The shoots were washed by placing them under tap water for 30–60 min then immersed in 1% Tween 20 (Sigma–Aldrich Ltd.)solution for 30 min and rinsed with sterile distilled water (SDW)5 times (5 min each rinse). After pretreatment, the shoots were surface sterilized by immersing them in 0.05% mercury chloride(HgCl2, Merck, Darmstadt, Germany) plus a few drops of Tween-20 for 10 min, followed by 5–6 rinses in SDW (5 min each rinse).After sterilization, the damaged surface of explants caused by chemicals was sliced off with a tissue culture blade (BB510, Aes-culap AG & Co. KG AM, Tutlingen, Germany) and shoot size was reduced by removing several leaves. Finally, the size of the donorexplant used as the in vitro explant was approximately 0.4 cm in length (Fig. 1A). The explants were then cultured in PLB initiationmedium.