Abstract
The Brazilian biomes (Amazon, Atlantic Forest, Cerrado, Caatinga, Pantanal, and Pampa) comprise one of the highest levels of plant diversity in the world; however, non-sustainable practices, deforestation, and land use have resulted in significant losses and fragmentation of the native forests. These ecosystems are now threatened and protection of their native plants through ex situ conservation is an urgent necessity. Cryopreservation and in vitro conservation are complementary options for securing and protecting Brazil’s native plant species because their potential economic value is critically important to develop strategies that will (1) support their sustainable utilization, (2) protect against the over-exploitation of species growing in natural habitats, and (3) conserve the genetic diversity of germplasm from species of different provenances. Biotechnological approaches will help to address future economic and environmental demands placed upon already at-risk species. Conserving seed germplasm ex situ provides an additional safeguard against the risks (e.g., loss due to disease, climate change) of field conservation. Moreover, seed banks and cryobanks permit the long-term conservation of a wider genetic base; this offsets the labor and space intensive costs of conserving in the active growing state. This paper is a compilation of the current status of strategies applied for conserving Brazilian native plant species.
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Introduction
The six major biomes of Brazil are the Amazon, Atlantic Forest, Pantanal, Cerrado, Caatinga, and Pampa. Totaling 8.5 million km2, they comprise one of the greatest measures (20%) of biodiversity in the world (Ministério do Meio Ambiente, Brazil 2010; http://www.mma.gov.br/sitio/). Table 1 summarizes data on their location, areas in relation to the total country, actual remaining areas, and the approximate number of native plant species, climate, and vegetation. The Amazon and Pantanal are the most preserved areas (82–83% remaining), followed by Cerrado, Caatinga, and Pampa (41–45% remaining areas) and the Atlantic Forest (of which only 7.26% remains). In terms of species richness, the Amazon and the Atlantic Forest together contain >50% of native Brazilian species. Deforestation and burning of the Amazon forest are serious concerns for environmentalists. From 2000 to 2003, 77,000 km2 of the Amazon forest was destroyed. In the last two decades, the Amazon forest contributed from 14% to 85% of the total wood used in Brazil (Ministry of Environment/The Brazilian Institute for Environment and Renewable Natural Resources (MMA/Ibama) 2003). The Atlantic Forest is the most endangered ecosystem due to urban occupation and exploitation as 70% of the Brazilian population lives in this biome (Instituto Brasileiro de Geografia e Estatística 2010; http://www.ibge.com.br). In the north of Parana state (south Brazil), forests of valuable hardwood have been devastated for coffee plantations (MMA/Ibama 2003). According to Ratter et al. (1997), Cerrado (center Brazil) covers about 2 million km2, and as an endangered biome (Palazetti and Shepherd 1999), it is estimated that it will be completely degraded in 30 yr (Centro Nacional Recursos Genéticos, Embrapa, Brazil 2010; http://www.cenargen.embrapa.br). Caatinga is a center of genetic diversity for aromatic species and several fruit trees, which are exported. Pantanal, located in the western states of Mato Grosso and Mato Grosso do Sul, is considered the largest wetland in the world by the MMA (Vieira 1999).
Brazilian Plant Species at Risk and Under Threat of Extinction
In 2005, Ibama and the Biodiversitas Foundation revised the red list of Brazilian native species in extinction or threatened by extinction; in 2008, a new list was published and 1,495 species were considered threatened by extinction. Subsequently, Ibama and MMA representatives evaluated 472 species on the verge of extinction and prioritized them for public political attention in Brazil (Biodiversitas Foundation 2010; http://www.biodiversitas.org.br). According to the Biodiversitas Foundation, the species considered vulnerable or at risk are distributed across Brazilian biomes as follows: 60 in the Amazon, 713 in the Atlantic rainforest, ten in Pantanal, 531 in Cerrado, 179 in Caatinga, and 61 in Pampa. The data in Table 2 were compiled from the official list of the Brazilian native species in danger of extinction published by the Ministry of Environment in 2008 (Instituto Brasileiro do Meio Ambiente 2010; http://www.ibama.gov.br/recursos-florestais/documentos/lista-oficial-de-especies-brasileiras-ameacadas-de-extincao/).
