Elimination of macro elements from inoculation and co-cultivation media enhances the efficiency of Agrobacterium-mediated transformation in Petunia
- 55 Downloads
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.
KeywordsMacro elements Co-cultivation Agrobacterium tumefaciens Transformation Petunia hybrida
- LF Alvan
Large Flower Alvan
Cauliflower mosaic virus
This work was supported by Bu-ali Sina University, Hamedan, Iran, and Novin Giti Gene Biotech. Co. Biotechnology Incubator Center of National Institute of Genetic Engineering and Biotechnology (NIGEB), Tehran, Iran.
- Azadi P, Chin DP, Kuroda K, Khan RS, Mii M (2010a) a) Macro elements in inoculation and co-cultivation medium strongly affect the efficiency of Agrobacterium-mediated transformation in Lilium. Plant Cell Tissue Organ Culture 101:201–209. https://doi.org/10.1007/s11240-010-9677-9 CrossRefGoogle Scholar
- Chen P, Wang ChK, Soong ShCh (2003) Complete sequence of the binary vector pBI121 and its application in cloning T-DNA insersion from transgenic plants. Molecular Breeding. Inst Bioagric Sci 11:287–293Google Scholar
- Doyle JJ, Doyle JL (1987) A rapid DNA isolation from small amount of fress tissue. Phytochem Bull 19:11–15Google Scholar
- Drummond RS, Martinezsanchez NM, Janssen BJ, Templeton KR, Simons JL, Quinn BD, Karunairetnam S, Snowden KC (2009) Petunia hybrida carotenoid clieavage dioxygenase7 is involved in the production of negative and positive branching signals in Petunia. Plant Physiol 151:1867–1877CrossRefPubMedPubMedCentralGoogle Scholar
- Li F, Li Ch, Li M, Yu M, Fand Ch, Wang Sh (2013) Microspores and Agrobacterium-mediated transient expression of β-glucuronidase (GUS) reporter gene. Int J Agric Biol 15:1098–1104Google Scholar
- Phelep M, Petit A, Martin L, Duhoux E, Tempe J (1991) Transformation and regeneration of a nitrogen-fixing tree, Allocasuarina verticillata Lam. Biotechnology 9:461–466Google Scholar
- Valizadeh Kaji B, Ershadi A, Tohidfar M (2013) Agrobacterium-Mediated Transformation of Pomegranate (Punica granatum L.) ‘Yousef Khani’ Using the gus Reporter Gene. Int J Hortic Sci Technol 1:31–41Google Scholar
- Wylie SJ, Tjokrokusumo D, MCCOMB JA (2003) Transformation of Petunia hybrida by the Agrobacterium suspension drop method. Mol Methods Plant, Analysis Vol, p 23Google Scholar