Directory UMM :Data Elmu:jurnal:T:Tree Physiology:Vol15.1995:
Tree Physiology 15, 457--465
© 1995 Heron Publishing----Victoria, Canada
Effects of gibberellin A 4/7, root pruning and cytokinins on seed and
pollen cone production in black spruce ( Picea mariana)
RON SMITH1 and MICHAEL GREENWOOD2
1
Canadian Forest Service--Maritimes Region, P.O. Box 4000, Fredericton, N.B. E3B 5P7, Canada
2
College of Forest Resources, University of Maine, Orono, Maine 04469, USA
Received May 31, 1994
Summary Stem injections of 8--9-year-old black spruce
(Picea mariana (Mill.) B.S.P.) trees with gibberellin A4/7
(GA4/7) alone and in combination with root pruning increased
both seed and pollen cone production in both 1991 and 1992.
Combining GA4/7 and root pruning produced a larger increase
in reproductive bud production than either treatment alone, but
the complementary effects were less during the warm dry
summer in 1991 than during the wet summer in 1992. Cytokinin applications reduced the stimulatory effects of GA4/7 and
root pruning, and the degree of reduction varied with the
amount and timing of application. Reduced reproductive bud
production was attributed to cytokinin countering the effects
of root pruning and natural drought on the GA4/7 response.
Keywords: drought, flowering, reproduction.
Introduction
Increased cone production in conifers in response to exogenous gibberellic acid (GA) is well documented (Pharis et al.
1987, Bonnet-Masimbert and Zaerr 1987, Pharis 1991); however, despite recent refinements to methods of applying GA,
trees must be predisposed to respond, i.e., GA applications by
themselves often do not induce flowering in reproductively
immature (juvenile) trees, in recalcitrant genotypes, or when
other environmental conditions are unfavorable (see reviews in
Owens and Blake 1985, and Bonnet-Masimbert 1987). Several
cultural treatments, including fertilizing, root pruning, and
drought and heat stress, significantly increase tree responsiveness to GA (Tompsett and Fletcher 1979, Philipson 1983,
Owens and Blake 1985, Ross et al. 1985, Pharis et al. 1987,
Ross 1988, Owens and Simpson 1988, Owens et al. 1992), but
the mechanism(s) whereby cultural treatments increase GA
effectiveness remain largely unresolved.
In Picea, responsiveness to GA treatments is only increased
when root pruning, drought and soil heat treatments are timed
to coincide approximately with vegetative bud burst, early
shoot elongation and cessation of shoot elongation, respectively (Ross et al. 1985, Ross 1988), indicating that the treatments affect different stages of bud development. Root pruning
may delay bud development and thereby increase bud developmental synchrony such that a greater proportion of buds will
be responsive to GA4/7 at any particular time (Owens et al.
1992). Drought may increase the concentrations of cone-inducing GAs, e.g., GA4/7 and GA9 (Moritz et al. 1989, Moritz
and Odén 1990), by retarding their conjugation or hydroxylation or both (Chalupka et al. 1982, Dunberg et al. 1983, Pharis
et al. 1989). Soil heat treatments may increase flowering by
reducing export from the roots of a flowering antagonist
(Philipson 1983). Drought and soil heat stress could also induce changes in the metabolism of both GA and cytokinin
(Imbault et al. 1988, Pilate et al. 1990).
Cytokinin concentrations in xylem exudate are positively
correlated with water availability and root growth (Itai and
Vaadia 1971, Marschner 1986, Thimann 1992), and negatively
correlated with flower initiation in many herbaceous species.
Because the principal site of cytokinin synthesis is in the roots,
treatments such as irrigation that promote root growth and, by
inference, cytokinin production would be expected to reduce
or inhibit the response to GA4/7 (Kinet et al. 1993). The first
objective of this study was to test the hypothesis that the
increase in efficacy of applied GA4/7 with root pruning results
from reduced cytokinin export from the roots.
Although sexual zonation within conifer tree crowns is generally acknowledged (Marquard and Hanover 1984a, 1984b,
Ross and Pharis 1987), distribution patterns of reproductive
structures within the crown (Powell 1977, Caron 1987, Tosh
and Powell 1991, Caron and Powell 1992) and along and
around shoots (Tosh and Powell 1991, Caron and Powell 1993)
have received limited attention. Furthermore, although crown
position can have a profound effect on response to cone induction treatments, there are few reports on the effects of cone
induction treatments on seed and pollen cone distribution
patterns (Marquard and Hanover 1984a, 1984b, Philipson
1987).
The second objective of this study was to test the effects of
root pruning, ammonium nitrate (NH4NO3) and cytokinins on
the numbers and within-crown distribution of seed and pollen
cones produced by black spruce (Picea mariana (Mill.) B.S.P.)
in response to GA4/7.
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SMITH AND GREENWOOD
Materials and methods
Study area and treatments
The study area is a black spruce seedling seed orchard located
70 km northeast of Fredericton, New Brunswick, Canada, that
was planted in 1983 by the New Brunswick Department of
Natural Resources and Energy. Soils are well-drained loamy
sands to sandy loams with moderate fertility (data on file).
In 1991, two experiments were established. Experiment 1
was a complete factorial and included controls (C), a fertilizer
treatment (F) in which 300 g of ammonium nitrate (34-0-0,
N,P,K) was applied in a band around the crown dripline, a
gibberellin treatment (GA) in which 10 mg of GA4/7 in 0.1 ml
of 95% ethanol was injected into the stem just below the 1989
whorl, and a root-pruning (RP) treatment in which tree roots
were cut to a depth of 25 cm on two sides of the stem at the
edge of the crown dripline. Experiment 2 was designed to test
the hypothesis that applying cytokinins (benzylaminopurine
(BAP), zeatin (Z) and zeatin riboside (ZR)) would reduce or
negate the GA-induced flowering response. The treatments
were C, GA, RP, Z1, ZR1, BAP7, GA + Z1, GA + ZR1, GA +
BAP7, GA + RP + BAP1, GA + RP + BAP3, and GA + RP +
BAP7 where the numeral after BAP represents the amount of
cytokinin injected in mg per 0.1 ml of 95% ethanol. Within
both experiments, one tree from each of 10 families was treated
on June 18--19, 1991, approximately 1 week after vegetative
bud burst.
In 1992, three experiments were established to evaluate the
effects of rate and time of cytokinin application on the response to GA4/7 and root pruning. In Experiment 1, the treatments were C, GA, RP, T1BAP1, T1BAP3, T1BAP7, T2BAP1,
T2BAP3, T2BAP7, GA + RP, GA + RP + T1BAP1, GA + RP
+ T1BAP3, GA + RP + T1BAP7, GA + RP + T2BAP1, GA +
RP + T2BAP3, and GA + RP + T2BAP7 where T1 and T2
represent early lateral shoot elongation (June 19--23) and cessation of shoot elongation (July 20) stages at which treatments
were applied, respectively. The same experimental design was
used to test the effects of zeatin and zeatin riboside; however,
for these trials only 0.1, 0.5 and 1.0 mg cytokinin were used,
and the application of cytokinin without GA + RP was omitted
on July 20. Injections were made just below the 1989 whorl.
