Forest Sci.,
Vol.
24,
No.
2, 1978, pp. 297-303
Isoenzyme Activities Differ in Compatible
and Incompatible Douglas..fir Graft Unions
DONALD
L. COPES
ABSTRACT. Unusually dark-stained peroxidase bands at Rm (relative mobility) 0.77 to
0.
80 and esterase bands at Rm 0.50 to 0.
56 were found in tissues of incompatible Douglas­
fir graft unions. Ninety- to 100-percent accuracy in identifying incompatible grafts was
achieved with union tissues 10 to 15 months after grafting (February to May). A practical
graft testing program is possible using electrophoretic methods for detecting changes in
stain intensity in bands from incompatible graft unions. Enzyme activity of bands with
other Rm values was not correlated, or was only weakly correlated, with graft incompati­
bility. Enzyme similarity of stock and scion tissues could not be used to determine poten­
tial compatibility of stock-scion combinations before grafting. No correlation with graft
incompatibility was found with enzyme activity or the presence or absence of isoenzyme
bands of catalase, acid phosphatase, or leucine aminopeptidase.
ADDITIONAL KEY WORDS.
FoREST Scr. 24:297-303.
Vegetative propagation, Pseudotsuga menziesii.
PLANT TISSUES formed in graft unions are composed of cells similar to those found
in areas previously subjected to wounding or injury. New graft unions in conifers
are typically composed of irregularly shaped parenchyma cells (callus) and irregu­
larly shaped and oriented tracheids and phloem elements. Although the cell types in
unions and wounds are similar, one condition present in unions is very different
from that found in wounds-the contiguous contact of cells of unlike genotypes.
Altered starch metabolism patterns have already been detected in graft tissues (Mel­
nick and others 1964), but studies of enzyme activity in graft unions of conifers
have not been reported.
A good test organism for enzymatic study of graft unions should exhibit a high
degree of cellular antagonism when genetically dissimilar individuals are grafted.
Douglas-fir (Pseudotsuga menziesii [Mirb.] Franco) meets this criterion-more
than one-third of the grafts made with Douglas-fir stocks and scions of unlike geno­
types soon show visible signs of between-cell antagonism. The first visible symptom
is the development of suberized zones between some cells of the stock and scion 3 to
4 months after grafting (Copes 1970). Necrotic areas develop later in and around
the suberized areas in about 35 percent of the unions. These grafted trees ultimately
die of bark girdles and are said to have been incompatible grafts. The necrotic areas
closely resemble the zones of controlled necrosis, described by Berryman ( 1972).
Such zones occurred in plants as a result of the "hypersensitive reaction" mecha­
nism. In many cases, hypersensitive reactions are initiated as defense reactions to
pathogen attack. Graft-induced between-cell antagonism in Douglas-fir may result
from activation of its hypersensitive reaction mechanism. The incompatible reaction
may be triggered by the ability of contiguous cells of unlike genotypes to recognize
The author is principal plant geneticist, Forestry Sciences Laboratory, Pacific Northwest
Forest and Range Experiment Station, USDA Forest Service, Corvallis, Oregon.
Manuscript
received 13 October 1977.
VOLUME
24, NUMBER 2, 1978 I 297
the adjacent Douglas-fir cells as foreign bodies and, thus, to react to prevent inva­
sion. Anatomical evidence supports this hypothesis (Copes 1970).
Activation of hypersensitive systems has been correlated with elevated activity of
some oxidative enzymes and increased synthesis of phenolic compounds (Berryman
1972). If antagonism in Douglas-fir unions is caused by a hypersensitive reaction
system, then the peroxidase, or polyphenol oxidase, enzyme systems should be
examined. Legrand and others (1976) found the phenylpropanoid pathway directly
linked to hypersensitive necrosis reactions in tobacco plants. High levels of phenolic
compounds in the stocks and scions of cherry trees were correlated with graft failure
( Yu 1972). Peroxidases exert direct control over the level of polyphenols in plants
by the catabolism of phenylpropanoid compounds (Konarev 1972).
Indirect evidence for a role of oxidative enzymes upon intercellular antagonism
was seen in bean plants where high peroxidase levels precede leaf abscission (Poo­
vaiah and Rasmussen 1973). Protective layers of cells in abscission zones closely
resemble graft rejection zones found in incompatible Douglas-fir unions. Peroxi­
dase activity correlates with the level of endogenous indole-3-acetic acid (Galston
and Davies 1969) and interacts with cytokinins and gibberellins (Lee 1972)­
which is further evidence that peroxidase enzymes are good monitors of physiologi­
cal responses.
