Orchis sancta'nın in vitro asimbiyotik çimlenmesi, fide oluşumu ve tüberizasyonu

Öz

Bitki doku kültürü teknikleri, son zamanlarda, nadir ve nesli tükenmekte olan türlerin çoğaltılması ve ekonomik açıdan önemli doğal ürünlerinin üretilmesi için ilgi çekici araçlar haline gelmiştir. Bu çalışmada, Türkiye'de doğal olarak yetişen yerli bir sahleb orkidesi türü olan Orchis sancta’nın fide ve yumrularının in vitro koşullarda etkin, hızlı ve güvenilir bir üretim protokolü oluşturulmuştur. Buna istinaden, olgun tohumların asimbiyotik çimlenmesi, fide oluşumu, yumru oluşumu ve yumru köklerin glukomannan içerikleri üzerine bazal ortam ve bitki büyüme düzenleyicilerinin etkinlikleri değerlendirilmiştir. Değerlendirilen tüm bazal ortamlar arasında aktif kömür içeren Orchimax en iyi çimlenme oranını (%62.46) verdi. Bu ortamın 1.0 mg/L zeatin ile desteklenmesi en yüksek çimlenme oranı (%81.18) ile sonuçlanmıştır. En düşük thidiazuron konsantrasyonunun (0.25 mg/L) sürgün uzaması üzerinde, kinetin’in (0.5 ve 1.0 mg/L) ise yaprak oluşumu üzerinde daha etkili olduğu belirlenmiştir. Yine aynı bazal besi ortamının 1.0 mg/L ve 2.0 mg/L IBA ile desteklenmesiyle sırasıyla kök uzamasını (31.16 mm) ve kök oluşumunu (fide başına 4.5 adet kök) daha fazla teşvik etmiştir. Sitokininlerin, özelliklede 2.0 mg/L zeatin’in, yumru oluşumunu ve yumrulardaki glukomannan birikiminde daha etkili olduğu tespit edilmiştir. İn vitro koşullarda üretilen fideler kademeli bir şekilde toprak koşullarına adapte edilmiştir.

Anahtar Kelimeler

• Orchis sancta
• Glukomannan
• Mikroçoğaltım
• Asimbiyotik çimlenme

In vitro asymbiotic germination, seedling formation and tuberization of Orchis sancta

Öz

Plant tissue culture techniques have recently become attractive tools for propagating rare and endangered species and producing their economically important natural products. Here, we introduce a protocol for an effective, fast and reliable production route of plantlets and tubers from salep orchid, Orchis sancta, a native orchid species growing wild in Turkey. In the course of this study, the efficacy of basal media and plant growth regulators on asymbiotic germination of mature seed, seedling formation, tuberization and glucomannan contents of tubers were assessed. Amongst all basal media evaluated, Orchimax including activated charcoal gave the best germination rate (62.46%). Supplementing this medium with 1.0 mg/L zeatin resulted in the highest germination rate (81.18%). Lowest thidiazuron concentration (0.25 mg/L) was very effective on shoot elongation while kinetin (0.5 and 1.0 mg/L) promoted leaf formation. The aforesaid basal medium with IBA favored root extension (31.16 mm) and root formation (4.5 roots per plantlet) at 1.0 mg/L and 2.0 mg/L IBA, respectively. Cytokinins, zeatin (2.0 mg/L) in particular, promoted tuber formation and glucomannan accumulation. Plantlets were successfully adapted to soil step by step.

