Seeds are remarkable organs that are essential in the life of humans and animals since they ensure plant reproduction. They are the natural forms of preservation of higher plants, designed to perpetuate the species through the germination process. They are also used as direct sources of food or in various industries such as oil, starch, and flour manufacturing and malting. Seed storage is the best technology for preserving and conserving plant biodiversity. For agronomical use, seeds must be stored under conditions that maintain high seed quality (i.e., viability and vigor) ^{[1][2][3][4][5][6][7]}[1,2,3,4,5,6,7].

Decline in seed viability and seed quality (i.e., germinability and vigor) depends on their tolerance to dehydration and is regulated by three important factors: seed moisture content in equilibrium with the relative humidity of the atmosphere, the storage temperature, and the gaseous environment ^{[1][4][5][6][7][8][9][10][11][12]}[1,4,5,6,7,8,9,10,11,12]. For the majority of the species classified as orthodox (i.e., seeds that tolerate dehydration [13]), a study found that seed longevity increased when reducing the seed moisture content and decreasing the temperature ^{[1][2][7][10][11][14][15]}[1,2,7,10,11,14,15]. However, for species classified as recalcitrant, i.e., seeds that do not tolerate dehydration ^[13][14][16][13,14,16], seed longevity is generally short—from a few weeks to a few months. Such seeds must be stored at high moisture content (between a 20 and 70% fresh weight basis) and at a temperature between 7 and 17 °C and −3 and 5 °C for species of tropical origin and those of temperate climate origin, respectively [17].

Oxidative damage due to ROS’ (reactive oxygen species) reactivity towards cell macromolecules, including proteins, sugars, lipids, and nucleic acids, has been demonstrated to be associated with ageing ^{[1][3][7][12][18][19][20][21][22][23][24][25]}[1,3,7,12,18,19,20,21,22,23,24,25]. However, a better understanding of the cellular, biochemical, and molecular mechanisms associated with ageing could lead to the identification of new markers of the loss of viability during storage.

