Plant Cell, Tissue and Organ Culture 56: 1–7, 1999.

© 1999 Kluwer Academic Publishers. Printed in the Netherlands.

1

Somatic embryogenesis in Canary Island date palm

Le Thi Lan Huong1, Michela Baiocco1 , Bao Phan Huy2 , Bruno Mezzetti2∗ , Rodolfo
Santilocchi2 & Pasquale Rosati2
1 Azienda
Agraria Didattico Sperimentale – University of Ancona, Via Brecce Bianche, 60131 Ancona, Italy;
2 Dipartimentodi Biotecnologie Agrarie ed Ambientali – DIBIAGA, University of Ancona, Via Brecce Biance,
60131 Ancona, Italy (∗ request for offprints; fax: 71 220 4858; e-mail: Bruno@popcsi .unian.it)

Received 30 January 1997; accepted in revised form 10 March 1999

Key words: Phoenix canariensis, plant regeneration, shoot tip

Abstract
Zygotic embryo and shoot tip explants of Phoenix canariensis were cultured on MS (1962) basal medium sup-
plemented with 100 µM Picloram and 9.5 µM kinetin or 10.8 µM or 45.25 µM 2,4-dichlorophenoxyacetic
acid (2,4-D) and 9.8 µM N6 -(2-isopentenyl) adenine (2iP). These explants after 12 weeks in darkness at 28 ◦ C,
produced embryogenic callus with very compact, pale yellow, nodular structures. Proliferation and maintenance
of embryogenic callus was on MS basal medium with 2.26 µM 2,4-D, 0.83 µM kinetin and 2 µM abscisic acid
(ABA), with a regular subculture every 3–4 weeks. Somatic embryo development was promoted by two months
of culture on MS liquid medium enriched with 2 µM ABA, for torpedo stage development, then on liquid MS
medium with 1 µM N6 -benzyladenine (BA) and 0.46 µM kinetin, for shoot induction. Germinated embryos were
transferred to basal media enriched with 0.45 µM BA and 0.06 µM naphthaleneacetic acid (NAA) for root and
shoot induction and elongation. Viable plants were recovered on basal MS free of plant growth regulators (PGRs),
but percentages of plant conversion have to be improved.

Abbreviations: ABA – abscisic acid; 2,4-D – 2,4-dichlorophenoxyacetic acid; NAA – naphthaleneacetic acid;
BA – N6 benzyladenine; IAA – indole-3-acetic acid; Picloram (4-amino-3, 5, 6 trichloropicolinic acid); MS –
Murashige and Skoog salts and vitamins mixture (1962); 2iP – N6 -(2-isopentenyladenine); DAPI – 4, 6-diamidino-
2-phenylidole; PGR – plant growth regulator

