Vol.4, No.11A, 1-11 (2013) Agricultural Sciences
http://dx.doi.org/10.4236/as.2013.411A001
Histological analysis and transcription profiles on
somatic embryogenesis in interspecific hybrids of
Elaeis guineensis × E. oleifera#
Paula Cristina da Silva Angelo1*, Douglas André Steinmacher2,3, Ricardo Lopes1,
Raimundo Nonato Vieira da Cunha1, Miguel Pedro Guerra2,3
1Embrapa Western Amazon, Manaus, Brazil; *Corresponding Author:
2UFSC, Federal University of Santa Catarina—CCA, Graduate Program in Plant Genetic Resources, Physiology of Development and
Plant Genetics Laboratory, Florianópolis, Brazil
3Brazilian Council for Technological Development—CNPq, Brazil
Received 23 July 2013; revised 29 August 2013; accepted 19 September 2013
Copyright © 2013 Paula Cristina da Silva Angelo et al. This is an open access article distributed under the Creative Commons Attri-
bution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly
cited.
ABSTRACT
Elaeis guineensis (African oil palm) and E. oleife-
ra (American oil palm) are bred to attain high oil
yields, disease resistances, and decelerated
shoot elongation. We cultivated immature zy-
gotic embryos from backcrossed and F1 inter-
specific progenies on media cont aining 110, 150,
or 200 mg·l−1 2,4-diclorophenoxyacetic acid (2,4-
D) to obtain embryogenic cultures. These were
set to multiply on medium containing 8 mg·l−1
2,4-D or lower concentrations of auxins and fi-
nally we induced plantlets regeneration, from
each zygotic embryo, independently, in order to
have the clones organized according to their
respective origins. Reductions in auxins in-
duced cultures to revert from highly embryo-
genic into competent for embryogenesis and
finally to organogenic degenerated callus lines.
Histology and the expression of SOMATIC EM-
BRYOGENESIS RECEPTOR KINASE, DEHYDRIN,
DEFENSIN, TRANSPOSASE, and LIPID TRANS-
FER PROTEIN were analy zed on four callus lines
representative of morphological aspects con-
sistently observed. The highest number of em-
bryogenic cultures was obtained on 150 mg·l−1
2,4-D. Maturation and multiplication of somatic
embryos through secondary embryogenesis
occurred simultaneously on 8 mg·l−1 2,4-D. LIPID
TRANSFER PROTEIN expression was detected
in one of the embryogenic cultures and corre-
lated with protoderm onset. Three six-week cy-
cles on induction medium yielded 1.5 shoots
above 6 cm per poly-embryogenic complex,
which performed better than embryoids indi-
vidualized mechanically. Rooting was observed
for 77% and 82% of shoots from these two types
of explants, respectively. Rooted plantlets ready
for acclimatization were obtained nine months
after shoot induct ion had started.
Keywords: Amazon; Arecaceae; Dend ê; Caiaué;
Gene Expression
1. INTRODUCTION
Elaeis guineensis (Jacq.) tp. tenera cultivars are con-
sidered the best producers of oil, which is used in food,
biofuels and cosmetics [1-3]. In Latin America, tenera
plantations are harmed by the lethal yellowing anomaly.
However, it does not eliminate F1 hybrids between E.
guineensis and the Amazon native species, E. oleifera
(Kunth) Cortés, growing in the same impacted areas. In
addition, E. oleifera has a lower rate of annual shoot
growth and a higher content of unsaturated fatty-acids
[4]. To maintain high yields, guineens is × oleifera hy-
brids are backcrossed with E. guineensis [4], and the
progenies resulting from this complex breeding strategy
present variability. Cloning can facilitate comparison
among plants grown in different experimental areas. A
few thousand clones for each progeny—tens of clones
#Part of the Pos-Doctoral activities of the first author.
Cite this paper: Silva Angelo, P., Steinmacher, D., Lopes, R., Vieira da
Cunha, R. and Guerra, M. (2013) Histological analysis and transcrip-
tion profiles on somatic embryogenesis in interspecific hybrids of Ela-
eis guineensis × E. oleifera. Agricultural Sciences, 4, 1-11.
http://dx.doi.org/10.4236/as.2013.411A001

P. C. da Silva Angelo et al. / Agricultural Sciences 4 (2013) 1-11
2
for some hundreds of zygotic embryos per progeny—
with their individual identities preserved would guaran-
tee representation and equal distribution of the geno-
types/embryos in field trials.
In vitro culture of E. guineensis tissues was first re-
ported in the 1970s [5,6], and the techniques were im-
proved in the 1980s and 1990s [7-10]. Massive multipli-
cation was reported late in the 1990s and in the 2000s
[11-13]. Nevertheless, many aspects of in vitro research,
including induction and maintenance of embryogenic
cultures, are still poorly understood.
Analyses of gene expression have added knowledge to
this matter. One of the genes firstly associated with em-
bryogenesis was SOMATIC EMBRYOGENESIS RE-
CEPTOR KINASE (SERK) [14]. Among palms, expres-
sion of SERK was recently observed at the onset of em-
bryogenic “ear”-like structures in coconut palm [15].
