Plant Cell Tiss Organ Cult (2010) 100:59–64
DOI 10.1007/s11240-009-9619-6
ORIGINAL PAPER
Development of in vitro plant regeneration of Australian native
waxflowers (Chamelaucium spp.) via somatic embryogenesis
K. Ratanasanobon • K. A. Seaton
Received: 2 April 2009 / Accepted: 22 September 2009 / Published online: 1 October 2009
Ó Springer Science+Business Media B.V. 2009
Abstract Waxflowers (Chamelaucium spp.) are native to
Australia and now are grown for the cut flower industry
worldwide. As part of an effort to achieve somatic hybrid-
ization between the species to improve flower quality,
somatic embryogenesis was achieved for Chamelaucium
uncinatum and C. repens. Somatic embryos from young
leaves of C. uncinatum and C. repens were induced in vitro
on Murashige and Skoog (MS) agar medium containing
20 g/l sucrose and 2,4-dichlorophenoxyacetic acid (2,4-D).
For C. uncinatum, up to 4% of explants developed somatic
embryos at 20 lM 2,4-D and for C. repens, up to 3%
developed somatic embryos at 5 lM 2,4-D. Somatic
embryos of C. uncinatum were also induced from immature
seeds—a maximum of 6% of seed explants producing
somatic embryos on MS medium containing 0.05 lM
6-benzyladenine (BA) and 0.5 lM Naphthalene acetic acid
(NAA). Somatic embryo cultures maintained on MS med-
ium supplemented with 0.1 lM 2,4-D were induced to
develop into plantlets after transfer to a hormone-free
medium under light.
Keywords Waxflower
Chamelaucium spp.

