RT Journal Article
SR Electronic
T1 Efficient Genetic Transformation of <em>Vitis vinifera</em> L. Mencía using a Hypervirulent Strain of <em>Agrobacterium tumefaciens</em> and qPCR Determination of Transgene Copy Number
JF American Journal of Enology and Viticulture
JO Am J Enol Vitic.
FD American Society for Enology and Viticulture
SP 0750020
DO 10.5344/ajev.2024.24012
VO 75
IS 2
A1 Martínez, Óscar
A1 Palomo-Ríos, Elena
A1 Rey, Manuel
A1 González, María Victoria
YR 2024
UL http://www.ajevonline.org/content/75/2/0750020.abstract
AB Background and goals Agrobacterium-mediated transformation efficiency is generally low in grapevine and varies significantly among cultivars, making it necessary to optimize the protocol for each grapevine cultivar.Methods and key findings Different strategies to select potentially transformed material were tested by combining different kanamycin concentrations at different time points in solid medium supplemented with 250 mg/L timentin. The additional selection step of 50 mg/L kanamycin in liquid medium allowed recovery of completely transformed plant material with up to 19.8% transformation efficiency.qPCR copy number estimates of the transgenes nptII and uidA using the ΔCq method were inconsistent and depended on the endogenous single-copy control gene (NCED2 or chi) used to perform the calculations. Modifying the analysis by including the geometric average of the two control genes resulted in equivalent estimated copy numbers in all but two lines. The highest qPCR expression level of the uidA gene was observed in the line with the most integrated transgene copies, although there was no correlation between these two measures in lines with a high transgene copy number.Conclusions and significance An efficient transformation protocol for the grapevine cultivar Mencía was established for the first time, using somatic embryo aggregates cocultured with the hypervirulent Agrobacterium tumefaciens strain AGL1 harboring the binary vector pBINUbiGUSInt. A robust qPCR analysis of transgene copy number allowed early and easy selection of the most interesting transgenic lines using very little plant material.