Each treatment was given to 10 trees, one from each of 10
families.
Growth measurements
In 1991, growth of the leader (L), one lateral shoot located in
the middle one-third of the previous year’s leader (LB), the
terminal shoot (WL), and one first-order lateral branch (WB)
from one 1990 whorl branch were measured twice per week.
In 1992, weekly observations were made on 30 trees, one tree
from each of the 30 families used. Total tree height and diameter at the hormone injection point were measured on all trees.
Cone counts and data analyses
Numbers of seed and pollen cones, by position within the
crown, were determined in both 1992 and 1993. Count data
were normalized by the square root (count + 1) transformation
(Snedecor and Cochrane 1980). Tree damage and mortality
resulted in dropping some plot trees leading to a small data
imbalance (n = 8 to 10 per treatment); therefore, Type III sums
of squares (SS) (GLM procedure, SAS Institute Inc., Cary,
NC) were reported. Standard analysis of covariance (ANCOVA), with tree diameter as the covariate, was used after
testing for homogeneity of slopes (Hendrix et al. 1982). Partitioning of main treatment effects and corresponding hypothesis testing was done by contrasts (Snedecor and Cochrane
1980). The specific hypotheses tested are reported in the ANCOVA tables.
Results
Effects on cone production
Exogenous GA4/7 induced 7-, 4- and 9-fold increases in seed
cone production in the 1991 fertilization, 1991 cytokinin and
1992 BAP trials, respectively (Figures 1A, 2A and 3A). In the
1992 zeatin and zeatin riboside trials, the more than 50-fold
increase in seed cone production in response to GA4/7 was
partly due to the small numbers (mean < 2) of seed cones
produced by the control trees (Figures 3C and 3E). Gibberellin
increased the numbers of pollen cones by two to six times
(Figures 1B, 2B, 3B, 3D and 3F). Based on ANCOVA, GA4/7
effects on seed cone production were significant for both years,
whereas GA4/7 effects on pollen cone production were only
significant in the 1991 cytokinin and 1992 BAP experiments
(Tables 1 and 2).
In both years, root pruning alone had little effect on seed or
pollen cone production, although root-pruned trees in the 1991
fertilization and 1992 BAP trials produced more pollen cones
than the controls (Figures 1B and 3F). The response to the
combined RP + GA treatment differed between 1991 and
1992. In 1991, trees in the RP + GA treatment produced the
same or slightly more seed and pollen cones than trees receiving GA4/7 alone, whereas in 1992, except for seed cones in the
1992 zeatin riboside trial, trees in the RP + GA treatment
produced significantly more cones of both sexes than trees
receiving GA4/7 alone (Figures 1--3). Applying ammonium
nitrate with GA reduced the stimulatory effect of GA4/7 on
pollen cone production (Figure 1B).
In 1991, applying cytokinin alone had no significant effect
on seed cone production, whereas injecting as little as 1 mg of
zeatin or zeatin riboside with GA4/7 significantly reduced pollen cone production relative to trees receiving GA4/7 alone
(Figure 2B). All three BAP treatments reduced the increase in
pollen cone production in response to the combined root pruning + GA treatment in 1991 (Figure 2B). In 1992, adding BAP
or zeatin riboside negated the synergistic increase in pollen
cone production in response to the combined RP + GA treatment (Figure 3). The reductions in seed and pollen cone production were greater when cytokinin application coincided
with the cessation of shoot elongation (T2) than with the time
of bud flush (T1). The mean numbers of seed and pollen cones
were not significantly different between trees receiving the
GA + RP + Z treatment and trees receiving the combined RP
+ GA, confirming the lack of effect of zeatin on flowering in
SEED- AND POLLEN-CONE PRODUCTION IN BLACK SPRUCE
Figure 1. Mean numbers of and standard errors for (A) seed and (B)
pollen cones produced in 1992 following fertilizer application (F),
root pruning (RP) and GA4/7 injection (GA) in 1991.
1992. Pollen cone production was significantly reduced in all
three BAP treatments and at both T1 and T2, and seed cone
production decreased with increasing amounts of BAP applied. The differences in numbers of seed cones were statistically significant for 3 and 7 mg BAP at T2.
In both 1992 and 1993, control trees produced either the
same or a slightly greater number of pollen cones than seed
cones. Pooling the data from all the experiments indicated that
applying GA4/7 alone more consistently increased numbers of
seed cones than of pollen cones, altering the sex ratio (female
to male) from 1/1 to 3/1. The combined RP + GA treatment
resulted in sex ratios of 2.5/1 and 1/1 in 1992 and 1993,
respectively.
Effects on cone distribution
In control trees, seed cones were produced predominantly on
first-order laterals of the previous year’s leader and its subtending whorl, whereas pollen cones were located on the whorl and
internodal whorl branches immediately below. Applying GA4/7
459
Figure 2. Mean numbers of and standard errors for (A) seed and (B)
pollen cones produced in 1992 for control trees (C) and trees receiving
stem injections in 1991 of 10 mg GA4/7 (GA) alone and in combination
with root pruning (RP) and cytokinins (1 mg zeatin (Z1), 1 mg zeatin
riboside (ZR1), and 1, 3 and 7 mg benzylaminopurine (B1, B3 and B7,
respectively)).
increased the proportions of buds within the upper crown
zones developing reproductively but did not affect the locations within the crown where seed and pollen cones were borne
(Figures 4A and 4C). In 1991, root-pruning increased the
magnitude of the response to GA4/7, but did not affect cone
distribution. In 1992, root pruning not only increased the
numbers of cones produced in response to GA4/7, but expanded
the zone producing significant numbers of pollen cones upward relative to control and GA4/7-treated trees (Figure 4C).
Cytokinins, especially BAP, decreased the numbers of cones
of both sexes above the injection point. The magnitude of
reduction decreased upward from the injection point, hence
pollen cone production was reduced proportionally more than
seed cone production. Reductions in numbers of cones in
response to zeatin and zeatin riboside, although smaller than
those observed for BAP, also diminished acropetally from the
460
SMITH AND GREENWOOD
Figure 3. Mean numbers of
and standard errors for seed
(A, C and E) and pollen
cones (B, D and F) produced
in 1993 for control trees (C)
and trees receiving stem injections in 1992 of 10 mg
GA4/7 (GA) alone and in
combination with root pruning (RP) and benzylaminopurine (BAP) (A and B),
zeatin (Z) (C and D), and
zeatin riboside (ZR) (E and
F). Cytokinin was injected
on either June 19--23
(TIME 1) or July 30
(TIME 2).
