Gametosomatic hybridization between egg cell protoplast and mesophyll protoplast of Petunia hybrida

Gametosomatic hybridization has so far been achieved by the fusion between male-gamatic (microspore tetrad or young-stage pollen) protoplasts and somatic protoplasts but no successful results have been reported on the use of female gamete so far. In this paper, we demonstrate for the first time the successful gametosomatic hybridization by using female gametophyte (egg cell) as the gametic-haploid partner instead of male gametophyte based on the method established for isolation of female germ units from ovules in Petunia in our previous report. Using P. hybrida strains. each single protoplast from egg cell and mesophyll cell were manually collected by a In micropump-connected microcapillary and were then fused together as an individual pair by electrofusion. Each heterokaryon thus produced was transferred into Millicell culture plate placed in a Petri dish containing nurse cells, where cell division and microcolony formation took place. Among the fusion combinations tested, only fusants between mesophyll protoplast and egg cell protoplast from the same Petunia strain could regenerate and develop into the complete plants, whereas inter-varietal combinations failed to grow after developing into microcolonies or to regenerate plants. The flowers of a triploid hybrid line (2n=3x=21), confirmed by flow cytometric analysis and chromosome observation, were smaller and shorter than those of the parent with original color, and showed male sterility. These results indicate the intactness of isolated female gametophyte protoplasts, which might enable not only to produce triploid plants with various genomic combinations through gametosomatic hybridization but also to conduct fundamental studies on in vitro fertilization with isolated sperm cell protoplasts.