Successful storage of protocorm-like bodies of hybrid Cymbidium (Orchidaceae) under low temperature conditions

Low temperatures result in lower metabolic cellular activity, thus slowing down cell division and growth. This is advantageous where a plant scientist might seek to store important germplasm without the risks associated with low temperature storage. In this study, two cold temperatures above freezing, namely 4 and 10 °C, were tested to assess for how long PLBs could be preserved without a significant loss in regeneration ability (i.e., the ability to form neo-PLBs). Control treatments were cultured at 25 °C on Teixeira Cymbidium (TC) medium at a 16-h photoperiod at a photosynthetic photon flux density (PPFD) of 45 μmol m⁻² s⁻¹. For the cold treatments, each was replicated in the dark and at low light intensity (12-h photoperiod and a PPFD of 10 μmol m⁻² s⁻¹). All cultures were sub-cultured six times onto fresh medium every 60 days, for approximately 1 year. On the 7th subculture, all neo-PLBs were prepared uniformly and replated onto standard TC medium under light conditions described above for the control. 45 days after the 7th subculture and just before subcultures 1–6, the number of neo-PLBs per half-PLB was measured. The number of neo-PLBs that formed under different treatments depended strongly on the temperature and light conditions with most neo-PLBs forming under control conditions, although that number dropped significantly as the temperature was dropped to 10 °C and then even more to 4 °C, the same trend being observed when explants were cultured and subcultured under dim light, with organogenesis being more strongly negatively influenced in darkness. For all low-temperature treatments, as well as the dimmed light and darkness treatments, the number of neo-PLBs increased significantly when recultured, on the 7th subculture, onto control TC medium under control environmental conditions, almost as high as the control values. In contrast, the control values decreased, with significantly fewer neo-PLBs by the 7th subculture relative to the control, indicating that new PLBs should be induced from shoot cultures at least once a year to maintain their vitality.