Summary

The genetic regulation of the development of the female gametophyte and early embryo of flowering plants is an area of considerable scientific interest. Cloning genes that are determinant in megagametogenesis and embryogenesis will be a major challenge for the future.

Using an enzymatic maceration technique, intact and viable embryo sacs have been isolated from mature ovules of Petunia hybrida. Total RNA was prepared and used as a template for the synthesis of cDNA. Due to the small amounts of material obtained, the cDNA was amplified, using the polymerase chain reaction (PCR), prior to cloning into λZAPII.

Using degenerate oligonucleotides, targeting conserved motifs in protein kinases expressed both maternally and zygotically in other organisms, ovule and embryo sac cDNA from P. hybrida was subjected to PCR amplification. cDNA fragments of about 160 bp were amplified and cloned. Two classes of ‘mini-clones’ have been characterised and shown to encode, respectively peptide sequences similar to the subdomains VI, VII and VIII of the protein kinases shaggy/zeste-white 3 and MAP/ERK (mitogen activated protein kinase or extracellular signal-regulated kinase). Probes derived from the above ‘mini-clones’ were used to screen cDNA libraries and several clones have been isolated and are being characterised.

In other systems, MAP and shaggy/zeste-white 3 kinases have been associated respectively with mitotic stimulation and establishment of embryo segment polarity. By analogy it might be possible that the homologous plant kinases are required for developmental processes during megagametogenesis and embryogenesis.