In vitro culture of isolated living sunflower (Helianthus annuus L.) embryo sacs

Ovules of sunflower tubular florets were observed histologically by serial sectioning and clearing to study the correlation between flower morphology and developmental stage. On the basis of these data, florets with visible stigma and receptive "curling" surfaces were chosen for embryo sac (ES) isolation. In such florets, similar to20% of the ovules were unfertilized; fertilized zygote-stage ES (similar to75% of ovules) or ES containing two-celled proembryo occurred in the remaining ovules. ES were dissected manually using needles under a stereomicroscope or were treated with enzymes for 4-5 h after manual isolation. The viability of ES isolated without enzymes was more than 90% as assessed by fluorescein diacetate staining, and decreased to similar to3% after 72 h of culture. The use of enzymes during isolation resulted in diminished ES viability, suggesting that removal of the protective layers of ovular cells may be part of the problem. Another factor affecting ES viability is medium osmolality. Living ES were then cultured in vitro. On liquid medium containing 9% sucrose, globular embryos developed in similar to10% of zygote-stage ES, but further growth of the embryos was abnormal and callus was produced. Transfer of embryo-derived callus to solid Murashige-Skoog medium supplemented with 1-naphtaleneacetic acid and kinetin resulted in organogenesis. When ES were co-cultured with androgenetic microspores and microspore-derived embryos of Brassica napus, similar to11% of the ES showed growth and development. Embryological study of the cultured ES revealed outgrowth of endothelium.