Optimal hydration status for cryopreservation of intermediate oily seeds: Citrus as a case study

Abstract

Background and aims: The purpose of this study was to investigate the basis of the optimal hydration status for cryopreservation of intermediate oily seeds using Citrus as a model.

Methods: The relationships between equilibrium relative humidity (RH), seed water content, presence of freezable water as determined by DSC analysis, and germination percentage after immersion in liquid nitrogen (LN) were investigated in Citrus aurantifolia, C. grandis, C. madurensis and C. reticulata. The relationship between the lipid content of seeds and their unfrozen water content was also investigated.

Key results: Independent of their level of seed desiccation tolerance, the optimal desiccation RH for seed tolerance to LN exposure was 75-80 % in the four species studied. This optimal hydration status always coincided with that at which presence of frozen water could not be detected in seed tissues during the cooling/thawing process. The unfrozen water content of seeds was variable between species and negatively correlated to seed lipid content. Using the present data, those obtained previously in seven coffee species and those reported by other authors for five other species, a significant linear relationship was found between the lipid content and the unfrozen water content of seeds.

Conclusions: This study provides additional evidence that intermediate oily seeds do not withstand the presence of freezable water in their tissues during the cooling/warming process. Moreover, it offers two important applied perspectives: (1) independent of their level of desiccation tolerance, testing germination of seeds of a given oily seed species after equilibration in 75-80 % RH at 25 degrees C and LN exposure, gives a rapid and reliable evaluation of the possibility of cryopreserving whole seeds of this given species; (2) it is now possible to calculate the interval of water contents in which non-orthodox oily seeds of a given species are likely to withstand LN exposure as a function of their lipid content.

Fig. 1.

Evolution of water content of C. reticulata seeds with dehydration time under three different relative humidities. Each point corresponds to one seed.

Fig. 2.

Sorption isotherms of the four Citrus species studied at 25 °C. The line corresponds to the fitted pattern using the simplified D'arcy and Watt model proposed in Dussert et al. (1999).

Fig. 3.

Relationship between desiccation RH (%) and germination percentage after desiccation (open circles) or desiccation and LN exposure (filled circles) in C. aurantifolia, C. grandis, C. madurensis and C. reticulata.

Fig. 4.

DSC heating thermograms of (A) seed samples of C. reticulata at various water contents and of (B) a seed sample of C. grandis at 0·07 g H₂O g⁻¹ d. wt and a sample of total lipids extracted from dry C. grandis seeds.

Fig. 5.

Determination of the unfrozen water content of seeds of the four Citrus species studied as calculated by the x-intercept of the intersection between the two lines of regression which best fitted the relationship between water content and enthalpy of the melting transitions of seed samples dried to various water contents.

Fig. 6.

Relationship between the lipid content and the unfrozen water content of seeds of the four Citrus (open circles) species analysed in the present study, seven coffee (filled circles) species (Dussert et al., 2001), Azadirachta indica (Sacandé et al., 2000; filled square), pea (Vertucci, 1989a; open diamond), Quercus rubra (Pritchard and Manger, 1998; open triangle), Quercus robur (Sun, 1999; open square) and soybean (Vertucci, 1989a; filled triangle).