Enhancement of rhamnetin production in Vernonia anthelmintica (L.) Willd. cell suspension cultures by eliciting with methyl jasmonate and salicylic acid.

Vernonia anthelmintica (L.) Willd. belongs to the family Asteraceae and has anthelmintic, anti-diabetic, diuretic and anti-asthmatic properties. Present study describes the production of rhamnetin: an O-methylated flavonol with anti-oxidant and anti-inflammatory activities; in cell suspension cultures of V. anthelmintica. Suspension cultures were established from cream coloured friable calli initiated from leaf explants and cultured in liquid MS medium supplemented with combinations of IAA (1.5 mg L-1) and BAP (1.5 mg L-1). After 20 days of cell suspension culture, the initial inoculum size of 0.5 g was increased to 1.05 g 100 mL-1 dry weight at exponential phase. When the culture was treated with 0.8 mM methyl jasmonate for 6 days; 2.2 fold accumulation of rhamnetin production was obtained on a dry weight basis compared to unelicited control. Likewise, the elicitation with salicylic acid 1 mM for 6 days brought about 2.7 fold rhamnetin production compared to unelicited control. The rhamnetin content in dried leaf tissue of field grown plants was found to be 0.143 mg g-1. Elicitation with methyl jasmonate and salicylic acid brought about 7.7 and 9.4 fold increments in rhamnetin production, compared to leaf tissue of field grown plants on a dry weight basis.

Effect of elicitation and precursor feeding on accumulation of 20-hydroxyecdysone in Achyranthes aspera Linn. cell suspension cultures.

20-Hydroxyecdysone is one of the most common ecdysteroids in plants with potential therapeutic applications. In this study, cell suspension cultures of Achyranthes aspera were raised in shake flasks to investigate the production of 20-hydroxyecdysone. The quantification and characterization of 20-hydroxyecdysone in the cultures were done by High performance liquid chromatography (HPLC) and Liquid Chromatography-quadrupole time-of- flight mass spectrometry (LC-Q-TOF) analyses. For raising the suspension, calli initiated from in vitro grown leaf explants were cultured in liquid Murashige and Skoog (MS) medium augmented with combinations of 2, 4-dichlorophenoxyacetic acid (1 mg L-1) and α-naphthaleneacetic acid (1 mg L-1). Maximum growth index of the cell suspension was 9.9, which was achieved during 20th day of culture (final phase of exponential growth). At this stage, the biomass accumulated was 1.09 ± 0.09 g dry weight (DW) and the 20-hydroxyecdysone concentration was 0.24 mg g-1 DW. Eliciting the cultures with 0.6 mM Methyl jasmonate for 6 days; enhanced the production of 20-hydroxyecdysone production to 0.35 mg g-1 DW. By augmenting the cultures with the precursors namely cholesterol (10 mg L-1) and 7-dehydrocholesterol (10 mg L-1), production of 20-hydroxyecdysone was boosted to 0.31 mg g-1 DW and 0.28 mg g-1 DW respectively.

Influence of methyl jasmonate and salicylic acid on Rhamnetin production in adventitious root cultures of Baccharoides anthelmintica (L.) Moench.

We developed adventitious roots from the leaf explants of Baccharoides anthelmintica (L.) Moench (syn. Vernonia anthelmintica) cultured on Murashige and Skoog (MS) medium. We tested MS and White's media fortified with different Indole Butyric Acid concentrations for their effect on root biomass production. MS medium supplemented with 1 mg L-1 IBA produced the highest biomass of adventitious roots compared to White's medium. HPLC and LC-Q-TOF analyses confirmed the ability of the adventitious roots of B. anthelmintica to produce rhamnetin. When the 15-day old root cultures were treated with 0.6 mM methyl jasmonate, on the 6th-day, rhamnetin content was enhanced to 0.443 mg g-1DW, which was 1.5 folds compared to untreated roots. Similarly, Salicylic acid also improved rhamnetin production to 0.36 mg g-1 DW, which was 1.2 folds increment compared to untreated roots. Rhamnetin content in the roots of field-grown plants was 0.112 mg g-1DW.

Penicillium setosum, a new species from Withania somnifera (L.) Dunal.

Medicinal plants are considered as sources of novel and unexplored groups of endophytic microorganisms. A study on endophytic fungal species from the medicinal plant Withania somnifera (L.) Dunal resulted in the isolation of a Penicillium isolate (WSR 62) with antibiotic activity. Phylogenetic analysis showed that the isolate belongs to section Lanata-divaricata, and it is most closely related to P. javanicum. Subsequent detailed phylogenetic analyses using partial ß-tubulin (BenA), calmodulin (CaM) and DNA-dependent RNA polymerase II (RPB2) gene sequences of a larger number of related strains revealed the distinctiveness of the isolate in the P. javanicum-clade. The isolate grows fast on Czapek yeast autolysate agar (CYA) and malt extract agar (MEA) incubated at 25°C, 30°C and 37°C. The obverse colony colour is dominated by the conspicuous production of cleistothecia and is greyish yellow on CYA and yellowish brown on MEA. Production of cleistothecia containing prominent spinose ascospores was present on all tested agar media. Based on the phylogenetic analysis and the phenotypic characterisation, strain WSR 62 from Withania is described here as a novel species named Penicillium setosum.

Endophytic fungal assemblage in Achyranthes aspera Linn. revealed by internal transcribed spacer region of nuclear ribosomal RNA genes.

Only scanty reports are available on endophytic fungal associations in Achyranthes aspera Linn. Hence in this study a total of 504 isolates belonging to ten different species of fungi were isolated from asymptomatic, surface sterilised segments of leaf, stem and root of A. aspera collected from different locations of Kerala, India. Among the isolates ascomycetes were most prevalent. Colonisation rate of fungal endophytes was high in leaf tissue (95%) followed by stem (77.75%) and root segments (33.33%). The most frequent and dominant coloniser of the host plant were Colletotrichum sp., which was isolated from all locations. Scanning electron microscopy demonstrated the presence of hyphae in the intra and intercellular spaces of the plant tissue. Morphological and phylogenetic analyses using internal transcribed spacer (ITS) sequences of nuclear rRNA genes showed that the fungi recovered belonged to the lineages of Sordariomycetes, Dothideomycetes, Eurotiomycetes and Tremellomycetes. A maximum likelihood tree revealed the relationship between the obtained sequence data and the closest sequences retrieved from the GenBank.

Genetic identification and phylogenetic relationships of Indian clariids based on mitochondrial COI sequences.

Mitochondrial cytochrome C Oxidase I (COI) sequence variation among the clariid fishes of India (Clarias magur, C. dussumieri and C. gariepinus) and their relationship with other representative clariids was studied in this work. Three species were sampled and together with 23 COI sequences from GenBank were used to reconstruct phylogenetic relationships in the family Clariidae. The study revealed two clades: one consisting of the African species with C. dussumieri, and the other of Asian species suggesting the prevalence of intra-continental diversification of catfishes. This study further revealed that the genus Clarias is monophyletic. For the COI gene, the interspecies genetic divergence ranged from 0.056 to 0.182. The mean genetic difference between C. dussumieri and other selected African species in this study is 12.1%. It was also observed that the morphological similarity of C. dussumieri and C. magur was not replicated in the genetic level. Clarias dussumieri was more close to African catfish C. gariepinus thus indicating the utility of COI phylogeny to identify the well-known African-Asian relationships within catfishes. The results also showed that C. magur and C. batrachus are genetically distinct from each other.