The Effect of Seed Cryopreservation on the Development of Protocorms by the Hybrid Orchid Bratonia

The aim of this work was to study the influence of storage in liquid nitrogen on the viability of seeds of the hybrid orchid Bratonia and further development of its protocorms in vitro. Seeds were frozen in ampoules by direct immersion in liquid nitrogen and stored in the cryobank for a month. The germination rates of cryopreserved and control (nonfrozen) seeds did not differ and remained as high as 100%. The protocorms derived were cultured on the agar-solidified Murashige and Skoog nutrient medium (MS), half-strength MS and Knop media and also in Morel liquid medium. During the first 45 days of culturing, protocorms derived from cryopreserved seeds grew faster than control protocorms on the MS and half-strength MS media but, at longer culturing (496 days), the size of control protocorms was significantly larger. After 639 days of culturing, there was no difference in the amount of perished, budding, and newly formed protocorms obtained from cryopreserved and control seeds, except half-strength MS medium where the number of budding protocorms in the case of cryopreserved seeds was a little greater than in the control treatment. After seed cryopreservation, the frequency of budding and newly formed protocorms was greater on the agarized MS and in liquid Morel media. Cryopreservation had little effect on the subsequent growth of protocorms in vitro. The preferable nutrient media for culturing the protocorms have been suggested.     

Photochemical activities of two photosystems in Bratonia orchid protocorms cryopreserved by vitrification method

Functional activities of two photosystems in orchid-specific embryos (protocorms) of a tropical hybrid orchid Bratonia were investigated before and after their cryopreservation by vitrification method. The kinetics of light-induced absorbance changes at 830 nm was analyzed as indicator of P700 redox conversions; changes in the variable chlorophyll fluorescence served to indicate the oxidation-reduction changes of the primary acceptor Q_A. Untreated protocorms exhibited low photochemical activity of photosystem II (PSII). In freeze-treated Bratonia protocorms, examined immediately after thawing, photosynthetic electron transport was strongly inhibited. Nevertheless, the cells retained activities of noncyclic electron flow and of alternative electron transport pathways related solely to PSI. However, Bratonia protocorms subjected to deep-freezing lost the capability of P700 photooxidation during the first day of reculturing. Deep freezing of protocorms had virtually no effect on the kinetics of dark relaxation of chlorophyll variable fluorescence, when measurements were made immediately after thawing. Unlike chlorophyll fluorescence, the kinetics of dark reduction of P700⁺ in protocorms exposed to freezing-thawing was substantially modified compared to untreated protocorms. Two exponential components with half-decay times of 27 and 310 ms were distinguished in the kinetics of P700⁺ reduction in treated samples, whereas the absorbance relaxation attributed to P700⁺ reduction in untreated samples followed an exponential decay with a half-decay time of 24 ms. Despite the appearance of additional slow component in the kinetics of P700⁺ reduction, the dark relaxation of variable fluorescence remained unaltered after deep freezing of protocorms. This observation indicates that the freezing-thawing procedure caused partial disorders in linear electron transport between PSII and PSI. Apparently, the functional interactions among carriers in the electron-transport chain were disturbed between the plastoquinone pool and the PSI reaction center. It is concluded that the vitrification method applied to protocorm cryopreservation did not cause their immediate death, but the protocorms died later, on the first day after reculturing.