Abstract
The use of plant systems as factories for recombinant protein production became a prominent alternative for pharmaceutical industries due to their high potential for protein accumulation. In the last decades, the application of plants for protein production has gained more attention, as plants represent an economic strategy that leads to high levels of purified and active proteins for the pharmaceutical sector. Currently, FDA approval of the first generation of recombinant proteins produced in carrot cells, taliglucerase alfa, demonstrated that plant cells have a significant capacity to express complex proteins for therapeutic use. Although plant systems still have technical and economic barriers that require improvements in future years, the optimization of upstream and downstream components affecting protein accumulation is considered a key feature in the development of new pharmaceutical proteins. Therefore, an improvement of critical features, including transcription, translation and post-translational modifications, in plant cells could make plant systems a safe and economical alternative for biopharmaceutical production. Hence, in this review, the most recent advances influencing the upstream and downstream processes involved in recombinant protein accumulation in plant cells are described. We also discuss how plant systems are becoming the benchmark for the production of several biopharmaceuticals.
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References
Abdel-Ghany SE, Day I, Heuberger AL, Broeckling CD, Reddy ASN (2013) Metabolic engineering of Arabidopsis for butanetriol production using bacterial genes. Metab Eng 20:109–120. doi:10.1016/j.ymben.2013.10.003
Aebi M (2013) N-linked protein glycosylation in the ER. Biophys Acta 1833(11):2430–2437 doi:10.1016/j.bbamcr.2013.04.001
Al-Rubeai M (2011) Antibody expression and production. Springer Netherlands, Dordrecht
Alvarez ML, Topal E, Martin F, Cardineau GA (2010) Higher accumulation of F1-V fusion recombinant protein in plants after induction of protein body formation. Plant Mol Biol 72(1–2):75–89. doi:10.1007/s11103-009-9552-4
Amin N, Liu AD, Ramer S, Aehle W, Meijer D, Metin M, Wong S, Gualfetti P, Schellenberger V (2004) Construction of stabilized proteins by combinatorial consensus mutagenesis. Protein Eng Des Sel 17(11):787–793. doi:10.1093/protein/gzh091
Amirsadeghi S, McDonald AE, Vanlerberghe GC (2007) A glucocorticoid-inducible gene expression system can cause growth defects in tobacco. Planta 226(2):453–463. doi:10.1007/s00425-007-0495-1
Atsmon J, Brill-Almon E, Nadri-Shay C, Chertkoff R, Alon S, Shaikevich D, Volokhov I, Haim KY, Bartfeld D, Shulman A, Ruderfer I, Ben-Moshe T, Shilovitzky O, Soreq H, Shaaltiel Y (2015) Preclinical and first-in-human evaluation of PRX-105, a PEGylated, plant-derived, recombinant human acetylcholinesterase-R. Toxicol Appl Pharmacol 287(3):202–209. doi:10.1016/j.taap.2015.06.004
Azhakanandam K, Weissinger SM, Nicholson JS, Qu R, Weissinger AK (2007) Amplicon-plus targeting technology (APTT) for rapid production of a highly unstable vaccine protein in tobacco plants. Plant Mol Biol 63(3):393–404. doi:10.1007/s11103-006-9096-9
Baur A, Reski R, Gorr G (2005) Enhanced recovery of a secreted recombinant human growth factor using stabilizing additives and by co-expression of human serum albumin in the moss Physcomitrella patens. Plant Biotechnol J 3(3):331–340. doi:10.1111/j.1467-7652.2005.00127.x
Benchabane M, Goulet C, Rivard D, Faye L, Gomord V, Michaud D (2008) Preventing unintended proteolysis in plant protein biofactories. Plant Biotechnol J 6(7):633–648. doi:10.1111/j.1467-7652.2008.00344.x
Biłas R, Szafran K, Hnatuszko-Konka K, Kononowicz AK (2016) Cis-regulatory elements used to control gene expression in plants. PCTOC 127(2):269–287. doi:10.1007/s11240-016-1057-7
Bocobza SE, Aharoni A (2014) Small molecules that interact with RNA: riboswitch-based gene control and its involvement in metabolic regulation in plants and algae. Plant J 79(4):693–703. doi:10.1111/tpj.12540
Bornke F, Broer I (2010) Tailoring plant metabolism for the production of novel polymers and platform chemicals. Curr Opin Plant Biol 13(3):354–362. doi:10.1016/j.pbi.2010.01.005
Brest P, Lapaquette P, Souidi M, Lebrigand K, Cesaro A, Vouret-Craviari V, Mari B, Barbry P, Mosnier JF, Hebuterne X, Harel-Bellan A, Mograbi B, Darfeuille-Michaud A, Hofman P (2011) A synonymous variant in IRGM alters a binding site for miR-196 and causes deregulation of IRGM-dependent xenophagy in Crohn’s disease. Nat Genet 43(3):242–245. doi:10.1038/ng.762
Buttner-Mainik A, Parsons J, Jerome H, Hartmann A, Lamer S, Schaaf A, Schlosser A, Zipfel PF, Reski R, Decker EL (2011) Production of biologically active recombinant human factor H in Physcomitrella. Plant Biotechnol J 9(3):373–383. doi:10.1111/j.1467-7652.2010.00552.x
Callis J, Fromm M, Walbot V (1987) Introns increase gene expression in cultured maize cells. Genes Dev 1(10):1183–1200
Cheah MT, Wachter A, Sudarsan N, Breaker RR (2007) Control of alternative RNA splicing and gene expression by eukaryotic riboswitches. Nature 447(7143):497–500. http://www.nature.com/nature/journal/v447/n7143/suppinfo/nature05769_S1.html
