24Background: Gene expression analyses are conducted using multiple approaches and 25 increasingly research has been focused on assessing gene expression at the level of a 26 tissue or even single-cells. To date, methods to assess gene expression at the single-cell in 27 plant tissues have been semi-quantitative, require tissue disruption, and/or involve laborious, 28 possibly artifact-inducing manipulation. In this work, we used grape berries (Vitis vinifera L. 29 Zinfandel) as a model in order to examine the validity and reproducibility of an in-situ gene 30 expression analysis method combining a cell pressure probe (CPP) with quantitative PCR 31 (qPCR).32 Results: We developed a method to directly assess gene expression levels via qPCR from 33 cellular fluids sampled in-situ with a CPP. Cellular fluids, with volumes in the picoliter range, 34 were collected from intact berries with a CPP at various depths across skin and mesocarp 35 tissues. The expression of a key anthocyanin biosynthetic gene, UDP-glucose: flavonoid 3-36 O-glucosyltransferase (VviUFGT), was analyzed as a test case since its expression is 37 restricted to cells producing anthocyanins in grape berry skins during ripening. The method 38 identifies samples contaminated with significant levels of genomic DNA by amplifying a 39 region of VviUFGT that spans an intron. Therefore false positives were discarded which 40 occurred in 28% of the samples tested. Shallow probing of skin cells showed high VviUFGT 41 expression as expected while deeper probing of mesocarp cells resulted in no VviUFGT 42 expression. 43 Conclusions: The clear correspondence of VviUFGT expression to the targeted cell 44 samples suggests that the in-situ gene expression analysis using a CPP is reliable and does 45 not result in contamination as the probe moves through tissues. This method can be paired 46 to single-cell transcriptomic analyses in the future. We conclude that this technique 47 235 exons flanking the intron. The expression of VviUFGT was analysed by these primer pairs: 236 VviUFGT-forward (5'-GCAGGGCCTAACTCACTCTC-3'), VviUFGT-reverse (5'-237 AAATTGAGCAGCTCGTCTTCA-3'), and VviUbi was analyzed according to (Castellarin et 238 al. 2007a). The final PCR products obtained from the VviUFGT amplification were analyzed 239 9 in 1.5 % agarose gel. The samples that amplified two bands, 178bp (VviUFGT gDNA) and 240 101bp (VviUFGT mRNA) bands, were discarded because of the gDNA comtamination. The 241 samples with only a 101bp band were considered to be positive samples and included in the 242 data analysis. VviUFGT gene expression is reported relative to VviUbi. 243 244 Declarations 245