Suman Kumaria

Start Codon Targeted (SCoT) marker reveals genetic diversity of Dendrobium nobile Lindl., an endangered medicinal orchid species.

Genetic variability in the wild genotypes of Dendrobium nobile Lindl. collected from different parts of Northeast India, was analyzed using a Start Codon Targeted (SCoT) marker system. A total of sixty individuals comprising of six natural populations were investigated for the existing natural genetic diversity. One hundred and thirty two (132) amplicons were produced by SCoT marker generating 96.21% polymorphism. The PIC value of the SCoT marker system was 0.78 and the Rp values of the primers ranged between 4.43 and 7.50. The percentage of polymorphic loci (Pp) ranging from 25% to 56.82%, Neis gene diversity (h) from 0.08 to 0.15 with mean Neis gene diversity of 0.28, and Shannons information index (I) values ranging from 0.13 to 0.24 with an average value of 0.43 were recorded. The gene flow value (0.37) and the diversity among populations (0.57) demonstrated higher genetic variation among the populations. Analysis of molecular variance (AMOVA) showed 43.37% of variation within the populations, whereas 56.63% variation was recorded among the populations. Cluster analysis also reveals high genetic variation among the genotypes. Present investigation suggests the effectiveness of SCoT marker system to estimate the genetic diversity of D. nobile and that it can be seen as a preliminary point for future research on the population and evolutionary genetics of this endangered orchid species of medicinal importance.

Genetic stability and phytochemical analysis of the in vitro regenerated plants of Dendrobium nobile Lindl., an endangered medicinal orchid

An efficient genetically stable regeneration protocol with increased phytochemical production has been established for Dendrobium nobile, a highly prized orchid for its economic and medicinal importance. Protocorm like bodies (PLBs) were induced from the pseudostem segments using thidiazuron (TDZ; 1.5 mg/l), by-passing the conventional auxin–cytokinin complement approach for plant regeneration. Although, PLB induction was observed at higher concentrations of TDZ, plantlet regeneration from those PLBs was affected adversely. The best rooting (5.41 roots/shoot) was achieved in MS medium with 1.5 mg/l TDZ and 0.25% activated charcoal. Plantlets were successfully transferred to a greenhouse with a survival rate of 84.3%, exhibiting normal development. Genetic stability of the regenerated plants was investigated using randomly amplified polymorphic DNA (RAPD) and start codon targeted (SCoT) polymorphism markers which detected 97% of genetic fidelity among the regenerants. The PIC values of RAPD and SCoT primers were recorded to be 0.92 and 0.76 and their Rp values ranged between 3.66 and 10, and 4 and 12 respectively. The amplification products of the regenerated plants showed similar banding patterns to that of the mother plant thus demonstrating the homogeneity of the micropropagated plants. A comparative phytochemical analysis among the mother and the micropropagated plants showed a higher yield of secondary metabolites. The regeneration protocol developed in this study provides a basis for ex-situ germplasm conservation and also harnesses the various secondary metabolite compounds of medicinal importance present in D. nobile.

Single primer amplification reaction (SPAR) reveals inter- and intra-specific natural genetic variation in five species of Cymbidium (Orchidaceae).

A total of 53 primers belonging to three SPAR methods, viz. RAPD, ISSR and DAMD, collectively produced 456 polymorphic amplicons with 96.6% polymorphism at inter-specific level in five species of Cymbidium, viz. C. aloifolium, C. mastersii, C. elegans, C. eburneum and C. tigrinum, whereas at intra-specific level, the observed polymorphism ranged from 51.2% to 77.1% among them. Three SPARs collectively revealed 25 unique species-specific amplicons; most of them were amplified with RAPD and DAMD primers besides few bands which were either missed (absent) or lost (heterozygosity). UPGMA clustering evidently distinguished the representatives of C. aloifolium and C. tigrinum, with distinct genetic distance, which may be due to their entirely different habitats as well as discrete morphological characteristics. Upon analysis of the data generated, all the three SPAR methods, either independently and/or in combination, revealed wide range of genetic variation between and within five species of Cymbidium. Comparison of matrix of individual SPAR method revealed that analysis of natural genetic variation using combination of SPAR methods, rather than an isolated approach, is highly effective. The critical analyses of the amplicon data are indicative of DAMD as the most powerful SPAR method by showing highest resolving power (Rp) followed by ISSR and RAPD. Alternatively, the total polymorphic information content was highest in case of RAPD followed by other two SPAR methods. Thus, the present investigation for the first time provides a valuable baseline data for genetic variation at inter- and intra-specific levels in horticultural Cymbidiums and also addresses conservation concerns.

Phyto-molecular profiling and assessment of antioxidant activity within micropropagated plants of Dendrobium thyrsiflorum: a threatened, medicinal orchid

The escalating loss of biological diversity throughout the world has become a major concern for the conservation biologists. Like other threatened plant species, the natural populations of the orchids are also severely threatened. Dendrobium thyrsiflorum is one such representative of the family Orchidaceae whose natural populations are getting destroyed at an alarming rate and deserves special conservation attention. Both direct shoot organogenesis (DSO) and indirect shoot organogensesis (ISO) pathways were experimented and the highest regeneration frequency for DSO and ISO pathways were found to be 86.2 and 96 % respectively. The regenerated shoots were best rooted in half-strength MS medium supplemented with 1 mg/l indole butyric acid (IBA) and 0.5 mg/l phloroglucinol. The genetic stability of the acclimatized plants derived from ISO and DSO pathways was assessed using Inter Simple Sequence Repeats (ISSR) and Start Codon Targeted (SCoT) molecular markers. SCoT proved to be a superior marker over ISSR in detecting clonal variability. The phytochemical analysis of the micropropagated plants also revealed a comprehensively higher yield of various secondary metabolites with significantly higher antioxidant potentials in both ISO- and DSO-derived plants over the mother plant. However, the ISO-derived plants were more phytochemically enriched compared to the DSO-plants. The rapid multiplication rate, higher genetic stability and secondary metabolite production ensures the utility of this micropropagation method for D. thyrsiflorum in the ex situ conservation and commercial exploitation of other important orchid species.

