ARS | Publication request: Antioxidant and Anti-stress Compounds Improve Regrowth of Cryopreserved Rubus Shoot Tips

Submitted to: In Vitro Cellular and Developmental Biology - Plants
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: May 21, 2010
Publication Date: July 20, 2010
Citation: Uchendu, E.E., Muminova, M., Gupta, S., Reed, B.M. 2010. Antioxidant and Anti-stress Compounds Improve Regrowth of Cryopreserved Rubus Shoot Tips. In Vitro Cellular and Developmental Biology - Plants. 46:246-256.

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 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, and high molecular weight and other compounds. 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 (more than 40%), however all the tested antioxidants except one compound 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: Regrowth of plants after cryopreservation varies and resulting regrowth ranges from poor to excellent. Oxidative stress is a potential cause of damage in plant tissues. Antioxidants and anti-stress compounds may improve regrowth by preventing or repairing the damage. Lipoic acid (LA), glutathione (GSH), glycine betaine (GB) and polyvinylpyrrolidone (PVP) were tested on cryopreservation of shoot tips using the plant vitrification solution 2 (PVS2) protocol. Two in vitro grown blackberry cultivars were cold acclimated, and then cryopreserved in liquid nitrogen (LN). The antioxidant and anti-stress compounds were added at 4 critical steps of the protocol: pretreatment, loading, rinsing, and regrowth. Three out of the 4 compounds significantly improved regrowth of cryopreserved shoot tips. Regrowth ranged from 40-50% for controls to >80% for treated shoot tips. LA (4-8 mM) produced high regrowth at pretreatment, loading and rinsing for ‘Chehalem’ and at all steps for ‘Hull Thornless’. Recovery improved at all steps with GSH (0.16 mM) and GB (10 mM). PVP had a neutral or negative impact on regrowth. Overall addition of LA, GSH and GB improved regrowth by ~25% over the shoot tips cryopreserved using the regular PVS2 protocol (control). This study shows that adding antioxidants and anti-stress compounds during the PVS2-vitrification protocol improves regrowth of shoot cultures following cryopreservation.