New Forests

DOI 10.1007/s11056-014-9420-1

Hormone level analysis on adventitious root formation

in Eucalyptus globulus

Naoki Negishi • Katsuhiko Nakahama • Nobuaki Urata •

Mikiko Kojima • Hitoshi Sakakibara • Akiyoshi Kawaoka

Received: 16 July 2013 / Accepted: 6 March 2014

Ó Springer Science+Business Media Dordrecht 2014

Abstract Eucalyptus globulus is the one of the most economically important trees for
pulp and paper industries due to its fast growth and short harvesting cycle. However, E.
globulus is well known as a woody plant that is hard to propagate vegetatively. In this
study, we found wide distribution of rooting ability among seven independent genotypes
we tested. Analysis of the mechanism of adventitious rooting by using the auxin transport
inhibitor, N-naphthylphthalamic acid, revealed that polar auxin transport is crucial in root
formation. The gravitropism of the stem was highly correlated to the percentage of
adventitious root formation. Hormonal analysis showed that the levels of indole-3-acetic
acid (IAA) were the same between difficult-to-root and easy-to-root genotypes, whereas
levels of IAAsp (indole-3-acetyl aspartic acid) were higher in the difficult-to-root geno-
type, indicating that IAA metabolism might play an important role in adventitious root
formation in this species. Levels of several cytokinins exhibited differences between
genotypes that varied in their cutting performance and rooting ability. Furthermore, higher
level of cytokinin in elongating shoots were correlated with rooting percentage. Taken
together, our results indicate that both auxins and cytokinins play a role in adventitious root
formation in E. globulus, and that a complex interplay between the levels of auxins and
cytokinins and their metabolism might result in root formation in this commercially
important plant.

Keywords Eucalyptus globulus  Adventitious root formation  Auxin  Cytokinin

Abbreviations
BA 6-Benzyl amino purine
IAA Indole-3-acetic acid

N. Negishi  K. Nakahama  N. Urata  A. Kawaoka (&)

Agri-Biotechnology Research Laboratory, Nippon Paper Industries Co. Ltd., 5-21-1 Oji, Kita-ku,
Tokyo 114-0002, Japan
e-mail: akawaoka@nipponpapergroup.com

M. Kojima  H. Sakakibara
RIKEN Center for Sustainable Resources Science, Tsurumi, Yokohama, Kanagawa 230-0045, Japan

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IAAsp Indole-3-acetyl aspartic acid

IBA Indole-3-butyric acid
NPA N-1-naphthylphthalamic acid

Introduction

The preservation and multiplication of desirable genotypes through clonal propagation is a

useful tool for increasing resistance to pests, diseases and drought, and for improving the
economic potential of plant species and their hybrids. Adventitious rooting is a key step in
this process (De Klerk et al. 1999), as it provides good attachment to the substrate and
affords efficient uptake of water and nutrients from the soil. The evaluation of improved
propagation methods requires attention not only to the rooting percentage, but also to
parameters such as the quantity and length of roots, all of which are relevant for the
establishment of clonal forests.
Adventitious rooting can be divided into three phases: induction, formation and
expression (Kevers et al. 1997). In practical terms, the latter two phases may be considered
as a single root formation phase (Fett-Neto et al. 2001). Induction is characterized by the
molecular and biochemical events that occur within undifferentiated cells without mor-
phological changes, while formation is characterized by cell division, root primordium
organization, and root growth. Many environmental and endogenous factors regulate the
rooting process. Auxins exert direct or indirect effects on root development (Blakesley
1994) and are required for root induction, but inhibit the process during the formation
phase (De Klerk et al. 1999). The endogenous concentration of auxins depends on their
biosynthesis, conjugation, transport and catabolism (Blakesley 1994; Normanly et al. 1995;
Garrido et al. 2002). Indole-3-acetic acid (IAA) is the main endogenous auxin, but it is
relatively labile. Indole-3-butyric acid (IBA) is a more stable endogenous auxin (Epstein
and Lavee 1984), and is also more efficiently transported than IAA in an acropetal
direction (Ludwig-Muller 2000), which is the main pathway of polar auxin transport.
Because of these differences, each of the auxins may have different effects on the quantity
and quality of adventitious root induction. In addition to IAA, IAA conjugates such as
IAAsp are known to influence rooting (Zelena and Fuksova 1991). Evidence from studies
on Arabidopsis also points a role for cytokines in adventitious rooting (Kuroha and Satoh
2007).
In this study, we selected Eucalyptus globulus elite candidate trees from a Western
Australia tree plantation and investigated auxin transport and endogenous levels of other
hormones in selected easy-to-root and difficult-to-root genotypes. Our results indicate that
the changes in the levels of both auxins and cytokinins, as well as their metabolism, play an
important role in the adventitious root formation in this species.

