Scientia Horticulturae 86 (2000) 323±333

In vitro shoot regeneration from mature tissue

of wild Cyclamen persicum Mill.
Nabila S. Karam*, Mohannad Al-Majathoub
Faculty of Agriculture, Jordan University of Science and Technology, PO Box 3030, Irbid, Jordan
Received 22 September 1999; received in revised form 13 March 2000; accepted 20 March 2000

Abstract

In vitro shoot regeneration of wild Cyclamen persicum Mill. was studied using mature tissue.
Leaf disc, petiole, petal, and peduncle explants were cultured on 13 MS medium supplemented with
6-benzyladenine (BA) at 0, 1, 2, or 3 mg l 1 or thidiazuron (TDZ) at 0, 0.0022, 0.022, 0.22, or
2.2 mg l 1. The effect of etiolation on shoot regeneration was also studied. Tubers were maintained
in light or dark for 6 weeks before etiolated or non-etiolated petiole explants were cultured on MS
media containing BA or TDZ of the above concentrations.
No shoot formation was observed in media containing BA regardless of concentration, explant
type, or etiolation. Leaf discs did not exhibit regenerative potential regardless of TDZ
concentration. The greatest percent shoot formation was obtained from peduncle explants cultured
on media supplemented with 0.022 or 0.22 mg l 1 TDZ and petal explants on media containing
0.022 mg l 1 TDZ. Peduncle explants cultured on media containing 0.22 mg l 1 TDZ produced the
greatest number of shoots. Explant type and TDZ concentration signi®cantly affected callus growth.
Cultures of etiolated petioles were free of contamination, had superior shoot regenerative potential,
and produced more vigorous callus compared to those of non-etiolated petioles. Etiolated petiole
explants exhibited the greatest percent shoot formation (74%) and number of shoots when cultured
on media containing 0.022 mg l 1 TDZ. # 2000 Elsevier Science B.V. All rights reserved.

Keywords: Cyclamen persicum; Cyclamen; Micropropagation; Organogenesis; Thidiazuron

1. Introduction

Wild Cyclamen persicum Mill. is an endangered species in Jordan. This fact

mandates a move towards conserving the germplasm of this species while

*
Corresponding author. Tel.: ‡962-2-709-5111, ext: 22206; fax: ‡962-2-709-5123.
E-mail address: karam@just.edu.jo (N.S. Karam).

0304-4238/00/$ ± see front matter # 2000 Elsevier Science B.V. All rights reserved.
PII: S 0 3 0 4 - 4 2 3 8 ( 0 0 ) 0 0 1 6 0 - 6
324 N.S. Karam, M. Al-Majathoub / Scientia Horticulturae 86 (2000) 323±333

maintaining its genotype. In vitro conservation provides a low maintenance and

reliable means of preservation of plant genetic material. To be able to accomplish
in vitro conservation of wild Cyclamen, an ef®cient and reliable system for
propagation of this plant in vitro should be available.
Different types of explants have been used for shoot regeneration in Cyclamen
cultivars including leaf discs (Dillen et al., 1996), petioles, blades, ovaries (Wicart
et al., 1984), and tubers (Loewenberg, 1969; Takamura et al., 1996). Tuber tissue
has been reported to have comparatively higher regenerative potential than other
plant parts such as leaf discs, petioles, and anthers (Geier, 1977). However, the
use of tuber tissue is restricted due to the frequent contamination caused by
internal microorganisms when explants are obtained from tuber tissue (Geier
et al., 1990). Geier (1977) demonstrated that contamination could be controlled
effectively by pretreatment of tuber segments with antibiotics. However,
antibiotics may result in reduced regeneration and induced mutations (Pierik,
1987). One other problem facing clonal propagation of Cyclamen is that excision
of tuber tissue would severely damage the stock plant. In contrast, the use of leaf
discs, petioles, peduncles, or petals would leave the stock plant intact. Ando and
Murasaki (1983) suggested the use of etiolated stock plants as a means of
controlling contamination caused by internal microorganisms. The authors
reported that cultures of etiolated petioles were free of contamination and exhi-
bited superior regenerative ability compared to those of non-etiolated petioles.
Etiolated petioles of Cyclamen were also suggested by Murasaki and Tsurushima
(1988) for controlling contamination and achieving high regeneration rate.
Although in vitro regeneration of several Cyclamen cultivars has been
attempted (Geier et al., 1990), to our knowledge, in vitro propagation of wild
Cyclamen is not documented. Therefore, this study was initiated to determine the
effect of explant type, etiolation, and growth regulator type and concentration on
shoot regeneration from mature tissue of wild Cyclamen.

