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For half a century, the limitations of obtaining cross-combinations in lilies because of the incompatibility and incongruity between different varieties have been known. Somatic hybridization is one of the most powerful tools for achieving distant interspecific hybrids. For this purpose, protoplast preparation is a first and important step in efficient system for the regeneration of plants from protoplasts. Protoplast isolation method was previously developed in Lilium ledebourii (Baker) Boiss. In this study, several valuable experiments were done based on completely randomized design with 3 replications and also each experiment was repeated twice. The results revealed that cell wall and colony formation were better in a liquid medium than those on a semi-solid medium. The highest plating efficiency (1.34×10⁶ per gr FW) and callus formation was obtained by using a medium containing 1 mg L^-1 2,4-D, 0.2 mg L^-1 Kin and 2 g L^-1 Yeast extract. Micro calli were formed after one month of culture. Many plantlets were formed on the calli after transfer of the proliferated calli to regeneration medium. The highest plantlet regeneration (91.66%) was obtained by using a medium containing 0.5mg L^-1 NAA, 1.5 mg L^-1 BA. Means comparison revealed that the semi- solid MS medium containing 0.5 mg L^-1 NAA and 1.5 mg L^-1 BA had the highest percentage of regeneration (91.66%), bulb number (8.83), and length (0.7366 cm), root length (0.421cm) and leaf number (13.66) and length (0.5052cm).

Keywords: Callus formation, In-vitro culture, medium, Proliferated calli

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