Materials and methods

Plant materials and shoot multiplication

The plant materials of E. globulus with superior growth rate were selected from a 5-year-
old plantation near Bunbury, Western Australia (Nippon Paper Resources Australia Pty.

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Ltd.). Branches with mature leaves were grafted to seed-grown 6 month-old E. globulus
seedlings as a root stock to generate new lateral shoots with juvenile leaves. After
3 months, shoots were sterilized by immersion in 2 % (v/v) sodium hypochlorite, then
70 % (v/v) ethanol, and rinsed three times with sterile water. After removal of wounded
tissue, explants were placed on a solid medium (15 mL) in 25 9 120 mm test tubes. The
solid medium consisted of modified Murashige and Skoog (MS) medium (half concen-
tration of NH4NO3 and KNO3) containing 2 % sucrose, 0.25 % gellan gum and
0.5 mg L-1 6-benzyl amino purine (BA). The pH of the medium was adjusted to 5.8 before
autoclaving and introduction of explants. Cultures were incubated in a culture room
(SANYO Electric Co. Ltd., Japan) at 25 °C and 60 % humidity. Cold cathode fluorescent
lamps (45 lmol m-2 s-1; Harison Toshiba Lighting Co., Japan) were used as a light
source for 16 h per day. After 4 weeks, generated shoots were transferred to the modified
MS plant mixture (Wako Pure Chemical Industries Co., Ltd, Japan) with 0.02 mg L-1 BA
for shoot elongation. Induced multiple shoots were subcultured on fresh medium every
6 weeks. Polycarbonate culture box (65 9 65 9 105 mm, Asahi Glassplant, Kumamoto,
Japan) was used for subcultures.

Rooting condition

The clonal E. globulus plants were micropropagated by a photoautotropic culture method

(Nagae et al. 1996). Seven independent genotypes of E. globulus (VN71, MZ12, MA45,
ML19, MO21, NA29 and MG44) were tested for rooting percentage at 1,000 lmol mol-1
of CO2. A plant growth chamber with a CO2 concentration regulator (Sanyo Electric,
Osaka, Japan) was used for rooting experiments. For rooting treatments, elongated shoots
([30 mm) with three pairs of leaves that had been grown for 4 weeks (Fig. 1c) were
transferred to the phenol resin foam (15 9 15 9 35 mm 9 25 cubes, Oasis LC-288;
Smithers-Oasis North America, Kent, OH, USA) with liquid B5 medium containing
2 mg L-1 (24.6 lM) IBA (Sigma, St Louis, USA) in a culture box (92 9 92 9 100 mm,
Asahi Glassplant, Kumamoto, Japan), which had two holes (8 mm of diameter) sealed with
a membrane filter (MilliSeal, Millipore Japan, Tokyo, Japan) under a cycle of 16 h light of
cold cascade fluorescent lamp (CCFL, 30 lmol m-2 s-1, Harison Toshiba Lighting Co.,
Imabari, Japan) that specially prepared as a light-strength ratio of red (680 nm) to blue
(470 nm) at 8:2 (Fig. 1d).

Measurements of root growth

Percent rooting, roots per shoot, and mean length of the longest root (root length) were
measured 3 weeks after transfer to the rooting medium (Fig. 1e). Each experiment was
repeated at least three times.

