PHYSIOLOGY Antioxidant and anti-stress compounds improve regrowth of cryopreserved Rubus shoot tips Esther E. Uchendu & Magfrat Muminova & Sandhya Gupta & Barbara M. Reed Received: 4 August 2009 /Accepted: 24 May 2010 /Published online: 9 June 2010 / Editor: D. T. Tomes # The Society for In Vitro Biology 2010 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 poly- vinylpyrrolidone (PVP) were tested during 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 four critical steps of the protocol: pretreatment, loading, rinsing, and regrowth. Three out of the four compounds significantly improved regrowth of cryopreserved shoot tips. Regrowth ranged from 40% to 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 non-vitamin antioxidants and anti-stress compounds during the PVS2-vitrification protocol improves regrowth of shoot cultures following cryopreser- vation. We recommend inclusion of antioxidants as part of standard cryopreservation protocols. Keywords Blackberry . Lipoic acid . Germplasm . Glutathione . Glycine betaine . Long-term storage . Oxidative stress . Polyvinylpyrrolidone Introduction The development of efficient cryopreservation protocols for vegetatively propagated species is of key importance to the long-term conservation of clonally propagated germplasm collections (Reed 2008). Cryopreservation provides stable, long-term, low-cost storage of plants safe from diseases or environmental damage. Storing plants at low temperatures does have some deleterious effects. Chilling and freezing injury promote many sub-lethal changes such as metabolic uncoupling, which can then lead to increased production of E. E. Uchendu Department of Horticulture, Oregon State University, 4017 Ag Life Sciences Bldg., Corvallis, OR 97331-7304, USA M. Muminova Department of Biotechnology, Tashkent Chemical-Technology Institute, 23 Navoyi str., Tashkent 100011, Uzbekistan S. Gupta Tissue Culture and Cryopreservation Unit, National Bureau of Plant Genetic Resources, Pusa Campus, New Delhi 110 012, India B. M. Reed (*) United States Department of Agriculture-Agricultural Research Service, National Clonal Germplasm Repository, 33447 Peoria Rd, Corvallis, OR 97333-2521, USA e-mail: Barbara.Reed@ARS.USDA.GOV Present Address: E. E. Uchendu Department of Plant Agriculture, Ontario Agriculture College, University of Guelph, E.C. Bovey Bldg. RM 4227, Guelph, ON N1G 2W1, Canada In Vitro Cell.Dev.Biol.—Plant (2010) 46:386–393 DOI 10.1007/s11627-010-9292-9