A SPAD-502 meter (Soil Plant Analysis Development, Minolta,
Japan), which uses measurements of transmitted radiation in the
red and near-infra red wavelengths, was used to provide numerical
values which are related to chlorophyll concentration (Minolta,
1989). Measurements were made at 2-week intervals between
January and June 2002 at four locations on the leaves used for gas
exchange studies for all species. To determine chlorophyll
concentration from SPAD values, calibration curves were constructed
using the acetone extraction method (Schaper and
Chacko, 1991) for leaves representing the full colour range from
young deep green leaves to old leaves showing signs of chlorosis.
1 g samples of fresh leaf tissue were macerated in 40 ml of 80%
acetone using a mortar and pestle, made up to 100 ml with 80%
acetone and centrifuged at 6000 rpm for 3 min before determining
absorbance of the supernatant at 647 and 664 nm (Spectronic 401,
Milton Roy, New York, USA).