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Introduction

 In lilies, scale bulblets have been used mainly for the conventional propagation method so far. Inadequate availability of healthy planting materials and slow multiplication rates limit the use of this method (van Aartrijk et al. 1990). Micropropagation provides an alternative to conventional propagation methods (van Aartrijk et al. 1990). Shoot regeneration of Lilium has been successfully achieved using various explants including pseudo-bulblets (Nhut 1998), bulb scales (Liu and Yang 2012), stems (Bacchetta et al. 2003), receptacles (Nhut 2003), and leaf segments (Kim et al. 2005; Liu and Yang 2012; Xu et al. 2009) among these, leaf segments are most often used because they are easy to obtain and their availability is independent of the season. With leaf segments, shoot regeneration has been reported in Lilium longiflorum (Bacchetta et al. 2003), L. Oriental hybrid (Bacchetta et al. 2003; Kim et al. 2005; Liu and Yang 2012) L. davidii var. unicolor (Xu et al. 2009), However, a genotypespecific response is very common and represents a key problem in lily regeneration. Therefore, development of a procedure applicable to shoot regeneration of diverse genotypes of Lilium would be useful.

 For regenerants produced by in vitro culture techniques, an important concern is their genetic integrity with respect to the mother plant (Martins et al. 2004; Modgil et al. 2005) because genetic variation may occur during the tissue culture process (George and Davies 2008). Furthermore, as the basic objective of micropropagation is the production of trueto- type plants, it is important to retain and certify the clonal fidelity for large-scale multiplication of true-to-type plants, and also for Agrobacterium-mediated transformation because phenotypic change of transformants can be caused by polyploidization. However, variations may occur in tissue culture-raised plants due to micromutations occurring in somatic cells and use of PGRs and supplements.

 The objective of the present study was to develop a procedure for shoot regeneration of Lilium oriental hybrid ‘Casa Blanca’ using leaf segments and to assess the genetic stability of the regenerants with flow cytometry analysis.

Materials and Methods

Plant material

 Lily bulbs of the Oriental hybrid ‘Casa Blanca’ were planted in a planting bed containing a mixture of soil and peat moss (1:1) in a greenhouse for plant growth. Greenhouse temperatures were maintained using a 25-28℃ day and 20-22℃ night heat set point. Before the plants shifted to the flowering stage, the leaves and stems (with four or five nodes) were collected from the uniform and healthy plants planted in the greenhouse. Then, these were thoroughly washed under running tap water. Thereafter, they were subsequently surface sterilized with a 70% (v/v) ethanol solution for 30 sec and a 20% commercial bleach (0.7% sodium hypochlorite) plus Tween 20 for 5 min followed by rinses five times with sterilized deionized water, after which the sterilized leaves and stems were used as explant sources.

Shoot regeneration

 The basal and apical parts from the disinfected leaves were cut into ca. 0.5-1 cm long sections. The remaining middle parts of the leaves were also transversely cut into ca. 1-cmlong sections. The leaf segments (apical, middle, and basal parts) were adaxially and firmly placed on the regeneration media. Similarly, the nodal (with a single node) and internodal segments from the sterilized stems were cut into ca. 0.5 cm long sections and vertically cultured on the same regeneration media. Regeneration was induced with MS medium (Murashige and Skoog 1962) supplemented with different concentrations of naphthaleneacetic acid (NAA), 6-benzyladenine (BA), and thidiazuron (TDZ) combinations, 3% sucrose, and 0.8% agar. All culture media used were adjusted to pH 5.8 before autoclaving at 121℃ for 15 min. The cultures were initially incubated a 25℃ for 30 days in the dark, and later exposed to fluorescent lights (60-100 μmol · m^-2 · s^-1) with a 16 hrs light and 8 hrs dark photoperiod without transferring to fresh medium.

Experimental design, collection of data, and statistical analysis

 In this experiment, a completely randomized design was used to test the combined effects of five NAA concentrations (0, 0.5, 1.0, 1.5, and 2.0 mg · L^-1) and four BA concentrations (0.1, 0.5, 1.0, and 2.0 mg · L^-1) on regeneration. A plant growth regulator (PGR) free control medium was also used for regeneration. There were three replicates, and each replicate consisted of 10 explants. Cultures were observed at weekly intervals for up to 45 days. The frequency of shoots and the number of shoots per explant were recorded at 45 days after culture initiation. The data were statistically analyzed with the SAS program. Differences among the means were tested by Duncan’s multiple range test.

Shoot growth, rooting, and acclimatization

 The explants with a shoot cluster were transferred onto half MS medium supplemented with 2.5 mg · L^-1 BA, 0.5 mg · L^-1 NAA, 1 g · L^-1 activated charcoal, 3% sucrose, and 0.8% agar for shoot growth and rooting. After 4 weeks of culture, well grown and rooted plantlets were transferred to a 500 mL of culture vessel containing the same media composition. Subsequently, the regenerated plantlets that reached a size of 6-8 cm were maintained at low temperature (4℃) for 4 weeks, and then the agar of the root was cleaned and the plantlets were transplanted into pots containing vermiculite and soil (1:2, v/v). The plants were then transferred to the greenhouse.

