Agrobacterium‐mediated transformation of Vitis Cv. Monastrell suspension‐cultured cells: Determination of critical parameters

Abstract

Although some works have explored the transformation of differentiated, embryogenic suspension-cultured cells (SCC) to produce transgenic grapevine plants, to our knowledge this is one of the first reports on the efficient transformation of dedifferentiated Vitis vinifera cv Monastrell SCC. This protocol has been developed using the sonication-assisted Agrobacterium-mediated transformation (SAAT) method. A construct harboring the selectable nptII and the eyfp/IV2 marker genes was used in the study and transformation efficiencies reached over 50 independent transformed SCC per gram of infected cells. Best results were obtained when cells were infected at the exponential phase. A high density plating (500 mg/dish) gave significantly better results. As selective agent, kanamycin was inefficient for the selection of Monastrell transformed SCC since wild type cells were almost insensitive to this antibiotic whereas application of paromomycin resulted in very effective selection. Selected eyfp-expressing microcalli were grown until enough tissue was available to scale up a new transgenic SCC. These transgenic SCC lines were evaluated molecularly and phenotypically demonstrating the presence and integration of both transgenes, the absence of Agrobacterium contamination and the ability of the transformed SCC to grow in highly selective liquid medium. The methodology described here opens the possibility of improving the production of valuable metabolites. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:725-734, 2016.

The state-of-the-art of grapevine biotechnology and new breeding technologies (NBTS)

“…This was shown to be particularly interesting for the production of bioactive compounds (Martínez-Márquez et al, 2015). To improve Agrobacterium-mediated transformation efficiency of dedifferentiated V. vinifera cv Monastrell cells, Chu et al (2016) used the Sonication-Assisted Agrobacteriummediated Transformation (SAAT) method that was previously developed for soybean transformation (Trick and Finer, 1997). This method significantly increases transformation efficiency (Chialva et al, 2016).…”

Development and Use of Biotechnology Tools for Grape Functional Analysis

“…Transformation and regeneration of grapevine plants have been achieved via organogenesis [84], embryogenesis [83,93], or from nonembryogenic cell cultures [86,94], demonstrating that grapevine is not recalcitrant to Agrobacterium infection but this approach is time consuming and takes several months to produce transgenic plants suitable for analyses. In some cases, the production of whole transgenic organisms may not be needed if a large number of cells within a plant can be uniformly and consistently transformed.…”

“…Sonication-assisted Agrobacterium-mediated transformation (SAAT) was first reported by Trick and Finer [98], who used this technique for the production of transgenic soybean (Glycine max) and Ohio buckeye (Aesculus glabra) plants from SAAT-treated embryogenic cell suspensions. Chu et al [94] recently reported the effect of different times in a sonication bath while infecting dedifferentiated nonembryogenic grapevine suspension cell cultures with A. tumefaciens. Plant tissue damaged by sonication allows the tissue to be much more easily transformed by A. tumefaciens.…”