Plant Cell, Tissue and Organ Culture (PCTOC)

, Volume 128, Issue 1, pp 25–41 | Cite as

Plant regeneration from calli in Japanese accessions of Miscanthus

  • Wataru TakahashiEmail author
  • Shin-ichi Tsuruta
  • Masumi Ebina
  • Makoto Kobayashi
  • Tadashi Takamizo
Original Article


We evaluated the ploidy levels and tissue culture responses of 16 Japanese Miscanthus accessions, which are registered and vegetatively maintained in the National Agriculture and Food Research Organization GeneBank, Japan, to screen suitable genotypes for the molecular breeding of Miscanthus species. A ploidy analysis showed that most M. sinensis and M. sinensis var. condensatus (var. condensatus) were putative diploids, but one accession identified as M. sinensis was unexpectedly a putative tetraploid. Additionally, M. sacchariflorus and its hybrid accessions were putative tetraploids. The deoxyribonucleic acid levels in var. condensatus were significantly higher than those in the diploid M. sinensis. Of the accessions, 10, including M. sinensis and var. condensatus, could induce plant regenerable embryogenic calli from apical meristems. We selected three of these M. sinensis accessions for further experiments because their calli growth rates were faster than those of the var. condensatus accessions. Tissue culture experiments with the selected accessions indicated that the frequencies of callus and green shoot formation strongly correlated with genotype. The broad-sense heritabilities of the embryogenic callus and green shoot formation frequencies in the selected accessions were 0.75 and 0.65, respectively, indicating that the cultures’ responses were mainly controlled by genetic factors. Thus, we further selected one accession that had the highest efficiencies in callus and green shoot formation, and we observed that light during callus culturing significantly inhibited calli growth, but promoted plant regeneration from calli in the selected accession.


Callus Miscanthus Ploidy level Regeneration Tissue culture 



We express our gratitude to the NARO GeneBank, Japan, for providing the Miscanthus accessions used in this study. We would like to thank Mr. Y. Nishimura (Chiba Industrial Technology Research Institute) and Dr. K. Sugawara (Institute of Livestock and Grassland Science, NARO) for their support in taking SEM-images. We thank Ms. S. Sasaki (Institute of Livestock and Grassland Science, NARO) for her prominent assistance in tissue culture. We thank Ms. L. J. Ackley (Joytalk Co., Ltd.) for supporting and giving us advice on manuscript writing.

Authors’ contributions

Conceived the experiments: WT, and TT. Designed the experiments: WT, ST, ME, and MK. Conducted the experiments: WT, ST, and ME. Analyzed the data: WT. Contributed materials: WT, ST, and MK. Wrote the paper: WT.


