Abstract
Protoplasts are a versatile and powerful cell-based system to study different plant processes in vivo, due to their ability to maintain cell identity and carry out reactions and metabolic processes similar to intact plants. In rice, despite numerous reports, difficulties are encountered in protoplast isolation and transfection. These include insufficient numbers of protoplasts isolated and inefficient transfection. Such difficulties limit the use of this simple yet useful technology. The need to use protoplasts is particularly important when similar experiments may not work in yeast or Pichia, due to differences in functionally essential protein post-translation modifications. In this chapter, we describe a rice protoplast isolation and transfection method.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Cocking EC (1960) A method for the isolation of plant protoplasts and vacuoles. Nature 187:927–929
Sheen J (2001) Signal transduction in maize and Arabidopsis mesophyll protoplasts. Plant Physiol 127:1466–1475
Potrykus I, Shillito RD, Saul M, Paszkowski J (1985) Direct gene transfer: state of the art and future perspectives. Plant Mol Biol Rep 3:117–128
Fromm M, Taylor LP, Walbot V (1985) Expression of genes transferred into monocot and dicot plant cells by electroporation. Proc Natl Acad Sci U S A 82:5824–5828
Hillmer S, Gilroy S, Jones R (1992) Visualizing enzyme secretion from individual barley (Hordeum vulgare) aleurone protoplasts. Plant Physiol 102:279–286
Yoo SD, Cho YH, Sheen J (2007) Arabidopsis mesophyll protoplasts: a versatile cell system for transient gene expression analysis. Nat Protoc 2(7):1565–1572
Chen S, Tao L, Zeng L, Vega-Sanchez ME, Umemura K, Wang GL (2006) A highly efficient transient protoplast system for analyzing defense gene expression and protein-protein interactions in rice. Mol Plant Pathol 7(5):417–427
De Sutter V, Vanderhaeghen R, Tilleman S, Lammertyn F, Vanhoutte I, Karimi M, Inze D, Goossens A, Hilson P (2005) Exploration of jasmonate signaling via automated and standardized transient expression assays in tobacco cells. Plant J 44:1065–1076
Kapila J, De Rycke R, Van Montagu M, Angenon G (1997) An Agrobacterium-mediated transient gene expression system for intact leaves. Plant Sci 122:101–108
Worley CK, Zenser N, Ramos J, Rouse D, Leyser O, Theologis A, Callis J (2000) Degradation of Aux/IAA proteins is essential for normal auxin signaling. Plant J 21:553–563
Wang S, Tiwari SB, Hagen G, Guilfoyle TJ (2005) Auxin-response factor 7 restores the expression of auxin-responsive genes in mutant Arabidopsis leaf mesophyll protoplasts. Plant Cell 17:1979–1993
Kovtun Y, Chiu WL, Tena G, Sheen J (2000) Functional analysis of oxidative stress-activated mitogen-activated protein kinase cascade in plants. Proc Natl Acad Sci U S A 97:2940–2945
Tena G, Asai T, Chiu WL, Sheen J (2001) Plant mitogen-activated protein kinase signaling cascades. Curr Opin Plant Biol 4:392–400
Hwang I, Sheen J (2001) Two-component circuitry in Arabidopsis cytokinin signal transduction. Nature 413:383–389
Sheen J (1996) Ca2+-dependent protein kinases and stress signal transduction in plants. Science 274:1900–1902
Zhang Y, Su J, Duan S, Ao Y, Dai J, Liu J, Wang P, Li Y, Liu B, Feng D, Wang J, Wang H (2011) A highly efficient rice green tissue protoplast system for transient gene expression and studying light/chloroplast-related processes. Plant Methods 7:30–43
Dixit S, Biswal AK, Min A et al (2015) Action of multiple intra-QTL genes concerted around a co-localized transcription factor underpins a large effect QTL. Sci Rep 5:15183
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2021 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Trinidad, J.L., Longkumer, T., Kohli, A. (2021). Rice Protoplast Isolation and Transfection for Transient Gene Expression Analysis. In: Bandyopadhyay, A., Thilmony, R. (eds) Rice Genome Engineering and Gene Editing. Methods in Molecular Biology, vol 2238. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1068-8_21
Download citation
DOI: https://doi.org/10.1007/978-1-0716-1068-8_21
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-0716-1067-1
Online ISBN: 978-1-0716-1068-8
eBook Packages: Springer Protocols