Regeneration of tobacco and petunia plants from protoplasts and culture of corn protoplasts.

Abstract

Protoplasts isolated enzymatically from leaves of tobacco (Nicotiana tabacum), stem callus of petunia (Petunia hybrida) and roots of corn (Zea mays) seedings were grown aseptically in microchambers, liquid droplets, or in stationary suspension cultures. Cell wall regenration occurred within 2 days in petunia, 6 to 7 days in tobacco, and 10 days in corn protoplasts. In petunia protoplasts, cell divisions ensued immediately after cell wall formation, and a colony of 30 to 40 cells was produced in 10 to 15 days after culture. Tobacco protoplasts took a longer time for the initiation of cell division activity and the formation of a cell colony. Plating of cell colonies derived from petunia and tobacco protoplasts, separately, resulted in larger sized cell masses, each of which formed a callus tissue when rransferred to fresh nutrient media. Auxins and cytokinins were essential for the differentiation, of shoots from protoplast-derived callus tissues of petunia and tobacco. Transfer of individual shoots to simpler nutrient media resulted in the formation of adventitious roots. Such plantlets of petunia and tobacco have flowered and set seeds in the greenhouse. Complete abortion of pollen was seen in plants derived from protoplasts of cytoplasmic male sterile plants of petunia. Corn root protoplasts elongated following cell wall regeneration, accumulated a few large lipid globules, and remained viable for over 10 weeks in culture. Cell wall regeneration and a low percentage of cell division were seen in protoplasts isolated from corn leaves. Adjacent protoplasts occasionally fused to form two-nucleate protoplasts.