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Tnt1 retrotransposon as an efficient tool for development of an insertional mutant collection of Lotus japonicus

Abstract

The Tnt1 retrotransposon of tobacco (Nicotiana tabacum) has proven to be a very efficient mutagen for the model legume Medicago truncatula ecotype 108 and cultivar Jemalong 2HA and for economically important plants, such as soybean and potato. In this study, the activity of Tnt1 in the model legume Lotus japonicus L. was tested. First, a new regeneration and transformation protocol was developed for L. japonicus that represents a new tool for legume mutagenesis and reverse genetics. Using this protocol, the Tnt1 retrotransposon was introduced into L. japonicus by Agrobacterium tumefaciens-mediated transformation, and primary transgenic lines, named starter lines, were constructed. In vitro regeneration via indirect somatic embryogenesis using starter lines harboring two to eight copies of the transgene resulted in new Tnt1 transposition events. The Tnt1 retrotransposon remained inactive during plant growth and in the T1 progeny, indicating that it is well suited for insertional mutagenesis in L. japonicus.

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Acknowledgments

This study was supported by a grant from the Ministry of Education and Science of Republic Bulgaria (project Do 02-268). The authors are grateful to Kety Krastanova for the valuable technical assistance. The sequencing work was supported by a grant from the Agence Nationale de la Recherche (ANR) Blanc International SVSE 6.2010.1 (LEGUMICS) to Pascal Ratet.

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Correspondence to Anelia Iantcheva.

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Editor: Mark Jordan

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Figure S1

Sequences of IPCR fragments obtained from regenerant R6 and T1 of St lines 3 and 12. (A) R6, (B) T1 St3, and (C) T1 St12. The Tnt1 region corresponding to the 5′ LTR sequence of the retroelement is highlighted in yellow. The Tnt1 region corresponding to the 3′ LTR sequence of the retroelement is highlighted in green. The EcoRI (GAATTC), Mfe1 (CAATTG), and hybrid MfeI/EcoRI (CAATTC) restriction sites, corresponding to the ligation site of the IPCR fragments, are noted with red letters. Each sequence is indicated as being a self-ligated Tnt1, a Tnt1-vector junction, or an insertion in the Lotus genome. (DOC 32 kb)

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Iantcheva, A., Revalska, M., Zehirov, G. et al. Tnt1 retrotransposon as an efficient tool for development of an insertional mutant collection of Lotus japonicus . In Vitro Cell.Dev.Biol.-Plant 52, 338–347 (2016). https://doi.org/10.1007/s11627-016-9768-3

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  • DOI: https://doi.org/10.1007/s11627-016-9768-3

Keywords

  • Tnt1 retrotransposon
  • Lotus japonicus
  • Insertional mutagenesis
  • Starter lines
  • Regeneration
  • Embryogenesis