Antifreeze Protein Improves the Cryopreservation Efficiency of Hosta capitata by Regulating the Genes Involved in the Low-Temperature Tolerance Mechanism

In this study, whether the addition of antifreeze protein (AFP) to a cryopreservative solution (plant vitrification solution 2 (PVS2)) is more effective in reducing freezing injuries in Hosta capitata than PVS2 alone at different cold exposure times (6, 24, and 48 h) is investigated. The upregulation of C-repeat binding factor 1 (CBF1) and dehydrin 1 (DHN1) in response to low temperature was observed in shoots. Shoots treated with distilled water (dH2O) strongly triggered gene expression 6 h after cold exposure, which was higher than those expressed in PVS2 and PVS2+AFP. However, 24 h after cold exposure, gene expressions detected in dH2O and PVS2 treatments were similar and higher than PVS2 + AFP. The expression was highest in PVS2+AFP when the exposure time was extended to 48 h. Similarly, nitric reductase activities 1 and 2 (Nia1 and Nia2) genes, which are responsible for nitric oxide production, were also upregulated in low-temperature-treated shoots, as observed for CBF1 and DHN1 expression patterns during cold exposure periods. Based on the gene expression patterns, shoots treated with PVS2+AFP were more likely to resist cold stress, which was also associated with the higher cryopreservation efficiency of PVS2+AFP compared to PVS2 alone. This finding suggests that the improvement of cryopreservation efficiency by AFP could be due to the transcriptional regulation of CBF1, DHN1, Nia1, and Nia2, which might reduce freezing injuries during cryopreservation. Thus, AFP could be potentially used as a cryoprotectant in the cryopreservation of rare and commercially important plant germplasm.

Fall Ethephon Application Enhances the Freezing Tolerance of Magnolia wufengensis During Overwintering

Magnolia wufengensis L.Y. Ma et L. R. Wang, a rare species which has been introduced and cultivated from southern China to northern China, frequently suffers from freezing injuries. To figure out the influence of ethephon (ETH) application on the cold tolerance during the natural overwintering of M. wufengensis, one-year shoots subjected to ethephon application at four concentrations (0, 700, 1000, and 1500 mg·L−1) were collected to measure the physiological and biochemical changes from September 2017 to 1 April 2018. The fall ETH application increased the freezing tolerance of M. wufengensis, and the optimum concentration for M. wufengensis was 1000 mg·L−1 (T2), which not only improved the shoot freezing tolerance by 1.4 times, but also led to a 25.0% faster cold acclimation rate and 13.7% slower de-acclimation rate. Moreover, 1000 mg·L−1 ETH delayed bud-burst in the spring by 10. 7 d, which was helpful for effectively avoiding cold spells in the spring, and improved the rates of bud survival by 47.1%. The improved freezing tolerance under exogenous ETH application was associated with an increased dehydration and accumulation of proline content. It seems that exogenous ETH application may be used on M. wufengensis grown in northern China to protect against freezing tolerance during the overwintering period.

Freezing injuries to flower buds and their influence on yield of apricot (Prunus armeniaca L.) and peach (Prunus persica L.)

Szymajda M., Pruski, K., Żurawicz, E. and Sitarek, M. 2013. Freezing injuries to flower buds and their influence on yield of apricot ( Prunus armeniaca L.) and peach ( Prunus persica L.). Can. J. Plant Sci. 93: 191–198. Each spring, for 3 consecutive years, 2009–2011, the degree of freezing injury to overwintering flower buds of selected cultivars and clones of apricot and peach was evaluated at the Dąbrowice Research Institute of the Horticulture E$xperimental Orchard (District of Skierniewice, Poland). Apricot flower buds were damaged less severely than those of peach during the winter of 2009/2010 when the lowest temperatures (maximum drop in temperature) coincided with the full dormancy stage of the trees. Conversely, peach flower buds were less damaged than buds of apricot when the maximum drop in temperature occurred after the late winter thaw in both 2008/2009 and 2010/2011. Under Polish climatic conditions, peach proved to be more reliable than apricot in consistency to produce fruit. Results have shown that the most hardy apricot and peach cultivars can develop very strong winter hardiness allowing the trees to survive temperatures as low as −28°C, as long as there are no severe temperature fluctuations during late winter.