  1. Adati S, Shiotani I (1962) The cytotaxonomy of the genus Miscanthus and its phylogenic status. Bull Fac Agric Mie Univ 25:1–24Google Scholar
  2. Asad S, Arshad M, Mansoor S, Zafar Y (2009) Effect of various amino acids on shoot regeneration of sugarcane (Sacchrum officinarum L.) Afr J Biotechnol 8:1214–1218Google Scholar
  3. Atienza SG, Ramirez MC, Martin A (2003a) Mapping QTLs controlling flowering date in Miscanthus sinensis Anderss. Cereal Res Commun 31:265–271Google Scholar
  4. Atienza SG, Satovic Z, Petersen KK, Dolstra O, Martín A (2003b) Identification of QTLs associated with yield and its components in Miscanthus sinensis Anderss. Euphytica 132:353–361Google Scholar
  5. Atienza SG, Satovic Z, Petersen KK, Dolstra O, Martín A (2003c) Identification of QTLs influencing combustion quality in Miscanthus sinensis Anderss. II. Chlorine and potassium content. Theor Appl Genet 107:857–863Google Scholar
  6. Atienza SG, Satovic Z, Petersen KK, Dolstra O, Martín A (2003d) Influencing combustion quality in Miscanthus sinensis Anderss.: identification of QTLs for calcium, phosphorus and sulphur content. Plant Breed 122:141–145Google Scholar
  7. Cho M-J, Jiang W, Lemaux PG (1998) Transformation of recalcitrant barley cultivars through improvement of regenerability and decreased albinism. Plant Sci 138:229–244CrossRefGoogle Scholar
  8. Clifton-Brown J, Chiang Y-C, Hodkinson TR (2008) Miscanthus: genetic resources and breeding potential to enhance bioenergy production. In: Vermerris W (ed) Genetic improvement of bioenergy crops. Springer, New York, pp 273–294Google Scholar
  9. Cochran WG (1943) Analysis of variance for percentages based on unequal numbers. J Am Stat Assoc 38:287–301CrossRefGoogle Scholar
  10. Dalton SJ (2013) Biotechnology of Miscanthus. In: Jain SM, Dutta Gupta S (eds) Biotechnology of neglected and underutilized crops. Springer, Dordrecht, pp 243–294Google Scholar
  11. Deuter M (2000) Breeding approaches to improvement of yield and quality in Miscanthus grown in Europe. In: Lewandowski I, Clifton-Brown JC (eds) European Miscanthus improvement—final report september 2000. Institute of Crop Production and Grassland Research, University of Hohenheim, Stuttgart, pp 28–52Google Scholar
  12. Engler D, Jakob K (2013) Genetic engineering of Miscanthus. In: Paterson AH (ed) Genomics of the Saccharinae, vol 11. Plant genetics and genomics: crops and models. Springer, New York, pp 255–301CrossRefGoogle Scholar
  13. Feltus FA, Vandenbrink JP (2012) Bioenergy grass feedstock: current options and prospects for trait improvement using emerging genetic, genomic, and systems biology toolkits. Biotechnol Biofuels 5:80Google Scholar
  14. Głowacka K, Jeżowski S, Kaczmarek Z (2010) The effects of genotype, inflorescence developmental stage and induction medium on callus induction and plant regeneration in two Miscanthus species. Plant Cell Tissue Organ Cult 102:79–86CrossRefGoogle Scholar
  15. Hanson WD (1963) Heritability. In: Hanson WD, Robinson HF (eds) Statistical genetics and plant breeding. National Academy of Science-National Research Council, Washington, DC, pp 125–140Google Scholar
  16. Hodkinson TR, Chase MW, Renvoize SA (2002) Characterization of a genetic resource collection for Miscanthus (Saccharinae, Andropogoneae, Poaceae) using AFLP and ISSR PCR. Ann Bot 89:627–636CrossRefPubMedPubMedCentralGoogle Scholar
  17. Holme IB, Krogstrup P, Hansen J (1997) Embryogenic callus formation, growth and regeneration in callus and suspension cultures of Miscanthus × ogiformis Honda Giganteus’ as affected by proline. Plant Cell Tissue Organ Cult 50:203–210CrossRefGoogle Scholar
  18. Hwang O-J, Cho M-A, Han Y-J, Kim Y-M, Lim S-H, Kim D-S, Hwang I, Kim J-I (2014a) Agrobacterium-mediated genetic transformation of Miscanthus sinensis. Plant Cell Tissue Organ Cult 117:51–63Google Scholar
  19. Hwang O-J, Lim S-H, Han Y-J, Shin A-Y, Kim D-S, Kim J-I (2014b) Phenotypic characterization of transgenic Miscanthus sinensis plants overexpressing Arabidopsis phytochrome B. Int J Photoenergy 2014:501016Google Scholar
  20. Kanda Y (2013) Investigation of the freely available easy-to-use software ‘EZR’ for medical statistics. Bone Marrow Transplant 48:452–458CrossRefPubMedGoogle Scholar
  21. Lewandowski I, Clifton-Brown JC, Scurlock JMO, Huisman W (2000) Miscanthus: European experience with a novel energy crop. Biomass Bioenerg 19:209–227Google Scholar
