Made in the United States of America Reprinted from FOREST SCIENCE Vol. 21, No.1, March 1975 pp. 63-67
Clonal Variation in Monoterpene Hydrocarbons
of Vapors of Douglas ..fir Foliage
M. A. RADWAN­
W. D. ELLIS
Abstract.
Monoterpene hydrocarbons in foliage vapors from four Douglas-fir clones,
two resistant and two susceptible to deer browsing, were compared by gas chromato­
graphic analyses. Foliage was collected during the winter. Many chemical components
were separated, but only 10 monoterpenes were identified. Different methods of sample
preparation and vapor collection affected total volatiles and peak ratios,but with all
methods tested foliage resistant to browsing produced more terpenes than that from
susceptible clones.
Results may have application' in breeding Douglas-fir resistant to
.
browsing. . Forest Sci. 21 :63-67.
Additional key words. Pseudotsuga menziesii, animal damage, genetic variation.
INHERENT differences in susceptibility to
browsing have long been observed among
different seed sources of individual conifer
species (Bates 1927, Squillace and Silen
1962). Such differences recently have been
demonstrated among Douglas-fir (Pseudo­
tsuga menziesii (Mirb.) Franco) genotypes
(Dimock 1974).
Studies of chemical factors affecting
relative susceptibility (or resistance) are
necessary for a better understanding of
plant-animal relationships and for devising
methods of alleviating animal damage to
forest trees. Previously, we reported differ­
ences in digestibility and chemical com­
position of Douglas-fir foliage with varying
susceptibility to browsing (Radwan 1972).
We did not differentially compare volatiles
which could be important in determining
palatability (Longhurst et at. 1968).
The present investigation deals with
Douglas-fir volatiles which may influence
olfaction and, hence, initial selection by
animals. We used four clones previously
rated for susceptibility to browsing by
black-tailed deer (Odocoileus hemionus
We sampled
columbianus Richardson).
foliage during winter to minimize metabolic
changes and compared monoterpene hydro­
carbons in foliage vapors by gas liquid
chromatography (GLC). Because methods
of processing foliage may influence results
(Flath et at. 1969), we experimented with
cut and uncut tissue and collected vapor in
presence of air or nitrogen.
Materials and Methods
Foliage was obtained from 15-yr-old grafted
Douglas-fir trees grown at a seed orchard
'
in western Washington. Both susceptible
(SD-19 and SD lO) and resistant (SD-22
and SD-13) clones, as determined during
winter with tree cuttings and captive deer,
were used. Average difference in relative
deer preference between susceptible and
resistant clones was approximately 44 per­
cent (adapted from Dimock 1974). Peri­
odically, 100-g composite samples were
collected from each clone in e rly morning
during the 1972-73 dormant season. Each
sample consisted of 5-cm tips of secondary
laterals cut from all sides of five trees,
about 1.5 m above ground. Samples were
individually sealed in precooled jars and
brought to the laboratory. There were 10
sample collections during the season.
The authors are, respectively, Principal Plant
Physiologist and Chemist, Pacific Northwest For­
est and Range' Experiment StatiOIi , USDA Forest
Service,
Olympia,
Wash.
p
Manuscri t
received
March 27, 1974.
volume 21, number 1, 1975
/63
In the laboratory, 15- and 25-g sub­
samples were take'n for vapor collection
from cut and uncut tissue, respectively.
Because it was not possible to complete all
analyses of each sampling the same day,
the 15-g subsamples were stored at -15°C
until the following day when they were
thawed and cut into small pieces prior to
vapor 'collection.
Vapors were collected in 200-ml flasks
fitted with ground-glass stoppers having
outlet stopcocks .and straight tips sealed
with serum caps. Tissue (fresh, uncut, or
thawed and cut) was introduced in the
flasks and allowed to equilibrate with its
vapors at 35°C for 1. 5 hours. At alternate
sampling dates, samples were gassed with
nitrogen prior to vapor collection to mini­
mize enzymatic reactions. Thus, for each
clone, vapors were collected from uncut
and cut tissues, each with and without
nitrogen; each of these four sample prepa­
ration and vapor collection methods was
represented by five vapor samples.
Vapors for OLC were withdrawn from
the flasks with a gas syringe, and analyses
were carried out with a gas chromatograph
equipped with flame ionization detectors
and two capillary, stainless steel columns.
Columns were 61 m X 0.05 cm (ID) and
each coated with either a mixture of 95
percent Carbowax 20M plus 5 percent Ige­
pal CO-880 or 95 percent SF-96(50) plus
5 percent Igepal CO-880. Operating con­
ditions were: injection port, 150°C; detec­
tor, 250°C; column, isothermal at 70°C;
and He flow, 5 ml/min. Vapor samples
were 0.25 ml from cut tissue with direct
injection on the column and 8 ml from
uncut tissue introduced on the column by
a modification of the precolumn technique
of Maarse and Kepner (1970).
