Seedlings with 2-3 leaves were used for shoot induction,
which were obtained in vitro from the MS culture medium
(Murashige and Skoog, 1962) at a half salts concentration
(½ MS), supplemented with 20.0 g L-1 sucrose, 20.0 mg L-1
adenine and 2.6 g L-1 phytagel. For shoot induction, the
same basal medium was used with the addition of the
growth regulator 6-benzylaminopurine (BAP), alone or in
combination with kinetin (KIN) and indole-3-acetic acid
(AIA); the basal medium without growth regulators was
used as the control treatment (Tab. 1). The cultures were
kept at 23±2ºC under lighted conditions. The medium pH
was adjusted to 5.75 with NaOH or HCl 1 N solutions. The
culture media were sterilized at 121ºC and 15 psi (103,42
kPa) for 20 min. The number of shoots was observed
through a stereoscope (Olympus SZ40, Tokyo, Japan) and
counted at 60 dai.