Open Journal of Forestry
2012. Vol.2, No.2, 54-58
Published Online April 2012 in SciRes (http://www.SciRP.org/journal/ojf) http://dx.doi.org/10.4236/ojf.2012.22007
Copyright © 2012 SciRes.
Transient GUS and GFP Expression in Spanish Red Cedar
(Cedrela odorata L.) Somatic Embryos. Optimization
of Bombardment Conditions and Evaluation of
Selective Agent Lethal Dose
Yuri J. Peña-Ramírez1*, Max M. Apolinar-Hernández1, Oscar A. Gómez-y-Gómez1,
Luisa A. López-Ochoa2, Aileen O’Connor-Sánchez2
1Instituto Tecnológico Superior de Acayucan, Acayucan, México
2Centro de Investigación Científica de Yucatán, Mérida, México
Received October 2nd, 2011; revised January 14th, 2012; accepted January 21st, 2012
Cedrela odorata is a tropical tree widely appreciated for its wood. Commercial plantations are frequently
hampered by the attack of the meliacea borer, Hypsipyla grandella, and the lack of resistant varieties. C.
odorata traditional breeding would consume very long periods of time, thus direct transfer of entomotoxic
coding genes to generate resistant varieties is a promising alternative. There are two prerequisites for gene
manipulation of this species: 1) to set the conditions for transgene delivery and 2) to have a way to select
regenerating transformed plants. In this paper, we report the optimal biolistics conditions for transient ex-
pression of uidA and gfp reporter genes in C. odorata somatic embryos and the selective doses for kana-
mycin, spectinomycin, phosphinotrycin and hygromycin to screen transformed cells.
Keywords: Biolistics; Genetic Transformation; Tree Genetic Modification; Tropical Wood
The establishment of commercial plantations of some tropi-
cal hardwood species native to the Americas, such as Spanish
red cedar (Cedrela odorata), mahogany (Swietenia macro-
phylla), and ipé (Tabebuia spp.), among others, faces serious
limitations because of the lack of domesticated varieties able to
sustain cultivated plantations (Merkle & Nairn, 2005). C. odo-
rata (Meliaceae) is the second most valued wood tropical
product, thus it has a huge economical importance as a crop,
however, its high susceptibility to the attack of the borer Hyp-
sipyla grandella (Lepidoptera: Pyralidae) has hindered the ef-
forts to grow this species as a managed crop (Pérez-Salicrup &
Esquivel, 2008). Among all the existing strategies for C. odo-
rata improvement, breeding through modern genetic manipula-
tion remains to date a pending issue. Direct transfer of a desir-
able gene into the trees could generate novel traits without any
significant modification of their genetic background, one of
those traits could be pest resistance (Campbell et al., 2003).
Gene manipulation of tree species has already been employed
as a tool for basic studies, for manipulation of lignin/cellulose
content, for improving wood quality or kraft pulping, for modi-
fication of flowering time and tree architecture, to confer
abiotic stress tolerance, and for developing pest-resistant tree
varieties (Giri et al., 2004).
In order to move forward to the genetic manipulation of C.
odorata, our group has recently developed an efficient regen-
eration system of juvenile material via somatic embryogenesis
using immature zygotic embryos as initial explants (Peña-
Ramírez et al., 2011). However, to establish a reliable gene
transfer protocol, it is also necessary to find an efficient gene
transfer procedure and a methodology to select transformed
cells. Nowadays, gene transfer mediated by biolistic bombard-
ment is a widely used tool, though the efficiency of this proce-
dure varies depending on a number of physical and biological
factors, such as the amount of DNA loaded onto the projectiles,
the projectile’s speed, their size and density (Sanford et al.,
1993), the helium pressure used (Able et al., 2001; Casas et al.,
1993; Ikea et al., 2003; Tadesse et al., 2003), the target explant
characteristics, and the osmotic pressure in the medium (Klein
& Jones, 1999). Therefore, the optimum efficiency of het-
erologous DNA transfer into plant cells can only be achieved
by a fine balance between the factors involved in bombardment
efficiency and factors related to target-tissue damage (Tadesse
et al., 2003; Zuker et al., 1995). Optimizing these conditions is
frequently a tedious and time consuming issue, mainly when a
stable expression of the genes used for detection and selection
is required. These limitations can be circumvented by assessing
transient expression of reporter genes, allowing almost imme-
diate detection of transformed cells (Hunold et al., 1994). Tran-
sient expression assays have proven useful to find the optimal
conditions for transformation (Able et al., 2001; Heim & Tsein,
1996; Jeoung et al., 2003). The genes that are most frequently
employed as reporters for transient gene expression are: 1) the
bacterial gene uidA (GUS) coding for β-glucuronidase, which
catalyses the hydrolysis of X-Gluc (Jefferson et al., 1987), and
2) the gfp gene coding for the green fluorescent protein (GFP)
