The Oriental hybrids originate from intra- or interspecific hybridization among at least 5 species (i.e., L. alexandrae, L. auratum, L. rubellum, L. speciosum, L. henryi) of the Archelirion section. Oriental cultivars have pink, white, or yellow flowers with a sweet fragrance, and about 2000 cultivars have been registered since 1990 (Leslie, 1982-2005). Due to their large flowers, bright colors, elegance and aromatic fragrance, the Oriental hybrids, deeply loved by consumers, have the largest cultivated area of all the lilies cut flowers. However, these hybrids are sensitive to virus and fusarium oxysporum, making it necessary to breed new Oriental cultivars with high disease resistance.

To breed new disease-resistant Oriental varieties, combing genetic engineering with normal breeding techniques is the most direct and efficient way. The basis of genetic engineering is the stable and highly efficient plant regeneration system. Through many studies on Lilium longiflorum in vitro culture, researchers can now get plant regeneration from anther (Han et al., 1997), pedicel (Tribulato et al., 1997), receptacle (Nhut et al., 2001a), leaf (Kato and Yasutake, 1977), stem (Nhut, 1998), and bulb scale (Simmonds and Cumming, 1976a; Simmonds and Cumming, 1976b) by organogenesis and somatic embryogenesis. The system of organogenesis and somatic embryogenesis using thin cell layer (TCL) culture was established by Nhut DT and his associates (Nhut et al., 2001c; Nhut et al., 2002; Nhut et al., 2001d; Nhut et al., 2001b; Nhut et al., 2006). Since then, TCLs have been extensively used to study lily differentiation. Though successful manipulation of morphogenic programs has established it as a model system in Lilium longiflorum (Xi et al., 2012; Teixeira da Silva, 2003), no one has induced adventitious bud using organogenesis from bulblet-scale TCL of Oriental lily cultivar ‘Constanta’.

To fill this gap, we studied the influence of 6-BA and TDZ concentration on adventitious bud induction of thin cell layers from bulblet-scale of Oriental lily cultivar ‘Constanta’. An efficient regeneration system for TCL tissue culture was created to study the culture condition and its influence on the desired regeneration system. The high efficiency regeneration system may also be useful for Agrobacterium-mediated or particle gun-mediated genetic transformation and large scale micropropagation for the important bulbous crops.
 
1 Results
1.1 Establishment of tissue culture
After being cultured on the initial medium for two weeks, the bulbscales turned green. Three to four weeks later, regeneration started, with 1~3 small bulb-like initials appearing on each scale surface. They grew gradually and formed small bulbs with roots with leaves appearing on some of them. The morphological changes and patterns for this variety were similar to those observed in Lilium rhodopaeum Delip (Stanilova et al., 1994). The small bulblets grew rapidly after being transferred to the bulblet culture medium. Their circumferences reached 3~4 cm after being subcultured twice (Figure 1A).

Figure 1 Plant regeneration of Oriental lily cultivar ‘Constanta’

1.2 Influence of 6-BA concentration on adventitious bud induction
The effects of 6-BA concentration on adventitious bud differentiation from bulblet-scale TCL explants of Oriental lily cultivar ‘Constanta’ were optimized (Table 1). All bulblet-scale TCL explants showed swelling as well as early signs of adventitious bud differentiation after being cultured on the adventitious bud induction medium for 8 days. After 30 days, adventitious buds were observed on the bulblet-scale TCL explants. At low 6-BA concentrations (0.0~0.8 mg/L), the adventitious bud induction frequency increased with increasing 6-BA concentration increased. However, hyperhydrated adventitious buds were observed when the 6-BA concentration was 1.0 mg/L. For the same plant growth regulator concentration, the adventitious bud induction frequency and the average number of adventitious buds were higher in the dark than that in the light condition. The results showed that 6-BA at 0.8 mg/L were the best concentration for adventitious bud differentiation.