SEED- AND POLLEN-CONE PRODUCTION IN BLACK SPRUCE
461
Table 1. Mean squares and significance levels for the 1991 trials. Model sums of squares were partitioned as follows: FERT91 = complete factorial
with main effects (C, FERT, GA, RP), two-way interactions (FERT × GA, GA × RP, FERT × RP) and three-way interaction (FERT × GA × RP);
CYTO91 = main effects (C, GA, RP, Z1, ZR1, BAP7), GA × CYTO (GA + Z1, GA + ZR1, GA + BAP7), GA + RP(BAP) (GA + RP + BAP1,
GA + RP + BAP3, GA + RP + BAP7), and the two contrasts, GA versus (GA × CYTO) and GA versus GA + RP(BAP) where the number after
cytokinin represents mg of cytokinin injected. The contrast ‘GA versus others’ within themain treatments is provided.
Treatment
Seed cones
MS
P
MS
P
1
3
3
1
77
85
292.65
226.56
136.81
61.42
18.31
0.0001
0.0001
0.0001
ns
140.72
13.49
13.57
56.91
13.48
0.0018
ns
ns
ns
1
5
(1)
2
2
1
1
99
111
21.92
52.09
245.54
58.7
26.61
82.12
569.6
15.31
ns
0.0070
0.0001
0.0249
ns
0.0226
0.001
13.31
22.96
97.02
3.54
2.33
90.18
768.39
9.95
ns
0.0499
0.0024
ns
ns
0.0033
0.001
df
FERT91
Diameter (covariate)
Main effects
Two-way
FERT × GA × RP
Error
Total
CYTO91
Diameter (covariate)
Main effect
GA versus others
GA × CYTO
GA + RP(BAP)
GA versus (GA × CYTO)
GA versus GA + RP(BAP)
Error
Total
Pollen cones
Table 2. Mean squares and significance levels for the 1992 trials. Sums of squares (SS) were partitioned as follows: BAP92 = main effect (C, GA,
RP, T1BAP1, T1BAP3, T1BAP7, T2BAP1, T2BAP3, T2BAP7), GA + RP(BAP) (GA + RP + T1BAP1, GA + RP + T1BAP3, GA + RP + T1BAP7,
GA + RP + T2BAP1, GA + RP + T2BAP3, GA + RP + T2BAP7), and the contrast GA versus GA + RP, where T1 and T2 are time of cytokinin
application (June 19--23 and July 20, 1992, respectively) and the number after BAP represents mg of BAP injected. The same coding and
partitioning of SS was used for Z92 and ZR92 except that the T2 applications of cytokinin without GA + RP were not included.
Treatment
Seed cones
Pollen cones
df
MS
P
MS
P
BAP92
Diameter (covariate)
Main effect
GA versus others
GA + RP(BAP)
GA versus GA + RP
Error
Total
1
8
(1)
6
1
143
159
439.37
57.27
404.79
136.55
291.55
14.36
0.0001
0.0003
0.0001
0.0001
0.0001
106.17
111.41
100.27
14.55
432.70
14.82
0.0083
ns
0.0103
ns
0.0001
Z92
Diameter (covariate)
Main effect
GA versus others
GA + RP(Z)
GA versus GA + RP
Error
Total
1
5
(1)
6
1
117
130
100.12
40.4
198.92
60.40
464.98
11.94
0.0001
0.0069
0.0001
0.000
0.0001
176.74
1.55
0.61
53.23
150.00
10.79
0.0001
ns
ns
0.00
0.0001
ZR92
Diameter (covariate)
Main effect
GA versus others
GA + RP(ZR)
GA versus GA + RP
Error
Total
1
5
(1)
6
1
112
125
102.36
96.02
470.60
66.31
226.02
16.79
0.0150
0.0001
0.0001
0.00
0.0001
89.06
24.10
96.36
43.08
261.69
29.24
0.0837
ns
0.0
ns
0.0001
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SMITH AND GREENWOOD
Figure 4. Mean numbers of seed and pollen cones in 1992 (A and B) and 1993
(C and D) by crown position for control
trees (C) and trees receiving stem injections of 10 mg of GA4/7 (GA) alone and
in combination with root pruning (RP)
and RP + GA plus 1, 3 or 7 mg benzylaminopurine (B1, B3 and B7, respectively). Crown position coding: 1990
whorl branches = W90, internodal
branches between the 1990 and 1991
whorls = IB90-91, etc. Point of hormone
injection is indicated by an arrow.
injection point (Figures 4B and 4D).
Effects on growth
In 1991, treatments did not affect either the final length of any
of the shoots measured or their rate of elongation, although
mean total height, diameter and leader length were less for the
trees receiving zeatin than for trees in the other treatments.
Tree height, diameter and final shoot lengths were positively
correlated. There were no significant differences in mean shoot
length measurements between experiments.
The pattern and duration of first-order lateral shoot growth
were similar to those reported for black spruce: a 4-week
period of rapid shoot elongation (mid-June to mid-July), followed by 1 week of slow growth (Caron 1987, Ho 1988, 1991).
Cessation of shoot elongation progressed acropetally within
the crown and along shoots, with leaders stopping elongation
growth by August 15. Time and duration of shoot growth
stages were similar between the 2 years, although the period of
rapid shoot growth was approximately 1 week longer in 1992
than in 1991.
Relationships between weather, growth and cone production
Five to 10 times more seed and pollen cones were produced by
control trees in 1992 than in 1993. There were two main
differences in weather patterns between 1991 and 1992: (1)
total degree days (5 °C base) were higher in 1991 than in 1992
throughout most of June and July, and differences were greatest at the time of cessation of lateral shoot elongation in midto late-July; and (2) there was approximately twice the amount
of precipitation from mid-June to early-August in 1992 than in
1991.
In the 1991 trials, there was little variation within and among
the 20 families in lateral shoot growth phenology. Treatment
efficacy was not significantly correlated with any of the growth
measurements or developmental observations. Similarly, differences among families in seed and pollen cone production
were not related to phenology but rather to tree size; cone
production was positively correlated with tree height and diameter (Table 3), and presumably with numbers of shoots/buds
(potential flowering sites).
Discussion
Injections of GA4/7 significantly increased the proportions of
buds developing reproductively above the injection point.
Similar results have previously been reported for Picea (Marquard and Hanover 1984a, Philipson 1987). Comparable increases in numbers of seed cones were observed in both years.
However, in 1992, GA4/7 was injected one whorl lower than in
1991, thereby including branches within the pollen-cone-bearing zone and producing a concomitant increase in pollen cone
production, which is contrary to the preferential GA4/7 induc-
SEED- AND POLLEN-CONE PRODUCTION IN BLACK SPRUCE
Table 3. Pearson correlation coefficients between tree height and
diameter and seed and pollen cone production for all trees in the 1991
(n = 198) and 1992 (n = 417) trials.
