Establishment and optimization of agrobacterium-mediated transformation in blueberry (Vaccinium species)

Abstract

Vaccinium species such as blueberry, cranberry and bilberry are widely planted in the world and have high economic values. Current varieties are mostly generated by conventional breeding methods, and application of biotechnology especially genetic modification in the breeding process is seldom. Moreover, mechanistic studies of orphan genes in Vaccinium species are more and more important to facilitate the improvement of yield, quality, resistance to abiotic and biotic stresses in breeding, which requires efficient genetic transformation systems. In this study, we select 19 Vaccinium varieties to investigate the effects of variety specificity, explant types and culture schemes on callus development, adventitious bud regeneration and transformation efficiency. The wood plant medium (WPM) contains sufficient nutrients for the effective callus induction from mature leaves of plants grown in the field. Both zeatin and indole-3-butyric acid with the concentration of 2.0 mg L−1 and 1.0 mg L−1, respectively, are critical for adventitious bud regeneration from a variety of Vaccinium leaves. Different Vaccinium varieties have distinct regeneration efficiencies from leaf discs, and the diploid wild Vaccinium reticulatum ‘Red Button’ has the highest efficiency of callus formation and adventitious bud regeneration. Furthermore, we establish an efficient Agrobacterium-mediated genetic transformation system using ‘Red Button’, in which the transformation efficiency of green fluorescent protein (GFP) encoding gene is about 12.82%. Taken together, our study establishes an efficient tissue culture and regeneration system from leaves of Vaccinium varieties, and identify the diploid material ‘Red Button’ as an ideal variety for genetic transformation, which would greatly accelerate the mechanistic studies and breeding program of Vaccinium genus using emerging plant biotechnologies in the future.