Abstract
GABA, a non-proteinogenic amino acid with anti-hypertensive properties, holds health-beneficial potential when enriched in crops. Previous studies have established that targeted disruption of the calmodulin-binding domain (CaMBD) of the tomato glutamate decarboxylase 3 (SlGAD3) enhances GABA biosynthesis. In this study, we used CRISPR/Cas9-mediated gene editing to precisely modify the CaMBD coding sequence of SlGAD3 in three elite tomato varieties (SFT1, SFT2, and SFT3). Under our experimental conditions, targeted editing of SlGAD3 led to substantial accumulation of GABA in all three varieties without compromising key agronomic traits such as fruit size and number. Although flowering was delayed in SFT2 and SFT3 mutants, SFT1 mutants had higher GABA levels but also maintained a wild-type flowering time. This result highlights the critical importance of selecting specific varieties, such as SFT1, to minimize pleiotropic effects. By identifying varieties that can accumulate high levels of GABA without major reductions in growth and yield potential, this work bridges a critical gap between plant metabolic-engineering research and practical applications in commercial crop-improvement programs. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s42994-025-00249-w.