Bromeliaceae, Cactaceae, and Orchidaceae are the families with the highest number of endangered species. The following genus/species of economically important plants are on the verge of extinction: Pfaffia (Amarantaceae, medicinal); Myracrodruon urundeuva (Anacardiaceae); Araucaria angustifolia (Araucariaceae); Bactris, Butia, and Euterpe edulis (Arecaceae); Lychnophora (Asteraceae, medicinal); Jacaranda and Tabebuia (Bignoniaceae, medicinal); Dyckia, Cryptanthus, and Vrisea (Bromeliaceae); Maytenus (Celastraceae); Dicksonia (Dicksoniaceae); Caesalpinia echinata, Dalbergia, Machaerium, and Mimosa (Fabaceae); Sinningia (Gesneriaceae); Ocotea (Lauraceae, medicinal); Bertholletia excelsa (Brazilian nut tree; Lecythidaceae); Cariniana (Lecythidaceae); Tibouchina (Melastomataceae); Swietenia macrophylla (mahogany; Meliaceae); Eugenia and Myrcia (Myrtaceae); Cattleya (Orchidaceae); Passiflora (Passifloraceae); and Pilocarpus (Rutaceae, medicinal) (Instituto Brasileiro do Meio Ambiente 2010; http://www.ibama.gov.br/recursos-florestais/documentos/lista-oficial-de-especies-brasileiras-ameacadas-de-extincao/).
Constraints in Plant Conservation Practices
A great constraint in the amelioration of species loss in Brazil is the problem of establishing strategies for forest seed germplasm conservation, exacerbated by limited information available on seed storage behavior in the scientific literature. Practical restrictions arise for certain species that are difficult to collect, especially when the trees are located in the forest and for which there is an insufficient seed supply for conservation experiments. Tree scarcity in certain areas is due to intensive exploitation, and this also contributes to low seed availability and collection. There is a lack of information on flowering and seed production of certain Brazilian native forest trees, and seed production is irregular in different yr. For these reasons, most research on the conservation of Brazilian native species is performed on orthodox seeds. However, in the last decades, major efforts have been made by Brazilian researchers to focus their studies on the collection and behavior characterization of seeds of native species from the Amazon and Atlantic Forest, their priority being the long-term conservation of species producing recalcitrant seeds (Table 3). Barbedo and Bilia (1998) estimated that 70% of the native species (e.g., Inga uruguensis, Theobroma cacao, Hevea brasiliensis, B. excelsa, E. edulis, Paullinia cupana) from humid forests are recalcitrant or intermediate. Thus, the overall aims of this paper are to review the status of the different strategies currently applied for conserving Brazil’s native plant species and to highlight the potential role that in vitro conservation may play in the future.
Overview of Plant Germplasm Conservation Strategies in Brazil
Although the Brazilian biomes are under strong environmental and anthropogenic pressure, the in situ conservation of native species in their natural environment is the most predominant approach for biodiversity protection and germplasm conservation. In situ conservation involves conservation in units (genetic reserves and national parks). Ibama (Instituto Brasileiro do Meio Ambiente 2010; http://www.ibama.gov.br) report a total of 728 distributed among Amazon, Atlantic Forest, Cerrado, Caatinga, Pantanal, and Pampa. Of these, only 126 conservation units are completely protected and 602 are for sustainable use. The conservation units comprise 74 national forests, 62 national parks, 29 biological reserves, 50 extractivist reserves, 32 ecological stations, 31 environmental protect areas, and 429 private reserves; the Atlantic Forest has ca. 860 conservation units and Caatinga has 53.
Brazilian Agricultural Research Corporation (Embrapa) created the National Center for Genetic Resources and Biotechnology (Cenargen) which is responsible for ex situ conservation through active germplasm banks, seed banks, DNA banks, and in vitro germplasm banks. The Plant Genetic Resources National Network created by Embrapa integrates germplasm conservation programs and expands the genetic basis for plant improvement carried out by the Embrapa Centers and other Brazilian institutions (Centro Nacional Recursos Genéticos, Embrapa, Brazil 2010; http://www.cenargen.embrapa.br). The Brazilian System Information of Genetic Resources (Sibrargen)–Colbase Embrapa is an internet access information system-based data bank developed in 1996 by Embrapa Genetic Resources and Biotechnology. It has registered 214 genus, 677 species, and 109,154 accessions (http://www.cenargen.embrapa./recgen/sibrargen/sibrargen.html—accessed February 3, 2010). The network comprises a considerable number of plant germplasm banks, biological collections, and conservation nuclei, distributed in the north, northeast, center-west, southeast, and south Brazil. The germplasm banks have a diverse genetic base and they primarily sustain genetic improvement programs for developing new Brazilian cultivars of native tropical fruit trees, medicinal and aromatic plants, forest trees, palms, and other non-native economically important species.
