Somatic proembryo formation and transient expression of a luciferase gene in Douglas fir and loblolly pine protoplasts

Somatic pro-embryos were regenerated from morphogenic protoplasts of cell suspension cultures of Douglas fir and loblolly pine. Morphogenic protoplasts were obtained from suspension cultures of embryonal cells grown in a modified basal medium high in myo-inositol. After 11–12 weeks, microcolonies of embryonal-suspensor masses (ESMs) were recovered from agarose-embedded protoplasts. Somatic pro-embryos were regenerated after 18–20 weeks by somatic polyembryogenesis from new ESMs on agar plates. The luciferase (luc) gene was successfully introduced into fir and pine protoplasts by electroporation. While viability of protoplasts was reduced from 90% to 45–55% by electroporation, the transient expression of the luc gene was detected in protoplasts surviving 36 h after electroporation. Gene expression was improved by the addition of polythylene glycol (PEG) to the electroporation mixture.