Coexpression of octopine and succinamopine Agrobacterium virulence genes to generate high quality transgenic events in maize by reducing vector backbone integration
- 59 Downloads
Agrobacterium-mediated transformation is a complex process that is widely utilized for generating transgenic plants. However, one of the major concerns of this process is the frequent presence of undesirable T-DNA vector backbone sequences in the transgenic plants. To mitigate this deficiency, a ternary strain of A. tumefaciens was modified to increase the precision of T-DNA border nicking such that the backbone transfer is minimized. This particular strain supplemented the native succinamopine VirD1/VirD2 of EHA105 with VirD1/VirD2 derived from an octopine source (pTi15955), the same source as the binary T-DNA borders tested here, residing on a ternary helper plasmid containing an extra copy of the succinamopine VirB/C/G operons and VirD1. Transformation of maize immature embryos was carried out with two different test constructs, pDAB101556 and pDAB111437, bearing the reporter YFP gene and insecticidal toxin Cry1Fa gene, respectively, contained in the VirD-supplemented and regular control ternary strains. Molecular analyses of ~ 700 transgenic events revealed a significant 2.6-fold decrease in events containing vector backbone sequences, from 35.7% with the control to 13.9% with the VirD-supplemented strain for pDAB101556 and from 24.9% with the control to 9.3% with the VirD-supplemented strain for pDAB111437, without compromising transformation efficiency. In addition, while the number of single copy events recovered was similar, there was a 24–26% increase in backbone-free events with the VirD-supplemented strain compared to the control strain. Thus, supplementing existing VirD1/VirD2 genes in Agrobacterium, to recognize diverse T-DNA borders, proved to be a useful tool to increase the number of high quality events in maize.
KeywordsAgrobacterium-mediated transformation T-DNA borders Octopine virulence genes VirD1 VirD2 EHA105
We would like to acknowledge Justin Komnick, Tatyana Minnicks and Heather Robinson for maize transformation. Authors are grateful to Cory Christensen and Otto Folkerts for their critical review of the manuscript, and to Rodrigo Sarria for his support.
Compliance with ethical standards
Conflict of interest
NS, SF, HW, and MG are the inventors on a US patent filing for the above listed work.
- Ainley M, Armstrong K, Belmar S, Folkerts O, Hopkins N, Menke MA, Pareddy D, Petolino JF, Smith K, Woosley A (2004) Regulatory sequences for transgenic plants. US Patent 6699984Google Scholar
- Cowen NM, Armstrong K, Smith KA (2007) Use of regulatory sequences in transgenic plants. US Patent 7179902Google Scholar
- De Framond AJ, Barton KA, Chilton MD (1983) Mini-Ti: a new vector strategy for plant genetic engineering. Biotechnology 1:262–269Google Scholar
- Kuraya Y, Ohta S, Fukuda M, Hiei Y, Murai N, Hamada K, Ueki J, Imaseki H, Komari T (2004) Suppression of transfer of non-T-DNA ‘vector backbone’ sequences by multiple left border repeats in vectors for transformation of higher plants mediated by Agrobacterium tumefaciens. Mol Breed 14:309–320CrossRefGoogle Scholar
- Merlo DJ, Russell SM, Retallack DM, Woosley AT, Meade T, Narva KE (2017) Method of increasing plant transformation frequency using modified strains of Agrobacteria. US Patent 9617551Google Scholar
- Olszewski N, Tzafrir I, Somers DA, Lockhart B, Torbert KA (2002) Sugarcane bacilliform virus promoter. US Patent 6489462Google Scholar
- Shagin DA, Barsova EV, Yanushevich YG, Fradkov AF, Lukyanov KA, Labas YA, Semenova TN, Ugalde JA, Meyers A, Nunez JM, Widder EA, Lukyanov SA, Matz MV (2004) GFP-like proteins as ubiquitous metazoan superfamily: evolution of functional features and structural complexity. Mol Biol Evol 21:841–850CrossRefGoogle Scholar
- Stuive MH, Ponstein AS, Ohl SA, Goddijn OJM, Simons LH, Dekker BMM, Hoekstra S, Tigelaar H, Elzinga N (2006) Plasmids for plant transformation and method for using the same. US Patent 7029908Google Scholar
- Woosley A, Worden S (2015) Increased protein expression in plants. US Patent application US 2015/0203857Google Scholar
- Ye X, Williams EJ, Shen J, Johnson S, Lowe B, Radke S, Strickland S, Esser JA, Petersen MW, Gilbertson LA (2011) Enhanced production of single copy backbone-free transgenic plants in multiple crop species using binary vectors with a pRi replication origin in Agrobacterium tumefaciens. Transgenic Res 20:773–786CrossRefGoogle Scholar