Reproduced by permission fl'om the CANADIAN JOURNAL OF FOREST RESEARCH
Vol. 5,
No.
Depart
nt of Agr.icu1ture, for official use.
2, June 1975,
pages 281-284,
by the FOREST SERVICE,
U.S.
281
Genotype and Season Influence Chlorogenic Acid Content in Douglas-fir Foliage M. A. RADWAN
Forestry Scie//ces LlI!J,)t'atory, Pacijic Northwest Forestlill" Rlilige Experime//t Statio//,
U.S. Forest Sen'ice, OIYlllpill, Washing tOil
Received October 30. 1974
Accepted February 4, 1975
RADWAN, M, A. 1975. Genotype and season influence chlorogenic acid content in Douglas-fir
foliage. Can. J. For. Res. S. 281-284.
Chlorogenic acid content was determined in foliage of six grafted clones and seven F, full-sib
progenies at three ditTerent times duJing 1971. Detectable amounts of the compound were found
in all foliage tissues examined. but levels varied among clones. among progenic , and by season of
sample collection. Chlorogenic acid levels in the progeny appeared to be inherited and influenced
by both parents. However. average content was substantially higher in the parents than in
progenies, reflecting a possible age effect. Results corroborate previous findings with respect to
genotypic differences of chlorogenic acid in Douglas-fir foliage and its positive association with
susceptibility to deer browsing.
RADWAN. M. A. 1975. Genotype and season influence chlorogenic acid content in Douglas-fir
foliage. Can. J. For. Res. 5. 281-284.
On a mesure la teneur en acide chlorogenique du feuillage de six clones greffes et de sept
descendances biparentales de Douglas, 11 trois periodes au coms de 1971. On a trouve des
quantites decelables du compose. dans tOllS les tissus foliaires examines, mais Ie, teneurs var­
iaient entre les clones. entre les provenances. et selon la periode d'echantillonnage. Les niveaux
d'acide chlorogenique des descendants semblent etre hereditaires et influences par les deux
parents. Cependant. Ie contenu moyen etait substantiellement plus eleve chez les parents que
chez les descendants. refletant un effet possible de i'age. Les resultats corroborent les ob­
servations signa lees anterieurement sur les differences genotypiques de la teneur en acide
chlorogenique du feuillage du Douglas et son association positive avec la susceptibilite au
broutage par Ie cerf.
[Traduit par Ie journal]
Introduction Chlorogenic acid (CGA) is widely dis­
tributed among angiosperms (2, 6, 17), In
these plants, many studies have shown that
concentrations of the compound differ with
various plant and environmental factors (1, 4,
10,14,20,21). Further, other investigations
have suggested important roles by CGA in
several physiological functions of plants (5,
12, 18, 22).
There is little published information about
CGA in gymnosperms. Early reports indicate
that the compound occurs in only two genera,
Podocarpus and Gnetlll7l (6, 8). Recently,
using a non-specific assay method, Habermann
(7) reported a 'chlorogenie acid value' for only
one species (Tsuga canadensis) out of six species belonging to three conifer families, However, in 1972 I had positively identified
CGA in the foliage of one conifer, Douglas-fir
(Pseudotsuga menziesii) (13). I also reported
an association of higher concentrations of eGA
Can. J. For. Res., 5, 2St (1975)
with greater susceptibility of different clones of
Douglas-fir to deer browsing, and suggested
that eGA content may be used, instead of con­
ventional bioassays, to screen Douglas-fir
breeding stock for resistance to deer browsing.
This paper is a further investigation of eGA
in foliage of Douglas-fir designed to compare
CGA content in different clones and progenies
at different times during the year, and to esti­
mate whether levels of the compound are
heritable.
Materials and Methods
Plant Material
Foliage samples were collected from six different
grafted 910nes and seven f ull-sib F, progenies ob­
tained by controlled matings among the clones. Clones
and progenies were randomly replicated with other
trees of the same age class in two separate but
adjacent plantations at the Olympic National Forest's
Dennie Ahl Seed Orchard in western Washington. At
time uf sampling in 1971, parent and progeny trees
were 14 and 6 years old, respectively. Four of the
six clones used here had been analyzed earlier
these were again assayed for comparison.
(13);
282
CAN. J. FOR. RES. VOL. 5. 1975
TABl.E I.
