Cellular and molecular aspects of brassica napus microspore embryogenesis

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Although embryogenesis is an organism's central development process, in plants the early events are difficult to study because they occur within the ovule. It is also a challenge to obtain sufficient quantities of early stage embryo tissue to study the process at cellular and molecular levels. In the last 20 years microspore (immature pollen) culture systems have been developed for several plant species which produce large numbers of embryos that are very similar to zygotic embryos in their composition, morphology and function. In addition, in microspore cultures, all stages of the process are readily accessible, the starting material is single-celled and the culture medium is liquid (and easily modified). For most microspore culture systems the developmental trigger is a simple stress treatment. For example, embryo development in Brassica napus microspore cultures is induced by a mild heat treatment. For these reasons microspore cultures are good model systems for studying the early stages of embryogenesis in plants. In addition, microspore cultures are widely used to produce double haploid populations for plant breeding programs. In the last five years our group has developed flow cytometric assays to identify cells that begin the embryogenic program as soon as 3 days after the heat treatment and we have developed cell sorting procedures to obtain pure populations of embryogenic cells. Our studies have shown that embryogenic cells become basic compared to those that remain in a pollen development pathway and we have identified 100 genes that are upregulated in embryogenic cells. The objective of the proposed work will be to determine how the redirection of development, from pollen maturation to embryogenesis, is initiated and sustained in microspore cultures at cellular and molecular levels. The information gained from the proposed studies will contribute to the basic understanding of the earliest stages of plant embryogenesis and could be applied to increase the responsiveness of genotypes that are currently recalcitrant to microspore culture but are desirable for plant improvement purposes.