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Technique for the identification of osmophores in flowers of herbarium material (TIOFH)

  • Marcelo P. Hernández
  • Liliana KatinasEmail author
New Methods in Cell Biology


The histochemical studies that search for osmophores differ in the protocol they follow for fixation and discoloration of the samples, and also in the type and number of stains utilized. Despite these differences, all the studies have one point in common: the use of fresh material either collected directly in the field or cultivated in botanical gardens and greenhouses. This is an obvious limitation for the studies of osmophores. Flower parts of herbarium specimens of different dates of collection and different plant families were exposed to variable times and percentages of discoloration agents and under different stains for finding if it is possible to positively test osmophores in this type of material. We obtained positive results discoloring the samples with ethyl alcohol 96° and sodium hypochlorite, and staining with Lugol, Oil Red O, and Neutral Red (TIOFH). A protocol (TIOFH3) for osmophore testing that combines these three stains into a single procedure is proposed.


Floral osmophores Protocol Herbarium material Histochemical technique 



We acknowledge Jorge Crisci and the curators of herbaria for loan of specimens.


This study was funded by the CONICET (grant PIP 0729) and the ANPCyT (grant PICT 2012-1683), Argentina.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.


  1. Aliscioni SS, Achler AP, Torretta HP (2017) Floral anatomy, micromorphology and visitor insects in three species of Aristolochia L. (Aristolochiaceae). New Zeal J Bot 55:496–513. CrossRefGoogle Scholar
  2. Almeida-Soares S, Polatto LP, Dutra JCS, Torezan-Silingardi HM (2010) Pollination of Adenocalymma bracteatum (Bignoniaceae): floral biology and visitors. Neotrop Entomol 39:941–948. CrossRefGoogle Scholar
  3. Arai T (1994) Volatile compounds of Narcissus tazetta var. chinensis flowers. Nippon Koryo Kyokai 184:105–111Google Scholar
  4. Bröderbauer D, Díaz A, Weber A (2012) Reconstructing the origin and elaboration of insect-trapping inflorescences in the Araceae. Am J Bot 99:1666–1679. CrossRefGoogle Scholar
  5. Cabral de Melo M, Leite Borba E, Sousa Paiva EA (2010) Morphological and histological characterization of the osmophores and nectaries of four species of Acianthera (Orchidaceae: Pleurothallidinae). Plant Syst Evol 286:141–151. CrossRefGoogle Scholar
  6. Cocucci AA (1996) El osmóforo de Cyphomandra (Solanaceae): Estudio con microscopio electrónico de barrido. Darwiniana 34:145–150. Google Scholar
  7. Cosa MT, Dottori N, Stiefkens L, Hadid M, Matesevach M, Delbón N, Wiemer P, Machado S, Cabrera V, Costa C, Pérez A, Trenchi A (2014) Aplicación de técnicas de histología vegetal a la resolución de diversos problemas. Laboratorio de Morfología Vegetal, Doctorado en Ciencias Biológicas, Facultad de Ciencias Exactas, Físicas y Naturales, Universidad Nacional de Córdoba, CórdobaGoogle Scholar
  8. Cousins SR, Witkowski ETF (2012) African aloe ecology: a review. J Arid Environ 85:1–17. CrossRefGoogle Scholar
  9. Curry KJ, McDowell LM, Judd WS, Stern WL (1991) Osmophores, floral features, and systematics of Stanhopea (Orchidaceae). Am J Bot 78:610–623. CrossRefGoogle Scholar
  10. Drawert H (1968) Vitalfdrbung und Vitalfluorochromierung pflanzlicher Zellen und Gebebe. Protoplasmatologia 2:6–734Google Scholar
  11. Dubrovsky JG, Guttenberger M, Saralegui A, Napsucialy-Mendivil S, Voigt B, Baluška F, Menzel D (2006) Neutral red as a probe for confocal laser scanning microscope studies of plant roots. Ann Bot 97:1127–1138. CrossRefGoogle Scholar
  12. Fahn A (1979) Secretory tissues in plants. Academic Press, LondonGoogle Scholar
  13. Gomes da Silva A (1990-1992) Osmóforos: Retrospectiva dos últimos 20 anos de pesquisa. Rodriguésia 42/44:7–20. CrossRefGoogle Scholar
  14. Guilliermond A (1930) La vacuome des cellules végétales. Protoplasma 9:133–174. CrossRefGoogle Scholar
  15. Hadacek F, Weber M (2002) Club-shaped organs as additional osmophores within the Sauromatum inflorescence: odour analysis, ultrastructural changes and pollination aspects. Plant Biol 4:367–383. CrossRefGoogle Scholar
  16. Holmgren PK, Holmgren NH, Barnett LC (1990) Index Herbariorum, Part I: The herbaria of the world, 8th edn. New York Botanical Garden, BronxGoogle Scholar
  17. Kerstiens G (2006) Water transport in plant cuticles: an update. J Exp Bot 57:2493–2499. CrossRefGoogle Scholar
  18. Kowalkowska AK, Kozieradzka-Kiszkurno M, Turzyński S (2015) Morphological, histological and ultrastructural features of osmophores and nectary of Bulbophyllum wendlandianum (Kraenzl.) Dammer (B. section Cirrhopetalum Lindl., Bulbophyllinae Schltr., Orchidaceae). Plant Syst Evol 301:609–622. CrossRefGoogle Scholar