Chebolu S, Daniell H (2009) Chloroplast-derived vaccine antigens and biopharmaceuticals: expression, folding, assembly and functionality. Curr Top Microbiol Immunol 332:33–54. doi:10.1007/978-3-540-70868-1_3
Chen Q, Lai H, Hurtado J, Stahnke J, Leuzinger K, Dent M (2013a) Agroinfiltration as an effective and scalable strategy of gene delivery for production of pharmaceutical proteins. Adv Tech Biol Med 1(1):103. doi:10.4172/atbm.1000103
Chen Z, He Y, Shi B, Yang D (2013b) Human serum albumin from recombinant DNA technology: challenges and strategies. Biochim Biophys Acta 1830(12):5515–5525. doi:10.1016/j.bbagen.2013.04.037
Chikwamba RK, Scott MP, Mejia LB, Mason HS, Wang K (2003) Localization of a bacterial protein in starch granules of transgenic maize kernels. Proc Natl Acad Sci USA 100(19):11127–11132. doi:10.1073/pnas.1836901100
Circelli P, Donini M, Villani ME, Benvenuto E, Marusic C (2010) Efficient Agrobacterium-based transient expression system for the production of biopharmaceuticals in plants. Bioeng Bugs 1(3):221–224 doi:10.4161/bbug.1.3.11722
Coego A, Brizuela E, Castillejo P, Ruíz S, Koncz C, del Pozo JC, Piñeiro M, Jarillo JA, Paz-Ares J, León J, The TC (2014) The TRANSPLANTA collection of Arabidopsis lines: a resource for functional analysis of transcription factors based on their conditional overexpression. Plant J 77(6):944–953. doi:10.1111/tpj.12443
Cardi T, Lenzi P, Maliga P (2010) Chloroplasts as expression platforms for plant-produced vaccines. Expert Rev Vaccines 9(8):893–911. doi:10.1586/erv.10.78
Clark DP, Pazdernik NJ (2013) Regulation of transcription in eukaryotes molecular biology, 2nd edn, Chap. e17. Academic Press, Boston, pp e374–e378
Coleman JR, Papamichail D, Yano M, Garcia-Suarez Mdel M, Pirofski LA (2011) Designed reduction of Streptococcus pneumoniae pathogenicity via synthetic changes in virulence factor codon-pair bias. J Infect Dis 203(9):1264–1273. doi:10.1093/infdis/jir010
Conrad U, Fiedler U (1998) Compartment-specific accumulation of recombinant immunoglobulins in plant cells: an essential tool for antibody production and immunomodulation of physiological functions and pathogen activity. Plant Mol Biol 38(1–2):101–109
Croft MT, Moulin M, Webb ME, Smith AG (2007) Thiamine biosynthesis in algae is regulated by riboswitches. Proc Natl Acad Sci USA 104(52):20770–20775 doi:10.1073/pnas.0705786105
da Cunha NB, Murad A, Vianna G, Rech E (2014) Recombinant biosynthesis of functional human growth hormone and coagulation factor IX in transgenic soybean seeds. BMC Proceed 8(4):P112 doi:10.1186/1753-6561-8-s4-p112
Daniell H, Lee SB, Panchal T, Wiebe PO (2001) Expression of the native cholera toxin B subunit gene and assembly as functional oligomers in transgenic tobacco chloroplasts. J Mol Biol 311(5):1001–1009. doi:10.1006/jmbi.2001.4921
del Campo EM (2009) Post-transcriptional control of chloroplast gene expression. Gene Regul Syst Biol 3:31–47
Doran PM (2006) Foreign protein degradation and instability in plants and plant tissue cultures. Trends Biotechnol 24(9):426–432. doi:10.1016/j.tibtech.2006.06.012
dos Reis M, Savva R, Wernisch L (2004) Solving the riddle of codon usage preferences: a test for translational selection. Nucleic Acids Res 32(17):5036–5044. doi:10.1093/nar/gkh834
Dufourmantel N, Tissot G, Garcon F, Pelissier B, Dubald M (2006) Stability of soybean recombinant plastome over six generations. Transgenic Res 15(3):305–311. doi:10.1007/s11248-005-5262-0
Duret L (2002) Evolution of synonymous codon usage in metazoans. Curr Opin Genet Dev 12(6):640–649
Egelkrout E, Rajan V, Howard JA (2012) Overproduction of recombinant proteins in plants. Plant Sci 184:83–101. doi:10.1016/j.plantsci.2011.12.005
Evans TC Jr, Xu MQ, Pradhan S (2005) Protein splicing elements and plants: from transgene containment to protein purification. Annu Rev Plant Biol 56:375–392. doi:10.1146/annurev.arplant.56.032604.144242
Evron T, Geyer BC, Cherni I, Muralidharan M, Kilbourne J, Fletcher SP, Soreq H, Mor TS (2007) Plant-derived human acetylcholinesterase-R provides protection from lethal organophosphate poisoning and its chronic aftermath. The FASEB J 21(11):2961–2969. doi:10.1096/fj.07-8112com
Farrell RE (2007) The regulation of gene expression in plants and animals. In: Bassett CL (ed) Regulation of gene expression in plants: the role of transcript structure and processing. Springer, Boston, pp 1–38
Faye L, Gomord V (2010) Success stories in molecular farming-a brief overview. Plant Biotechnol J 8(5):525–528. doi:10.1111/j.1467-7652.2010.00521.x
Fernandez-San Millan A, Mingo-Castel A, Miller M, Daniell H (2003) A chloroplast transgenic approach to hyper-express and purify human serum albumin, a protein highly susceptible to proteolytic degradation. Plant Biotechnol J 1(2):71–79. doi:10.1046/j.1467-7652.2003.00008.x
Fischer R, Emans N (2000) Molecular farming of pharmaceutical proteins. Transgenic Res 9(4–5):279–299 (discussion 277)
Fischer R, Schumann D, Zimmermann S, Drossard J, Sack M, Schillberg S (1999) Expression and characterization of bispecific single-chain Fv fragments produced in transgenic plants. Eur J Biochem 262(3):810–816
Fischer R, Schillberg S, Hellwig S, Twyman RM, Drossard J (2012) GMP issues for recombinant plant-derived pharmaceutical proteins. Biotechnol Adv 30(2):434–439. doi:10.1016/j.biotechadv.2011.08.007
