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Scientia Horticulturae 116 (2008) 169–175
www.elsevier.com/locate/scihorti
Development of zygotic and somatic embryos of
Phoenix dactylifera L. cv. Deglet Nour: Comparative study
Besma Sghaier *, Mouna Bahloul, Radhia Gargouri Bouzid, Noureddine Drira
Laboratoire des Biotechnologies Végétales Appliquées à l’Amélioration des Cultures, Faculté des Sciences de Sfax, B.P. 802, 3018 Sfax, Tunisia
Received 9 April 2007; received in revised form 22 October 2007; accepted 14 November 2007
Abstract
In order to improve somatic embryogenesis production in date palm Phoenix dactilyfera L. cv. Deglet Nour (DN), a comparative study between
somatic (SE) and zygotic (ZE) embryos developments was carried out. The data showed that ZE maturation occurred from 10 to 19 weeks after
pollination (WAP). During this period, the fresh weight (FW) and the dry weight (DW) of ZE increased progressively to reach a maximum level at
19 WAP. SE development occurred in three distinct stages. The DW remained constant during the two first stages, and declined slightly during the
third and final stage. Embryo protein analysis revealed significant differences between ZE and SE. The ZE total protein level was initially low and
increased to the maximum at mature stage. However, no significant change in total protein was detected during SE development. SDS-PAGE
analysis showed a poor protein profile for SE, compared to that of ZE. In the latter, a 22 kDa protein was identified by N-terminal sequencing as a
glutelin. This protein was accumulated rapidly during early development and remained at a relatively constant level during ZE development, and
then declined progressively 12 days after embryo germination (DG). This protein seems to be absent in SE.
# 2007 Published by Elsevier B.V.
Keywords: Zygotic embryo; Somatic embryo; Phoenix dactylifera L. cv. DN; Total protein; Glutelin
1. Introduction
Date palm (Phoenix dactylifera L.) is one of the most
economically important fruit crop and is cultivated across
North Africa and the Middle-East. Its nutritive value and
widespread cultivation underscore the need for improving
propagation methodologies, especially in vitro techniques
(Al-Khayri and Al-Bahrany, 2004). Clonal propagation via
somatic embryogenesis – the production of embryos without
recourse to sexual reproduction – is the most promising
approach to improve propagation in date palm. This technique
has been widely and successfully applied to many cultivated
plant species (Shah et al., 2000). In vitro protocols are already
available for date palm, which permit the production of many
synchronous somatic embryos in liquid culture (Fki et al.,
Abbreviations: DG, days of germination; DN, Deglet Nour; DW, dry
weight; ELISA, enzyme-linked immunosorbent assay; FW, fresh weight;
MW, molecular weight; OPD, orthophenylene diamine; PBS, phosphate buffered saline; PBS-T, phosphate buffered saline tween; SE, somatic embryo;
WAP, weeks after pollination; ZE, zygotic embryo.
* Corresponding author. Tel.: +216 22 716 844; fax: +216 74 274 437.
E-mail address: sghaierbesma@yahoo.fr (B. Sghaier).
0304-4238/$ – see front matter # 2007 Published by Elsevier B.V.
doi:10.1016/j.scienta.2007.11.009
2003). Date palms derived from somatic embryos are less
susceptible to somatic variation compared to those derived
from organogenesis (Ammirato, 1987; Merkle et al., 1990;
Osuga et al., 1999). Although somatic embryo production is a
well-established process for date palm (Al-Khayri, 2005),
there are still possibilities for improvement. For example, a
number of biochemical approaches have been proposed to
distinguish embryogenic calli (Al-Khayri, 2005). The somatic
embryo (SE) differs from the zygotic embryo (ZE), as it lacks
a dormancy phase, and it also lacks a seed integument and an
endosperm, both of which are required for seed survival and
germination (Brownfield et al., 2007). This may explain why
SE seedlings are less vigorous than those raised from true
seed. More knowledge is required concerning SE physiology,
and especially of the differences in protein content between
SE and ZE, which may improve the quality of SE and SEderived seedlings. The poor vigour of SE-derived seedlings
seems to be related to their incomplete maturation under
standard in vitro conditions (Roberts et al., 1990). Major
changes take place in the latter stages of ZE maturation,
including a switch from cell specification over to the
accumulation of carbohydrates, thus preparing the embryo
for full seed development (Yadegari and Goldberg, 1997).
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B. Sghaier et al. / Scientia Horticulturae 116 (2008) 169–175
Therefore maturation is considered as a critical step in embryo
development.
