186
Genetics of Graft Rejection in Douglas-fir
DONALD L. COPES1
Pacific Nol'/hll'est Forest and Rangl! E.\'periment Station, U.S.D.A. Forl!sl Sen·ice, Port/am/, Orl!gon97208
Received September 17, 1973
Accepted January 8, 1974
CoPES, D. L. 1974. Genetics of graft rejection in Douglas-fir. Can. J. For. Res. 4,
186-!92.
Graft rejection in Douglas-fir was controlled primarily by additive genes. Heritability
of graft incompatibility was 0.81. No significance for specific combining ability was
found. A breeding program to develop highly compatible stocks should enable tree im­
provement workers to substantially reduce incompatibility losses within clonal orchards.
Highly incompatible clones may have different or additional rejection factors which less
incompatible clones do not exhibit.
CoPES, D. L. 1974. Genetics of graft rejection in Douglas-fir. Can. J. For. Res. 4,
186-192.
Le rejet des greffes chez Ps£•udotsuga menziesii fut surtout dO a !'action de genes addi­
tifs, et l'heritabilite de cette incompatibilite se chiffra a 0.81. On obtint aucun resultat
significatif quant a Ia capacite specifique d'union. Un programme d'amelioration visant
a developper des porte-greffes fortement compatibles devrait permettre aux ameliorateurs
forestiers de diminuer les pertes dans les pares a clones, dues a cette incompatibilite. Les
clones fortement incompatibles peuvent montrer certains signes de rejet differents ou
ajoutes, lesquels passent inapergus chez les clones plus compatibles.
[Traduit par le journal]
Introduction
Breeding programs to develop highly com­
patible stocks for clonal Douglas-fir (Pseudo­
tsuga menziesii [Mirb.] Franco) seed orchards
are needed, but some knowledge of the type of
inheritance involved in tissue rejection is re­
quired before an efficient program can be ini­
tiated. Although graft rejection in plants has
been studied for hundreds of years, the under­
lying mechanisms are still poorly understood.
Horticulturists have indicated that close genetic
relationship between stock and scion generally
increased the likelihood of compatible unions
(Bregger 1948, Grasselly 1968, Kester et al.
1965). Incompatibility factors in almonds were
transferred from parent to offspring (Kester
1970). In this case, both quantitative and
qualitative inheritance were evident.
Graft rejection mechanisms in forest conifers
are even less well understood, although the
transfer of incompatibility factors from parent
to progeny appears to occur. Some gain in
1Present address: Forestry Sciences Laboratory,
Pacific Northwest Forest and Range Experiment Sta­
tion, U.S.D.A. Forest Service, Corvallis, Oregon
97331.
Can. J. For. Res., 4, 186(1974)
compatibility resulted from grafting parental
clones on related stocks in Pinus taeda (Lantz
1970), P. caribaea (Slee and Spidy 1970),
and Pseudotsuga menziesii (Copes 1973).
Additive gene action was thought to be the
primary type of inheritance present in Douglas­
fir graft rejection, but one of the 12 clones stu­
died was found to deviate greatly from the
expected response. The incidence of incom­
patibility was more than twice as great when
grafted on related stocks as when grafted on
unrelated stocks (Copes 1973).
No previous compatibility study has yielded
accurate estimates of additive and nonadditive
gene effects nor have any estimates of herit­
ability been made. The following grafting study
utilized scions from six Douglas-fir parents and
their progeny to obtain the estimates for gen­
eral and specific combining ability and for the
heritability of graft rejection.
