2.5. Chlorophyll extraction
Samples were taken from plants with ten fully expanded leaves
after 4 weeks of all treatments. All leaves were cut into small pieces
(five leaf discs per plant), weighed, and placed separately in plastic
tubes containing 10 mL of 96% ethanol wrapped in aluminum foil
to prevent photodegradation of chlorophyll. Full extraction of
chlorophyll was achieved when the sample was discolored. After
the incubation in the water bath at 80 8C the samples were
centrifuged at 12,000 g for 10 min and the supernatant was
transferred to a separate tube. Using 96% ethanol as a blank, the
absorbance of the chlorophyll extract was recorded on a UV/VIS
LKB Ultraspec II spectrophotometer (Pharmacia LKB Biotechnology,
Uppsala, Sweden) at 665 and 649 nm wavelengths (Wintermans
and De Mots, 1965). Samples were diluted with 96% ethanol
when necessary to obtain an absorbance value less than 1.5. Total
chlorophyll concentration (Chla+b) was calculated from the
equations previously reported (Wintermans and De Mots, 1965)
expressed as mg Chl per g of fresh weight (mg g1 FW).
2.5. Chlorophyll extraction
Samples were taken from plants with ten fully expanded leaves
after 4 weeks of all treatments. All leaves were cut into small pieces
(five leaf discs per plant), weighed, and placed separately in plastic
tubes containing 10 mL of 96% ethanol wrapped in aluminum foil
to prevent photodegradation of chlorophyll. Full extraction of
chlorophyll was achieved when the sample was discolored. After
the incubation in the water bath at 80 8C the samples were
centrifuged at 12,000 g for 10 min and the supernatant was
transferred to a separate tube. Using 96% ethanol as a blank, the
absorbance of the chlorophyll extract was recorded on a UV/VIS
LKB Ultraspec II spectrophotometer (Pharmacia LKB Biotechnology,
Uppsala, Sweden) at 665 and 649 nm wavelengths (Wintermans
and De Mots, 1965). Samples were diluted with 96% ethanol
when necessary to obtain an absorbance value less than 1.5. Total
chlorophyll concentration (Chla+b) was calculated from the
equations previously reported (Wintermans and De Mots, 1965)
expressed as mg Chl per g of fresh weight (mg g1 FW).
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