LIPID TRANSFER PROTEIN was expressed in carrot
pro-embryogenic masses and somatic embryos [16]. Ex-
pressed sequence tags (ESTs) identified as LIPID
TRANSFER PROTEIN [17], DEFENSIN and DEHY-
DRIN were preferentially expressed in E. guineensis em-
bryogenic cultures when compared with nonembryogenic
cultures. However, the expression rates of these two last
genes diverged among callus lines with different geno-
types and it was concluded that transcription had been
modulated principally by stress [18]. At last, a TRANS-
POSASE was among the ESTs expressed exclusively in
actively proliferating E. guineensis embryogenic cell cul-
tures and was not expressed in nonmultiplying cultures
[19].
In the present study, embryogenic cultures were in-
duced from single immature zygotic embryos randomly
taken from backcrossed and F1 progenies of E. guineen-
sis × E. oleifera. Cultures were multiplied on 8 mg·l−1
2,4-D, and we also tested multiplication in lower con-
centrations of auxins. Four of the callus lines obtained
during the trials were selected for further analyses, be-
cause they were representative of aspects observed re-
peatedly and also reported elsewhere for E. guineensis
and other palm species. We applied histological tech-
niques and reverse transcription polymerase chain reac-
tion (RT-PCR) to analyze the expression of SERK, DE-
HYDRIN, DEFENSIN, LIPID TRANSFER PROTEIN and
TRANSPOSASE genes, and examined the relationships
among the morphology and histology of those four callus
lines and the results of RT. The expression of those genes
has been investigated in E. guineensis, but the associa-
tions between gene expression and histology have not yet
been studied for most of them. We tested different media
to regenerate shoots from somatic embryos individual-
ized mechanically or clustered in poly-embryogenic
complexes (as defined in [8]). We applied for the first
time more than one six-week cycles in two different me-
dia for shoot induction and also compared the rates of
rooting for shoots obtained from individual embryoids or
poly-embryogenic complexes.
2. MATERIAL AND METHODS
2.1. Plant Material
Pollination was conducted at Rio Urubu Experimental-
Station (Embrapa Western Amazon), Rio Preto da Eva,
AM, Brazil (2˚35ʹS, 59˚28ʹW, 200 m above sea level)
and fruits were collected 106 days after this. Progeny
NFEC SN585 (shortly RC585) was generated backcros-
sing [(E. guineensis tp. tenera × E. oleifera) × E.
guineensis tp. tenera]. Progeny F1 (NFA CN1384, shortly
F1) was generated crossing E. guineensis tp. tenera × E.
oleifera. E. guineensis tp. tenera is obtained breeding tp.
dura × tp. pisifera. Zygotic embryos used for reverse
transcription were collected 90 days after pollination.
2.2. Induction and Maintainance of
Embryogenic Cultures
Fruit tissues were removed and seeds washed with de-
tergent and 50% bleach. One day after collection, zygotic
embryos were excised from the endosperm, transferred
for an aseptic flow chamber, immersed in 5% bleach (ap-
proximately 0.1% active chlorine) for 5 min, and washed
three times in autoclaved water. We cultivated 75 em-
bryos from RC585 and 100 embryos from the F1 progeny.
Ten embryos/90 mm Petri dish were set on 20 - 25 ml of
basal medium containing MS salts and vitamins [20], 1
mM Na+ provided as sodium phosphate, Phytagel (2.2
g·l−1), polyvinylpyrrolidone (500 mg·l−1), cysteine.HCl
(500 mg·l−1), hydrolyzed casein (500 mg·l−1), activated
charcoal (2.5 g·l−1), sucrose (30 g·l−1), what was supple-
mented with 2,4-diclorophenoxyacetic acid (2,4-D) at
110 (452 µM from [9]), 150 or 200 mg·l−1 to be desig-
nated as embryogenesis induction medium. Each embryo/
calli represented one experimental unit. Treatments were
compared by χ2.
Embryogenic sectors on primary calli were transferred
for multiplication/maturation medium: same as the basal
medium supplemented with 8 mg·l−1 (40 µM) 2,4-D [21].
Cultures were multiplied in this medium for at least 12
months at 25˚C ± 2˚C in the dark, with transfers for fresh
medium every two months. We also tested alternative
multiplication/maturation medium supplemented with
reduced concentrations of 2,4-D (1 or 3 mg·l−1) or 2.4
mg· l −1 (10 µM) Picloram.
Around the eighth month, embryogenic cultures were
transported from the Laboratory of Plant Biotechnology
at Embrapa Western Amazon, Manaus, for the Labora-
tory of Developmental Physiology and Plant Genetics, in
the University of Santa Catarina, Florianópolis, Brazil.
Thereafter, they were kept on multiplication/maturation

P. C. da Silva Angelo et al. / Agricultural Sciences 4 (2013) 1-11 3
medium modified to contain vitamins described in [22]
instead of those from [20] plus glutamine (1 g·l-1) and
with no cysteine.
