RESUMO
Camptothecin (CPT), a monoterpene indole alkaloid, is a potent inhibitor of DNA topoisomerase I and has applications in treating ovarian, small lung and refractory ovarian cancers. Stem wood tissue of Nothapodytes nimmoniana (Graham) Mabb. (family Icacinaceae) is one of the richest sources of CPT. Since there is no genomic or transcriptome data available for the species, the present work sequenced and analysed transcriptome of stem wood tissue on an Illumina platform. From a total of 77,55,978 reads, 9,187 transcripts were assembled with an average length of 255 bp. Functional annotation and categorization of these assembled transcripts unraveled the transcriptome architecture and also a total of 13 genes associated with CPT biosynthetic pathway were identified in the stem wood tissue. Four genes of the pathway were cloned to full length by RACE to validate the transcriptome data. Expression analysis of 13 genes associated with CPT biosynthetic pathway in 11 different tissues vis-à-vis CPT content analysis suggested an important role of NnPG10H, NnPSLS and NnPSTR genes in the biosynthesis of CPT. These results indicated that CPT might be synthesized in the leaves and then perhaps exported to stem wood tissue for storage.
RESUMO
Nothapodytes nimmoniana is an endangered medicinal tree and regarded most convenient natural source for large scale isolation of anticancer alkaloid camptothecin. In order to meet pharmaceutical market demand for camptothecin and conserve natural population of the species, in vitro culture techniques can be employed for mass propagation and production of the alkaloid. Seed germination is one of the difficult challenges in the establishment of the aseptic in vitro cultures. Pre-germination treatments of soaking in water, GA3 solution and aseptic excision of the embryo axis were employed to enhance seed germination and seedling recovery respectively for further in vitro studies. Seed soaking in water or GA3 solution for 24 hours enhanced the germination capacity with better response obtained when seeds were soaked in GA3 (2.6 μM) solution and germinated on half strength MS medium supplemented with the GA3. Removal of the outer, inner seed coat and inoculation of the embryo axis on medium amended with or without GA3 reduced seedling recovery time to 3-4 weeks. Seedlings obtained on medium fortified with GA3 showed vigorous growth with complete plantlet development than on PGR-free medium. The strategy can be employed to reduce seedling recovery time to within 3-4 weeks compared to 7-10 weeks required for germinating the seeds in vitro.