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Ankara Üniversitesi Fen Bilimleri Enstitüsü Doktora ANKARA ÜN ĐVERS ĐTES Đ FEN B ĐLĐMLER Đ ENST ĐTÜSÜ DOKTORA TEZ Đ GENTIANA OLIVIERI GRISEB.’DE KALLUS VE SÜSPANS ĐYON KÜLTÜRLER ĐNDE BULUNAN BAZI SEKONDER METABOL ĐTLER Canan YA ĞCI TÜZÜN BĐYOLOJ Đ ANAB ĐLĐM DALI 2011 ANKARA Her hakkı saklıdır TEZ ONAYI Canan YA ĞCI TÜZÜN tarafından hazırlanan “ Gentiana olivieri Griseb.’de Kallus ve Süspansiyon Kültürlerinde Bulunan Bazı Sekonder Metabolitler” adlı tez çalı şması 02/12/2011 tarihinde aşağıdaki jüri tarafından oy birli ği ile Ankara Üniversitesi Fen Bilimleri Enstitüsü Biyoloji Anabilim Dalı’nda DOKTORA TEZ Đ olarak kabul edilmi ştir. Danı şman : Prof. Dr. M. Cihat TOKER Eş danı şman : - Jüri Üyeleri : Ba şkan: Prof. Dr. Rukiye TIPIRDAMAZ Hacettepe Üniversitesi Fen Fakültesi Biyoloji Bölümü Üye: Prof. Dr. M. Cihat TOKER Ankara Üniversitesi Fen Fakültesi Biyoloji Bölümü Üye: Doç. Dr. Gül Nilhan TU Ğ Ankara Üniversitesi Fen Fakültesi Biyoloji Bölümü Üye: Yrd. Doç. Dr. H. Nurhan BÜYÜKKARTAL Ankara Üniversitesi Fen Fakültesi Biyoloji Bölümü Üye: Yrd. Doç. Dr. Hatice ÇÖLGEÇEN Zonguldak Karaelmas Üniversitesi Fen-Edebiyat Fakültesi Biyoloji Bölümü Yukarıdaki sonucu onaylarım Prof. Dr. Özer KOLSARICI Enstitü Müdürü 2 ÖZET Doktora Tezi GENTIANA OLIVIERI GRISEB.’DE KALLUS VE SÜSPANS ĐYON KÜLTÜRLER ĐNDE BULUNAN BAZI SEKONDER METABOL ĐTLER Canan YA ĞCI TÜZÜN Ankara Üniversitesi Fen Bilimleri Enstitüsü Biyoloji Anabilim Dalı Danı şman: Prof. Dr. M. Cihat TOKER Flavonoit, iridoit ve alkaloit içeren Gentiana olivieri Griseb. (Gentianaceae), antidiyabetik, antidepresan ve sindirime yardımcı olarak kullanılmaktadır. Flavonoit (izoorientin ile izoviteksin) ve iridoitlerin (gentiopikrozit ile svertiamarin) varlı ğı G. olivieri kallus ve süspansiyon kültürlerinde ara ştırıldı. G. olivieri tohumları 0.1 mM GA 3 içeren Woody Plant Medium (WPM)’da çimlendirildi. Kallus üretmek için ilk deneme için yaprak ve kök eksplantları, ikincisi için sadece yaprak eksplantı kullanıldı. Her iki denemede de eksplantlar 60 günlük aseptik fidelerden alındı. Eksplantlar 1 mg/l Naftalen Asetik Asit (NAA) ile 0.5 mg/l Benzil Amino Purin (BAP) veya 0.2-0.5 mg/l Kinetin içeren Murashige ve Skoog (MS) ile WPM ortamlarına ekildi. Kültürler ilk denemede karanlıkta; ikincide ise 16/8 aydınlık/karanlık fotoperiyotta 3000 lux ı şık şiddetinde inkübe edildi. Đkinci denemeden elde edilen kalluslardan süspansiyon kültürleri kuruldu. Kallus kültürlerinden 3 alt kültür ve süspansiyon hücrelerinden 25 gün boyunca üretilen sekonder metabolitler; kalitatif ve kantitatif olarak ara ştırıldı. Sekonder metabolit üretiminde süspansiyon kültürü, kallus kültürüne göre daha verimli bulundu. 1 mg/l NAA ve 0.5 mg/l BAP içeren WPM ortamında 20. günde süspansiyon kültüründe geli şen hücrelerde izoviteksin miktarı G. olivieri ekstresinden 10 kat fazla bulundu (0.408 mg/g). 1 mg/l NAA ve 0.5 mg/l Kinetin içeren WPM ortamında 1. alt kültürde geli şen kallus hücreleri izoorientin üretimi için en iyi sonucu verdi (0.492 mg/g). Đridoit yapıda oldu ğu dü şünülen fakat gentiopikrozit ve svertiamarin ile e şle şmeyen maddeler üretildi. Aralık 2011, 124 sayfa Anahtar Kelimeler: Gentiana olivieri , kallus kültürü, hücre süspansiyon kültürü, bitki sekonder metabolitlerinin üretimi i ABSTRACT Ph. D. Thesis SOME SECONDARY METABOLITES OF CALLUS AND SUSPENSION CULTURES OF GENTIANA OLIVIERI GRISEB. Canan YA ĞCI TÜZÜN Ankara University Graduate School of Natural and Applied Sciences Department of Biology Supervisor : Prof. Dr. M. Cihat TOKER Gentiana olivieri Griseb. (Gentianaceae), which contains flavonoid, iridoid and alkaloid, has been used as antidiabetic, antidepressant and digestive aid. Existence of flavonoids (isoorientin and isovitexin) and iridoids (gentiopicroside and swertiamarin) were investigated in callus and suspension culture of G. olivieri . Seeds of G. olivieri were germinated in Woody Plant Medium (WPM) with 0.1 mM GA 3. Leaf and root explants were used for the first experiment while only leaf explant was used for the second to produce callus. In both experiments, explants were obtained from 60 days old aseptic plantlets. Explants were sown onto Murashige & Skoog Medium (MS) and WPM including 1 mg/l Naphtalane Acetic Acid (NAA) with 0.5 mg/l Benzyl Amino Purine (BAP) or 0.2-0.5 mg/l Kinetin. Cultures were incubated in the dark in the first experiment while the second was in 3000 lux at 16/8h light/dark photoperiod. Suspension cultures were established from calli which obtained from the second experiment. Secondary metabolites, which were produced from callus cultures during 3 sub-culture and from suspension cultures during 25 days, were investigated qualitatively and quantitatively. Suspension cultures were found more productive than callus cultures in terms of secondary metabolite production. Quantity of