Seed cone
Pollen cone
1992 Cones
Height
Diameter
0.212*1
0.150*
0.238***
0.177*
1993 Cones
Height
Diameter
0.182***
0.222***
0.133***
0.142**
1
*** = Significant at 0.001, ** = significant at 0.01, * = significant
at 0.05.
tion of seed over pollen cones reported by others (Ross and
Pharis 1987, Ho 1988, Ross 1990, Greenwood et al. 1991, Ho
1991). Our results support the hypothesis that GA4/7 has a role
in determining sexual versus vegetative development
(Chalupka 1980), although they do not preclude the possibility
that sex determination is controlled by other factors, e.g., the
ratio of gibberellins to auxins (Pharis and Kuo 1977).
Control trees produced seed cones almost exclusively on
lateral branches along 1-year-old shoots. Caron and Powell
(1993) reported similar findings for plantation-grown black
spruce trees aged 9 to 11 years. Exogenous GA4/7 alone had
little effect on the location of seed- and pollen-cone-bearing
branches within the tree crown, whereas the combined RP +
GA treatment resulted in appreciable overlapping of the male
and female zones on the internodal branches (IB) two whorls
back (IB89-90 and IB90-91 in 1992 and 1993, respectively)
(Figures 4A and 4C). Sexual zonation is generally less pronounced in good flowering years and in response to successful
cone induction treatments than in poor flowering years (Ross
and Pharis 1987).
Tompsett and Fletcher (1979) proposed a bud vigor gradient
hypothesis for Picea sitchensis (Bong.) Carr. in which, in order
of decreasing vigor, buds would develop into strong vegetative,
female, intermediate vegetative, male, and weak vegetative
structures, and that for young trees in which strong vegetative
shoots predominate, stress treatments would favor seed over
pollen cone induction (i.e., a downward shift of one vigor
class). We found that root pruning increased the response to
GA4/7 for both seed and pollen cone production, although in
1993, the pollen cone production response was greater than the
seed cone production response, indicating a possible shift from
intermediate vegetative to male buds. Vegetative shoot vigor in
conifers generally decreases in a basipetal direction between
branches within the crown, shoots along branches, and buds
along shoots (Powell 1977, Remphrey and Powell 1984, Caron
1987). Although bud vigor based on relative position along
shoots may help describe sexual zonation within a tree crown,
it does not explain how cone induction treatments affect the
correlative development between buds along shoots.
Reduced root activity is often related to successful flower
induction (Bonnet-Masimbert et al. 1982, Zaerr and BonnetMasimbert 1987). Although root pruning increased the magni-
463
tude of the response to GA4/7, it did not increase seed cone
production in either year, or pollen cone production in 1992.
The failure of the root pruning treatment to elicit a response in
1991 may be attributed to a combination of root pruning and
dry weather during most of the active shoot growth period in
1991 resulting in a water stress that exceeded what is optimal
for flower induction. A combined root pruning + heat stress
treatment had an adverse effect on both seed and pollen cone
production in potted black spruce (Ho 1991).
In 1992, cytokinins countered the complementary effect of
root pruning on the GA4/7 response. This effect differs from the
effects found for Sitka spruce (Tompsett 1977) and Douglas-fir
(Ross and Pharis 1976, Zaerr and Bonnet-Masimbert 1987). In
1991, seed and pollen cone production decreased in response
to increasing amounts of cytokinin applied with or without
GA4/7, whereas in 1992, trees receiving cytokinin alone produced similar numbers of cones of both sexes as the control
trees. Flowering was greater in 1992 than in 1993, as a result
of the warm dry weather in 1991.
Morphogenetic transitions, such as the shift from vegetative
to reproductive development, are usually associated with specific changes in gene expression (Bernier 1989, Meeks-Wagner et al. 1989). The expression of flowering genes following
induction treatments is reversible, e.g., proliferated and bisexual cones in black spruce (Caron and Powell 1990, 1991),
indicating that environmental conditions must continue to be
favorable for the continued expression of these genes. In 1992,
application of cytokinin either at the same time as GA4/7 and
root pruning, or 4 weeks later, reduced seed and pollen cone
production, although the magnitude of the response varied
with the type and amount of cytokinin used. The presumed
expression of flowering genes was reversible up to cessation of
shoot elongation, the time of bud determination in black spruce
(Ho 1988) and Picea in general (Owens and Blake 1985). Our
observations provide circumstantial support for the hypothesis
that the concentration of GA4/7 to which cells within developing buds are exposed and the duration of that exposure determine if the bud develops reproductively. Applying exogenous
GA4/7 increases the potential for cells within buds to be exposed to the ‘cone-inducing’ form of GA. Root pruning reduces, at least temporarily, cytokinin export from the roots,
thereby either (1) slowing cell cycling within developing buds
and increasing their period of receptivity to both endogenous
and exogenously applied GA4/7, or (2) relieving the bud of the
presence of cytokinins, if inhibitory to flowering.
None of the treatments affected shoot growth in the year of
treatment, which confirms other studies in which shoot elongation and flowering responded independently (Webber et al.
1985, Pharis et al. 1987). Although flowering was positively
correlated with reduced shoot elongation in Douglas-fir (Pilate
et al. 1990), the effects of girdling, root pruning and drought
were probably manifested as a temporary delay in shoot elongation and bud development (Tompsett 1978, Owens et al.
1985, Owens 1987) rather than a reduced amount of growth.
In Sitka spruce, the best response to cone induction treatments
occurred when apical mitotic indices were retarded, thereby
shifting differentiation back to coincide with cessation of shoot
464
SMITH AND GREENWOOD
elongation (Owens and Simpson 1988). Root pruning may
inhibit or enhance shoot elongation depending on whether GA
(Ross 1991a, 1991b) or other hormone production from regenerated roots exceeds that from roots before pruning.
In 1991, fertilization with NH4NO3 did not increase either
seed or pollen cone production in 1992, contrary to results with
black spruce (Smith 1986, 1987, 1993) If NH4NO3 increases
flowering at least partly through a rooting effect, then a lack of
flowering response to root pruning in 1991 would be expected.
The slight reduction in pollen cone production for trees receiving fertilizer and GA4/7 compared with GA4/7 alone may be
partly due to increased root growth (Marschner 1986) and
increased cytokinin production in the roots following fertilization. Betula pendula Roth. seedlings grown under nitrogen
deficient conditions exhibit lower concentrations of endogenous cytokinin than seedlings grown under nondeficient
conditions (Horgan and Wareing 1980).
Conclusion
We obtained circumstantial evidence that cultural treatments
increase efficacy of GA4/7 applications by temporarily reducing cytokinin export from the roots. Reduced cytokinin export
may result in a reduction in cell division of the developing buds
with the slowly cycling cells being more receptive to GA4/7,
and reduced cytokinin concentrations within the developing
bud may ensure continued expression of the flowering genes.
Acknowledgments
The authors thank Ms. K. Tosh of the New Brunswick Department of
Natural Resources and Energy for cooperation in providing access to
the orchard trees, and Dr. Y.S. Park, Dr. C.H.A. Little, Dr. J. Loo and
Mr. J.D. Simpson for reviewing the manuscript. Special thanks to Ms.
L. Yeates for technical assistance during all phases of this work.
References
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Reproduction: From Floral Induction to Pollination. Eds. E. Lord
and G. Bernier. Am. Soc. Plant Physiologists Symp. Series, Vol. 1,
pp 42--50.
