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Kocaeli Üniversitesi Fen Bilimleri Enstitüsü Biyoloji

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Kocaeli Üniversitesi Fen Bilimleri Enstitüsü Biyoloji KOCAELİ ÜNİVERSİTESİ FEN BİLİMLERİ ENSTİTÜSÜ BİYOLOJİ ANABİLİM DALI DOKTORA TEZİ KİTOSAN İLE BAZI BİTKİ BÜYÜME DÜZENLEYİCİLERİNİN SERAPIAS VOMERACEA’NİN IN VITRO FİZYOLOJİSİ ÜZERİNE ETKİLERİNİN KARŞILAŞTIRILMASI ARDA ACEMİ KOCAELİ 2018 ÖNSÖZ VE TEŞEKKÜR Bu tezde model organizma olarak ele alınmış olan Serapias vomeracea üzerinde kitosan biyopolimerinin doğal bir bitki büyüme artırıcısı olarak orkidelerde sebep olabileceği fizyolojik değişikliklerin yaygın olarak kullanılan bitki büyüme düzenleyicilerinin etkileri ile kıyaslanarak ortaya çıkarılması hedeflenmiştir. Bitki yetiştiriciliğinde giderek artan kimyasal madde kullanımına alternatif, çevre dostu ve güvenilir bitki büyüme artırıcılarına olan ihtiyaç ileriki zamanlarda kitosan ve benzer biyopolimerlerin önemini ortaya çıkaracaktır. Yükseköğrenimim boyunca bana sonsuz bir çalışma alanı özgürlüğü tanıyan danışmanım Prof. Dr. Fazıl Özen’e, ilk laboratuvar deneyimimi kendisinin desteğiyle kazandığım Prof. Dr. İbrahim Özkoç’a, kendisinden kısa zamanda çok fazla bilgi edindiğim Dr. Öğr. Üyesi Sevgi Türker-Kaya’ya, bu tezde kullandığım kromatografik tekniklerin tamamını kendisinden öğrendiğim merhum Doç. Dr. Nour Eddine El Gueddari’ye, tez izleme ve tez savunmasındaki katkılarından dolayı Prof. Dr. Yasemin Özdener Kömpe ve Doç. Dr. Özlem Aksoy’a, akademik hayatımda bana katkı sağlayan diğer tüm hocalarıma, desteklerinden dolayı aileme, yol arkadaşım Ruhiye Kıran’a, yolculuklarda büyüdüğüm şehirlerarası yollara ve geleceğe dair hala kurmakta olduğum tüm hayallere içtenlikle teşekkür ederim. Haziran - 2018 Arda ACEMİ i İÇİNDEKİLER ÖNSÖZ VE TEŞEKKÜR ............................................................................................. i İÇİNDEKİLER………………….……………………………………………………ii ŞEKİLLER DİZİNİ ...................................................................................................... v TABLOLAR DİZİNİ ................................................................................................. vii SİMGELER VE KISALTMALAR DİZİNİ ............................................................... ix ÖZET............................................................................................................................ x ABSTRACT ................................................................................................................ xi GİRİŞ ........................................................................................................................... 1 1. GENEL BİLGİLER ................................................................................................. 5 1.1. Tezde Kullanılan Bitki Materyali ...................................................................... 5 1.1.1. Ochidaceae familyası üyelerinin genel özellikleri................................... 5 1.1.2. Serapias vomeracea’nin biyolojik özellikleri.......................................... 6 1.1.3. Serapias vomeracea’nin ekonomik değeri .............................................. 8 1.2. Bitki Doku Kültürü Bilimi ................................................................................ 9 1.2.1. Bitki büyüme düzenleyicileri (BBD) ....................................................... 9 1.2.2. Diğer besiyeri katkı maddeleri .............................................................. 15 1.3. Fourier Dönüşümlü Kızılötesi (FTIR) Spektroskopisi .................................... 18 2. MALZEME VE YÖNTEM.................................................................................... 22 2.1. Bitki Materyali ................................................................................................ 22 2.1.1. Tohum canlılığının belirlenmesi ............................................................ 22 2.2. Besiyerlerinin Hazırlanması ............................................................................ 23 2.3. Bitki Büyüme Düzenleyicileri Stok Çözeltilerinin Hazırlanması ................... 25 2.4. Diğer Besiyeri Katkı Maddelerinin Hazırlanması ........................................... 25 2.5. Kitosan Oligomerleri ve Polimerinin Karakterizasyonu ................................. 26 2.6. Doku Kültürü Denemeleri ............................................................................... 27 2.6.1. Tohumların yüzeysel sterilizasyonu ...................................................... 27 2.6.2. Özgün yüzeysel sterilizasyon protokolünün geliştirilmesi .................... 28 2.6.3. Tohumların besiyerlerine ekimi............................................................. 29 2.6.4. Tohumların sayımı ve değerlendirme .................................................... 30 2.6.5. Protokormların besiyerine aktarılması................................................... 