Plant Cell Tiss Organ Cult (2010) 102:251–258
DOI 10.1007/s11240-010-9714-8
RESEARCH NOTE
Characterisation of a cyclin-dependent kinase (CDKA) gene
expressed during somatic embryogenesis of coconut palm
Mayra Montero-Cortés • Francisco Rodrı́guez-Paredes •
Caroline Burgeff • Teresa Pérez-Nuñez • Iván Córdova •
Carlos Oropeza • Jean-Luc Verdeil • Luis Sáenz
Received: 23 October 2009 / Accepted: 11 February 2010 / Published online: 26 February 2010
Ó Springer Science+Business Media B.V. 2010
Abstract The CDKA gene is linked to the cellular con-
trol. This gene was isolated from coconut palm (Cocos
nucifera L) and a detailed expression analysis was done
during somatic embryogenesis. Analysis of the deduced
amino acid sequence showed the most important residues
to be conserved. The highest homology was with Picea
abies (96% similarity). Expression of the putative
CnCDKA gene steadily increased during embryogenic
callus formation phase when embryogenic competence is
attained. In situ hybridization specified the localization of
the transcripts as being mainly in a few cell layers within
the meristematic centres in embryogenic calli at 90 day
cultures. Analysis of CnCDKA expression at different
somatic embryo formation stages showed that the expres-
sion was decreased progressively with the lowest expres-
sion in germinated somatic embryos.
Keywords Cocos nucifera  Control of cell division 
Somatic embryogenesis
M. Montero-Cortés  F. Rodrı́guez-Paredes  C. Burgeff 
T. Pérez-Nuñez  I. Córdova  C. Oropeza  L. Sáenz (&)
Biotechnology Unit, Centro de Investigación Cientı́fica de
Yucatán, Calle 43 No. 130. Colonia Chuburna de Hidalgo, 97200
Mérida, Yucatán, México
e-mail: vyca@cicy.mx
J.-L. Verdeil
Centre de Coopération Internationale en Recherche
Agronomique pour le Développement, Montpellier Cedex 5,
France
J.-L. Verdeil
Institut de Recherche pour le Développement, Montpellier
Cedex, France
Introduction
Coconut palm (Cocos nucifera L.) is a major crop in most
tropical areas, often generating income and subsistence
for small-scale producers. The coconut palm provides a
number of raw materials that are used to produce different
value added products in the coconut industry. Coconut oil
that is extracted from the solid endosperm, is one of the
most valuable products of the coconut palm. It is a
valuable option to substitute the fossil diesel that has
already been practiced in the Philippines (Tan et al.
2004).
Clonal production of coconut is a promising plant
breeding tool (Chan et al. 1998) and the efficiency of clonal
propagation of coconut palm has been improved signifi-
cantly (Pérez-Nuñez et al. 2006). Nonetheless, coconut
remains a recalcitrant species for in vitro culture, that is
mainly reflected by slow in vitro morphogenesis (e.g.
3–4 months to embryogenic calli formation). Histocyto-
logical studies of in vitro cultured coconut explants have
also revealed the frequent existence of nuclei with con-
densed chromatin, which is typical of quiescent cells
(Buffard-Morel et al. 1992; Verdeil et al. 1994). Coconut
tissues that have been used as explants for in vitro culture
exhibit a low mitotic index. The immature leaves contained
less than 1% of actively dividing cells (Jesty and Francis
1992). Furthermore, a progressive slowing of the growth
was determined during in vitro culture in the G0/G1 phase
by flow cytometric analysis (Sandoval et al. 2003). Con-
trolling the orientation of coconut cells towards a meri-
stematic state (i.e. cells beginning active division) is clearly
challenging.
Plant morphogenesis involves close control and coordi-
nation of proliferative activity through regulation of the cell
cycle in meristematic tissues (Sugiyama 1999; Planchais
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252
et al. 2000). Artificial initiation and maintenance of cell
division is a prerequisite for generation and establishment of
the dedifferentiated, targeted meristematic cell. In cell cul-
tures, dividing cells can follow alternative developmental
pathways such as unorganized callus growth, root and shoot
initiation or somatic embryo formation. In the latter, division
of somatic or dedifferentiated cells generates a cellular state
similar to that of the zygote formed after egg cell fertiliza-
tion (Fehér et al. 2003).
