Conservation perspective - In-situ and Ex-situ
Concept of base and active collections
Long-Term and short-term storage of field crops
Conservation needs: Physiological and allied requirements
Recent years have seen an increasing global concern over the loss of genetic resources of crop plants. Future progress in crop improvement and our food security depends, to a great extent, on immediate conservation of the rapidly vanishing crop gene resources and their effective utilisation by plant breeders. In this context, a great deal has been accomplished in the last decade to safeguard the plant genetic wealth which constitutes the natural heritage. However, much still remains to be done in improving the conservation strategies and upgrading the collections, which encompass a wide range of diversity comprising wild relatives, primitive cultivars and landraces, weedy forms, unimproved and modern cultivars, and genetic testers.
The fundamental objective of genetic resources conservation is the maintenance of broad based genetic diversity within each of the species (i.e., intra-specific genetic diversity) with a known or potential value in order to ensure availability for exploitation by present and future generations.
International efforts aimed at collecting and conservation of plant genetic resources have been coordinated since 1974 by the International Board for Plant Genetic Resources (IBPGR). By and large, the national/global network of genetic resources is now (getting) well established. Some of these aspects are highlighted here.
There are broadly speaking two basic approaches to genetic resources conservation, namely, in-situ and ex-situ conservation. In-situ means the setting aside of natural reserves, where the species are allowed to remain in their ecosystems within a natural or properly managed ecological continuum. The natural biosphere reserve is a useful solution for species that are endangered and nearly on the point of extinction (Prescott-Allen, 1981). However, for species more widely distributed, the conservation of total genetic diversity of (that) species in-situ is difficult. Although species conserved in their natural habitats have the potential for continued evolution of a particular trait within the species and are subject to natural selection, there are indeed many problems in establishing this type of reserve, for example, cost, size and maintenance aspects, political and social issues and the danger of genetic wipe out as a result of natural disasters, fire, etc. In particular, this method of conservation is of significance to the wild relatives of crop plants and a number of other crops, especially tree crops and forest species where there are limitations on the effectiveness of ex-situ methods of conservation. The crops of immediate interest for in-situ conservation are the perennials that are vegetatively propagated (Hawkes, 1975) and those with seeds that cannot survive cold storage (King and Roberts, 1979; Hawkes, 1982). Wild species maintain their original characteristics best in the habitat to which they are adapted, which necessitates the formation of nature reserves in appropriate climatic, altitudinal and latitudinal zones.
The ex-situ form of conservation includes, in a broad sense, the botanic gardens and storage of seed or vegetative material in genebanks. The field genebanks where clonal materials are maintained as living collections in a field/orchard or plantation also represent ex-situ form of conservation. However, field genebanks have the potential risk of germplasm being lost due to disease, stress or disaster, and large amount of space and labour are required to maintain a small proportion of diversity. Cryogenic preservation of vegetative material is another mode of ex-situ conservation and it holds promise, especially for base collections.
Efforts to conserve genetic resources ex-situ in seed genebanks have accelerated in the past decade. In the genebank, the aim is to provide ideal storage conditions so that the mean viability period of the seeds is greatly extended by reducing the life processes to a low level. Successful seed storage depends on effective control of several factors including temperature, seed moisture content, storage atmosphere, etc. in response to storage conditions. Seeds within heterogenous germplasm accessions frequently deteriorate at different rates thereby causing selection within the samples to favour genotypes more amenable to given storage conditions. The selection within the germplasm accession during seed conservation and subsequent regeneration has a strong influence on the genetic composition of an accession. This is one aspect of ex-situ conservation in genebanks that makes it desirable to ensure indefinite maintenance of some wild populations of most crops in-situ. In-situ conservation involves its own set of risks and difficulties but can function as an evolutionary insurance for long-term germplasm availability.
The choice of in-situ and ex-situ conservation is sometimes seen in terms of exclusive alternate strategies but the two alternatives may be more constructively viewed as mutually complementary activities and each can play an important part in safeguarding particular plant populations. It would be an ideal situation where both may be used to best advantage to ensure both long-term species survival and an adequate supply of germplasm for improvement of related crops.
Efforts have been made here to deal in detail with the ex-situ conservation of seed germplasm of agri-horticultural crops. The National Bureau of Plant Genetic Resources (NBPGR) has taken a lead in this direction. A National repository for plant germplasm resources has been set up and conservation activities are being coordinated.
