Location: National Clonal Germplasm RepositoryTitle: Vitamins C and E Improve Regrowth and Reduce Lipid Peroxidation of Blackberry Shoot Tips Following Cryopreservation Author
Submitted to: Plant Cell Reports
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 10/15/2009
Publication Date: 2/15/2010
Citation: Uchendu, E., Leonard, S.W., Traber, M.G., Reed, B.M. 2010. Vitamins C and E Improve Regrowth and Reduce Lipid Peroxidation of Blackberry Shoot Tips Following Cryopreservation. Plant Cell Reports. 29:25-35. Interpretive Summary: Stresses such as cold temperatures can decrease the survival of plant tissues. Antioxidants could improve plant recovery following freezing. We studied the effects of vitamin E (Vit E) and vitamin C (Vit C) added at four points during the process of freezing shoot tips in liquid nitrogen (-320 F) (pretreatment, loading, rinsing, recovery medium). Shoot tips of two blackberry cultivars were cryopreserved. Vit E added to the pretreatment medium for 48 h prior to liquid nitrogen exposure or to the rinse solution following rewarming improved recovery of shoots compared to those without Vit E. Recovery of shoot tips treated with Vit C was better than untreated tissues. Recovery of shoot tips on standard regrowth medium with Vit C was reduced compared to the recovery of untreated shoot tips. Combinations of Vit E and Vit C was no different from treatment with Vit C alone. We recommend adding Vit C to the pretreatment medium. This is the first use of antioxidant vitamins to improve recovery of plants from freezing.
Technical Abstract: Oxidative processes involved in cryopreservation protocols may be responsible for the reduced viability of tissues after liquid nitrogen (LN) exposure. Antioxidants that counteract these reactions should improve recovery. This study focused on oxidative lipid injury and the effects of exogenous vitamin E (tocopherol, Vit E) and vitamin C (ascorbic acid, Vit C) treatments on regrowth at four critical steps of the PVS2 vitrification cryopreservation technique; pretreatment, loading, rinsing, and regrowth. Initial experiments showed that Vit E at 11-15 mM significantly increased regrowth at all 4 steps. There was significantly more malondialdehyde (MDA) (P<0.001), a lipid peroxidation product, at each of the steps than in fresh untreated shoot tips (control). Vit E uptake was assayed at each step and showed significantly more a- and '-tocopherols in treated shoots than those without added Vit E (P<0.001). Vit E added at each step significantly reduced MDA formation and improved shoot regrowth. Vit C (0.14-0.58 mM) also significantly improved regrowth of shoot tips at each of the steps compared to the controls. A combination of Vit E (11 mM) and Vit C (0.14 mM) produced significantly higher regrowth than the control or Vit E alone. Vit C alone was not significantly different from the combination, suggesting that the benefit was mostly due to Vit C. We recommend adding Vit C (0.14 mM) to the pretreatment medium, the loading solution or the rinse solution as the most convenient for the PVS2 vitrification process. This is the first report of the application of vitamins for improving cryopreservation of plant tissues by minimizing oxidative damage.