The eukaryote cell cycle is controlled by ordered action
of cyclin-dependent kinases (CDKs). These serine/threo-
nine kinases are activated by binding positive regulators,
the cyclins which appear periodically during the cellular
cycle and determine activity as well as substrate specificity,
as well as localization and controlled stability of cell
division partners (Roudier et al. 2000). Plants contain a
number of CDK-related genes and several lines of evidence
suggest that various related genes are involved in different
phases of the cellular cycle (Burssens et al. 1998).Up to
date, five CDK genes have been described in plants based
on the cyclin-binding motif present in each type (Joubès
et al. 2000) and the five classes of mitotic cyclins (Dewitte
and Murray 2003). They clearly have potential in regula-
tion of the cell cycle in plants. Four types of CDKs have
been described in Arabidopsis thaliana (Dewitte and
Murray 2003). The CDKA carries the PSTAIRE amino acid
hallmark, while other CDK classes contain divergent
motifs: PPTALRE or PPTTLRE motif, PITAIRE, N(I/
F)TALRE and SPTAIRE (Joubès et al. 2000).
Complementation of temperature-sensitive mutants in
yeast cdc2/cdc28 genes, the first evidence for plant CDK
functionality, was only successful with plant CDK from the
CDKA group (Colasanti et al. 1991; Ferreira et al. 1991).
In Saccharomyces cerevisiae, the CDC28 protein alone is
involved in the G1/S and G2/M controls (Mendenhall and
Hodge 1998), whereas in animals, CDK1, -2 and -3 (all
belonging to the same group as plant CDKA) ensure dis-
tinct functions from G1 to mitosis (Morgan 1997). At the
transcript and protein levels, plant CDKA does not fluc-
tuate significantly during the cell cycle and is present at
low levels in non-dividing tissues. This suggests the dual
function of plant CDKA during both S and M phase pro-
gression (Colasanti et al. 1991; Ferreira et al. 1991; Hirt
et al. 1993), and their involvement in cell proliferation and
maintenance of cell division competence in differentiated
tissues during plant development (Martinez et al. 1992;
Hemerly et al. 1993).
The objective of the present study was to analyze a
putative CnCDKA gene from coconut palm, which is
cloned and characterized for the first time. This gene is
involved in control of the cellular cycle and will provide
insight into this process in this specie.
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Plant Cell Tiss Organ Cult (2010) 102:251–258
Materials and methods
Plant material
Coconut fruit were harvested 12–14 months after con-
trolled pollination of 15 year-old Malayan dwarf palms.
Endosperm cylinders (2 cm diameter) containing embryos
were excised in the field, placed in a 0.6% NaClO (w/v)
solution and rinsed three times with sterile distilled water
during collection. Under aseptic laboratory conditions, the
cylinders were washed in 70% ethanol for 3 min, rinsed
three times with sterile distilled water for 5 min each,
washed again in a 6% NaClO solution for 20 min and
finally rinsed three times with sterile water. The embryos
were excised from the endosperm and washed in a 0.6%
NaClO solution for 10 min before rinsing with sterile
distilled water three times. The plumules were excised
from these embryos under a stereoscopic microscope and
placed directly on culture medium.
Media preparation
Media preparation and culture conditions were done
according to Pérez-Nuñez et al. (2006). Media I and II,
each prepared using Y3 medium (Eeuwens 1976) were
supplemented with 3 g l-1 Gelrite and 2.5 g l-1 charcoal
(acid-washed, plant cell culture tested). Medium I con-
tained 600 lM 2,4-dichlorophenoxyacetic acid (2,4-D),
while Medium II contained 6 lM 2,4-D and 300 lM
benzyladenine (BA). All chemicals were reagent grade
(Sigma, St. Louis, MO, USA). Medium pH was adjusted to
5.75 with KOH before autoclaving for 20 min at 120°C.
