Directory UMM :Data Elmu:jurnal:T:Tree Physiology:Vol14.1994:

Tree Physiology 14, 1303-1312
© 1994 Heron Publishing----Victoria, Canada

Floral induction in Eucalyptus nitens
M. W. MONCUR1 and O. HASAN2
1

CSIRO, Division of Forestry, P.O. Box 4008, Canberra, ACT 2600, Australia

2

Cooperative Research Center for Temperate Hardwood Forestry and Department of Plant Science,
University of Tasmania, GPO Box 252C, Hobart, Tasmania 7001, Australia

Received January 3, 1994
Summary
Eucalyptus nitens (Deane & Maiden) Maiden takes at least five years to initiate flower buds from seed
and is an infrequent and light flowerer. Because this behavior constitutes a major impediment to breeding
programs, we examined the mechanisms controlling floral induction in E. nitens, with the long-term aim
of reducing generation time and increasing seed yield.
Application of paclobutrazol reduced the concentration of endogenous gibberellic acid (GA) in apical

tissue and enhanced the reproductive activity of grafted trees maintained outside over winter in Canberra,
Australia. Grafts maintained in a warm greenhouse over winter did not produce flower buds, despite the
paclobutrazol-induced reduction in GA concentration of the apical tissue. Exposing untreated grafts,
which had been maintained over winter in a warm greenhouse, to low temperature the following spring
reduced growth but did not induce flower bud production. Addition of GA3 to paclobutrazol-treated grafts
reduced the effect of paclobutrazol on reproductive activity.
Keywords: flowering, generation time, gibberellins, low temperature, paclobutrazol, seed yield.

Introduction
Tree breeding and seed orchard production depend on early and consistent flowering,
neither of which is characteristic of forest trees in general. Species of the Eucalyptus
genus are important for forestry both in Australia and overseas, and several genetic
improvement programs are in progress. However, flower induction in Eucalyptus is
not well understood. Eucalyptus species produce flower buds in the leaf axils of new
growth in spring following a cold winter (Tibbits 1989), suggesting that changing
day length or exposure to low temperature may be involved in the flower induction
process. A number of Australian native species have a low temperature requirement
for initiation of flowering (King et al. 1992). Eucalyptus lansdowneana F. Muell. &
J. Brown initiates flower buds when plants are transferred from a warm to a cold
regime for 4--6 weeks and then back to a warm regime, regardless of day length

(Moncur 1992), whereas Bolotin (1975) reported that Eucalyptus occidentalis Endl.
seedlings, less than one year old, flowered precociously when grown in a long day
regime of 16 h or longer. Floral initiation in Eucalyptus nitens (Deane & Maiden)
Maiden occurs in spring following a cold winter (Moncur, unpublished data), but the
mechanisms controlling flowering have yet to be determined.
Growth retardants can enhance flowering and control vegetative growth in horticultural crops (Jones et al. 1989) and Eucalyptus (Hetherington and Jones 1990,

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Hetherington et al. 1991, Griffin et al. 1993, Moncur et al. 1994). Moncur et al.
(1994) reported that a single application of paclobutrazol resulted in reduced endogenous GA concentrations in E. nitens plants six to eight weeks before flower buds
were first visible.
We have examined the effects of low temperatures and paclobutrazol on flower bud
production by E. nitens grafts. We used a single clone to avoid provenance variation,
and grafts were used because E. nitens, in common with many Eucalyptus species,
has a well defined juvenile period (Jacobs 1955). Although there have been no reports
of reversion to juvenility following grafting in eucalypts, this possibility was tested
by the use of different aged grafts.

We investigated the association between the concentration of active GA in apical
tissues at the time of flower bud initiation and the number of flower buds observed
12 weeks later. The hypothesis that paclobutrazol enhances flowering by reducing
GA concentrations was tested by treating paclobutrazol-treated material with a
commercially available GA with very similar structure and activity to endogenous
GA1.

