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Somatic Embryogenesis from the Etiolated Petiole of Cyclamen (Cyclamen persicum Mill.)

The induction of somatic embryos from etiolated petioles was examined in five commercial cyclamen (Cyclamen persicum Mill.) cultivars. Somatic embryogenesis was only observed in ‘Anneke’. The optimal medium was Murashige and Skoog medium with 5.0μM 2, 4-D and 0.5μM kinetin, and the optimal incubation condition was complete dark at 25°C. A much higher percentage of explants forming embryoids and also larger number of embryoids were recorded in the explants from a third distal end of 5-10cm petioles than from other explants. This study suggests that somatic embryogenesis from etiolated petiole tissues was effectively utilized for the micropropagation of cyclamen.


Introduction
Cyclamen (Cyclamen persicum Mill.) is one of the most popular pot plants and it is commercially propagated by seeds. In horticultural practice, vegetative propagation is desirable because of the heterogeneity in seedlings of the most cultivars.
It is, however, difficult to propagate cyclamen by division, cutting and grafting. Consequently, the micropropagation of cyclamen has been expected.
One of the serious problems encountered in the micropropagation of cyclamen is microbial contamination. Furthermore, conventional micropropagation methods through axillary or adventitious bud systems has not been economical in cyclamen1) because of the low multiplication rate.
Therefore, micropropagation via somatic embryogenesis without microbial contamination are desired. Though it has already been reported that microbial contamination was prevented by the use of etiolated petioles ( Fig. 1) in the organogenesis of cyclamen2.3), the somatic embryogenesis from the etiolated petiole tissues has never been reported. We, therefore, examined the induction Bar=2cm of somatic embryos from the etiolated petioles of mature plants in commercial cyclamen cultivars.

Materials and Methods
Five cyclamen cultivars, i, e.'Anneke','Bonfire', 'Golden Boy','Lilac' and 'Pure White', were used as the plant materials. The etiolation treatment on the mother plants and the surface sterilization procedures were in accordance with Ando and Murasaki2). Effects of plant growth regulators were initially examined. After surface sterilization, the distal quarters of etiolated petioles which had elongated to 10 cm in length were resected and about 5mm of the both ends were removed. Then, they were cut into 2mm lengths and used as explants. The experiments were repeated at least twice. In all experiments, the media were adjusted to pH 5.8 and autoclaved at 121C for 20 minutes. The cultures were transferred to MS medium without plant growth regulator after 6 weeks of culture.
After 5 weeks of subculture, the number of cultures forming embryoids and the number of embryoids formed were recorded. Fifty plantlets obtained in the optimal condition for somatic embryogenesis were planted in the greenhouse and allowed to flower.
Some calli and embryoids were collected and then embedded in paraffin (melting point 58-60C) as in previous reports". The materials embedded in paraffin blocks were sectioned at 10um and stained with 0.25% Hidenhein's iron hematoxylin for microscopic examination.

Results and Discussion
Microbial contamination was prevented in all the explants of etiolated petioles except three explants in 'Lilac', whereas 12.5 to 85% of explants were contaminated in the explants of leaf segments ( Table 1). The three contaminated etiolate petiole explants in 'Lilac' might be due to the infection with bacterium in the mother plant, since contamination percentage of the leaf segments in 'Lilac' were also much higher than that in the other cultivars and the 'Lilac' plant died of a bacterial disease during the cultivation after the etiolated treatment.
In every experiment, somatic embryogenesis was observed only in 'Anneke'. Two types of calli were formed in the petiole culture of this cultivar, one was transparent and friable and the other was opaque white and compact. The former produced somatic embryoids (Fig. 2) as reported by Otani and Shimada5). The somatic embryoids developed through the heart-and torpedo-shaped  In embryogenesis from the petiole tissues of cyclamen seedling, it has been reported that the number of somatic embryoid and percentage of explants forming embryoids were higher at 25C than at 20C or 30C6). Similarly, 25C was the optimal temperature for the embryogenesis from the etiolated petioles (Table 3). It was reported that the maturation of somatic embryos of Daucus and caraway proceeded more normally in complete darkness than under light condition7.8). In seedling tissue culture of cyclamen; a much higher number of embryoids were obtained in the dark than under light condition6) a In the etiolated petiole of cyclamen, a much higher number of embryoids and percentage of explants forming somatic embryoids occurred in the dark than under light condition.
It has been reported that the number of explants producing embryogenic callus was higher in the explants from the top portion of the hypocotyl than in the explants from the middle and bottom portions of hypocotyl in Solanum melongena9. In cyclamen, the percentage of explants forming embryoids and the number of embryoids produced varied according to the portions and length of etiolated petioles ( Table 4). The somatic embryogenesis was not observed in the explants from the bottom portions of the etiolated petioles (bottom explants) and much higher percentage of explants forming embryolds and number of embryoids were recorded in the explants from the top portions of etiolated petioles (top explants) than the middle portions (middle explants). The highest percent age of explants forming embryoids was recorded in the top explants of 5 cm etiolated petioles and the highest number of embryoids was recorded in the top explants of 5 and 10 cm etiolated petioles. These results suggest the importance of petiole length and the portion used as explants, which has   4).
The elimination of microbial contamination was hardly mentioned in the reports on the micro propagation of cyclamen via somatic embryogenesis5, 1-13). In contrast, the use of aseptic seedling was beneficial for micropropagation through somatic embryogenesis without microbial contamination6). However, propagation of true-to-type plants may not be sure in the aseptic seedling tissue culture because of the heterogenous nature of the seedlings in many of the cultivars commercially available.
In the present study, we confirmed that the etiolated petioles of cyclamen were scarcely infected by microorganisms. As we can obtain the etiolated petioles as the explants after observation of the characteristics of flowering plants, somatic embryogenesis from etiolated petiole tissues should be an effective method for the micropropagation of true-to-type cyclamen cultivars.
Varietal differences were previously reported in the efficiency of somatic embryogenesis from the aseptic seedlings of cyclamen14). Somatic embryogenesis was observed only in 'Anneke' although 5 cultivars were examined in this study. The studies of genetic effects on the somatic embryogenesis is required for further application of tissue culture for commercial micropropagation of cyclamen.