Shoot organogenesis and somatic embryogenesis in leaf tissue of Pulsatilla tongkangensis Y.N. Lee & T.C. Lee

Leaf material explants of Pulsatilla tongkangensis Y. N. Lee & T. C. Lee were used to regenerate plants of this endangered species by somatic embryogenesis and organogenesis from meristematic nodules, induced by MS medium supplemented with zeatin (Zn) and indole-3-acetic acid (IAA). Globular structures were induced on the surface of the explants after 2 weeks and after 6–7 weeks of culture, multiple shoots developed from the nodules. Morpho-histological analysis of light green globular, heart-shaped structures resembling somatic embryos revealed, however, that these were organogenic, with strongly vacuolated parenchymatous cells surrounded by a single layer of epithermal cells, and tracheid elements, but no root pole. Milky-white callus also developed around the nodules after 4–6 weeks. Morpho-histological analysis of the globular, heart-, and torpedo-shaped stages of regenerants present in this callus confirmed the development of somatic embryos in the milky-white structures, characterized by deeply staining, small cells with rich cytoplasm, very little vascular tissue in the developing embryos, and no vascular connection with the surrounding callus. The highest rooting frequency (93.33%) was achieved on MS medium containing 1.5 mg/l NAA. Plantlets were acclimatized and successfully transferred to pots. Our results provide a plant regeneration system with potential for germplasm conservation of endangered plants and the rapid propagation and molecular breeding of P. tongkangensis .