An effective nutrient medium for asymbiotic seed germination and large-scale in vitro regeneration of Dendrobium hookerianum, a threatened orchid of northeast India.

Submergence inhibits photosynthesis by terrestrial wetland plants, but less so in species that possess leaf gas films when submerged. Floodwaters are often supersaturated with dissolved CO2 enabling photosynthesis by submerged terrestrial plants, although rates remain well-below those in air. This important adaptation that enhances survival in submerged conditions is reviewed.

Assessment of phylogenetic inter-relationships in the genus Cymbidium (Orchidaceae) based on internal transcribed spacer region of rDNA.

Sequence data obtained from nrITS region were used to assess phylogenetic inter-relationships and infrageneric classification of ten Cymbidium species collected from north-east India. The final aligned data matrix of combined ITS 1, 5.8S and ITS 2 yielded 684 characters. The ITS 1 and ITS 2 regions showed variable sequence lengths and G+C content (%). The 5.8S region was found to be more conserved (98.71%) followed by ITS 1 (86.12%) and ITS 2 (69.40%). ITS 2 recorded highest percentage of parsimony informative sites (7.46%), high sequence divergence with indels (24.63%), high number of transitions and transversions. ITS sequence data determined the phylogeny of Asiatic Cymbidiums with high bootstrap values. All three proposed subgenera could be distinguished clearly by all four (MP, ML, NJ, and BI) phylogenetic methods. This study validates the utility of ITS rDNA region as a reliable indicator of phylogenetic relationships, especially ITS 2 as probable DNA barcode at higher levels and can serve as an additional approach for identification of broader range of plant taxa especially orchids.

Applicability of ISSR and DAMD markers for phyto-molecular characterization and association with some important biochemical traits of Dendrobium nobile, an endangered medicinal orchid

Dendrobium nobile is an important medicinal orchid having profound importance in traditional herbal drug preparations and pharmacopeias worldwide. Due to various anthropogenic pressures the natural populations of this important orchid species are presently facing threats of extinction. In the present study, genetic and chemical diversity existing amongst 6 natural populations of D. nobile were assessed using molecular markers, and the influence of genetic factors on its phytochemical activity especially antioxidant potential was determined. Molecular fingerprinting of the orchid taxa was performed using ISSR and DAMD markers along with the estimation of total phenolics, flavonoids and alkaloid contents. Antioxidant activity was also measured using DPPH and FRAP assays which cumulatively revealed a significant level of variability across the sampled populations. The representatives from Sikkim in Northeast India revealed higher phytochemical activity whereas those from Mizoram showed lesser activity. Analysis of molecular variance (AMOVA) revealed that variation amongst the populations was significantly higher than within the populations. The data generated by UPGMA and Bayesian analytical models were compared in order to estimate the genetic relationships amongst the D. nobile germplasm sampled from different geographical areas of Northeast India. Interestingly, identical grouping patterns were exhibited by both the approaches. The results of the present study detected a high degree of existing genetic and phytochemical variation amongst the populations in relation to bioclimatic and geographic locations of populations. Our results strongly establish that the cumulative marker approach could be the best suited for assessing the genetic relationships with high accuracy amongst distinct D. nobile accessions.

Multiple shoot induction from axillary bud cultures of the medicinal orchid, Dendrobium longicornu

The present investigation was undertaken to propagate D. longicornu, a medicinally important orchid using axillary bud segments. This approach could also help in conserving other threatened orchids as well.

A simple and efficient protocol for the mass propagation of Cymbidium mastersii: an ornamental orchid of Northeast India

The present investigation was undertaken to mass propagate Cymbidium mastersii, an ornamental orchid of Northeast India by in vitro propagation method. This approach could also help for the conservation as well as commercialization of C. mastersii and other threatened and ornamental orchids.

Single primer amplification reaction (SPAR) methods reveal subsequent increase in genetic variations in micropropagated plants of Nepenthes khasiana Hook. f. maintained for three consecutive regenerations.

The genetic fidelity of in vitro-raised plants of three successive regenerations of Nepenthes khasiana Hook. f. was assessed using three different single primer amplification reaction (SPAR) methods, viz., random amplified polymorphic DNA (RAPD), inter-simple sequence repeat (ISSR) and direct amplification of minisatellite DNA region (DAMD) markers. Out of 80 RAPD primers screened, 14 primers reflected a genetic variation of 4.1% in the first regeneration which was increased to 9.4% in the third regeneration. In the case of ISSR, out of 36 primers screened for assessment of genetic homogeneity of the regenerated plantlets, 12 primers showed an increase of genetic variation from 4.3% to 10% from the first to the third regenerations. In DAMD profiling, 15 primers were used for the evaluation of genetic fidelity where 8.47% of polymorphism was observed in the first regeneration which was increased to 13.33% in the third regeneration. The cumulative analysis reflected a genetic variation of 5.65% in the first regeneration which increased subsequently to 7.77% in the second regeneration and 10.87% in the third regeneration. The present study demonstrates SPAR technique to be an efficient tool for the assessment of clonal fidelity of in vitro-raised plants.