xa0μM). Shoot buds were transferred to proliferation medium containing 2.2xa0μM BA, 2.3xa0μM Kn, and 3.9xa0μM phloroglucinol (PG) for further growth and development of shoot system. Elongated shoots were rooted using a two-step procedure involving pulse treatment of 7xa0days in a medium containing 71.6xa0μM choline chloride (CC) and 3.9xa0μM PG and then transferred to rooting medium containing ½ MS, 1.2xa0μM IBA, 3.6xa0μM PAA, and 14.3xa0μM CC for 3xa0weeks. Well-rooted plants were transferred to a greenhouse for hardening and further growth. Random amplification of polymorphic DNA (RAPD) showed monomorphic bands in all the plants thereby confirming clonality of the regenerants. Thin layer chromatography (TLC) showed the presence of withanolides in the regenerated plants. Quantification through reverse-phase HPLC revealed increased concentration of withanolides in the regenerated plants compared to the field-grown mother plant. Accumulation of withaferin A and withanolide A increased up to twofold and that of withanone up to tenfold. Direct regeneration via leaf explants will be useful for Agrobacterium-mediated genetic transformation, and will facilitate pathway manipulation using metabolic engineering for bioactive withanolides.

Micropropagation of Withania coagulans (Stocks) Dunal: A Critically Endangered Medicinal Herb

An efficient micropropagation protocol has been developed for Withania coagulans, a highly endangered medicinal herb and an important natural source of withanolides. Prolific multiplication of axillary buds occurred from the nodal segments taken from adult plant, and cultured on MS medium enriched with BA (0.5 mg l−1), Kn (0.5 mg l−1) and PG (0.5 mg l−1). Nodal segments and shoot tips of elongated microshoots also behaved the same way in cultures and formed multiple shoots through axillary bud multiplication. Addition of PG (0.5 mg l−1) in the regeneration medium significantly improved induction and elongation of shoot buds. Elongated shoots were placed on filter paper bridges soaked in MS medium with CC (10 mg l−1) and PG (0.5 mg l−1) for the initial 7 days’ pulse treatment and thereafter, they were transferred to rooting medium containing IBA (0.25 mg l−1) + PAA (0.5 mg l−1) + CC (2 mg l−1). This protocol has the capacity of producing 1000 plants from one nodal segment after 4 subcultures of 2 weeks each.

Optimization of factors influencing microprojectile bombardment-mediated genetic transformation of seed-derived callus and regeneration of transgenic plants in Eleusine coracana (L.) Gaertn

Microprojectile bombardment mediated genetic transformation parameters have been standardized for seed derived callus of Eleusine coracana. Plasmid pCAMBIA 1381 harboring hygromycin phosphotransferase (hptII) as selectable marker gene and β-glucuronidase (gus A) as reporter gene, was used for the optimization of gene transfer conditions. The transient GUS expression and survival of putative transformants were taken into consideration for the assessment of parameters. Optimum conditions for the microprojectile bombardment mediated genetic transformation of finger millet were 1,100xa0psi rupture disk pressure with 3xa0cm distance from rupture disk to macrocarrier and 12xa0cm microprojectile travel distance. Double bombardment with gold particles of 1.0xa0μm size provided maximum transient GUS expression and transformation efficiency. Osmotic treatment of callus with 0.4xa0M sorbitol enhanced efficiency of particle bombardment mediated genetic transformation. Regenerative calli were bombarded at optimum conditions of bombardment and placed on regeneration medium with hygromycin to obtain transformed plants. The integration of hptII and gus A genes was confirmed with PCR amplification of 684 and 634xa0bp sizes of the bands respectively from putative transformants and Southern blot hybridization using PCR amplified DIG labeled hptII gene as probe. PCR analysis with hptII gene specific primers indicated the presence of transgene in T1 generation plants. Thus a successful genetic transformation system was developed using particle bombardment in E. coracana with 45.3% transformation efficiency. The protocol will be helpful for the introgression of desired genes into E. coracana.

Micropropagation of Pithecellobium dulce (Roxb.) Benth—a multipurpose leguminous tree and assessment of genetic fidelity of micropropagated plants using molecular markers

An efficient and reproducible protocol has been developed for in vitro propagation of Pithecellobium dulce (Roxb.) Benth (a multipurpose leguminous tree) from field grown nodal segments (axillary bud). Shoot bud induction occurred from nodal explants of 15-years-old tree on Murashige and Skoog (MS) basal medium supplemented with 4.4xa0μM 6-benzyladenine (BA) and multiplication was achieved on MS medium supplemented with 4.4xa0μM BA + 0.73xa0μM phenylacetic acid (PAA) i.e. up to 7 shoot buds in the period of 5–6xa0weeks. Addition of adenine sulphate (AdS) to this medium further enhanced the number of shoot buds up to 10. Proliferating shoot cultures were established by repeatedly subculturing primary culture on fresh medium (MS + 4.4xa0μM BA + 0.73xa0μM PAA) after every 25xa0days. In vitro rooting was achieved on MS medium supplemented with 2.46xa0μM Indole-3-butyric acid (IBA) + 41.63xa0μM activated charcoal (AC). The micropropagated shoots with well developed roots were acclimatized in green house in pots containing sand, soil and manure (1:1:1). Genetic stability of micropropagated clones was evaluated using Random amplified polymorphic DNA (RAPD) and Inter simple sequence repeat (ISSR) markers. The amplification products were monomorphic in micropropagated plants and similar to those of mother plant. No polymorphism was detected revealing the genetic uniformity of micropropagated plants. This is the first report of an efficient protocol for regeneration of P. dulce through organogenesis, which can be used for further genetic transformation and pharmaceutical purposes.

