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).