Abstract
Plant protoplasts are derived by controlled enzymatic digestion that removes the plant cell wall without damaging the cell membrane. Protoplasts represent a true single-cell system and are useful for various biochemical and physiological studies. Protoplasts from several agriculturally important crop species can be regenerated into a fertile whole plant, extending the utility of protoplasts from transient expression assays to the generation of stable transformation events. Here we describe procedures for transient and stable transformation of leaf mesophyll protoplasts obtained from axenic shoot cultures of canola (Brassica napus). Key steps including enzymatic digestion for protoplast release, density gradient-based protoplast purification, PEG-mediated transfection, bead-type culturing (sea-plaque agarose and sodium alginate), and the recovery of putative transgenic canola plants are described. This method has been used for double-stranded DNA break-mediated genome editing and for the routine generation of stable transgenic canola events at commercial scale.
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References
Yoo S-D, Cho Y-H, Sheen J (2007) Arabidopsis mesophyll protoplasts: a versatile cell system for transient gene expression analysis. Nat Protoc 2:1565–1572
Chen J, Yi Q, Song Q, Gu Y, Zhang J, Hu Y, Hanmei L, Yinghon Y, Huang Y (2015) A highly efficient maize nucellus protoplast system for transient gene expression and studying programmed cell death-related processes. Plant Cell Rep 34:1239–1251
Fujikawa Y, Nakanishi T, Kawakami H, Yamasaki K, Sato MH, Tsuji H, Matsuoka M, Kato N (2014) Split luciferase complementation assay to detect regulated protein-protein interactions in rice protoplasts in a large-scale format. Rice 7:11
Li C, Lin H, Dubcovsky J (2015) Factorial combinations of protein interactions generate a multiplicity of florigen activation complexes in wheat and barley. Plant J 84:70–82
Cao J, Yao D, Lin F, Jiang M (2014) PEG-mediated transient gene expression and silencing system in maize mesophyll protoplasts: a valuable tool for signal transduction study in maize. Acta Physiol Plant 36:1271–1281
Nakashima K, Yamaguchi-Shinozaki K, Shinozaki K (2014) The transcriptional regulatory network in the drought response and its crosstalk in abiotic stress responses including drought, cold, and heat. Front Plant Sci 5:170
Pruneda-Paz JL, Breton G, Nagel DH, Kang SE, Bonaldi K, Doherty CJ, Ravelo S, Galli M, Ecker JR, Kay SA (2014) A genome-scale resource for the functional characterization of Arabidopsis transcription factors. Cell Rep 8:622–632
Cardoza V, Stewart CN (2004) Agrobacterium-mediated transformation of canola. In: Curtis IS (ed) Transgenic crops of the world – essential protocols. Kluwer, Dordrecht, pp 379–387
Facciotti MT, Bertain PB, Yuan L (1999) Improved stearate phenotype in transgenic canola expressing a modified acyl–acyl carrier protein thioesterase. Nat Biotechnol 17:593–597
Knutzon DS, Hayes TR, Wyrick A, Xiong H, Davies HM, Voelker TA (1999) Lysophosphatidic acid acyltransferase from coconut endosperm mediates the insertion of laurate at the sn-2 position of triacylglycerols in lauric rapeseed oil and can increase total laurate levels. Plant Physiol 120:739–746
Stoutjesdijk PA, Hurlestone C, Singh SP, Green AG (2000) High-oleic acid Australian Brassica napus and B. juncea varieties produced by co-suppression of endogenous delta 12-desaturases. Biochem Soc Trans 28:938–940
Katavic V, Freisen W, Barton DL, Gosen KK, Giblin EM, Luciw T, An J, Zou JT, MacKenzie SL, Keller WA, Males D, Taylor DC (2001) Improving erucic acid content in rapeseed through biotechnology: what can the Arabidopsis FAE1 and the yeast SLC1-1 genes contribute? Crop Sci 41:739–747
Oelck MM, Phan CV, Eckes P, Donn G, Rakow G, Keller WA (1991) Field resistance of canola transformants (Brassica napus L.) to ignite (phosphinothricin). Proc GCIRC Int Rapeseed Congr 8:292–297
Falco SC et al (1995) Transgenic canola and soybean seeds with increased lysine. Nat Biotechnol 13(6):577–582
Zhang ZB, Kornegay ET, Radcliffe JS, Wilson JH, Veit HP (2000) Comparison of phytase from genetically engineered Aspergillus and canola in weanling pig diets. J Anim Sci 78:2868–2878
Ravanello MP, Ke D, Alvarez J, Huang B, Shewmaker CK (2003) Coordinate expression of multiple bacterial carotenoid genes in canola leading to altered carotenoid production. Metab Eng 5(4):255–263
Voelker TA, Hayes TR, Cranmer AM, Turner JC, Davies HM (1996) Genetic engineering of a quantitative trait: metabolic and genetic parameters influencing the accumulation of laurate in rapeseed. Plant J 9:229–241
