Climatic conditions during seed growth significantly
influence oil content and quality in winter and spring
evening primrose crops (
Oenothera
spp.)
Andrew F. Fieldsend
a,*, James I.L. Morison
baScotia Pharmaceuticals Ltd,Plant Technology Centre,Writtle College,Chelmsford CM1 3RR, UK bDepartment of Biological Sciences,Uni6ersity of Essex,Wi6enhoe Park,Colchester CO4 3SQ, UK
Accepted 31 March 2000
Abstract
Evening primrose (Oenotheraspp) seed is an important source ofg-linolenic acid, a relatively rare fatty acid with
value as a pharmaceutical and nutritional supplement. The influence on oil content and quality of climatic conditions during seed growth was investigated in three years of field trials comparing crops sown in the late summer and overwintered with crops spring-sown the following year. At the onset of oil accumulation, palmitic acid, linoleic acid and a-linolenic acid were the predominant fatty acids in the seeds and g-linolenic acid was hardly present. At
maturity, linoleic acid constituted 70 – 75% of the oil,g-linolenic acid content ranged from 8.0 to 9.9% anda-linolenic
acid was almost undetectable. In all years, seeds from the overwintered plants of cv. Merlin contained more oil than did seeds from the equivalent spring-sown plants, but theg-linolenic acid content of the oil was lower. The rate of
increase in seed oil content was faster in the overwintered crops but the duration of oil accumulation was shorter. Oil content at seed maturity in cv. Merlin was positively correlated with both mean daily temperature (r2, 0.59) and mean
daily incident solar radiation (r2, 0.71) during the main period of seed filling. Strong negative correlations existed
between the final g-linolenic acid content of the oil and both climatic variables during the final phase of oil
accumulation (r2, −0.78 and −0.83, respectively). Temperature was probably the primary determinant of the final
g-linolenic acid content but it was unclear which variable most influenced final seed oil content. Differences in oil
content and seed size also existed between seeds harvested from different parts of the same plant. © 2000 Elsevier Science B.V. All rights reserved.
Keywords:Evening primrose;Oenotheraspp.; Seed oil content;g-linolenic acid; Solar radiation; Temperature
www.elsevier.com/locate/indcrop
1. Introduction
In common with most major commercial oil-seeds, the seeds of evening primrose (Oenothera spp) contain significant amounts of 9c,12c-linoleic acid (C18:2) in the oil. Linoleic acid is the main
* Corresponding author. Present address: Semundo Ltd., Abbots Ripton, Huntingdon, PE28 2PH, UK. Tel.: + 44-1487-773595; fax: +44-1487-773532.
E-mail address: andrew.fieldsend@swseed.se (A.F. Fieldsend)
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 sufficientg-linolenic acid in this way, the conver-sion of linoleic acid tog-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 officommer-cially 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
andg-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 andg-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
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) Analyti-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 BF3 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
3.1. Oil and g-linolenic acid contents at G.S. 5,95
3.2. Oil and fatty acid accumulation during seed filling
The pattern of fatty acid accumulation as a proportion of total oil content observed in year 2 (Fig. 1) was representative of all years. At the onset of oil accumulation, palmitic acid, linoleic acid and a-linolenic acid were the predominant fatty acids, butg-linolenic acid was almost unde-tectable. Subsequently, the proportion of linoleic acid in the oil increased very rapidly, constituting 70 – 75% of the oil at G.S. 5,95 in all treatments. By contrast, the proportion ofa-linolenic acid in the oil declined rapidly, to just 0.2 – 0.4% in most treatments at G.S. 5,95. In the late spring-sown treatment in year 3, 0.7% of the oil wasa-linolenic acid (data not shown). The proportions of palmitic and oleic acids also declined. The concen-tration of g-linolenic acid in the oil increased rapidly during the first 10 – 20 days of oil accumu-lation, then slowed markedly. The lowg-linolenic acid content of the oil of cv. Peter was associated with a particularly high oleic acid content.
In all treatments, the oil content of the seeds
increased until the crop reached approximately G.S. 5,95, the year 3 results (Fig. 2) being repre-sentative of all treatments. Each of the five major fatty acids increased as a proportion of total seed weight but, after an initial increase, the concentra-tion of a-linolenic acid in the seeds declined. At G.S. 5,95, the polyunsaturated fatty acids (pri-marily linoleic acid) represented approximately 19 – 23% of seed mass whilst palmitic, stearic and oleic together constituted 5% or less.