Fletcher SP, Muto M, Mayfield SP (2007) Optimization of recombinant protein expression in the chloroplasts of green algae. Adv Exp Med Biol 616:90–98. doi:10.1007/978-0-387-75532-8_8
Franklin S, Ngo B, Efuet E, Mayfield SP (2002) Development of a GFP reporter gene for Chlamydomonas reinhardtii chloroplast. Plant J 30(6):733–744
Gaspar P, Moura G, Santos MAS, Oliveira JL (2013) mRNA secondary structure optimization using a correlated stem–loop prediction. Nucleic Acids Res. doi:10.1093/nar/gks1473
Giddings G, Allison G, Brooks D, Carter A (2000) Transgenic plants as factories for biopharmaceuticals. Nat Biotechnol 18(11):1151–1155. doi:10.1038/81132
Gion WR, Davis-Taber RA, Regier DA, Fung E, Medina L, Santora LC, Bose S, Ivanov AV, Perilli-Palmer BA, Chumsae CM, Matuck JG, Kunes YZ, Carson GR (2013) Expression of antibodies using single open reading frame (sORF) vector design: demonstration of manufacturing feasibility. mAbs 5(4):595–607. doi:10.4161/mabs.25161
Giritch A, Marillonnet S, Engler C, van Eldik G, Botterman J, Klimyuk V, Gleba Y (2006) Rapid high-yield expression of full-size IgG antibodies in plants coinfected with noncompeting viral vectors. Proc Natl Acad Sci USA 103(40):14701–14706. doi:10.1073/pnas.0606631103
Gisby MF, Mellors P, Madesis P, Ellin M, Laverty H, O’Kane S, Ferguson MW, Day A (2011) A synthetic gene increases TGFbeta3 accumulation by 75-fold in tobacco chloroplasts enabling rapid purification and folding into a biologically active molecule. Plant Biotechnol J 9(5):618–628. doi:10.1111/j.1467-7652.2011.00619.x
Gitzinger M, Parsons J, Reski R, Fussenegger M (2009) Functional cross-kingdom conservation of mammalian and moss (Physcomitrella patens) transcription, translation and secretion machineries. Plant Biotechnol J 7(1):73–86. doi:10.1111/j.1467-7652.2008.00376.x
Gould N, Hendy O, Papamichail D (2014) Computational tools and algorithms for designing customized synthetic genes. Front Bioeng Biotechnol 2:41. doi:10.3389/fbioe.2014.00041
Goulet C, Benchabane M, Anguenot R, Brunelle F, Khalf M, Michaud D (2010) A companion protease inhibitor for the protection of cytosol-targeted recombinant proteins in plants. Plant Biotechnol J 8(2):142–154. doi:10.1111/j.1467-7652.2009.00470.x
Grabowski GA, Golembo M, Shaaltiel Y (2014) Taliglucerase alfa: an enzyme replacement therapy using plant cell expression technology. Mol Genet Metab 112(1):1–8. doi:10.1016/j.ymgme.2014.02.011
Gray BN, Ahner BA, Hanson MR (2009) High-level bacterial cellulase accumulation in chloroplast-transformed tobacco mediated by downstream box fusions. Biotechnol Bioeng 102(4):1045–1054. doi:10.1002/bit.22156
Greer AL (2015) Early vaccine availability represents an important public health advance for the control of pandemic influenza. BMC Res Notes 8(1):191. doi:10.1186/s13104-015-1157-1
Gustafsson C, Govindarajan S, Minshull J (2004) Codon bias and heterologous protein expression. Trends Biotechnol 22(7):346–353. doi:10.1016/j.tibtech.2004.04.006
Habibi P, Grossi de Sa MF, Makhzoum A, Malik S, da Silva ALL, Hefferon K, Soccol CR (2017) Bioengineering hairy roots: phytoremediation, secondary metabolism, molecular pharming, plant-plant interactions and biofuels. In: Lichtfouse E (ed) Sustainable agriculture reviews. Springer, Cham, pp 213–251
Hauptmann V, Weichert N, Menzel M, Knoch D, Paege N, Scheller J, Spohn U, Conrad U, Gils M (2013) Native-sized spider silk proteins synthesized in planta via intein-based multimerization. Transgenic Res 22(2):369–377. doi:10.1007/s11248-012-9655-6
He J, Lai H, Brock C, Chen Q (2012) A novel system for rapid and cost-effective production of detection and diagnostic reagents of West Nile virus in plants. J Biomed Biotechnol 2012:106783. doi:10.1155/2012/106783
Hefferon KL (2012) Plant virus expression vectors set the stage as production platforms for biopharmaceutical proteins. Virology 433(1):1–6. doi:10.1016/j.virol.2012.06.012
Heitzer M, Eckert A, Fuhrmann M, Griesbeck C (2007) Influence of codon bias on the expression of foreign genes in microalgae. Adv Exp Med Biol 616:46–53. doi:10.1007/978-0-387-75532-8_5
Hennecke M, Kwissa M, Metzger K, Oumard A, Kroger A, Schirmbeck R, Reimann J, Hauser H (2001) Composition and arrangement of genes define the strength of IRES-driven translation in bicistronic mRNAs. Nucleic Acids Res 29(16):3327–3334
Herz S, Fussl M, Steiger S, Koop HU (2005) Development of novel types of plastid transformation vectors and evaluation of factors controlling expression. Transgenic Res 14(6):969–982. doi:10.1007/s11248-005-2542-7
Hieno A, Naznin HA, Hyakumachi M, Sakurai T, Tokizawa M, Koyama H, Sato N, Nishiyama T, Hasebe M, Zimmer AD, Lang D, Reski R, Rensing SA, Obokata J, Yamamoto YY (2014) ppdb: plant promoter database version 3.0. Nucleic Acids Res 42(database issue):D1188–D1192. doi:10.1093/nar/gkt1027
Horvath H, Huang J, Wong O, Kohl E, Okita T, Kannangara CG, von Wettstein D (2000) The production of recombinant proteins in transgenic barley grains. Proc Natl Acad Sci USA 97(4):1914–1919. doi:10.1073/pnas.030527497
Houdebine LM, Attal J (1999) Internal ribosome entry sites (IRESs): reality and use. Transgenic Res 8(3):157–177
Huang Z, Chen Q, Hjelm B, Arntzen C, Mason H (2009) A DNA replicon system for rapid high-level production of virus-like particles in plants. Biotechnol Bioeng 103(4):706–714. doi:10.1002/bit.22299
