[Show abstract][Hide abstract] ABSTRACT: Marker-free transgenic white poplar (Populus alba L., cv ‘Villafranca’) plants, expressing the PsMT
gene from Pisum sativum for a metallothionein-like protein, were produced by Agrobacterium tumefaciens-mediated transformation. The 35SCaMV-PsMT
-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
-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.
[Show abstract][Hide abstract] ABSTRACT: Transgenic white poplar (Populus alba L.) plants expressing a novel Arabidopsis thaliana cysteine proteinase inhibitor (Atcys) gene have been produced using Agrobacterium tumefaciens-mediated gene transfer. Internodal stem segments of cv. Villafranca were co-cultivated with the EHA105 pBI-Atcys A.tumefaciens strain. Sixteen putative transgenic plant lines were regenerated from different calli with a transformation efficiency of 11%. The integration and expression of the cysteine proteinase inhibitor (Atcys) gene into the plant genome was confirmed by Southern and northern blot analyses. Papain inhibitory activity was detected in poplar transgenic tissues by means of a specific invitro assay. Such activity was sufficient to inhibit most of the digestive proteinase activity of chrysomelid beetle (Chrysomela populi L.) and confer resistance to C.populi larvae on selected transgenic plants. A close correspondence between the inhibition of papain and resistance to poplar leaf beetle was observed in all tested transgenic lines. Our results indicate that Atcys could be succesfully employed in breeding programmes aimed at the selection of new poplar genotypes resistant to major insect pests.