Cucurbit Genetics Cooperative Report 8:69-70 (Article 26) 1985
Rapid TLC and HPLC Test for Cucurbitacins
Gorski, P. M.*, A. Jaworski, S. Shannon and R. W. Robinson
Department of Horticultural Science, New York State Agricultural Experiment Station, Geneva, NY 14456
Recently, there have been reports (2,4) of serious illness and litigation due to a low incidence of a gene for bitter fruit in summer squash. In some cases, the frequency was estimated to be one plant in 10,000 with bitter fruit.
Such a low frequency makes it difficult for a seedsman to determine if a seed lot is free of the deleterious gene. In cucumber, plants with non-bitter fruit due to the bi
gene can be detected in the seedling stage by tasting the cotyledons. Classification for the cu
gene in Cucurbita pepo
can also be made in the seedling stage by tasting the cotyledons or by feeding tests with Diabrotica
beetles (3). While these methods are useful for breeding squash resistant to cucumber beetles, these seedling tests are not reliable methods to test for the frequency of Bi, the gene for bitter fruit. The cu gene reduces the cucurbitacin concentration in cotyledons sufficiently to prevent them from having a bitter taste but, unlike the bi
gene of cucumber, it does not prevent cucurbitacins and bitterness from developing in the fruit.
Existing methods for analyzing cucurbitacins are too cumbersome for screening large squash populations for Bi, since a person can analyze less than 50 samples per day. A rapid test involving the application of crude chloroform extracts to filter paper with antimony bichloride was reported by Andeweg and de Bruyn (1) to fluoresce under U.V. light when cucurbitacin C was present. However, we found that results of this test did not agree with those obtained by tasting cotyledons in a cucumber F2 population segregating for bit The test did not work even when applied to cucurbitacin C, for which the test was specifically developed. We also found that the cucurbitacin concentration in C. pepo
cv Blackjack cotyledons quantified with this test showed no correlation with cucurbitacin concentration determined by the HPLC method reported by Ferguson (3).
We developed a new test in which leaf, fruit, or cotyledon tissues are sampled simply by pressing the tissue against a TLC plate (5x20 silica gel with fluorescent indicator) so Juice is expressed on the absorbent. Fifteen to twenty samples can be placed 1.5 cm from the bottom of the long dimension of the plate. Sampling can be done in the field. The plates are developed about 3 cm with a methanol:water (45:55) solvent (5). After drying, the chromatograms are observed under a 254 nm U.V. lamp. (It is best to view the plates in subdued light while wearing protective lenses to protect eyes from exposure to U.V. radiation). Most interfering compounds, including chlorophyll which also absorbs U.V. light, remain at the starting point. All cucurbitacins move near the solvent front and appear as dark spots due to quenching of fluorescence. Pure standards of cucurbitacins B, D, E and I and the glycoside of E can be detected at 1 nanogram per spot. Leaf and cotyledon tissues usually contain other fluorescnet-quenching substances and the TLC test can be used as a rough preliminary method for selection of high and low cucurbitacin individuals. However, fruit placental tissue contains only traces of interfering compounds, and sap expressed from this tissue can be used for selection of more precise classes of cucurbitacin concentrations.
The TLC test can be used for screening large populations in breeding programs and for varietal purity tests. Several hundred samples can be screened each day at a materials cost of 2-3 cents per sample.
Fruit placental sap samples can also be used for quantitative HPLC determinations of cucurbitacins. Since there are very low levels of interfering compounds, it is only necessary to add methanol to sap (8:2), and filter to remove precipitated materials. The filtered solution can be injected directly into the HPLC. Although not as fast as the TLC method, this method can be used for rapid and accurate estimates of different cucurbitacins in fruit. The number of analyses is limited by the rate of HPLC output.
- Andeweg, J. M. and J. W. de Bruyn. 1959. Breeding of non-bitter cucumbers.
- Ferguson, J. E., D. C. Fischer, and R. L. Metcalf. 1983. A report of
cucurbitacin poisonings in humans. Cucurbit Genetics Coop. Report
- Ferguson, J. E., E. R. Metcalf and A. M. Rhodes. 1983. Influence of
cucurbitacin content in cotyledons of Cucurbitaceae
feeding behavior of Diabrotica
Entom. Soc. Amer. 76:47-51.
- Rymal, K. S., O. L. Chambliss, M. D. Bond, and D. A. Smith. 1984. Squash
containing toxic cucurbitacin compounds occurring in California and
Alabama. J. Food Protection 47:270-271.
- Stoewsand, G. S., A. Jaworski, S. Shannon and R. W. Robinson. 1985.
Toxicologic response in mice fed Cucurbita
fruit. J. Food Protection
* On leave from Dept. of Biochemistry, Institute of Soil Science and Plant
Cultivation, 24-100, Pulawy, Poland.