The seeds of B. variegata were procured from
Afforestation Division, Hattisar, Kathamandu, Nepal
and were carried to the laboratory of Institute of
Pharmacognosy, Vienna, Austria and were preserved
at 4º C until subsequent experimental use. The healthy
seeds were initially washed in water containing few
drops of Teepol detergent solution. The seeds were
then soaked in distilled water for an hour prior to
sterilization. The soaked seeds were washed with
sterilized distilled water five times. The seeds were
then surface sterilized with 30 % ethanol for 10 min,
followed by treatment with sodium hypochlorite
solution (10%) for 10 minutes and then rinsed three
times with sterilized distilled water. The seeds were
again sterilized in 70 % alcohol for one minute and
washed with sterile water three times. The seeds were
then inoculated on Murashige and Skoog (1962)
medium containing 3% sucrose. The pÇ of the medium
was adjusted to 5.8 ±1 before autoclaving at 121ºC for
20 minutes. The media were solidified by adding 0.8 %
agar (Bacteriological). The cultures were maintained at
25 ± 4ºC with a photoperiod of 16h (40 µ mol.m– ²s-1
supplied by OSRAM Biolux tubes) and relative
humidity of 70 % in a growth chamber.
young seedlings and sub-cultured on MS medium
supplemented with 0.5µM BAP to obtain sufficient
explants needed for subsequent experiments. The
explants from these cultures were then used for
inoculating MS media having different concentrations
of BAP and NAA.