Methods
Early in 1964, six 20- to 25-year-old Douglas-fir
parent trees were selected from a native population
in the western foothills of the Oregon Cascade
Range. Parent trees were separated from each other
by a minimum of 100 m and were selected solely for
187
COPES: GRAFT REJECTION IN DOUGLAS-FIR
their ability to produce seeds. In order to determine
the average incompatibility of each parent, scions
from each tree were collected in March 1968 and
cleft-grafted that same spring on the terminals of
7-year-old Douglas-fir stocks growing in a plantation
near Corvallis, Oregon. The stocks were random,
nursery-run seedlings from a low elevation Willa­
mette Valley seed source. Each parent clone was
grafted on 50-66 different stocks. In September 1969,
the grafts were severed from the stocks and the
unions preserved in 50% alcohol. The unions were
sectioned into transverse sections on a sliding micro­
tome and stained with safranin 0 and fast green
(Copes 1967). Microscope preparations were made
and examined for the presence (incompatible graft)
or absence (compatible graft) of wound-xylem areas
in the xylem tissues joining the stock and scion
(Copes 1970). A percentage incompatibility value,
based on the 50- to 66-graft sample, was then de­
termined for each parent tree (number incompatible
unions + total number unions X 100).
In April 1964, the six Douglas-fir parent trees were
control-pollinated in a 6 X 6 diallel design. A Model
I design, where the reciprocal crosses and self-polli­
nation combinations were omitted, was used (Griffing
1956). Fifteen diallel families resulted from this de­
sign. Wind-pollinated families were also collected
from each parent tree when the control-pollinated
seed was gathered in September 1964. Seedlings were
grown from 1965-1967 at Corvallis, Oregon, in cold
frames in a randomized block design. In the winter
of 1967, they were then planted within the boundaries
of the original parent stand.
Fifty scions from each diallel and wind-pollinated
family were collected in March 1970. Each scion was
cut from a different 5-year-old seedling, so no seed­
ling genotype was represented more than once in the
study. The scions were cleft grafted in April 1970 on
lateral branch tips of 50 9-year-old stocks. Grafting
was done by proceeding systematically down each
row of stock trees. Each consecutive group of 10
stocks was regarded as a block. Each stock tree held
21 different grafts on 21 of its branch tips: one graft
from each of the 15 control-pollinated families and
the six wind-pollinated families. Graft technique
failures were regrafted in April 1971. The stocks
were all part of the same plantation where the six
parents had previously been grafted. Grafts were
sacrificed 18 months after grafting for microscope
study. The staining techniques and incompatibility
detection method used for parental grafts were also
used for seedling grafts.
Heritability of graft rejection was determined
directly from the progeny-midparent regression.
Diallel analysis of control-pollinated seedling grafts
used the procedure described by Griffing as Model I,
Method 4 (Griffing 1956). The model used was as
follows:
xiJ = J.1 + g1 + g1 + siJ + (1/b) E, LJ eiJkt
where: X1J = ijth observation;
= study mean;
g1 and g1 = general combining ability (male and female); SIJ = specific combining ability; eukt = error; and
b = number blocks.
Percentage data were transformed to arcsins prior to
sum of squares analysis.
Results
Incompatibility percentages of the six parent
clones and their progenies are given in Table 1.
The mean for all parents was 38.3%, only
slightly higher than the 35% mean for a large
number of randomly selected clones. In gen­
eral, crosses of more compatible parents gave
progeny which were more likely to form com­
patible grafts than were progeny derived from
crossing more incompatible parent trees. Cor­
relation between midparent and progeny was
significant (r = 0.89), as was the regression of
progeny on midparent ( b - 0.81). This b
value is also a heritability estimate for graft
rejection by the six parents under the condi­
tions of this study. In the diallel analysis, the
effect of general combining ability (GCA) was
highly significant; the effect of specific combin­
ing ability (SCA) was nonsignificant (Table 2).
In most cases GCA ranking of parents cor­
related closely with rankings of parents by their
incompatibility values. But, the actual incom­
patibility values of the seedling grafts from
five of the six parents average 4.9% lower than
their parents. SCA values ranged from +10.9
to -15.7% and GCA values ranged from 26.7
to 39.5% (Table 1). The three largest SCA
values all came from crosses of parent 1 1.
If results from this study are to be applicable
to _seed orchard conditions, it is important that
the stocks be similar to those grafted in the
orchards. In order to determine if they were
similar, the average incompatibility of each
stock was calculated from the 2 1 unions
grafted .on each stock. Each stock value was
plotted into one of 10 incompatibility classes
and a histogram drawn (Fig. 1). The histo­
gram illustrated approximately the same distri­
bution pattern encountered in numerous past
tests from orchards when many clones were
grafted on unselected stocks. In addition, the
mean incompatibility value from this study
was approximately the same as the average
normally encountered in orchards with larger
·
......
