Numerous environmental and endogenous factors affect the level of genetic diversity in natural populations. Genetic variability is the cornerstone of evolution and adaptation of species. However, currently, more and more plant species and local varieties (landraces) are on the brink of extinction due to anthropopression and climate change. Their preservation is imperative for the sake of future breeding programs. Gene banks have been created worldwide to conserve different plant species of cultural and economic importance. Many of them apply cryopreservation, a conservation method in which ultra-low temperatures (−135 °C to −196 °C) are used for long-term storage of tissue samples, with little risk of variation occurrence. Cells can be successfully cryopreserved in liquid nitrogen (LN) when the adverse effect of ice crystal formation and growth is mitigated by the removal of water and the formation of the so-called biological glass (vitrification). This state can be achieved in several ways. The involvement of key cold-regulated genes and proteins in the acquisition of cold tolerance in plant tissues may additionally improve the survival of LN-stored explants. The present review explains the importance of cryostorage in agronomy and presents an overview of the recent works accomplished with this strategy. The most widely used cryopreservation techniques, classic and modern cryoprotective agents, and some protocols applied in crops are considered to understand which parameters provide the establishment of high quality and broadly applicable cryopreservation. Attention is also focused on the issues of genetic integrity and functional genomics in plant cryobiology.
Development of polyploidy in Dendrobium 'Sonia' using colchicine and oryzalin is described. Five-month-old plantlets derived from protocorm-like bodies produced in vitro were used. These plantlets were immersed into a bubble bioreactor containing different concentrations of colchicine (0.05, 0.10 and 0.15% for 3 days) and oryzalin (14.40 and 28.90 µM for 4 days) together with Nonidet (P-40) as a surfactant. Plantlets were aerated to prevent hypoxia. Tetraploidy was successfully induced by 0.10 and 0.15% colchicine. Polyploidy levels were estimated using flow cytometry and then confirmed by chromosome counting, and through anatomical, cytological and morphological studies. The highest percentage (26.60%) of polyploids was induced with 0.15% colchicine. Oryzalin treatment did not yield tetraploid plantlets. The relative fluorescence intensity emitted by propidium iodide (PI), a fluorescent intercalating agent or a DNA-binding dye, was almost twofold higher in tetraploids and nearly fourfold higher in octaploid as compared to diploids. Plantlets with changed chromosome number were obtained at 0.05-0.15% colchicine concentrations (tetraploid induction) and 14.40 µM oryzalin concentration (mixoploid induction). The chromosome number was 2n = 38 in diploids and 2n = 76 in the tetraploids. The tetraploid and diploid plantlets exhibited significant differences in morphological, anatomical and cytological parameters. Stomata of tetraploid plantlets were longer than diploids. Survival rates from treatments were above 70%. In all treatments with colchicine and oryzalin, 0.10 and 0.15% of colchicine proved the most effective for generation of polyploid plantlets. This is beneficial for further breeding purposes of D. 'Sonia.' Oryzalin was found lethal. New ornamental varieties with improved traits can be produced by polyploidy using colchicine.
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