Microspores were isolated from wheat (Triticum aestivum L.) spikes by means of a micro-blender immediately following their removal from the donor plants. Isolated microspores were then subjected to a chemical treatment consisting of 0.18 mM 2-hydroxynicotinic acid, minerals and 9% maltose in the dark at 25掳C for 38-52 h. Following purification via filtration and gradient centrifugation on 21% maltose, the microspores were cultured in the presence of excised ovaries in liquid medium at 27掳C. Embryoid yield, percentage of green plants, and the frequency of spontaneously doubled haploids ranged from 360 to 4,914 embryoids per spike, 15% to 95%, and 39% to 78%, respectively. Other compounds that were effective in maintaining viability and triggering microspore embryogenesis were benzotriazole-5-carboxylic acid and violuric acid monohydrate. This system has proven to be highly efficient for producing doubled haploids over a range of genotypes.
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