A cytological study of pretreatments used to improve isolated microspore cultures of wheat (Triticum aestivum L.) cv. Chris

Isolated microspores of wheat can be induced in vitro to switch their devel opment from the gametophytic pollen pathway to a sporophytic pathway, resul ting in embryoid or callus formation. The influence of cold or mannitol pre treatment on karyokinesis and cytokinesis in isolated microspore culture re sponses were investigated. Anthers were pretreated in mannitol for 7 d at 2 8 degrees C; spikes at 4 degrees C for 28 d. Microspores often completed th e Ist mitotic nuclear division during pretreatment while cytokinesis was de layed. During mannitol pretreatments, the 1st mitotic nuclear division was mostly symmetrical while only asymmetric Ist nuclear divisions were seen du ring or after cold pretreatment. Following the symmetrical division, the tw o similar nuclei often appeared to fuse to form a diploid nucleus. Subseque ntly, these nuclei underwent rapid nuclear divisions to form multinucleate, and later, multicellular structures in induction medium. Cold pretreatment s also induced muticellular structures but frequencies were lower than afte r mannitol. A novel pretreatment of spikes, combining 0.4 M mannitol soluti on at 4 degrees C for 4 d, delayed the Ist nuclear division, keeping all mi crospores in a haploid uni-nucleate stage and resulted in higher induction frequencies. The proportion of embryos larger than 2 mm that developed into green plants was as high as 70% when transferred to regeneration media. Ni nety-five percent of the plantlets transferred from culture to soil survive d. The improved pretreatment enhanced the potential of isolated microspore culture in wheat for plant breeding by producing large numbers of plants an d for gene transformation by maintaining a uniform population of haploid un i-nucleate stage microspores as targets.