ANNOUNCEMENTS
7.1 Genetic diversity analysis
Genetic diversity analysis has direct implications in conservation of endangered medicinal and aromatic plant resources as it enables identification of areas and regions representing maximum genetic variations enabling conservation of population from these areas at utmost priority (Shanker and Ganeshaiah 1997). Bearing this in mind, Inter Simple Sequence Repeat (ISSR) marker assay was employed for analysing genetic diversity of S. chirayita collected from the temperate Himalayan regions of India. The study was conducted on two data sets. A population of S. chirayita from Mandal region of Uttaranchal state in India comprised data Set I. Collections of S. chirayita from different geographical locales, herbal dealers and commercial plantations comprised data Set II. Allied species of S. chirayita were used in both the studies as outliers. Nineteen UBC primers generated a total of 315 ISSR bands in data set I, revealing 98.73% polymorphism among the genotypes assayed. This was reduced to 42.59% when the outliers were excluded. The binary data was employed to generate Jaccard‟s similarity matrix and different clustering algorithms, such as, Complete Linkage (CL), Single Linkage (SL), Unweighted Pair Group Method with Arithmetic Averages (UPGMA) and Weighed Pair Group with Arithmetic Averages (WPGMA) and PCO analysis were used to reveal the groupings in the data set. The results revealed a high genetic diversity within the genotypes, which can be correlated to the cross-pollinating behaviour of the plant. The results of the study emphasised the need to conserve the available genetic diversity as well as the need to protect the habitat of the plant. In data set II, 246 polymorphic bands, revealing cent percent polymorphism were produced with eighteen UBC primers. On excluding the outliers, the polymorphism was reduced to 97.84% only. The high level of polymorphism revealed can be attributed to the heterogeneity of the samples analysed in the data set. The statistical analysis of the data set revealed a clear-cut separation between the authentic chiretta and its substitutes / adulterants that were either sold at herbal markets or grown in commercial plantations in lieu of the authentic herb. The study highlights the need to define core collection of genuine chiretta, which can be used for providing quality planting material to commercial growers.
7.2 Micropropagation of Swertia chirayita
In vitro culture is an efficient method for ex situ conservation of plant biodiversity and multiplication of the endangered species. Hence, a cost effective micropropagation protocol for S. chirayita was standardised by employing axillary proliferation. The cultures were initiated from seedling derived nodal explants. Seeds were treated with GA3 and seed sterilisation was performed using 1% sodium hypochlorite. The sterilised seeds were inoculated on MS medium for germination. Four-week old aseptic seedlings were used as a source of nodal explants, which were inoculated on cytokinin supplemented MS medium. In the present study, a 4.5 fold multiplication was obtained every four weeks on MS medium supplemented with 4 μM BAP + 1.5 μM 2iP. The shoots obtained from these cultures were used to optimise rooting. A hundred percent rooting response was obtained on MS ½ medium supplemented with 1 M NAA + 500 mgl-1 activated charcoal. Hardening of the rooted plants was tried both under in vitro and in vivo conditions and survival rate of 94.5% was obtained in both the cases. Ex vitro performance of the plants was studied. The plants were transplanted to field at Mandal (1500 m asl), where a survival percentage of 65.3 were obtained. A regeneration protocol of the herb was also established using leaf explants derived from in vitro cultures. Leaf, root and stem explants of the in vitro plantlets were tested for regeneration and only the leaf explants responded favourably. Seventy eight percent leaf explants responded to regeneration on MS ½ medium supplemented with 0.5 mgl-1 of 2,4,5-T and 2.0 mgl-1 of BAP, producing an average of ten shoots per explant. Most of the shoots developed roots on the MS ½ basal medium itself and were successfully hardened.
7.3 Clonal fidelity of micropropagated plants
Somaclonal variations have been defined as genetic and phenotypic variations among clonally propagated plants of a single donor clone and these are manifested as somatically or meiotically stable events. However, these genetic variations are not sought-after when clonal propagation or transformation is the ultimate aim. Also, somatic variations may result in decline in vigour and regenerability of the cultures over time. Therefore, clonal fidelity analysis of the micropropagated plantlets was established using ISSR assay. S. chirayita plantlets were collected at 10th and 15th multiplication passage. Seven UBC primers were used and they amplified a total of 106 bands between the size ranges of 200 bp to 2 Kb. Primer UBC 841 amplified the highest number of 19 bands and also deciphered maximum polymorphism between the S. chirayita genotypes and the outliers. Primer UBC 868 on the other hand, amplified the lowest number of 11 bands in the data set. In continuation of the above study, plantlets were also collected at 42nd culture passage (after 168 weeks of subculture) and screened for the clonal uniformity with reference to samples from the above study. In this data set a total of sixteen ISSR primers were used and these amplified 150 bands. The banding profile obtained among the tissue culture raised progeny was completely uniform suggesting a high level of genetic fidelity even after the prolonged sub-culturing. Clonal fidelity of S. chirayita plantlets regenerated from leaf explants was also assessed. The plants were found to be clonally uniform to the donor plant as revealed by employing seven ISSR primers, which generated a total of fifty-three amplification products. Thus, in the three data sets analysed, no genetic variation was detected, irrespective of the culture duration and method of regeneration.
7.4 Quantitative determination of swertiamarin
Biochemical analysis of medicinal plants is an essential prerequisite for determining the phyto-chemical biodiversity of the target species as it renders greater control over quality and supply of the raw material. HPLC analysis for swertiamarin content, one of the important active principles of Swertia chirayita, was conducted in the present study. Reverse phase C-18 column was used for HPLC analysis and elutions were carried out under isocratic conditions using acetonitrile: water (10: 90) at a flow rate of 1.0 ml/min. The analysis of in vitro grown plantlets was carried out and comparative analysis of swertiamarin content in roots, leaves and stem portions was done separately after six months, one year and two years of plant growth in the field. Swertiamarin content in root derived callus cultures as well as in three species of Swertia and two market samples of chiretta was also assayed. Whereas 5.07% swertiamarin was revealed in in vitro plantlets of chiretta, marked variation was observed in different parts of the micropropagated plants after their transplantation to the field. Callus cultures of chiretta were established from root explants. In contrast to a comparably higher swertiamarin content in in vitro plantlets of S. chirayita (5.07%), the root derived callus cultures contained almost negligible amounts of swertiamarin. These results show that shoot cultures of S. chirayita can serve as a viable alternative for production of swertiamarin. Three species of Swertia, namely S. cordata, S. paniculata and S. purpurascens were also analyzed and S. paniculata was found to contain maximum amount of the active principle. The market samples revealed a highly variable chromatographic profile. These results clearly demonstrate the levels of substitution and adulteration in trade of chiretta.
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