Micropropagation of a Casuarina hybrid - Citrus Research and ...

Plant Cell Tiss Organ Cult
DOI 10.1007/s11240-009-9502-5
RESEARCH NOTE
Micropropagation of a Casuarina hybrid (Casuarina equisetifolia
L. 3 Casuarina glauca Sieber ex Spreng) following facilitated seed
germination
Xiuli Shen Æ William S. Castle Æ Frederick G. Gmitter Jr
Received: 29 October 2008 / Accepted: 29 December 2008
Ó Springer Science+Business Media B.V. 2009
Abstract A suitable protocol for micropropagation of
Casuarina hybrid, Casuarina equisetifolia L. 9 Casuarina
glauca Sieber ex Spreng (C. e. 9 C. g.), was developed.
When seeds without seed coats were cultured on 4 germination
media, the optimal seed germination percentage
(91%) was obtained on 0.8% agar solidified water medium.
Shoot multiplication was achieved by culturing 2-cm long
epicotyls, excised from germinated seedlings, on MS
(Murashige and Skoog 1962) basal medium supplemented
with BA (6-benzylaminopurine) at 4.4, 8.8, 17.8 and
35.6 lM. The greatest percentage of axillary bud sproutings
(87.5%), mean number of sprouts per explant (3.8), and
shoot length (3.2 cm) were achieved on MS medium supplemented
with 17.8 lM BA. MS medium supplemented
with 4 different concentrations of IBA (indole-3-butyric
acid) (4.3, 8.7, 13.0 and 17.4 lM) were used for rooting
of in vitro grown shoots. The highest rooting percentage
(65.6%), mean number of roots per explant (2.5) and mean
length of roots per explant (1.6 cm) was achieved at
13.0 lM IBA. Rooted shoots grew well after transfer to a
substrate of peat and pinebark (7:3) in the greenhouse.
Keywords Casuarina hybrid (C. e. 9 C. g.)
Micropropagation Seed germination
Abbreviations
BA 6-Benzylaminopurine
C. e. 9 C. g. Casuarina equisetifolia L. 9 Casuarina
glauca Sieber ex Spreng
X. Shen W. S. Castle (&) F. G. Gmitter Jr
Citrus Research and Education Center (CREC),
Institute of Food and Agricultural Sciences (IFAS),
University of Florida (UF), 700 Experiment Station Road,
Lake Alfred, FL 33850-2299, USA
e-mail: bcastle@ufl.edu
IBA Indole-3-butyric acid
MS Murashige and Skoog (1962)
PGRs Plant growth regulators
Casuarina species of the family Casuarinaceae are tropical
and subtropical trees native to Australia, southeastern Asia
and islands of the western Pacific Ocean. Among other
uses, Casuarina trees are widely used as windbreaks for
rehabilitating and stabilizing dunes, as ornamental trees,
and for timber and firewood production (Beadle 1981;
El-Lakany 1983a, b; Kondas 1983; Turnbull 1990; Midgley
et al. 1983, National Academy of Science 1984; Castle
2008a, b).
Seed propagation of Casuarina, as with many plants
reproduced by seed, requires rapid, uniform germination to
achieve efficient nursery production. Seeds of Casuarina
have germination percentages that range from 10 to 50%
(El-Lakany and Shepherd 1983; Turnbull and Martensz
1982; El-Lakany et al. 1989; Goh et al. 1995, Boland et al.
1996). The reasons for poor seed performance have been
attributed to the existence of a large number of shrunken,
insect damaged, or empty seeds in seed lots, and to the
barrier imposed by the seed coat (Sivakumar et al. 2007).
In our earlier study of the causes of poor germination and
techniques to improve the germination of a Casuarina
hybrid and C. cunninghamiana Miq., we also experienced
poor germination (unpublished data). Seeds classified as
filled had the highest germination rate. Filled seeds generally
sank when seeds were separated by a petroleum ether
technique. We hypothesized that the germination performance
of Casuarina seeds could be further improved by
culturing only filled seed with their seed coat removed.
In vitro seed germination offers an alternative means to
propagate recalcitrant seeds and it has the potential to
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overcome all limitations of traditional seed propagation
(Fay 1991, 1992). Only a few studies on in vitro propagation
of Casuarina have been reported due to its
recalcitrant nature (Duhoux et al. 1986; Parthiban et al.
1997; Seth et al. 2007). There are no reports of in vitro
micropropagation of Casuarina hybrids. Therefore, the
objective of this study was to establish a protocol for
efficient seed germination and micropropagation of a
Casuarina hybrid (C. equisetifolia L. 9 C. glauca Sieber
ex Spreng) and examine the effects of BA and IBA plant
growth regulators (PGRs) on shoot multiplication and root
formation, respectively.