Vapor components were identified by
comparison of their retention data on the
two columns and infrared spectra' with
those of known compounds and by peak
enrichment. Quantitative data were ex­
pressed as peak heights, because peaks
were very sharp and because differences
rather than absolute amounts were impor­
tant (Maarse and Kepner 1970). Data
were calculated on per-gram basis, and
total terpenes were obtained by summing
64 /Forest
Science
all values (cm/g) including those of un­
knowns.
Because samples were collected at differ­
ent times during winter, there were no true
replications and, thus, results were not
statistically analyzed. This condition was
necessary because emphasis was on fresh
samples; the number of such samples which
could be analyzed shortly after collection
was limited.
Results and Discussion
Approximately 28 peaks were separated,
but many were present in small or trace
amounts. Monoterpenes identified were
a-pinene, camphene, ,B-pinene, sabinene, 3­
cal'ene, myrcene, limonene, ,B-phellandrene,
y-terpinene, and terpinolene. Heating sam­
ples to 35°C increased concentration of
volatiles in the vapors. This temperature
apparently did not produce detectable arti­
facts or changes in relative amounts of the
components because similar chromato­
graphic patterns were obtained when vapor
was collected at room temperature.
Quantitative data (Tables 1 and 2) show
wide ranges within clones for both vapor
components and total terpenes due to col­
lection of samples at different times during
winter. Within samplings, however, trends
of differences among clones seldom varied
from those shown by the means.
For all clones, cutting and nitrogen,
separately and together, increased emis­
sions of volatiles (Tables 1 and 2). Aver­
age increases due to cutting were much
greater than those caused by nitrogen. In
addition, with both processing methods,
components contributed unequally to total
increase in volatiles, and this changed many
peak ratios. Cutting and nitrogen, however,
produced different changes, and resulting
ratios also varied by clone.
Increases in volatiles by cutting were
probably due to rupture of resin ducts and
escape of volatiles via cut surfaces rather
than through the more restrictive normal
release mechanisms of intact tissues. This
view is supported by effects of physical
structure of fruit samples on results of
vapor analysis (Flath et al. 1969). Nitro­
gen, on the other hand, could also have an
injurious but subtle effect on tissue. Ras­
TABLE 1.
Monoterpenes of vapors from whole foliage of Douglas-fir.n
Peak height (cm/g)·
Clone SD-19
Component"
Range
Mean
Clone SD-22
Clone SD-1O
Range
Mean
Clone SD-13
Range
Mean
Range
Mean
130-202
162
219-296
250
A. In presence of air
a-pinene
Camphene
/:l-pinene
123-163
4-9
128-170
135
7
150
Sabinene
12-49
33
,1,-3-carene
41-68
51
Myrcene
1-18
6
Limonene
8-12
10
42-52
47
1-3
2
37-50
6-50
100-120
6-11
8
4-15
8
275
166-243
198
12-63
42
. 22-169
114
48-64
57
60-133
98
42
227-339
33
109
1-10
4
1-3
2-4
3
10-18
2
1-68
27
13
6-15
11
/:l-phellandrene
2-6
4
2-6
4
4-8
6
5-16
10
'Y-terpinene
2-10
5
2-10
6
3-9
6
6-32
20
Terpinolene
4-8
6
Total terpenes
372-484
430
a-pinene
114-232
173
6-10
269-303
6-10
8
280
521-720
8
16-31
24
610
679-974
805
166
206-410
266
B. In presence of nitrogen
Camphene
5-13
/:l-pinene
102-262
8
43-80
2-4
173
30-74
Sabinene
44-59
53
42-96
,1,c3-carene
18-84
54
83-187
Myrcene
14-26
19
1-65
Limonene
12-20
14
66
152-208
3
6--12
9
55
162-38
269
162-406
222
5-13
8
74
27-106
56
63-296
153
142
24-106
79
62-248
131
26
2-29
12
1-13
6
3-7
5
13-24
18
12-20
15
14
/:l-phellandrene
4-8
6
4-10
6
5-8
6
11-22
'Y-terpinene
2-13
6
2-17
6
2-11
6
8-36
23
Terpinolene
6-13
8
12-20
14
7-14
11
21-48
29
Total terpenes
685-1005
800
507-740
671
688-1116
924
874-1780
1126
• Year-old foliage sampled during winter.
SD-19 and SD-I0 susceptible to browsing, SD-22 and SD-13
resistant.
I> S eparated on a capillary column. Total terpenes include all unknowns .
• Figures, rounded to nearest cm, represent five composite samples.
mussen (1970) obtained more volatiles
from foliage disturbed by wind than from
undisturbed samples.
Whether changes by cutting or nitrogen
were due to release of compounds already
contained in tissue or were a result of
enzymatic reactions, isomerization, and
other factors after processing cannot be
ascertained. However, injury of plant tis­
sue has been shown to produce artifacts
(Major et al. 1963).
