Optimized flow cytometric protocol and genome size estimation of Sabah snake grass (Clinacanthus nutans)

Abstract

Clinacanthus nutans is an economically important medicinal plant that can be found grown in many countries in the Asian region. Useful medicine properties such as anti-cancer, anti-bacteria, and anti-viral, backed by its high content of phytochemical compounds such vitexin, isovitexin, stigmasterol and lupeol has increased the demand for C. nutans in the market. Extensive work had been carried out on its content and pharmacological activity using fresh samples but limited studies for in vitro cultures of C. nutans . No genome size estimation has been done for C. nutans . Objective of this study was to analyse nuclear DNA content of C. nutans using flow cytometry. Preparation of different nuclei isolation buffer and stoichiometric DNA staining using propidium iodide was carried out. The genome size of C. nutans was estimated using Glycine max cv. Polanka as internal standard and its genome size was compared with in vitro plantlets of C. nutans. Flow cytometry analysis revealed that nuclear 2C DNA of C. nutans content is estimated at 1.75 ± 0.006 pg. Coefficient of variation in flow cytometric analysis was within the limit of 5% implying that the results were reliable with the Tris.MgCl 2 being the best nuclei isolation buffer. No significant difference was observed from field grown and in vitro C. nutans . This finding will assist further in genome size evolution analysis of Clinacanthus spp. and to determine polyploids for increased active compounds and biomass. replicate being performed on different days. In all the experiments, the fluorescence of at least 5000 nuclei to 10000 nuclei was measured. Conversion from picograms (pg) to base pair numbers was done as follows: 1 pg DNA is equivalent to 0.978 × 10 9 bp (Dolezel et al., 2007). The results were analysed using one way analysis of variance (ANOVA) using SPSS version 25. For significant effect, Tukey’s pairwise comparison was carried out with p value ≤ 0.05..