Development of highly efficient genetic transformation protocols for table grape Sugraone and Crimson Seedless at low Agrobacterium density

Abstract

Highly efficient genetic transformation protocols and the regeneration of transgenic plants of Sugraone and Crimson Seedless grapevines (Vitis vinifera L.) were achieved from embryogenic calli co-cultured with low Agrobacterium tumefaciens densities. The sensitivity of embryogenic cultures to kanamycin, as well as the effect of Agrobacterium strains, C58(pMP90) or EHA105, and the bacterial concentration (0.06 or 0.2 at Optical Density OD 600 ) on transformation efficiency were studied. Embryogenic cultures showed different kanamycin sensitivities and the total suppression of embryo differentiation at 20 and 50 mg/l kanamycin for Crimson Seedless and Sugraone, respectively. sgfp gene expression was evaluated in callus co-cultured with each bacterial strain. Although GFP transient expression was higher with A. tumefaciens EHA105 in both cultivars at the beginning of the culture, there were no significant differences 28 days post-inoculation. However, the concentration of Agrobacterium did affected transformation efficiency: 0.06 OD 600 being more effective for the transformation of Crimson Seedless and 0.2 OD 600 for Sugraone. By following the optimised procedure, 21 and 26 independent transgenic plants were generated from Sugraone and Crimson Seedless respectively, three to five months post-infection. PCR analyses were carried out to verify the integration of the sgfp and nptII genes into grapevine genome and the stable integration of the sgfp gene was confirmed by Southern blot.

Factors affecting Agrobacterium-mediated genetic transformation of embryogenic callus of Parthenocissus tricuspidata Planch

“…In our experiments, both EHA105 and C58 could be used for transformation, but EHA105 seems to be better than C58, which is a hypervirulent bacterium that has been used successfully in the transformation of various plant species (Holland et al 1997;Li et al 2007a, b;López-Pérez et al 2008;Akcay et al 2009). …”

Vascular-specific expression of GUS and GFP reporter genes in transgenic grapevine (Vitis vinifera L. cv. Albariño) conferred by the EgCCR promoter of Eucalyptus gunnii

“…Simple integration pattern was found in four of the five lines tested. Different authors [7,27,29,39] have reported variable insertion numbers ranging from 1 to 3 in genomic blottings with different gene probes in other cultivars of transformed grapevine via Agrobacterium. In our study a high proportion of a unique number of gene insertions in the transgenic cv.…”

“…Moreover, there is a debate going on in the scientific press, about the risks of using the CaMV35S promoter regarding its "unpredictable" recombinogenic potential [13]. Constitutive promoters, such as CaMV35S, are typically used for grapevine transformation [15,29,30,39,42]. In order to avoid such problems, the use of tissue-specific promoters is an attractive alternative since gene expression is restricted to tissues of interest and at given developmental stages.…”

Enhanced resistance to Botrytis cinerea in genetically-modified Vitis vinifera L. plants over-expressing the grapevine stilbene synthase gene

“…Agrobacterium tumefaciens EHA105-pKSTS706 at OD 600 = 0.2 were co-cultured with embryogenic calli for 2 days following the plant regeneration protocol described by López-Pérez et al (2008) and Dabauza and Velasco (2012): calli were washed with MS ? 900 mg l -1 cefotaxime for 20 min and cultured on MSAC [modified MS (half strength of macroelements MS), 100 mg l -1 myoinositol, 30 g l -1 sucrose and 2.5 g l -1 of activated charcoal (AC)] (López-Pérez et al 2005, 2006 with 300 mg l -1 cefotaxime for embryo differentiation.…”