Journal
FRONTIERS IN PLANT SCIENCE
Volume 13, Issue -, Pages -Publisher
FRONTIERS MEDIA SA
DOI: 10.3389/fpls.2022.860971
Keywords
bialaphos; CRISPR-Cas9; helper plasmid; tissue culture; Zea mays
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Funding
- National Science Foundation Plant Genome Research Program [1917138]
- USDA NIFA Hatch project [IOW04714]
- State of Iowa funds
- Crop Bioengineering Center of Iowa State University
- Plant Transformation Facility at the Donald Danforth Plant Science Center
- Division Of Integrative Organismal Systems
- Direct For Biological Sciences [1917138] Funding Source: National Science Foundation
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This paper presents an advanced B104 transformation protocol that can generate transgenic plants in a quicker and simplified manner, suitable for genome editing applications. The success rate of this protocol is high, making it a valuable tool for researchers in the field.
For maize genome-editing and bioengineering, genetic transformation of inbred genotypes is most desired due to the uniformity of genetic background in their progenies. However, most maize inbred lines are recalcitrant to tissue culture and transformation. A public, transformable maize inbred B104 has been widely used for genome editing in recent years. This is primarily due to its high degree of genetic similarity shared with B73, an inbred of the reference genome and parent of many breeding populations. Conventional B104 maize transformation protocol requires 16-22 weeks to produce rooted transgenic plants with an average of 4% transformation frequency (number of T0 plants per 100 infected embryos). In this Method paper, we describe an advanced B104 transformation protocol that requires only 7-10 weeks to generate transgenic plants with an average of 6.4% transformation frequency. Over 66% of transgenic plants carried CRISPR/Cas9-induced indel mutations on the target gene, demonstrating that this protocol can be used for genome editing applications. Following the detailed and stepwise procedure described here, this quick and simplified method using the Agrobacterium ternary vector system consisting of a T-DNA binary vector and a compatible helper plasmid can be readily transferable to interested researchers.
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