2. Materials and methods

The basic culture medium used in this study was a one-third-strength MS

(Murashige and Skoog, 1962) medium solidi®ed with 0.8% agar and containing
3% sucrose and 0.1 mg l 1 naphthaleneacetic acid (NAA) at pH 5.8.

2.1. Regeneration from leaf discs, petioles, petals, and peduncles

Wild Cyclamen plants at the ¯owering stage were collected from the native
habitat in Jordan, transplanted into a mixture of 1 peat:1 perlite (v/v), and
maintained under glasshouse conditions. Leaf discs, petioles, petals, and
peduncles were excised from a single Cyclamen plant, washed with water for
 N.S. Karam, M. Al-Majathoub / Scientia Horticulturae 86 (2000) 323±333 325

10 min, dipped in 70% ethanol for 30 s, surface sterilized in 4% sodium

hypochlorite solution for 15 min, and ®nally rinsed with sterile distilled water.
Leaf discs and petals were aseptically sectioned into 5 mm5 mm sections and
petioles and peduncles into 10 mm segments. Explants were cultured into 9.0 cm
plastic Petri dishes containing 30 ml of MS medium supplemented with different
concentrations of 6-benzyladenine (BA) (0, 1, 2, or 3 mg l 1) or 1-phenyl-3-
(1,2,3-thiadiazo-5-yl)urea (thidiazuron) (TDZ) (0, 0.0022, 0.022, 0.22, or
2.2 mg l 1). Petiole and peduncle explants were placed horizontally and leaf
disc and petal explants were placed with the abaxial surface in contact with the
medium. Cultures were maintained in dark at 2218C.

2.2. Regeneration from etiolated petioles

Tubers of wild Cyclamen were planted in a mixture of 1 peat:1 perlite (v/v),

maintained at 208C for 6 weeks, and watered as needed to keep them moist. A
number of tubers were maintained in dark and used as a source of etiolated
petioles, other tubers were maintained under light conditions and used as a source
of non-etiolated petioles. Etiolated and non-etiolated, 10 cm-long petioles were
excised from the tubers, washed with water for 10 min, dipped in 70% ethanol for
30 s, surface sterilized in 4% sodium hypochlorite solution for 15 min, and ®nally
rinsed with sterile distilled water. Petioles were aseptically sectioned into 10 mm
segments. Explants were cultured into 9.0 cm plastic Petri dishes containing
30 ml of MS medium supplemented with different concentrations of BA (0, 1, 2,
or 3 mg l 1) or TDZ (0, 0.0022, 0.022, 0.22, or 2.2 mg l 1). Explants were placed
horizontally on the surface of the medium. Cultures were maintained in dark at
2218C.

2.3. Experimental design, data collection, and statistical analysis

The experimental design in each experiment was split-plot with explant type as
the main-plot factor and growth regulator concentration as the subplot factor.
Each explant typegrowth regulator concentration treatment consisted of ®ve
replicates (Petri dishes) that were completely randomized. For each replicate,
four explants were placed on the medium. Cultures in each experiment were
evaluated after 8 weeks for shoot regeneration, number of shoots, and callus size
and weight. Callus growth was also assessed 8 weeks after it had been cultured on
fresh media (16 weeks after initiation of experiments). For each experiment, data
were subjected to analysis of variance (ANOVA) and regression analysis by the
General Linear Models Procedure using SAS (Statistical Analysis System, 1995,
SAS Institute, Cary, NC). Mean separation was performed using the least
signi®cant difference (LSD) method. Percent data were arcsine transformed
before performing ANOVA.
326 N.S. Karam, M. Al-Majathoub / Scientia Horticulturae 86 (2000) 323±333

Fig. 1. Shoots developing on a wild Cyclamen peduncle explant (A) and petal explant (B) on MS
media containing 0.1 mg l 1 NAA and 0.022 mg l 1 TDZ. A colour version of Fig. 1 is available as
an electronic supplement at the journal's home page: http://www.elesevier.nl/locate/scihorti.

