Abstract
Insertion mutant collections are powerful tools for genetic studies in plants. Although large-scale insertional mutagenesis using T-DNA is not feasible in legumes, the Tnt1 tobacco retrotransposon can be used as a very efficient mutagen in the Medicago truncatula R108 genotype. In this article, we show that Tnt1 can also be exploited to create insertional mutants via transformation and/or regeneration in the reference cultivar Jemalong. Tnt1 insertional mutagenesis in Jemalong following Agrobacterium tumefaciens-mediated transformation was found to be very efficient, with an average of greater than 15 insertions/line. In contrast, regeneration using low-copy transgenic starter lines resulted in a highly variable rate of new Tnt1 insertions. With the goal of increasing the number of additional Tnt1 insertions during regeneration of starter lines, we have compared the insertion frequencies for a number of different regeneration protocols. In addition, we have been able to show that sucrose-mediated osmotic shock preceding regeneration significantly increases the transposition frequency. Under optimal conditions, 95% of the regenerated Jemalong plants possess new insertions.
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Abbreviations
- 2,4D:
-
2,4-Dichlorophenoxyacetic acid
- BAP:
-
6-Benzylaminopurine
- NAA:
-
α-Naphthaleneacetic acid
- IBA:
-
Indol-3-butyric acid
- TDZ:
-
N-Phenyl-N 1-1,2,3,-thiadiazol-5-ylurea, thidiazuron
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Acknowledgments
This work was financed in the framework of the European FP6 (FOOD-CT-2004-506223 GRAIN LEGUMES IP, GLIP program). Members of the GLIP WP5.2 (Tnt1 mutagenesis) are acknowledged for their helpful discussions and suggestions during this work.
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Communicated by R. Schmidt.
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Supplemental Figure S1
Scheme of the lineage of starter plants A and B (TIFF 4015 kb)
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Iantcheva, A., Chabaud, M., Cosson, V. et al. Osmotic shock improves Tnt1 transposition frequency in Medicago truncatula cv Jemalong during in vitro regeneration. Plant Cell Rep 28, 1563–1572 (2009). https://doi.org/10.1007/s00299-009-0755-6
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DOI: https://doi.org/10.1007/s00299-009-0755-6