Materials and methods
In October 1989 and 1990, scions from mature E. nitens trees of a single clone were
grafted onto E. nitens seedling root stock originating from the Associated Pulp and
Paper Mills (now North Forest Products) breeding program at Ridgley, Tasmania.
Eighty grafts were potted in 460 or 380 mm containers filled with a 3/1/1/1 (v/v) mix
of composted tan bark, perlite, vermiculite and rice hulls, and grown outdoors in
Canberra, Australia. The grafts were fertilized with Osmocote, irrigated and sprayed
to control insects as required. Grafts were repotted at the end of the first year to avoid
root constriction.
Effects of temperature and paclobutrazol on 6- and 18-month-old E. nitens grafts
In April 1991, paclobutrazol was applied at the rate of 0.02 g of active ingredient per
mm of stem circumference. The required dose was added to 400 ml of water for
18-month-old grafts or 200 ml of water for 6-month-old grafts and applied as a root

drench. Five grafts of each age were then placed in a naturally lit greenhouse with a
day (0800--1600 h)/night temperature of 25/17 °C. The remaining grafts were grown
outside (see Figure 2). A further five grafts of each age were transferred to the
greenhouse in June 1991 and September 1991. Grafts were held in these conditions
until early December 1991, when flower bud production is normally completed, and
then all grafts were moved outdoors until December 1992.
In early October 1991 at the time flower buds would have been initiating, apical
shoot material, consisting of meristematic tissue and developing leaves, was sampled
from the 18-month-old grafts for determination of GA concentrations. Four to eight
grams of tissue were removed from each of the five replicated grafts of each
treatment, combined, frozen in liquid nitrogen and held at − 26 °C until processed.

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Extracts were purified by C18 Sep-Pak chromatography, anion-exchange chromatography and high performance liquid chromatography (Hasan et al. 1994). Both GA1
and GA20 were subsequently quantified by gas chromatography--mass spectrometry
in the selected ion monitoring mode (Hasan et al. 1994).
Relationship between endogenous GA concentrations and flower bud production

in espaliered grafts of E. nitens
In April 1989, one-year-old grafts maintained in the Canberra espalier orchard were
treated with one of three concentrations of paclobutrazol by either trunk injection or
soil drench (Moncur et al. 1994). During late September 1992, samples of apical
shoot tissue, including meristematic apices and developing leaves, were harvested
from the four-year-old grafted trees and quantified for GA1 concentrations according
to the methods described by Hasan et al. (1994). Flower buds were recorded in
mid-December 1992.
Interaction between GA3 and paclobutrazol on growth and flower bud
development on E. nitens grafts
Grafts were established in October 1991 (Figure 1). Each graft was potted in a
300 mm container filled with a 1/1/1/3 (v/v) mixture of rice hulls, perlite, vermiculite
and composted tan bark. In April 1992, 90 grafts were placed in a factorial combination of paclobutrazol with two concentrations of GA3. Paclobutrazol was applied at
20 mg of active ingredient per mm of stem circumference in 200 ml of water as a root
drench. An aqueous stock solution of 0.3 mM GA3 was applied as a soil drench on

Figure 1. Schedule for Experiment 1. G = time of grafting; NAT = outside conditions at Canberra (see
Figure 2); +P = time when paclobutrazol was applied; broken lines with arrows indicate time of transfer
of grafts to greenhouse.

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June 5 and July 2, 1992 at two rates (High-GA3 = 50 ml, Low-GA3 = 10 ml). Grafts
were grown outdoors until December 1992 when flower bud production was complete.
Effects of continuous and cyclic temperatures on floral induction in E. nitens
Scions were grafted onto seedling root stock and grown at Ridgley, Tasmania.
Twenty nine-month-old grafts were transferred to Canberra and placed in the CSIRO
Phytotron. Grafts were potted in 250 mm containers filled with a 1/1 (v/v) mix of
perlite and vermiculite. Each pot was watered with Hoagland’s nutrient solution
twice a week, increasing to three times a week after Week 10, and tap water as
required. Pots were placed in a greenhouse with a day (0830-1630 h)/night temperature of 21/16 °C. The photoperiod was extended to 16 h by use of incandescent
lighting from 0430--0830 and 1630--2030 h.
After two weeks, 10 grafts were placed in a warm diurnal regime (24/19 °C) in an
open greenhouse, and another 10 grafts were subjected to a cyclic temperature
treatment consisting of one week in a naturally lit cabinet at 15/10 °C, followed by
six weeks at 7/4 °C, one week at 15/10 °C, four weeks at 24/19 °C and finally, one
week at 15/10 °C. This treatment was repeated until Week 25 when grafts were too
large for the cabinet and were transferred to a larger cabinet with a temperature

regime of 10/5 °C. Both treatments received a photoperiod extended to 16 h with
incandescent lighting.
Treatments were terminated after 35 weeks and the grafts were then transplanted
to 300 mm pots and grown outside (Figure 2). Five grafts remained outdoors at
Ridgley for comparison with the experimental grafts.

