Characteristics of the Tetraploid Plant Derived as a Somaclonal Variation in Lilium longiflorum

Somaclonal variation that possibly occurs during the process of plant tissue culture is considered to provide a source of new germplasm. Polyploidy is one of the most frequent incidents among the somaclonal variations. In the present study, we performed cytological and morphological characterization of tetraploid plants that were obtained from long-term subcultured callus culture of Lilium longiflorum. Friable green calli of L. longiflorum were induced from in vitro leaves on solidified MS medium supplemented with 30 g L sucrose and 1 mg L picloram and maintained for 3 years by subculturing routinely on the same medium at 30-day intervals. High regeneration ability was observed after transferring the calli to half-strength MS medium without plant growth regulator, and approximately 6.7% of the regenerated plantlets tested were tetraploid (2n=4x=48). Karyotypic analysis of the control plant (a bulblet-derived plant) showed diploid chromosome number (2n=2x=24) with 4-large submetacentric, 12-medium acrocentric, 4-medium telocentric, 2-small acrocentric and 2-small telocentric chromosomes (L4 + M12 + M4 + S2 + S2). Whereas karyotypic pattern of a tetraploid (code no. V18) revealed the completely duplicated set of chromosomes with 8-large submetacentric, 24-medium acrocentric, 8-medium telocentric, 4-small acrocentric and 4-small telocentric chromosomes (L8 + M24 + M8 + S4 + S4). The average number of stomata in leaves of somaclonal tetraploids was less than in control plants, but thicker leaves with greenish color and the alteration of the shape of guard cells were detected in these tetraploids. INTRODUCTION In addition to micropropagation, plant cell and tissue culture has been extensively reviewed as a potential source for somatic variability for use in plant breeding. The process of dedifferentiated callus and suspension cultures could induce chromosomal changes (Syono, 1965). This has been known in terms of somaclonal variation (Larkin and Scowcroft, 1981) that may present alterations in phenotype, physiology, cytology and molecular characteristics from the original plants. On account of the genetic instability of plant cell culture, high possibility of the variations probably occurs in plantlets Proc. IX Intl. Symp. on Flower Bulbs Eds.: H. Okubo, W.B. Miller and G.A. Chastagner Acta Hort. 673, ISHS 2005 168 regenerated from long-term subculturing cell suspension or callus cultures. These are seen as various types of somaclonal variations, especially in chromosomal changes, i.e. polyploidy, aneuploidy, chromosome breakage, deletions, translocation, etc. (Bajaj, 1990). Cytological variations in garlic (Allium sativum L.) resulted in a mixture of tetraploid, aneuploid and mixoploid individuals. These somaclones subsequently produced higher bulb weights than the parental clone (Novák, 1980). Triploid banana (Musa spp.) derived from somaclonal variation were taller, had more and larger fruit, and had deeper color (Vuylsteke et al., 1991). Diploid somaclones of grapevine (Vitis vinifera ‘Podarok Magaracha’) were found to resist fungal pathogens (Botrytis cinerea and Plasmopora viticola) and/or have larger fruits and bunches than the initial plants (Kuksova et al., 1997). Sexual reproduction is also affected, a complete sterility of Pisum sativum was found in somaclones derived from somatic embryogenesis (Griga, 2000). In Easter lily (Lilium longiflorum), a commercial ornamental bulb, Sheridan (1974) reported chromosomal stability of calli-derived plant regenerated after a 7-year callus culture. Priyadarshi and Sen (1992) reported no morphological or chromosomal variations of regenerants from 3-year mass propagation of callus culture in L. longiflorum. However, Supaibulwatana and Mii (1998) reported somaclonal variations in long-term subculturing suspension cultures of friable green calli using flow cytometric analysis. Thereafter, Purwantoro et al. (1999) confirmed a high frequency of somaclonal variation at the DNA level using RAPD (Random Amplified Polymorphic DNA), although no variation was observed by karyotypic and flow cytometric analysis. In this present report, in vitro plantlets of L. longiflorum derived from friable-green calli mained for 3 years were screened for somaclonal variations. The characteristics of the somaclones were confirmed at molecular and cytological (using flow cytometry, squash technique, karyotypic analysis) levels. The anatomy and morphology of somaclones were also observed in comparison to control. MATERIALS AND METHODS In Vitro Culture System of L. longiflorum for Selection of Somaclonal Variations In vitro culture of L. longiflorum ‘Georgia’ was modified according to Supaibulwatana and Mii (1998). Callus was induced and maintained by regular subculture (every 30 days) onto 0.7% (w/v) agar-solidified MS medium (Murashige and Skoog, 1962) containing 1 mg L picloram, 3% (w/v) sucrose and pH 5.7. The cultures were incubated at 25±2°C. Shoot regeneration was induced after 3 years of culture by transferred the calli to regeneration medium (half strength-macronutrient MS medium supplemented with 0.1 mg L NAA, 0.25 mg L BA and 1.5% sucrose). Plantlets with complete shoot/root and bulblet formation were regularly proliferated on MS medium supplemented with 1 mg L NAA, 2 mg L BA and 3% sucrose. Somaclonal variations were then investigated in comparison with control plant obtained from ex vitro bulbscale. Characterization of Somaclonal Variations 1. Cytological Analysis. Flow cytometric (FCM) analysis was performed (Mishiba and Mii, 2000) using a Partec PA cytometerTM. In order to release nuclei, leaf tissue was chopped in solution-A of Plant High Resolution DNA kit type P (Partec GmbH-MunsterTM) in a plastic Petri dish. After incubating the crude nuclei suspension for 5 min at room temperature, staining solution (10 mM Tris, 50 mM sodium citrate, 2 mM MgCl2, 1 % (w/v) PVP, 0.1 % (v/v) Triton X-100 and 2 mg L 4’,6’-diamidino-2-phenylindol (DAPI), pH 7.5) was added and filtered through a 30 μm nylon mesh. After 5 min staining, nuclei suspensions were injected to the cytometer. The peak height can be considered as the relative number of nuclei at each ploidy level. The chromosome numbers and structures were investigated using the squash technique modified from Kurata and Omura (1978). Root tips of regenerated plants were excised prior to immerse into 8-hydroxyquinoline for 14 h, and then treated with absolute ethanol:glacial acetic acid (3:1) for 30 min. The chromosome-fixed root tips were