Anahtar Kelimeler

• Orchis sancta
• Glucomannan
• Micropropagation
• Asymbiotic germination

Kaynakça

1. Aktar S, Nasiruddin KM, Huq H (2007). In vitro root formation in dendrobium orchid plantlets with IBA. Journal of Agriculture and Rural Development 5: 48-51.
2. Bektaş E, Cüce M, Sökmen A (2013). In vitro germination, protocorm formation and plantlet development of Orchis coriophora (Orchidaceae), a naturally growing orchid species in Turkey. Turkish Journal of Botany 37: 336-342.
3. Bektaş E, Sökmen A (2016). In vitro seed germination, plantlet growth, tuberization and synthetic seed production of Serapias vomeracea (Burm. F.) Briq. Turkish Journal of Botany 40: 584-594.
4. Black M, Bewley JD, Fountain D (1974). Lettuce seed germination and cytokinins: their entry and formation. Planta 117: 145-152.
5. Chua M, Chan K, Hocking TJ, Williams PA, Perry CJ, Baldwin TC (2012). Methodologies for the extraction and analysis of konjac glucomannan from corms of Amorphophallus konjac K. Koch. Carbohydrate Polymers 87: 2202-2210.
6. Crafts CB, Miller CO (1974). Detection and identification of cytokinins produced by mycorrhizal fungi. Plant Physiology 54: 586-588.
7. Díaz MSS, Álvarez CC (2009). Plant regeneration through direct shoot formation from leaf cultures and from protocorm-like bodies derived from callus of Encyclia mariae (Orchidaceae), a threatened Mexican orchid. In Vitro Cellular and Developmental Bilogy-Plant 45: 162-170.
8. Eymar E, Alegre J, Toribio M, López-Vela D (2000). Effect of activated charcoal and 6-benzyladenine on in vitro nitrogen uptake by Lagerstroemia indica. Plant Cell Tissue and Organ Culture 63: 57-65.

9. Fernie AR, Willmitzer L (2001). Update on tuber formation, dormancy and sprouting: molecular and biochemical triggers of potato tuber development. Plant Physiology 127: 1459-1465.
10. Gaspar T, Kevers C, Penei C, Greppin H, Reid DM, Thorpe TA (1996). Plant hormones and plant growth regulators in plant tissue culture. In vitro Cellular and Developmental Biology-Plant 32: 272-289.
11. Haberer G, Kieber JJ (2002). Cytokinins: New insights into a classic phytohormone. Plant Physiology 128: 354-362.
12. Jackson SD (1999). Multiple signaling pathways control tuber induction in potato. Plant Physiology 119: 1-8.
13. Malmgren S (1992). Large-scale asymbiotic propagation of Cypripedium calceolus plant physiology from a surgeon’s point of view. Botanic Gardens Micropropagation News 1: 59-63.
14. Paek KY, Murthy HN (eds) (1977). Orchid Biology: Reviews and Perspectives. Temperate Oriental Cymbidium species. Dordrecht: Kluwer Academic Publishers.
15. Pedroso MC, Pais MS (1992). Minituber production from immature seed suspension culture of Orchis papilionacea. In Vitro Cellular and Developmental Biology 28: 183-186.
16. Sarkar D (2008). The signal transduction pathways controlling in plant tuberization in potato: an emerging synthesis. Plant Cell Reports 27: 1-8.
17. Sezik E (1984). Orkidelerimiz. Sandoz Kültür Yayınları, pp 166.
18. Sgarbi E, Grimaudo M, Del Prete C (2009). In vitro asymbiotic germination and seedling development of Limodorum abortivum (Orchidaceae). Plant Biosystems 143: 114-119.
19. Skoog F, Miller CO (1957). Chemical regulation of growth and organ formation in plant tissues cultured in vitro. Symposia of the Society for Experimental Biology 11: 118-130.
20. Stewart LS, Kane ME (2006). Asymbiotic seed germination and in vitro seedling development of Habenaria macroceratitis (Orchidaceae), a rare Florida terrestrial orchid. Plant Cell Tissue and Organ Culture 86: 147-158.
21. Van Waes JM, Debergh PC (1986). In vitro germination of some Western European Orchids. Physiologia Plantarum 67: 253-261.
22. Vesjadova H (2006). Factors affecting seed germination and seedling growth of terrestrial orchids cultured in vitro. Acta Biologica Cracoviensia Series Botanica 48: 109-113.
23. Vreugdenhil D, Sergeeva LI (1999). Gibberellins and tuberization in potato. Potato Research 42: 471-481. Werbrouck SPO, Debergh PC (eds) (1994). Plant Cell Culture - A Pratical Approach. Applied aspects of plant regeneration (micropropagation). New York: Oxford University Press.