Myrtaceae
Somatic embryogenesis
Plant regeneration
Abbreviations
2,4-D 2,4-Dichlorophenoxyacetic acid
BA 6-Benzyladenine
K. Ratanasanobon (&)
K. A. Seaton
Innovative Plant Products Project, Horticulture Division,
Department of Agriculture and Food, Western Australia,
3 Baron-Hay Court, South Perth, WA 6151, Australia
e-mail: kanokwan.ratanasanobon@agric.wa.gov.au
K. A. Seaton
e-mail: kevin.seaton@agric.wa.gov.au
IBA Indole-3-butyric acid
MS Murashige and Skoog
NAA Naphthalene acetic acid
PGR Plant growth regulator
Introduction
Geraldton waxflower (Chamelaucium uncinatum, family
Myrtaceae, tribe Chamelaucieae) is one of the main Aus-
tralian native cut flowers for export to Japan, Europe and
USA. Approximately 600 million stems are produced per
annum worldwide and major international production areas
apart from Australia include USA (California), Israel,
South American and South Africa. Besides its significance
as a cut flower, waxflower plants are drought tolerant and
are adapted to low soil nutrient conditions. These charac-
teristics make them ideal for amenity and garden plants in
dry climates. There are 293 taxa recognized within the
Chamelaucium alliance, which has members with a range
of flower colors, yellow, red, pink and white, flowering
times from late fall to late summer, a number of different
flower types, and plant forms from small shrubs 0.5 m high
to tall shrubs 2 m high. This diversity provides a diverse
genetic resource for breeding novel varieties for cut flowers
and amenity plants.
The Myrtaceae comprises more than 3,000 species
including several with commercial importance. However,
somatic embryogenesis has been only reported in euca-
lyptus (Glocke et al. 2006; Muralidharan et al. 1989;
Nugent et al. 2001; Pinto et al. 2008; Watt et al. 1991),
pineapple guava (Cangahuala-Inocente et al. 2007), myrtle
(Canhoto et al. 1999) and Brazilian grape tree (Motoike
et al. 2007). There are no reports of somatic embryogenesis
123
60
from taxa within the Chamelaucium alliance. In this
study we report a plant regeneration system via somatic
embryogenesis using immature seeds and young leaves
producing somatic embryos, and maintaining and con-
verting them to plantlets.
Materials and methods
Plant materials and culture conditions
In vitro shoots
Young shoot tips of C. uncinatum (line 583) and C. repens
(a single selection No. 1), grown in a nursery at the
Department of Agriculture and Food Western Australia,
South Perth, were surface sterilized by submerging in 10%
commercial bleach (containing 3.5% calcium hypochlorite)
with 0.01% Tween 80 under vacuum for 1 min. These
shoots remained submerged in bleach solution for 20 min
with gentle shaking and then were washed three times with
sterile deionised water. Shoots were patted dry before
placing them in MS basal medium (Murashige and Skoog
1962) supplemented with 0.3 lM BA and 0.1 lM Indole-
3-butyric acid (IBA) and 20 g/l sucrose. The medium was
solidified with 0.8% agar and the pH adjusted to 6.0 before
autoclaving. Shoots were cultured at 25 ± 1°C under a
16 h photoperiod (40 lmol m-2 s-1) and transferred to
fresh medium every 4 weeks.
Immature seeds
Fruits of C. uncinatum (line 583) were collected from
open-pollinated shrubs about 8 weeks after flowers opened.
These were surface sterilized using the procedure described
Fig. 1 Embryogenic callus was induced from young leaves of
C. repens on MS base media containing 5 lM 2,4-D and somatic
embryos emerged after the callus was transferred to MS base media
containing 0.1 lM 2,4-D (a), then developed to cotyledonary
123
Plant Cell Tiss Organ Cult (2010) 100:59–64
above. After sterilization, fruits were dissected under a
microscope to isolate intact immature seeds.
Somatic embryogenesis induction
The effect of plant growth regulators (PGRs) on somatic
embryogenesis was investigated. The youngest three pairs
of uppermost leaves of C. uncinatum and C. repens from in
vitro shoots at 2 weeks after subculture were cut into pie-
ces 0.5 cm in length and cultured on MS basal media
supplemented with various concentrations of 2,4-D: 0, 1, 5,
10 and 20 lM for C. uncinatum and 0, 1, 5 and 10 lM for
C. repens. Immature seeds of C. uncinatum were placed on
MS basal media supplemented with 0.1, 0.5, 1, 5 or 10 lM
of 2,4-D as well as with 0.5 lM NAA in combination with
0.05, 0.5, 1 or 5 lM of BA. All MS media contained 20 g/l
sucrose, solidified with 0.8% agar and adjusted to pH 6.0
before autoclaving. Once globular embryos developed, the
calli were transferred to MS01 (MS medium containing
0.1 lM 2,4-D and 30 g/l sucrose, solidified with 0.8% agar
and adjusted to pH 6.0 before autoclaving). Five explants
were used per replicate and at least three replicates were
tested per treatment. Petri dishes were sealed with parafilm
and incubated in the dark at 25 ± 1°C.
Maturation and germination of somatic embryos
All embryogenic calli were multiplied and maintained on
MS01 in sealed Petri dishes with monthly subculture period.
For germination, somatic embryos were transferred to MS
basal medium without PGR and cultures incubated under
light (40 lmol m-2 s-1) with 16 h photoperiod. Plant con-
version rate was calculated from number of plantlets
obtained from three replicates of five embryogenic calli
each.
embryos (b) and plantlet (c) after they were transferred to MS base
media without PGR under light. Bar represented 1 mm in length
unless otherwise indicated
Plant Cell Tiss Organ Cult (2010) 100:59–64
Results
Two weeks after culturing, calli were observed from the cut
ends of all C. repens leaf explants on MS media containing
2,4-D and after 4 weeks the calli morphology had differ-
entiated. They appeared compact and yellow on MS med-
ium with 1 lM 2,4-D and were more friable and yellow
on media with 5 and 10 lM 2,4-D. No calli were observed
in explants on control medium lacking 2,4-D. Of the calli
produced in these different media, only one explant, cul-
tured on 5 lM 2,4-D supplemented MS medium, had a
white mass that appeared at the lower edge 6 weeks after
induction. When this callus was transferred to MS01
Fig. 2 Somatic embryogenesis in C. uncinatum. White friable calli
were induced from leaves on MS base media containing 2,4-D (a).
Somatic embryos (arrowed) emerged underneath the leaf-derived
callus induced on MS base media containing 2,4-D 20 lM (b).
Repetitive secondary embryos (arrowed) budded directly from
surface of leaf-derived embryo (c). Callus induced from zygotic
61
medium, it produced somatic embryos after 8 weeks
