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.
2.3. Inoculation into a hormone-free mediumThe suspension was ready for inoculation into ahormone-free medium 8–12 days after the initiationof a fresh culture. Eighty milliliters of theculture were filtered through a nylon sieve with amesh diameter 150 mm, and then centrifuged for 5min at 100g. The pellet was resuspended in amedium of the same composition but without2,4D. Cells were washed in this manner threetimes. After resuspension in a liquid medium (at adensity of 1103 cells per ml) cells and the aggregateswere placed in a Petri dish (10 cm in diameter,4 ml liquid culture each). The culture was thenplaced in a growth chamber with a light intensityof about 200 Lx (LF PhillipsTLD36W:33), temperature26°C and a 16 h photoperiod. SE werehand 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.
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