Table 1 The effect of 6-BA concentration on adventitious bud induction of thin cell layers from bulblet-scale of Oriental lily cultivar ‘Constanta’

1.3 Influence of TDZ concentration on adventitious bud induction
In order to explore the effect of TDZ concentration on adventitious bud differentiation from bulblet-scale TCL explants, low concentration of TDZ were tested in the present study. Adventitious bud formation was observed on the bulblet-scale TCL when the bulblet-scale TCL explants were cultured on the adventitious bud induction medium for 25 days. Green adventitious buds and some roots were differentiated from explants in the light condition (Figure 1D), and white adventitious buds and more roots were differentiated from explants in the dark condition (Figure 1C). Results displayed in table 2 showed that the adventitious bud induction frequency increased as the concentration of TDZ increased from 0.000 mg/L to 0.004 mg/L. However, hyperhydrated adventitious buds and callus were observed when the TDZ concentrations were 0.006 mg/L and 0.008 mg/L. The adventitious bud induction frequency was higher in the dark than that in the light condition while maintaining the same TDZ concentration. Thus, we had decided that MS basal medium supplemented with 0.004 mg/L TDZ and 0.2 mg/L 2, 4-D was the optimal medium for adventitious buds formation from bulblet-scale TCL. The differentiation frequency and the average number of adventitious buds reached 98.89% and 6.53, respectively in the dark condition (Table 2).

Table 2 The effect of TDZ concentration on adventitious bud induction of thin cell layers from bulblet-scale of Oriental lily cultivar ‘Constanta’

1.4 Growth in the greenhouse
These bulblets began germination 15 days after planting and all sprouted after 26 days. The survival percentage reached 96% after six weeks. Having the same rapid growth rate, more than 80% of the bulblets had born stems with heights ranging from 20~30 cm. All these plants grew normally in the greenhouse.

2 Discussion
Plant hormone plays an important role in organog- enesis under tissue culture conditions. For example, the types, ratio and concentration of cytokinin and auxin regulate the direction of organogenesis. Bacchetta and his associates concluded that 6-BA was most effective for Asiatic hybrids organ differentiation (Bacchetta et al., 2003). However, the result of our study indicated that, with appropriate concentration, TDZ was significantly more effective than 6-BA for inducting adventitious bud from the bulblet-scale TCL of Oriental lily cultivar ‘Constanta’.

Just as plant hormone, lighting condition has obvious influence on organogenesis. In their research on Fritillaria thunbergii bulb in vitro regeneration, Paek and Murthy found that light condition was more advantageous to bulb regeneration (Paek and Murthy, 2002), but our study, on the contrary, showed that dark conditions were conducive to adventitious bud induction from the bulblet-scale TCL of Oriental lily, which was similar to the result of Niimi and Onozawa’s study of lilium (Niimi and Onozawa, 1979).

Cold treatment is an effective method to break lily bulb dormancy. Paek and Murthy succeeded in breaking Fritillaria thunbergii bulblet dormancy by a 5℃ cold treatment for 5 weeks (Paek and Murthy, 2002). Similar to their results, our study showed that the dormancy of bulblets that were 3~4 cm in circumference could be broken by 4℃ cold treatment for 8 weeks. These bulblets then germinated neatly and developed normally in the greenhouse.

The main advantage of the regenerative system we have established is its efficiency. One bulblet which is 3~4 cm in circumference can be cut into 30 to 60 1 mm thick bulblet-scale TCL explants, which in turn will be inoculated in the adventitious bud induction medium. On average, one bulblet-scale TCL can generate 6.5 adventitious buds. This means that in just two months, a bulblet of 3~4 cm in circumference can regenerate 195~390 adventitious buds. Having established this high efficiency regeneration system of Oriental lily cultivar ‘Constanta’, our next study will be focused on the genetic stability of the bulbs regenerated and the genetic transformation of such cultivar.

3 Materials and methods
3.1 Plant materials
Dormant bulbs of Oriental lily cultivar ‘Constanta’ were bought from the Luxin Flower Company, Yunnan Province, China. Bulbs were stored in plastic bags at 4℃ until being used as the initial explants for the experiments.

3.2 Explant preparation and sterilization
The dormant bulbs were rinsed with tap water. The bulb-scales were hand-peeled from the bulbs, submerged in detergent solution for 30 min, and followed by rinsing continuously for 35 min with running tap water. Under sterile conditions in a flowcabinet, the bulb-scales were surface sterilized in 75% (v/v) ethanol for 1 min, in 0.1% HgCl₂ (w/v) for 15 min, and then washed 5~6 times with sterile distilled water. Finally, the bulb-scales were transversely cut into two parts and the basal bulb- scales were cultured horizontally on initial medium.