There were no qualitative differences
among volatiles of different clones. This
finding is consistent with results of studies
on effects of variety on volatiles from fruits
and seeds (Flath et al. 1969, Hougen et al.
1971) .
Quantitative differences between clones
were apparent in some components. For
example, vapors of SD-lO, SD-13, and
SD-22 were highest in 3-carene, sabinene,
and ,B-pinene, respectively. However, data
did not indicate specific components to
distinguish resistant from susceptible clones.
Rather, in all analyses, the major compo­
nents together were the deciding factor.
These compounds were emitted in various
amounts and, hence, materially contributed
to differences in total volatiles produced by
foliage of different browsing susceptibility.
Thus, with all methods of sample prepara­
tion and vapor collection tested, mean
values of total volatiles. emitted by SD-13
or SD-22 were consistently greater than
those from SD-19 or SD-lO (Tables 1 and
2). Foliage resistant to browsing produced
more terpenes than that from susceptible
clones. This result supports unpublished
volllme 21, /lllmber 1, 1975
/65
TABLE 2.
Monoterpenes of vapors from cut foliage of Douglas-fir."
Peak height (em/g)'
Clone SD-19
Co mp onentb
Range
Mean
1908-4243
3315
Clone SD-IO
Range
Clone SD-22
Mean
Range
Clone SD-13
Mean
Range
Mean
3234
2207-3624
3025
A. In presence of air
a-pinene
Camphene
240-602
433
1290-2708
9 1- 197
1923
1866-4477
146
/1-pinene
2 1 1 1-4946
4008
922-3006
1937
Sabinene
2940
123-330
222
2665-7931
4933
2537-4456
3633
848-2356
1566
2601-4541
3578
59-117
88
442-1247
885
704-4520
<l"3-carene
59-197
132
197-757
489
101-384
232
165-485
3 12
Myrcene
59- 171
128
64-224
152
9 1-320
193
96-240
Limonene
43-160
108
16-69
46
80-288
171
53- 117
170
88
/1-phellandrene
27-85
61
27-85
61
37-155
94
5-11
8
1-48
31
1 1-27
17
240
'Y-terpinene
T erpinolene
48-202
129
Total terpenes
5207-11886 9511
a-pinene
3219-4307
202-778
558
4403-12648 8500
117-373
6 140- 12499 11179
48-107
2 1-37
79
31
320-773
542 8299-14722 11786 B. In presence of nitrogen
Camphene
426-613
3850
519
1375-2558
80-187
2121
3 134-6396
192-565
157
4234
313
/1-pinene
3347-6865
4737
1482-2878
2262
4626-14071 7424
Sabinene
757-1364
1106
2110-4307
3462
A-3-carenC'
160-298
218
373-837
632
1258-3006
256-554
Myi-cene
144-256
123-224
183
187-314
Limonene
117-245
177
164
37-80
60
/1-phellandrene
Total terpenes
85-139
3283-6396
3262-6396
224-730
245
96-192
126
109
93
43-10 1
75
91-144
113
11-43
31
1-27
17
85-165
32-64
277
560-922
9205-14631 11478
432-890
197-352
670
6353-12062 10069
4 16
181-293
18
196
4430
4290
176-378
1-53
107-261
3902
1 12
247
228
69-133
'Y-terpinene
Terpinolene
1942
399
3070-4946
10537-25691 15550
42
699
11422-21245 14943
• Year-old foliage sampled during winter.
SD-19 and SD-I0 susceptible to browsing, SD-22 and SD-13
resistant.
b Separated on a capillary column.
Total terpenes include all unknowns.
• Figures, rounded to nearest em, represent five composite samples.
data we obtained with the same clones dur­
ing 1971-72 winter using a packed chro­
matography column.
Conclusions
Results demonstrate the usefulness of vapor
analysis in contrasting Douglas-fir of dif­
ferent browsing susceptibility. Although
methods of sample preparation and vapor
collection produced different vapor profiles,
all methods were about equal in distinguish­
ing resistant from susceptible foliage by
total volatiles emitted. Any of the methods
tested could be used to evaluate relative
susceptibility of foliage to browsing. How­
ever, work with fresh whole tissue and
vapor collection in the presence of air
should be preferred, if only to avoid the
66 /Forest Science
possibility of artifact formation and to
obtain vapor profiles more closely approxi­
mating those of the trees.
Prospects for a program of breeding
Douglas-fir resistant to browsing based on
indirect selection are enhanced by results
obtained and by heritability of terpene
level and composition in conifers (Hanover
1966, Squillace 1971, von Rudloff 1972).
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Varietal differences. J
Forest 25:610.
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How likely and how soon?
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Reproduced by ti,e FOREST SERViCE U. S. Department of Agriculture For Official Use About this file: This file was created by scanning the printed publication. Some mistakes introduced by scanning may remain.
\'oitllll1' 21. /lilli/be/' 1, 1975 / 67