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In Vitro Propagation of Cyathea Atrovirens (Cyatheaceae): Spore Storage and Sterilization Conditions

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In Vitro Propagation of Cyathea Atrovirens (Cyatheaceae): Spore Storage and Sterilization Conditions In vitro propagation of Cyathea atrovirens (Cyatheaceae): spore storage and sterilization conditions Isabel Beatriz de Vargas1 & Annette Droste1,2* 1. Laboratório de Biotecnologia Vegetal, Universidade Feevale, Rodovia RS 239, 2755, CEP 93352-000, Novo Hamburgo-RS, Brazil; [email protected] 2. Programa de Pós-Graduação em Qualidade Ambiental, Universidade Feevale, Rodovia RS 239, 2755, CEP 93352- 000, Novo Hamburgo-RS, Brazil; [email protected] * Correspondence Received 05-XII-2012. Corrected 20-VII-2013. Accepted 30-VIII-2013. Abstract: Cyathea atrovirens occurs in a wide range of habitats in Brazil, Paraguay, Uruguay and Argentina. In the Brazilian State of Rio Grande do Sul, this commonly found species is a target of intense exploitation, because of its ornamental characteristics. The in vitro culture is an important tool for propagation which may contribute toward the reduction of extractivism. However, exogenous contamination of spores is an obstacle for the success of aseptic long-term cultures. This study evaluated the influence of different sterilization methods combined with storage conditions on the contamination of the in vitro cultures and the gametophytic development of C. atrovirens, in order to establish an efficient propagation protocol. Spores were obtained from plants collected in Novo Hamburgo, State of Rio Grande do Sul, Brazil. In the first experiment, spores stored at 7oC were surface sterilized with 0.5, 0.8 and 2% of sodium hypochlorite (NaClO) for 15 minutes and sown in Meyer’s culture medium. The cultures were maintained in a growth room at 26±1ºC for a 12-h photoperiod and photon flux density of 100µmol/m2/s provided by cool white fluorescent light. Contamination was assessed at 60 days, and gametophytic development was scored at 30, 60, 120 and 130 days of in vitro culture, analyzing 300 individuals for each treatment. There was no significant difference in culture contamination among the different sodium hypochlorite concentrations tested, and all treatments allowed for the development of cordiform gametophytes at 130 days of culture. In the second experiment, spores stored at 7 and -20°C were divided into two groups. Half of the spores were surface sterilized with 2% of NaClO for 15 minutes and the other half was not sterilized. All spores were sown in Meyer’s medium supplemented with one of the following antibiotics: nystatin, Micostatin® and actidione. The culture conditions and the procedures used for evaluating contamination and gametophytic development were the same described for the first experiment. No contamination was observed in spores stored at -20°C and treated with NaClO and actidione. In all treatments, cordiform gametophytes presenting antheridia were observed at 120 days. The percentages of these gametophytes increased from 120 to 130 days and no significant differences were observed among treatments. Archegonia were observed on cordiform gametophytes at 130 days. The findings provide data relevant to in vitro propagation procedures of this species, which may increase the availability of plants for ornamental purposes, therefore contributing to the reduction of the exploi- tation of endangered tree ferns species. Rev. Biol. Trop. 62 (1): 299-308. Epub 2014 March 01. Key words: actidione, antibiotic, gametophyte, germination, in vitro culture, surface sterilization, tree fern. Tree ferns are an important component of & Fisch.) Domin (Cyatheaceae) occurs in a tropical rainforests and among them Cyatheace- wide range of habitats in Brazil, Paraguay, ae is a noteworthy family, being represented by Argentina (Ponce, 1996) and Uruguay (Mar- approximately 170 species in the Neotropics quez & Brussa, 2011). In the Brazilian State of (Tryon & Tryon, 1982) and 23 species in South Rio Grande do Sul, the species is commonly and Southeastern regions of Brazil (Windisch & found in open or moderately shaded humid or Santiago, 2013). Cyathea atrovirens (Langsd. swampy places and in areas largely affected by Rev. Biol. Trop. (Int. J. Trop. Biol. ISSN-0034-7744) Vol. 62 (1): 299-308, March 2014 299 human action, such as roadsides (Lorscheit- ethylic alcohol and compounds with chlorine, ter, Ashraf, Windisch & Mosbrugger, 1999). like calcium hypochlorite and sodium hypo- Cyathea atrovirens forms 6m high caudices, chlorite (Wu, Ping-Tin, Li-Ping & Long-Qing, with a sheath of adventitious roots at the per- 2009). Recently, the sodium dichloroisocyan- sistent petiole bases. Petioles and leaves are, urate was described as an efficient sterilizing respectively, up to 1.10 and 3m long, and the agent for spores (Barnicoat, Cripps, Kendon laminas are bipinnate-pinnatifid