Somatic embryogenesis and plantlet development in Pinus sylvestris and Pinus pinaster on medium with and without growth regulators

To initiate somatic embryogenesis in Pinus sylvestris and Pinus pinaster, i mmature seeds were collected from June to August and the developmental stag e of the zygotic embryos was determined. Four developmental stages were dis tinguished and the response of the zygotic embryos at each of the four deve lopmental stages was compared intra- and inter-species. For this study, mod ified Litvay's medium (LM), with or without growth regulators, was chosen. Somatic embryogenesis was initiated and maintained on both media but the tw o species displayed different propensities. In P, sylvestris, the highest i nitiation frequency was obtained with intact megagametophytes containing em bryos at the four-cell stage to the stage of cleavage polyembryony (up to 2 2 and 9%, respectively). The culture medium had no significant effect on th e initiation and proliferation of embryogenic cultures. In P, pinaster, how ever, the best response occurred from excised zygotic embryos at the stage prior to elongation of cotyledon primordia (up to 40%, explants responded), on medium with growth regulators. Another characteristic distinguishing th e two species in culture was that in some embryogenic cell lines of P, sylv estris, somatic embryos matured spontaneously when initiated and maintained on medium without growth regulators, Some of these embryos developed into plantlets on the same medium at the frequency of 40%, Therefore, in P, sylv estris all the stages of somatic embryogenesis were achieved on the medium without growth regulators. However, in both species, maturation of a large number of somatic embryos was greatly improved on medium containing high co ncentration of gellan gum (Gelrite 10 g l(-1)) and abscisic acid (60 mu M). Cotyledonary somatic embryos subsequently germinated (72 and 80% for P, sy lvestris and P, pinaster, respectively) and developed into plantlets (48 an d 29%, for P, sylvestris and P, pinaster, respectively). This represents a significant improvement in plantlet recovery from somatic embryos of both s pecies.