In Vitro Cell. Dev. Biol.ÐPlant 37:190±198, March±April 2001 DOI: 10.1079/IVP2000119 q 2001 Society for In Vitro Biology 1054-5476/01 $10.0010.00 QUERCUS SUBER L. SOMATIC EMBRYO GERMINATION AND PLANT CONVERSION: PRETREATMENTS AND GERMINATION CONDITIONS GEMMA GARCI  A-MARTI  N 1 *, M. ELENA GONZA  LEZ-BENITO 2 ², and J. A. MANZANERA 3 1 Instituto Madrilen Ä o de Investigacio Ân Agraria, AutovõÂa de Arago Ân Km.36, Aptdo.Correos 127, Alcala  de Henares, Madrid 28800, Spain 2 Departamento de BiologõÂa Vegetal, Escuela Universitaria de IngenierõÂa Te Âcnica Agrõ Âcola, Ciudad Universitaria, 28040 Madrid, Spain 3 Departamento de Silvopascicultura, Universidad Polite Âcnica de Madrid, Ciudad Universitaria, 28040 Madrid, Spain (Received 11 April 2000; accepted 31 July 2000; editor T. A. Thorpe) Summary Osmoticum (high sucrose concentration) and chilling (48C) treatments were assayed in order to promote germination (radicle elongation) and plant conversion of Quercus suber somatic embryos. Those treatments were applied before embryos were set to germinate on Murashige and Skoog (MS) liquid medium. Chilling for 2 mo. improved germination rate (best results close to 100%). On the other hand, that treatment increased plant conversion, especially when high sucrose concentration was used. The best results were obtained with 150 g l 21 sucrose (75% of cultured embryos converted to plants). Incubation conditions during germination were also studied. Although there were no significant differences on final results, warmer temperatures and darkness increased germination rate. The influence of the different treatments on shoot morphology was also studied. The highest percentages of shoots with normal leaves obtained were close to 25%. Key words: chilling; cork oak; plantlet; priming; in vitro. Introduction Somatic embryogenesis has been studied in several species of the genus Quercus. Gingas and Lineberger (1989) induced somatic embryogenesis without growth regulators in Q. robur and Q. alba zygotic embryos. For maturation purposes they used osmotica obtaining only 5% shoot elongation and with an increased mortality. Q. robur somatic embryos were also obtained from immature zygotic embryos (Chalupa, 1990). Seedling development was low, despite the use of cytokinins in the medium. Quercus suber somatic embryogenesis has been achieved from immature tissues and anthers (Bueno et al., 1992, 1997; Manzanera et al., 1993). In the latter report, radicle elongation was promoted by chilling and epicotyl elongation by cytokinins. Cork oak somatic embryo radicles elongated to a high percentage (Bueno et al., 1992). However, plumule elongation percentage was quite low. Besides, many of those plantlets showed abnormal morphology. Some showed cotyledon-like leaves, which could indicate the co-existence of maturation and germination processes (Finkelstein and Crouch, 1984). Adequate plantlet development (plant conversion) should be achieved in a relatively high embryo percentage and synchronously, to truly apply this micropropagation system to plant improvement programs, together with other biotechnology procedures in cork oak. Seed priming (the incubation of seeds in high osmotic potential solutions prior to germination) has been successfully applied to some species to synchronize and reduce germination time (Bradford, 1986). Although the use of an osmoticum (sorbitol) has been tested with cork oak somatic embryos without success (Manzanera et al., 1993), this procedure has been further studied in the present work using sucrose. Priming agent, treatment duration and water potential could be of great importance for the final results (Frett et al., 1991). Osmotica and temperatures under the required minimum to trigger radicle elongation can accelerate that process after seeds are incubated in adequate conditions (Tarquis and Bradford, 1992). Cold treatments (chilling) to seeds and somatic embryos improved germination in several species (Takeno et al., 1983; Deng and Cornu, 1992), including Q. suber, as has been stated before (Bueno et al., 1992; Manzanera et al., 1993; Ferna Ândez-Guijarro et al., 1995). Light and temperature during germination can also influence germination rate and final percentages. Darkness favored Q. robur acorns and Picea sp. somatic embryo germination (Attree et al., 1990; Finch-Savage and Clay, 1994). Sucrose concentration in germination medium could affect germination and plumule elongation of somatic embryos, as has been demonstrated in Picea sp. (Flinn et al., 1993). Low concentrations could induce an autotrophic metabolism and favor normal shoot morphology. It was considered important to determine if cork oak somatic embryos were capable of germinating in a sucrose-free culture medium, thus increasing the possibility of direct embryo germination in ex vitro conditions. This paper shows the results of different studies carried out in order to increase Quercus suber somatic embryo germination and appropriate plant conversion. Treatments were applied prior and at the time of setting embryos to germinate. The effect of sucrose 190 *Present address: Instituto Tecnolo Âgico de Desarrollo Agrario, Ronda de Atocha no. 17, Madrid 28012, Spain. ²To whom correspondence should be addressed: Email egonzalez@ agricolas.upm.es