3. Results

3.1. Regeneration from leaf discs, petioles, petals, and peduncles

No shoot formation was observed in media containing BA regardless of

concentration or explant type. However, in media containing TDZ, all explants
except leaf discs exhibited the capacity for shoot formation (Fig. 1). There were
signi®cant effects of explant type, TDZ concentration, and their interaction on
percent shoot formation and number of shoots per explant (Table 1). Analyses of
variance performed separately on data for each explant type indicated signi®cant
 N.S. Karam, M. Al-Majathoub / Scientia Horticulturae 86 (2000) 323±333 327

Table 1
Shoot regeneration and callus growth in wild Cyclamen cultures as in¯uenced by explant type and
TDZ concentration (mg l 1) in MS media
Treatment Shoot Number Callus growth after
formation (%) of shoots
8 weeks 16 weeks
2
Weight (g) Size (cm ) Weight (g) Size (cm2)
Explant type (E)
Peduncle 54aa 2.13a 0.14a 0.63b 0.59a 1.29a
Petal 38a 1.07b 0.18a 0.98a 0.81a 2.49a
Petiole 7b 0.16c 0.02b 0.03c 0.92a 2.04a
Leaf disc ±b ± ± ± ± ±
TDZ concentration (C)
0 0 0 0.06 0.43 0.02 0.02
0.0022 10 0.12 0.11 0.46 0.40 0.81
0.022 75 2.39 0.10 0.59 0.71 1.72
0.22 69 2.22 0.20 1.00 0.98 2.91
2.2 0 0 0.09 0.48 1.25 3.57
Significancec
** *** *** ***
E NS NS
*** *** * * *
C NS
*** **
EC NS NS NS NS
a
Means within columns followed by different letters are signi®cantly different by LSD at
P  0.05.
b
No shoot formation occurred on leaf discs.
c
NS,*,**,*** Ð non-signi®cant or signi®cant at P  0.05, 0.01 or 0.001, respectively.

effects (P0.001) of TDZ concentration on percent shoot formation and shoot

number for peduncle and petal explants only (Fig. 2). No shoot formation was
observed on any explant in media free of TDZ or on petiole explants except those
cultured on media containing 0.22 mg l 1 TDZ. Furthermore, shoot formation
was completely suppressed in media containing 2.2 mg l 1 TDZ regardless of
explant type. The greatest percent shoot formation was obtained from peduncle
explants cultured on media containing 0.022 or 0.22 mg l 1 TDZ or petal
explants on media containing 0.022 mg l 1 TDZ, where 94±100% shoot
formation was achieved. Peduncle explants cultured on media supplemented
with 0.22 mg l 1 TDZ produced the greatest average number of shoots (4.3
shoots per explant).
Callus was also formed on all types of explants. Callus growth as indicated by
changes in weight and size was dependent on explant type and TDZ
concentration, with peduncle and petal explants or media containing 0.22 mg l 1
TDZ exhibiting the greatest callus growth after 8 weeks of culture (Table 1).
328 N.S. Karam, M. Al-Majathoub / Scientia Horticulturae 86 (2000) 323±333

Fig. 2. Shoot regeneration in wild Cyclamen as in¯uenced by explant type and TDZ concentration.
Regressions for percent shoot formation (transformed data): Yˆ 0.28‡159.13X 723.02X2‡
295.80X3 (r2ˆ0.96) (peduncle); Yˆ0.28‡148.04X 696.03X2‡285.77X3 (r2ˆ0.89) (petal). Regres-
sions for shoot number: Yˆ 0.33‡175.20X 771.15X2‡314.35X3 (r2ˆ0.69) (peduncle); Yˆ 0.04
‡162.06X 765.81X2‡314.62X3 (r2ˆ0.67) (petal), where X is the TDZ concentration in mg l 1.

However, the only signi®cant effect observed after 16 weeks of culture was that
of TDZ concentration on callus weight.