Gravitropic response

Short stems (ca. 3 cm length), with shoot apex and four leaves cultivated by tissue cul-
tivation, were horizontally placed in phenol resin (15 9 15 9 35 mm 9 25 cubes, Oasis
LC-288) containing the rooting medium without IBA (Fig. 4). At 48 h after transfer to high
CO2 conditions, the curvature of the bend stem was measured.

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Fig. 1 Photographs of E. globulus micropropagation. Bars indicate 10 mm. a Newly generated axillary
shoots of E. globulus VN71 in MS medium containing 0.02 mg L-1 BA concentration, 2 % (w/v) sucrose
and 0.25 % (w/v) gellan gum. b Transplanted explants for shoot elongation. c Root formation in phenol
resin with liquid B5 medium and 2.0 mg L-1 IBA. d Rooting stage under CCFL. e Adventitious root
formation of E. globulus VN71. Bars 1 cm

Measurement of hormone levels

Highly sensitive and high-throughput analysis of plant hormones was carried out by liquid
chromatography–tandem mass spectrometry (Kojima et al. 2009). Stem samples [100 mg
(fresh weight) of basal part] were used and measurements were taken at 0, 4 and 8 days
after the rooting treatment on a rooting medium.

Results

Variation of rooting percentage among genotypes

After 4 weeks of shoot elongation on the medium containing 0.02 mg L-1 BA, shoots
were transferred to a rooting medium. Among the seven genotypes tested, four genotypes,
VN71, MA45, ML19 and MO21 showed over 60 % of rooting. Rooting percentages of
NA29 and MG44 genotypes were quite low (Fig. 2a). These also showed less number of
roots that were appreciably shorter compared to the other five genotypes (Fig. 2b, c). Thus,
a wide variation in the ability to form adventitious roots was observed in these genotypes.
For further analysis, we used VN71 as the easy-to-root genotype and NA29 as the difficult-
to-root genotype.

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a b c
100 40 10

Root length (mm)

80
30

Number of root
Rooting (%)

60 6
20
40 4

10
20 2

0 0 0
VN71 NA29 MZ12 MA45 ML19 MO21 MG44 VN71 NA29 MZ12 MA45 ML19 MO21 MG44 VN71 NA29 MZ12 MA45 ML19 MO21 MG44

Fig. 2 Variations in the ability to form adventitious roots in several E. globulus genotypes. After 4 weeks
of shoot elongation on the medium containing 0.02 mg L-1 BA, shoots were transferred to a rooting
medium. a Rooting percentage, b mean of the longest root length, and c number of roots 3 weeks after
transfer to rooting medium at 1,000 lmol mol-1 CO2. Each data point represents the average of three
replicates, and over 50 explants were used for each experiment. Error bars represent mean ± SE

Correlation between gravitropism response and adventitious root formation

Gravitropism is very common in these genotypes, affecting their growth. Abundant evi-
dence suggests that gravitropic response of stems is due to regulated movements of auxin,
its concentration and transport (Epel et al. 1992; Fukaki et al. 1996; Friml et al. 2002;
Swarup et al. 2005; Haga and Iino 2006). This finding that rooting performance and stem
curvature were positively correlated may simply suggest both processes are regulated by
the same hormonal pathway. We looked at the correlation between the curvature of the
stems and the percentage of adventitious roots in this species and found a high positive
correlation (R2 = 0.87) at higher CO2 condition (1,000 lmol mol-1) (Fig. 3).
We then investigated if a correlation existed between auxin transport and adventitious
root formation by inhibiting auxin transport using N-1-naphthylphthalamic acid (NPA), a
known auxin transport inhibitor. Various concentrations of NPA were added to the rooting
medium and rooting percentages were observed in VN71 and NA29 genotypes at higher
CO2 condition. Emergence of adventitious roots was inhibited 3 weeks after rooting
treatment with NPA, as seen in the decrease in rooting percentages in VN71 and NA29
genotypes with increasing concentrations of NPA (Fig. 4). This result indicates that polar
auxin transport is necessary for adventitious root formation.