cloned from jellyfish Aequorea victoria (Elliott et al., 1999),
which allows a simple detection by visualizing its expression in
Y. J. PEÑA-RAMÍREZ ET AL.
intact tissues, without adding exogenous substrates. In addition
to an optimized DNA-delivery system, to achieve a successful
plant transformation requires an efficient selection of the trans-
formed cells, to allow only the regeneration of transgenic plants,
preventing the proliferation of non-transformed sectors (Pérez-
Barranco et al., 2009). The main goals in the present work were
to optimize the conditions to transform C. odorata somatic
embryos by biobalistics, using transient expression of uidA and
gfp as reporter genes, and to determine the inhibitory doses of
four selective agents to be used for T0 plant selection. Both
achievements will lead to the establishment of an efficient C.
odorata transformation protocol.
Materials and Methods
Embryogenic calluses were initiated and maintained accord-
ing to the methodology previously reported by our group (Peña-
Ramírez et al., 2011). For biolistic experiments, calli were
subcultured one week prior to transformation. Each shoot was
performed over approximately 0.1 g of embryogenic calli type
II-A located inside of a 3 cm-diameter circle at the center of
petri dishes containing full-strength MS (Murashige and Skoog
1962) medium, 80 mM sucrose, 13.4 µM dicamba and 2.5%
(w/v) GelRite® (PhytoTechnology Laboratories, Lenexa, KS),
pH 5.7. For lethal-dose experiments, 0.2 g of embryogenic
clusters were spread in each Petri dish containing the same
basal medium supplemented with the appropriate antibiotics
Plasmid Preparation and Biolistics
The plasmids pCAMBIA1201 and pCAMBIA1302 (www.
cambia.org) carrying either uidA or gfp gene driven by the
constitutive CaMV 35S promoter, previously cloned in E. coli
DH5αF’, were extracted using QIAGEN (Germantown, MD)
Maxi Prep kits. The concentration of the plasmid DNA was cal-
culated using a spectrophotometer (SmartSpec, BioRad, Her-
cules, CA). Embryo bombardment was carried out using a
PDS-1000/He Biolistic Particle Delivery System (BioRad). The
plasmid DNA was coated onto gold particles as described by
the manufacturer (BioRad). Six microlitres of the DNA-mi-
crocarrier suspension (see Table 1) were dispensed onto each
macrocarrier membrane and allowed to dry. The standard
bombardment procedure was performed using manufacturer’s
instructions. In the first treatment, variables were set as fol-
lows: 6 cm of shooting distance, 1 µm particle diameter, 6 µg
of DNA, 0.1 M spermidine, and vector pCAMBIA1302. To
assess the subsequent parameters, further variables were set
according to the optimal condition previously determined.
Each treatment was repeated 3 times, each consisting of 5
Evaluati on o f GUS and GFP Transient Expression
Transient expression was analyzed 36 hr after bombardment.
GUS assays were carried out using the protocol reported by
Jefferson et al. (1987) with minor modifications. GUS expres-
sion was tested by immersing explants in 5-bromo-4-chloro-3-
indolyl-β-d-glucuronic acid (X-Gluc) buffer overnight, at 37˚C
in the dark, with a subsequent wash for 24 h in absolute ethanol.
The number of blue spots (foci) on explants was observed un-
der a stereomicroscope using a white light source. For GFP
analysis, calli were observed under an Olympus microscope
equipped with a GPP-2 filter and ultraviolet illumination source.
For both systems, the number of foci per petri dish was quanti-
fied by analyzing digital photographs with the assistance of the
software Quantity One® (BioRad).
