Studies of barley and maize indicate that chromosome doubling occurs via nuclear fusion during an early stage of microspore embryogenesis, but the time and mechanism by which chromosome doubling occurs in bread wheat (Triticum aestivum) remains undetermined. The purpose of this study was to determine the relative time during induction culture when chromosome doubling may occur in wheat, and to identify early indicators for doubled haploid microspores. Microspore nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI) and observed under a fluorescent microscope on the day of isolation, three days after isolation, and six days after isolation. The change in the percentage of microspores containing a single small nucleus, two small nuclei, a single enlarged nucleus, and three or more nuclei was then tracked throughout the six-day period. Ploidy levels were estimated by determining the cross-sectional area and number of nucleoli in microspores containing small and large nuclei then comparing the results of each respective celltype. The percentage of microspores containing enlarged nuclei increased throughout the six-day test period, and the percentage of binucleated microspores containing small nuclei decreased. Comparison of the changes in average percentage of microspores containing a single small nucleus, binucleated microspores, microspores containing a single large nucleus, and multinucleate microspores on days 0, 3, and 6 indicates that nuclei classified as "small" are likely haploids and nuclei classified as "large" are doubled haploids. The percentage of microspores with enlarged nucleus (nuclei) during the first six days of induction culture could be used as an early indicator for the frequency of chromosome doubling in wheat microspore culture.

Doubled haploid production via microspore culture is a technique known to accelerate crop breeding by shortening the breeding cycle through achieving homozygosity in one generation. Prior research observed that some embryogenic microspores aborted their development before reaching the embryoid stage. Such embryogenic abortion reduces embryoid yield, making microspore cultures less efficient. The present research aims at identifying stages during which microspore development is susceptible to embryogenic abortion. Information gained through delineation of the developmental dynamics of microspores in culture could be used to improve the efficiency of microspore culture. Embryogenic microspores were isolated from stress-treated wheat (Triticum aestivum L.) tillers and cultured in liquid medium. The development of embryogenic microspores was monitored over a 35 day period. At day 7, 10, 14, 21, 28, and 35, the developing microspores were counted and categorized into multicellular structures, pre-embryoids, immature embryoids and mature embryoids. The results showed that 44% -62% of embryogenic microspores halted their development before the mature embryoid stage. Of these aborted embryogenic microspores, 21% -33% terminated as multicellular structures, 16% -19% arrested their development as pre-embryoids, and 7% -10% halted development as immature embryoids. Identifying factors that are responsible for embryogenic abortion and finding remedy to the issue will help improve the efficiency of doubled haploid production.

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