In vitro culture of immature zygotic embryos of white clover [Trifolium repens L.]

On the basis of EC6 medium, specific conditions were established to improve its properties as a nutrient medium for isolated, immature zygotic embryos of white clover. The highest frequency of embryo development occurred on medium with 117 mM sucrose concentration and 30% (v/v) coconut water for globular-stage (23.3%) and heart-stage embryos (78.3%). The development of globular-stage embryos was abnormal, however. The embryos callused after two weeks of culture. From the callus produced, plantlets and then plants were regenerated. A double-layer culture system, with the top layer having higher osmolarity than the bottom layer, enabled proembryos smaller than 60 µm to be cultured. For in ovulo embryo culture, Nitsch medium supplemented with 10% (v/v) cucumber juice proved most suitable. On this medium, approximately 13% of the ovules containing few-celled embryos germinated and produced seedlings.