The study was conducted to obtain basic information for producing somatic hybrid as clarifying factors influencing protoplast isolation and optimal growth regulators for cell division, callus formation from cell colony, and organogenesis in Lycopersicon esculentum × L. pimpinelifolium ev. Tiny Tim.
Results obtained were summarized as follows. Cold treatment at 4℃ for 12 to 14 hours was more effective to high protoplast yield from mesophyll, compared with no cold treatment.
The highest yield and viability of protoplasts from mesophyll cultured in vivo were obtained in 3.0% cellulase and 2.0% macerozyme solution containing 0.4M mannitol as osmoticum.
Protoplast yield and viability were remarkably increased by addition of 0.3% BSA, 0.01M MES and 1.0% PDS to enzyme solution, but protoplast yield was extremely decreased by addition of PVP to above soluton. The optimal pH value was identified as pH 6.3, but protoplast yield was decreased in the higher pH values. The highest protoplast yield was produced by the one step harvest method with 0.6M sucrose after 3 hours´ digestion.
The recommended solution for protoplast isolation from in vitro cultured material was formulated as 1.5% cellulase and 0.3% macerozyme solution for cotyledon and hypocotyl and 2.0% cellulase and 0.5% macerozyme solution for stem tissue, and optimal osmoticum in enzyme solution was 0.3 M sucrose.
Cell division and cell colony formation after plating protoplasts with 5 × 10⁴/㎖ protoplast density were highly stimulated in modified 8p-KM medium containing 0. 3㎎/ℓ 2, 4-D, 1.0㎎/ℓ NAA and 0.5㎎/ℓ BA at 29℃ under 1,000lux, and growth of callus was the best in Murashige-Skoog´s agar solidified medium enriched with 1.0㎎/ℓNAA, 0.2㎎/ℓ 2, 4-D and 0.5㎎/ℓ BA. When transplanted these calli to another medium for organogenesis, organ was not differentiated. While callus with chlorophyll was continuously proliferated in 2.0㎎/ℓ 2ip combined with 0 to 1.5㎎/ℓ IAA or 2.0㎎/ℓ BA combined with 0. 1 to 1.0㎎/ℓ NAA.
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