Tree Physiology 15, 419--426

1995 Heron Publishing----Victoria, Canada

From flower induction to seed production in forest tree orchards

M. BONNET-MASIMBERT1 and J. E. WEBBER2
1

INRA, Station dAmlioration des Arbres Forestiers, Ardon 45160, France

British Columbia Ministry of Forests, Glyn Road Research Station, 1320 Glyn Road, Victoria, B.C. V8W 3E7, Canada

Received August 10, 1994

Keywords: flowering, fruit development, gibberellins, hormones, pollen, seed orchard.

Introduction
Seed orchards are widely used to produce superior seed for
breeding programs and silvicultural purposes. However, classical seed orchard designs suffer from many constraints including high initial investment costs with a slow incorporation of
genetic gain because of the long juvenile period and inconsistent flowering. Even in clonal orchards established with sexually mature scions, significant seed production does not
normally occur for 6--8 years. The initial wide spacing of
clonal orchards makes roguing difficult without severely affecting the distribution and parental contribution of the remaining clones. Finally, poor or irregular flowering is often
observed in orchards that are not located on good flowering
sites, and even on good sites, micro-site influences are important (Sweet 1992).
In an attempt to overcome the limitations of classical seed
orchards, several alternative orchard designs have been developed including meadow or hedged orchards (Carson et al.
1992, Sweet et al. 1992) and indoor or container orchards.
Container orchards have been used successfully for birch
breeding and seed production (Tyystjarvi and Pirttila 1984)
and are under development for some conifers (Greenwood et
al. 1979, Ross et al. 1986, Eysteinsson et al. 1993 ). All of the
alternative orchard designs have the potential for fast incorporation of genetic gain and flexibility in introducing improved
genotypes. The container orchards exhibit more consistent
flowering than classical orchards because it is possible to
accelerate maturation in them, by rapidly increasing crown

size and complexity, and to control temperature and water

availability during induction, pollination and seed cone maturation. It should also be easier to study environmental effects
on parental development (Johnsen 1989a, 1989b), reproductive development and fertilization (Johnsen et al. 1995) in
container orchards than in classical orchards. In addition to
orchard design, specific improvements to orchard seed production include the use of induction treatments, supplemental
mass pollination and crop protection (frost and insect control).
Seed orchards are not the only delivery tool for tree improvement. Asexual propagation techniques, including micropropagation (e.g., Sutton et al. 1993 for white spruce (Picea glauca
(Moench) Voss)), have been developed, but the cost per
propagule is high. Both rooted cuttings (Ritchie 1991), and the
more exotic emblings obtained from somatic embryogenesis
(e.g., Webster et al. 1990 on Picea and Lelu et al. 1994 on
Larix) are close to operational production.
Stimulation of flowering
Our ability to manipulate cone and seed production is still
determined largely by juvenility and environmental conditions. Furthermore, the mechanisms underlying cone induction
techniques remain largely unknown. A few studies have detailed the role of specific hormones in flowering (Moritz 1989,
Pharis 1991), but the mechanism controlling the first few steps
of flowering has not been elucidated (Pharis et al. 1987).
There are many examples of successful flower induction
treatments in conifers (Wheeler et al. 1985, Ross and Bower
1989, Wheeler and Bramlett 1991, Ross 1991). Flower induction treatments range from stem girdling, root pruning, heat
treatments (within controlled environment facilities) and general fertilizer treatments to specific hormone applications (see
reviews by Bonnet-Masimbert 1987, Owens 1991). Eucalyptus sp. exhibit a flowering response to anti-GAs, like paclobutrazol (Cauvin 1992, Griffin et al. 1993).
It has been suggested that roots play a key role in flower
induction (Bonnet-Masimbert et al. 1982, Bonnet-Masimbert
and Zaerr 1987) because root pruning promotes flower induction, and other flower induction treatments, including stem
injection of GAs, reduce root activity (Figure 1). However,
little has been done to characterize the effects of rootstock on
flowering (Schmidtling 1983), a task complicated by the fact

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Summary We review developments in cone and seed production research during the past decade. We conclude that
although cone induction techniques, including hormonal and
cultural procedures, have been refined, they still do not fully
overcome juvenility or noninductive environmental effects.
The role pollen plays in enhancing seed production and the
genetic quality of seed is discussed. Techniques for handling
pollen are described, in vitro viability assays are reviewed, and
we also examine recent developments in pollen collection and
its artificial ripening.

