Asymbiotic seed germination and in vitro seedling development of the threatened orchid Hoffmannseggella cinnabarina

Hoffmannseggella cinnabarina has not been found in the wild for the last 70?yr in the State of S?o Paulo and, therefore, wild populations of this native orchid are thought to be extinct. This investigation studied seed storage at a low temperature, in vitro germination, and seedling development of H. cinnabarina in order to establish an optimized protocol for propagation, and thus assure species conservation. Seeds of different ages were incubated on Knudson C (KC), Murashige and Skoog, and Vacin and Went media with or without 1???M of N⁶-benzyladenine (BA) and exposed to either 12 or 16?h of light (30???mol?m^?2?s^?1 at 26?±?2°C). Seed surface sterilization was deleterious to 3-mo-old seeds and severely reduced the viability of the 4-mo-old seeds. More mature seeds were not affected by the sterilization procedure. In general, the germination of 4-, 5-, 6-, and 7-mo-old seeds increased when BA was added to the culture medium especially under 16?h of light. Germination rates were highest with 8- and 9-mo-old seeds, and application of BA failed to enhance germination rates further. Developmental studies revealed that this cytokinin reduced seed and protocorm mortality rates; however, protocorm development was negatively affected in its presence. Seedling development was more pronounced when KC medium with 16?h of light was used. Long-term seed storage at 4°C did not provide promising results. The protocol described in this study proved to be efficient and relevant to in vitro seed germination and initial development of H. cinnabarina, and thus will contribute to conservation of this orchid species.