3.2. Regeneration from etiolated petioles

No shoot formation was observed in media containing BA regardless of

concentration or etiolation. However, in media containing TDZ, both etiolated
 N.S. Karam, M. Al-Majathoub / Scientia Horticulturae 86 (2000) 323±333 329

Table 2
In¯uence of leaf petiole etiolation and TDZ concentration (mg l 1) on shoot regeneration and callus
growth in wild Cyclamen culture on MS media
Treatment Shoot Number Callus growth after
formation (%) of shoots
8 weeks 16 weeks
2
Weight (g) Size (cm ) Weight (g) Size (cm2)
Explant type (E)
Etiolated 32 0.86 0.31 1.40 1.40 3.21
Non-etiolated 7 0.16 0.02 0.03 0.92 2.04
TDZ concentration (C)
0 0 0 0.12 0.63 0.31 0.17
0.0022 26 0.58 0.18 0.99 0.86 1.59
0.022 52 1.41 0.13 0.80 1.19 3.37
0.22 38 1.06 0.29 1.23 2.05 5.09
2.2 0 0 0.30 0.98 3.19 7.33
Significancea
* ** *** ***
E NS NS
** ** * *** ***
C NS
EC NS NS NS NS NS NS
a *,**,***
NS, Ð non-signi®cant or signi®cant at P  0.05, 0.01, or 0.001, respectively.

and non-etiolated petiole explants exhibited the capacity for shoot formation
(Table 2). Contamination was negligible when etiolated petioles were used as
a source of explants, whereas the incidence of contamination was greater with
non-etiolated petioles. There were signi®cant effects of etiolation and TDZ
concentration on frequency of shoot formation and number of shoots per
explant (Table 2). Averaged over TDZ concentration, etiolated petioles exhibited
a higher shoot regenerative potential than non-etiolated ones. Averaged over
explant type, 0.022 mg l 1 TDZ resulted in the greatest percent shoot formation
and number of shoots among all concentrations tested. The greatest percent
shoot formation (74%) and average number of shoots (2) were obtained from
etiolated explants cultured on media containing 0.022 mg l 1 TDZ (Fig. 3).
Non-etiolated petiole explants did not exhibit shoot regenerative potential except
when cultured on media with 0.22 mg l 1 TDZ, at which shoot formation did
not exceed 25%.
Although callus formed on both etiolated and non-etiolated explants, callus
growth was signi®cantly greater on etiolated than non-etiolated explants after 8
weeks of culture (Table 2). However, after 16 weeks of culture, etiolation had no
effect on callus growth, which was affected by TDZ concentration with
2.2 mg l 1 resulting in the greatest callus growth.
330 N.S. Karam, M. Al-Majathoub / Scientia Horticulturae 86 (2000) 323±333

Fig. 3. Percent shoot regeneration and number of shoots per explant as in¯uenced by etiolation of
petioles and concentration of TDZ in MS media. Solid lines represent percent explants forming
shoots. Broken lines represent number of shoots per explant. Regressions for etiolated petiole:
percent shoot formation (transformed data)ˆ0.41‡99.12X 472.63X2‡194.31X3 (r2ˆ0.35); shoot
numberˆ0.27‡89.19X 421.25X2‡173.02X3 (r2ˆ0.33), where X is the TDZ concentration in
mg l 1.

4. Discussion

The regeneration potential of different explants from mature tissue of wild

Cyclamen was examined using different types and concentrations of growth
regulators. Although it was reported to be effective for shoot formation from
various Cyclamen explants (Loewenberg, 1969; Ando and Murasaki, 1983;
Wainwright and Harwood, 1985; Hawkes and Wainwright, 1987; Dillen et al.,
1996), BA failed to induce shoot regeneration in the current study. In contrast,
TDZ, a cytokinin-like compound of non-purine structure was highly effective in
promoting shoot formation at very low concentrations. The biological activity of
TDZ was reported to be higher than or comparable to that of the most active
cytokinin (Mok et al., 1982; Kerns and Meyer, 1986; Chvojka et al., 1992;
Bretagne et al., 1994). Furthermore, TDZ induced shoot organogenesis when
other cytokinins failed (Jordan et al., 1996) and dramatically increased
 N.S. Karam, M. Al-Majathoub / Scientia Horticulturae 86 (2000) 323±333 331

