Cryopreservation Protocols and the Associated Ultrastructural Changes in Dormant Buds of Vitis amurensis

Abstract

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 Vitis amurensis 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 V. amurensis 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 V. amurensis varieties.

Keywords: Vitis amurensis; conservation of resources; dormant buds; structural changes; viability.

Figure 1

The lowest temperature profile at the orchard of the Jilin Agricultural University (latitude 43°86′; longitude 125°28′), Changchun, China from November 2017 to March 2018.

Figure 2

The average temperature at the orchard of the Jilin Agricultural University (latitude 43°86′; longitude 125°28′), Changchun, China. (A) The average temperature from November 2017 to March 2018. (B) The average temperature of January in 2019, 2020, and 2021.

Figure 3

Cryopreservation process of the dormant twigs of V. amurensis. (A) Twig desiccation at 5 °C in a refrigerator; (B) desiccated twigs were vacuumed packed with aluminum-foil bag; (C) desiccated twigs frozen in a CryoMed controlled rate freezer; (D) desiccated twigs cryopreserved in LN; (E) cryopreserved twigs after thawing; (F) twigs that survived after grafting for two months.

Figure 4

Effects of different drying times on the moisture content in twigs of ‘Shuanghong’ collected in January 2018 and the viability of the DBs after the cryopreservation of V. amurensis. Note: Mean values in each point with the same letter were not significantly different at p < 0.05 by the Duncan’s multiple range test.

Figure 5

Effects of different thawing methods on the viability of dormant buds of ‘Shuanghong’ collected in January 2018 after cryopreservation. Note: Mean values with the same letter were not significantly different at p < 0.05 by the Duncan’s multiple range test.

Figure 6

Ultrastructural observations of meristem cells in V. amurensis buds. (A,B) The buds before desiccation; (C,D) the buds desiccated for 28 d; (E,F) the buds after cryopreservation. Abbreviations: cm: cell membrane; cw: cell wall; lb: lipid body; m: mitochondrion; n: nucleus; nu: nucleolus; p: proplastid; s: starch; v: vacuole.