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BONNET-MASIMBERT AND WEBBER

Figure 1. Effects of GA4 and GA7, with or without stem girdling, on

root growth of 6-year-old Douglas-fir cuttings. Root growth is expressed as the number of actively growing roots at Ti over its number
at bud burst (T0). Bars with an asterisk (*) are significantly different
at P < 0.05 from the control.

Figure 2. Effects of GA4/7 spray, stem girdling or root flooding on the

isopentenyladenine concentration of shoots and roots of 4-year-old
Douglas-fir cuttings (adapted from Lucas et al. 1991 and Hattier,
unpublished data).

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that grafting stimulates flowering independently of the quality

of the rootstock.
Nitrogen fertilization is widely used but we do not know
whether nitrogen is important for induction, bud development,
or facilitating the steps from flower to fruit or seed maturation.
Daoudi et al. (1991, 1994) studied the effects of nitrate fertilization (N) with or without a stem injection of GA4/7 on flowering in 6- to 8-year-old cuttings of Douglas-fir (Pseudotsuga
menziesii (Mirb.) Franco). No control trees flowered, and the
maximum flowering response was observed in the N + GA4/7
treatment. As observed in previous studies, N increased total
amino acids, especially arginine (e.g., Barnes and Bengston
1968, Ebell and McMullan 1970) and putrescine (e.g., Barnes
and Bengston 1968, Cabanne et al. 1977, Rohozinski et al.
1986). The combined N + GA4/7 treatment had a synergistic
effect on the increase in arginine, proline, putrescine, spermidine and spermine content of the shoots. The increased content
of nitrogenous compounds was observed for up to 10 weeks
after the combined treatments were applied, including the
period when flower-bud initiation occurred.
There is increasing evidence that specific GAs and their
metabolites are involved in flowering (Moritz 1989, Pharis
1991). A mixture of the less polar GAs, GA4 and GA7, induces
flowering in Pinaceae species, whereas GA3 is commonly used
for Cupressaceae and Taxodiaceae species. Doumas et al. (unpublished results, cited by Pharis 1991) observed higher concentrations of GA4 and GA7 in shoots and primordia of
flowering Douglas-fir than in similiar organs of nonflowering
Douglas-fir. Primordia with a high potential to flower have 50
to 80 times more GA7 than primordia on nonflowering
Douglas-fir trees (16 ng g 1 dry weight (DW) for control trees).
Root pruning applied in the absence of an exogenous application of GA4/7increased the amount of GA7 present in primordia
(188 versus 831 ng g 1 DW, respectively). When both root
pruning and GA4/7 treatments were applied together, a strong
synergistic increase in GA7 was observed (1264 ng g 1 DW).
Gibberellin treatments are normally applied in combination
with other induction treatments because the combination often
results in a synergistic response.
Other phytohormones, including cytokinins may also be

involved in flowering. Induction of flowering by root flooding

with or without GA4/7 stem injection results in an increase in
the concentration of endogenous isopentenyladenine (iPA) in
Douglas-fir shoots 3--6 weeks after bud burst (Imbault et al.
1988, Pilate et al. 1990). Moreover, an exogenous application
of iPA stimulates the female flowering response on lateral
shoots of potted Douglas-fir trees (Imbault et al. 1988). Single
stem girdling, root flooding or GA4/7 spray treatment applied
to 4-year-old cuttings of Douglas-fir at bud burst decreased the
iPA concentration of roots and increased the iPA concentration
of lateral shoots compared to the controls (Figure 2) (Lucas et
al. 1991, Hattier, unpublished observations), suggesting either
modified cytokinin biosynthesis and metabolism, or modified
transport.
Treatments that stimulate flowering usually increase, by 30
to 75%, the number of developing axillary meristems of all
types: latent, sexual or vegetative (e.g., Bonnet-Masimbert
1988). Similar increases in axillary meristems have been observed between good and poor flowering years (Allen and
Owens 1972). These observations support Greenwoods
(1981) conclusion that the first effect of flower induction
treatments is to stimulate development of lateral apices. The
second step is the growth of these apices and their conversion
to sexual buds. Greenwoods conclusion and the finding that
combinations of hormonal and cultural treatments are often
synergistic suggest that the hormonal and cultural treatments
affect different stages of the flowering process. Some cultural
treatments might create a delay between vegetative development (shoot elongation and possibly root growth) and bud
development. Owens et al. (1986) observed that, in Douglasfir, root pruning retards the rate of axillary bud development
during shoot elongation. Normal bud activity resumed about

