2. 74 S.C. Fernando et al. / Industrial Crops and Products 86 (2016) 73–78
There are challenges to be overcome in order to maximize the
profitability of oil mallees. Optimal growth rates require that plants
invest photosynthate astutely between the root system, lignotuber,
stems and leaves. This investment pattern will be different for dif-
ferent harvesting frequencies, given the need for sufficient stored
carbon and nutrients to be present in the roots and the lignotu-
ber to initiate rapid coppice regeneration following harvest (Noble,
2001). Essential oil yield in turn depends on the growth rate of
the above ground parts, as well as the proportion of biomass allo-
cated to leaves and the amount of oil in each leaf. Many of these
attributes have been shown to be highly variable, even between
closely related individuals (Davis, 2002; Doran, 2002). For example,
King et al. (King et al., 2004) found that blue mallee plants within a
single, naturally occurring population varied in foliar essential oil
content from less than 1% to greater than 5% of fresh weight. Simi-
larly, plants showed considerable quantitative variation in major
oil constituents, growth rates, leaf morphology and water-use-
efficiency (King et al., 2004).
This natural variation provides significant scope for develop-
ing improved mallee lines with regard to biomass and essential oil
production. Some progress has been made in improving mallees
through conventional selective breeding (Doran, 2002), but given
that it typically takes several years for mallees to flower and that
several generations are needed to realize significant selection gains,
this approach will likely prove unsatisfactorily slow. Goodger et al.
(Goodger et al., 2008; Goodger and Woodrow, 2008) adopted a
more rapid approach involving seed production from a series of
elite clones, which were selected for their exceptionally high foliar
oil content, desirable oil quality and high growth rate. Their strategy
was based on findings that oil and growth traits are highly her-
itable (Goodger and Woodrow, 2012; Barton et al., 1991; Doran
and Matheson, 1994), and genetic correlations between oil and
growth traits can be neutral or even slightly positive depending
on the genotype (King et al., 2006). Clonal forestry approaches of
this kind involving other species have produced good results. For
example, in a related Myrtaceous species (Melaleuca alternifolia),
clonal seed orchards yielded significant selection gains in terms of
tea tree oil production at both the leaf and plantation level (Butcher
et al., 1996; Doran et al., 2006). Preliminary analyses of large-scale
plantations derived from seed from the elite mallee clones have
shown a marked improvement in foliar oil yield and growth rates
relative to natural populations (I. Woodrow, unpublished results).
Establishment of a clonal seed orchard by Goodger et al.
(Goodger and Woodrow, 2009) relied on the development of an
in vitro plant regeneration protocol for blue mallee, given that this
species is not amenable to efficient propagation from potted stem
cuttings (Slee, 2007). This method involves proliferation of axilliary
buds from lignotuber-derived explants, with subsequent rooting of
shoots (Goodger et al., 2008). Ramets developed using this proto-
col have shown very close similarity to the ortet in all important
attributes, including foliar oil quality and quantity (Goodger and
Woodrow, 2009). Nevertheless, because of the difficulty of genetic
transformation, a protocol involving proliferation of axilliary buds
is unlikely to be sufficient to underpin many future improvement
strategies in blue mallee as a crop. Moreover, an efficient genetic
transformation protocol will be required for investigations of the
genetic and physiological processes underlying growth, develop-
ment and secondary metabolism. Blue mallee is a useful target for
these latter studies given that none of the model plant species share
some of the most remarkable attributes of this species, includ-
ing exceptionally large, sub-dermal foliar secretory cavities. The
implementation of forward and reverse genetics approaches, such
as transposon-insertional mutagenesis (Fladung and Polak, 2012),
activation tagging (Busov et al., 2011), gene and enhancer trapping
(Groover et al., 2004), and targeted genome engineering (Maggio
and Goncalves, 2015), in blue mallees, will require an efficient sys-
Table 1
Composition of media used for E. polybractea organogenesis.