2.3. Origin and Morphology of
Representative Callus Lines
The callus lines designated as 509, and 511A and 511B
were raised from single zygotic embryos designated as
509 and as 511, respectively, from progeny RC585. The
callus line designated as 002 was raised from zygotic
embryo 002 from progeny F1.
 Callus line 511A was a derivative of the highly em-
bryogenic culture 511. Following 14 months on mul-
tiplication/maturation medium containing 8 mg·l−1
2,4-D, part of that culture was transferred for 1 mg·l−1
2,4-D in basal medium free of charcoal. Two months
after this, we observed the reversion for calli bear-
ing “ear”-like embryogenic structures (as defined in
[15]), which were transferred to 3 mg·l−1 2,4-D for
additional two months.
 Callus lines 509 and 002 were highly embryogenic
cultures collected from multiplication/maturation me-
dium supplemented with 8 mg·l−1 2,4-D 18 months
after the introduction of zygotic embryos in vitro.
 Callus line 511B was obtained by transferring “ear”-
like embryogenic structures excised from 511A for
2.4 mg·l−1 Picloram in basal medium free of charcoal
[23] for two months followed by additional three
months on basal medium free of growth regulators
and charcoal.
Immediately after registration of the morphological
aspects through high definition digital images, biological
material (calli and/or more friable cultures) was organ-
ized as individual 100 mg samples, immediately frozen
in liquid nitrogen and preserved at −80˚C.
2.4. Histological Analysis
Samples from callus lines 511A, 509, 002, and 511B
were defrosted overnight at 8˚C inside 3% paraformal-
dehyde in 0.1 M phosphate buffer (pH 6.8). The material
went through a dehydration series (30 to 96.5˚ ethanol)
and was embedded in historesin (Leica) according to the
instructions of the manufacturer. Tissues were sliced in 8 -
10 µm sections and stained with 0.05% toluidine blue in
0.1 M phosphate buffer (pH 6.8) for analyses using an
Olympus light microscope/documentation system.
2.5. Transcription Profiles
Total RNA was extracted from individual samples of
100 mg following disruption in a tissue lyser (Precellys)
using the RNeasy Plant Mini kit (QIAGEN). The number
of samples extracted from each callus line (511A, 509,
002 and 511B) was defined by the amount of RNA ob-
tained from each one of them. The sum of the aliquots
should reach approximately 13 µg to allow replication.
RNA was treated with DNase I, purified in RNeasy Mini
Spin columns (QIAGEN), quantified by spectrophotome-
try (Nanodrop) and evaluated through denaturing agarose
gel electrophoresis. Extraction was repeated from 90
days old immature zygotic embryos as well.
RNA aliquots of 4 µg (biological replicas) from callus
lines or 2 µg from immature zygotic embryos were used
for 20 µl standard reactions of first strand polymerization
with the Long Range 2-Step RT-PCR kit and oligo-dT
primers (QIAGEN). From those, 3 µl were used for sec-
ond-step PCR amplification reactions (technical repli-
cates) using specific primers for SERK (5’-GGCGTCGT
CGTAAGCCGCAAG-3’ and 5’-TGGCCAAGCCAAAG
TCTCCAACA-3’), designed from conserved regions in
the coconut SERK mRNA (GI90891655). Primers for
DEFENSIN (5’-TGGGGACGAAGGTGGCGGA-3’ and
5’-AAAGCATCGGCGCCGGACT-3’), DEHYDRIN (5’-
GACGAGTACGGCAACCCGATC-3’ and 5’-CTTTTCC
ATCATCCCCTTCTT-3’) and TRANSLATION ELON-
GATION FACTOR 1, the internal reference gene
(5’-GGTGTGAAGCAGATGATTTGC-3’ and 5’-CCTG
GATCATGTCAAGAGCC-3’) were reported in [24].
Primers for the LIPID TRANSFER PROTEIN (5’-TG-
GAGAAAGGTGCACCGAAGCCGT-3’ and 5’-CCGG-
CCAGTCTTACAGCCTGCACA-3’) and the TRANS-
POSASE (5’-GCCTTGCTAGATCTAGGAGCTA GT-3’
and 5’-CCGTAGCTAAGAATGGTCTTCCT-3’) were
designed for conserved regions in corresponding E.
guineensis ESTs (GI193220058/DW248433 and
GI221144290, respectively). All the cDNA amplicons
were produced simultaneously using a step-down proce-
dure [25] of −0.8˚C·s−1 from 78˚C to 54˚C for annealing
in the first 13 PCR cycles. Anneling temperature was
fixed in 54˚C for the next 32 cycles. Synthesis was fixed
in 68˚C for 1 minute and denaturation was fixed in 93˚C
for 10 seconds. Amplicons were stained with GelRed
(Biotium), resolved in 1.5% agarose gels and exposed to
UV illumination. The images (TIF) were used to calcu-
late the calibrated quantity of fluorescence using linear
regression equations developed with Quantity One 4.6
(BioRad) taking the ELONGATION FACTOR 1 as cali-
brator. Reverse transcription (with three biological repli-
cas), cDNA amplification, electrophoresis and quantifi-
cation (in three technical replicas) were performed for
each callus line. The identity of the amplicons was con-
firmed by the proper size in electrophoresis, sequencing
(MegaBace) and, for the LIPID TRANSFER PROTEIN,
by amplification using nested (5’-TGCTGCAGTGACC
TGACACGGGCT-3’ and 5’-TCGCAGGGGCTAAGAG
CGTCGGA-3’) primers. The mean values for the cali-
brated quantity of fluorescence for the five amplicons in

P. C. da Silva Angelo et al. / Agricultural Sciences 4 (2013) 1-11
4
the four callus lines were compared using ANOVA and
Tukey tests.
