Swasti S. Swain

Production of Triterpenoid Anti-cancer Compound Taraxerol in Agrobacterium-Transformed Root Cultures of Butterfly Pea (Clitoria ternatea L.)

Independent transformed root somaclones (rhizoclones) of butterfly pea (Clitoria ternatea L.) were established using explant co-cultivation with Agrobacterium rhizogenes. Rhizoclones capable of sustained growth were maintained under low illumination in auxin-free agar-solidified MS medium through subcultures at periodic intervals. Integration of TL-DNA rolB gene in the transformed rhizoclone genome was verified by Southern blot hybridization, and the transcript expression of TR-DNA ags and man2 genes was ascertained by reverse transcription polymerase chain reaction analysis. The major compound isolated and purified from the transformed root extracts was identified as the pentacyclic triterpenoid compound taraxerol using IR, 1H-NMR, and 13C-NMR spectroscopy. The taraxerol yield in cultured hairy roots, as quantified by HPTLC analysis, was up to 4-fold on dry weight basis compared to that in natural roots. Scanning of bands from cultured transformed roots and natural roots gave super-imposable spectra with standard taraxerol, suggesting a remarkable homology in composition. To date, this is the first report claiming production of the cancer therapeutic phytochemical taraxerol in genetically transformed root cultures as a viable alternative to in vivo roots of naturally occurring plant species.

Evaluation of genetic diversity among commercial cultivars, hybrids and local mango (Mangifera indica L.) genotypes of India using cumulative RAPD and ISSR markers

An assessment of genetic diversity studies was undertaken to understand the level and pattern of diversity in 65 mango (Mangifera indica L.) genotypes of India including 20 commercial cultivars, 18 hybrids, 25 local genotypes and two exotic cultivars based on qualitative and quantitative fruit characters as well as RAPD and ISSR profiles. A considerable variation was observed in respect of three important qualitative characters namely table quality, fruit attractiveness and storage life of ripe fruits and potentially superior genotypes for the above traits were identified. A wide variation was noticeable regarding metabolite composition of pulp of ripe mango fruit with respect to total soluble solids, total sugar, reducing sugar, acidity, sugar:acid ratio, ascorbic acid and phenolic content. Fifteen RAPD primers yielded 27 monomorphic and 129 polymorphic bands with percent polymorphism averaging 82.7%. Of a total 70 ISSR bands generated from eight ISSR primers, 60 bands (85.71%) were found to be polymorphic. Cumulative band data from these two methods precisely arranged accessions into eight clusters which correspond well with their pedigree relationship. UPGMA dendrograms drawn using RAPD, ISSR and cumulative data showed highly similar grouping of genotypes on the basis of their parental origin. No clear-cut geographical separation was revealed among East, West, North and South Indian mango cultivars by neither of these molecular markers nor their combinations. This supports the common gene pool origin of mango as well as operation of similar selection pressure as the cultivar preferences in these areas are largely similar.

ED-XRF spectrometry-based trace element composition of genetically engineered rhizoclones vis-à-vis natural roots of a multi-medicinal plant, butterfly pea (Clitoria ternatea L.)

The energy dispersive X-ray fluorescence set-up incorporating a molybdenum secondary exciter was used for quantitative determination of major and minor elements in genetically transformed root somaclones (rhizoclones) of butterfly pea (Clitoria ternatea L.) which had been established via explant co-cultivation with Agrobacterium rhizogenes. The multi-elemental composition of these transformed rhizoclones was compared with that of the naturally grown in vivo donor plant. Trace elements namely Cr, Mn, Fe, Co, Ni, Cu, Zn, Se, Rb, Sr and Pb in addition to two macro-elements K and Ca were identified and quantified in root tissues of both sources. The elemental content of transformed root cultures was found to be at par with that of the natural roots of in vivo grown plants of the same species. These findings are implicated on the context of utilization of such Agrobacterium-mediated genetically transformed root cultures as a viable alternative to natural roots, the former being a fast-proliferating renewable resource of medicinally useful minerals essential for designing of effective drugs, besides providing an ex situ means for plant conservation.

Agrobacterium rhizogenes-mediated transformation of a multi-medicinal herb, Boerhaavia diffusa L.: optimization of the process and anti-microbial activity against bacterial pathogens causing urinary tract infections

Hairy root cultures (HRCs) of Boerhaavia diffusa L., an ethno-medicinally important herb, were established via explant co-cultivation with Agrobacterium rhizogenes. Several key factors influencing transformation efficiency were optimized. Rhizoclones showing prolific growth were maintained through successive subcultures in solidified Murashige & Skoogs medium without growth regulators (MS0) under diffused illumination. PCR analysis of the rhizoclones demonstrated positive amplification of rolB and rolC genes of pRi TL-DNA. Reverse transcription-PCR (RT-PCR) and opine assay revealed the presence and expression of the pRi TR-DNA borne ags gene. The methanol and dichloromethane (M:DM) extracts of hairy roots (HRs) and natural roots (NRs) were screened against a wide range of bacterial pathogens causing human urinary tract infections (UTIs). The MIC and MBC values of HRs were markedly lower when compared to NRs against microorganisms causing UTIs. Extracts of HRs were more effective than those of NRs not only against wild type strains but also, and more importantly, against multi-drug resistant strains. Establishment of fast-proliferating renewable HRCs of B. diffusa would provide a direct source of anti-microbial phytocompounds against several pathogenic bacteria, including resistant strains, causing UTIs.

Quantification of β-Sitosterol in Hairy Root Cultures and Natural Plant Parts of Butterfly Pea (Clitoria ternatea L.)

A simple normal-phase high-performance thin-layer chromatography (HPTLC) method has been developed for the quantification of β-sitosterol in hairy root cultures of Clitoria ternatea vis-à-vis natural plant parts. Chromatographic separation was achieved on normal-phase TLC plates by using optimized mobile phase of n-hexane. acetone (8:2, v/v). Densitometric scanning was performed after postchromatographic derivatization of the plate at 414 nm. The limits of detection and quantification were found to be 40 ng and 100 ng spot−1, respectively. A linear response of calibration line was observed over the range of 100 to 500 ng spot−1 with a correlation coefficient of r2 = 0.999. Recovery values from 97.22 to 98.51% with small coefficient of variation showed excellent accuracy of the method. The method was validated according to the International Conference on Harmonization (ICH) protocol. The method enables excellent separation and accurate quantification of β-sitosterol in different plant parts of the two floral varieties of C. ternatea along with their cultured roots derived through genetic transformation. β-Sitosterol content was comparable between the blue-flowered and white-flowered plants; roots had a higher content than that in stem and leaf regardless of the two floral varieties. Transformed root cultures had a lower content of β-sitosterol compared to natural roots.