Isolation, culture, and induction of embryogenesis in protoplasts from cell-suspensions ofAtropa belladonna

Summary

Protoplasts isolated from actively growing cell-suspensions ofAtropa belladonna have been induced to divide repeatedly, and to undergo embryogenesis. An optimal protoplast yield of up to 80% was obtained in 4–5 hours by treating cell-suspensions with an enzyme mixture of cellulase R 10 (1%) and macerozyme R 10 (0.5%) in 0.6 M sorbitol at 30 °C. The protoplasts cultured at a density of 6 · 10⁴/ml in a modifiedMurashige andSkoog's (1962) liquid medium supplemented with NAA (2 mg/l), kinetin (0.1 mg/l) and 0.5 M sorbitol, and incubated in the dark at 28 °C regenerated cell walls within 48 hours. They underwent first division in 3–4 days and formed cell clumps and colonies in 10 days, which when plated on an agar-solidified medium developed into masses of calli. After transfer to an auxin-free liquid medium these calli underwent embryogenesis within the next two weeks and eventually developed into plantlets.