Brown cones ca. 11 months old were collected on
March 5, 2008 from a naturalized mature hybrid
(C. e. 9 C. g.) tree of unknown age. The tree was located
about 15 km west of Vero Beach, Florida (Lat. 27.70123,
Long. 80.50877) and growing along a ditch bank within a
citrus grove. The hybrid nature of the plant was determined
by AFLP (amplified fragment length polymorphism)
analysis (pers. comm., John Gaskin, USDA-ARS). Cones
were allowed to dehisce at room temperature (20°C) for
2 days. Seeds and cones were placed in plastic bags and
shaken to ensure that all seeds were collected. Seeds of
Casuarina are a tiny (\5 mm in length) dry, indehiscent
samara with a single wing.
In a preliminary experiment, the optimal treatment for
seed sterilization and seed coat softening was immersion of
seeds in 5 N NaOH (sodium hydroxide) for 15 min followed
by soaking in sterile water for 48 h. The media tested
for seed germination were: (1) water; (2) MS (Murashige
and Skoog 1962) basal medium; (3) MS basal medium plus
3% sucrose; and (4) MS basal medium supplemented with
3% sucrose and 1.6 g l -1 NH4NO3 (ammonium nitrate).
Media were adjusted to pH 5.8 with 0.1 N NaOH prior to the
addition of 8 g l -1 TC agar (Fisher BioReagents, Fair
Lawn, New Jersey) and autoclaved at 1.2 kg cm -2 for
30 min. Seed coats were carefully removed under an optical
microscope (Leica Zoom 2000, model Z45L, Leica Inc.
Buffalo, NY, U.S.A.). Only filled, fully developed seeds were
used for germination (Fig. 1). They were generally larger and
softer with a light creamy color compared to shrunken or
presumed insect damaged seeds. Seeds were cultured in
100 9 15 mm 2 Petri dishes containing 20 ml of medium.
There were 5 seeds per Petri dish and 10 replicate dishes per
treatment. Once seeds germinated, they were transferred to
GA-7 vessels (Magenta Corporation, Chicago, U.S.A.) containing
the same medium for another 4 weeks of growth.
Seedlings were kept in a culture room at a temperature of
22 ± 3°C with 12 h dark and 12 h light photoperiod at
40 lmol m -2 s -1 provided by cool white fluorescent lamps
(Lithonia Lighting F40 W/SS, Georgia, U. S. A.). The
number of germinated seeds was recorded at the end of
2 weeks and the germination percentage calculated.
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Plant Cell Tiss Organ Cult
Fig. 1 Casuarina hybrid (C. e. 9 C. g.) seed types as observed
without seed coats. From left to right: insect-damaged, shrunk and
filled seeds. Only filled seeds were used in the present study.
Bar = 1000.00 lm
Seed germination was the best on 8 g l -1 agar solidified
water medium (Table 1). Only seedlings germinated on this
medium were used for the shoot multiplication experiment
to avoid any carry-over effect of germination media on
shoot multiplication. Epicotyls about 2 cm in length were
severed and cultured on shoot multiplication media which
included MS basal medium plus 3% sucrose and supplemented
with BA at 4.4, 8.8, 17.8 and 35.6 lM. Medium free
of BA was used as the control. There were 4 explants per
GA-7 vessel and 8 replicate GA-7 vessels for each treatment.
Cultures were kept in a culture room under the same
conditions of seed germination. After 6 weeks of culture,
data on the number of explants with sprouted axillary buds,
the number of sprouts per explant and shoot length were
taken and the percentage of axillary bud sprouting was
calculated.
