Regenerants coming from different embryogenic events, randomly chosen from three different explants cultured on different media, were compared to the mother plant for evaluation of genetic stability. Plant DNA was isolated from fresh leaves (100 mg) using the cetyltrimethylammonium bromide (CTAB) method as described by Doyle and Doyle [53]. The isolated genomic DNA was used for RAPD and ISSR analyses in order to assess mutations (Supplementary Table S1). The RAPD analysis of the grapevine genotypes was performed using eight random decamer primers—OPAT14, OPH15, OPM04, UBC219, UBC234, UBC239, UBC247, and UBC251 [54,55]. Seven ISSR primers—UBC834, UBC841, UBC848, UBC851, UBC855, ENEA7–9, and ENEA12 were used to amplify the genomic DNA [41,48,50]. For both molecular markers, polymerase chain reaction (PCR) amplification and analysis were performed as described by Carra et al. [48]. Only in those cases where the flow cytometry histograms revealed that the ploidy level of regenerated plants was different from that of the mother plant, an additional set of seven ISSR and nine simple sequence repeats (SSR) was tested for more accurate genetic stability evaluation. The additional ISSR analysis was carried out with ISSR2 + 2b ((AC)8YG; Ta 49 °C), ISSR 3 + 3b ((AG)8YC; Ta 49 °C), ISSR11 + 11b ((GA)8YC; Ta 56 °C), ISSR1-6 ((CA)8RG; Ta 49 °C), ENEA21 ((GA)8GG; Ta 49 °C), ENEA34 ((ACC)6CC; Ta 52 °C), and ENEA36 (CC(ATG)6; Ta 56 °C) [41,56,57]. SSR analysis was carried out using nine primer pairs—VVMD7, VVMD24, VVMD25, VVMD27, VVIb01, VVIh54, VVIp31, VVIp60, and VVIq52, distributed homogenously along the 19 chromosomes of diploid genome (475 Mbp and 2n = 38 chromosomes) of the grapevine (Supplementary Table S1). The SSR-PCR reactions were followed as described by De Michele et al. [58]; amplicons of each primer on all individuals were scored by an external service (Eurofins Genomics, Germany), and SSR allelic size was determined using the Gene Mapper v. 5.0 software. To confirm the reproducibility of the banding patterns, all analyses were repeated twice.

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