A Rapid Agrobacterium-Mediated Transformation Method Using Maize B104 Immature Embryos
- Minjeong Kang1,2,3,
- Mercy K. Azanu1,2,3,
- Keunsub Lee1,3 and
- Kan Wang1,3,4
- 1Department of Agronomy, Iowa State University, Ames, Iowa 50011, USA
- 2Interdepartmental Plant Biology, Iowa State University, Ames, Iowa 50011, USA
- 3Crop Bioengineering Center, Iowa State University, Ames, Iowa 50011, USA
- ↵4Correspondence: kanwang{at}iastate.edu
Abstract
Maize genetic transformation is a critical tool for functional genomics and crop improvement. Many laboratories, however, continue to face multiple challenges in attempting to achieve routine genetic transformation of maize inbred genotypes. Here, we describe a rapid and robust maize B104 transformation method using immature embryos as explants. This method uses an Agrobacterium ternary vector system, which includes a conventional T-DNA binary vector (pCBL101-RUBY) and a compatible ternary helper plasmid (pKL2299) that carries extra copies of essential virulence genes. The T-DNA binary vector carries the neomycin phosphotransferase II (NptII) gene for selection and a betalain biosynthesis marker, RUBY, for visual screening. We provide step-by-step instructions for immature embryo explant preparation, Agrobacterium infection, tissue culture procedures, and greenhouse care for acclimatization of regenerated plantlets.
Footnotes
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From the Maize collection, edited by Candice N. Hirsch and Marna D. Yandeau-Nelson. The entire Maize collection is available online at Cold Spring Harbor Protocols and can be accessed at https://cshprotocols.cshlp.org/.
