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A procedure has been developed for the cryopreservation of wheat female gametes. The procedure involves loading the cells with 25% concentrated vitrification solution consisting of 30% glycerol, 10% sucrose, 120 mM ascorbic acid (AA) and 5% propylene glycol (PG), dehydration in 80% concentrated vitrification solution, droplet vitrification and storage in liquid nitrogen, unloading and rehydration of the cells by gradual addition of isolation solution. Supplementation with AA significantly increased the proportion of viable egg cells after deand rehydration. During the early phase of rehydration AA reduced the probability of membrane damage caused by rapid water uptake. Maintaining the temperature of the cells at 0℃ during the de- and rehydration processes increased cell survival. Microscopic examination of the semi-thin sections of untreated and viable cryopreserved cells revealed that the vitrification process might cause changes in cell structure.
Zygotes fertilized in planta developed into fertile plants in vitro. Microspore cultures and ovaries derived from the same species were tested as nurse cells. With both types of feeder system about 20% of the isolated zygotes were able to regenerate into plants. The morphology, cytological properties and development rhythm of the zygotes resembled those of the in vivo course of zygotic development, except that the first division appeared symmetrical in contrast to the asymmetrical division observable in planta. The results indicate that ovaries may have the same nurse effect as microspores on zygotes cultured in vitro. Using ovaries as a nurse system is much less time-consuming than using isolated microspore cultures.
Moderate heat stress may provide protection against a subsequent severe high temperature stress in plants. However, the exact mechanisms of heat acclimation of wheat are still poorly understood. In the present work, two wheat varieties Ellvis and Soissons were exposed to a moderate elevated temperature at 30 °C, and the changes of certain protective mechanisms were investigated. Although the differences in the proline level between the genotypes were not substantial, it was approx. 2–3 times higher in the heat-treated plants than in the controls. After exposure to moderate elevated temperature, the activities of ascorbate peroxidase and catalase were also induced. Similarly, the amount of the free salicylic acid also increased after moderate heat stress, independently on the genotypes. The amount of the main polyamines, namely, putrescine, spermidine, and spermine either did not change or decreased after the same period. However, heat acclimation increased the level of 1,3-diaminopropane, in parallel with a polyamine oxidase gene, TaPAO. While the expression level of the peroxisomal polyamine oxidase gene TaperPAO hardly changed, TaPAO showed a substantial increase after 1 day, especially in Soissons, and at the end of the heat treatment was still significantly higher than in the controls. These suggest that signalling processes related to polyamine metabolisms or salicylic acid-related processes might also contribute to the higher heat tolerance induced by moderate heat stress. The variations in recorded measurements were mainly temperature dependent, and the effect of genotype was less pronounced than the effect of moderate heat treatment itself.
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