(Fig. 1a). Embryos developed to the globular and torpedo
stages. Embryogenic calli were subcultured and maintained
on MS01 medium for more than a year without losing their
capacity to develop into plantlets. Embryos converted into
plantlets after transfer to MS basal medium under light.
They developed into cotyledonary embryos and plantlets
(Fig. 1b, c) at an average conversion rate of 31.1 ± 8.3%.
Induction of somatic embryogenesis of C. uncinatum
was carried out using two types of explants: young leaves
and immature seeds. Young leaf explants were placed on
MS solid media supplemented with 0–20 lM 2,4-D. They
gave friable white calli for each 2,4-D treatment (Fig. 2a)
immature embryo on MS base media containing higher BAP in
combination of BAP and NAA (d). Somatic embryos (arrowed)
emerged from embryogenic callus induced from immature seeds on
MS base media containing BAP:NAA at 0.05:0.5 lM (e). Plantlets
regenerated from leaf-derived somatic embryos (f). Bar represented
1 mm in length unless otherwise indicated
123
62
but gave more compact and smaller calli with brown pig-
ments in 20 lM 2,4-D (Fig. 2b). When these calli were
transferred to MS01 medium after 8 weeks (two subcul-
tures), white embryos emerged underneath a brown callus
(Fig. 2b). These somatic embryos gave rise to secondary
embryos in MS media with 0.1 or 0.5 lM 2,4-D (Fig. 2c).
Results for C. uncinatum were similar to those from
C. repens, where somatic embryos could be converted to
plants (Fig. 2f), when they were transferred to MS without
PGR under light condition at an average rate of 53.3 ±
13.3%.
C. uncinatum immature seeds accumulated and released
black compounds into the media when they were cultured
on media containing 0.1–10 lM 2,4-D. After 6 weeks, the
explants surviving on 2,4-D supplemented medium were
less than those on BA/NAA supplemented medium
(Table 2). The surviving explants either germinated to
shoots or differentiated to grey calli which did not give
rise to somatic embryos after being transferred to MS01
medium.
Calli induced from immature seeds on MS media
containing BA and NAA had different morphology after
4 weeks. On MS media with low BA concentrations (0.05
and 0.5 lM) and 0.5 lM NAA, calli were loosely friable and
yellow, while calli on media with higher BA (1 and 5 lM)
concentrations with 0.5 lM NAA had green spots with the
appearance of leaf primordia (Fig. 2d) and some had shoots
with roots. After 6 weeks of induction on BA/NAA media,
calli without shoots and roots were then transferred to
MS01 media. Only calli induced on a BA:NAA ratio of
0.05:0.5 lM developed into somatic embryos at a rate of
6.00 ± 3.05% (Table 2). This process occurred after three
subcultures of 4 weeks each on MS01 medium (Fig. 2e).
Embryogenic calli were maintained in MS solid media with
0.1 lM 2,4-D for 6 months without loss in their regeneration
capacity. Somatic embryos developed into plants when they
were transferred to MS without PGR at an average rate of
37.4 ± 6.1%.
Somatic embryos of C. uncinatum that were initiated
from different type of explants with different PGR treat-
ments could be distinguished by their morphology, their
nature of giving rise to repetitive secondary somatic
embryos and also the response to be cultivated in suspen-
sion culture. Somatic embryos obtained from immature
seeds had pale yellow color while leaf-derived somatic
embryos were a turbid white color. Repetitive secondary
somatic embryos emerged directly from immature seed-
derived somatic embryos bodies or indirectly via calli
while repetitive secondary somatic embryos emerged
directly from leaf-derived somatic embryos bodies only.
Leaf-derived somatic embryos released dark purple com-
pound to liquid media when they were shaken with media
solution to make cell suspension culture and low amount
123
Plant Cell Tiss Organ Cult (2010) 100:59–64
of cell aggregates were initiated. After three months, very
little growth had occurred. On the other hand somatic
embryos derived from immature seeds gave good yields of
new cell aggregates in suspension culture with the same
media and could be established embryogenic suspension
culture stock for protoplast isolation (data not presented).
The characteristic of somatic embryos initiated from leaf
explants treated on medium containing high 2,4-D (20 lM)
which gave direct repetitive secondary embryos without
passing through the callus phrase would be more efficient
for rapid plant propagation.
Plantlets of both Chamelaucium species which grew
from somatic embryos regardless the source of explant
they were derived from, gave rise to secondary somatic
embryos (Fig. 3a). Plantlets transferred to MS with double
strength agar with no PGR gave no more secondary
embryos. Root-like organs (Fig. 3b) were initiated from
induced calli in media containing 2,4-D or higher ratio of
NAA in combination with BA (Tables 1, 2) and did not
develop further.
Discussion
In this study, a plant regeneration system via somatic
embryogenesis of leaf and immature seed tissues was
developed for C. repens and C. uncinatum. Somatic
embryos induced from young leaf explants of both species
using MS media supplemented with 2,4-D. Three-four
percent of explants gave rise to somatic embryos. In the
Myrtaceae, induction of somatic embryogenesis from
vegetative explants appears to be difficult. In Eucalyptus
globulus they could not be induced from leaf, stem and
hypocotyl explants (Pinto et al. 2002), while Nugent et al.
(2001) reported induction of somatic embryos from young
hypocotyls and cotyledons in the same species at 0.3–1.4%
but these failed to develop into plantlets. A high percent
(70%) of hypocotyl explants which gave rise to somatic
embryos was reported in E. nitens but somatic embryos
obtained grew only to the globular stage with no further
development (Ruaud et al. 1997). In myrtle (Myrtus com-
munis) 4% hypocotyls and 8% cotyledons gave rise to
somatic embryos which could develop into full plants
(Parra and Amo-Marco 1998).
Transition from vegetative cells to embryogenic cells
requires cell dedifferentiation and differentiation (see
review by (Fehér et al. 2003)) indicating that the devel-
opmental stage of the explant used is critical. In the two
Chamelaucium species studied, the youngest three pairs of
uppermost leaves from 2-week-old subcultures of in vitro
shoots were used for initiating somatic embryos. Optimi-
zation of leaf explant age chosen for somatic embryo
induction might improve efficiency.
Plant Cell Tiss Organ Cult (2010) 100:59–64 63

