Seed Germination and Development of Serapias vomeracea (Burm ...

Research Journal of Biotechnology Vol. 7 (3) August (2012)Res. J. BiotechSeed Germination and Development of Serapiasvomeracea (Burm.fil.) Briq. ssp. orientalis Greuter inTissue CultureGumus Cevdet 1 * and Ellialtıoglu Sebnem 21. Bartin Vocational School, Bartin University, Bartin, TURKEY2. Department of Horticulture, Faculty of Agriculture, Ankara University, Ankara, TURKEY*cgumus42@hotmail.comAbstractSerapias vomeracea (Burm. fil.) Briq. ssp. orientalisGreuter plants were obtained via asymbioticgermination under in vitro conditions. For thegermination of seeds, different doses of gibberellicacid (GA 3 ) (0, 0.1 and 0.5 mg/L) were added into abasic nutrient media consisting of half-strength MS (½MS), Van Waes and DeBergh (VW and DB), KnudsonC and non-inorganic nitrogen Knudson C (KC-N).Cultures were incubated in darkness for the first 3months and thereafter transferred to a light/darkphotoperiod of 16:8. At the end of the third month,protocorm rates were identified. They weresubcultured every 4 weeks and ½ MS medium wasused as a transfer medium. The highest germinationrate, protocorm rate and plant growth rate wereobtained from the Knudson C medium. Thegermination rate and number of plantlets wereaffected negatively by adding GA 3 .Keywords: Orchid, germination, Serapias vomeracea,medium.IntroductionSerapias vomeracea (Burm. fil.) Briq. ssp. orientalis is aspecies belonging to the Orchidaceae family and isconsidered to be in the group of terrestrial orchids. Theplant is 10-30 cm in height with lanceolate, flaring leaves.Bracts are in the form of wide lanceolate with variouscolors from purple to silvery-green. The flowers are big,generally 3-6 on each spike, in colors ranging fromyellowish-green, reddish-brown, to dark purple. Serapiasvomeracea is native to dry and damp meadows, scrubs androad sides. It blooms between the end of March and May.They are tuber orchids. The tubers, gathered from theirhabitats to produce salep, generally spread along thecoastline regions of Turkey 5,19 .These orchids generally reproduce through the productionof seeds in their natural habitat, although some orchidspecies are known to reproduce vegetative. The seeds ofthis species are very tiny and have a dust like structure.They have no endosperm and lose embryo viability quitequickly; thus, less than 5% of them are able to germinate intheir habitat. It takes an extremely a long time, 2-16 years,for the orchids to become mature after germination.*Author for correspondence(4)The tubers produce just 1 fresh tuber each year and as thenew tuber grows, it supersedes the older one andeventually, the older one vanishes. Serapias vomeracea hasbecome an endangered species due to low propagation andsenseless harvesting. Although senseless harvesting stillcontinues, these plants are sparsely found in nature due tothe low germination rate of their seeds and slowreproduction of their tubers 19 .To maintain their lifecycle, terrestrial orchids would notnecessarily have a symbiotic relationship with fungi 17 .Germination of orchid seeds in dark conditions isappropriate; however, some require both light andphotoperiodic conditions and some others germinate at thesame rate in both conditions 1 . Hormone applications haveno significant effect on the germination of orchid seeds 2 .The nutrient media composition plays an important role inorchid seed germination. Although epiphytic orchidsneeded intensive nutrient media, terrestrial orchidsgerminate better in a diluted media 2,8 . Similarly, Pierik 17proposed that germination of orchid seeds was possible ona simple medium containing minerals and sugars. Özkoç 16and Gönülşen et al 6 reported that removing the nitrogensource from the nutrient medium positively affected thegermination of orchid seeds. According to Taiz andZeiger 20 , gibberellins play a role in several aspects of seedgermination and gibberellic acid (GA 3 ) can also breakcertain kinds of dormancies in seeds. In contrast, there aresome references indicating no positive affect of GA 3 on thegermination of orchid seeds. On the other hand, Arditti andErnst 3 explained that there is a possibility of a negativeeffect of GA 3 applications on the germination of orchidseeds. The addition of GA 3 into the nutrient media had nopositive effect on protocorm growth and seed germinationin Calanthe discolor Lindl. 12 . Önal 14 noticed that after theaddition of 0.2mg/L of GA 3 to the Knudson C nutrientmedium, there was no germination observed in O. laxiflora.In his study on symbiotic and asymbiotic germination ofSerapias vomeracea subsp, laxiflora and Orchis laxiflora,Özkoç 15 argued that oat medium with some additives is themost appropriate medium for symbiotic cultures. Thehighest rate of germination in oat medium with fungalisolates was 30.6%, whereas it was 9.1% in the modifiedoat medium. During in vitro germination trials, the highestgermination rate was obtained from the media notcontaining inorganic nitrogen of the modified mediums ofMS, Knudson and VWD. Among the media, the highest

