ISSN 1021-4437, Russian Journal of Plant Physiology, 2006, Vol. 53, No. 4, pp. 548–555. © MAIK “Nauka /Interperiodica” (Russia), 2006. Original Russian Text © N.A. Moiseeva, V.N. Serebryakova, L. Nardi, S. Lucretti, R.G. Butenko, 2006, published in Fiziologiya Rastenii, 2006, Vol. 53, No. 4, pp. 612–620. 548 INTRODUCTION Citrus embryogenic cultures hold a special place by their morphological, anatomic, and physiological char- acteristics. Several reports describe Citrus sinensis embryogenic cell lines of different origin, which con- sist of meristematic tissue structured as spherical parti- cles (bodies) associated in clusters [1–3.] Button et al. [1] considered such cell lines to be totally formed by globular proembryoids and distinguished two simulta- neous processes occurring in such cultures. The first is adventitious embryogenesis, i.e., repeated cycles of new globular proembryoid development from the exist- ing ones resulting in the growth of embryogenic cul- tures; the second is direct morphogenesis of globular proembryoids resulting in the development of differen- tiated somatic embryos, i.e., somatic embryos with morphologically distinct cotyledons (designated fur- ther as differentiated embryos). Differently to the majority of embryogenic cultures, in the case of citrus embryogenic cell cultures, the abil- ity to realize these processes is independent on the pres- ence of exogenous growth regulators and is usually kept for many years while culturing on agar medium containing sucrose as carbohydrate source [1, 4]. Stud- ies on the optimization of regeneration conditions have showed that introduction of gibberellin or adenine into nutrient medium exhibited a normalizing effect on the morphogenesis of globular proembryoids [1]. Replace- ment of sucrose with other carbon sources, namely, galactose, lactose, and glycerol, stimulated the process of newforming of spherical bodies [5, 6]. Dunstan et al. [7] concluded that existing protocols for plant regener- ation in citrus tissue culture consist usually of two steps, which, in their opinion, was the main reason for the extremely low frequency of plant regeneration from somatic embryos. It seems natural that, in studies on somatic hybridization between different citrus geno- types, the main problem was not to obtain truly hybrid cells but to regenerate the whole plants [8–10]. In most of the reports on hybrid or transgenic citrus plants, regeneration in vitro was performed by stem morpho- genesis, which usually assumes subsequent plantlet rooting [8] or grafting the plantlets on the fast-growing stocks [9–12]. Obviously, any stage of somatic embryo develop- ment may serve a bottleneck of somatic embryogenesis in vitro, including both initial developmental stages (initial cell–globular-shaped embryoid–heart-shaped embryoid–torpedo-shaped embryoid) and later devel- Organization of Initial Stages of Somatic Embryogenesis in Tissue Culture of Citrus sinensis cv. Tarocco at the Organismal Level N. A. Moiseeva a , V. N. Serebryakova a , L. Nardi b , S. Lucretti b , and R. G. Butenko †a a Timiryazev Institute of Plant Physiology, Russian Academy of Sciences, Botanicheskaya ul. 35, Moscow, 127276 Russia; fax: 7 (495) 977 8018; e-mail: b Italian National Agency for New Technologies, Energy and the Environment, Rome, Italy Received September 7, 2005 Abstract—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 proce- dure 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 sim- ulating somatic embryogenesis by dividing this process into three partially independent steps: PEM glob- ular 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 spher- ical 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. DOI: 10.1134/S1021443706040182 Key words: Citrus sinensis - in vitro - somatic embryogenesis - embryo - developmental stages Abbreviations: CU—cultured unit; MT—Murashige and Tucker nutrient medium; PEM—proembryogenic cell mass. † Deceased.