2.3. Inoculation into a hormone-free medium
The suspension was ready for inoculation into a
hormone-free medium 8–12 days after the initiation
of a fresh culture. Eighty milliliters of the
culture were filtered through a nylon sieve with a
mesh diameter 150 mm, and then centrifuged for 5
min at 100g. The pellet was resuspended in a
medium of the same composition but without
2,4D. Cells were washed in this manner three
times. After resuspension in a liquid medium (at a
density of 1103 cells per ml) cells and the aggregates
were placed in a Petri dish (10 cm in diameter,
4 ml liquid culture each). The culture was then
placed in a growth chamber with a light intensity
of about 200 Lx (LF PhillipsTLD36W:33), temperature
26°C and a 16 h photoperiod. SE were
hand harvested at the following growth stages:
globular (Fig. 2A), early heart, late heart (Fig.
2B), early cotyledons (Fig. 2C) and late cotyledons
(Fig. 2D). Plant material was kept frozen (
76°C) until ABA extraction and analysis.