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Climatic conditions during seed growth significantly influence oil content and quality in winter and spring evening primrose crops (Oenothera spp.)
Industrial Crops and Products 12 (2000) 137 – 147 www.elsevier.com/locate/indcrop
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 b a b
Scotia Pharmaceuticals Ltd, Plant Technology Centre, Writtle College, Chelmsford CM1 3RR, UK Department of Biological Sciences, Uni6ersity of Essex, Wi6enhoe Park, Colchester CO4 3SQ, UK Accepted 31 March 2000
Abstract Evening primrose (Oenothera spp) seed is an important source of g-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% and a-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 the g-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 (r 2, 0.59) and mean daily incident solar radiation (r 2, 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 (r 2, − 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; Oenothera spp.; Seed oil content; g-linolenic acid; Solar radiation; Temperature
1. Introduction * Corresponding author. Present address: Semundo Ltd., Abbots Ripton, Huntingdon, PE28 2PH, UK. Tel.: +441487-773595; fax: + 44-1487-773532. E-mail address: [email protected] (A.F. Fieldsend)
In common with most major commercial oilseeds, 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
0926-6690/00/$ - see front matter © 2000 Elsevier Science B.V. All rights reserved. PII: S 0 9 2 6 - 6 6 9 0 ( 0 0 ) 0 0 0 4 9 - 2
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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-6desaturase. Although healthy adults will obtain sufficient g-linolenic acid in this way, the conversion 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 oilseeds 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 commercially 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 content can lead to significant reductions in extraction 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 College, Chelmsford UK, has produced several cultivars 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 (Harris et al., 1978). Several studies on evening primrose (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 conditions 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-linolenic acid accumulation during seed growth. Differences 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° 47%N, 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 overwintered plots of cv. Peter. Details of the site, trial
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design, crop establishment and harvest methods were reported by Fieldsend and Morison (forthcoming). 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, humidity and wind data were obtained from the Meteorological 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 occasions during seed growth. The penultimate harvest was taken at the optimal growth stage for swathing, i.e. when 95% of the spike length bears capsules containing non-white seeds, designated 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 resonance analyser (Newport 4000, Oxford Analytical Instruments, Abingdon, UK) calibrated with two reference standards (defatted seed and pure oil). The fatty acid composition of the oil was determined by gas chromatography of methyl esters. 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 contents were thoroughly mixed before being centrifuged 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-
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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 The oil content of seeds from plants of cv. Peter grown in year 2 was lower than that of seeds from the equivalent cv. Merlin treatment and the oil contained less g-linolenic acid (Table 1), reflecting commercial experience with these cultivars (Scotia, unpublished data). Cv. Peter also produced the largest seeds. Across the cv. Merlin treatments a wide range of results were obtained: the highest oil content was 21% higher than the lowest, whilst the highest g-linolenic acid content was 27% higher than the lowest. The highest oil contents were achieved in year two and the highest g-linolenic acid contents were produced in year 3. Seeds produced in year 1 were lowest in both oil and g-linolenic acid content. Thus, the crops with high oil contents were not necessarily those which produced oil with the highest g-linolenic acid contents. In all years, seeds from the overwintered plants of cv. Merlin contained more oil than did seeds from the equivalent spring sown plants, but the g-linolenic acid content of the oil was lower. Only the early spring-sown plots in year three (the earliest maturing of all spring treatments) gave similar oil and g-linolenic acid content results to the overwintered plots. The weight of the seeds from the overwintered treatments was relatively consistent (thousand seed weight, TSW, 0.346–0.354 g) but the spring-sown treatments produced a wider range of mean seed sizes (TSW, 0.300– 0.375 g). There was no significant relationship between seed size and either the oil content of the seeds or the g-linolenic acid of the oil.
