Polyploid plants, such as hexaploid Solanum nigrum, present new challenges and opportunities for trait improvement due to their complex genome structures and homoeologous gene interactions. Gene editing of homoeologs provides a powerful tool to investigate the inheritance and phenotypic effects of mutations in polyploid systems. Here, we investigated the inheritance and phenotypic traits of homoeolog-edited triple and double mutants in hexaploid S. nigrum. Triple mutant genotypes (sns and snsp) were used for analyzing inheritance patterns through backcrossing and self-pollination, revealing independent segregation of homoeologous variants and consistent genotypic ratios across generations. Yield and related traits were evaluated in fixed double mutants (sns1 sns2, sns1 sns3, sns2 sns3) under greenhouse and open-field conditions. Open-field cultivation significantly enhanced yield, primarily through increased fruit weight per inflorescence, whereas greenhouse cultivation showed more modest improvements. Other traits such as plant weight and Brix values remained largely unchanged. Yield improvements were stable across F3 to F5 generations, with minimal genotype-specific variability. These findings highlight the potential of homoeolog-targeted editing in polyploid plants to achieve stable and environment-specific yield enhancements.

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