Analysis of ploidy level

 In vitro rooted shoots derived from different explant sources were randomly selected from the rooting media to determine their ploidy levels. About 20 mg of leaf tissue per plantlet was chopped with a sharp razor blade in a plastic petri dish containing 500 mL of nuclei extraction buffer (Partec, GmbH, Munster, Germany) to get a fine suspension. The samples were filtered through a nylon mesh (30 μm). Subsequently, 2 mL of staining buffer (Partec, GmbH, Münster, Germany) was added and the suspension was supplemented with 4’, 6-diamidino-2-phenylindole; then, the ploidy level from each sample was measured with a flow cytometer (Partec, GmbH, Munster, Germany). Leaf tissue from the mother plants planted in the greenhouse was used as a diploid reference and the flow cytometer was adjusted. All the experiments were done at least three times.

Results

Shoot induction from leaf explants

 After 15 days of culture, most leaf explants showed elongation and enlargement. Subsequently, the cut edges of the explants exhibited a brown color. Direct shoot formation was observed in all parts of the tested leaves after 30 days of culture. Shoot regeneration started mainly from the basal cut edges of the basal parts of the leaves (Fig. 1A). No shoot formation was observed on the PGR-free medium (control, data not shown). When the leaf segments were cultured on media containing NAA in combination with different BA or TDZ concentrations, the shoot regeneration frequency, and the number of adventitious shoots per leaf segment were higher in with the combination of NAA and BA than with the combination of NAA and TDZ. However, the number of adventitious shoots per leaf segment significantly decreased from 3.1 to 1.2 as the BA levels in the medium were increased from 0.1 to 1.0 mg · L^-1. Further increases in the BA concentration resulted in decreased numbers of shoots (Table 1). Therefore, the combination of 0.1 mg · L^-1 NAA with 0.1 mg · L^-1 BA was considered optimal for shoot regeneration. It is noteworthy that the BA at lower concentrations seemed to be effective for shoot formation from the basal parts of the leaves.

Fig. 1. Plant regeneration from the basal leaf segments of L. longiflorum hybrid ‘Casa Blanca’ (A) Direct shoot formation from the basal parts of the leaves at dark condition, (B) Direct shoot formation under a regular 16-h photoperiod after 4 weeks of culture, (C) Rooted plantlet with the formation of scale bulblets.

Table 1. Effect of combining NAA with different BA and TDZ concentrations on shoot regeneration from the basal leaf segments of L. Oriental hybrid ‘Casa Blanca’.

 Leaf position did not influence the shoot regeneration frequency, but significantly affected the number of shoots per segment (Table 2). The lower two parts of the leaves (middle and basal) produced significantly more shoots than the older ones (Upper).

Table 2. Effect of leaf position on shoot regeneration from various kinds of leaf positions in L. Oriental hybrid ‘Casa Blanca’.

 Compared with the other lengths of the leaf segments, the shoot regeneration frequency was much lower for the 0.4-cm leaf segments (Table 3). No differences in the shoot regeneration frequency were found among the segments with lengths ranging from 0.6 to 1.0 cm (Table 3). The number of shoots significantly increased from 2.2 to 3.1 as the length of the leaf segments increased from 0.4 to 0.6, reached the maximum (about 3.6) for the 0.8 cm leaf segments, and then decreased significantly for the 1.0 cm leaf segment.

Table 3. Effect of explant size on shoot regeneration from the basal leaf segments of L. Oriental hybrid ‘Casa Blanca’.

 The light regime significantly influenced the number of shoots, although not the shoot regeneration frequency (Table 4). The dark treatment, applied for either 1 or 2 weeks of the initial culture period, induced root formation (data not shown) and resulted in a significant reduction in shoot number (Table 4). The greatest number of shoots was obtained from the leaf segments cultured under a consistent 16 hrs photoperiod without any initial dark treatment (Fig. 1B).

Table 4. Effect of the duration of dark treatment at the initial stage on shoot regeneration from the basal leaf segments of L. Oriental hybrid ‘Casa Blanca’.

Shoot growth, rooting, and acclimatization

 When transferred to half MS medium supplemented with 2.5mg · L^-1 BA, 0.5mg · L^-1 NAA, and 1 g · L^-1 activated charcoal, the shoots reached a size of 2-3 cm with more than 5 roots after 4 weeks of culturing. Subsequently, they grew up to 6-8 cm in size with many roots at 4 weeks after transfer to a larger culture vessel containing the same media composition (Fig. 1C). Plantlets were then transferred into pots containing vermiculite and peat-based soil (1:2, v/v) with a survival rate of 95%.

Analysis of ploidy level

 To analyze whether the in vitro plants could have ploidy variation, the ploidy level of the mother plant was confirmed first. Typical flow cytometric profiles are shown (Fig. 2A and B), indicating the same ploidy level between the regenerated plants and the mother plants planted in the greenhouse.

Fig. 2. (A) Flow cytometer analysis of 4’, 6-diamidino-2-phenylindole (DAPI)-stained nuclei in the leaves of the control plant, (B) leaves of the in vitro plants regenerated from the basal parts of the leaf segments.