2.6. Plantlet Regeneration
For shoot induction, we used nondisrupted poly-em-
bryogenic complexes (156 explants) or mechanically in-
dividualized pear-shaped somatic embryos (180 explants)
taken from callus lines 509 and 002. Cultivation was
conducted on basal medium free of casein, glutamine,
and charcoal, supplemented with 0.56 mg·l−1 (3 µM)
naphthaleneacetic acid (NAA) and 2.25 mg·l−1 (10 µM)
6-benzyladenine (BA) [6] that was designated as NB, or
with 0.08 mg·l−1 (0.44 µM) NAA and 5.00 mg·l−1 (24.6
µM) 2-isopentenyladenine (2-iP) [26], designated as NI.
Explants were exposed to 16 h of photoperiod under 40 -
50 μmol·m−2·s−1 irradiance. Poly-embryogenic com-
plexes were submitted to three cycles of shoot induction,
six weeks long each. Comparison between percentages
of leaf-like structures obtained from each type of explant
was performed only at the end of the first cycle because
individualized embryos were not submitted to more than
one cycle of shoot induction. Shoots were allowed to
reach 6 cm on in half-strength basal medium supple-
mented with 30 g·l−1 sucrose before mechanical indi-
vidualization for rooting.
Rooting was tested on 76 shoots from poly-embryo-
genic complexes and on 38 shoots from mechanically
individualized somatic embryos, in basal medium free of
casein or glutamine, containing half the concentration of
iron, supplemented with 30 or 60 g·l−1 sucrose [6] and
NAA (11.1 mg·l−1 or 16.7 mg·l−1). Explants were culti-
vated on 50 ml medium/500 ml flask or 15 ml medium/
22.5 × 2.5 cm culture tubes. Treatments were under.
All the statistical analyses (χ2 tests, Z tests, ANOVA
and Tukey tests) were performed using SigmaPlot, ver-
sion 11.2, P ≤ 0.05.
3. RESULTS
3.1. Induction of Embryogenesis
E. guineensis × E. oleifera immature zygotic embryos
were maintained for more than 12 months on induction
medium, because embryogenesis in palms often depends
on long-term cultivation [27,28]. By the sixth month,
20% and 39% of the cultures from F1 progenies and
RC585, respectively, were active and responsive to
changes in the cultivation conditions. The explants were
primary compact calli or calli that had given raise to em-
bryogenic cultures, which were first observed in the
second month (Table 1). Up to this point, discards were
caused by oxidation, hardening, rooting, or contamina-
tion (around 5%), in this order. The use of 150 mg·l−1
2,4-D was valuable in inducing embryogenesis for both
Table 1. Classification of calli induced on immature zygotic
embryos from two progenies of Elaeis guineensis × E. oleifera,
six months after inoculation on induction media supplemented
with different concentrations of 2,4-D.
Calli (%)
Progeny n 2,4-D (mg·l−1) Primary Embryogenic
110 25.0 5.0
150 45.0 15.0 F1 100
200 5.0 5.0
110 31.0 3.4
150 17.3 24.1 RC58575
200 20.7 3.5
progenies but it was significantly superior for RC585 (χ2
= 7.16; P = 0.0278).
3.2. Morphology and Histology of
Representative Callus Lines
Callus line 511A derived from 511 following a reduc-
tion in 2,4-D concentration. Two months were enough
for the reversion of the cultures from friable and highly
embryogenic back to compact calli displaying superficial
“ear”-like embryogenic structures (Figure 1A). This
structures had already been observed during the transi-
tion from primary to embryogenic calli, in the first
months of in vitro cultivation, more than a year before.
The highly embryogenic features of 511 was not main-
tained on 1 mg·l−1 2,4-D or recovered on 3 mg·l−1 2,4-D,
but we could still observe displacement of internal mer-
istematic cores for the surface of the embryogenic struc-
tures, where segmentation and spatial reorientation of the
external cell layers took place (Figure 1B). Simultane-
ously, there was the extrusion of tiny pro-embryos with a
small number of cells (Figure 1C), which would possi-
bly maturate and emerge through or detach from the sur-
face of the calli (Figure 1D).
Lines 509 (Figure 1E) and 002 (Figure 1I) diverged
in important aspects by the time of collection despite
both were highly embryogenic. Somatic embryos in cal-
lus line 509 erupted in clusters from a compact basal
callus. Some of them were intermediary, translucent and
elongating (Figure 1E, arrow), with protoderm ongoing
differentiation (Figure 1F, arrows). This phase of de-
velopment was observed solely in this callus line. Other
sectors in the same calli and culture produced pear-
shaped somatic embryos with procambium and periderm,
which entered secondary embryogenesis, characterized
by the arising of multiple clumps of meristematic cells at
the junctions to the basal callus (Figure 1H).
