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 development from the gametophytic pollen pathway to a sporophytic pathway, resulting in embryoid or callus formation. The influence of cold or mannitol pretreatment on karyokinesis and cytokinesis in isolated microspore culture responses were investigated. Anthers were pretreated in mannitol for 7 d at 28 degrees C; spikes at 4 degrees C for 28 d. Microspores often completed the Ist mitotic nuclear division during pretreatment while cytokinesis was delayed. During mannitol pretreatments, the 1st mitotic nuclear division was mostly symmetrical while only asymmetric Ist nuclear divisions were seen during or after cold pretreatment. Following the symmetrical division, the two similar nuclei often appeared to fuse to form a diploid nucleus. Subsequently, these nuclei underwent rapid nuclear divisions to form multinucleate, and later, multicellular structures in induction medium. Cold pretreatments also induced muticellular structures but frequencies were lower than after mannitol. A novel pretreatment of spikes, combining 0.4 M mannitol solution at 4 degrees C for 4 d, delayed the Ist nuclear division, keeping all microspores 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. Ninety-five percent of the plantlets transferred from culture to soil survived. The improved pretreatment enhanced the potential of isolated microspore culture in wheat for plant breeding by producing large numbers of plants and for gene transformation by maintaining a uniform population of haploid uni-nucleate stage microspores as targets.