Kinetics of Douglas-fir (Pseudotsuga menziesii) somatic embryo development

Douglas-fir (Pseudotsuga menziesii (Mirb.) Franco) is one of the most economically important softwood species in the Pacific Northwest region. Somatic embryogenesis is a potential mass propagation technology for increasing the productivity of existing forest acreage. Combined with traditional breeding methods and recent advances of genetic engineering in plant species, somatic embryos can shorten the elite clone selection process significantly. Somatic embryo culture of Douglas-fir involves three stages: maintenance, abscisic acid (ABA) singulation, and maturation. At the beginning of all stages of culture, the population of cells with embryogenic potential is increased through the weekly subcultured maintenance stage; transfer into the ABA singulation stage initiates embryo development, while cotyledonary embryos are formed in the maturation state. The first two stages were carried out in submerged suspension culture, while during the maturation stage the developing embryos were placed on a polyester pad in a Petri dish. The growth kinetics in these stages were investigated. Fresh and dry weights were observed to double in the maintenance stage, while a smaller increase occurred in the ABA singulation stage. NH4⁺ was consumed preferentially to NO₃^- in all culture stages. Sucrose, the primary carbon source, was hydrolyzed to glucose and fructose rapidly. During cultivation, glucose and fructose were consumed simultaneously. The hydrolysis of sucrose resulted in a slight osmolarity increase at the beginning of all culture stages, while the subsequent consumption of glucose and fructose coincided with a gradual decrease in osmolarity. This dynamic osmolarity pressure profile is most profound in the maturation stage, in which the initial high osmotic pressure of 600 ± 20 mOsm/kg (mean ± SD) increased to 700 ± 27 mOsm/kg after sucrose was hydrolyzed but decreased to 350 ± 14 mOsm/kg after the depletion of sugars at the end of cultivation. The complete process of embryo development, from the week-long maintenance culture, through the weekly subcultured ABA singulation culture, to the maturation of embryos took between 70 and 80 days. Each millilitre of culture present at the onset of maintenance culture gave rise to approximately 420 mature embryos. During that same time period, the biomass increased approximately 100 times. Prolonging the cultivation time failed to increase the yield of mature embryos. These results give a more complete view of the kinetic behavior of developing Douglas-fir embryos and will aid in the optimization and scale-up of this important process.