Can Biospecimen Science Expedite the Ex Situ

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Can Biospecimen Science Expedite the Ex Situ Conservation of Plants in Megadiverse Countries? A Focus on the Flora of Brazil a

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Keith Harding , Erica E. Benson , Eduardo da Costa Nunes , Fernanda Kokowicz Pilatti , c

Juliane Lemos & Ana Maria Viana

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Damar Research Scientists , Damar, Drum Road, Cuparmuir, Cupar, Fife , KY15 5RJ , Scotland , United Kingdom b

EPAGRI, Estação Experimental de Urussanga , Caixa Postal 49, Urussanga , SC , 88840-000 , Brazil c

Departamento de Botânica, Centro de Ciências Biológicas , Universidade Federal de Santa Catarina , Florianópolis , SC , 88049-070 , Brazil Published online: 03 Jul 2013.

To cite this article: Keith Harding , Erica E. Benson , Eduardo da Costa Nunes , Fernanda Kokowicz Pilatti , Juliane Lemos & Ana Maria Viana (2013) Can Biospecimen Science Expedite the Ex Situ Conservation of Plants in Megadiverse Countries? A Focus on the Flora of Brazil, Critical Reviews in Plant Sciences, 32:6, 411-444, DOI: 10.1080/07352689.2013.800421 To link to this article: http://dx.doi.org/10.1080/07352689.2013.800421

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Critical Reviews in Plant Sciences, 32:411–444, 2013 C Taylor & Francis Group, LLC Copyright ISSN: 0735-2689 print / 1549-7836 online DOI: 10.1080/07352689.2013.800421

Can Biospecimen Science Expedite the Ex Situ Conservation of Plants in Megadiverse Countries? A Focus on the Flora of Brazil Keith Harding,1 Erica E. Benson,1 Eduardo da Costa Nunes,2 Fernanda Kokowicz Pilatti,3 Juliane Lemos,3 and Ana Maria Viana3 1

Downloaded by [2.96.18.97] at 00:25 05 July 2013

Damar Research Scientists, Damar, Drum Road, Cuparmuir, Cupar, Fife, KY15 5RJ, Scotland, United Kingdom 2 EPAGRI, Estaça˜ o Experimental de Urussanga, Caixa Postal 49, Urussanga, SC, 88840-000, Brazil 3 Departamento de Botânica, Centro de Ciências Biológicas, Universidade Federal de Santa Catarina, Florianópolis, SC, 88049-070, Brazil

Table of Contents I.

GENERAL INTRODUCTION ...................................................................................................................................................................... 412 A. Plant Conservation, Global Agendas and Biodiversity Loss ......................................................................................................... 413 1. The Convention on Biological Diversity and Sustainable Development Post 2015 ........................................................ 413 B. Megadiverse Countries, Biodiversity Hotspots and Centres of Plant Diversity ...................................................................... 414 C. The Green Economy: Integrating Conservation with Sustainable Practices ............................................................................ 415 D. Plant Conservation Science in Practice ................................................................................................................................................. 416

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RELEVANCE OF BIOSPECIMEN SCIENCE FOR BIODIVERSITY CONSERVATION ............................................ 416 A. What Is Biospecimen Science? ................................................................................................................................................................ 417 1. In Vitro Conservation and Biospecimen Science Research ....................................................................................................... 417 2. Genetic Resources and Environmental Sample Process Mapping ......................................................................................... 417

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CONTEXTUALIZING BIOSPECIMEN SCIENCE FOR IN VITRO PLANT CONSERVATION AND ENVIRONMENTAL RESEARCH ............................................................................................................................................................................ 419 A. Intercalating Biospecimen Science across Environmental Biobanks and Genebanks .......................................................... 419 B. Biospecimen Science and Problematic Storage .................................................................................................................................. 422

IV.

IN VITRO PLANT CONSERVATION PRESENTS SPECIAL CHALLENGES FOR MEGADIVERSE COUNTRIES .......................................................................................................................................................................................................................... 422 A. Complex Life Cycles and Germplasm Procurement ........................................................................................................................ 425 B. Species Interactions, Axenicity, and Phytosanitary Control .......................................................................................................... 426 1. Species Associations, Biopreservation and Molecular Analyses ............................................................................................ 426 C. Megadiversity Loss, Habitat Erosion, and Climate Change ........................................................................................................... 427

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APPLICATIONS OF IN VITRO TECHNOLOGIES FOR THE CONSERVATION OF BRAZIL’S NATIVE FLORA ....................................................................................................................................................................................................................... 427 A. Taxonomic Overview of In Vitro Techniques Applied to Brazil’s Native Flora ..................................................................... 427

Referee: Marcos Edel Mart´ınez Montero, Head of Group, Conservation of Plant Genetic Resources, Bioplantas Centre (University of Ciego ´ de Avila), Carretera a Morón km 10, CP 69450, Ciego de Avila, Cuba. Address correspondence to Dr. Keith Harding, Damar Research Scientists,Damar, Drum Road, Cuparmuir, Cupar, Fife, Scotland KY15 5RJ, UK. E-mail: [email protected]

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EVALUATING THE POTENTIAL USE OF BIOSPECIMEN SCIENCE TOOLS FOR THE IN VITRO CONSERVATION OF PLANTS FROM MEGADIVERSE COUNTRIES ........................................................................................... 430 A. Quality Management and Biospecimen Science Principles ........................................................................................................... 430 B. The Standard Preanalytical Code: SPREC ........................................................................................................................................... 431 1. Calibrating SPREC for In Vitro Plant Conservation Protocols ................................................................................................ 431 C. Modern Manufacturing Principles .......................................................................................................................................................... 432

VII. MOLECULAR TECHNOLOGIES AND GENETIC DIVERSITY ASSESSMENTS: APPLICATIONS FOR THE CONSERVATION OF BRAZIL’S NATIVE FLORA ............................................................................................................... 433 A. Molecular Technology Applications: Biospecimen Science, Stability and Risk Mitigation .............................................. 433 B. Examples of Molecular Genetic Technologies Applied to the Diversity Assessment and Conservation of Plants Native to Brazilian Forest Biomes ............................................................................................................................................................ 434 VIII. CONCLUSIONS: CAN BIOSPECIMEN SCIENCE EXPEDITE THE EX SITU CONSERVATION OF PLANTS IN MEGADIVERSE COUNTRIES? ....................................................................................................................................... 437 ACKNOWLEDGMENTS


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I. Increasing the number of species conserved ex situ in Megadiverse countries is a major task exacerbated by many intricate factors including: biome complexity, wide range of biodiversity and an incomplete knowledge of life cycles, reproductive strategies, adaptations and species interactions. Although, establishing safe reserves is a crucial conservation measure their security and effective maintenance can be unfavourably compromised by climate change and the risks incurred by socioeconomic instability and changes in land use. Anthropogenic impacts, non-sustainable practices and habitat erosion have motivated current international efforts which focused on Brazil as host of ‘Rio+20’ the United Nation’s twentieth anniversary conference on sustainable development. The revised targets of the Global Strategy for Plant Conservation (GSPC) are responses to species decline and realizing Target 8, which concerns ex situ conservation, places the heaviest burdens on countries that are custodians of the highest levels of global biodiversity. At the scientific level, ex situ conservation of endemic species in genebanks is often hindered by a lack of information about molecular genetics and problematic (recalcitrant) storage behaviors that restrict the preservation of flora native to Megadiverse countries. The potential for applying the ‘Biospecimen Science’ paradigm in expediting conservation in biodiversity-rich biomes is considered using Brazil as an exemplar of a Megadiverse country. The impacts of process chains on the quality of preserved plant germplasm and using evidence-based research to improve conservation outcomes, risk and quality management systems are appraised. The Biospecimen Science approach is not intended to displace conventional conservation practices but rather, to enhance their effectiveness in terms of the scale and efficiency of their scientific and technical operations. Keywords

biodiversity, genetic resources, germplasm, preservation, sustainability

GENERAL INTRODUCTION This review investigates the applicability of the Biospecimen Science paradigm and how it may potentially contribute to the molecular, biotechnological and biopreservation technologies used for the ex situ conservation and sustainable utilization of bioresources in species-rich countries. The authors propose that some of the methods used in Biospecimen Science may have particular relevance for the exploitation of plant diversity in ‘green economies.’ Almost all Megadiverse countries are entirely or partially located in or near to sub-tropical, tropical, and equatorial regions, this presents special conservation challenges which are exacerbated by the task of preserving an enormous range of species that are distributed across ecologically distinct biomes. Such an undertaking is particularly difficult for poorer countries which have limited infrastructures and economic capacity. Realizing global conservation targets thus places the heaviest burdens on countries with the greatest levels of biodiversity, for which Brazil ranks, highest in terms of the diversity of its endemic flora (Embrapa, 2009). Pilatti et al. (2011) summarizes the country’s main challenges associated with plant conservation: • biome (environmental) complexity, • huge array of species richness (biodiversity), • intricate species interactions and co-dependencies and a lack of understanding about their biology, • limited fundamental knowledge on plant life cycles, adaptations and reproductive strategies, • lack of baseline information on preservation and in vitro conservation behaviors,

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• germplasm storage recalcitrance, • limited basic knowledge of species biology, population and molecular genetics. These are deleteriously influenced by anthropogenic factors such as climate change, non-sustainable practices, habitat erosion and changes in land use. The preservation of Brazil’s plant genetic resources in ex situ genebanks and the use of biomolecular and biopreservation technologies to facilitate their conservation is limited to a few socio-economic species (Embrapa, 2009). Nevertheless, over the last twenty years encouraging progress has been made in the in situ and ex situ conservation of flora endemic to Brazil (Viana et al., 1999; Pilatti et al., 2011). As presented in the second Food and Agricultural Organization (FAO) national report on the ‘State of Brazil’s Plant Genetic Resources” (Embrapa, 2009), Brazil has made considerable advances in the conservation and use of its plant genetic resources, largely for those involved in food production. In the case of in situ conservation, the creation and establishment of conservation units has supported the preservation of relict native vegetation that is disappearing, with the largest conservation units being established in the Amazon biome (Embrapa, 2009). Embrapa’s Genetic Resources and Biotechnology program coordinates genetic resources conservation across Brazil’s National Genetic Resources Platform which involves some 350 Active Germplasm Banks and Base collections maintained in cold chambers, the field and in vitro (Embrapa, 2009). With respect to expanding utility of in vitro conservation for native species, this review evaluates the modern concept of Biospecimen Science (Riegman et al., 2008) in the context of environmental sustainability and megadiversity conservation. It expands on previous studies of the authors (Nunes et al., 2012; Harding and Benson, 2012; Pilatti et al., 2011) and places emphasis on ex situ/in vitro techniques, biomarker and molecular genetics technologies. The question is thus posed, Can Biopreservation Science expedite the ex situ conservation of plants in Megadiverse countries? A.

Plant Conservation, Global Agendas and Biodiversity Loss International agendas support the conservation of biodiversity within the framework of poverty alleviation, sustainability and international development. The revised 2011-2020 targets of the Global Strategy for Plant Conservation (GSPC) responds to the rapid rate of species decline. Objective 2, Target 8 states: “Plant diversity is urgently and effectively conserved. At least 75% of threatened plant species in ex situ collections, preferably in the country of origin and at least 20% available for recovery and restoration programmes” (Jackson and Kennedy, 2009; Sharrock, 2011; Sharrock et al., 2010). The GSPC was updated and endorsed by the World’s governments at the 2010 Convention on Biological Diversity Conference of Parties (COP10 Nagoya, Japan). The GSPC is aligned with international agendas and conventions including the Convention on Biological

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Diversity (CBD) Strategic Plan for Biodiversity (2011-2020), the Aichi Biodiversity Targets (Secretariat of the Convention on Biological Diversity, 2010) and the Nagoya Protocol (Secretariat of the Convention on Biological Diversity, 2011) which concerns the fair and equitable sharing of benefits arising from the utilization of genetic resources and the conservation and sustainable use of biodiversity. The implementation of the Strategic Plan for Biodiversity is facilitated by the UN which declared 2011-2020 as the “Decade of Biodiversity.” In 2012, the twentieth anniversary of the UN’s ‘Earth Summit’ was convened in Rio de Janeiro, Brazil; the 1992 Conference of Parties (COP) was the first of its kind to address, at the global level, the issue of sustainable development in the context of the environment, a significant outcome was ‘Agenda 21’. This is the plan of action adopted by governments across the world to address how humanity impacts on the environment at local, national and global levels. During the 20 years since, several Multilateral Environmental Agreements (MEAs) entered into force, including the United Nations Framework Convention on Climate Change (UNFCCC), the CBD and the United Nations Convention to Combat Desertification (UNCCD).

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The Convention on Biological Diversity and Sustainable Development Post 2015 The Rio+20 United Nations Conference on Sustainable Development focused on the ‘Green Economy,’ sustainable development and poverty eradication. Core objectives included renewing international political commitment to sustainable development, a progress review, identification of implementation gaps and addressing new and emerging challenges. The primary outcome of Rio+20 was the resolution adopted by the UN’s General Assembly (September 2012; 66/288) “The Future We Want” which was drawn up by the Heads of State of Governments and high-level representatives (United Nations General Assembly, 2012). The resolution renewed their commitment to sustainable development, ensuring the promotion of an economically, socially and environmentally sustainable future for the planet. Poverty eradication was identified as the greatest global challenge facing the world today (United Nations General Assembly, 2012). ‘Article 61’ of the resolution recognizes the urgent need to deal with unsustainable patterns of production and consumption and that these must address environmental sustainability and promote conservation and the sustainable use of biodiversity and ecosystems, including natural resources regeneration and the promotion of sustained, inclusive, and equitable global growth. ‘Article 111’ reaffirms the necessity to conserve plant and animal genetic resources, biodiversity and ecosystems and enhance resilience to climate change and natural disasters. It also recognizes the need to maintain natural ecological processes to support food production. ‘Article 197’ which is dedicated to biodiversity reaffirms the intrinsic value of biological diversity including its ecological, genetic, social, economic, scientific, educational, cultural, recreational, and aesthetic value. The critical


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role of biodiversity in maintaining ecosystems services for sustainable development and human well-being is acknowledged. Support for the three objectives of the CBD is reiterated in ‘Article 198,’ they are: (i) the conservation of biological diversity, (ii) the sustainable use of its components and (iii) the fair and equitable sharing of benefits arising from the utilization of genetic resources. The call for urgent actions that effectively reduce the rate of, halt, and reverse the loss of biodiversity are imbedded in the Strategic Plan for Biodiversity 2011–2020, the Aichi Biodiversity Targets (Secretariat of the Convention on Biological Diversity, 2010) and the Nagoya Protocol on ‘Access to Genetic Resources and the Fair and Equitable Sharing of Benefits Arising from their Utilization’ (Secretariat of the Convention on Biological Diversity, 2011). Encapsulated in “The Future We Want” (United Nations General Assembly, 2012) is a primary commitment (Articles 245–251) to develop new goals that will be focused on sustainable development (i.e., ‘Sustainable Development Goals’) these will build upon the internationally agreed Millennium Development Goals (MDGs) which concern poverty alleviation and for which the target to achieve their objectives is 2015. The outcomes and follow up activities of Rio+20 provide the guidance, vision and framework for the post-2015 period. The UN’s Post 2015 Task Team (United Nations Task Team, 2012) identified increased human activity as a critical threat to the Earth’s carrying capacity and its growing environmental footprint which is now threatening ecosystem services and biodiversity loss. Examples of which are, the loss of ∼50% of the Earth’s forests, an accelerated loss of biodiversity and a 40% increase in global CO2 emissions during 1990–2008. Consequently, a major result of Rio+20 will be the Member States constituting an open working group to guide the process of preparing new sustainable development goals that will build upon the MDGs during the post 2015 period. B.

