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 parental stocks.

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 at 30°C.

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.