In the present Douglas-fir study, isoenzymes of peroxidase, catalase, leucine
aminopeptidase, acid phosphatase, and esterase were studied in bark tissues of
unions of known compatible and incompatible stock-scion combinations, in wounds,
and in nonunion areas of stock and scions. Tissues were sampled at 17 different
graft ages over a 3-year period. Consistent differences between the enzymes from
compatible and incompatible grafts support the hypothesis that hypersensitive reac­
tions were responsible for between-cell antagonism in Douglas-fir graft unions.
METHODS
Douglas-fir grafts were made on 11- and 12-year-old stock trees during April 1973
and April 1974. Three trees (S-1, S-2, LW-2) 20 to 30 years old were selected as
sources of scions for homoplastic grafts (grafts with scion and stocks of the same
species but of unlike genotypes). Grafts were placed on branch tips of eight stock
trees in 1973 and on five different stock trees in 1974. At least 20 grafts of each
scion-clone were made on each rootstock. Twenty autoplastic grafts (grafts with
scion and stock of identical genotypes) of each of the five rootstocks were made in
1974, but none were grafted in 1973.
Plant collections for electrophoresis were made 1 to 4 days prior to electropho­
resis. Plant material was stored in plastic bags at about 2 °C until used. One graft
of each scion-stock combination was examined in each electrophoretic run. Grafts
were examined from 1 to 27 months after grafting. Twenty-one electrophoresis
runs were made from May 1973 to October 1975.
Samples of bark included all tissues from the periderm to inner cambium. Two
samples were cut from nongrafted areas of the scion and stock (2.5 em above and
5.0 em below the graft union). Another sample was cut from a wound area near the
union but not in the area where stock and scion tissues merged. The fourth sample
was cut from the narrow union zone where the stock and scion cells grew together.
The latter sample was a 2- to 3- by 10-mm piece of bark which was cut from the
graft with a surgical scalpel. The exact perimeter of the union zone could easily be
identified through the first two growing seasons but became increasingly difficult
during the third growing season. For half the trees an additional sample was made
by mixing equal weights of tissue from nonunion areas of both scion and stock. This
sample was made to determine if changes in bands could occur if two different
298 I FOREST SCIENCE
genotypes were mixed in vitro vs. the in vivo condition found in unions. The com­
posite sample was substituted for wound samples. For all samples, 100- to 250-mg
of tissue was macerated just prior to the start of each electrophoretic run in 200 to
500 p.l of gel buffer solution containing 4.5 percent polyvinylpyrrolidone (MW
40,000). A ratio of 100 mg of tissue to 200 p.l of gel buffer was maintained. Fluid
extracts from the macerated tissues were absorbed on 5- by 13-mm paper wicks cut
from Whatman No. 1 chromatography paper and immediately inserted into the
starch gels at the origin slit. Four sample wicks from each graft (one each from the
stock, union, scion, and combined scion-stock or wound) were inserted side by side
in the same gel.
Starch gel electrophoresis generally followed the methods outlined by Conkle
(1972) and modified by Copes (1975). Gels (12 percent and pH 8.0) were
poured into molds 20- by 13- by 1.6-cm. Each gel contained 461 ml of gel buffer
solution (417 ml tris-citrate [0.5 M tris and 0. 1 M citric acid, pH 8.3] and 50 ml
lithium borate [0.03 M lithium hydroxide and 0. 17 M boric oxide, pH 7.4]).
Lithium borate (0. 03 M lithium hydroxide and 0.17 M boric oxide) was used as
the electrode buffer solution. Constant current of 75 rna was applied until a bromo­
phenol blue marker dye had moved 8 em from the origin slit. Voltage averaged
about 400 V (220-540 V) and gel temperatures averaged 8°C (5°- 14°C) during
an average 4- to 4.5-hour run.
After electrophoresis, each gel was sliced horizontally and different slices of each
gel were stained for peroxidase, catalase, leucine aminopeptidase, esterase, and acid
phosphatase. The esterase, acid phosphatase, and leucine aminopeptidase were
stained by procedures described by Scandalios (1969). Peroxidase (polyphenol
oxidase) was tested with three different hydrogen donors: benzidine (Scandalios
1969), 3, 3-dimethoxy-benzidine (Brewbaker and others 1968), and N, N-dimethyl­
p-phenylenediamine (Molnar and LaCroix 1972). Catalase was tested by the Shaw
and Prasad ( 1970) recipe for bacteria.
