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Typhonium Flagelliforme Lodd.) Based on Morphology, RAPD, and GC-MS Markers View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Binus University Repository Pertanika J. Trop. Agric. Sci. 40 (1): 185 - 202 (2017) TROPICAL AGRICULTURAL SCIENCE Journal homepage: http://www.pertanika.upm.edu.my/ Analysis of Gamma Irradiated-Third Generation Mutants of Rodent Tuber (Typhonium flagelliforme Lodd.) Based on Morphology, RAPD, and GC-MS Markers Sianipar, N. F.1*, Purnamaningsih, R.2, Gumanti, D. L.3, Rosaria3, and Vidianty, M.3 1Research Interest Group Food Biotechnolgy, Bina Nusantara University, Jl. Jalur Sutera Barat Kav. 21, Alam Sutera, 15325 Tangerang, Indonesia 2Indonesian Center for Agricultural Biotechnology and Genetic Resources Research and Development (BB-Biogen), 16111 Bogor, Indonesia 3Alumni of Biology Department, Faculty of Science and Technology, Pelita Harapan University, 15811 Tangerang, Indonesia ABSTRACT Rodent tuber is an anticancer plant. The natural genetic diversity of rodent tuber is low due to vegetative propagation. It is important to increase the plant’s genetic diversity in order to obtain plants with a high amount of anticancer compounds. In vitro calli were irradiated with gamma rays to increase its genetic diversity. Seventeen clones of the first generation of vegetative mutants in a green house (MV1) were propagated until MV3. This research aimed to analyse the stability of mutation in MV3 based on morphology, RAPD and GC-MS markers. Clone 6-1-2 had the highest increase of shoots and leaf number than the control and the other MV3 clones while clone 6-1-3-4 had the highest fresh and dry weight. RAPD analysis using 15 primers produced 67 polymorphic DNA bands and showed four main clusters at the similarity coefficient cut-off of 0.87. The GC-MS showed that MV3 contained at least eight types of anticancer compound in the leaves and six types in the tubers; these were higher than in the control. MV3 leaves and tubers contained at least eight new anticancer compounds that were not found in the control. This research proved that rodent tuber MV3 clones were solid mutants and had a high potential for being ARTICLE INFO developed into anticancer drugs. Article history: Received: 26 May 2016 Accepted: 10 November 2016 Keywords: anticancer compounds, gamma irradiation, E-mail addresses: GC-MS, morphology, RAPD, Typhonium flagelliforme [email protected] ; [email protected] (Sianipar, N. F.), Lodd [email protected] (Purnamaningsih, R.), [email protected] (Gumanti, D. L.), [email protected] (Rosaria), [email protected] (Vidianty, M.) * Corresponding author ISSN: 1511-3701 © Universiti Putra Malaysia Press Sianipar, N. F., Purnamaningsih, R., Gumanti, D. L., Rosaria and Vidianty, M. INTRODUCTION might increase the probability of generating Rodent tuber (Typhonium flagelliforme plant clones that contain a higher amount Lodd.) is a herbal plant from the Araceae of anticancer compounds. Mutation can family (Essai, 1986) that contains be induced by irradiation with physical detoxification and anticancer compounds. mutagens such as gamma ray. Rodent tuber is a plant native to Indonesia Genetic diversity can be analysed using and has been used as traditional medicine molecular markers such as RAPD, RFLP, for years. Bioactive compounds of rodent AFLP and SSR (Powell et al., 1996). The tuber are alkaloids, saponins, steroids and Randomly Amplified Polymorphic DNA glycosides (Syahid, 2007). Anticancer (RAPD) marker is able to detect genetic compounds can be found in all parts of the diversity of a plant whose genome has rodent tuber plant, including the root, tuber, not been sequenced yet (McClelland et stem and leaf (Choo et al., 2001). al., 1994), such as rodent tuber. Genetic Rodent tuber has been reported to be characterisation of a plant’s germplasm cytotoxic against cancer of the lung, breast is essential for harnessing the maximum (Lai et al., 2010), liver (Lai et al., 2008), potency of the plant’s genetic diversity blood (leukemia) (Mohan et al., 2010), (Rout, 2006). colon, prostate gland and cervix (Hoesen, Embryogenic calli of rodent tuber 2007). Rodent tuber extract has also been plant have been induced, proliferated and reported to be able to prevent breast and regenerated using the single node culture cervical cancers (Syahid & Kristina, 2007). method (Sianipar et al., 2011). Rodent tuber Rodent tuber hexane extract was toxic mutant clones have also been successfully against Artemia salina (Sianipar et al., generated by combining the effects of 2013a). Other biological activities of rodent gamma irradiation with somaclonal in vitro tuber have been found to be antibacterial and culture variation. A somatic cell population antioxidant (Mohan et al., 2008) and able to (calli) of rodent tuber was