  19. Kowalkowska AK, Pawlowicz M, Guzanek P, Krawczyńska AT (2018) Floral nectary and osmophore of Epipactis helleborine (L.) Crantz (Orchidaceae). Protoplasma 255:1811–1825. CrossRefGoogle Scholar
  20. Küster E (1939) Vital-staining of plant cells. Bot Rev 5:351–370CrossRefGoogle Scholar
  21. Lallana M, Billard CE, Elizalde JH, Lallana VH (2006) Breve revisión sobre características de la cutícula vegetal y penetración de herbicidas. Cien Doc Tecnol 17I:229–241Google Scholar
  22. Lillie RD, Ashburn LL (1943) Supersaturated solutions of fat stains in dilute isopropanol for demonstration of acute fatty degeneration not shown by Herxheimer’s technique. Arch Pathol 36:432Google Scholar
  23. Méndez M, Obeso JR (1992) Influencia del osmóforo en la producción de infrutescencias en Arum italicum Miller (Araceae). An Jardin Bo Madrid 50:229–237Google Scholar
  24. Miguez MB, Amela García MT (2015) Biología reproductiva de Sagittaria montevidensis Cham. et Schlech (Alismataceae). B Soc Argent Bot 48 (Supl:109)Google Scholar
  25. Miguez MB, Grohar M, Aquino D, Bouza A, Herrera Cano A, Lafuente Díaz M, Scorza V, Chamer M, Amela García MT, Hoc PS (2013) Biología floral de Lantana camara L. (Verbenaceae). B Soc Argent Bot 50 (Supl:88Google Scholar
  26. Novikoff AB, Novikoff PM, Rosen OM, Rubin CS (1980) Organelle relationships in cultured 3T3-L1 preadipocytes. J Cell Biol 87:180–196. CrossRefGoogle Scholar
  27. Pansarin LM, Pansarin ER, Sazima M (2008) Reproductive biology of Cyrtopodium polyphyllum (Orchidaceae): a Cyrtopodiinae pollinated by deceit. Plant Biol 10:650–659. CrossRefGoogle Scholar
  28. Piechulla B, Pott MB (2003) Plant scents: mediators of inter- and intraorganismic communication. Planta 217:687–689. CrossRefGoogle Scholar
  29. Plachno BJ, Faber J, Jankun A (2005) Cuticular discontinuities in glandular hairs of Genlisea St.-Hil. in relation to their functions. Acta Bot Gallica 152:125–130CrossRefGoogle Scholar
  30. Plachno BJ, Świątek P, Stpiczyńska M, Fernandes Oliveira Miranda V (2018) Flower palate ultrastructure of the carnivorous plant Genlisea hispidula Stapf with remarks on the structure and function of the palate in the subgenus Genlisea (Lentibulariaceae). Protoplasma 255:1139–1146. CrossRefGoogle Scholar
  31. Proescher F (1927) Oil red O pyridin, a rapid fat stain. Stain Technol 2:60–61CrossRefGoogle Scholar
  32. Rodrigues Alves G, Peruchi A, Agostini K (2010) Polinização em área urbana: O estudo de caso de Jacaranda mimosifolia D. Don (Bignoniaceae). Bioikos 24:31–41Google Scholar
  33. Ruzin SE (1999) Plant microtechnique and microscopy. Oxford University Press, OxfordGoogle Scholar
  34. Sazima M, Vogel S, Cocucci A, Hausner G (1993) The perfume flowers of Cyphomandra (Solanaceae): pollination by euglossine bees, bellows mechanism, osmophores, and volatiles. Pl Syst Evol 187:51–88. CrossRefGoogle Scholar
  35. Singh Y, van Wyk AE, Baijnath H (1996) Floral biology of Zantedeschia aethiopica (L.) Spreng. (Araceae). S Afr J Bot 62:146–150. CrossRefGoogle Scholar
  36. Stadelmann EJ, Kinzel H (1972) Vital staining of plant cells. In: Prescott DM (ed) Methods in cell physiology, vol 5. Academic Press Inc, New York, pp 325–372Google Scholar
  37. Stern WL, Curry KJ, Whitten, WM (1986) Staining fragrance glands in orchid flowers. Bull Torrey Bot Club 113:288–297.
  38. Stern WL, Curry KJ, Pridgeon AM (1987) Osmophores of Stanhopea (Orchidaceae). Am J Bot 74:1323–1331. CrossRefGoogle Scholar
  39. Vogel S (1963) Duftdrüsen im Dienste der Bestäubung: Über Bau und Funktion der Osmophoren. Abhandl Math-Naturwiss Kl Akad Wiss Mainz 10:600–763Google Scholar
  40. Vogel S (1990) The role of scent glands in pollination, first edn. Smithsonian Institution, Washington DCGoogle Scholar
  41. Weryszko-Chmielewska E, Chwill M, Sawidis T (2007) Micromorphology and histochemical traits of staminal osmophores in Asphodelus aestivus Brot. flower. Acta Agrobot 60:13–23. CrossRefGoogle Scholar
  42. Weryszko-Chmielewska E, Sulborska A (2012) Diversity in the structure of the petal epidermis emitting odorous compounds in Viola x wittrockiana Gams. Acta Sci Pol- Hortoru 11:155–167Google Scholar
  43. Wiemer AP, Moré M, Benitez-Vieyra S, Cocucci AA, Raguso RA, Sérsic AN (2009) A simple floral fragrance and unusual osmophore structure in Cyclopogon elatus (Orchidaceae). Plant Biol 11:506–514. CrossRefGoogle Scholar
  44. Zarlavsky GE (2014) Histología Vegetal: Técnicas simples y complejas. Sociedad Argentina de Botánica, CórdobaGoogle Scholar

Copyright information

© Springer-Verlag GmbH Austria, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Laboratorio de Morfología Comparada de Espermatófitas (LAMCE), Facultad de Ciencias Agrarias y ForestalesUNLPLa PlataArgentina
  2. 2.División Plantas Vasculares, Museo de La Plata, Facultad de Ciencias Naturales y MuseoUNLPLa PlataArgentina

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