Conservation of seed propagated plants is relatively easy for seeds with orthodox type of storage behaviour, i.e., the viability can be maintained by drying the seeds and storing these at low temperature. For orthodox or desiccation tolerant seeds, lower seed moisture content is associated with an increase in storage life of a sample within certain limits. Most agri-horticultural crop plants have seeds that can tolerate desiccation. Many crops like cacao and rubber, most tropical fruits and many timber species have seeds with recalcitrant behaviour. These are desiccation sensitive and are generally killed if dried below a critical moisture content value, usually between 12 and 35 percent moisture. Research on physiology of desiccation sensitive seeds is very limited and many problems remain to be solved, especially on the effects of desiccation on the cell and the cause of damage and deterioration. The critical level of desiccation sensitivity in these recalcitrant seeds is also unclear and the effect of partial desiccation on seed viability may be worth consideration for storage. Recalcitrant seeds are relatively short lived (few weeks to months) even under high moisture conditions and require different storage techniques (Stanwood, 1985). In-vitro cultures and cryogenic preservation offer promising avenues to overcome the recalcitrant characteristics. However, the genetic stability of in-vitro cultures has yet to be fully ascertained before an entire collection is committed to this storage technique. These aspects are separately dealt with in Chapters 10 and 11.
For orthodox seeds, large scale mechanical refrigeration systems, which hold seeds at temperatures down to -20° C, have greatly increased the storage life of a seed sample making ex-situ conservation of seed germplasm an easy and safe method of conservation. However, deterioration and loss of viability can still occur with increasing time in storage. The longevity of seeds or the maintenance of seed viability is a balance between extrinsic and intrinsic deleterious factors and repair or protective mechanism. Depending on the particular mechanism(s) involved and external factors, such as storage temperature, seed moisture content and oxygen availability, the life span of a seed sample may be shortened or extended.
Currently, two main types of collections are held at most genetic resources conservation centres.
Base collection: It is held under conditions which retain viability for long periods of time. This component of the system has the sole purpose of acting as a conservation measure. This is not drawn upon except for viability testing and subsequent regeneration, is normally restricted in distribution, and acts as a back-up to an active collection. The IBPGR Seed Storage Committee (1985) established requirements for long-term storage in base collections as follows:
1. Temperature of -10 to -20° C, generally -18° C has been specified arbitrarily as preferred standard because this storage environment is technically achievable at reasonable cost, whilst providing good storage conditions in which the loss in viability for all orthodox seeds occur extremely slowly.Active collection: Accessions are stored for short to medium periods of time (generally up to 30 years) as is often the case for breeder's collections, for regeneration, evaluation, research and distribution to end users. Active collections are generally held at temperatures between 0 and 10° C. Storage conditions for active collections are often less stringent than for base collections for economic and practical reasons.
2. Seed moisture content of 5 ± 1 percent (wb).
3. Hermetically sealed airtight containers.
Under the same conditions of storage, the seeds of different species will have different periods of longevity. Thus, it is difficult to define precisely the period envisaged for active collections. The base and active collections are defined based on their functions of collections rather than on storage conditions. Centres may maintain both active and base collections, while others may be concerned exclusively with one type. The processing of germplasm accessions for active and base collections could be done in a similar manner. However, the type of storage containers in the two categories would be generally different. Also, the sample size of accessions in active collections are bigger than for base collections. At centres which maintain both types of collection, the two are linked by its documentation system.
Even within the group of orthodox seeds, there is considerable variation between species in length of storage period which can be achieved under any given set of conditions, varying from comparatively long periods for many of the major cereals through intermediate periods for some of the grain legumes and relatively short periods for some grasses and several vegetable species. For example, barley, wheat, maize and pea are inherently long-lived species, whereas onion, sugarcane, coffee and soybean are inherently short-lived species. In some species, as in lettuce, barley and maize, there may be a considerable genotypic variation in the storage potential between cultivars.
Although a controlled atmosphere is essential for safe storage of seeds, both for long-term and short-term periods, the conditions for long-term storage are more exacting because the seed viability is to be preserved as long as it is possible. The operational cost of storage facility per unit seed stored increases considerably as the requirements of temperature and relative humidity become more stringent. Therefore, prior to storing seeds, a decision must be made on the type of storage needed. This can be related to the time period for which storage is expected and the storage characteristics of the species. In general, base collections are held in long-term storage while active or working collections are held in short/medium-term storage. However, there is no technical basis except economy in operational cost of the facilities. Each species has its own 'safe' seed equilibrium moisture content for a given storage temperature and relative humidity. For example, safflower and sesame seeds containing 4 percent seed moisture showed essentially the same germination after two years of sealed storage at all the temperatures these were tested. At 7 percent moisture content, safflower and sesame seeds in sealed metal cans did not store safely at temperatures above 10° C (Bass et al., 1963). Cotton seeds stored in sealed containers with seed moisture content below 13 percent showed no deterioration in 15 years at 0.6° C (Simpson, 1953), and with 7, 9 and 11 percent moisture, there was no deterioration in 19 years (Simpson, 1957). Good quality cotton seeds can be stored in sealed containers upto 38 years without complete loss of viability if the temperature is held at 0.6° C and the seed moisture content does not exceed 11 percent (Pate and Duncan, 1964).
