Efficient production of protocorm-like bodies and plant regeneration from flower stalk explants of the sympodial orchid Epidendrum radicans

Abstract

A simple and efficient micropropagation method was established for direct protocorm-like body (PLB) formation and plant regeneration from flower stalk internodes of a sympodial orchid, Epidendrum radicans. Small transparent tissues formed on surfaces and cut ends of flower stalk internodes on a modified half-strength Murashige and Skoog basal medium with or without thidiazuron (TDZ) after 1–2 wk of culture. In the light, the transparent tissues enlarged and turned into organized calluses on most of the explants. However, PLBs formed only on a medium supplemened with 0.45 μM TDZ within 2 mo. of culture. Sucrose, NH4NO3, and KNO3 were used in media to test their effects on PLB proliferation and shooting. The best response on number of PLBs per tube was 23.6 at 40 gl−1 sucrose, 825 mgl−1 NH4NO3, and 950 mgl−1 KNO3, and the highest number of PLBs with shoots was found at 10 gl−1 sucrose, 825 mgl−1 NH4NO3, and 950 mgl−1 KNO3. Homogenized PLB tissues produced by blending were used to test the effects of four cytokinins [TDZ, N6-benzyladenine (BA), zeatin-riboside, and kinetin] on PLB proliferation and shoot formation. The best responses on number of PLBs per tube, proliferation rate, and number of PLBs with shoots per tube were obtained at 4.44 μM BA, 0.28 μM zeatin-riboside, and 1.39 μM kinetin, respectively. Normal plantlets converted from PLBs on the same TDZ-containing medium after 1 mo. of culture. The optimized procedure required about 12–13 wk from the initiation of PLBs to plantlet formation. The regenerated plants grew well with an almost 100% survival rate when acclimatized in a greenhouse.