The existing national protected area systems are often a valuable starting point for a network of conservation stands of a particular species. Indeed there are likely to be several thousand tree species which only occur within the existing protected area network. However, the security of forest protected areas remains a major concern, especially in developing nations. It has been argued that only a minor percentage of protected areas can be truly regarded as secure and more than half face threats to their integrity and existence in the medium term. On a positive note, FRA 2010 found that the area of forest designated for conservation of biological diversity increased by about 6.3 million hectares per year during the last decade and a similar increase occurred in the area of forest in protected areas. In both cases the increase is equivalent to nearly two percent per year over the last decade. The role of protected areas in conserving FGR has been reviewed in Thomson and Theilade (2004). These authors also discuss ways to enhance the FGR conservation role of existing protected areas, and emphasized the imperative of involving local people in protected area conservation measures.
FGR in agroforestry systems, including trees on farms
The work of ICRAF and national partners in the development of context-specific agroforestry systems, integrating traditional knowledge and scientific advances, and based on diverse, adapted tree germplasm, offers one of the most promising solutions to addressing problems of over-population and limited land base. FAO estimates that 1.2 billion people use trees on farms to generate food and cash (http://www.fao.org/forestry/livelihoods accessed November 2012), with almost half of the agricultural land in the world, or more than 1 billion hectares, having a tree cover of more than 10 percent (Zomer et al., 2009). There has been an increasing appreciation of the importance of using appropriate, matching, diverse and improved germplasm in agroforestry systems over the past two decades, including appropriate seed and seedling production and dissemination systems. This has included the domestication of many different indigenous fruit and nut tree species to provide a source of nutrition and income for rural households through meeting identified, different market opportunities. Simmons and Leakey (2004) have coined the term of agroforestry tree products (AFTP) for these new products. The R&D and extension efforts into agroforestry led by ICRAF, and many national, donor and NGO organization partners have borne and will continue to bear fruit as long as the genetic diversity on which they rely is both conserved and accessible. There has also been an important spill over of knowledge on the importance of, selection and improvement of germplasm in conventional plantation forestry to agroforestry R&D. An example of the evolution of use of tree germplasm in modern agroforestry is provided from Fiji and the South Pacific Island nations. Through the mid-1970s and early 1980s, the official promotion of village forestry mainly consisted of distributing seedlings of Caribbean pine (Pinus caribaea), including those leftover from P. caribaea planting programs. During the 1980s the emphasis moved to alley cropping systems with fast-growing nitrogen fixing exotic trees, through the Fiji-German Forestry Project: red calliandra (Calliandra calothyrsus) was shown to be well-suited but the systems were not adopted by farmers. During the mid-1990s and 2000s the AusAID-funded SPRIG (South Pacific Regional Initiative on Forest Genetic Resources) project worked with national partners to develop and domesticate a much broader selection of native tree species and a few key exotics, such as big-leaf mahogany (Swietenia macrophylla) and teak (Tectona grandis), which are now being incorporated into a diverse range of agroforestry systems, including modified traditional polycultural systems, in Fiji and other South Pacific Nations. Species such as the extremely cyclone tolerant, multipurpose timber tree malili (Terminalia richii) which had been reduced to scattered trees by mid-1980, is now being widely planted by smallholder farmers and tree growers in Samoa.
1.1.6 Role of ex situ conservation
Role of genebanks:
The primary aim of ex situ conservation has always been to ensure the survival of genetic resources which otherwise would have disappeared. For forest genetic resources, ex situ conservation has generally been referred to storage as seed, when practical, usually under conditions of low moisture content, and where species are intolerant of these conditions, it has been necessary to rely on field or glasshouse collections. However, such collections are costly to maintain, are at risk from pest and disease outbreaks and climate variability and extremes, and therefore are not as safe a long-term option as seed storage. For these reasons in vitro technology has been proposed as an alternative strategy. Conventional seed storage is believed to be a safe, effective and inexpensive method of conservation for seed-propagated species. Successful long-term conservation through seed storage relies on determining the factors that regulate seed viability and vigour, as well as continuous monitoring of viability with re-collection or regeneration whenever the viability drops below an acceptable level. Seeds can be categorized according to their storage behaviour, which is a reflection of the seed moisture content. The final moisture content in the seeds depends on the species and the external environment. Orthodox seeds dry out to 5-10% during maturation; these seeds are shed in a highly hydrated condition, endure a chilling period during maturation and are therefore adapted to the low temperatures used for orthodox seed storage. They can be stored for long periods of time at seed moisture contents of 3-7% on a fresh weight basis at -18oC or cooler (Theilade and Petri 2003).