The medium- and long-term strategies for ex situ conservation are supported by the Embrapa’s seed germplasm bank, created in 1976; it has 107,000 accessions of 661 species; and its objective is to ensure the availability of genetic resources for food and agriculture. The main strategies for ex situ conservation are currently the storage of orthodox seeds at −20°C and minimal growth of in vitro collections applied to vegetatively propagated or highly heterozygotic species. Protocols are currently being established for the in vitro culture and cryopreservation of vegetative and reproductive structures derived from species producing storage recalcitrant or intermediate seeds. DNA conservation of endangered species ensures the availability of “molecular genetic diversity” which can be used to study phylogeny, molecular systematics, comparative genomics, and gene prospecting (Centro Nacional Recursos Genéticos, Embrapa, Brazil 2010; http://www.cenargen.embrapa.br). As a large number of native Brazilian species are classified as vulnerable or at risk of extinction, the following species are targeted by Embrapa Genetic Resources for the establishment of DNA banks: Cedrela fissilis and Amburana cearensis (Fabaceae), Caesalpinia equinata (Caesalpiniaceae), Dalbergia nigra (Fabaceae), Cedrela odorata (Meliaceae), Manikara multifida (Sapotaceae), Ilex paraguariensis (Aquifoliaceae), A. angustifolia (Araucariaceae), Butia eriospatha (Arecaceae), Ocotea porosa (Lauraceae), B. excelsa (Lecythidaceae), and Hymenaea courbaril (Caesalpiniaceae; Centro Nacional Recursos Genéticos, Embrapa, Brazil 2010; http://www.cenargen.embrapa.br).
Progress and Incentives for Recalcitrant Seed Conservation in Brazil
Barbedo and Bilia (1998) analyzed the progress of recalcitrant seed research in Brazil. It was initiated in 1950, focusing on recalcitrant seed identification and assessment of tolerance and responses to different storage parameters (low temperatures, humidity, packaging, chemical composition, viability, fungicide efficacy). Until the 1980s, the Brazilian scientific literature reported on only the few economically important recalcitrant-seeded species, including H. brasiliensis, A. angustifolia, and T. cacao (Table 3). Barbedo and Bilia (1998) report the study of recalcitrant seeds from the Brazilian native species: Inga edulis, Inga striata, A. angustifolia, T. cacao, E. edulis, H. brasiliensis, Virola surinamensis, Ocotea sp., Nectandra sp., and the Brazilian Palm family. From the 1990s, the classification of native species by seed behavior was stimulated by demand for plants to regenerate degraded areas and aid ecosystem conservation. This research has increased in recent years and fostered studies on seed storage and conservation using the protocol of Hong and Ellis (1996). Wielewick et al. (2006) studied 27 forest trees native to the Atlantic Forest, reporting seeds of Myrcianthes pungens as recalcitrant, as qualified by a water content of 39.6% on collection and dehydration intolerance. Carvalho et al. (2006) classified the seed storage behavior of 39 tree species native to the Atlantic Forest (see Table 3 for examples).
