Summary

Maize is a flowering plant well suited for biological research on reproduction processes. In addition to interesting sexual traits, there is in this species a large amount of data regarding genetics and cytogenetics. The embryo sac and the female germ unit have been characterized, and precise studies have been developed on male gametes. Finally, the time course of double fertilization has recently been determined. Nevertheless, the intimate mechanisms of double fertilization are still unknown. A new way to approach this phenomenon is to develop systems of “in vitro fertilization.” In this chapter, the technical procedures to isolate and manipulate male and female gametes in maize are described. The embryo sac and egg cells are prepared with the help of an enzymatic treatment. Sperm cells or male nuclei from viable pollen grains are released in an acidic medium and selected from a specific layer of a Percoll gradient after centrifugation steps. With these cellular tools, two in vitro methods of fertilization have been developed: (1) spontaneous fusion of male and female gametes in the presence of 5 mM of calcium and (2) microinjection of male nuclei either into the egg or into the central cell of a mature embryo sac. Culture of the artificial zygotes and microinjected embryo sacs is in progress. All these techniques provide a new window for investigating the first steps of fertilization and early embryogenesis at the cellular and molecular levels.

Introduction

Camerarius (1694, reviewed in Ducker and Knox 1985) was the pioneer in plant reproductive biology.