Morphological and anatomical insights into de novo shoot organogenesis of in vitro ‘Delite’ rabbiteye blueberries

Blueberries are valued for their taste and their high nutritional benefits, including their antioxidant and anti-inflammatory properties. In vitro culturing is an alternative method for clonal propagation, and has been used in many biotechnological studies. Most blueberry research is concentrated on highbush and lowbush taxa (Vaccinium corymbosum and Vaccinium angustifolium respectively), with only limited investigations of rabbiteye cultivars (Vaccinium virgatum) that are more suitable for subtropical climates and regions with warmer winters as a result of climate change. There is therefore a need to determine in vitro protocols for that species and group of cultivars. We examined here adventitious shoot regeneration in the ‘Delite’ rabbiteye blueberry cultivar. Leaf explants were cultured in vitro in Woody Plant Medium (WPM), and the effects of different thidiazuron (TDZ) concentrations, the orientation of the leaf (adaxial or abaxial surface in contact with the medium), and two portions of the leaf segment (basal or apical) were examined. De novo shoot development was studied using light and scanning electron microscopy. All concentrations of TDZ used showed similar survival and regeneration rates; 0.5 μM TDZ showed high efficiency in regenerating adventitious shoots (100%, with 57 adventitious shoots/explant), as did the adaxial surface in contact with the medium using either the apical or the basal portion of the leaf (97% shoot regeneration, 47.5 adventitious shoots/explant). Anatomical analyses showed direct and indirect organogenesis. The shoots developed leaf primordia with stomata, trichomes, and well-developed vascular tissues, with further elongation and rooting of the plants. We therefore describe here a high-efficiency regeneration method through de novo shoot organogenesis using TDZ in foliar explants of rabbiteye blueberry, with direct and indirect organogenesis.