Euphytica 123: 49–56, 2002. © 2002 Kluwer Academic Publishers. Printed in the Netherlands. 49 Colchicine, trifluralin, and oryzalin promoted development of somatic embryos in Ilex paraguariensis (Aquifoliaceae) H.Y. Rey 1,∗ , P.A. Sansberro 1 , M.M. Collavino 1 , J.R. Daviña 2 , A.M. Gonz` alez 1 & L.A. Mroginski 1 1 IBONE, Facultad de Ciencias Agrarias (UNNE), C.C. 209 (3400) Corrientes, Argentina; 2 Facultad de Ciencias Exactas Qu´ ımicas y Naturales (UNaM), Rivadavia 2370, (3300) Posadas, (Misiones) Argentina; ( ∗ author for correspondence) Received 23 February 2000; accepted 22 February 2001 Key words: antimicrotubule agents, embryo culture, plant regeneration, ploidy Summary A protocol for somatic embryogenesis and plant regeneration of Ilex paraguariensis St. Hil. from embryos cultures was developed. Heart stage zygotic embryos were removed from seeds of immature, light green fruit and treated with antimicrotubule agents (0.1; 0.2, and 0.5% colchicine for 24 and 48 h; 1; 10, and 20 μM of either trifluralin, ααα- trifluoro- 2,6-dinitro-N,N- dipropyl-p-toluidine, or oryzalin, 3,5-dinitro-N 4 , N-dipropylsulphate during 48 h). The embryos were cultured aseptically on quarter-strength Murashige and Skoog medium containing 3% sucrose, 0.65% agar (1/4MS), and 0.46 μM zeatin. Cultures were incubated in darkness at 27 ± 2 ◦ C. All the treatments provoked a diminution of the number of germinated embryos and in some of the treated embryos somatic embryo- genesis was induced. Somatic embryo maturation and conversion into whole plants could be achieved by culturing the embryos on 1/4MS lacking hormones and incubated at 27 ± 2 ◦ C, 14 h photoperiod (116 μmol m -2 s -1 ). Most of the plants regenerated from somatic embryos appeared morphologically normal and grew under greenhouse conditions. Only 2 plants out of 152 studied contained the tetraploid number of the chromosomes (2n = 4x = 80), meanwhile the rest of the plants had the normal diploid number of chromosomes (2n = 2x = 40). Somatic embryos with abnormal morphology were also observed. Introduction ‘Maté’ or ‘Yerba Mate’ (Ilex paraguariensis St. Hil.) is a South American tree belonging to the Aquifoli- aceae family. The leaves are used to prepare a stimu- lating beverage very appreciated in some regions of Uruguay, Argentina, Brazil and Paraguay (Giberti, 1995). Plant tissue culture of I. paraguariensis permits plant regeneration through in vitro culture of both, nodal segments (Rey & Mroginski, 1988; Sansberro et al., 1999) and immature embryos (Sansberro et al., 1998). However, plant regeneration via somatic embryogenesis han not been accomplished. Somatic embryogenesis is important to the genetic improvement of plants using biotechnology for two major reasons. First, this system offers the capabil- ity to produce unlimited propagules and second, the embryogenic system could be used for genetic trans- formation. The importance of medium composition, especially growth regulators, in callus induction and plant regeneration through somatic embryogenesis has been reported in many plant species (Ammirato, 1989; Dunstan et al., 1995). Treatments with some antimicrotubule agents, such as colchicine, trifluralin and oryzalin have been widely employed in order to induce chromosome doubling of several plant species by using various in vitro systems such as, anther (Alemanno & Guider- doni, 1994; Redha et al., 1998), isolated microspore (Hansen & Andersen, 1998a,b; Zhao et al., 1996), ovule (Hansen et al., 1998), callus (Song et al., 1997) and cell suspension cultures (Hunault, 1997). In some