Elucidation of the fertilization process of Petunia hybrida. Analysis of the genetical, molecular and cell biological aspects of gamete transfer, recognition and fusion.
The project indicates that in vitro fusion of isolated gametes may become a powerful biotechnological tool in plant breeding for the realization of interspecific hybridization. Results to date are:
messenger ribonucleic acid (mRNAs) were extracted from isolated Petunia embryo sacs and ovules;
cell biological aspects of isolated embryo sacs were studied;
an in vitro culture system for pollen tubes is being developed for the isolation of sperm cells;
the in vitro fusion system is installed and tested;
the testing of gamete specific monoclonal antibodies is organized;
Petunia mutants are collected to be analyzed for fertilization defects;
the bioassay for directed pollen tube growth gametophyte interaction is being developed;
the isolation gametophytic mRNA from isolated embryo sacs for the construction of a complementary deoxyribonucleic acid (cDNA) library which strongly enhances the prospects for successful characterization of gametophyte and gamete specific gene expression.
mRNAs are collected from isolated embryo sacs and sperm cells of Petunia hybrida, in order to construct representative cDNA libraries. From these libraries gamete specific genes will be selected, the expression of which in developing male and female gametophytes will be analyzed by in situ hybridization. Proteins from isolated embryo sacs and sperm cells are used to produce monoclonal antibodies. Gamete specific monoclonals will be selected by immunolabelling, and used for the immunolocalization of gamete specific protein synthesis.
Plasma membrane specific antibodies will be used in in vitro fertilization experiments with isolated egg and sperm cells, to identify membrane bound recognition and fusion proteins. Sperm and egg cell development will be analyzed ultrastructurally to elucidate the cytological aspects of gamete differentiation. This will provide the cytological basis for the analysis of specific gene expression by in situ hybridisation and the analysis of specific protein synthesis by immunolabelling. Fertilization defective mutants of Petunia hybrida will be searched for, to be used as experimental tools to establish possible functions of selected genes and gene products in gamete transfer, recognition and fusion.