Bonnet-Masimbert, M. 1987. Floral induction in conifers: a review of
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© 1995 Heron Publishing----Victoria, Canada
Effects of gibberellin A 4/7, root pruning and cytokinins on seed and
pollen cone production in black spruce ( Picea mariana)
RON SMITH1 and MICHAEL GREENWOOD2
1
Canadian Forest Service--Maritimes Region, P.O. Box 4000, Fredericton, N.B. E3B 5P7, Canada
2
College of Forest Resources, University of Maine, Orono, Maine 04469, USA
Received May 31, 1994
Summary Stem injections of 8--9-year-old black spruce
(Picea mariana (Mill.) B.S.P.) trees with gibberellin A4/7
(GA4/7) alone and in combination with root pruning increased
both seed and pollen cone production in both 1991 and 1992.
Combining GA4/7 and root pruning produced a larger increase
in reproductive bud production than either treatment alone, but
the complementary effects were less during the warm dry
summer in 1991 than during the wet summer in 1992. Cytokinin applications reduced the stimulatory effects of GA4/7 and
root pruning, and the degree of reduction varied with the
amount and timing of application. Reduced reproductive bud
production was attributed to cytokinin countering the effects
of root pruning and natural drought on the GA4/7 response.
Keywords: drought, flowering, reproduction.
Introduction
Increased cone production in conifers in response to exogenous gibberellic acid (GA) is well documented (Pharis et al.
1987, Bonnet-Masimbert and Zaerr 1987, Pharis 1991); however, despite recent refinements to methods of applying GA,
trees must be predisposed to respond, i.e., GA applications by
themselves often do not induce flowering in reproductively
immature (juvenile) trees, in recalcitrant genotypes, or when
other environmental conditions are unfavorable (see reviews in
Owens and Blake 1985, and Bonnet-Masimbert 1987). Several
cultural treatments, including fertilizing, root pruning, and
drought and heat stress, significantly increase tree responsiveness to GA (Tompsett and Fletcher 1979, Philipson 1983,
Owens and Blake 1985, Ross et al. 1985, Pharis et al. 1987,
Ross 1988, Owens and Simpson 1988, Owens et al. 1992), but
the mechanism(s) whereby cultural treatments increase GA
effectiveness remain largely unresolved.
In Picea, responsiveness to GA treatments is only increased
when root pruning, drought and soil heat treatments are timed
to coincide approximately with vegetative bud burst, early
shoot elongation and cessation of shoot elongation, respectively (Ross et al. 1985, Ross 1988), indicating that the treatments affect different stages of bud development. Root pruning
may delay bud development and thereby increase bud developmental synchrony such that a greater proportion of buds will
be responsive to GA4/7 at any particular time (Owens et al.
1992). Drought may increase the concentrations of cone-inducing GAs, e.g., GA4/7 and GA9 (Moritz et al. 1989, Moritz
and Odén 1990), by retarding their conjugation or hydroxylation or both (Chalupka et al. 1982, Dunberg et al. 1983, Pharis
et al. 1989). Soil heat treatments may increase flowering by
reducing export from the roots of a flowering antagonist
(Philipson 1983). Drought and soil heat stress could also induce changes in the metabolism of both GA and cytokinin
(Imbault et al. 1988, Pilate et al. 1990).
Cytokinin concentrations in xylem exudate are positively
correlated with water availability and root growth (Itai and
Vaadia 1971, Marschner 1986, Thimann 1992), and negatively
correlated with flower initiation in many herbaceous species.
Because the principal site of cytokinin synthesis is in the roots,
treatments such as irrigation that promote root growth and, by
inference, cytokinin production would be expected to reduce
or inhibit the response to GA4/7 (Kinet et al. 1993). The first
objective of this study was to test the hypothesis that the
increase in efficacy of applied GA4/7 with root pruning results
from reduced cytokinin export from the roots.
Although sexual zonation within conifer tree crowns is generally acknowledged (Marquard and Hanover 1984a, 1984b,
Ross and Pharis 1987), distribution patterns of reproductive
structures within the crown (Powell 1977, Caron 1987, Tosh
and Powell 1991, Caron and Powell 1992) and along and
around shoots (Tosh and Powell 1991, Caron and Powell 1993)
have received limited attention. Furthermore, although crown
position can have a profound effect on response to cone induction treatments, there are few reports on the effects of cone
induction treatments on seed and pollen cone distribution
patterns (Marquard and Hanover 1984a, 1984b, Philipson
1987).
The second objective of this study was to test the effects of
root pruning, ammonium nitrate (NH4NO3) and cytokinins on
the numbers and within-crown distribution of seed and pollen
cones produced by black spruce (Picea mariana (Mill.) B.S.P.)
in response to GA4/7.
458
SMITH AND GREENWOOD
Materials and methods
Study area and treatments
The study area is a black spruce seedling seed orchard located
70 km northeast of Fredericton, New Brunswick, Canada, that
was planted in 1983 by the New Brunswick Department of
Natural Resources and Energy. Soils are well-drained loamy
sands to sandy loams with moderate fertility (data on file).
In 1991, two experiments were established. Experiment 1
was a complete factorial and included controls (C), a fertilizer
treatment (F) in which 300 g of ammonium nitrate (34-0-0,
N,P,K) was applied in a band around the crown dripline, a
gibberellin treatment (GA) in which 10 mg of GA4/7 in 0.1 ml
of 95% ethanol was injected into the stem just below the 1989
whorl, and a root-pruning (RP) treatment in which tree roots
were cut to a depth of 25 cm on two sides of the stem at the
edge of the crown dripline. Experiment 2 was designed to test
the hypothesis that applying cytokinins (benzylaminopurine
(BAP), zeatin (Z) and zeatin riboside (ZR)) would reduce or
negate the GA-induced flowering response. The treatments
were C, GA, RP, Z1, ZR1, BAP7, GA + Z1, GA + ZR1, GA +
BAP7, GA + RP + BAP1, GA + RP + BAP3, and GA + RP +
BAP7 where the numeral after BAP represents the amount of
cytokinin injected in mg per 0.1 ml of 95% ethanol. Within
both experiments, one tree from each of 10 families was treated
on June 18--19, 1991, approximately 1 week after vegetative
bud burst.
In 1992, three experiments were established to evaluate the
effects of rate and time of cytokinin application on the response to GA4/7 and root pruning. In Experiment 1, the treatments were C, GA, RP, T1BAP1, T1BAP3, T1BAP7, T2BAP1,
T2BAP3, T2BAP7, GA + RP, GA + RP + T1BAP1, GA + RP
+ T1BAP3, GA + RP + T1BAP7, GA + RP + T2BAP1, GA +
RP + T2BAP3, and GA + RP + T2BAP7 where T1 and T2
represent early lateral shoot elongation (June 19--23) and cessation of shoot elongation (July 20) stages at which treatments
were applied, respectively. The same experimental design was
used to test the effects of zeatin and zeatin riboside; however,
for these trials only 0.1, 0.5 and 1.0 mg cytokinin were used,
and the application of cytokinin without GA + RP was omitted
on July 20. Injections were made just below the 1989 whorl.
