Quantification of Colchicine Identification of the colchicines was done by comparing the retention time of the sample with that of the standard obtained from Sigma. Water HPLC systems equipped with a binary pump 1525 (Max. Pressure: 6000 psi.) and a porous silica with 5μm diameter C18 4.6 × 150 mm column was used for separation. The mobile phase consisted of Acetonitrile: 3% Acetic acid (60:40), at a flow rate of 1mL min-1 and an injection volume of 20 μL. The peaks eluted were detected at 245 nm and identified with authentic standards. Extraction of genomic DNA DNA was isolated using a modified cetyltrimethylammonium bromide (CTAB) method 7. For each population, about 5 g of bulked leaf tissue collected from five plants each was ground to a fine powder using liquid nitrogen and then suspended in 20 mL of extraction buffer 20 mM EDTA (pH 8.0), 100 mMTris–HCl (pH 8.0), 1.4 M NaCl, 2% CTAB, and 1% β-mercaptoethanol. The suspension was mixed well, incubated at 60°C for 45 min, followed by chloroform isoamyl alcohol (24:1) extraction and precipitation with 2/3 of the volume of isopropanol at -20°C for 1 h. The DNA was pelleted down by centrifugation at 12,000 rpm for 10 min and suspended in TE buffer 10 m MTris-HCl, 1 mM EDTA (pH 8.0). The DNA was purified from RNA and proteins by standard procedures 8 and DNA concentration was estimated by agarose gel electrophoresis and staining with ethidium bromide. Primer screening Thirty decamer primers from Operon, Advanced Biotechnologies Inc., Almeda, USA were initially screened using one individual clone to determinate the suitability of each primer for the study. Primers were selected for further analyses based on their ability to detect distinct clearly resolved and polymorphic amplified products within the population. To ensure reproducibility the primers generating no, weak, or complex patterns were discarded. PCR amplification PCR amplification was performed in a total volume of 25 μL containing 10 × Taq buffer (2.5 μL) (Genei, Bangalore), dNTPs (2.5 μL) (10 Mm each) (Genei, Bangalore), Taq DNA polymerase (0.5 μL) (3 U/μL) (Genei, Bangalore), Primer (1 μL) (40 μM/μL) (Sigma Aldrich, Bangalore), template DNA (0.5 μL) (40 ng/μL) and sterile nanopure water (18 μL) in Eppendorf Master Cycler (AG 22331 Harmburg , Germany) using the following conditions: (a) initial denaturation at 94°C for 5 min; (b) 45 cycles each consisting of denaturation step at 94°C for 1 min, annealing step at 35°C for 1 min, amplification at 72°C for 2 min step; and (c) final extension at 72°C for 5 min followed by arresting the reaction at 4°C for infinite period. Control reactions without template DNA (negative control) were also run in the experiments. All the experiments were repeated thrice to ensure reproducibility. PCR amplified products (12.5 μl) were subjected to electrophoresis in a 1.5% agarose gel in 1X Tris-borate-EDTA (TBE) buffer at 100 V for 3.5 h using submarine electrophoresis unit. The DNA profile was photographed using biorad gel documentation system. Data scoring and statistical analysis of RAPD data Data of RAPD marker analysis (band size of all amplification products, estimated from the gel by comparison with standard marker) were scored as discrete variables using (+) to indicate presence and (-) to indicate Proceedings of the International Conference on Climate Change, Biodiversity and Ecosystem Services for the 61 Sustainable Development Goals (SDGs): Policy and Practice 27-29 June 2016, Cha-am, Phetchaburi, Thailand
Proceedings of International Conference on Climate Change, Biodiversity and Ecosystem Services for the Sustainable Development Goals : Policy and Practice 27-29 June 2016 at the Sirindhorn International Environmental Park, Cha-am, Phetchaburi, Thailand
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