Genes encoding lysozyme (T4L) from T4 bacteriophage and attacin E (attE) from Hyalophora cecropia were used, either singly or in combination, to construct plant binary vectors, pLDB15, p35SAMVT4, and pPin2Att35SAMVT4, respectively, for Agrobacterium-mediated transformation of `Galaxy' apple, to enhance resistance to Erwinia amylovora. In these plasmids, the T4L gene was controlled by the cauliflower mosaic virus 35S promoter with duplicated upstream domain and the untranslated leader sequence of alfalfa mosaic virus RNA 4, and the attE gene was controlled by the potato proteinase inhibitor II (Pin2) promoter. All transgenic lines were screened by polymerase chain reaction (PCR) for T4L and attE genes, and a double-antibody sandwich enzyme-linked immunosorbent assay for neomycin phosphotransferase II. Amplification of T4L and attE genes was observed in reverse transcriptase-PCR, indicating that these genes were transcribed in all tested transgenic lines containing each gene. The attacin protein was detected in all attE transgenic lines. The expression of attE under the Pin2 promoter was constitutive but higher levels of expression were observed after mechanical wounding. Some T4L or attE transgenic lines had significant disease reduction compared to nontransgenic `Galaxy'. However, transgenic lines containing both attE and T4L genes were not significantly more resistant than nontransgenic `Galaxy', indicating that there was no in planta synergy between attE and T4L with respect to resistance to E. amylovora.
Kisung Ko, John L. Norelli, Jean-Paul Reynoird, Herb S. Aldwinckle, and Susan K. Brown
Kisung Ko, Susan K. Brown, John L. Norelli, and Herb S. Aldwinckle
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.
John L. Norelli and Herb S. Aldwinckle
Regeneration from apple (Malus × domestica Borkh.) M.26 leaf tissue was completely inhibited by (μg·ml-1) 1 geneticin, 5 kanamycin, 10 to 25 paromomycin, and 100 neomycin. nptII-transgenic M.26 had an increased tolerance to all four of the antibiotics tested, with inhibition of regeneration occurring at (μg·ml-l) 2.5 geneticin, 100 kanamycin, 375 paromomycin, and 375 neomycin. Paromomycin (100 to 250 μg·ml-l) and neomycin (250 μg·ml-1) significantly increased the amount of regeneration from nptII-transgenic M.26 apple leaf tissue. p35SGUS-INT, a plasmid with a chimeric b -glucuronidase gene containing a plant intron, was useful for studying the early events of apple transformation by eliminating GUS expression from Agrobacterium tumefaciens. It was used to determine that the optimal aminoglycoside concentrations for the selection of nptII-transgenic M.26 cells were (μg·ml-1) 2.5 to 16 kanamycin, 63 to 100 neomycin, and 25 to 63 paromomycin. Geneticin was unsuitable as a selective agent.
Yi Tan, Baisha Li, Yi Wang, Ting Wu, Zhenhai Han, and Xinzhong Zhang
recent years for apple transformation using leaves from in vitro shoots ( Dai et al., 2013 ; Wang et al., 2017 ). Another important reason for using leaves from in vitro shoots as source of explants for regeneration is the excellent uniformity between