3.3. Climatic influence on oil and g-linolenic acid
content
In all years, oil accumulation commenced on or after day of year 200 and took place during a period of declining light levels and temperatures (Fig. 3). As should be expected, there was a close, positive relationship between incident photosyn-thetically active radiation (PAR) levels and tem-perature. The spring-sown crops matured during cooler, duller conditions than did the overwin-tered crops. Gompertz growth model functions of the form
Table 1
Day of year of harvest (DOY), mean oil content of seeds,g-linolenic acid (C18:3v6) content of seed oil and thousand seed weight (TSW) of seeds from evening primrose plants harvested at growth stage G.S. 5,95 in 3 yearsa
Year Treatment G.S. 5,95(DOY) Oil (%) C18:3v6 (%) TSW (g) Anthesis (DOY) 95% Oil (DOY)
Overwintered 246 24.6a
1 8.02a 0.354a 191 236
Spring-sown 288 23.0a 9.41b 0.375b 226 275
0.866 0.057 0.010 SED
0.155 B0.001 0.032 P-value
Overwintered (P) 239 7.68c 0.411b 197 234
Spring-sown 273 24.6b 9.55a 0.338a 223 267
SED 0.320 0.083 0.018
B0.001 B0.001 0.014 P-value
Overwintered 237 26.3a 9.02a 0.346a 195 235
3
Early spring-sown 259 27.1a 9.02a 0.374a 210 253
23.1b
287 0.300b 232 279
Late spring-sown 9.94b
0.643
SED 0.187 0.013
P-value 0.002 0.004 0.004
aAll treatments are cv. Merlin except (P) cv. Peter. Also shown are estimated day of year of anthesis and date when seed oil
Fig. 1. Variation over time in the fatty acid composition of the seed oil in overwintered evening primrose cv. Merlin (a) and cv. Peter (b) and spring-sown evening primrose cv. Merlin (c) in year 2 (1997). The scale for linoleic acid content (C18:2) appears on the right hand side of the figure. Error bars represent91 SE. Downward arrows indicate the date by which the crops had reached growth stage 5,95. Short vertical lines indicate the date at which oil content reached 85 and 95% of the final figure, as estimated from Gompertz growth model functions.
the spring-sown crops (0.56 – 0.76% day−1). It was
also lower in cv. Peter than in the equivalent cv. Merlin treatment. In all of the overwintered crops (including cv. Peter), the duration of oil accumu-lation in the seeds (i.e. between 5% and 95% of final oil content) ranged only from 28 to 32 days. It was longer in the spring-sown treatments,
rang-Fig. 2. Variation over time in the fatty acid content of the seeds of overwintered (a) and early spring-sown (b) and late spring-sown (c) evening primrose cv. Merlin in year three (1998). Error bars represent91 SE. Downward arrows indi-cate the date by which the crops had reached growth stage 5,95. The fitted curves indicate seed oil content as estimated from Gompertz growth model functions and the horizontal dotted lines indicate 5, 80, 85 and 95% of final seed oil content.
y=a×exp {−exp[-(x−x0)/b]}
Fig. 3. Variation over time in the oil content of the seeds of evening primrose (solid lines) and theg-linolenic acid (GLA) content of the oil (dotted lines) as estimated from Gompertz growth model functions fitted to data points in (a) year 1, (b) year 2 and (c) year 3. Closed circles, overwintered cv. Merlin; closed triangles, overwintered cv. Peter; open circles, spring-sown cv. Merlin (years 1 and 2), early spring-spring-sown cv. Merlin (year 3); open diamonds, late spring-sown cv. Merlin. Error bars represent91 SE. Downward arrows indicate the date by which the crops had reached growth stage 5,95. Also (upper dashed lines) daily mean temperature and (lower solid lines) incident daily photosynthetically-active radiation (5 day smoothed averages).
was lower in the year two spring-sown crop, where it coincided with a period of particularly high temperatures (Fig. 3b).
