Induction of Mutation in Toothbrush Orchids using Ethylmethane Sulphonate (EMS) and Detection of Genetic Variation by SSR (Simple Sequence Repeat) Marker
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Abstract
The toothbrush orchid is a monocotyledonous plant. It is classified in the Dendrobium genus, which is an important and outstanding economic flowering plant. It is an orchid that is sold both domestically and internationally due to its overall toothbrush-like design, both in the flower arrangement and stem. Recently, there are fewer toothbrush orchids now. Therefore, the objective of this study was to study the effects of Ethylmethane sulfonate (EMS) and detection of genetic variation in toothbrush orchids using simple sequence repeat (SSR) marker. EMS solution was used to soak a 0.5 cm piece of the protocorm like bodies (PLBs) at concentrations of 0.5, 1.0, and 2.0%. Thereafter, they were cultured on VW medium supplemented with 1 mg/l BA, 20 g/l sucrose, adjusted to pH 5.7 and solidified with 0.2 % phytagel. The cultures were maintained at 26 ±2°C under light at intensity of 3,000 lux for 14 hours per day. After culturing for 30 days, the results showed that PLBs immersed in EMS solution at 1.7% for 90 minutes gave the highest average survival rates at 50% (LD50). For the development of PBLs, PBLs were derived with immersed in 0.5 % EMS for 60 minutes gave the highest PLBs induction (7.37%) and average number of PLBs (1.41 PLBs/explant) after culturing for 8 weeks. For detection of genetic variation, a total of 9 SSR primers were used, including EgCIR0409, EgCIR0905, EgCIR0781, EgCIR0446, EgCIR1772, EgCIR0337, EgCIR0337, EgCIR0243, mEgCIR0465, and mEgCIR008. The results found that only one primer gave polymorphic banding. EgCIR0905 primer gave polymorphic banding at 50% and specific DNA banding size at 200 bp.
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