Chromosome Doubling and In Vitro Regeneration of Southern Highbush Blueberry (Vaccinium spp.) from Leaves by Colchicine Treatment

Chromosome doubling is a conventional and effective strategy to enhance genetic variation and facilitate the breeding of high-quality, stress-tolerant varieties. Optimizing an efficient adventitious shoot regeneration system is a critical prerequisite for the stable in vitro induction of polyploids. In this study, leaf explants of the blueberry cultivar ES were inoculated onto woody plant medium supplemented with 2.0 mg·L −1 zeatin and 0.05 mg·L −1 indole-3-butyric acid, resulting in a maximum adventitious shoot regeneration frequency of 85.58%. Furthermore, following 5 days of dark pretreatment and subsequent cultivation under standard growth conditions (16-h photoperiod) for 30 days, the shoot regeneration frequency of leaf explants reached 98.52%, indicating that a defined period of dark pretreatment enhanced the shoot regeneration efficiency. Using the established efficient leaf regeneration system, leaf explants from two tetraploid southern blueberry cultivars, Legacy and O’Neal, were treated with 0.0375% (w/v) colchicine for up to 48 h. Four octoploid ‘Legacy’ plantlets and two ‘O’Neal’ plantlets were confirmed via flow cytometry and chromosome counting analysis. Compared with the tetraploid plants, the octoploid plants exhibited significantly reduced plant height and stomatal density, while the size of guard cells was significantly increased. Furthermore, stem diameter, vascular bundle diameter, and leaf thickness were all increased. These newly identified octoploid plantlets represent a valuable germplasm resource for the development of high-quality blueberry varieties.