Fig. 3 Secondary embryos

(arrowed) grew from plantlets
regenerated from somatic
embryos of C. repens (a). Root-
alike organs (arrowed) emerged
from calli induced on MS base
media containing 2,4-D (b)

Table 1 Effect of PGRs on differentiation into root-like organs and somatic embryo of C. repens and C. uncinatum leaf explants
2,4-D concentration (lM) C. repens leaves C. uncinatum leaves
No. of root-like organs Percentage of explant No. of root-like organs Percentage of explant
per explant with somatic embryos per explant with somatic embryos

0 0b 0b 0b 0b
1 0.03 ± 0.03 b 0b 0b 0b
5 0.67 ± 0.22 a 3.33 ± 3.33 a 1.04 ± 0.23 a 0b
10 0.13 ± 0.08 b 0b 0.36 ± 0.11 b 0b
20 N/A N/A 0.64 ± 0.18 a 4±4a
Values are means ± SE. Same alphabet letter showed no significance difference at the 0.05 level by Tukey HSD

Table 2 Effect of PGRs on differentiation into root-like organs, shoots and somatic embryos of C. uncinatum immature seeds
PGR treatment C. uncinatum immature seeds
No. of root-like organs No. of shoots and roots Percentage of explant survived Percentage of explant
per explant per explant after 6 weeks (*) with somatic embryos

2,4-D concentration (lM)

0.1 1.9 ± 0.64 a 0.1 ± 0.1 a 50 0
0.5 0b 0.3 ± 0.15 a 30 0
1 1.7 ± 0.82 a 0a 80 0
5 0.7 ± 0.37 a 0.2 ± 0.13 a 60 0
10 1.9 ± 1.06 a 0a 30 0
BA:NAA (lM)
0 0b 0.70 ± 0.15 a 70 0b
0.05:0.5 2.30 ± 0.99 a 0.10 ± 0.10 b 90 6.00 ± 3.05 a
0.5:0.5 1.60 ± 0.52 a 0b 90 0b
1.0:0.5 0b 0.60 ± 0.16 a 100 0b
5.0:0.5 0b 0.60 ± 0.16 a 100 0b
Values are means ± SE. Same alphabet letter showed no significance difference at the 0.05 level by Tukey HSD. (*) indicates that data in that
column was not statistically analysed

Indirect somatic embryogenesis was induced from BA in the medium supplemented with NAA at 0.5 lM was
zygotic embryos of immature seeds of C. uncinatum. The essential and increased survival rate and somatic embryos
efficiency was higher (6%) than leaf explants. Presence of were obtained in media of BA/NAA at 0.05: 0.5 lM. In