Bands from isoenzymes were recorded in photographs and diagrams. Stain inten­
sity was recorded in addition to relative migration distance. Bowling and Crowden
( 1973) found a good correlation between peroxidase stain intensity in gels and that
obtained by regular assay methods on soluble peroxidases; thus, major increases or
decreases in intensity of staining of individual isoenzymes were used in the present
study to indicate possible quantitative differences in enzyme activity. Because of the
inexactness of this method, only isoenzymes with pronounced visible differences
were recorded as having significantly increased or decreased activity. Within-tree
comparisons were made between tissues from union and nonunion areas, as well as
between-tree comparisons of tissues taken from compatible and incompatible
unions. Bands were measured to the closest mm and transformed into relative
mobility units (Rm) : the distance the isoenzyme traveled from the origin slit
toward the anode, divided by the distance a bromophenol blue marker dye had
moved from the origin slit toward the anode.
The part of each graft union that remained after electrophoresis analysis was
preserved in 50-percent ethanol for later anatomical study. The preserved graft
unions were cross-sectioned, stained, and microscopically examined to determine
graft compatibility (Copes 1970). These anatomical data were then correlated
with the isoenzyme data to determine which isoenzymes might be useful to detect
incompatible or compatible grafts.
RESULTS
Anatomical tests of unions of homoplastic grafts showed 50 percent in 1973 and 53
percent in 1974 to be incompatible. No incompatibility symptoms were detected in
VoLUME
24, NuMBER 2, 1978 1 299
TABLE
Percent accuracy achieved in correctly identifying compatible and
1.
incompatible graft unions of increasing age with electrophoresis methods.
Unions correctly identified
1973 grafts•
Peroxidase
Month
Graft age
Month s
1974 grafts•
Peroxidase
Esterase,
Esterase,
Rm0.27
Rm0.44
Rm0.77
Rm0.50
Rm0.27
Rm0.44
Rm0.77
Rm0.50
and0.35
and0.49
and0.80
and0.56
and0.35
and0.49
and0.80
and0.56
. _______ --_____________ . _____________________
_c
-Percent_____------__________________________---______
May
1
0
0
0
June
2
0
0
0
0
July
3
0
0
0
65
August
4
0
63
0
60
September
5
0
85
0
65
November
7
0
0
0
0
0
0
0
65
0
0
100
96
0
0
100
100
0
0
93
90
0
0
100
85
0
0
95
90
February
10
April
12
May
13
July
August
October
18
0
0
77
76
November
19
0
0
79
71
0
0
94
15
0
0
96
92
16
71
0
92
67
96
January
21
0
0
87
62
February
22
83
55
83
75
April
24
0
75
83
79
July
27
0
0
87
65
•
0
= 24 graft unions sampled each month.
• = 20 graft unions sampled each month.
c
=
not sampled.
autoplastic grafts. Two scion clones (LW-2 and S-2) were both compatible or both
incompatible when grafted on the same stocks, but the third scion clone (S- 1)
always had the opposite compatibility of LW-2 and S-2.
Similarity of nonunion scion and stock tissues had no predictive value in deter­
mining which combinations would form compatible graft unions. Scion clones and
stock trees which formed compatible graft unions were no more similar than scions
and stocks which formed incompatible combinations. The presence or absence of
particular bands had no relationship to whether a graft union would be compatible
or incompatible.
Noticeable differences in enzyme activity were evident in comparisons of tissues
from compatible and incompatible unions. Certain bands from tissues of incom­
patible unions stained much darker than the same bands from tissues of compatible
unions or from nonunion areas. These unusually dark-stained bands occurred at
Rm 0.44, 0.49, 0.77, and 0.80 for peroxidase and Rm 0.50 and 0.56 for esterase.
The percentage of trees correctly identified at different graft ages as having incom­
patible graft unions is shown in Table 1. Correct identification was made when
electrophoretic results corresponded with anatomical test results.
Incompatible grafts could be accurately identified by electrophoretic techniques
only during specific months or at certain graft ages. At other times, high general
activity in all union tissues made detection of incompatible-correlated increases dif­
ficult. Increases in enzyme activity in union tissues at Rm 0. 77 and Rm 0.80 were
not correlated with graft incompatibility until February, 10 months after grafting.
300 I FOREST SCIENCE
A B
FIGURE 1.
C
0
A
8
C
0
A
B
C
D
A
B
C
0
The two arrows point to location of Rm 0.44 and 0.49 peroxidase bands from
tissues of two incompatible graft unions. The other bands were from two compatible unions
and from nonunion areas of the scion and stock (A, stock; 8, union; C, scion; D, scion +
stock).
