Establishment of Miscanthus sinensis with decreased lignin biosynthesis by Agrobacterium–mediated transformation using antisense COMT gene
- 105 Downloads
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.
KeywordsAgrobacterium-mediated transformation Antisense caffeic acid O-methyl-transferase (COMT-AS) Lignin biosynthesis Lignocellulosic complex Miscanthus sinensis
Caffeic acid O-methyltransferase
Antisense caffeic acid O-methyltransferase
Cetyl trimethyl ammonium bromide
High-performance liquid chromatography
Miscanthus induction callus medium
Miscanthus sinensis co-culture medium
Miscanthus sinensis elongation medium
Miscanthus sinensis rooting medium
Miscanthus sinensis shoot regeneration medium
- npt II
Neomycin phosphotransferase gene
Scanning electron microscope
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.
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.
Compliance with ethical standards
Conflict of interest
There are no conflicts of interest to declare.
- 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 226:1117–1129CrossRefPubMedGoogle 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 8:71–87CrossRefPubMedPubMedCentralGoogle Scholar
- Jang MS (2012) Variation in lignin, cellulose, and hemicellulose contents of Miscanthus species at different collection sites and harvest times. Dissertation, Konkuk UniversityGoogle Scholar
- Lee YN, Lee SB, Lee JD (2011) Characteristics of lignin removal in cellulosic ethanol production process. Appl Chem Eng 22:77–80Google 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 130:1675–1685CrossRefPubMedPubMedCentralGoogle 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 5:71CrossRefPubMedPubMedCentralGoogle 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 3:322–332CrossRefGoogle 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 UniversityGoogle 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 15:285–290Google Scholar