Research Journal of Biotechnology Vol. 7 (3) August (2012)Res. J. BiotechNutrientmediaTable 1The effects of MS or KC media and the addition of 0.1 mg/L GA 3 on the germination,protocorm formation and plant growth of Serapias vomeracea.Germination rate(%)Protocormformation rate(%)½ MS 30.55 c 22.81 bc 15.14 b½ MS + 0.1GA 3 52.38 b 36.02 b 20.02 bKC 76.85 a 63.93 a 42.80 aKC + 0.1GA 3 32.72 c 15.63 c 11.14 bPlantlet growth rate (%)Table 2The effects of KC or KC-N media and the addition of0.5 mg/L GA 3 on the germination, protocorm formationand plant growth of Serapias vomeracea.NutrientmediaGerminationrate(%)Protocormformation(%)Plantgrowth(%)KC 71.98 a 59.21 a 40.24 aKC + 0.5GA 3 34.34 bc 18.39 c 12.94 bcKC - N 47.51 b 32.42 b 17.54 bKC - N + 0.5GA 3 30.41 c 15.04 c 11.02 c3. Arditti J. and Ernst R., Physiology of germinating orchid seeds,Bot. Rev., 33, 1-197 (1984)4. Çağlayan K., Özavcı A. and Eskalen A., In VitroMultiplication of Salep Orchids, Growing Commonly in the EastMediterranean Region of Turkey, Turkish J. of Agriculture andForestry, 22(2), 187-191 (1998)Table 3The effects of the ½ MS, VW and DB and KC nutrientmedia and the addition of 0.1 or 0.5 mg/L of GA3 on thegermination, protocorm formation and plant growth ofSerapias vomeracea.NutrientmediaGerminationrate(%)Protocormformation(%)Plantgrowth(%)½ MS 31.98 cd 25.49 b-d 17.68 b-d½ MS + 0.1 GA 3 51.70 b 35.14 b 20.31 bc½ MS + 0.5 GA 3 21.26 ef 15.94 cd 11.32 cdVW & DB 29.37 de 24.36 b-d 20.01 bcVW & DB + 0.1 GA 3 31.47 cd 27.65 b 24.37 bVW & DB + 0.5 GA 3 19.16 f 14.90 d 10.28 dKC 83.44 a 72.75 a 57.65 aKC + 0.1 GA 3 46.16 b 26.22 bc 21.71 bKC + 0.5 GA 3 41.56 bc 24.35 b-d 19.52 b-dFigure 1: a) Germination of seeds b) protocorms that turned green under lightc) plant growth in KC medium and d) a plantlet at the beginning of the acclimatization stage(7)

Research Journal of Biotechnology Vol. 7 (3) August (2012)Res. J. Biotech5. Davis P.H., Flora of Turkey and the East Aegan Islands, 8,Edinburgh at the University Press (1984)6. Gönülşen N., Önal K., Ercan N., Yıldızgördü K., Şekeroğlu E.,Biçici M. and Eskalen A., In vitro Propagation of Some Speciesfrom Orchidaceae Family Existing in the Natural Flora of Aegeanand East Mediterranean Region, TÜBİTAK Proje No: TBGAG-52 (1996)7. Harley, The Biology of Mycorriza, L. Hill, London, 2ndedition, 1-334 (1969)8. Harvais G., Growth requirements and development ofCypripedium reginae in axenic culture, Can. J. Bot., 51, 327-332(1973)9. Kısakürek Ş., Arpacı B.B., Özdemir A., Dalfesoğlu K., ErgunN. and Kaya Y., Kahramanmaraş Doğal Florasında Yetişen veSalep Üretiminde Kullanılan Bitkilerin Kültüre AlınabilmeOlanakları, TAGEM Projesi Sonuç Raporu, KahramanmaraşTarımsal Araştırma Enstitüsü (2009)10. Knudson L., A new nutrient solution for the germination oforchid seed, Amer. Orchid. Soc. Bull., 15, 214-217 (1946)11. Lauzer D., St. Arnaud M. and Barabe D., Tetrazolium stainingand in vitro germination of mature seeds of Cypripedium acaule(Orchidaceae), Lindleyana, 9(3), 197-204 (1994)14. Önal K., In vitro propagation of some species fromOrchidaceae family existing in the natural flora of aegean region,Turkish J. of Agriculture and Forestry, 23(5), 1057-1064 (1999)15. Özkoç İ., Serapias vomeracea (Burm fil.) Briq. subsp.laxiflora (Soo) Gölz et. Reinhard ve Orchis laxiflora Lam.(Orchidaceae) Tohumlarının Simbiyotik ve AsimbiyotikKültürlerde Çimlenme ve Gelişmesi Üzerinde Araştırılması(doktora tezi), Ondokuz Mayıs Üniversitesi, Fen BilimleriEnstitüsü, Samsun (1991)16. Özkoç I. and Dalcı M., Germination of the Seeds of Orchislaxiflora Lam. (Orchidaceae) Through Asymbiotic CultureTechniques, Turkish Journal of Botany, 18, 461-464 (1994)17. Pierik R.L.M., In vitro Culture of Higher Plants, 149-158(1987)18. Pierık R.L.M., Steegmans H.H.M., Verhaegh J.A.M. andWouters A.N., Effect of cytokinins and cultivar on shootformation of Gerbera jamesonii in vitro, Neth. J. Agric. Sci., 30,341-346 (1982)19. Sezik E., Orkidelerimiz, Sandoz Kültür Yayınları, 6, 166s(1984)20. Taiz L. and Zeiger E., Plant Physiology, Sinauer Associates,Sunderland, MA (1998)12. Miyoshi K. and Mii M., Phytohormone pre-treatment for theenhancement of seed germination and protocorm formation by theterrestrial orchid, Calanthe discolor (Orchidaceae), in asymbioticculture, Scientia Hort., 63, 263-267 (1995)13. Murashige T. and Skoog F., A revised medium for rapidgrowth and bioassays with tobacco cultures, Physiol. Plant, 15,473-497 (1962)*****21. Van Waes J.M. and Debergh P.C., In vitro germination ofsome western European Orchids, Physilogia Plantarum., 67(2),253-261 (1986).(Received 30 th November 2011, accepted 15 th June 2012)(8)