Alterations in nptII and gus Expression following Micropropagation of Transgenic M.7 Apple Rootstock Lines

Abstract

Seven nptII and gus transgenic lines of the apple (Malus ×domestica Borkh.) rootstock Malling 7 (M.7) were examined by glucuronidase (GUS) histochemical testing and a double-antibody sandwich enzyme-linked immunosorbent assay (ELISA). These lines had different amounts of neomycin phosphotransferase II (NPTII). The amounts of NPTII among lines was positively correlated with the ability of the transgenic lines to regenerate in the presence of kanamycin, paromomycin, or geneticin. Regenerants derived from transgenic lines also varied greatly in GUS expression. The apical portion of regenerant stem tissues had stronger GUS staining than the basal portion of stem. All regenerated tissue of T1, a transgenic line originally classified as a uniform GUS staining line, showed non-GUS staining, while the regenerated tissues of chimeric transgenic lines showed nonstaining, chimeric staining, or uniform GUS staining, indicating the potential to select uniform GUS staining lines from chimeras. Polymerase chain reaction (PCR) indicated the gus gene was present in GUS negative (nonstaining) lines. Negative PCR results with primers derived from vir G of Agrobacterium tumefaciens, and failure to isolate A. tumefaciens from M.7 transgenics indicated that PCR and GUS staining results were not due to A. tumefaciens. A modified PCR methylation assay (MPMA) indicated that methylation of cytosines of the CCGG site in the gus gene, and in the border between the CaMV35S promoter and the gus gene, was positively correlated with complete gus gene silencing (nonstaining lines). However, the MPMA indicated that methylation was not always associated with variable GUS expression, suggesting that chimeric staining could be due to a mixture of transformed and nontransformed cells in some transgenic lines.