Megadiverse Countries, Biodiversity Hotspots and Centres of Plant Diversity The idea of designating countries of megadiversity primarily concerns prioritizing world conservation efforts and raising awareness about habitat destruction and species extinctions. They are defined by the criterion of endemism, as related to the ranking of a taxonomic level (species, genus and family) occurring in one area and nowhere else (Mittermeier, 1988; Mittermeier et al., 1997). The global distribution of biodiversity is unbalanced with only a few countries, largely situated in the sub-tropical, tropical, and equatorial regions, containing the greatest number of species (Gaston and Spicer, 2004). Based on the original designations of Mittermeier (1988) these comprise seventeen countries: Brazil, Indonesia, Colombia, Mexico, Australia, Madagascar, China, Philippines, India, Peru, Papua New Guinea, Ecuador, USA, Venezuela, Malaysia, South Africa and the Democratic Republic of Congo. At the species level of biodiversity categorization, Brazil contains: 50,000 to 56,000 plants, >3,000 fresh water fish, 517 amphibians, 468 reptiles, 1,622

birds, and 525 mammals (Gaston and Spicer, 2004; Mittermeier et al., 1997). According to the United Nations Environmental Programme (UNEP) World Conservation Monitoring Centre (WCMC) “A-Z of Areas of Biodiversity Importance” (UNEP-WCMC, 2010a), and as précised from Mittermeier et al. (1997) the concept of assigning Megadiversity country status is based on four premises: 1. Biodiversity of each and every nation is critically important to that nation’s survival, and must be a fundamental component of national/regional development strategies. 2. Biodiversity is not evenly distributed across the planet, some countries (especially in the tropics) have far greater concentrations of biodiversity than others. 3. Some of the richest, most diverse countries have ecosystems under the most severe threat. 4. To achieve maximum impact with limited resources, it is necessary to concentrate (but not exclusively) on countries that are richest in diversity/endemism and that are the most severely threatened; with investment being roughly proportional to their overall contribution to global biodiversity. Various international schemes bring attention to the need to prioritize the conservation of habitats and species at high risk, one of the most significant being the creation of the Earth’s twenty five ‘Biodiversity Hotspots’ (Myers et al., 2000; Gaston and Spicer, 2004), of which two, the Cerrado and the South Atlantic Forest, are in Brazil. Biodiversity hotspots contain exceptionally high levels of endemic species that are located in areas with elevated levels of threat to extinction and habitat loss. The concept, developed by Myers and colleagues is based on source data from more than 100 scientists with direct experience of the countries and regions concerned (Myers et al., 2000). The main criteria used to designate biodiversity ‘hotspots’ are: 1. Number of endemic species: an area must contain at least 0.5% or 1,500 of the world’s plant species as endemics, based on the fact that plants are essential to almost all life forms on Earth and their conservation is crucial to every ecosystem. 2. Extent of threat of habitat loss: to qualify as a hotspot, an area should have lost 70% or more of its primary vegetation (Myers et al., 2000). Brazil is considered to have the richest flora on the planet, represented by at least 50,000 species and about one sixth of the total number of plant species identified on Earth. The Brazilian South Atlantic Forest ranks in the top five leading hotspots (Myers et al., 2000). The plant kingdom is one of the most influential in defining the eligibility of a Megadiverse country, which must have at least 5,000 of the world’s plants as endemic (Mittermeier, 1988; Mittermeier et al., 1997). In recognition of the vital role that plants play in supporting biodiversity, environmental systems and human wellbeing, the International Union for the Conservation of Nature (IUCN) and the World Wildlife Fund (WWF) initiated the ‘Centres of Plant Diversity’ (CPD) project. This highlights

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global sites of botanical importance, as précised from UNEPWCMC (2010b) the project aims to:


1. Identify global areas that, if conserved, would protect the greatest number of plant species. 2. Document the economic and scientific benefits that the conservation of these areas would bring to society. 3. Outline the potential value of each CPD to sustainable development. 4. Draw up strategies for designated conservation areas. Centres of Plant Diversity are grouped into regions comprising: Europe, Asia, SE Asia, the Middle East, Asia, Australasia, the Pacific and the Americas; they cover 234 priority sites in various countries. The CPD guide (WWF and IUCN, 1994–97; UNEP-WCMC, 2010b) adopts criteria for the selection of sites and vegetation types based mainly on the requirement that each area must have one or both of the following two characteristics: (i) evidently species rich, even though the number of species present may not be accurately known and (ii) known to contain a large number of endemic species. As summarized from UNEP-WCMC (2010b) and based on extensive consultations (WWF and IUCN, 1994–97) with experts in the world’s major regions several selection criteria are used to identify CPD sites: 1. They contain an important plant gene pool of value, or potential use to humans. 2. They contain a diverse range of habitat types. 3. They contain a significant proportion of species adapted to special edaphic conditions; 4. They are threatened or under imminent threat of large-scale devastation. As archived by the Department of Botany, National Museum of Natural History, Smithsonian Institution (Smithsonian Institution, 2012) South American (SA) Brazilian centers of plant diversity comprise: SA4: Transverse Dry Belt; SA5: Manaus Region; SA6: Upper Rio Negro Region; SA12: Atlantic Moist Forest of S Bahia; SA13: Tabuleiro Forests of N Espirito Santo; SA14: Cabo Frio Region; SA15: Mountain Ranges of Rio de Janeiro; SA16: Serra do Japi; SA17: Juréia-Itatins Ecological Station; SA19: Caatinga of NE Brazil; SA20: Espinhaço Range Region; SA21: Distrito Federal and SA22: Gran Chaco. The practical steps that are needed to halt biodiversity loss are addressed by UNEP (2011) in ‘The Living Planet Index’ (based on monitoring ca. 8,000 populations of >2,500 vertebrate species) which reflects how changes in earth ecosystem health have altered deleteriously, especially in the tropical regions which have experienced a 30% decline in biodiversity since 1992. This is indicative of severe ecosystem degradation caused by high rates of deforestation in primary forests and transformation into agricultural land and pasture. Satellite images reveal that large areas of rainforest have been cleared on the southern boundary of the Amazon Basin with the Brazilian states of Rondônia, Para and Mato Grosso having the largest losses, as reported April 29, 2010 by the Instituto

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Nacional de Pesquisas Espaciais (National Institute for Space Research), (UNEP, 2011). This situation was worsened by the severe droughts of 2005 and 2010 which increased the frequency of fire, substantiating concerns that the Amazon is reaching a ‘tipping point’ where large tracts of forest could be replaced by a more savanna-like ecosystem (Lewis et al., 2011). General threats to global plant diversity are summarized by UNEP (2011) which reports that approximately a quarter of all plant species are estimated to be threatened with extinction, in some groups this rises to >60% of species being considered as threatened. C.

The Green Economy: Integrating Conservation with Sustainable Practices Acknowledging the connectivity between biodiversity preservation and the utilization of biological resources is important for nations that are the custodians of megadiversity. The CBD devised the ecosystem approach to implement its objectives by integrating the management of land, water and living resources to promote their conservation, sustainable and equitable use. Whilst conservation practitioners have evolved different perspectives, for practical reasons ecosystem approaches need to be integrated with the ex situ conservation practices that service the green economies of biodiversity-rich countries. To this end, the Cancún Declaration (Cancún Declaration, 2002) of like-minded Megadiversity countries (Brazil, China, Colombia, Costa Rica, Ecuador, India, Indonesia, Kenya, Mexico, Peru, South Africa and Venezuela) was put in place to reaffirm the sovereign rights of the States over their own natural resources according to the provisions of the CBD. The Declaration acknowledges the importance of the countries’ natural heritage (representing ∼70% of the Earth’s biodiversity) which is associated with cultural wealth and diversity and, which must be preserved and used in a sustainable manner. The Cancún Declaration recognizes the need to enable like-minded Megadiverse countries to participate in new economies that link the use of biological diversity with biotechnology which also has a role in genetic resources conservation and sustainable development (Benson, 1999; Noor et al., 2011; Rao, 2004). Building a sustainable green economy is thus one of the key inter-governmental processes of relevance for post-2015 UN development agenda preparations (United Nations Task Team, 2012), and for which two priorities have been identified: as (i) evolving the green economy in the context of sustainable development and poverty eradication and (ii) providing institutional frameworks for sustainable development. Allen and Clouth (2012) and Allen (2012) have produced guidebooks that survey green economy principles and collate resources and information related to developing and assessing the progression of green economies in different countries. They summarized that one of the most common green economy principles concerns the protection of biodiversity and ecosystems and that the Rio+20 outcome document affirms that green economy policies should be implemented in accordance with all Rio Principles (Allen, 2012; United Nations General Assembly, 2012).


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Critical issues surrounding the fulfilment of the Rio principles for emerging green economies concern addressing the matter of scale (conserving a large number of species) and pace (ensuring that robust practices are in place to offset the accelerating risk of biodiversity loss and species extinction). The challenges of progressing towards a sustainable society and developing Brazil’s green economy, defined by UNEP as that which “results in improved human well-being and social equality, while significantly reducing environmental risks and ecological scarcity” have been addressed by Gramkow and Prado (2011) and colleagues as a prequel to the Rio+20 UN conference on sustainable development. They state that the transition to a green economy could benefit Brazil in various ways, particularly as the country has an abundance of natural resources, is a major contributor to global ecosystem services and is custodian to some of the richest biodiversity on the planet. These natural assets enable Brazil’s transition to a global green economy, including the integration of conservation in the sustainable exploitation of plant diversity. As a member of the like-minded Megadiverse countries (Cancún Declaration, 2002) Brazil highlighted the need to balance plant diversity conservation agendas with the interests of national agronomic, green and industrial economies (Jacobi et al., 2011; Versieux, 2011), this will require concerted actions as outlined by Embrapa (2009). D.

Plant Conservation Science in Practice Coates and Dixon (2007) encourage debate across the plant conservation community regarding the role of science and research in different conservation practices, suggesting that although significant advances have been made, the pace and scale at which the global conservation crisis is addressed needs to be increased. Volis and Blecher (2010) similarly propose that the unification of ex situ and in situ conservation is necessary; ranging from the wild sampling of plants to the point of their restoration. This has been demonstrated by the authors of this review in a project which identifies the need to harmonize South Atlantic forest tree seed cryostorage with the practices of end users in remote field stations that are without access to specialist facilities (Higa et al., 2011). Such practical examples encapsulate the ‘adaptive management’ approach which is the delivery of effective and timely science-based conservation solutions within operational settings (Coates and Dixon, 2007). The research-to-development continuum is similarly embraced by the Consultative Group on International Agricultural Research (CGIAR) which encourages custodianship and benefit sharing of international public goods using roadmaps that involve: problem definition, research design, research implementation, translation of research results into ideas and technologies for development, dissemination, adoption, adaptation and application (CGIAR, 2006). Future advances in plant conservation science are also dependent upon maintaining and accessing the cumulative knowledge (Jorge et al., 2010) that captures ‘prior art’ and sustains contemporary research. Similarly, best practice guidelines and quality and risk management systems linked

to the, ex situ/in vitro conservation of crop genetic resources (Benson et al., 2011c; Jorge et al., 2010; SGRP, 2010) provide a knowledge platform on which to build initiatives for a wider taxonomic range of plant diversity. It is therefore imperative that sustainable practices are supported by the restoration and strengthening of at risk populations of species in their natural environment as well as conservation ex situ. The in vitro preservation of plants that are rare, difficult to conserve and exhibit storage recalcitrance behaviors is especially challenging and knowledge of natural plant adaptations and life cycle strategies contributes to realising satisfactory preservation outcomes. Coates and Dixon (2007) consider that there is a fundamental link between off-site protection and germplasm preservation using traditional and contemporary biotechnological conservation approaches. They identify the ‘science-operational continuum’ which concerns making scientific research outcomes more relevant and accessible to conservationists. This framework concurs with the Biospecimen Science paradigm.

II.

RELEVANCE OF BIOSPECIMEN SCIENCE FOR BIODIVERSITY CONSERVATION Advances in medical molecular and biomarker technologies have caused a shift in preservation priorities, the ultimate goals of which are safeguarding biospecimen safety, quality, and usability. Biospecimen Science emerged as a new discipline in its own right to meet the exacting demands of clinical researchers and healthcare practitioners working in personalized, translational and regenerative medicine, all of which have a prerequisite for high-quality specimens that satisfy critical performance indicators (PIs) (Riegman et al., 2008). Processing, tracking, and preserving huge numbers of healthcare samples from diverse sources using the most robust, efficient, safe, and effective means possible is at the core of Biospecimen Science. This paradigm is now being used by non-medical sectors including environmental biobanks that are mandated to conserve, on a very large-scale, thousands to millions of diverse samples (Pugh et al., 2008). Therefore, it is timely to consider the merits of Biospecimen Science for the ex situ conservation of plants from regions of megadiversity. A central objective of Biospecimen Science is to address the lack of scientific data concerning the effects of process chain variables, including storage behaviors and regimes on biological samples. This concept is generic to all preserved samples because it defines the precise relationship between how a sample is handled and preserved by placing emphasis on performance after retrieval from a biorepository, culture collection, or genebank (Riegman et al., 2008). Implicitly, Biospecimen Science supports the three principles: authenticity, purity, and stability of the modern Biological Resource Centre, a framework created by the Organisation for Economic Cooperation and Development (OECD) to promote Best Practices (BPs) in culture collections servicing the biotechnology sector (OECD 2007).

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The same principles are now being adopted by culture collections and environmental repositories (Stacey and Day, 2007) and plant genebanks (Benson, 2008; Benson et al., 2011 a, b, c; Jorge et al., 2010; SGRP, 2010).


A.

What Is Biospecimen Science? The term biospecimen defines preserved biological samples that comprise viable, non-viable, replicable, and nonreplicable specimens and their derivatives (strains, cell, tissue and organ cultures, germplasm, genetic resources, cell extracts, DNA/RNA). It is distinguished from other types of conservation research because it specifically studies specimens and bioresources from the perspective of the technical, quality and process management operations (e.g., as undertaken in BRCs, biorepositories, biobanks, genebanks, culture collections). Biospecimen Science involves understanding the effects of process chains on the stability and quality of biomaterials and translating research outcomes into evidence-based BPs, Standard Operating Procedures (SOPs) and risk and quality management guidelines (Harding and Benson, 2012; Benson et al., 2011a-d; Betsou et al., 2010; Jorge et al., 2010; Lehmann, 2012; SGRP, 2010). The lack of well annotated, uniform, biospecimen processing procedures was identified as an impediment to clinical biorepository quality systems management. Consequently the ultimate goal is to safeguard biospecimen safety, quality, and usability using practices that are underpinned by robust information systems (Mills and Brooks, 2010). This also includes the identification, annotation and tracking of pre-analytical variables using Standard Pre-analytical Variable codes (SPRECs) (Benson et al., 2011d; Betsou et al., 2010, Lehmann et al., 2012), proficiency testing, methods validation and evidence-based QC tools (Betsou et al., 2013). 1.

In Vitro Conservation and Biospecimen Science Research The ex situ conservation of plants by means of in vitro techniques, including cryopreservation has mainly been achieved on a ‘case-by-case’ basis using empirical approaches underpinned by fundamental research (Benson, 2008; Lakshmanan et al., 2011). As most Megadiverse countries encompass sub-tropical, tropical, and equatorial regions it is necessary to generate knowledge about cold and desiccation stresses incurred before, during and after preservation, particularly as many tropical species exhibit problematic germplasm storage behaviors (Berjak et al., 2011; Noor et al., 2011). Proteomics technologies provide valuable insights into how certain tropical crops respond to cryopreservation and systems biology is used to study cryopreservation recalcitrance (Carpentier et al., 2005, 2007, 2008; Noor et al., 2011). These investigative approaches parallel, and are analogous to, the exploitation of molecular/omics technologies, analytical processing and systems biology in medical biobanks and microbial BRCs (Wang and Lilburn, 2009). Albeit, the scale of preservation practices and the use of molecular diagnostics and biomarkers in clinical biobanks requires high throughput processing and more stringent management (Betsou et al., 2010;

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Downey and Peakman, 2008; Riegman et al., 2008). Fundamental research and empirical-methods testing are resource intensive and often outside the capacity of conservationists tasked to preserve a wide range of plant diversity for which priority has to be given to those of the most socioeconomic importance (Pilatti et al., 2011). Expanding ex situ conservation to at risk endemics and improving the sustainable utilization of overexploited plant taxa is made more difficult because of the lack of baseline scientific knowledge. Conservation may thus be facilitated by research strategies that specifically focus on increasing the accuracy of process chain documentation, the efficiency of preservation practices and the quality of their outcomes. This approach is in line with the ideas presented by Kaczmarczyk et al. (2011) regarding the cryopreservation of at risk Australian flora. Expressly, the logistical problems of developing storage methods for the rising numbers of endangered plant species, for which the time-consuming empirical approaches used in protocol development are no longer feasible or timely options. 2.

