Effect of proline pretreatment on grapevine shoot-tip response to a droplet-vitrification protocol

Proline has been shown to accumulate in plants in response to biotic and abiotic stresses. Exogenous proline has thus been used for improving some plant cryopreservation protocols. Further enhancement of cryopreservation efficiency for in vitro grapevines could be expected if stresses linked to cryopreservation procedures could be reduced. We therefore studied the possible beneficial effect of proline in grapevine cryopreservation. Single-node explants from in vitro grown grapevine plantlets (Vitis vinifera L. cv Portan) were cultured on shooting media (half-strength MS + 1 μM BAP) con- taining no proline (control) or 50, 500, or 2000 μM filter-sterilized L-proline. Shoot tips excised from these micro- shoots were subjected to a PVS2-based droplet-vitrification procedure. Control and rewarmed explants were grown on a recovery medium containing 1 μM BAP. Shoot development on control medium and lower proline concentrations did not notably differ whereas the highest concentration of proline inhibited shoot development. Carry-over effects were observed since lower survival and regrowth were obtained both for non-frozen or LN-treated explants excised from micro-shoots obtained on the 2000 μM proline medium. No significant differences in survival and regrowth were ob- served for non-frozen explants subjected to pretreatment without LN exposure. A slightly enhancing effect (although non-significant) on post-cryopreservation survival was observed for explants derived from shoots developed on 50 or 500 μM proline, but no significant improvement of regrowth percentage was observed for these two conditions. Al- though a slight increase in survival could be observed, no significant beneficial effect of proline pretreatment on post- cryoconservation regrowth could be evidenced in our conditions. However, the 2-week period before explant excision could have allowed at least partial metabolism and catabolism of exogenous proline; the results observed could thus have been the consequence of complex interactions. Shorter proline treatments applied closer to the actual LN exposure step might produce different results and allow for clearer interpretation