Histocytological observations indicated that storage protein
accumulation in SE is poor compared to ZE (Aberlenc-Bertossi
et al., 1999; Sané et al., 2006; Zouine et al., 2005). In
Eucalyptus nitens, the cells of SE and ZE contain similar lipidrich globular bodies, except in the meristematic regions. SE
have slightly lower levels of storage proteins in their
cotyledonary cells compared to ZE (Saumitra and John,
2000). These carbohydrate reserves play a vital role in seedling
survival and development, as they sustain the seedling until
photosynthesis (Galau et al., 1986). The storage proteins of
dicots are predominantly albumins and globulins, whereas
prolamin and glutelin are the major ones in monocots
(Derbyshire et al., 1976). In the case of rice seeds, glutelin
is the predominant storage protein, reaching 80% of total
endosperm proteins (Takaiwa et al., 1999).
In the present report, a comparative study between protein
content of date palm ZE and SE is given. The evolution of the
glutelin as an important known storage protein during embryo’s
development was also investigated. This latter protein may be a
useful biochemical marker.
2. Materials and methods
2.1. Plant material and embryo collection
ZE were collected from date palm kernels (cv. Deglet Nour),
beginning 10 weeks after pollination (WAP) when the embryo
endosperm had hardened and finishing after 19 weeks. Fifty ZE
were collected at each harvest. Mature seeds were imbibed in
water to induce germination and raised at 25 1 8C in the dark
for 25 days. Every 3 days 20 seeds were collected.
Somatic embryos were obtained from embryogenic suspension cultures derived from the DN variety as previously
described (Fki et al., 2003). Before being structured, samples
of 0.5 g of embryos were removed from their maintenance
media (M3) and transferred on a fresh medium. Embryos
were subcultured weekly until maturation (structured
embryos). Three development stages of SE were identified
(Fig. 1B).
2.2. Protein extraction
The embryos were frozen in liquid nitrogen and ground into
a fine powder and mixed with 50 mM Maleate Tris Buffer (pH
8.3) containing 2% SDS, 0.5 mM EDTA, 2 mM PMSF, 1 mM
DTT and 2 mM b-mercaptoethanol. After centrifugation, the
quantity of soluble proteins in the supernatant was estimated
using Bradford’s Method (Bradford, 1976). Proteins were then
separated by SDS-PAGE according to Laemmli (1979) and
stained with Coomassie Brilliant Blue-R250. The molecular
weights (MW) were obtained by comparison to standard
protein markers (SDS-PAGE Standards, 161-0304, Bio-Rad).
The protein N-terminal sequencing was performed using an
Applied Biosystems Procise 492 equipped with 140 C HPLC
systems.
Fig. 1. Date palm seed cv. DN incised in the middle showing the position of the
ZE (A). The three stages of date palm SE (B): stage 1: ovoid embryo; stage 2:
longed embryo and stage 3: structured embryo.
2.3. Immunological analysis
After SDS-PAGE electrophoresis, the glutelin protein band
was cut from the gel and used as antigen for rabbit
immunization. The gel slice was dissolved in 0.5 ml of PBS
and the mixture was injected into a rabbit in presence of
complete Freund’s Adjuvant (F-5881, SIGMA). Four subsequent injections were performed every 10 days in presence of
incomplete Freund’s Adjuvant (F-5506, SIGMA). The rabbit
immunological reaction was tested by ELISA using an antirabbit serum (1/1000) conjugated to the peroxidase. The optical
density was measured at 492 nm. The obtained rabbit antiserum
was used for protein gel blot analysis as follows: after
electrophoresis in SDS-PAGE, the proteins were electroblotted
on a nitrocellulose membrane using 20 mM Tris Buffer
containing 150 mM glycine (pH 8.5) at 70 V for 1 h. The
fresh membrane was blocked for 2 h with PBS buffer 3% milk
containing rabbit polyclonal antiserum (1/1000) of date palm
glutelin. The membrane was then incubated with an anti-rabbit
IgG conjugated with peroxidase for 2 h. The reaction was
revealed using the ECL Plus Western Blotting Detection
Reagents from Amersham Biosciences, as described by the
Supplier.