Genetic Resources and Environmental Sample Process Mapping Process chains meticulously chart the manipulations that biospecimens or germplasm are exposed to as they progress through sequential handling stages. Thus, there is the opportunity to apply Biospecimen Science to in vitro conservation practices that may be integrated with seed and environmental biobanks. The aim of process mapping is to improve handling efficiency and precision and help pinpoint those procedures and operations that may affect the quality of biospecimens and biological resources (Holland et al., 2003). The size of a facility is immaterial as even the smallest conservation laboratory, culture collection, or genebank benefits from well optimized and thoroughly documented procedures. Processing depends upon specimen or germplasm type and can extend from the point of collection, to storage and recovery, to downstream analyses. These can range from simple viability testing to omics technologies and the application of the cryobionomics which concerns the fitness-for-purpose of plants recovered from cryopreserved germplasm and their reintroduction/introduction into the natural environment (Harding, 2004). Figure 1 indicates where selected Biospecimen Science tools (e.g., identifying pre-analytical variables in SPREC, biomarkers); concepts (e.g., cryobionomics, BRC principles) and quality management measures (e.g., the application of Taguchi statistics) may be incorporated in the operations of environmental biorepositories and genebanks. The technical components of process chains depend upon the sample, and whilst facilities will differ, most need to track common generic steps. Mapping processes helps curators, research managers and end users to plan evidence-based decisions regarding biorepository management, infrastructure, and operations including: (i) regulatory, legal, ethical oversight; (ii) risk management, and mitigation; (iii) quality management, control and assurance and (iv) inventory, tracking, and

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FIG. 1. Road map demonstrating the potential for integrating in vitro conservation with seed bank and environmental biobank operations that are organized within Biospecimen Science and Quality Management (QM) frameworks. Abbreviations: BRC = Biological Resource Centre; A = Authenticity; P = Purity; S = Stability; IVAG = In Vitro Active Genebank; IVBG = In Vitro Base Genebank; MMP = Modern Manufacturing Principles; SPREC = Standard Pre-analytical Code.

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knowledge management. These needs highlight the potential role of process chains in helping to understand the relationship between how a sample is handled and stored within quality and risk management frameworks that adopt BPs, SOPs and guidelines, and that conform to performance criteria acceptable to end users (Benson et al., 2011, a, b, c; ISBER, 2012; OECD, 2007; Moore et al., 2011; SGRP, 2010). Furthermore, stringent process mapping is required for validation, proficiency testing and third party accreditation.


III.

CONTEXTUALIZING BIOSPECIMEN SCIENCE FOR IN VITRO PLANT CONSERVATION AND ENVIRONMENTAL RESEARCH Biospecimen Science involves a conceptual change that gives more attention to research that is specifically directed at enhancing germplasm storage processing efficiency, genebank quality control (QC), and risk management. It involves streamlining and optimizing the technical operations of biobanks and genebanks and this enhances the overall capacity and pace at which biospecimens and germplasm can be processed and conserved. One advantage of Biospecimen Science is that it could potentially help to offset the financial and logistical burdens of ex situ conservation at the species level, which are considerable, but necessary to build the green economy of nations with high levels of biological diversity (Gramkow and Prado, 2011). The approach may also benefit conservationists that are overwhelmed by the acceleration in numbers of plant species that are being placed at risk, and for which limited availability of germplasm constrains efforts to optimize storage protocols (Kaczmarczyk et al., 2011). Heywood and Iriondo (2003) discuss how to balance ex situ conservation with ecosystem approaches and Donaldson (2009) encourages botanic gardens to use living collections more effectively in global-change research by linking biodiversity conservation to securing ecosystem services benefits. These concepts share much in common with environmental biobanks (Pugh et al., 2008), making Megadiverse countries well-positioned to conserve genetic resources and provide reference specimens for environmental research, impact assessments and biotechnology programs within analogous and where feasible, shared or interconnected research infrastructures that cross-cut disciplines (Figure 1). A.

Intercalating Biospecimen Science across Environmental Biobanks and Genebanks Biospecimen Science has been pioneered in environmental biobanks that hold diverse collections of organisms, environmental and biological samples (Pugh et al., 2008) providing a proven precedent for in vitro plant conservationists and environmental biobanks to cooperate, by creating a bridge between conservation in genebanks with a knowledge of ecosystem services and environmental health (Donaldson, 2009; Pugh et al., 2008). In such a model (Figure 1) the harmonization of technical operations and quality systems may be desirable, albeit this would potentially require the coordination of different standards

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and quality management guidelines (ISBER, 2012, Genebank Standards, 1994; SGRP, 2010) to meet established criteria, for example in active and base collections (Benson et al., 2011 a, b, c). Enhancing the connectivity between environmental biorepositories and the genebank sector (Figure 1) has advantages, including economy of scale and the sharing of resources, complementary expertise and knowledge. This is especially relevant for regions that have a high level of biodiversity and that are also at risk from climate change, thus genetic resources of wild species may be strategically banked during different climatic episodes or following environmental incidents. Climate change and habitat erosion impinge on the environmental signals that trigger plant life cycles, even small disturbances in these cues can deleteriously interfere with the timing of flowering, seed and fruit production, and maturation. Environmental factors and adaptive behaviors have downstream consequences for the optimization of plant germplasm storage regimes (Benson, 2008; Gale et al., 2013; Lynch et al., 2012;) and they may critically affect the conservation of storage-recalcitrant germplasm (Benson, 2008; Berjak et al., 2011). Genebank operations could thus benefit from the experiences of environmental repositories as extrapolated and adapted from Pugh et al. (2008): 1. Conservation operations are closely affiliated with environmental research programmes. 2. Technical operations for conservation and environmental services are harmonized. 3. Selection of samples is based on both conservation and environmental research criteria. 4. Technical protocols are designed and tracked to ensure quality is maintained throughout the process chain from germplasm and sample collection to storage. 5. Materials are banked on the basis that they provide a resource for conservation as well as materials for ongoing and retrospective monitoring of the environment. 6. Parallel samples may need to collected and processed for the analysis of real-time status. Procedures for collecting, handling, and transporting environmental samples, germplasm donors, and genetic resources (e.g., whole plants, seeds, vegetative propagules, herbarium specimens) are stringently standardized and documented (Table 1, Figure 1). Thus limiting the undesirable consequences of sample degradation/deterioration, deficient recording of provenance data and poor annotation, the consequences of which greatly devalue biospecimen or germplasm utility and as such could be considered as elements in risk management programs (Benson et al., 2011d; Betsou et al., 2010; Pugh et al., 2008; SGRP, 2010). The physiological status of donor plants, tissues and explants exposed to deleterious or variable environmental episodes or storage treatments can interfere with their natural, adaptive life cycle programs (Benson, 2008; Ahuja et al., 2010; Nicotra et al., 2010). These factors can affect sample quality, germplasm totipotency and storage behavior and may even predispose recovering germplasm to post-storage selection.

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ELEMENT 1 Mother Plant – Sampling Choice 1 Sampling for optimal quality is a precondition for viability-longevity; this element introduces the process chain history: by annotating the details of the mother plant (donor) for phenology and collection procedures. Capturing donor profile and sampling parameters is important as they can affect in vitro and storage manipulations later in the process chain. ELEMENT 2 Collecting Method 1 Method dictated by tree, access, phenology and environmental factors. Recalcitrant seeds deteriorate rapidly and are less amenable to storage when suboptimally collected, annotated collecting history informs later processing decisions.

SEC

SEB

X Z PCA PCB PCC X Z SA

TSB

X Z TSA

DSI DSM CMA CMB CMC

IFI IFT IFS

EXAMPLES OF CODE ELEMENT VARIABLES CODE

Indehiscent Fruits (∗ example code = IF) • Development stage 1 immature (example code = I) • Development stage 2 transitional maturity • Development stage 3 mature & ∗ Shed from tree (example code = S) Dehiscent Fruits (seeds) • Pre-dehiscence (immature) • Post-dehiscence (mature) Collection Method from ground (∗∗ example code = CM) Collection method by climbing (∗∗ example code = CM option A) Collection method after dispersal (∗∗ example code = CM option B. . . thereafter C,D,E) Unknown Other 1–2 days controlled environment transport ELEMENT 3 Transit Time & Stabilization Time between collection to processing at primary facility. 2Options are 25◦ C, 80–95% RH intermediate holding in environment-controlled vehicles, field site >1 week controlled environment transport facilities; 1stabilization is critical for recalcitrant/immature seeds; 25◦ C, 80–95% RH antimicrobials can be applied to offset spoilage; in vitro collecting Unknown processes may be used. Other Immediate processing: ELEMENT 4 Post Collection Processing & Interim Storage 1,2 This element can be a critical factor, options vary dependent upon 1 month 20◦ C 60% RH fruit development/type, seed storage behavior3–8 and type of 3 months 5◦ C 60% RH facility. Include details of seed moisture content status as appropriate Unknown (dry wt/f wt and/or g/g water). Other Wash seeds in detergent/water, sterilize 10% (v/v) hypochlorite solution, ELEMENT 5 Sterilization - Explant Excision - Initiation 3–9 Options dependent on explant (whole seed, excised zygotic 10 min, rinse 50% (v/v) ethanol 1 min × 3 SPW washes, transfer to embryo/embryonic axis, shoot) optimal disinfection, surface HF-MS medium, germinate. sterilization, culture initiation medium requirements, growth Wash seeds detergent/water, apply fungicide solution 20 min, sterilize in conditions. Special treatment options may be included: 10% (v/v) hypochlorite solution for 15 min × 3 SPW washes, excise antioxidants,5,9 free radical scavengers, (DMSO) activated charcoal, embryonic axes, transfer to MS medium + 0.2 mg/L BA and culture. including timed intervals to allow explants/propagules to recover from Wash seeds in detergent & water, sterilize 10% (v/v) hypochlorite sterilization and dissection before commencing the storage protocol solution for 10 min, rinse with 50% (v/v) ethanol 1 min × 3 SPW process chain. washes, excise embryo and transfer to HF-MS medium for 16 h to recover from dissection injury.

SPREC ELEMENT (1-7) ELEMENT DESCRIPTOR (Notes)

TABLE 1 Prototype template for Standard Pre-Analytical Code (SPREC) variables adapted for the in vitro conservation of germplasm using technical examples from studies undertaken on tree species from the megadiverse countries Brazil, Malaysia and South Africa


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Excise apical shoots from 20–30 day old in vitro seedlings, encapsulate in MS medium with 2% sucrose + 4% sodium alginate, transfer to 0.4% water-agar, store for 6-8 months, recover under standard culture regimes for 1–2 subculture cycles. Transfer shoots of 2-week-old subcultures to MS medium + 0.3% mannitol store at 15◦ C for 6–8 months, recover under standard culture regimes for 1–3 subcultures. Intermediate-recalcitrant seed excised embryos, apply loading solution, 0.4 M sucrose, 2 M glycerol for 3 h (25◦ C) remove, apply PVS2 for 1h (25◦ C) place in cryovials, plunge into (liquid phase) LN, rewarm at 45◦ C, 1 min, recover on MS medium. Recalcitrant seed, excised embryos, alginate encapsulation, apply PVS2 (25◦ C) for time = 10–60 min, place in cryovials, plunge into (liquid phase) LN, rewarm at 45◦ C, 1 min, unload in 1.2 M sucrose, recover on MS medium. Excised shoot meristems of 4-week old shoot cultures derived from in vitro-germinated recalcitrant seed; alginate encapsulation-PVS2 (25◦ C) for time = 10–30 min, place in cryovials, plunge directly into (liquid phase) LN, rewarm at 45◦ C, 1 min, unload in 1.2 M sucrose, wash in liquid MS medium, recover on solid MS medium.

CRC

CRB

CRA

MTB

MTA

Intermediate-recalcitrant-seed: excised embryos from surface-sterilized IVA seeds, preculture on 0.75M sucrose for 3 days. Nodal cuttings from germinated seedling (micropropagated) cultured on IVB HF-MS proliferation medium, subcultured every 4–6 weeks. Nodal cuttings from germinated embryo seedling, cultured IVC (micropropagated) on MS proliferation medium (+ 0.5 mg/L BA & 0.2 mg/L IAA), subcultured every 6–8 weeks.

Examples of elements include: variables for donor, sample collection, transfer, surface sterilisation, culture initiation and excision, in vitro manipulations and options for medium and long-term storage (7th Element) An inconsistent or unknown is coded X; a known option that does not corresponded to a standard procedure is coded Z. Examples of how element options can be coded are demonstrated (bold letters) for Elements 1∗ and 2∗∗ . In Element 1 depicted as initials of the sample/process e.g. to produce the code IFI (∗ example code = Indehiscent Fruits = IF, immature [I] or mature and shed [S]). Sequential coding options are depicted in Element 2∗∗ starting with the initials of the process e.g. CM = collecting method; with options listed as A,B,C and thereafter to produce the code CMA (∗∗ example code = Collection Method from ground [CM] and climbing option [A]). Elements have been constructed using different protocol components adapted from: 1Schmidt (2000); 2Marzalina et al. (1999); 3Nashatul et al. (2007); 4Higa et al. (2011); 5 Naidoo et al. (2011); 6Nunes et al. (2003); 7,8Nadarajan et al. (2007, 2008); 9Benson and Harding (2012); 10Benson (1999). Abbreviations: BA = benzyl adenine; DMSO = dimethyl sulphoxide; HF = hormone free; IAA = indole acetic acid; LN = liquid nitrogen; MS = Murashige and Skoog medium; PVS2 = plant vitrification solution number 2; RH = relative humidity; SPW = sterile purified water.

ELEMENT 7 Option 2 Long-Term Storage (LTS) Cryopreservation 3–10 Element options/details dependent on optimized parameters: germplasm choice (seed, embryo/embryonic axis, shoot meristem), preculture and pretreatments, unloading and loading solutions, cryoprotectants, mode of cryopreservation, vitrification, controlled cooling, ultra rapid freezing, cooling, thawing, rewarming, recovery regimes. Moisture content status (e.g., as dry wt/f wt and/or g/g water) may be included to define storage parameters/behavior. SPRECs provide valuable annotations of sample history for germplasm conserved in cryobanks for extended periods.

ELEMENT 6 In Vitro Pretreatments & Culture 3–9 Element options and details dependent on which stage the in vitro treatment is applied (e.g., ranging from sterile excision of embryos prior to cryostorage to long-term micropropagation). Details for optimal culture initiation and proliferation medium, and conditions are required.9 Detail of code options is species-specific and may include: culture medium composition, culture regime (temperature, light, photoperiod, subculture time) and special treatments (e.g., activated charcoal, antioxidants). ELEMENT 7 Option 1 Medium-Term Storage (MTS) Culture 6,10 Element options/details dependent on optimized parameters (growth retardants, osmotica e.g., mannitol, sucrose, low temperatures/light proliferation media, culture regimes).


