Optimization of in vitro asymbiotic seed germination protocol for Serapias vomeracea

Open access

Abstract

Serapias vomeracea is an economically important orchid species which is over-collected from nature, because of its glucomannan-rich tubers. Thus, optimization of in vitro culture methodology in this species is required to meet industrial needs and to secure its populations in nature. This study aimed to optimize the surface sterilization protocol for S. vomeracea seeds and to select the optimal seed germination medium by comparing the commonly used media in in vitro orchid culture. During seed surface sterilization, ethyl alcohol (EtOH) pre-treatment prior to sodium hypochlorite (NaOCl) treatment increased the disinfection success and viable seed yield when examined using the triphenyl tetrazolium chloride (TTC) seed viability test. Also, low-g force centrifugation as an additional step in the surface sterilization method separated the seeds without embryo from the viable seeds and thereby decreased potential counting errors after incubation. Comparison of media showed that solid Knudson C (KN) medium induced the highest number of germinated seeds. However, seed germination success of Lindemann (LN) and Vacin & Went (VW) media was found to be higher when the media was used in liquid form. Half-strength liquid VW was the only medium that induced higher germination success than the other full-strength media. The highest number of ungerminated seeds was found when using KN medium whereas liquid VW medium gave the lowest number. In general, protocorm formation was triggered when the media were used in liquid form. However, rhizoid elongation was suppressed in liquid media. These findings suggest that this optimized seed surface sterilization method offers a simple and effective alternative to classical methods. Additionally, solid KN medium may be considered as a cost-effective and reliable alternative to other commonly-used complex media in S. vomeracea cultures.

1. Bellusci F, Pellegrino G, Musacchio A. Different levels of inbreeding depression between outcrossing and selfing Serapias species (Orchidaceae). Biol Plant 2009; 53: 175-178.

2. Acemi A, Çobanoğlu Ö, Türker-Kaya S. FTIR-based comparative analysis of glucomannan contents in some tuberous orchids, and effects of pre-processing on glucomannan measurement. J Sci Food Agric 2019; 99: 3681–3686.

3. Arvill A, Bodin L. Effect of short-term ingestion of konjac glucomannan on serum cholesterol in healthy men. Am J Clin Nutr 1995; 61: 585-589.

4. Sood N, Baker LW, Coleman CI. Effect of glucomannan on plasma lipid and glucose concentrations, body weight, and blood pressure: Systematic review and meta-analysis. Am J Clin Nutr 2008; 88: 1167-1175.

5. Arditti J. Micropropagation of orchids 2nd ed., Blackwell Publishing, UK, 2008.

6. Rasmussen HN, Dixon KW, Jersáková J, Těšitelová T. Germination and seedling establishment in orchids: a complex of requirements. Ann Bot 2015; 116: 391-402.

7. Paul S, Kumaria S, Tandon P. An effective nutrient medium for asymbiotic seed germination and large-scale in vitro regeneration of Dendrobium hookerianum, a threatened orchid of northeast India, AoB Plants 2012; plr032.

8. Yeung EC, Park J, Harry IS. Orchid seed germination and micro-propagation I: Background information and related protocols; pp. 101-125 in Lee YI; Yeung ET (Eds) Orchid Propagation: From laboratories to greenhouses—Methods and protocols. New York, Springer Protocols Handbooks, Humana Press, 2018.

9. Katsalirou E, Gerakis A, Haldas X, Deconninck G. Optimal disinfection times for seeds of Mediterranean orchids propagated on nutrient media. Eur J Environ Sci 2017; 7: 119-124.

10. Özkoç I, Dalcı M. Germination of the seeds of Orchis laxiflora Lam. (Orchidaceae) through asymbiotic culture techniques. Turk J Bot 1994; 18: 461-464.

11. Gümüş C, Ellialtıoğlu Ş. Seed germination and development of Serapias vomeracea (Burm. fil.) Briq. ssp. orientalis Greuter in tissue culture. Res J Biotechnol 2012; 7: 4-8.

12. Knudson L. A new nutrient solution for germination of orchid seed. Amer Orchid Soc Bull 1946; 15: 214-217.

13. Vacin E, Went FW. Some pH changes in nutrient solutions. Bot Gaz 1949; 110: 605-613.

14. Lindemann EG, Gunckel JE, Davidson OW. Meristem culture of Cattleya, Amer Orchid Soc Bull 1970; 39: 1002-1004.

15. Yamazaki J, Miyoshi K. In vitro asymbiotic germination of immature seed and formation of protocorm by Cephalanthera falcata (Orchidaceae), Ann Bot 2006; 98: 1197-1206.

