Ceviz (Juglans regia L.) bitkisinin in vitro mikroçoğaltımı üzerine kültür tiplerinin etkileri
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Dosyalar
Tarih
2021
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Yayıncı
Ege Üniversitesi, Fen Bilimleri Enstitüsü
Erişim Hakkı
info:eu-repo/semantics/openAccess
Özet
Bu çalışmanın amacı, ceviz (Juglans regia L.) bitkisinde 'Paradox' anaç çeşidine ait nod eksplant kökenli in vitro çoğaltım için farklı kültür tiplerinin (yarı katı, sıvı ve çift-fazlı) etkilerinin belirlenmesi ve mikroçoğaltım için gerekli tüm basamakların gerçekleştirilerek etkili bir mikroçoğaltım protokolünün oluşturulmasıdır. Çalışmanın ilk aşamasında uygun sterilizasyon yöntemini belirlemek amacıyla, Ulu Ceviz firmasından temin edilen nod eksplantları, farklı sterilizasyon yöntemlerine maruz bırakıldıktan sonra DKW (Driver and Kuniyuki, 1984) ve Rugini (Rugini, 1984) temelli yarı katı besin ortamlarında kültüre alınmışlardır. En etkili sterilizasyon yöntemi olan ST2 yöntemiyle, 15 dk deterjanlı su ve arkasından 15 dk akar su altında bekletilen eksplantlar, kabin içerisine alınarak 5 dk %70 alkol uygulandıktan sonra 5 dk birkaç damla Tween 20 ilave edilmiş %0,2 HgCl2 içerisinde çalkalanmıştır. Daha sonra üç defa steril sudan geçirilerek otoklavlanmış besin ortamlarına transfer edilmişlerdir. Bu sterilizasyon işlemi, eksplantlarda en az kararmanın (%4) gözlemlendiği yöntem olarak belirlenmiştir. Çalışmanın ikinci aşamasında yüzey sterilizasyonu sağlanan nod eksplantlarından sürgün indüksiyonu ve çoğaltımını gerçekleştirmek amacıyla, farklı bitki büyüme düzenleyicilerinin (IBA ve BAP) ve farklı kimyasal bileşiklerin (adenin hemisülfat ve kazein hidrolizat) kullanıldığı farklı besin ortamlarında (DKW, Rugini, Modifiye Rugini), farklı kültür tiplerinde (yarı katı, sıvı, çift-fazlı) kültüre alınmışlardır. Çalışmada elde edilen sonuçlara göre, birim eksplant başına düşen en yüksek sürgün sayısı (1,05 sürgün/eksplant) yarı katı SP4 (4 mg/L BAP+ 0.001 mg/L IBA + 200 mg/L kazein hidrolizat+ 50 mg/L adenin hemisülfat içeren DKW) besin ortamında elde edilmiştir. Ayrıca birim eksplant başına düşen ortalama nod sayısı (7,95 adet/eksplant) ve ortalama sürgün uzunluğu (1,6 cm) bakımından en başarılı ortam yarı katı SP2 (2 mg/L BAP + 0.001 mg/L IBA+ 200 mg/L kazein hidrolizat+ 50 mg/L adenin hemisülfat içeren DKW) olmuştur. Çalışmanın üçüncü aşamasında çoğaltımı gerçekleştirilen sürgün eksplantlarının köklendirilmesi amacıyla iki aşamalı bir in vitro köklendirme işlemi gerçekleştirilmiştir. Köklendirme çalışmasının ilk aşaması olan kök indüksiyonu için; sürgün eksplantları, farklı sükroz konsantrasyonlarının (30 ve 40 g/L) ve FeEDDHA (119 mg/L)'nın denenmesi amacıyla 4 mg/L IBA içeren farklı yarı katı besin ortamlarında (Modifiye MS, ½ Modifiye MS, Modifiye DKW, ½ Modifiye DKW) 7 gün boyunca karanlık koşullar altında kültüre alınmışlardır. Çalışmadan elde edilen sonuçlara göre en iyi kök indüksiyonu; 4 mg/L IBA içeren Modifiye yarı güç MS ortamında sağlanmıştır. Kök indüksiyonu gerçekleştirilen sürgün eksplantları daha sonra kök oluşumunun ve uzamasının sağlanması amacıyla; jelleştirici ajan olarak 2.4 mg/L Gelrite içeren, hormonsuz ve vermikulit ile karıştırılan (1:1 h/h) farklı besin ortamlarında (Modifiye DKW, ½ Modifiye DKW) 21 gün boyunca 16 saatlik fotoperiyotta (3500 lüks) kültüre alınmıştır. Kök oluşumu için en etkili ortam DKW temelli ¼ DFe olarak belirlenmiş ve %45,45 köklendirme oranı elde edilmiştir. Araştırmamızda, sıvı ve çift-fazlı besin ortamlarında kültüre alınan eksplantlar kararma ve kontaminasyon nedeniyle kaybedilmiştir. Sürgün rejenerasyonu ve köklenme bakımından yarı katı besin ortamları daha başarılı bulunmuştur.
