Using GFP as a scorable marker in walnut somatic embryo transformation

Somatic embryogenesis is the foundation of genetic transformation in several economically important tree species. In the Juglans regia L. (Persian walnut) somatic embryogenesis-based transformation system, a major limiting factor is the selection of non-chimeric transgenic embryos in tissue culture. We transformed Persian walnut somatic embryos with the S65T synthetic green fluorescent protein (GFP) gene in order to assess the effect of this visual marker gene on embryo viability and the selection of transgenic embryos. Following a 10 d period of transient GFP expression in all inoculated embryos, stable fluorescent sectors were apparent in several embryos, allowing efficient and rapid visual selection of primary transgenic embryos. Two chimeric embryos were selected 40 d after transformation, and these two embryos gave rise to 13 stable transgenic embryo lines and 44 whole plants. GFP-expressing walnut plants and embryos developed normally and transformation was verified by GUS analysis. Our analysis suggests that the use of GFP as a selectable marker can significantly reduce labour, cost, and time in the walnut somatic embryogenesis-based (C) 2000 Annals of Botany Company.