Evaluation of rapid molecular diagnostics for differentiating medicinal Kaempferia species from its adulterants.

Accurate detection of unique herbs is crucial for herbal medicine preparation. Zingiberaceae species, which are important in Ayurvedic medicine of India, are often misidentified in Northeast (NE) Indian herbal markets. Kaempferia galanga (Zingiberaceae) is one of the major components of popular Ayurvedic drugs used for rheumatic diseases (i.e., "Gandha Thailam" and "Rasnairandadi Kashayam"), contusions, fractures, and sprains. In NE India, herbal healers often misidentify plants from the Marantaceae family (e.g., Calathea bachemiana and Maranta leuconeura) as Kaempferia, which leads to adulteration of the medicinal herb. This misidentification of herbs occurs in NE India because Zingiberaceae plant barcoding information is inadequate. As a consequence, herbal medicine is not only therapeutically less effective but may also cause adverse reactions that range from mild to life-threatening. In this study, we used eight barcoding loci to develop "fingerprints" for four Kaempferia species and two species frequently mistaken for Kaempferia. The PCR and sequencing success of the loci matK, rbcL and trnH-psbA were found to be 100%; the combination of matK, rbcL, and trnH-psbA proved to be the ideal locus for discriminating the Kaempferia species from their adulterants because the combined loci showed greater variability than individual loci. This reliable tool was therefore developed in the current study for accurate identification of Kaempferia plants which can effectively resolve identification issues for herbal healers.

Gene expression analysis of volatile-rich male flowers of dioecious Pandanus fascicularis using expressed sequence tags.

Pandanus fascicularis is dioecious with the female plant producing a non-scented fruit while the male produces a flower rich in volatiles. The essential oil extracted from the flowers is economically exploited as a natural flavouring agent as well as for its therapeutic properties. Molecular dissection of this distinct flower for identifying the genes responsible for its aroma by way of expressed sequence tags (ESTs) has not been initiated in spite of its economic viability. A male flower-specific cDNA library was constructed and 977 ESTs were generated. CAP3 analysis performed on the dataset revealed 83 contigs (549 ESTs) and 428 singlets, thereby yielding a total of 511 unigenes. Functional annotation using the BLAST2GO software resulted in 1952 Gene ontology (GO) functional classification terms for 621 sequences. Unknown proteins were further analysed with InterProScan to determine their functional motifs. RNA gel blot analysis of 26 functionally distinct transcripts potentially involved in flowering and volatile generation, using vegetative and reproductive tissues of both the sexes, revealed differential expression profiles. In addition to an overview of genes expressed, candidate genes with expression that are modulated predominantly in the male inflorescence were also identified. This is the first report on generation of ESTs to determine the subset of genes that can be used as potential candidates for future attempts aimed towards its genetic and genome analysis including metabolic engineering of floral volatiles in this economically important plant.

Growth inhibitory activity of fatty acid methyl esters in the whole seed oil of madagascar periwinkle (Apocyanaceae) against Helicoverpa armigera (Lepidoptera: Noctuidae).

Crude hexane and water extracts of Catharanthus roseus Linn. (Syn: Vinca rosea) (Apocyanaceae) stem, leaf, and seed exhibited pesticidal activity against Helicoverpa armigera (Hübner) (Lepidoptera Noctuidae). The extracts differed significantly in their efficacy, with the crude hexane extract of whole seed being the most effective in curtailing pupal survivorship to 18% followed by the hexane extracts of leaf (21%) and stem (24%). Average pupal weight (68.2 mg) and length (1.5 cm) in the whole seed treatment were drastically reduced, compared with the controls (415.2 mg and 2.72 cm), subsequently reducing adult emergence to 15.7%. SiO2 column purification yielded eight fractions of which fraction 1 exhibited 90% larval mortality, with severe reduction of the larvae weight (12.7 mg) and length (1.5 cm). Gas chromatographic-mass spectrometric analysis of fraction 1 suggested the presence of 16 compounds, among which oleic, linoleic, palmitic, and margaric acids were detected as major constituents. Presence of the alkane hydrocarbons triacontane, tetracosane, and heptacosane also was noted.

An oxidative and salinity stress induced peroxisomal ascorbate peroxidase from Avicennia marina: molecular and functional characterization.