TREE FLOWERING FROM INITIATION TO SEED PRODUCTION

mid-July, and trees that exhibited the most severe retardation

in bud development had the heaviest flowering.
Pollen

Pollen collection
Pollen is collected by either hand-picking cones or artificially
maturing cut branches. Mass pollen collection using vacuum
systems has been successfully used in Douglas-fir orchards
(Copes et al. 1991). One person using this device can collect
1.5 l of pollen per hour from 10--15-m tall trees. Pollen viability of the vacuum-collected pollen is as good or better than that
of naturally shed pollen. An expensive system developed by
Philippe and Baldet (1992) for Larix sp. is useful for species
with limited pollen production and for species for which it is
difficult to force unripe male buds. The entire tree is tightly
enclosed in a tank fixed to a tractor. Branches are whipped by
blasting compressed air in semirigid pipes, and the pollen
shaken from male strobili is sucked through a filtering system.
Because the collection only takes about 1.5 min for a 4-m tall
tree, several collections can be made during the pollen maturation phase of the tree.

Artificial pollen ripening

There are a few studies on the effects of artificial ripening on
pollen yield and fertility. Colangeli and Owens (1991) studied
the cytological effects on forcing western hemlock (Tsuga
heterophylla (Raf.) Sarg.) pollen buds in water at room temperature. Forcing buds too early (i.e., before meiotic activity)
resulted in reduced yields and reduced fertility potential. Ross
(1988) also found lower yields per cone, increased proportions
of underdeveloped pollen and poorer pollen quality when
pollen cone development was hastened by 21 days by subjecting potted Engelmann spruce (Picea englemannii Parry ex
Engelm.) stock to elevated temperature and humidity in a
closed polyethylene-covered greenhouse. Philipson et al.
(1990) exposed potted Sitka spruce (Picea sitchensis (Bong.)
Carr.) grafts to an elevated temperature when the pollen-cone
buds were at their premeiotic stage and found that the temperature treatment hastened cone-bud development but reduced
both pollen yield and viability. They attributed the poor pollen
viability to abnormalities in the latter stages of pollen development (not meiosis) and the poor seed development to retarded
development of the distal portion of the cones.
An important step in evaluating the effects of forcing pollen
has been the development of reliable methods for assessing the
amount of extracted pollen. Extraction efficiency or the ratio
of extracted pollen volume to pollen-cone bud volume is the
most widely used method. Extraction efficiency values range
from 5--6% for larch, 4--6% for Douglas-fir, 10--15% for
lodgepole pine (Pinus contorta var. latifolia Engelm. ex S.
Wats.), and up to 20% for spruce (Webber, unpublished observations). However, the actual volumes realized depend on the
stage of elongation of the catkins or strobili. Generally, the
greater the space between the microsporangia, the greater the
volume extracted; however, the characteristics of pollen grains
and the amount that can be expected from one male bud are
species specific. Table 1, adapted from Stanley and Linskens
(1974), gives examples of such variation.
When defining conditions for artificially maturing catkins to
dehiscence, it is important to consider temperature, relative
humidity, light, shoot water relations and nutrients, either from
the shoot or within the culture solutions for cut branches
(Stanley and Linskens 1974). In an experiment (summarized
in Table 2) by Mellerowicz (1983), the effects of two environmental conditions (greenhouse and laboratory) with different
values and amplitude of temperature and relative humidity
were assessed in terms of pollen viability and amount of

Table 1. Characteristics of pollen size and pollen yield for some conifer and broad-leaved species (adapted from Stanley and Linskens 1974).
Species