micropropagation ef®ciency of many plant species (Huetteman and Preece,

1993). In a study with apple (Malus domestica Borkh.) and aspen (Populus
tremula L.), Chvojka et al. (1992) observed more ribosomes and polysomes in
cytoplasm of cells on media containing TDZ compared to media containing BA
indicating more intensive protein synthesis and higher cell activity with TDZ.
In this study, a signi®cant effect of TDZ concentration on shoot regeneration
was detected, indicating the importance of the ratio of TDZ to NAA in the culture
medium. Omitting TDZ from the media resulted in failure of shoot formation
presumably due to low levels of endogenous cytokinin. Shoots were formed when
TDZ was incorporated into the media at low concentrations, where adventitious
meristems were regenerated. However, high concentration of TDZ drastically
inhibited shoot regeneration, which is in agreement with other studies. Potential
liabilities with using TDZ for organogenesis include problems with shoot
fasciation (Huetteman and Preece, 1993), a phenomenon that did not occur at the
low concentrations of TDZ used in this study.
A signi®cant difference in regenerative potential was detected among explants,
which may be explained by the degree of cell sensitivity towards growth
regulators due to explant origin, endogenous growth regulator levels, and auxin
and cytokinin oxidases activity (Tran Thanh and Trinh, 1990). No shoot
regeneration occurred on leaf disc explants regardless of TDZ concentration,
which may be due to the age of the donor plant. Shoot regeneration capacity in
Cyclamen was shown to be greatest in young leaves, to decline with advanced
leaf age, and to be suppressed in explants from leaves of more than 30 mm length
(Geier, 1977; Geier et al., 1990). In the current study, peduncle explants were
relatively highly regenerative, which is in agreement with results of other studies
on Cyclamen (Schwenkel and Grunewaldt, 1988; Dohm et al., 1989).
The use of etiolated stock plants as a means of controlling contamination
caused by internal microorganisms was suggested by Ando and Murasaki (1983).
The authors observed complete elimination of microbial growth and higher
regeneration potential in explants from etiolated petioles (12.5% contamination)
as compared to those from non-etiolated petioles (83% contamination). In fact,
the authors obtained negligible shoot formation from non-etiolated plants as was
the case in the current study where 74% of etiolated petioles produced shoots as
compared to 25% of non-etiolated petioles. Etiolated petioles were also suggested
by Murasaki and Tsurushima (1988) to be promising explants for controlling
contamination and achieving high regeneration rate in Cyclamen cultures.
In the current study, shoots from peduncle and etiolated petiole cultures were
rooted on a medium containing 1.0 mg l 1 NAA. In vitro-rooted shoots were
removed from culture tubes, cleaned from agar, transplanted into 1 peat:1 perlite
(v/v), and successfully acclimatized. Plants were then grown under greenhouse
conditions. The plants appeared normal and did not exhibit morphological
abnormalities.
332 N.S. Karam, M. Al-Majathoub / Scientia Horticulturae 86 (2000) 323±333

In this study, formation of callus was observed, which was reported to retain its
capacity to differentiate shoots (Geier et al., 1990; Dillen et al., 1996) even after
more than 6 years of subculture (Loewenberg, 1969). Callus size was higher with
etiolated than non-etiolated petioles, which is in agreement with observations of
Ando and Murasaki (1983).

5. Conclusion

To achieve appreciable results in propagation of wild Cyclamen in vitro,

peduncles or petals could be used for shoot regeneration when cultured on 13 MS
medium containing 0.1 mg l 1 NAA and 0.022 mg l 1 TDZ. Etiolated petioles
cultured on the same medium were shown to retain a higher regenerative potential
than non-etiolated petioles. The employment of etiolated petioles could also
avoid the problem of contamination.

Acknowledgements

We acknowledge the Deanship for Scienti®c Research at Jordan University of

Science and Technology (JUST) for funding this project. Sincere appreciation
goes to Dr. Hani Ghosheh (JUST) for his guidance in statistical analysis.

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