skip to main content
DOE PAGES title logo U.S. Department of Energy
Office of Scientific and Technical Information

Title: Generation, analysis, and transformation of macro-chloroplast Potato (Solanum tuberosum) lines for chloroplast biotechnology

Abstract

Abstract Chloroplast biotechnology is a route for novel crop metabolic engineering. The potential bio-confinement of transgenes, the high protein expression and the possibility to organize genes into operons represent considerable advantages that make chloroplasts valuable targets in agricultural biotechnology. In the last 3 decades, chloroplast genomes from a few economically important crops have been successfully transformed. The main bottlenecks that prevent efficient transformation in a greater number of crops include the dearth of proven selectable marker gene-selection combinations and tissue culture methods for efficient regeneration of transplastomic plants. The prospects of increasing organelle size are attractive from several perspectives, including an increase in the surface area of potential targets. As a proof-of-concept, we generated Solanum tuberosum (potato) macro-chloroplast lines overexpressing the tubulin-like GTPase protein gene FtsZ1 from Arabidopsis thaliana . Macro-chloroplast lines exhibited delayed growth at anthesis; however, at the time of harvest there was no significant difference in height between macro-chloroplast and wild-type lines. Macro-chloroplasts were successfully transformed by biolistic DNA-delivery and efficiently regenerated into homoplasmic transplastomic lines. We also demonstrated that macro-chloroplasts accumulate the same amount of heterologous protein than wild-type organelles, confirming efficient usage in plastid engineering. Advantages and limitations of using enlarge compartments in chloroplast biotechnologymore » are discussed.« less


Citation Formats

Occhialini, Alessandro, Pfotenhauer, Alexander C., Frazier, Taylor P., Li, Li, Harbison, Stacee A., Lail, Andrew J., Mebane, Zachary, Piatek, Agnieszka A., Rigoulot, Stephen B., Daniell, Henry, Stewart, Jr, C. Neal, and Lenaghan, Scott C.. Generation, analysis, and transformation of macro-chloroplast Potato (Solanum tuberosum) lines for chloroplast biotechnology. United Kingdom: N. p., 2020. Web. https://doi.org/10.1038/s41598-020-78237-x.
Occhialini, Alessandro, Pfotenhauer, Alexander C., Frazier, Taylor P., Li, Li, Harbison, Stacee A., Lail, Andrew J., Mebane, Zachary, Piatek, Agnieszka A., Rigoulot, Stephen B., Daniell, Henry, Stewart, Jr, C. Neal, & Lenaghan, Scott C.. Generation, analysis, and transformation of macro-chloroplast Potato (Solanum tuberosum) lines for chloroplast biotechnology. United Kingdom. https://doi.org/10.1038/s41598-020-78237-x
Occhialini, Alessandro, Pfotenhauer, Alexander C., Frazier, Taylor P., Li, Li, Harbison, Stacee A., Lail, Andrew J., Mebane, Zachary, Piatek, Agnieszka A., Rigoulot, Stephen B., Daniell, Henry, Stewart, Jr, C. Neal, and Lenaghan, Scott C.. Thu . "Generation, analysis, and transformation of macro-chloroplast Potato (Solanum tuberosum) lines for chloroplast biotechnology". United Kingdom. https://doi.org/10.1038/s41598-020-78237-x.
@article{osti_1730964,
title = {Generation, analysis, and transformation of macro-chloroplast Potato (Solanum tuberosum) lines for chloroplast biotechnology},
author = {Occhialini, Alessandro and Pfotenhauer, Alexander C. and Frazier, Taylor P. and Li, Li and Harbison, Stacee A. and Lail, Andrew J. and Mebane, Zachary and Piatek, Agnieszka A. and Rigoulot, Stephen B. and Daniell, Henry and Stewart, Jr, C. Neal and Lenaghan, Scott C.},
abstractNote = {Abstract Chloroplast biotechnology is a route for novel crop metabolic engineering. The potential bio-confinement of transgenes, the high protein expression and the possibility to organize genes into operons represent considerable advantages that make chloroplasts valuable targets in agricultural biotechnology. In the last 3 decades, chloroplast genomes from a few economically important crops have been successfully transformed. The main bottlenecks that prevent efficient transformation in a greater number of crops include the dearth of proven selectable marker gene-selection combinations and tissue culture methods for efficient regeneration of transplastomic plants. The prospects of increasing organelle size are attractive from several perspectives, including an increase in the surface area of potential targets. As a proof-of-concept, we generated Solanum tuberosum (potato) macro-chloroplast lines overexpressing the tubulin-like GTPase protein gene FtsZ1 from Arabidopsis thaliana . Macro-chloroplast lines exhibited delayed growth at anthesis; however, at the time of harvest there was no significant difference in height between macro-chloroplast and wild-type lines. Macro-chloroplasts were successfully transformed by biolistic DNA-delivery and efficiently regenerated into homoplasmic transplastomic lines. We also demonstrated that macro-chloroplasts accumulate the same amount of heterologous protein than wild-type organelles, confirming efficient usage in plastid engineering. Advantages and limitations of using enlarge compartments in chloroplast biotechnology are discussed.},
doi = {10.1038/s41598-020-78237-x},
journal = {Scientific Reports},
number = 1,
volume = 10,
place = {United Kingdom},
year = {2020},
month = {12}
}