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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, but g-linolenic acid was almost undetectable. 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 of a-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 was a-linolenic acid (data not shown). The proportions of palmitic and oleic acids also declined. The concentration of g-linolenic acid in the oil increased rapidly during the first 10 – 20 days of oil accumulation, then slowed markedly. The low g-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 representative of all treatments. Each of the five major fatty acids increased as a proportion of total seed weight but, after an initial increase, the concentration of a-linolenic acid in the seeds declined. At G.S. 5,95, the polyunsaturated fatty acids (primarily 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 photosynthetically active radiation (PAR) levels and temperature. The spring-sown crops matured during cooler, duller conditions than did the overwintered 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)
1
Overwintered Spring-sown
246 288
SED P-value 2
Overwintered Overwintered (P) Spring-sown
Overwintered Early spring-sown Late spring-sown SED P-value a
24.6a 23.0a 0.866 0.155
239 239 273
SED P-value 3
Oil (%)
27.8a 25.1b 24.6b 0.320 B0.001
237 259 287
26.3a 27.1a 23.1b 0.643 0.002
C18:3v6 (%) 8.02a 9.41b 0.057 B0.001 8.70b 7.68c 9.55a 0.083 B0.001
TSW (g)
Anthesis (DOY)
95% Oil (DOY)
0.354a 0.375b
191 226
236 275
197 197 223
234 234 267
195 210 232
235 253 279
0.010 0.032 0.349a 0.411b 0.338a 0.018 0.014
9.02a 9.02a 9.94b
0.346a 0.374a 0.300b
0.187 0.004
0.013 0.004
All treatments are cv. Merlin except (P) cv. Peter. Also shown are estimated day of year of anthesis and date when seed oil content was 95% of final oil content. Data bearing the same letter(s) within a column and year are not significantly different at P, 0.05. SED is the standard error of the difference between means for a one-way ANOVA for each year.
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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 accumulation 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. 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 represent9 1 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.
y= a× exp {−exp[-(x −x0)/b]} were fitted to the data. The rate of oil accumulation was at a maximum between 5 and 80% of final oil content (Fig. 2) and this rate was reasonably constant across the overwintered cv. Merlin treatments (0.97–1.15% day − 1) but was lower in
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 represent 91 SE. Downward arrows indicate 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.
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Fig. 3. Variation over time in the oil content of the seeds of evening primrose (solid lines) and the g-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, springsown cv. Merlin (years 1 and 2), early spring-sown cv. Merlin (year 3); open diamonds, late spring-sown cv. Merlin. Error bars represent 9 1 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).
ing from 38 days in the year 3 late spring-sown crop to 51 days in the year 1 crop (Fig. 3). The rate of increase in the percentage of g-linolenic acid in the oil was also relatively constant, but
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 (r 2, 0.59) between final oil content, as estimated by the Gompertz growth model functions, and mean daily temperature during the period from 5 to 95% of final oil content (Fig. 4a). However, a stronger correlation (r 2, 0.71) was recorded between oil content and mean daily incident PAR (data not shown). The correlation 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 (r 2, 0.79, Fig. 4b), but that between final oil content and mean daily temperature during the same period was low (r 2, 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 temperature prior to day 250 was relatively high (Fig. 3b). The final g-linolenic acid content of the oil was negatively correlated with the mean daily temperature during the period from 5 to 95% of final oil content (r 2, − 0.59) and also with mean daily PAR during this period (r 2, − 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 temperature and PAR were weaker (r 2, −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 (r 2, − 0.78, Fig. 4c) and mean daily PAR (r 2, − 0.83) during this period. The
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time between anthesis (estimated from the sampling date when plants were producing new flowers) and 95% of final oil content ranged from 37 to 49 days (Table 1).
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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 primary 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-linolenic contents were similar throughout the plant. In cv. Peter the upper primary branches contributed 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. Similarly, 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 significant (P, 0.052) for the upper primary branches.
4. Discussion
Fig. 4. The relationship in evening primrose cv. Merlin between (a) final oil content of the seeds and mean daily temperature during the period from 5 to 95% of final oil content, (b) final oil content of the seeds and mean daily incident photosynthetically-active radiation during the period from 5 to 80% of final oil content and (c) final g-linolenic acid (GLA) content of the oil and mean daily temperature during the period from 85 to 95% of final oil content of the seeds. Oil and g-linolenic acid contents were estimated from Gompertz growth model functions. Closed symbols, overwintered crops; open symbols: spring crops; circles, year 1; squares, year 2; triangles, year 3.
