Abstract
Limitations in tissue culture and regeneration techniques present significant barriers to the development of improved blueberry (
Vaccinium
spp.) cultivars, restricting the adoption of tissue culture in commercial production. This challenge emphasizes the urgent need for a standardized regeneration protocol. Addressing this, we developed enhanced in vitro regeneration protocols for six highbush blueberry (
Vaccinium corymbosum
L.) cultivars—Emerald, NC5288, Rowan (NC3104), Legacy, Pinnacle, and Jewel—each with varying levels of tissue culture recalcitrance. We evaluated the effects of auxins, specifically α-Naphthaleneacetic Acid, cytokinins [including trans-Zeatin (ZT), 1-(2-Chloro-4-pyridyl)-3-phenylurea 98.0+%, TCI America™ (CPPU), and 6-γ-γ-[Dimethylallylamino]-purine (2iP)], and Thidiazuron (TDZ), which exhibits both auxin- and cytokinin-like activities, in various plant species cultured in vitro, on callus induction and regeneration from leaf explants. Media supplemented with ZT + 2iP (PZ9, PZ10) consistently outperformed all other treatments, enhancing regeneration significantly across all cultivars with high genotype-independent reproducibility, particularly in treatment PZ10. In contrast, TDZ-based treatments yielded highly variable results and were ineffective in recalcitrant cultivars such as Jewel and Pinnacle. ZT + CPPU treatments performed moderately across all metrics and may offer utility for cultivars with intermediate responsiveness. These findings provide critical insights into refining tissue culture techniques, offering a foundation for improving blueberry cultivar development and efficient genome editing. This work represents the first published report of in vitro regeneration for the highbush blueberry cultivars Rowan (NC3104), NC5288, and Pinnacle, expanding the scope of possibilities for advancing blueberry crop improvement.