Most agri-horticultural crop seeds which fall in the 'orthodox' category can be stored safely under the storage conditions recommended by the IBPGR for base and active collections. However, over long periods of storage under current preservation standards of -18° C and 4-6 percent moisture content (IBPGR, 1985), metabolism still takes place and viability eventually declines. For example, under storage conditions imposed at the NSSL, Fort Collins, Colorado, seeds of many species declined in viability over time (Stanwood, 1985). Tomato seeds considered to store well showed an average drop in germination of 0.4 percent per year while the germination of wheat and sorghum seeds declined on an average of 0.8 percent and 1 percent per year respectively.
Cryopreservation of seeds in liquid nitrogen (- 196° C) provides improved maintenance of seeds over long periods. This aspect and the conservation of vegetatively propagated crops have been dealt with separately in Chapter 10.
Good quality seeds, i.e., high viability accessions of species with good storage characteristics, such as the cereals, forage legumes and low oil content grain legumes store satisfactorily for many years, if they are dried to about 5 percent moisture content and hermetically sealed and stored between 10 and -20° C. In addition to inherent seed-to-seed variation in any constant storage environment, the actual longevity can be affected by the genotype, various environmental factors that affect seed quality before storage (e.g. ripening, harvesting, drying and processing) and the conditions under which the seeds are stored. The genotype and pre-storage environment affect storage potential, and the storage conditions modify that potential. Within a species, difference in longevity among accessions, which may result from genotype and/or maternal and post-harvest environment, affect the relationship between storage period and percentage viability through their effect on initial quality. These effects on initial quality affect the absolute longevity of accessions but not the relative effects of temperature and moisture content on longevity.
Seed moisture content and storage temperature are the two primary factors that can be practically controlled. Relationship among seed moisture and longevity is logarithmic and varies significantly among species. The major difference in the sensitivity of different species to moisture content appears to be correlated with seed oil content.
Different approaches are required to control seed deterioration and reduce routine maintenance process, such as monitoring and re-growing of deteriorated samples. The absolute storage life for each seed lot under genebank conditions will depend on its initial viability when the seeds are placed in genebank. To retain the viability at high levels, the seed samples should be dried in the field itself, even though this may often be impractical and difficult. In addition to drying it quickly, it is important to reduce the moisture content to a reasonably low level as small difference in moisture content can have large effects on longevity. The collections should be regularly despatched to less damaging conditions, such as controlled environment drying facility currently recommended by IBPGR. If too much time is spent between harvest and receipt at the seed bank, then loss of viability in the seed lot can be expected. In warm humid tropics, significant losses of viability can be expected relatively early. The increasing period between maturity and harvest will necessarily result in a greater accumulation of damage prior to harvest. Harvesting grain before it is ripe also reduces its longevity. The reduction in the initial viability will then be reflected in a reduced lifespan under constant storage conditions. Maximum longevity is achieved by harvesting as soon as possible after physiological maturity. Even minute drop in viability can have large effect on potential longevity. Seeds should be placed as soon as possible in a cool low humidity environment in order to further arrest viability loss till they are processed for final storage in genebank. The collector/potential donor must care for the following guidelines while sending the material to the genebank for storage:
1. Cleaned seeds are preferred over inflorescences or unthreshed material still covered with floral structures like hull or glumes.Drying, which is beneficial to the maintenance of viability, involves forced ventilation of seed lots with air at low temperature. Optimally, these conditions should be 15° C and 15 percent relative humidity. Use of 15° C and 15 percent RH is a compromise between cost of operating, drying facility, rate of drying and loss in viability. The drying method used and the environmental conditions prevailing during desiccation affect seed longevity even in orthodox species, and the effect is critical in species with desiccation sensitive seeds. Below the critical moisture content, the loss of viability is more fully understood, being an integration of moisture content, temperature and time according to the viability equations of Roberts (1973).
2. Accessions should contain at least 4000 seeds for long-term storage but ideally 12,000 seeds are required, especially for heterogenous materials, to fully represent the variation in the original sample and to allow sufficient seeds for viability monitoring during storage and subsequent regeneration. Norms/standards on seed numbers for wild species need to be established.
3. Seed/inflorescence after harvest should be dried (not using heated air driers) to around 10-12 percent moisture content and then packed for transport to genebank.