Culture conditions for the formation of embryogenic
callus and somatic embryos
Embryogenic structures were excised from embryogenic
calli obtained from plumule explants and subcultured in
Medium I to induce new embryogenic callus. This was
repeated twice so three cycles of embryogenic callus mul-
tiplication were carried out. The embryogenic structures
obtained from the calli of the third cycle were used then on as
explants for the experiments reported here. Callus induction
was done by culturing each explant for 90 days in a 35 ml
glass vessel containing 10 ml Medium I. Cultures were kept
under complete darkness at 27 ± 2°C without subculturing
(conditions I). Somatic embryos were induced by subcul-
turing the calli in a 100 ml glass vessel containing 25 ml
Medium II. Cultures were kept under a 16 h photoperiod
(45–60 lmol m-2 s-1 PPFD) at 27 ± 2°C, and explants
subcultured once every 2 months (conditions II). Embryo
germination occurred in the same medium and conditions.
Plant Cell Tiss Organ Cult (2010) 102:251–258
Histology
Histological procedures were done according to Buffard-
Morel et al. (1992), with slight modifications. Tissue
samples were fixed in 4% paraformaldehyde in phosphate
buffer (pH 7.2) for 24 h under negative pressure. Samples
were dehydrated using a series of aqueous ethanol solutions
(30, 50, 70, 80, 90, 95 and 100%) for 1 h in each solution.
This was followed by impregnation with JB-4R resin
(Polyscience, Warrington, PA, USA). Five-micrometer
sections were obtained from the resin-impregnated tissues
using a microtome (HM 325, MICROM) equipped with
steel blades. Sections were double-stained with periodic
acid-Schiff (PAS) reagent and protein specific naphthol
blue black (NBB). PAS stains starch reserves and walls in
pink, while NBB specifically stains soluble or reserve
proteins dark blue (Fisher 1968).
Cloning of CDKA cDNAs
A randomly primed (a32)-labelled cdc2a cDNA probe of A.
thaliana (Jim Murray, Institute of Biotechnology, Cam-
bridge) was used to screen approximately 5 9 105 clones
from a cDNA library built in pBluescript SK vector with
poly (A?) RNA isolated from plumules of zygotic coconut
embryos cultured in vitro in media without growth regu-
lators. Hybridisations were done at 40°C, and the mem-
branes washed twice for 5 min in 29 SSC/0.1% (w/v) SDS
at ambient temperature and twice in 19 SSC/0.1% (w/v)
for 15 min SDS at 40°C. Selected positive clones were
purified by screening round and the cDNA inserts excised
in vivo following manufacturer instructions. The cDNA of
the positive clones was sequenced with an external agent
on both strands.
Sequence comparison
Sequence analysis was carried out at the National Center for
Biotechnology Information (NCBI, http://www.ncbi.nlm.nih.
gov/) using the BLAST service. Sequence alignments were
done using the ClustalW2 program (Larkin et al. 2007) pro-
vided by the EMBL-EBI server http://www.ebi.ac.uk/
Tools/clustalw2/index.html).
Nucleic acid extraction and cDNA synthesis
The calli and somatic embryos were immediately frozen in
liquid nitrogen. Total RNA was extracted using 0.1 M
Tris–HCl (pH 8.0); 0.2 mM EDTA (pH 8.0); 2% SDS; and
0.15% a-monothiolglycerol followed by a phenol-chloro-
phorm (1:1) extraction. The mixture was then incubated at
253
70°C for 5 min, centrifuged and the supernatant was col-
lected. RNA was precipitated by incubating with cold
isopropanol at -20°C for 10 min, followed by centrifug-
ing. The pellet was washed with 75% ethanol. Each RNA
sample was analyzed by 1% agarose gel electrophoresis.
DNA from the samples was eliminated with a DNAse I
(Ambion, Foster city, CA, USA) treatment. RNA was
quantified with the Ribogreen RNA Quantitation Kit
(Invitrogen, Carlsbad, CA, USA). First-strand cDNA was
prepared using random hexamers (Invitrogen) and 1 lg
RNA in conjunction with reverse-transcriptase (Superscript
II, Invitrogen), according to the directions of the
manufacturer.
Expression studies
Real-time PCR was performed using platinum SYBR green
qPCR superMix-UDG (Invitrogen) and analyzed in an
iCycler IQ real-time PCR detection system (BIO-RAD,
Hercules, CA, USA) following manufacturer instructions.