2. Biological Categories of Seeds

There exist two main biological types of seeds that have been termed “orthodox” and “recalcitrant” seeds [13]. The majority of seeds referred to as orthodox require desiccation tolerance during their development, allowing them to be stored for long periods under air-dry storage ^[3][26][3,26]. On the other hand, recalcitrant seeds have a high moisture content at shedding and do not tolerate desiccation; therefore, they are able to be stored in a dry state ^{[18][27][28][29]}[18,27,28,29]. More recently, a third category of seeds, named “intermediate”, has been defined ^[30][31][30,31]; they survive the loss of water, but they become damaged and die during dry storage at low temperatures. The Compendium of Information on Seed Storage Behaviour ^[17][32][17,32] recognizes these three biological types. “Orthodox” seeds generally undergo dehydration prior to shedding; they are desiccation tolerant and can be stored successfully in a dehydrated state at a temperature below freezing for very long periods (decades or longer). At maturity (or harvest), they contain no more than 10–15% water and survive drying to very low moisture content (3–5%). They therefore tolerate subsequent storage at sub-zero temperatures [33]. Orthodox seeds acquire desiccation tolerance relatively early during their development and usually before the maturation drying phase. Several metabolic changes occur with respect to the protection of seed cells against dehydration damage ^{[18][26][33][34][35]}[18,26,33,34,35]. In particular, carbohydrate metabolism ^[36][37][36,37] and specific proteins (dehydrins, late embryogenesis abundant proteins: LEA, and heat shock proteins: HSPs) [33] seem to be involved in this process. Some soluble sugars, such as sucrose and oligosaccharides (raffinose, stachyose, and verbascose), might also play an important part in this process by facilitating the stabilization of lipids and proteins in cell membranes or by promoting the vitrification of water and then the protection of cytosolic structures ^[35][37][38][35,37,38]. “Recalcitrant” seeds are desiccation intolerant. They are highly hydrated at shedding and do not survive drying below a moisture content between 30–65% depending on the species [39]. They include seeds mostly produced by tropical or subtropical species from fruit crops: Litchi chinensis (litchi), Euphorbia longan (longan), Garcinia mangostana (mangosteen), Mangifera indica (mango), and Nephelium lappaceum (rambutan), species used as beverages: Cacao theobroma (cocoa) and Coffea robusta (coffee), plantation crops: Hevea brasiliensis (rubber), Elaeis guineensis (oil palm), and Cocos nucifera (coconut), and many timber species belonging to the Dipterocarpaceae family (Table 1) ^{[27][39][40][41]}[27,39,40,41]. They also concern temperate species such as Quercus spp. (oak), Juglans spp. (walnut), Castanea spp. (chestnut), Corylus avellana (filbert), and Salix spp. (willow) (Table 1). These species are highly hydrated at shedding and cannot survive drying below a certain critical moisture content ^[28][42][28,42] (Table 2, ^{[43][44][45][46][47][48][49][50][51][52][53][54][55][56][57][58][59][60]}[43,44,45,46,47,48,49,50,51,52,53,54,55,56,57,58,59,60]). Sensitivity to dehydration is often different for the whole seed and isolated embryos. For example, seeds of Araucaria angustifolia die when the water content decreases to 25% [46], while isolated embryos die when the water content decreases to about 40% [47]. In natural conditions, these seeds rapidly die if they cannot germinate as soon as they fall to the ground. Such seeds cannot be stored by means of conventional methods and place constraints on our ability to store them, both in the long term for germplasm conservation and in the short term for seed trade or storage from one season to the next ^[61][62][61,62]. recommend −3 °C to maintain viability and avoid germination. The storage temperature must not be below −3 °C for silver maple and sycamore. Another characteristic of recalcitrant seeds, in particular seeds of tropical origin, is that many of them are chilling sensitive [63]. Table 3 shows that tropical recalcitrant seeds of Hevea brasiliensis, Hopea odorata, Mangifera indica, Shorea roxburghii, and Symphonia globulifera do not tolerate temperatures below 12–15 °C, while seeds of Dryobalanops aromatica and Shorea talura are less sensitive to low temperatures and tolerate 5 °C. In the case of temperate species, the high moisture content of the seeds does not allow storage at negative temperatures. Sycamore seeds are completely killed at a temperature below −26 °C, while acorns of pedunculate oak with a moisture level of 40–45% fresh weight basis are killed at −7–−9 °C. Suszka et al. [55] “Intermediate” seeds: The term intermediate is used for seeds that do not behave exactly like orthodox or recalcitrant seeds. They tolerate a greater degree of dehydration than typical recalcitrant seeds but are less tolerant to dehydration than usual orthodox seeds. When dry, such seeds lose viability more rapidly at 0 °C or −20 °C than at warmer temperatures such as those around 15 °C ^[30][31][30,31]. Coffea spp. ^[30][31][30,31], oil palm (Elaieis guineensis) [64], Carica papaya [65], and Azadirachtaidica (neem) [66] seeds belong to this category. It is important to underline that there is wide variation in the desiccation sensitivity of intermediate seeds that has been evaluated or quantified by the water content at which half of the initial viability is lost, i.e., within the genera Coffea ^[67][68][67,68]. The repartition of the three categories of seeds (orthodox, intermediate, and recalcitrant) depends on the family. At the family level, the percentage of recalcitrant seeds varies from 77.1–80.0% in the Lauraceae and the Fagaceae to less than 2% in the Melastomataceae, with it being around 65.4% in the Sapotaceae, 42.1–48.8% in the Clusiaceae and the Moraceae, and 25.8–31.0% in the Arecaceae and Sapindaceae ^[1][69][1,69]. All of these families also present orthodox seeds: 3.8% (Sapotaceae), 17.4% (Fagaceae), 27.8% (Aracaceae), 50–51.2% (Maraceae and Rutaceae), 57.9% (Clusiaceae), 63.1% (Sapindaceae), and 83.9% (Myrtaceae). The majority of angiosperms (Asteraceae, Brassicaceae, Caryophyllaceae, Cyperaceae, Ericaceae, Gentianaceae, Poaceae, Ranunuculaceae, Scrophylariaceae, and Solanaceae) have 95–100% orthodox seeds [69]. The intermediate seeds characterize some species such as the Arecaceae, the Rutaceae, the Sapotaceae, and the Zingiberaceae [69].

