24. Willow seeds viability after imbibition, freezing and freeze-drying treatments

Abstract

The conservation of labile biomolecules/structures in biological, pharmaceutical and food sciences is generally performed in frozen or dehydrated systems. Vegetal germplasm is usually conserved as seeds, which are naturally dehydrated systems. Orthodox seeds (the most common) tolerate dehydration at low water contents (wc), and remain viable for several years upon conservation at 20 °C or at 4–8 °C. Willow seeds are orthodox but show some recalcitrant characteristics: the longevity does not follow the dehydration tolerance as is shown for orthodox seeds, and they lose viability in a few weeks at 20 °C. In practice, collected seeds are usually dehydrated at 20 °C for 3 h from wc 70% to 10–12% (dry basis) and then kept at −70°C, which highly increases storage time-dependant costs. The purpose of the present work was to analyze the conservation of willow (Salix nigra L.) seeds through freezing and freeze-drying. Seed imbibition with 45%w/v trehalose or polyethylene glycol 400 – PEG – or water were conducted for 16 h at 4 °C prior to freezing or freeze-drying, in order to introduce cryo- or dehydro-protective agents in the seeds by imbibition. A comparison with vacuum drying seeds was also performed. The germination rate was measured after each treatment. Water content, water activity (aw), and differential scanning calorimetry (DSC) determinations were conducted. The control seed pool (aw of 0.745; 13%db wc) retained 77% of its original germination capacity. Priming effect (Chojnowski et al., Seed Science Research 7 (1997) 323–331) was observed during seed imbibition with water and trehalose, but only 70% of the imbibed seeds with PEG germinated after imbibition, which can be related to a certain degree of PEG toxicity toward the seeds. After freezing, a great germination capability loss was observed in trehalose and water imbibed seeds; instead, PEG seeds maintained their ability to germinate. Water crystallization was observed in all the imbibed seeds (aw ∼ 0.97), and followed the increasing order: PEG Water crystallization was not observed in control seeds. Their thermograms showed a broad lipid melting (between −40 and 10 °C) and protein denaturation (between 55 and 110 °C). Since there are no major changes in protein denaturation after freezing of imbibed seeds, membrane damage could be the major responsible of the lower germination capacity observed after freezing and freeze-drying. After freeze-drying, a 30% decrease of the germination capability was observed in the PEG imbibed seeds in comparison to the frozen ones, arriving at aw of 0.269 and wc of 5%db. Water and especially trehalose imbibed seeds subjected to vacuum drying showed better germination capability with respect to the freeze-dried ones, revealing that both the amount of crystallized water and the presence of adequate protective agents were the main critical factors involved in willow seed conservation.