3.3 Culture medium and culture conditions
The experiments used three types of culture media: the initial medium, the culture bulblet medium, and the adventitious bud induction medium. The initial medium in the experiments used the Murashige and Skoog (MS) basal medium (Murashige and Skoog, 1962)., and the culture bulblet medium used the MS basal medium containing 8% (w/v) sucrose, while adventitious bud induction medium used the MS basal medium supplemented with growth regulators at various levels of concentrations: (a) 6-benzyladenine (6-BA) at 0.0 mg/L, 0.2 mg/L, 0.4 mg/L, 0.6 mg/L, 0.8 mg/L, or 1.0 mg/L; (b) Thidiazuron (TDZ) at 0.000 mg/L, 0.001 mg/L, 0.002 mg/L, 0.004 mg/L, 0.006 mg/L or 0.008 mg/L; (c) 2,4-dichlorophenoxyacetic acid (2,4-D) at 0.2 mg/L.

Unless otherwise stated, the media contained 0.65% (w/v) agar and 3% (w/v) sucrose, and were adjusted to pH 5.8 before being autoclaved at 121℃ for 16 min. All cultures were incubated at (25±2)℃ under cool white fluorescent lights (80 μmol.m-2.s-1) with a 14 h photoperiod and 60% relative humidity.

3.4 Adventitious bud induction
The bulblet-scales were peeled aseptically from 3~4 cm bulblets (Figure 1A), excised longitudinally (approximately 1.0 mm thick) and placed on the adventitious bud induction medium. The culture conditions included light and dark culture. Each Petri dish were inoculated with fifteen bulblet-scale TCL explants with 20 mL to 25 mL medium in each 15×90 mm plate and were sealed with a double layer of Parafilm (Figure 1B). Each treatment comprised 2 Petri dishes of total 30 bulblet-scale TCL explants and the experiment was repeated three times. The experiments were completely randomized. The data from the bulblet-scale TCL were recorded after culturing for two months. We evaluated significant differences between means by analyzing variance in all the data.

3.5 Hardening and acclimatization
Adventitious buds elongated rapidly when they were transferred to the initial medium (Figure 1E). Then the plantlets were grown on the bulblet culture medium until the circumferences of the bulblets reached 3~4 cm (Figure 1A), when they were transferred to cold storage (4℃) to break dormancy. After 8 weeks, the bulblets were gently removed from the bottles. The medium residuals remaining on the bulblets and roots were rinsed off with tap water. Exactly 100 bulblets were transferred to a mixture with peat to perlite ratio of 7 to 3 in the greenhouse of Nanjing Forestry University (China) for acclimatization. The greenhouse was maintained under natural light conditions, temperature of 15~29℃, and relative humidity of 60%~80%.

Acknowledgments
The authors wish to thank Professor Zhongen Xi (Chongqing University of Posts and Telecommunications, China) for his valuable advices on the manuscript. The authors also appreciate the financial support from the National Natural Science Foundation of China (30972407), Jiangsu Agriculture Science and Technology Innovation Fund CX(12)2018, and fund from A Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions.

References
Bacchetta L., Remotti P.C., Bernardini C., and Saccardo F., 2003, Adventitious shoot regeneration from leaf explants and stem nodes of Lilium, Plant Cell Tissue Organ Cult, 74: 37-44
 http://dx.doi.org/10.1023/A:1023315321931
 
Han D.S., Niimi Y., and Nakano M., 1997, Regeneration of haploid plants from anther cultures of the asiatic hybrid lily `connecticut king', Plant Cell Tissue Organ Cult, 47: 153-158
 http://dx.doi.org/10.1007/BF02318951
 
Kato Y., and Yasutake Y., 1977, Plantlet formation and differentiation of epidermal tissue in green callus cultures from excised leaves of Lilium, Phytomorphology, 27: 390-396
 
Leslie A.C., 1982-2005, The international lily register (including supplements), The Royal Horticultural Society, London
 
Murashige T., and Skoog F., 1962, A revised medium for rapid growth and bioassays with tobacco tissue cultures,Physiol Plant, 15: 473-497
 http://dx.doi.org/10.1111/j.1399-3054.1962.tb08052.x
 
