The nodal segments were surface sterilized by agitating in a
solution of sodium troclosene dihydrate (CTX-200/GR®; CTX S.A.U.,
Barcelona, Spain) at a concentration of 3.5 g l1, containing 0.1% (v/
v) Tween-20 for 2 h in a laminar flow hood.
Shoot cultures were
established in vitro and subcultured monthly on culture medium
for 3 months.
The medium (T0) was composed of Murashige and
Skoog (MS) medium (Murashige and Skoog,1962), 3% (w/v) sucrose
and 0.7% (w/v) Plant Propagation Agar (Pronadisa®) in 300 ml
culture vessels, each containing 100 ml culture medium. The pH
was adjusted to 5.7 using 0.1 N KOH prior to autoclaving at 122 C
(1.1 kg cm2) for 16 min. Shoots were cultured in climatic chamber
at 25 ± 1 C and with a 16 h light period (45 mmolm2 s1; Sylvania
Gro-lux fluorescent tubes).