Towards in vitro fertilization, gametosomatic cybridization and DNA transfer in perennial ryegrass (Lolium perenne L.)

In this thesis, research towards invitro fertilization, gametosomatic cybridization andDNAtransfer in perennial ryegrass ( Loliumperenne L.), the most important forage grass of North West Europe is described. Techniques for in vitro fertilization and gametosomatic cybridization were aimed at developing a new regeneration technique allowingDNAmodification of perennial ryegrass byDNAtransfer or by transfer of cytoplasmic traits. For this, isolation and storage techniques for sperm cells and egg cells were developed. The isolated sperm cells proved to be haploid and exhibited a moderate cytological variability. They contain only a small number ofDNAcontaining organelles, which renders these cells suitable as nucleus donors in cybridization experiments. The morphology of isolated egg cells was comparable to the morphology of egg cells in situ of other monocotyledons and angiosperms in general. The most likely option to obtain in vitro fertilization and gametosomatic cybridization between, respectively, egg cells and sperm cells and sperm cells and cytoplasts, will be one to one fusion under microfusion conditions. Furthermore, to analyze whether promoters of rice genes are adequate for transgene expression in perennial ryegrass a method for direct gene transfer was developed. Stable transformation was obtained using biolistic bombardment of a non embryogenic cell suspension culture. The constitutive promoter of the rice gene GOS2 proved suitable for expression of a transgene in perennial ryegrass. For production of transgenic plant material in the near future, biolistic bombardment of an embryogenic cell suspension or of other regenerative tissues, will be a more realistic option than via in vitro fertilization as regeneration technique inDNAtransfer experiments. The performed research has created a broad basis for further research aimed at both cybridization, using sperm cells as nucleus donors, and direct gene transfer in perennial ryegrass, as well as at topics such as gamete interaction, zygote formation and early embryogenesis of higher plants at the cellular and molecular level.