Elimination of macro elements from inoculation and co-cultivation media enhances the efficiency of Agrobacterium-mediated transformation in Petunia

Aso Nobakht Vakili; Hedayat Bagheri; Pejman Azadi

doi:10.1007/s12298-018-0553-0

Elimination of macro elements from inoculation and co-cultivation media enhances the efficiency of Agrobacterium-mediated transformation in Petunia

Physiol Mol Biol Plants. 2018 Jul;24(4):703-710. doi: 10.1007/s12298-018-0553-0. Epub 2018 May 31.

Authors

Aso Nobakht Vakili¹, Hedayat Bagheri², Pejman Azadi³

Affiliations

¹ 1Department of Biotechnology, Faculty of Agriculture, Bu Ali-Sina University, Hamedan, 6517838695 Iran.
² 2Department of Plant Biotechnology, Faculty of Agriculture, Bu Ali-Sina University, Hamedan, 6517838695 Iran.
³ 3Department of Genetic Engineering, Agricultural Biotechnology Research Institute of Iran (ABRII), Agricultural Research, Education and Extension Organization (AREEO), Karaj, Iran.

Abstract

In order to evaluate the effect of inoculation and co-cultivation media elements on transformation frequency in Petunia hybrida, modified MS media with different elements were tested on Alvan and Large Flower Alvan (LF Alvan), two local cultivars. Leaf explants of both cultivars were inoculated with Agrobacterium tumefaciens strain LBA4404 (pBI121) containing neomycin phosphotransferase (nptII) and an intron-containing β-glucuronidase (gus) genes. When medium lacking KH₂PO₄, NH₄NO₃, KNO₃, and CaCl₂ was used as inoculation and co-cultivation medium, a higher frequency of transformation for Alvan (22%) and LF Alvan (16%) was obtained. Kanamycin resistant plantlets were stained blue by GUS assay. Furthermore, polymerase chain reaction (PCR) analysis revealed the presence of both gus and nptII genes in all putative transformants. Finally, southern blot hybridization confirmed insertion of 1-4 copies of gus gene in transgenic plants.

Keywords: Agrobacterium tumefaciens; Co-cultivation; Macro elements; Petunia hybrida; Transformation.