Huang Z, Phoolcharoen W, Lai H, Piensook K, Cardineau G, Zeitlin L, Whaley KJ, Arntzen CJ, Mason HS, Chen Q (2010) High-level rapid production of full-size monoclonal antibodies in plants by a single-vector DNA replicon system. Biotechnol Bioeng 106(1):9–17. doi:10.1002/bit.22652
Ikemura T (1981) Correlation between the abundance of Escherichia coli transfer RNAs and the occurrence of the respective codons in its protein genes: a proposal for a synonymous codon choice that is optimal for the E. coli translational system. J Mol Biol 151(3):389–409
Irwin B, Heck JD, Hatfield GW (1995) Codon pair utilization biases influence translational elongation step times. J Biol Chem 270(39):22801–22806
Ishikura K, Takaoka Y, Kato K, Sekine M, Yoshida K, Shinmyo A (1999) Expression of a foreign gene in Chlamydomonas reinhardtii chloroplast. J Biosci Bioeng 87(3):307–314. doi:10.1016/S1389-1723(99)80037-1
James EA, Wang C, Wang Z, Reeves R, Shin JH, Magnuson NS, Lee JM (2000) Production and characterization of biologically active human GM-CSF secreted by genetically modified plant cells. Protein Expr Purif 19(1):131–138. doi:10.1006/prep.2000.1232
Kang HG, Fang Y, Singh KB (1999) A glucocorticoid-inducible transcription system causes severe growth defects in Arabidopsis and induces defense-related genes. Plant J 20(1):127–133
Kang MS, Prasanta KS, Baisakh N, Priyadarshan PM (2008) Crop breeding methodologies: classic and modern. In: Kang MS, Priyadarshan PM (eds) Breeding major food staples. Wiley, Hoboken, pp 5–40
Keasling JD (2012) Synthetic biology and the development of tools for metabolic engineering. Metab Eng 14(3):189–195. doi:10.1016/j.ymben.2012.01.004
Kim M-Y, Yang M-S, Kim T-G (2009) Expression of dengue virus E glycoprotein domain III in non-nicotine transgenic tobacco plants. Biotechnol Bioprocess Eng 14(6):725–730 doi:10.1007/s12257-009-3011-6
Kim HJ, Lee SJ, Kim HJ (2010) Optimizing the secondary structure of human papillomavirus type 16 L1 mRNA enhances L1 protein expression in Saccharomyces cerevisiae. J Biotechnol 150(1):31–36. doi:10.1016/j.jbiotec.2010.07.032
Kim Y, Lee G, Jeon E, Sohn EJ, Lee Y, Kang H, Lee DW, Kim DH, Hwang I (2014) The immediate upstream region of the 5′-UTR from the AUG start codon has a pronounced effect on the translational efficiency in Arabidopsis thaliana. Nucleic Acids Res 42(1):485–498. doi:10.1093/nar/gkt864
Kircheis R, Halanek N, Koller I, Jost W, Schuster M, Gorr G, Hajszan K, Nechansky A (2012) Correlation of ADCC activity with cytokine release induced by the stably expressed, glyco-engineered humanized Lewis Y-specific monoclonal antibody MB314. mAbs 4(4):532–541. doi:10.4161/mabs.20577
Kohli N, Westerveld DR, Ayache AC, Verma A, Shil P, Prasad T, Zhu P, Chan SL, Li Q, Daniell H (2014) Oral delivery of bioencapsulated proteins across blood-brain and blood-retinal barriers. Mol Ther 22(3):535–546. doi:10.1038/mt.2013.273
Komarova TV, Baschieri S, Donini M, Marusic C, Benvenuto E, Dorokhov YL (2010) Transient expression systems for plant-derived biopharmaceuticals. Expert Rev Vaccines 9(8):859–876. doi:10.1586/erv.10.85
Koul B, Yadav R, Sanyal I, Sawant S, Sharma V, Amla DV (2012) Cis-acting motifs in artificially synthesized expression cassette leads to enhanced transgene expression in tomato (Solanum lycopersicum L.). PPB 61:131–141 doi:10.1016/j.plaphy.2012.09.014
Kozak M (1999) Initiation of translation in prokaryotes and eukaryotes. Gene 234(2):187–208
Kubo M, Imai A, Nishiyama T, Ishikawa M, Sato Y, Kurata T, Hiwatashi Y, Reski R, Hasebe M (2013) System for stable β-estradiol-inducible gene expression in the moss Physcomitrella patens. PLoS ONE 8(9):e77356. doi:10.1371/journal.pone.0077356
Kunes YZ, Gion WR, Fung E, Salfeld JG, Zhu RR, Sakorafas P, Carson GR (2009) Expression of antibodies using single-open reading frame vector design and polyprotein processing from mammalian cells. Biotechnol Prog 25(3):735–744. doi:10.1002/btpr.182
Kwon K-C, Chan H-T, León IR, Williams-Carrier R, Barkan A, Daniell H (2016) Codon optimization to enhance expression yields insights into chloroplast translation. Plant Physiol 172(1):62–77. doi:10.1104/pp.16.00981
Laguía-Becher M, Martín V, Kraemer M, Corigliano M, Yacono ML, Goldman A, Clemente M (2010) Effect of codon optimization and subcellular targeting on Toxoplasma gondii antigen SAG1 expression in tobacco leaves to use in subcutaneous and oral immunization in mice. BMC Biotechnol 10(1):1–14. doi:10.1186/1472-6750-10-52
Lai H, Chen Q (2012) Bioprocessing of plant-derived virus-like particles of Norwalk virus capsid protein under current good manufacture practice regulations. Plant Cell Rep 31(3):573–584. doi:10.1007/s00299-011-1196-6
Lai H, Engle M, Fuchs A, Keller T, Johnson S, Gorlatov S, Diamond MS, Chen Q (2010) Monoclonal antibody produced in plants efficiently treats West Nile virus infection in mice. Proc Natl Acad Sci USA 107(6):2419–2424. doi:10.1073/pnas.0914503107
Lakshmi PS, Verma D, Yang X, Lloyd B, Daniell H (2013) Low cost tuberculosis vaccine antigens in capsules: expression in chloroplasts, bio-encapsulation, stability and functional evaluation in vitro. PLoS ONE 8(1):e54708. doi:10.1371/journal.pone.0054708