In contrast relatively high moisture content, generally greater than 40-50%, is maintained in recalcitrant seeds. A distinction has been made between those seeds that are temperate-recalcitrant and tropical-recalcitrant. Species which fall into the former group can be stored at near freezing temperatures for several years but are intolerant of drying. For example, Quercus species can be stored for 3-5 years as long as a high (35-40%) seed moisture content is maintained. Seeds from tropical-recalcitrant species require the same gas and moisture levels but are very sensitive to low temperatures. For example, species from the genus Shorea, Hopea and several tropical fruit trees will lose viability at 10-15oC (Phartyal et al. 2002). Many forest tree species from temperate and especially tropical regions produce recalcitrant seeds. An intermediate category has been identified where seeds are partly tolerant to dehydration and cold. Longevity of intermediate seeds is quite short, a significant constraint for conservation in a number of species, which include a large proportion of tropical forest trees (Joët et al. 2009). Generally seed behaviour is probably best considered as a progression from orthodox to recalcitrant, and the number of species identified with non-orthodox behaviour is increasing, and its basis more complex than initially envisaged (Berjak and Pammenter 2008).
Before the seed from any species can be considered for storage the behaviour of that seed to desiccation and chilling must be determined. Variable success has been achieved globally with seed drying such that some species considered as recalcitrant have later been identified as orthodox. For example, European beech (Fagus sylvatica) and two tropical species, lemon (Citrus limon) and African oil palm (Elaeis guineensis) fall into this category (Phartyal et al. 2002) A further complexity occurs when species within a genera show both orthodox and recalcitrant behaviour, for example, Acer spp. (Phartyal et al. 2002) and Shorea spp. (Theilade and Petri, 2003). Infrequently apparent seed storage behaviour may vary geographically within the same species as in yang-na (Dipterocarpus alatus; with populations from drier zones having more desiccation tolerant seeds) and New Caledonian sandalwood (Santalum austrocaledonicum; Thomson 2006) and even depending on the stage of maturation at collection, storage and rehydration regimes in neem (Azadirachta indica; Sacandé and Hoekstra 2003).
Short to medium term storage of recalcitrant seeds can be achieved by maintaining the seeds at the lowest temperature they will tolerate, under conditions that do not allow water loss. However, these conditions will encourage the growth of micro-organisms and therefore appropriate action, such as fungicide treatment has to be used (Berjak and Pammenter, 2008). Fungicide treatment was effective in extending the storage life of kongu (Hopea parviflora; Sunilkumar and Sudhakara 1998). Problems with seed handling and storage affect the implementation of conservation programmes. The Millennium Seed Bank Project (MSBP) is the largest ex situ conservation project in the world; the project aims to conserve 25% of wild plant species by 2020. The number of tree species conserved by the MSBP at this stage is difficult to predict, however the research being conducted by the MSBP into the challenges of seed banking, such as post-harvest handling (including seed sensitivity to drying) will significantly expand existing possibilities for conservation of forest genetic resources. To date the seeds of more than 20 important palm species and around 100 dryland species have been tested for tolerance to drying (http://www.kew.org/science-conservation/save-seedprosper/millennium-seed-bank/seed-research-problem-solving/index.htm).
Seed storage duration varies across different plant species, hence the MSBP is evaluating various factors such as structure of the seed embryo and climate conditions during seed development and ripening, which can impact on seed storage duration. Baseline data on the desiccation tolerance and longevity of tree seeds is very limited (Hong et al. 1998; Dickie and Pritchard 2002). Similarly, more information is needed on the control of tree seed germination, including the method by which dormancy can be alleviated. Species of the temperate and tropical highland zones possess a range of varying degrees of dormancy with dormancy conditions within a species differing according to factors such as time of collection and climatic conditions. Under the MSBP, a unique seed database has been established, which provides information on a wide range of functional traits or characters including seed desiccation tolerance, germination and dormancy etc. Seeds are classified according to the different storage categories, at the same time the lack of knowledge that exists for tropical species is acknowledged. The World Agroforestry Centre maintains an Agroforestree database, which provides storage information for a total of 670 agroforestry tree species.
In vitro conservation
More than 70% of commercially valuable tropical tree species are estimated to have recalcitrant or intermediate seeds (Ouédraogo et al. 1999), as such long term conservation using conventional seed storage is not possible. For this reason significant effort has gone into establishing in vitro approaches for conserving forest genetic resources. However, woody species are often difficult to establish in vitro, with problems occurring at any one of the multiple stages of shoot culture establishment.