Oliveira and Valio (1992) report that seeds of Hancornia speciosa (Apocynaceae), a native of the Cerrado and economically important for fruit production, lost viability when dehydrated to moisture content <30%. José et al. (2007) carried out studies on the desiccation tolerance and storage of five tree species of the Atlantic Forest to support future ex situ conservation programs for Miconia argyrophylla (Melastomataceae), Allophylus edulis (Sapindaceae), Ixora warmingii (Rubiaceae), Aulomyrcia venulosa (Myrtaceae), and Metrodorea stipularis (Rutaceae). These species grow near small rivers and streams and are important for preserving local biodiversity. According to their studies, A. edulis, I. warmingii, and A. venulosa were classified as recalcitrant, as they completely lost viability when desiccated to moisture contents, respectively, of <16% (A. edulis) and <25% (I. warmingii and A. venulosa), whereas M. argyrophylla and M. stipularis tolerated storage at −20°C and had a moisture content of <5%. Carvalho et al. (2008) investigated the Lauraceae family, showing recalcitrant seed behavior in Nectandra grandiflora, Nectandra lanceolata, Nectandra oppositifolia, Ocotea corymbosa, and Ocotea pulchella. Species from the Lauraceae family occur in the Brazilian Amazon and Atlantic Forests. Some such as Nectandra micranthera, Persea pedunculosa, Ocotea cryptocarpa, Ocotea cymbarum, and Ocotea serrana are in danger of extinction (Instituto Brasileiro do Meio Ambiente 2010; http://www.ibama.gov.br/recursos-florestais/documentos/lista-oficial-de-especies-brasileiras-ameacadas-de-extincao/). The Nectandra and Ocotea are economically important for the production of valuable hardwood, and many species of these genera are in danger of extinction due to over-exploitation.
V. surinamensis (Myristicaceae), a native of the Amazon forest, is one of the urgent priorities in the Brazilian genetic resources conservation program, and its storage in germplasm banks is required. Rodrigues (1980) reports that V. surinamensis seeds must be kept hydrated in water immediately after harvesting to maintain viability for up to 4 mo. If the seeds dehydrate in their natural environment, viability is lost after 20 d, indicative of recalcitrant behavior. Cunha et al. (1992) showed that seed initial moisture content was ca. 25.4%, but when exposed to drying at 22°C and 53% relative humidity (RH) for 60 min, moisture content decreased to 20%, for which a maximum germination rate (69%) was achieved. However, a drying period ≥90 min decreased the moisture content to 18.8%, and germination rate decreased to 45%, reaching zero after 5 h dehydration. These studies confirm low desiccation tolerance and the need for in vitro conservation strategies. By comparison, Martins et al. (1999, 2000) and Carvalho and Muller (1998) reported that recalcitrant seeds of Euterpe espiritosantensis, E. edulis, and Bactris gasipaes, native palm species from the Atlantic Forest and Amazon, could be dehydrated to 46.6%, 33%, and 39%, respectively, without a decrease in germination rates.
According to Maluf and Pisciottano-Ereio (2005), the Myrtaceae family is one of the most economically important. It includes the genus (e.g., Eugenia, Campomanesia, Psidium, and Myrciaria) of many edible fruit trees native to the Atlantic Forest. Delgado and Barbedo (2007) confirm Eugenia species as particularly important for fruit production; they are of medicinal importance in the pharmaceutical industries. However, there are constraints as to their use due to lack of information on seed management. Kohoma et al. (2006) showed that a seed moisture content <43.1% reduced germination rate for Eugenia brasiliensis, which is a native fruit tree from the Atlantic Forest. However, seeds with 48.9% moisture content could be stored for 180 d at 7°C with 60% viability. Gentil and Ferreira (1999) and Delgado and Barbedo (2007) studied several other Eugenia species (Table 3), concluding that, in general, seeds did not tolerate moisture contents <45% with a loss in their viability occurring at <15%. Similarly, Cruz (2007) evaluated desiccation tolerance in Theobroma grandiflorum, a native tree of the Amazon and economically important for the production of edible fruits, the seeds of which could be desiccated to 37.8% moisture content without viability loss. Inga vera subsp. affinis produces recalcitrant seeds unable to withstand dehydration to levels <35% (Faria et al. 2004). Similar seed behavior was reported for I. uruguensis (Bilia et al. 1999). However, Bonjovani and Barbedo (2008) showed that mature embryos of I. vera subsp. affinis desiccated to a hydric potential of −4 MPa became tolerant to −2°C, offering another approach for conservation. Magnolia ovata (Magnoliaceae) is a native forest tree used for recuperation of areas near rivers; it produces recalcitrant seeds (23.5% water content) that lose viability after 30 d storage. In contrast, viability was maintained for 60 d (starting at 30%, decreasing to 23%, compared to initial germination rate) in seeds dehydrated to 10.9% water content and stored at 15°C or 20°C (Pupim et al. 2009). Another Magnoliaceae, Talauma ovata, was also classified as recalcitrant by Carvalho et al. (2006). B. excelsa produces recalcitrant seeds; it is the “Brazil nut tree” native to the Amazon and, as the main export product of the region, is in danger of extinction due to over-exploitation. Silva et al. (2009) reported seeds of B. excelsa with a 14% moisture content before storage in wet sand; they were able to maintain viability when their water content was increased to 35% after 175 d of storage. Problems associated with the preservation of at risk and endangered Brazilian native species and especially those producing recalcitrant seeds (Table 3) have stimulated the exploration of in vitro and cryopreservation as alternative and complementary conservation strategies.