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Such outcomes may be significant for germplasm sampled from provenances that are affected by climatic extremes and episodic (seasonal) environmental cues and stresses (Gale et al., 2013; Lynch et al., 2007, 2012). Uniformity across collecting and processing chains (Figure 1) is also required for biomarker studies (Harding and Benson, 2012; Holland et al., 2005) as suboptimal practices can put at risk the accurate interpretation of research findings (Figure 1, Tables 2 and 3). Similarly it is worthwhile to consider the identification and development of evidence-based quality control (QC) tools in the environmentalbiodiversity sector (Betsou et al., 2013). Testing the feasibility of intercalating Biospecimen Science (see Section VI) into already existing ex situ conservation best practices may be one step towards evaluating the applicability of the paradigm.


B.

Biospecimen Science and Problematic Storage Storage problems cross-cut all types of biorepository and it is useful to define storage recalcitrance in a way that differentiates predetermined (genetic/epigenetic) responses from those that are attributed to suboptimal processing which exacerbates storage sensitivity in materials, that otherwise could potentially survive. Intrinsic, genetically determined recalcitrance may be attributed to natural, evolutionary adaptations that are incompatible with storage treatments (Benson, 2008). These would normally confer advantages in the native habitat but cause the eventual demise of germplasm exposed to preservation treatments. Intrinsic storage recalcitrance arises because the innate qualities of germplasm are consistently proven incompatible with usual preservation treatments (e.g., intolerance to desiccation, low temperatures in tropical species). The feasibility of developing in vitro conservation for intrinsically recalcitrant germplasm is reliant on understanding adaptive physiologies and using empirical and evidence-based approaches to devise solutions that reduce storage stress. Success is limited by two main responses, the inability to survive treatments and failure to sustain recovery; all too often the resumption of normal growth is not sustained despite a promising initial level of survival (Harding et al., 2009). In contrast, recalcitrance caused by extrinsic factors (i.e., treatment-determined) occurs as a consequence of suboptimal processing and storage and poor QC, although in practice both are interconnected (Benson, 2008). Extrinsic factors thus reside in the domain of Biospecimen Science, which is distinguished from fundamental research because it specifically focuses on understanding the effects of process chain variables on the quality of preserved biomaterials and germplasm. It is thus judicious to study recalcitrance using several approaches: 1. Accumulating fundamental knowledge to elucidate the stress/adaptive responses involved and how they are affected by the process chain. 2. Empirical testing of different biopreservation strategies. 3. Use of properly validated and proficiency-tested protocols. 4. Effective technology transfers.

5. Formulation of robust guidelines, BPs and SOPs. 6. Adherence to risk and quality management systems. 7. Adoption of rigorous QC measures. A combined strategy incorporating the basic principles of Biospecimen Science (see Sections II and VI) may help to offset laborious, time-consuming empirical testing and/or undertaking fundamental research which leads to unworkable, impractical outcomes at the stage of technology transfer. There is a shift in focus towards the rigorous testing and validation of methods and QC measures (e.g., proficiency testing, PIs) and the production of well-documented SOPs and BPs that ensure protocols are precisely followed and achieve consistently, reliable storage outcomes (Benson et al., 2011c; Betsou et al., 2013; ISBER, 2012; Paltiel et al., 2012). This may be hard to establish for at endangered and recalcitrant germplasm, in which case Biospecimen Science approaches (see Section VI) may help to identify critical point factors that differentiate between extrinsically determined, e.g., related to suboptimal processing, poor QC and ‘true’ intrinsic (genetically determined) recalcitrance (Benson, 2008). IV.

IN VITRO PLANT CONSERVATION PRESENTS SPECIAL CHALLENGES FOR MEGADIVERSE COUNTRIES Regions of megadiversity are usually represented by subtropical, tropical and equatorial biomes that are more likely to be prone to species extinction (Ghazoul and Sheil, 2010), examples of factors that could potentially contribute to their vulnerability include: • • • •

high levels of specialism and endemism, occupancy of distinctly narrow niches, space and resource competition, critical dependencies on other species for nutrition and reproduction, • specialist symbiotic and commensal associations, • complex parasitic, pathogenic and allelopathic interactions, • lack of resilience to anthropogenic pressures. In Brazil, certain in situ cultivation strategies have been integrated with biodiversity conservation, a case in point is the ‘cabruca system’ in which an understory of native Atlantic forest is cleared and the canopy thinned to provide appropriate shade conditions for cultivating cocoa trees. Although studies have shown that the role of the system in supporting biodiversity enrichment may be more limited than originally thought (Rolim and Chiarello, 2004). Harmonizing ex situ-in vitro and in situ conservation thus inevitably presents challenges that are restricted by a lack of baseline information regarding complex life cycles, particularly when they have been deleteriously influenced by human interventions and climate change (Ghazoul and Sheil, 2010; Pilatti et al., 2011). Similarly, species interactions and adaptive behaviors at the ecosystem-level can influence the

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Type of Technique, Analysis or Biomarker and Application Context (Reference)

Environmental Environmental biobanking (Pugh et al., 2008) phenotypes, genetic diversity, habitats, Genetic diversity of Brazilian tree species in natural in situ populations (Alves et al., 2007; Azevedo et al., 2007; ecological factors, and climate change Lemes et al., 2002, 2003, 2010; Tarazi et al., 2010) Genetic diversity of Brazilian tree species in anthropogenic-impacted environments (Lemes et al., 2007a) Glutathione system as a stress marker in plant ecophysiology (Tausz et al., 2005) Induced emission of plant volatile organic compounds (Holopainen and Gershenzon, 2010) MicroRNA networks and developmental plasticity in plants (Rubio-Somoza and Weigel, 2011) Molecular responses to environmental stresses of climate change (Ahuja et al., 2010) Plant phenotypic plasticity in a changing climate (Nicotra et al., 2010) Protein/hormone ecophysiology biomarkers of plant stress (Kosakivska, 2008) Baseline Knowledge DNA banks providing novel options for genebanks (de Vicente and Andersson, 2006) tropical species genetic diversity and DNA barcode for land plants and the discovery of unknown species (CBOL, 2009) life cycles (in relation to genebanking) DNA Barcoding markers for species identification (Sass et al., 2007) genebank management DNA Barcoding of Amazonium tree species (Gonzalez et al., 2009) Geneflow in fragmented populations of Brazilian forest species (Seoane et al., 2005; Martins et al., 2008) Evolving role of genebanks in field of molecular genetics (de Vicente, 2004) Molecular markers for genebank management (Spooner et al., 2005; Moretzsohn et al., 2002) Plant conservation biotechnology (Pilatti et al., 2011; Noor et al., 2011; Lakshmanan et al., 2011) Recalcitrance Viability and cryoinjury seed and in vitro storage problems DSC application to cryopreserve recalcitrant seed producing tropical trees (Nadarajan et al., 2008) Functional genomics/proteomics applications to plant cryopreservation (Volk 2010) Growth, vigor, protein synthesis, TTC-respiratory responses of zygotic embryos to partial dehydration and cryopreservation (Naidoo et al., 2011; Sershen et al., 2011a) Growth/regeneration of cryopreserved shoot tips (Varghese et al., 2009) Musa spp. acclimation to osmotic stress and cryopreservation (Carpentier et al., 2005; 2007; 2008; Zhu et al., 2006) Biophysical markers in cryopreserved seeds/pollen of numerous species (Walters, 2004; Buitink et al., 2000) Viability/ultrastructure of zygotic embryos to cryopreservation (Sershen et al., 2011b) Viability-FDA of cryopreserved pollen (Chaudhury et al., 2010) (Continued on next page)

Research Application

TABLE 2 Examples of the use of molecular technologies, analytical techniques and biomarkers that are applicable for the conservation of plant diversity


424 Type of Technique, Analysis or Biomarker and Application Context (Reference)

Examples are selected from different disciplines to indicate the relevance of different molecular and analytical techniques for the conservation of plant species from Megadiverse countries, (applications to Brazilian plant diversity are highlighted); review citations include various analytical techniques. Abbreviations: DSC = differential scanning calorimetry; FDA = fluorescein diacetate; ROS = reactive oxygen species; TTC = triphenyl tetrazolium chloride.

Stress Physiology Oxidative stress adaptive and in vitro stress physiology Antioxidants, ROS and epigenetic changes during in vitro culture (Johnston et al., 2005, 2006, 2007, 2009, 2010) Headspace volatile markers for cryopreserved somatic embryos (Fang et al., 2008) Metabolic and proteomic generic markers for oxidative stress (Shulaev and Oliver, 2006) Protein markers for ROS in cryopreserved embryonic axes (Whitaker et al., 2010) Programmed/cryopreservation induced cell death Glutathione a universal stress marker of programmed cell death in seeds (Kranner et al., 2006) Mitochondrial markers for delayed onset cell death/apoptosis in cryopreservation failure (Baust et al., 2007) Physiological factors in cryopreservation failure (Harding et al., 2009) Stability DNA methylation/siRNAs epigenetic communication from plant to progeny (Mosher and Melnyk, 2010) biomolecular epigenetic, genetic Epigenetics of plant cells cultured in vitro and somaclonal variation (Miguel and Marum, 2011; Scowcroft, 1984) Genetic/epigenetic stability of plant cryopreservation systems (Harding 1996; 1999; 2004; Harding et al., 2000; 2009; Johnston et al., 2005; 2010); effects of storage on biomarkers (Balasubramanian et al., 2010; Kugler et al., 2011) MicroRNA misregulation an overlooked factor generating somaclonal variation (Rodriguez-Enriquez et al., 2011) Microsatellite markers of cryopreserved somatic embryos (Fang et al., 2009)

Research Application

TABLE 2 Examples of the use of molecular technologies, analytical techniques and biomarkers that are applicable for the conservation of plant diversity (Continued)


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TABLE 3 The application of molecular, analytical and biomarker techniques applicable to Biological Resource Centre (BRC) operating principles contextualized for ex situ - in vitro plant conservation BRC Operating Principles Authenticity


Purity

Stability

Type and Application of Technique, Analysis or Biomarker (Reference) Tropical plant germplasm characterization: molecular markers (Ayad et al., 1997; Karp et al., 1997; de Vicente, 2004, 2006; Oliveira and Silva, 2008; Sass et al., 2007) Taxonomy, characterization and conservation: molecular markers (INIBAP 2006; Schlögl et al., 2007) Genebank accession characterization: molecular markers (Ayad et al., 1997; de Oliveira and Silva, 2008; de Oliveira et al., 2007b, 2010; Ribeiro et al., 2010; Spooner et al., 2005) Contaminants, microbial hazards in plant tissue and cell cultures (Leifert and Cassells, 2001; Lucero et al., 2011; de Oliveira, 2000) Detection of pathogens: ELISA and PCR methods (Rao, 2004; Sugii, 2011) Diagnostics for plant pathogens and detection arrays (Lievens et al., 2005) Detection of endophytic bacteria in tissue cultures (Diekmann and Putter, 1996; Van den Houwe and Swennen, 2000) Mutualistic and symbiotic associations (Salifah et al., 2011) Biophysical Stability: Thermal analyses: Differential Scanning Calorimetry (DSC); molecular mobility (Faltus et al., 2008; Gale et al., 2013; Nadarajan et al., 2008; Walters, 2004) to assess the biophysical stability of the cryopreserved state (e.g. glass stabilization, vitrification/de-vitrification, glass relaxation, critical Tgs during cooling and warming) Viability: Parameters for viability assays: accuracy, precision, specificity, sensitivity and standardization (Bank and Schmehl, 1989) Viability prediction: electrolyte leakage, TTC, spectroscopy staining and MDA techniques (Verleysen et al., 2004) Survival/biochemical assays: TTC, chlorophyll, protein and peroxidases (Yin et al., 2011) Phenotype/genotype: Phenotypic variation: shoot/root in vitro morphology (Okere and Adegeye, 2011) Somaclonal variation: molecular markers RAPD, AFLP, cytological studies (Fang et al., 2009; Msogoya et al., 2011; Rodriguez-Enriquez et al., 2011; Sahijram et al., 2003; Scowcroft, 1984) Genetic stability: (e.g., electrophoretic isozyme patterns, AFLP, PCR, RAPD) molecule genetic profiling (Scocchi et al., 2004; Volk, 2010); DNA adducts (Johnston et al., 2005; 2010) epigenetic stability: DNA methylation (Harding et al., 2000; Johnston et al., 2005, 2009; Kaity et al., 2008) Growth/development: Field performance, epi/genetic integrity: morphological, RAF, AMP markers (Harding et al., 2009; Kaity et al., 2008, 2009) Field performance (Cˇote et al., 2000; Mart´ınez-Montero et al., 2002)

Examples are selected from various disciplines to demonstrate the relevance of using different molecular and analytical techniques for the conservation of tropical plants and species from Megadiverse countries within the framework of BRC operating principles. Abbreviations: AFLP = amplified fragment length polymorphism; AMP = amplified DNA methylation polymorphism; MDA = malondialdehyde; PCR = polymerase chain reaction; RAF = randomly amplified DNA fingerprinting; RAPD = randomly amplified polymorphic DNA; TTC = triphenyl tetrazolium chloride.

technical practices involved in germplasm preservation (Benson, 2008). For example, a lack of baseline knowledge can impair the effective in vitro propagation and conservation of plants such as bromeliads that are of both commercial and ecological significance (Paiva et al., 2009). Encouragingly, tissue culture protocols optimized for the conservation and mass propagation of a bromeliad (Vriesea reitzii) threatened by extinction in the Brazilian Atlantic Forest have proved highly successful, producing 100% survival of acclimatized shoots that have been optimally micropropagated (Filho et al., 2005). Such studies are proven cases as to how in vitro technologies can support both conservation and the sustainable livelihoods that are dependent upon Brazil’s native flora. Different field, in vitro and cryostor-

age conservation approaches may be used as ‘test-systems’ on which to develop preservation strategies for more problematic species and germplasm, for example Mentha spp. may be used as a model candidate (Silva et al., 2006) as it is amenable to in vitro conservation. A.

Complex Life Cycles and Germplasm Procurement The functional traits of plants are often under the control of specific ecological conditions, consequently habitat erosion and fragmentation can disrupt life cycles and accelerate the demise of species, particularly those that have evolved specialized associations (Sugii, 2011); susceptible examples include the cycads (Donaldson, 2004; Litz et al., 2004) and orchids (Kauth


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et al., 2011). The subtle environmental cues, ecological pressures and species associations that are involved in regulating plant life cycles and reproduction are easily disturbed. Some of the most complex are found in tropical and equatorial rainforest tree species which have reproductive strategies involving mass flowering and dependencies on other species for pollination and seed dispersal. Seed production occurs sporadically and in some cases only after many years thus, idiosyncratic reproductive strategies have major disadvantages for ex situ conservation because the window of opportunity for procuring and preserving germplasm is limited. The time frame in which seed storage protocols can be optimized is also restricted because many tropical plants produce ‘intrinsically,’ storage-recalcitrant seeds which deteriorate rapidly following collection (Marzalina and Krishnapillay, 1999; Mansor, 2012). Accordingly, complex life cycles, idiosyncratic seed production and intrinsic storage recalcitrance are the main motivations for using in vitro techniques to conserve plants from Megadiverse regions, as exemplified by rainforest and tropical crop species (Benson et al., 1996; Benson, 2008; Nadarajan et al., 2006; 2007; 2008; Nashatul et al., 2007; Viana et al., 1999; Mansor, 2012; Noor et al., 2011). For the same reasons, Biospecimen Science tools, including Taguchi analysis, the precise annotation of sample processing history (see Section VI) and molecular technologies (see Section VII) applied in quality management frameworks are especially relevant for the conservation of species that have complex life cycles and intrinsic, recalcitrant storage behaviors. B.

