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Gene delivery into the plant mitochondria via organelle-specific peptides

  • Trevor MacMillan
  • Alicja Ziemienowicz
  • Fengying Jiang
  • François Eudes
  • Igor Kovalchuk
Original Article
  • 450 Downloads

Abstract

We report a method for genetically engineering the mitochondria of plant cells. Several peptides selected from sorting signal sequences of plant mitochondrial proteins were tested for cell-penetrating, specific organelle-targeting and nucleic acid-binding properties. Selected sequences were named mitochondrial targeting peptides (mTPs). Five of them were used to deliver a linear dsDNA gene construct into AC Ultima spring triticale (X. Triticosecale Wittmack) protoplasts and microspores. The mitochondrial transient expression of the aadA:gfp reporter gene was qualitatively determined with confocal microscopy and quantitatively measured using qRT-PCR. Significant aadA:gfp transcript abundance was reported in protoplasts from deliveries of all five mTPs, while this transcript abundance remained low in microspores. mTP1 transfected microspores were cultured to produce green plantlets and screened for aadA:gfp by endpoint PCR. Targeted gene integration into the mitochondrial genome of eight regenerated haploid triticale lines was confirmed by sequencing. This represents the first report of a successful in vivo transfection of crop plant somatic and germ cells via mitochondrial peptides and a method that can be used to study transient gene expression and to stably deliver genes into the mitochondria of a plant.

Keywords

Microspores Mitochondria mTPs Protoplasts Transformation Triticale 

Notes

Acknowledgements

We express our appreciation for the contributions made by Bernie Genswein for his computational assistance as well as the Kovalchuk Laboratory, Eric Amundsen and Victoria Hodgson for their technical assistance in the lab.

Supplementary material

11816_2018_502_MOESM1_ESM.docx (20 mb)
Supplementary material 1 (DOCX 20477 KB)

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Copyright information

© Crown 2018
corrected publication 2018

Authors and Affiliations

  1. 1.Agriculture and Agri-Food CanadaLethbridge Research and Development CentreLethbridgeCanada
  2. 2.Department of Biological SciencesUniversity of LethbridgeLethbridgeCanada

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