From the point of view of their preservation, two types of seeds are usually distinguished: orthodox and recalcitrant. Orthodox seeds can be desiccated down to moisture contents of 4-7% and even ultradesiccated down to contents of 1-3%. In this way, the moisture factor can be used without restrictions to achieve a good longevity. Recalcitrant seeds do not tolerate desiccation well, thus making other procedures necessary. However, no clear limits between both types of seeds exist and there are cases showing an intermediate behaviour.
Low moisture, low temperature, low ethylene concentration and probably low oxygen concentration are the most relevant factors to have in mind for an efficient long term seed preservation. Some interrelations among them exist.
Low moisture and low temperature have been the traditional factors considered. Still, in the practice of the past few decades, low temperature has comparatively received much more attention, while an efficient control of low moisture has been largely neglected. However, in order to guarantee an efficient long term preservation of orthodox seeds, a good control of seed moisture content is essential. At this respect, two major principles should be taken into account:
The container should be perfectly tight, preventing water vapour intake. If the container is not perfectly vapour tight, the seeds will tend to get balanced with the external air humidity. In this way, any potential benefit of low temperature will be offset by an increase in seed moisture. It must be noticed that relative humidity inside any uncontrolled cold room is usually very high. Further to this, it should be emphasised that dried seeds are highly hygroscopic and – even if often very slowly - there is time enough ahead for moisture to enter the container. Unfortunately, the utilisation of inadequate containers has been commonplace in seed genebanks (Gómez-Campo, 2002).
Ultradrying down to a moisture content of 1-3% might extend the seed life span perhaps 4-16 times (Harrington, 1972). Ultradrying has been considered harmful by some authors (Walter & Engels, 1998; Hu, C. & al. 1998), while others have defended that it is not damaging to orthodox seeds (Ellis, 1998; Hong & al. 2005). The debate might last for years until the subtle factors behind these opposite opinions are fully understood. However, under the conditions utilised in the Universidad Politécnica de Madrid (UPM) genebank, ultradry seeds preserved during 40 years have maintained their viability intact (average = 98,4%, not significantly different from 100% ; Pérez-García & al., 2007). As far as we know, these results are unmatched by any other seed genebank and set a solid base to develop improved preservation methods. This has moved the author to prepare the present condensed guide.
When dealing with ultradry seeds, enormous savings of energy can be made by avoiding the use of too low temperatures during storage. Temperature may be the key factor when seeds are merely dried but it loses relative importance for ultradried seeds (Pérez-García & al., 2007). Independently, very low temperatures, close to that of liquid nitrogen (-196 ºC), provide an alternative method for the preservation of seeds and tissues (cryopreservation) that might play an important role in the future, especially for recalcitrant material.
A third factor - the presence or absence of oxygen - has been the source of a contradictory literature over the years and, perhaps for this reason, it has largely been neglected from the practical point of view. However, recent research by Ellis & Hong (2007) suggests that ultradry seeds may be sensitive to oxygen; therefore, it is a factor that, at least tentatively, should be taken into account. Perhaps, the success of the UPM bank partly resides in the fact that air was originally substituted by CO2 in the atmosphere within the containers.
Ethylene and other toxic gases that slowly evolve during seed aging should be removed in order to extend the seed life span.
For many years it has been widely admitted that containers which are appropriate for cold drinks or to store chips for a few months, are also appropriate for long term seed storage. This is very far from reality. Results of a survey on 40 different containers - some of them widely used in many genebanks - showed that 36 of them (90%) allowed moisture inside in less than three years (Gómez-Campo, 2002, 2006a).
All plastic containers allowed moisture inside. The water molecule is small enough to get through the pores of the polymers used to make the plastics and in the medium and long term the fact is unavoidable – although not noticeable in the short term. Polyvinyl bags are especially permeable to humidity. An additional reason for the failure of containers with a lid is based on the fact that both pieces are usually made of different materials, therefore expanding or contracting in a different way with changes in temperature. This creates fissures allowing humidity to get inside the container. This last mechanism appears not only in plastic containers, but also in most glass containers with twist-off or screw lid. Even plasticized twist-off type jam jars often show oxidation stains inside after some time has elapsed.