The first stage of establishing cultures derived from mature forest trees can be challenging because of high levels of contamination and/or high secretion of polyphenols and tannins. A review of the progress made in establishing tissue cultures of threatened plants (Sarasan et al. 2006) highlights a range of methods that have been developed to initiate cultures of often recalcitrant plants of limited number. The same review explores different approaches to managing tissue and medium browning. Successful initiation of in vitro cultures is not the only challenge; of key importance is the establishment of stabilized shoot cultures to provide a stock of plants that are more reproducible and stable to those found in the field or greenhouse. Despite progress in this area, in vitro shoot growth stabilization, that is a culture with uniform and continuous shoot growth, is not well understood (McCown and McCown 1987). However, rejuvenation is undoubtedly a major contributing factor with explants derived from juvenile sources easier to establish in vitro than adult plants of the same genotype. The use of juvenile tissue has been successful with a number of species for example, mangium (Acacia mangium), ear-pod wattle (A. auriculiformis), teak (Tectona grandis), jelutong (Dyera costulata), sentang (Azadirachta excela), agarwood (Aqualaria malaccense) and rotan manau (Calamus manan) (Krishnapillay 2000).
Episodic species, such as many of the nut trees and conifers, are highly problematic in tissue culture compared to the sympodial species which show continuous seasonal shoot growth, such as many of the pioneer trees. Episodic trees tend to maintain their episodic growth pattern in culture, so that random flushes of growth are followed by periods of inactivity during which the cultures deteriorate. Success using in vitro approaches is generally found in non-episodic species, for example Eucalyptus and Populus (McCown, 2000). However, two approaches to culturing highly episodic species have been successful. One approach utilizes the generation of shoots de novo; the actual induction of adventitious meristems being a rejuvenation process in itself. This approach has been very successful with conifers (Ahuja, 1993). The second approach focuses on rejuvenation either of the stock source or of the tissue cultured tissues (Greenwood 1987; McComb and Bennett 1982). Multiplication and rooting of shoot culture systems for tree species can be demanding with very specific requirements depending on the species and often the variety. For example, multiplication and rooting of the endangered tree ginkgo (Ginkgo biloba) was promoted by incorporating the endosperm from mature seeds of the same species in the culture medium (Tommasi and Scaramuzzi 2004). Sarasan (2003) reported the use of supporting materials such as Florialite and Sorbarods to improve rooting and the quality of roots produced from the critically endangered tree Saint Helena ebony (Trochetiopsis ebenus). A recent publication (Pijut 2012) reviewed in vitro culture of tropical hardwood tree species from 2001 to 2011. The publication provides outlines of methods used for a wide range of species of this commercially important group. Only once an efficient and effective system for generating stabilized shoot cultures is established should there be any attempt to develop an in vitro storage protocol. In vitro conservation technology provides two options, that of restricted or minimal growth conditions or cryopreservation. Minimal growth storage can be achieved in a number of ways. The most popular are modification of the culture medium, reduction of the culture temperature or light intensity. Minimal growth storage has been reported for several species such as flooded gum (Eucalyptus grandis; Watt et al. 2000), lemon-scented gum (E. citriodora; Mascarenhas and Agrawal 1991) and Populus spp. (Hausman et al. 1994). In vitro conservation of kokum (Garcinia indica) with subculture duration of up to 11 months has been reported after the establishment of cultures from adventitious bud derived plantlets (Malik et al. 2005).
Minimal growth culture is generally only considered as a short-to-medium term conservation approach, because of problems in the management of collections even if the intervals between transfers are extended and also because of concerns of genetic instability caused by somaclonal variation. In addition, it is generally very difficult to apply one protocol to conserve genetically diverse material. A study conducted into in vitro storage of African coffee germplasm, which included 21 diversity groups showed large variability in the response: losses occurred in some groups whereas others were safely conserved (Dussert et al. 1997a). Technical guidelines are available on establishing and maintaining in vitro germplasm collections, although not specifically of forest genetic resources (Reed et al. 2004).
Cryopreservation offers an additional in vitro methodology for long-term conservation of forest genetic resources. Cryopreservation is the storage of biological material at ultra-low temperatures (usually that of liquid nitrogen, -196oC). At this temperature all cellular divisions and metabolic processes are stopped, and therefore the material can be stored without alteration or modification for theoretically an unlimited period of time. In addition, cultures are stored in a small volume, are protected from contamination and require very little maintenance (Engelmann 2004). One of the disadvantages of minimal growth storage is the possibility of somaclonal variation occurring. Cryopreservation reduces this possibility because the metabolism of the plant cells is suspended and subculturing is not part of the process. However it has to be acknowledged that the cryoprotocol exposes plant tissues to physical, chemical and physiological stresses which can all cause cryoinjury. However, although the number of studies conducted to determine the risk of genetic and epigenetic alterations is limited, there is no clear evidence that morphological, cytological or genetic alterations take place due to cryopreservation (Harding, 2004). For example, the genetic fidelity of white cedar (Melia azedarach) after cryopreservation was confirmed using isoenzyme analysis and RAPD markers (Scocchi et al. 2004). Cryopreservation is particularly useful for conserving embryogenic cultures of conifers where regular subculturing with conventional in vitro storage could affect the growth and embryogenic potential of the cultures.
