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Establishment of Miscanthus sinensis with decreased lignin biosynthesis by Agrobacterium–mediated transformation using antisense COMT gene

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Abstract

This study was to determine a transformation system for Miscanthus sinensis, and to optimize factors and conditions required for expression of an antisense caffeic acid O-methyltransferase gene in the M. sinensis (MsCOMT-AS). Transformation of callus derived from seeds and immature inflorescences of M. sinensis was established by using Agrobacterium tumefaciens strain LBA4404 harboring a binary vector pMBP1. In order to establish the stable transformation system, several transformation factors such as explant type, strain, co-culture periods, acetosyringone concentration, and selective markers were assessed. In this study, seven putative transgenic plants were obtained from callus transformation and plantlet regeneration. Various tests including PCR analysis and RT-PCR were used to detect the transgenic insert. The transgenic plants were also characterized for their agronomic and morphological characteristics, expression of MsCOMT-AS gene, and variation in lignocellulosic content. Biomass related traits such as plant height, number of leaves, length of leaf, stem diameter, fresh weight, dry weight, and cell size of the control plants were superior to transgenic plants. Total lignin content of transgenic plants was lower than that of the control plant due to reduced caffeic acid O-methyltransferase (COMT) gene expression related to lignin production. Cellulose and hemicellulose content in transgenic plants were not increased. Variation in cellulose and hemicellulose content had no correlation with variation in lignin content of transgenic plants. In conclusion, transgenic M. sinensis was obtained with down-regulated COMT gene. Lignin synthesis was decreased what offers possibility of crop modification for facilitated biofuel production.

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Abbreviations

AS:

Acetosyringone

COMT :

Caffeic acid O-methyltransferase

COMT-AS :

Antisense caffeic acid O-methyltransferase

CTAB:

Cetyl trimethyl ammonium bromide

HPLC:

High-performance liquid chromatography

Km:

Kanamycin

LB:

Luria–Bertani medium

MIC:

Miscanthus induction callus medium

MS-COM:

Miscanthus sinensis co-culture medium

MS-GSM:

Miscanthus sinensis elongation medium

MS-RM:

Miscanthus sinensis rooting medium

MS-SRM:

Miscanthus sinensis shoot regeneration medium

npt II:

Neomycin phosphotransferase gene

SEM:

Scanning electron microscope

References

  • Acker RV, Vanholme R, Storme V, Mortimer JC, Dupree P, Boerjan W (2013) Lignin biosynthesis perturbations affect secondary cell wall composition and saccharification yield in Arabidopsis thaliana. Biotechnol Biofuels 6:46

    Article  PubMed  PubMed Central  Google Scholar 

  • Barkat A, Carels N, Bernardi G (1997) The distribution of genes in the genome of Gramineae. Proc Natl Acad Sci USA 94:6857–6861

    Article  Google Scholar 

  • Chen L, Auh CK, Dowling P, Bell J, Lehmann D, Wang ZY (2004) Transgenic down-regulation of caffeic acid O-methyltransferase (COMT) led to improved digestibility in tall fescue (Festuca arundinacea L.). Funct Plant Biol 31:235–245

    Article  CAS  Google Scholar 

  • Danalatos NG, Archontoulis SV, Mitsios I (2007) Potential growth and biomass productivity of Miscanthus × giganteus as affected by plant density and N-fertilization in central Greece. Biomass Bioenergy 31:145–152

    Article  Google Scholar 

  • Do CT, Pollet B, Thevenin J, Sibout R, Denoue D, Barriere Y, Lapierre C, Jouanin L (2007) Both caffoyl coenzyme A 3-O-methyltransferase 1 and caffeic acid O-methyltransferase 1 are involved in redundant functions for lignin, flavonoids and sinapoyl malate biosynthesis in Arabidopsis. Planta 226:1117–1129

    Article  CAS  PubMed  Google Scholar 

  • Gray KA, Zhao LS, Emptage M (2006) Bioethanol. Curr Opin Plant Biol 10:141–146

    Article  CAS  Google Scholar 

  • Guillaumie S, Goffner D, Barbier O, Martinant JP, Pichon M, Barrière Y (2008) Expression of cell wall related genes in basal and ear internodes of silking brown-midrib-3, caffeic acid O-methyltransferase (COMT) down-regulated, and normal maize plants. BMC Plant Biol 8:71–87

