Plant Cell, Tissue and Organ Culture (PCTOC)

, Volume 122, Issue 2, pp 363–371 | Cite as

Genetic transformation of endo-1,4-β-glucanase (Korrigan) for cellulose enhancement in Eucalyptus tereticornis

  • Diwakar Aggarwal
  • Anil Kumar
  • M. Sudhakara Reddy
Original Paper


KORRIGAN plays an important role in cellulose biosynthesis and other physiological processes like plant growth, development, cell division and expansion. In the present study, a full length cDNA of Korrigan (PdeKOR) encoding 619 amino acids was amplified and characterized from Populus deltoides. Sequence analysis revealed the presence of glycosylation, phosphorylation sites and a single transmembrane domain (between amino acids 72 and 94) suggesting that PdeKOR belongs to GH9A subclass from glycosyl hydrolase family 9 (GH9) of proteins. The PdeKOR gene was successfully transformed into Eucalyptus tereticornis by Agrobacterium mediated genetic transformation. Efficient regeneration of transformed shoots from the explants following co-cultivation was achieved on MS medium supplemented with 25 µM α-naphthaleneacetic acid. Transgenic lines showed over expression of the PdeKOR gene as determined by qPCR analysis. Maximum expression of the PdeKOR gene was observed in the tissues of stem compared to leaves and shoot tips. This is the first report where an elite clone of E. tereticornis was successfully transformed with Korrigan gene, which can help in understanding its function in wood formation.


Cellulose Endoglucanses Populus deltoides Wood formation Eucalyptus tereticornis 



The authors are thankful to Council of Scientific and Industrial Research (CSIR), Govt. of India, New Delhi for providing financial support through Scheme No. 38(1158)/07/EMR-II). Authors are also thankful to TIFAC-CORE, Thapar University, Patiala.

Conflict of interest

The authors declare that they have no conflict of interest.


