Plant Regeneration from Leaf Mesophyll Protoplasts of Selected Ornamental Nicotiana Species1

Abstract

Shoot tips of Nicotiana aiata Link & Otto, N. forgetiana Sander and N. sanderae W. Wats seedlings were established on modified Murashige and Skoog (MS) salts and vitamins medium to provide leaf material as the cell source for protoplasts. Viable protoplasts (8.0 × 10⁵ to 1.5 × 10⁶/ml per g fresh weight) were enzymatically isolated from N. alata in 2.5% Driselase plus 4.0–4.7% mannitol or sorbitol in cell protoplast wash solution (CPW). An enzyme mixture of 1.0% Driselase, 1.0% Macerozyme R-10, 1.0% Cellulase R-10, 0.5% potassium dextran sulfate and 4.0% mannitol in CPW released 4.0 × 10⁵ to 1.0 × 10⁶ and 7.6 × 10⁵ to 8.5 × 10⁵/ml protoplasts per g leaf tissue respectively from N. forgetiana and N. sanderae. Only 4 of 13 tested culture media, also exhibiting species selectivity, promoted sustained cell division of protoplasts to the macroscopic callus stage in 28-35 days. Optimum plating efficiency ranged from 15 to 37% when the protoplasts were cultured at 5.0 × 10⁴/ml in Cool White light. Plating efficiency did not increase when cultures were placed under Gro-Lux light. Macroscopic callus was readily regenerated to shoots in 2 months on MS medium + 1.0 mg/liter zeatin (Z) or MS + 2.0 mg/liter idoleacetic acid (I A A) + 1.0 mg/liter benzy lamino purine (BA). Rhizogenesis occurred in hormone-free MS medium. Regenerated plants flowered in the greenhouse and exhibited minor variation in flower form and pigmentation.