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Abstract AgrobacteriumT‐DNA integration into the plant genome is essential for the process of transgenesis and is widely used for genome engineering. The importance of the non‐homologous end‐joining (NHEJ) protein DNA polymerase Θ, encoded by thePolQgene, for T‐DNA integration is controversial, with some groups claiming it is essential whereas others claim T‐DNA integration inArabidopsisand ricepolQmutant plant tissue. Because of pleiotropic effects of PolQ loss on plant development, scientists have previously had difficulty regenerating transgenicpolQmutant plants. We describe a protocol for regenerating transgenicpolQmutant rice plants using a sequential transformation method. This protocol may be applicable to other plant species.
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Agrobacterium T‐DNA integration in somatic cells does not require the activity of DNA polymerase θ
Summary Integration ofAgrobacterium tumefacienstransferred DNA (T‐DNA) into the plant genome is the last step required for stable plant genetic transformation. The mechanism of T‐DNA integration remains controversial, although scientists have proposed the participation of various nonhomologous end‐joining (NHEJ) pathways. Recent evidence suggests that inArabidopsis, DNA polymerase θ (PolQ) may be a crucial enzyme involved in T‐DNA integration.We conducted quantitative transformation assays of wild‐type andpolQmutantArabidopsisand rice, analyzed T‐DNA/plant DNA junction sequences, and (forArabidopsis) measured the amount of integrated T‐DNA in mutant and wild‐type tissue.Unexpectedly, we were able to generate stable transformants of all tested lines, although the transformation frequency ofpolQmutants was c.20% that of wild‐type plants. T‐DNA/plant DNA junctions from these transformed rice andArabidopsis polQmutants closely resembled those from wild‐type plants, indicating that loss of PolQ activity does not alter the characteristics of T‐DNA integration events.polQmutant plants show growth and developmental defects, perhaps explaining previous unsuccessful attempts at their stable transformation.We suggest that either multiple redundant pathways function in T‐DNA integration, and/or that integration requires some yet unknown pathway.
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- Award ID(s):
- 1725122
- PAR ID:
- 10398555
- Publisher / Repository:
- Wiley-Blackwell
- Date Published:
- Journal Name:
- New Phytologist
- Volume:
- 229
- Issue:
- 5
- ISSN:
- 0028-646X
- Page Range / eLocation ID:
- p. 2859-2872
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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