Results
Paclobutrazol suppressed height growth and leaf production (Table 1), but flowering
occurred in the paclobutrazol-treated grafts that were exposed to low temperatures
over winter (Figure 2, Table 2). By December 1992 (Year 2), all grafts had experienced two winters outdoors and those treated with paclobutrazol produced flower
buds (Table 2). Two 18-month-old and two 6-month-old paclobutrazol-treated grafts
that initiated flower buds in Year 1 (Table 2) were grown in a greenhouse (25/18 °C)
during the second winter. In contrast to the grafts maintained outdoors over the
second winter, the grafts in the warm greenhouse failed to produce flower buds the
following season.
At the time of sampling the espalier orchard in late September, the concentration
of GA1 in the apical meristematic tissue was related to the number of flower buds
produced in December (Figure 3). Gibberellin concentrations greater than approximately 0.6 ng gDW−1 in the apical meristematic tissue were associated with a marked
reduction in subsequent flower bud production. However, lower GA1 concentrations
were associated with both high and low flowering responses, suggesting that low

concentrations of active GA were not the sole requirement for enhanced flowering

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Figure 2. Monthly mean maximum and minimum temperatures at Canberra. s = maximum temperature;
d = minimum temperature; G indicates when gibberellin samples were obtained; F indicates when
flower bud numbers were counted.

responses. Treatment with paclobutrazol can reduce GA concentrations (Table 3)
without a flowering response (Table 2).
Application of GA3 by soil drench significantly increased stem diameter (P <
0.001), indicating that applied GA3 was assimilated by the grafts. The stem thickening response has been noted as the most dramatic effect of GA3 application on
vegetative tissues of apples (Mauk et al. 1990), which may explain its detection in
the absence of any significant effect on height increment (P = 0.863) (Table 4b).
Flower bud numbers were reduced by up to 40% following application of GA3. There
were more grafts with less than 25 flower buds in the paclobutrazol + Low-GA3 and
paclobutrazol + High-GA3 treatments than in the paclobutrazol treatment (data not
shown), indicating a reduction in reproductive activity following GA3 application.

Growth of untreated 6- and 18-month-old grafts was continuous in the greenhouse,
but growth was suppressed when grafts were grown outside at low temperatures
(Table 1). The height and number of leaves of grafted trees increased with increasing
exposure to warm greenhouse conditions (Table 1). No flower buds were produced
by untreated grafts in Year 1; however, in Year 2, one untreated graft produced flower
buds (Table 2).
Height increment and leaf production of grafts grown at 24/19 °C in the Phytotron
increased over time. When grafts were transferred to low temperatures (cyclic
treatment), growth virtually ceased, but growth resumed when the grafts were
returned to 24/19 °C. When grafts were returned to 24/19 °C, leaves were produced

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Table 1. Effects of paclobutrazol and duration of cold treatment on height increment and number of leaves
of 6- and 18-month-old grafts of E. nitens grown at Canberra.

TreatmentPaclobutrazolControl

HeightNo. of leavesHeightNo. of leaves

Oct1Dec2OctDecOctDecOctDec
18-Month-old grafts
April3202206 7.811.232839224.632.0
June204213 6.810.628536421.828.6
September17718110.612.221027115.423.8
Outside201214 8.415.619423810.217.8
6-Month-old grafts
April 90 98 8.213.419830525.833.0
June 97101 7.2 9.419726924.633.0
September 94 98 8.0 8.412018115.626.6
Outside 92 98 8.2 9.011217614.223.0
ANOVAHeightNo. of leaves

OctDecOctDec
at a faster rate than for grafts grown continuously at 24/19 °C. Leaf production in
both treatments declined with age. All new leaves were vegetatively adult, as
indicated by their alternate and petiolate appearance. No flower buds were observed
under either regime. By late November, following transfer of grafts outdoors, flower

buds were observed in 20% of the surviving grafts with no significant difference
between initial treatments. Grafts at Ridgley produced adult leaves, but no flower
buds.
There was no indication of reversion to juvenility, as demonstrated by the production of juvenile foliage, following treatment of grafted material with paclobutrazol.
The effect of paclobutrazol on flowering was similar for both the 6- and 18-monthold grafts, indicating that juvenility was not a confounding factor.
Discussion
Under natural conditions, E. nitens produces flower buds at about age five years, and

FLORAL INDUCTION IN EUCALYPTUS NITENS

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Table 2. Effects of paclobutrazol and duration of cold treatment on flower bud production in 6- and
18-month-old grafts of E. nitens grown at Canberra.