The CnCDKA primers FW 50 -TGGATCAGTATGA
GAAGGTGGAGAAGAT-30 and RV 50 -TCGATCTCAG
AATCTACAGGAAACAGT-30 were designed to quantify
CnCDKA expression levels, which were normalized to
those of the 18S rRNA gene (FW 50 -CGGCTACCACA
TCCAAGGAA-30 ; and RV 50 -GCTGGAATTACCGC
GGCT-30 ) in each sample. PCR conditions were one cycle
of 52°C for 2 min and 95°C for 10 min, followed by 35
cycles of 94°C for 2 min, 62°C for 40 s and 72°C for 40 s).
Each quantitative PCR experiment was conducted in
duplicate, and the entire procedure was repeated three
times. Real-time PCR results were analyzed using the
2-DDCt method (Livak and Schmittgen 2001) with appro-
priate validation experiments performed beforehand (Real-
time PCR. Applications Guide BIO-RAD). Amplified
cDNAs were cloned and sequenced to check amplification
product specificity.
In situ hybridisation analysis
The pCRII-TOPO plasmid containing the CnCDKA cDNA
(1,323 bp) was used to synthesize both sense and antisense
DIG-labelled RNA probes, using T7 or SP6 RNA poly-
merase, respectively, and following manufacturer instruc-
tions (DIG RNA labelling kit SP6/T7, Roche, Germany).
Riboprobes were digested to an average length of 300
nucleotides by alkaline hydrolysis. The overall in situ
hybridization procedure was done according to Burgeff
et al. (2002). Briefly, embryogenic calli explants were fixed
overnight at 4°C in 4% paraformaldehyde in phosphate
buffer at pH 7.2 under negative pressure. The fixed tissues
were then dehydrated in a series of alcohol solutions,
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embedded in paraffin (Paraplast, Sigma), sectioned at
10 lm and mounted on poly-L-lysine-coated slides (Poly-
Prep, Sigma). Sections were rehydrated and incubated
30 min at 37°C with 1 lg ml-1 proteinase K (Sigma).
Hybridisations were done overnight at 50°C with approx-
imately 2.5 ng digested riboprobe per slide diluted in
hybridization buffer (50% formamide, 69 SSC, 1% Den-
hardt, 100 lg ml-1, tRNA, 100 lg ml-1). After washing
twice with 0.1 SSC and 0.1% SDS at 50°C, the slides were
subjected to an RNAse A treatment (10 lg ml-1) for
30 min. The sections were then washed twice with SSC 29
at 50°C and with TBS 5 min at room temperature, and
incubated 60 min in blocking solution (Roche). After
incubation, the slides were washed with a solution of 0.1%
BSA and 0.3%Triton X-100 in TBS buffer for 30 min, and
incubated with anti-DIG sheep antibody coupled to alka-
line phosphatase (150 U) for 90 min (Roche), diluted
1:3,000 in blocking solution). The sections were washed
three times in the previous solution (20 min each washing),
rinsed in staining buffer (100 mM Tris–HCl pH 9.5;
100 mM NaCl; 50 mM MgCl2; Roche (Germany)
and incubated overnight in staining buffer supplemented
with 4-nitroblue tetrazolium chloride (0.4 mg ml-1) and
5-bromo-4-chloro-3-indolyl-phosphate toluidine salt
(0.19 mg ml-1). Stained sections were photographed with
an Axioplan microscope (Carl Zeiss, Jena, Germany).
Statistical analysis
Data represent the means ± standard deviation (SD) from
the results of quantitative PCR analysis of three indepen-
dent biological samples (each by duplicate) by treatment.
An analysis of variance (ANOVA) and a Student–New-
man–Keuls test was performed to test the differences
between the means.
Fig. 1 Embryogenic callus formation during in vitro culture: initial
explant (a) and changes occurring at 30 days (b), 60 days (c), 75 days
(d) and 90 days (e) of culture in induction medium; and non
embryogenic callus (f). Formation of somatic embryos: callus with
123
Plant Cell Tiss Organ Cult (2010) 102:251–258
Results
Morphogenic development
The initial explants were embryogenic structures
excised from a previous embryogenic calli as described
by Pérez-Nuñez et al. (2006) (Fig. 1a). After 30 days of
culture initial callus formed, this callus was beige in
colour and 2–3 mm in diameter (Fig. 1b). At 60 days,
the callus exhibited translucent structures and had
attained 4–5 mm diameter (Fig. 1c). At 75 days, trans-
lucent structures developed, embryogenic structures
began to form, and the callus was *5–6 mm diameter
(Fig. 1d). At 90 days, the callus measured 6–9 mm
diameter and showed clear embryogenic structures
(Fig. 1e). During the development of explants, callus
that did not show embryogenic structure formation and
somatic embryo were referred to as non embryogenic
callus (NEC). They were yellowish and smaller than the
embryogenic callus (Fig. 1f).