In contrast, line 002 from F1 hybrids had friable,
pearly colored sectors (Figure 1I, arrow) that were finely

P. C. da Silva Angelo et al. / Agricultural Sciences 4 (2013) 1-11 5
fragmented (Figure 1J), and multiplied fast. This char-
acteristic was exclusive of this callus line. In comparison
to 509, by the time they were collected for histological
and reverse transcription analyses, 002 displayed fewer
sectors of fully developed/clustered somatic embryos on
compact basal calli.
Callus line 511B was raised from “ear”-like embryo-
genic structures excised from 511A, cultivated in re-
duced auxin concentration (2.4 mg·l−1 Picloram) and in
absence of growth regulators. Concomitant with reduce-
tions in auxin concentration inflicted to 511 (from 110
for 8 mg·l−1), again to 511A (from 8 to 3 mg·l−1) and fi-
nally to 511B (as stated above) we observed the con-
tinuous degeneration of a highly embryogenic culture
until reach the sectored, hairy, calli displaying root-like
structures and embryogenic sectors, producing a few
peripheral yellowish pear-shaped somatic embryos (Fig-
ure 1M). These would be associated with residual mer-
istem segmentation (Figure 1N). Xylem elements pre-
senting secondary wall deposition (Figure 1P) were pre-
sent but no organized vascular bundle was observed.
As stated for 511A, lines 509 and 002 also produced
very small isolated pro-embryos, several of them ob-
served as four-celled structures in the histological sec-
tions, surrounded by extra-cellular matrix and thickened
outer walls (Figures 1D, G and K). These barriers would
promote the isolation of pro-embryos in coconut and date
palm [28,29] and contribute to maturation. A thickened
surrounding matrix can be observed surrounding larger
portions of cells which are presumably in course of or-
ganization to differentiate in somatic embryos in callus
line 002 as well (Figures 1J and L). This process was
not observed in callus line 509. It is part of the difference
between 509 and 002 with possible repercussions to the
origin of the embryos coming from each one of these
callus lines: from big compact basal callus in 509 and
from more friable meristematic cores in 002.
3.3. Transcription Profiles
We detected the expression of the targeted genes
through the presence of cDNA amplicons of expected
sizes. Except for the LIPID TRANSFER PROTEIN,
which was observed only in callus line 509 (Figure 2A,
LT), amplicons coming from the other genes were pre-
sent in all the callus lines and also in immature zygotic
embryos (Figure 2B). There was no statistically signifi-
cant difference among callus lines (Tukey tests, P > 0.05)
regarding the mean values of calibrated quantity of fluo-
rescence recorded for SERK, DEHYDRIN, DEFENSIN or
TRANSPOSASE transcripts. The highest quantity of
fluorescence was recorded for DEHYDRIN, followed by
DEFENSIN, TRANSPOSASE, SERK and finally LIPID
TRANSFER PROTEIN in callus line 509, which was ap-
proximately half that of SERK (Figure 2C).
We obtained good quality sequence reads covering
432 of the 579 base pairs (bp) expected for the SERK
amplicon, including the catalytic domain of the corre-
spondent enzyme. When that read was used to screen the
GenBank, the closest match [E−value = 5e−143 and 91%
(368/408) identity] was to a Cocos nucifera SERK
mRNA (AY791293.2/GI 90891655), as expected since at
that moment there were no entries designated as SERK
from Elaeis in the nonredundant nucleotide collection.
The similarity among sequences from different palm
species had already been reported [24].
Accordingly, the next best hit was a SERK from Areca
catechu (GU584945.1), another Arecaceae. The third and
fourth closest matches were, respectively, to a not yet
named mRNA and to an mRNA for a SERK-FAMILY
RECEPTOR-LIKE KINASE (AB188247.1), both from
Oryza sativa, which is a monocot. SERK primers also
prompted the amplification of a 550 bp band (Figures
2A and B), which we could not isolate and was consid-
ered an artifact of the PCR.
The sequenced portion of the cDNA amplicon corre-
sponding to the LIPID TRANSFER PROTEIN was 80%
identical to the E. guineensis DW248433 (GI 193220058)
mentioned in [17]. The reads covered a domain (cd-
00010-AAI/LTSS) conserved in proteins involved in
plant defense against insects and pathogens, lipid trans-
port between intracellular membranes and, nutrient stor-
age. This same domain is present in the polypeptide de-
duced from DW248433 when translated in frame +1
from the first ATG codon. The amplicon produced by
nested primers had around 156 bp (result not shown).
The nucleotide sequences obtained for DEHYDRIN,
DEFENSIN and TRANSPOSASE were around 97% simi-
lar to those published for E. guineensis.