Lee ER, Baker JL, Weinberg Z, Sudarsan N, Breaker RR (2010) An allosteric self-splicing ribozyme triggered by a bacterial second messenger. Science 329(5993):845–848. doi:10.1126/science.1190713
Leelavathi S, Gupta N, Maiti S, Ghosh A, Reddy VS (2003) Overproduction of an alkali- and thermo-stable xylanase in tobacco chloroplasts and efficient recovery of the enzyme. Mol Breed 11(1):59–67. doi:10.1023/A:1022168321380
Li H, Yang J, Chen Y, Guan L, Du L, Guo Y, Wang W, Wang L, Li H, Jiang C, Li X (2014) Expression of a functional recombinant oleosin-human hyaluronidase hPH-20 fusion in Arabidopsis thaliana. Protein Expr Purif 103:23–27. doi:10.1016/j.pep.2014.03.007
Lico C, Chen Q, Santi L (2008) Viral vectors for production of recombinant proteins in plants. J Cell Physiol 216(2):366–377. doi:10.1002/jcp.21423
Lindgreen S (2012) Codon evolution: mechanisms and models. Trends Evol Biol 4(1):8. doi:10.4081/eb.2012.e8
Liu Q, Xue Q (2005) Comparative studies on codon usage pattern of chloroplasts and their host nuclear genes in four plant species. J Genet 84(1):55–62
Lopez-Lastra M, Rivas A, Barria MI (2005) Protein synthesis in eukaryotes: the growing biological relevance of cap-independent translation initiation. Biol Res 38(2–3):121–146
Lu J, Sivamani E, Azhakanandam K, Samadder P, Li X, Qu R (2008) Gene expression enhancement mediated by the 5′ UTR intron of the rice rubi3 gene varied remarkably among tissues in transgenic rice plants. Mol Genet Genom 279(6):563–572. doi:10.1007/s00438-008-0333-6
Luke GA, Roulston C, Tilsner J. Ryan MD (2015) Growing uses of 2A in plant biotechnology. In: Ekinci D (ed) Biotechnology. InTech Croatia, Rijeka, pp 165–193
Mallory AC, Parks G, Endres MW, Baulcombe D, Bowman LH, Pruss GJ, Vance VB (2002) The amplicon-plus system for high-level expression of transgenes in plants. Nat Biotechnol 20(6):622–625. doi:10.1038/nbt0602-622
Mann DGJ, King ZR, Liu W, Joyce BL, Percifield RJ, Hawkins JS, LaFayette PR, Artelt BJ, Burris JN, Mazarei M, Bennetzen JL, Parrott WA, Stewart CN (2011) Switchgrass (Panicum virgatum L.) polyubiquitin gene (PvUbi1 and PvUbi2) promoters for use in plant transformation. BMC Biotechnol 11:74–74. doi:10.1186/1472-6750-11-74
Mao Y, Liu H, Liu Y, Tao S (2014) Deciphering the rules by which dynamics of mRNA secondary structure affect translation efficiency in Saccharomyces cerevisiae. Nucleic Acids Res. doi:10.1093/nar/gku159
Michelet L, Lefebvre-Legendre L, Burr SE, Rochaix JD, Goldschmidt-Clermont M (2011) Enhanced chloroplast transgene expression in a nuclear mutant of Chlamydomonas. Plant Biotechnol J 9(5):565–574. doi:10.1111/j.1467-7652.2010.00564.x
Mohammadzadeh S, Roohvand F, Ajdary S, Ehsani P, Hatef Salmanian A (2015) Heterologous expression of hepatitis C virus core protein in oil seeds of Brassica napus L. Jundishapur. J Microbiol 8(11):e25462. doi:10.5812/jjm.25462
Monreal-Escalante E, Banuelos-Hernandez B, Hernandez M, Fragoso G, Garate T, Sciutto E, Rosales-Mendoza S (2015) Expression of multiple taenia solium immunogens in plant cells through a ribosomal skip mechanism. Mol Biotechnol 57(7):635–643. doi:10.1007/s12033-015-9853-6
Morandini P (2013) Control limits for accumulation of plant metabolites: brute force is no substitute for understanding. Plant Biotechnol J 11(2):253–67. doi:10.1111/pbi.12035
Morello L, Gianì S, Troina F, Breviario D (2011) Testing the IMEter on rice introns and other aspects of intron-mediated enhancement of gene expression. J Exp Bot 62(2):533–544. doi:10.1093/jxb/erq273
Moustafa K, Makhzoum A, Tremouillaux-Guiller J (2015) Molecular farming on rescue of pharma industry for next generations. Crit Rev Biotechnol. doi:10.3109/07388551.2015.1049934
Mozes-Koch R, Gover O, Tanne E, Peretz Y, Maori E, Chernin L, Sela I (2012) Expression of an entire bacterial operon in plants. Plant Physiol 158(4):1883–1892. doi:10.1104/pp.111.186197
Mueller S, Coleman JR, Papamichail D, Ward CB, Nimnual A, Futcher B, Skiena S, Wimmer E (2010) Live attenuated influenza virus vaccines by computer-aided rational design. Nat Biotechnol 28(7):723–726. doi:10.1038/nbt0.1636
Muller K, Siegel D, Rodriguez Jahnke F, Gerrer K, Wend S, Decker EL, Reski R, Weber W, Zurbriggen MD (2014) A red light-controlled synthetic gene expression switch for plant systems. Mol Biosyst 10(7):1679–1688. doi:10.1039/c3mb70579j
Naqvi S, Farre G, Sanahuja G, Capell T, Zhu C, Christou P (2010) When more is better: multigene engineering in plants. Trends Plant Sci 15(1):48–56. doi:10.1016/j.tplants.2009.09.010
Niederkrüger H, Dabrowska-Schlepp P, Schaaf A (2014) Suspension culture of plant cells under phototrophic conditions industrial scale suspension culture of living cells. Wiley, New York, pp 259–292
Nochi T, Takagi H, Yuki Y, Yang L, Masumura T, Mejima M, Nakanishi U, Matsumura A, Uozumi A, Hiroi T, Morita S, Tanaka K, Takaiwa F, Kiyono H (2007) Rice-based mucosal vaccine as a global strategy for cold-chain- and needle-free vaccination. Proc Natl Acad Sci USA 104(26):10986–10991. doi:10.1073/pnas.0703766104
Nuttall J, Vine N, Hadlington JL, Drake P, Frigerio L, Ma JK (2002) ER-resident chaperone interactions with recombinant antibodies in transgenic plants. Eur J Biochem 269(24):6042–6051