irradiated with induce apoptosis of cancer cells (Lai et al., 6-Gy gamma rays. The irradiated in vitro 2008). plantlets exhibited various growth responses The development of Indonesian rodent (Sianipar et al., 2013b). Those irradiated in tuber into anticancer drugs was inhibited by vitro plantlets (mutant) and control plants its low genetic diversity, which is caused were found to have genetic differences by the conventional clonal vegetative based on analysis with RAPD molecular propagation method. Low genetic diversity markers (Sianipar et al., 2015a). is followed by low bioactive compound Thirty-seven clones of first generation diversity in rodent tuber (Syahid, 2008). vegetative mutant of rodent tuber induced in Mutation induction of in vitro somatic cell a green house (MV1) were analysed based population (calli) or shoot culture could on morphological and RAPD markers. increase its genetic diversity, which in turn MV1 clones had various morphological 186 Pertanika J. Trop. Agric. Sci. 40 (1) 186 - 202 (2017) Rodent Tuber (Typhonium flagelliforme Lodd.) Mutant Analysis characteristics (Sianipar et al., 2013c). Out height and fresh and dry weight of the of those 37 MV1 clones, there were 17 rodent tuber control and mutant plants. that had a diversified genetic profile and Morphological characteristics were analysed showed genetic differences from the control using the NTSYS DIST coefficient and plants based on RAPD molecular analysis UPGMA. (Sianipar et al., 2015b). Genetic mutation might also affect the Molecular Analysis with RAPD Marker relative abundance of bioactive compounds. DNA isolation was done based on Doyle Gas Chromatography-Mass Spectrometry and Doyle (1987). In this method, 2.5 g of (GC-MS) is a method for analysing the leaf sample was homogenised with PVP metabolomic profile of an organism. 0.1% and liquified nitrogen. A volume of GC utilises gas as the mobile phase to 2 mL of CTAB buffer (CTAB 10% b/v, separate chemical compounds. GC is able EDTA 0.5 M pH 8.0, Tris-HCl 1M pH 8.0, to separate a lot of compounds in one run NaCl 5M) and 10 μL of 1-merkaptoetanol and can be combined with MS to identify 1% (b/v) was added to the sample powder. the compounds based on the database The sample was homogenised with vortex, (Kayser & Quax, 2007). Rodent tuber MV1 incubated at 60°C for 20 min and cooled clones were propagated and regenerated at room temperature. About 750 μl of into MV3 clones. This research aimed to chloroform:isoamyl alcohol (24:1) solution analyse the third generation of vegetative was added to the sample and then vortexed. mutant clones of rodent tuber (MV3) based The sample was centrifuged at 11.000 on morphological analysis, molecular rpm for 10 min. A supernatant was added marker with RAPD profiling and GC-MS to to 1 mL of chloroform:isoamyl alcohol identify the relative abundance of bioactive (24:1) and centrifuged at 11.000 rpm for compounds. 10 min. About 750 μL of cold isopropanol, homogenised and stored at -20°C for one MATERIALS AND METHODS night was added to the supernatant. The Plant Material sample was centrifuged at 11.000 rpm for This research analysed the third generation 10 min. A DNA precipitate was dried in a of vegetative mutant clones of rodent tuber vacuum for one h. The dried DNA sample (MV3) (in the patenting process). Rodent was solubilised in 200 μL of buffer TE tuber mother plants were obtained from (Tris-HCl 1M pH 8.0; EDTA 0.5M pH 8.0). Bogor (West Java, Indonesia). About 200 μL of DNA solution was added to 20 μL of RNase (10 mg/mL) and incubated at 37°C for one h. DNA was incubated Morphological Characterisation at 4°C for one night. DNA solution was The parameters of observation were the stored at -20°C. The DNA sample was number of shoots, number of leaves, plant amplified with 15 decamer primers using Pertanika J. Trop. Agric. Sci. 40 (1): 187 - 202 (2017) 187 Sianipar, N. F., Purnamaningsih, R., Gumanti, D. L., Rosaria and Vidianty, M. the Thermal Cycler Gene PCR (ABI 9700). with a sonificator and incubated at room The composition of 1x PCR reaction was 5 temperature for 48 h. The extracts were µL of the DNA template (5 ng/µL), 0.2 µL filtered with Whatman filter paper and of dNTP 0.2 mM, 2.5 µL of PCR buffer + analysed with a GC-MS detector. A volume MgCl2 (1x), 1 µL of 10 pmol primer, 0.2 of 5 µL of extract was injected into the µL of 1U Taq polymerase and 16.1 µL of column at a split ratio of 5:1 at 250°C. ddH2O with a total volume of 25 µL. The Helium was used as a carrier gas with a PCR reaction thermal cycle was repeated flow rate of 0.8 µL/minute. The initial 45 times in stages as follows: 94°C for one column oven temperature was 70°C. It was min, 36°C for one min, 72°C for 2 min then increased at a rate of 5°C/min until the and 72°C for 4 min.
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