Length
(m)

Width
(m)

Volume of 1 million
pollen grains (10 3 ml)

Weight of 1 million
pollen grains (10 3 g)

Volume of pollen per

100 catkins or strobili (ml)

Pinus
Pseudotsuga
Larix
Alnus
Betula
Fagus

41.5
84.8
76.0
26.4
10.1
55.1

45.9
81.1
72.0
22.8
10.1
40.5

35.5
219.2
180.2
4.4
2.9
50.3

37.0
188.8
176.3
1.4
0.8
26.0

150
2
0.3
4
12
1.3

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Pollen is a powerful tool for manipulating the genetic composition of production seed lots. To exercise maximum control
over contributing pollen parents, some form of pollen handling
ex situ is necessary. There are several steps in handling pollen
ex situ that are critical to maintaining pollen fertility (Stanley
and Linskens 1974, Franklin 1981, Owens and Blake 1985,
Blackmore and Knox 1990, Webber 1991, Jett et al. 1993,
Bramlett et al. 1993, Webber and Painter 1995). Both stored
and freshly collected pollen can be used in small-lot breeding
crosses or large volume supplemental mass pollination (SMP)
programs in seed orchards. However, competing pollen cloud
density, application technique and pollen viability all affect
SMP success (Webber 1995).
The advantages of increasing or controlling pollen supply to
open pollinated orchards have been well documented (Bridgwater and Trew 1981, Bridgwater et al. 1993). Supplemental
mass pollination has been used to improve genetic efficiency
(El-Kassaby and Ritland 1986a, Askew 1992, Bridgwater et al.
1993) and to reduce the effect of contaminating pollen (ElKassaby and Ritland 1986b). However, where paternal analyses have been completed, the effectiveness of SMP has been
generally low (Wheeler and Jech 1985, El-Kassaby et al. 1993,
Eriksson et al. 1994, Webber 1995).

421

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BONNET-MASIMBERT AND WEBBER

Table 2. Effects of environmental conditions on maturation and shedding of Alnus glutinosa pollen (from Mellerowicz 1983): G = Greenhouse
(temperature variation (T) = 13.9 and 24.8 C (average 16.8 C), relative humidity variation (RH) = 35 to 70%), L = Laboratory (T = 14.4 to 19.2 C
(average 17.4 C), RH = 41 to 53% (average 46.5%)), FCR+ = positive response to fluorochromatic viability test.
Conditions during
catkin elongation

Conditions during
pollen dehiscence

Amount of pollen
per catkin (mg)

Viability
(% FCR+)

G
L
G
L

G
G
L
L

17.7
20.3
9.7
3.3

77.4
77.9
88.0
85.1

Pollen testing
In vitro pollen viability is a useful measure of the potential of
pollen to set seed in a control cross-pollination. However, the
in vitro pollen viability of individual pollen lots used within a
pollen mix (i.e., polymix mating) cannot be used to predict
mating probabilities of individual lots. The assumptions that
all male parents have the same probability of mating and that
paternal contributions to a polymix mating are equal are seldom valid (Moran and Griffin 1985, Schoen and Cheliak 1987,
Wiselogel and van Buijtenen 1988). Differential male success
occurs and can have both a physiological and a genetic basis
(Apsit et al. 1989). Even for equally viable pollen lots, the
importance of prezygotic events affecting the competitive ability of pollen during germination within the micropyle, tube
penetration of the nucellus and egg cells, and archegonial
development must be considered (Nakamura and Wheeler
1992, Crook and Friedman 1992). Little is known about either
paternal--maternal interactions or environmental effects on fertilization and seed development in conifers.
Pollen competition has been described in various angiosperm species (Snow and Spira 1991), and variability in paternal performance can lead to nonrandom mating. Because of the
pollen population size, gametophytic selection may influence
traits such as tolerance to biotic and abiotic stresses and plant
vigor (Ottaviano and Mulcahy 1989). It may be that pollen
competition and gametophytic selection do not occur in conifers because few pollen grains actually compete at the micropyle or archegonial level; however, when paternal
genotypes compete at the tree or orchard level, the number of
pollen grains involved is large.
There are numerous tests of in vitro pollen viability (see
Stanley and Linskens 1974) and some tests provide a direct
measure of pollen fertility (Moody and Jett 1990, Webber and
Bonnet-Masimbert 1993). Assays include germination on an
artificial medium, conductivity measurements of pollen
leachates (Ching and Ching 1976), respiration of pollen in an