In Vitro Culture, Conservation, and Sustainability of Native Brazilian Plants
In vitro studies on native species have been mainly carried out in universities and research institutions and published primarily in Brazilian journals (Table 4). While much of this literature concerns micropropagation and plant production, it has important significance for the in situ and ex situ conservation of Brazil’s native plant species, particularly those that are overexploited and at risk in their native habitats. In vitro and seed conservation strategies address three main issues. First, seed storage, seedling production, vegetative production, and micropropagation support the sustainable and managed cultivation of commercially important genotypes of native species. This offsets the need to continually source plants direct from the wild, a practice that has caused the over-exploitation of many native Brazilian species, leading to genetic erosion, biodiversity loss, and extinction. Second, knowledge of seed storage behavior and vegetative physiologies can be applied to develop short- and medium-term conservation measures, which, coupled with seedling propagation in nurseries and micropropagation, increases plant production capacity for habitat restoration and endangered species reintroduction. Third, experience gained from developing plant tissue culture protocols for economically important native Brazilian species can be used to assist their in vitro conservation. This is highly practicable because the technical challenges of tissue culture are common to all in vitro procedures. They involve the optimization of stepwise manipulations (see Table 4) used in commercial and conservation scenarios: mother plant status, explant selection, surface sterilization, in vitro germination, axenic culture, micropropagation (organogenesis, somatic embryogenesis), plant regeneration, rooting, acclimatization ex vitro, and issues pertaining to genetic stability. Callus and cell suspension cultures are used for somatic embryogenesis micropropagation and in various biotechnological applications to exploit the economic value of native plant biodiversity, all of which have a requirement for in vitro conservation, especially cryopreservation.
In vitro culture and conservation are being increasingly applied to endemic Brazilian species, but steps forward remain limited compared to crop species. Embrapa reports protocols for pineapple, yam, sweet potato, vanilla, oregano, strawberry, potato, cassava, mint, banana, asparagus, grape, and Stevia. For example, in vitro conservation of sugarcane germplasm for 12 mo was achieved for apical buds cultured on 20 g/L sucrose, abscisic acid at 15°C (Lemos et al. 2002). A summary of in vitro culture technologies applied to native and/or economically important Brazilian plants is shown in Table 4. It is important to highlight the relevance of using “collective know-how” gained from economic and conservation plant biotechnologies to underpin economically significant and sustainable practices and in vitro plant conservation. In vitro germination, micropropagation, somatic embryogenesis, zygotic embryo culture, and callus culture systems have been developed successfully for a substantial number of native Brazilian species. It is anticipated that knowledge gained from these systems can be potentially used to further in vitro germplasm conservation studies.
A focus on the Meliaceae: constraints and successes.
The successful application of in vitro conservation protocols for native Brazilian plant species remains restricted to a few cases. This is largely due to a lack of species-specific physiological knowledge, particularly regarding germplasm behavior, a problem that is exacerbated by the wide range of plant diversity under study and limited resources. The authors have placed particular emphasis on developing in vitro conservation protocols for the Meliaceae. C. fissilis is a valuable, fast-growing timber tree, and as a member of Meliaceae native to the Brazilian Atlantic Forest, it is at risk in its native habitat. Nunes et al. (2002, 2003) reported the establishment of a consistent micropropagation protocol and the successful optimization of plant recovery and rooting from calcium alginate-encapsulated C. fissilis shoot tips, cotyledonary, and epicotyledonary nodal segments stored on substrate containing 0.4% (w/v) agar, at 25°C. This protocol is used for in vitro short-term storage for up to 6 mo.