Monsanto (2002) Safety assessment of Roundup Ready canola event GT73 http://www.monsanto.com/monsanto/content/our_pledge/roundupcanola_product.pdf
Stewart CN, Adang MJ, All JN, Raymer PL, Ramachandran S, Parrott WA (1996) Insect control and dosage effects in transgenic canola containing a synthetic Bacillus thuringiensis cry1Ac gene. Plant Physiol 112:115–120
Grison R, Grezesbesset B, Schneider M, Lucante N, Olsen L, Leguay JJ, Toppan A (1996) Field tolerance to fungal pathogens of Brassica napus constitutively expressing a chimeric chitinase gene. Nat Biotechnol 14:643–646
Gupta M, DeKelver RC, Palta A, Clifford C, Gopalan S, Miller JC, Novak S, Desloover D, Gachotte D, Connell J, Flook J, Patterson T, Robbins K, Rebar EJ, Gregory PD, Urnov FD, Petolino JF (2012) Transcriptional activation of Brassica napus β-ketoacyl-ACP synthase II with an engineered zinc finger protein transcription factor. Plant Biotechnol J 10(7):783–791
Lawrenson T, Shorinola O, Stacey N, Li C, Østergaard L, Patron N, Uauy C, Harwood W (2015) Induction of targeted, heritable mutations in barley and Brassica oleracea using RNA-guided Cas9 nuclease. Genome Biol 16:258
Radke SE, Andrews BM, Moloney MM, Crouch ML, Krid JC, Knauf VC (1988) Transformation of Brassica napus L. using Agrobacterium tumefaciens: developmentally regulated expression of a reintroduced napin gene. Theor Appl Genet 75:685–694
De Block M, De Brower D, Tenning P (1989) Transformation of Brassica napus and Brassica oleracea using Agrobacterium tumefaciens and the expression of the bar and neo genes in the transgenic plants. Plant Physiol 91:694–701
Moloney MM, Walker JM, Sharma KK (1989) High efficiency transformation of Brassica napus using Agrobacterium vectors. Plant Cell Rep 8:238–242
Fry J, Barnason A, Horsch RB (1987) Transformation of Brassica napus with Agrobacterium based vectors. Plant Cell Rep 6:321–325
Pua EC, Mehra Palta A, Nagy F, Chua NH (1987) Transgenic plants of Brassica napus L. Biotechnology 5:815–817
Hervé C, Rouan D, Guerche P, Montané MH, Yot P (1993) Molecular analysis of transgenic rapeseed plants obtained by direct transfer of 2 separate plasmids containing, respectively, the cauliflower mosaic virus coat protein gene and a selectable marker gene. Plant Sci 91:181–193
Spangenberg G, Koop H-U, Lichter R, Schweiger HG (1986) Microculture of single protoplasts of Brassica napus. Physiol Plant 66:1–8
Souvre A, Jardinaud MF, Alibert G (1996) Transformation of rape (Brassica napus L.) through the haploid embryogenesis pathway. Acta Soc Bot Polon 65:194–195
Jones-Villeneuve E, Huang B, Prudhomme I, Bird S, Kemble R, Hattori J, Miki B (1995) Assessment of microinjection for introducing DNA into uninuclear microspores of rapeseed. Plant Cell Tissue Organ Cult 40:97–100
Fukuoka H, Ogawa T, Matsuoka M, Ohkawa Y, Yano H (1998) Direct gene delivery into isolated microspores of rapeseed (Brassica napus L.) and the production of fertile transgenic plants. Plant Cell Rep 17:323–328
Nehlin L, Möllers C, Bergman P, Glimelius K (2000) Transient β-gus and gfp expression and viability analysis of microprojectile bombarded microspores of Brassica napus L. J Plant Physiol 156:175–183
Menczel L, Nagy F, Kiss Z, Maliga P (1981) Streptomycin-resistant and sensitive somatic hybrids of N. tabacum + N. knightiana: correlation of resistance to N. tabacum plastids. Theor Appl Genet 59:191–195
Spangenberg G, Potrykus I (1996) In: Potrykus I, Spangenberg G (eds) Polyethylene glycol-mediated direct gene transfer to tobacco protoplasts and regeneration of transgenic plants: gene transfer to plants. Springer-Verlag, Berlin, Heidelberg, New York, pp 59–65
Sambrook J, Russell DW (2006) Estimation of cell number by hemocytometry counting. Cold Spring Harb Protoc. https://doi.org/10.1101/pdb.prot4454
Huang CN, Corenjo MJ, Bush DS, Jones RL (1996) Estimating viability of plant protoplasts using double and single staining. Protoplasma 135:80–87
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Sahab, S., Hayden, M.J., Mason, J., Spangenberg, G. (2019). Mesophyll Protoplasts and PEG-Mediated Transfections: Transient Assays and Generation of Stable Transgenic Canola Plants. In: Kumar, S., Barone, P., Smith, M. (eds) Transgenic Plants. Methods in Molecular Biology, vol 1864. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-8778-8_10
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DOI: https://doi.org/10.1007/978-1-4939-8778-8_10
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