O’Brien KM, Schufreider AK, McGill MA, O’Brien KM, Reitter JN, Mills KV (2010) Mechanism of protein splicing of the Pyrococcus abyssi lon protease intein. Biochem Biophys Res Commun 403(3–4):457–461. doi:10.1016/j.bbrc.2010.11.055
O’Keefe BR, Murad AM, Vianna GR, Ramessar K, Saucedo CJ, Wilson J, Buckheit KW, da Cunha NB, Araújo ACG, Lacorte CC, Madeira L, McMahon JB, Rech EL (2015) Engineering soya bean seeds as a scalable platform to produce cyanovirin-N, a non-ARV microbicide against HIV. Plant Biotechnol J 13(7):884–892. doi:10.1111/pbi.12309
Obembe OO, Popoola JO, Leelavathi S, Reddy SV (2011) Advances in plant molecular farming. Biotechnol Adv 29(2):210–222. doi:10.1016/j.biotechadv.2010.11.004
Orellana-Escobedo L, Rosales-Mendoza S, Romero-Maldonado A, Parsons J, Decker EL, Monreal-Escalante E, Moreno-Fierros L, Reski R (2015) An Env-derived multi-epitope HIV chimeric protein produced in the moss Physcomitrella patens is immunogenic in mice. Plant Cell Rep 34(3):425–433. doi:10.1007/s00299-014-1720-6
Osbourn AE, Field B (2009) Operons. Cell Mol Life Sci 66(23):3755–3775. doi:10.1007/s00018-009-0114-3
Palidwor GA, Perkins TJ, Xia X (2010) A general model of codon bias due to GC mutational bias. PLoS ONE 5(10):e13431. doi:10.1371/journal.pone.0013431
Park C, Chen X, Yang J-R, Zhang J (2013) Differential requirements for mRNA folding partially explain why highly expressed proteins evolve slowly. Proc Natl Acad Sci USA 110(8):E678–E686. doi:10.1073/pnas.1218066110
Parsons J, Altmann F, Arrenberg CK, Koprivova A, Beike AK, Stemmer C, Gorr G, Reski R, Decker EL (2012) Moss-based production of asialo-erythropoietin devoid of Lewis A and other plant-typical carbohydrate determinants. Plant Biotechnol J 10(7):851–861. doi:10.1111/j.1467-7652.2012.00704.x
Paul M, van Dolleweerd C, Drake PM, Reljic R, Thangaraj H, Barbi T, Stylianou E, Pepponi I, Both L, Hehle V, Madeira L, Inchakalody V, Ho S, Guerra T, Ma JK (2011) Molecular pharming: future targets and aspirations. Hum Vaccin 7(3):375–382
Paul MJ, Teh AY, Twyman RM, Ma JK (2013) Target product selection: where can molecular pharming make the difference? Curr Pharm Des 19(31):5478–5485
Penchovsky R, Stoilova CC (2013) Riboswitch-based antibacterial drug discovery using high-throughput screening methods. Expert Opin Drug Discov 8(1):65–82. doi:10.1517/17460441.2013.740455
Phoolcharoen W, Bhoo SH, Lai H, Ma J, Arntzen CJ, Chen Q, Mason HS (2011) Expression of an immunogenic Ebola immune complex in Nicotiana benthamiana. Plant Biotechnol J 9(7):807–816. doi:10.1111/j.1467-7652.2011.00593.x
Pillay P, Schluter U, van Wyk S, Kunert KJ, Vorster BJ (2014) Proteolysis of recombinant proteins in bioengineered plant cells. Bioengineered 5(1):15–20. doi:10.4161/bioe.25158
Plotkin JB, Kudla G (2011) Synonymous but not the same: the causes and consequences of codon bias. Nat Rev Genet 12(1):32–42
Pogue GP, Vojdani F, Palmer KE, Hiatt E, Hume S, Phelps J, Long L, Bohorova N, Kim D, Pauly M, Velasco J, Whaley K, Zeitlin L, Garger SJ, White E, Bai Y, Haydon H, Bratcher B (2010) Production of pharmaceutical-grade recombinant aprotinin and a monoclonal antibody product using plant-based transient expression systems. Plant Biotechnol J 8(5):638–654
Poirier Y (2001) Production of polyesters in transgenic plants. In: Babel W, Steinbüchel A (eds) Biopolyesters. Springer, Berlin, pp 209–240
Rademacher T, Sack M, Arcalis E, Stadlmann J, Balzer S, Altmann F, Quendler H, Stiegler G, Kunert R, Fischer R, Stoger E (2008) Recombinant antibody 2G12 produced in maize endosperm efficiently neutralizes HIV-1 and contains predominantly single-GlcNAc N-glycans. Plant Biotechnol J 6(2):189–201. doi:10.1111/j.1467-7652.2007.00306.x
Ramirez N, Lorenzo D, Palenzuela D, Herrera L, Ayala M, Fuentes A, Perez M, Gavilondo J, Oramas P (2000) Single-chain antibody fragments specific to the hepatitis B surface antigen, produced in recombinant tobacco cell cultures. Biotechnol Lett 22(15):1233–1236. doi:10.1023/A:1005605214703
Reski R, Parsons J, Decker EL (2015) Moss-made pharmaceuticals: from bench to bedside. Plant Biotechnol J 13(8):1191–1198. doi:10.1111/pbi.12401
Rigano MM, Alvarez ML, Pinkhasov J, Jin Y, Sala F, Arntzen CJ, Walmsley AM (2004) Production of a fusion protein consisting of the enterotoxigenic Escherichia coli heat-labile toxin B subunit and a tuberculosis antigen in Arabidopsis thaliana. Plant Cell Rep 22(7):502–508. doi:10.1007/s00299-003-0718-2
Ronald PC (2007) Plant-pathogen interactions: methods and protocols, vol 354. Human Press, Totowa
Rosales-Mendoza S, Soria-Guerra RE, Moreno-Fierros L, Alpuche-Solis AG, Martinez-Gonzalez L, Korban SS (2010) Expression of an immunogenic F1-V fusion protein in lettuce as a plant-based vaccine against plague. Planta 232(2):409–416. doi:10.1007/s00425-010-1176-z
Rose AB (2008) Intron-mediated regulation of gene expression. Curr Top Microbiol Immunol 326:277–290
Rose AB, Emami S, Bradnam K, Korf I (2011) Evidence for a DNA-based mechanism of intron-mediated enhancement. Front Plant Sci 2:98. doi:10.3389/fpls.2011.00098
Schillberg S, Zimmermann S, Voss A, Fischer R (1999) Apoplastic and cytosolic expression of full-size antibodies and antibody fragments in Nicotiana tabacum. Transgenic Res 8(4):255–263. doi:10.1023/A:1008937011213