Species Interactions, Axenicity, and Phytosanitary Control Conserving species interactions facilitates environmental restoration, but these associations present special issues for plants preserved in tissue culture and cryostorage as their successful maintenance may be influenced by symbiotic or commensal partnerships. One advantage of in vitro conservation research is that it allows the study of complex symbioses under controlled conditions (Kauth et al., 2011; Salifah et al., 2011). Axenicity (the term that describes a culture free from contaminating organisms) is a fundamental principle of most biorepositories and genebanks. However, it requires careful consideration for plant germplasm that is associated with beneficial organisms, especially if the partnership is from an at risk habitat. In these scenarios it may be crucial to conserve both the plant and its associated, beneficial, microbial flora in order to sustain later reintroductions or introduction into a new habitat. Maintaining the purity of in vitro cultures is essential to avoid cross contamination and microbial flora contribute to seed spoilage and reduce viability. Loss of cultures and germplasm through contamination and the spread of nuisance and pathogenic microflora within and outside genebanks is important for species under phytosanitary regulation. This may be problematic for certain native species, especially those from tropical and equatorial regions that have a propensity for harbouring diverse microbial flora, species associations and sym-

biotic partnerships. They present technical problems as demonstrated by Thomas et al. (2007) who isolated fourteen distinct endophytic bacterial clones from surface-sterilized papaya shoots. Similarly, Sugii (2011) reports that the rigorous optimization of disinfestation protocols is required to establish native Hawaiian plants in vitro. Axenicity may also be considered a pre-analytical variable in certain cases (Benson et al., 2011d) as the presence of contaminating or benign microbial flora in stored germplasm could potentially affect positive storage outcomes and confound molecular data interpretation (Benson, 2008; Stacey, 2004). The predilection of species interactions occurring in plants indigenous to regions of megadiversity requires deliberation regarding the management of germplasm axenicity, non-axenicity and phytosanitary control in ex situ genebanks. The potential for surface and systemic microbial flora to be present should be assumed throughout all stages of germplasm procurement, handling and storage (Benson, 2008). Within the Biospecimen Science framework, explant surface-sterilization and stringent asepsis control may be considered as pre-analytical variables in SPRECs (Table 1). Furthermore the presence of associated microorganisms in seeds and vegetative propagules can affect biomarker analysis (Table 2) and impair the realization of BRC operating principles (Table 3). Conversely, species interactions and symbioses can have positive benefits as some microorganisms contribute to plant health. Seeds germinated on tissue culture medium may require the maintenance of mycorrhizal associations that are vital for the sustained proliferation of in vitro grown plants once they are returned to the wild. Arnold and Lutzoni (2007) consider tropical leaves as ‘biodiversity hotspots’ and emphasize the ecological significance of cryptic symbionts and fungal endophytes, they found that plants respond to these partnerships in ecologically significant ways. Interestingly, Márquez et al. (2007) characterized mutualistic associations between a fungal endophyte, a virus and a tropical grass that allows the plant and the fungus to tolerate high soil temperatures. Salifah et al. (2011) found that the germination of Grammatophyllum speciosum improved in the presence of cultured isolates of mycorrhizae. 1.

Species Associations, Biopreservation and Molecular Analyses Clean cultures are an essential prerequisite for in vitro germplasm conservation in most plant genebanks, however, in special cases the in vitro propagation and cryopreservation of non-axenic cultures may be desirable (Sommerville et al., 2008). It is foreseeable that the issue of axenicity may need increasingly careful consideration for the in vitro conservation of plants that are dependent upon species associations and, as these are more prevalent in regions of high biodiversity it may influence the success of in situ restoration programs. Arnold and Lutzoni (2007) suggest that the ecological relevance of species associations requires the classification of taxonomic units based on molecular analyses that can estimate species boundaries among plant endophytes. Lucero et al. (2011) characterized seed-borne

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systemic endophytes in Atriplex spp. grasses grown in vitro using a range of molecular techniques, concluding that whilst these ‘microbiome’ analyses are enabled by high throughput sequencing there are some limitations. Identifying molecular biomarkers (Harding and Benson, 2012) is pivotal to the study of native species interactions especially in the context of in vitro plant conservation, reintroduction and restoration in complex biomes. On the other hand, axenicity is crucial for molecular genetics and omics research because the presence of associated and covert contaminants can jeopardize the interpretation and authenticity of data (Stacey, 2004). Assessing the conditions of axenicity and non-axenicity with respect to the stabilizing complex associations as they pass through biopreservation process chains is a likely future requirement. This may necessitate the development of biomarkers to assess the authenticity and stability of individual ‘axenic partners’ and their complex species associations with respect to their differential responses to tissue culture and cryopreservation and down-stream use. C.

Megadiversity Loss, Habitat Erosion, and Climate Change The main threats to in situ populations of plants from regions of high biodiversity are habitat destruction caused by agriculture, plantation forestry, agroforestry, urbanization, environmental modifications, and climate change. Habitat erosion and changes in land use are the main threats to biodiversity loss, especially in tropical regions and these are coupled to non-sustainable practices and over-exploitation (Berjak et al., 2011). To alleviate their affects, ex situ conservation is best integrated with traditional in situ measures (Pilatti et al., 2011; Sugii 2011). Loss of species is exacerbated by anthropogenic pressure on mutualistic associations, predators and parasites, consequently extinctions are accelerated by an overall decline in biodiversity (Ghazoul and Sheil, 2010). Co-evolved species interactions and mutualisms are deleteriously impacted by habitat fragmentation as this disrupts the complex interconnections of food webs, nutrient cycles and ultimately tropical ecosystem services (Ghazoul and Sheil, 2010). In vitro conservation can potentially offset some loss of plant diversity by the creation of genebanks that provide an insurance against the demise of species at high risk in their native habitats. In vitro genebanks also support the sustainable utilization of biodiversity in regions prone to habitat erosion, as they support sustainable management practices and provide plants for restoration (Berjak et al., 2011; Krishnan et al., 2011; Marriott and Sarasan, 2011; Pilatti et al., 2011). V.

APPLICATIONS OF IN VITRO TECHNOLOGIES FOR THE CONSERVATION OF BRAZIL’S NATIVE FLORA The revised Target 8 of the GSPC (see Section I) states that at least 75% of threatened plant species should be preserved in ex situ collections, preferably in the country of origin (Sharrock, 2011; Sharrock et al., 2010) the scale of this task is formidable for Megadiverse countries that have high levels of endemic

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species. It is also hindered by the fact that plants from tropical and equatorial ecosystems often produce recalcitrant seeds, or orthodox seed that have idiosyncratic storage behaviors. In these cases the development of new storage regimes can often require the laborious and empirical optimization of speciesspecific tissue culture and cryogenic treatments. Whilst in vitro conservation is the preferred option for problematic germplasm this is not without its drawbacks as tissues exposed to storage treatments can succumb to contamination (see Section IVB), hypersensitive responses, oxidative stress, and lethal in vitro and cryo-injuries (Benson, 2008; Berjak et al., 2011; Harding et al., 2009; Naidoo et al., 2011; Normah et al., 2011). Notably, progress has been made using in vitro storage in active and slow growth states and in the use of vitrification-based cryostorage protocols, although a large proportion of germplasm from plants indigenous to Brazil, have yet to be tested as suitable candidates for in vitro conservation (Pilatti et al., 2011). Several government policies have been successfully established to enable the in situ conservation of Brazil’s native plants but the application of in vitro conservation remains restricted to a few species e.g. Cedrela fissilis (Meliaceae) (Nunes et al., 2003), Ocotea catharinensis (Lauraceae) (Santa-Catarina et al., 2004), Hancornia speciosa (Apocynaceae) (Soares et al., 2007), Acmella oleracea (Compositae) (Malosso et al., 2007), Piper hispidinervum and P. aduncum (Piperaceae) (Silva and Scherwinski-Pereira 2011). To date, tissue culture has been mainly used for economically significant native plants, with a view to exploiting their biotechnological potential in Brazil’s green economy. It is timely for conservationists in Megadiverse countries to capture prior art related to crop plant conservation (see Jorge et al., 2010) and use this existing knowledge base to extend the taxonomic range to endangered and at-risk endemic species. Therefore, the aim of this section is to compile a taxonomic bibliography of tissue culture manipulations (in vitro germination, micropropagation, somatic embryogenesis, zygotic embryo culture, and callus culture) that are available to inform the in vitro conservation of plant species indigenous to Brazil. These examples represent some of the country’s most diverse and at risk biomes (see Section IB), specifically, the Caatinga, Cerrado and the Amazon and South Atlantic Forests. A.

Taxonomic Overview of In Vitro Techniques Applied to Brazil’s Native Flora Biotechnological approaches can help to progress the ex situ conservation of plant germplasm by helping to overcome problematic storage behaviors. To raise awareness as to their applications for the sustainable utilization of endemic plants the following bibliographic section collates selected taxonomic case studies that describe in vitro techniques relevant to conserving Brazil’s indigenous plants. Examples are given for specific phylogenetic groups based on biome distribution, socio-economic/medicinal importance, and their threatened or at risk status. Technical details are included to illustrate the current state-of-the-art and future scope for using different


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in vitro manipulations to aid the sustainable utilization and biopreservation of Brazilian flora. Emphasis is placed on species that are at risk in their native habitat and vulnerable to overexploitation. Anacardiaceae. Myracrodruon urundeuva is an important medicinal tree which occurs in the Caatinga, Cerrado and Atlantic Forest biomes. It is on the verge of extinction and on the official list of Brazil’s threatened flora and is classified as vulnerable. Andrade et al. (2000) developed protocols for M. urundeuva, including, in vitro seed germination, shoot proliferation, rooting, and acclimatization. Germination was achieved on half strength MS (Murashige and Skoog) medium (Murashige and Skoog, 1962) supplemented with 30 g/L sucrose, 4.5 g/L agar. Shoots were produced through the proliferation of pre-existing buds from nodal and apical segments excised from axenic seeds and cultured on half strength MS medium supplemented with 4.5 μM BAP (6-benzylaminopurine), rooted on modified MS medium supplemented with 4.8 μM NAA (α- naphthaleneacetic acid). Apocynaceae. Aspidosperma polyneuron is a native tree valuable for its hardwood, it is at risk of extinction and is on the list for ex situ and in situ conservation programs in Brazil and Venezuela. Ribas et al. (2005) report irregular fruit production and difficult seed harvesting due to the large tree size and unsynchronized germination. The successful development of micropropagation and acclimatization protocols will improve in vitro conservation for medium-term germplasm conservation. Hancornia speciosa is a tree native to the Cerrado biome, it is very popular in NE Brazil where it is important for fruit production and export. According to Pinheiro et al. (2001) its seeds are recalcitrant and germination is inhibited by fruit pulp. These authors designed a protocol that supported high germination rates by using seeds (without teguments) cultured on sucrosefree MS medium supplemented with 0.1 mg/L gibberellic acid. The micropropagation protocol of Grigoletto (1997) involves apical and nodal segments from axenic seedlings being inoculated on MS medium supplemented with BAP and IBA (indole3-butyric acid) at 1.28 mg/L. This method was used successfully by Machado et al. (2004) to propagate 11 genotypes that were used for in vitro plant production on a large scale. Soares et al. (2007) optimized the micropropagation protocol using 3 and 5 mg/L BAP to stimulate effective shoot proliferation from node segments, after which 3 mg/L IBA was applied to induce 20% micro-shoot rooting. Mandevilla velutina, a medicinal plant used to treat snake bites and as an anti-inflammatory is native to the Cerrado and in danger of extinction by overexploitation. Biondo et al. (2007) micropropagated this species using nodal segments excised from axenic seedlings germinated in vitro and inoculated on MS medium with 0.44 μM BAP, for shoot multiplication, and on half strength MS with 26.85 μM NAA for rooting. In vitro storage was successfully achieved using micro-shoots cultured on MS medium supplemented with 2% (w/v) sucrose, 13.8 mM spermidine, 2% (w/v) sorbitol and 2% (w/v) dextrose. Seedling production via micropropagation

is now available for reforestation programs and genotypes are maintained in the Germplasm Bank “Cerrado in vitro.” Aquifoliaceae. Ilex paraguariensis is a native tree from the forests of S Brazil, it is economically important for the production of tea. Quadros (2009) developed a protocol for ex vitro rooting, using 1000 ppm IBA and acclimatization of in vitro–produced micro-shoots. Seeds of I. paraguariensis demonstrate dormancy that can be overcome by zygotic embryo culture (Hu, 1975); in vitro–raised seedlings are important sources of micro-shoots that can be easily rooted for large scale production. In vitro protocols can now be used for germplasm bank maintenance. Asteraceae. Lychnophora pinaster is a medicinal plant native to the Cerrado, it is used for anti-inflammatory and healing purposes and due to intense exploitation is classified as being vulnerable to extinction. Souza et al. (2003) optimized a protocol using zygotic embryo germination and in vitro seedling growth on one-fourth strength MS supplemented with 0.6% (w/v) agar. In vitro multiplication was achieved by culturing nodal segments (containing two axillary buds) excised from the axenic seedling and inoculating them on one-fourth strength MS supplemented with 7.5 g/L sucrose and 0.25 or 0.5 mg/L BAP. This micropropagation protocol may be adapted for the in vitro conservation of L. pinaster. Bignoniaceae. Tabebuia impetiginosa, is native to the Cerrado, on the verge of extinction and listed for ex situ conservation by the Instituto Florestal de São Paulo, it is an important medicinal tree. Cabral et al. (2003) report that seeds of Tabebuia species, including T. impetiginosa have short longevity which limit their conservation in seed banks and hinder seedling production. Martins et al. (2009) developed protocols for in vitro seed germination and shoot proliferation using nodal segments excised from axenic seeds cultured on Woody Plant Medium (WPM) (Lloyd and McCown, 1981) supplemented with 30 g/L sucrose and 6 g/L agar and 1.0 mg/L BAP Bromeliaceae. Ananas lucidus is an ornamental Bromeliace native to the Caatinga, it is economically important in NE Brazil and is exported to the United States and Europe. De Oliveira et al. (2007a) produced a consistent micropropagation protocol using liquid MS medium supplemented with 30 g/L sucrose and 1-4 mg/L BAP for shoot proliferation; high plantlet survival was achieved. The richest biome for the Bromealiaceae is the Atlantic Forest of which Cryptanthus sinuosus is a native in SE Brazil, it is under increasing risk of selective extraction due to the supply and demand of the expanding ornamental market, these destructive activities may lead to its rapid extinction. The micropropagation protocol of Arrabal et al. (2002) is relevant for plant production and in vitro conservation as high regeneration rates were achieved on MS liquid medium without growth regulators. Alves et al. (2004) have characterized natural populations of Bromeliads native to the Atlantic forest. Vriesea reitzii is an endangered Bromeliaceae from the Atlantic Forest for which Alves et al. (2006) produced a reproducible protocol for large scale plant production by inducing