    Article  PubMed  PubMed Central  Google Scholar 

  • Hisano H, Nandakumar R, Wang ZY (2009) Genetic modification of lignin biosynthesis for improved biofuel production. In Vitro Cell Dev Biol-Plant 45:306–313

    Article  CAS  Google Scholar 

  • Hwang OJ, Cho MA, Han YJ, Kim YM, Lim SH, Kim DS, Hwang I, Kim JI (2014) Agrobacterium-mediated genetic transformation of Miscanthus sinensis. Plant Cell Tiss Organ Cult 117:51–63

    Article  CAS  Google Scholar 

  • Jang MS (2012) Variation in lignin, cellulose, and hemicellulose contents of Miscanthus species at different collection sites and harvest times. Dissertation, Konkuk University

  • Kim SJ, Lee JY, Kim YM, Yang SS, Hwang OJ, Hong NJ, Kim KM, Lee HY, Song PS, Kim JI (2007) Agrobacterium-mediated high efficiency transformation of creeping bentgrass with herbicide resistance. J Plant Biol 50:577–585

    Article  CAS  Google Scholar 

  • Lee YN, Lee SB, Lee JD (2011) Characteristics of lignin removal in cellulosic ethanol production process. Appl Chem Eng 22:77–80

    CAS  Google Scholar 

  • Li X, Hu D, Luo M, Zhu M, Li X, Luo F, Li J, Yan J (2013) Nuclear DNA content variation of three Miscanthus species in China. Genes Genom 35:13–20

    Article  Google Scholar 

  • Murray MG, Thompson WF (1980) Rapid isolation of high molecular weight plant DNA. Nucleic Acids Res 8(19):4321–4325

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Piquemal J, Chamayou S, Nadaud I, Beckert M, Barriere Y, Mila I, Lapierre C, Rigau J, Puigdomenech P, Jauneau A, Digonnet C, Boudet AM, Goffner D, Pichon M (2002) Downregulation of caffeic acid O-methyltransferase in maize revisited using a transgenic approach. Plant Physiol 130:1675–1685

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Seong ES, Yoo JH, Kil HY, Lee JG, Yu CY (2010) Establishment of a regeneration system by callus induction from explants of Miscanthus sinensis. J Korean Soc Appl Biol Chem 53(6):661–667

    Article  Google Scholar 

  • Seong ES, Yoo JH, Lee JG, Kim HY, Hwang IS, Heo K, Kim JK, Lim JD, Sacks EJ, Yu CY (2013) Antisense-overexpression of the MsCOMT gene induces changes in lignin and total phenol contents in transgenic tobacco plants. Mol Biol Rep 40:1979–1986

    Article  CAS  PubMed  Google Scholar 

  • Tschaplinski TJ, Standaert RF, Engle NL, Martin MZ, Sangha AK, Parks JM, Smith JC, Samuel R, Jiang N, Pu Y, Ragauskas AJ, Hamilton CY, Fu C, Wang ZY, Davison BH, Dixon RA, Mielenz JR (2012) Down-regulation of the caffeic acid O-methyltransferase gene in switchgrass reveals a novel monolignol analog. Biotechnol Biofuels 5:71

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Vanholme R, Morreel K, Ralph J, Boerjan W (2008) Lignin engineering. Curr Opin Plant Biol 11:278–285

    Article  CAS  PubMed  Google Scholar 

  • Wang X, Yamada T, Kong FJ, Abe Y, Hoshino Y, Sato H, Takamizo T, Kanazawa A, Yamada T (2011) Establishment of an efficient in vitro culture and particle bombardment-mediated transformation systems in Miscanthus sinensis Anderss., a potential bioenergy crop. GCB Bioenergy 3:322–332

    Article  CAS  Google Scholar 

  • Weiting N, Nancy LP, Richard AD (1994) Reduced lignin in transgenic plants containing a caffeic acid O-methyltransferase antisense gene. Transgenic Res 3:120–126

    Article  Google Scholar 

  • Xi Y, Fu C, Ge Y, Nandakumar R, Hisano H, Bouton J, Wang ZY (2009) Agrobacterium-medaited transformation of Swichgrass and inheritance of the transgenes. Bioenerg Res 2:275–283

