Cucurbit Genetics Cooperative Report 4:36 (article 18) 1981
Non-Destructive Fatty Acid Analysis of Cucurbit Seed
Allen Gathman and W. P. Bemis
University of Arizona,
Tucson, AZ 85721
In breeding for higher linoleic acid content in the seed
oil of the buffalo gourd, Cucurbita foetidissima,
we have found that this character varies greatly within
seeds from the same cross, due to heterozygosity of the
We have developed a technique for half-seed analysis of
fatty acid composition so that the phenotypes of individual
embryos may be established non-destructively.
Seeds are imbibed in water overnight, then dipped in 10%
bleach solution. The seed coat is incised around the edge
with a scalpel and removed, and the nucellar membrane is
stripped off with forceps. The embryo is then cut in half.
The anterior portion is supported on glass wool in a vial
containing an inorganic nutrient solution. Vials are incubated
The posterior half of the cotyledons is used for fatty
acid analysis. It is ground on a small piece of 400 grit
sandpaper with a spatula, then scraped off into a small
reaction vial and agitated in 1 ml of hexane. The hexane
is evaporated under a stream of nitrogen. 0.2 ml toluene
and 0.5 ml of 5% H2SO4 in methanol are added and the tube
is tightly covered with a teflon lined screw cap. The mixture
is heated for 3 hrs at 100°C, cooled, and transferred
to a 10x17 mm test tube. 0.5 ml hexane and 1.0 ml distilled
water are added and the tubes are centrifuged 15 min at
2000 rpm. Using a dropping pipet the top layer is transferred
to another tube and the solvents are evaporated under nitrogen.
The remaining fatty acid methyl esters are redissolved in
0.1-0.2 ml hexane.
This method provides more than enough fatty acid methyl
esters for gas chromatographic analysis from about 10-20
mg of seed material. Germination is not significantly reduced
from that of whole seeds. After a week of growth in the
vials, selected seedlings are potted in a soil medium and
transferred to the greenhouse, then transplanted to the
field after 3-6 weeks.