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nodule clusters and regenerating plants from young basal leaf segments cultured on MS medium supplemented with 2,4-D and kinetin. This regime was followed by culture on MS supplemented with 2iP (6-γ ,γ -dimethylallyamino-purine) and NAA and transfer to MS medium free of plant growth regulators to achieve plant development. Cactaceae. Arrojadoa spp. is an ornamental Cactaceae native to Brazil and an endemic of the Caatinga biome, it is on the verge of extinction in Minas Gerais state and as the plants are a vital part of bird food chains this places both flora and fauna at risk (Mendonça and Lins 2000). Dias et al. (2008) developed an efficient protocol for in vitro seed germination and seedling growth using growth regulator–free MS medium supplemented with 2.5 g/L activated charcoal. These authors report that the species can be seed-propagated but germination under natural conditions is inhibited by mucilage, as in vitro culture overcomes this problem their protocol is particularly applicable for ex situ conservation. Caryocaraceae. Caryocar brasiliense is an economically important native tree from the Cerrado biome, it has medicinal properties and produces edible fruits and oils that are used in cosmetics. Its seeds are strongly dormant, germination is low dropping drastically after 4 months of storage, there are no studies on effective seed storage and high costs are associated with seedling production. Oliveira (2000) established in vitro culture and somatic embryogenesis protocols for this species, but further studies need to be carried out to optimize the system. For the purposes of plant regeneration, Landa et al. (2000) devised a protocol for callus culture of C. brasiliense. Callus proliferated from leaf segments cultured on WPM medium supplemented with 30 g/L sucrose, 0.65% (w/v) agar, 5.37 μM NAA and 1.11 μM BAP and 500 mg/L malt extract. Care is required when adapting protocols involving dedifferentiated tissue cultures for conservation purposes as potentially they can generate genetic instability through Somaclonal Variation (Harding, 2004; Scowcroft, 1984). Cochlospermaceae. Cochlospermum regium is a medicinal species, a native of the Cerrado and Caatinga biomes and one of the priorities in conservation programs of central and NE Brazil. Camillo et al. (2009) have produced a culture protocol using seedlings from in vitro germinated seeds of C. regium, yielding 100% survival after 3 months storage on half-strength MS medium at 20◦ C. Clusiaceae. Hypericum brasiliense is a native of the Amazon forest, it has pharmacological potential and produces xantones and other secondary metabolites of interest. Velloso et al. (2009) developed an in vitro protocol to study gene expression and secondary metabolite production. Axillary buds are excised from adult plants in the greenhouse and cultured on a filter paper bridge in liquid MS medium supplemented with 3% (w/v) sucrose in the absence of growth regulators. Velloso et al. (2009) successfully raised seedlings that could be exposed to culture medium containing elicitors that enhance secondary metabolite production. Kielmeyera coriacea is a medicinal plant and im-

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portant for wood production, it is from the biome Cerrado and due to intense exploitation has become extinct in some regions. Pinto et al. (1994) report propagation of K. coriacea by seeds but non-uniform plants are produced and vegetative propagation is not viable. The authors consequently established a successful micropropagation protocol using nodal segments from axenic seedlings cultured on MS medium supplemented with 30 g/L sucrose and 0.5 mg/L BAP. Compositae. Acmella oleracea is a medicinal tree from the Amazon forest; according to Malosso (2007) its seeds fail to germinate in vitro. Nodal segment explants from greenhousegrown plants were thus cultured on MS medium supplemented with 0.1 mg/L kinetin, resulting in high levels of proliferating shoots capable of rooting and the production of complete plants. For in vitro conservation, shoots were inoculated on MS medium supplemented with 20 g/L sucrose and 40 g/L sorbitol and maintained at 18 ± 2◦ C, 60% RH and 16 h photoperiod for 6 months. Fabaceae. Anadenanthera colubrina is a native of semi-arid regions, it is important for forest management but is at risk of extinction; seed propagation produces high heterogeneity amongst seedlings. Nepomuceno et al. (2009) applied an in vitro germination protocol to establish seedlings using WPM medium supplemented with 0.7% (w/v) agar and 58 mM sucrose. Vigorous seedlings were obtained that could be used as a source of explants in micropropagation systems. Parkinsonia aculeata, which is native to the Caatinga is a multipurpose tree also at risk of extinction. To ensure seedling production for micropropagation, Gomes (2007) developed an in vitro germination protocol for this species in which seeds were inoculated on MS medium supplemented with 6 g/L agar; achieving 94% germination. Pterodon pubescens. a native of the Cerrado, is a medicinal plant that is important for reforestation programs, its seeds have low germination rates, motivating Coelho et al. (2001) to design an efficient protocol for in vitro germination. Thus, teguments are removed from the seeds and then cultured on a filter bridge on liquid MS medium supplemented with 2% (w/v) sucrose. Gesneriaceae. Sinningia allagophylla is a perennial herb of the Cerrado, plant growth and production from seeds is very slow and attempts to propagate via tuber cuttings in the greenhouse have been unsuccessful. Consequently, in vitro initiation and micropropagation have protocols been established by Palazetti and Shepherd (1999). Malpighiaceae. Byrsonima intermedia is indigenous to the Cerrado, it is a medicinal species used to treat diarrhea; seed germination is low and seedling emergence slow. Nogueira et al. (2004) used an in vitro protocol for seed and zygotic embryo culture, highest seed germination occurred on half-strength MS supplemented with 0.7% (w/v) agar; highest zygotic embryo germination was observed on one-half WPM supplemented with 0.7% (w/v) agar. Meliaceae. Aniba rosaeodora is a native tree of the Amazon forest and economically important for its oil which is used in the


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cosmetics industries. It can be propagated vegetatively via root cuttings, but there are constraints associated with seedling propagation as seed production is irregular, producing low yields. To help alleviate this problem, Handa et al. (2005) established a protocol for zygotic embryo culture (53% germination rate) in which embryos were cultured on MS medium supplemented with 5% (w/v) sucrose and 1.8 g/L Phytagel. In a parallel study, apical and axillary bud cultures excised from seedlings grown in the greenhouse achieved 48% survival (using MS medium with 300 mg/L Agrymicin, 3% (w/v) sucrose and 0.7% (w/v) agar) however, antibiotics were required to control systemic contamination. In vitro techniques have also been applied to the endangered Amazonian tree species Swietenia macrophylla (mahogany) and Cedrela fissilis an at-risk tree species native to the S Atlantic Forest (Couto, 2002;, Nunes et al., 2007, 2003, 2012). Moraceae. Brosimum gaudichaudii is a medicinal plant of the Cerrado. Fidelis et al. (2000) developed an in vitro protocol for seed germination and seedling growth on MS medium supplemented with 0.6% (w/v) agar. Myrtaceae. Eugenia dysenterica is a medicinal fruit tree and a native of the Cerrado. Martinotto et al. (2007) classified its seeds as recalcitrant, although they do exhibit dormancy. They report a protocol for its in vitro germination using scarified seeds inoculated on MS medium supplemented with 30 g/L sucrose, 0.7% (w/v) agar; achieving 88.25% germination after 71 days in vitro culture. Eugenia pyriformis is native to the Atlantic Forest, for which seedling production is restricted by short seed storage longevity, as this species is a valuable hardwood and produces edible fruits alternative conservation measures are required to offset overexploitation. Protocols for E. pyriformis seed in vitro germination, shoot proliferation, rooting and plantlet acclimatization have been tested by Nascimento et al. (2008). Shoots proliferated from nodal segments excised from axenic seedlings that had been cultured on WPM medium (supplemented with 1.0 mg/L BAP, 30 g/L sucrose and 0.7% (w/v) agar), rooting was achieved using WPM supplemented with 1.0 mg/L IBA. Palmae. Euterpe edulis (heart palm) is native to the Atlantic rainforest in which natural populations have been reduced due to commercial exploitation. Saldanha (2007) optimized somatic embryogenesis protocols for immature zygotic embryos which were successfully cultured on MS medium supplemented with 30–40% (w/v) sucrose; somatic embryos proliferated from the cotyledonary nodes of immature zygotic embryos. Complete plantlets were produced, presenting the possibility of developing protocols for medium-term in vitro conservation in the future. Acrocomia aculeata is a native palm tolerant to dry seasons, the fruits and seeds of which have a high oil content that is exploited for biodiesel production. Like other palms, this species presents difficulties regarding asexual reproduction, and commercial interest has lead to overexploitation because of the reliance on natural populations. Moura et al. (2008) established a somatic embryogenesis system for A. aculeata using zygotic embryos cultured on medium supplemented with picloran. Whilst plant

regeneration via somatic embryogenesis was not achieved, the study provides a useful starting point for sustainable plant production and germplasm conservation. Quillajaceae. Quillaja brasiliensis is a native species from southern Brazil and economically important for the production of bioactive saponins. Fleck et al. (2009) devised micropropagation and acclimatization protocols for this species which may also have potential applications for in vitro conservation. They comprise multiple shoot production and rooting from nodal segments cultured on MS medium supplemented with BAP or IAA (indole-3-acetic acid), respectively. Corresponding bibliographic information on the wider taxonomic application of in vitro technologies for the conservation of the Araucariaceae, Arecaceae, Fabaceae, Lauraceae, Meliaceae, Myrtaceae, Piperaceae, Rubiaceae, and Sterculiaceae has been collated by Nunes et al. (2012). Collectively, these case studies highlight the complex issues associated with problematic seed storage, vegetative propagule production and the tissue culture of species native to Brazil’s biomes. Since physiological behaviors do not always concur with traditional tissue culture and seed recalcitrance classifications the development of optimal storage protocols requires further investigation (Benson, 2008; Higa et al., 2011). The relevance of Biospecimen Science tools (see Sections VI and VII) for conserving a broader taxonomic range focuses on improving the overall efficiency and reliability of conservation measures particularly by instigating quality and risk management procedures (Benson, 2011a,b,c; SGRP, 2010). Molecular and biomarker technologies pinpoint and help to ameliorate the stresses incurred during biopreservation, and they are used as performance indicators to assess the stability and fitness-for-purpose (see Section VII) of germplasm recovered from in vitro culture and cryostorage (Harding, 2004; Harding et al., 2009). VI.

EVALUATING THE POTENTIAL USE OF BIOSPECIMEN SCIENCE TOOLS FOR THE IN VITRO CONSERVATION OF PLANTS FROM MEGADIVERSE COUNTRIES This section considers the potential use of quality management regarding selected biospecimen science approaches to facilitate the ex situ conservation of flora from Megadiverse regions with a focus on species native to Brazil. A.

Quality Management and Biospecimen Science Principles The management of crop plant germplasm collections is based on well-established operating principles (Genebank Standards, 1994) that have evolved internationally over several decades to meet the changing needs of contemporary end users (Benson et al., 2011a, b, c; Dulloo et al., 2010; Khoury et al., 2010; Garming et al., 2010; SGRP, 2010). These principles are underpinned by processes that sustain genebank quality management practices, which ultimately may lead to external, thirdparty accreditation (Engels and Visser, 2003). Albeit, this may

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be outside the capacity of many conservation organizations, in which case the development of quality management systems is an aspirational process, progressed in line with feasible best options. The remit of Biospecimen Science is to bridge the gap between biopreservation and end users by implementing procedures within quality assurance and risk management frameworks, these drive up quality standards and, inherently support storage best practices (Pugh et al., 2008; ISBER, 2012; OECD, 2007; Riegman et al., 2008). In a different circumstance, Kaczmarczyk et al. (2011) identify the problem that many plant species are difficult to cryopreserve using current procedures and they consider that plant conservation is at a ‘cross-roads’ regarding how to cost-effectively conserve a wide range of species. Although Biospecimen Science has emerged from the clinical sector and appears to have little in common with sustaining environmental systems or plant conservation in Megadiverse countries, there are some remarkable similarities that suggest it has a wider applicability: • • • • • • • • • • •

diversity of sample types often limited knowledge of pre-storage history recalcitrant and/or variable storage behavior complex physiologies and metabolic states constraints in sample availability constraints in sample size/number rare, unique and one-time-only samples restricted/irregular supply chains paucity of information about factors affecting stability limited knowledge of biomolecular characteristics Limited knowledge of how process chains impact on biopreservation outcomes

Coates and Dixon (2007) propose that conservation science research needs to make significant advances to address the pace and scale of the global conservation crisis (see Section IIAi). Because Biospecimen Science links the science of sample collection, processing, and preservation with quality management, QC, proficiency testing, validation and the use of performance indicators, it has the potential to enhance the efficiency, capacity, and the rapidity of conservation practices. This has advantages when they are applied to a wide taxonomic range that would otherwise require species-specific optimization. Morrison et al. (2006) offer another perspective, and caution that one of the main challenges for high-throughput omics technologies used in ecological and environmental studies relates to the concept of sample and how it is derived for informatics purposes. In the same way, the stringent annotation of sample process chain history in SPRECs helps to establish quality management systems and, as such may have a wider applicability for in vitro conservation. B.

The Standard Preanalytical Code: SPREC The International Society for Biological and Environmental Repositories (ISBER) Working Group on Biospecimen Science developed the Standard Preanalytical Code (acronym ‘SPREC’)

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for clinical specimens (Betsou et al., 2010; Lehmann et al., 2012). The motivation being, the more precise the record of a sample’s history the more accurate and explicit the information that can be gained when it is used. Preanalytical processes take place between the point of sample collection and their use; variations are attributed to the specimen process chain and are a result of handling procedures, they are not related to the intrinsic properties of samples. Employing a SPREC enhances sample traceability and by definition preanalytical variables must be within the control of biobank or genebank (Benson et al., 2011d; Betsou et al., 2010; Lehmann et al., 2012; Palmirotta et al., 2011). The code is created as a simple handwritten cipher or digitised as a ‘supermarket-style’ electronic barcode which is intercalated with already established data management systems (e.g. passport data, certification records, accession numbers, records for seed sourcing, collection, handling and testing and taxonomic information). Although the SPREC was designed by clinical biobankers (Betsou et al., 2010, Lehmann et al., 2012) it has relevance for culture collections and environmental biobanks (Benson et al., 2011d; Pugh et al., 2008). A prototype SPREC template is presented (Table 1) which has been adapted for the in vitro conservation of forest tree germplasm using examples of published technical procedures applied to species from some Megadiverse countries. The code gives retrospective insights into how preanalytical process chain variables might affect germplasm and biospecimen storability, stability, and quality. The need to stabilize storage-recalcitrant tropical plant germplasm is not a new issue, with hindsight the early innovations used to stabilize recalcitrant seeds during their transport from the rainforest to the laboratory concurs with the modern notion of ‘standardizing’ process chain variables (Mansor, 2012; Marzalina et al., 1999; Marzalina and Krishnapillay, 1999). Capturing a basic knowledge (Table 1) of how germplasm samples are handled in a well-annotated process chain history is significant as pre-analytical variables can affect storage protocol work-up and optimization as well as germplasm stability and quality. Thus, using SPREC in in vitro plant conservation benefits both preservation and post-storage analyses, for example in cases where germplasm is used for genetic, omics, and systematics research. Preanalytical coding also has advantages for large-scale collaborative infrastructures which require uniformity of germplasm/sample processing, as would be necessary for molecular genetic studies, protocol validations, collaborative experiments, and establishing duplicate collections (Benson et al., 2011d, Betsou et al., 2010; Palmirotta et al., 2011; SGRP, 2010). 1.

