Summary

The ability to isolate angiosperm gametes has opened new experimental avenues, including in vitro fertilization. The use of biotechnological methods such as micromanipulation and single cell culture has led to the technique of in vitro fertilization at the single cell level. In this chapter, a description is given of (a) in vitro fusion techniques using single isolated egg and sperm cell protoplasts of maize and (b) the subsequent development of the fusion product, the zygote, in individual culture. The electrofusion of the gametic protoplasts leads to zygotic embryogenesis and fertile hybrid plants. Furthermore, a nonelectrical alternative technique to fuse isolated higher plant gametes using a fusiogenic medium is given and its relevance to studies of adhesion, recognition, and fusion of these gametes is discussed. The experimental experiences obtained are so far limited to maize. These micromanipulation techniques and their possible application for fundamental and applied studies are described.

Introduction

Biotechnological methods have been applied in addition to sexual crossings in breeding programs for a number of years. For example, cell and tissue culture techniques such as somatic cell genetics, anther and microspore culture, and methods of ovule culture are extensively used. Since the first successful in vitro pollination/fertilization of excised ovules with mature pollen of Papaver somniferum L. was performed (Kanta et al. 1962), techniques of embryo rescue (Stewart 1981) and in vitro pollination/fertilization of flower explants, ovaries, and ovules have been used to overcome cases of self- and cross-incompatibility (for example, Rangaswamy and Shivanna 1967, 1971; Rangaswamy 1977; Zenkteler 1990, 1992). Meiotic and pollen tetrad protoplasts were used in fusion experiments (Ito and Maeda 1973; Deka et al. 1977).