One of the best-known species of the Meliaceae is S. macrophylla (mahogany), a native tree from the Amazon, which is now on the verge of extinction in the wild because it has been intensively exploited for its valuable hardwood. Couto (2002) developed a protocol for the in vitro germination of seeds from this species as well as protocols for callus culture and shoot formation from pre-existing buds. Shoot production was achieved when hypocotyl segments comprising original axillary buds were cultured on half-strength MS (Murashige and Skoog 1962) medium supplemented with 6-benzylaminopurine.
Aniba rosaeodora is a native tree from the Amazon forest, important economically for its oil which is used in the cosmetics industries. It can be propagated by seeds and vegetatively via root cuttings; however, seed production is low and irregular. To help overcome these constraints, Handa et al. (2005) developed an alternative protocol for A. rosaeodora zygotic embryo culture and achieved a germination rate of 53%. This required embryos to be cultured on MS medium supplemented with 5% (w/v) sucrose and 1.8 g/L Phytagel. The survival of apical and axillary bud cultures was in contrast, 48% on MS medium supplemented with 300 mg/L agrymicin, 3% (w/v) sucrose, and 0.7% (w/v) agar. The buds were excised from seedlings grown in the greenhouse, and antibiotics were necessary to control endogenous contamination in explants.
Cryopreservation of Economically Important Non-native and Native Brazilian Plant Species
The development of cryostorage protocols for plants native to Brazil remains limited, with most efforts targeted at economically important species. Endangered and at-risk plant groups present special challenges due to recalcitrance, limited germplasm supply and lack of knowledge pertaining to seed storage behavior, adaptive physiology, and culture responses. In vitro manipulation and cryoconservation of intermediate and orthodox seed germplasm provide examples of the most noteworthy advances.
Intermediate seed cryopreservation.
The Rubiaceae and Palmae families both demonstrate intermediate seed behavior; cryopreservation combined with in vitro zygotic embryo culture can be successfully and consistently employed for their germplasm conservation. This section will overview the cryopreservation of two economically important, non-native crops cultivated in Brazil, experience of which can be applied to native species.
Rubiaceae—Coffea arabica, a native of Africa, is one of the most important economic species of Brazil. Santos et al. (2002) reported seed tolerance to dehydration (5–7% moisture content) as applied in conventional storage, but sensitivity to low temperatures (−18°C). C. arabica seeds are thus classified as intermediate. However, seeds can be conserved for a maximum of 24 mo when partially dehydrated and stored at 20°C (Naidu and Sreenath 1999). Germplasm conservation of Coffea species in Brazil is carried out for vulnerable germplasm banks in the field; thus, Santos et al. (2002) developed a simple protocol for cryopreserving embryonic axes without using chemical cryoprotectants or a programmable freezer. Seeds were immersed in liquid nitrogen (LN) for 17 h, thawed rapidly in a water bath at 40 ± 2°C for 3 min, surface-sterilized, and immersed in water for 48 h, after which their embryonic axes were removed and cultured on Wood Plant Medium. High survival rates were observed in embryos removed from seeds with 12.6–22% moisture content; this decreased at water contents of 29.4%. The protocol is a viable alternative for long-term C. arabica germplasm conservation. Eira et al. (2005) successfully achieved cryostorage for 24 mo for coffee seeds after their partial dehydration to 20% moisture content (concurring with Dussert et al. 1997, 1998). Thawing was achieved in a water bath at 40°C. Dehydration was carried out according to Eira et al. (1999a, b) and involved applying a NaCl solution (75.5% RH) at 15°C. Eira et al. (2005) report that their protocol maintains seed viability throughout storage in contrast to previous literature (Dussert et al. 1997, 1998; Eira et al. 1999a). The method of Eira and colleagues was successfully applied to 30 C. arabica cultivars, endorsing the method for the long-term conservation of Brazilian and other Coffea species.
Palmae—Elaeis guineensis (oil palm), a native of Africa, has been incorporated in genetic improvement programs by Embrapa in Brazil, in the Amazon basin. According to Camillo et al. (2009a), maintenance of field genebanks is expensive and new strategies are needed. Oil palm seeds are intermediate according to Ellis et al. (1991). Camillo et al. (2009a) developed a protocol for seed cryopreservation of five E. guineensis genotypes in which seeds were immersed in LN, stored for 7 d, and slow thawed at room temperature (25 ± 3°C) for 12 h. The seed tegument was removed following LN storage; the zygotic embryos were excised and inoculated on half-strength MS medium supplemented with 2 mg/L pantothenic acid, 3% (w/v) sucrose, and 0.25% (w/v) Phytagel. After 28 d, in vitro zygotic embryo germination rates varied from 73% to 100%, depending on the genotype. The advantages of this protocol are that cryoprotection is not necessary, and seeds can be cryopreserved with intact teguments.
