An Acad Bras Cienc (2021) 93(1): e20191133 DOI 10.1590/0001-3765202120191133 Anais da Academia Brasileira de Ciências | Annals of the Brazilian Academy of Sciences Printed ISSN 0001-3765 I Online ISSN 1678-2690 www.scielo.br/aabc | www.fb.com/aabcjournal CROP SCIENCES Post-seminal development and cryopreservation Running title: POST-SEMINAL of endemic or endangered bromeliads DEVELOPMENT AND CRYOPRESERVATION OF BROMELIADS SIMONE S.S. SILVA, EVERTON H. SOUZA, FERNANDA V.D. SOUZA, DANIELA A.S. MAX, MONICA L. ROSSI & MARIA A.P.C. COSTA Academy Section: CROP Abstract: Vriesea bahiana, Hohenbergia castellanosii and Encholirium spectabile are endemic Brazilian species that are considered endemic or endangered. Development SCIENCES of strategies to conserve these species is important to prevent irreversible genetic erosion. The objective of this study was to evaluate the post-seminal development and seed cryopreservation of three endemic or in danger of extinction bromeliad species in e20191133 Brazil, to obtain a protocol that can safeguard the genetic variability of these species. In the seed cryopreservation assay, we evaluated fi ve desiccation periods. The seeds in the cryotubes were taken from the desiccator and immediately plunged into liquid 93 nitrogen. For the analysis of post-seminal development, seeds in different germination (1) stages were collected and evaluated by light and scanning electron microscopy. Vriesea 93(1) bahiana seeds frozen in liquid nitrogen presented almost 100% germination, indicating dormancy break of this species. Vriesea bahiana can be cryopreserved with 5.9% water DOI content after being dried for 24 hours. Hohenbergia castellanosii and E. spectabile 10.1590/0001-3765202120191133 seeds did not need to be desiccated before being cryopreserved. The most relevant morphological traits for differentiation of genera and subfamilies of Bromeliaceae are the shape and type of seed appendages. In this study, all three species presented well- differentiated size and shape of their structures. Key words: Encholirium spectabile, Hohenbergia castellanosii, Vriesea bahiana, Brome- liaceae, Seed desiccation, Germplasm conservation. INTRODUCTION severe fragmentation caused by destruction of habitat by farming and logging, as well as heavy The family Bromeliaceae Juss. contains a large exploitation of these plants for ornamental number of species, and IS considered to be one purposes (Jorgensen 2004, Pereira et al. 2008). of the most diversifi ed families in the world, with Despite the ecological importance of the 78 genera and 3,659 species (Gouda et al. 2021), family and its wide diversity, little is known about of which 1,178 are endemic to Brazil (Forzza et al. the propagation structures and germination 2020). Despite being present in all the country’s behavior of seeds in response to environmental ecosystems, the greatest diversity of species is factors. Studies of the morphological structures found in the Atlantic Forest biome (Forzza et of seeds are very important to differentiate al. 2012, 2013). Some of these species grow in taxonomic groups, as well as to support adequate very restricted environmental conditions and storage and better understand the germination occur only as small and isolated populations processes (Moura et al. 2010, Pêgo et al. 2011). (Benzing 2000). Besides this, many bromeliads Observation of post-seminal development is are considered to be endangered due to important to differentiate plant species and to An Acad Bras Cienc (2021) 93(1) SIMONE S.S. SILVA et al. POST-SEMINAL DEVELOPMENT AND CRYOPRESERVATION OF BROMELIADS understand the dynamics of the establishment species (Voronkova & Kholina 2010, Kaya et al. of natural populations, since the emergence 2013, Rodrigues et al. 2014, Ferrari et al. 2016, from seeds is a critical stage in their life cycle Pullman et al. 2019, Streczynski et al. 2019). (Fontenelle et al. 2007, Silva & Môro 2008). Nevertheless, additional studies are Furthermore, seeds can be ideal structures necessary to determine the best cryogenic to store the gene pool of a determined species, to storage conditions for seeds, such as cooling guarantee the conservation of genetic variability and thawing rates, physical and chemical (Gosling 2003). Therefore, development of properties, and most importantly, the quantity methods for secure storage of seeds contributes of water in seeds (González-Benito et al. 2003). to the preservation of genetic diversity, which is In all types of preservation, the moisture of crucial in the case of species that are vulnerable seeds is an important factor: in conventional or endangered. conservation, too much water can cause Despite the existence of various ex situ deterioration and attract insect attacks, while conservation