16. Yeung EC. A perspective on orchid seed and protocorm development. Bot Stud 2017; 58: 33.

17. Kauth PJ, Dutra D, Johnson TR, Stewart SL, Kane ME, Vendrame W. Techniques and applications of in vitro orchid seed germination; pp. 375–391 in Teixeira da Silva, J.A. (Ed.) Floriculture, Ornamental and Plant Biotechnology: Advances and Topical Issues. Isleworth, UK, Global Science Book, 2008.

18. Ponert J, Vosolsobê S, Kmecová K, Lipavská H. European orchid cultivation – from seed to mature plant. Eur J Environ Sci 2011; 1: 95-107.

19. Ercole E, Rodda M, Girlanda M, Perotto S. Establishment of a symbiotic in vitro system between a green meadow orchid and a Rhizoctonia-like fungus. Bio-protocol 2015; 5(10): e1482.

20. Barsberg S, Rasmussen HN, Kodahl N. Composition of Cypripedium calceolus (Orchidaceae) seeds analyzed by attenuated total reflectance ir spectroscopy: In search of understanding longevity in the ground. Am J Bot 2013; 100(10): 2066–2073.

21. Bektaş E, Cüce M, Sökmen A. In vitro germination, protocorm formation and plantlet development of Orchis coriophora (Orchidaceae), a naturally growing orchid species in Turkey. Turk J Bot 2013; 37: 336-342.

22. Bektaş E, Sökmen A. In vitro seed germination, plantlet growth, tuberization, and synthetic seed production of Serapias vomeracea (Burm.f.) Briq. Turk J Bot 2016; 40: 584-594.

23. Zahara M, Datta A, Boonkorkaew P, Mishra A. The effects of Different media, sucrose concentrations and natural additives on plant-let growth of Phalaenopsis hybrid ‘Pink’. Braz Arch Biol Technol 2017; 60: e17160149.

24. De Long JR, Swarts ND, Dixon KW, Egerton-Warburton LM. Mycorrhizal preference promotes habitat invasion by a native Australian orchid: Microtis media. Ann Bot 2012; 111: 409-18.

25. Yoder JA, Zettler LW, Stewart SL. Water requirements of terrestrial and epiphytic orchid seeds and seedlings, and evidence for water uptake by means of mycotrophy. Plant Sci 2000; 156: 145-150.

26. Tsai W-T, Chu C-Y. Static liquid culture of Doritaenopsis seedlings. Hortscience 2008; 43: 206–210.

27. Edwin FG. Plant propagation by tissue culture. Exegetics Ltd., Edington Wilts, UK, 1993.

28. Gogoi K, Kumaria S, Tandon P. Ex situ conservation of Cymbidium eburneum Lindl.: a threatened and vulnerable orchid, by asymbiotic seed germination. 3 Biotech 2012; 2: 337.

29. Alexander C, Alexander IJ, Hadley G. Phosphate uptake by Goody-era repens in relation to mycorrhizal infection. New Phytol 1984; 97: 401-411.

30. Rodrigues DT, Novais RF, Alvarez VH, Dias JMM, Villani EMA. Orchid growth and nutrition in response to mineral and organic fertilizers. Rev Bras Cienc Solo 2010; 34: 1609-1616.

31. Fochi V, Chitarra V, Kohler A, Voyron S, Singan VR, Lindquist EA, Barry KW, Girlanda M, Grigoriev IV, Martin F, Balestrini R, Perotto S. Fungal and plant gene expression in the Tulasnella calospora – Serapias vomeracea symbiosis provides clues about nitrogen pathways in orchid mycorrhizas. New Phytol 2017; 213: 365-379.

32. Kauth PJ, Vendrame W, Kane ME. In vitro seed culture and seedling development of Calopogon tuberosus. Plant Cell Tissue Organ Cult 2006; 85: 95-102.

33. Das AK, Das J, Gogoi HK, Srivastava RB. Mass propagation of orchids through in vitro seed culture technology. J Cell Tissue Cult Res 2008; 8: 1585-1588.

34. Philip RJ, Kakati JP, Sebestinraj J, Suriya K. In vitro seed germination of Cymbidium aloifolium (L.) Sw., a potential medicinal orchid from Eastern Ghats of Tamil Nadu, India. J Plant Biotechnol 2017; 44: 343-348.

Journal Information

Metrics

All Time Past Year Past 30 Days
Abstract Views 0 0 0
Full Text Views 40 40 40
PDF Downloads 34 34 34