Nhut D.T., 1998, Micropropagation of lily (Lilium longiflorum) via in vitro stem node and pseudo-bulblet culture, Plant Cell Rep, 17: 913-916
 http://dx.doi.org/10.1007/s002990050508
 
Nhut D.T., Bui V.L., Fukai S., Tanaka M., and Tran Thanh Van K., 2001c, Effects of activated charcoal, explant size, explant position and sucrose concentration on plant and shoot regeneration of Lilium longiflorum via young stem culture, Plant Growth Regul, 33: 59-65
 http://dx.doi.org/10.1023/A:1010701024963
 
Nhut D.T., Bui V.L., Minh N.T., Teixeira da Silva J.A., Fukai S., Tanaka M., and Tran Thanh Van K., 2002, Somatic embryogenesis through pseudo-bulblet transverse thin cell layer of Lilium longiflorum, Plant growth Regul, 37: 193-198
 http://dx.doi.org/10.1023/A:1020532511081
 
Nhut D.T., Bui V.L., Silva D.A., and Aswath C.R., 2001d, The cell layer culture system in Lilium:regeneration and transformation perspectives, In Vitro Cell Dev Biol-Plant, 37: 516-523
 http://dx.doi.org/10.1007/s11627-001-0090-2
 
Nhut D.T., Bui V.L., Tanaka M., and Tran Thanh Van K., 2001a, Shoot induction and plant regeneration from receptacle tissue of Lilium longiflorum, Sci Hortic, 87: 131-138
 http://dx.doi.org/10.1016/S0304-4238(00)00163-1
 
Nhut D.T., Bui V.L., and Tran Thanh Van K., 2001b, Manipulation of the morphogenetic pathways of Lilium longiflorum transverse thin cell layer explants by auxin and cytokinin. In Vitro Cell Dev. Biol .- Plant, 37: 44-49
 http://dx.doi.org/10.1007/s11627-001-0009-y
 
Nhut D.T., Hanh N.T.M., Tuan P.Q., Nguyet L.T.M., Tram N.T.H., Chinh N.C., Nguyen N.H., and Vinh D.N., 2006, Liquid culture as a positive condition to induce and enhance quality and quantity of somatic embryogenesis of Lilium longiflorum, Sci Hortic, 110: 93-97
 http://dx.doi.org/10.1016/j.scienta.2006.05.015
 
Niimi Y., and Onozawa T., 1979, In vitro bulblet formation from leaf segments of lilies, especially Lilium rubellum Baker, Sci Hort, 11: 379-389
 http://dx.doi.org/10.1016/0304-4238(79)90023-2
 
Paek K. Y., and Murthy H. N., 2002, High frequency of bulblet regeneration from bulb scale sections of Fritillaria thunbergii, Plant Cell Tissue Organ Cult, 68: 247–252
 http://dx.doi.org/10.1023/A:1013952803887
 
Simmonds J.A., and Cumming B.G., 1976a, Propagation of Lilium hybrids. I.Dependence of bulblet production on time of scale removal and growth substances, Sci Hortic, 5: 77-83
 http://dx.doi.org/10.1016/0304-4238(76)90026-1
 
Simmonds J.A., and Cumming B.G., 1976b, Propagation of Lilium hybrids. II. Production of plantlets from bulb-scale callus cultures for increased propagation, Sci Hortic, 5: 161-170
 http://dx.doi.org/10.1016/0304-4238(76)90078-9
 
Stanilova M.I., Ilcheva V.P., and Zagorska, N.A., 1994, Morphogenetic potential and in vitro micropropagation of endangered plant species Leucojum aestivum L. and Lilium rhodopaeum Delip, Plant Cell Rep, 13: 451-453
 http://dx.doi.org/10.1007/BF00231965
 
Teixeira da Silva J.A., 2003, Thin Cell Layer technology in ornamental plant micropropagation and biotechnology, Afr. J. Biotechnol, 2: 683-691
 
Xi M.L., Sun L.N., Qiu S., Liu J.J., Xu J., and Shi J.S., 2012, In vitro mutagenesis and identification of mutants via ISSR in lily (Lilium longiflorum), Plant Cell Rep, 31: 1043-1051
 http://dx.doi.org/10.1007/s00299-011-1222-8
 PMid:22228557