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 acceptable to the market. Seeds from overwintered crops tended to contain more oil, but with a lower g-linolenic acid content, than seeds from springsown crops (Table 1). The use of improved cultivars 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 and g-linolenic acid content can occur between years.
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Seed sourcea
WM-M B U
Year 1b
Year 2b
% of yield
Oil (%)
C18:3v6(%)
TSW (g)
% of yield
Oil (%)
C18:3v6(%)
TSW (g)
% of yield
Oil (%)
79 9 12
25.0a 24.7a 21.5b
8.09a 8.41a 7.46b
0.360a 0.323a 0.323a
68 21 11
28.1a 27.3a 27.3a
8.70a 8.70a 8.62a
0.358a 0.350a 0.293b
94 0 6
26.3a
0.257 0.027
0.018 0.130
0.139 0.797
0.019 0.026
7.95a 8.26a 7.58a
0.468a 0.413a 0.396a
0.294 0.147
0.032 0.146
9.54a
0.339a
SED P-value
1.166 0.044
WP-M B U
0.458 0.194 22 2 76
SED P-value SM-M B U SED P-value LM-M B U SED P-value
Year 3b
24.0a 23.0a 25.5b 0.497 0.007
54 32 14
23.6a 22.4b 21.6b 0.386 0.005
9.41a 9.38a 9.46a
0.386a 0.375a 0.340b
0.085 0.640
0.010 0.011
94 0 6
24.7a 23.2b 0.279 0.013
9.52a
0.316a
0.105 0.843
0.039 0.356
26.5a 0.504 0.692
65 23 11
27.5a 26.5a 25.9a 0.647 0.117
72 14 14
23.1a 23.9a 22.2a 0.958 0.305
C18:3v6(%) 9.03a
TSW (g) 0.348a
8.99a
0.301b
0.119 0.789
0.016 0.011
8.88a 9.34a 9.24a
0.387a 0.367ab 0.320b
0.155 0.056
0.020 0.043
9.85a 10.19b 10.27b
0.313a 0.266a 0.241a
0.100 0.012
0.023 0.052
a Seed 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. b Data bearing the same letter(s) within a column and year are not significantly different at P, 0.05. SED is the standard error of the difference between means for a one-way ANOVA for each year.
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Table 2 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
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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 Stem type
Capsule colour
Oil (%)
C18:3v6(%)
TSW (g)
Main
Brown Green
24.90a 21.45b
9.33a 9.54a
0.390a 0.380a
0.692 0.016
0.134 0.211
0.012 0.459
22.72a 22.04a
9.35a 9.37a
0.380a 0.371a
0.866 0.493
0.107 0.864
0.006 0.212
21.28a 21.53a
9.09a 9.57a
0.318a 0.353a
0.760 0.761
0.155 0.052
0.031 0.341
SED P-value Basal primary branches
Brown Green
SED P-value Upper primary branches
Brown Green
SED P-value
a Data bearing the same letter(s) within a column and year are not significantly different at P, 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 percentage of a-linolenic acid (Fig. 1). This percentage declines as the percentage of g-linolenic acid increases, 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, expressed 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 membrane 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 seeds g-linolenic acid is incorporated mainly into triacylglycerols.
When comparing analyses of seed oils from plants growing in the wild at different geographical locations (e.g. Lotti et al., 1978), it is very difficult to separate temperature effects from those of daylength, PAR, soil type and other biotic influences. Interpretation of results from a single location and/or relatively stress-free environments, such as field crops, can be easier, but temperature and PAR effects remain difficult to separate. Our data showed a positive relationship between final seed oil content in evening primrose and mean daily temperature during seed filling (Fig. 4a). The existence of such a relationship would be explained if the synthesis of energy-rich lipids was favoured over other biochemical processes by higher, but not extreme, temperatures. Alternatively, it may be that the amount of incident PAR during oil accumulation (particularly the period of rapid, almost linear increase in oil content), rather than temperature, is the major determinant of final seed oil content. The basis of such a relationship could be simple: higher incident PAR should result in more assimilate being available to be partitioned into storage compounds, i.e. oil. If this is the case, it might be expected that the seeds would be larger and a positive correlation between oil content and TSW
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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 relatively quickly (Fig. 1) and it is surprising, therefore, that temperatures during the earlier part of the seed filling period appear to have little influence 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 percentage 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 subsequently. 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 differences 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-linolenic acid contents present within a seed stock will be reduced by the cleaning process as small seeds from the later-formed capsules are removed (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 extraction 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 analysis.
