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Börülce (Vigna unguiculata L.)’de doku kültürü ve gen aktarım çalışmaları

Diğer Başlık: In vitro shoot regeneration and gene transfer in cowpea (Vigna unguiculata L.)

Oluşturulma Tarihi: 2010

Niteleme Bilgileri

Tür: Tez

Alt Tür: Doktora

Yayınlanma Durumu: Yayınlanmış

Dosya Biçimi: PDF

Dil: Türkçe

Konu(lar): BİLİM, Tarım (Genel), Tarla mahsulleri,

Yazar(lar): AASIM, Muhammad (Yazar),

Emeği Geçen(ler): ÖZCAN, Sebahattin (Tez Danışmanı),


Yayınlayan: Ankara Üniversitesi Fen Bilimleri Enstitüsü Tarla Bitkileri Anabilim Dalı Yayın Yeri: Ankara Yayın Tarihi: 2010 Yayınlandığı Sayfalar: 133 s.


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Anahtar Kelimeler

Agrobacterium Tumefaciens, Bacillus Thuringiensis, Börülce, Cry Genleri, Kanamisin, Transgenik, Vigna Unguiculata L, Cowpea,  Cry Genes, Kanamycin, Transgenic


Özet

   Börülce (Vigna unguiculata L.) önemli bir yemeklik baklagil bitkisi olup, birçok ülkede çeşitli şekilde tüketilmektedir. Bu çalışmada  farklı konsantrasyonlarda sitokinin ve oksin içeren MS besin ortamı kullanılarak Akkız ve Karagöz börülce çeşitlerinin tam ve yarım kotiledon boğum, sürgün ucu, olgunlaşmamış kotiledon, plumula ve embriyonik eksen eksplantlarından sürgün rejenerasyonu elde edilmiştir. Eksplant başına en fazla (10.8 adet) sürgün embriyonik eksenden 1.00 mg/l BAP ve 0.1 mg/l NAA içeren MS ortamından elde edilmiştir. Genel olarak ortama NAA ilavesi sürgün rejenerasyon oranını ve eksplant başına sürgün sayısını olumsuz etkilerken, sürgün uzunluğunu olumlu şekilde etkilenmiştir. Elde edilen sürgünler 0.5 mg/l IBA içeren MS ortamında köklendirilerek serada dış şartlarda alıştırılıp tohum elde edilmiştir. Çalışmanın 2. aşamasında markör genleri (NPTII ve GUS), heRbisitlere (bar) ve böceklere dayanıklılık (cry 1C, cry ICST, cry 2A, cry 2 AST, cry 1AB ve hibrit SN19) genlerini taşıyan Agrobacterium tumefaciens’in GV2260, LBA 4404 ve AG10 hatları kullanarak transgenik bitkiler seleksiyon ortamında seçilmiştir. Farklı genleri taşıyan çok sayıda seçilmiş transgenik aday sürgünler 0.5 mg/l IBA, 50 mg/l kanamisin ve 500 mg/l Augmentin içeren MS ortamda köklendirilmiştir. Köklendirilen bitkiler serada dış şartlarda alıştırılıp tohum elde edilmiştir. Değişik primerler kullanarak PCR analizi ile transgenik bitkiler teyit edilmiştir.

Abstract

   Cowpea (Vigna unguiculata L.), an important food grain legume crop, is consumed in many countries in different ways. The study reports in vitro shoot regeneration from halved cotyledon node, shoot meristem, immature cotyledon, plumule and embryonic axis explants of cv. Akkiz and Karagöz of cowpea on MS medium containing various concentrations of cytokinins and auxins. Highest number of 10. 08 shoots per explant were recorded on MS medium containing 1.00 mg/l BAP and 0.1 mg/l NAA from embryonic axis. Inclusion of NAA in the culture medium generally had negative effect on frequency of shoot regeneration and number of shoots per explant but had positive effect on shoot length. Regenerated shoots were rooted on MS medium containing 0.50 mg/l IBA and acclimatized in the greenhouse, where they set seeds. In the second phase of study, Agrobacterium tumefaciens strains GV2260,  LBA4404 and AG10 containing markör (NPT II ve GUS),  herbicide resistant (bar) and insect resistant (cry 1C, cry 1CST, cry 2A, cry 2AST, cry 1AB and  hybrid SN19 genes were used to select putative transgenic plants in medium containing kanamycin. Putative transgenic shoots regenerated on different media from various explants were rooted on MS selection medium containing 0.50 mg/l IBA, 50 mg/l kanamisin and 500 mg/l Augmentin. Rooted plants were acclimatised in the greenhouse and seeds were obtained. Putative transgenic plants were confirmed by PCR analysis using different primers.


İçindekiler

ÖZET .......................................................................................................................

i

ABSTRACT ...............................................................................................................

ii

TEŞEKKÜR.........................................................................................................................

iii

İÇİNDEKİLER ..................................................................................................................

iv

SİMGELER DİZİNİ...........................................................................................................

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ŞEKİLLER DİZİNİ ...........................................................................................................

viii

ÇİZELGELER DİZİNİ .....................................................................................................

xi

1.GİRİŞ .....................................................................................................................

1

2. KURAMSAL TEMELLER ...........................................................................................

8

2.1 Yemeklik Tane Baklagillerde Doku Kültürü Çalışmaları .......................................

8

2.2 Yemeklik Tane Baklagillere Gen Aktarım Çalışmaları ..........................................

17

2.3 Bt Genleriyle Böceklere Dayanıklı Transgenik Bitkilerin geliştirilmesi..................

23

3. MATERYAL ve YÖNTEM ...........................................................................................

30

3.1 Materyal ....................................................................................................

30

3.1.1 Bitki materyali .............................................................................................

30

3.1.2 Rejenerasyon için kullanılan eksplantlar ................................................................

30

3.1.3 Büyüme Düzenleyicilerinin  Çözücüleri ve Saklama Koşulları ............................

32

3.1.4 Bakteri materyali ..........................................................................................

32

3.2 Yöntem .................................................................................................................

