Development of marker-free transgenic sorghum [Sorghum bicolor (L.) Moench] using standard binary vectors with bar as a selectable marker
- 458 Downloads
We report an Agrobacterium-mediated transformation system that can generate marker-free transgenic sorghum [Sorghum bicolor (L.) Moench] from a public line [P898012] using standard binary vectors with bar as a selectable marker. Eight co-cultivation conditions were examined for their effect on transformation. The average transformation frequencies were 0.4 and 0.7% for pZY101-TC2 and pZY101-SKRS, respectively, derived from binary vector pZY102 and containing bar and target gene(s) in separate T-DNA regions. A low selection pressure (2.5 mg l−1 DL-phosphinothrithin, PPT) was deployed during callus induction in combination with rapid selection to generate plants from 80 independent events, all but three of which were fertile and set seed. PCR and Southern analyses showed that 36 out of 80 events contained both bar and the target gene(s) (an average co-transformation frequency of 45%). Seedlings of the T1 generation transmitted T-DNAs with target gene(s) and bar gene independently, generating a fraction of progeny with only the target gene(s).
KeywordsSorghum bicolor Agrobacterium bar Binary vector Marker-free Transformation
The authors thank: Scanlon S and other colleagues from Dr. Folk’s lab for their assistance; Z. Zhao, N. Wang, S. Zheng and H. Cline (Pioneer Hi-Bred Intl. Inc.) for helpful suggestions and for P898012 and Drs. G. Liang and Z. S. Gao (Kansas State University) for helpful comments; Aventis CropScience (Research Triangle Park, NC) for providing glufosinate-ammonium and Liberty® as generous gifts; Dr. Seth D. Findley (University of Missouri, Columbia, MO) for critical review of the manuscript. All sorghum transformation experiments were conducted in the Plant Transformation Core Facility at the University of Missouri. Financial support was provided by the University of Missouri Agricultural Experiment Station, the Provost’s office and the University of Missouri Food for the twenty-first Century Eminence Program.
- Able JA (1998) Transformation of sorghum using the particle inflow gun (PIG). Int Sorghum Millets Newsl 39:98–100Google Scholar
- Cai T, Pierce DA, Tagliani LA, Zhao ZY (2002) Agrobacterium mediated transformation of sorghum. US Patent 6369298Google Scholar
- Carvalho CHS, Zehr UB, Gunaratna N, Anderson JM, Kononowicz HH, Hodges TK, Axtell JD (2004) Agrobacterium-mediated transformation of sorghum: factors that affect transformation efficiency. Genet Mol Biol 27:259–269Google Scholar
- Casas AM, Kononowicz AK, Hann TG, Zhang L, Tomes DT, Bressan RA, Hasegawa PM (1997) Transgenic plants obtained after microprojectile bombardment of immature inflorescence. In Vitro Cell Dev Biol-Plant 33:92–100Google Scholar
- Folk WR (2003) How to improve plant protein quality: err on the side of goodness. ISB News Rep (http://www.isb.vt.edu)
- Lee BK, Kennon AR, Chen X, Jung TW, Ahn BO, Lee JY, Zhang Z (2007) Recovery of transgenic events from two highly recalcitrant maize (Zea mays L.) genotypes using Agrobacterium-mediated standard-binary-vector transformation. Maydica 52:457–469Google Scholar
- Zhao ZY, Glassman K, Sewalt V, Wang N, Miller M, Chang S, Thompson T, Catron S, Wu E, Bidney D, Kedebe Y, Jung R (2003) Nutritionally improved transgenic sorghum. In: Vasil IK (ed) Plant biotechnology 2002 and beyond. Proceedings of the 10th IAPTC & B Congress. Kluwer Academic Publishers, pp 413–416Google Scholar
- Zhu H, Muthukrishana S, Krishnaveni S, Jeoung JM, Liang GH (1998) Biolistic transformation of sorghum using a rice chitinase gene. J Genet Breed 52:243–252Google Scholar