Introduction adaptation to temperate climates and with resistance to

Date palms belong to the Arecaceae (palmae) family
(McCurrach, 1960). Some palms are important also
as ornamental plants. The Canary Island date palm
(Phoenix canariensis) is a prominent feature of the
landscape and most well known seaside resorts of
the Canary Islands. Plant adaptation is mainly linked
to climatic conditions and to the spread of specific
highly aggressive diseases. Among lethal diseases of
the palm, Fusarium wilt disease affects the genus
Phoenix. The soil borne fungus (Fusarium oxysporum
f. sp. canariensis) causes this disease and has des-
troyed millions of palms (Gary, 1995). The selection
and propagation of new genetic material with a better
the major diseases is important.
Conventional vegetative propagation of the Canary
Island date palm (Phoenix canariensis) is not pos-
sible, the ability to propagate pure lines to propagate
through seeds is also impossible because this palm is
dioecious. Currently there are no reliable protocols
for micropropagation of this species through in vitro
proliferation of axillary buds.
Somatic embryogenesis is currently applied to a
wide range of genera and species (Ammirato, 1993)
as a tool to obtain rapidly a large number of elite or
disease resistant plants. Furthermore, a well controlled
somatic embryogenesis procedure would be useful for
in vitro selection of disease resistant plants and/or ge-
2
netic transformation (Merkle et al., 1990; Parrott et
al., 1991). So far, propagation through somatic em-
bryogenesis has already been reported in the date palm
(Eenwens, 1978; Dreza and Benbadis, 1985; Gabr and
Tisserat, 1985; Brondon and Blake, 1989), but be-
cause of its agronomic importance in date production,
most emphasis is on Phoenix dactylifera (Merkle et
al., 1990; Parrott et al., 1991).
The aim of our work, was the development of an
efficient method for plant propagation of Canary Is-
land Date Palm (Phoenix canariensis) through somatic
embryogenesis. Starting from two types of explants
(zygotic embryos and shoot tip), factors affecting so-
matic embryo induction, development and maturation
were studied.
Material and methods
Plant material and embryogenic callus induction
Zygotic embryos and shoot tips used for explants ori-
ginated from seeds of Phoenix canariensis, collected
from plants located in a central area along the Ad-
riatic coast. Zygotic embryos, excised from mature
seeds, and shoot tips, about 0.5 cm long, excised
from dissected shoots, were surface disinfested for
30 min in 2.6% sodium hypochlorite solution, then
rinsed 5 times in sterile distilled water. Disinfested
explants were placed in Petri dishes, 90 mm diameter,
containing 10 ml of a basal medium of Murashige
and Skoog’s (MS-1962) salts and vitamins, 30 g l−1
sucrose, 7.5 g l−1 B&V commercial agar (Parma,
Italy) and supplemented with different concentrations
of 2,4-D combined with 2iP or of Picloram combined
with kinetin (Table 1). The highest 2,4-D and Picloram
concentrations were also combined with 0.3% activ-
ated charcoal. Media pH was adjusted to 5.8 before
autoclaving at 121 ◦ C for 20 min.
The effect of each PGR combination on the in-
duction of embryogenic callus was evaluated in two
subsequent replications of 5 Petri dishes (90 mm dia)
containing 6 explants each. All cultures were incub-
ated darkness in a growth chamber at 28 ◦ C. The
percentage of explants forming embryogenic callus
was recorded after 12 weeks of culture. Significant
differences among means of explants forming em-
bryogenic callus were calculated using the Student
Neuman Keuls test, p<0.05.
Proliferation, maintenance and maturation of somatic
embryos
Embryogenic callus obtained from explants on media
with PGR combinations promoting the highest induc-
tion, mainly 10.08 µM 2,4-D and 2.46 µM 2iP, were
subcultured every 3–4 week onto media containing the
same PGR combinations or media with lower auxin
contents 0, 1, 2 or 3 µM 2,4-D + 0 or 1 µM kin-
etin + 0, 1 or 2 µM ABA. Globular somatic embryos
produced from embryogenic callus were transferred to
liquid MS basal medium containing various concen-
trations (0.45, 1, 1.5 or 2 µM) of BA, 2iP, kinetin,
NAA and IAA. Liquid cultures were in 150 ml Er-
lenmeyer flasks placed on a rotary shaker at 120 rpm
and subcultured every 2 weeks. Somatic embryo mat-
uration was observed after 2 months of culture. Cell
response to the different media and culture conditions
was evaluated in two subsequent replications of 5 Petri
dishes (90 mm dia), for solid media, or 5 flasks for
liquid media.
Results and discussion
Induction and prolifertation of embryogenic callus
lines
Among the two original explants zygotic embryos had
a greater potential to produce embryogenic callus than
shoot tip. Media containing the highest concentrations
of 2,4-D (Table 1) completely inhibited the production
of embryogenic callus regardless of the explant source.
The highest percentage of embryogenic callus
formation from zygotic embryos explants was in-
duced by media supplemented with 10.8 µM 2,4-D
+ 2.46 µM 2iP or with 100 µM Picloram + 9.5 µM
kinetin (Table 1).
Zygotic embryo explants began to swell at the
mesocotyl region after two weeks of culture on all
different callus induction media. Some of these PGR
combinations induced compact-pearly coloured em-
bryogenic callus. After 12 weeks the callus pieces
reached approximately 1 cm dia and formed many
nodular structures.
This embryogenic callus transferred to media with
reduced auxins produced clumps of globular embryos
from the nodular structures (Figures 1, 2).
The percentage of shoot tip explants forming so-
matic embryos was lower than from zygotic embryos
explants, varing from 4% to 45% depending on the
auxin type and concentration. The highest percentage
 3

Figure 1. Compact, yellow embryogenic callus with nodular structure induced from zygotic embryo explant (25×).

Table 1. Effect of plant growth regulators (PGRs) on the induction of embryogenic callus
lines from the two tipes of explants (seed embryos and shoot tip) of Phoenix canariensis,
observed after 12 weeks of culture. Means calculated from 60 explants (2 replications of
5 Petri dishes).