Each treatment was given to 10 trees, one from each of 10
families.
Growth measurements
In 1991, growth of the leader (L), one lateral shoot located in
the middle one-third of the previous year’s leader (LB), the
terminal shoot (WL), and one first-order lateral branch (WB)
from one 1990 whorl branch were measured twice per week.
In 1992, weekly observations were made on 30 trees, one tree
from each of the 30 families used. Total tree height and diameter at the hormone injection point were measured on all trees.
Cone counts and data analyses
Numbers of seed and pollen cones, by position within the
crown, were determined in both 1992 and 1993. Count data
were normalized by the square root (count + 1) transformation
(Snedecor and Cochrane 1980). Tree damage and mortality
resulted in dropping some plot trees leading to a small data
imbalance (n = 8 to 10 per treatment); therefore, Type III sums
of squares (SS) (GLM procedure, SAS Institute Inc., Cary,
NC) were reported. Standard analysis of covariance (ANCOVA), with tree diameter as the covariate, was used after
testing for homogeneity of slopes (Hendrix et al. 1982). Partitioning of main treatment effects and corresponding hypothesis testing was done by contrasts (Snedecor and Cochrane
1980). The specific hypotheses tested are reported in the ANCOVA tables.
Results
Effects on cone production
Exogenous GA4/7 induced 7-, 4- and 9-fold increases in seed
cone production in the 1991 fertilization, 1991 cytokinin and
1992 BAP trials, respectively (Figures 1A, 2A and 3A). In the
1992 zeatin and zeatin riboside trials, the more than 50-fold
increase in seed cone production in response to GA4/7 was
partly due to the small numbers (mean < 2) of seed cones
produced by the control trees (Figures 3C and 3E). Gibberellin
increased the numbers of pollen cones by two to six times
(Figures 1B, 2B, 3B, 3D and 3F). Based on ANCOVA, GA4/7
effects on seed cone production were significant for both years,
whereas GA4/7 effects on pollen cone production were only
significant in the 1991 cytokinin and 1992 BAP experiments
(Tables 1 and 2).
In both years, root pruning alone had little effect on seed or
pollen cone production, although root-pruned trees in the 1991
fertilization and 1992 BAP trials produced more pollen cones
than the controls (Figures 1B and 3F). The response to the
combined RP + GA treatment differed between 1991 and
1992. In 1991, trees in the RP + GA treatment produced the
same or slightly more seed and pollen cones than trees receiving GA4/7 alone, whereas in 1992, except for seed cones in the
1992 zeatin riboside trial, trees in the RP + GA treatment
produced significantly more cones of both sexes than trees
receiving GA4/7 alone (Figures 1--3). Applying ammonium
nitrate with GA reduced the stimulatory effect of GA4/7 on
pollen cone production (Figure 1B).
In 1991, applying cytokinin alone had no significant effect
on seed cone production, whereas injecting as little as 1 mg of
zeatin or zeatin riboside with GA4/7 significantly reduced pollen cone production relative to trees receiving GA4/7 alone
(Figure 2B). All three BAP treatments reduced the increase in
pollen cone production in response to the combined root pruning + GA treatment in 1991 (Figure 2B). In 1992, adding BAP
or zeatin riboside negated the synergistic increase in pollen
cone production in response to the combined RP + GA treatment (Figure 3). The reductions in seed and pollen cone production were greater when cytokinin application coincided
with the cessation of shoot elongation (T2) than with the time
of bud flush (T1). The mean numbers of seed and pollen cones
were not significantly different between trees receiving the
GA + RP + Z treatment and trees receiving the combined RP
+ GA, confirming the lack of effect of zeatin on flowering in
SEED- AND POLLEN-CONE PRODUCTION IN BLACK SPRUCE
Figure 1. Mean numbers of and standard errors for (A) seed and (B)
pollen cones produced in 1992 following fertilizer application (F),
root pruning (RP) and GA4/7 injection (GA) in 1991.
1992. Pollen cone production was significantly reduced in all
three BAP treatments and at both T1 and T2, and seed cone
production decreased with increasing amounts of BAP applied. The differences in numbers of seed cones were statistically significant for 3 and 7 mg BAP at T2.
In both 1992 and 1993, control trees produced either the
same or a slightly greater number of pollen cones than seed
cones. Pooling the data from all the experiments indicated that
applying GA4/7 alone more consistently increased numbers of
seed cones than of pollen cones, altering the sex ratio (female
to male) from 1/1 to 3/1. The combined RP + GA treatment
resulted in sex ratios of 2.5/1 and 1/1 in 1992 and 1993,
respectively.
Effects on cone distribution
In control trees, seed cones were produced predominantly on
first-order laterals of the previous year’s leader and its subtending whorl, whereas pollen cones were located on the whorl and
internodal whorl branches immediately below. Applying GA4/7
459
Figure 2. Mean numbers of and standard errors for (A) seed and (B)
pollen cones produced in 1992 for control trees (C) and trees receiving
stem injections in 1991 of 10 mg GA4/7 (GA) alone and in combination
with root pruning (RP) and cytokinins (1 mg zeatin (Z1), 1 mg zeatin
riboside (ZR1), and 1, 3 and 7 mg benzylaminopurine (B1, B3 and B7,
respectively)).
increased the proportions of buds within the upper crown
zones developing reproductively but did not affect the locations within the crown where seed and pollen cones were borne
(Figures 4A and 4C). In 1991, root-pruning increased the
magnitude of the response to GA4/7, but did not affect cone
distribution. In 1992, root pruning not only increased the
numbers of cones produced in response to GA4/7, but expanded
the zone producing significant numbers of pollen cones upward relative to control and GA4/7-treated trees (Figure 4C).
Cytokinins, especially BAP, decreased the numbers of cones
of both sexes above the injection point. The magnitude of
reduction decreased upward from the injection point, hence
pollen cone production was reduced proportionally more than
seed cone production. Reductions in numbers of cones in
response to zeatin and zeatin riboside, although smaller than
those observed for BAP, also diminished acropetally from the
460
SMITH AND GREENWOOD
Figure 3. Mean numbers of
and standard errors for seed
(A, C and E) and pollen
cones (B, D and F) produced
in 1993 for control trees (C)
and trees receiving stem injections in 1992 of 10 mg
GA4/7 (GA) alone and in
combination with root pruning (RP) and benzylaminopurine (BAP) (A and B),
zeatin (Z) (C and D), and
zeatin riboside (ZR) (E and
F). Cytokinin was injected
on either June 19--23
(TIME 1) or July 30
(TIME 2).