Endogenous hormone levels in basal parts of the stem in VN71 and NA29 genotypes

Next, we measured the endogenous concentrations of some of the auxins [such as IAA and
indole-3-acetyl aspartic acid (IAAsp)], abscisic acid (ABA), and cytokinins [such as trans-
zeatin (tZ), tZ-riboside (tZR), tZR 50 -monophosphate (tZRMP), cis-zeatin (cZ), cZ riboside
(cZR), cZR 50 -monophosphate (cZRMP), N6-(D2-isopentenyl)adenine (iP), iP riboside
(iPR) and iPR 50 -monophosphate (iPRMP)] in the basal parts of the stems of VN71 and
NA29. Measurements were made by ultra-performance liquid chromatography (UPLC)
coupled with a tandem quadrupole mass spectrometer (qMS/MS) equipped with an elec-
trospray interface (ESI) (Kojima et al. 2009) at 0, 4 and 8 days after rooting treatment at
higher CO2 condition.
The IAA levels showed significant increase during the course of development in both
VN71 and NA 29. At 0 day, the IAA level in VN71 was significantly higher than that in
NA29. Result indicates the poor-rooting genotype, NA29 produces IAA and then transports
IAA to the basal stem location at 4 and 8 days. The IAAsp conjugate, however, showed an

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0h 48 h e
a b 90
R2=0.87

Shoot curvatures
VN71 70
g
50
c d
30
NA29
g 10

0 20 40 60 80 100
Rooting (%)

Fig. 3 Correlation between adventitious root formation and gravitropism. a, b VN71, c, d NA29 genotypes.
e Correlation between shoot curvatures and rooting in seven independent genotype genotypes NA29, MG44,
MZ12, ML19, MA45, MO21 and VN71. Shoot curvatures were measured at 48 h after the treatment. Each
data point represents the average of three replicates, and over 50 explants were used for each experiment.
Error bars represent mean ± SE. Bars 1 cm

NPA
(µM) 0 2 10 50
a b c d i
100
VN71 VN71
80 NA29
Rooting (%)

60
e f g h 40

NA29 20

0
0 2 10 50
5 mm NPA (µM)

Fig. 4 Effect of NPA on adventitious root formation in an easy-to-root genotype (VN71) and a difficult-to-
root genotype (NA29) of Eucalyptus. Various concentrations of NPA (2–50 lM) were added into the
rooting medium. a–d VN71, e–h NA29 and i Addition of NPA decreases rooting. Each data point represents
the average of three replicates, and over 50 explants were used for each experiment

increase only in the easy-to-root genotype VN71 and not in the NA29 (Fig. 5). The lower
concentrations of IAAsp in NA29 indicate that the auxin metabolism is compromised in
this genotype, coinciding with poor adventitious root formation. ABA concentration
increased gradually from 0 to 8 days after rooting treatment in NA29, but in VN71, the
level of ABA increased significantly 4 days after treatment and then declined by day 8.
Thus, ABA levels at 8 days were quite different between VN71 and NA29 (Fig. 5).

Levels of endogenous cytokinins

In general, similar to auxin levels, the cytokinin concentrations at basal part of stem were
higher in the easy-rooting genotype VN71. The levels of zeatin derivatives, tZ, tZR,
tZRMP, cZ, cZR, and cZRMP in VN71 were relatively higher in VN71 compared to the
NA29 during the course of development (especially day 8). The iP derivatives, iP, iPR and
iPRMP levels also showed such a difference between VN71 and NA29 (Fig. 5).

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(pmol/gFW) IAA (pmol/gFW) IAAsp (pmol/gFW) ABA

1000 3000
800 1200

600 2000 * 800

#
400
1000 400 #
200 #
0 0 0
0 4 8 0 4 8 d 0 4 8 0 4 8 d 0 4 8 0 4 8 d
VN71 NA29 VN71 NA29 VN71 NA29
(pmol/gFW) tZ tZR (pmol/gFW) tZRMP
(pmol/gFW)
0.8 2.5
0.4
0.6 * * 0.3
2.0 *
#
* 1.5
0.4 0.2
* 1.0
0.2 0.1 0.5
0 0 0
0 4 8 0 4 8 d 0 4 8 0 4 8 d 0 4 8 0 4 8 d
VN71 NA29 VN71 NA29 VN71 NA29
(pmol/gFW) cZ (pmol/gFW) cZR (pmol/gFW) cZRMP
0.6 4.0
* 0.6 # #
3.0 *
0.4
0.4

