Stone pine (Pinus pinea L.) is a Mediterranean forest species used for the commercial harvesting of its edible seeds, known as pine nuts. Plant regeneration by somatic embryogenesis on semi-solid medium has been achieved in stone pine, but suspension culture systems for the large-scale production of somatic embryos need to be developed. Suspension cultures were established from several embryogenic lines. Growth parameters as settled cell volume (SCV) and dry weight (DW) in suspension cultures were influenced by genotype, initial inoculum density, orbiting speed and type of carbohydrate in the culture medium. A higher linear growth (DW) up to week four was obtained using 20 mg mL−1 inoculum. The orbiting speed affected the SCV as well as the morphology of embryogenic cultures. Growth parameters decreased when orbiting speed was increased from 50 rpm to 150 rpm. Cultures at 50 rpm most commonly grew as clusters of embryonal cell masses, whereas at 150 rpm there were large numbers of single cells. Cultures grew slowly in medium with maltose instead of sucrose, but growth was not affected when plant growth regulators (PGRs) concentration was decreased. A sustainable suspension culture could be maintained by transferring 250 mg of filtered cell mass to 25 mL of liquid medium at 3-week intervals. Culture scaling-up in liquid medium resulted in a 23-fold DW increase in 6 weeks, compared with 12 fold increase on semi-solid medium. Cotyledonary embryos were obtained on semi-solid maturation medium from lines proliferated in suspension cultures. Increased somatic embryo production was obtained when proliferation was performed at 100 rpm orbiting speed in liquid medium with low PGRs concentration and sucrose instead of maltose.