References Canvin, D.T., 1965. The effect of temperature on the oil content and fatty acid composition of the oils from several oil seed crops. Can. J. Bot. 43, 63 – 69. Cisowski, W., Zielinska-Stasiek, M., Luczkiewicz, M., 1993. Fatty acids and triacylglycerols of developing evening primrose (Oenothera biennis) seeds. Fitoterapia LXVI, 155 – 162. Court, W.A., Hendel, J.G., Pocs, R., 1993. Determination of the fatty acids and oil content of evening primrose (Oenothera biennis L.). Food Res. Int. 26, 181 – 186. Fieldsend, A.F., Morison, J.I.L., 1999. Manipulating light capture and seed yield in winter and spring evening primrose (Oenothera spp.). Aspects Appl. Biol. 56, 233 – 240. Fieldsend, A.F., Morison, J.I.L., forthcoming. Contrasting growth and dry matter partitioning in winter and spring evening primrose crops (Oenothera spp.) Submitted to Field Crops Res. Harris, H.C., McWilliam, J.R., Mason, W.K., 1978. Influence of temperature on oil content and composition of sunflower seed. Aust. J. Agric. Res. 29, 1203 – 1212. Horrobin, D.F., 1990. GLA: an intermediate in essential fatty acid metabolism with potential as an ethical pharmaceutical and as a food. Dev. Contemp. Pharmacother. 1, 1 – 45. Levy, A., Palevitch, D., Ranen, C., 1993. Increasing gamma linoleic acid in evening primrose grown under hot temperatures by breeding early cultivars. Acta. Hort. 330, 219 – 225. Lotti, G., Izzo, R., Landi, M.G., 1978. Influenza del clima e
A.F. Fieldsend, J.I.L. Morison / Industrial Crops and Products 12 (2000) 137–147 delle concimazioni sull’olio di semi di Oenothera biennis L., La Rivista della Societa` Italiana di Scienza dell. Alimentazione 7, 361 – 368. Mukherjee, K.D., Kiewitt, I., 1987. Formation of g-linolenic acid in the higher plant evening primrose (Oeothera biennis L.). J. Agric. Food Chem. 35, 1009–1012. Reiner, H., Marquard, R., 1988. Investigations in cultivation
.
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abilities and seed quality of Oenothera biennis L. Fat Sci. Technol. 90, 1 – 7. Simpson, M.J.A., 1994. A description and code of development of evening primrose (Oenothera spp.). Ann. Appl. Biol. 125, 391 – 397. Simpson, M.J.A., Fieldsend, A.F., 1993. Evening primrose: harvest methods and timing. Acta Hort. 331, 121 – 128.
