The Application of Ooubled Haploid Plants to Population ... - MSpace

5-6
represented one sample from C, and C2.
DNA Extrachon
Leaf material was fiozen with Iiquid nitrogen, lyophiiized in a freeze dtier for a
minimum of 48 hours and then stored at - 20°C. DNA was isolated from the lyophilized
tissue (200 mg) suspended in a buffer solution containing cetyftrimethylammonium
bromide (CTAB). This procedure was based on the method reported by Kidwell and
Osbom (1992). Two chforofon extradions were perfomed to remove insoluble debris
and soluble proteins. DNA was precipitated with ethanol (-20°C) and a pellet was
recovered and washed hMce with 75% ethanol (-20°C) and 10 mM ammonium aœtate.
The pellet was dried and dissolved in 300 pl 10 mM Tris/ 1 mM EDTA (TE) bufier.
DNA extracted from plants grown under controlled and field conditions yielded
approximately 114 to 2280 pg and 105 to 2164 pg of DNA respedively. The amount of
time required to dissolve the DNA samples obtained from the field samples was several
hours compared to several minutes with growth chamber plants. The field samples
were taken at a more advanced stage of plant development than the growth chamber
sarnples and therefore they may have contained more impurities, such as
carbohydrates. as demonstrated by some 2601280 ratios recorded with the UV
spectrophotometer falling outside of the acceptable range of 1.8-2.0. Reduced DNA
yields from Brassica were also found by Kidwell and Osbom (1992) when they sampled
leaf tissue of different maturity and stress levels.
The DNA concentration was measured using an ultraviolet spectrophotometer at