ORIGINAL PAPER
Plant regeneration through callus cultures derivedfrom immature-cotyledon explants of oleaster(Elaeagnus angustifolia L.)
Omid Karami Æ Khosro Piri Æ Reza Bahmani
Received: 27 January 2008 / Revised: 2 September 2008 / Accepted: 11 September 2008 / Published online: 21 October 2008
� Springer-Verlag 2008
Abstract Efficient plant regeneration was achieved from
callus derived from immature-cotyledon explants of ole-
aster (Elaeagnus angustifolia L.). Calli were obtained on
MS media containing 3% sucrose and different concen-
trations of TDZ. The highest rate of green, compact and
nodular callus was formed on MS medium supplemented
with 1 mg/l of TDZ. Shoot organogenesis was achieved
when the callus was transferred onto MS media containing
3% sucrose and BA alone (05–4 mg/l) or BA (0.5 and
1 mg/l) combined with NAA or IAA (0.5 and 1 mg/l).
Maximum organogenesis was obtained with 1 mg/l BA in
combination with 0.5 mg/l NAA. Rooting of the shoots
was achieved on MS medium supplemented with 0.2 mg/l
IBA. Regenerated plantlets were acclimatized and success-
fully transplanted to soil.
Keywords Oleaster � Plant regeneration � Callus
Abbreviations
BA 6-Benzyladenine
2,4-D 2,4-Dichlorophenoxyacetic acid
MS Murashige and Skoog (1962) basal medium
NAA a-Naphthalene acetic acid
IBA Indole-3-butyric acid
TDZ N-phenyl-N-1,2,3-thidiazol-5-yiurea
IAA Indole-3-acetic acid
Introduction
Oleaster is an Eurasian tree which has been naturalized and
invaded zones along watercourses in many arid and semi-
arid regions of the world. These habitats are characterized
by vertical environmental gradients, so the plant has
adapted to a wide range of regional conditions (Klich
2000). Oleaster plays a very important role in maintaining
ecosystem function in the arid areas, because of its toler-
ance to severe drought, high salinity and alkalinity of soils
(Zhang and Zhao 1996). This plant has also various
medicinal uses. The ripe fruits of oleaster have been used
to treat amoebic dysentery (Perry 1980). There is a general
belief that leaves and fruits of the plant have antipyretic
effect (Zargari 1990). In folk medicine, oleaster fruit or
flower preparations are used for treating nausea, vomiting,
jaundice, asthma, and flatulence (Mirhydar 1998). An
infusion of the oleaster fruit has been used as an analgesic
agent for alleviating pain in rheumatoid arthritis patiensts
in Iranian traditional medicine. Its flower has been tradi-
tionally used for treating tetanus (Hosseinzadeh et al.
2003).
Oleaster can be propagated from seeds, but seed propa-
gation is complicated due to poor germination (Iriondo et al.
1995). Hence, there is a need to develop an in vitro pro-
pagation technique for this medicinal plant. Shoot regene-
ration in vitro has already been reported for oleaster
(Economou and Spanoudaki 1988; Economou and Maloupa
1995; Iriondo et al. 1995; Li et al. 2004). However, there is
only one report on callus cultur of this plant (Lucchesini and
Communicated by O. Junttila.
O. Karami � K. Piri (&)
Department of Biotechnology, Faculty of Agriculture,
Bu-Ali Sina University, Hamadan, Iran
e-mail: [email protected]
R. Bahmani
Department of Horticulture, Faculty of Agriculture,
Bu-Ali Sina University, Hamadan, Iran
123
Trees (2009) 23:335–338
DOI 10.1007/s00468-008-0281-0
Mensuali Sodi 1996). No report has been found in the liter-
ature on plant regeneration from immature-cotyledons of
oleaster. In this study, we were able to induce callus from
immature-cotyledons of oleaster and to develop an efficient
regeneration system for this plant.