    Article  Google Scholar 

  • Yan J, Chen WL, Luo F, Ma HZ, Meng AP, Li XW, Zhu M, Li SS, Zhou HF, Zhu WX, Han B, GE S, Li JQ, Sang T (2012) Variability and adaptability of Miscanthus species evaluated for energy crop domestication. GCB Bioenergy 4:49–60

    Article  Google Scholar 

  • Yi SY, Kim JH, Joung YH, Lee S, Kim WT, Yu SH, Choi D (2004) The pepper transcription factor CaPF1 confers pathogen and freezing tolerance in Arabidopsis. Plant Physiol 136:2862–2874

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Yoo JH (2016) Establishment, expression and characterization of Miscanthus sinensis transformed by Agrobacterium tumefaciens using COMT gene related to lignin biosynthesis. Dissertation, Kangwon National University

  • Yuan JS, Tiller KH, Al-Ahmad H, Stewart NR, Stewart NC (2008) Plants to power: bioenergy to fuel the future. Trends Plant Sci 13:421–429

    Article  CAS  PubMed  Google Scholar 

  • Yun JS, Park JS, Kim IH, Hong EY, Yun T, Lee CH, Jeong JH, Yang DC (2007) Agrobacterium-mediated transformation of PAT into Platycodon grandifloirum A. De. Candolle. Korean J Med Crop Sci 15:285–290

    Google Scholar 

Download references

Acknowledgements

This study was funded by U.S. Department of Energy Office of Science, Office of Biological and Environmental Research (BER), Grant Nos. DE-SC0006634 and DE-SC0012379. This work was carried out with the support of “Cooperative Research Program for Agricultural Science & Technology Development (Project No. PJ012883), Republic of Korea. In part, this work was supported by the Bioherb Research Institute, Kangwon National University, Republic of Korea.

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Authors and Affiliations

  1. Bioherb Research Institute, Kangwon National University, Chuncheon, 24341, South Korea

    Ji Hye Yoo, Kweon Heo & Chang Yeon Yu

  2. Department of Bio-Resource Sciences, Kangwon National University, Chuncheon, 24341, South Korea

    Kweon Heo & Chang Yeon Yu

  3. Department of Medicinal Plant, Suwon Women’s University, Suwon, 16632, South Korea

    Eun Soo Seong

  4. Department of Applied Bioscience, Konkuk University, Seoul, 05029, South Korea

    Bimal Kumar Ghimire

  5. Department of Agronomy and the Key Laboratory of Crop Germplasm Resource of Zhejiang Province, Zhejiang University, Hangzhou, 310058, China

    Xiaoli Jin

  6. Field Science Center for Northern Biosphere, Hokkaido University, Sapporo, Hokkaido, 060-0810, Japan

    Toshihiko Yamada

  7. Department of Crop Sciences, University of Illinois at Urbana-Champaign, 1201 W. Gregory Drive, Urbana, IL, 61802, USA

    Lindsay V. Clark & Erik J. Sacks

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  1. Ji Hye Yoo
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  2. Eun Soo Seong
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  3. Bimal Kumar Ghimire
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  4. Kweon Heo
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  5. Xiaoli Jin
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  6. Toshihiko Yamada
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  7. Lindsay V. Clark
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  8. Erik J. Sacks
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  9. Chang Yeon Yu
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Contributions

JHY and ESS performed the experiments for isolation of gene, transformation of Miscanthus, and analysis of lignocellulosic contents in transgenic plants. KH, BKG and CYY prepared and collected samples (Miscanthus) from fields. XJ, TY and EJS made helpful comments on our work and manuscript. JHY wrote the manuscript and LVC correct the content of it. CYY conceived the study, and all authors read and approved the final manuscript.

Corresponding author

Correspondence to Chang Yeon Yu.

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There are no conflicts of interest to declare.

Additional information

Communicated by Sergio J. Ochatt.

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Yoo, J.H., Seong, E.S., Ghimire, B.K. et al. Establishment of Miscanthus sinensis with decreased lignin biosynthesis by Agrobacterium–mediated transformation using antisense COMT gene. Plant Cell Tiss Organ Cult 133, 359–369 (2018). https://doi.org/10.1007/s11240-018-1389-6

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  • DOI: https://doi.org/10.1007/s11240-018-1389-6

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