TreatmentYear 1Year 2

PaclobutrazolControlPaclobutrazolControl

Pot1Graft2PotGraftPotGraftPotGraft
18-Month-old grafts
April30/5 00/505/5111.60/5 0
June0/5 00/505/5 19.60/5 0
September0/5 00/50na4nanana
Outside4/555.00/503/3 92.30/5 0
6-Month-old grafts

Figure 3. Relationship between flower bud production and GA1 in an E. nitens espaliered orchard.
Concentration of GA1 was detected in apical tissue by the method of Hasan et al. (1994). Regression: y =
1990.674 × 10 − 1.321x, r2 = 0.798.

production increases with age. Although the mechanisms for induction are not
known, trees growing in natural environments will experience a series of cold
periods, changing day lengths and, in many cases, variations in water status. The
effect of these stimuli, either singly or in combination, may be cumulative, resulting
in a flowering threshold being surpassed after several potentially inductive events.

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Table 3. Effects of paclobutrazol and duration of cold treatment on endogenous GA concentrations
(pg gFW− 1) in apical tissue of 18-month-old grafts of E. nitens growing at Canberra.

TreatmentGA1GA20

ControlPaclobutrazolControlPaclobutrazol
April1 597 64 76520
June 639117 89363
Exposure to low temperatures in the absence of paclobutrazol treatment did not
produce a floral response in Year 1, although a few grafts produced a small number
of flower buds after a second winter outside. The apparent inability of changing day
length to induce flowering in combination with paclobutrazol, but in the absence of
maximum cold treatment, suggests that photoperiod is not a strong stimulus of
flowering in E. nitens. This contrasts with the results of Bolotin (1975) for E. occidentalis and, may be indicative of a species-specific response within the genus.
All grafts that flowered had low GA concentrations. The action of paclobutrazol
in stimulating reproductive activity by reducing net biosynthesis of endogenous GA
was also partially substantiated by the finding that reproductive activity in paclobutrazol-treated grafts partially reverted to the untreated state following treatment with
GA3. Enhanced growth in GA3-treated grafts may have redirected assimilates away
from reproductive centers, resulting in a reduction in reproductive activity. The
application of GA generally inhibits flowering in woody angiosperms. Most notably,
GA3 and GA4/7 inhibit or suppress flowering in a wide variety of fruit trees (references cited by Pharis and King 1985).
Our data indicate that the concentration of active GA is not the sole factor
determining the induction of floral primordia in E. nitens. Paclobutrazol-treated
material exposed to different low temperature treatments had similar GA concentrations, although only material receiving maximum cold exposure produced flower
buds. This response may resemble the vernalization response in annual plants,
though its function in a woody perennial species is not clear. A period of cold could
result in destruction of a flowering inhibitor, a change in inter-organ competition, or
both. Metzger (1985) suggests that vernalization activates one or more steps in GA
biosynthesis such that a specific GA accumulates above a threshold concentration.
The promotion of flowering by paclobutrazol constitutes a practical method of
promoting flowering for breeding and seed production in several eucalypt species.
This finding is particularly important in E. nitens, because domestication of this
species has been limited by shortage of seed. Detailed characterization of the
environmental stimuli of flowering could further enhance the promotive effects of
paclobutrazol application, as well as indicating ideal sites to conduct and establish
seed orchards.

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Table 4. Effects of applied GA31 on (a) flower bud production and GA concentrations, and (b) height,
leaves, diameter and volume2 in grafts of E. nitens.
(a)

TreatmentNumber of potsMean flowerEndogenous GA concentration
with flower budsbuds per pot(pg gFW−1)
GA1GA20
Control 10.701.710.58
Paclobutrazol (P)1033.300.440.14
P + Low-GA31218.700.550.21
P + High-GA3 917.800.520.18
Control + Low-GA3 00.001.570.56
Control + High-GA3 10.301.760.93
ANOVA
P< 0.001
GA0.293
P × GA0.342
s.e.d
P4.65
GA5.70
P × GA8.06
(b)

TreatmentIncrement increase (April--December)
HeightLeavesDiameterStem volume
(mm)(mm)(cm3)
Control56713.806.2355.09
Paclobutrazol (P)1494.673.3915.36
P + Low-GA31454.873.6116.41
P + High-GA31516.534.3621.07
Control + Low-GA357514.875.7138.94
Control + High-GA359812.807.6569.35

Acknowledgments
We are grateful to Associated Pulp and Paper Mills, Ridgley, Tasmania for supplying the clones and
financial assistance to conduct the Phytotron work. We thank CSIRO, Division of Plant Industry staff at
the Phytotron for maintaining the trees utilized in the second experiment and J. Turner for technical
assistance with the remaining experiments. R.P. Pharis gave valuable advice with GA3 applications.
R. King, P. Kriedemann and D. Sheriff made many valuable comments on an earlier draft.

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