Materials and methods
Plant material and culture conditions
Immature green fruits were collected from a 40-year-old
oleaster tree grown in a natural forest in the eastern part of
Hamadan, Iran, on July 2007. Immature seeds were gently
separated from the green fruits and surface-sterilized using
70% ethanol for 30 s plus 2% sodium hypochlorite solution
for 15 min followed by three rinses with sterile distilled
water. After surface sterilisation, immature embryos were
isolated from the seeds and cotyledons were excised and
used as explants for callus induction.
The pH of the culture media were adjusted to 5.8 using
NaOH (1 N) before adding gelling agent (Agar–Agar,
Merck). All culture media were sterilized by autoclaving at
121�C for for 15 min.
Induction of callus
For callus induction, the explants were placed on MS
medium containing 3% sucrose and different concentra-
tions (0.5, 1, 2, 3, 4 and 6 mg/l) of 2,4-D, NAA, BA,
kinetin or TDZ. All cultures were incubated at 26�C under
16 h photoperiod under 50 lmol/m2 per s illumination
provided by cool white fluorescent lamps. Almost 80–85
explants and/or 200 mg callus were considered for each
treatment and all experiments were repeated twice. The
ANOVA was performed for analysis of the data obtained
for each experiment and the means were tested using the
LSD test (P \ 0.05).
Shoot organogenesis from callus
Calli were transferred onto culture media containing 3%
sucrose plus different concentrations of BA (0.5, 1, 2, 3 and
4 mg/l) alone or BA (0.5 and 1 mg/l) combined with NAA
or IAA (0.5 and 1 mg/l), for shoot organogenesis. All
cultures were incubated in the condition as used for callus
induction. The number of originated shoots were recorded
5 weeks after culture. About 400 mg callus were used for
each treatment and the experiment was conducted with six
replicates. The data were presented as mean ± SE of two
repeated experiment. The ANOVA was performed for
analysis of the data obtained for each experiment and the
means were tested using the LSD test (P \ 0.05).
Plantlet formation and plant acclimatization
Regenerated shoots were separated from calli and planted
for rooting onto MS medium containing 3% sucrose and
0.2 mg/l IBA. Rooted shoots were transferred into plastic
pots containing an autoclaved mixture of soil, sand, and
compost (1:1:1 v/v) and kept for 3 weeks, then transplanted
into plastic pots containing garden soil and grown in the
growth room (20 ± 2�C, 16 h photoperiod, 40 lmol/m2
per s illumination). Plants were finally acclimatizated in a
greenhouse at 28�C for 5 weeks before they were moved to
a greenhouse without temperature control.
Results and discussion
Induction of callus
Among of the different plant growth regulators, which
were used, only TDZ produced callus. Callus initiation
started from the cut edges of the explants within
2–3 weeks. Two types of callus were observed: type I
callus was green, compact (Fig. 1a), while type II callus
was yellowish-green and soft (Fig. 1b). In oleaster, callus
cultures from seedlings were established on modified LS
medium with 1 mg/l 2,4-D plus 0.5 mg/l kinetin by
Lucchesini and Mensuali Sodi (1996). However, produc-
tion of type I callus of oleaster with high capacity has not
been reported earlier.
There was a high frequency of type II callus. Formation
of type I callus was dependent on the concentration of
TDZ, and the highest proportion of explants with type I
callus was obtained at 2 mg/l TDZ (Fig. 2). Shoot organ-
ogenesis was not observed in cultures grown on media with
TDZ. TDZ can be substituted with adenine-type cytokinins
in various culture systems, including callus and micro-
propagation of many woody species (Lu 1993), but this
was not tested with oleaster.
TDZ has been shown to have cytokinin-like effects also
on cucumber cotyledons (Visser et al. 1995). Victor et al.
