cultivar: Chardonnay genotype/variation: wild type tissue: Whole berries
Treatment protocol
Stable transformation of grapevines: A pART7 vector containing the VvMYBA1 cDNA sequence between a Cauliflower Mosaic Virus 35S promoter and an octopine synthase gene (OCE) transcriptional terminator (Gleave, 1992), had been previously constructed (Walker et al., 2007). This expression cassette was excised from pART7 using a NotI restriction enzyme and ligated to the plant expression vector pART27 (Gleave, 1992) to create the 35S:VvMYBA1 construct[kos036 1] . Primers A13pfxho and A13prasp (containing XhoI and Asp178 restriction sites respectively, Table S6) were used to amplify a 400 bp sense fragment from the 3’ end of the VvMYBA1 gene, and primers A13pfxba and A13prcla (containing XbaI and ClaI restriction sites respectively, Table S6) amplified the same fragment in the anti-sense direction by PCR (RED TAQ®, Invitrogen, USA). PCR cycling conditions were as follows: 94oC for 3 min followed by 35 cycles of 94oC for 30 s, 58oC for 30 s and 72oC for 2 min, then final extension at 72oC for 10 min. Fragments were ligated to pDrive (Qiagen, Netherlands), from which the sense and anti-sense fragments were excised using XhoI/Asp178 and XbaI/ClaI restriction enzymes respectively. These fragments were then sequentially ligated into the pN6 vector. The silencing cassette was excised from pN6 using a NotI restriction enzyme and this fragment was ligated into the pART27 vector to create the VvMYBA silencing (VvMYBAsi) construct. pART27 VvMYBA expression or silencing constructs were inserted into the Agrobacterium tumefaciens strain EHA105 which was used to transform embryogenic callus using the method of (Iocco et al., 2001). Callus was initiated from anthers of immature flowers of Shiraz (BVRC12) and Chardonnay (I10V1) growing in the Coombe Vineyard, Adelaide University, Urrbrae, South Australia. Shiraz was initiated onto PIV medium and Chardonnay onto HT medium. Embryogenic callus from both cultivars was maintained on C1 and in culture for 12-20 months prior to transformation. Transformed callus was selected using 100 µg/mL kanamycin. Germinated embryos were cut to remove the roots and the tips, comprising the cotyledons supported on approximately 5 mm of hypocotyl, were transferred to shooting medium containing 10 µM benzylaminopurine (BAP) for shoot development. Rooted plantlets, whose leaves tested positive by PCR to the transgene were deflasked into potting mix and hardened off over 3 weeks in the glasshouse under shade and with a gradual reduction in humidity.
Growth protocol
Transgenic Chardonnay/Shiraz and non-transformed WT controls were all grown in the same glasshouse in CSIRO Plant Industry, Adelaide (Australia) in ambient light with a night break. Day and night temperatures were approximately 27oC and 22oC respectively.
Extracted molecule
total RNA
Extraction protocol
Whole berries were sampled from three independent transgenic Chardonnay and four from transgenic Shiraz lines, resulting in three and four biological replicates respectively. the samples were immediately frozen in liquid nitrogen. For skin samples, the skins were first removed from fresh berries then frozen in liquid nitrogen. All samples were stored at -80oC. Total RNA was extracted using a modified perchlorate method previously described in Boss et al. (2001).
Label
Cy3
Label protocol
cDNA synthesis and labeling reactions were performed according to the NimbleGen Arrays User’s Guide (V 3.2).
Hybridization protocol
Hybridization and washing procedures were performed according to the NimbleGen Arrays User’s Guide (V 3.2).
Scan protocol
Each microarray was scanned using a Axon GenePix 4400 A at 532 nm (Cy-3 absorption peak) and GenePix Pro7 software (Molecular Devices, Sunnyvale, CA, USA) according to the manufacturers' instructions.
Description
Whole berries of wild type Vitis vinifera cv Chardonnay (control), replicate 1
Data processing
Images were analyzed using NimbleScan v2.5 software (Roche), background correction and standard RMA normalization were selected.