123
64
Myrtus communis L. and Eucalyptus globulus Labill.,
somatic embryos were induced from zygotic embryonic
tissues on media containing 2,4-D and NAA as auxin
sources (Canhoto et al. 1999; Parra and Amo-Marco 1998;
Pinto et al. 2002). However, with C. uncinatum, on MS
media containing 2,4-D no somatic embryos were
obtained.
In conclusion, a plant regeneration system via somatic
embryogenesis was achieved for C. repens and C. unci-
natum. Somatic embryos from both species gave rise to
repetitive secondary embryos which can be maintained for
long-term use. In C. uncinatum, two types of somatic
embryos were induced and their different nature of pro-
ducing secondary embryos can be used in different aspect.
The direct secondary embryos production could be suitable
for rapid plant propagation whereas the indirect secondary
embryos with their embryogenic calli could be in good use
for generating cell suspension stock for protoplast isolation
in somatic hybridization research. Findings from this study
have application for development of somatic hybridization
techniques for Chamelaucium species to improve flower
quality.
Acknowledgments The research team thanks Horticulture Australia
Limited (HAL) for Voluntary Contribution Funding for this research.
The authors thank Dr Stephen Wylie for review of this manuscript,
Mr Christopher McMullan for plant materials and Ms Leah Chong for
technical assistance.
References
Cangahuala-Inocente GC, Dal Vesco LL, Steinmacher D, Torres AC,
Guerra MP (2007) Improvements in somatic embryogenesis
protocol in Feijoa (Acca sellowiana (Berg) Burret): induction,
123
Plant Cell Tiss Organ Cult (2010) 100:59–64
conversion and synthetic seeds. Sci Hortic 111:228–234. doi:
10.1016/j.scienta.2006.10.030
Canhoto J, Lopes M, Cruz G (1999) Somatic embryogenesis and plant
regeneration in myrtle (Myrtaceae). Plant Cell Tissue Org Cult
57:13–21. doi:10.1023/A:1006273128228
Fehér A, Pasternak TP, Dudits D (2003) Transition of somatic plant
cells to an embryogenic state. Plant Cell Tissue Org Cult 74:
201–228. doi:10.1023/A:1024033216561
Glocke P, Collins G, Sedgley M (2006) Effects of auxins on organogen-
esis and somatic embryogenesis from juvenile explants of Euca-
lyptus erythronema, E. stricklandii, and two inter-specific hybrids.
J Hortic Sci Biotechnol 81:1009–1014
Motoike S, Saraiva E, Ventrella M, Silva C, Salomão L (2007)
Somatic embryogenesis of Myrciaria aureana (Brazilian grape
tree). Plant Cell Tissue Org Cult 89:75–81. doi:10.1007/s11240-
007-9210-y
Muralidharan EM, Gupta PK, Mascarenhas AF (1989) Plantlet
production through high frequency somatic embryogenesis in
long term cultures of Eucalyptus citriodora. Plant Cell Rep
8:41–43. doi:10.1007/BF00735775
Murashige T, Skoog FA (1962) A revised medium for rapid growth and
bioassays with tobacco tissue cultures. Physiol Plant 15:473–497.
doi:10.1111/j.1399-3054.1962.tb08052.x
Nugent G, Chandler SF, Whiteman P, Stevenson TW (2001) Somatic
embryogenesis in Eucalyptus globulus. Plant Cell Tissue Org
Cult 67:85–88. doi:10.1023/A:1011691110515
Parra R, Amo-Marco JB (1998) Secondary somatic embryogenesis
and plant regeneration in myrtle (Myrtus communis L.). Plant
Cell Rep 18:325–330. doi:10.1007/s002990050580
Pinto G, Santos C, Neves L, Araújo C (2002) Somatic embryogenesis
and plant regeneration in Eucalyptus globulus Labill. Plant Cell
Rep 21:208–213. doi:10.1007/s00299-002-0505-5
Pinto G, Park Y-S, Silva S, Neves L, Araújo C, Santos C (2008)
Factors affecting maintenance, proliferation, and germination of
secondary somatic embryos of Eucalyptus globulus Labill. Plant
Cell Tissue Org Cult 95:69–78. doi:10.1007/s11240-008-9417-6
Ruaud J-N, Churchill K, Pepper S (1997) Somatic embryogenesis
initiation in Eucalyptus nitens. Acta Hortic (ISHS) 447:185–186
Watt MP, Blakeway F, Cresswell CF, Herman B (1991) Somatic
embryogenesis in Eucalyptus grandis. S Afr For J 157:59–65