At that time, 100 percent of the incompatible grafts were identified with either
peroxidase or esterase bands. Between 90- and 1 00-percent accuracy in detecting
incompatible grafts was achieved from that time until the following May for both the
Rm 0.77 and 0.80 peroxidase bands and the Rm 0.50 and 0.56 esterase bands, and
until August if only the peroxidase bands were used. After that time, the detection
accuracy obtained with peroxidase and esterase averaged 84 percent and 74 percent
for the remaining 12 months of the study (Table 1).
A general increase in activity of Rm 0.44 and 0.49 peroxidase was noted in
wounds and in both autoplastic and homoplastic graft tissues. This increase may
have resulted from the numerous callus or parenchyma cells in wound and graft
tissues, and was not correlated with graft incompatibility. An even darker staining
of these same bands was noted 5 months after grafting. At that time 85 percent of
the incompatible unions could be identified by the unusually dark-stained Rm 0.44
and 0.49 peroxidase bands (Fig. 1, Table 1). These bands also showed correlation
with incompatibility 4 and 22 months after grafting.
Bands other than the Rm 0.50 and 0.56 esterase and the Rm 0.44, 0.49, 0.77,
and 0.80 peroxidase from union tissues occasionally stained a darker color than
bands with the same relative mobility from nonunion tissues, but their presence had
little or no correlation with graft incompatibility. Such bands were found at Rm
0.27, 0.35, 0.40, 0.66, and 0.71 for peroxidase and at Rm 0.37, 0.69, 0.75, 0.80,
0.83, and 0.87 for esterase.
DISCUSSION
Incompatible graft unions were identified most accurately by electrophoresis 10 to
15 months after grafting. At that time, 90 to 100 percent of the incompatible
unions were detected using the Rm 0.50 and 0.56 esterase and the Rm 0.77 and
0.80 peroxidase bands. Detection accuracy decreased after the 15th month because
tissues cut from older grafts contained too much nonunion scion or stock tissue. A
program to detect incompatible stock-scion combinations by electrophoretic tech­
niques is possible if union tissues of the proper age are tested, but similarity of iso­
enzymes of stock trees and scion clones is useless in predicting compatibility prior
to grafting.
Electrophoretic detection of incompatible grafts has several advantages over the
anatomical test method (Copes 1970) currently used. First, the unions need not be
destroyed since only a thin slice of bark from the area where the stock and scion
tissues join is reguired. Thus, only one graft is needed on each stock. A second
advantage is that reliable electrophoretic testing can be done 10 months after graft­
ing, whereas reliable anatomical testing is not possible until the grafts are at least 16
to 17 months old.
VOLUME 24, NUMBER 2, 1978 I 301
The actual cause of between-cell antagonism was not found, but the increase in
peroxidase activity suggests that the hypersensitive reaction mechanism had been
activated. Reliable electrophoretic identification of incompatible graft unions was
not possible until after visible symptoms of between-cell antagonism were seen
through a microscope. Minor isoenzyme differences, detected from 3 to 7 months
after grafting, correlated somewhat with the appearance of suberized and necrotic
tissues in graft unions of incompatible combinations (Copes 1970). The character­
istic darker staining of peroxidase bands from tissues in incompatible graft unions
may have been associated with cell necrosis. A similar role of phenols and cell
necrosis was demonstrated in hypersensitive reactions of tobacco plants (Legrand
and others 1976). Increases in esterase activity have been reported in spruce
needles and birch leaves injured by fluoride emissions (Yee-Meiler 1975). The
altered esterase bands found in this study may also be caused from cellular antago­
nism (necrosis).
Comparisons of zymograms from union and nonunion tissues graphically demon­
strate union and wound enzyme activities to be different than enzyme activity seen
in nongrafted or healthy tissues. The most significant difference was darkly stain­
ing bands at Rm 0.27, 0.35, 0.44, and 0.49, indicating an increase in peroxidase
activity in practically all graft unions and wound areas.
No isoenzyme differences between compatible and incompatible grafts could be
detected with leucine aminopeptidase, acid phosphatase, or catalase.
LITERATURE CITED
BERRYMAN, A. A.
1972. Resistance of conifers to invasion by bark beetle-fungus associations.
Bioi Sci 22:59 8-602.
BowLING, A. C., and R. K. CROWDEN.
1973.
Peroxidase activity and lignification in the pod
membrane of Pisum sativum L. Aust J Bioi Sci 26:679-684.
BREWBAKER, J.L., M. D. UPADHYA, Y. MAKINEN, and T. MACDONALD.
1968.
Isoenzyme poly­
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Plant 21:930-940.
CoNKLE, M. T.
1972.
Analyzing genetic diversity in conifers ... isoenzyme resolution by
starch gel electrophoresis.
