Origin and development of secondary somatic embryos in transformed embryogenic cultures of Medicago sativa

Non-transformed and transformed embryogenic cultures of alfalfa (Medicago sativa L. cv. Zaječarska 83), long-term maintained on growth regulator-free medium, were histologically analyzed. In all examined cultures, somatic embryos at various stages of development were observed and secondary embryos were formed in the cotyledonary, hypocotylary and radicular region of the primary embryos. Detailed histological analysis of the torpedo shape somatic embryo revealed that secondary somatic embryos arose directly from single epidermal cells of hypocotylary axis after an unequal periclinal division. Bipolar proembryos were composed of one smaller cytoplasm rich cell and one larger more vacuolated cell. Further cell division pattern was similar for both non-transformed and transformed embryos. However, multicellular origin of secondary embryos in a direct process and even from callus can not be excluded.