ISSN 1021-4437, Russian Journal of Plant Physiology, 2010, Vol. 57, No. 5, pp. 720–731. © Pleiades Publishing, Ltd., 2010. Original Russian Text © N.A. Moiseeva, V.N. Serebryakova, L. Nardi, S. Lucretti, A.M. Nosov, 2010, published in Fiziologiya Rastenii, 2010, Vol. 57, No.5, pp. 771–782. 720 INTRODUCTION Investigations of somatic embryogenesis in vitro aim at producing high-quality somatic embryos capa- ble to properly complete their development. Although in vitro somatic embryogenesis in the representatives of the genus Citrus was described long ago, conditions that would ensure mass regeneration of appropriate somatic embryos have not been worked out [1–4]. In many respects, this depends on the fact that realiza- tion of the program of embryonic development in vitro is not synchronous; therefore, the population of somatic embryos thus produced is permanently heter- ogeneous. Such a situation is common in polyembry- onic seeds of citrus plants where numerous somatic embryos develop from the initial cells of nucellus along with sexual embryo [5, 6]. It is generally accepted that because of competition for space and nutrition, devel- opment of the majority of embryos is suppressed at early stages and only some of them become differenti- ated. The mature polyembryonic seeds (MPS) of sweet orange comprise 2 to 9 embryos of various size, with a sexual embryo may be present among them. The size of both nucellar and sexual embryos occurring in MPS of citrus plants depends on the extent of development of their cotyledons and ranges from 2 to 9 mm [7]. A negative correlation was shown between the number and size of the embryos present in one seed: the more numerous are the embryos, the smaller they become RESEARCH PAPERS Morphogenetic Status of Somatic Embryos of Citrus sinensis from Mature Polyembryonic Seeds and Those Produced In Vitro N. A. Moiseeva a , V. N. Serebryakova a , L. Nardi b , S. Lucretti b , and A. M. Nosov a a Timiryazev Institute of Plant Physiology, Russian Academy of Sciences, Botanicheskaya ul. 35, Moscow, 127276 Russia; fax: 7 (495) 977-8018; e-mail: morphogenesis@mail.ru b Italian National Agency for New Technologies, Energy and the Environment, Rome, Italy Received March 3, 2009 Abstract—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 orga- nization, 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 cotyle- dons, 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 characteris- tically 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. Key words: Citrus sinensis, somatic embryogenesis in vitro, polyembryonic seeds, cotyledons, cell number, cell size, ultrastructure, protein bodies. DOI: 10.1134/S102144371005016X Abbreviations: EC1—embryos of class 1 from mature polyembry- onic seeds; EC2—embryos of class 2 from mature polyembryonic seeds; EC3—embryos of class 3 from mature polyembryonic seeds; MPS—mature polyembryonic seeds; SE-in vitro— somatic embryos produced in vitro.