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Background: The extension of the blueberry industry in China is restricted by the low performance of proliferation coefficient, transplanting survival rate and the long cycle production. Hypothesis: We hypothesized the suitable medium with optimal concentration and type combination would improve the survival rate of Vaccinium corymbosum. Species studied: Vaccinium corymbosum L. cultivar ‘O’Neal. Study site and years of study: Our study was conducted in Yunnan Breeding and Cultivation Research and Development Center of Endangered and Daodi Chinese medicinal materials, Yunnan University of Chinese Medicine since November 2015. Methods: Efficient protocol of plant regeneration about Southern highbush blueberry (cultivar ‘O’Neal) was established using annual shoots via single factor and orthogonal experiments. Results: Olive medium with 2.0 mg·L-1 zeatin was found to be most suitable for initiation culture. The highest callus induction and bud formation rate were determined with 93.67 % and 95.57 %, respectively. Furthermore, as the germination of axillary buds, numerous shoots were formed with the proliferation coefficient (> 60). Well-developed shoots were obtained using rejuvenation culture on half-strength Olive medium supplemented with combination of 2.0 mg·L-1 indole-3-butyric acid, 1.0 mg·L-1 naphthaleneacetic acid, 0.01 mg·L-1 chlormequat chloride and 0.5 g·L-1 activated charcoal. The rooting rate could reach to 100 % using 2 cm length of well-developed shoots transferred into the 5:1 sterilized peat:perlite, which was enhanced from 27.61 up to 95 % after transplanting to the field. Conclusions: An efficient system for plant regeneration from bud induction to transplanting to the field was established to maintain the characteristics of southern highbush blueberry varieties.