00
00
TABLE 1.
Estimates of percentage incompatibility (IC) for each parental clone, midparents, control, and wind-pollinated families and their general (GCA) and
specific (SCA) combining abilities
Parent clone
numbers
Average
IC' of
parent
clone
Percentage incompatibility
5
56
32
58
9
35
8
27
11
29
10
25
Mean
38.3
Parent clone numbers
5
9
8
11
10
Average of
mid parents
Average of
wind-pollinated
progeny
39.5
38.3
36.5
30.6
28.8
26.7
33.4
45.4
46.2
37.0
33.8
34.7
33.0
38.3
36
43
34
33
22
33
35.3 ()
>
z
X
44
45
34
45
30
Estimate of specific combining abilityc
5
X
32
+5.1
9
+7.0
8
-1.1
II
+10.9
10
-3.1
aJc denotes incompatjbility. 32
Average of
progeny with
common
parent (GCA)"
X
41
43
40
X
37
29
23
34
X
+3.6
+8.5
+6.5
-9.5
X
14
27
X
18
X
:--
'11
0
:00
;:d
til
:"
<
0
r-
::;;
-..1
X
+3.4
-3.7
+2.4
"""
X
-15.7
-1.6
X
-9.8
bGCA denotes general combining ability (average incompatibility value of the five progeny families per parent clone). <Calculated as individual cross incompatibility values-(GCA o + GCA )/2. X
189
COPES: GRAFT REJECTION IN DOUGLAS-FIR TABLE 2. Analysis of variance for graft rejection in control-pollinated families. Percentages were
transformed to arcsins before analysis
Source of variation
Total
Blocks
Families
General combining ability
Specific combining ability
Error
5
Degrees of
freedom
74
4
(14) 5
9
56
Mean squares
Fvalues
487.225
365.093
620.253
223.337
184.437 2.64**
1.97*
3,36**
1.2"·'·
Coefficient variation 41%
25
a;
Q.
::E
0
u
i!!:
:I: u
<
w
i!!:"'
"' "'
,.,..:
u-' ou ...
"'
u.
0
"' "'
z
w
u
0:
w
..
0
0
90
10%
100
INCOMPATIBILITY CLASSES
FIG. 1. Histogram showing the distribution of 50 stocks according to their average incom­
patibility values.
samples (33.4 versus 35%, respectively).
Stocks grafted in this study were typical of the
type of stock normally grafted in seed orchards
at this time.
Preliminary inspection of the data indicated
that the relative incompatibilities of stock and
scion might affect their interaction with each
other. Stocks, therefore, were separated for
analysis into five groups of 10 trees according
to their incompatible values. The incompatibil­
ity ranges within the groups were 0-19%,
20-30%, 30-38%, 38-48%, and 48-72%.
Grafts on stocks in the. range 0-48% incom­
patibility exhibited similar inheritance patterns
-progenies of the most compatible parents
pr<'duced the most compatible grafts (Table 3,
Fig. 2). But a different response was observed
on stocks of the most incompatible group ( 48­
72% ), where progenies of the most incom­
patible parents produced the more compatible
grafts.
Variation between blocks was found to be
highly significant, but I believe this to have
been merely a chance occurrence. Examina­
tions of average compatibility of stocks within
blocks revealed that the fourth block was
responsible for the significance noted. Two of
the 10 stocks in that block were highly incom­
patible. If more stocks per block had been
used, much of the block variation would have
been removed.
Discussion
Incompatibility symptoms in Douglas-fir
(Copes 1970) are identical to the 'contact
type' described by Mosse in 1962. Fruit tree
studies (Mosse 1962) and my own work with
Douglas-fir (unpublished data) all indicate
that reciprocal grafts, in species typefied by the
contact type of incompatibility, yield identical
compatibility tests. It makes little difference
whether a clone is grafted as the stock or as
the scion. Thus, compatibility values deter­
mined in this study from grafting parental
CAN. J. FOR. RES. VOL. 4, 1974
190
TABLE 3.