33

3.2.1 Besin ortamları ve kültür koşulları .........................................................................

33

3.2.2 Börülce  tohumlarının  yüzey sterilizasyonu ..........................................................


33

3.2.3 Börülce  tohumlarının  in vitro’da çimlendirilmesi ................................................

34

3.2.4 Eksplant izolasyonu ve kültüre alınması..................................................................

34

3.2.5 Sürgünlerin köklendirilmesi ve alıştırılması ..........................................................

 35

3.2.6 Bakteri kültürlerinin büyütülmesi ...........................................................................

   35

3.2.7 Bakterinin uzun süreli korunması ...........................................................................

36

3.2.8 Agrobacterium tumefaciens ile gen aktarım ............................................................

36

3.2.9 Histokimyasal GUS analizi .......................................................................................

37

3.2.10 DNA izolasyonu ...............................................................................................

37

3.2.11 CTAB  tampon hazırlanması .................................................................................

38

3.2.12 TE tampon hazırlanması ........................................................................................

38

3.2.13 DNA izolasyon .................................................................................

38


3.2.14 Primer dizileri ................................................................................................

39

3.2.15 PCR reaksiyon koşulları .........................................................................................

40

3.2.16 PCR Programı  .............................................................................................

40

3.2.17 Örneklerin agaroz jel elektroforezi .......................................................................

40

3.2.18 Verilerin istatistiksel değerlendirilmesi ................................................................

41

4. BULGULAR ...................................................................................................................

42

4.1 Plumula Yaprağı Ve Embriyonik Hipokotil Eksplantlarından Sürgün Rejenerasyonu.............................................................................................................


42

4.2 Yaprak ve Yaprak Sapı Eksplantlarından Sürgün Rejenerasyonu.........................

45

4.3 Birinci ve İkinci Koltuk Altı Meristemi, Modifiye Petiol ve Gövde Eksplantlarından Sürgün Rejenerasyonu ................................................................


46

4.4 Sürgün Uçlarından Sürgün Rejenerasyonu........................ .......................................

46

4.4.1 Farklı BAP ve NAA dozlarının sürgün  rejenerasyonuna etkisi ..........................

46

4.4.2 Farklı TDZ dozlarının sürgün  rejenerasyonuna etkisi..........................................

52

4.5 Olgunlaşmamış Embriyo ve Kotiledon Eksplantlarından Sürgün Rejenerasyonu

58

4.6 Kotiledon Boğum Eksplantlarından Sürgün Rejenerasyonu ..................................

65

4.7 Plumula ve Embriyonik Eksen Eksplantından Sürgün Rejenerasyonu ................

69

 4.7.1 Farklı BAP ve NAA dozlarının plumula eksplantlarından sürgün rejenerasyonuna etkisi...

69

4.7.2 Farklı BAP ve NAA dozlarının embriyonik eksen eksplantından sürgün rejenerasyonuna etkisi ..........................................................................................


71

4.7.3 Farklı TDZ dozlarının plumula ve embriyonik eksen  eksplantlarından sürgün rejenerasyonuna etkisi ..........................................................................................


73

4.8 İn Vitro Köklendirilen Sürgünlerin Diş Şartlara Adaptasyonu ...........................

74

4.9  Agrobacterium tumefaciens  Aracılığıyla Börülceye Gen Aktarımı.........................

77

4.9.1 A. tumefaciens GV2260 p35S GUS-INT hattı aracılığıyla börülceye gen aktarımı ...............................................................................................................


77

4.9.2 A. tumefaciens LBA4404 pRGG BAR hattı aracılığıyla börülceye gen aktarımı

81

4.10 A. tumefaciens Aracılığıyla  Böceklere Dayanıklılık Genlerinin Börülceye Aktarılması ...............................................................................................................


85

4.10.1 Plumula ve embiyonik eksen eksplantlarına gen aktarımı..................................

85

4.10.2 Sürgün ucu ve yarım kotiledon boğum eksplantlarına gen aktarımı..................

89


   4.11 Böceklere dayanıklı Transgenik Adayı Börülce Bitkilerinin PCR ile Teyit Edilmesi .......................................................................................................................

90

4.11.1 cry1C geni aktarılan transgenik adayı bitkilerinin PCR ile teyit edilmesi ........

90

4.11.2 Cry1CST  geni aktarılan  transgenik adayı bitkilerinin PCR ile teyit edilmesi .

93

4.11.3  Cry2Aa2 geni içeren  transgenik adayı bitkilerinin PCR ile belirlenmesi .........

96

4.11.4 Cry2AST geni aktarılan  transgenik adayı bitkilerinin PCR ile teyit edilmesi ..

99

4.11.5 Cry 1Ab  geni içeren  transgenik adayı bitkilerinin PCR ile belirlenmesi ..........

102

4.12 Transgenik Adayi Bitkilerin Adaptasyon ve Alıştırılması ....................................

103

5. TARTIŞMA VE SONUÇ..............................................................................................

106

5.1 Börülcede Sürgün Rejenerasyonu ..............................................................................

106

5.2 A. tumefaciens Aracılığıyla Börülceye Gen Aktarımı ...............................................

112

KAYNAKLAR ....................................................................................................................

117

ÖZGEÇMİŞ ...........................................................................................................

130


Açıklamalar



Haklar



Notlar



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