Schünmann PHD, Surin B, Waterhouse PM (2003) A suite of novel promoters and terminators for plant biotechnology. II. The pPLEX series for use in monocots. Funct Plant Biol 30(4):453–460. doi:10.1071/FP02167
Serres-Giardi L, Belkhir K, David J, Glemin S (2012) Patterns and evolution of nucleotide landscapes in seed plants. Plant Cell 24(4):1379–1397. doi:10.1105/tpc.111.093674
Sharp PM, Li WH (1987) The codon adaptation index: a measure of directional synonymous codon usage bias, and its potential applications. Nucleic Acids Res 15(3):1281–1295
Sharp PM, Tuohy TM, Mosurski KR (1986) Codon usage in yeast: cluster analysis clearly differentiates highly and lowly expressed genes. Nucleic Acids Res 14(13):5125–5143
Shinmyo A, Kato K (2010) Molecular farming: production of drugs and vaccines in higher plants. J Antibiot 63(8):431–433
Sijmons PC, Dekker BM, Schrammeijer B, Verwoerd TC, van den Elzen PJ, Hoekema A (1990) Production of correctly processed human serum albumin in transgenic plants. Nat Biotechnol 8(3):217–221
Silverman IM, Li F, Gregory BD (2013) Genomic era analyses of RNA secondary structure and RNA-binding proteins reveal their significance to post-transcriptional regulation in plants. Plant Sci 205–206:55–62. doi:10.1016/j.plantsci.2013.01.009
Simon AE, Miller WA (2013) 3′ cap-independent translation enhancers of plant viruses. Annu Rev Microbiol 67:21–42. doi:10.1146/annurev-micro-092412-155609
Specht EA, Mayfield SP (2014) Algae-based oral recombinant vaccines. Front Microbiol 5:60. doi:10.3389/fmicb.2014.00060
Specht E, Miyake-Stoner S, Mayfield S (2010) Micro-algae come of age as a platform for recombinant protein production. Biotechnol Lett 32(10):1373–1383. doi:10.1007/s10529-010-0326-5
Staub JM, Garcia B, Graves J, Hajdukiewicz PT, Hunter P, Nehra N, Paradkar V, Schlittler M, Carroll JA, Spatola L, Ward D, Ye G, Russell DA (2000) High-yield production of a human therapeutic protein in tobacco chloroplasts. Nat Biotechnol 18(3):333–338. doi:10.1038/73796
Stavolone L, Kononova M, Pauli S, Ragozzino A, de Haan P, Milligan S, Lawton K, Hohn T (2003) Cestrum yellow leaf curling virus (CmYLCV) promoter: a new strong constitutive promoter for heterologous gene expression in a wide variety of crops. Plant Mol Biol 53(5):663–673. doi:10.1023/B:PLAN.0000019110.95420.bb
Stern DB, Goldschmidt-Clermont M, Hanson MR (2010) Chloroplast RNA metabolism. Annu Rev Plant Biol 61:125–155. doi:10.1146/annurev-arplant-042809-112242
Stoger E, Fischer R, Moloney M, Ma JK (2014) Plant molecular pharming for the treatment of chronic and infectious diseases. Annu Rev Plant Biol 65:743–768. doi:10.1146/annurev-arplant-050213-035850
Sun Y, Ye SH, Lu HW (2012) Study of RNA secondary structure prediction algorithms. In: Advanced materials research, vol 393. Trans Tech Publications, Zurich, pp 955–960
Sunil Kumar GB, Ganapathi TR, Srinivas L, Revathi CJ, Bapat VA (2006) Expression of hepatitis B surface antigen in potato hairy roots. Plant Sci 170(5):918–925. doi:10.1016/j.plantsci.2005.12.015
Suzuki H, Saito R, Tomita M (2004) The ‘weighted sum of relative entropy’: a new index for synonymous codon usage bias. Gene 335:19–23. doi:10.1016/j.gene.2004.03.001
Suzuki K, Yang L, Takaiwa F (2012) Transgenic rice accumulating modified cedar pollen allergen Cry j 2 derivatives. J Biosci Bioeng 113(2):249–251. doi:10.1016/j.jbiosc.2011.10.005
Tacket CO, Pasetti MF, Edelman R, Howard JA, Streatfield S (2004) Immunogenicity of recombinant LT-B delivered orally to humans in transgenic corn. Vaccine 22(31–32):4385–4389. doi:10.1016/j.vaccine.2004.01.073
Takagi H, Hiroi T, Yang L, Tada Y, Yuki Y, Takamura K, Ishimitsu R, Kawauchi H, Kiyono H, Takaiwa F (2005) A rice-based edible vaccine expressing multiple T cell epitopes induces oral tolerance for inhibition of Th2-mediated IgE responses. Proc Natl Acad Sci USA 102(48):17525–17530. doi:10.1073/pnas.0503428102
Takagi H, Hiroi T, Hirose S, Yang L, Takaiwa F (2010) Rice seed ER-derived protein body as an efficient delivery vehicle for oral tolerogenic peptides. Peptides 31(8):1421–1425. doi:10.1016/j.peptides.2010.04.032
Takaiwa F (2009) Allergen-specific immunotherapy with plant-based oral vaccines. Immunotherapy 1(4):517–9. doi:10.2217/imt.09.28
Takaiwa F (2011) Seed-based oral vaccines as allergen-specific immunotherapies. Hum Vaccin 7(3):357–366
Tang L, Cui T, Wu JJ, Liu-Mares W, Huang N, Li J (2010) A rice-derived recombinant human lactoferrin stimulates fibroblast proliferation, migration, and sustains cell survival. Wound Repair Regen 18(1):123–131. doi:10.1111/j.1524-475X.2009.00563.x
Thomas DR, Walmsley AM (2015) The effect of the unfolded protein response on the production of recombinant proteins in plants. Plant Cell Rep 34(2):179–187. doi:10.1007/s00299-014-1680-x
Thuenemann EC, Meyers AE, Verwey J, Rybicki EP, Lomonossoff GP (2013) A method for rapid production of heteromultimeric protein complexes in plants: assembly of protective bluetongue virus-like particles. Plant Biotechnol J 11(7):839–846. doi:10.1111/pbi.12076
Torrent M, Llompart B, Lasserre-Ramassamy S, Llop-Tous I, Bastida M, Marzabal P, Westerholm-Parvinen A, Saloheimo M, Heifetz PB, Ludevid MD (2009) Eukaryotic protein production in designed storage organelles. BMC Biol 7:5. doi:10.1186/1741-7007-7-5