Calibrating SPREC for In Vitro Plant Conservation Protocols Each biospecimen is assigned a seven-element long code (handwritten or digitised) that corresponds to the technical steps of its process chain history (e.g., collection method, pretreatment, cryoprotectant, cooling, and storage). Code elements are


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defined by letters (usually three, but may be less) in a specific order separated by hyphens, where possible the code intercalates with preexisting data management systems; web-based, digitized templates can be constructed to produce the code (Lehmann et al., 2012). The SPREC tracks the step-wise sequence of a biospecimen or germplasm sample as it passes through a process chain. If the preanalytical option is unknown or inconsistent a letter ‘X’ is used, if the variable is known but does not fit a standard option ‘Z’ is designated. The code is annotated to the germplasm sample throughout its entire processing history including cryostorage and recovery. To illustrate how the SPREC could be applied to enable the preservation of plants from Megadiverse countries, the elements of a ‘pilot’ SPREC have been calibrated using generalised variables derived for tropical/subtropical forest tree species (Table 1). The code tracks the germplasm sample from the point of in situ collection to its in vitro conservation, putative codes have been constructed using examples of logical annotations (IV for in vitro codes; CR for cryogenic codes) and/or thereafter sequentially (A, B, C). Where elements have the same starting code letter it is important to decipher the code using an alphanumeric format. Code elements usually comprise 1, 2, or 3 letters. Examples demonstrating how a preanalytical code is constructed and calibrated using generic seed sampling and processing elements (see Table 1) for Medium Term Storage (MTS) and Long-term Storage (LTS) which is considered equivalent to cryopreservation (cryostorage) are shown as follows: Code for a 7-ELEMENT SPREC: Medium-Term Storage Option 1 Element 1 = Mother Plant-sampling choice = IFT Element 2 = Collecting method = CMB Element 3 = Transit time and stabilization = TSA Element 4 = Post-collection processing and interim storage = PCA Element 5 = Sterilization-explant excision/initiation = SA Element 6 = In vitro preteatments and culture = IVB Element 7 = Medium-term storage culture = MTA Code for a 7-ELEMENT SPREC: Long-Term Storage Option 2 Element 1 = Mother Plant-sampling choice = DSM Element 2 = Collecting method = CMA Element 3 = Transit time and stabilization (unknown option) =X Element 4 = Post-collection processing and interim storage = PCB Element 5 = Sterilization-explant excision-initiation = SEC Element 6 = In vitro pretreatments and culture = IVA Element 7 = Long-term storage cryopreservation = CRB The hyphenated-letter codes are constructed in shorthand abbreviations as follows: Medium-Term Storage 7-code string = IFT-CMB-TSAPCA-SA-IVB-MTA

Long-Term Storage 7-code string = DSM-CMA-X-PCBSEC-IVA-CRB Preanalytical variables attributed to storage parameters are considered by the wider biobanking sector as important for assuring the quality and interpretation of molecular research, this is because pre-analytical variables can affect DNA quality and stability and influence the interpretation of data derived from biomarker analyses. Standard pre-analytical codes improve quality, efficiency, and experimental rigor in biobanks (Balusubramanian et al., 2010; Benson et al., 2011d; Betsou et al., 2010; Kugler et al., 2011; Lehmann et al., 2012; Palmirotta et al., 2011) thus, designing SPRECs for in vitro plant conservation may offer similar benefits. They can be used to compare successes and failures associated with critical processing elements (Table 1) and could potentially identify factors connected to intrinsic and extrinsic storage recalcitrance (see Section IIIB). Pre-analytical codes can inform risk mitigation strategies for precious samples that exhibit problematic storage behaviors exacerbated by suboptimal and non-conforming sampling and handling procedures (Benson, 2008; Benson et al., 2011d). Tracking preanalytical variables using a code that allows the robust characterization and reporting of elements (Table 1) could enhance positive storage outcomes and improve the overall quality of genebank management practices. The pre-analytical code is relevant to tropical plant germplasm conservation because of sensitivities to collecting regimes, transportation, storage, and in vitro manipulations as these contribute to the success or failure of protocols (Marzalina et al., 1999, Benson 2008; Benson et al., 1996) and affect the quality of derivatives (DNA, RNA) used in molecular, omics and biomarker analyses (Harding and Benson, 2012). C.

Modern Manufacturing Principles The scale of biospecimen processing within the clinical sector is enormous and can involve the collection and storage of millions of sample aliquots. This has necessitated the creation of automated, industrial-scale procurement and process systems, consequently some biobanks have adopted modern manufacturing principles (MMP) to facilitate high throughputs and ensure sample quality conforms to performance criteria (Downey and Peakman, 2008). Clinical biobanks now exploit Taguchi design manufacturing principles to test quality and performance across their process chains, including cryostorage (Downey and Peakman, 2008). Taguchi analyses were originally designed for the statistical testing of quality control parameters in the manufacturing industry; they use the optimum treatment combination that maximises a signal to noise ratio (SNR) dependent on whether a response variable is: (i) as small as possible, (ii) close to a nominal value, or (iii) as large as possible. The basis of Taguchi principles is to standardize processes using statistical tools to achieve a reduced variation in manufacturing processes. Although this approach has only recently been taken up by the clinical biobank community (Downey and Peakman 2008), Staines et al. (1999) were the first to


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apply Taguchi analyses for the conservation of seed germplasm from trees of Malaysia’s tropical rainforest. They tested the suitability of Taguchi principles to aid the development of cryopreservation protocols for Malaysian rainforest tree species by re-constructing seed storage experiments to test protocol work up parameters using seedling performance indicators as response variables. The Taguchi approach was explored in more detail (Muthusamy et al., 2005; Nadarajan et al., 2006) to aid the development of cryopreservation protocols for Malaysian plant species representing different types of germplasm and storage behaviors. Kaczmarczyk et al. (2011) explain that the limited availability of vegetative material from at risk, native Australian plants makes testing of larger numbers of samples in multifactorial experiments difficult, and too slow to realistically support future ex situ conservation programs. Taguchi-designed experiments may thus help to develop in vitro preservation protocols for plant genetic resources limited by sample size and germplasm collecting constraints. As such, these statistical approaches have considerable relevance for conserving endangered species, for which it is necessary to ‘do more’ with less available germplasm (Kaczmarczyk et al., 2011).

VII.

MOLECULAR TECHNOLOGIES AND GENETIC DIVERSITY ASSESSMENTS: APPLICATIONS FOR THE CONSERVATION OF BRAZIL’S NATIVE FLORA There have been a significant advances in the use of molecular genetics and omics technologies for the improvement, sustainable exploitation and conservation of plants in situ and in the context of climate change (Ahuja et al., 2010), as well as the preservation of plant genetic resources ex situ (de Vicente, 2004). Consequently, there is a substantive knowledge base on which to test the utility of molecular tools within a Biospecimen Science framework. In clinical and environmental biobanks molecular technologies (e.g., biomarkers, PIs) are an integral component of biopreservation operations. They facilitate the setting of standards and support risk mitigation by ensuring that conserved genetic resources are fit-for-purpose. It is also important that biomolecular data associated with samples/germplasm is well documented and authentic (Benson et al., 2011d; Riegman et al., 2008). The OECD’s working party on biotechnology report concerning policy issues for the development and use of biomarkers in health OECD (2011) identifies challenges related to the ‘genomics revolution’ and personalized medicine. The first is to develop infrastructures that produce and manage molecular biomarker knowledge more efficiently; the second concerns biomarker development/application in clinical settings that have an evidence base. Specifically, in which the utility and validity of biomarker-based tests may be assessed and their quality and efficacy can be assured by identifying evidence-based biospecimen QC tools (Bestou et al., 2013). In the context of biodiversity conservation the molecular and biomarker technology elements of Biospecimen Science (Tables 2 and 3) have a wider

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significance, ranging from supporting conservation genetics at the population level to in vitro preservation and enabling the sustainable exploitation of plants and their products. These applications are collated in Tables 2 and 3 and examples as to how they may be exploited include: 1. Informing (evidence based) decisions as to what should be conserved. 2. Supporting genebanking quality management operations. 3. Supporting the biotechnological exploitation of biological products from species that are conserved ex situ. Cooke and O’Connor (2010) propose the concept of ‘conservation physiology’ which recognizes that physiological tools and knowledge have the potential to inform conservation policy makers because they focus on rigorous experimental approaches and cause-effect relationships. These researchers, put forward the idea of a ‘conservation physiology toolbox’ that ensures a thorough understanding of the use of physiological markers as specifically applied in conservation efforts. Whilst Cooke and O’Connor (2010) concerns ecology-based conservation, their proposed, nascent discipline of conservation physiology resonates with the principles of Biospecimen Science. The application of molecular technologies in megadiversity conservation is analogous to using physiological parameters as biomarkers in this newly proposed subject. The following section investigates how molecular technologies may be used by Megadiverse countries for the conservation and sustainable exploitation of plant taxa. Two of the world’s most at-risk forest biomes, the Brazilian South Atlantic and Amazon forest (see Nunes et al., 2012), are used as exemplars. A.

Molecular Technology Applications: Biospecimen Science, Stability and Risk Mitigation The scope of molecular technology applications for plant conservation practice and research is shown in Table 2, ranging from elucidating the basis of adaptive and stress physiologies, to genetic diversity assessments in genebank accessions. Knowledge of species variation and genetic structure is critical for effective biodiversity conservation and molecular tools are employed to evaluate variation within species and their wild populations. They help prioritize which genetic representatives of species’ populations should be targeted for conservation. Molecular biomarkers are used as ecological indicators in environmental impact monitoring and to elucidate the effects of anthropogenic activities, including climate change (Harding and Benson, 2012; Nunes et al., 2012; Kramer and Havens, 2009). There is an intrinsic connection between using molecular tools for biodiversity conservation and the biomolecular analyses and procedures that are employed in Biospecimen Science to establish robust quality and risk management systems (e.g. preanalytic variables/biospecimen analysis). Similarly, biomolecular PIs corroborate the BRC operating principles (Table 3) authenticity, purity, and stability, for which applications for the in vitro conservation of plant species are shown in Table 3. The


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need for robust molecular tools in conservation biology is reiterated by Morrison et al. (2006) with respect to defining the concept of ‘sample’ in omics technologies that are applied in ecological and environmental frameworks. In Biospecimen Science, a process or technique that causes genetic alterations is a quality issue, and molecular techniques and biomarkers are used as PIs in risk assessment and mitigation and QC (Tables 2 and 3). Genetic stability and the maintenance of trueness-to-type characters are major considerations when using in vitro technologies for plant conservation (Harding, 1996, 1999, 2004; Harding et al., 2000, 2009). Somaclonal variation (Rodriguez-Enriquez et al., 2011) and ‘off types’ are undesirable because they do not conform to the in vitro conservation principle of maintaining stability during storage (Scowcroft, 1984; Msogoya et al., 2011). In cryopreservation the concept of cryobionomics has been proposed as a framework for hypothesis driven research to examine the connection between cryoinjury and germplasm stability. Cryobionomics helps to assess the risks of cryopreservation and its associated in vitro manipulations and mitigate against them. Because it links the performance of plants regenerated from cryopreserved germplasm to their reintroduction into the natural environment cryobionomics has particular value for the restoration of flora in regions with high levels of biological diversity (Harding and Benson, 2012). The potential for integrating cryobionomics studies in different conservation models is shown in Figure 1, noting such studies require phenotypic, cytological, biochemical, and molecular biological knowledge of plants in order to assess cellular/biochemical damage, impairment of metabolism and the loss of reproductive functionality. In this respect, the tools used to study cryobionomics are comparable to those exploited in clinical biospecimen science research. Specifically biomarkers (Tables 2 and 3) ascertain those treatments or processes that could incur cryoinjury and predispose germplasm to epigenetic and genetic factors that alter stability by affecting the quality of preserved germplasm (Table 3). Their reintroduction following storage may also influence adaptive capacity, stress physiology and epigenetic status and this may be especially the case for storage-recalcitrant species (Sershen et al., 2010, 2011a, b) and in vitro–conserved germplasm (Miguel and Marum, 2011). B.

Examples of Molecular Genetic Technologies Applied to the Diversity Assessment and Conservation of Plants Native to Brazilian Forest Biomes The rationale for focusing on forest species is in recognition of Brazil’s international role in conserving species endemic to the Amazon and South Atlantic Forests. These are two of the planet’s major ecosystems; only 82% of the original Amazon Forest and 69%. E. oleracea conserved in Embrapa’s germplasm collection had a high level of genetic differentiation within the provenances and high genetic variability. When provenances were represented by a few accessions they did not express the genetic variability existing in the sites of collection. De Oliveira and Silva, (2008) suggest that new harvesting is needed to ensure that provenances will be well represented in the germplasm collection. De Oliveira et al. (2007b) evaluated 116 accessions conserved in the Embrapa’s Amazon germplasm collection using 28 primers revealing 263 polymorphic RAPD loci representing a wide genetic diversity among the accessions. Subsequently, de Oliveira et al. (2010) assessed genetic variability among 116 E. oleracea accessions from the same collection using microsatellite (SSR) markers showing high diversity between the accessions. At least four highly divergent accessions were identified as potentially useful for establishing nuclear collections and genetic improvement. Euterpe edulis is an important economic species due to ‘heart-of-palm’ being its edible apical meristem, it is over exploited and at risk of extinction in the highly fragmented Atlantic Forest (Seoane et al., 2005). Using 18 microsatellite loci, Gaiotto et al. (2003) analyzed the genetic structure, mating system and the long distance gene flow in E. edulis representing two populations of the gallery forests of Cerrado. They observed a low, but significant level of genetic variation between populations indicating high levels of gene flow and they recommended establishing managed in situ reserves as well as ex situ conserved collections, further advising they should contain several hundreds of open-pollinated maternal families from the few distant populations. Seoane et al. (2005) studied the effect of forest fragmentation on the genetic structure of two populations of E. edulis, one from a fragmented area and the other from continuous forest. Seeds, seedlings and adult trees were analyzed using microsatellite markers, revealing that genetic diversity levels of the fragmented forest were similar to those of species from continuous forest, but that the level of inbreeding was higher in fragmented forests. These authors surmised that the higher genetic divergence of continuous forest populations was due to the large number of genes coming in from outside the sampled populations which is restricted in fragmented forests. Because the morphological similarity between seeds makes it difficult for the seed technologists and plant breeder to iden-

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tify species. Martins et al. (2007) used isozymes to study seed differentiation in Euterpe spp. their studies showed that the most effective isozymes were phosphoglucomutase, phosphoglucose isomerase and peroxidase, each producing distinctive electrophoretic profiles. Conte et al. (2008) examined the genetic structure and mating system of 10 populations of E. edulis using allozymes and microsatellite markers as applied to seedlings, saplings and adult trees, they surmised that microsatellite markers had a higher degree of polymorphism than allozymes. However, as estimates of genetic structure and multilocus out-crossing rates were similar for both markers the authors concluded that allozymes are still useful markers to estimate population genetic parameters. Studies on the population structure of E. edulis in south Bahia were carried out by Silva et al. (2009) revealing a demographic structure similar to that found in surveys of E. edulis originating from S/SE Brazil. Celastraceae. Maytenus aquifolium and Maytenus ilicifolia are popularly used in Brazil for their anti-spasmodic, contraceptive, anti-ulcerous, diuretic, wound healing and analgesic properties. M. aquifolium is native to the mixed ombrophile forest in S Brazil and it also occurs in the fragmented Atlantic Forest, because of over-exploitation its natural populations are endangered. Southwest Brazil is considered the centre of specific diversity as it has the highest number of species. Using RAPD molecular markers, Mossi et al. (2009) analyzed the genetic variability of 18 native populations of M. ilicifolia from south and midwest regions of Brazil using M. aquifolium and M. evonymoidis for interspecific comparisons. Their data showed the efficiency of RAPD markers in the differentiation of the three species studied and revealed genetic variability within M. ilicifolia. Markers demonstrated genetic similarity in M. ilicifolia populations and allowed the separation of subpopulations related to the distinct environments in which they occurred in S Brazil’s Araucaria Forest and in the Mountains of Mato Grosso do Sul, differences were attributed to the fragmentation of small populations. The authors suggest that the genetic variability detected in the three distinct groups of M. ilicifolia should be used to develop conservation programs. Ribeiro et al. (2010) applied AFLP markers to assess genetic diversity in M. ilicifolia germplasm bank accessions that comprised 20 genotypes from south Brazil. Their results indicated that the accessions represent a large number of alleles providing coverage of the M. ilicifolia genome; genetic diversity detected within populations was greater than that found between populations. Sahyun et al. (2010) profiled genetic variability in three natural populations of Maytenus aquifolium using RAPD markers that revealed 21.77% genetic variation among populations, they indicated that all should be conserved. Fabaceae. This family includes several economically important wood producing species which are at risk from forest fragmentation. Copai´ıfera langsdorffi a native to both the Amazon and Atlantic Forests has most of its populations fragmented in the Atlantic Forest of San Paulo state. Forest fragmentation causes a decrease in the population size and loss of alleles,


