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United States Department of Agriculture

Agricultural Research Service

Research Project: MANAGEMENT OF TEMPERATE FRUIT NUT AND SPECIALTY CROP GENETIC RESOURCES

Location: National Clonal Germplasm Repository (Corvallis, Oregon)

Title: Improving Shoot Tip Cryopreservation with Antioxidant and Antistress Compounds

Authors
item Reed, Barbara
item Uchendu, Esther -
item Muminova, Magfrat -
item Traber, Maret -

Submitted to: In Vitro Cellular and Developmental Biology - Plants
Publication Type: Abstract Only
Publication Acceptance Date: February 15, 2010
Publication Date: June 10, 2010
Citation: Reed, B.M., Uchendu, E., Muminova, M., Traber, M. 2010. Improving Shoot Tip Cryopreservation with Antioxidant and Antistress Compounds. In Vitro Cellular and Developmental Biology - Plants. 46:S280.

Interpretive Summary: Storage in liquid nitrogen (cryopreservation) is successful for many plant types and techniques have improved so that now many germplasm collections are being stored. Multiple stresses are involved in cryopreservation protocols and can reduce regrowth after warming. Adding antioxidants that counteract these stresses should increase regrowth. Shoot tips of blackberries were cryopreserved and antioxidants were added at four critical steps. Vitamin E , vitamin C , and several other antioxident chemicals were tested . Regrowth of blackberry shoot tips treated with the compounds had increased regrowth at all four steps. Shoot tip regrowth of both blackberry cultivars increased from 45% for the untreated controls to 92% with vitamin C treatments. Adding antioxidants protected the cultures from oxidative stress during cryopreservation. We recommend adding vitamin C during the cryopreservation process for improved regrowth after cryopreservation.

Technical Abstract: Cryopreservation techniques have improved so that now many germplasm collections are being stored. Multiple stresses are involved in cryopreservation protocols and can reduce cryopreservation success. Adding antioxidants that counteract the oxidation induced by these stresses should increase regrowth. Shoot tips of in-vitro grown blackberry cultivars were cryopreserved using a PVS2 vitrification protocol. Antioxidants were added at four critical steps: pretreatment, loading, rinsing, and regrowth. Vitamin E (Vit E), ascorbic acid (AA), polyvinylpyrrolidone (PVP), glutathione (GSH), lipoic acid (LA) and glycine betaine (GB) were tested over a range of concentrations. At each step a lipid peroxidation product, malondialdehyde (MDA), was detected at significantly higher concentrations (P<0.001) than in fresh untreated shoot tips. Shoot tips treated with Vit E had the same low MDA concentrations as the control shoot tips and contained significantly more Vit E. Regrowth of shoot tips treated with Vit E (11.04 & 14.72 mM), AA (0.14-0.58 mM), GB (10 mM) or GSH (0.16 mM) had significantly increased regrowth at all four steps. LA (4 mM) was very effective in the first three steps. 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 AA treatments. Increases in regrowth were highest with AA, however all except PVP improved regrowth by 25 to 30%. Adding antioxidants protected in vitro cultures from oxidative stress during cryopreservation. We recommend adding AA (0.28 mM) to the pretreatment or loading stage for improved regrowth after cryopreservation.

Last Modified: 7/30/2014
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