Graft incompatibility of seedling scions grafted on stocks which were I (0-19%), II (20-30%), III
(30-38%), IV (38-48%), and V (48-72%) incompatible
Stock group
number
Parent
clone
nu mber
Percentage incompatible
I
5
II
III
IV
v
Average incompatibility of
progeny with common
parent (GCA)
5
9
8
11
10
X
32
9
8
11
10
30
10
0
5
32
9
8
11
10
X
44
11
60
40
30
0
22
X
5
32
9
8
11
10
50
30
70
38
50
5
32
9
8
11
10
70
78
50
60
33
5
32
9
8
11
10
32
X
X
10
70
30
30
33
X
10
11
20
11
X
33
56
67
25
X
40
22
38
33
X
50
70
50
22
X
71
56
33
30
X
11
0
0
X
10
0
14
X
40
30
44
X
30
30
30
X
90
80
80
clones and seedling families as scions can be
used directly to approximate how those same
individuals would react as stocks.
It appears possible for breeding programs to
develop highly compatible stocks which can be
used with scions from most plus trees. With
mainly additive gene effect (heritability was
0.81), considerable gains in compatibility can
be made through stringent parent selection
and cross-breeding among the best parents. A
likely area to begin this approach is in crossing
the most highly compatible orchard clones. I
started a program of this type in 1971 and
information on the resulting stock compatibility
should be known about 1975.
The most incompatible trees in the popula­
tion may have rejection factors not found in
X
0
0
X
0
13
X
0
30
X
10
30
X
67
67
X
0
X
0
X
11
X
10
X
60
(Mean= 8%)
X
18.7
14.3
6.1
2.0
12.5
4.3
(Mean= 26%)
X
40.8
48.9
19.6
20.4
23.9
15.4
(Mean= 35%)
X
47.9
37.0
36.0
32.7
22.2
34.0
(Mean = 43%)
X
58.3
53.1
42.9
38,0
32.0
25.0
(Mean = 55%)
X
34.7
37.8
78.7
61.7
55.3
54.2
less incompatible trees. Results from grafting
on stocks with known incompatibility values
suggest that rejection factors in about 80%
of the stocks (0-48% incompatible) can be
overcome by grafting on stocks grown from
seed of crosses of moderately compatible
parents.
It should be remembered that none of the
six diallel parents had extremely high com­
patibilities; the best clone was only 75%
compatible. When the 10 most incompatible
stocks were selected out of a population of 50,
the incompatibility factors of the stocks may
have exceeded the compatibility limits of the
parents. If two 95% compatible parents had
been included in the study and crossed, their
progeny may have had a broad enough com­
COPES: GRAFT REJECTION IN DOUGLAS-FIR
100
90
>t:
...J
iii
::.
0
u
!i:
..:
0:
(!)
..:
u
(!)
Gr o up
1
stocks
Group 4
Group 2 Group
3
stocks
191
Group 5 stocks
stocks
stocks
80
70
60
50
40
30
20
10
0
0
70
Group 1
moan
mean
mean
mean
80
mean
AVERAGE STOCK INCOMPATIBILITY
(%)
FIG. 2. General combining ability (GCA) of the six parents on five classes of increasingly
incompatiblt< stocks (Groups 1-V).
patibility spectrum to encompass the rejection
factors of most Douglas-fir trees.
Ortet age appeared to have an effect on
average incompatibility. Grafts from 5-year­
old ortets were uniformly lower in average
incompatibility than their 24- to 29-year-old
midparents (33.4 versus 38.3% ). A similar
case of lowered incompatibility associated with
juvenility was noted for grafts of peach on
plum stocks (Herrero and Tabuenca 1969).