32 2.6.6. Büyüme parametrelerinin değerlendirilmesi ......................................... 32 2.6.7. Kültür şartları ......................................................................................... 33 2.7. FTIR Spektroskopisi Çalışmaları .................................................................... 33 2.7.1. FTIR için doku örneklerinin hazırlanması ............................................ 33 2.7.2. FTIR spektrumlarının eldesi .................................................................. 33 2.7.3. FTIR spektrumlarının analizi................................................................. 34 ii 2.7.4. Hiyerarşik kümeleme analizi (HCA) ..................................................... 34 2.8. Verilerin Değerlendirimesi ve İstatistiksel Analiz .......................................... 35 3. BULGULAR .......................................................................................................... 36 3.1. Tohum Canlılığının Belirlenmesine Ait Bulgular ........................................... 36 3.2. Kitosanların Karakterizasyonuna Ait Bulgular ............................................... 36 3.3. Yüzeysel Sterilizasyon Denemelerine Ait Bulgular ........................................ 38 3.4. Faz 1 Besiyerlerinde Çimlendirme Denemelerine Ait Bulgular ..................... 38 3.4.1. Katı besiyerlerine ait bulgular ............................................................... 38 3.4.2. Sıvı besiyerlerine ait bulgular ................................................................ 41 3.5. Faz 2 Besiyerlerinde Çimlendirme Denemelerine Ait Bulgular ..................... 43 3.5.1. Muz homojenatı denemelerine ait bulgular ........................................... 43 3.5.2. Sükroz denemelerine ait bulgular .......................................................... 44 3.5.3. Hindistan cevizi suyu denemelerine ait bulgular ................................... 45 3.5.4. Aktif kömür denemelerine ait bulgular ................................................. 46 3.5.5. Sükroz yerine muz homojenatı kullanımına ait bulgular ...................... 47 3.5.6. Sükroz yerine hindistan cevizi suyu kullanımına ait bulgular ............... 48 3.5.7. 6-benzilaminopürin denemelerine ait bulgular ...................................... 49 3.5.8. Kinetin denemelerine ait bulgular ......................................................... 51 3.5.9. İndol-3-asetik asit denemelerine ait bulgular ........................................ 53 3.5.10. İndol-3-bütirik asit denemelerine ait bulgular ..................................... 55 3.5.11. Jasmonik asit denemelerine ait bulgular .............................................. 57 3.5.12. Oligomerik kitosan denemelerine ait bulgular .................................... 59 3.5.13. Polimerik kitosan denemelerine ait bulgular ....................................... 61 3.6. Faz 2 Besiyerlerinde Protokorm Oluşumlarına Ait Bulgular .......................... 65 3.7. Bitki Büyüme Verilerine Ait Bulgular ............................................................ 68 3.7.1. Kontrol besiyerine ait bulgular .............................................................. 68 3.7.2. Muz homojenatı denemelerine ait bulgular ........................................... 68 3.7.3. Hindistan cevizi suyu denemelerine ait bulgular ................................... 70 3.7.4. 6-benzilaminopürin denemelerine ait bulgular ...................................... 71 3.7.5. Kinetin denemelerine ait bulgular ......................................................... 74 3.7.6. İndol-3-asetik asit denemelerine ait bulgular ........................................ 76 3.7.7. İndol-3-bütirik asit denemelerine ait bulgular ....................................... 78 3.7.8. Jasmonik asit denemelerine ait bulgular ................................................ 80 3.7.9. Oligomerik kitosan denemelerine ait bulgular ...................................... 82 3.7.10. Polimerik kitosan denemelerine ait bulgular ....................................... 84 3.7.11. Bitki büyüme verilerinin toplu karşılaştırması .................................... 86 3.8. FTIR Verilerine Ait Bulgular .......................................................................... 92 3.8.1. Yaprak spektrumlarına ait bulgular ....................................................... 94 3.8.2. Kök spektrumlarına ait bulgular......................................................... 108 3.9. Hiyerarşik Kümeleme Analizi (HCA) Verilerine Ait Bulgular .................... 115 4. TARTIŞMA ......................................................................................................... 120 4.1. Doku Kültürü Çalışmaları ............................................................................. 120 4.1.1. Yüzeysel sterilizasyon ........................................................................
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