aqueous solution (Binder and Ballantyne 1975), and the

fluorochromatic test (Heslop-Harrison and Heslop-Harrison
1970). For most of these tests, the pollen must be preconditioned (i.e., hydrated) because a minimum pollen water content is critical for membrane continuity and enzyme activity.
Under natural conditions, pollen hydration is essential as the
first step toward germination (Heslop-Harrison 1987). Pollen
prehydration is necessary before in vitro germination occurs in
Douglas-fir (Charpentier and Bonnet-Masimbert 1983) or loblolly pine (Pinus taeda L.) (Jett and Frampton 1990). In
Douglas-fir, pollen prehydration improves conductivity measurements (lowers response), but it has little effect on respiration tests (Webber and Bonnet-Masimbert 1993). Actual assay
responses to prehydration effects vary with pollen water content (Charpentier and Bonnet-Masimbert 1983, Jett and
Frampton 1990) and are probably species specific.
There are several difficulties associated with interpreting the
results of in vitro pollen germination tests as exemplified in a
study of hydrated and nonhydrated Scots pine (Pinus
sylvestris L.) pollen (Figures 3A and 3B). Four germination
media were used: 10% Brewbaker and Kwack (1963) solution
used alone (BK10), BK10 with the addition of two concentrations of polyethylene glycol-4000 (P), 10% (BK10 + P10) or
20% (BK10 + P20), and BK10 with 10% sucrose (BK10 +
S10). Pollen was hydrated for 4 h at 20 C in an atmosphere
with 100% relative humidity. The nonhydrated pollen had an
ambient water content of about 8%. Three methods of germinating pollen (Stanley and Linskens 1974) in a dark incubator
at 20 C were compared (Figure 3C): (1) a spot test in which
pollen was dusted on the top of a drop (75 l) of medium on a
slide placed in a petri dish, (2) a hanging drop test made by
dusting the pollen on a drop as for method (1) and then
inverting the slide, and (3) a hanging drop (150 l) test in an
inverted Eppendorf tube.
After 24 h, pollen germination was determined by counting
those pollen grains having a tube length that was at least twice
the diameter of the hydrated, ungerminated grain. The morphology of pollen tube growth was classified in four groups
(Figure 3C): (1) pollen tubes that were straight and clear
without any branching (considered normal), (2) pollen tubes
that were clear but showed some branching, (3) pollen tubes
that were opaque (dark grains that are likely starch in the tubes)
and often had some branching, and (4) pollen tubes that were
straight and clear but exhibited swelling of the subterminal
part.
The results shown in Figure 3 demonstrate that the assay

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extracted pollen. The treatments were applied to cut branches

of Alnus glutinosa (L.) Gaertn. that bore male catkins at either
the elongation (ripening) or dehiscence phase. The more variable greenhouse conditions resulted in about six times more
pollen being extracted than the less variable laboratory conditions. Some clonal variability for the response to environmental conditions was also observed. The treatments had little
effect on pollen viability (measured by the fluorochromatic
test, Heslop-Harrisson and Heslop-Harrisson 1970).

TREE FLOWERING FROM INITIATION TO SEED PRODUCTION

423

fertility. For angiosperms, in which thousands of pollen grains

can adhere to the stigma, a different approach to relating in
vitro assay response to actual fertility is required than for
conifers where only a few grains can be accommodated within
the micropyle. Regardless of species, it should be possible to
relate each assay type to seed yield and thereby establish a
threshold value of pollen viability above which satisfactory
seed yields are predicted.
Pollen drying and storage