Somatic embryo formation generally began at approxi-
mately 30 days culture in medium II; under conditions
II (Fig. 1g), proembryos were observed as early as 15–
25 days culture (Fig. 1h). At 30–60 days globular (Fig. 1i)
and coleoptilar (Fig. 1j) stage embryos were observed. The
coleoptilar embryos began to germinate at 60–90 days
culture (Fig. 1k).
CDKA gene cloning and sequence analysis
To identify genes with a potential CnCDKA-related gene, a
cDNA bank from germinating plumules was screened with
a heterologous CDKA gene from A. thaliana. One CDKA-
related gene was isolated: CnCDKA. The open reading
frame (ORF) of CnCDKA consists of 882 bp with the
somatic embryos at different stages (g), piece of callus with
proembryos (h), globular embryo (i), coleoptilar embryo (j) and
germinating embryo (k). All bars = 1 mm, except K = 5 mm. TS
translucid structures, ESt embryogenic structures
Plant Cell Tiss Organ Cult (2010) 102:251–258 255

potential to encode a 294 aa (GenBank GQ924086). This
ORF is flanked by 317 and 606 bp in the 50 and 30
untranslated regions respectively. The predicted protein
contains five functionally important regions that were
characteristics for CDKA. They are cyclin-binding domain
(residues 44–56) containing the PSTAIRE motif (residues
44–56), threonine and tyrosine in positions 14 and 15
phosphorylation of which inhibits this enzyme’s activity,
T-loop area (residues 147–172) centred around threonine a
phosphorylation of which stabilizes the cyclin-binding
(Joubès et al. 2000), T-loop flanking Asp-146 that was
involved in positioning of the bound ATP required for
kinase activity and SUC/CKS-binding motif (residues 207–
In situ expression in the embryogenic callus
In situ localization of the CnCDKA gene RNA transcripts
was identified in embryogenic calli at 90 days. Transcripts
were identified in periphery of the meristematic nodules in
the calli (Fig. 4a). Only a few cell layers of the meriste-
matic nodules had transcripts, while the protodermis and
inner cells emitted no signals (Fig. 4b). A similar
embryogenic callus doubled-stained with NBB and PAS
a

Relative expresssion units

244; Fig. 2). The CDKA amino acid sequences were gen- 4
erally highly conserved with some variation in the extreme
b
30 region (Fig. 2).
3

CnCDKA expression during embryogenic callus

formation 2
bc bc

The CnCDKA gene expression profile was analysed using 1 c

quantitative Real-Time PCR during 90 days culture in the
embryogenic calli formation phase, when calli acquired
0
embryogenic competence. CnCDKA gene expression was 30 60 75 90 NEC
lowest in the early days of culture and began to increase at Days of culture
day 75 (Fig. 3). By day 90 when embryogenic structures
Fig. 3 CnCDKA expression profile during embryogenic callus
appeared, CnCDKA expression level was four fold the level
formation. Figure represents means ± standard deviation from three
at day 30 (Fig. 3). Non embryogenic structures showed different biological samples. Values marked with different letters are
similar levels of expression of initial callus (Fig. 3). significantly different (P \ 0.05). NEC non embryogenic callus

Fig. 2 Alignment of deduced

amino acid sequences of
different CDKA genes.
Sequence of Picea abies
(GenBank X77680), Pinus
contorta (GenBank X80845),
Zea mays (NCBI
NM_001111872) and
Arabidopsis thaliana (Genbank
M59198). Sequences were
aligned with the CLUSTALW
program. Identical and
conserved amino acid residues
are boxed in black and grey,
respectively. The different
functional domains are
indicated by black lines above
the corresponding sequences

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256
A B
MN
Pd
1 mm
Fig. 4 Localization of CnCDKA transcripts by in situ hybridization
in embryogenic calli. Sections were probed using RNA antisense
probes (a, b). a Overview of embryogenic calli at 90 day culture
showing a high specific signal with the antisense CnCDKA probe. b
Higher magnification shows zones with a higher CnCDKA signal. c
exhibited meristematic nodules containing small meriste-
matic-like cells with the characteristic features of irregu-
larly shape, visible nucleus and a vaguely defined
nucleolus. The cells also contained densely stained cyto-
plasm indicating high metabolic activity and the proto-
dermis is clearly defined (Fig. 4c). With the sense probe,
no signal was detected in any region of the calli (Fig. 4d).
