GREEN SEED COAT COLOUR RETENTION IN LENTIL - University ...

during the time taken to remove the seed coat that little chlorophyll degradation
occurred. After the seed coats were dry they were weighed to the nearest 0.1 mg
and placed into a test tube with 4 mL of an 80% acetone and 20 % 0.1 M TRIS HCl
buffer solution with a pH of 7.0. The samples were placed on a shaker in a dark box
for 90 hours for chlorophyll extraction. Then the samples were centrifuged and 2
ml of the supernatant was removed and placed into a quartz cuvette and the
absorbance measured with a spectrophotometer (Ocean Optics, Dunedin, Florida) as
a percentage. It was calibrated with the quartz cuvette filled with the acetone TRIS
buffer solution. The equations used to calculate chlorophyll content yielded
chlorophyll amount (mg) / volume of acetone buffer solution (ml). To calculate the
amount of chlorophyll a and chlorophyll b in the seed coat, the following equations
were used (Porra, 2002):
Chlorophyll a (µg/mL) = 12.25 (A663.6) – 2.55 (A646.6) and, (4.3)
Chlorophyll b (µg/mL) = 20.31 (A646.6) – 4.91 (A663.6), (4.4)
where,
A646.6 is the percentage of light absorbance measured at 646.6 nm and,
A663.6 is the percentage of light absorbance measured at 663.6 nm.
The same chlorophyll extraction method was used on seed coats of a second
set of four genotypes: 1294M-23, CDC Plato, CDC Greenland, and CDC Meteor.
The seed originated from plants grown in three different phytotron environments
when all four genotypes were grown simultaneously. All four genotypes were used
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