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 persicumMill. was studied using mature tissue. Leaf disc, petiole, petal, and peduncle explants were cultured on1
3MS medium supplemented with
6-benzyladenine (BA) at 0, 1, 2, or 3 mg lÿ1
or thidiazuron (TDZ) at 0, 0.0022, 0.022, 0.22, or 2.2 mg lÿ1. 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 lÿ1
TDZ and petal explants on media containing 0.022 mg lÿ1TDZ. Peduncle explants cultured on media containing 0.22 mg lÿ1TDZ 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 lÿ1
TDZ.#2000 Elsevier Science B.V. All rights reserved.
Keywords: Cyclamen persicum; Cyclamen; Micropropagation; Organogenesis; Thidiazuron
1. Introduction
WildCyclamen 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: [email protected] (N.S. Karam).
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 lÿ1 naphthaleneacetic acid (NAA) at pH 5.8.
2.1. Regeneration from leaf discs, petioles, petals, and peduncles
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 lÿ1) or 1-phenyl-3-(1,2,3-thiadiazo-5-yl)urea (thidiazuron) (TDZ) (0, 0.0022, 0.022, 0.22, or 2.2 mg lÿ1). 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 lÿ1
) or TDZ (0, 0.0022, 0.022, 0.22, or 2.2 mg lÿ1
). 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
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 Fig. 1. Shoots developing on a wild Cyclamen peduncle explant (A) and petal explant (B) on MS media containing 0.1 mg lÿ1
NAA and 0.022 mg lÿ1
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 lÿ1
TDZ. Furthermore, shoot formation was completely suppressed in media containing 2.2 mg lÿ1 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 lÿ1 TDZ or petal explants on media containing 0.022 mg lÿ1 TDZ, where 94±100% shoot formation was achieved. Peduncle explants cultured on media supplemented with 0.22 mg lÿ1 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 lÿ1 TDZ exhibiting the greatest callus growth after 8 weeks of culture (Table 1). Table 1
Shoot regeneration and callus growth in wild Cyclamen cultures as in¯uenced by explant type and TDZ concentration (mg lÿ1) in MS media
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
Means within columns followed by different letters are signi®cantly different by LSD at
P0.05.
b
No shoot formation occurred on leaf discs.
c
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 Fig. 2. Shoot regeneration in wild Cyclamen as in¯uenced by explant type and TDZ concentration. Regressions for percent shoot formation (transformed data): Yÿ0.28159.13Xÿ723.02X2 295.80X3(r20.96) (peduncle);Y0.28148.04Xÿ696.03X2285.77X3(r20.89) (petal). Regres-sions for shoot number:Yÿ0.33175.20Xÿ771.15X2314.35X3(r20.69) (peduncle);Yÿ0.04 162.06Xÿ765.81X2314.62X3(r20.67) (petal), whereXis the TDZ concentration in mg lÿ1
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 lÿ1
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 lÿ1 TDZ (Fig. 3). Non-etiolated petiole explants did not exhibit shoot regenerative potential except when cultured on media with 0.22 mg lÿ1 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 lÿ1resulting in the greatest callus growth.
Table 2
In¯uence of leaf petiole etiolation and TDZ concentration (mg lÿ1
) on shoot regeneration and callus growth in wild Cyclamen culture on MS media
Treatment Shoot
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
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 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)0.4199.12Xÿ472.63X2194.31X3(r20.35); shoot number0.2789.19Xÿ421.25X2173.02X3 (r20.33), where X is the TDZ concentration in mg lÿ1
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 lÿ1
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 on13MS medium containing 0.1 mg lÿ1
NAA and 0.022 mg lÿ1
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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