In these initial experiments, field-grown plants of inbred Ohio 43 were used. The tassels of plants at the appropriate stage were heat shocked by maintaining them between 41 C and 44 C in the field for two hours, using a system of plastic jackets containing heated water; a control tassel was harvested from an untreated sibling of the same stage in the same accession. Upon harvest, tassels were fixed immediately in FAA using vacuum infiltration and shaken overnight, then stored in 70% ethanol at -20 C. Spikelets were dehydrated, embedded, and processed for in situ hybridization as in the seedling study.
Based on our initial observations, vigorous and localized shsp
transcript accumulation in cross-sections of heat shocked
spikelets is seen in both early prophase and late prophase/early
uninucleate anthers. The strongest signal is in the dense
cytoplasm of the tapetum, but transcript accumulation also
appears to occur in the developing microsporocytes, and also to
some degree in the immature vascular bundles of the anthers.
Interestingly, when the lower-level developmental accumulation
that exists in untreated material is examined using spikelets from
the control plant, it appears to be localized to the same regions.
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