Aynur KARADAĞ, Ahmet Murat ÖZGEN, Melahat BİRSİN AVCI

Transfer of a β-glucuronidase marker gene to Triticale (xTriticosecale Wittmack) via particle bombardment (Biolistic) method

Ankara Üniversitesi Ziraat Fakültesi Tarla Bitkileri Bölümü biyoteknoloji laboratuvarında yürütülen bu çalışmanın amacı; Tritikale (xTriticosecale Wittmack )’nin olgun embriyo ve yaprak eksplantlarına partikül bombardımanı (Biyolistik) tekniği ile gen aktarmada kullanılabilecek en uygun parametreleri belirlemektir. Tritikale (x Triticosecale Wittmack )’den elde edilen olgun embriyo ve yaprak eksplantları, b-glukuronidaz (GUS: uidA) işaret genini içeren pBI221.23 plazmidi ile kaplanmış ve hızlandırılmış altın ve tungsten partikülleri ile bombardıman edilmiştir. Hedef eksplantlara olan farklı bombardıman mesafeleri (6, 9, 12, cm) ve farklı kırılma-diski basınçları (900, 1100, 1550 psi) fiziksel parametreler olarak kullanılmıştır. b-glukuronidaz gen ifadesinin olduğu mavi hücreler histokimyasal bir analiz ile belirlenmiştir. Bombalanan explantlardaki mavi nokta sayılarındaki varyasyon gen transfer etkinliğinin belirlenmesinde ölçüt olarak kullanılmıştır. Tritikale’ye, partikül bombardımanı tekniği ile doğrudan gen aktarımında; olgun embriyoların yapraklara oranla, altının ise tungstene oranla daha iyi olduğu belirlenmiştir. Denemede en iyi sonucu veren olgun embriyolara, bombardıman mesafesinin 6 cm, kırılma-diski basıncının ise 1100 psi olduğu saptanmıştır.

Partikül bombardımanı (Biyolistik) yöntemi ile Tritikaleye (xTriticosecale Wittmack) β-glukuronidaz işaret geni aktarımı

This study was carried out to investigte the biotechnology laboratory of Department of Field Crops, Ankara University with the aim of investigating the optimum parameters of gene transfer to mature embryo and leaf segment explants of Triticale (x Triticosecale Wittmack) via particle bombardment (Biolistic) method. Mature embryo and leaf segment explants obtained from Triticale (x Triticosecale Wittmack) were obtained via bombardment with accelerated gold and tungsten particles coated with the plasmid pBI221.23 containing the b-glucuronidase (GUS: uidA) marker gene. Different bombardment distances from the stopping plate (6, 9, 12, cm) and different rupture disk pressures (900, 1100, 1550 psi ) were used as physical parameters. Blue spots expressing the b-glucuronidase gene were detected by using a histochemical assay. The variation in the number of blue spots in bombarded explants was used to determine the gene transfer efficiency. The mature embryos of triticale were found to be more amenable for the direct delivery of foreign gene by the particle bombardment technique than the leaf segments. Also the gold particles were more suitable than tungsten particles in the gene transfer efficiency. The highest gene transfer results were obtained in mature embryos when 6 cm bombardment distance and 1100 psi rupture disk pressure were used.

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