Determination of Antibiotic Lethal Dose
Wild type embryogenic calli were cultured on MS medium
prepared as previously described and supplemented with dif-
ferent concentrations of Kanamycin (KAN) (0.0, 20.0, 50.0,
100.0, 150.0, 200.0, 350.0, 500.0, or 750.0 µM); hygromycin
(HYG) (0.0, 1.0, 2.0, 5.0, 10.0, 15.0, 20.0, 35.0, or 50.0 µM);
Spectinomycin (SPE) (0.0, 20.0, 50.0, 100.0, 150.0, 200.0,
500.0, 750.0, or 1000.0 µM); or phosphinotricin (PPT) (0.0, 0.5,
1.0, 2.0, 3.5, 5.0, 10.0, 15.0, 20.0, 35.0, or 50 µM) (Phy-
toTechnology Laboratories, Lenexa, KS). Antibiotics were
filter-sterilized and added to the autoclaved medium at 45˚C. 10
calli, 20 mg each, were used for each treatment and the proce-
dure was repeated 3 times. After three weeks, plant material
was transferred to an antibiotic-free medium. Viability was
assessed 3 weeks later as the ability of embryos to proliferate
(measured as weight gain) on an antibiotic-free medium.
Each parameter was evaluated by using an experimental
sample of five Petri dishes and repeated twice. The data were
analyzed using standard ANOVA procedures. The differences
between the means were determined by Fisher’s least signifi-
cant difference (LSD) tested with the assistance of Statistica®
software package (StatSoft, Tulsa, OK).
Results and Discussion
To find the optimal conditions for microparticle delivery, 16
treatments were analyzed, which involved different membrane
rupture pressures, shooting distances, microcarrier diameters,
plasmid DNA amounts, spermidine concentrations, and two
pCAMBIA vectors. Treatment efficiency was evaluated by the
number of transient expression foci produced. As can be seen in
Table 1, the results show significant differences between the
different pressures assayed. 1200 psi produced the best results,
followed by 1600 and 900 psi. These acceleration pressures
might be considered relatively high for this kind of explants,
according to Rasco-Gaunt et al. (1999) who found that bom-
bardment pressures over 900 psi cause a drastic drop in tran-
sient expression. However, other authors have reported similar
results for transformation of embryogenic calli (Abdollahi et al.,
2009; Tee & Maziah, 2005).
Shooting distance also had a significant effect. 9 cm resulted
the best, followed by 7.5 and 6 cm; longer distances caused an
abrupt decrease in the number of foci. Tee and Maziah (2005)
and Abdollahi et al., (2009) have found that short distances
combined with high shooting pressures might cause an increase
in cell damage or injury, resulting in low regeneration rates. We
observed this to happen in the case of C. odorata regeneration
index, which was significantly affected in treatments 5, 6, and 7,
where high shooting pressures were combined with short dis-
tances. In those cases the regeneration rate dropped by nearly
Copyright © 2012 SciRes. 55
Y. J. PEÑA-RAMÍREZ ET AL.
Copyright © 2012 SciRes.
Optimization of biolistic parameters.
Treatment Membrane rupture
diameter (µm)DNA amount (µg)Spermidine
addition pCambia vector Number of foci/100 mg
of embryogenic calli
1 400 6 1 6 + 1302 21.0 ± 1.1c
2 600 6 1 6 + 1302 28.6 ± 1.9d
3 900 6 1 6 + 1302 49.4 ± 1.5e
4 1100 6 1 6 + 1302 49.2 ± 1.0fg
5 1550 6 1 6 + 1302 45.6 ± 1.9ef
6 1100 3 1 6 + 1302 15.0 ± 2.0b
7 1100 4.5 1 6 + 1302 19.6 ± 1.5c
8 1100 7.5 1 6 + 1302 56.0 ± 1.4h
9 1100 9 1 6 + 1302 60.6 ± 2.6i
10 1100 12 1 6 + 1302 12.4 ± 2.4ab
11 1100 9 0.6 6 + 1302 32.2 ± 1.1d
12 1100 9 1.6 6 + 1302 10.4 ± 1.1ab
13 1100 9 1 2 + 1302 32.2 ± 0.9c
14 1100 9 1 10 + 1302 67.2 ± 0.7j
15 1100 9 1 10 - 1302 8.8 ± 1.2ab
16 1100 9 1 10 + 1201 57.6 ± 1.2h
The average value of the number of foci per treatment is presented ± Mean Standard Error. Cursive letters next to values correspond to significant difference levels by LSD
test at p ≤ 0.05; n = 5 × 3.