Across all cv. Merlin treatments, there was a positive correlation (r2, 0.59) between final oil
content, as estimated by the Gompertz growth model functions, and mean daily temperature dur-ing the period from 5 to 95% of final oil content (Fig. 4a). However, a stronger correlation (r2,
0.71) was recorded between oil content and mean daily incident PAR (data not shown). The correla-tion between final oil content and mean daily PAR between 5 and 80% of final oil content, i.e. when the rate of increase in oil content of the seeds was virtually linear, was stronger still (r2,
0.79, Fig. 4b), but that between final oil content and mean daily temperature during the same pe-riod was low (r2, 0.32). The result for the year 2
spring-sown crop was a major cause of this low correlation: the final oil content of the seeds was relatively low, as was the mean daily temperature between 80 and 95% of final oil content (day of year 250 to day 267), but the mean daily tempera-ture prior to day 250 was relatively high (Fig. 3b). The finalg-linolenic acid content of the oil was negatively correlated with the mean daily temper-ature during the period from 5 to 95% of final oil content (r2,
−0.59) and also with mean daily PAR during this period (r2, −0.67, data not
shown). During the period between 5 and 80% of final oil content the correlations between final g-linolenic acid content and mean daily tempera-ture and PAR were weaker (r2, −0.27 and −
0.50, respectively). The result for the year 2 spring-sown crop was again a notable outlier; the final g-linolenic acid content being much higher than would be expected from the mean daily temperature during this period. Also, the final g-linolenic acid of the year one overwintered crop was lower than might have been expected. The rate of oil accumulation slowed rapidly after 85% of final oil content had occurred (Fig. 2) and the period between 85 and 95% ranged from 8 – 9 days in the overwintered crops to 10 – 15 days in the spring-sown crops. There was a strong correlation between final g-linolenic acid content and mean daily temperature (r2, −0.78, Fig. 4c) and mean
daily PAR (r2, −0.83) during this period. The
time between anthesis (estimated from the sam-pling date when plants were producing new flow-ers) and 95% of final oil content ranged from 37 to 49 days (Table 1).
3.4. Variation in oil and g-linolenic acid contents
in seeds from different parts of the plant
In all cv. Merlin treatments, most seed was produced on the main stems (Table 2). In years 1 and 2, the main stem seeds contained a higher percentage of oil than seeds from the upper pri-mary branches, but in year 3 the differences in oil content were not significant. Seeds from the upper primary branches had the lowest TSW, but g-lino-lenic contents were similar throughout the plant. In cv. Peter the upper primary branches con-tributed 76% of total seed yield and this seed contained significantly more oil than did seeds from the main stems or basal primary branches.
For the year one spring crop, seed samples from earlier (brown) and later (green) ripening capsules were analysed. A capsule was counted as brown if at least half of its length was brown. Seeds from the early maturing main stem capsules contained 16% more oil than seeds from the later capsules (Table 3). The differences in oil content between the earlier and later maturing seeds on the primary branches were not significant. Simi-larly, there were no significant differences in seed size between early and later maturing seeds on a stem. The oil in the seeds from the later maturing capsules tended to have the highest g-linolenic acid content, but the difference was only signifi-cant (P, 0.052) for the upper primary branches.
4. Discussion
Our results show that oil and g-linolenic acid content of evening primrose seeds can be strongly influenced by the climatic conditions prevailing during seed growth, such that on occasions the quality of the harvested seed is below that accept-able to the market. Seeds from overwintered crops tended to contain more oil, but with a lower g-linolenic acid content, than seeds from spring-sown crops (Table 1). The use of improved culti-vars such as cv. Merlin can reduce the risk of producing low-quality seed but, even for a given cultivar and time of sowing, large differences in oil andg-linolenic acid content can occur between years.
A
Distribution of yield, mean oil content of seeds,g-linolenic acid content (C18:3v6) of seed oil and thousand seed weight (TSW) of seeds from evening primrose plants harvested at growth stage G.S. 5,95 in 3 yearsa
79 25.0a 8.09a 0.360a 28.1a 8.70a 0.358a 94 26.3a 9.03a 0.348a
WM-M
27.3a 8.70a 0.350a 0 21
24.7a
B 9 8.41a 0.323a
11
12 21.5b 7.46b 0.323a 27.3a 8.62a 0.293b 6 26.5a 8.99a 0.301b
U
0.458 0.139 0.019 0.504 0.119 0.016
1.166 0.257 0.018 SED
0.194 0.797 0.026 0.692 0.789 0.011
0.027
0.044 0.130
P-value
22 24.0a 7.95a 0.468a WP-M
24.7a 9.54a 0.339a 65 27.5a
0.386a 8.88a
SM-M 54 23.6a 9.41a 94 0.387a
32 22.4b 9.38a 0 23 26.5a 9.34a 0.367ab
B 0.375a
23.2b 9.52a 0.316a 11 25.9a 9.24a
U 14 21.6b 9.46a 0.340b 6 0.320b
0.279 0.105 0.039 0.647
0.010 0.155 0.020
SED 0.386 0.085
0.013 0.843 0.356 0.117 0.056 0.043
0.005 0.640 0.011 P-value
LM-M 72 23.1a 9.85a 0.313a
14 23.9a 10.19b 0.266a B
14 22.2a 10.27b 0.241a U
SED 0.958 0.100 0.023
0.305 0.012 0.052 P-value
aSeed source: WM=overwintered cv. Merlin; WP=overwintered cv. Peter; SM=spring-sown cv. Merlin; LM=late spring-sown cv. Merlin; M=main stem; B=basal primary branch; U=upper primary branch.
