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A cytological study of barley microspores during pretreatment of the uninucleate stage to the early culture stage was conducted utilizing six genotypes. Among the three main pretreatments investigated, microspores completed the first mitotic division during 28 d cold pretreatment of spikes, with or without leaf sheath attached, and during 0.3 M mannitol pretreatment of anthers at 25 degrees C. However, during a 4 d pretreatment in 0.3 M mannitol at 4 degrees C this first mitotic division was blocked or delayed and subsequently most often occurred during the first day on culture medium. The first mitotic division of most microspores pretreated in 0.3 M mannitol was mostly symmetrical (55-60%), whereas it was asymmetric (94%) during the 28 d cold pretreatment of spikes. Following the first mitotic division during the mannitol pretreatment at 25 degrees C, closely associated daughter nuclei often appeared to fuse via membrane coalescence, leading to a high frequency of large uninucleate microspores. Based upon nuclear size, the frequencies of fused uninucleate microspores in genotypes GBC 778, GBC 777 and Igri were estimated to be 87%, 54% and 75%, respectively, after a 4 d mannitol pretreatment at 25 degrees C. Chromosome numbers in dividing nuclei and relative densitometry measurements of nuclear DNA in microspores from cv. Igri confirmed the apparent fused nature of large nuclei in uninucleate microspores. The high frequency of fused nuclei indicates that nuclear fusion occurred between both symmetric and asymmetric nuclei. Microspores of cv. Igri cultured on filter paper following three different pretreatments provided an average of about 12 000 embryo-like structures (ELS) per plate. In samples, 85-97% of these ELS regenerated green shoots. The frequency of doubled haploids (74-83%) following all pretreatments was similar to the frequencies of fused nuclei. The pretreatment of spikes in 0.3 M mannitol at 4 degrees C for 4 d is preferred as it appears to provide genotype independent induction and suspension of nuclear division, as well as regenerating green plants in a shorter time than cold alone.

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°C; spikes at 4°C for 28 d. Microspores often completed the 1st mitotic nuclear division during pretreatment while cytokinesis was delayed. During mannitol pretreatments, the 1st mitotic nuclear division was mostly symmetrical while only asymmetric 1st 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°C for 4 d, delayed the 1st 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.Key words: wheat, pretreatment, karyokinesis, embryogenesis, microspore, cold, mannitol.

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Herbicide tolerant crops provide farmers access to a new weed control option of nonselective herbicide such as Roundup1 A wheat transgenic event 33391 was produced via Agrobacterium‐mediated transformation of a donor cultivar Bobwhite wheat (Triticum aestivum L.) and was identified as a commercial candidate to develop Roundup Ready wheat2 The objective of this study was to assess field efficacy of the transgenic event in spring wheat production regions in North America. Transgenic event 33391 was tested in field trials at 14 locations in 1999, 13 locations in 2000, and 14 locations in 2001. All trials were split‐plot designs with multiple rates of Roundup treatment. No vegetative or reproductive damage was observed with the application of 4 L ha−1 Roundup at the 3‐ to 5‐leaf stages. No yield reduction was observed with Roundup treatment. The transgenic event with or without Roundup application yielded as high as the nontransgenic Bobwhite. These results indicate that the wheat transgenic event 33391 has at least 2× tolerance to the nonselective herbicide Roundup.