Journal Article:
Free Publicly Available Full Text
Publisher's Version of Record
https://doi.org/10.1038/s41598-020-78237-x

Save / Share:

Works referenced in this record:

High-frequency plastid transformation in tobacco by selection for a chimeric aadA gene.
journal, February 1993

  • Svab, Z.; Maliga, P.
  • Proceedings of the National Academy of Sciences, Vol. 90, Issue 3
  • DOI: 10.1073/pnas.90.3.913

Validation of leaf enzymes in the detergent and textile industries: launching of a new platform technology
journal, February 2019

  • Kumari, Uma; Singh, Rahul; Ray, Tui
  • Plant Biotechnology Journal, Vol. 17, Issue 6
  • DOI: 10.1111/pbi.13122

Non-photosynthetic plastids as hosts for metabolic engineering
journal, February 2018

  • Mellor, Silas Busck; Behrendorff, James B. Y. H.; Nielsen, Agnieszka Zygadlo
  • Essays in Biochemistry, Vol. 62, Issue 1
  • DOI: 10.1042/ebc20170047

Single Walled Carbon Nanotubes Exhibit Dual-Phase Regulation to Exposed Arabidopsis Mesophyll Cells
journal, September 2010


MoChlo: A Versatile, Modular Cloning Toolbox for Chloroplast Biotechnology
journal, January 2019

  • Occhialini, Alessandro; Piatek, Agnieszka A.; Pfotenhauer, Alexander C.
  • Plant Physiology, Vol. 179, Issue 3
  • DOI: 10.1104/pp.18.01220

Stable Plastid Transformation in PEG-treated Protoplasts of Nicotiana tabacum
journal, January 1993

  • Golds, Timothy; Maliga, Pal; Koop, Hans-Ulrich
  • Nature Biotechnology, Vol. 11, Issue 1
  • DOI: 10.1038/nbt0193-95

Ftsz Ring Formation at the Chloroplast Division Site in Plants
journal, April 2001

  • Vitha, Stanislav; McAndrew, Rosemary S.; Osteryoung, Katherine W.
  • The Journal of Cell Biology, Vol. 153, Issue 1
  • DOI: 10.1083/jcb.153.1.111

Visualization of an FtsZ Ring in Chloroplasts of Lilium longiflorum Leaves
journal, June 2001

  • Mori, Toshiyuki; Kuroiwa, Haruko; Takahara, Manabu
  • Plant and Cell Physiology, Vol. 42, Issue 6
  • DOI: 10.1093/pcp/pce095

Field production and functional evaluation of chloroplast-derived interferon-?2b
journal, July 2007


Growth retardation of plants transformed by overexpression of NtFtsZ1-2 in Tobacco
journal, June 2002

  • Jeong, Won Joong; Jeong, Suk Won; Min, Sung Ran
  • Journal of Plant Biology, Vol. 45, Issue 2
  • DOI: 10.1007/bf03030292

Plastid Biotechnology: Food, Fuel, and Medicine for the 21st Century
journal, January 2011


β-Carboxysomal proteins assemble into highly organized structures in Nicotiana chloroplasts
journal, June 2014

  • Lin, Myat T.; Occhialini, Alessandro; Andralojc, P. John
  • The Plant Journal, Vol. 79, Issue 1
  • DOI: 10.1111/tpj.12536

Chloroplast Division in Higher Plants Requires Members of Two Functionally Divergent Gene Families with Homology to Bacterial ftsZ
journal, December 1998

  • Osteryoung, Katherine W.; Stokes, Kevin D.; Rutherford, Stephen M.
  • The Plant Cell, Vol. 10, Issue 12
  • DOI: 10.1105/tpc.10.12.1991

The Engineered Chloroplast Genome Just Got Smarter
journal, October 2015


The plastid genome as a chassis for synthetic biology-enabled metabolic engineering: players in gene expression
journal, July 2018

  • Schindel, Heidi S.; Piatek, Agnieszka A.; Stewart, C. Neal
  • Plant Cell Reports, Vol. 37, Issue 10
  • DOI: 10.1007/s00299-018-2323-4

Stable plastid transformation of rice, a monocot cereal crop
journal, September 2018

