Location: National Clonal Germplasm RepositoryTitle: Antioxidants improve regrowth of cryopreserved in vitro shoot tips Author
Submitted to: Society for Cryobiology Meeting
Publication Type: Abstract Only
Publication Acceptance Date: 6/15/2009
Publication Date: 12/15/2009
Citation: Uchendu, E., Muminova, M., Traber, M., Reed, B.M. 2009. Antioxidants improve regrowth of cryopreserved in vitro shoot tips. Society for Cryobiology Meeting. 59:386. Interpretive Summary: Cryopreservation, storing plant tissues in liquid nitrogen (-320°F), causes many types of stress for the cells. The addition of antioxidants that counteract oxidation induced by these stresses should improve plant regrowth. In this study we added antioxidants at four critical steps of the cryopreservation process using shoots of two tissue-cultured blackberry cultivars. Antioxidants tested were vitamin E, vitamin C, polyvinylpyrrolidone, glutathione, lipoic acid and glycine betaine. Regrowth of liquid-nitrogen stored blackberry shoots after rewarming increased from ~45% for standard cryopreservation to as high as 92% when antioxidant were added. Increases in regrowth were best with ascorbic acid (40%), however all the tested antioxidants except polyvinylpyrrolidone improved regrowth of the cryopreserved shoot tips. These data present experimental evidence to support the concept that antioxidants protect shoot cultures from oxidative damage during cryopreservation.
Technical Abstract: Multiple stresses involved in cryopreservation protocols reduce the regrowth of shoot tips after rewarming. The addition of antioxidants that counteract oxidation induced by these stresses should improve plant regrowth. This study focused on the effects of exogenous antioxidant treatments at four critical steps of the PVS2 vitrification technique (Pretreatment, Loading, Rinsing, and Regrowth). Shoots of two in vitro grown blackberry cultivars were cold acclimated prior to cryopreservation in liquid nitrogen and tips (0.8 -1 mm) cryopreserved using a PVS2 vitrification protocol. Antioxidants tested were vitamin E (Vit E), ascorbic acid (AA), polyvinylpyrrolidone (PVP), glutathione (GSH), lipoic acid (LA) and glycine betaine (GB). Vit E (11.04 & 14.72 mM) significantly increased regrowth (P<0.001) following cryopreservation at all the steps tested. Malondialdehyde (MDA), a lipid peroxidation product, was detected in shoot tips at significantly higher concentrations at each of the steps compared to fresh untreated shoot tips. Shoot tips treated with Vit E had significantly higher (P<0.001) quantities of Vit E than control shoot tips and low MDA concentrations, similar to control shoot tips. AA (0.14-0.58 mM) significantly improved regrowth of shoot tips at each of the steps. AA in the standard regrowth medium decreased regrowth when compared with the controls, however, iron-free regrowth medium with AA produced improved regrowth similar to the other steps. GSH (0.16 mM) significantly increased regrowth at all four steps. LA (4 mM) in the pretreatment, loading and rinsing solutions were very effective but addition at regrowth was not. GB (10 mM) also improved regrowth at all four steps compared to the controls. Only PVP had a negative or neutral effect at all steps. Shoot tip regrowth of both blackberry cultivars increased from ~45% for standard PVS2 vitrification to 92% with antioxidant treatments. Increases in regrowth were highest with AA, however all the tested antioxidants except PVP significantly improved regrowth of the cryopreserved shoot tips. These data present experimental evidence to support the concept that exogenous antioxidants protect in vitro cultures from oxidative stress during cryopreservation.