Concenlration of chlorogenic acid in foliage of
dill'erent clones and p rog e n i es of Douglas-fir at three
dill'crent sampling dates during 197,1
Clone or
family
number"
Clones
Chlorogenic acid,
July"
732
Sep tember"
527
372
284
546
SD-IO
SD-8
SD-22
172
209
108
SD-13
Average"
89
349 x
65
261 y
Progenies
10 x 2
10 x 8
90
51
8
64
22 x 8
8 x 22
13 x 8
43
37
27
13 x 2
Average"
41
50 x
19
x
dry weight
December"
599
461
291
261
SD-19
SD-2
274
280
Ilg/g
101
76
619 a
460
283
250
137
76
131
75
303
99 a
70 b
83
77
71
53
52
38
62
66
46
54
71
b
c
d
e
f
z
106
73
54
53
40
50
64 y
Average"
b
c
c
d
48 c
z
'Numbers arc those assigned clones and progenies at the U.S. Forest
Service Seed Orchard. Shelton. Washington.
bValues are means of two composite samples.
'Within each of the two sections of the table, averages followed by the
same Jetter designation, a through d or f in vertical sequence, or x through
z in horizontal sequence. are not significantly dilIerent (P = 0.05) by Tukey's
test.
Sampling and Design
trations in the tissues were calculated on a dry-weight
In July, September, and December two composite
100-g samples were collected in early morning from
each genotype. Each sample was taken from five
basis using two replicates with three chromatograms
per replicate. Data were subjected to analysis of
trees selected at random from available replicates,
and the same trees were used at all three sampling
dates.
Samples,
taken from current year's growth,
consisted of 5-cm tips of secondary laterals cut from
all sides of the trees. Parent and progeny trees were
sampled at heights of 1.5 and 0.6 m. respectively.
Samples were individually sealed in glass containers
and brought to the laboratory in a portable cooler.
The experiment, therefore, had a randomized
block, split-plot design, with plantations at the
orchard, genotypes, and months as blocks, whole
plots, and split plots, respectively.
Chemical Analysis
In the laboratory, subsamples were taken for mois­
ture determination. Remaining tissue was extracted
as outlined earlier (13). Due to the presence of large
amounts of extraneous matter, extracts were purified
before chromatography. Purification consisted of pre­
cipitation of phenols with lead acetate followed by
regeneration with H,S. Solutions were then filtered
and filtiates evaporated to dryness. Phenolic com­
pounds in the residues were taken up in 80%
methanol, and resulting solutions were used for paper
chromatography. Separations of phenolics present,
and detection, identification, and spectrophotometric
quantification of eGA followed standard procedures
as previously outlined (13). Average CGA concen­
variance,
and
means were separated according to
Tukey's test (16). Correlation coefficient (r) between
midparent and progeny CGA average values was
also calculated to estimate effect of parents on CGA
content of the progeny.
Results and Discussion
Detectable amounts of CGA were found in
all foliage tissues examined (Table O. Amounts,
however, varied significantly (P = 0.05) among
clones, among progenies, and by season of
sample collection.
Clones versus Progenies
ChI orogenic acid in each progeny was much
less than that of either parent. Average CGA
level, therefore, was substantially higher in
clones (304/1g/g) than in progenies (62 flg/g).
This contrast cannot be attributed to factors
already known to influence CGA concentrations
in plants, such as environmental conditions (1,
20), or to tissue age (4, 10, 15); all trees were
grown under the same environ mental condi­
tions, and foliage tissues of both clones and
progenies were of approximately the same age
RADWAN: CHLOROGENIC ACID IN DOllGLAS-FIR
at each of the sampling dates. Similarly, method
of propagation (vegdative in clones· verslis
sccd in progcllies) cannot account for the Jif­
fcrcncc since our unpublished data with seed­
propagated parents and opcn-poliinated prog­
eny show thc same difTerence between the
two groups of trees as that reported here. Dif­
fercnces between clones ancl progenks in eGA
content, thereforc, suggest a tree-age efIect­
older trees (clones) appear to have grenter
ability to synthesize CGA than younger trees
(progeny). The difference also corroborates
my previous conclusion with respect to positive
association of CGA with susceptibility to
browsing ( 13). During the winter, recent bio­
assay tests with penned black-tailccl deer
showed that year-old foliage of the older clones
was much more susceptible to browsing than
that from the younger progeny trees (3).
Genotype Effects
Averaged over all sampling dates, CGA
content varied significantly (P = 0.05) among
all six clones. In general, concentrations were
highest in SD-19 and SD-2, intermediate in
SD-IO and SD-8, and lowest in SD-22 and
SD-13. Variations also occurred among prog­
enies but were less pronounced than among
clones, possibly as a result of the younger aged
progenies' limited ability to synthesize the com­
pound. Among the seven progenies, highest
CGA levels were in lOx 2, followed by lOx 8
and 19 X 8, then 22 X 8, 8 X 22, and 13 X 2,
and finally 13 X 8.
