Seeds of C. morifolium cv. Chuju were surface sterilized in 0.1% (w/v) mercury dichloride (HgCl2), followed by rinsing at least three times with sterile water and placed on half-strength (1/2) MS medium containing 0.8% (w/v) agar and 1.5% (w/v) sucrose. The sterilized seeds were vernalized for 48 h at 4°C in darkness, and were then cultured in a growth chamber at 25°C with a photoperiod of 14 h light/10 h dark (light intensity of 200 μmol m-2 s-1). After 10 days (d) of germination, the seedlings were transferred into pots with soils (3:1:1, clay:vermiculite:perlite). Soils were sterilized by autoclaving at 120°C for 1 h before using it.