to pinatilobate & Sarasan, 2011). Addition of antimicrobial (Sehnem, 1978; Fernandes, 1997). The spores agents to the culture medium has also been are tetrahedral-globose and present a triangular tested. Among antibiotics that can be used to equatorial limb with prominent rounded angles avoid the in vitro contamination of plants (Kyte and straight to slightly depressed sides (Lors- & Kleyn, 1996), Micostatin® (Ranal, 1991), cheitter et al., 1999). nystatin (Dyer, 1979; Simabukuro et al., 1998; Habitat destruction and fragmentation are Quintanilla, Amigo, Pangua & Pajaron, 2002) the main factors related to the reduction of tree and streptomycin (Cox, Bhatia & Ashwath, fern populations. In addition, tree ferns are 2003) were tested for ferns, although without exploited because of their gardening potential total elimination of the microorganisms from (Windisch, 2002). Leaves (Tryon & Tryon, the cultures. The treatment of plant tissues with 1982) and entire plants (Schmitt & Windisch, sodium hypochlorite, followed by the addition 2012) of C. atrovirens are used for ornamen- of the fungicide Benlate® (Benomil - Dupont) tal purposes. Caudices of older plants, when to the culture medium has also been used for presenting a sheath of adventitious roots at the fern species (Brum & Randi, 2006; Begnini & base, are used to manufacture fiber handicrafts Randi, 2009; Viviani & Randi, 2008; Santos, (Fernandes, 2000). Lehmann, Santos & Randi, 2010). However, The in vitro culture is an important tool for this fungicide had its manufacture discontin- plant propagation which may contribute toward ued since 2001 (Dupont, 2012). Unfortunately, the reduction of exploitation of natural popula- most surface sterilization treatments may also tions (Caldecott, Jenkins, Johnson & Groom- reduce fern spore germination (Hamilton & bridge, 1996; Giudice, Giacosa, Luna, Yañez Chaffin, 1998; Simabukuro et al., 1998). & de la Sota, 2011). Spore germination is Storage conditions under different temper- considered the most efficient method of in vitro atures have been tested focusing on the survival culture of ferns and lycophytes (Pence, 2008) and germination of spores (Simabukuro et al., and studies with different species can be cited 1998; Rogge, Viana & Randi, 2000; Quintanil- (e.g. Kiss & Kiss, 1998; Bertrand, Albuerne, la et al., 2002; Begnini & Randi, 2009; Santos Fernandéz, González & Sánchez-Tamés, 1999; et al., 2010). The viability of spores from dif- Fernandéz, Bertrand & Sánchez-Tamés, 1999; ferent species has been investigated after cold Kuriyama, Kobayashi & Maeda, 2004; Souza, storage (for a revision, see Ranker & Haufler, Medeiros & Mendes, 2007). 2008), and low temperature was considered as In general, studies analyzing the viabil- minimizing bacterial and fungal contamination ity of the spores and the initial ontogenetic without decreasing spore viability (Quintanilla development of gametophytes do not refer to et al., 2002). contamination (Bertrand, Albuerne, Fernández, The aim of this study was to test the González & Sánchez-Tamés, 1999; Fernandéz influence of different sterilization methods et al., 1999; Chen, Cheng, Liu & Jiao, 2008), combined with storage conditions on the con- despite exogenous contamination of spores is tamination of the in vitro cultures and the an obstacle for the success of aseptic long-term gametophytic development of C. atrovirens, in cultures (Dyer, 1979; Simabukuro, Dyer & order to contribute to the establishment of an Felippe, 1998). The substances most common- efficient propagation protocol contributing to ly used for plant tissue surface sterilization are management programs of this species. 300 Rev. Biol. Trop. (Int. J. Trop. Biol. ISSN-0034-7744) Vol. 62 (1): 299-308, March 2014 MATERIAL AND METHODS fungi and bacteria. The grids were placed sepa- rately under each dish, covering the entire area, Fertile leaves of C. atrovirens were collec- and the number of fields presenting macrosco- ted in the Parque Municipal Henrique Luis pically visible contamination was counted. Roessler (29°40’54” S - 51º06’56” W, at 16.4m Gametophytic development was scored at in elevation), which is a conservation area 30, 60, 120 and 130 days of in vitro culture. of the Rio dos Sinos basin in the municipali- Three slides (one slide per dish) from each ty of Novo Hamburgo, State of Rio Grande treatment were analyzed, and 100 individuals do Sul, Brazil. The leaves were wrapped in (spores or young gametophytes) were counted smooth paper and kept at room temperature on each slide (300 individuals per treatment). to induce dehiscence of sporangia. Spores Individuals were classified according to their were filtered through lens cleaning tissue and developmental stage (Rechenmacher, Schmitt stored. Two different experiments were carried & Droste, 2010) in the following classes: out as follows. non-germinated spores (NG), gametophyte with
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