The aim of this study is to determine the effects of different culture types (semi solid, liquid, double-phase) and to create an effective micropropagation protocol performing all the steps required for in vitro reproduction of nodal explant origin in "Paradox" rootstock cultivar in walnut (Juglans regia L.) plant. In the first phase of the study, in order to determine the appropriate sterilization method, nodal explants obtained from Ulu Ceviz company were cultured in DKW (Driver and Kuniyuki, 1984) and Rugini (Rugini, 1984) based nutrient semi solid media after exposure to different sterilization methods. With the ST2 method, which is the most effective sterilization method, explants kept in detergent water for 15 minutes and then under running water for 15 minutes and then taken into the cabin and 70% alcohol was applied for 5 minutes, and after, shaked for 5 minutes with 0.2% HgCl2 added a few drops of Tween 20. Later, it was passed through sterile water three times and transferred to autoclaved nutrient media. This sterilization method was determined as the method in which the least blackening (4%) observed in the explants. In the second stage of the study, different plant growth regulators (IBA and BAP) and different chemical compounds (adenine hemisulphate and casein hydrolyzate) were used in different nutrient media (DKW, Rugini, Modified Rugini) and different culture types (semi solid, liquid, double-phase) in order to perform shoot induction and production from node explants with surface sterilization. According to the results obtained in the study, the highest number of shoot per unit (1.05 shoots / explant) was obtained in semi solid SP4 (4 mg/L BAP + 0.001 mg/L IBA + 200 mg/L casein hydrolyzate + 50 mg/L adenine hemisulphate containing DKW) in nutrient medium. In addition, the most successful medium in terms of the the avarege number of nodes per explant (7.95 nodes / explant) and avarage shoot length (1.6 cm) was semi solid SP2 (DKW with 2 mg/L BAP + 0.001 mg/L IBA + 200 mg/L casein hydrolyzate + 50 mg / L adenine hemisulphate). In the third stage of the study, a two-step in vitro rooting process was carried out in order to root of shoot explants produced. For root induction, which is the first stage of rooting study; shoot explants were cultured in different semi solid nutrient media containing 4 mg/L IBA (Modified MS, Modified MS, Modified DKW, ½ Modified DKW) to test the effect of different sucrose concentrations (30 and 40 g / L) and FeEDDHA (119 mg / L) under dark conditions during the 7 day. According to the results from the study, the best root induction was provided from half strength Modified MS medium with 4 mg/L IBA. Shoot explants, which root induction was performed, later in order to ensure root formation and elongation were cultured in different nutrient media (Modified DKW, ½ Modified DKW) containing 2.4 mg/L Gelrite as a gelling agent, without hormones and mixed with vermiculite (1:1 v/v) for 21 days in a 16-hour photoperiod (3500 lux). The most effective medium for root formation rate (45.45%) was determined as DKW based ¼ DFe. In our study, explants that were cultured in liquid and double-phase nutrient media were lost due to browning and contamination. Semi-solid nutrient media were found more successful in terms of shoot regeneration and rooting.
The aim of this study is to determine the effects of different culture types (semi solid, liquid, double-phase) and to create an effective micropropagation protocol performing all the steps required for in vitro reproduction of nodal explant origin in "Paradox" rootstock cultivar in walnut (Juglans regia L.) plant. In the first phase of the study, in order to determine the appropriate sterilization method, nodal explants obtained from Ulu Ceviz company were cultured in DKW (Driver and Kuniyuki, 1984) and Rugini (Rugini, 1984) based nutrient semi solid media after exposure to different sterilization methods. With the ST2 method, which is the most effective sterilization method, explants kept in detergent water for 15 minutes and then under running water for 15 minutes and then taken into the cabin and 70% alcohol was applied for 5 minutes, and after, shaked for 5 minutes with 0.2% HgCl2 added a few drops of Tween 20. Later, it was passed through sterile water three times and transferred to autoclaved nutrient media. This sterilization method was determined as the method in which the least blackening (4%) observed in the explants. In the second stage of the study, different plant growth regulators (IBA and BAP) and different chemical compounds (adenine hemisulphate and casein hydrolyzate) were used in different nutrient media (DKW, Rugini, Modified Rugini) and different culture types (semi solid, liquid, double-phase) in order to perform shoot induction and production from node explants with surface sterilization. According to the results obtained in the study, the highest number of shoot per unit (1.05 shoots / explant) was obtained in semi solid SP4 (4 mg/L BAP + 0.001 mg/L IBA + 200 mg/L casein hydrolyzate + 50 mg/L adenine hemisulphate containing DKW) in nutrient medium. In addition, the most successful medium in terms of the the avarege number of nodes per explant (7.95 nodes / explant) and avarage shoot length (1.6 cm) was semi solid SP2 (DKW with 2 mg/L BAP + 0.001 mg/L IBA + 200 mg/L casein hydrolyzate + 50 mg / L adenine hemisulphate). In the third stage of the study, a two-step in vitro rooting process was carried out in order to root of shoot explants produced. For root induction, which is the first stage of rooting study; shoot explants were cultured in different semi solid nutrient media containing 4 mg/L IBA (Modified MS, Modified MS, Modified DKW, ½ Modified DKW) to test the effect of different sucrose concentrations (30 and 40 g / L) and FeEDDHA (119 mg / L) under dark conditions during the 7 day. According to the results from the study, the best root induction was provided from half strength Modified MS medium with 4 mg/L IBA. Shoot explants, which root induction was performed, later in order to ensure root formation and elongation were cultured in different nutrient media (Modified DKW, ½ Modified DKW) containing 2.4 mg/L Gelrite as a gelling agent, without hormones and mixed with vermiculite (1:1 v/v) for 21 days in a 16-hour photoperiod (3500 lux). The most effective medium for root formation rate (45.45%) was determined as DKW based ¼ DFe. In our study, explants that were cultured in liquid and double-phase nutrient media were lost due to browning and contamination. Semi-solid nutrient media were found more successful in terms of shoot regeneration and rooting.
Açıklama
Anahtar Kelimeler
Juglans Regia L., Ceviz, Paradox, İn Vitro, Kültür Tipleri, Yarı Katı, Sıvı, Çift-Fazlı, Mikroçoğaltım, Nod Eksplantı, Sürgün, Juglans Regia L., Walnut, Paradox, In Vitro, Culture Types, Semi Solid, Liquid, Double-Phase, Micropropagation, Node Explants, Shoot