Glutelin protein was quantified in crude protein extract by
indirect ELISA. Proteins (20 ng) extracted from embryos were
diluted in PBS buffer. Immunoplate wells were coated with
100 ml of protein solution for 24 h at 4 8C. The plate wells were
then blocked with 3% milk in PBS for 2 h at 37 8C. Polyclonal
B. Sghaier et al. / Scientia Horticulturae 116 (2008) 169–175
antibodies were diluted to a suitable concentration in blocking
solution (1% milk-PBS) and incubated in the plate wells for 2 h
at 37 8C. The plate wells were then washed with PBS-T (1%)
solution. The second antibody (anti-rabbit IgG conjugated to
peroxidase previously diluted to 1:5000 in PBS milk) was
added to each well and incubated for 1 h at 37 8C. Plates were
washed with PBS-T and then 100 ml of OPD (SIGMA, P-5412),
prepared in substrate buffer (Bio-Rad, 2J0431), was added to
each well. After incubation for 30 min at room temperature, the
absorbance was measured at 492 nm.
3. Results
3.1. Fresh, dry weights and water content of zygotic and
somatic embryos
The ZE (Fig. 1A) were extracted from seeds of date palm cv.
DN after the transition stage (see section 2). The regular
determination of the dry and fresh weights, at different times
after pollination showed that they increased until maturation
(Fig. 2A). At 12 WAP the water content started to decrease from
an initial level of 76.25% to reach 28% in the mature embryo.
SE showed rapid in vitro-development, with morphological and
shape changes occurring within 1 month. The embryos were
then classified according to their developmental stages:
globular (stage 1), elongated (stage 2) and structured (stage
3) (Fig. 1B). The dry weight of the SE remained constant during
Fig. 2. Variations in dry weight and water content during development of
zygotic (A) and somatic (B) embryos. Data represent means standard error
for three replicates from different extracts.
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stages 1 and 2, then decreased slowly at stage 3 while the water
content remained high during all stages reaching 90% at the
latest one (Fig. 2B). In contrast, the ZE water content is low at
the end of seed development.
3.2. Comparison of the protein content in mature zygotic
and somatic embryos
The quantitative analysis of date palm ZE protein content
showed that it was approximately 20-fold higher than SE, with
120.4 and 5.28 mg g 1 FW, respectively. This important
difference in protein content can be explained by the presence
of a much higher number of proteins in ZE than in SE, as shown
by SDS-PAGE analysis (Fig. 3). The ZE major bands with MW
ranging from 16 to 82 kDa were named P1 to P13. The P1, P2
and P3 protein bands of high MW ranged between 55 and
82 kDa, showed the most darkly stained bands.
The comparison of protein profiles between zygotic and
somatic embryos showed that they share five common bands
(P3, P4, P5, P8 and P9). The other bands P1, P2, P6, P10, P11,
P12 and P13 were only present in the ZE profile. However, a
30 kDa band was detected only in the SE protein profile
(Fig. 3). The P3 protein band was more abundant in the ZE
profile.
To investigate further the differences between the ZE and SE
protein profiles, the three protein bands corresponding to P1,
Fig. 3. SDS-PAGE analysis of total proteins from mature zygotic and somatic
embryos of date palm (cv. DN). MM: molecular mass standards, numbers
indicate molecular weight of protein markers in kDa; ZE: zygotic embryos; SE:
somatic embryos. Proteins were resolved in a 12% polyacrylamide gel and
stained with Coomassie Brilliant Blue.
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Table 1
Comparison of amino acid sequences of P1, P11 and P12 bands with those from other species (EMBL data base)
The N-terminal sequences of bands P1, P11 and P12 are in bold.
P11 and P12 in the ZE profile were excised from the gel and
their N-terminal were sequenced by EDMAN sequencing
method (Hewick et al., 1981). Amino acid sequence
comparisons in the EMBL data base (Table 1) showed that
the N-terminal sequence of the 22 kDa ZE band (P12)
displayed high identity (82%) with an oil palm glutelin (Elaeis
guineensis: accession number AAF69015). This known storage
protein was then used as a potential marker to follow embryonic
maturation. The N-terminal sequences of P11 and P1 bands
displayed identities with the 1-cys-peroxyredoxin of Medicago
truncatula (68%) and a phenylalanine ammonia-lyase from
Populus tremuloides (80%), respectively.
3.3. Analysis of the protein contents during ZE and SE
development
In order to define specific markers characterizing distinct
embryogenesis phases, the accumulation of total proteins was
monitored during ZE development, from the moment of
solidification of the endosperm (10 WAP) until the maturation
stage (19 WAP). The data showed that total protein level
increased from an initial of 49.76 mg g 1 FWup to 120.3 mg g 1
FW at embryo maturity. The protein level remained constant
from 17 WAP until dormancy (around 110 mg g 1 FW).