It is hypothesized that Douglas-fir tissue
becomes more hypersensitive to foreign tissues
in developing from the seedling to older ju­
venile or adult stages and, as a result, exhibits
a decrease in overall graft compatibility. Ana­
tomical examinations of 5- to 10-year-old
grafts have indicated that the compatibility of
unions between old scions and young stocks do
not change as the stocks and scions grow older
(unpublished data). Thus, some chemical
adjustment or immune response between con­
tiguous stock and scion cells may occur through
the years which allows them to maintain viable
cell contact even after the noncontiguous stock
and scion cells become physiologically incom­
patible. At this later date, grafts made between
noncontiguous stock and scion tissues develop
into incompatible grafts (unpublished seed
orchard data).
There appears to be a difference between
clones in the amount of compatibility change
caused by maturation. If it can be assumed
that GCA values are fair estimates of parental
clone performance when young, it appears that
the most incompatible clones become more
incompatible with increased age than do the
less incompatible clones. A comparison of
difference between GCA and parent incom­
patibility values illustrates this point. Clones
C-5 and C-32 were the most incompatible
parents and the difference between their GCA
and parent values was much greater than the
differences for the other four less incompatible
parents and progenies.
Grafting tests of stocks grown from wind­
pollinated seeds can be used to obtain usable
GCA estimates for selecting parents for breed­
ing. But in wind-pollinated progeny, it is not
known if only one or two different trees or if
many trees contributed their genes to a ma­
jority of the progeny. Even with this limita­
tion, the wind-pollinated GCA values were all
within 7% of those obtained with diallel seed­
lings.
BREGGER, J. T. 1948. Peach variety incompatibilities on
seedlings of a Yunnan understock. Proc. Am. Soc.
Hortic. Sci. 52, 141-142.
CoPES, D. L. 1967. A simple method for detecting incom­
patibility in 2-year-old grafts of Douglas-fir. U .S.D.A.
For. Serv., Pac. Northwest For. Range Exp. Stn.,
Portland, Oreg. Res Note PNW-70.
-- 1970. Initiation and development of graft incom­
patibility symptoms in Douglas-fir. Silvae Genet. 19,
101-107.
192
CAN. J. FOR. RES. VOL. 4, !974
1973. Inheritance of graft compatibility in
Douglas-fir. Bot. Gaz. 134(1), 49-52.
GRASSELLY, C. 1968. The plums Prunus domestica root­
stocks for peach trees. Study and improvement of graft­
ing compatibility of their seedlings. (Fr.) Ann. Amer­
lior. Plantes 18(1), 59-73. In Bioi. Abstr. SO, 133870.
GRIFFING, B. 1956. Concept of general and specific com­
bining ability in relation to diallel crossing systems.
Aust. J. Bioi. Sci. 9, 463-493.
HERRERO, J., and TABUENCA, M. C. 1969. Incom­
patibiJidad entre patron e injerto. X. Comportamiento
de Ia combinacion melocotonero/mirobohin injertado
en estado cotiledonar. (Incompatibility between root­
stock and scion. X. Behavior of the combination
peach/myrobalan grafted at the cotyledonary stage.)
An. Estac. Exp. Auld Dei. 10, 937-945. In Hortic.
Abstr. 41, 5898.
KESTER, D. E. 1970. Graft-compatibility of almond seed­
ling populations to Marianna 2624 plum rootstock.
(Abstr.) Hortic. Sci., Sec. 2, 5(4), 349.
KESTER, D. E., HANSEN, C. J., and PANETSOS, C. 1965,
Effect of scion and interstock variety on incompatibil­
ity of almond on Marianna 2624 rootstock. Proc. Am.
Soc. Hortic. Sci. 86, 169-177.
LANTZ, C. W. 1970. Graft incompatibility in loblolly pine.
Ph.D. thesis, N.C. State Univ. at Raleigh. Univer.
Microfilms, Ann Arbor, Mich., Int. Diss. Abstr. 32(1),
16-B.
MossE, B. 1962. Graft incompatibility in fruit trees. Com­
monw. Bur. Hortic. Plant. Crops, Tech. Commun. 28.
SLEE, M. U., and SPIDY, T. 1970. The incidence of graft
incompatibility with related stock in Pinus caribaell
Mor. var. hondurensis B. et. G. Silvae Genet. 19(5-6),
184-187.