Plant regeneration via direct and callus-mediated organogenesis from leaf explants of Chirita swinglei (Merr.) W. T. Wang

Plant regeneration via direct shoot organogenesis and callus-mediated organogenesis from leaf explants of Chirita swinglei (Merr.) W. T. Wang was studied. Cytokinins played a crucial role in the direct formation of adventitious shoots, which originally appeared as small nodular protuberances and developed into adventitious shoots as culture period increased. These protuberances were similar to somatic embryos in external morphology, but their anatomical structure confirmed that these were shoots and not somatic embryos. The highest frequency of shoot buds was induced by 2.0 mu M thidiazuron (TDZ) and 2.5 mu M 6-benzyladenine (BA). Leaf explants produced the highest frequency of shoot buds (100%) on MS medium supplemented with both 2.0 mu M TDZ and 2.5 mu M BA. In the presence of 2.0 mu M TDZ, leaf explants became swollen after culture for 15 d. Some shoot buds were observed after 20 d of culture. Shoot buds were clearly visible as culture period was extended from 35 to 45 d. Histological analysis revealed the presence of meristematic tissues coincident with shoot tips. Callus could also be induced from leaves when alpha-naphthaleneacetic acid (NAA) was used alone or in combination with TDZ and BA. Three types of callus, pink and friable, white and compact, and green-yellow and compact, formed, but only the latter two could differentiate into plantlets. Over 90% of plantlets survived after transplanting into sand or a mixture of sand, loessal clay, and vermiculite (1:1:1, v/v). This protocol provides an efficient method via two organogenic pathways, to mass produce and conserve C. swinglei, an attractive ornamental plant and important medicinal herb.