Aksenik jüvenil sakız ağacı (Pistacia lentiscus L.) eksplantlarından kallus kültürlerinin başlatılması ve optimizasyonu
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Tarih
2018-8-04
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Batman Üniversitesi Fen Bilimleri Enstitüsü
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info:eu-repo/semantics/openAccess
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Attribution-ShareAlike 3.0 United States
Özet
Pistacia lentiscus L.'ta kallus kültürlerinin başlatılması ve optimizasyonu için etkili bir protokol geliştirmek amacıyla yüzey sterilizasyonu yapılan tohumlar, 1 mg/l IBA destekli MS besi ortamında çimlendirilmiş, sonrasında elde edilen aksenik apikal sürgünler 1 mg/l BAP + 0.5 mg/l GA3 içeren MS besi ortamında çoğaltılmıştır. İn vitro çoğaltılmış kültürlerden gelen aksenik yapraklar ve tohumların in vitro çimlendirilmesiyle elde edilen kökler, kallus indüksiyonu için eksplant kaynağı olarak kullanılmıştır. Kallus oluşumuna, her biri 0.25, 0.5, 1.0 ve 2mg/l olacak şekilde farklı oksin (IAA, IBA, NAA ve 2,4-D) ve sitokinin (BAP, Kin, TDZ ve 2İP) kombinasyonlarının etkisi ile yine her biri 1/1 kuvvette hazırlanan farklı besiyeri tiplerinin (MS, WPM, SH, B5) etkileri test edilmiştir. Bunların yanı sıra kallus kültürlerinin gelişimlerinin optimizasyonları üzerine farklı şeker tipi (glukoz ve sukroz) ve konsantrasyonları (15, 30, 50 mg/l), farklı pH (4.5, 5, 5.8, 6.5, 7), farklı ışık yoğunluğu (20,40,80 μmol/s), farklı sıcaklık (10, 20, 25, 30, 35°C) uygulamaları ile farklı besiyeri tipleri (MS, WPM, SH, B5) ve konsantrasyonlarının (0.25, 0.5, 1 ve 2 mg/l) etkileri test edilmiştir. Kallus kültürlerinin başlatılması çalışmalarında besiyeri tipi olarak hem kök (%80) hem yaprak (%84) eksplantları için, 1/1 MS besi ortamının, farklı BBD tipleri bakımından hem kök (%80) hem yaprak (%80) eksplantları için 1mg/l Kin ve 1 mg/l 2,4-D içeren MS besi ortamının en iyi sonuçlar verdiği tespit edilmiştir. Kallus kültürlerinin gelişimleri üzerine optimizasyon çalışmalarında ise, farklı besiyeri tiplerinin araştırıldığı denemede kök (%80) ve yaprak (%100) eksplantlarında 1/1 MS besi ortamının, farklı şeker tip ve konsantrasyonlarının araştırıldığı denemede, kök ve yaprak eksplantlarında 15 mg/l sukroz ortamının, farklı pH uygulamaları arasında kök (%96) ve yaprak (%100) eksplantlarında pH 5.8 ortamının, farklı ışık şiddeti uygulamaları arasında kök eksplantları için 20 μmol (%100), yaprak eksplantları için 80 μmol (%100) ışık uygulamalarının, farklı sıcaklık uygulamaları arasından ise hem kök (%76) hem yaprak (%100) eksplantlarında 25°C sıcaklık uygulamasının en iyi sonuçları verdiği tespit edildi. Özetle bu çalışma, değerli sekonder metabolitlerin P. lentiscus L. kallus kültürleri yoluyla daha fazla miktarlarda üretilmesine ışık tutacak, aynı zamanda kallus kültürlerinin başlatılması ve optimizasyonu için rutin olarak kullanılabilecek bir protokol geliştirilmesine olanak sağlamıştır.