A special mention should be made to aluminium foil bags because of their still widespread use in genebanks of cultivated species. It’s been a long time since bi-laminated bags (coated with plastic only on one side) were rejected on the basis of their inefficiency. However, tri-laminated bags (coated on both sides) are still widely accepted. According to our experience a maximum of 80% of a given set keeps vapour tight after 3-6 years. It is evident that to base the whole operation of a genebank on a material with these characteristics is risky. To put an example forwards, a collection of 100.000 samples where we learn that 20% of them (20.000) won’t keep their tightness creates a really awful scenario which is now quite common in genebanks. Additionally, it is not possible to see from the outside in which ones the problem is. Massive germination tests or even measurements of moisture in all the samples, are not practical solutions. Let’s add to it that, being sealed with plastic, they carry on the long term the disadvantages of this material.
The aforementioned experiment showed that only four containers remained vapour tight (Figure 1):
Those vials with a rubber lid fastened by an aluminium cover, used some years ago to contain penicillin, were not tested. However, users of this container refer to its behaviour positively.
It is very possible that other appropriate containers could be found in the future. However, to do reliably so, perhaps several years might be necessary. On the other hand, models very similar in appearance - case (C) and also some (B) - can behave very differently and it is advisable to test them before choosing a particular one. The method there described might serve to test other containers.
Considering that most banks around the world have too often used non vapour-proof containers, this experiment probably shows the main reason of failure in seed preservation – especially for banks of cultivated species. It also shows some possible solutions. Solution (B), in particular, provides a larger volume and opens a way to the use of the silica gel method in seed genebanks of cultivated species – a method already used successfully since 1966 with wild species (Gómez-Campo, 1972).
On the other hand, it is obvious that the containers tightness is linked, in a practical way, to the need for frequent or only occasional handling of the seed lots. For very long term collections of valuable materials addressed to the future (“black-box” collections), there is no doubt that the flame-sealed glass vials (Figures 1A and 2a) are the best option. Those can also be of help for other material used more often, especially from wild species or any small sized species, by placing smaller quantities of seeds and making more vials, so that one of them can be broken when necessary.
For base (long term) collections, Kilner jars (Figures 1 B and 3) are also advisable and so are the other options already mentioned. Active collections (for distribution), require more frequent access and need containers capable of being opened and closed. Kilner jars may also be used here if being fast when taking the sub-samples and, whenever possible, doing it in a dry environment.
For active collections of species with small seeds, tubes with a screwed plastic lid (Figure 2b) might be of interest. They are easy to use but not entirely reliable.
Transparency of the containers becomes a key quality when wishing to take advantage of the benefits of the silica gel method for an easy and direct monitoring of the samples from the outside.
To this date, the most commonly used procedure has consisted of drying the seeds with different procedures until reaching a moisture content of approximately 5-7%. After this, the seeds are placed in containers whose vapour tightness is most often untested and the success is mainly trusted to low temperatures. Ultradrying has been very rarely used, perhaps by no more than 50 banks – mostly of wild species in botanical gardens - out of more than 1 500 existing in the world. The UPM’s results have proved beyond doubt the efficiency of ultradrying in orthodox seeds at least under anaerobic conditions and also prove the comparatively lesser importance of temperature for ultradried seeds.
It should be noted that moisture content of 4-5% may be enough for the efficient conservation of some orthodox seeds such as those of many legumes (Ellis & al. 1988). However, as this author points out, these seeds do not suffer when ultradesiccated to 1-3%. In a genebank, with thousands of accessions being handled, the need to individualise the methods should be avoided. In this light, a general benefit to the use of ultradrying can be attributed - always speaking of orthodox seeds.
Ultradrying could be achieved following different ways, but perhaps the most practical one might consist of storing the seeds in balance with dehydrated silica gel. Leaving some gel afterwards with the stored seeds inside the tight container is most useful because the coloured indicator would show any accidental entry of moisture.
