Chloroplast-selective gene delivery and expression in planta using chitosan-complexed single-walled carbon nanotube carriers


Plant genetic engineering is an important tool used in current efforts in crop improvement, pharmaceutical product biosynthesis and sustainable agriculture. However, conventional genetic engineering techniques target the nuclear genome, prompting concerns about the proliferation of foreign genes to weedy relatives. Chloroplast transformation does not have this limitation, since the plastid genome is maternally inherited in most plants, motivating the need for organelle-specific and selective nanocarriers. Here, we rationally designed chitosan-complexed single-walled carbon nanotubes, utilizing the lipid exchange envelope penetration mechanism. The single-walled carbon nanotubes selectively deliver plasmid DNA to chloroplasts of different plant species without external biolistic or chemical aid. We demonstrate chloroplast-targeted transgene delivery and transient expression in mature Erucasativa, Nasturtiumofficinale, Nicotianatabacum and Spinaciaoleracea plants and in isolated Arabidopsisthaliana mesophyll protoplasts. This nanoparticle-mediated chloroplast transgene delivery tool provides practical advantages over current delivery techniques as a potential transformation method for mature plants to benefit plant bioengineering and biological studies.

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Fig. 1: Schematic illustration of chloroplast-targeted delivery of pDNA by using SWNTs in a plant leaf.
Fig. 2: The optimal pDNA loading on SWNTs for high-efficiency trafficking of the nanocarriers into the chloroplast.
Fig. 3: Release of DNA from DNA–chitosan-complexed SWNT conjugates in vitro and transgene expression in isolated protoplasts.
Fig. 4: Chloroplast-targeted gene delivery and transient YFP expression in mature arugula plants.
Fig. 5: Chloroplast-targeted gene delivery and transient YFP expression in mature plants.

Data availability

The data that support the plots within this paper and other findings of this study are available from the corresponding author upon reasonable request.


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This research was supported by the National Research Foundation (NRF), Prime Minister’s Office, Singapore under its Campus for Research Excellence and Technological Enterprise (CREATE) program. The Disruptive & Sustainable Technology for Agricultural Precision (DiSTAP) is an interdisciplinary research group (IRG) of the Singapore MIT Alliance for Research and Technology (SMART) Centre. We also acknowledge support of Sime Darby Malaysia. T.T.S.L. and M.H.W. were supported on a graduate fellowship by the Agency of Science, Research and Technology, Singapore. V.B.K. was supported by The Swiss National Science Foundation (project no. P300P2_174469). The authors are grateful for helpful discussion with J. P. Giraldo and thank M. M. R. Ambavaram of Yield10 Bioscience for technical guidance and C. Xu of Yield10 Bioscience (now at Jounce Therapeutics) for technical assistance.

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S.-Y.K. and T.T.S.L. co-wrote the paper. S.-Y.K., T.T.S.L. and M.S.S. conceived and designed the experiments. C.J.S. assisted with the preparation and characterization of the chitosan-complexed SWNTs. V.B.K. performed AFM analysis. M.H.W. assisted with experimental design. K.D.S., K.B.-T. and J.S.S. constructed plasmid DNA. K.D.S. and K.B.-T. contributed the plastid DNA construct pMBX1120. J.S.S. and N.-H.C. constructed the nuclear DNA construct pBA-GFP-NL. All authors have revised the manuscript and given their approval of the final version.

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Correspondence to Michael S. Strano.

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Kwak, S., Lew, T.T.S., Sweeney, C.J. et al. Chloroplast-selective gene delivery and expression in planta using chitosan-complexed single-walled carbon nanotube carriers. Nat. Nanotechnol. 14, 447–455 (2019).

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