CAHIERS DE LA SOCIÉTÉ FRANÇAISE D’ORCHIDOPHILIE N°9 – 2018 18th European Orchid Council Conference and Exhibition Proceedings What future for orchids? Cah. Soc. Fr. Orch., n° 9 (2018) – Proceedings of the 18th EOCCE - What future for orchids? Proceedings of the 18th European Orchid Council Conference and Exhibition Scientific conference What future for orchids? 24-24 March 2018 Paris Event Center, Paris On behalf of L’orchidée en France Conference organizing committee: Alain Benoît, Richard Bateman, Pascale Besse, Yves Henry, Jana Jersákowá, Ray Ong, Daniel Prat, Marc-Andre Selosse, Tariq Stevart Cover photography from Philippe Lemettais Proceeding edition: Daniel Prat Cah. Soc. Fr. Orch., n° 9 (2018) – Proceedings of the 18th EOCCE - What future for orchids? Cahiers de la Société Française d’Orchidophilie, N° 9, Proceedings of the 18th European Orchid Council Conference and Exhibition – Scientific conference: What future for orchids? ISSN 0750-0386 © SFO, Paris, 2018 Certificat d’inscription à la commission paritaire N° 55828 Proceedings of the 18th European Orchid Council Conference and Exhibition – Scientific conference: What future for orchids? SFO, Paris, 2018, 100 p. Société Française d’Orchidophilie 17 Quai de la Seine, 75019 Paris Meiotic chromosome analysis in tropical orchid genus Sobralia José MORA1, Stefano ALBERTAZZI, Pablo BOLAÑOS-VILLEGAS1,* 1 Fabio Baudrit Agricultural Research Station, University of Costa Rica, La Garita de Alajuela, Alajuela 20102, Costa Rica * pablo.bolanosvillegas@ucr.ac.cr Abstract – Tropical orchid genus Sobralia comprises terrestrial plants with elongated, cane-like stems and large symmetrical, yet ephemeral flowers. This genus is distributed throughout Central and South America and may hold horticultural potential. Nonetheless, little is known about the reproductive viability of species that comprise this genus. In this project male meiocytes have been examined by light microscopy to determine chromosome numbers, frequency of chromosome segregation defects and the frequency of normal tetrads at the end of meiosis II. The species sampled belong to an ex situ collection managed by the Lankester Botanical Garden of the University of Costa Rica. The species were: S. amparoae, S. artropubescens, S. boucheri, S. bradeorum, S. carazoi, S. crispissima, S. danjanzenii, S. fenzliana, S. geminata, S. helleri and S. rosea. Our results indicate that meiocytes from these species have a variable diploid (2n) chromosome number of 24, 30 and 32, that segregation defects are rare and that tetrad formation rates exceed 80%, suggesting efficient meiotic progression and high pollen viability. Taken together our results suggest that at least in Costa Rica the populations sampled are reproductively healthy and amenable to horticultural breeding. Keywords: Sobralia, orchid, meiosis, chromosome analysis, Costa Rica. INTRODUCTION called the Lankester Botanical Garden. Within the garden a greenhouse is used to grow and Little is known about how geographical study plants from orchid tribe Sobraliae. isolation and the absence of pollinators impacts Sobraliae is polyphyletic neotropical the reproduction of tropical orchids (Waterman orchid tribe from the Americas that comprises and Bidartondo, 2008), Nonetheless it has been about 200 species from genera Elleanthus, hypothesized that in Central America at any Epilyna, Sertifera and Sobralia (Neubig et al., given time the number of fertile orchid plants 2011). These plants are often terrestrial plants is low, seed production is limited, and that with cane like stems and in the case of genus therefore gene flow is also severely restricted Sobralia, large flowers (Neubig et al., 2011). (Tremblay and Ackerman, 2001). This set of Flowers are symmetrical and beautiful, conditions may lead to high selection pressures however little to no plant breeding has been and the creation of new species (Tremblay et performed in Costa Rica using Sobralias, a al., 2005). situation that makes little commercial sense To test these hypotheses experimental considering the availability of native species, comparisons of populations are required, but and its ease of cultivation and propagation, usually this is not possible due to slow factors that have been determinant in the reproductive cycles, few flowers and little or success of breeding programs elsewhere no divergence in the genome of the individuals (Kamemoto and Kuehnle, 1996). sampled (Lahaye et al., 2008). One approach For this study Sobralia