Protoplast-to-plant regeneration of American elm (Ulmus americana)

This study describes a protocol for regeneration of plants from cell suspension-derived protoplasts of American elm (Ulmus americana). Efficient protoplast isolation was achieved from a two-phase culture system through the incorporation of 100 mu M 2-aminoindan-2-phosphonic acid, with a yield of approximately 2 x 10(6) protoplasts/ml packed cell volume. Isolated protoplasts failed to survive in liquid or alginate bead culture systems but initiated and continued to divide when embedded in low melting point agarose beads. Protoplast-derived callus proliferated and differentiated into shoot buds in response to 10 or 20 mu M thidiazuron. Differentiated buds elongated and continued to proliferate on elm shoot medium supplemented with 3.0 mu M GA(3). The protoplast-derived shoots rooted and acclimatized to greenhouse conditions and continued to grow. This system provides the first protoplast-to-plant regeneration system for American elm and provides a framework for the development of protoplast fusion or genome editing technologies.