Cryopreservation of Shoot Tips: Antioxidant Investigations with Rubus and Protocols for Mentha and Vaccinium

approved: ____________________________________ Barbara M. Reed Oxidative stress that occurs during cryopreservation may be reduced by the addition of antioxidants. Vitamin E (Vit E), Vitamin C (Vit C), glutathione (GSH), lipoic acid (LA), glycine betaine (GB) and polyvinylpyrrolidone (PVP) were applied at four steps of the PVS2 vitrification protocol (pretreatment, loading, rinsing, and regrowth). Shoot tips of in-vitro grown blackberry cultivars „Chehalem‟ and „Hull Thornless‟ were cryopreserved. Malondialdehyde (MDA), a lipid peroxidation product was higher in shoot tips at each step compared to controls. Shoot tips treated with Vit E had low MDA similar to the controls. GB, GSH, Vit E and Vit C significantly increased regrowth at all steps. Vit C added to regrowth medium with double Murashige and Skoog iron concentration decreased recovery; however, in iron-free medium Vit C improved regrowth. LA was not very effective in regrowth medium. PVP was not effective at any step. Regrowth was highest (up to 92%) with Vit C. This is the first report of vitamins C, and E, GB, and LA use in protecting cultures from oxidative damage during cryopreservation. Mentha species [M. canadensis L., M. australis R.Br., Mentha x piperita citrata (Ehrh.) Briq. and M. cunninghamii Benth,] were cryopreserved using controlled cooling (CC), encapsulation dehydration (ED), and PVS2 vitrification (VIT). Recovery of Mentha x piperita citrata and M. australis showed significant differences among protocols with CC > VIT > ED. M. canadensis and M. cunninghamii recovery with CC and ED was significantly better than VIT. All species responded to CC (93%) better than ED (71%) or VIT (73%). Cranberry (Vaccinium macrocarpon Aiton) cultivars „Wilcox‟ and „Franklin‟ and blueberry (Vaccinium corymbosum L.) „Berkeley‟, „O‟Neal‟ and „Brigitta‟ were tested for desiccation tolerance and recovery after cryopreservation. Blueberry cultivars were desiccation tolerant after 7 h of drying under laminar air flow while cranberry cultivars were mostly dead by 3 h. Cryopreserved blueberry cultivars had 83% to 92% regrowth with ED. The results varied from 33% to 87% for VIT and from 50% to 67% for CC. Cranberry cultivars had poor regrowth with these protocols but improved by 20-30% with Vit C added at any of the four PVS2 steps.