Materials and methods Directory UMM :Data Elmu:jurnal:I:Industrial Crops and Products:Vol12.Issue2.Aug2000:

dietary essential fatty acid for humans where, to be fully utilised by the body, it must be converted into 6c,9c,12c-linolenic acid C18:3v6, g-linolenic acid, a reaction catalysed by the enzyme delta-6- desaturase. Although healthy adults will obtain sufficient g-linolenic acid in this way, the conver- sion of linoleic acid to g-linolenic acid can be very slow in individuals suffering from a number of common diseases Horrobin, 1990. Substantial clinical improvements can be produced in patients suffering from diseases such as atopic eczema or breast pain by administering a regular exogenous supply of g-linolenic acid. Whereas in many oil- seeds the desaturation of linoleic acid gives rise to 9c,12c,15c-linoleic acid C18:3v3, a -linolenic acid, in evening primrose the product of linoleic acid desaturation is g-linolenic acid. Although there are other sources of g-linolenic acid, both plant and fungal, evening primrose oil appears to be clinically the most effective Horrobin, 1990. Hence, evening primrose oil has become commer- cially significant in recent years, being officially recognised as a prescription pharmaceutical in several countries and sold as a dietary supplement in many more. Compared to mainstream oilseed crops, the oil content of evening primrose seed is relatively low approximately 25 and an increase in oil con- tent can lead to significant reductions in extrac- tion costs. A g-linolenic acid content of 9 has become the minimum acceptable standard for the nutritional supplements industry. Seed containing less than 9 g-linolenic acid in the oil can be of considerably reduced value, or even unmarketable in years of oversupply. Hence, both the total oil and g-linolenic acid contents of evening primrose seed are of considerable economic importance. A plant breeding programme at Writtle Col- lege, Chelmsford UK, has produced several culti- vars which yield improved oil and g-linolenic acid contents, including cv. Peter and cv. Merlin. However, in addition to genotype, the oil content of oilseeds is known to be affected by a number of environmental factors, including water stress, temperature, disease and nitrogen nutrition Har- ris et al., 1978. Several studies on evening prim- rose e.g. Lotti et al., 1978; Reiner and Marquard, 1988; Court et al., 1993 suggest that a positive correlation exists between seed oil content and temperature during seed filling, although in some instances the results are not conclusive. On the other hand, it has long been known that in many oilseed crops e.g. oilseed rape, sunflower and flax the extent of desaturation in the fatty acid composition of the seed oil is inversely related to temperatures prevailing during seed maturation Canvin, 1965. This seems to be the case with evening primrose, too e.g. Levy et al., 1993 and crops grown at warmer latitudes tend to produce oil with lower g-linolenic acid content Simpson and Fieldsend, 1993. In the UK, evening primrose crops ripen during a period of reducing daylengths, light levels and temperatures and, at a given location, climatic conditions can influence oil and g-linolenic acid content in two ways. Firstly, conditions will differ between years. Secondly, owing to a difference in maturity date of several weeks, crops which are sown in late summer and overwintered and crops sown in the spring will experience different condi- tions during ripening. Through three seasons of field studies, this paper investigates the effect of climatic conditions in eastern England, a major evening primrose growing area, on oil and g-lino- lenic acid accumulation during seed growth. Dif- ferences in oil and g-linolenic acid content were found to occur both between seeds from different parts of plants and between overwintered and spring-sown crops harvested at the optimal growth stage. Final oil content and oil quality were shown to be determined at different stages during seed growth.

2. Materials and methods

2 . 1 . Site, treatments and weather During three seasons, field experiments were conducted on a commercial farm at Hatfield Peverel, near Chelmsford, Essex, UK latitude 51° 47N, longitude 0° 31’E, altitude 50 m. The year 1 1995 – 96 trial compared overwintered and spring-sown evening primrose cv. Merlin, whilst the year 2 1996 – 1997 trial also included over- wintered plots of cv. Peter. Details of the site, trial design, crop establishment and harvest methods were reported by Fieldsend and Morison forth- coming. Equivalent details for the year three 1997 – 1998 trial, which compared overwintered and early and late spring-sown evening primrose cv. Merlin, were reported by Fieldsend and Morison 1999. All trials consisted of four blocks. Daily incident solar radiation, maximum and minimum temperature, precipitation, humid- ity and wind data were obtained from the Mete- orological Office approved climatological station at Writtle College, Chelmsford, approximately 10 km from the experimental site. From each plot, representative samples of plant material were harvested by hand on four or five occa- sions during seed growth. The penultimate har- vest was taken at the optimal growth stage for swathing, i.e. when 95 of the spike length bears capsules containing non-white seeds, desig- nated by Simpson 1994 as growth stage G.S. 5,95. 2 . 2 . Laboratory analyses Seed samples were dried overnight at 80°C in a forced-draught oven and were then allowed to cool in a desiccator. The oil content of the seeds was measured directly by a nuclear magnetic res- onance analyser Newport 4000, Oxford Analyti- cal Instruments, Abingdon, UK calibrated with two reference standards defatted seed and pure oil. The fatty acid composition of the oil was de- termined by gas chromatography of methyl es- ters. For each sample, 0.1 g of seed was placed in a Pyrex sample tube and 2 ml of HPLC grade toluene and 2 ml of BF 3 methanol were added. The tubes were then transferred to a heating block set at 90°C for 40 min, after which they were allowed to cool. To each tube 0.9 sodium chloride solution was then added and the con- tents were thoroughly mixed before being cen- trifuged for 15 min at 3000 rpm. The layer of solvent containing the fatty acid methyl esters was transferred to a clean vial and the solvent was evaporated off under a stream of nitrogen, following which 1 ml of n-hexane was added to each sample. An aliquot of this sample was in- jected into the gas chromatography column. A 30 m Supelcowax 10 column Supelco was used, operating at 165°C for 2 min, then 165 – 190°C at 3°C min − 1 , then 190°C for 5 min, then 190 – 220°C at 3.5°C min − 1 , then 220°C for 10 min. The temperature of the injector of the Hewlett Packard gas chromatograph was 220°C, the flame ionisation detector temperature was 250°C and the carrier gas was nitrogen.

3. Results