The investigation of the protein profile evolution during ZE
development showed that important changes in protein pattern
started after 13th WAP, with the appearance of the P2 band and a
number of other proteins ranging between P4 and P12 (Fig. 4A).
The P1 and P3 content increased whereas other bands remained
at the same level throughout all development stages (particularly
P4, P9 and P12). The protein level for SE during the various
developmental stages was low and remained steady (5.5 mg g 1
FW), and the SDS-PAGE showed a similar protein composition
without any modification in protein profile (Fig. 4C).
During the germination process, the protein content started
to be similar to that of mature ZE until 9 days of germination
(DG). During this period, the protein profile remained stable
and similar to that of mature ZE, then decreased abruptly
between the 9th and 12th DG (Fig. 5A). There were notable
changes in the number and intensity of protein bands after 12
DG. The P4 band increased in strength while other bands
disappeared gradually especially P1, P2, P3, P9 and P12
(Fig. 4B). This decrease in quantity and number of protein
bands coincided with the emergence of the cotyledonary leaf
(Fig. 5B). The P12 protein remained constant during ZE
development and disappeared during germination. This protein
could also be a storage protein since it shares 82% of identity
with the glutelin of Elaeis guineensis, so we have considered it
as a biochemical marker of the mature phase.
3.4. Glutelin as a biochemical marker of the developmental
process
In order to follow the evolution of glutelin content during
development of ZE and SE, a specific antiserum was produced
by injection of the purified protein into a rabbit. These
antibodies were able to recognize the target proteins (P12) from
a crude extract of ZE (Fig. 6). An ELISA test on crude protein
extracts obtained from different stages of ZE maturation
showed that the glutelin remained constant (OD 0.02). P12
decreased rapidly in germinating embryos and disappeared
after 12 DG (Fig. 7). In contrast, no glutelin signal was detected
throughout the three stages of SE development, suggesting a
lack of this storage protein in this kind of embryo which
confirms our previous results. They correlate also with the SDSPAGE analysis showing that glutelin level remained stable
during ZE development but it rapidly disappeared during
germination.
4. Discussion
4.1. Comparative study between zygotic and somatic
embryos
The comparison of ZE and SE described in this report
showed that ZE reach maturity after 23 WAP, whereas SE
B. Sghaier et al. / Scientia Horticulturae 116 (2008) 169–175
173
Fig. 5. Accumulation of total soluble proteins of zygotic embryos of date palm
cv. DN during germination. Data represent means standard error for three
determinations from different extracts (A). Date palm seeds during germination
(B), DG: days of germination (numbers of days are indicated).
Fig. 4. SDS-PAGE analysis of total proteins of zygotic embryos date palm cv.
DN during development (A), germination (B) and during somatic embryos
development (C). MM: molecular mass standards, numbers indicate molecular
mass of protein markers in kDa; ZE: zygotic embryos. Proteins were resolved in
a 12% polyacrylamide gel and stained with Coomassie Brillant Blue.
maturation took 1 month, and can be classified into three stages.
These data are in agreement with those reported by Sané et al.
(2006), who observed such difference between both kinds of
embryos. They were also able to distinguish three maturation
stages 1, 2 and 3 in date palm SE. ZE water content showed a
decrease during maturation, which is known to induce
suppression of precocious germination in Arabidopsis. Water
depression is required for acquisition of desiccation tolerance
and induction of dormancy (Koorneef and Karssen, 1994).
However, the high water content of SE contributes to its rapid
germination, without undergoing any dormancy process.
Consequently, the regenerated plants will be less vigorous
than those obtained through zygotic embryogenesis (Dodemann et al., 1997).
Fig. 6. Protein gel blot analysis of zygotic embryo crude extraction with antiglutelin, MM: molecular mass standards, numbers indicate molecular mass of
protein markers.
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carbohydrate and protein accumulation in SE during maturation. Date palm ZE continually accumulate proteins until
maturity. Some of these may be storage proteins, as they rapidly
disappear during germination. Protein content changed and
declined during germination, especially between 9 DG and 12
DG, which corresponds to the emergence of the cotyledonary
leaf. Since this process needs the hydrolysis of storage proteins,
we suggest that these proteins could be classified as storage
proteins. These results are in agreement with those found by Lai
and McKersie (1994), who showed that storage proteins and
starch reserves are rapidly hydrolysed following germination of
alfalfa embryos.
Fig. 7. ELISA tests for ZE during development and germination.