Table 1. Advantages of the use of silica gel
Table 1 offers a summary of the advantages of using silica gel with an indicator. Besides those related to ultradrying itself, it is worth noting the last one (number 6), which refers to a totally independent protection mechanism. In seed samples stored within a closed container, damaging gases are generated during the aging process and as they accumulate, they prove deathly for the entire sample. The mortality, which might have been supposed a priori probabilistic for every individual seed, becomes accelerated at the end. Being capable of absorbing these gases, silica gel significantly delays aging and postpones the moment of death. Ethylene is an important component of this mixture of gases (Lee, & al. 2001).
Silica gel is simply silicic anhydride, SiO2, though amorphous (non crystalline) obtained through an industrial process. It is granular in texture, white and very porous. It is this last characteristic which gives its absorbent properties. When it is well dehydrated and placed on a scale, it can be observed to absorb up to 20% of its own weight of water. If placed in a closed container such as a Kilner jar, it balances itself with the confined atmosphere until this reaches approximately 10-12% of relative humidity.
The name "gel" is here improperly used. We understand by gel some substance of a colloidal nature, which is not this case. However, the use of this term for this product has long been used and is now strongly established.
The colour usually shown by silica gel is due to an indicator, added to see directly when it is dehydrated and when it has absorbed moisture. For many years, cobalt chloride (Cl2Co) has been used. This substance gives the dehydrated gel a strong blue colour and a pale pink colour to the gel having absorbed moisture. Recently, the European Union banned its use because of considering it carcinogen through inhalation. A search for new alternatives led to some iron salts, where the change in colour can be poorly distinguished. At present, the most advisable alternative is methyl violet, which gives the dehydrated gel an orange colour and a green colour to the hydrated gel.
The gel is manufactured in granules of different sizes. This size is not irrelevant because, to be used in Kilner jars, a gel with a size > 4 mm can be more convenient, while a 2-3 mm size can be better for glass vials. By hydrating with water coarse silica gel granules (be careful, heat is produced!) the grains get fragmented, and it is possible to screen it in fractions with smaller granule sizes.
Hydrated silica gel can be regenerated through heating. When it is accompanied with cobalt or iron salts, this is done at approximately 220 0C, thus a standard cooking oven can be enough. The process may last one or two hours - depending on the mass to be regenerated. With an organic indicator (e.g. methyl violet) a temperature of 110-120°C must not been surpassed and the process will require comparatively more time.
Freeze drying (“liophilisation”) has been successfully used to obtain ultradry levels of moisture. This procedure consists in freezing the seed water and sublimating the ice afterwards. This technique was developed in the National Centre of Natural History (Paris) in the 1980s for long term storage of pollen grains (Schoenke & Bey, 1981. The Conservatoire Botanique National Méditerranéen (CBNM) started using it for seeds in Porquerolles in 1987 (Figure 6).
It is assured that freeze dried seeds do not suffer any decrease in their viability because of the process. However, it does not seem to be the case for every species and the details when applying this technique must be optimized for every species (Anonymous, 2006). As it will be explained, freeze drying may have the advantage of achieving a direct ultradrying instead of one done in stages – as is generally the case with silica gel which needs to be regenerated every time it is used.
In comparison, freeze drying needs a rather expensive machine (Hu, X. & al. 1998). In turn, silica gel reaches lower water contents. Also, considering that silica gel is going to play an essential role afterwards in keeping a low moisture and monitoring the samples, it may be more practical a direct drying with gel. In other words, while freeze drying only ultradries, silica gel not only ultradries but also provides an efficient monitoring during subsequent storage.
The use of low temperatures has been widespread in long term seed preservation. Standards set by the Food and Agriculture Organization of the United Nations (FAO) and the International Plant Genetic Resources Institute (IPGRI, now Biodiversity) advise a storage temperature of -18 0C or lower (FAO/IPGRI, 1994) and almost everybody now planning a genebank instinctively thinks of a cold room capable of at least reaching -20 0C. Localisation of the new installations of the Nordic Seed Bank in a region of "permafrost" responds to the same idea.
Unfortunately, emphasis on low temperatures and neglecting low moisture (either because of not drying enough or because of the use of inappropriate containers) has brought that a significant number of the actual genebanks are worried with the burdensome need to rejuvenate their prematurely aged material.
