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: hiva@basu.ac.ir

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.

References

Economou AS, Maloupa EM (1995) Regeneration of Elaeagnusangustifolia from leaf segments of in vitro derived shoots. Plant

Cell Tissue Organ Cult 40:285–288. doi:10.1007/BF00048135

Economou AS, Spanoudaki MJ (1988) Regeneration in vitro of

oleaster (Elaeagnus angustifolia L.) from shoot tips of mature

tree. Acta Hortic 227:363–368

Hosseinzadeh H, Ramezani M, Namjo N (2003) Muscle relaxant

activity of Elaeagnus angustifolia L. fruit seeds in mice.

J Ethnopharmacol 84:275–278. doi:10.1016/S0378-8741(02)

00331-8

Iriondo M, Delaiglesia M, Perez A (1995) Micropropagation of Ela-eagnus angustifolia from mature trees. Tree Physiol 15:

691–693

Klich MG (2000) Leaf variations in Elaeagnus angustifolia related to

environment heterogeneity. Environ Exp Bot 44:171–183. doi:

10.1016/S0098-8472(00)00056-3

Koroch A, Juliani HR, Kapteyn J, Simon JE (2002) In vitro

regeneration of Echinacea purpurea from leaf explants. Plant

Cell Tiss Org Cult 69:79–83. doi:10.1023/A:1015042032091

Li Y, Ma L, Ozaki H, Hisajima S (2004) Micropropagation of

Elaeagnus angustifolia by rebroductive organ culture in vitro.

Jpn J Trop Agric 48(1):23–24

Lu CY (1993) The use of thidiazuron in tissue culture. In Vitro Cell

Dev Biol 29:92–96

Lucchesini M, Mensuali Sodi A (1996) Regeneration of oleaster

(Elaeagnus angustifolia L.) was achieved from different types of

explants. Gartenbauwissenschaft 61:276–280

Mirhydar H (1998) Encyclopedia of plants: indications of plants in

the prevention and treatment of disease,vol 2. Islamic Farhang,

Tehran, pp 163–164

Murashige T, Skoog FA (1962) Revised medium for rapid growth and

bioassays with tobacco tissue cultures. Physiol Plant 154:73–479

Murthy BNS, Murch SJ, Saxena PK (1995) Thidiazuron induced

somatic embryogenesis in intact seedlings of peanut (Arachishypogaea L.): endogenous growth regulator levels and signifi-

cance of cotyledons. Physiol Plant 94:268–276. doi:10.1111/

j.1399-3054.1995.tb05311.x

Pereira AM, Bertoni BW, Appezzato-da-Gloria B, Araujo ARB,

Januario AH, Lourenco MV et al (2000) Micropropagation of

Pothomorphe umbellate via direct organogenesis from leaf

explants. Plant Cell Tiss Org Cult 60:47–53. doi:10.1023/A:

1006409807719

Perry LM (1980) Medicinal plants of the East and Southeast Asia.

MIT Press, London, p 131

Victor JMR, Murch SJ, Krishnaraj S, Saxena PK (1999) Somatic

embryogenesis and organogenesis in peanut: the role of thidi-

azuron and N6-benzylaminopurine in the induction of plant

morphogenesis. Plant Growth Regul 28:9–15. doi:10.1023/A:

1006274615736

Visser C, Fletcher RA, Saxena PK (1995) Thidiazuron stimulates

expansion and greening in cucumber cotyledons. Physiol Mol

Biol Plants 1:21–25

Xie DY, Hong Y (2001) Regeneration of Acacia mangium through

somatic embryogenesis. Plant Cell Rep 20:34–40. doi:10.1007/

s002990000288

Zargari A (1990) Medicinal plants, vol 4. Tehran University Press,

Tehran, pp 275–277

Zhang BZ, Zhao KF (1996) Study on salt tolerance in Robinia andElaeagnus angustifolia. Shandong Sci 9:53–55

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

338 Trees (2009) 23:335–338

123