3.4. Plantlet Regeneration
Haustorium-like structures were not included in Table
2, despite the presence of chlorophyll. The two types of
explants—poly-embryogenic complexes and somatic em-
bryos individualized mechanically from the complexes
Table 2. Percentage of Elaeis guineensis × E. oleifera indi-
vidualized somatic embryoids (ISE) and poly-embryogenic
complexes (PEC) producing shoots only, roots only or display-
ing no reaction after a period of six weeks on induction media
supplemented with NAA and BA (NB) or NAA and 2-iP (NI)
and submitted to a 16 h photoperiod.
Explant nIM Shoots Roots No reaction
90NB 11.5 21.5 0
ISE 90NI 16.5 34.0 1.5
78NB 15.5 5.5 7.5
PEC 78NI 33.0 2.5 14.0

P. C. da Silva Angelo et al. / Agricultural Sciences 4 (2013) 1-11
6
Figure 1. Morpho-histological aspects of callus lines obtained from Elaeis guineensis × E. oleifera immature zy-
gotic embryos. A-D, callus line 511A. A, superficial “ear”-like embryogenic structures (bar = 1.0 mm). B, strands
of cells spatially re-oriented reaching the callus surface and tissue segmentation (bar = 200 µm). C, organization
and extrusion of pro-embryos (arrows); the largest one shows a thickened outer wall (bar = 50 µm). D, cluster of
isolated pro-embryos covered with extracellular material (arrow) (bar = 200 µm). E-H, callus line 509. E, sector of
callus with somatic embryos at intermediary developmental phases (arrow) observed only in this line (bar = 2.0
mm). F, protoderm differentiation (arrow) in intermediary developed somatic embryos (bar = 50 µm). G, isolated
pro-embryo (arrow) covered with extracellular material (bar = 200 µm). H, meristematic cells clumps at the base of
a somatic embryo undergoing secondary embryogenesis (bar = 200 µm). I-L, callus line 002. I, sector of very fri-
able tissue (arrow) observed only in this line (bar = 2.5 mm). This sector comprised meristematic cores undergoing
recurrent fragmentation as detailed in J (bar = 200 µm). K, isolated pro-embryos (arrows) covered with extracellu-
lar material (bar = 200 µm). L, organization of pro-embryos and fragmentation of meristems observed simultane-
ously (bar = 100 µm). M-P, callus line 511B. M, compact callus displaying hairy sectors, roots, and somatic em-
bryos (bar = 2 mm). N, reminiscent meristematic layer fragmentation (bar = 50 µm). O, trichomes and phenolic in-
clusion that were observed only in this line (bar = 200 µm). P, disorganized vascular elements (bar = 50 µm). m =
meristematic tissue; pc = procambium; ph = phenolic inclusion; r = root; se = somatic embryo; tr = trichome; v =
vascular element.
—had reached different (z = 3.758; P < 0.001) rates of
shoot formation at the end of one cycle of cultivation in
shoot induction media (IM) and exposition to light, but
poly-embryogenic complexes produced more shoots. A
difference between NB and NI was observed too. For the
first cycle of induction (Table 2), the produced with two
cycles of induction (204/241), even when compared with
ne and three cycles taken together (37/241). It is o

P. C. da Silva Angelo et al. / Agricultural Sciences 4 (2013) 1-11 7
Figure 2. Profile of gene expression in Elaeis guineensis × E. olei fera hybrids. A, amplicons
for callus lines 511A, 509, 002 and 511B. B, amplicons for immature zygotic embryos, col-
lected 90 days after pollination. C, calibrated quantity of fluorescence to the amplicons for
each callus line. Amplicons were produced by RT-PCR and fluorescence was calibrated by
that of the internal reference gene. Columns represent mean values for three replicates and
bars represent the standard deviations of the means. SK = SERK (somatic embryogenesis re-
ceptor kinase); DH = DEHYDRIN ; DF = DEFENSIN; LT = LIPID TRANSFER PROTEIN;
TR = TRANSPOSASE; EF = ELONGATION FACTOR 1, the internal reference gene. M = 1
kb Plus Ladder (Invitrogen).
interesting to note that individualized pearshaped em-
bryos often produced multiple shoots too (Figures 3C
and D), probably as consequence of secondary embryo-
genesis (Figure 1H), and this feature contributed to the
final scores. We obtained 47 shoots above 6 cm from
individualized somatic embryos (26% in 180, Table 2)
and 241 from poly-embryogenic complexes (1.54 shoots/
explant in average; 1.46 for callus line 509 and 1.56 for
callus line 002).
Some shoots were used for rooting experiments (Fig-
ure 3E) following separation from the complexes. De-
spite media supplemented with 60 g·l−1 of sucrose and
16.7 mg·l−1 NAA promoted rooting in higher frequencies,
the concentrations of sugars and NAA tested did not dif-
fer statistically. Percentages of rooting were 77 and 82
for shoots coming from poly-embryogenic complexes or
individualized embryoids, respectively.
