Cryopreservation Protocols and the Associated Ultrastructural Changes in Dormant Buds of .

There is an urgent need for the cryopreservation of dormant buds to conserve the genetic resources of woody plants, particularly fruit trees, as this method is less time-consuming and relatively inexpensive. In the present study, three different cryopreservation protocols were tested on dormant buds from three varieties of Rupr. The explants were collected between November 2017 and March 2018. Twig segments harvested from field-grown plants, each containing one dormant bud, were desiccated in a low-temperature test chamber at -5 °C. The viability of the buds was highest (45%) after 28-30 days of desiccation, when the moisture content was approximately 25-30%. Cryopreservation using the CP3 protocol (which involves decreasing the temperature at a rate of 0.1 °C/min to -30 °C and holding this temperature for 24 h, followed by a 0.5 °C/min decline to -80 °C, a 1 °C/min decline to -180 °C, and finally reaching -196 °C in a CryoMed controlled rate freezer) significantly enhanced the viability (66.67%) when the samples were packed in aluminum-foil bags. Additionally, immersing the twigs in ice-cold (4 °C) water for 24 h in a refrigerator during thawing proved to be more conducive to viability. The dormant buds of all three varieties collected in January exhibited the highest viability after cryopreservation, followed by those collected in February and December. In contrast, the dormant buds collected in November and March showed the lowest viability after cryopreservation. The average viability of twigs of 'Shuanghong', 'Zuoshanyi', and 'Shuangfeng' collected between 2019 and 2021 all exceeded 60%. After the cryopreservation process, the outer multilayered cells in the buds were completely damaged; however, the inner cells exhibited moderate damage and were able to resume growth after thawing. Therefore, based on graft viability and histological observations, the dormant bud cryopreservation protocols tested in this study could be applicable to these three varieties.