Components Culture media
M1 M2 M3 M4
Woody plant basal salt mixturea
(g l−1
) 2.3 2.3 2.3
Murashige & Skoog modified vitamins
(×1000) (ml l−1
)
1.0 2.0 1.0 1.0
Sucrose (g l−1
) 25 30 25 25
2iP (M) 3.0
IBA (M) 100
TDZ (M) 3.0
NAA (M) 0.1
BAP (M) 4.4
Agar (g l−1
) 7.0 7.0 7.0
Gelrite (g l−1
) 2.5
a
Lloyd & McCown woody plant basal salt mixture (Austratec, Australia).
tem for proliferating cloned plants from genetically transformed
cells.
Here, we outline a highly efficient protocol for regeneration of
blue mallee clones through indirect organogenesis and use it to
develop the first genetically transformed plants of this species.
Successful genetic transformation and regeneration, via indirect
organogenesis or somatic embryogenesis, has been reported for a
sizeable number of commercially important (largely for hardwood
timber) Eucalyptus species (Chauhan et al., 2014). Nevertheless,
several challenges need to be overcome before transformation and
regeneration of healthy, stable transgenic plants can be undertaken
routinely. First, because target trees are generally identified at the
adult stage, protocols must work efficiently with adult explant
material. It is noteworthy in this context that most of the research
on eucalypt transformation and in vitro regeneration has involved
seedling-derived explants, with some notable exceptions (Mullins
et al., 1997; Chen et al., 1996, 2001; Spokevicius et al., 2005). Sec-
ond, protocols need to function independently of genotype. It has
generally been found that aspects of both the transformation and
in vitro regeneration protocol need to be modified for each new
genotype (Chauhan et al., 2014). In this paper, we outline a protocol
for in vitro regeneration of blue mallee that is applicable to adult-
derived explants and shows exceptionally high efficiency across
several genotypes. We used one genotype to show that geneti-
cally transformed plants can be successfully regenerated using this
protocol.
2. Materials and methods
2.1. Plant material
In vitro cultures of three clones (clones 1, 2 and 3) originating
from nodal cultures of three separate adult trees were used for this
study. These adult trees were from separate populations on private
land (several kilometres apart) near Inglewood, Victoria, Australia,
and each had relatively high amounts of foliar essential oil (>4%
fresh weight). The nodal cultures were established according to
Goodger et al. (Goodger et al., 2008) using explants taken from
young (<20 cm in length) coppice shoots. Large numbers of ram-
ets of each of other blue mallee clones have been produced from
similar in vitro cultures, and these have been shown to have excep-
tional similarity with regard to essential oil quantity and quality
as well as growth performance (Goodger and Woodrow, 2008).
This indicates that, at least with regard to these traits, the occur-
rence of somaclonal variation is relatively improbable. Permission
for the collection of plant material was granted by the land owner
—FGB Natural Products. Cultures were maintained in the dark in
a medium containing 2iP (M1) (Table 1) by sub-culturing at two
monthly intervals.
3. S.C. Fernando et al. / Industrial Crops and Products 86 (2016) 73–78 75
2.2. Explant preparation
Elongated shoots were rooted in medium M2 as described else-
where (Goodger et al., 2008) (Table 1). Immediately after root
initiation, plantlets were transferred to light (16 h photoperiod).
Young expanded leaves (4-6 leaves of 5-7 mm length from each
plantlet) of 6-8 week old plantlets were then used as explants. After
trimming the bases and tips, leaflets were cut into halves perpen-
dicular to the midrib and both halves were used for subsequent
experiments.
2.3. Callogenesis and plant regeneration
Ten explants per Petri dish (9 cm diameter) were cultured in
25 ml of callusing medium M3 (Table 1), with the abaxial side in
contact with the medium for 6-8 weeks without light at 25 ◦C. Cul-
tures were transferred to fresh media of the same composition at
two-weekly intervals. Initiated calli were transferred to the regen-
eration medium M1 or M4 (Table 1). Cultures were maintained in
the dark, and they underwent four to five cycles of three-weekly
sub-culturing into freshly prepared media. Initiated shoots were
elongated (30-40 mm) in medium M1 in the dark and rooted in
M2 (Table 1). After four to five weeks in the latter medium, rooted
plantlets were potted in a mixture of top soil, sand, vermiculite, and
perlite at a ratio of 1:1:1:1, and they were acclimatized by grad-
ually decreasing humidity and increasing light intensity over 30
days. Plants were then grown in a naturally lighted glasshouse for
6 months to monitor survival, growth, development and foliar 1,8-
cineole content. For the latter, fully expanded leaves were ground
to a fine powder in liquid nitrogen, extracted in hexane and ana-
lysed using gas chromatography as described by King et al. (King
et al., 2004).
