Effect of the desiccation tolerance and cryopreservation methods on the viability of Citrus limon L. Burm cv. Eureka seeds

Cryopreservation of the germplasm for long-term periods is of great importance to maintain the genetic resource. Argentina is one of the world's highest lemon producing country. The performance of different cooling/warming rates in the cryopreservation method of Citrus limon L. Burm cv. Eureka seeds and their influence on the interval of optimal moisture content in the desiccation stage were analyzed. Water sorption isotherm was determined and modeled using D'Arcy & Watt equation; it provided important information concerning the amounts of water associated to strong, weak and multimolecular binding sites along the sorption isotherm. Seeds tolerated a wide range of desiccation conditions (0.1<a_w<0.85) showing a high viability (>80%), however desiccation to 0.0526 g H₂O g^?1 d.b. (a_w = 0.0901) produced a significant loss of viability. Differential Scanning Calorimetry was used to identify the thermal transitions of lipids and water in the seed; enthalpies were used to calculate the unfrozen water fraction (0.19 g H₂O g^?1 d.b. corresponding to a_w = 0.64). Two cooling/warming rates were tested on desiccated seeds (0.11<a_w<0.85): i) 200 °C min^?1 (reached with seeds placed inside a closed cryogenic vial); ii) 1000 °C min^?1 (reached with aluminum-foiled seeds placed in a perforated cryogenic vial). For both methods, viability was maximum (83.3%) at a_w = 0.64. Lethal ice formation was responsible for the loss of viability at a_w>0.64 corresponding to the unfrozen water fraction. The use of higher cooling/warming rates enables a wider range of desiccation conditions (0.33<a_w<0.76) in cryopreservation procedures. This work contributes to the optimization of cryopreservation methods of economically important germplasm.