  • Wang, Yunpeng; Wei, Zhengyi; Xing, Shaochen
  • Biochemical and Biophysical Research Communications, Vol. 503, Issue 4
  • DOI: 10.1016/j.bbrc.2018.06.164

A faster Rubisco with potential to increase photosynthesis in crops
journal, September 2014

  • Lin, Myat T.; Occhialini, Alessandro; Andralojc, P. John
  • Nature, Vol. 513, Issue 7519
  • DOI: 10.1038/nature13776

Arabidopsis FtsZ2-1 and FtsZ2-2 Are Functionally Redundant, But FtsZ-Based Plastid Division Is Not Essential for Chloroplast Partitioning or Plant Growth and Development
journal, November 2009

  • Schmitz, Aaron J.; Glynn, Jonathan M.; Olson, Bradley J. S. C.
  • Molecular Plant, Vol. 2, Issue 6
  • DOI: 10.1093/mp/ssp077

A Large Population of Small Chloroplasts in Tobacco Leaf Cells Allows More Effective Chloroplast Movement Than a Few Enlarged Chloroplasts
journal, April 2002

  • Jeong, Won Joong; Park, Youn-Il; Suh, KyeHong
  • Plant Physiology, Vol. 129, Issue 1
  • DOI: 10.1104/pp.000588

Primer3—new capabilities and interfaces
journal, June 2012

  • Untergasser, Andreas; Cutcutache, Ioana; Koressaar, Triinu
  • Nucleic Acids Research, Vol. 40, Issue 15
  • DOI: 10.1093/nar/gks596

Stable genetic transformation of tomato plastids and expression of a foreign protein in fruit
journal, September 2001

  • Ruf, Stephanie; Hermann, Marita; Berger, Irving J.
  • Nature Biotechnology, Vol. 19, Issue 9
  • DOI: 10.1038/nbt0901-870

Expression of Brassica oleracea FtsZ1-1 and MinD alters chloroplast division in Nicotiana tabacum generating macro- and mini-chloroplasts
journal, December 2011

  • Chikkala, Veera R. N.; Nugent, Gregory D.; Stalker, David M.
  • Plant Cell Reports, Vol. 31, Issue 5
  • DOI: 10.1007/s00299-011-1212-x

Plant cell-made protein antigens for induction of Oral tolerance
journal, November 2019


Colocalization of Plastid Division Proteins in the Chloroplast Stromal Compartment Establishes a New Functional Relationship between FtsZ1 and FtsZ2 in Higher Plants
journal, December 2001

  • McAndrew, Rosemary S.; Froehlich, John E.; Vitha, Stanislav
  • Plant Physiology, Vol. 127, Issue 4
  • DOI: 10.1104/pp.010542

Transgene containment by maternal inheritance: Effective or elusive?
journal, April 2007


A galinstan expansion femtosyringe for microinjection of eukaryotic organelles and prokaryotes
journal, September 1999

  • Knoblauch, Michael; Hibberd, Julian M.; Gray, John C.
  • Nature Biotechnology, Vol. 17, Issue 9
  • DOI: 10.1038/12902

Cold chain and virus‐free oral polio booster vaccine made in lettuce chloroplasts confers protection against all three poliovirus serotypes
journal, January 2019

  • Daniell, Henry; Rai, Vineeta; Xiao, Yuhong
  • Plant Biotechnology Journal, Vol. 17, Issue 7
  • DOI: 10.1111/pbi.13060

Efficient metabolic pathway engineering in transgenic tobacco and tomato plastids with synthetic multigene operons
journal, February 2013

  • Lu, Y.; Rijzaani, H.; Karcher, D.
  • Proceedings of the National Academy of Sciences, Vol. 110, Issue 8
  • DOI: 10.1073/pnas.1216898110

High-efficiency generation of fertile transplastomic Arabidopsis plants
journal, February 2019


Introduction and expression of foreign DNA in isolated spinach chloroplasts by electroporation
journal, October 1996


Chloroplast-selective gene delivery and expression in planta using chitosan-complexed single-walled carbon nanotube carriers
journal, February 2019

  • Kwak, Seon-Yeong; Lew, Tedrick Thomas Salim; Sweeney, Connor J.
  • Nature Nanotechnology, Vol. 14, Issue 5
  • DOI: 10.1038/s41565-019-0375-4

Stable transformation of plastids in higher plants.
journal, November 1990

  • Svab, Z.; Hajdukiewicz, P.; Maliga, P.
  • Proceedings of the National Academy of Sciences, Vol. 87, Issue 21
  • DOI: 10.1073/pnas.87.21.8526

Manipulation of starch granule size distribution in potato tubers by modulation of plastid division
journal, January 2006


Genome-Wide Analysis of Plastid Gene Expression in Potato Leaf Chloroplasts and Tuber Amyloplasts: Transcriptional and Posttranscriptional Control
journal, June 2009