3. The Lifespan of Seeds

The lifespan or longevity of seeds varies greatly depending on the species; some survive for very long periods of time in the soil or under ambient conditions whereas others die rapidly—surviving less than 3 years. Ewart [70] in a treatise entitled “On the longevity of seeds” arbitrarily divided seeds into three classes according to their period of survival under natural conditions: macrobiotic, mesobiotic, and microbiotic seeds. Macrobiotic seeds are defined as seeds capable of surviving for more than 15 years in the soil or under ambient conditions, but seeds with longevity exceeding 50 or 100 years are far from rare. They are often hard seeds (i.e., seeds with seed coats impermeable to water). This has been shown by Becquerel [71] who succeeded in germinating very old seeds from the collections of the Natural History Museum of Paris and estimated their longevity as 55 years (Melilotus lutea), 63–68 years (Cytisus austriacus, Lavatera pseudo-olbia, Ervum lens, and Trifolium arvense), 81–86 years (Mimosa glomerata, Cytisus biflorus, and Astragalus massiliensis), and a period of 100–158 years (Cassia multijuga and Dioclea pauciflora) [1]. Evidence from hundreds to thousands of years of seeds has been described [1]. Lotus (Nelumbo nucifera, the Indian lotus) seeds are also well known for their longevity of several hundred years [1]. For example, Dum [72] also achieved the germination of Chenopodium album and Spergula arvensis seeds that were about 1700 years old. The literature reveals that the record is held by Lupinus arcticus seeds which successfully germinated despite being more than 10,000 years old [73]. However, such long longevity must be regarded with skepticism without direct dating of the seeds ^[1][34][1,34]. Radiocarbon dating allows us to determine the age of seeds at about 2000 years for the date (Phoenix dactylifera) [74], at 1300 years for lotus (Nelumbo nucifera) [75], and at 600 years for canna (Canna compacta) [76]. The myth concerning the longevity of mummy grains discovered in Egyptian tombs and supposed to remain able to germinate is in fact a mistake or a hoax ^[1][34][1,34]. Mesobiotic seeds have a lifespan of 3 to 15 years. The great majority of the species fall under this category ^[1][77][1,77]. They include species with a longevity of 3–5 years, such as rape, bean, pea, carrot, cyclamen, and nasturtium, or longer (5–15 years) such as celery, sugar-beet, cabbage, chicory, and cereals (wheat, oat, and barley). Microbiotic seeds survive at most 3 years under natural conditions. All recalcitrant seeds (see Table 1) and orthodox oleaginous seeds exhibit such short longevity. In this group, we can cite seeds of vegetables (onion, leek, fennel, and parsley) or ornamental species (dahlia, delphinium, petunia, and viola) ^[1][77][78][1,77,78]. Although this classification gives information concerning the putative survival behavior of numerous species, it is debatable because it does not take into account the main factors of storage (temperature and seed moisture content). Indeed, seed survival is both genetically and environmentally controlled. Depending on the conditions of storage, microbiotic seeds could become mesobiotic or macrobiotic ones.

