Donor plants of Hordeum vulgare L. cv. Igri were grown in a conditioned environment to minimise fluctuations in the composition of the microspore population. After isolation different types of microspores were identified within each population, amongst others an embryogenic subpopulation. It was shown that the optimum plating density is achieved by adjusting the density to 2×10(4) embryogenic microspores per ml, with a lower threshold at 5×10(3) per ml. By increasing the osmolality of the pretreatment solution to 440 mOs.kg(-1) and that of the culture medium to 350 mOs.kg(-1), up to 15% of the population developed into embryo-like structures. When microspores of cv. Igri were cultured under the optimized conditions, the ratio of green/albino plants increased from 1∶1 to 34∶1, and 50 green plants per anther were formed.
Intra-nucleosomal cleavage of DNA into fragments of about 200 bp was demonstrated to occur in developing anthers, in which microspores had developed into the mid-late to late uni-nucleate stage in situ, i.e. at the verge of mitosis. The same was observed, but to a much larger extent, if these anthers were pre-treated by a hyper-osmotic shock. Pretreatment of anthers before the actual culture of microspores was required for optimal androgenesis of microspores. The use of the TUNEL reaction, which specifically labels 3' ends of DNA breaks, after intra-nucleosomal cleavage of DNA, revealed that DNA fragmentation mainly occurred in the loculus wall cells, tapetum cells and filament cells. TUNEL staining was absent or infrequently observed in the microspores of developing anthers in situ. Electron microscopy studies showed condensed chromatin in nuclei of loculus wall cells in the developing anthers. These observations at the chromatin and DNA level are known characteristics of programmed cell death, also known as apoptosis. Features of apoptosis were infrequently found in microspores from freshly isolated mature anthers. However, most tapetum cells had disappeared in these anthers and the remaining cell structures showed loss of cellular content. The viability of microspores in pre-treated anthers was comparable to those in freshly isolated anthers and almost four times higher than in anthers from control experiments. This observation was correlated with three to four times less microspores showing TUNEL staining and a two times higher level of ABA in the anther plus medium samples than in controls. Addition of ABA to the controls enhanced the viability and lowered the occurrence of apoptosis linked characteristics in the microspores. These data suggest that pre-treatment is effective in stimulating androgenesis because it leads to an increase in ABA levels which protects microspores from dying by apoptosis.
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