CDKA expression during somatic embryo formation
During somatic embryo formation CnCDKA expression
gradually decreased. The relative expressions of proglob-
ular, globular and coleoptilar embryos were about 60, 50
and 40% (correspondingly) of the expression in embryo-
genic callus. In germinating somatic embryos expression
was the lowest at nearly 0% (Fig. 5).
Discussion
There is evidence supporting that cell division and differ-
entiation are related developmental processes. For example
in whole plants, rhizobium-induced formation of root
a
EC: embryogenic callus
Pro: pro-embryo stage
1.0
Relative expression units
Glo: globular stage
clp: coleoptilar stage
Gm: germinated
0.8
b
c
0.6
d CDKA gene family from the coconut palm Cocos nucifera.
0.4
0.2
0.0
EC Pro Glo Clp
Fig. 5 CnCDKA expression profile during somatic embryo forma-
tion. Figure represents means ± standard deviation from three
different biological samples. Values marked with different letters
are significantly different (P \ 0.05)
123
Plant Cell Tiss Organ Cult (2010) 102:251–258
C D
MN Pd
MN
0.5 mm 0.5 mm 1 mm
Embryogenic callus stained with naphthol blue–black and periodic
acid-Schiff showing densely-stained cells. d Embryogenic callus
hybridized with sense probe as a negative control. MN meristematic
nodules, Pd protodermis
nodule primordia in alfalfa can only be initiated in cortical
cells in G0/G1 stage (Yang et al. 1994); and in Arabidopsis
lateral root development, only G2-arrested pericycle cells
respond to auxin treatment forming lateral root primordium
(Beeckman et al. 2001). Similarly in tobacco cultures under
sucrose starvation stress microspores accumulated in G2
phase and in these embryogenic development occurred
(Touraev et al. 1996). There also observations linking
particular cell division regulatory proteins and develop-
ment. In Arabidopsis, leaf aging and differentiation were
associated with increased expression of the ICKI, a CDKA
inhibitor protein (Wang et al. 1998). In maize leaf, the
regions of older differentiated cells contain more Rb pro-
tein than younger tissues (Huntley et al. 1998).
Cyclin-dependent kinases (CDKs) are proteins involved
in the regulation of cellular cycle progression (Mironov
et al. 1999). Type-A CDKs are expressed in all phases of
the cellular cycle (Hemerly et al. 1993). Early studies in A.
thaliana led to the conclusion that AtCDKA expression is a
critical factor in cell division and in establishing a certain
degree of competence for the division (Hemerly et al.
1993; Martinez et al. 1992). Joubès et al. (2000) built a
comprehensive phylogenetic tree for CDK-related kinases
in plants, analyzing 31 CDKA from 22 different species.
They found that the most important residues were con-
served and that sequence clustering was strictly linked to
taxonomy. In the angiosperms, eight cluster sequences
represent two monocotyledon families (graminae and lili-
aceae) and six dicotyledon families.
We isolated and characterized a single member of the
It was found that the CnCDKA gene had highest homology
with Picea abies (96% similarity), medium homology with
a homologous gene from the genus Pinus (Pinus contorta
e
and P. abies) and a lower homology with a homologous
Gm gene from the monocotyledon family (Z. mays). The ana-
lyzed CDKA sequences differed mainly at the extreme 30
region however, generally confirmed the claim of Joubès
et al. (2000) indicating the CDKA family contains a highly
conserved amino acid sequence with 89% similarity.
Plant Cell Tiss Organ Cult (2010) 102:251–258
In vitro culture reduces the tissue division rate (Win-
kelmann et al. 1998), which holds true in coconut tissues
(Jesty and Francis 1992; Sandoval et al. 2003). In vitro
cultured coconut explants have a high proportion of cells in
the G0/G1 phase (*90%; Sandoval et al. 2003), high-
lighting the slowness of the morphogenesis process during
in vitro regeneration of coconut palm: in most explants,
calli develop 3–4 months after inoculation on the callo-
genesis medium (Buffard-Morel et al. 1992; Verdeil et al.