Plant growth regulators Responding explant

2,4-D 2iP Picloram Kinetin Activated Seed embryo Shoot
(µM) (µM) (µM) (µM) charcoal (%) tip
(%) (%)

4.25 0.98 37.5b 0c

10.08 2.46 86.7a 0c
18.1 2.46 53.3ab 0c
27.14 4.9 45.8ab 8.3b
36.20 4.9 33.3b 37.5a
45.25 9.8 0c 45.8a
90.50 14.7 0c 4.1bc
226.50 14.7 0.3 0c 0c
452.50 14.7 0.3 0c 0c
25 2.32 29.1b 0c
50 4.60 50ab 0c
100 9.5 74.9a 24.9ab
150 9.5 54.1ab 20.8ab
200 9.5 46.7ab 0
200 9.5 0.3 0 0

Means followed by the same letter are not significantly different. Student Neuman Keuls
test, p<0.05.
4
Figure 2. Direct somatic embryos formation from shoot tip explant (25×).
of somatic embryogenesis was obtained from shoot tip
on media enriched with 45.25 µM 2,4-D and 9.5 µM
kinetin. This type of differentiation started with glob-
ular structures and after 12 weeks formed somatic
embryos directly.
Secondary embryogenesis was observed using li-
quid media containing 2.26 µM 2,4-D and 2 µM ABA
(Figure 3). This interesting property of somatic em-
bryos suggests it has potential for application as a mass
cloning technique (Reynolds and Murashige, 1979;
Sharma et al., 1980; Vasil, 1982; Merkle et al., 1990;
Parrot et al., 1991).
On both explants, 2,4-D induced a higher per-
centage of explants forming somatic embryos than
Picloram (Table 1). Activated charcoal, which acts as
an adsorbent, has been used in some species, includ-
ing Phoenix dactylifera, to stimulate embryogenesis
(Ammirato,1983; Gabr and Tisserat, 1985). On the
contrary, with Phoenix canariensis the presence of
activated charcoal, combined with the highest auxin
concentration, completely inhibited the induction of
embryogenic callus.
Embryogenic callus of some species requires con-
tinuous exposure to auxin to produce repetitive so-
matic embryos, while in others, a pulse of auxin as
short as a few days is sufficient to induce repetitive
embryogenesis that will continue for years on basal
medium (Merkle et al., 1990; Parrot et al., 1991).
In our work, the presence of 2,4-D was a prerequis-
ite for embryogenic callus induction and subsequently
for their proliferation and maintenance. In order to re-
duce as much as possible abnormal development of
somatic embryos, media with low 2,4-D were stud-
ied. The most suitable medium for embryogenic callus
proliferation and maintenance was MS basal medium
enriched with 2.26 µM 2,4-D + 0.83 µM kinetin + 2
µM ABA.
Somatic embryos maturation
On solid media, the development of somatic embryos
was very slow, taking 1 or 2 months in vitro. There-
fore, liquid culture media with the same composition
were tested to stimulate (somatic) embryo develop-
ment. Using MS liquid medium supplemented with 2
µM ABA, the development of somatic embryos from
the globular to torpedo stages was observed within
two months. A longer time on this medium induced
root but not shoot formation. Thus, at the torpedo
stage, they were transferred to liquid medium in the
 5

Figure 3. Secondary embryogenesis induction in liquid culture with 2,4-D 2.26 µM and ABA 2 µM (15×).

Figure 4. Germination of somatic embryos with shoot and root on MS liquid media supplemented with BA 1 µM and Kinetin 0.46 µM (15×).
6
Figure 5. Phoenix canariensis date palm plant development on MS medium free of PGRs.
presence of cytokinins for shoot development (Fig-
ure 4). In liquid medium containing BA, kinetin or 2iP
alone, embryos did not develop. Embryo development
was promoted by the combination of 1 µM BA and
0.46 µM kinetin. In this case, about 76% of torpedo
shaped embryos gave rise to a single shoot and root.
In general, the growth of somatic embryos in liquid
medium gave better results than solid medium with
the same composition. This, confirms results obtained
with other studies on date palm (Phoenix dactylifera)
(Omar and Novak, 1990; Veramendi and Navarro,
1996). Germinated embryos were transferred to MS
basal medium containing 0.45 µM BA and 0.06 µM
NAA for shoot and root elongation. When these em-
bryos were exposed to light, they turned green after
two weeks, but in most cases the shoot apex failed
to develop further or became hyperhydric. There-
fore, viable plant regeneration was obtained on MS
basal without PGRs (Figure 5), but at the low rate
of 5%. Further studies on factors affecting this stage
of plant development could improve the efficieny of
this system and demonstrate the potential of somatic
embryogenesis technology in the clonal propagation
of Phoenix canariensis.
Acknowledgements
This work has been supported by the local govement
of San Benedetto del Tronto and Grottammare (AP-
Italy) for germplasm collection and breeding of orna-
mental plants adapted to the Mediterranean coasts.
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