plants from ex situ to tackle these problems is to study collection of the Lankester Botanical Garden chromosomes (Kao et al., 2007). were sampled and pollinia were collected to Chromosomes are complexes of nucleic acids determine the chromosome number and to and proteins whose number and morphology observe and record chromosome segregation varies across species thus allowing for patterns during meiosis, including the evolutionary studies (Kao et al., 2007; Lee et formation of tetrads, which is the end stage of al., 2011). Fortunately, the University of Costa meiosis (Mercier et al., 2015). Rica runs a dedicated orchid ex situ collection 118 MATERIALS AND METHODS groups of Sobralias are observed in disturbed habitats across Costa Rica (personal Pollinia from flower buds before anthesis communication, Dr. Robert Dressler). It is also were collected from the ex situ orchid known that most Sobralias are pollinated by collection located at the Lankester Botanical Euglossine bees and hummigbirds, which are Garden, University of Costa Rica. Pollinia common pollinators of Central American were processed according to Lee and Chung orchids (personal communication, Dr. Mario (2010). Briefly, they are placed in 2 mM Blanco), and that these plants show 8-hydroquinoline (Sigma-Aldrich) solution for synchronous gregarious flowering (Dressler, 5 hrs at 25° C, fixated in ethanol/glacial acetic 1990). Therefore, availability of either a acid solution (3:1, v/v) for 12 hrs and then pollinator or a flowering partner does not frozen at -20° C. Samples were digested appear to be a problem in these plants. enzymatically with 6% cellulase and pectinase solution (Sigma-Aldrich) dissolved in 75 mM KCl at a pH of 4.0, for 1 hr at 37° C. Table 1. Formation of tetrads is normal in Digested samples were macerated in a Sobralia species, suggesting high pollen drop of 40% acetic acid solution and then viability. Results are the mean of three stained with fluorescent DNA stain biological and three technical samples, n=100 4′,6-diamidine-2′-phenylindole dihydrochloride plus the standard deviation, nd: not (DAPI, Sigma-Aldrich). Images were obtained determined. with a BX53 epifluorescence microscope (Olympus, Tokyo) connected to a ColorQ5 Species Normal Chromosome CCD camera (Olympus, Tokyo) and a Dell Tetrads number (2n) Precision Tower T7810 computer (Dell, Round (%) Rock, TX). Images were analyzed with Adobe S. amparoae 89,3±8,5 30 Photoshop CS5 (Adobe Systems, San José, S. artropubescens 80,0±6,6 nd CA). S. boucheri 88,4±5,3 nd S. bradeorum 89,7±1,8 nd RESULTS AND DISCUSSION S. carazoi 85,7±5,1 nd S. crispissima 83,1±10,2 nd Analysis of meiotic cell cycle progression S. danjanzenii 93,5±0,7 32 across all species suggested that there are no S. fenzliana 87,5±1,0 nd apparent defects during synapsis and pairing S. geminata 89,5±1,0 32 (zygotene and pachytene) (Mercier et al., S. helleri 86,0±2,1 nd 2015), and no defects during the alignment and S. rosea 93,1±2,1 24 segregation of bivalents (metaphase, anaphase and telophase I and II) (Mercier et al., 2015), as observed in Figure 1. Our results also indicate that wild Counts of normal tetrads (regular tetrads) individuals of Sobralia may be amenable to suggest that formation is normal (Table 1), hybridization and plant breeding. Work is suggesting normal chromosome segregation, scheduled to continue until 2019 and may and possibly formation of viable pollen after involve propagation by tissue culture. mitosis I and II. Preliminary chromosome counts during metaphase I and anaphase I Acknowledgements suggest a variable chromosome number of 24, 30 and 32. Sampling will continue with frozen This work was possible thanks to grant samples. B5A49 from the office of the Vice Chancellor These preliminary results indicate that for Research and a travel permit to attend the unlike in Puerto Rican Lepanthes (Tremblay EEOCE Paris 2018 from the Chancellor of the and Ackerman, 2001), sexual viability in Costa University of Costa Rica. The authors would Rican Sobralia is normal, suggesting like to thank the following students for their reproductive success, effective population contribution to this project: Y. Corrales, M.A. sizes, competitive ability, or ecological Vega, K. Scott, K. Scott, and S. Castro- tolerance (Levin, 2002). For instance, sizeable Pacheco. The authors also thank the scientific 119 Figure 1. Cell division during male meiosis is normal in Sobralia species. 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