  22. Liu C, Moon K, Honda H, Kobayashi T (2001) Enhanced regeneration of rice (Oryza sativa L.) embryogenic callus by light irradiation in growth phase. J Biosci Bioeng 91:319–321Google Scholar
  23. Moon Y-H, Cha Y-L, Choi Y-H, Yoon Y-M, Koo B-C, Ahn J-W, An G-H, Kim J-K, Park K-G (2013) Diversity in ploidy levels and nuclear DNA amounts in Korean Miscanthus species. Euphytica 193:317–326CrossRefGoogle Scholar
  24. Murashige T, Skoog F (1962) A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol Plant 15:473–497Google Scholar
  25. Nadir M, Tanaka A, Kuwabara S, Matuura H, Yamada T, Stewart JR, Nishiwaki A (2014) Comparison of relative DNA content estimated using DAPI and PI-FCM in Miscanthus sinensis, Miscanthus sacchariflorus, and their hybrids. J Warm Reg Soc Anim Sci Jpn 57:53–57Google Scholar
  26. Nieves N, Sagarra F, González R, Lezcano Y, Cid M, Blanco MA, Castillo R (2008) Effect of exogenous arginine on sugarcane (Saccharum sp.) somatic embryogenesis, free polyamines and the contents of the soluble proteins and proline. Plant Cell Tissue Organ Cult 95:313–320CrossRefGoogle Scholar
  27. Nishiwaki A, Mizuguti A, Kuwabara S, Toma Y, Ishigaki G, Miyashita T, Yamada T, Matuura H, Yamaguchi S, Rayburn AL, Akashi R, Stewart JR (2011) Discovery of natural Miscanthus (Poaceae) triploid plants in sympatric populations of Miscanthus sacchariflorus and Miscanthus sinensis in southern Japan. Am J Bot 98:154–159CrossRefPubMedGoogle Scholar
  28. Öztürk L, Demir Y (2002) In vivo and in vitro protective role of proline. Plant Growth Regul 38:259–264CrossRefGoogle Scholar
  29. Pyter R, Voigt T, Heaton E, Dohleman F, Long S (2007) Giant miscanthus: biomass crop for Illinois. In: Janic J, Whipkey A (eds) The 6th National New Crops Symposium, San Diego, California, Octorber 14–18 2007. Issues in New Crops and New Uses. ASHS Press, Alexandria, pp 39–42Google Scholar
  30. Rayburn AL, Crawford J, Rayburn CM, Juvik JA (2009) Genome size of three Miscanthus species. Plant Mol Biol Rep 27:184–188CrossRefGoogle Scholar
  31. Rikiishi K, Matsuura T, Maekawa M, Takeda K (2008) Light control of shoot regeneration in callus cultures derived from barley (Hordeum vulgare L.) immature embryos. Breed Sci 58:129–135.CrossRefGoogle Scholar
  32. Sacks E, Juvik JA, Lin Q, Stewart JR, Yamada T (2013) The gene pool of Miscanthus species and its improvement. In: Paterson AH (ed) Genomics of the Saccharinae, vol 11. Plant genetics and genomics: crops and models. Springer, New York, pp 73–101Google Scholar
  33. Slavov G, Allison G, Bosch M (2013) Advances in the genetic dissection of plant cell walls: tools and resources available in Miscanthus. Front Plant Sci 4:217CrossRefPubMedPubMedCentralGoogle Scholar
  34. Snedecor GW, Cochran WG (1980) Statistical methods. 7 edn. Iowa State University Press, AmesGoogle Scholar
  35. Takahashi W, Takamizo T (2012) Molecular breeding of grasses by transgenic approaches for biofuel production. In: Çiftçi YO (ed) Transgenic plants—advances and limitations. In Tech, Rijeka, pp 91–116Google Scholar
  36. Takahashi W, Takamizo T (2013) Plant regeneration from embryogenic calli of the wild sugarcane (Saccharum spontaneum L.) clone ‘Glagah Kloet’. Bull NARO Inst Livest Grassl Sci 13:23–32Google Scholar
  37. Takahashi W, Komatsu T, Fujimori M, Takamizo T (2004) Screening of regenerable genotypes of Italian ryegrass (Lolium multiflorum Lam.) Plant Prod Sci 7:55–61CrossRefGoogle Scholar
  38. Takahashi W, Takamizo T, Kobayashi M, Ebina M (2010) Plant regeneration from calli in giant reed (Arundo donax L.). Grassl Sci 56:224–229CrossRefGoogle Scholar
  39. Wang X, Yamada T, Kong F-J, 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 3:322–332Google Scholar
  40. Yi Z, Zhou P, Chu C, Li X, Tian W, Wang L, Cao S, Tang Z (2004) Establishment of genetic transformation system for Miscanthus sacchariflorus and obtaining of its transgenic plants. High Technol Lett 10:27–31Google Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2016

Authors and Affiliations

  • Wataru Takahashi
    • 1
    Email author
  • Shin-ichi Tsuruta
    • 1
    • 2
  • Masumi Ebina
    • 1
  • Makoto Kobayashi
    • 1
  • Tadashi Takamizo
    • 1
  1. 1.Division of Forage Crop Research, Institute of Livestock and Grassland ScienceNational Agriculture and Food Research Organization (NARO)NasushiobaraJapan
  2. 2.Tropical Agriculture Research FrontJapan International Research Center for Agricultural SciencesIshigakiJapan

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