(1999) showed that regeneration of peanut was enhanced
both by cytokinins and TDZ, but their morphological
effects were different. They concluded that, in peanut, TDZ
appears to fulfill both the role of auxin and cytokinin for
regeneration. In fact, treatment with TDZ has been shown
to increase endogenous levels of both auxin and cytokinins
in peanut (Murthy et al. 1995).
Shoot organogenesis from callus
Shoots were induced within 1–2 weeks from type I calli
(Fig. 1c) when they were transferred to media containing
different concentrations of BA alone or in combination
336 Trees (2009) 23:335–338
123
with NAA or IAA (Table 1). No shoot induction occurred
on type II calli. Significant differences among different
concentrations of BA alone and BA in combination with
NAA and IAA on number of shoots regenerated from type I
calli is shown in Table 1. The maximum number of shoots
was obtained from 400 mg callus on media containing
1 mg/l BA in combination with 0.5 mg/l NAA.
Lucchesini and Mensuali Sodi (1996) have reported the
induction of buds from oleaster callus cultured on the LS
medium with 10 mg/l BA, but in our experiments, high
frequency of regeneration was obtained from type I calli on
MS medium containing 1 mg/l BA in combination with
0.5 mg/l NAA. In the present study, BA in combination
with NAA gave better results than BA alone, indicating a
synergistic effect of auxin and cytokinin on shoot regen-
eration in oleaster, as has been shown in several other
systems (Pereira et al. 2000; Xie and Hong 2001; Koroch
et al. 2002).
Plantlet formation and acclimatization
About 85% of shoots were rooted in medium containing
IBA within 3–4 weeks (Fig. 1d). A high percentage
(approximately 85%) of plantlets were successfully trans-
ferred into soil and they developed into normal plants in the
greenhouse with 90% survival. All acclimatized plants
were finally transferred to field conditions and grew
Fig. 1 a Induction of type I calli on MS medium containing 2 mg/l of
TDZ 5-week after culture. b Induction of type II calli on MS medium
containing 2 mg/l of TDZ 5-week after culture. c Shoot
organogenesis from type I calli on medium containing 2 mg/l of
BA 2-week after culture. d Formation of root from shoots on MS
medium containing 0.2 mg/l of IBA after 4 weeks
Fig. 2 Effect of different concentrations of TDZ on induction of type
I callus from immature-cotyledon explants of oleaster on MS basal
medium containing 3% sucrose 6 weeks after culture. Ten explants/
replicate, six replicate/treatment and experiment repeated twice
Trees (2009) 23:335–338 337
123
normally in the natural environment. Phenotypic variability
was not observed in plants in this experiment.
Conclusions
In conclusion, this study provides a protocol for an efficient
regeneration system of oleaster from callus. Induction of
callus is achieved with TDZ, shoot formation with BA, and
root formation with IBA. Rooted seedlings can be success-
fully transplanted into soil. Establishment of conditions that
are required for high frequency of regeneration would facil-
itate genetic transformation and mass propagation in oleaster.
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Table 1 The effect of different concentrations of BA alone or in
combination with NAA or IAA on the mean number of shoots formed
on type I callus in E. angustifolia 5 weeks after culture
BA
(mg/l)
NAA
(mg/l)
IAA
(mg/l)
Number of shoots
induced on type I
callus (mean ± SE)f
0.5 – – 23 ± 1.5d
1 – – 34 ± 2.5c
2 – – 30 ± 2c
4 – – 9 ± 1.3e
0.5 0.5 – 41 ± 3b
1 1 – 43 ± 3.2b
1 0.5 – 58 ± 4.3a
0.5 1 – 21 ± 1.6d
0.5 – 0.5 29 ± 2.3c
1 – 1 33 ± 3c
1 – 0.5 44 ± 3.6b
0.5 – 1 10 ± 0.6e
Cultures were grown in light on MS basal medium containing 3%
sucrosea–e Means having the same letter in column were not significantly
different by LSD test (P \ 0.05)f About 400 mg callus/replicate, six replicate/treatment and experi-
ment repeated twice
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