In-situ seed production after pollination with in-vitro-matured,isolated pollen

Immature tobacco (Nicotiana tabacum L.) pollen has been isolated from anthers in three distinct stages of development, including the microspore stage. In in-vitro cultures, fully functional, mature pollen was obtained. In a germination medium, this pollen produced pollen tubes. After application to stigmas in situ, the in-vitro-matured pollen fertilized ovules, and seeds were produced. Genetic tests with seedlings obtained from pollinations with in-vitro-matured pollen from a transgenic plant revealed normal Mendelian segregation of two marker genes, the neomycin-phosphotransferase II gene and the nopaline-synthase gene. These results are of interest with respect to the control of self-incompatibility, cytoplasmic male sterility and pollen-allergen formation, and it offers an alternative route for gene transfer in those plants which cannot be regenerated in vitro.Abbreviations cms cytoplasmic male sterility; AMGLU, MS, M2S, MR26 - GK culture media, see Material and methods