Acta bot. bras. 21(4): 777-783. 2007 Germinability after desiccation, storage and cryopreservation of seeds from endemic Encholirium Mart. ex Schult. & Schult. f. and Dyckia Schult. & Schult. f. species (Bromeliaceae) Erika Tarré1, Bárbara Balzana Mendes Pires1, Ana Paula Mançano Guimarães1, Leonardo Alves Carneiro1, Rafaela Campostrini Forzza2 and Elisabeth Mansur1,3 Received: September 22, 2006. Accepted: February 9, 2007 RESUMO – (Germinabilidade de sementes de espécies endêmicas de Encholirium Mart. ex Schult. & Schult. f. e Dyckia Schult. & Schult. f. (Bromeliaceae) após dessecação, armazenamento e criopreservação). O armazenamento de sementes requer a determinação prévia das condições ótimas de temperatura e luz para a germinação, assim como da tolerância à dessecação e a baixas temperaturas. O objetivo deste trabalho foi o estudo do efeito da dessecação, armazenamento a baixa temperatura e criopreservação de seis espécies de Encholirium e duas de Dyckia, selecionadas de acordo com critérios de vulnerabilidade. A germinabilidade de sementes recém-coletadas variou entre 35 e 95%. As sementes apresentaram comportamento fotoblástico, uma vez que a presença de luz foi necessária para induzir a germinação ou aumentar sua eficiência. A dessecação não afetou significativamente a germinabilidade das sementes testadas, com exceção de E. heloisae e E. scrutor. Sementes dessecadas e armazenadas durante um ano a 4 e -20 °C não apresentaram alterações na germinabilidade, exceto em E. pedicellatum. A germinabilidade após congelamento em nitrogênio líquido foi maior que ou similar à obtida no controle, em todas as espécies estudadas. Entretanto, em E. pedicellatum a tolerância ao congelamento só foi obtida após dessecação das sementes a um conteúdo hídrico de 2,5%. Considerando a tolerância à dessecação e ao armazenamento em baixas temperaturas, as sementes estudadas podem ser classificadas como ortodoxas e conservadas ex situ. Palavras-chave: Cadeia do Espinhaço, conservação de sementes, criopreservação, germinação, sementes ortodoxas ABSTRACT – (Germinability after desiccation, storage and cryopreservation of seeds from endemic Encholirium Mart. ex Schult. & Schult. f. and Dyckia Schult. & Schult. f. species (Bromeliaceae)). Seed storage procedures require previous determination of optimal temperature and light conditions for germination, as well as of tolerance to desiccation and low temperatures. The aim of this paper was to study the effects of desiccation, storage at low temperatures and cryopreservation on the germinability of seeds of six Encholirium and two Dyckia species, which were selected according to vulnerability criteria. Initial germinability of newly harvested seeds varied from 35 to 95%. Seeds presented photoblastic behaviour since light was necessary to induce or increase germination. Except for E. heloisae and E. scrutor, desiccation did not affect significantly the germinability of tested seeds. Storage for one year at 4 and -20 °C did not affect the germinability of desiccated seeds, except for E. pedicellatum. Germinability after freezing in liquid nitrogen was higher than or similar to control seeds for all species. However, freezing tolerance of E. pedicellatum seeds was only achieved after desiccation to 2.5% moisture content. As regards tolerance to desiccation and to storage at low temperatures, the seeds studied here can be classified as orthodox and conserved ex situ. Key words: cryopreservation, Espinhaço mountain range, germination, orthodox seeds, seed conservation Introduction vegetation (Forzza & Wanderley 1998), in which the Bromeliaceae family plays an important part. Surveys Pitcairnioideae is one of the three subfamilies of of the area have demonstrated the existence of a high Bromeliaceae, including 16 genera that are represented diversity and level of endemism, especially among in Brazil by two endemic taxa, Dyckia and members of Pitcairnioideae. Thus, bromeliad species Encholirium (Forzza & Wanderley 1998). Great part occurring in this region are important for a better of the Brazilian species of this subfamily is understanding of genus delimitation and phylogenetic concentrated in Espinhaço mountain range (Minas lines (Giulietti et al. 1987). However, as a consequence Gerais, Brazil), a region characterized by rupestral field of several impacts, such as selective exploitation, fire 1 Universidade do Estado do Rio de Janeiro, Departamento de Biologia Celular e Genética, Laboratório de Micropropagação e Transformação de Plantas, Rua São Francisco Xavier 524 - PHLC, sala 505, 20550-013 Rio de Janeiro, RJ, Brasil 2 Instituto de Pesquisa Jardim Botânico do Rio de Janeiro, Rua Pacheco Leão 915, 22460-030 Rio de Janeiro, RJ, Brasil 3 Corresponding Author: [email protected] 778 