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    Correspondence between flowers and leaves in terpenoid indole alkaloid metabolism of the phytoplasma-infected Catharanthus roseus plants
    (Springer, 2014) Srivastava, Suchi; Pandey, Richa; Kumar, Sushil; Nautiyal, Chandra Shekhar
    Several plants of Catharanthus roseus cv 'leafless inflorescence (lli)' showing phenotype of phytoplasma infection were observed for symptoms of early flowering, virescence, phyllody, and apical clustering of branches. Symptomatic plants were studied for the presence/absence and identity of phytoplasma in flowers. Transcription levels of several genes involved in plants' metabolism and development, accumulation of pharmaceutically important terpenoid indole alkaloids in flowers and leaves and variation in the root-associated microbial flora were examined. The expression profile of 12 genes studied was semi-quantitatively similar in control leaves and phytoplasma-infected leaves and flowers, in agreement with the symptoms of virescence and phyllody in phytoplasma-infected plants. The flowers of phytoplasma-infected plants possessed the TIA profile of leaves and accumulated catharanthine, vindoline, and vincristine and vinblastine in higher concentrations than leaves. The roots of the infected plants displayed lower microbial diversity than those of normal plants. In conclusion, phytoplasma affected the biology of C. roseus lli plants multifariously, it reduced the differences between the metabolite accumulates of the leaves and flowers and restrict the microbial diversity of rhizosphere.
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    Isolation of microsatellites from Catharanthus roseus (L.) G. Don using enriched libraries
    (Springer, 2009) Bhatia, Sabhyata; Shokeen, Bhumika
    Catharanthus roseus is an indispensable source of the anticancerous alkaloids-vincristine and vinblastine, even though they are produced in trace amounts in vivo. In order to increase the yield of alkaloids, in vitro tissue culture studies are carried out which result in a large number of lines/cultures. For identification and characterization of the in vitro cultures, microsatellites in the form of STMS (Sequenced Tagged Microsatellite Sites) markers are used for identification of genetic polymorphism. STMS markers are also used for assessment of genetic diversity within natural populations as well as for construction of genetic linkage maps. Isolation of microsatellites and development of STMS markers typically involves library construction and screening, DNA sequencing, polymerase chain reaction (PCR) primer design, and PCR optimization. This chapter details two approaches for the isolation of microsatellite loci. The first approach is based on PCR using microsatellite containing primers which also have degenerate bases at the 5¢-end that act as anchors preventing the primers from slippage to the 3¢-end and the subsequent loss of polymorphism. The multi-locus PCR amplified product is cloned and sequenced. Though this method generates a large number of microsatellites, the major drawback is the high redundancy observed in this method. The second approach described in this chapter is based on the construction of a microsatellite enriched library which involves preferential cloning of the microsatellite enriched fraction of genomic DNA. This method therefore necessitates the isolation of microsatellites through hybridization with biotin labeled oligoprobe followed by their capture with streptavidin-coated magnetic beads. In comparison to the first approach, this approach yields less redundant clones with high microsatellite enrichment. Moreover enriched libraries are 40–60 times more efficient than the conventional small insert genomic libraries.