Agrobacterium tumefaciens kullanilarak, kimerik bir Bt geni olan cry1X'i eksprese eden transgenik cv. TMV- 2 yer fistigi bitkileri (Arachis hypogeae) elde edildi. Bu calismada, A. tumefaciens'de apikal meristemi hedefleyen ve doku kulturunden bagimsiz bir transformasyon metodu olan in planta metodu kullanildi. Bu protokol, cimlenmekte olan tohumlarin embriyo eksenlerinin in planta inokulasyonunu ve ex vitro olarak fide haline getirilmelerini icermektedir. PCR analizi, T1 nesli bitkilerin ongorulen transgenik dogasini gosterdi. Biyoassayler, bazi T1 bitkilerinin yer fistiginin iki buyuk zararlisi Helicoverpa armigera ve Spodoptera litura larvalarina karsi iyi performans sergiledigini ortaya cikardi. Calisma T1 bitkilerinin %22'sinin bu transgeni icerdigini gosterdi. Bir sonraki nesle devam etmelerine izin verildiginde 27 T1 bitkisinin tohumlari, test edilen bitkilerin cogunda ilgili genin etkisini artirdi. ELISA kullanilarak yuksek oranda eksprese eden bitkiler belirlendi. Quickstix'de protein bandinin gorunmesi kimerik Bt toksininin ekspresyonunu onaylamaktadir. 10 yuksek oranda eksprese eden bitkinin Southern analizi transgenin entegrasyonunu onaylamaktadir. Bu sonuclar, Bt geninin transgenik yer fistiginda islevsel oldugunu ve eksprese edildigini gostermektedir. Calisma ayrica, cry1X genini iceren yer fistigi bitkilerinin, onemli iki yaprak zararlisina karsi direncli oldugunu gosterdi.
The transgenic plants of the groundnut (Arachis hypogeae) cv. TMV-2 expressing a chimeric Bt gene, cry1X, were generated using an Agrobacterium tumefaciens - mediated transformation system. A tissue culture-independent transformation method, in planta which targets the A. tumefaciens to the apical meristem was used in this study. The protocol involves in planta inoculation of the embryo axes of the germinating seeds and allowing them to grow into seedlings ex vitro. PCR analysis indicated the putative transgenic nature of the T1 generation plants. Bioassays against two major pests of the groundnut, Helicoverpa armigera and Spodoptera litura revealed several T1 plants that perform well against both the larvae. This revealed that 22% of T1 plants harbor the transgene. The seeds of 27 T1 plants when allowed to continued into the next generation amplified the gene of interest in most of the plants tested. Enzyme Linked-Immuno Sorbent Assay (ELISA) was used to identify the high expressing plants. The appearance of the protein band in the quickstix confirmed the expression of the chimeric Bt toxin. Southern analysis of 10 high expressing plants confirmed the integration of the transgene. These results suggest that the chimeric Bt gene was functional in the transgenic groundnut and was being expressed. The study also showed that the groundnut plants harboring the cry1X gene were resistant to two major insect defoliators of the groundnut.
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