Articles
TRANSFORMATION OF AUTHENTIC M.26 APPLE ROOTSTOCK FOR ENHANCED RESISTANCE TO FIRE BLIGHT
Article number
489_44
Pages
259 – 266
Language
Abstract
A previous report erroneously indicated the transformation of M.26 apple rootstock with the lytic protein, attacin E, and a consequent increase in resistance to fire blight, when in fact a mislabeled source of explant material had resulted in transformation of M.7. We now report the transformation of authentic M.26, whose correct identity had been confirmed by RAPD analysis.
Plasmid binary vectors containing the gene for the lytic protein, attacin E (attE), under the control of the enhanced Cauliflower Mosaic Virus 35S (CaMV 35S) promoter, with a) the Alfalfa Mosaic Virus translation enhancer sequence (AMV) and a signal peptide, b) only AMV, and c) without AMV or signal peptide; and vectors with attE under control of the potato proteinase inhibitor II promoter, with the T4 lysozyme gene (T4L) under control of CaMV 35S, and with both the latter constructs were used for transformation of M.26 leaf tissue by Agrobacterium tumefaciens. Transformation was confirmed by ELISA for the NPTII selectable marker, and by PCR analysis for the transgenes and for absence of bacterial virG. Attacin E expression is being evaluated by western analysis, and T4 lysozyme by ELISA. Own-rooted plants of several transgenic lines have been obtained and are being evaluated for resistance to fire blight by inoculation with Erwinia amylovora.
Plasmid binary vectors containing the gene for the lytic protein, attacin E (attE), under the control of the enhanced Cauliflower Mosaic Virus 35S (CaMV 35S) promoter, with a) the Alfalfa Mosaic Virus translation enhancer sequence (AMV) and a signal peptide, b) only AMV, and c) without AMV or signal peptide; and vectors with attE under control of the potato proteinase inhibitor II promoter, with the T4 lysozyme gene (T4L) under control of CaMV 35S, and with both the latter constructs were used for transformation of M.26 leaf tissue by Agrobacterium tumefaciens. Transformation was confirmed by ELISA for the NPTII selectable marker, and by PCR analysis for the transgenes and for absence of bacterial virG. Attacin E expression is being evaluated by western analysis, and T4 lysozyme by ELISA. Own-rooted plants of several transgenic lines have been obtained and are being evaluated for resistance to fire blight by inoculation with Erwinia amylovora.
Publication
Authors
E.E. Borejsza-Wysocka, J.L. Norelli, H.S. Aldwinckle, K. Ko
Keywords
Agrobacterium tumefaciens, Erwinia amylovora, genetic engineering
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