Optimising vitrification of human oocytes using multiple cryoprotectants and morphological and functional assessment

Oocyte vitrification is a clinical practice that allows preservation of fertility potential in women. Vitrification involves quick cooling using high concentrations of cryoprotectants to minimise freezing injuries. However, high concentrations of cryoprotectants have detrimental effects on oocyte quality and eventually the offspring. In addition, current assessment of oocyte quality after vitrification is commonly based only on the morphological appearance of the oocyte, raising concerns regarding its efficiency. Using both morphological and functional assessments, the present study investigated whether combinations of cryoprotectants at lower individual concentrations result in better cryosurvival rates than single cryoprotectants at higher concentrations. Surplus oocytes from IVF patients were vitrified within 24 h after retrieval using the Cryotop method with several cryoprotectants, either individually or in combination. The morphological and functional quality of the vitrified oocytes was investigated using light microscopy and computer-based quantification of mitochondrial integrity, respectively. Oocyte quality was significantly higher using a combination of cryoprotectants than vitrification with individual cryoprotectants. In addition, the quality of vitrified oocyte varied depending on the cryoprotectants and type of combination used. The results of the present study indicate that observations based purely on the morphological appearance of the oocyte to assess the cryosurvival rate are insufficient and sometimes misleading. The outcome will have a significant implication in the area of human oocyte cryopreservation as an important approach for fertility preservation.

Frost Dehardening and Rehardening of Hydrangea macrophylla Stems and Buds

Hydrangea macrophylla is a popular and commercially important flowering shrub, but frost injury of buds and current-year shoots is a common problem in some of its cultivars. As a result of climate warming, temperate winters are becoming progressively milder, and temperature patterns are becoming increasingly irregular with an increased frequency of warm spells. Warm spells may induce premature dehardening, increasing the risk of subsequent freezing injuries. This study investigated cold-hardiness of stems and buds of Hydrangea macrophylla ssp. macrophylla (Thunb.) Ser. ‘Alma’ during dehardening in response to simulated warm spells and subsequent rehardening in January and early March. Plants were acclimated in the field and dehardened in the greenhouse at controlled warm temperatures for various durations. Dehardened plants were rehardened for up to 12 days in an unheated greenhouse (January) or in the field (March). Buds of H. macrophylla were slightly less cold-hardy than stems. In both stems and buds, the dehardening resistance and the rate of dehardening were influenced by temperature, but buds appeared to be less resistant to dehardening and dehardened faster than stems. In stems, dehardening proceeded faster in March than in January, and the capacity of the stems to reharden seemed reduced, indicating that both dehardening and rehardening were influenced by the progression of winter. Results of this study indicate that buds of H. macrophylla are more sensitive to frost injury than stems and the vulnerability of stems to frost injuries, caused by an unstable temperature regime, changes during the winter season.

Freezing injuries in the embryos of Piaractus mesopotamicus

SummaryCryopreservation of mammal embryos has been technically feasible for many years, but morphological injuries still persist in fish embryos during cryopreservation. Thus, the objective of the present study was to describe these freezing injuries in Piaractus mesopotamicus embryos. Two hundred and twenty-five embryos were collected at the post-gastrula stage and assigned into four treatments of sucrose at 8.5, 17.0, 25.0 or 34.0% plus 9.0% methanol. The control was prepared with distilled water only. The gradual decrease in the temperature was 0.5°C/min. After the seeding stage, the fish embryos were stored in liquid nitrogen at −33°C. Thereafter, they were thawed for evaluating per cent hatching, and the samples collected from every treatment were submitted to scanning electron microscopy for morphological analysis. The micrographic images showed that there was substantial alterations in embryo morphology under the highest concentrations of sucrose. These solutions did not prevent the formation of ice crystals, which lead to deformities and killed the embryos, but the observed reduced level of morphological structure in these embryos when treated with 17.0% sucrose plus 9.0% methanol is a compelling argument for additional studies.