The best shoot multiplication was achieved on the MS
medium supplemented with 17.8 lM BA (Table 2), and
only shoots produced on this treatment were used for the
Table 1 Seed germination of a Casuarina hybrid (C. e. 9 C. g.)on4
media after 2 weeks culture at 22°C with a 12/12 h light/dark
photoperiod
Medium Germination (%) a
H2O 91 ± 0.3a
MS 45 ± 0.5b
MS ? 3% sucrose 33 ± 0.5bc
MS ? 3% sucrose ? 1.6 g/l NH4NO3 21 ± 0.4c
a Means followed by the same letter in each column are not significantly
different at 0.05 level. Data are means of 10 replicates and 5
samples per replicate

Plant Cell Tiss Organ Cult
Table 2 Effect of BA concentration on shoot proliferation after
6 weeks of culturing Casuarina hybrid (C. e. 9 C. g.) epicotyls on
MS medium
BA
concentration
(lM)
Sprouted
axillary
bud (%)
Mean number
of sprouts/
explant ± SE a
Shoot
length
(cm) ± SE
0 40.7 ± 0.1c 1.4 ± 0.2d 2.3 ± 0.1b
4.4 62.5 ± 0.1b 2.0 ± 0.2c 2.3 ± 0.1b
8.8 78.1 ± 0.1ab 2.4 ± 0.2c 2.5 ± 0.1b
17.8 87.5 ± 0.1a 3.8 ± 0.2a 3.2 ± 0.1a
35.6 84.3 ± 0.1a 3.2 ± 0.2b 3.0 ± 0.1a
a Means followed by the same letter in each column are not significantly
different at 0.05 level. Data are means of 8 replicates and 4
samples per replicate
rooting experiment. Proliferated shoots were removed from
the GA-7 vessels and cultured on root induction media
made of MS basal medium plus 3% sucrose and IBA at 4.3,
8.7, 13.0 and 17.4 lM. Medium without IBA served as the
control. There were 4 shoots per GA-7 vessel and 8 replicate
GA-7 vessels for each treatment. Cultures were kept in the
same culture room as described above. The number of
shoots forming roots, root number, and the length of longest
root of each shoot were recorded after 6 weeks induction
and the rooting percentage was calculated. For acclimatization,
plantlets (shoots with roots) longer than 3 cm with at
least 2 or 3 needles (branchlets) were removed from GA-7
vessels and carefully rinsed to remove any residual
medium. Plantlets were transplanted individually into trays
of 50 cells with each 5.0 9 5.0 9 10 cm 3 cell containing a
mixture of peat and pinebark (7:3). Plantlets were covered
for the first 2 weeks with a clear plastic tray to maintain
high humidity. All plantlets were maintained in a greenhouse
under natural photoperiod (10–14.5 h light) at a
temperature range of 20–31°C and hand watered as needed.
Experiments were established in a completely randomized
design. All data were subject to analysis of variance
using SAS (SAS Institute Inc 1999). Mean separation was
achieved by the Least Significant Difference test at 95%
level.
We did not observe any contamination following sterilization
of the Casuarina seeds. Seed coats were very easily
removed under the optical microscope after the NaOH
soaking and rinsing with sterile water. Radicles emerged
within 3 days and cotyledonary leaves formed within a
week among filled seeds with their coats removed (Fig. 2).
However, culture medium composition had a significant
effect on seed germination percentage. The greatest seed
germination (91%) was obtained on agar solidified water
medium, followed by 45% on MS, 33% on MS plus 3%
sucrose, 21% on MS plus 3% sucrose and 1.6 g/l NH4NO3
medium (Table 1). In nature, all Casuarina species form
Fig. 2 Seed germination stages in a Casuarina hybrid (C. e. 9 C. g.).
Seeds were germinated on 0.8% agar solidified water medium for
2 weeks at 22 ± 3°C under 12/12 h light/dark photoperiod: a naked,
filled seeds; b radicle development and emergence of cotyledonary
leaves; c, d developing seedlings; and e a fully developed seedling
with root and expanded cotyledonary leaves
root nodules with the soil actinomycete Frankia which fixes
atmospheric nitrogen and stimulates tree growth (Kang
1996; Mark et al. 1998; Zimpfer et al. 2004). However,
when the MS medium was supplemented with 1.6 g/l
NH4NO3 which doubled the N content compared to MS
medium alone, there were no beneficial effects on seed
germination. During seed germination, external nutrients in
the medium were apparently not required. The germination
rate decreased as the medium nutrient supplement increased
(Table 2). The most critical factor was water content, i.e.,
increased media water content resulted in higher germination
percentages. The results suggest that the nutrients
stored in the cotyledons were sufficient to support seed
germination.
Casuarina seeds have a low germination percentage,
often \50%, under commercial nursery conditions with
intact seeds planted in soil (Boland et al. 1996; Jerlin and
Srimathis 1997). In our previous study with intact seeds of
the Casuarina hybrid (C. e. 9 C. g.), seed germination was
only about 1% in soil and 4% on agar solidified medium
when intact seeds were tested (unpublished data). In the
current study, the highest seed germination percentage
through in vitro culture was 91%. There are two explanations
for this improvement: (1) only fully developed, filled
seeds were cultured. Seeds with low expectancy for germination
(shrunken, insect damaged and empty) were
identified under the optical microscope and eliminated; and
(2) seed coats were removed, thus, reducing their impediment
to germination.
