Relative DNA Content of Fused Protoplasts and Colony Cells in Sesamum indicum

Abstract

Protoplasts were isolated from hypocotyl tissues of seedlings of cultivated sesame, Sesamum indicum (2n=26). The isolation medium used was CPW inorganic salts solution containing 2.0% Cellulase Onozuka R-10, 0.4% Macerozyme R-10, 0.5% potassium dextran sulfate and 0.7 M mannitol at pH 5.7 with 2hr reciprocal shaking at 25°C.
The 95%. of freshly isolated protoplasts were regenerated the cell wall within 72hr and began to divide in modified MS medium containing 5g/l agar, 2g/l casein hydrolysate, 20g/l sucrose, 109g/l mannitol, 0.3mg/l NAA and 3.0 mg/l BA after 5 days of culture. The some of divided protoplasts developed to colonies of about 1 mm in diameter within 5 weeks of culture in 1% of plating efficiency.
Relative DNA content of nucleus of both protoplasts and colonies was determined microspectrophotometrically with DAPI staining. After 24 hr of culture, the most of the nuclei in protoplasts showed to 2C and 4C in their DNA content. From 3 days of culture, various DNA content of nuclei was observed showing nuclear fusion and DNA synthesis. After 5 weeks of culture, the 84.4% of developed colonies indicated the normal diploid level of nuclear DNA content, whereas the 7.4% indicated to be tetraploid nuclear DNA content which may probably caused on nuclear fusion in the protoplasts, remainder 4.2% indicated the abnormal heteroploidal DNA content between 2C and 4C.