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In tissue culture of sweet orange (Citrus sinensis (L.) Osbeck, cv. Tarocco), we obtained mass regeneration of somatic embryos with two morphologically distinct cotyledons about 3 mm in length, their numbers amounting to 110–150 embryos per petri dish and 60 to 80% of the population. The morphogenetic state of somatic embryos was compared using the embryos with the cotyledons of different size (from 3 to 10 mm) from mature polyembryonic seeds as a test system and the cell number, size, and ultrastructural organization, and the number of protein bodies in the cotyledon cells as morphological and biochemical criteria. Cell number in the cotyledons of different size was related to the content of protein bodies therein. Typical protein bodies where 33 kD polypeptide of storage proteins was identified were detected only in the cotyledons, which size was identical to that of embryonic cotyledons from monoembryonic seeds of citrus plants. In the cells of smaller cotyledons, we detected protein-accumulating vacuoles with electron-dense inclusions that irrespective of their size, shape and structure accumulated the gold label. The number of the cells with protein depositions in vacuoles decreased when the cotyledons became smaller. Irrespective of the origin of embryos (in vivo or in vitro), lipids were the major storage metabolites in the cells of 3-mm cotyledons. As the cotyledon-forming cells became smaller and less numerous, their metabolic activity tended to decrease in line with the fragmentation of endoplasmic reticulum, the absence of polysomic complexes, and indistinct inner organization of mitochondria and plastids. It was concluded that somatic embryos developing in vivo and in vitro were physiological dwarfs, that is, the structures with diminutive storage organ with characteristically incomplete cell differentiation. Apparently such forms emerged due to the suppression of cotyledon growth at the initial stages of their organogenesis; as a result, the cell population could not properly realize both organo- and histogenesis.

Four-step protocol was established for the in vitro regeneration of Citrus sinensis cv. Tarocco somatic embryos that were morphologically similar to small somatic embryos in vivo. The regeneration procedure comprises a mechanical destruction of embryogenic culture to obtain proembryogenic cell masses (PEMs) (step 1) followed by culturing on three different media (steps 2–4). The approach developed allows in vitro simulating somatic embryogenesis by dividing this process into three partially independent steps: PEM → globular somatic embryo → heart-shaped somatic embryo → somatic embryo with developed cotyledons. The highest frequency of morphogenetic stage transition was 64, 40, and 26%, respectively. It was shown that the first step (PEM → globular embryo) was associated with the formation of heterogeneous population of spherical bodies 50–500 μm in diameter, among which about 40% were somatic embryos at globular stage. The scheme is offered of alternative pathways for the development of spherical bodies in vitro, and interrelations between their sizes and ability to direct morphogenesis are discussed.