STABLE TRANSFORMATION OF PICEA-GLAUCA BY PARTICLE-ACCELERATION

By defining the somatic embryo developmental stage which expressed beta-glucuronidase (GUS) at a high level yet was also competent to form embryogenic callus at a high frequency under selection, we obtained transformed Picea glauca (white spruce) embryogenic callus, embryos and seedlings expressing GUS in all cells. Plasmid DNA, containing three chimeric constructs [enhanced cauliflower mosaic virus (CaMV) 35s-GUS, nopaline synthase-neomycin phosphotransferase (NPTII), and CaMV 35s-Bacillus thuringiensis (B.t.) cryIA endotoxin] was introduced into four developmental stages of white spruce somatic embryos by particle acceleration. Transient expression was observed in all of the stages of somatic embryos tested, but transformed embryogenic callus was induced only from the two most advanced. Embryogenic callus was identified by histochemical staining for GUS as early as 6 weeks following particle acceleration. All GUS positive embryogenic callus lines also showed NPTII activity. Incorporation of the introduced genes into the genome was confirmed by PCR and Southern blot analysis of embryogenic callus and regenerated transformed plants. Plants derived from several transformed embryogenic callus lines are currently undergoing acclimatization in the greenhouse. Spruce budworm (Chorisoneura fumiferana) feeding trials with embryogenic callus and transformed seedlings indicate a low, sublethal level of Bt. expression.