4. DISCUSSION
In this work, the goal was to clone E. guineensis × E.
oleifera zygotic embryos through somatic embryogene-
Figure 3. Overview of in vitro regenerated plantlets of Elaeis
guineensis × E. oleifera hybrids. A-B, shoots emerging from
poly-embryogenic complexes. C-D, shoots produced from in-
dividualized somatic embryos. E, shoots transferred for rooting
media.
sis. Histological and reverse transcription techniques
were used to complement morphology as support for

P. C. da Silva Angelo et al. / Agricultural Sciences 4 (2013) 1-11
8
decisions, going deeper on the comprehension of em-
bryogenesis and multiplication/maturation of embryo-
genic cultures, and to institute molecular markers to as-
sist selection of the best callus lines, maybe among
thousands if numerous progenies are going to be cloned.
An example would be the CYCLIN DEPENDENT
KINASE from coconut palm, which increased expression,
marked the emergence of “ear”-like embryogenic struc-
tures [30].
“Ear”-like structures were reported decades ago for E.
guineensis [8] and observed in coconut [15] and Bactris
[23]. In the present study, they became visible during the
initial months of cultivation and again following the re-
duction in auxin concentration, which induced culture
511 to revert throughout the route for embryogenesis to
produce callus line 511A. For this last callus line, histo-
logical analysis revealed the migration of meristematic
activity from the center of the calli to the periphery in the
“ear”-like embryogenic structures, “where cells became
spatially organized in strands, and the tissues entered
segmentation. Rearrangements like these could be the
prognostication of the development of “clusters of pro-
embryos, which would remain fused to form poly-em-
bryogenic complexes” [8]. In addition, rare clusters of
isolated globular pro-embryos, with a reduced number of
cells were also observed. These results all together indi-
cated the occurrence of more than one route for embryo-
genesis in callus line 511A. Somatic embryos would ori-
ginate from one or a few cells [28], or from the segmen-
tation of sectors of meristematic “cells re-oriented to
divide anticlinally, rather than periclinally”, what was
suggested to occur in barley too [8, 31]. Nevertheless, all
the somatic embryos promoted to the experimental phase
of shoot regeneration in this work came from poly-em-
bryogenic complexes and not from spontaneously iso-
lated somatic embryo.
Multiple meristematic cores were observed in 511A
and the other callus lines, certainly resulting from the
fragmentation of an initial meristem in each zygotic em-
bryo. The importance of cellular injuries inflicted to the
external meristematic layers and/or the central and pe-
ripheral zones of the shoot apical meristem (SAM) to
induce its segmentation and the organization of multiple
meristem cores has been demonstrated [32]. These pro-
cesses possibly occurred in association with the appear-
ing of “ear”-like embryogenic structures. Periclinal cellu-
lar divisions would trigger radial expansion in detriment
of somatic embryos multiplication/maturation. Radial ex-
pansion could impel meristematic activity from the inner
cores of the calli towards its surface, finally causing the
mechanical rupture of the external cell layers, inducing
fragmentation, followed by spatial reorganization and
tissue segmentations. It is plausible to suppose that under
ideal conditions the equilibrium between multiplication,
fragmentation and reorganization of peripheral meris-
tematic cells is achieved and the proper number of cellu-
lar layers and rows is settled in individual neighbor api-
cal meristems. Thereafter, photomorphogenesis and dif-
ferentiation of multiple shoots from poly-embryogenic
complexes become possible. The entrance in equilibrium
would be interesting to mark in order to avoid unneces-
sary exposition of the cultures to high concentrations of
auxins.
Differing from Bactris [26], isolated “ear”-like struc-
tures could not be induced to display secondary embryo-
genesis by cultivation in medium supplemented with 2.4
mg· l −1 Picloram. Isolation and sequential reductions in
the concentration of auxins resulted in a degenerated
callus line with organogenic characteristics. This demons-
trated that at least 8 mg·l−1 of 2,4-D were essential to
maintain the highly embryogenic features observed in
callus lines 511 (not shown) and 509, at least, which
were multiplied for more than a year. Once transferred
back for media containing 8 mg·l−1, calli from 511A re-
sumed the characteristics observed in 511, produced so-
matic embryos and plantlets (results not show).
Cultivation in medium free of growth regulators dur-
ing the stage of multiplication of the embryogenic cul-
tures would be essential to obtain normal regenerated
plantlets, when this stage is to be maintained for very
long periods, as 20 years [13]. In the present work the
best condition for multiplication-maturation of somatic
embryos was 8 mg·l−1 2,4-D applied for a little more than
a year [21]. Characteristics resembling the description of
fast growing cultures, that would be associated with the
mantled anomaly were only observed in callus line 002.
In this reason, we considered that if plants affected are to
be expected they will be found among regenerants from
that callus line.
Expression of SERK genes has been related to the ac-
quisition of cellular totipotency, which is essential for
embryogenesis [33] and can be followed by pluripotency
and finally organogenesis. Indeed, highly embryogenic
features, competence for embryogenesis, or embryogene-
sis observed simultaneously to organogenesis could ex-
plain SERK transcription in the callus lines we studied.
These conditions were observed in callus lines 509 and
002, in callus line 511A, which was very similar to the
coconut calli expressing SERK in peripheral embryo-
genic structures [15] and finally in 511B, respectively.