Discussion

 Genotype independent response has been a bottleneck for the wide application of in vitro orgonogenesis of Lilium to micropropagation (Nhut et al. 2001), genetic transformation (Nhut et al. 2001), cryopreservation (Matsumoto et al. 1995), and cryotherapy for virus eradication (Wang and Valkonen 2009). There have been only a few studies on the development of widely applicable protocols for the organogenesis of Lilium. Bacchetta et al. (2003) attempted to develop a shoot regeneration protocol for Lilium and found that direct adventitious shoots could regenerate from in vitro leaf segments of Asiatic hybrid ‘Elite’ and Oriental hybrid ‘Star Gazer’ when cultured on the same medium, but leaf segments of L. longiflorum ‘Snow Queen’ failed to develop any shoots on this medium. When different concentrations of BA and NAA were used, formation of shoots was observed from the leaf explants (basal and middle parts of the leaf). This result was contrary to Wang et al. (2002), who reported that shoots cannot be induced from the leaf segments of L. davidii var. unicolor on media containing different concentrations of BA and NAA, and all the leaf segments died after 45 days of incubation. The shoots in this study always occurred directly from the leaf explants (basal and middle parts of leaf) without forming callus. Ling Fei et al. (2009) previously reported similar results for L. davidii var. unicolor. Loretta et al. (2003) also mentioned direct shoot formation from the middle part of leaf for the lilies ‘Elite’ and ‘Star Gazer’, however, they reported that shoot formation remained restricted to the basal part of the leaf in the lily hybrids but no explanation was provided.

 The explants (the basal parts of the leaves) appeared to be more responsive in media containing lower concentrations of BA alone or in combination with NAA, whereas the addition of NAA to the media showed less frequency of shoot formation.

 Niimi and Onozawa (1979) earlier reported the stimulatory effect of lower concentrations of BA and the inhibitory effect of NAA on shoot formation from the leaf segment in L. rubellum Baker. However, the middle parts of the leaves promoted the highest number of shoots on media containing NAA and BA combinations. A similar result has been reported by Mi and Liu (2008) for plant regeneration from the leaf segment of L. longiflorum hybrid ‘Reizah’. No shoot formation was observed in the apical parts of the leaves, which was similar to the finding of the lily hybrids ‘Star Gazer’, ‘Snow Queen’, ‘Elite’, and ‘Pollyanna’ (Loretta et al. 2003). We discovered that shoot formation was determined by the ratio of auxin and cytokinin, and the position of the explant in leaves.

 An explanation for the differences would be that there might have been relatively different contents of endogenous plant hormones or less organogenic competent along with the various parts of the leaves. Probably, the apical parts of the leaves contained less auxin and cytokinin, so they were difficult to induce shoots. The middle parts of the leaves, which required NAA to initiate shoots, contained less auxin. Because NAA inhibited shoot formation from the basal parts of the leaves, there was more auxin. However, the content of cytokinin contained in the basal and middle parts of the leaves might be same because the basal parts of the leaves induced shoots well enough on media containing 1.0 mg · L^-1 BA alone, while the middle parts of the leaves produced shoots on medium containing 1.5 mg · L^-1 NAA and 1.0 mg · L^-1 BA.

 The effects of BA alone or in combination with NAA on shoot formation from the stem explants (nodal and internodal segments) have been reported in some lilies (Kapoor et al. 2008; Loretta et al. 2003; Nhut 1998). However, Nhut (1998) and Loretta et al. (2003) reported that shoot formation occurred directly from the explants, and no callus formation was observed when BA alone was used. Kapoor et al. (2008) mentioned that direct shoot formation was observed from the internodal and nodal segments cultured on the media with a combination of NAA and BA. In our study, shoots can be induced by neo organogenesis directly or indirectly from the intermodal and nodal segments. The explants induced indirect shoots mostly based on the hormone (auxin/cytokinin) balance in the medium, which might favor shoot initiation through callus formation. The internodal segment required NAA to induce callus when BA alone was not effective for shoot formation, and they produced shoots mainly from callus formation, although direct shoot formation was observed. On the contrary, in the case of the nodal segment, indirect shoot formation was less noted, while direct shoot formation was found on all the media including the PGR-free medium. This result strongly suggests that the nodal segments have more organogenic potential for shoot formation than that of the internodal segments. The possible explanation would be that the nodal segments can induce shoots from both the area adjacent to the region between the stem and leaf, and at the wound of the cut surfaces. Another reason would be that this may be the result of different distributions of endogenous hormones in the stem explants (internodal and nodal explants).

 Aida and Shibita (2002) reported a high frequency of polyploidization in the regenerated plants of Kalanchoe blossfeldiana cv. Tetra Vulcan. Chorabik (2009) also reported the occurrence of somaclonal variation in the embryogenic culture of sliver fri (Abies alba Mill.). In this study, in vitro regenerated plants were genetically stable with respect to the ploidy levels both under in vivo and in vitro conditions without any gross chromosomal abnormalities during regeneration.