SEED- AND POLLEN-CONE PRODUCTION IN BLACK SPRUCE
461
Table 1. Mean squares and significance levels for the 1991 trials. Model sums of squares were partitioned as follows: FERT91 = complete factorial
with main effects (C, FERT, GA, RP), two-way interactions (FERT × GA, GA × RP, FERT × RP) and three-way interaction (FERT × GA × RP);
CYTO91 = main effects (C, GA, RP, Z1, ZR1, BAP7), GA × CYTO (GA + Z1, GA + ZR1, GA + BAP7), GA + RP(BAP) (GA + RP + BAP1,
GA + RP + BAP3, GA + RP + BAP7), and the two contrasts, GA versus (GA × CYTO) and GA versus GA + RP(BAP) where the number after
cytokinin represents mg of cytokinin injected. The contrast ‘GA versus others’ within themain treatments is provided.
Treatment
Seed cones
MS
P
MS
P
1
3
3
1
77
85
292.65
226.56
136.81
61.42
18.31
0.0001
0.0001
0.0001
ns
140.72
13.49
13.57
56.91
13.48
0.0018
ns
ns
ns
1
5
(1)
2
2
1
1
99
111
21.92
52.09
245.54
58.7
26.61
82.12
569.6
15.31
ns
0.0070
0.0001
0.0249
ns
0.0226
0.001
13.31
22.96
97.02
3.54
2.33
90.18
768.39
9.95
ns
0.0499
0.0024
ns
ns
0.0033
0.001
df
FERT91
Diameter (covariate)
Main effects
Two-way
FERT × GA × RP
Error
Total
CYTO91
Diameter (covariate)
Main effect
GA versus others
GA × CYTO
GA + RP(BAP)
GA versus (GA × CYTO)
GA versus GA + RP(BAP)
Error
Total
Pollen cones
Table 2. Mean squares and significance levels for the 1992 trials. Sums of squares (SS) were partitioned as follows: BAP92 = main effect (C, GA,
RP, T1BAP1, T1BAP3, T1BAP7, T2BAP1, T2BAP3, T2BAP7), GA + RP(BAP) (GA + RP + T1BAP1, GA + RP + T1BAP3, GA + RP + T1BAP7,
GA + RP + T2BAP1, GA + RP + T2BAP3, GA + RP + T2BAP7), and the contrast GA versus GA + RP, where T1 and T2 are time of cytokinin
application (June 19--23 and July 20, 1992, respectively) and the number after BAP represents mg of BAP injected. The same coding and
partitioning of SS was used for Z92 and ZR92 except that the T2 applications of cytokinin without GA + RP were not included.
Treatment
Seed cones
Pollen cones
df
MS
P
MS
P
BAP92
Diameter (covariate)
Main effect
GA versus others
GA + RP(BAP)
GA versus GA + RP
Error
Total
1
8
(1)
6
1
143
159
439.37
57.27
404.79
136.55
291.55
14.36
0.0001
0.0003
0.0001
0.0001
0.0001
106.17
111.41
100.27
14.55
432.70
14.82
0.0083
ns
0.0103
ns
0.0001
Z92
Diameter (covariate)
Main effect
GA versus others
GA + RP(Z)
GA versus GA + RP
Error
Total
1
5
(1)
6
1
117
130
100.12
40.4
198.92
60.40
464.98
11.94
0.0001
0.0069
0.0001
0.000
0.0001
176.74
1.55
0.61
53.23
150.00
10.79
0.0001
ns
ns
0.00
0.0001
ZR92
Diameter (covariate)
Main effect
GA versus others
GA + RP(ZR)
GA versus GA + RP
Error
Total
1
5
(1)
6
1
112
125
102.36
96.02
470.60
66.31
226.02
16.79
0.0150
0.0001
0.0001
0.00
0.0001
89.06
24.10
96.36
43.08
261.69
29.24
0.0837
ns
0.0
ns
0.0001
462
SMITH AND GREENWOOD
Figure 4. Mean numbers of seed and pollen cones in 1992 (A and B) and 1993
(C and D) by crown position for control
trees (C) and trees receiving stem injections of 10 mg of GA4/7 (GA) alone and
in combination with root pruning (RP)
and RP + GA plus 1, 3 or 7 mg benzylaminopurine (B1, B3 and B7, respectively). Crown position coding: 1990
whorl branches = W90, internodal
branches between the 1990 and 1991
whorls = IB90-91, etc. Point of hormone
injection is indicated by an arrow.
injection point (Figures 4B and 4D).
Effects on growth
In 1991, treatments did not affect either the final length of any
of the shoots measured or their rate of elongation, although
mean total height, diameter and leader length were less for the
trees receiving zeatin than for trees in the other treatments.
Tree height, diameter and final shoot lengths were positively
correlated. There were no significant differences in mean shoot
length measurements between experiments.
The pattern and duration of first-order lateral shoot growth
were similar to those reported for black spruce: a 4-week
period of rapid shoot elongation (mid-June to mid-July), followed by 1 week of slow growth (Caron 1987, Ho 1988, 1991).
Cessation of shoot elongation progressed acropetally within
the crown and along shoots, with leaders stopping elongation
growth by August 15. Time and duration of shoot growth
stages were similar between the 2 years, although the period of
rapid shoot growth was approximately 1 week longer in 1992
than in 1991.
Relationships between weather, growth and cone production
Five to 10 times more seed and pollen cones were produced by
control trees in 1992 than in 1993. There were two main
differences in weather patterns between 1991 and 1992: (1)
total degree days (5 °C base) were higher in 1991 than in 1992
throughout most of June and July, and differences were greatest at the time of cessation of lateral shoot elongation in midto late-July; and (2) there was approximately twice the amount
of precipitation from mid-June to early-August in 1992 than in
1991.
In the 1991 trials, there was little variation within and among
the 20 families in lateral shoot growth phenology. Treatment
efficacy was not significantly correlated with any of the growth
measurements or developmental observations. Similarly, differences among families in seed and pollen cone production
were not related to phenology but rather to tree size; cone
production was positively correlated with tree height and diameter (Table 3), and presumably with numbers of shoots/buds
(potential flowering sites).
Discussion
Injections of GA4/7 significantly increased the proportions of
buds developing reproductively above the injection point.
Similar results have previously been reported for Picea (Marquard and Hanover 1984a, Philipson 1987). Comparable increases in numbers of seed cones were observed in both years.
However, in 1992, GA4/7 was injected one whorl lower than in
1991, thereby including branches within the pollen-cone-bearing zone and producing a concomitant increase in pollen cone
production, which is contrary to the preferential GA4/7 induc-
SEED- AND POLLEN-CONE PRODUCTION IN BLACK SPRUCE
Table 3. Pearson correlation coefficients between tree height and
diameter and seed and pollen cone production for all trees in the 1991
(n = 198) and 1992 (n = 417) trials.
