The Isolation of Sperm Cells, Their Microinjection into the Egg Apparatus and Methods for Structural Analysis of the Injected Cells

Abstract

This chapter deals with the study of the fertilization process in plants using micromanipulation techniques in both the experimental and the subsequent structural analytical phase of the experiments. Therefore we directly inject isolated sperm cells into egg cells and synergids. The main reasons to develop and use micromanipulation techniques are: firstly, to study the wound reaction of cells after injection with micropipettes of rather wide diameter, secondly to investigate the fate of isolated intra- and interspecific sperm cells after artificial injection into cells of the egg apparatus of intact ovules and thirdly, in case of a succesfull fertilization, obtaining interspecific hybrids. It must be mentioned that the third goal can be more easily reached by isolating not only the sperm cells ( = protoplasts), but also the egg cells, followed by (protoplast-)fusion of both cells (Kranz et al., 1991). However, in the latter case the fusion product ( = mainly the egg cell) has been taken out of the ovule, i.e. its natural environment for fertilization and embryogenesis, so that subsequent ultrastructural studies may reveal phenomena that are more the result of the in vitro situation than when working with intact ovules.