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 b a b
Scotia Pharmaceuticals Ltd, Plant Technology Centre, Writtle College, Chelmsford CM1 3RR, UK Department of Biological Sciences, Uni6ersity of Essex, Wi6enhoe Park, Colchester CO4 3SQ, UK Accepted 31 March 2000
Abstract Evening primrose (Oenothera spp) seed is an important source of g-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% and a-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 the g-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 (r 2, 0.59) and mean daily incident solar radiation (r 2, 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 (r 2, − 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; Oenothera spp.; Seed oil content; g-linolenic acid; Solar radiation; Temperature
1. Introduction * Corresponding author. Present address: Semundo Ltd., Abbots Ripton, Huntingdon, PE28 2PH, UK. Tel.: +441487-773595; fax: + 44-1487-773532. E-mail address: [email protected] (A.F. Fieldsend)
In common with most major commercial oilseeds, 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
0926-6690/00/$ - see front matter © 2000 Elsevier Science B.V. All rights reserved. PII: S 0 9 2 6 - 6 6 9 0 ( 0 0 ) 0 0 0 4 9 - 2
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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-6desaturase. Although healthy adults will obtain sufficient g-linolenic acid in this way, the conversion 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 oilseeds 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 commercially 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 content can lead to significant reductions in extraction 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 College, Chelmsford UK, has produced several cultivars 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 (Harris et al., 1978). Several studies on evening primrose (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 conditions 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-linolenic acid accumulation during seed growth. Differences 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° 47%N, 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 overwintered plots of cv. Peter. Details of the site, trial
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design, crop establishment and harvest methods were reported by Fieldsend and Morison (forthcoming). 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, humidity and wind data were obtained from the Meteorological 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 occasions during seed growth. The penultimate harvest was taken at the optimal growth stage for swathing, i.e. when 95% of the spike length bears capsules containing non-white seeds, designated 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 resonance analyser (Newport 4000, Oxford Analytical Instruments, Abingdon, UK) calibrated with two reference standards (defatted seed and pure oil). The fatty acid composition of the oil was determined by gas chromatography of methyl esters. 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 contents were thoroughly mixed before being centrifuged 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-
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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 The oil content of seeds from plants of cv. Peter grown in year 2 was lower than that of seeds from the equivalent cv. Merlin treatment and the oil contained less g-linolenic acid (Table 1), reflecting commercial experience with these cultivars (Scotia, unpublished data). Cv. Peter also produced the largest seeds. Across the cv. Merlin treatments a wide range of results were obtained: the highest oil content was 21% higher than the lowest, whilst the highest g-linolenic acid content was 27% higher than the lowest. The highest oil contents were achieved in year two and the highest g-linolenic acid contents were produced in year 3. Seeds produced in year 1 were lowest in both oil and g-linolenic acid content. Thus, the crops with high oil contents were not necessarily those which produced oil with the highest g-linolenic acid contents. In all years, seeds from the overwintered plants of cv. Merlin contained more oil than did seeds from the equivalent spring sown plants, but the g-linolenic acid content of the oil was lower. Only the early spring-sown plots in year three (the earliest maturing of all spring treatments) gave similar oil and g-linolenic acid content results to the overwintered plots. The weight of the seeds from the overwintered treatments was relatively consistent (thousand seed weight, TSW, 0.346–0.354 g) but the spring-sown treatments produced a wider range of mean seed sizes (TSW, 0.300– 0.375 g). There was no significant relationship between seed size and either the oil content of the seeds or the g-linolenic acid of the oil.
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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, but g-linolenic acid was almost undetectable. 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 of a-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 was a-linolenic acid (data not shown). The proportions of palmitic and oleic acids also declined. The concentration of g-linolenic acid in the oil increased rapidly during the first 10 – 20 days of oil accumulation, then slowed markedly. The low g-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 representative of all treatments. Each of the five major fatty acids increased as a proportion of total seed weight but, after an initial increase, the concentration of a-linolenic acid in the seeds declined. At G.S. 5,95, the polyunsaturated fatty acids (primarily 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 photosynthetically active radiation (PAR) levels and temperature. The spring-sown crops matured during cooler, duller conditions than did the overwintered 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)
1
Overwintered Spring-sown
246 288
SED P-value 2
Overwintered Overwintered (P) Spring-sown
Overwintered Early spring-sown Late spring-sown SED P-value a
24.6a 23.0a 0.866 0.155
239 239 273
SED P-value 3
Oil (%)
27.8a 25.1b 24.6b 0.320 B0.001
237 259 287
26.3a 27.1a 23.1b 0.643 0.002
C18:3v6 (%) 8.02a 9.41b 0.057 B0.001 8.70b 7.68c 9.55a 0.083 B0.001
TSW (g)
Anthesis (DOY)
95% Oil (DOY)
0.354a 0.375b
191 226
236 275
197 197 223
234 234 267
195 210 232
235 253 279
0.010 0.032 0.349a 0.411b 0.338a 0.018 0.014
9.02a 9.02a 9.94b
0.346a 0.374a 0.300b
0.187 0.004
0.013 0.004
All treatments are cv. Merlin except (P) cv. Peter. Also shown are estimated day of year of anthesis and date when seed oil content was 95% of final oil content. Data bearing the same letter(s) within a column and year are not significantly different at P, 0.05. SED is the standard error of the difference between means for a one-way ANOVA for each year.