0.2
2.0 *
0.2 1.0

0 0 0
0 4 8 0 4 8 d 0 4 8 0 4 8 d 0 4 8 0 4 8 d
VN71 NA29 VN71 NA29 VN71 NA29

(pmol/gFW) iP (pmol/gFW) iPR (pmol/gFW) iPRMP

1.0 #
1.2 #
12.0
0.8
* 0.8 *
0.6
0.4
* 8.0

0.4 4.0
0.2
0 0 0
0 4 8 0 4 8 d 0 4 8 0 4 8 d 0 4 8 0 4 8 d
VN71 NA29 VN71 NA29 VN71 NA29

Fig. 5 Endogenous levels of various hormones. Each data point represents the average of three replicates.
Asterisk or sharp in VN71 indicate significant difference from NA29 values at the same time as p \ 0.01 or
p \ 0.05, respectively. Values represent mean ± SE

Higher cytokinin level promotes adventitious root formation

Next, we investigated whether exogenous cytokinin would promote adventitious root

formation. In this experiment, we used MG44 and added 0.1 mg L-1 of BA into the shoot
elongation medium. The micropropagated shoots by tissue culture were then transferred
and cultivated for 4 weeks at higher CO2 condition. At this time, no difference in phe-
notype between high cytokinin concentration and ordinary culture condition could be
observed. These shoots were subsequently used for further rooting treatment.
As shown in Fig. 6, the rooting percentage increased with the addition of exogenous
cytokinin BA (p \ 0.05). However, the addition of exogenous cytokinin did not result in
increase in the length or the number of adventitious roots.

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100 60 7
a b c
6
80 50

Root length (mm)

5

Number of root
40
Rooting (%)

60
4
30
40 3
20
2
20
10 1

0 0 0
0.02 0.1 0.02 0.1 0.02 0.1
BA (mg/L) BA (mg/L) BA (mg/L)

Fig. 6 Effect of exogenous cytokinin, BA into the shoot formation medium for adventitious root formation.
a Rooting percentage, b mean of longest root length, and c number of roots 3 weeks after transfer to rooting
medium at 1,000 lmol mol-1 CO2. Each data point represents the average of three replicates, and over 50
explants were used for each experiment. Asterisk indicates significant difference of the mean at p \ 0.05.
Values represent mean ± SE

Discussion

In this study, we selected several genotypes of E. globulus that are amenable to micro-
propagate into shoots and roots (Fig. 1). We observed high variation in the ability to form
adventitious roots in E. globulus genotypes at high CO2 levels (Fig. 2). This wide variation
may be due to several reasons, such as the differences in auxin biosynthesis, polar auxin
transport, etc. Auxin plays a crucial role in adventitious root formation (De Klerk et al.
1999) and is often applied exogenously to promote the adventitious root formation of stem
cuttings of difficult-to-root genotypes. However, exogenous auxin is not always effective,
and a better understanding of the physiological mechanisms through which it regulates
adventitious rooting is needed in order to improve the rooting of recalcitrant genotypes. In
this study, we compared physiological functions of adventitious root formation between
selected difficult-to-root and easy-to-root genotypes of E. globulus grown in Western
Australia.
We observed a positive correlation between gravitropism of stem and adventitious root
formation. Gravitropism is the growth response whereby a plant orients itself with respect
to the gravity vector. Stem gravitropic response is thought to involve polar auxin transport
(Moore 2002). Polar auxin transport moves auxin from cell to cell across the intervening
cell wall by sequential uptake and subsequent efflux via an active carrier. Our results
suggest that the gravitropic response could be due to the polar auxin transport as the polar
auxin transport inhibitor NPA inhibits adventitious root formation in both in the difficult-
to-root as well as easy-to-root genotypes (Fig. 4). NPA inhibits auxin efflux, and abolishes
gravitropic growth response, but not auxin uptake. While the endogenous auxin IAA is
mainly synthesized in shoot apex and leaf (Fujita and Syono 1996; Da Rocha Correa et al.
2012), adventitious roots are generated at the basal part of the cut stem. Taken together, our
results suggest that polar auxin transport is important in adventitious root formation in this
species.
The analysis of endogenous hormone levels during adventitious root formation between
the easy-to-root and difficult-to-root genotypes provided much useful information. With

