Agroinfiltration of grapevine leaves for fast transient assays of gene expression and for long-term production of stable transformed cells
Articolo
Data di Pubblicazione:
2008
Citazione:
Agroinfiltration of grapevine leaves for fast transient assays of gene expression and for long-term production of stable transformed cells / M. Zottini, E. Barizza, A. Costa, E. Formentin, C. Ruberti, F. Carimi, F. Lo Schiavo. - In: PLANT CELL REPORTS. - ISSN 0721-7714. - 27:5(2008), pp. 845-853.
Abstract:
Agrobacterium-mediated transient assays for
the analysis of gene function are used as alternatives to
genetic complementation and stable plant transformation.
Although such assays are routinely performed in several
plant species, they have not yet been successfully applied
to grapevines. We explored genetic background diversity
of grapevine cultivars and performed agroinfiltration into
in vitro cultured plants. By combining different genotypes
and physiological conditions, we developed a protocol for
efficient transient transformations of selected grapevine
cultivars. Among the four cultivars analyzed, Sugraone and
Aleatico exhibited high levels of transient transformation.
Transient expression occurred in the majority of cells
within the infiltrated tissue several days after agroinfiltration
and, in a few cases, it later spread to a larger portion of
the leaf. Three laboratory strains of Agrobacterium tumefaciens
with different virulence levels were used for
agroinfiltration assays on grapevine plants. This method
promises to be a powerful tool to perform subcellular
localization analyses. Grapevine leaf tissues were transformed
with fluorescent markers targeted to cytoplasm
(free GFP and mRFP1), endoplasmatic reticulum
(GFP::HDEL), chloroplast (GAPA1::YFP) and mitochondria
(b::GFP). Confocal microscope analyses demonstrated
that these subcellular compartments could be easily visualized
in grapevine leaf cells. In addition, from leaves of
the Sugraone cultivar agroinfiltrated with endoplasmic
reticulum-targeted GFP-construct, stable transformed cells
were obtained that show the opportunity to convert a
transiently transformed leaf tissue into a stably transformed
cell line.
Tipologia IRIS:
01 - Articolo su periodico
Keywords:
Agrobacterium; Plant cell cultures; Subcellular localization; Transformation; Vitis vinifera
Elenco autori:
M. Zottini, E. Barizza, A. Costa, E. Formentin, C. Ruberti, F. Carimi, F. Lo Schiavo
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