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Journal of Experimental Botany, Vol. 52, No. 364, pp. 2089-2095, November 1, 2001
© 2001 Oxford University Press

Original Papers

An improved procedure for production of white spruce (Picea glauca) transgenic plants using Agrobacterium tumefaciens

Van Quy Le, Julie Belles-Isles, Mathieu Dusabenyagasani and Francine M. Tremblay1

Centre de Recherche en Biologie Forestière, Pavillon Charles-Eugène Marchand, Université LAVAL, Québec, QC, Canada, G1K 7P4

An efficient and reproducible procedure for the transformation of white spruce (Picea glauca [Moench] Voss) embryogenic tissues was developed using A. tumefaciens-mediated gene transfer. Rapidly dividing white spruce embryogenic tissues were co-cultivated with disarmed A. tumefaciens strains containing additional copies of the virulence regions from plasmid PToK47. The plasmid pBi121, containing the neomycin phosphotransferase II (nptII) gene providing kanamycin resistance as a selectable marker and the ß-glucuronidase (uidA) reporter gene, was used as binary vector. The highest frequency of transformation (15 transformed tissues g-1 FW of treated embryogenic tissue) was obtained with 5-d-old tissues grown in liquid medium and co-cultivated with Agrobacterium for 2 d in the same medium but containing 50 µM acetosyringone. Recovery of kanamycin-resistant tissues was improved when tissues were first grown for 10 d on a timentin-containing medium (400 mg l-1), to prevent bacterial overgrowth, before application of the selection pressure. After 6 weeks on kanamycin-selection medium, resistant tissues were obtained and showed stable uidA expression. The presence of the transgenes was demonstrated by PCR analysis and their integration into the genome was confirmed by Southern hybridization. Transgenic plants were regenerated from transformed tissues within 4 months after co-culture.

Key words: Agrobacterium tumefaciens, embryogenic tissue, transformation procedure, transgenic plants, uidA expression, white spruce.


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