  1. Aggarwal D, Kumar A, Reddy MS (2011) Agrobacterium tumefaciens mediated genetic transformation of selected elite clones of Eucalyptus tereticornis. Acta Physiol Plant 33:1603–1611CrossRefGoogle Scholar
  2. Aggarwal D, Kumar A, Sharma J, Reddy MS (2012) Factors effecting micro propagation and acclimatization of an elite clone of Eucalyptus tereticornis. In Vitro Cell Dev Biol-Plant 48:521–529CrossRefGoogle Scholar
  3. Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25:3389–3402PubMedCentralPubMedCrossRefGoogle Scholar
  4. Bhandari S, Fujino T, Thammanagowda S, Zhang D, Xu F, Joshi CP (2006) Xylem-specific and tension stress-responsive coexpression of KORRIGAN endoglucanase and three secondary wall-associated cellulose synthase genes in aspen trees. Planta 224:828–837PubMedCrossRefGoogle Scholar
  5. Brummell DA, Catala C, Lashbrook CC, Bennett AB (1997) A membrane-anchored E-type endo-1,4-beta-glucanase is localized on Golgi and plasma membranes of higher plants. Proc Natl Acad Sci-USA 94:4794–4799PubMedCentralPubMedCrossRefGoogle Scholar
  6. Chang S, Puryear J, Cairney J (1993) A simple and efficient method for isolating RNA from pine trees. Plant Mol Biol Report 11:113–116CrossRefGoogle Scholar
  7. de la Torre F, Rodriguez R, Jorge G, Viller B, Alvarez-Otero R, Grima-Pettenati J, Gallego PP (2014) Genetic transformation of Eucalyptus globulus using the vascular specific EgCCR as an alternative to constitutive CaMV35S promotor. Plant Cell Tiss Organ Cult 117:77–84CrossRefGoogle Scholar
  8. Du Q, Wang L, Yang X, Gong C, Zhang D (2015) Populus endo-β-1,4-glucanases gene family: genomic organization, phylogenetic analysis, expression profiles and association mapping. Planta. doi: 10.1007/s00425-015-2271-y Google Scholar
  9. Dutt D, Tyagi CH (2011) Comparison of various Eucalyptus species for their morphological, chemical, pulp and paper making characteristics. Indian J Chem Technol 18:145–151Google Scholar
  10. Endler A, Persson S (2011) Cellulose synthases and synthesis in Arabidopsis. Mol Plant 4:199–211PubMedCrossRefGoogle Scholar
  11. Hall TA (1999) BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symp Ser 41:95–98Google Scholar
  12. Heid CA, Stevens J, Livak KJ, Williams PM (1996) Real time quantitative PCR. Genome Res 6:986–994PubMedCrossRefGoogle Scholar
  13. Henrissat B (1991) A classification of glycosyl hydrolases based on amino acid sequence similarities. Biochem J 280(Pt 2):309–316PubMedCentralPubMedGoogle Scholar
  14. Ho CK, Chang SH, Tsay JY, Tsai CJ, Chiang VL, Chen ZZ (1998) Agrobacterium tumefaciens-mediated transformation of Eucalyptus camaldulensis and production of transgenic plants. Plant Cell Rep 17:675–680CrossRefGoogle Scholar
  15. Holsters M, De Waele D, Depicker A, Messens E, Van Montagu M, Schell J (1978) Transfection and transformation of Agrobacterium tumefaciens. Mol Gen Genet 163:181–187PubMedCrossRefGoogle Scholar
  16. Hong YB, Liu SP, Zhu YP, Xie C, Jue DW, Chen M, Kaleri HA, Yang Q (2013) Expression of the MSI-99m gene in transgenic potato plants confers resistance to Phytophthora infestans and Ralstonia solanacearum. Plant Mol Biol Rep 31:418–424CrossRefGoogle Scholar
  17. Hood EE, Gelvin SB, Melchers LS, Hoekema A (1993) New Agrobacterium helper plasmids for gene transfer to plants. Trans Res 2:208–218CrossRefGoogle Scholar
  18. Jefferson RA, Kavanagh TA, Bevan MW (1987) GUS fusions: beta-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J 6:3901–3907PubMedCentralPubMedGoogle Scholar
  19. Lane DR, Wiedemeier A, Peng LC, Hofte H, Vernhettes S, Desprez T, Hocart C, Birch R, Baskin T, Burn J, Arioli T, Betzner A, Williamson R (2001) Temperature-sensitive alleles of rsw2 link the Korrigan endo-1,4-β-glucanase to cellulose synthesis and cytokinesis in Arabidopsis. Plant Physiol 126:278–288PubMedCentralPubMedCrossRefGoogle Scholar
  20. Letourneur F, Klausner RD (1992) A novel di-leucine motif and a tyrosine-based motif independently mediate lysosomal targeting and endocytosis of CD3 chains. Cell 69:1143–1157PubMedCrossRefGoogle Scholar
  21. Lopes ALL, Oliveira Y, Costa JL, Mudry CS, Procopiuk M, Scheidt NG, Brondani EG (2011) Preliminary results for genetic transformation of shoot tip of Eucalyptus saligna Sm. Via Agrobacterium tumefaciens. J Biotech Biodiver 2:1–6Google Scholar
  22. Maloney VJ, Samuels AL, Mansfield SD (2012) The endo-1,4-beta-glucanase Korrigan exhibits functional conservation between gymnosperms and angiosperms and is required for proper cell wall formation in gymnosperms. New Phytol 193:1076–1087PubMedCrossRefGoogle Scholar
  23. Master ER, Rudsander UJ, Zhou W, Henriksson H, Divine C, Denman S, Wilson DB, Teeeri TT (2004) Recombinant expression and enzymatic characterization of PttCel9A, a KOR homologue from Populus tremula x tremuloides. Biochem 43:10080–10089CrossRefGoogle Scholar
