Culture initiation
Around 2,500 leaf explants (1 cm in width) per spear were cultured on solid MS basal medium [15]
supplemented with 5% (w/v) sucrose, 2,4Dichlorophenoxyacetic acid (2,4-D) and Naphtalene Acetic Acid (NAA),
and incubated in dark room to induce callus and embryos [4]. Room temperature and Relative Humidity was
maintained at 28±2 oC and below 60% [16, 17]. Subculture was performed using the same medium every three
months to induce callus, and every two months to regenerate callus into embryos. Leaf explants were incubated for 1
year to induce callus, and callus also incubated for 1 year to regenerate into embryo.