2.4. Agrobacterium-mediated genetic transformation
The transformation experiments made use of the hyper-virulent
Agrobacterium tumefaciens strain AGL1 harboring the binary vec-
tor pMDC204 (Tair Accession-vector: 1009003763). The vector
contained the green fluorescent protein (mgfp6) and hygromycin
phosphotransferase (hpt) genes. These genes were under the con-
trol of the dually-enhanced CaMV35S promoter (CaMV35Sx2). The
transformed strain was maintained at 28 ◦C in liquid LB medium
containing rifampicin (50 mg l−1) and kanamycin (50 mg l−1).
One hundred young leaf explants of clone 2 (prepared as
described in the Section 2.2) were pre-cultured in medium M3
(Table 1) supplemented with 100 M acetosyringone for two days
to improve vir gene induction. The explants were then inoculated
with the Agrobacterium (the optical density at 600 nm (OD600) = 0.8-
1.0) for 10 min and co-cultivated in M3 supplemented with 100 M
acetosyringone at 25 ◦C in the dark. After three days, the explants
were rinsed with 500 mg l−1 cefotaxime and 500 mg l−1 carbeni-
cillin solution and transferred to M3 supplemented with 500 mg l−1
cefotaxime and 500 mg l−1 carbenicillin for four days and then
to M3 supplemented with 500 mg l−1 cefotaxime and 10 mg l−1
hygromycin. The medium was refreshed biweekly. After four
cycles, explants were maintained in a medium supplemented
with 10 mg l−1 hygromycin only. Developed callus was trans-
ferred to medium M4 supplemented with 10 mg l−1 hygromycin
to promote regeneration. Cultures at callusing and shoot regener-
ation stages were examined under a fluorescent stereomicroscope
equipped with a 480 nm excitation filter to detect the presence
of GFP. Regenerated shoots were elongated in M1 supplemented
with 10 mg l−1 hygromycin and rooted in the absence of antibi-
otics. The plants were acclimatized successfully. Several elongated
shoots were tested for the presence of the hpt gene by extract-
ing DNA using Extract-N-AmpTM Plant PCR Kit (Sigma-Aldrich,
Table 2
Plant regeneration from E. polybractea calli.
Genotype Plant regeneration (%)
Medium M1 Medium M4
Clone1 93a
93a
Clone 2 27b
87a
Clone 3 30b
90a
Plant regeneration percentages superscripted with different letters within and
across the columns indicate a significant difference (P < 0.001). Significant differ-
ences due to genotype (P < 0.001) and medium type (P < 0.001) were detected, as
well as an interaction between both factors (P < 0.001).
St. Louis, USA) and amplifying the 308-bp fragment of hpt using
oligo-nucleotide primers: 5 -CTATTTCTTTGCCCTCGGACG-3 and
5 -CTTGTATGGAGCAGCAGACGC-3 . The amplified products were
separated by electrophoresis on a 1.5% agarose and bands were
visualized with ethidium bromide.
2.5. Statistical analysis
Thirty explants per clone per treatment were cultured. Data on
callogenesis and plant regeneration was collected after eight and
23 weeks of culture initiation. Data was analyzed with two-way
ANOVA using IBM SPSS Statistics 22 (IBM Corporation, Armonk,
USA).
3. Results
3.1. Indirect organogenesis
Leaf explants of the three different clones of blue mallee showed
varying responses to culture conditions. After two weeks of cul-
ture in a medium supplemented with TDZ and NAA, explants of
clones 1 and 2 remained visibly unchanged while those of clone 3
enlarged, discoloured and initiated callus along the cut edges. How-
ever, after four weeks of culturing approximately 95% of explants
of all three clones had produced callus, with some differences in
morphology between clones. For clone 1, 100% of calli were com-
pact and possessed small nodular structures (Fig. 1A). Clone 2 calli,
by contrast, were mostly friable and mucilaginous (63%; Fig. 1B),
with the remainder appearing similar to clone 1. Clone 3 produced
more callus per explant, compared to other two genotypes, and the
calli, like clone 1, were compact but the nodular structures were
somewhat larger. Initially, all calli were of pale yellowish colour,
but with time developed patches of whitish, reddish or brownish
colour.