4. Material is not to be treated with chemicals.
5. A duplicate set of accessions, well documented, must be provided to an alternate centre.
6. The passport and characterization data must accompany each accession.
Research has demonstrated that there is a considerable improvement of the longevity after drying to very low moisture content, i.e., below 5 percent. True seeds of potato can be easily dried to 2.5 percent moisture content which resulted in an approximate tenfold increase in subsequent longevity compared to seed at 5 percent moisture content. In sesame, a reduction in seed moisture content for storage from 5 to 2 percent increased seed longevity by a factor of 40 (Ellis et al., 1986). However, some caution is necessary before advocating storage at ultra low moisture contents without apparent damage. Firstly, it can be difficult to dry some species (e.g. the grain legumes) to very low moisture contents (2-3 percent). There may be problems with severe cracking of the seed coat at such low moisture contents. Secondly, potential problems of imbibition injury, due to rapid entry of water when seeds are subsequently germinated, are likely to occur. This can be largely overcome by careful humidification in a saturated atmosphere before imbibition.
Maintenance of seeds under good storage conditions is an important conservation aspect. The necessity for specifying good storage conditions stems from the following facts.
1. Loss of viability in storage is asociated with a considerable amount of genetic change, and thus regeneration should be undertaken as soon as viability has begun to fall significantly, i.e., as soon as viability has fallen by 5-10 percent.In view of the relatively high cost of processing and conserving germplasm collections, monitoring is necessary to ensure the maintenance of viability of stored seed germplasm. The determination of the quality of seed by periodically testing viability is of greatest importance for the effective management of a genebank in relation to the period for which the seeds can withstand storage under specified condition before reaching an acceptable level of deterioration. The assessment of the initial quality of the seed at the time of packaging plays an important role in predicting that period and, consequently, it determines to a large extent the frequency of monitoring the quality of the seeds stored in the gene-bank. The cost of monitoring is two-fold. Firstly, the value of seeds destroyed in the monitoring test must be considered, and secondly, the cost of the monitoring test which involves considerable labour.
2. The costs of regeneration are high, and the difficulties and dangers are considerable. Moreover, loss of purity can occur through mechanical mixing and cross pollination and, in genetically heterogenous samples, selection can occur depending on the environmental conditions in which the regeneration is carried out.
3. The capital and running costs of providing really good conditions are not much greater than for providing storage conditions of inferior quality. Also, storage at the preferred standards require less detailed management than those providing poorer storage.
Genebank managers should set regeneration standards for each crop species. Essentially, the level of viability chosen will be a compromise between the risk of valuable gene loss and the effect of ageing on the physiological performance of surviving seeds against the high cost and difficulties of regeneration. Potential genetic shift through environment, pest and disease pressures during regrowth have been known to result in a change of the original gene content (Allard, 1970). Preliminary findings stress that it is the integrity of the genetic complement of an accession that should be conserved rather than its component genes. In view of the value and importance of the material held in genebank, a standard of 85 percent viability for regeneration level for all species has been recommended to encourage good quality seeds. However, it is noted that a number of wild species, forestry and vegetable species, e.g., lettuce, cucumber, fenugreek, bottlegourd and others have characteristically low viability. Setting an 85 percent regeneration standard may appear highly optimistic for these species. In general, the regeneration standard should be set on a crop to crop basis and according to the characters of a particular species, and should be as high as practicable.
There is a need for establishing better procedures for monitoring the viability of the material in store and for appropriate regeneration methods, especially for wild species and other difficult materials for maintenance of allelic frequencies and genetic integrity during multiplication. Recent research has indicated that sequential germination tests are as reliable as fixed sample size test, while at the same time requiring considerably fewer seeds to determine when seed regeneration should take place (Ellis and Wetzel, 1983).
Management of facility
Management of collections
Management of information
The organisation of various steps of conservation process in a coordinated sequence of events leading to a well documented, preserved germplasm is the essence of the genebank. Essentially, the procedure in the genebank entails the acquisition, storage and documentation of new material; the monitoring and maintenance of existing collections; and the dissemination of information and germplasm to the users.
Since genebanks are long-term investments, highly efficient work practices should be adopted to hold annual operation cost to acceptable levels. Stringent operation standards for carrying out genebank activities, large number of collections, heavy workload and shortage of funds and resources are the factors imposing need for the use of sound management principles in genebank. Efficient management is vital without which many problems can develop. More importantly, these result in the loss of invaluable and irreplaceable genetic resources.
The genebank management is a mission-oriented management. It should evolve through numerous changes based upon past experience, new technologies and broadened national and international collaboration, attempting to identify the work trends and needs for improvement.