  • Valkov, Vladimir T.; Scotti, Nunzia; Kahlau, Sabine
  • Plant Physiology, Vol. 150, Issue 4
  • DOI: 10.1104/pp.109.140483

Generation of homoplasmic plastid transformants of a commercial cultivar of potato (Solanum tuberosum L.)
journal, June 2005


Release of Proteins from Intact Chloroplasts Induced by Reactive Oxygen Species during Biotic and Abiotic Stress
journal, June 2013


Transformation of Agrobacterium Using the Freeze-Thaw Method
journal, December 2006

  • Weigel, D.; Glazebrook, J.
  • Cold Spring Harbor Protocols, Vol. 2006, Issue 30
  • DOI: 10.1101/pdb.prot4666

Participation of Leaky Ribosome Scanning in Protein Dual Targeting by Alternative Translation Initiation in Higher Plants
journal, January 2009

  • Wamboldt, Yashitola; Mohammed, Saleem; Elowsky, Christian
  • The Plant Cell, Vol. 21, Issue 1
  • DOI: 10.1105/tpc.108.063644

Engineering Plastid Genomes: Methods, Tools, and Applications in Basic Research and Biotechnology
journal, April 2015


Imaging of multiple fluorescent proteins in canopies enables synthetic biology in plants
journal, December 2020

  • Rigoulot, Stephen B.; Schimel, Tayler M.; Lee, Jun Hyung
  • Plant Biotechnology Journal
  • DOI: 10.1111/pbi.13510

Strategies for metabolic pathway engineering with multiple transgenes
journal, March 2013


Engineered RNA-binding protein for transgene activation in non-green plastids
journal, April 2019


Direct cell to cell transfer of organelles by microinjection
journal, January 1992

  • Verhoeven, HarrieA.; Blaas, Jan
  • Plant Cell Reports, Vol. 10, Issue 12
  • DOI: 10.1007/bf00232381

Investigational new drug enabling angiotensin oral-delivery studies to attenuate pulmonary hypertension
journal, March 2020


Division and Dynamic Morphology of Plastids
journal, April 2014


A ubiquitin carboxyl extension protein secreted from a plant-parasitic nematode Globodera rostochiensis is cleaved in planta to promote plant parasitism
journal, February 2013

  • Chronis, Demosthenis; Chen, Shiyan; Lu, Shunwen
  • The Plant Journal, Vol. 74, Issue 2
  • DOI: 10.1111/tpj.12125

Phenomics: the next challenge
journal, November 2010

  • Houle, David; Govindaraju, Diddahally R.; Omholt, Stig
  • Nature Reviews Genetics, Vol. 11, Issue 12
  • DOI: 10.1038/nrg2897

The Molecular Machinery of Chloroplast Division
journal, October 2017

  • Chen, Cheng; MacCready, Joshua S.; Ducat, Daniel C.
  • Plant Physiology, Vol. 176, Issue 1
  • DOI: 10.1104/pp.17.01272

Chloroplast Division and Morphology Are Differentially Affected by Overexpression of FtsZ1 and FtsZ2 Genes in Arabidopsis
journal, December 2000

  • Stokes, Kevin D.; McAndrew, Rosemary S.; Figueroa, Rubi
  • Plant Physiology, Vol. 124, Issue 4
  • DOI: 10.1104/pp.124.4.1668

Transgenic tobacco plants with improved cyanobacterial Rubisco expression but no extra assembly factors grow at near wild-type rates if provided with elevated CO 2
journal, December 2015

  • Occhialini, Alessandro; Lin, Myat T.; Andralojc, P. John
  • The Plant Journal, Vol. 85, Issue 1
  • DOI: 10.1111/tpj.13098

Stable chloroplast transformation in potato: use of green fluorescent protein as a plastid marker
journal, July 1999


Terpene metabolic engineering via nuclear or chloroplast genomes profoundly and globally impacts off-target pathways through metabolite signalling
journal, March 2016

  • Pasoreck, Elise K.; Su, Jin; Silverman, Ian M.
  • Plant Biotechnology Journal, Vol. 14, Issue 9
  • DOI: 10.1111/pbi.12548

Validation of leaf and microbial pectinases: commercial launching of a new platform technology
journal, February 2019

  • Daniell, Henry; Ribeiro, Thuanne; Lin, Shina
  • Plant Biotechnology Journal, Vol. 17, Issue 6
  • DOI: 10.1111/pbi.13119

High-Frequency Transformation of Undeveloped Plastids in Tobacco Suspension Cells
journal, May 2004

  • Langbecker, Camri L.; Ye, Guang-Ning; Broyles, Debra L.
  • Plant Physiology, Vol. 135, Issue 1
  • DOI: 10.1104/pp.103.035410

Chloroplast Vector Systems for Biotechnology Applications
journal, December 2007