FINDINGS: We optimized the assembly of a Hevea bark transcriptome based on 16 Gb Illumina PE RNA-Seq reads using the Oases assembler across a range of k-mer sizes. We then assessed assembly quality based on transcript N50 length and transcript mapping statistics in relation to (a) known Hevea cDNAs with complete open reading frames, (b) a set of core eukaryotic genes and (c) Hevea genome scaffolds. This was followed by a systematic transcript mapping process where sub-assemblies from a series of incremental amounts of bark transcripts were aligned to transcripts from the entire bark transcriptome assembly. The exercise served to relate read amounts to the degree of transcript mapping level, the latter being an indicator of the coverage of gene transcripts expressed in the sample. As read amounts or datasize increased toward 16 Gb, the number of transcripts mapped to the entire bark assembly approached saturation. A colour matrix was subsequently generated to illustrate sequencing depth requirement in relation to the degree of coverage of total sample transcripts.
CONCLUSIONS: We devised a procedure, the "transcript mapping saturation test", to estimate the amount of RNA-Seq reads needed for deep coverage of transcriptomes. For Hevea de novo assembly, we propose generating between 5-8 Gb reads, whereby around 90% transcript coverage could be achieved with optimized k-mers and transcript N50 length. The principle behind this methodology may also be applied to other non-model plants, or with reads from other second generation sequencing platforms.
METHODS: The experimental approach in the present study was based on a bioassay-guided fractionation. The crude methanol and fractionated extracts (hexane, chloroform and water) from different parts of A. scabra (leaves, rhizomes, roots and pseudo stems) were prepared prior to the cytotoxicity evaluation against human ovarian (SKOV-3) and hormone-dependent breast (MCF7) carcinoma cells. The identified cytotoxic extracts were then subjected to chemical investigations in order to identify the active ingredients. A normal human lung fibroblast cell line (MRC-5) was used to determine the specificity for cancerous cells. The cytotoxic extracts and fractions were also subjected to morphological assessment, DNA fragmentation analysis and DAPI nuclear staining.
RESULTS: The leaf (hexane and chloroform) and rhizome (chloroform) extracts showed high inhibitory effect against the tested cells. Ten fractions (LC1-LC10) were yielded after purification of the leaf chloroform extract. Fraction LC4 which showed excellent cytotoxic activity was further purified and resulted in 17 sub-fractions (VLC1-VLC17). Sub-fraction VLC9 showed excellent cytotoxicity against MCF7 and SKOV-3 cells but not toxic against normal MRC-5 cells. Meanwhile, eighteen fractions (RC1-RC18) were obtained after purification of the rhizome chloroform extract, of which fraction RC5 showed cytotoxicity against SKOV-3 cells with high selectivity index. There were marked morphological changes when observed using phase-contrast inverted microscope, DAPI nuclear staining and also DNA fragmentations in MCF7 and SKOV-3 cells after treatment with the cytotoxic extracts and fractions which were indicative of cell apoptosis. Methyl palmitate and methyl stearate were identified in the hexane leaf extract by GC-MS analysis.
CONCLUSIONS: The data obtained from the current study demonstrated that the cell death induced by cytotoxic extracts and fractions of A. scabra may be due to apoptosis induction which was characterized by apoptotic morphological changes and DNA fragmentation. The active ingredients in the leaf sub-fraction VLC9 and rhizome fraction RC5 may lead to valuable compounds that have the ability to kill cancer cells but not normal cells.
METHODS: The capabilities of callus, shoot, and root formation were evaluated by culturing both explants on Murashige and Skoog (MS) medium supplemented with various PGRs at the concentrations of 0, 1, 3, 5, and 7 mg/L.
RESULTS: Medium supplemented with 3 mg/L indole-3-butyric acid (IBA) showed the optimal callogenesis from both leaf and stem explants with (72.34 ± 19.55)% and (70.40 ± 14.14)% efficacy, respectively. IBA was also found to be the most efficient PGR for root induction. A total of (50.00 ± 7.07)% and (77.78 ± 16.47)% of root formation were obtained from the in vitro stem and leaf explants after being cultured for (26.5 ± 5.0) and (30.0 ± 8.5) d in the medium supplemented with 1 and 3 mg/L of IBA, respectively. Shoot formation was only observed in stem explant, with the maximum percentage of formation ((100.00 ± 0.00)%) that was obtained in 1 mg/L zeatin after (11.0 ± 2.8) d of culture.
CONCLUSIONS: Callus, roots, and shoots can be induced from in vitro leaf and stem explants of L. pumila through the manipulation of types and concentrations of PGRs.