1994, Chan et al. 1998; Pérez-Nuñez et al. 2006).
Embryogenic competence is induced by 2,4-D in in vitro
cultured coconut explants (Chan et al. 1998; Pérez-Nuñez
et al. 2006). In the alfalfa leaf protoplast system, high 2,4-
D concentration or stress-induced embryogenic compe-
tence have been associated with small cell size and earlier
cell cycle activation (Pasternak et al. 2002). In carrot cell
cultures, a 2,4-D concentration-dependent switch between
elongation and division has been identified, and it was
found that 2,4-D indirectly inhibited cell elongation as a
consequence of promoting cell division (Lloyd et al. 1980).
In the present study, the CnCDKA-related gene isolated
from coconut palm was further characterized during
embryogenic callus formation and somatic embryo for-
mation and germination. CnCDKA expression was found to
increase steadily during embryogenic calli formation,
beginning at 75 days culture when the calli were acquiring
embryogenic competence. Expression was at its highest at
90 days culture, when the calli exhibited embryogenic
structures that eventually developed into somatic embryos
(Sáenz et al. 2006). The non embryogenic callus showed
low levels of expression, this could be related to an
abundance large and vacuolated cells whereas that did not
seem to be in active division (data not shown). In the only
other study addressing CDKA-related gene expression
during somatic embryogenesis, Footitt et al. (2003) repor-
ted that P. abies exhibited lower Cdc2Pa expression during
proliferation of cells which promoted pro-embryogenic
masses (PEMs). This is similar to the lower CnCDKA
expression during embryogenic calli formation in coconut
at 60 days culture observed in the present study. When the
PEMs in P. abies transitioned to somatic embryos, Cdc2Pa
expression increased, which is analogous to the increased
CnCDKA expression observed here in the coconut calli at
60–90 days. This suggests that cellular cycle progression is
a prerequisite to somatic embryo formation.
During embryogenic callus formation in coconut,
embryogenic structures consisting of small cells forming
meristematic centres developed at the callus periphery.
CnCDKA transcripts were detected in only a few cell layers
within the meristematic centres, while none were detected
in the callus interior. This suggests that cells in the meri-
stematic centres are actively dividing or competent to
divide. Presence of CnCDKA in these structures coincides
257
with the presence of RNA of the homologous SERK
(denominated CnSERK; Pérez-Núñez et al. 2009), meaning
that these cell layers may be dividing actively and have
already initiated the embryogenic program. In contrast,
CnCDKA expression progressively decreased during
somatic embryo formation. Decreased cell doubling time
has been associated with the beginning of somatic
embryogenesis (Warren 1980). This may indicate that
embryo cells have stopped dividing and/or have become
incompetent to divide, and have begun to enlarge and
differentiate. This decrease in CnCDKA expression coin-
cides with a steady decrease in Cdc2Pa expression during
somatic embryo formation and maturation in P. abies
(Footitt et al. 2003).
In conclusion, a putative CnCDKA gene was isolated
from coconut palm and characterised. The deduced amino
acid sequence contained most of the CDKA family con-
served motif. The CnCDKA expression profile during
embryogenic calli formation was highest in embryogenic
calli stage, coinciding its in situ localization with meri-
stematic cell centres from which somatic embryos devel-
oped, and on the other hand CnCDKA expression
progressively decreased with embryo development, possi-
bly indicating that coconut somatic embryo cells are
incompetent to divide but can enlarge and differentiate.
Therefore, the study of the expression profile of genes that
control the cellular cycle such as CDKA and those that are
involved in induction of somatic processes such as SERK,
could be a useful tool to help us to identify the meriste-
matic potential of tissues cultured in vitro that seems to be
linked to embryogenic competence.
Acknowledgments L. Saenz received a postdoctoral fellowship
from the Université Montpellier 2 (France) and continued support
from the Centro de Investigación Cientı́fica de Yucatán (CICY). The
authors also thank J-L Chan for assistance with the in vitro culture.
This research was partially funded by CONACYT, México (Grant no.
43834-Z) and a scholarship awarded M. Montero (no. 183253).
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