4. Loss of Seed Viability

4.1. Change in Viability during Storage: Viability Equations

At harvest, the initial seed viability is a product of the seed history through development on the mother plant ^[1][34][1,34]. Subsequent seed longevity depends on post-harvest treatments (drying, cleaning, sorting, coating, etc.) and the conditions of storage (temperature, moisture content, and oxygen availability) [34]. The viability equations are based on fitting a negative cumulative normal distribution to viability percentages. The conversion of the negatively sigmoidal curve obtained to probits linearizes the curve ^{[1][2][34][78]}[1,2,34,78] (Figure 1). Roberts and Coll ^{[13][79][80][81]}[13,79,80,81] developed and generalized a descriptive equation, known as the “viability equation”, taking into account the conditions of seed storage on the loss of viability: where v represents the probit % germination after p days of storage; Ki is the probit of initial germinability for the seed lot; K_E, C_w, C_H, and C_Q are constants depending on the species; m is the seed moisture content (expressed on a fresh weight basis); and t is the storage temperature in °C. This equation indicates that longevity increases as the storage temperature decreases. Dickie et al. [82] working on eight species (barley, chickpea, cowpea, soybean, elm, mahogany, terb, and lettuce) placed over a wide range of storage conditions (temperature from −13 to 90 °C; moisture content from 1.8 to 25% fresh weight) demonstrated that the temperature coefficients (C_H and C_Q) of the equation do not differ significantly between these species, with them being equal to 0.0329 and 0.000478, respectively, i.e., the effect of temperature on longevity appears to be similar. In contrast, the effect of moisture content on longevity evaluated by the coefficient C_W differs between species ^[83][84][85][83,84,85]. This variation in C_W results in part from differences in seed composition; for example, the higher the seed oil content, the lower the value of C_W ^[1][2][1,2]. Data obtained by Ellis et al. ^[84][85][84,85] indicate that longevity is doubled for each 8.4–8.7% reduction in seed equilibrium in relative humidity between 90 and 10% [2]. Roberts ^[13][79][13,79] derived a simple mathematical equation, allowing us to calculate the P₅₀ value or half-viability period. where P₅₀ is the time taken for 50% of the seed population to lose viability, m is the moisture content expressed on a fresh weight basis, t is the temperature in °C and K_v, and C₁ and C₂ are constants. Figure 1 shows a theoretical curve of loss of viability in probit during ageing, and Table 4 gives some examples of an estimated half-viability period of seeds from various cultivated species stored under open storage conditions in a temperate climate with the mean temperature at about 10 °C and the average RH at about 60–75% ^[1][86][1,86]. Under these conditions, the P₅₀ value varies from 3.4–4.1 years (parsley, parsnip, and celery) to 10.5–15.9 years (lucerne, French bean, garden pea, and broad bean) and 24.5 years for tomato. Generally, seeds containing a high amount of starch (e.g., cereals) can be stored well (7.2–12.9 years), but this is not the case for rye (4.5 years). Seeds containing high levels of oil are often considered to be relatively short-lived, but this link between the amount of seed oil and P₅₀ is debatable: for example, tomato seeds that contain a high level of oil (more than 30%) have high longevity (P₅₀ = 24.5 years) whilst onion seeds that are poor in oil (often less than 10%) are difficult to store (a P₅₀ of about 5.4 years). On the other hand, oily seeds such as soybean and sunflower are characterized by a short P₅₀ (3.4 and 5.4 years, respectively), while the P₅₀ of rape seeds is about 13.9 years. It is often difficult to compare the data concerning seed longevity across species and seed lots because the conditions of storage are different depending on the laboratory. For example, when seeds were stored at 5 °C with 5 ± 2% water in the USDA National Seed Storage Laboratory, the P₅₀ calculated [86] was higher than that obtained in open storage (as indicated in Table 4). It reaches 53 years for Helianthus annuus (sunflower), 36 years for Secale cereale (rye), 30 years for Glycine max (soybean), 22 years for Lactuca sativa (lettuce), and 18 years for Allium cepa (onion) and is longer than 80 years for Medicago sativa (lucerne), Pisum sativum (garden pea), and Lycopersicon esculentum (tomato). Hay et al. ^[87][88][87,88] discussed and described the most widely adopted protocols that are used to measure seed longevity, in particular, a “comparative longevity protocol” established at the Millennium Seed Bank (MSB) of the Royal Botanic Gardens, Kew [89]. This protocol suggests choosing 60% RH and 45 °C; these conditions allow one to obtain data within an acceptable length of time.

4.2. Modulation of Viability by Storage Conditions

As indicated by the “improved viability equation”, loss of seed viability is mainly regulated by seed moisture content and temperature during storage. The storage of orthodox seeds follows two rules ^{[1][2][4][7][34][79][80]}[1,2,4,7,34,79,80]:

-

For each 1–2% decrease in seed moisture content (when the MC ranges between 5 and 14%), the seed storage life is doubled;

-

For each 10 °F (5.6 °C) decrease in seed storage temperature (between 0 °C and 50 °C), the seed storage life is doubled.

However, maximum seed longevity is achieved at a critical low moisture content limit for the application of the viability equation, and drying the seeds below this critical value does not improve seed longevity ^{[8][30][84][85][90][91]}[8,30,84,85,90,91]. It varies among species and depends on the seed composition, in particular the lipid content and temperature. It is for example about 2% (fresh weight basis) in Arachis hypogea [85] and 6.2% in Pisum sativa [84], and it varies in an inverse relationship with the lipid content ^[8][84][85][8,84,85]. Maximum survival after 4–5 years at ambient temperature was determined to be in the range of 1.8–2.5% for sesame, 4.3–5% for soybean, and 7.6–9.7% for wheat [92]. In addition, this critical moisture content also decreases with increasing temperature, with it ranging between 3 and 4% at high temperatures and 4 and6% at ambient temperature [93]. The availability of oxygen (i.e., hermetic vs. open storage) can also influence seed viability during storage ^[94][95][96][94,95,96]. Oxygen is generally detrimental to seed viability maintenance, but the beneficial effect of low oxygen or anaerobic conditions depends on the conditions of ageing. Ellis and Hong [95] indicate that this negative effect increases as the seed moisture content decreases. Storage in N₂ (i.e., in the absence of oxygen) is advantageous for pea, broad beans, and barley [97], but it has no significant effect on cabbage, onion, and red clover [98]. In the case of non-primed and primed lettuce seeds stored in low RH (33%), longevity was extended in an anaerobic environment, but this effect was less under storage in controlled deterioration (75% RH, 50 °C) [96]. In onion, this beneficial effect of anaerobic conditions is only observed in primed seeds [96].