Shoot proliferation readily occurred on multiplication
media (Fig. 3a) and was significantly affected by the
medium BA concentration (Table 2). Although 40.7% of
the explants sprouted axillary buds without BA, the number
123

Fig. 3 Micropropagation of a Casuarina hybrid (C. e. 9 C. g.): a
shoot multiplication of epicotyls excised from germinated seeds and
cultured on MS medium supplemented with 17.8 lM BA for 6 weeks
at a temperature of 22 ± 3°C with 12/12 h light/dark photoperiod; b
roots induced on MS medium supplemented with 13.0 lM IBA from
of sprouts per explant was very low, only 1.4. With the
addition of BA to the medium at increasing concentrations,
significant increases in the percentage of sprouted axillary
buds and mean number of sprouts per explant occurred at
all BA concentrations. However, shoot length was only
significantly increased at the 2 highest BA concentrations.
It is well established that BA is the best cytokinin for
stimulating shoot multiplication in a broad range of species
(Gaspar et al. 1996). Our greatest shoot multiplication, i.e.,
87.5% sprouted axillary buds, 3.8 sprouts per explant and
an average shoot length of 3.2 cm, was obtained on MS
supplemented with 17.8 lM BA. In the absence of BA,
explants responded poorly. In other reports, 11.1 lM BA
was optimal for sprouting of axillary buds (5.78 buds per
explant) from immature female inflorescence explants in
C. equisetifolia (Duhoux et al. 1986), and 4.44 lM BA
produced 73.8% axillary bud sprouting and 3.81 sprouts
per explant in a study of clonal propagation of C. equisetifolia
using shoot tips as explants (Seth et al. 2007).
These results suggest that optimal BA concentration for
shoot multiplication depends on species and type of explant
tissue.
Rooting is the biggest hindrance to the propagation of
woody plants (Murashige 1974; Zimmerman 1988, Hartmann
et al. 1990, Thorpe and Harry 1990). This is
confirmed by our experience in several attempts to root
cuttings of C. cunninghamiana through conventional mist
bed and water culture techniques (Castle 2007).
In the current study, rooting performance was greatly
improved by the presence of IBA in the medium (Fig. 3-b).
No root formation was observed on the control medium
without IBA and most plants became yellow and eventually
died over a period of 6 weeks. Rooting percentage,
root number per explant and root length per explant significantly
increased as IBA concentration increased
123
proliferated shoots after 6 weeks culture under the same conditions as
shoot multiplication; and, c plantlets transplanted to a potting mixture
of peat and pinebark (7:3) in plastic trays placed in a greenhouse
(Bar = 1 cm)
Table 3 Effect of IBA concentration on root development in Casuarina
hybrid (C. e. 9 C. g.) explants after 6 weeks of culture on MS
medium
IBA
concentration
(lM)
Rooting
(%)
Plant Cell Tiss Organ Cult
Mean number
of roots/
explant ± SE a
Mean length
of roots/
explant ± SE
0 0c 0c 0d
4.3 37.5 ± 0.1b 1.1 ± 0.1b 0.6 ± 0.1c
8.7 46.9 ± 0.1b 1.3 ± 0.1b 0.6 ± 0.1c
13.0 65.6 ± 0.1a 2.5 ± 0.1a 1.6 ± 0.1a
17.4 53.1 ± 0.1ab 1.5 ± 0.1b 1.0 ± 0.1b
a Means followed by the same letter in each column are not significantly
different at 0.05 level. Data are means of 8 replicates and 4
samples per replicate
(Table 3). The highest rooting percentage (65.6%) was
obtained on MS supplemented with 13.0 lM IBA. The
largest root number (2.5) and root length per explant
(1.6 cm) were also produced at this concentration, but
declined at the higher concentration.
All regenerated plantlets grew well under greenhouse
conditions (Fig. 3c). Therefore, we demonstrated that in
vitro root induction appears to be an alternative means for
propagation of difficult-to-root species. Inclusion of auxin
in the medium is a common and effective method to
stimulate adventitious root formation in vitro (Gavidia
et al. 1996; De Klerk 2002). Plants have been divided into
3 classes according to their dependence on auxin for
rooting (Noiton et al. 1992; Pruski et al. 2000). Casuarina
seems to belong to the class in which exogenous auxin is
required and can stimulate adventitious root formation.
However, the type and concentration of auxin that determine
optimal rooting response depends mainly on species,
type of explant tissue and the developmental stage of the
plant (Abdullah et al. 1989; De Klerk et al. 1997).

Plant Cell Tiss Organ Cult
In conclusion, a protocol for in vitro propagation of
Causarina hybrid (C. e. 9 C. g.) following facilitated seed
germination was established. The protocol consists of 4
steps, namely seed germination, shoot multiplication,
rooting and acclimatization. Seed germination, shoot multiplication
and rooting performance can be optimized by
manipulation of the culture medium. This in vitro culture
protocol provides an alternative means for propagation of
Casuarina and has a potential for large scale propagation on
a year-round basis.
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