Materials and methods
Tuberous rhizomes of Nymphaea nouchali var. versicolor ‘Bua Phuean’ were collected from natural pond in Pathum Thani province, Thailand. After flowers, leaves and roots had been removed (Figure 1), rhizomes were washed thoroughly in running tap water to remove unwanted matter and soaked in soapy water for 5 minutes, followed by rinsing in running tap water for 30 minutes. After that, rhizomes were dipped in 50% (v/v) ethanol for 1 minute and rinsed with sterile distilled water for 5 minutes.
Subsequently, rhizomes were immersed for 15 minutes in 0.1% (w/v) mercuric chloride to which 2 drops of Tween-20 were added and washed in sterile distilled water for 5 minutes. Afterward, they were disinfested by immersing for 10 minutes in 15% (v/v) Clorox (a commercial bleach solution containing
5.25%, w/w, sodium hypochlorite as available chlorine) mixed with 2 drops of Tween-20 and washed 3 times with sterile distilled water each for 5 minutes.