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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 accumulation 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. 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 represent9 1 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.
y= a× exp {−exp[-(x −x0)/b]} were fitted to the data. The rate of oil accumulation was at a maximum between 5 and 80% of final oil content (Fig. 2) and this rate was reasonably constant across the overwintered cv. Merlin treatments (0.97–1.15% day − 1) but was lower in
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 represent 91 SE. Downward arrows indicate 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.
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Fig. 3. Variation over time in the oil content of the seeds of evening primrose (solid lines) and the g-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, springsown cv. Merlin (years 1 and 2), early spring-sown cv. Merlin (year 3); open diamonds, late spring-sown cv. Merlin. Error bars represent 9 1 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).
ing from 38 days in the year 3 late spring-sown crop to 51 days in the year 1 crop (Fig. 3). The rate of increase in the percentage of g-linolenic acid in the oil was also relatively constant, but
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 (r 2, 0.59) between final oil content, as estimated by the Gompertz growth model functions, and mean daily temperature during the period from 5 to 95% of final oil content (Fig. 4a). However, a stronger correlation (r 2, 0.71) was recorded between oil content and mean daily incident PAR (data not shown). The correlation 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 (r 2, 0.79, Fig. 4b), but that between final oil content and mean daily temperature during the same period was low (r 2, 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 temperature prior to day 250 was relatively high (Fig. 3b). The final g-linolenic acid content of the oil was negatively correlated with the mean daily temperature during the period from 5 to 95% of final oil content (r 2, − 0.59) and also with mean daily PAR during this period (r 2, − 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 temperature and PAR were weaker (r 2, −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 (r 2, − 0.78, Fig. 4c) and mean daily PAR (r 2, − 0.83) during this period. The
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time between anthesis (estimated from the sampling date when plants were producing new flowers) and 95% of final oil content ranged from 37 to 49 days (Table 1).
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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 primary 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-linolenic contents were similar throughout the plant. In cv. Peter the upper primary branches contributed 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. Similarly, 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 significant (P, 0.052) for the upper primary branches.
4. Discussion
Fig. 4. The relationship in evening primrose cv. Merlin between (a) final oil content of the seeds and mean daily temperature during the period from 5 to 95% of final oil content, (b) final oil content of the seeds and mean daily incident photosynthetically-active radiation during the period from 5 to 80% of final oil content and (c) final g-linolenic acid (GLA) content of the oil and mean daily temperature during the period from 85 to 95% of final oil content of the seeds. Oil and g-linolenic acid contents were estimated from Gompertz growth model functions. Closed symbols, overwintered crops; open symbols: spring crops; circles, year 1; squares, year 2; triangles, year 3.
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 acceptable to the market. Seeds from overwintered crops tended to contain more oil, but with a lower g-linolenic acid content, than seeds from springsown crops (Table 1). The use of improved cultivars 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 and g-linolenic acid content can occur between years.
