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Item Predominance of the serpentine route in monoterpenoid indole alkaloid pathway of Catharanthus roseus(Indian National Science Academy, 2008) Singh, Digvijay; Rai, Sanjay Kumar; Pandey-Rai, Shashi; Srivastava, Suchi; Mishra, Raghvendra Kumar; Sharma, Srikant; Kumar, SushilSingle runs of RP-HPLC procedure were used for simultaneous and organ-wise quantification of the terpenoid indole alkaloids (TIAs) serpentine, ajmalicine, vindoline, catharanthine, vincristine and vinblastine in whole adult plants of Catharanthus roseus, sampled from 81 accessions and F2 generation of a cross. The average total TIA content was 159 mg distributed among stem, root, leaf and inflorescence organs in 13:8:3:1 proportion. The root and stem barks, leaf-petiole and corolla petals were several fold TIA-richer than root and stem woods, leaf–lamina and rest of flower organs, respectively. TIAs were undetectable in seed but present in the seedling. Quantitatively, serpentine (+ ajmalicine), serpentine and catharanthine, vindoline, serpentine, catharanthine and vinblastine (+ vincristine) were the principal TIAs detected in stem, root, leaf and inflorescence organs, respectively. Significant amounts of vinblastine, vindoline and catharanthine were found present in the non-chlorophyllous corolla petals. Correlation studies showed that contents of catharanthine and serpentine in roots, catharanthine, vindoline and vinblastine in leaves, serpentine in roots and stems and stems and leaves were interdependent. Serpentine could be involved in suppressing the accumulation of other TIAs in leaves. The observed inter- and intra- organ contents of end-products from serpentine, catharanthine and vindoline TIA branch pathways and correlations between them showed predominance of the serpentine branch.Item Transcript profiling of terpenoid indole alkaloid pathway genes and regulators reveals strong expression of repressors in Catharanthus roseus cell cultures(Springer, 2007) Dutta, Ajaswrata; Singh, Digvijay; Kumar, Sushil; Sen, JayantiThe understanding of the complexities and molecular events regulating genes and the activators involved in terpenoid indole alkaloid (TIA) metabolism is known to a certain extent in cell cultures of an important TIA yielding plant, Catharanthus roseus, though it is not yet complete. Recently, the repressors of early TIA pathway genes have also been identified. However, their roles in the regulation of TIA pathway in C. roseus cell cultures remains yet unknown. We have made a comparative profiling of genes catalyzing the important steps of 2-C methyl-D-erythritol-4- phosphate (MEP), shikimate and TIA biosynthetic pathways, their activator and repressors using macro- array, semiquantitative RT-PCR and northern analyses in a rotation culture system of C. roseus comprising differentiated and proliferated cells. Our results demonstrate that TIA biosynthetic pathway genes and their activators show variable expression pattern, which was correlated with the changes in the cellular conditions in these systems. Under similar conditions, TIA pathway repressors show strong and consistent expression. The role of repressors in the complex regulation of the TIA pathway in C. roseus cell cultures is discussed. The results were supported by HPLC data, which demonstrated that the molecular program of cellular differentiation is intimately linked with TIA pathway gene expression and TIA production in C. roseus cell cultures.Item Volatile compounds of leaves and flowers of periwinkle Catharanthus roseus (L.) G. Don from New Delhi(Wiley-Blackwell, 2006) Pandey-Rai, Shashi; Mallavarapu, Gopal Rao; Naqvi, A. A.; Yadav, Anju; Rai, Sanjay Kumar; Srivastava, Suchi; Singh, Digvijay; Mishra, Raghvendra; Kumar, SushilThe hydrodistilled waxy oils of the leaves and flowers of Catharanthus roseus (L.) G. Don cv. Nirmal plants grown at New Delhi were analysed by GC and GC–MS. A total of 52 compounds were identified, accounting for the 91.3% of the leaf oil.Among these the main constituents were as follows: (E,E )-2,4-hexadienal (7.7%), citronellol (7.9%), geraniol (7.9%), p-cresol (4.7%), (Z,E )-pentadecanal (6.6%), hexadecanoic acid (4.9%), palmitic acid (4.9%) and phytol (6.4%). A total of 41 compounds were identified, accounting for 99.4% of the flower oil, whose main constituents were heneicosane (20.8%), tricosane (37.9%), tetracosane (6.1%) and docosane (4.3%). A total of 24 compounds occurred in both the leaf and flower oils.Item Growth and terpenoid indole alkaloid production in Catharanthus roseus hairy root clones in relation to left- and right-termini-linked Ri T-DNA gene integration(Springer, 2004) Batra, Jyoti; Dutta, Ajaswrata; Singh, Digvijay; Kumar, Sushil; Sen, JayantiHairy root cultures of Catharanthus roseus var. Prabal were established by infecting the leaves with Agrobacterium rhizogenes agropine-type A4 strain. Two hundred and fifty independent root clones were evaluated for growth, morphology, number of integration of Ri T-DNA genes and alkaloid contents. On the basis of growth pat- tern, type of branching and number of lateral roots we were able to separate the hairy root clones into four categories. However based on the integration of the Ri TL-DNA and TR-DNA genes, there were only three different categories of independent hairy root clones–C1 (rolA&B+/ags+), C2 (rolA&B-/ags+) and C3 (rolA&B+/ags ). Southern hybridization analysis revealed both single and multiple copies of T-DNA integration in the root clones. The accumulation of considerable amounts of the root-specific alkaloids ajmalicine and serpentine was observed in the presence of both the TL-DNA and TR-DNA genes (C1) and the TL-DNA gene (C3) alone. Two rolA&B but ags+ clones (C2) accumulated much less or only very negligible amounts of ajmalicine. The possible role of the TL-DNA and TR-DNA genes on growth and alkaloid accumulation in these root clones is discussed