Seed cone
Pollen cone
1992 Cones
Height
Diameter
0.212*1
0.150*
0.238***
0.177*
1993 Cones
Height
Diameter
0.182***
0.222***
0.133***
0.142**
1
*** = Significant at 0.001, ** = significant at 0.01, * = significant
at 0.05.
tion of seed over pollen cones reported by others (Ross and
Pharis 1987, Ho 1988, Ross 1990, Greenwood et al. 1991, Ho
1991). Our results support the hypothesis that GA4/7 has a role
in determining sexual versus vegetative development
(Chalupka 1980), although they do not preclude the possibility
that sex determination is controlled by other factors, e.g., the
ratio of gibberellins to auxins (Pharis and Kuo 1977).
Control trees produced seed cones almost exclusively on
lateral branches along 1-year-old shoots. Caron and Powell
(1993) reported similar findings for plantation-grown black
spruce trees aged 9 to 11 years. Exogenous GA4/7 alone had
little effect on the location of seed- and pollen-cone-bearing
branches within the tree crown, whereas the combined RP +
GA treatment resulted in appreciable overlapping of the male
and female zones on the internodal branches (IB) two whorls
back (IB89-90 and IB90-91 in 1992 and 1993, respectively)
(Figures 4A and 4C). Sexual zonation is generally less pronounced in good flowering years and in response to successful
cone induction treatments than in poor flowering years (Ross
and Pharis 1987).
Tompsett and Fletcher (1979) proposed a bud vigor gradient
hypothesis for Picea sitchensis (Bong.) Carr. in which, in order
of decreasing vigor, buds would develop into strong vegetative,
female, intermediate vegetative, male, and weak vegetative
structures, and that for young trees in which strong vegetative
shoots predominate, stress treatments would favor seed over
pollen cone induction (i.e., a downward shift of one vigor
class). We found that root pruning increased the response to
GA4/7 for both seed and pollen cone production, although in
1993, the pollen cone production response was greater than the
seed cone production response, indicating a possible shift from
intermediate vegetative to male buds. Vegetative shoot vigor in
conifers generally decreases in a basipetal direction between
branches within the crown, shoots along branches, and buds
along shoots (Powell 1977, Remphrey and Powell 1984, Caron
1987). Although bud vigor based on relative position along
shoots may help describe sexual zonation within a tree crown,
it does not explain how cone induction treatments affect the
correlative development between buds along shoots.
Reduced root activity is often related to successful flower
induction (Bonnet-Masimbert et al. 1982, Zaerr and BonnetMasimbert 1987). Although root pruning increased the magni-
463
tude of the response to GA4/7, it did not increase seed cone
production in either year, or pollen cone production in 1992.
The failure of the root pruning treatment to elicit a response in
1991 may be attributed to a combination of root pruning and
dry weather during most of the active shoot growth period in
1991 resulting in a water stress that exceeded what is optimal
for flower induction. A combined root pruning + heat stress
treatment had an adverse effect on both seed and pollen cone
production in potted black spruce (Ho 1991).
In 1992, cytokinins countered the complementary effect of
root pruning on the GA4/7 response. This effect differs from the
effects found for Sitka spruce (Tompsett 1977) and Douglas-fir
(Ross and Pharis 1976, Zaerr and Bonnet-Masimbert 1987). In
1991, seed and pollen cone production decreased in response
to increasing amounts of cytokinin applied with or without
GA4/7, whereas in 1992, trees receiving cytokinin alone produced similar numbers of cones of both sexes as the control
trees. Flowering was greater in 1992 than in 1993, as a result
of the warm dry weather in 1991.
Morphogenetic transitions, such as the shift from vegetative
to reproductive development, are usually associated with specific changes in gene expression (Bernier 1989, Meeks-Wagner et al. 1989). The expression of flowering genes following
induction treatments is reversible, e.g., proliferated and bisexual cones in black spruce (Caron and Powell 1990, 1991),
indicating that environmental conditions must continue to be
favorable for the continued expression of these genes. In 1992,
application of cytokinin either at the same time as GA4/7 and
root pruning, or 4 weeks later, reduced seed and pollen cone
production, although the magnitude of the response varied
with the type and amount of cytokinin used. The presumed
expression of flowering genes was reversible up to cessation of
shoot elongation, the time of bud determination in black spruce
(Ho 1988) and Picea in general (Owens and Blake 1985). Our
observations provide circumstantial support for the hypothesis
that the concentration of GA4/7 to which cells within developing buds are exposed and the duration of that exposure determine if the bud develops reproductively. Applying exogenous
GA4/7 increases the potential for cells within buds to be exposed to the ‘cone-inducing’ form of GA. Root pruning reduces, at least temporarily, cytokinin export from the roots,
thereby either (1) slowing cell cycling within developing buds
and increasing their period of receptivity to both endogenous
and exogenously applied GA4/7, or (2) relieving the bud of the
presence of cytokinins, if inhibitory to flowering.
None of the treatments affected shoot growth in the year of
treatment, which confirms other studies in which shoot elongation and flowering responded independently (Webber et al.
1985, Pharis et al. 1987). Although flowering was positively
correlated with reduced shoot elongation in Douglas-fir (Pilate
et al. 1990), the effects of girdling, root pruning and drought
were probably manifested as a temporary delay in shoot elongation and bud development (Tompsett 1978, Owens et al.
1985, Owens 1987) rather than a reduced amount of growth.
In Sitka spruce, the best response to cone induction treatments
occurred when apical mitotic indices were retarded, thereby
shifting differentiation back to coincide with cessation of shoot
464
SMITH AND GREENWOOD
elongation (Owens and Simpson 1988). Root pruning may
inhibit or enhance shoot elongation depending on whether GA
(Ross 1991a, 1991b) or other hormone production from regenerated roots exceeds that from roots before pruning.
In 1991, fertilization with NH4NO3 did not increase either
seed or pollen cone production in 1992, contrary to results with
black spruce (Smith 1986, 1987, 1993) If NH4NO3 increases
flowering at least partly through a rooting effect, then a lack of
flowering response to root pruning in 1991 would be expected.
The slight reduction in pollen cone production for trees receiving fertilizer and GA4/7 compared with GA4/7 alone may be
partly due to increased root growth (Marschner 1986) and
increased cytokinin production in the roots following fertilization. Betula pendula Roth. seedlings grown under nitrogen
deficient conditions exhibit lower concentrations of endogenous cytokinin than seedlings grown under nondeficient
conditions (Horgan and Wareing 1980).
Conclusion
We obtained circumstantial evidence that cultural treatments
increase efficacy of GA4/7 applications by temporarily reducing cytokinin export from the roots. Reduced cytokinin export
may result in a reduction in cell division of the developing buds
with the slowly cycling cells being more receptive to GA4/7,
and reduced cytokinin concentrations within the developing
bud may ensure continued expression of the flowering genes.
Acknowledgments
The authors thank Ms. K. Tosh of the New Brunswick Department of
Natural Resources and Energy for cooperation in providing access to
the orchard trees, and Dr. Y.S. Park, Dr. C.H.A. Little, Dr. J. Loo and
Mr. J.D. Simpson for reviewing the manuscript. Special thanks to Ms.
L. Yeates for technical assistance during all phases of this work.
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