APX (EC, 1.11.1.11) has a key role in scavenging ROS and in protecting cells against their toxic effects in algae and higher plants. A cDNA encoding a peroxisomal ascorbate peroxidase, Am-pAPX1, was isolated from salt stressed leaves of Avicennia marina (Forsk.) Vierh. by EST library screening and its expression in the context of various environmental stresses was investigated. Am-pAPX1 contains an ORF of 286 amino acids coding for a 31.4 kDa protein. The C-terminal region of the Am-pAPX1 ORF has a putative transmembrane domain and a peroxisomal targeting signal (RKKMK), suggesting peroxisomal localization. The peroxisomal localization of Am-pAPX1 was confirmed by stable transformation of the GFP-(Ala)(10)-Am-pAPX1 fusion in tobacco. RNA blot analysis revealed that Am-pAPX1 is expressed in response to salinity (NaCl) and oxidative stress (high intensity light, hydrogen peroxide application and excess iron). The isolated genomic clone of Am-pAPX1 was found to contain nine exons. A fragment of 1616bp corresponding to the 5' upstream region of Am-pAPX1 was isolated by TAIL-PCR. In silico analysis of this sequence reveals the presence of putative light and abiotic stress regulatory elements.

Over expression of cytosolic copper/zinc superoxide dismutase from a mangrove plant Avicennia marina in indica rice var Pusa Basmati-1 confers abiotic stress tolerance.

Antioxidant enzymes play an important role in conferring abiotic stress tolerance. Superoxide dismutase (SOD) is the first enzyme in the enzymatic antioxidative pathway. Halophytic plants like mangroves have been reported to have a high level of SOD activity, which plays a major role in defending the mangrove species against severe abiotic stresses. We had previously reported the isolation of Sod1, a cDNA encoding a cytosolic copper zinc superoxide dismutase from the mangrove plant Avicennia marina and its mRNA expression pattern during various oxidative and abiotic stresses. The present study is an extension of the previous study in further characterizing the Sod1 cDNA by transforming it into rice and analysing the transgenic plants for abiotic stress tolerance. Southern hybridization of A. marina genomic DNA using Sod1, revealed that this gene in A. marina genome is present as a single copy. The cDNA was cloned into a binary vector (pCAMBIA 1300) and transformed into indica rice var Pusa Basmati-1. Southern hybridization analysis of transgenic rice plants revealed stable integration of the Sod1 transgene in the rice genome. The mRNA transcript of Sod1 was detected by Northern hybridisation in the transgenic rice plants. SOD isozyme assay of the transgenic rice plants revealed the stable expression of the transgenic Sod1 protein. The transgenic plants were more tolerant to methyl viologen mediated oxidative stress in comparison to the untransformed control plants. The transgenic plants also withstood salinity stress of 150 mM of NaCl for a period of eight days while the untransformed control plants wilted at the end of the stress treatment in hydroponics. Pot grown transgenic plants could also tolerate salinity stress better than the untransformed control plants, when irrigated with saline water. The transgenic plants also revealed better tolerance to drought stress in comparison to untransformed control plants.

Monitoring expression profiles of antioxidant genes to salinity, iron, oxidative, light and hyperosmotic stresses in the highly salt tolerant grey mangrove, Avicennia marina (Forsk.) Vierh. by mRNA analysis.

Plant photosynthesis results in the production of molecular oxygen. An inevitable consequence of this normal process is the production of reactive oxygen species (ROS) by the transfer of electrons to molecular oxygen. Plants are adequately protected by the presence of multiple antioxidative enzymes in different organelles of the plant such as chloroplasts, cytosol, mitochondria and peroxisomes. Under high light and CO(2) limiting conditions caused by environmental stress like salinity, these antioxidative enzymes play an important role in scavenging toxic radicals. To investigate the functions of antioxidative enzymes in a mangrove plant, we isolated three cDNAs encoding cytosolic Cu-Zn SOD (Sod1), catalase (Cat1) and ferritin (Fer1) from Avicennia marina cDNA library. Sod1, Cat1 and Fer1 cDNA encoded full-length proteins with 152, 492 and 261 amino acids respectively. We studied the expression of these antioxidant genes in response to salt, iron, hydrogen peroxide, mannitol and light stress by mRNA expression analysis. Cat1, Fer1 showed short-term induction while Sod1 transcript was found to be unaltered in response to NaCl stress. A decrease in mRNA levels was observed for Sod1, Cat1 while Fer1 mRNA levels remained unaltered with osmotic stress treatment. Sod1, Cat1 and Fer1 mRNA levels were induced by iron, light stress and by direct H(2)O(2) stress treatment, thus confirming their role in oxidative stress response.