  24. Mølhøj M, Pagant S, Höfte H (2002) Towards understanding the role of membrane-bound Endo-1,4-glucanases in cellulose biosynthesis. Plant Cell Physiol 43:1399–1406PubMedCrossRefGoogle Scholar
  25. Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15:473–497CrossRefGoogle Scholar
  26. Nicol F, His I, Jauneau A, Vernhettes S, Canut H, Hofte H (1998) A plasma membrane–bound putative endo-1, 4-beta-D-glucanase is required for normal wall assembly and cell elongation in Arabidopsis. EMBO J 17:5563–5576PubMedCentralPubMedCrossRefGoogle Scholar
  27. Pena L, Sequin A (2001) Recent advances in the genetic transformation of trees. Trends Biotechnol 19:5000–5506CrossRefGoogle Scholar
  28. Porto MS, Pinheiro MP, Batista VG et al (2014) Plant promoters: an approach of structure and function. Mol Biotechnol 56:38–49PubMedCrossRefGoogle Scholar
  29. Prakash MG, Gurumurthi K (2009) Genetic transformation and regeneration of transgenic plants from precultured cotyledon and hypocotyl explants of Eucalyptus tereticornis Sm. using Agrobacterium tumefaciens. In Vitro Cell Dev Biol Plant 45:429–434CrossRefGoogle Scholar
  30. Sato S, Kato T, Kakegawa K, Ishii T, Liu YG, Awano T, Takabe K, Nishiyama Y, Kuga S, Sato S, Nakamura Y, Tabata S, Shibata D (2001) Role of the putative membrane-bound endo-1,4-β-glucanase Korrigan in cell elongation and cellulose synthesis in Arabidopsis thaliana. Plant Cell Physiol 42:251–263PubMedCrossRefGoogle Scholar
  31. Shani Z, Dekel M, Tsabary G, Shoseyov O (1997) Cloning and characterization of elongation specific endo-1,4-β-glucanase (cel1) from Arabidopsis thaliana. Plant Mol Biol 34:837–842PubMedCrossRefGoogle Scholar
  32. Shani Z, Dekel M, Roiz L, Horowitz M, Kolosovski N, Lapidot S, Alkan S, Koltai H, Tsabary G, Goren R, Shoseyov O (2006) Expression of endo-1,4-β-glucanase (cel1) in Arabidopsis thaliana is associated with plant growth, xylem development and cell wall thickening. Plant Cell Rep 25:1067–1074PubMedCrossRefGoogle Scholar
  33. Somerville CR (2006) Cellulose synthesis in higher plants. Annu Rev Cell Dev Biol 22:53–78PubMedCrossRefGoogle Scholar
  34. Szyjanowicz PMJ, McKinnon I, Taylor NG, Gardiner J, Jarvis MC, Turner SR (2004) The irregular xylem 2 mutant is an allele of korrigan that affects the secondary cell wall of Arabidopsis thaliana. Plant J 37:730–740PubMedCrossRefGoogle Scholar
  35. Takahashi J, Rudsander UJ, Hedenström M, Banasiak A, Harholt J, Amelot N, Immerzeel P, Ryden P, Endo S, Ibatullin FM, Brumer H, Campillo ED, Master ER, Scheller HV, Sundberg B, Teeri TT, Mellerowicz EJ (2009) KORRIGAN1 and its aspen homolog PttCel9A1 decrease cellulose crystallinity in Arabidopsis stems. Plant Cell Physiol 50:1099–1115PubMedCrossRefGoogle Scholar
  36. Tamura K, Stecher G, Peterson D, Filipski A, Kumar S (2013) MEGA6: molecular evolutionary genetics analysis version 6.0. Mol Biol Evol 30:2725–2729PubMedCentralPubMedCrossRefGoogle Scholar
  37. Tournier V, Grat S, Marque C, El Kayal W, Penchel R, de Andrade G, Boudet AM, Teulieres C (2003) An efficient procedure to stably introduce genes into an economically important pulp tree (Eucalyptus grandis × Eucalyptus urophylla). Trans Res 12:403–411 Google Scholar
  38. Turnbull JW (1999) Eucalyptus plantations. New For 17:37–52Google Scholar
  39. Updegraff DM (1969) Semimicro determination of cellulose in biological materials. Anal Biochem 32:420–424PubMedCrossRefGoogle Scholar
  40. Urbanowicz BR, Bennett AB, Del Campillo E, Catala C, Hayashi T, Henrissat B, Hofte H, McQueen-Mason SJ, Patterson SE, Shoseyov O, Teeri TT, Rose JKC (2007) Structural organization and a standardized nomenclature for plant endo-1,4-beta-glucanases (cellulases) of glycosyl hydrolase family 9. Plant Physiol 144:1693–1696PubMedCentralPubMedCrossRefGoogle Scholar
  41. Yu L, Sun J, Li L (2013) PtrCel9A6, an Endo-1,4-β-glucanase, is required for cell wall formation during xylem differentiation in Populus. Mol Plant 6:1904–1917PubMedCrossRefGoogle Scholar
  42. Yu L, Chen H, Sun J, Li L (2014) PtrKOR1 is required for secondary cell wall cellulose biosynthesis in Populus. Tree Physiol. doi: 10.1093/treephys/tpu020 PubMedGoogle Scholar
  43. Zhang JW, Xu L, Wu YR, Chen XA, Liu Y, Zhu SH, Ding WN, Wu P, Yi KK (2012) OsGLU3, a putative membrane-bound endo-1,4-beta-glucanase, is required for root cell elongation and division in rice (Oryza sativa L.). Mol Plant 5:176–186PubMedCrossRefGoogle Scholar
  44. Zuo JR, Niu QW, Nishizawa N, Wu Y, Kost B, Chua NH (2000) Korrigan, an Arabidopsis endo-1,4-β-glucanase, localizes to the cell plate by polarized targeting and is essential for cytokinesis. Plant Cell 12:1137–1152PubMedCentralPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2015

Authors and Affiliations

  • Diwakar Aggarwal
    • 1
  • Anil Kumar
    • 1
  • M. Sudhakara Reddy
    • 1
  1. 1.Department of BiotechnologyThapar UniversityPatialaIndia

Personalised recommendations