Torres E, Vaquero C, Nicholson L, Sack M, Stoger E, Drossard J, Christou P, Fischer R, Fischer R, Perrin Y (1999) Rice cell culture as an alternative production system for functional diagnostic and therapeutic antibodies. Transgenic Res 8(6):441–449. doi:10.1023/A:1008969031219
Tregoning JS, Nixon P, Kuroda H, Svab Z, Clare S, Bowe F, Fairweather N, Ytterberg J, van Wijk KJ, Dougan G, Maliga P (2003) Expression of tetanus toxin fragment C in tobacco chloroplasts. Nucleic Acids Res 31(4):1174–1179. doi:10.1093/nar/gkg221
Twyman RM, Schillberg S, Fischer R (2013) Optimizing the yield of recombinant pharmaceutical proteins in plants. Curr Pharm Des 19(31):5486–5494
Twyman RM, Stoger E, Schillberg S, Christou P, Fischer R (2003) Molecular farming in plants: host systems and expression technology. Trends Biotechnol 21(12):570–578. doi:10.1016/j.tibtech.2003.10.002
Ullrich KK, Hiss M, Rensing SA (2015) Means to optimize protein expression in transgenic plants. Curr Opin Biotechnol 32:61–67. doi:10.1016/j.copbio.2014.11.011
Vamvaka E, Arcalis E, Ramessar K, Evans A, O’Keefe BR, Shattock RJ, Medina V, Stöger E, Christou P, Capell T (2016a) Rice endosperm is cost-effective for the production of recombinant griffithsin with potent activity against HIV. Plant Biotechnol J 14(6):1427–1437. doi:10.1111/pbi.12507
Vamvaka E, Twyman RM, Murad AM, Melnik S, Teh AY-H, Arcalis E, Altmann F, Stoger E, Rech E, Ma JKC, Christou P, Capell T (2016b) Rice endosperm produces an underglycosylated and potent form of the HIV-neutralizing monoclonal antibody 2G12. Plant Biotechnol J 14(1):97–108. doi:10.1111/pbi.12360
Wachter A (2014) Gene regulation by structured mRNA elements. Trends Genet 30(5):172–181. doi:10.1016/j.tig.2014.03.001
Wachter A, Tunc-Ozdemir M, Grove BC, Green PJ, Shintani DK, Breaker RR (2007) Riboswitch control of gene expression in plants by splicing and alternative 3′ end processing of mRNAs. Plant Cell 19(11):3437–3450. doi:10.1105/tpc.107.053645
Wan XF, Xu D, Kleinhofs A, Zhou J (2004) Quantitative relationship between synonymous codon usage bias and GC composition across unicellular genomes. BMC Evol Biol 4:19. doi:10.1186/1471-2148-4-19
Wang L, Wessler SR (2001) Role of mRNA secondary structure in translational repression of the maize transcriptional activator Lc(1,2). Plant Physiol 125(3):1380–1387
Wang DM, Zhu JB, Peng M, Zhou P (2008) Induction of a protective antibody response to FMDV in mice following oral immunization with transgenic Stylosanthes spp. as a feedstuff additive. Transgenic Res 17(6):1163–1170. doi:10.1007/s11248-008-9188-1
Weise A, Rodriguez-Franco M, Timm B, Hermann M, Link S, Jost W, Gorr G (2006) Use of Physcomitrella patens actin 5′ regions for high transgene expression: importance of 5′ introns. Adv Appl Microbiol 70(3):337–345. doi:10.1007/s00253-005-0087-6
Winkler WC, Nahvi A, Roth A, Collins JA, Breaker RR (2004) Control of gene expression by a natural metabolite-responsive ribozyme. Nature 428(6980):281–286
Wolfson W (2013) Grow your own: protalix biotherapeutics produces drugs in carrot cells. Chemistry Biology 20(8):969–970. doi:10.1016/j.chembiol.2013.08.003
Woodrow KA, Bennett KM, Lo DD (2012) Mucosal vaccine design and delivery. Annu Rev Biomed Eng 14:17–46. doi:10.1146/annurev-bioeng-071811-150054
Xie P (2015) Dwell-time distribution, long pausing and arrest of single-ribosome translation through the mRNA duplex. Int J Mol Sci 16(10):23723–23744. doi:10.3390/ijms161023723
Yang J, Barr LA, Fahnestock SR, Liu ZB (2005) High yield recombinant silk-like protein production in transgenic plants through protein targeting. Transgenic Res 14(3):313–324
Yang J, Guan L, Guo Y, Du L, Wang F, Wang Y, Zhen L, Wang Q, Zou D, Chen W, Yu L, Li H, Li X (2015) Expression of biologically recombinant human acidic fibroblast growth factor in Arabidopsis thaliana seeds via oleosin fusion technology. Gene 566(1):89–94. doi:10.1016/j.gene.2015.04.036
Youm JW, Jeon JH, Kim H, Kim YH, Ko K, Joung H, Kim H (2008) Transgenic tomatoes expressing human beta-amyloid for use as a vaccine against Alzheimer’s disease. Biotechnol Lett 30(10):1839–1845. doi:10.1007/s10529-008-9759-5
Zhang D, Nandi S, Bryan P, Pettit S, Nguyen D, Santos MA, Huang N (2010) Expression, purification, and characterization of recombinant human transferrin from rice (Oryza sativa L.). Protein Expr Purif 74(1):69–79. doi:10.1016/j.pep.2010.04.019
Zimran A, Brill-Almon E, Chertkoff R, Petakov M, Blanco-Favela F, Munoz ET, Solorio-Meza SE, Amato D, Duran G, Giona F, Heitner R, Rosenbaum H, Giraldo P, Mehta A, Park G, Phillips M, Elstein D, Altarescu G, Szleifer M, Hashmueli S, Aviezer D (2011) Pivotal trial with plant cell-expressed recombinant glucocerebrosidase, taliglucerase alfa, a novel enzyme replacement therapy for Gaucher disease. Blood 118(22):5767–5773. doi:10.1182/blood-2011-07-366955
Zingaro KA, Papoutsakis ET (2012) Toward a semisynthetic stress response system to engineer microbial solvent tolerance. MBio. doi:10.1128/mBio.00308-12
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Habibi, P., Prado, G.S., Pelegrini, P.B. et al. Optimization of inside and outside factors to improve recombinant protein yield in plant. Plant Cell Tiss Organ Cult 130, 449–467 (2017). https://doi.org/10.1007/s11240-017-1240-5
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DOI: https://doi.org/10.1007/s11240-017-1240-5