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Efficient Protoplast Isolation and DNA Transfection for Winter Oilseed Crops, Pennycress (Thlaspi arvense ) and Camelina (Camelina sativa )

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Protoplast Technology

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2464))

Abstract

Pennycress (Thlaspi arvense) and camelina (Camelina sativa) are nonfood winter oilseed crops that have the potential to contribute to sustainable biofuel production. However, undesired agronomic traits of pennycress and camelina currently hinder broad cultivation of these plants in the field. Recently, genome editing using the CRISPR-Cas technology has been applied to improve poor agronomic traits such as the weedy phenotype of pennycress and the oxidation susceptible lipid profile of camelina. In these works, the CRISPR reagents were introduced into the plants using the Agrobacterium-mediated floral dipping method. For accelerated domestication and value improvements of these winter oilseed crops, DNA-free genome editing platform and easy evaluation method of the CRISPR-Cas reagents are highly desirable. Cell wall-free protoplasts are great material to expand the use of gene engineering tools. In this chapter, we present a step-by-step guide to the mesophyll protoplast isolation from in vitro culture-grown pennycress and soil-grown camelina. The protocol also includes procedures for DNA transfection and protoplast viability test using fluorescein diacetate. With this protocol, we can isolate an average of 6 × 106 cells from pennycress and 3 × 106 cells from camelina per gram of fresh leaf tissues. Using a 7.3 kb plasmid DNA carrying green and red fluorescent protein marker genes, we can achieve an average transfection rate of 40% validated by flow cytometry for both plants.

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Acknowledgments

The authors thank Oluwatoyosi Akintayo of Dr. Madan Bhattacharyya lab for providing camelina plantlets and Shawn Rigby of the Flow Cytometry Facility for technical assistance. This project was partially supported by National Science Foundation Plant Genome Research Program Grants 1917138, to K.W., by the USDA NIFA Hatch project #IOW04714, by State of Iowa funds, and by the Crop Bioengineering Center of Iowa State University.

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Authors and Affiliations

  1. Department of Agronomy, Iowa State University, Ames, IA, USA

    Minjeong Kang, Keunsub Lee & Kan Wang

  2. Crop Bioengineering Center, Iowa State University, Ames, IA, USA

    Minjeong Kang, Keunsub Lee & Kan Wang

  3. Interdepartmental Plant Biology Major, Iowa State University, Ames, IA, USA

    Minjeong Kang

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  1. Minjeong Kang
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  2. Keunsub Lee
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Correspondence to Kan Wang .

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Editors and Affiliations

  1. Department of Agronomy, Iowa State University, Ames, IA, USA

    Kan Wang

  2. Department of Plant and Microbial Biology, University of Minnesota, Saint Paul, MN, USA

    Feng Zhang

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Kang, M., Lee, K., Wang, K. (2022). Efficient Protoplast Isolation and DNA Transfection for Winter Oilseed Crops, Pennycress (Thlaspi arvense ) and Camelina (Camelina sativa ). In: Wang, K., Zhang, F. (eds) Protoplast Technology. Methods in Molecular Biology, vol 2464. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2164-6_12

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  • DOI: https://doi.org/10.1007/978-1-0716-2164-6_12

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-2163-9

  • Online ISBN: 978-1-0716-2164-6

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