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.
The effect of fructose, glucose, maltose and sucrose at various concentrations on Brassica napus embryos was studied. The morphogenetic reaction depended on the type and concentration of sugar in the medium. The frequency of developing embryos was highest on sucrose, followed by maltose and glucose. Fructose did not stimulate embryo growth. Spectrophotometry of autoclaved fructose showed an absorbance rise between 260-320 nm that could correspond to the formation of furfural derivatives. Autoclaving-induced toxicity probably inhibited embryo growth; heart-shaped stage embryos developed on filter-sterilized fructose. The frequency of developing embryos increased with sugar concentration, but normal embryogenesis occurred only on 1% sucrose and maltose; at higher concentrations callus and/or shoots were formed. On media with 6% sucrose and 12% maltose, shoots and somatic embryos were produced.