Location: National Clonal Germplasm RepositoryTitle: Vitamins C and E Improve In Vitro Recovery of Cryopreserved Blackberry Shoot Tips) Author
Submitted to: CryoLetters
Publication Type: Abstract only
Publication Acceptance Date: 8/16/2009
Publication Date: 12/14/2009
Citation: Uchendu, E., Reed, B.M. 2009. Vitamins C and E Improve In Vitro Recovery of Cryopreserved Blackberry Shoot Tips. CryoLetters. 31:180. 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.
Technical Abstract: Regrowth of blackberry, Rubus, shoot tips after cryopreservation is often less than optimal. Oxidative processes involved in cryopreservation protocols may be responsible for the reduced viability of tissues after liquid nitrogen (LN) exposure. We hypothesized that recovery would be improved by antioxidants that counteract these reactions. We studied four critical steps of the PVS2 vitrification cryopreservation technique; pretreatment, loading, rinsing, and regrowth to determine the amount of oxidative lipid injury and the effects of vitamins on shoot regrowth. Two blackberry cultivars, ‘Chehalem’ and ‘Hull Thornless’, were selected for study because they had moderate (40-60%) regrowth after standard PVS2 vitrification. Shoot tips treated with Vit E and assayed at each step showed greatly increased a- and '-tocopherols compared to those without added Vit E or for control in vitro or field grown plants. Vitamin E (tocopherol, Vit E) at 11-15 mM added at each of the 4 steps increased regrowth after cryopreservation by 20-30%. Lipid peroxidation measured by the production of malondialdehyde (MDA) was 2 to 4 times higher at each of the first three steps than in fresh untreated shoot tips (control). Shoot tips treated with Vit E had low MDA formation, similar to the controls, at each step and improved shoot regrowth after cryopreservation (80%) compared to the controls (50%). Vitamin C (ascorbic acid, Vit C) (0.14-0.58 mM) when added at each of the steps also significantly improved regrowth of cryopreserved shoot tips (90%) compared to the controls (50%). Shoot tips recovered on standard Rubus regrowth medium that contained double MS iron and with Vit C had significantly reduced recovery (<20%) compared to untreated shoot tips (50%). Regrowth medium without iron and with Vit C improved regrowth to levels similar to the first three steps. Treatments with a combination of Vit E (11 mM) and Vit C (0.14 mM) produced significantly higher regrowth than the untreated control or Vit E alone, but Vit C alone was not significantly different from the combination. Both blackberry cultivars had improved recovery with the vitamin treatments. These studies with Vit E determined that lipid peroxidation is involved in the death of plants during the cryopreservation process. This is the first application of antioxidant vitamins for improving cryopreservation of plant tissues. 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.