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under these conditions gene flow via pollen and seeds can be restricted due to small and isolated populations being formed, this increases endogamy and decreases genetic diversity. The survival and maintenance of fragmented populations depends on establishing appropriate strategies for the management of remaining forests. Martins et al. (2008) evaluated the effect of forest fragmentation on the genetic structure of this taxa using six microsatellites to analyse 30 regenerant individuals and 30 adult trees in three areas, they revealed high levels of genetic diversity in both groups. This indicated that forest fragmentation had not yet caused allele losses, however their study also showed that forest fragmentation is reducing gene flow between populations as compared to continuous forest. To address this issue, Martins et al. (2008) propose an alternative strategy in which agroforestry systems managed by farmers are created to facilitate pollen and seed dispersion between forest fragments. Caesalpinia echinata Lam. (Leguminosae-Caesalpinioideae) was the first native Brazilian species from the Atlantic Forest to be used commercially on a large scale. It has been intensively exploited since Brazil was discovered by the Portuguese 500 years ago; it is now at risk of extinction as only small populations occur in forest fragments. Neto et al. (2005) studied the mating system of C. echinata trees from an arboretum by using allozyme analysis of progeny arrays and mixed-mating and correlated-mating models and emphasized the need for integrating ex situ and in situ conservation. They showed a high level of endogamy amongst adult trees, allogamy was the predominant reproduction system and the species is not auto-incompatible. It is recommended that C. echinata seeds harvested from arboreta could be used for reforestation so long as they are collected from a high number of different trees. Melo et al. (2007) developed nuclear microsatellite markers to study mating systems, gene flow, population structure, and paternity in natural populations of C. echinata, and were able to differentiate between individuals, an outcome which could be potentially used to advise conservation programs. Hymenaea courbaril var. stilbocarpa is an at risk tropical tree from the Atlantic Forest in SE Brazil for which ex situ conservation has been established by Feres et al. (2009). These researchers analyzed genetic diversity in a genebank of H. courbaril using six nuclear microsatellite loci. Data revealed respectively 79 and 91 alleles in 65 seed-trees and their 176 offspring and they reported that an effective population size of 170 can retain a substantial part of the present genetic diversity of H. courbaril. This study confirms that the genebank holds substantial genetic variability which can be used to conserve this threatened species. Schizolobium parahyba is a fast growing tree that is important for reforestation programs, it is native to the Atlantic Forest and has suffered high levels of fragmentation. Freire et al. (2007) used RAPD markers with five highly polymorphic primers showing 32 dominant markers to estimate genetic diversity within and among 74 individuals of five populations. Their results demonstrated a total genetic diversity of 89% within and 11% of genetic diversity among populations, revealing a high

level of polymorphism and genetic diversity. This study emphasizes the need to consider levels of molecular genetic variation within and among populations that are used for conservation. Lauraceae. Tarazi et al. (2010) assessed genetic diversity in four natural populations of Ocotea catharinensis, a native tree of the Atlantic Forest that is in danger of extinction. Eighteen allozyme loci were used to analyze genetic diversity in two populations located in conservation units in continuous forest and from small isolated forest fragments. Results showed high genetic diversity leading the authors to recommend that a connection between fragments should be established to allow the gene flow between populations. They also emphasized the importance of integrating conservation planning through agroforestry systems. The percentage of polymorphic loci was 83.3% and the expected genetic diversity in adult trees was highly indicative that all studied populations had the potential for genetic conservation. Meliaceae. This family includes economically important species (Mahogany) from the Amazon and Atlantic Forests. Raposo et al. (2007) studied the genetic diversity of two populations of Carapa guianensis and compared their diversity with those of other populations. Their data indicates that the populations have similar diversity patterns, however the number of rare alleles differed between populations and those with a higher proportion were more susceptible to genetic diversity loss. The authors comment that complementary genetic analyses are needed to define in situ conservation strategies for this species. Lemes et al. (2002, 2003, 2007a) characterized 10 highly variable microsatellites loci for Swietenia macrophylla (now on the verge of extinction due to selective logging) from 126 sequenced clones (29% of these clones yielded useful microsatellite loci). They identified 158 alleles in 121 adult trees, concluding that microsatellite loci allow reliable evaluations of gene flow, mating system, and paternity which could be used to study natural population genetics and establish strategies for conservation. Lemes et al. (2007a) used microsatellite analyses to assess the mating system of the remaining trees of S. macrophylla of a logged population. Eight highly polymorphic microsatellite loci were identified for this species and used to analyze 25 adult trees and their 400 offspring. This population showed an out-crossing mating system leading the authors to conclude that the species was tolerant to environmental disturbances caused by logging as it sets fruits with out-crossed seeds even at low densities. The remaining trees from logged areas (or fragments) are thus important for long-term population recovery and genetic conservation programs. Lemes et al. (2007a) recommended that low intensity logging and conservation of a proportion of reproductive trees combined with forest conservation activities and regeneration activities may be the best strategy for combining conservation with the sustainable exploitation of this species. Lemes et al. (2010) used cpDNA microsatellites to study intra-specific variation and quantify/compare the genetic diversity of S. macrophylla populations in Central America and the


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Amazon. The objective being to determine if population-specific cpDNA haplotypes (a combination of alleles for different genes located closely together on the same chromosome that tend to be inherited together) are credible as regional DNA barcodes for monitoring timber harvests. Their results showed different regional patterns for S. macrophylla, spatial analysis identified a single Central American phylogroup and four Amazonian phylogroups. They revealed high haplotype diversity in Brazilian populations suggesting that cpSSRs can be used as DNA barcodes for regional timber certification. Piperaceae. Piper hispidinervum is a small Amazonian tree of commercial value, however, its seeds have short longevity in natural conditions. It is important for safrol production and is commercially used by the cosmetics and insecticide industries (Wadt, 2001). Wadt and Kageyama (2004) used RAPD markers to assess the genetic structure and mating system of the species, diversity was analyzed between and within natural populations of the western Amazon. Their data indicates that P. hispidinervum is allogamous and spatially structured according to a pattern of isolation by distance. Sapotaceae. Manilkara huberi is an intensively exploited hardwood from the Amazon Forest. Azevedo et al. (2007) characterized its’ spatial distribution, genetic structure and mating system using seven microsatellite loci applied to 481 adult trees and 810 seedlings from a natural population. The authors report that the species is predominantly allogamous with a pollen flow restricted to 47 m and suggest fragmentation may cause the loss of subpopulations, advising management for production and conservation of M. huberi should include large areas. Azevedo et al. (2007) suggest seed conservation should involve collecting from >175 maternal trees and because the species is widely spread across the Amazon Forest samples should include several populations to represent the highest possible level of genetic diversity. Sterculiaceae. Theobroma grandiflorum is a fruit producing tree native to the Amazon forest and is one of the most profitable crops of the region, it has the potential for agroforestry cultivation as its fruit pulp is used in juices, ice cream, and cosmetics. This species is now threatened by deforestation and its long-term conservation requires a knowledge of genetic diversity and population structure. Alves et al. (2007) assessed the genetic diversity of three natural populations of T. grandiflorum using 21 polymorphic microsatellite loci developed for T. cacao with 113 alleles. The genetic divergence between populations was high and as other neo-tropical species occur at low densities and have a low genetic diversity within populations endogamy is elevated. Due to the high genetic divergence among natural populations Alves et al. (2007) recommended that for in situ conservation the establishment of a large number of areas for medium- and long-term genetic diversity preservation would be necessary. For ex situ conservation germplasm collection should include many sites and sample various individuals within each site to cover maximum genetic diversity; 50 seeds from at least 25 trees per population should be sampled.

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T. sylvestre and T. subincanum are both from the Amazon, although they are less exploited than other Theobroma spp. and are cultivated on a smaller scale. Lemes et al. (2007b) studied cross-species amplification of 23 microsatellite markers developed for Theobroma cacao in three other Amazon Forest native species. They report the importance of using this set of polymorphic markers to study diversity, breeding and mating systems because they allow precise discrimination between species and individual trees. To summarize, molecular tools (Tables 2 and 3) may be applied for the conservation of Brazil’s native tree species in several contexts to: (i) evaluate diversity, genetic structures, mating, loci transferability, and gene flow in native populations; (ii) enable the study of phylogenetic relationships; (iii) facilitate studies to assess the impacts of natural population fragmentation and genetic erosion; and (iv) assess the diversity of genebank holdings. Thus, molecular-taxonomic studies can be used to inform conservation programs and support the sustainable exploitation of timber and non-timber forest products in the green economy. Further information concerning the application of molecular technologies for the conservation of tree species native to the Brazilian Amazon and South Atlantic Forests has been collated by Nunes et al. (2012). The outcomes of preexisting molecular/biomarker studies connected to traditional conservation practices (see Section VIIA) can be revisited from the perspective of Biospecimen Science, and give added value by facilitating the refinement of fitness-for-purpose criteria and performance indicators.

VIII.

CONCLUSIONS: CAN BIOSPECIMEN SCIENCE EXPEDITE THE EX SITU CONSERVATION OF PLANTS IN MEGADIVERSE COUNTRIES? Achieving Target 8 of the GSPC, “Plant diversity is urgently and effectively conserved. At least 75% of threatened plant species in ex situ collections, preferably in the country of origin and at least 20% available for recovery and restoration programmes” and addressing the sustainable development goals scheduled to emerge from the Rio+20 action plans (Allan, 2012; United Nations General Assembly, 2012) necessitates rigorous and harmonized conservation practices to: (i) counterbalance the unremitting loss of habitat and biodiversity and (ii) support the sustainable green economies of Megadiverse countries (Allan and Clouth, 2012; Cancún Declaration, 2002). To date, the scientific literature published on the conservation of Brazil’s native flora shows that most research has concentrated either on in situ conservation or on ex situ conservation through seed banks (Pilatti et al., 2011). However, despite the increasing use of tissue culture technologies, the volume of international literature concerning their applications in Brazilian native plant cryostorage and in vitro conservation remains limited. In view of the applications described in this review there is considerable scope for exploiting the Biospecimen Science paradigm to expedite the in vitro conservation of plant


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diversity in Megadiverse countries. However, the approach must be targeted in such a way that supports and brings added value to well-established conservation practices by building upon ‘prior art’ and pre-existing knowledge platforms (see taxonomic rankings in Sections IV and VII). This may be best achieved by facilitating established programs or supporting new initiatives that incorporate Biospecimen Science efficiency, quality, and risk management tools to address the major ‘megadiversity challenges’ of scale (conserving a large number of species) and pace (ensuring that efficient practices are in place to assuage biodiversity loss and species extinction) as stated by Coates and Dixon (2007). Similarly, biotechnological studies that focus on the molecular characterization of plants endemic to Megadiverse countries demonstrate the value of using molecular analyses and biomarkers to establish evidence-based in situ and ex situ conservation strategies (Harding and Benson, 2012; Nunes et al., 2012). Thus, Biospecimen Science may underpin the mandates of Megadiverse countries which, at the scientific level involves basic research on different taxa and the application of in vitro conservation for the sustainable utilization of endemic plants in the green economy (Allen and Clouth, 2012, Cancún Declaration, 2002; Gramkow and Prado, 2011). Khoury et al. (2010) describe trends in the ex situ conservation of plant genetic resources presenting the case that new technologies and research are required to develop and refine techniques for the in vitro conservation of vegetative crops for which robust genebank quality management systems are an overarching requirement. These have been encapsulated in the framework of ‘Global Public Goods’ as applied to plant genetic resources (Benson et al., 2011 a,b,c; Jorge et al., 2010; SGRP, 2010). Dulloo et al. (2010) report on the research and advances required to secure agrobiodiversity including: (i) developing BPs for genebank management; (ii) using enhanced technologies for promoting the utility of genetic diversity to end users; and (iii) ex situ conservation research concerning stability, variability and quality. Expanding the species range that can be conserved in Megadiverse countries will require intensive efforts regarding the translation of fundamental knowledge of complex adaptations, species interactions and problematic storage behaviors (Benson, 2008; Fang et al., 2008; Whitaker et al., 2010) into technically feasible procedures and robust and reliable storage protocols. Thus to conclude, “Can Biospecimen Science expedite the ex situ conservation of plants in Megadiverse countries?” Conceptually, this paradigm has yet to become a reality for the biodiversity conservation sector, however, it does have considerable relevance. This is because it concerns establishing routine procedures and tools that have been specifically created to: (i) enhance biopreservation efficiency and effectiveness for large-scale collections of biological resources and biospecimens, (ii) set genebank and culture collection standards, and (iii) instigate quality and risk mitigation procedures that help to ensure germplasm is conserved and recovered, fit-for-purpose from in vitro genebanks.

ACKNOWLEDGMENTS The authors acknowledge the support of the International Foundation for Science (Sweden), The British Council (UK), Capes (Ministry of Education, Brazil) and CNPq (National Research Council, Brazil). The authors are most grateful to Dr Marcos Edel Mart´ınez Montero, Bioplantas Centre, University of Ciego de Avila, Cuba, for his diligent review of their paper. The authors thank Dr Fotini Betsou (Integrated Biobank of Luxemburg for introducing them to the concept of Biospecimen Science. The authors greatly appreciate the encouragement of Dato’ Dr Marzalina Mansor (Forest Research Institute of Malaysia) and her kind consideration regarding the application of the Biospecimen Science paradigm for the conservation of biodiversity in Megadiverse countries. The authors gratefully acknowledge their ongoing collaborations with Dr Jayanthi Nadarajan, and previous collaborations with colleagues at the Forest Research Institute of Malaysia, and the Universities of the Witwatersrand and KwaZulu-Natal, South Africa. REFERENCES Ahuja, I., de Vos, R. C. H., Bones, A. M., and Hall, R. D. 2010. Plant molecular stress responses face climate change. Trends Plant Sci. 15: 664–674. Allen, C. 2012. A Guidebook to the Green Economy Issue 2: Exploring Green Economy Principles. United Nations Division for Sustainable Development, UNDESA. UN, New York. Allen, C., and Clouth, S. 2012. A Guidebook to the Green Economy Issue 1: Green Economy, Green Growth, and Low-Carbon Development – History, Definitions and a Guide to Recent Publications. United Nations Division for Sustainable Development, UNDESA. UN, New York. Alves, G. M., Dal Vesco, L. L., and Guerra, M. P. 2006. Micropropagation of the Brazilian endemic bromeliad Vriesea reitzii through nodule clusters culture. Sci. Hort. 110: 204–207. Alves, G. M., Rech Filho, A., Puchalski, A., Reis, M. S., and Nodari, R. O. 2004. Allozymic markers and genetic characterization of a natural population of Vriesea friburgensis var. Paludosa, a bromeliad from the Atlantic. Forest.Plant Gen. Resources 2: 23–28. Alves, R. M., Sebbenn, A. M., Artero, A. S., Clement, C., and Figueira, A. 2007. High levels of genetic divergence and inbreeding in populations of cupuassu (Theobroma grandiflorum). Tree Gen and Gen 3: 289–298. Andrade, M. W., Luz, J. M. Q., Lacerda, A. S., and Melo, P. R. 2000. Micropropagaça˜ o da aroeira (Myracrodruon urundeuva Fr. All). Ciênc. Agrotec. 24: 174–180. Arnold, A. E., and Lutzoni, F. 2007. Diversity and host range of foliar fungal endophytes: are tropical leaves biodiversity hotspots? Ecology 88: 541–549. Arrabal, R., Amancio, F., Carneiro, L. A., Neves, L. J., and Mansur, E. 2002. Micropropagation of endangered endemic Brazilian bromeliad Cryptanthus sinuosus (L.B. Smith) for in vitro preservation. Biodiver. Conserv. 11: 1081–1089. Ayad, W. G., Hodgkin, T., Jaradat, A., and Rao, V. R. 1997. Molecular Genetic Techniques for Plant Genetic Resources. Report of an IPGRI workshop, 9-11 Oct., 1995, IPGRI, Rome. Azevedo, V. C. R., Kanashiro, M., Ciampi, A. Y., and Grattapaglia, D. 2007. Genetic structure and mating system of Manilkara huberi (Ducke) A. Chev., a heavily logged Amazonian timber species. J. Heredity 98: 646–654. Balasubramanian, R., Müller, L., Kugler, K., Hackl, W., Pleyer, L., Dehmer, M., and Graber, A. 2010. The impact of storage effects in biobanks on biomarker discovery in systems biology studies. Biomarkers 15: 677–683. Bank, H. L., and Schmehl, M. K. 1989. Parameters for evaluation of viability assays: accuracy, precision, specificity, sensitivity, and standardization. Cryobiology 26: 203–211.


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