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experiments were maintained at 25 "C and under a 16)s h light/dark period. Plants
were grown in 15 cm diameter plastic pots in soil. Green explants were subcultured
onto a fresh medium containing cefotaxim (100 mgll) :~nd cyanamide (10 mMII) every
2 weeks for 2 months.
3 . 2 . 3 . 5 . 3 . 2 . 3 . Cyanamide tolerance assay
Cyanamide tolerance assays were performed as described (Zhang
et
al.,
2005).
Putative transformants and untransformed plants of 30 days-old were tested for the
expression of cah gene by applying 5% cyanamide solution as surface spray on the
leaves. Observation was made after 72 h of application.
3 . 2 . 3 . 5 . 3 . 2 . 4 . Cyanamide hydratase assay
Tobacco leaf tissues (100 mg) were frozen in liquid nitrogen, homogenized
with a pestle and mortar, diluted with 100 pl of
5
rnM sodium phosphate buffer (pH
8.0). vortexed for 5 min., and centrifuged at 8,000
g
for 5 min. The supernatant was
transferred, 2 p1 of 1 M cyanamide was added to the solution and incubated at 37 "C
for 12 h. To the 60 p1 of incubation mixture. 40 pl of sodium carbonate buffer
solution (pH 10.4) and
5
p1 of color reagent consisting of 4% (wlv) solution of
trisodium pentacyanoamineferroate (Eastern Chemicals, Smithtown, NY) were added.
The solution was incubated in dark at room temperature (28
"C)
for 10 min.
Absorbance at 530 nm (Steller
et al.,
1965) was measured to detect the reduction in
cyanamide concentration which is the indicative of the enzyme cyanamide hydratase.
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