Antimycobacterial activity of Citrullus colocynthis (L.) Schrad. against drug sensitive and drug resistant Mycobacterium tuberculosis and MOTT clinical isolates.

ETHNOPHARMACOLOGICAL RELEVANCEnCitrullus colocynthis (Cucurbitaceae), a folk herbal medicine and traditionally used natural remedy for tuberculosis in India has been studied to validate its antitubercular activity against drug sensitive and drug resistant (including multidrug resistant) Mycobacterium tuberculosis and Mycobacterium other than tuberculosis (MOTT) bacilli.nnnMATERIALS AND METHODSnInhibitory and bactericidal activities of crude extracts, fractions and compounds of Citrullus colocynthis plant, consisting of aerial parts and ripe deseeded fruits were performed against the drug sensitive standard strain of Mycobacterium tuberculosis H37Rv (ATCC 27294), 16 drug resistant strains of Mycobacterium tuberculosis and two MOTT strains, using radiometric BACTEC 460TB system.nnnRESULTSnMethanolic extract of ripe deseeded fruit of Citrullus colocynthis has shown good activity (MIC ≤ 62.5 µg/ml), whereas among the bioactive fractions, FC IX demonstrated the best activity (MIC 31.2 µg/ml) against Mycobacterium tuberculosis H37Rv. Bioactive FC III, IX and X also inhibited 16 clinical isolates of Mycobacterium tuberculosis consisting of seven non-multidrug resistants, eight multidrug resistants, one extensively drug resistant and two of MOTTs with MICs in the range of 50-125, 31.2-125 and 62.5-125 µg/ml, respectively. Ursolic acid and cucurbitacin E 2-0-β-d-glucopyranoside were identified as the main biomarkers active against Mycobacterium tuberculosis H37Rv (MICs 50 and 25 µg/ml respectively), as well as against the 18 clinical isolates. FC III and FC IX showed better inhibition of drug resistant and MOTT clinical isolates. Minimal bactericidal concentrations of extracts, fractions and compound C-2 were ≥ two-fold MIC values.nnnCONCLUSIONSnThe study provides a scientific rationale for the traditional use of Citrullus colocynthis fruit in the treatment of tuberculosis. In addition, the study elucidates a broad spectrum antimycobacterial action of Citrullus colocynthis fruit, which can contribute to the development of improved preparation of an antitubercular natural drug for the treatment of drug resistant tuberculosis and MOTT infection as well.

Regeneration and Agrobacterium-mediated genetic transformation of Terminalia bellerica Roxb.: a multipurpose tree species

An efficient in vitro transformation and plant regeneration protocol was developed for Terminalia bellerica using cotyledonary node cultures. High-frequency shoot bud proliferation was obtained on medium with 6-benzyladenine. Significant improvements in plant regeneration occurred using elevated levels of CuSO4 and CoCl2. Rooting occurred on a half-strength Murashige and Skoog medium containing indole-3-butyric acid. The rooted plants were acclimatized and transferred to field conditions. The genetic fidelity of the regenerated plants was confirmed using randomly amplified polymorphic DNA analysis. An Agrobacterium-mediated genetic transformation protocol was developed for Terminalia by varying several factors which influence T-DNA delivery. Southern blot analysis of regenerated plants confirmed selectable marker gene integration in transgenic plants. This transformation protocol can be utilized for further genetic manipulation of T. bellerica.

Micropropagtion of Terminalia bellerica from nodal explants of mature tree and assessment of genetic fidelity using ISSR and RAPD markers

The present study reports an efficient in vitro micropropagation protocol for a medicinally important tree, Terminalia bellerica Roxb. from nodal segments of a 30xa0years old tree. Nodal segments taken from the mature tree in March-April and cultured on half strength MS medium gave the best shoot bud proliferation response. Combinations of serial transfer technique (ST) and incorporation of antioxidants (AO) [polyvinylpyrrolidone, PVP (50xa0mgxa0l−1)u2009+u2009ascorbic acid (100xa0mgxa0l−1)u2009+u2009citric acid (10xa0mgxa0l−1)] in the culture medium aided to minimize browning and improve explant survival during shoot bud induction. Highest multiplication of shoots was achieved on medium supplemented with 6-benzyladenine (BA, 8.8xa0μM) and α-naphthalene acetic acid (NAA, 2.6xa0μM) in addition to antioxidants. Shoot elongation was obtained on MS medium containing BA (4.4xa0μM)u2009+u2009phloroglucinol (PG, 3.9xa0μM). Elongated shoots were transferred to half strength MS medium containing indole-3-butyric acid (IBA, 2.5xa0μM) for root development. The acclimatization of plantlets was carried out under greenhouse conditions. The genetic fidelity of the regenerated plants was checked using inter simple sequence repeats (ISSR) and randomly amplified polymorphic DNA (RAPD) analysis. Comparison of the bands among the regenerants and mother plant confirmed true-to-type clonal plants.