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respect to auxin and its derivatives, while IAA levels were almost the same in each
genotype, marked differences were seen in the levels of the IAA conjugate, IAAsp between
VN71 and NA29, and the easy-to-root genotype, VN71 demonstrated much higher levels
of the hormone after cutting treatment (Fig. 5b). IAA conjugates such as IAAsp and
indole-3-acetyl glutamic acid (IAGlu) are reported to not have an effect in plant cells
(Staswick et al. 2005). Exogeneous IAAsp was reported to enhance rooting percentage in
bean (Zelena and Fuksova 1991). However, in some species, no correlation between the
auxin levels and adventitious root formation has been observed. A recent study shows that
Arabidopsis transgenic lines ARF6-OX, ARF8-OX and MIR160c-OX, which demonstrated
increased adventitious rooting, showed no increases in IAA, IAAsp or IAGlu levels in
hypocotyl cuttings (Gutierrez et al. 2009). Endogenous auxin is often used to induce
adventitious root formation on stem cutting of different species (de Klerk et al. 1999; Geiss
et al. 2009; Li et al. 2009). In A. thaliana, it has been shown that several auxin-over-
producing mutants or transgenic plants, such as superroot1, superroot2 or yucca1, spon-
taneously develop numerous adventitious roots on the hypocotyle (Boerjan et al. 1995;
Delarue et al. 1998; Zhao et al. 2001). The SUR2 gene encodes the cytchrome P450
CYP83B1. Mutations in the SUR2 gene clearly induced an increase in the IAA pool size in
all tissues analyzed (Barlier et al. 2000). Endogenous levels of IAAsp and IAGlu are also
increased in the sur2 mutant (Barlier et al. 2000; Sorin et al. 2005). In this study, we
observed an increase in the free IAA levels after cutting treatment in both genotypes
(VN71 and NA29). The IAAsp conjugate, however showed an increase only in the VN71
(Fig. 5). These results suggest that the metabolism of IAA may be important for adven-
titious root formation. Interestingly, our results also showed that the ABA levels in the
difficult-to-root genotype, NA29 was higher than those in easy-to-root genotype, VN71
(Fig. 5). ABA acts as an inhibitor for lateral root formation (De Smet et al. 2003) and in the
present case, it is likely to negatively contribute towards adventitious root formation in
NA29.
The levels of the nine cytokinin species measured (tZ, tZR, tZRMP, cZ, cZR, cZRMP,
iP, iPR and iPRMP) were significantly increased after cutting treatment in VN71 geno-
types. However, no such increases in any of the cytokinin levels were observed in the
difficult-to-root NA29. Kuroha and Satoh (2007) reported that one of the cytokinin species,
ZR acted as an endogenous suppressor of hypocotyl adventitious root formation in planta.
Arabidopsis loss-of-function mutant leg3 (wol-3) that lacked a functional cytokinin
receptor exhibited stimulated adventitious rooting (Kuroha and Satoh 2007). Our results
suggest that the levels of the cytokinin species might have a positive regulation in
adventitious root formation as indicated by the increase in cytokinin levels in the VN71
and the lack of any significant change in the NA29. In addition, we also observed that the
addition of exogenous cytokinin BA into a shoot elongation medium resulted in the
increase in adventitious root formation (Fig. 6). These results indicate that the addition of
exogenous cytokinin during shoot development is effective in adventitious root formation.
In conclusion, results of our present study suggest that polar auxin transport has a
crucial role and several endogenous cytokinin species also contribute towards adventitious
root formation in this species. Evidently, the mechanism by which these hormones control
rooting appears complex, but our study has provided insight into the factors that could
influence adventitious rooting in this important commercial species.

Acknowledgments We thank Toshiaki Tanabe, Keiichi Shimizu, Noriko Kumazawa, Eiji Iwata, Kazunori
Hayashi and Chris Schiller for technical help.

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