Developing cryogenic storage protocols for orthodox seeds of native forest species.
Seed cryopreservation is advantageous for the long-term conservation of tropical and subtropical forest species biodiversity, as it circumvents problems related to embryo isolation and in vitro handling. Even for orthodox and intermediate seeds, cryostorage offers the advantages of seed longevity so long as LN levels are maintained. Storage of dry seeds at −20°C in seed banks for long periods might also lead to physiological and genetic damage in the very long term (Stanwood and Ross 1979; Stanwood and Bass 1981).
Table 5 summarizes cryogenic storage methods applied to orthodox seeds of native species. Wetzel et al. (2003) found that seeds of 13 species collected from the biome Cerrado (center Brazil) had initial seed moisture contents that varied from 5.41% (Mimosa setosa) to 25.17% (Qualea parviflora). After drying for 15 d in a drying oven at 20 ± 3°C and 12 ± 3% RH, seeds were tested for tolerance to dehydration before immersion in LN and after drying; moisture contents varied from 3.46% (M. setosa) to 8.46% (Q. parviflora). Although all seeds studied by Wetzel et al. (2003) were tolerant to LN, in the cases of Cassia ferruginea, Platypodium elegans, Sclerobium aureum, and Roupala montana, germination rates after LN immersion were lower than controls. However, successful cryostorage was achieved for most of the dehydration tolerant seeds.
Astronium urundeuva is a valuable wood and medicinal tree. Medeiros and Cavallari (1992) exposed their orthodox seeds to drying at 25°C and 10–15% RH for 24, 48, 72, 96, and 120 h before immersion in LN; thawing was at room temperature for 30 min. Seeds survived cryogenic storage even without desiccation (8.01% moisture content), but best germination was optimized by Medeiros et al. (1992) after 24, 72, and 96 h drying, after which the seeds reached respective moisture contents of 7.13%, 5.89%, and 5.96%.
Salomão (2002) studied the LN tolerance of orthodox seeds from 66 tropical species native to Cerrado and the Atlantic Forest and demonstrated cryopreservation as an alternative to conservation at −20°C, on the basis of enhancing longevity (Stanwood and Bass 1981). Salomão (2002) did not dehydrate seeds prior to their immersion in LN, in which they were stored for 3 d; thawing was carried out at room temperature (25°C) for 3 h. Hard coat seeds were scarified after LN exposure when failure to germinate in 30 d was observed; seed moisture contents varied from 3.0% to 15.0% on a fresh weight basis. All species tested were cryostorage tolerant; however, sensitivity to LN varied among species, and for some, seed viability was reduced as follows: Dimorphandra mollis and Bauhinia acuruana (from 95% to 65%), Bauhinia ungulata (from 98% to 82%), Dalbergia miscolobium (from 100% to 53%), Sterculia striata (from 100% to 85%), and Styrax camporum (from 90% to 36%). Salomão (2002) highlighted the need to optimize protocols for cryogenic storage for these species. In others, germination was apparently increased: Schinopsis brasiliensis (from 45% to 86%), Spondias mombin (from 11% to 53%), Bowdichia virgilioides (from 20% to 85%), Pterodon emarginatus (from 50% to 80%), Mimosa somnians var. viscida (from 65% to 90%), Stryphnodendron polyphyllum (from 50% to 90%), Apeiba tibourbou (from 22% to 54%), Crotalaria cf. spectabilis (from 73% to 94%), and Cariniana estrellensis (from 4% to 52%) after LN exposure. This interesting effect could be due to the LN and freeze/thaw cycles acting as a scarification treatment resulting in enhanced permeability and/or changes in seed physiology after cryogenic storage which result in an enhanced level of germination compared to controls.
