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Plant Cell, Tissue and Organ Culture (PCTOC)

, Volume 133, Issue 3, pp 359–369 | Cite as

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

  • Ji Hye Yoo
  • Eun Soo Seong
  • Bimal Kumar Ghimire
  • Kweon Heo
  • Xiaoli Jin
  • Toshihiko Yamada
  • Lindsay V. Clark
  • Erik J. Sacks
  • Chang Yeon Yu
Original Article
  • 354 Downloads

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.

Keywords

Agrobacterium-mediated transformation Antisense caffeic acid O-methyl-transferase (COMT-ASLignin biosynthesis Lignocellulosic complex Miscanthus sinensis 

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

Notes

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.

Author 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.

Compliance with ethical standards

Conflict of interest

There are no conflicts of interest to declare.

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Copyright information

© Springer Science+Business Media B.V., part of Springer Nature 2018

Authors and Affiliations

  1. 1.Bioherb Research InstituteKangwon National UniversityChuncheonSouth Korea
  2. 2.Department of Bio-Resource SciencesKangwon National UniversityChuncheonSouth Korea
  3. 3.Department of Medicinal PlantSuwon Women’s UniversitySuwonSouth Korea
  4. 4.Department of Applied BioscienceKonkuk UniversitySeoulSouth Korea
  5. 5.Department of Agronomy and the Key Laboratory of Crop Germplasm Resource of Zhejiang ProvinceZhejiang UniversityHangzhouChina
  6. 6.Field Science Center for Northern BiosphereHokkaido UniversitySapporoJapan
  7. 7.Department of Crop SciencesUniversity of Illinois at Urbana-ChampaignUrbanaUSA

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