Planning is the first step in the management process. The scientific and organisational mission should be well defined in terms of goals of the genebank, i.e., short, medium or long-term. The genebank should enlist the help of different specialists/advisory committees to deal with specialised subjects, such as individual crops, methods of conservation and documentation. Well defined delegation of responsibilities, long range plans and contingencies for emergencies or urgent needs must be prepared in advance. The importance of standard procedures and protocols in this regard should not be overlooked.
The genebank staff should be dedicated and exercise the service spirit to translate the decision into accepted responsibilities and effective action. Every operational facet should be backed up by other knowledgeable staff members to lend continuity to genebank activities in case of emergency. The genebank policies should be correctly interpreted and implemented. Communication among different units is essential for efficient working of a genebank. The genebank design and procedures followed should be such that there is maximum efficiency of operation and security.
Essentially, the genebank management involves management of facilities, management of collections and management of information. The flow of germplasm and associated information is illustrated in Fig. 1.
The genebank manager has the ultimate responsibility for operating the genebank and associated facilities. For long-term security and low maintenance cost, it is essential to manage the facility professionally and take precautions. Cost may be kept low by suitable selection of equipment in relation to the number of collections to be held.
Refrigeration equipment is costly to operate and sometimes difficult to maintain. To help overcome the vulnerability of germplasm collections to voltage fluctuations and equipment problems, standby duplicates of essential refrigeration equipment, voltage regulators as well as back-up generators are recommended. Security of genebank facilities should also be assessed in terms of continuity of organisational support, natural disasters, reliability of local power supply and availability of duplicate storage site. The engineering/ maintenance staff should maintain vigilance about minute details. It is advisable that the temperature and relative humidity within the medium-term cold store and the drying room are monitored and logged. Management involves all security concerns that are to be adequately addressed to the appropriate extent through testing to assure that cooling, safety and backup systems are fully operational.
Fig. 1. Flow of seed material in National Gene Bank
Management of collections aims at ensuring long-term security of conserved materials with a minimum loss of genetic variability while ensuring the availability of collections to plant breeders and other users. A collection may suffer great losses in viability if it is improperly monitored for viability or if rejuvenation is delayed. It is important to devise and implement management procedures to fulfil strict scientifically defined standards for viability monitoring and seed regeneration. Research to predict seed longevity for the purpose of providing a rational basis for an economical regime of monitoring test during storage should be given due emphasis. New improved approaches to monitoring viability test like sequential monitoring test should be adopted to minimise the depletion of seed during viability tests. Help of other appropriate institutions should be taken in the seed rejuvenation phase especially when the crop is unadapted to the site of the genebank. Duplication of base collection at a different site is essential. However, duplication within the genebank should be minimised.
Rationalisation of collections in order to reduce cost and unnecessary work and detection of gaps in the representation of collections are important aspects of management of collections.
At present, quantitative data for all species to the extent to which collections represent available variation is lacking. It is important to stress that maintenance of a wider spectrum of diversity is more important than the maintenance of large number of accessions per se.
In a genebank, which maintains many species, the best storage conditions should be provided for the accessions of those species with poor storage characters and those most likely to deteriorate rapidly in preference to accessions of species with good storage characteristics.
The information system is a tool in a genebank manager's hands to facilitate the capture, maintenance and processing of data relevant to the diverse activities of the genebank. This is essential so that management procedures are organised and performed in an efficient way. Monitoring the quality and quantity of seeds in store, preparing for regeneration, identification of samples to be evaluated and distribution of seeds are examples of procedures which require careful screening before the actual work begins. Proper documentation facilitates access to seed material held in the genebank. The ultimate effective use of germplasm is totally dependent upon the availability and quality of information associated with it.
There are four categories of descriptors associated with each accession in the genebank viz. passport data, characterisation data, evaluation data and accession management data.
In view of the large number of existing germplasm collections and their associated description, computers provide the only effective answer to data management problems. These provide an easy access and increased service to the users of germplasm. The nature of data flow in a genebank information handling system necessitates the use of the data base management concept. Due emphasis is now being given to the extensive documentation and data management of germplasm holdings in the medium and long-term collections.
There is increasing global awareness of the need for conserving genetic resources of crop plants for their current use and for posterity. This has led to the development of a network of plant genetic resources centres. In India, these activities are coordinated by the genebank at the National Bureau of Plant Genetic Resources. In the light of above developments, the principles and procedures of ex-situ conservation of orthodox seeds as active and base collections in the genebank are discussed. Emphasis is laid on genebank management to regulate and coordinate the activities involved in conservation of germplasm resources and dissemination of this information for the user scientists.
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