Cryopreservation of Malus Red Splendor Crabapple Shoots

  Objective   Cryopreservation technology was applied for the conservation of the ornamental Malus Red Splendor crabapple germplasm.

  Methods   Sucrose concentration and time for pretreatment as well as conditions for the subsequent loading, PVS2, and unloading treatments on the shoot tips of Malus spp. were studied. Under the selected preservation conditions, additional improvement on the post-freezing survival of the shoots by application of antioxidants (i.e., CAT, AsA, and GSH) and PCD inhibitors (i.e., Eth and SNP) was explored.

  Results   (1) The procedures employed for vitrification to preserve the shoots included cutting the histoponic seedlings into 4-5 mm segments containing terminal buds, inoculating the cut stems segments in a pre-culture medium containing 0.7 mol·L⁻¹ sucrose at 4 ℃ for 2 d, cutting the tips into approximately 1.5-2 mm in length and placing in a loading solution for 20 m at room temperature, treating with PVS2 solution for 90 m at 0 ℃ followed by immediate submerging in liquid nitrogen, removing the cryogenically frozen tips to rapidly thaw them in a water bath at 38 ℃ for 1 m, and rinsing twice with an unloading solution at room temperature with constant shaking for 10 m each time. The post-cryopreservation survival rate of the shoot tips was 66.58%, and 16.67% of them recovered to grow as normal. (2) The best improvement by the addition of various agents was found to be CAT at 200U·mL⁻¹, AsA at 600 μmol·L⁻¹, and GSH at 0.04 μmol·L⁻¹, which increased the post-freezing survival rate by 20.28%, 6.75%, and 27.61%, respectively, over control. On the other hand, the addition of Eth or SNP posted no significant effect on the tip survival.

  Conclusion   The survival rate of the post-cryopreservation Malus Red Splendor shoot tips could be further improved by the addition of GSH, CAT or AsA in appropriate concentration. GSH could most significantly raise the rate of viable plants after the cryopreservation from 16.67% to 41.39%.