USDA Forest Serv Res Note PSW-264, 5 p.
Pac Southwest
Forest and Range Exp Stn, Berkeley, Calif.
CoP Es, D.
1970.
Initiation and development of graft incompatibility symptoms in Douglas­
fir. Silvae Genet 19:10 1-107.
CoPEs, D. L.
1975.
Isoenzyme study of dwarf and normal Douglas-fir trees. Bot Gaz 136:
347-352.
GALSTON, A.W., and P. J. DAVIES.
1969.
Hormonal regulation in higher plants. Science
163:1 2 8 8- 1297.
KoNAREV, V. G.
1972.
Cytochemistry and histochemistry of plants (Tsitokhimiya i gisto­
khimiya rastenii). Translated by P. Harry.
LEE, T. T.
1972.
Jerusalem, Israel Program Sci Trans!, 260 p.
Interaction of cytokinin, auxin and gibberellin on peroxidase isoenzymes in
tobacco tissues cultured in vitro. Can J Bot 50:2471-2477.
LEGRAND, M. , B. FRITIG, and L. HmTH.
1976.
Enzymes of the phenylpropanoid pathway
and the necrotic reaction of hypersensitive tobacco to tobacco mosaic virus. Phytochemis­
try 15:1353-1359.
MELNICK, V. L., L. HOLM, and B. E. STRUCKMEYER.
1954.
Physiological studies on fruit
development by means of ovule transplantation in vivo. Science 145:609-61 1.
MoLNAR, J. M., and L. J.LACROIX. 1972.
macrophyl/a:
Studies of the rooting of cuttings of Hydrangea
enzyme changes. Can J Bot 40:31 5-322.
PooVAIAH, B. W., and H. P. RAsMUSSEN.
1973.
Peroxidase activity in the abscission zone of
bean leaves during abscission. Plant Physiol 52:263-267.
SCANDALJOS, J. G.
1969.
Genetic control of multiple molecular forms of enzymes in plants:
A review. Biochem Genet 3:37-79.
302 I FOREST SCIENCE
SHAW, C. R., and R.PRASAD.
1970.
Starch gel electrophoresis of enzymes-a compilation of
recipes. Biochem Genet 4:297-320.
YEE-MEILER, D.
1975.
On the suitability of phosphatase and esterase activity in spruce
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Forest Sci., Vol. 24, No. 2, 1978, pp. 303-308
Aspen Sucker Regeneration Following Burning and
Clearcutting on Two Sites in the Rocky Mountains
Note by George
ABSTRACT.
A. Schier and Robert B. Campbell
Aspen (Populus tremuloides Michx.) root suckers arising after a control burn
in Wyoming and clearcutting in Utah were studied to obtain information on depth and
diameter of parent roots producing suckers, occurrence of new roots, and the effects of
burn intensity on suckering. Compared with parent roots of aspen in the Lake States, those
of western aspen were deeper and slightly larger.
Clonal differences were found in the
depth and diameter of parent roots and in the ability to initiate new roots around the base
of suckers. Few suckers had well-developed independent root systems. A high burn inten­
sity increased the depth at which suckers were initiated. FoREST SCI. 24:303-308.
ADDITIONAL KEY WORDS.
Populus tremuloides, vegetative propagation, adventitious shoots,
roots.
FOLLOWING LOGGING OR FIRE, aspen
(Populus tremuloides
Michx.) reproduces vegeta­
tively from suckers (adventitious shoots) that develop from long, ropelike lateral roots.
Suckers occur on parts of the undulating roots that rise near the soil surface (Baker
1925,
Day
1944,
Sandberg
19511).
The only quantitative data describing the depth and diameter of roots that give rise to
suckers are from the Lake States (Sandberg
1951,
Farmer
1962).
This information may
not adequately describe regeneration in western aspen because of differences between
regions in climate and soil and aspen genotypes.
Therefore, when mature aspen was
clearcut in one area of the Rocky Mountains and control burned in another as part of a
research program on the ecology and management of aspen, we collected data on depth
and diameter of parent roots of aspen suckers. We also studied the formation of new
roots and the effect of burn intensity on aspen regeneration.
1
Sandberg, D. 1951.
Univ Minn.
The regeneration of quaking aspen by root suckering. M. F. Thesis,
172 p.
The authors are, respectively, Plant Physiologist and Biological Technician, Intermountain
Forest and Range Experiment Station, Forest Service, U.S. Department of Agriculture, Ogden,
Utah 84401, located at the Intermountain Station's Forestry Sciences Laboratory, Logan, Utah.
Manuscript received 15 July 1977.
VOLUME 24, NUMBER 2,
1978 I 303