After six to eight weeks, calli were transferred to a regenera-
tion medium. Early signs of shoot regeneration could be seen after
three cycles in regeneration medium M4, while the time taken for
regeneration in M1 was longer (five cycles). The number of calli
that regenerated shoots after five cycles of regeneration media is
given in Table 2. Clone 1 calli regenerated shoots at high frequency
(>90%) in cytokinin media containing either BA or 2iP. However,
clones 2 and 3 calli showed significantly different regeneration
ability between the two media. Accordingly, a highly significant
interaction between genotype and medium was detected in a two-
way ANOVA (Table 2). Interestingly, BA enriched medium (M4)
promoted an equally high (about 90%) regeneration rate (Fig. 1C),
which was independent of genotype.
When the shoot clusters (with 5 mm long shoots) regenerated
in M4 were transferred to M1, they elongated (Fig. 1D) and, within
six to eight weeks, were ready for multiplication or root initiation.
In the rooting medium M2, 60% of clone 1, 89% of clone 2 and 64%
of clone 3 shoots initiated roots (Fig. 1E). All plantlets were suc-
cessfully acclimatized in an environment with gradually decreasing
4. 76 S.C. Fernando et al. / Industrial Crops and Products 86 (2016) 73–78
Fig. 1. Plant regeneration from E. polybractea leaf explants. (A) Eight week old com-
pact and small nodular callus derived from clone 1 leaf (bar = 1.7 mm). (B) Eight week
old mucilaginous callus derived from clone 2 leaf (bar = 1.7 mm). (C) Plant regener-
ation from clone 3 callus maintained in medium supplemented with BA (medium
M4) for 12 weeks (bar = 3.8 mm). (D) Clone 2 shoot elongation in medium supple-
mented with 2iP (medium M1) (bar = 8 mm). (E) Clone 2 shoots rooted in medium
supplemented with IBA (medium M2) (bar = 14 mm). (F) Plants of clone 2 after five
months of acclimatization (bar = 107 mm).
humidity and increasing light intensity over a one month period.
Hardened plants were then transferred to a glasshouse (Fig. 1F)
where all survived and showed similar morphology, growth and
essential oil composition. As has been found for other blue mallee
clones produced using in vitro culture, 1,8-cineole was the domi-
nant terpene, making up over 80% of the total terpene in all ramets.
Meaningful comparison of essential oil with the ortets was not pos-
sible because it has been shown that adult oil quality and quantity
is not attained until trees are three to five years of age (Goodger
and Woodrow, 2009).
3.2. Genetic transformation
Of the 100 leaf explants incubated with Agrobacterium, six ini-
tiated callus that showed GFP fluorescence (Fig. 2A and B) when
grown on a medium supplemented with hygromycin. All six calli
regenerated plants in the presence of hygromycin and young shoots
also showed GFP fluorescence (Fig. 2C and D). Individual shoots
were selected on hygromycin containing medium and rooted in
antibiotic free medium. The rooted transgenic plants were all suc-
cessfully hardened and transferred to soil in pots. GFP fluorescence
Fig. 2. Genetic transformation of E. polybractea. (A) Callus initiated from a leaf inocu-
lated with Agrobacterium and grown on 10 mg l−1
hygromycin medium for 10 weeks
(bar = 1.2 mm). (B) Callus in A showing fluorescence of GFP (bar = 1.2 mm). (C) Shoot
regenerated from callus grown on 10 mg l−1
hygromycin medium (bar = 1.5 mm). (D)
Shoot in C showing fluorescence of GFP (bar = 1.5 mm). (E) Six months old transgenic
plants. (F) Presence of hpt gene (308 bp) in lanes 1-5 (5 transgenic plants) and lane
C (DNA extracted from Agrobacterium as positive control) and its absence in lane 6
(control plant) (L = Bioline EasyLadder l).
