Computer-Controlled Tracking of Single Cell Development in Nicotiana tabacum L. and Hordeum vulgare L. Protoplasts Embedded in Agarose/Alginate Films

Abstract

Summary Techniques for the identification of protoplast types capable of sustained division in culture are of great interest for workers in the field of plant cell biology. Such information can be of even greater importance for those species where low yields and limited response in culture are observed. Here we describe a new culture technique for embedding protoplasts in thin films of agarose or alginate using a polypropylene mesh as a support. When used in combination with a computer-controlled motorised microscope stage, the culture system allows individually selected protoplasts to be marked and traced, accurately and reproducibly. The application of the technique is illustrated by following the development of mesophyll protoplasts of tobacco and cell suspension protoplasts of barley. In barley we additionally illustrate that using such analyses we can identify subpopulations of protoplasts (based on size and morphology) which show superior response in culture. Additional benefits of agarose/alginate film culture are discussed with reference to improved protoplast culture.