Direct regeneration and morpho-histological study of de novo shoot development from leaf explants of Rhododendron mucronulatum Turcz.

Abstract

Abstract Rhododendron mucronulatum Turcz., a rare medicinal and ornamental deciduous multi-branched shrub native to northern parts of East Asia, is a valuable genetic resource for breeding and biotechnological producing bioactive metabolites. To create propagation system of R. mucronulatum, an efficient method of direct shoot regeneration from leaf explants based on thidiazuron (TDZ) and dark treatment was developed, and detailed histological analysis for revealing localization of cell competent to morphogenesis was carried out for the first time. The highest regeneration rate (75%) and number of shoots per explant (an average of 51.7) were achieved under 0.5 µM TDZ and dark treatment. The dark treatment was found to increase the regeneration rate more than two-fold compared to culturing under light conditions. The histological analysis showed direct organogenesis pathway. The competent to morphogenesis cells were located in epidermal tissue of leaf explants on adaxial side of petiole next to main vein. Two sites of beginning of TDZ-induced adventitious shoot formation were revealed: the shoot formation directly from epidermal tissues and shoot formation from de novoepidermis-derived protrusions. The formation of embryo-like structures was observed on the late morphogenesis stages. It was discovered that parenchymal and epidermal tissues of embryo-like structures were separated from explant tissue but its vascular bundle was connected with vascular bundle of explant. These structures gave rise to development of adventitious shoots. The result obtained can promote further establishment of efficient and stable systems for genetic transformation and biomass production of R. mucronulatum.