Cryopreservation is also a cost-effective conservation protocol compared to minimal growth storage. To date studies have only been conducted on crop plants but the annual maintenance of the cassava collection (about 5,000 accessions) at the International Centre for Tropical Agriculture (CIAT) is USD 30,000 for slow growth storage and USD 5,000 for cryopreservation (Engelmann 2010). Cryopreservation of biological tissue is only successful when the formation of intra-cellular ice crystals is avoided, since the crystals can cause irreparable damage to cell membranes, destroying their semi-permeability. In cryopreservation, crystal formation can be avoided through vitrification which significantly reduces cellular water through the formation of an amorphous or glassy state (non-crystalline) from an aqueous state. For cells to vitrify, a concentrated cellular solution and rapid freezing rates are required. Three categories of explants can be cryopreserved for woody species, shoot-tips for species that are vegetatively propagated, seeds or isolated embryos axes for those species which reproduce using seeds, and finally embryogenic callus.
Cryopreservation of hardwood trees has become increasingly successful since the introduction of PVS2 (plant vitrification solution), a solution containing penetrating and non-penetrating cryoprotectant solutions. Species where the vitrification/one-step freezing protocol (using PVS2) has been successful include Malus, Pyrus, Prunus and Populus spp. With these species survival rates higher than 50% have been achieved (Lambardi and De Carlo 2003). Over 90% survival rates have been reported for flowering cherry (Cerasus jamasakura; Niino et al. 1997) and white poplar (Populus alba; Lambardi et al. 2000). Vitrification has proved successful (71% recovery rate) with silver birch and morphology and RAPD analysis of regenerated plants in the greenhouse suggests that the genetic fidelity remains unchanged (Ryynanen and Aronen 2005a). Compared to shoot tips, cryopreservation of embryogenic callus and somatic embryos from hardwood trees however is limited. Success using the vitrification/one-step freezing protocol has been achieved with European chestnut (Castanea sativa; Correidoira et al. 2004), and cork oak (Quercus suber; Valladares et al. 2004).
Cryopreservation of embryogenic cultures of conifers is well advanced with successful application to a range of species including Abies, Larix, Picea, Pinus, and Pseudotsuga. Over 5,000 genotypes of 14 conifer species are cryostored in a facility in British Columbia (Cyr 2000). The technique used is mainly based on slow cooling technology where slow cooling to -40oC concentrates the intra-cellular solution sufficiently to vitrify upon plunging into liquid nitrogen. Other cryobank collections of tree species exist (Panis and Lambardi 2005):
2,100 accessions of apple (Malus spp.; dormant buds) at the National Seed Storage Laboratory, Fort Collins, USA;
Over 100 accessions of pear (Pyrus spp.; shoot-tips) at the National Clonal Germplasm Repository of Corvallis, USA;
Over 100 accessions of elm (Ulmus spp.; dormant buds) at the Association Forêt-cellulose (AFOCEL), France; and
About 50 accessions of mulberry (Morus spp.) at the National Institute of Agrobiological Resources, Japan.
In addition some tropical and sub-tropical species are being cryo-preserved:
80 accessions of oil palm (Elaeis guineensis) at the L'Institut de Recherche pour le Développement, France (Engelmann 2004); and
National Bureau of Plant Genetic Resources, India holds collections of Citrus spp., jackfruit (Artocarpus heterophyllus), almond (Prunus dulcis) and litchi (Litchi chinensis) (Reed 2001).
Despite the progress made with cryopreservation, only a limited number of truly recalcitrant seed species have been successfully cryopreserved. There are many reasons as to why progress is so slow. A relatively large number of species, many of which are wild species, have recalcitrant seeds. Little is known about their biology and seed storage behaviour. Seeds are difficult to cryopreserve because they tend to be large and have high moisture contents when shed; excised embryos or embryonic axes can be an option. However viable tissue culture protocols needed to regrow embryos and embryonic axes after freezing are often non-existent or not fully operational. In addition, significant variation is often found in the moisture content and maturity stage of seeds and embryos of recalcitrant species between provenances, between and among seed lots and between successive harvests, making cryopreservation difficult (Engelmann 2010). Despite these hurdles, various technical approaches are being explored by various groups throughout the world to better understand and control desiccation sensitivity, and to improve knowledge of the mechanisms which are responsible for seed recalcitrance.
To improve the ex situ conservation of forest genetic resources using seed storage, significant effort has to be spent in developing post-harvest technology for proper handling and identification of storage behaviour. Once seeds of a particular species have been classified, then strategies can be developed for their conservation according to their storage behaviour.
1.2 Values and importance of Forest Genetic Resources
This section reviews the immense value for humankind, and more generally to life on earth, that FGR represent.