Project 1: Construction of cell and tissue specific libraries from wheat meiocytes and components of the developing wheat grain

Develop a series of cell- and tissue-specific cDNA libraries for discovery of genes involved in gamete and grain development
Develop an expertise base in micro-cloning for targeted gene identification

cDNA libraries, from the stages from pre-meiotic interphase to late metaphase I for both male and female gametes of wheat and from the early stages of embryo and endosperm development, covering egg and central cell to about five days after fertilisation
Staff trained in the construction of cDNA libraries from micro-dissected cells and able to apply this technology to other plant cells and tissues.

The techniques used for cell preparation varies depending upon the target tissues. Cells at early meiosis are prepared from staged anthers (one anther from a floret is taken for staging and the remaining two are used for meiocyte isolation). For the early stages of endosperm and embryo development, micro-dissection is used to collect cells. The applicants are currently able to isolate embryos and endosperm cells of wheat from about 5 hours after fertilisation. The applicants have also developed systems for the in vitro fertilisation of embryo and endosperm using isolated egg, central and sperm cells. These cells can be taken through development in tissue culture. These techniques are still under development but they would provide a means of obtaining embryo and endosperm from the very first cell divisions to the stages that can be isolated by micro-dissection (5 hours after fertilisation). RNA preparation can be achieved using commercial micro-isolation kits and solid phase cDNA synthesis will be used to minimise losses through precipitation and handling. The cDNA is amplified via PCR and cloned using standard methods. This project will also conduct assessment and evaluation of the libraries, including EST sequencing and database searches.