Evaluating genetic diversity of geographically diverse populations of Embelia ribes Burm f., a highly medicinal woody liana from the Western Ghats of India, using random amplified polymorphic DNA (RAPD) and intersimple sequence repeats (ISSR) markers.

BACKGROUND: Embelia ribes Burm f. (Primulaceae) is a medicinal and vulnerable woody liana distributed throughout India. Embelin, a well-recognized active phytoconstituents in berries, is commonly used in ayurvedic formulations. Due to over-exploitation, the status of the plant is vulnerable. Previous studies on this species mainly focused on its phytochemical analysis, which led to overexploitation and loss of the germplasm. METHODS AND RESULTS: In the present study, 20 RAPD and 18 ISSR markers were employed to assess genetic divergence in 40 genotypes of E. ribes collected from different parts of the Western Ghats of India. In RAPD analysis, all 40 accessions with 20 RAPD primers amplified 282 fragments, with 83.91% average polymorphism and with an average of 14.10 bands per primer. The size of amplicons varied from 200 to 2500 bp. While, ISSR primers produced 203 fragments of which 161 were polymorphic with an average of 11.28 bands per primer with 73.25% average polymorphism. The size of amplicons ranges from 200 to 2500 bp. RAPD and ISSR markers were also assessed by calculating polymorphic information content (PIC) to discriminate the genotypes; the average PIC value for RAPD, ISSR, and combined RAPD + ISSR markers obtained was more than 0.50 suggesting the informativeness of markers. UPGMA analysis based on Jaccard's similarity coefficient for RAPD, ISSR, and RAPD + ISSR data reveals that 40 accessions of E. ribes were depicted in four clusters. The clustering pattern of all individuals in PCoA analysis agreed with the UPGMA dendrograms, which further confirms the genetic relationships explained by cluster analysis. AMOVA analysis of RAPD, ISSR, and combined marker system revealed variation within the population, ranging from 41 to 44%, and among the population, it ranged from 56 to 59%. CONCLUSION: The present study provides an optimized method for evaluating the genetic diversity of Embelia ribes using RAPD and ISSR markers which are useful for further sustainable utilization and conservation of natural populations in the Western Ghats of India.

Characterization of influx and efflux silicon transporters and understanding their role in the osmotic stress tolerance in finger millet (Eleusine coracana (L.) Gaertn.).

Over the last decade, silicon (Si) has been widely accepted as a beneficial element for plant growth. The advantages plant derives from the Si are primarily based on the uptake and transport mechanisms. In the present study, the Si uptake regime was studied in finger millet (Eleusine coracana (L). Gaertn.) under controlled and stress conditions. The finger millet can efficiently uptake Si and accumulate it by more than 1% of dry weight in the leaf tissues, thus categorized as a Si accumulator. Subsequent evaluation with the single root assay revealed a three-fold higher Si uptake under osmatic stress than control. These results suggest that Si alleviated the PEG-induced stress by regulating the levels of osmolytes and antioxidant enzymes. Further, to understand the molecular mechanism involved in Si uptake, the Si influx (EcoLsi1 and EcoLsi6) and efflux transporters (EcoLsi2 and EcoLsi3) were identified and characterized. The comparative phylogenomic analysis of the influx transporter EcoLsi1 with other monocots revealed conserved features like aromatic/arginine (Ar/R) selectivity filters and pore morphology. Similarly, Si efflux transporter EcoLsi3 is highly homologous to other annotated efflux transporters. The transcriptome data revealed that the expression of both influx and efflux Si transporters was elevated due to Si supplementation under stress conditions. These findings suggest that stress elevates Si uptake in finger millet, and its transport is also regulated by the Si transporters. The present study will be helpful to better explore Si derived benefits in finger millet.

An insight into the role of silicon on retaliation to osmotic stress in finger millet (Eleusine coracana (L.) Gaertn).

