Plant Cell, Tissue and Organ Culture 76: 121–130, 2004. © 2004 Kluwer Academic Publishers. Printed in the Netherlands. 121 Somatic embryogenesis from 20 open-pollinated families of Portuguese plus trees of maritime pine C´ elia Miguel 1,∗ , S´ onia Gonçalves 1 , Susana Tereso 1 , Liliana Marum 1 , João Maroco 2 & M. Margarida Oliveira 1,3 1 Instituto de Biologia Experimental e Tecnol´ ogica (IBET)/Instituto de Tecnologia Qu´ ımica e Biol´ ogica (ITQB), Grupo Pinus, Aptd. 12, 2781-901 Oeiras, Portugal; 2 Grupo de Etstat´ ıstica e Matem´ atica, Instituto Superior de Psicologia Aplicada, Rua Jardim do Tabaco 34, 1149-041 Lisboa, Portugal; 3 Departamento de Biologia Vegetal, Faculdade de Ciências de Lisboa, Bloco C2, Piso 1, Campo Grande, 1749-016 Lisboa, Portugal ( ∗ requests for offprints; Fax: +351-21-442-1161; E-mail: cmiguel@itqb.unl.pt) Received 31 July 2002; accepted in revised form 11 June 2003 Key words: breeding, clonal propagation, conifer, Pinus pinaster Abstract Immature zygotic embryos from 20 open-pollinated (OP) families of maritime pine (Pinus pinaster) plus trees were screened for their somatic embryogenic capacity. The best time for zygotic embryo collection was between 30th June and 16th July 1999 when most embryos were at a pre-cotyledonary stage of development. The somatic em- bryogenesis (SE) initiation frequency was highest on DCR basal medium with 13.6 μM 2,4-dichlorophenoxyacetic acid (2,4-D) and 4.4 μM 6-benzylaminopurine (BAP) supplemented with L-glutamine and casein hydrolysate. On this medium, initiation frequencies among OP families ranged from 4.6 to 49.1%. Initiation of embryogenic cell lines from all 20 OP families was possible only on DCR based medium, but the addition of L-glutamine and casein hydrolysate significantly increased the number of zygotic embryos producing SE. Most families showed a similar behaviour on different initiation media; however, a few exceptions were observed. Further development of somatic embryos on maturation medium, consisting of DCR with 120 μM abscisic acid (ABA), 100 g l −1 polyethylene glycol (PEG) and 10 g l −1 gellan gum, occurred in 29% of 896 embryogenic lines representing all 20 OP families. However, development into cotyledonary somatic embryos was observed in only 11% of the cell lines, but this still represented 18 OP families. Introduction Maritime pine (Pinus pinaster Sol. ex Ait.) covers more than 4 million hectares in southwestern Europe. In Portugal, this species occupies 33% of the forest area (1 million hectares). Due to its economic impor- tance, a pine breeding program integrating biotechno- logy and traditional breeding has been implemented to accelerate the tree improvement process initiated in the 1980s. A set of 60 plus-trees selected in 1964– 1966 from the Leiria Forest (Portugal) represents part of the breeding population for this program. This was the most vigorous provenance of Pinus pinaster under Western Australian conditions (Hopkins and Butcher, 1993). In vitro clonal propagation has the potential for fast multiplication of superior genotypes, allowing the ex- ploitation of maximum genetic gain achieved in the breeding program. When compared to other in vitro propagation methods, somatic embryogenesis (SE) offers several advantages in improving forest trees (Jain, 1999) including the fact that embryogenic tissue is readily cryopreserved. By using SE in conjunc- tion with cryopreservation, it is possible to develop high-value clonal varieties by retrieving from liquid nitrogen those clones that in genetic tests had been the best performers and by subsequently propagating these (Park et al., 1998). Elite clones can then be used for advanced breeding programmes and commercial forestry (Aitken-Christie, 2001).