was not evident in leaves or shoots of hardened plantlets at this
stage, but the presence of the transformed hpt gene was confirmed
by agarose gel electrophoresis of PCR products (Fig. 2F). The lack
of an evident GFP phenotype was most likely due to spontaneous
gene silencing, which has been observed in other species (e.g. Sohn
et al. (Sohn et al., 2011)). Selected hardened plants of transfor-
mants and non-transformed organogenesis-derived controls were
then grown for six months in a glasshouse. At this sapling stage,
GFP expression was still not evident in the transformants, but hpt
presence was confirmed in both root tip and shoot material, sug-
gesting that the plants were not chimeric. The transformed saplings
were morphologically similar to their untransformed counterparts
(Fig. 2E). Leaves of transformants and controls contained clearly vis-
ible secretory cavities and oil extracted from all leaves contained
1,8-cineole as the main constituent (>80% of monoterpenes).
4. Discussion
Here, we have demonstrated an efficient indirect organogenesis
protocol for E. polybractea using adult-derived explants. We found
that the BA enriched medium M4 promoted a very high (about
5. S.C. Fernando et al. / Industrial Crops and Products 86 (2016) 73–78 77
90%) shoot regeneration rate, which was independent of genotype.
Moreover, root production and subsequent plant hardening suc-
cess was approximately 60% for two genotypes and 90% for the
other. This protocol provided the platform for successful Agrobac-
terium mediated genetic transformation of one blue mallee clone;
we found that 6% of explants (callus and shoots) expressed the GFP
marker gene. Six-month-old transformed plants grew normally and
foliar secretory cavities contained comparable 1,8-cineole propor-
tions to control plants.
Successful plant regeneration through organogenesis or somatic
embryogenesis has been reported for a limited number of com-
mercially important, timber-producing species of Eucalyptus. These
studies have mainly used seedling explants such as cotyledons and
hypocotyls (Bandyopadhyay et al., 1999; Cid et al., 1999; Ouyang
et al., 2012; Matsunaga et al., 2012; Ahad et al., 2014) or leaves
from micropropagated seedlings (Tournier et al., 2003; Glocke et al.,
2006a; Dibax et al., 2010a; Mendonca et al., 2013). However, there
have been some studies involving material collected from field-
grown adult plants (Mullins et al., 1997; Laine and David, 1994;
Herve et al., 2001; Aggarwal et al., 2010). For example, Aggarwal
et al. (Aggarwal et al., 2010) cut back three 12-year-old E. tereticor-
nis clones and used coppice material (nodal explants) to regenerate
shoots through indirect organogenesis. We took a similar approach
here, except that we used adult plants from three separate blue
mallee populations that had been harvested as coppice bienni-
ally for at least 50 years. Nodal explants from these adults were
first propagated in vitro using axillary bud proliferation before leaf
explants were taken from these proliferated shoots for subsequent
work.
The efficiency of the organogenesis protocol for blue mallee is
relatively high compared to other studies involving explants from
adult trees. Some 90% of explants from each clone showed elon-
gated shoot production, compared to a maximum of 40% for an
E. tereticornis clone (Aggarwal et al., 2010), 65% for an E. grandis
clone (Laine and David, 1994), and only 9% for an E. gunnii clone
(Herve et al., 2001). Moreover, the rooting efficiency of the blue
mallee clones, which ranged from 60% to 90%, compares well to
other studies. For example, Laine and David (Laine and David, 1994)
showed that for one E. grandis clone, rooting efficiency was 85% but
shooting efficiency was only 30%. Higher shooting efficiencies in
the other clones were apparently associated with lower rooting
efficiencies. In our study, clone 2 was exceptionally amenable to
propagation with an efficiency of some 90% for both shoot and root
production, and over 95% for callus production from explants. This
is comparable to the best statistics reported for Eucalyptus tissue
culture, for any explant type or set of conditions (Mullins et al.,
1997; Aggarwal et al., 2010). Genotype dependent variability in
callus formation, organ differentiation and somatic embryogene-
sis has been observed in many plants and is due, at least in part, to
genetic factors (e.g. Pinto et al. (Pinto et al., 2008)).
