2.4. Explants and surface disinfection
Leaf explant (2nd and 3rd node from the apex) was collected from healthy
and vigorously growing mother stock plants. The excised explant
materials were initially rinsed under tap water for 30 min to remove the
dust particles from their surface. Then the explants were treated with
commercial detergent (Largo, Ethiopia) for 5 minutes and were rinsed
well with distilled water for three to five times. Under a clean Laminar
flow hood, the explants were subjected to 70% (v/v) ethanol for one
minute and rinsed with sterile distilled water three to four times.
Further the explant materials were then surface sterilized by 2.5% of
Local bleach (Berekina, Ethiopia) containing two drops of Tween 20 for
15min time of exposure. After that the explants were rinsed with
sterilized double distilled water for three to four times to remove the
residual effect of these sterilants. After sterilization process was
completed, individual leaf explant was trimmed aseptically into
approximately 1cm2 leaf disc segments and placed with
the adaxial side in contact with the MS basal medium (Kumar et
al ., 2010a).