Stable transformation and regeneration of transgenic plants of Pinus radiata D. Don

• Published in: Plant cell reports Vol. 17; no. 6/7; pp. 460 - 468
• Main Authors: Walter, C, Grace, L.J, Wagner, A, White, D.W.R, Walden, A.R, Donaldson, S.S, Hinton, H, Gardner, R.C, Smith, D.R
• Format: Journal Article
• Language: English
• Published: Berlin Springer 01-04-1998; Springer Nature B.V
• Subjects: Bacteria; biolistics; Biological and medical sciences; Biotechnology; clones; developmental stages; DNA; embryo (plant); Fundamental and applied biological sciences. Psychology; gene expression; gene transfer; Genetic engineering; Genetic technics; genetic transformation; genetic variation; genetic vectors; Genomes; histochemistry; Integration; localization; Methods. Procedures. Technologies; micropropagation; nptii genes; Pine trees; Pinus radiata; Polymerase chain reaction; promoter regions; Promoters; regenerative ability; Reporter gene; reporter genes; Somatic embryos; Studies; Transformation; Transgenic animals and transgenic plants; Transgenic plants; Trees; uida reporter genes; Microprojectile bombardment; Embryo; Vegetals; β-Glucuronidase; Genetic transformation; Enzyme; Transferases; Tissue culture; Pinus radiata; Gene expression; Kanamycin kinase; Regeneration; Reporter gene; Glycosidases; Gymnospermae; Softwood forest tree; Hydrolases; Coniferales; Spermatophyta; Transgenic plant; O-Glycosidases; Transformation; Biolistic; Embryogenic tissue; Key wordsPinus radiata
• ISSN: 0721-7714; 1432-203X
• DOI: 10.1007/s002990050426

A biolistic particle delivery system was used to genetically transform embryogenic tissue of Pinus radiata. The introduced DNA contained a uidA reporter gene under the control of either the tandem CaMV 35S or the artificial Emu promoter, and the npt II selectable marker controlled by the CaMV 35S promoter. The average number of stable, geneticin-resistant lines recovered was 0.5 per 200 mg fresh weight bombarded tissue. Expression of the uidA reporter gene was detected histochemically and fluorimetrically in transformed embryogenic tissue and in derived mature somatic embryos and regenerated plants. The integration of uidA and npt II genes into the Pinus radiata genome was demonstrated using PCR amplification of the inserts and Southern hybridisation analysis. The expression of both genes in transformed tissue was confirmed by Northern hybridisation analysis. More than 150 transgenic Pinus radiata plants were produced from 20 independent transformation experiments with four different embryogenic clones.