Tarré et al. (2007) developed an efficient protocol for the cryostorage of eight Bromeliaceae species (six from the genus Encholirium and two from the genus Dyckia) native to the Atlantic Forest. Seed moisture contents varied from 11.2% to 28.2%; their protocol did not include seed dehydration prior to the immersion in liquid nitrogen, except for Encholirium pedicellatum (seeds were desiccated to 2.5% moisture content). Thawing was undertaken at room temperature and the survival rates observed were similar to controls. This protocol is highly relevant as six Dyckia species are in the red list of the Brazilian species in danger of extinction.
Lima et al. (2008) studied tolerance to LN of 19 species belonging to 11 genus of the deciduous forest of Paraná river valley in Goiás (center Brazil). Their protocol did not include prior dehydration. The seeds were immersed directly in LN and stored for 3 d, after which they were thawed at room temperature (25°C). Seed moisture content varied from 5.7% (Jacaranda brasiliana) to 12% (M. urundeuva). All species were tolerant to cryopreservation, but viability was reduced in H. courbaril (5.8% moisture content) and Aspidosperma pyrifolium (6.8% moisture content).
Studies of extended seed storage in LN.
For the purpose of testing the cryogenic parameters required to achieve some level of survival, most research carried out on seed cryostorage in Brazil concerns short-term exposure (3 to 7 d) to LN (Table 5). This section reports outcomes for seeds maintained for longer periods in LN (see Table 6). C. echinata, a native of the Atlantic rainforest, was the first Brazilian tree to be exploited commercially; it is now on the verge of extinction. Its seeds are orthodox (9.7% moisture content), but they lose viability after 3 mo in their natural environment. Barbedo et al. (2002) showed that seeds tolerate desiccation to 7.6% moisture content and can be stored for 18 mo at 7°C, maintaining 80% viability. Hellmann et al. (2006) inform that seeds of C. echinata could be stored at −18°C for 24 mo, producing 80% normal seedlings; those stored at 7°C produced only 20% of normal seedlings. Zanotti et al. (2007) reported that seeds under cryostorage for 6 mo were viable and able to produce normal seedlings. Cryopreservation can thus expand storage time to ensure availability of seedlings for regeneration of natural populations.
Additional studies on seed cryostorage from 4 to 26 mo were carried out (Table 6) for Cecropia glazioui (Cecropiaceae); for the Bignoniaceae Cybistax antisyphilitica, Tabebuia impetiginosa, Tabebuia pentaphylla, Tabebuia roseo-alba, Tabebuia avellanedae, and Tabebuia heptaphylla; and for C. fissilis (Meliaceae). Initial seed moisture contents varied from 6.6% to 10.3%; seeds were not dehydrated before storage in LN, and thawing was achieved at room temperature (25°C) for 30 min. No significant differences in seed viability were detected between the treatments for most species except for C. antisyphilitica (Table 6). As its seeds tolerated 1 h immersion in LN without loss of viability, no significant differences were observed between the control seeds (46%) and those exposed to LN (44%), and a slight decrease in the germination did not indicate intolerance to cryostorage.
Studies performed on orthodox seed cryostorage will be used to progress the development of improved protocols for the more difficult to preserve intermediate and recalcitrant seed germplasm of Brazilian native species.
Conclusions
This review highlights the different strategies currently applied to conserve Brazil’s native flora. The value of experience gained from the tissue culture and seed cryopreservation of economically important native and non-native species is also considered in the Brazilian context. However, there is now an urgent need to expand conservation research to a greater number of native species. Although studies on in vitro germination, zygotic embryo excision, micropropagation, and somatic embryogenesis of endangered species have increased considerably, knowledge gaps still remain. The development of in vitro conservation and cryopreservation methods for Brazil’s endemic species producing recalcitrant seeds must be a research priority. Future studies requiring immediate attention concern the development of reproducible ex situ conservation protocols for the medium and long term (cryostorage) of critically endangered plants. In particular, it will be important to target those at high risk of over-exploitation through non-sustainable practices, climate change, deforestation, and habitat loss.
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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).
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Pilatti, F.K., Aguiar, T., Simões, T. et al. In vitro and cryogenic preservation of plant biodiversity in Brazil. In Vitro Cell.Dev.Biol.-Plant 47, 82–98 (2011). https://doi.org/10.1007/s11627-010-9302-y
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DOI: https://doi.org/10.1007/s11627-010-9302-y