Finger millet, a vital nutritional cereal crop provides food security. It is a well-established fact that silicon (Si) supplementation to plants alleviates both biotic and abiotic stresses. However, precise molecular targets of Si remain elusive. The present study attempts to understand the alterations in the metabolic pathways after Si amendment under osmotic stress. The analysis of transcriptome and metabolome of finger millet seedlings treated with distilled water (DW) as control, Si (10 ppm), PEG (15%), and PEG (15%) + Si (10 ppm) suggest the molecular alterations mediated by Si for ameliorating the osmotic stress. Under osmotic stress, uptake of Si has increased mediating the diversion of an enhanced pool of acetyl CoA to lipid biosynthesis and down-regulation of TCA catabolism. The membrane lipid damage reduced significantly by Si under osmotic stress. A significant decrease in linolenic acid and an increase of jasmonic acid (JA) in PEG + Si treatment suggest the JA mediated regulation of osmotic stress. The relative expression of transcripts corroborated with the corresponding metabolites abundance levels indicating the activity of genes in assuaging the osmotic stress. This work substantiates the role of Si in osmotic stress tolerance by reprogramming of fatty acids biosynthesis in finger millet.

Molecular Authentication of the Traditional Medicinal Plant "Lakshman Booti" (Smithia conferta Sm.) and Its Adulterants through DNA Barcoding.

BACKGROUND: Smithia conferta Sm. is an annual herb widely used in Indian traditional medical practice and commonly known as "Lakshman booti" in Sanskrit. Morphological resemblance among the species of genus Smithia Aiton. leads to inaccurate identification and adulteration. This causes inconsistent therapeutic effects and also affects the quality of herbal medicine. AIM: This study aimed to generate potential barcode for authentication of S. conferta and its adulterants through DNA barcoding technique. MATERIALS AND METHODS: Genomic DNA extracted from S. conferta and its adulterants was used as templates for polymerase chain reaction amplification of the barcoding regions. The amplicons were directed for sequencing, and species identification was conducted using BLASTn and unweighted pair-group method with arithmetic mean trees. In addition, the secondary structures of internal transcribed spacer (ITS) 2 region were predicted. RESULTS: The nucleotide sequence of ITS provides species-specific single nucleotide polymorphisms and sequence divergence (22%) than psbA-trnH (10.9%) and rbcL (3.1%) sequences. The ITS barcode indicates that S. conferta and Smithia sensitiva are closely related compared to other species. CONCLUSION: ITS is the most applicable barcode for molecular authentication of S. conferta, and further chloroplast barcodes should be tested for phylogenetic analysis of genus Smithia. SUMMARY: The present investigation is the first effort of utilization of DNA barcode for molecular authentication of S. conferta and its adulterants. Also, this study expanded the application of the ITS2 sequence data in the authentication. The ITS has been proved as a potential and reliable candidate barcode for the authentication of S. conferta. Abbreviations used: BLASTn: Basic Local Alignment Search Tool for Nucleotide; MEGA: Molecular Evolutionary Genetic Analysis; EMBL: European Molecular Biology Laboratory; psbA-trnH: Photosystem II protein D1- stuctural RNA: His tRNA gene; rbcL: Ribulose 1,5 bi-phosphate carboxylase/oxygenase large subunit gene.

Highly efficient in vitro regeneration, establishment of callus and cell suspension cultures and RAPD analysis of regenerants of Swertia lawii Burkill.

Highly efficient in vitro regeneration system has been developed for Swertia lawii Burkill, an important herb used as substitute for Swertia chirayita. Shoot tips explants were cultured on MS medium with various phytohormones for multiple shoot production. The best shoot production frequency (100%) and maximum shoots (10.4 ± 0.8) were obtained on MS media containing TDZ (3.0 mg l-1) in combination with IBA (0.3 mg l-1). Maximum callus induction (95 ± 4.8%) and callus growth (1.7 ± 0.4 gm) was achieved on MS medium with 2, 4-D (3.0 mg l-1). Cell suspension cultures were established and studied for their growth kinetics. Shoots were rooted best (22.1 ± 2.5) in 1/2 MS medium with IAA (3.0 mg l-1). The genetic uniformity of the micropropagated clones was assessed using RAPD markers. Out of 405 bands, 400 (98.76%) were monomorphic and rest 5 (1.24%) were polymorphic. High multiplication frequency and low risk of genetic instability ensures the efficacy of this protocol.