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Seed sourcea
WM-M B U
Year 1b
Year 2b
% of yield
Oil (%)
C18:3v6(%)
TSW (g)
% of yield
Oil (%)
C18:3v6(%)
TSW (g)
% of yield
Oil (%)
79 9 12
25.0a 24.7a 21.5b
8.09a 8.41a 7.46b
0.360a 0.323a 0.323a
68 21 11
28.1a 27.3a 27.3a
8.70a 8.70a 8.62a
0.358a 0.350a 0.293b
94 0 6
26.3a
0.257 0.027
0.018 0.130
0.139 0.797
0.019 0.026
7.95a 8.26a 7.58a
0.468a 0.413a 0.396a
0.294 0.147
0.032 0.146
9.54a
0.339a
SED P-value
1.166 0.044
WP-M B U
0.458 0.194 22 2 76
SED P-value SM-M B U SED P-value LM-M B U SED P-value
Year 3b
24.0a 23.0a 25.5b 0.497 0.007
54 32 14
23.6a 22.4b 21.6b 0.386 0.005
9.41a 9.38a 9.46a
0.386a 0.375a 0.340b
0.085 0.640
0.010 0.011
94 0 6
24.7a 23.2b 0.279 0.013
9.52a
0.316a
0.105 0.843
0.039 0.356
26.5a 0.504 0.692
65 23 11
27.5a 26.5a 25.9a 0.647 0.117
72 14 14
23.1a 23.9a 22.2a 0.958 0.305
C18:3v6(%) 9.03a
TSW (g) 0.348a
8.99a
0.301b
0.119 0.789
0.016 0.011
8.88a 9.34a 9.24a
0.387a 0.367ab 0.320b
0.155 0.056
0.020 0.043
9.85a 10.19b 10.27b
0.313a 0.266a 0.241a
0.100 0.012
0.023 0.052
a Seed 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. b Data bearing the same letter(s) within a column and year are not significantly different at P, 0.05. SED is the standard error of the difference between means for a one-way ANOVA for each year.
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Table 2 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
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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 Stem type
Capsule colour
Oil (%)
C18:3v6(%)
TSW (g)
Main
Brown Green
24.90a 21.45b
9.33a 9.54a
0.390a 0.380a
0.692 0.016
0.134 0.211
0.012 0.459
22.72a 22.04a
9.35a 9.37a
0.380a 0.371a
0.866 0.493
0.107 0.864
0.006 0.212
21.28a 21.53a
9.09a 9.57a
0.318a 0.353a
0.760 0.761
0.155 0.052
0.031 0.341
SED P-value Basal primary branches
Brown Green
SED P-value Upper primary branches
Brown Green
SED P-value
a Data bearing the same letter(s) within a column and year are not significantly different at P, 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 percentage of a-linolenic acid (Fig. 1). This percentage declines as the percentage of g-linolenic acid increases, 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, expressed 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 membrane 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 seeds g-linolenic acid is incorporated mainly into triacylglycerols.
When comparing analyses of seed oils from plants growing in the wild at different geographical locations (e.g. Lotti et al., 1978), it is very difficult to separate temperature effects from those of daylength, PAR, soil type and other biotic influences. Interpretation of results from a single location and/or relatively stress-free environments, such as field crops, can be easier, but temperature and PAR effects remain difficult to separate. Our data showed a positive relationship between final seed oil content in evening primrose and mean daily temperature during seed filling (Fig. 4a). The existence of such a relationship would be explained if the synthesis of energy-rich lipids was favoured over other biochemical processes by higher, but not extreme, temperatures. Alternatively, it may be that the amount of incident PAR during oil accumulation (particularly the period of rapid, almost linear increase in oil content), rather than temperature, is the major determinant of final seed oil content. The basis of such a relationship could be simple: higher incident PAR should result in more assimilate being available to be partitioned into storage compounds, i.e. oil. If this is the case, it might be expected that the seeds would be larger and a positive correlation between oil content and TSW
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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 relatively quickly (Fig. 1) and it is surprising, therefore, that temperatures during the earlier part of the seed filling period appear to have little influence 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 percentage 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 subsequently. 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 differences 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-linolenic acid contents present within a seed stock will be reduced by the cleaning process as small seeds from the later-formed capsules are removed (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 extraction 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 analysis.
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A.F. Fieldsend, J.I.L. Morison / Industrial Crops and Products 12 (2000) 137–147 delle concimazioni sull’olio di semi di Oenothera biennis L., La Rivista della Societa` Italiana di Scienza dell. Alimentazione 7, 361 – 368. Mukherjee, K.D., Kiewitt, I., 1987. Formation of g-linolenic acid in the higher plant evening primrose (Oeothera biennis L.). J. Agric. Food Chem. 35, 1009–1012. Reiner, H., Marquard, R., 1988. Investigations in cultivation
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