In vitro propagation of piper hispidum by somatic embryogenesis in leaf explants

Purpose: A protocol for regeneration of P. hispidum through somatic embryogenesis in leaf explants is described. Under aseptic conditions, the leaves were cut into 1.0 cm2 explants. These explants were inoculated into test tubes with 10.0 mL of an Murashige and Skoog (MS) basal culture medium supplemented with 30.0 g L-1 sucrose, 6.0 g L-1 agar and a factorial combination of the growth regulators 2,4-Dichlorophenoxyacetic acid (2,4-D) and 6-Benzylaminopurine (BA), both at 0.0, 1.0, 2.0, 3.0 and 4.0 mg L-1, totalizing 25 treatments. The pH was adjusted to 5.8 and the medium autoclaved at 121°C for 20 minutes. After 75 days, the average number of cotyledonary somatic embryos in each explant was recorded.Maximum number of 9.2 cotyledonary embryos per explant was observed with the combination of 2.0 mg L-1 BA + 2.0 mg L-1 2,4-D. The subculture of the cotyledonary embryos to a medium without growth regulators resulted in 95% conversion into plantlets, which were acclimatized with 100% survival.