Seeds which had been freeze dried in the Conservatoire Botanique National Mediterranéen (CBNM) of Porquerolles and then kept at room temperature for 10 years showed a slightly better behaviour than those kept under low temperature. In a much longer term, ultradry seeds with silica gel kept for 40 years at room temperature inside a closet (Pérez-García & al., 2007) did not behave very differently from those kept in the cold room. It appears hence clear that the main role attributed to low temperature during decades should now be replaced by a much more thorough attention to the moisture factor.
However, it is obvious that low temperature helps, and it would be irresponsible not to take it into consideration. In the last mentioned work, it is suggested that temperatures between -5 °C and +5 °C could very well be enough for orthodox ultradry seeds, thus saving considerable amounts of energy.
An alternative sometimes used consists of controlling moisture in the cold room itself. It is not a bad idea itself, although it shows some disadvantages. With a temperature of about 0 °C (more than sufficient as it has just been explained), to reach a relative humidity of approximately 25% inside the chamber is neither difficult nor expensive. Anyway, this would be far more efficient than having a chamber at -20 /-25°C with the containers tightness being neglected. However, controlling the moisture content within the containers themselves by using silica gel is, in the long term, much more practical and efficient, since it allows reaching more suitable ultradry levels and a better monitoring.
Ultradrying with silica gel can be achieved by putting the samples inside a closed container along with the dehydrated silica gel (Figures 2 and 3).
The use of flame-sealed vials, obtained from standard laboratory glass tubes of 20 ml is a system whose high efficiency has already been proven. If it cannot be generally recommended, is only on the grounds that it allows little volume – a maximum of 8 ml - for large accessions. However, it should be considered that 8 ml means some 160 seeds of wheat, 1 400 of cabbage and 40 000 of Diplotaxis siettiana (extinct in Nature). The method is almost perfect for especially valuable material such as rare, endemic or threatened species, special breeding material, “black-box” collections, etc. Also, the idea of preparing several vials for each accession is realistic and most often worthwhile.
In the following paragraphs, the different steps to create such ampoules according to the procedure used in the UPM genebank (Figure 5) are explained in detail:
When stretched with the flame and externally labelled, the vials should become of a final homogeneous size. In the UPM’s genebank they have traditionally been stored inside Kilner jars - long before finding that the last also were tight enough. Around 25 vials can be placed in each jar - thus obtaining a double security.
The described protocol might seem difficult and work demanding but this is not at all the case when the work is properly set up and organised. The seeds can thus be preserved without any evident viability loss for at least 40 years , which may well mean an efficient preservation for at least one century or probably more. The advantage and the disadvantage at the same time, is that the vials can only be opened by breaking them, unlike what happens with the Kilner jars.
The detection of sufficiently reliable larger containers as the Kilner jars (Figure 3) opened the way for the adaptation of the silica gel method to the larger samples that are common in crop genebanks. In this case, three important differential aspects should be taken into account
Firstly, drying in situ is trickier than it was with tubes. Since the seed sample may have an initial moisture content of approximately 8-12%), the gel will be hydrated by the sample and will change its colour. It will be necessary to replace this gel with a new, dehydrated one, and the operation might need to be repeated several times. Once the gel does not change colour, one more replacement is convenient. The seed will then be ultradesiccated and will remain indefinitely balanced with the dry gel if the container is kept tight. However, changing the gel repeatedly might be fastidious. Therefore, it is convenient to dry the seeds as much as possible by some other method (sun exposure, dry room, dry air currents or other drying agents such as Cl2Ca). In other words, it is advisable to start ultradrying with the seeds already as dry as possible. Drying speed will depend of the mass ratio between the desiccating agent and the seeds but once the equilibrium is reached, only a small amount of silica gel to act as indicator is enough (and also convenient, see later).
Plastic chambers (not long-term tight, but tight enough to perform this operation, see figure 5, can hold a series of cardboard boxes, some with gel and other with seeds. By changing the gel when necessary, it will be possible to desiccate inside a high number of samples in only a few weeks. Since most seed collecting missions at least in the Mediterranean area take place in June or July, it is possible to use the period of August - September to desiccate the seeds.
If there are funds available to purchase new equipment, and once the efficiency of the method becomes well established, a previous freeze drying (Figure 6) can take the seeds directly to moisture levels very close to the balance with dehydrated gel.
