Genetic transformation of black walnut (Juglans nigra)

Disarmed Agrobacterium tumefaciens strains with binary vectors carrying transgenes for kanamycin resistance (npt II) and beta-glucuronidase (GUS, uidA) were used for the genetic transformation of Eastern black walnut (Juglans nigra) somatic embryos. In total, explants from 16 embryo lines, representing 10 genotypes, were used and regeneration was compared both in the absence of kanamycin selection and with different levels of selection stringency (200-250 or 500 mg/L kanamycin). Histochemical GUS expression assays indicated that the rates of T-DNA transfer were high (75-100%), with gene transfer being demonstrated for 14/16 lines. Regeneration was observed for 15% of the infected embryos, compared with frequencies of 50% for the non-infected controls. The regeneration frequencies were nearly two-fold higher when kanamycin was excluded from the culture medium, but most of the secondary embryos appeared non-transgenic. When kanamycin was present, most of the secondary embryos were transgenic, but chimeric, consisting of mixtures of transgenic and non-transgenic cells; and few exhibited de novo growth following harvest, even on non-selective medium. Less than 10% of the initial secondary embryos were wholly transgenic, as assessed by GUS assay; however, these embryos appeared essential for the initiation of stable transgenic lines. The establishment of transgenic lines was also facilitated by the use of lower kanamycin concentrations (200-250 mg/L) and by the precocious proliferation of the initial secondary embryos.