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Rivista di Biologia Molecolare e Biotecnologie
Indexed/Abstracted in: EMBASE, Science Citation Index Expanded (SciSearch), Scopus
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Minerva Biotecnologica 2012 Giugno;24(2):53-7
Development of a tissue culture-independent Agrobacterium-mediated transformation method for wheat
Manan S., Hussain J., Tul Muntha S., Maroof Shah M.
Department of Environmental Sciences, COMSATS Institute of Information Technology, Abbottabad, Pakistan
Aim. Presently genetic modification of wheat is being carried out mainly through gene gun and Agrobacterium-mediated transformation methods; both involving tissue culture. This has been a bottleneck since wheat is a recalcitrant crop and hence tissue culture response of most of the varieties has been poor. The aim of current study was to develop an improved Agrobacterium-mediated transformation method that does not involve tissue culturing.
Methods. The seeds of wheat varieties were soaked in water and incubated overnight at room temperature. Imbibed seeds were wounded with the help of a fine needle and inoculated with Agrobacterium tumefaciens containing rolB gene in binary vector pLBR. After co-cultivation, the seeds were processed for regeneration. The seedlings were subjected to kanamycin selection and potential transformants were transferred to soil for further growth in natural environment.
Results. We have successfully obtained transformants on the basis of antibiotic resistance. Transformation efficiencies of the selected varieties used, i.e., khyber-87 and Inqilab-91 were 4% and 4.67%, respectively. Moreover, we also optimized the protocol for selection of transformants with kanamycin.
Conclusion. The protocol developed for wheat transformation is simple, inexpensive, and most importantly by-passes tissue culturing. The application of this protocol will be extremely beneficial in creating transgenic wheat with desirable traits.