Sunflower anthers placed on solid medium developed calli and embryos after 12 days. Embryogenesis was improved by the addition of 0.1% polyvinylpyrrolidone (PVP) that alleviated anther and medium browning. As in other species, genotypic variability was an important parameter in the anther response and a medium genotype interaction was suggested with a different PVP effect depending on the genotype. Embryo germination was largely increased by the successive use of germination media with decreasing sucrose concentrations (10% ffi> 6% ffi> 3 %). Histological examination of the anthers during the first ten days of culture showed that, under our conditions, the embryos were of somatic origin, arising directly from the anther wall on the outside or inside of the anther loculus, or indirectly from proliferating anther wall-or connective tissue-derived callus. Finally, the ploidy status of 78 embryo-derived plants was determined by Feulgen stain or flow cytometry : all plants were diploid (2n -34).Abbreviations: PVP -polyvinylpyrrolidone

Previously reported attempts to routinely produce certified doubled haploid sunflower plants were unsuccessful. Isolated microspore culture was assayed in order to overcome the high reactivity of somatic tissues such as anther wall, multicellular hair-type structures, anther connective and parenchymatous vascular bundle. Using filtration or density gradients in liquid medium (N6 supplemented with NAA 1 mg 1-1, BA 0.2 mg I-l and maltose 0.44 M), asymmetrical and symmetrical microspore divisions were obtained. However, contaminating cells or groups of cells coming from hairy type structures were very reactive, and able to develop into calluses. A second purification step was therefore necessary after two days in culture, to isolate a population consisting of highly viable swollen microspores. Using this procedure, besides avoiding somatic contaminants, the microspore response in terms of viability and initial division rate was increased, and sustained division and microcallus formation were achieved after addition of aminocyclopropane carboxylic acid, an ethylene precursor.Abbreviations: ACC -aminocyclopropane carboxylic acid, AOA -aminooxyacetic acid, DAPI -4,6 diamidino-2-phenyl indole

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