2.1. Plant Materials and Culture Initiation
Healthy Plectranthus amboinicus plants (about one month-old) were maintained in a net house for two weeks
prior to explants excision and establishment in vitro. Shoots and stem segments of plants collected were washed
in running tap water for 1 h. Three centimetre pieces of stem carrying vegetative buds were prepared from either
lateral shoots or apical shoots and washed with detergent (Teepol) solution for 30 min, followed by rinsing in
distilled water. The explants were transferred to a laminar air flow chamber where they were surface sterilized
with 10% - 20% Clorox® containing drops of Tween-20 for 5 - 20 min on a rotary shaker. After rinses with sterile
water, the explants (apical shoots and lateral shoots) were cut into 2 - 3 cm, and then cultured on Murashige
and Skoog (MS) medium supplemented with the plant growth regulators benzyl amino purine (BAP), 1-naphthaleneacetic
acid (NAA) and kinetin (KIN) at concentrations of 0, 0.5, 1.0, 3.0 or 5.0 mg/L. The basal MS medium
that also contained 30 g/L sucrose was adjusted to pH 5.7 to 5.8 before adding 3 g/L gelrite agar for gelling.
Sterilization of the culture medium was performed by autoclaving at 12˚C for 20 min. Explants were inoculated
on to 40 mL of medium contained in 150 mL flasks. The cultures were incubated in a plant growth
room at a temperature of 25˚C ± 1˚C with a 16 h photoperiod provided by cool-white fluorescent lamps (1000 -
2000 lux). The cultures were checked regularly for contamination and observations were recorded at weekly intervals.
Twenty replicated flasks were used in each treatment to compare the effects of the growth regulators.
Results were expressed as percent shoot induction, the number of shoots per culture and also the length of the
shoots after 45 days of culture.