Investigation of gene expression during plant gametogenesis by in situ hybridisation

Summary

Introduction

The study of the genes responsible for pollen and ovule development has long been frustrated by the fact that the cells concerned are embedded deep in the sporophytic tissue. The situation is further complicated by their investment by actively transcribing nurse tissues – the tapetum and nucellus. In situ hybridisation thus represents the only approach by which gene expression may be studied in these systems. However, the resolution offered by isotopic methods is insufficient to distinguish between members of the microscope tetrad, or individual cells of the developing embryosac. For this reason non-isotopic techniques have been investigated and a protocol devised for anthers. Enzymic detection systems can be used, but high endogenous levels of phosphatase makes blocking and other pretreatments often essential. Further, the methods normally employed to fix and embed material for in situ work do not give good results with reproductive cells, principally because of their small size and fragility. The use of resins as embedding media has therefore been explored.

The problems posed by plant reproductive systems

The cells involved in male and female gametogenesis in higher plants are unusual in a number of ways. Firstly, they are comparatively few in number and are surrounded by other tissues carrying out radically different functions. Secondly, they undergo a complex programme of differentiation and division leading to the formation of a number of highly differentiated daughter cells. Thirdly, this development takes place comparatively rapidly, with the cells passing from stage to stage in hours, rather than days as is normally the case for plants.