Cryopreservation of Indigenous Plums and Monitoring of Multiplication and Rooting Capacity of Shoots Obtained from Cryopreserved Specimens

Abstract

The objective of this study is to assess the suitability of vitrification cryo-plate (V cryo-plate) and dehydration cryo-plate (D cryo-plate) methods for the long-term conservation of eight autochthonous Prunus domestica L. genotypes originating from the Balkan Peninsula region. In vitro shoot tips were briefly pre-cultured for 1 day at 23 °C in the dark on a medium containing 0.3 M sucrose and then embedded in calcium alginate gel within the wells of the aluminum cryo-plates. In the V cryo-plate protocol, dehydration was carried out at room temperature using the following vitrification solutions: original plant vitrification solution 2 (PVS2) and 90% PVS2 solution (for 20 and 40 min) and plant vitrification solution 3 (PVS3) (for 60 and 80 min). In the D cryo-plate protocol, desiccation was performed for 2, 2.5, or 3 h over silica gel at 23 °C. The effect of different treatments was evaluated by monitoring the regrowth of both non-frozen and cryo-preserved explants. After cryo-preservation, five genotypes achieved regrowth rates over 40% in at least one of the applied protocols, while two genotypes showed regrowth rates of around 10%. A significant improvement in regrowth success for all genotypes using both cryo-plate methods was achieved by pre-culturing shoot tips for 7 days on a medium containing 0.5 M sucrose in complete darkness at 4 °C. Shoots regenerated from cryo-preserved explants were further monitored in vitro. By the third subculture, they had not only regained but had even exceeded the multiplication capacity (index of multiplication, length of axial, and lateral shoots) of shoots regenerated from dissection controls. Following multiplication, the cryo-preserved shoots were successfully rooted and rooting ability was assessed by monitoring the percentage of rooting, number and length of roots, and height of rooted plantlets.

Optimizing the Droplet-Vitrification Procedure by Balancing the Cryoprotection and Cytotoxicity of Alternative Plant Vitrification Solutions Based on the Nature of Donor Plant Vigor

“…These alternative PVSs have been applied in more than 30 pieces of literature with or without direct comparison with PVS2 and PVS3 from 2009 to 2023, as listed in Table 1 and summarized in Table 2. These alternative PVSs have been applied mainly to DV and a few cases of vitrification [47,[51][52][53], EV [49,54], and V cryo-plate methods [55][56][57][58]. The usefulness of alternative PVSs was confirmed in encapsulation-based procedures, too.…”