Tarré, Pires, Guimarães, Carneiro, Forzza & Mansur: Germinability after desiccation, storage and cryopreservation... and devastation of habitats by urbanization, many cases problems such as abnormal germination or death endemic species of Espinhaço mountain range are by internal injuries have been reported (Vertucci 1989). seriously threatened, requiring protection by These problems are generally associated with seed conservation programs. characteristics such as size, moisture content and As in situ conservation of species with restricted chemical composition (Belletti et al. 1990). Thus, the endemism relies on conservation practices that are not effect of freezing must be analysed on each species always rigorously applied or respected, complementary before proposing cryopreservation as a conservation strategies by ex situ germplasm banks are method. recommended. Seed storage is the most used method In the present work, six endemic species of for ex situ conservation, since seeds are the natural Encholirium Mart. ex Schult. & Schult. f. and two of structures of plant reproduction and each one may Dyckia Schult. & Schult. f. occurring at Espinhaço represent a potentially unique and genetically different mountain range were selected according to vulnerability plant. In addition to being a cost-effective method, criteria. The list of endangered species of Minas Gerais sampling and storage of seeds from natural populations State (Mendonça & Lins 2000) includes E. heloisae are important to support restoration programs (Vasquéz- (L.B. Sm.) Forzza & Wand. as vulnerable to extinction, Yanes & Aréchiga 1996; Slageren 2003). Although the adopting the IUCN categories. E. pedicellatum (Mez.) rupestral field vegetation of Espinhaço mountain range Rauh and Dyckia ursina L.B. Sm. were considered has been studied for many years, there is lack of as critically endangered. E. reflexum Forzza & Wand., information about seed biology of native species E. magalhaesii L.B. Sm., E. subsecundum (Baker) (Madeira & Fernandes 1999). Mez., E. scrutor (L.B. Sm.) Rauh and D. sordida The evaluation of seed storage behaviour is the Baker were not included in the list, but are also exposed first step for the establishment of storage conditions, to risk of genetic erosion or even extinction (Forzza including information on temperature and light et al. 2003). Germination, desiccation tolerance, requirements for germination, desiccation tolerance and germinability after storage, and cryocapability of seeds freezing sensitivity. However, seed germination is one of these species were investigated to explore the of the less studied aspects of bromeliad reproduction, possibilities for their ex situ conservation. as optimal temperatures and light requirement for germination were only evaluated in about 50 of the Material and methods 2700 species of the family (Downs 1963; Mercier & Guerreiro Filho 1990). Seed material – Seed harvest was performed according Maintenance of seed viability after storage can to the dispersion periods of the chosen species. Seeds be achieved by adjusting the moisture content to a of Encholirium reflexum, E. magalhaesii, critical level and by reducing the temperature. Since E. subsecundum, E. scrutor and E. pedicellatum insufficient drying may result in less than maximum were harvested at Diamantina city and E. heloisae, longevity, and over-drying may reduce seed Dyckia sordida and D. ursina at Serra do Cipó physiological quality (Walters et al. 1998), the moisture National Park, Espinhaço mountain range, Minas Gerais level at which viability of seeds can be maintained after State, in May and July 2003. The region presents a long periods must be evaluated. mesotermic climate (CwB according to the Köppen Cryostorage techniques have an important system) with a dry season from May to August and a application for preserving endangered species (Touchell rainy season from September to April, with an average & Dixon 1994), allowing seeds to be stored for precipitation around 1350 mm. The average annual indefinite periods of time at ultra-low temperatures that temperature ranges between 25 and 30 °C in the reduce metabolic rates and deterioration. In these summer and 8 to 18 °C in the winter (Madeira & systems, the problems of traditional seed storage, such Fernandes 1999). as DNA damages, requirement for periodic evaluations, In order to obtain representative seed samples of viability control, risk of loss by disease and the local population, 10-12 fruits per plant were environmental problems are reduced or even eliminated collected from at least ten individuals. Fruits with (Benson et al. 1996; Moukadiri et al. 1999; Pita et al. opened capsules were considered as mature. Samples 1998). Although cryogenic storage has been carried consisted of