Table 3
Mean oil content of seeds,g-linolenic acid (C18:3v6) content of oil and thousand seed weight (TSW) of seeds from earlier (brown) and later (green) ripening capsules from year one spring-sown plants of evening primrose cv. Merlin harvested at G.S. 5,95a
Oil (%)
Stem type Capsule colour C18:3v6(%) TSW (g)
Main Brown 24.90a 9.33a 0.390a
21.45b 9.54a
Green 0.380a
SED 0.692 0.134 0.012
P-value 0.016 0.211 0.459
22.72a
Basal primary branches Brown 9.35a 0.380a
Green 22.04a 9.37a 0.371a
0.866
SED 0.107 0.006
0.493 0.864 0.212
P-value
Upper primary branches Brown 21.28a 9.09a 0.318a
21.53a
aData bearing the same letter(s) within a column and year are not significantly different atP, 0.05. SED is the standard error of
the difference between means for a one-way ANOVA for each year.
The lipid fraction in newly-formed evening primrose seeds contains a relatively high percent-age of a-linolenic acid (Fig. 1). This percentage declines as the percentage of g-linolenic acid in-creases, and it has been suggested that during seed maturation a-linolenic acid is transformed into g-linolenic acid (work cited by Cisowski et al., 1993). This is unlikely, as no pathway for the conversion of a-linolenic acid to g-linolenic acid has been shown to occur in plants. In fact, ex-pressed as a percentage of seed weight, the amount of a-linolenic acid present is always low (Fig. 2). More probably, at the onset of seed filling, the concentration of cell membrane lipids in the analysed seed sample was high relative to the concentration of triacylglycerols, the storage lipids. Furthermore, the proportion of cell mem-brane lipids in our analysed seed samples will have declined as storage lipids accumulated. Mukherjee and Kiewitt (1987) demonstrated that a-linolenic acid is channelled almost exclusively into phospholipids and glycolipids, which are the major constituents of cellular membranes, but that the a-linolenic acid content of these lipid classes declines sharply with seed maturation. In evening primrose seedsg-linolenic acid is incorpo-rated mainly into triacylglycerols.
should be demonstrable. Our data do not show such a relationship.
Evening primrose seed oil attains virtually its final percentage content of g-linolenic acid rela-tively quickly (Fig. 1) and it is surprising, there-fore, that temperatures during the earlier part of the seed filling period appear to have little influ-ence on the final g-linolenic acid content of the oil. In a growth cabinet study, Levy et al. (1993) showed that final g-linolenic acid percent-age was most influenced by temperatures 30 – 40 days after anthesis. Similarly, we have shown that, in the field, temperatures during a 8 – 15 day period ending between 37 and 49 days after anthesis, as the rate of increase in oil content was slowing down, most strongly determined the final g-linolenic acid content of the oil. During rapid oil accumulation, the large amount of linoleic acid produced (Fig. 2) may mask any residual effect of temperatures during this period on the g-linolenic acid content of the oil. Only once the rate of accumulation has slowed might any temperature effects be detectable subse-quently.
Seeds formed on the main stem of evening primrose cv. Merlin had a higher oil content and were larger than seeds formed on the branches, particularly the upper primary branches. Similarly, on a main stem, at least, the earlier formed seeds tended have a higher oil content whereas the later formed seeds on a branch tended to have a higher g-linolenic acid content in the oil. These results agree with those of Court et al., (1993). However, these differ-ences may reflect the position of the seed on the plant in relation to supply of assimilate as well as any influence of climate. In any case, in a commercial situation the range of oil and g-lino-lenic acid contents present within a seed stock will be reduced by the cleaning process as small seeds from the later-formed capsules are re-moved (Simpson and Fieldsend, 1993).
5. Conclusion
Both the oil content and the percentage of g-linolenic acid in the oil of evening primrose
seeds were influenced by temperatures during seed growth and possibly also by incident PAR. However, final oil content and oil quality were determined at different stages during seed growth. With regard to crops grown in the UK, spring-sown crops were more likely to produce oil with a g-linolenic acid content of 9% or more. Seeds from overwintered crops tended to contain more oil, leading to savings in extrac-tion costs which may be attractive as long as the minimum acceptable g-linolenic acid content is achieved.
Acknowledgements
We thank Peter Martin of the Scotia Plant Technology Centre for carrying out the NMR analyses and sample preparation for GC analy-sis.
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