To develop an effective protocol for the initiation and optimization of callus cultures in Pistacia lentiscus L., the surface-sterilized seeds were germinated in an MS medium supplemented with 1 mg/l IBA and then the axenic apical shoots obtained were propagated in MS medium containing 1 mg/l BAP + 0.5 mg/l GA3. The roots obtained by in vitro germination of the seeds and the axenic leaves from in vitro propagated cultures were used as explant source for callus induction. The effect of the combination of different auxin (IAA, IBA, NAA and 2,4-D) and cytokinin (BAP, Kin, TDZ and 2IP) each one at 0.25, 0.5, 1.0 and 2 mg /l concentrations, respectively were tested. for callus formation. The effects of different nutrient media types (MS, WPM, SH, B5) prepared in 1/1 strength were also tested. In addition, different types of sugar (glucose and sucrose) and their different concentrations (15, 30, 50 mg / l), different pHs (4.5, 5, 5.8, 6.5, 7), different light intensities (20,40,80 μmol/s), and different temperature (10,20,25,30,35 ° C) effects were tested for callus development. Besides these experiments, different types of nutrient media (MS, WPM, SH, B5) prepared in (0.25, 0.5, 1 and 2 mg/l) strength were also tested. In the study of callus cultures initiation, it was found that the MS medium for both root (80%) and leaf (84%) explants gave the best results as nutrient medium type. In terms of different types of PGR, again an MS medium containing 1mg/l Kin ve 1 mg/l 2,4-D for both root (80%) and leaf (80%) explants gave the best results. In the optimization studies on callus cultures development, the highest root and leaf explants in application results of different sugar types and concentrations from 1/1 MS medium in root (80%) and leaf (100%) explants in which different food types were searched. The optimal value for root (96%) and leaf (100%) explants was different from pH 5.8 medium between different pH applications and 20 μmol (100%) for root explants among different light intensity applications from MS medium containing 15 mg/l sucrose 80% (100%) light applications for explants and the best result for different temperature applications was obtained from both roots (76%) and leaves (100%) at 25°C temperature. In the optimization studies on callus cultures development, in experiments investigating the different media types, the root (80%) and leaf (100%) of explants that 1/1 MS medium, different sugar types and their different concentrations the root and leaf of explants at 15 mg / l sucrose, different pH applications . root (96%) and leaf (100%) explants at pH 5.8, different light intensity applications the root (100%) at 20 μmol and leaf (100%) at 80 μmol, different temperature applications, both root (76%) and leaf (100%) explants at 25 ° C temperature gave the best results was determined. In summary, this study allows to develop a protocol that can routinely be used for the initiation and optimization of callus cultures, which at the same time will shed light on the production of valuable secondary metabolites by P. lentiscus L. callus cultures.
To develop an effective protocol for the initiation and optimization of callus cultures in Pistacia lentiscus L., the surface-sterilized seeds were germinated in an MS medium supplemented with 1 mg/l IBA and then the axenic apical shoots obtained were propagated in MS medium containing 1 mg/l BAP + 0.5 mg/l GA3. The roots obtained by in vitro germination of the seeds and the axenic leaves from in vitro propagated cultures were used as explant source for callus induction. The effect of the combination of different auxin (IAA, IBA, NAA and 2,4-D) and cytokinin (BAP, Kin, TDZ and 2IP) each one at 0.25, 0.5, 1.0 and 2 mg /l concentrations, respectively were tested. for callus formation. The effects of different nutrient media types (MS, WPM, SH, B5) prepared in 1/1 strength were also tested. In addition, different types of sugar (glucose and sucrose) and their different concentrations (15, 30, 50 mg / l), different pHs (4.5, 5, 5.8, 6.5, 7), different light intensities (20,40,80 μmol/s), and different temperature (10,20,25,30,35 ° C) effects were tested for callus development. Besides these experiments, different types of nutrient media (MS, WPM, SH, B5) prepared in (0.25, 0.5, 1 and 2 mg/l) strength were also tested. In the study of callus cultures initiation, it was found that the MS medium for both root (80%) and leaf (84%) explants gave the best results as nutrient medium type. In terms of different types of PGR, again an MS medium containing 1mg/l Kin ve 1 mg/l 2,4-D for both root (80%) and leaf (80%) explants gave the best results. In the optimization studies on callus cultures development, the highest root and leaf explants in application results of different sugar types and concentrations from 1/1 MS medium in root (80%) and leaf (100%) explants in which different food types were searched. The optimal value for root (96%) and leaf (100%) explants was different from pH 5.8 medium between different pH applications and 20 μmol (100%) for root explants among different light intensity applications from MS medium containing 15 mg/l sucrose 80% (100%) light applications for explants and the best result for different temperature applications was obtained from both roots (76%) and leaves (100%) at 25°C temperature. In the optimization studies on callus cultures development, in experiments investigating the different media types, the root (80%) and leaf (100%) of explants that 1/1 MS medium, different sugar types and their different concentrations the root and leaf of explants at 15 mg / l sucrose, different pH applications . root (96%) and leaf (100%) explants at pH 5.8, different light intensity applications the root (100%) at 20 μmol and leaf (100%) at 80 μmol, different temperature applications, both root (76%) and leaf (100%) explants at 25 ° C temperature gave the best results was determined. In summary, this study allows to develop a protocol that can routinely be used for the initiation and optimization of callus cultures, which at the same time will shed light on the production of valuable secondary metabolites by P. lentiscus L. callus cultures.
Açıklama
Bu tez çalışması TÜBİTAK tarafından 114Z842 nolu proje ile desteklenmiştir.
Anahtar Kelimeler
Pistacia Lentiscus L., Sakız, Kallus Kültürleri, Optimizasyon, Mastic, Callus Cultures, Optimization
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Künye
Demir, E. (2018). Aksenik jüvenil sakız ağacı (Pistacia lentiscus L.) eksplantlarından kallus kültürlerinin başlatılması ve optimizasyonu. (Yayınlanmamış Yüksek Lisans Tezi). Batman Üniversitesi Fen Bilimleri Enstitüsü, Batman.