Protein content of date palm ZE and SE showed that it was
higher in ZE compared to SE, as was reported by Sané et al.
(2006), after staining cytoplasm proteins by NBB. They
concluded that a fundamental difference between the
development of date palm SE and ZE is the very weak
accumulation of reserves during SE development. Similar
results were also found for alfalfa SE and mature ZE (Lekha
and Bewly, 1998). The cotyledonary stage SE displayed less
protein content when compared to ZE at the same developmental stage. These differences also concerned protein
profiles. The ZE was rich in protein, with the presence of
three darkly stained protein bands (P1, P2 and P3) with MW
between 55 and 82 kDa. These proteins have their homologues
in the oil palm ZE profile (Chandra and Demason, 1988).
Differences between ZE and SE were previously described for
white spruce (Joy et al., 1991). They are likely due to
differences in the embryonic environment. Contrary to the ZE,
SE may lack biochemical signals for synthesis and accumulation of storage carbohydrates. Moreover, the composition of
the culture medium may not be adequate for such massive
synthesis (Joy et al., 1991). These conditions are not in favour
of endosperm differentiation and suspensor tissue formation
as reported for carrot somatic embryos (Dodemann et al.,
1997).
ZE protein content showed that the date palm protein profile
did not show a significant variation during the first and the last
developmental stages. Important changes were evident in the
protein profiles during the intermediate phase (13 and 15 WAP).
According to these data, it seems that the 13–15th WAP
correspond to an important period during which significant
quantitative and qualitative changes occurred in ZE protein
content. Similar data were reported for avocado ZE (Carolina
et al., 2002), where noticeable changes in storage protein
content were observed during maturation. Several storage
proteins initiated their accumulation at early stages and then
remained constant during embryo development, while other
proteins seem to be associated with the latest maturation stages
(Carolina et al., 2002). In contrast to ZE, the protein level and
the SDS-PAGE profile of SE remained fairly constant during
the different developmental stages. Similar results were
reported by Sané et al. (2006). They found little evidence of
4.2. Glutelin as a biochemical marker of the developmental
process
The immunoblotting results showed that glutelin is present
during the first stage of ZE maturation. The presence of the
glutelin storage protein at an early developmental stage in ZE of
other angiosperms was reported (Rahman et al., 1982).
Raghavan (1997) showed that avocado embryos (Persea
americana Mill.) accumulated 40, 45 and 48 kDa albumins
at early developmental stages. The same patterns were
described in carrot, in which the synthesis of storage proteins
occurs as soon as embryogenesis is initiated and independently
of any maturation background (Dodemann, 1995). However, in
a standard developmental medium, date palm SE did not
accumulate glutelin at any stage. Thomas (1993) suggested that
the expression of seed storage proteins is tissue-specific, since it
occurs in embryo and endosperm but never in mature vegetative
tissues. The sequential biosynthesis and accumulation of
specific storage proteins showed that they are under a strict
developmental control (Goldberg et al., 1989). They were then
used as indicators of embryo developmental processes.
Low rate of protein accumulation observed in somatic
embryos could be due to a lack of precursors in the medium
(Misra, 1994; Nomuna and Komanine, 1995). It may also be the
result of the absence of inducing signals (e.g. hormones and/or
desiccation), which are required to stimulate the synthesis of
specific molecules. The absence of such storage protein
accumulation may explain the lack of maturation phases in SE
under the culture conditions used.
5. Conclusion
Zygotic and somatic embryogenesis are complex phenomena that have been widely described in the literature. In this
paper a comparative study between ZE and SE protein contents
was carried out. In ZE, the physical enclosure by the seed coat,
the gradual lowering of water content and the qualitative and
quantitative changes in protein content lead to the developmental arrest. However, in the case of SE, such programmed
and co-ordinate changes do not occur, as the embryonic
environment is different from that found in ZE. Thus, it is not
surprising to find that SE germinates precociously. The glutelin
was considered as a potential marker for ZE embryo
development.
B. Sghaier et al. / Scientia Horticulturae 116 (2008) 169–175
Acknowledgments
This work was supported by the ‘‘Ministère de l’Enseignement Supérieur, de la Recherche Scientifique et de la
Technologie’’ and the International Atomic Energy Agency,
under TC Project RAF/5/049. Thanks are due to Prof. Majdoub
Hafeth and Dr. Rouis Souad for their technical help. The
authors gratefully thank Dr. Mohamed Ali Borgi, Dr. Afif
Hassairi and Prof. Bruce Sutherland for their help with the
manuscript and their comments.
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