On the other hand, in Rosa hybrida cv. Linda two out of
four SERK genes analyzed were good markers for so-
matic embryogenesis but would not be exclusively re-
lated to embryogenesis, despite no conclusion about al-
ternative functions for those gene products had been
proposed [34].
The ubiquitous expression of DEHYDRIN and DE-
FENSIN was reputed to the stresses imposed equally to

P. C. da Silva Angelo et al. / Agricultural Sciences 4 (2013) 1-11 9
all the cultures as stated previously [18]. Dehydrins are
late embryogenesis abundant (LEA) proteins related to
the acquisition of desiccation tolerance but, in addition,
they have been clearly related to stress responses, as well
as defensins [24,35-38]. The differences among the ex-
pression profiles of those two genes and that of the
LIPID TRANSFER PROTEIN led us to conclude that
transcription of this last was not related to stress in our
experiments.
We propose that the differences in those patterns of
multiplication/maturation were the reasons for the exclu-
sive expression of the LIPID TRANSFER PROTEIN in
line 509. Callus line 509 presented mid-term pro-em-
bryos, going through protoderm organization, fixed to a
compact basal callus. Callus line 002 presented finely
fragmented meristematic sectors going through cyclic
multiplication. We considered the LIPID TRANSFER
PROTEIN transcription as a transient indicator of route
definition towards embryo maturation, probably going on
more intensely in the poly-embryogenic complexes of
callus line 509, in contrast with the prevalence of cyclic
multiplication and fragmentation of meristematic cores
in callus line 002. Reverse transcription was performed
using the maximum amount of total RNA (4 μg) recom-
mended for 20 μl standard reactions and a great number
of PCR cycles were used for cDNA amplification in each
biological replicate. These procedures assured that the
results captured the differential transcription of the
LIPID TRANSFER PROTEIN. Indeed, transcription in
callus lines 002, 511A and 511B, if there was any, was so
minimal that it could not be detected. In carrot, all the
cell masses with diameters between 30 and 50 µm that
produced somatic embryos were found to express the
LIPID TRANSFER PROTEIN, what was not observed in
the earliest stages, and expression was proposed to occur
in concert with protoderm formation [39]. We detected
transcription of the LIPID TRANSFER PROTEIN in 90
days old zygotic embryos, and this pointed out for the
possibility of an extended period of expression in Elaeis.
However, it was demonstrated in rice that expression of
LIPID TRANSFER PROTEIN changed spatially during
maturation from the protoderm, for the leaf primordium,
and finally for the vascular bundles [40]. The association
of a lipid transfer protein with the cytoplasm, cell walls,
and spaces between protodermal and subprotodermal
cells in embryogenic but not in meristematic cells of the
SAM was recently reported for a lipid transfer protein in
Arabidopsis thaliana. It was considered that some rela-
tion between the accumulation of the protein and the
differentiation/dedifferentiation—named together as chan-
ge of developmental direction—could exist [41]. The
absence in the SAM of a protein expressed when tissues
become committed to differentiation would be coherent
since it is expected to last nondifferentiated for long pe-
riods [33]. Finally, a correlation between the lipid trans-
fer protein and the accumulation of reserves in the em-
bryos cannot be completely discarded, neither some in-
fluence of the differences among the genotypes of the
callus lines on the intensity of gene expression, in reason
of the complex origin of the embryos.
Transposon activation via expression of TRANSPO-
SASE has been correlated with the chromatin demethyla-
tion and remodeling in presence of 2,4-D [42], which
was considered ultimately a stress factor that can influ-
ence chromatin conformation and induce embryogenesis
[43]. The presence of ESTs homologous to TRANSPO-
SASE was exclusively associated with the occurrence of
embryogenesis in E. guineensis [19] but transposons
were detected in tissues of tenera collected in and ex
vitro with no remarkable differences in distribution/lo-
calization [44].
For regeneration of plantlets, those poly-embryogenic
complexes that produced green, leaf-like structures were
transferred to half-strength basal medium free of growth
regulators for shoot elongation up to 6 cm, and those
which did not present such structures were submitted to
further cycles of shoot induction. The induction of shoots
on poly-embryogenic complexes was proven successful
[12,13] as proposed earlier for the interspecific hybrids
[45]. Cultivating poly-embryogenic complexes in me-
dium supplemented with abscisic acid [9] before shoot
induction might contribute to synchronization [46]. The
procedures described above reduced the manipulation
required to individualize embryos mechanically, contri-
buting to decrease contamination rates and to obtain
rooted plantlets ready for acclimatization in a year and
five months.
5. ACKNOWLEDGEMENTS
We are grateful to Embrapa (grant #02.09.03.005.00) for financial
support, to Aline Mabel Rosa for sectioning the tissues for histological
analyses, Alison Gonçalves Nazareno for assistance with the automatic
sequencer, Nelson Lourenço de Paula, Raimundo Oliveira do Nasci-
mento, and Raimundo César Pereira de Moraes for excising the zygotic
embryos and Raimundo Rocha for collects at Rio Urubu Experimental
Station.
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