Influence of auxin and cytokinin concentrations on callus induction, subculture growth, and somatic embryogenesis for plant regeneration in zoysiagrass (Zoysia spp.)

Genetic engineering and gene editing present transformative opportunities for turfgrass improvement by permitting the introduction of genetic variation that does not naturally exist within a species. However, the successful application of these approaches depends on the generation of efficient and reproducible tissue culture protocols. Optimizing these methodologies for warm-season turfgrass species is essential to ensure the scalability and reliability of genetic modification efforts. In this study, parameters affecting the efficiency of callus formation, growth, and somatic embryogenesis were studied in zoysiagrass ( Zoysia spp.). Seeds were evaluated under four auxin and four cytokinin concentrations for callus induction and subculture. The combination of 2,4-dichlorophenoxyacetic acid (2,4-D) = 1.5 mg·L −1 and 6-benzylaminopurine (6-BA) = 0.05 mg·L −1 was identified as the most effective treatment for callus induction with a 67% success rate. Results demonstrated that a moderate concentration of 2,4-D combined with minimal 6-BA significantly enhanced callus formation and net callus growth over time. Additionally, plant regeneration trials revealed that gibberellic acid (GA 3 ) concentrations influenced somatic embryo development, with high levels yielding the highest regeneration efficiency. A total of 1,387 plants were recovered from 1,536 calli. The optimized protocol described here provides a foundation for potential advancements in zoysiagrass breeding, enabling research on somaclonal variation, plant transformation, and genome-editing tools that will contribute to creating genetically modified turfgrasses with improved agronomic traits.