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
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-lino- lenic 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 con- sistent 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.
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 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 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 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 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 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 years
a
Year 95 Oil DOY
Treatment Anthesis DOY
TSW g C18:3v6
Oil G.S. 5,95DOY
Overwintered 246
24.6a 1
8.02a 0.354a
191 236
Spring-sown 275
226 0.375b
9.41b 23.0a
288 0.866
0.057 0.010
SED 0.155
B 0.001
0.032 P-value
197 0.349a
8.70b 27.8a
234 239
Overwintered 2
25.1b Overwintered P
7.68c 0.411b
197 234
239 Spring-sown
273 24.6b
9.55a 0.338a
223 267
SED 0.320
0.083 0.018
B 0.001
B 0.001
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
287 279
232 0.300b
Late spring-sown 9.94b
0.643 SED
0.187 0.013
P-value 0.002
0.004 0.004
a
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 letters 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.
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 represent 9 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.
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 represent 9 1 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 − x b]}
were fitted to the data. The rate of oil accumula- tion was at a maximum between 5 and 80 of
final oil content Fig. 2 and this rate was reason- ably constant across the overwintered cv. Merlin
treatments 0.97 – 1.15 day
− 1
but was lower in
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, spring-
sown 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.
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 dur- ing 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 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 r
2
, 0.79, Fig. 4b, but that between final oil content
and mean daily temperature during the same pe- riod 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 tempera-
ture 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 temper-
ature 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 tempera- ture 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 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
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