Ready-to-use desiccators containing silica gel which is automatically regenerated already exist in the market. They usually consider that the chamber where the samples would be placed will be provided by the user (Figure 7). When the chamber is a store room, even tiled, big or small, it is inevitable that humidity will come inside through the walls. This makes it difficult to achieve relative humidity levels below 15%, especially with low temperatures. Besides, the drier constitutes a dynamic system with several pieces, joints, etc., very far from the simplicity of the jar of Figure 3 (where <5% can be reached). Compact systems with built-in chamber to be placed on top of a laboratory bench are already present in the market. However, these cabinets never guarantee a relative humidity under 20%. R.H. under 15% could perhaps be reached in the future with well designed impermeable glass or metal chambers. However, if the final goal is to store the seeds in Kilner (Scotch) jars, a previous storage at 15-20% R.H. in the cold room may help to reduce the number of times the silica gel must be replaced.
Secondly, it is important to take into account that silica gel can absorb important amounts of water (up to 20% of its own weight) and lose part of its efficiency before changes in colour can be appreciated. This is particularly important with jars that are frequently opened and closed. To obviate this problem fully dehydrated silica gel should always be used and, once the equilibrium seed/gel has been obtained, to place only a small amount of gel (i.e. a small bag in the upper part) is sufficient. In this way, any moisture intake will be detected soon.
In the third place, it is convenient to minimise the presence of oxygen, either by filling the containers as much as possible with the seed material or by replacing the air with an inert gas, preferably CO2 because it is heavier and tend to occupy the lower parts of a volume as water do. A few open Kilner jars could be placed in an “ad hoc” box where CO2 is added. Otherwise, CO2 could be directly added from the cylinder if this is accompanied by a pressure reducing device. Also, unlike sealed vials, a Kilner jar may be opened, perhaps frequently, and this has to be taken into account by adding CO2 once in a while. It should be noted that the substitution does not need to be perfect because the aim is to reduce (not necessarily eliminate) the O2 present within the container.
The same system (Kilner jars with silica gel at the bottom) could be valid to stop aging in badly preserved seed collections. It would be enough to place the samples inside (in bulk, in their original containers or better in paper envelopes) and to regenerate the gel as many times as necessary until no further colour change would occur. An additional substitution of the gel will ensure that this is completely dehydrated. In the particular case of foil bags, several of them could be fitted inside each jar. Each jar would be afterwards given a number and the accessions position within the genebank then recorded in the database.
These seeds are characterised by their relatively high moisture content (15-25%) and by a clear intolerance to desiccation. (Berjak & Pammenter, 2004). Therefore, they cannot be preserved by desiccation.
Recalcitrant seeds can often be recognised morphologically because of their large size and smooth tegument. However, other seeds with a more normal appearance could also be recalcitrant. In the Mediterranean area they are not very spread but they can be found in woody species, climactic or not (Quercus, Castanea) or in riverbank species (Populus, Salix). In the rainforest, the species with recalcitrant seeds are dominant. Ecologically, they follow a strategy of lasting as seedlings, better than as seeds. That means that their seeds avoid getting too dry in a humid environment, where the very process of drying would be more difficult. Their lifespan is generally short, between a few weeks and 2-3 years.
If we make an exception of those species growing in riverbanks, seed size seems to be a key factor to determine the orthodox or recalcitrant behaviour of a species. This is because mechanical damage produced during drying is more severe in large seeds. For example, the seeds of pumpkin (Cucurbita sp.) and of some Phaseolus species (P. lunatus) can be damaged through drying while the smaller watermelon seeds (Citrullus moschatus) or those from green beans (Phaseolus vulgaris) are orthodox. The internal composition could also set differences. When working with small seeds, it is possible to be fairly confident that they will be orthodox, whereas, with an increase in size, exploratory tests should be carried out.
Being orthodox or recalcitrant is not an absolute character (Berjak & Pammenter,1994), since "semi-recalcitrant" seeds with an intermediate behaviour exist. These accept a partial desiccation, if only to a certain level. In any case, they should not be expected to tolerate ultradrying.