isovitexin, grown in WPM suspension cultures containing 1 mg/l NAA and 0.5 mg/l BAP in the 20th day, was found ten times more from G. olivieri extract (0.408 mg/g). Callus cells which grown in WPM containing 1 mg/l NAA and 0.5 mg/l Kinetin in the first sub-culture were found to be more succesful for isoorientin production (0.492 mg/g). Substances, which were considered to have iridoidal structure but had no match with gentiopicroside and swertiamarin, were produced. December 2011, 124 pages Key Words: Gentiana olivieri , callus culture, cell suspension culture, production of plant secondary metabolites ii TE ŞEKKÜR Bu tez, Ankara Üniversitesi Biyoteknoloji Enstitüsü tarafından desteklenen “ Gentiana olivieri Griseb.’de kallus olu şumu ve kallusta izoorientin miktarının tayini (2001-K- 120-240)” ile TÜB ĐTAK tarafından desteklenen “Bitkisel Kaynaklı Đlaç Etken Maddelerinin Biyoteknolojik Yöntemlerle Üretilmesi Üzerine Çalışmalar (1055343)” adlı projeler kapsamında yürütülmü ştür. Bu çalı şmayı yürütmemi sa ğlayan, fikirleriyle beni yönlendiren, ara ştırmalarımda ilgi, bilgi ve deste ğini esirgemeyen danı şman hocam Sayın Prof. Dr. M. Cihat TOKER ve çalı şmalarımda her zaman deste ğini gördü ğüm ve Gazi Üniversitesi Eczacılık Fakültesi Farmakognozi Ana Bilim Dalı’nda bulunan laboratuarında tüm imkanları sunan Sayın Hocam Prof. Dr. Gülnur TOKER’e sonsuz te şekkürlerimi sunarım. Kantitatif analizleri yürüttü ğüm Hacettepe Üniversitesi Eczacılık Fakültesi Farmakognozi Ana Bilim Dalı’nda görev yapan Sayın Hocalarım Prof. Dr. Đhsan ÇALI Ş, Prof. Dr. Tayfun ERSÖZ, Doç. Dr. Funda Nuray YALÇIN ba şta olmak üzere tüm personele güleryüzlerinden, verdikleri destek ve yardımlarından dolayı şükranlarımı sunarım. Hayatım boyunca attı ğım her adımda bana destek olan babam Emin Ali YA ĞCI, annem Ay şe YA ĞCI, abim Ayhan YA ĞCI’ya tüm kalbimle te şekkür ederim. Tezimi bitirmemde sonsuz manevi deste ğini gördü ğüm e şim Mehmet Murat TÜZÜN’e sonsuz te şekkür ederim. Canan YA ĞCI TÜZÜN Ankara, Aralık 2011 iii ĐÇĐNDEK ĐLER ÖZET ........................................................................................................................................................... i ABSTRACT ............................................................................................................................................... ii TE ŞEKKÜR .............................................................................................................................................. iii SĐMGELER D ĐZĐNĐ ................................................................................................................................ vii ŞEK ĐLLER D ĐZĐNĐ ................................................................................................................................ viii ÇĐZELGELER D ĐZĐNĐ ..............................................................................................................................x 1. G ĐRĐŞ .......................................................................................................................................................1 2. KAYNAK ÖZETLER Đ ...........................................................................................................................5 2.1 Gentianaceae Familyası ve G. olivieri ile Đlgili Sistematik Bilgiler ...................................................5 2.1.1 Gentianaceae familyası ......................................................................................................................5 2.1.2 Gentiana L. cinsi .................................................................................................................................5 2.1.3 Türkiye’de yayılı ş gösteren Gentiana türlerinin te şhis anahtarı (Davis 1978) ..............................6 2.1.4 Gentiana olivieri Grisebach ...............................................................................................................7 2.1.4.1 Sistematik bilgileri ..........................................................................................................................7 2.1.4.2 Genel özellikleri ve habitatı ...........................................................................................................7 2.1.4.3 Türkiye’deki yayılı şı .......................................................................................................................8 2.1.4.4 Sinonim ve yerel isimleri ................................................................................................................9 2.2 Gentiana L.’nin Kullanım Alanları .....................................................................................................9 2.2.1 Gentiana olivieri ’nin kullanım alanları .........................................................................................
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