50% (data not shown). With regard to particle size, the 1 µm
microprojectile resulted in the highest numbers of foci. Optimal
results were obtained when 10 µg of plasmid DNA were pre-
cipitated onto 2 mg of gold particles. In contrast, spermidine
depletion strongly decreased the number of foci, demonstrating
its importance for the adequate adsorption of the plasmid onto
the gold particle. These results coincide with the findings re-
ported by Rasco-Gaunt et al., (1999), who reported a drop of
uidA transient expression in samples without spermidine.
Moreover, the differences observed between pCAMBIA plas-
mids 1302 and 1201could be an effect of the image analysis,
because fluorescent foci can be quantified more efficiently than
blue ones. The lower contrast between expressed uidA foci and
their background, as well as the difficulty to get spots arrange-
ments in a single plane, making it hard to get good images for
foci quantification (Schöpke et al., 1997) and therefore the ana-
lysis might be less sensitive. Nonetheless it can be concluded
that both uidA and gfp can be successfully used as reporter
genes for transformation in C. odorata embryogenic calli (Fig-
Selective Dose Determination
To determine a selective concentration of antibiotics useful
to inhibit the proliferation of untransformed plant cells, four
selective agents were evaluated cultivating C. odorata type II-A
embryogenic calli in the presence of different concentrations of
KAN, HYG, SPE and PPT. As shown in Figure 1(d), callus
viability was affected by all the tested antibiotics following
similar patterns. PPT was the most toxic agent, requiring a
concentration of 5 µM to kill 100% of calli, followed by 20 µM
HYG, 500 µM SPE, and 1 mM KAN. Similar concentrations
have been reported to inhibit embryo regeneration of chestnut
(Rothrock et al., 2007), grape (Geier et al., 2008), oil palm
(Parveez et al., 1997), Eucalyptus (Sartoretto et al., 2002) and
poplar (Okumura et al., 2006). The obtained lethal doses could
be considered as common for several plant species, however it
was very important to establish them for C. odorata because
sometimes even slight variations in the physiological conditions
of a particular tissue or in its the genetic background may
change its susceptibility to the toxicity of a given selective
agent (Duke, 1996). Finally, it was observed a non linear sus-
ceptibility curve for all the tested antibiotics, which quickly
drops to around 20% to 25% of survival, followed by a weaker
slope to finally reach 0% of somatic embryo viability. A bi-
phasic behavior has also been reported for other embryogenic
cultures (Catlin, 1990; Parveez et al., 1997) and is in agreement
with classic data for cell culture systems with high mitotic ac-
tivity (Drewinko et al., 1974), thus it supports our previous
observations that suggest an unsynchronized and highly prolif-
erative nature of C. odorata embryogenic callus.
As far as we know, this work is the first report of an ap-
proach to establish a C. odorata gene manipulation protocol.
The optimized set of biolistic parameters and the determination
of lethal dosages for four antibiotics commonly used for selec-
tive screening of transformed plants, provide the necessary
foundations for future efforts to generate improved varieties of
C. odorata through modern genetic manipulation, including
those resistant to H. grandella. Most of the previous work re-
lated with genetic modification of Meliacea has been con-
stricted the genus Azadirachta (Morimoto et al., 2006) via
Agrobacterium-mediated gene delivery. This work provides
experimental data that could be used not only for C. odorata
Y. J. PEÑA-RAMÍREZ ET AL.
Transient expression in embryogenic calli and lethality curves. Bom-
barded embryogenic calli expressing transient foci of GUS (a) or GFP
under white (b) or UV light (c). Arrows in (a) point to foci clusters in a
representative bombarded callus. The white bar in a) corresponds to a
length of 50 µm whereas in (b) and (c) it is equivalent to 1 cm. d)
shows the lethality curves of PPT (×), HIG (●), SPE (▲), and KAN (■)
measured as viability of C. odorata embryogenic callus cultured under
several concentrations of selective agents. Bars at each point corre-
spond to the Mean Standard Error.
manipulation, but also lay the foundations to generate trans-
formed Meliacea trees of other genus via biolistic approach.
The authors thank to Virginia Herrera for training given to
MMAH. Financial support provided by CONACYT-CONA-
FOR C03-10013; SEP-CONACYT C01-53851and ITSA-DIC
2004-1 is gratefully acknowledged. MMAH and GJTL thank
CONACYT for undergraduate studentships.
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