4.3. Procedures for Long-Term Storage in Genebanks

Improving seed storage techniques is a major research focus of the International Plant Genetic Resources Institute (IPGRI). Guidelines for long-term conservation recommend storage at −20 °C ± 4 °C and 15 ± 3% RH, considered a conventional method [99]. Cryopreservation is also a possible technique to prolong the longevity of orthodox seeds with short lifespans [100]. In contrast, recalcitrant and intermediate seeds require cryopreservation ^{[7][101][102]}[7,101,102]. To achieve the appropriate moisture content, Kew recommends equilibrating the seeds to 15% RH at 15 °C (www.rbgkew.org.uk/, accessed on 1 November 2023), Ellis et al. ^[30][84][85][30,84,85] suggest equilibrating the seeds to 10% RH at 20 °C, and Vertucci and Roos ^[90][103][90,103] propose to equilibrate the seeds at 20–25% RH at storage temperature. Storing seeds in hermetically sealed containers to maintain their water content is also recommended. Most orthodox seeds also show long longevity when ultra-dried or freeze-dried. One advantage of ultra-drying or freeze-drying is that seeds can be stored at room temperature, but they must be maintained under vacuum in tightly closed containers or bags impermeable to water vapor. Table 5 shows that freeze-drying gives excellent results with the seeds of some vegetable species compared to storage under ambient conditions ^[62][77][62,77]. However, large seeds, such as pea, soybean, and bean, can be damaged by freeze-drying, with cracks in the cotyledons occurring at thawing or during seed re-imbibition. The results obtained with vacuum-dried seeds (seeds dried under vacuum for 3 days at 20 °C) and freeze-dried seeds (seeds immersed in liquid nitrogen and placed under vacuum for 3 days at 20°) and then stored under vacuum in the presence of silica gel for 12–19 months at 5, 20, and 30 °C clearly indicated that the responsiveness of ultra-dried seeds to storage depends on the species and the temperature of storage. Ultra-dried seeds of lettuce and leek remained viable after 12 months of storage although the germination rate of leek seeds and freeze–dried lettuce seeds was slightly reduced. In onion and lamb’s lettuce seeds, ultra-drying resulted in a marked decrease in seed viability after 12 months of storage, and this deleterious effect was reinforced by increasing the temperature of germination. In addition, ultra-dried seeds stored for 12 months were more sensitive to accelerated ageing treatment (40–45 °C, 100% RH) suggesting that the seeds became progressively less vigorous during storage.

Table 5. Comparison of the longevity of seeds from some vegetable species stored in the open air or freeze-dried and stored under vacuum at ambient temperature. C, control non-freeze-dried seeds; FD, freeze-dried seeds. Modified from ^[62][77][62,77].

Species			Germination (%) after Storage for
	0 (Harvest)		4 Years		8 Years		12 Years		20 Years
	C	FD	C	FD	C	FD	C	FD	C	FD
Allium cepa	92	92	8	72	2	74	2	76	0	68
(onion)
Asparagus officinale	93	96	0	62	0	60	2	35	0	25
(asparagus)
Cichorium intybus	96	90	4	100	0	68	0	65	0	85
(endive)
Foeniculum officinale	100	83	4	72	0	50	0	56	0	50
(fennel)
Lonicera caprifolium	90	92	60	100	50	78	38	71	32	96
(honeysuckle)
Papaver somnifera	92	90	80	100	16	72	0	60	0	80
(opium poppy)
Portulaca oleracea	84	100	100	100	69	96	7	95	0	80
(purslane)
Trifolium repens	100	100	100	100	90	88	82	81	100	100
(white clover)
Valerianella olitoria	88	96	4	100	0	80	0	84	0	72
(lamb’s lettuce)