To preserve recalcitrant seeds, there is not much to be done, except to put them in their lower tolerance limit as far as moisture content and temperature are concerned. As they inhabit wet places, it is usual that they die prematurely due to fungal or bacterial attack. This means that a treatment with an appropriate phyto-therapeutic product could perhaps extend their life span to some extent. Perhaps the most practical method whenever possible could be the establishment of gene banks of seedlings, since these seedlings use to be quite long lived when placed in low lighting conditions. For long term effects, it would be necessary to resort to the cryopreservation of their embryos.
It is not difficult to establish a protocol to set apart orthodox from recalcitrant seeds – based on the first ones being tolerant to desiccation. This could consist of the following steps: (1) An initial germination test, (2) the desiccation of an equivalent lot and (3) a new germination test of the desiccated seeds to show whether they are still alive or not. However, it should always be kept in mind that low values in the germination tests do not necessarily mean that the sample is dead or dying. These values could be originated by dormancy. If so, an immediate treatment of the seed (either by scarification, immersion in gibberellic acid solution or any other suitable procedure) should follow in order to overcome any possible dormancy. This is particularly important for wild species.
Before deciding whether a given seed is recalcitrant - especially in medium sized seeds - there is still a test to conduct, which may enable us to desiccate it and to preserve it in the long term. Some apparently recalcitrant seeds seem to tolerate drying and perhaps ultradrying when these are gradually carried out, without sudden changes. For these "pseudo-recalcitrant" seeds it is advisable a previous successive balance with atmospheres showing a decreasing relative humidity. If this occurs, the number of species which could be preserved in the long term by the silica gel method will considerably be enlarged.
It consists of storing the material at temperatures near that of liquid nitrogen (-196°C). Under these conditions all enzymatic processes are practically halted and it is thought that any type of biological material (in plants: meristems, calluses, embryos, pollen, seeds, somatic tissues, etc.) can thus be preserved for a potentially infinite period. Only cosmic rays could place a limit after a few millennia.
Orthodox seeds can easily be cryopreserved. However, doing this has not much meaning once the high efficiency of ultradrying has been demonstrated for them. Ultradrying is also much cheaper. It is with recalcitrant seeds that cryopreservation might play a role in the future.
For an efficient cryopreservation, it is fundamental to avoid the intracellular formation of ice crystals which are highly damaging for the cell internal structures. A rapid descent of temperature is a must in the process, and a previous desiccation or ultradesiccation of the material is highly helpful. Also cryoprotective treatments (osmotic agents and/or substances such as glycerol, proline, abscisic acid, etc.) are often used. Most commonly used methods today aim to obtain a transition between an aqueous solution and an amorphous "glassy" state by means of concentrated solutions of sucrose and some derivatives of glycerol.
In the case of recalcitrant seeds many difficulties appear with whole seeds (Engelmann, 1997) but these can be partly overcome by preserving the embryos only (Fu & al., 1993). Basically, the technique consists of an encapsulation of the embryo in calcium alginate followed by a desiccation in concentrated sucrose solutions and then a final immersion in liquid nitrogen.
Ultradrying and storage with silica gel of orthodox seeds in hermetic containers, preferably in an anaerobic atmosphere, with only moderately low temperatures, provide a tested and reliable method to keep the seeds alive for many decades and - likely - for centuries.
Efficient long term seed preservation saves much genetic material, and also much time, labour, electricity and money. The now widespread resignation over the need to rejuvenate the seed material in cycles of 25-35 years (in fact middle term preservation), could give path to the quite different situation of long term cycles of one or more centuries where the tremendous inconveniences of rejuvenation become minimised. Also, much of the time devoted to too frequent and tedious germination tests - so often only valid to check passively how the stored seeds are ageing and dying - can be devoted to research on more useful and stimulating subjects, such as the initial quality of the seed accessions, the possible dormancy in some of them and the adequate method to remove it, the orthodox, recalcitrant or pseudo-recalcitrant behaviour of others and so on (Gómez-Campo, 2006b).
This guide is an English free translation of the revised chapter devoted to seed preservation which will be included in the Spanish version of Manuale APAT, Roma. Thanks are due to M. Pilar Ballesteros who made valuable style suggestions.