ALTERATIONS IN NPTII AND GUS EXPRESSION FOLLOWING MICROPROPAGATION OFTRANSGENIC M7 APPLE ROOTSTOCK LINES

Seven nptII and gus transgenic lines of the apple (Malus xdomestica Bo rkh,) rootstock Malling 7 (M.7) were examined by glucuronidase (GUS) h istochemical testing and a double-antibody sandwich enzyme-linked immu nosorbent assay (ELISA). These lines had different amounts of neomycin phosphotransferase II (NPTII). The amounts of NPTII among lines was p ositively correlated with the ability of the transgenic lines to regen erate in the presence of kanamycin, paromomycin, or geneticin., Regene rants derived from transgenic lines also varied greatly in GUS express ion, The apical portion of regenerant stem tissues had stronger GUS st aining 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 tr ansgenic lines showed nonstaining, chimeric staining, or uniform GUS s taining, 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 fai lure to isolated. tumefaciens from M.7 transgenics indicated that PCR and GUS staining results were not due to A. tumefaciens. A modified PC R methylation assay (MPMA) indicated that methylation of cytosines of the CCGG site in the gus gene, and in the border between the CaMV35S p romoter and the gus gene, was positively correlated with complete gus gene silencing (nonstaining lines), However, the MPMA indicated that m ethylation was not always associated with variable GUS expression, sug gesting that chimeric staining could be due to a mixture of transforme d and nontransformed cells in some transgenic lines.