Recovery of transgenic trees after electroporation of poplar protoplasts

Abstract

Protoplasts from leaflets of in vitro cuttings were electroporated in osmotically adjusted and buffered solutions containing plasmid DNA: pABD1, carrying the nptlIgene for resistance to neomycin; pGH1, carrying a mutant acetolactate synthase gene, als, for resistance to sulfonylurea; and pGSFR781A, carrying a synthetic phosphinothricin acetyltransferase (pat) for resistance to phosphinothricin (Basta). Gene transfer was repeatedly efficient, without use of carrier DNA, in the range of one transformant for 105 to 104 protoplast-derived cell colonies. This was probably due to the high plating efficiency (30%) of protoplasts in our culture process. Selection for expression of foreign genes was applied in liquid medium and repeatedly achieved with 30 ~M paromomycin for NPTII, 200 nM chlorsulfuron for the mutant ALS of Arabidopsis and 25 ~tM phosphinothricin for PAT expression. Integration of foreign genes into genomic DNA of resistant poplar trees was demonstrated by Southern blot hybridizations, which revealed that for some transformants practically no other part of the vector plasmid than the selected gene was integrated.Effective processes for protoplast culture, efficient selection at the cell colony stage and gene transfer will provide new possibilities in poplar breeding.

Transformation as a Tool for Genetic Analysis in Populus

“…A variety of transformation methods have been successfully used in Populus, including biolistic approaches (McCown et al, 1991) electroporation of protoplasts (Chupeau et al, 1994), and cocultivation with Agrobacterium. Protoplast transformation is very rarely used, and biolistics is mainly used in specialized applications such as for rapid assessment of promoter::reporter expression in different tissues, transformation of highly recalcitrant genotypes, and transformation of plastids.…”

Current status and environmental impact of transgenic forest trees

“…Populus deltoides Han et al 1997 A. rhizogenes Han et al 2000 A. tumefaciens Populus nigra Wang et al 1996; and others b A. tumefaciens Populus tremuloides Tsai et al 1994 A. tumefaciens Populus tremula Tzfira et al 1996A. rhizogenes Tzfira et al 1997and others b A. tumefaciens Populus tricocarpa Han et al 1997 A. rhizogenes Populus hybrids b Fillatti et al 1987; and others b A. tumefaciens Han et al 1997;and others b A. rhizogenes Chupeau et al 1994 Electroporation Robinia pseudoacacia Han et al 1993 A. rhizogenes Igasaki et al 2000 A. tumefaciens Ulmus procera Gartland operational dosages was obtained in radiata pine, Norway spruce and Eucalyptus camaldulensis (Bishop-Hurley et al 2001;Harcourt et al 2000). The commercial potential of herbicide-tolerant trees has been demonstrated in various field trials during the past decade.…”

“…Research on the bar gene from Streptomyces hygroscopicus as a selectable marker for plant transformation has enabled engineering of tolerance to a third class of herbicides. Glufosinate tolerance was first achieved in poplar in the late 1980s(de Block 1990;Chupeau et al 1994), and more recently tolerance to…”

Genetic Transformation of Forest Trees

“…Protoplasts can be transformed by direct DNA uptake, following polyethylene glycol pretreatment, by microinjection or by electroporation. Although many studies have resulted in successful transient expression of a transgene in cell-derived protoplasts (Bekkaoui et al, 1995), very few have described the regeneration of transgenic trees (Chupeau et al, 1994). This is probably due to the lack of suitable methods to recover whole plants from protoplasts of most tree genotypes.…”