PARTIAL DEHYDRATION AND CRYOPRESERVATION OF CITRUS SEED.

N GRAIVER,; A CALIFANO,; N. E. ZARITZKY

Querétaro, México

Simposio; ISOPOW 11: 11th International Symposium on the Properties of Water; 2010

<!-- /* Style Definitions */ p.MsoNormal, li.MsoNormal, div.MsoNormal {mso-style-parent:""; margin:0cm; margin-bottom:.0001pt; mso-pagination:widow-orphan; font-size:12.0pt; font-family:"Times New Roman"; mso-fareast-font-family:"Times New Roman";} @page Section1 {size:612.0pt 792.0pt; margin:70.85pt 3.0cm 70.85pt 3.0cm; mso-header-margin:36.0pt; mso-footer-margin:36.0pt; mso-paper-source:0;} div.Section1 {page:Section1;} --> Three categories of seed storage behavior are generally recognized among plant species: orthodox, intermediate, and recalcitrant. Intermediate seeds cannot be stored in liquid nitrogen (LN) without a previous partial dehydratation process. The purpose of this study was to investigate the basis of the optimal hydration status for cryopreservation of citrus seeds (Citrus Sinensis and Citrus Paradisi) in liquid nitrogen. The water content (WC) of the seeds at the moment of immersion in LN must be regarded as the most critical factor in cryopreservation. To study tolerance to dehydration and LN exposure, seeds were desiccated by equilibration at 20ºC over seven saturated salt solutions providing a relative humidity (RH) spectrum between 11 and 95%. The WC of seeds at equilibrium (expressed as gH2O              g-1dry basis) was determined gravimetrically after oven-drying the seeds at 103ºC until constant weight. Sorption isotherms were determined and modeled using the simplified D´arcy and Watt model. Half of the seeds were directly tested for germination and the other seeds were wrapped in aluminum foil envelopes before direct immersion in LN. Frozen seeds were thawed during 5 min in a water-bath at 37ºC before the germination test. Germination was carried out placing the seeds on humid sand in closed plastic boxes at 25ºC. For each treatment, forty-eight seeds were used to assess seed survival. The lipid content of the seeds was measured by Soxhlet and the obtained values ranged between 33.6 and 36.4% weight (dry basis).   Seed desiccation sensitivity (WC50) was quantified by the quantal response model. Differential scanning calorimetry (DSC) thermograms were determined on cotyledon tissue of the tested species, at different moisture contents in the temperature range -120 to 100ºC. Enthalpies corresponding to ice melting peaks were measured in each case, and the unfrozen water content was determined. Samples of total seed lipid extract were analyzed in triplicate by DSC to confirm the identification of the endothermic lipid transitions in the thermograms.  The optimal desiccation RH for seed tolerance to LN exposure was 64-75% corresponding to water contents lower than 0.110 and 0.184 gH2O g-1dry weight for Citrus Sinensis and Citrus Paradisi respectively.