Another important finding in this study was that BA alone in the
culture medium (M4) yielded relatively high plant regeneration
efficiency irrespective of the clone, whereas 2iP showed signifi-
cant clonal dependence. To our knowledge, a comparison of the
effect of BA and 2iP on plant regeneration has not been reported
in Eucalyptus. However, such comparisons in Ribes magellanicum
(Arena and Pastur, 1997) and Campanulla glomerata (Joung et al.,
2002) showed a similar superior effect of BA compared to 2iP
on plant regeneration from leaves. Despite the clonal sensitivity,
this is the only report of successful use of 2iP for plant regen-
eration from Eucalyptus callus. Different combinations of BA and
NAA have been used for many eucalypts including E. camaldulensis
(Mendonca et al., 2013; Dibax et al., 2010b), E. nitens, E. globulus
(Bandyopadhyay et al., 1999), E. grandis and its hybrids with E. uro-
phylla (Hajari et al., 2006) and E. gunnii (Herve et al., 2001). Other
combinations of cytokinins (zeatin, kinetin, TDZ and N-phenyl-N’-
[6-(2-chlorobenzo-thaizol)-yl]) urea) and auxins (indole-3-acetic
acid, 2,4-dichlorophenoxyacetic acid and picloram) have also been
used for these and other species. Interestingly, some studies have
used the same hormonal combination for both callogenesis and
organogenesis. For example, a medium enriched only with BA was
used for plant regeneration from leaf explants of E. camaldulensis
(Mullins et al., 1997) and the ornamental species E. erythronema
and E. stricklandii and their hybrid (Glocke et al., 2006b). In the
present study, TDZ in combination with NAA was used to initiate
callus at very high frequency (>95%) but not a single shoot could be
regenerated by continuous maintenance of cultures in the callusing
medium. A similar finding was reported for E. grandis x E. urophylla
(Tournier et al., 2003; de Alcantara et al., 2011) and E. saligna (Dibax
et al., 2010a).
We made use of the excellent performance of clone 2 in tissue
culture to examine whether we could use this as a platform for effi-
cient genetic transformation of blue mallee. As with most previous
studies of eucalypts (Chauhan et al., 2014; Girijashankar, 2011), we
trialed Agrobacterium-mediated gene transfer and we used GFP as
a visual marker, hygromycin phosphotransferase (hpt) as a selec-
tion gene, and the dually-enhanced CaMV35S as a promoter for
both genes. This was a preliminary experiment and accordingly the
transformation frequency of 6% was in the range of previous stud-
ies (Matsunaga et al., 2012; Girijashankar, 2011; Aggarwal et al.,
2011; de la Torre et al., 2014). The challenge now is to increase this
frequency whilst not compromising the efficiency of the high rate
of shoot regeneration shown in our previous experiments. There
are many variables that are likely to impact the transformation
efficiency, including explant wounding to improve Agrobacterium
access to cells, co-cultivation conditions, the choice of Agrobac-
terium strain, explant, and genotype, as well as modifications to the
various stages of plant regeneration (Chauhan et al., 2014). Given
the high efficiency of the regeneration system for E. polybractea
reported here, it is likely that this system can be utilized as part of
an efficient genetic transformation protocol for E. polybractea.
In conclusion, we have outlined a protocol for in vitro regen-
eration of blue mallee that is applicable to adult-derived explants
and shows exceptionally high efficiency across several genotypes.
Moreover, we have used one genotype to show that genetically
transformed plants can be successfully regenerated using this pro-
tocol. This work will enable large scale cloning of adult blue mallee
plants with desirable properties and, after optimization of the
transformation protocol, provide an important platform for further
research on the biosynthesis of eucalyptus oils with the potential
to dramatically improve blue mallee as a crop.
Acknowledgements
The authors thank Mr. Peter Abbott of FGB Natural Products
for access to plantations and Ms. Marianne Weisser and Ms Edita
Ritmejeryte for research assistance. This research was funded by
grants from Australian Research Council (projects DP 1094530 and
LP150100798).
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