methods, storage in seed banks is in cryopreservation, water forms damaging ice most efficient for a wide range of plant species, crystals upon freezing, which at the extreme can mainly due to its easy application and the large cause cell death (Engelmann 1997). On the other number of species that can be preserved in a hand, larger reductions in seed water content small space (Gosling 2003). Hence, this is the can trigger automatic lipid oxidation processes most common conservation method in the and, consequently, generate free radicals that, world, such that seed banks represent nearly being very reactive, may inactivate enzymes and 90% of all genetic collections (FAO 1996). alter the integrity of cell membranes as well Although many other structures, types of tissues as causing unrivaled damage and reduction and even entire plants are preserved in genetic in seed viability (Harrington 1972). Therefore, banks, seed banks are the main repositories of it is necessary to remove the water through genes. The creation of the Svalbard Global Seed desiccation or to use cryoprotective substances Vault (SGSV) in 2008 is the leading example of to reduce these cell damages. This water this method. reduction may vary between species and even Cryopreservation can be used to preserve within the same species. various types of tissues, including meristematic The need for efficient conservation tips, pollen grains and seeds, among others strategies is relevant for endemic bromeliads (Benson 2008, Rodrigues et al. 2014, Kaya et al. species that are vulnerable to anthropic actions. 2016). It basically consists of maintenance of Cryopreservation of seeds obtained from natural plant tissues frozen in liquid nitrogen at ultra- populations of these species (Vriesea bahiana, low temperature (-196 °C). Since the metabolism Hohenbergia castellanosii and Encholirium is virtually stopped at this temperature, the spectabile) can be a valuable alternative, even if process of deterioration is nearly zero, as are only for security duplicates. the risks of loss through ailments or genetic Based on this assumption, the objective variations (Walters et al. 2004, Kaya & Souza of this study was to evaluate the post-seminal 2017). In recent decades, significant advances development and cryopreservation of three have been made in cryopreservation, providing endemic bromeliad species or in danger of alternatives for long-term preservation of extinction (V. bahiana, H. castellanosii and E. the valuable genetic resources of many plant spectabile) in Brazil, from three sub-families, to An Acad Bras Cienc (2021) 93(1) e20191133 2 | 16 SIMONE S.S. SILVA et al. POST-SEMINAL DEVELOPMENT AND CRYOPRESERVATION OF BROMELIADS obtain a protocol that can safeguard the genetic For the germination test, the seeds were variability of these species. first disinfected by immersion in 70% alcohol for one minute and then were rinsed three times in autoclaved distilled water. The seeds were MATERIALS AND METHODS placed to germinate in plastic boxes (Gerbox®) Genetic material at temperature of 27 ± 2 °C, light intensity of 36 -2 -1 Seeds of the following species were collected μmol m s and 16-hour photoperiod. The seeds in different locations (Table I). To obtain seed were evaluated daily for 40 days. samples representative of the populations, 40 Three germination substrates were used: ® ripe fruits (based on coloration) from different Germitest paper; the commercial substrate ® plants of each species were collected at random Vivato ; and washed sand. All were autoclaved from the three populations. beforehand at a temperature of 120 °C for 20 minutes. In the case of Germitest® paper, the Seed germination tests seeds were distributed evenly and the paper The seeds were removed from the fruits manually was moistened with autoclaved water (2.5 times and the appendage (V. bahiana) and mucilage the weight of the paper). For the commercial ® (H. castellanosii and E. spectabile) surrounding substrate Vivato and washed sand, 500 grams the seeds were removed with tweezers and of substrate was weighed and moistened with paper towels to prevent proliferation of 200 mL of autoclaved water. microorganisms. A digital caliper was used to The germination criterion was the measure the length, width and thickness of emergence of the first root (1 mm minimum 150 seeds of each species, chosen randomly, length), as described by Pereira et al. (2008). according to the method described by Pereira The germination percentage, germination speed et al. (2008). The weight of 1,000 seeds was index (GSI) and mean germination time (MGT) determined with a precision analytical balance, were calculated by the following formulas: G(%) according to the Brazilian Seed Analysis Rules = (N/A) × 100, where N