Selection and validation of suitable reference genes for qPCR expression analysis in young in vitro grown kohlrabi under cytokinin and sucrose treatments

Reference genes (RGs) commonly serve as internal controls for normalization of gene expression data in quantitative real-time polymerase chain reaction (qPCR) analyses. To ensure accurate and reliable normalization and interpretation of the results, a systematic validation of RG stability under particular experimental conditions is essential. Our research group previously established a suitable in vitro model system for kohlrabi (Brassica oleracea var. gongylodes) growth and regeneration, encompassing four early developmental stages under varying cytokinin and sugar treatments. As the stability of potential RGs had not yet been evaluated for kohlrabi, we selected and tested suitable RGs for our specific experimental setup. A total of 15 candidate RGs were analyzed using the RefFinder online tool, which integrates four statistical algorithms (geNorm, NormFinder, BestKeeper, and the comparative ΔCt method) to generate a comprehensive stability ranking. TUA2, ACT7 and SAND were identified as the most stable RGs across all experimental subsets, while GAPB and GLUR3.2 exhibited the lowest stability. These findings were validated by qPCR analysis of selected sucrose-related target genes. Our results highlight the importance of context-specific RG validation and provide a valuable resource for gene expression studies in non-model horticultural crops such as kohlrabi.