42 Micropropagation of Lilium “Gran cru” by Somatic Embryogenesis F. Fidalgo * • A. Santos Departamento de Botânica, Faculdade de Ciências and Instituto de Biologia Molecular e Celular (IBMC), Universidade do Porto, Rua do Campo Alegre, 823, 4150-180 Porto, Portugal Corresponding author: * ffidalgo@ibmc.up.pt Keywords: Asiatic lily hybrid, bulb-scales, plant regeneration, somatic embryos ABSTRACT An efficient method for regeneration of Lilium Asiatic hybrid “Gran cru” via somatic embryogenesis was developed. For this purpose bulb-scale segments were cultured on a modified Murashige and Skoog medium (Hussey 1982), supplemented with either 2.0 M 2,4- dichlorophenoxyacetic acid (2,4-D) or 2.0 M 4-amino-3,5,6-trichloro-picolinic acid (picloram). Induction of embryogenic callus was obtained at 2.0 M picloram while the auxin 2,4-D failed to induce an embryogenic response. Embryogenic callus predominantly formed from both the basal and middle segments of bulb-scales. Embryo-like structures developed on the embryogenic calli surface. Somatic embryo maturation was achieved when the produced bipolar structures were cultured on similar basic medium devoid of auxin, on which mature embryos developed into normal plantlets. Regenerated plants were transplanted to trays and were successfully acclimatized in glasshouse conditions. 1. INTRODUCTION Lilium is one of about 220 genera belonging to the family Liliaceae, and it is a very important bulbous plant that is cultivated for the production of its attractive and fragrant flowers. Lilium ranks seventh among the cut flowers auctioned in the world (Anonymous 2000), the Asiatic and Oriental hybrids of Lilium largely being traded in the international market. One of the main constraints in conventional propagation of lilies is the low and slow multiplication rate of natural propagation hindering to meet the present market demands. According to Varshney et al. (2000) the conventional method of scaling yields at most 3-5 bulbs from each scale, depending on the species, variety and bulb-scale size. To achieve rapid and large-scale propagation of lilies, in vitro techniques were used and some in vitro culture systems were established. In vitro propagation of Lilium has been reported for several species and Asiatic hybrids, from a range of explants, mainly bulb-scales (Van Aartrijk and Blom-Barnhoorn 1981, Maesato et al. 1994, Niimi 1995, Kim et al. 1996, Varshney et al. 2000, Lian et al. 2003). Recently, somatic embryogenesis from bulb-scales and regeneration of whole plants has been reported for some lily hybrids (Haensch 1996) and also for L. longiflorum (Tribulato et al. 1997, Nhut et al. 2002). The present report describes the induction of somatic embryogenesis on bulb-scales of Lilium Asiatic hybrid “Gran cru” and the regeneration of whole plants. The Asiatic lily hybrid “Gran cru” has beautiful and attractive yellow flowers with a bold red center and pleasant fragrance, characteristics that make this cultivar highly valued in cut-flower commerce. 2. MATERIALS AND METHODS 2.1. Plant material, explant source and disinfection Bulbs of Lilium Asiatic hybrid “Gran cru”, of diameter 4.5-5.0 cm, were obtained from a commercial source (DeLeeuw Flower Bulb B.V., Holland). After removing the roots and damaged outer-scales, the bulbs were carefully washed with soap and rinsed with tap water for 60 min. Healthy scales were isolated from the bulbs and soaked for 3 h in a fungicidal mixture containing 0.3% (v/v) Previcur N (propamocarb 66% a.i. w/w), 0.16% (w/v) Derosal (carbendazim a.i. 60% w/w) and 0.2% (w/v) Rhodax (folpet a.i. 25% w/w and fosetyl 50% a.i. w/w). Bulb-scales were then disinfected with 80% ethanol for 3 min, followed by 20% commercial bleach plus 0.003% (w/v) of Tween 20, for 30 min; finally, bulb-scales were washed several times with sterilized deionised water. Afterwards bulb-scales were cut transversally into apical, middle and basal segments (1x1 cm 2 ). Each segment was cultured with its basipetal cut-surface in contact with the culture medium. 2.2. Nutrient media and culture conditions Bulb-scale segments were cultured on a modified Murashige and Skoog medium (Hussey 1982). This basic medium was supplemented with either 2.0 M 2,4-D (ML1) or 2.0 M picloram (ML2). Both media were adjusted to pH 5.7-5.8 and then 0.6% (w/v) of agar was added before