Abstract
Marker-free transgenic white poplar (Populus alba L., cv ‘Villafranca’) plants, expressing the PsMT A1 gene from Pisum sativum for a metallothionein-like protein, were produced by Agrobacterium tumefaciens-mediated transformation. The 35SCaMV-PsMT A1 -NosT cassette was inserted into the ipt-type vector pMAT22. The occurrence of the abnormal ipt-shooty phenotype allowed the visual selection of transformants, while the yeast site-specific recombination R/RS system was responsible for the excision of the undesired vector sequences with the consequent recovery of normal marker-free transgenic plants. Molecular analyses confirmed the presence of the 35SCaMV-PsMT A1 -NosT cassette and transgene expression. Five selected lines were further characterized, revealing the ability to withstand heavy metal toxicity. They survived 0.1 mM CuCl2, a concentration which strongly affected the nontransgenic plants. Moreover, root development was only slightly affected by the ectopic expression of the transgene. Reactive oxygen species were accumulated to a lower extent in leaf tissues of multi-auto-transformation (MAT)-PsMTA1 plants exposed to copper and zinc, compared to control plants. Tolerance to photo-oxidative stress induced by paraquat was another distinctive feature of the MAT-PsMTA1 lines. Finally, low levels of DNA damage were detected by quantifying the amounts of 8-hydroxy-2′-deoxyguanosine in leaf tissues of the transgenic plants exposed to copper.
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Acknowledgments
This research was supported by funds from the Italian Ministry for Education, University and Scientific Research, to D. Carbonera (PRIN 2005055337), to S.Botti (PRIN 2005055337_005), to M. Racchi (PRIN 2005055337_004) and to G. Lingua (PRIN 2005055337_003). Authors gratefully acknowledge Dr Keiko Yamada (Nippon Paper Industries, Tokyo, Japan) for supplying the pMAT22 vector.
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Balestrazzi, A., Botti, S., Zelasco, S. et al. Expression of the PsMT A1 gene in white poplar engineered with the MAT system is associated with heavy metal tolerance and protection against 8-hydroxy-2′-deoxyguanosine mediated-DNA damage. Plant Cell Rep 28, 1179–1192 (2009). https://doi.org/10.1007/s00299-009-0719-x
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DOI: https://doi.org/10.1007/s00299-009-0719-x