Within each of the sampling months, there
were significant (P = 0.05) differences in CGA
level among clones and between progenies.
Those differences, however, wcre not consistcnt
and, consequently, ranking according to CGA
contcnt changed some from one sampling date
to the next, especially among progenies.
On dry-weight basis, the December data re­
ported hcre for clones SD-10, SD- 13, SD- 19,
and SD-22 are lower than those rcported
earlier for the same clones ( 13). Changes in
environmental conditions, metabolic demands,
or methods of preparing extracts for analysis
may explain this discrcpancy. Howevcr, it is
interesting to note that both sets of data pro­
duced exactly the same ranking for the four
cloncs.
Variations of CGA with genotype in Douglas-
283
fir parallel earlier observations with cultivars
of herbaceous dicots (9, I I, 14). Clearly, CGA
content is among the chcmical traits which
show genetic vari tioll in plants.
Scason Effccts
Averages of all clones and all progenies ovcr
each of the sampling dates show that in each
of the two groups of trccs, eGA content varied
according to month of foliage collcction. With·
only one exception, average concentrations of
eGA in the clones were highest in July, inter­
mediate in December, and lowest in Septcmber.
In the progenies, on the other hand, CGA
incrcased continuously from July until the final
sampling in December. Again, thesc difTerent
trends probably resultcd from differcnces in
age and, consequcntly, differences in rates of
growth and mctabolic activities between the
two groups of trces.
Seasonal variations in CGA in foliage of
Douglas-fir were expected. Changes with time
during growth (4) and even diurnal fluctua­
tions ( 19) have been rcported in other species.
Clearly, as with other plants, CGA is subject
to much metabolic turnover in Douglas-fir
foliage.
Parent Influence
Chlorogenic acid in the progenies was
affected by levcls of the compound in the parent
trees. The correlation coefficient (r) bctween
midparent clnd progcny averagc CGA values
W3S 0.98, significant at the 0.0 1 level.
In progenies resulting from crosses of dif­
ferent maternal trees with the same paternal
parent, CGA levels were generally related to
its concentration in the maternal parents. Thus,
higher levels of the compound wcre consistently
found in lOx 8 and 19 X 8 than in 22 X 8
and 13 x 8, and in lOx 2 than in 1 3 X 2.
Similarly, in crosses of the same female parent
with differcnt pollen trces, concentrations varied
according to lcvels in the paternal parents. For
example, eGA contents of lOx 2 and 13 X 2
were always higher than those in lOx 8 and
13 X 8, respectivcly. Chlorogenic acid levels
in foliage of the progenies studied, therefore,
varicd in patterns suggesting inlluence by both
parents. Thc parcnt-progeny data, howcvcr,
do not clearly indicate relative influence by
each parent. Thus, whereas similar CGA levels
in 22 X 8 and the rcciprocal cross 8 X 22
284
CAN. J. FOR. RES. VOL. 5, 1975
suggest cqual effects by the parents. the small
differences between lOx 8 and 19 x 8 may
indicate a low maternal parent effect.
Because of the small number of genotypes
used in this study. additional work with a
larger number of parents and a more complete
mating scheme would be required to strengthen
the parent-progeny relationships suggested by
the data,
Conclusions
Results from this study substantiate earlier
findings (13) with respect to genotypic differ­
ences of chlorogenic acid in Douglas-fir foliage
and its positive association with susceptibility
to deer browsing. Further, the data, indicating
that levels of the compound were heritable,
enhance prospects of using eGA content as an
indirect indicator of resistance to select desired
trees in breeding Douglas-fir for resistance to
browsing. Demonstrated differcnees due to tree
age and seasonal fluctuations in eGA content,
however, suggest that comparative analyses
should be conducted among trees of similar
age and preferably during the dormant season.
Similarly, the biparent influence on eGA con­
centration in the progeny shown here implies
that breeding programs should give equal con­
sideration to parents of both sexes. Also,
initiation of such programs should probably
await additional information on eGA in
Douglas-fir and its relation to browsing against
different physiological backgrounds, especially
when the trees are young and most vulnerable
to damage by animals.
Whether deer select directly for high levels
of eGA in Douglas-fir or use other associated
compound(s) remains to be determined. Al­
though such information would be interesting
and useful, it is not essential to further bio­
chemical studies to develop the compound as
an indicator of resistance to browsing, and
hopefully, its use in practical programs to
alleviate deer damage on Douglas-fir.
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