conditions had large effects on the in vitro germination response. There were significant interactions between germination technique (the spot test was the best), pollen prehydration
(Figures 3A and 3B) and germination medium. Pollen grains
in medium BK10 + S10 exhibited a high proportion of nonnormal tubes. Pollen germinated by the hanging drop technique
exhibited normal tube growth, whereas pollen germinated in
the Eppendorf tubes exhibited a high proportion of nonnormal
tubes.
Based on these observations, we conclude that the in vitro
germination response must be correlated to either in vivo
germination or seed set before it can be used reliably to predict
fertility. In loblolly pine, Moody and Jett (1990) found a high
correlation (R2 = 0.82) between in vitro pollen germination on
agar and in vivo pollen germination as well as a high coefficient of determination (r2 = 0.79) between in vitro germination
and percent filled seed per cone. Even if the germination
conditions are optimized (i.e., for tube growth) for each species, the germination response may not necessarily reflect
actual fertility under field conditions. Similar high correlations
were found for three viability assays, including germination,
and seed set for Douglas-fir (Webber and Bonnet-Masimbert
1993).
For conifers, the pollination mechanism, which can be wet
or dry, active or passive (Owens and Blake 1985), must be
considered when relating the results of a viability test to actual

Conclusion
The techniques for controlling flowering have been refined
during the past decade, despite a lack of fundamental knowledge about flowering mechanisms. However, there is increasing concern about the genetic quality and adaptability of seed
produced by seed orchards. Efforts are now being directed
toward understanding the interactions between environment
and genotypes at all stages of the reproductive process.

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Figure 3. Effects of germination medium and germination test on the

germination of hydrated (A) and nonhydrated (B) Scots pine pollen.
(C) Effects of germination medium on pollen tube morphology (BK10
= Brewbaker and Kwack solution diluted at 10%, BK10 + P10 = BK10
+ 10% polyethylene glycol, BK10 + S10 = BK10 + 10% sucrose) (C.
Bastien, INRA, unpublished results).

Pollen of most conifer species can be stored for at least 3 years

provided handling ex situ or during forcing does not adversely
affect viability. Recommended protocol includes the reduction
of pollen water content to 7--8%, the placement of pollen in
air-tight bottles or laminated foil packages, preferably under
vacuum or nitrogen (Webber 1991, Webber and Painter 1995),
and the storage of pollen at temperatures below 20 C or in
liquid nitrogen (see Stanley and Linskens 1974, Owens and
Blake 1985, Webber 1987, Copes 1987, Jett et al. 1993).
Drying pollen cone buds to a constant water content is
relatively easy using internal air-conditioning systems
(Eriksson 1993, Webber and Painter 1995). Drying extracted
or shed pollen is more difficult. For any given species, water
content can vary quickly depending on the atmosphere, the
temperature and the size of the pollen lot. For small quantities
of pollen, drying over silica gel (Figure 4AI) is common.
However, this technique is difficult because the time to reach
a desired water content (7.5%) depends on the initial water
content (Figure 4AII), and the technique does not allow for
continuous monitoring of water loss.
A better method is to dry pollen under controlled conditions
as described in the schematic drawing shown in Figure 4B. Air
is humidified by bubbling it through water (1) and then mixed
with the appropriate volume of dry air (2) to obtain the desired
humidity. This system allows control over both temperature
and humidity (3) and was tested on various Douglas-fir pollen
lots that differed in initial water content and volume. At a given
temperature, the final pollen water content depends only on the
relative humidity of the air provided the pollen is exposed
under the set conditions long enough to equilibrate (Figure
4BII).
Table 3 shows the effects of various air relative humidities
at 20 C on Douglas-fir pollen water content. We recommend
that the pollen be exposed to a relative humidity of 45% at an
air temperature of 20 C for 24 h. Extended exposure periods
(i.e., 3 days) had no detrimental effect on pollen viability and
did not result in increased water content.

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BONNET-MASIMBERT AND WEBBER

Table 3. Effect of equilibrating Douglas-fir pollen for 24 h at various

relative humidities on pollen water content (on a fresh-weight basis).
Relative humidity of the air (%)

Pollen water content (%)

65
60
55
50
45

12.0
11.3
10.0
9.2
7.6

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Figure 4. Systems for drying pollen (AI and BI) and drying curves
for Douglas-fir pollen (AII and
BII); R.H. = relative humidity.

TREE FLOWERING FROM INITIATION TO SEED PRODUCTION

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