MICRONUCLEATION IN DEVELOPING SPATHIPHYLLUM WALLISII REGEL MICROSPORES FOR MICROPROTOPLAST PREPARATION

Abstract

Fusion of protoplasts and microprotoplasts is a known method for producing asymmetric somatic hybrids. One prerequisite for microprotoplast induction is use of an efficient tool to form micronuclei in somatic or gametic cells. We chose to use pollen mother cells as starting material. Preliminary results indicated that micronucleation is restricted to a particular flower stage. Dissected full anthers of genotype 6526 of Spathiphyllum wallisii were treated in a culture room with different concentrations of oryzalin, a spindle toxin, at concentrations of 10, 20, 50 and 100 µM in half-strength liquid MS media. Observations of pollen in various developmental stages were made at 24, 48, 72 and 96-hour intervals after treatment. The observations were made using a fluorescent microscope after staining the anthers with 4' 6-diamidino-2-phenylindole (DAPI). The micro-nucleation index (percentage of cells with additional micronuclei) was measured. The number of nuclei per cell varied between 1 and 9. This fragmentation was observed in pollen mother cells, dyads, and tetrads as well as in mature pollens. At a concentration of 100 µM oryzalin, formation of mature pollen was significantly inhibited. The highest micro-nucleation index (0.93) was observed after 72 h culture of anthers treated with 10 µM oryzalin. The lowest micro-nucleation index (0.13) was observed 24 h after treatment with 100 µM oryzalin. These results can lead to development of protocols for obtaining protoplasts containing 1 to a few chromosomes for microprotoplast mediated chromosome transfer (Doherty and Fisher, 2003). This research may form the basis for introgression of chromosomes between Araceae species.