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Agrobacterium tumefaciens mediated genetic transformation of selected elite clone(s) of Eucalyptus tereticornis

Acta Physiologiae Plantarum volume 33, pages 1603–1611 (2011)Cite this article

Abstract

Procedure for the Agrobacterium tumefaciens mediated T-DNA delivery into the elite clone(s) of Eucalyptus tereticornis using leaf explants from microshoots has been developed. Amongst two strains of A. tumefaciens namely, EHA105 and LBA4404 (harbouring pBI121 plasmid), strain EHA105 was found to be more efficient. Pre-culturing of tissue (2 days) on medium supplemented with 100 μM acetosyringone, before bacterial infection significantly increased transient expression of reporter gene (GUS). Co-cultivation period of 2 days and a bacterial density of 0.8 OD600 resulted in higher transient GUS expression. Method of injury to tissue, presence of acetosyringone in co-cultivation medium and photoperiod during co-cultivation also influenced the expression of transient GUS activity. Amongst the three clones tested, maximum transient GUS activity was recorded in clone ‘CE2’ followed by clone ‘T1’. Regeneration of transformed shoots was achieved on modified Murashige and Skoog medium (potassium nitrate was replaced with 990 mg/l potassium sulphate and ammonium nitrate with 392 mg/l ammonium sulphate, and mesoinositol concentration was increased to 200 mg/l). Stable transformation was confirmed on the basis of GUS activity and PCR amplification of DNA fragments specific to uidA and nptII genes. The absence of bacteria in the stable transformed tissues was confirmed by PCR amplification of fragment specific to 16S rRNA of bacteria.

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Abbreviations

2,4-D:

2,4-Dichlorophenoxyacetic acid

BA:

Benzyladenine

CaMV 35S :

35S promoter of the cauliflower mosaic virus

GUS:

β-Glucuronidase

MS:

Murashige and Skoog (1962)

NAA:

α-Naphthaleneacetic acid

Nos :

Nopaline synthase

nptII :

Neomycin phosphotransferase

OD600 :

Optical density at 600 nm

PCR:

Polymerase chain reaction

X-Gluc:

5-bromo-4-chloro-3-indolyl-β-d-glucuronic acid

References

  • Aggarwal D, Kumar A, Reddy MS (2010) Shoot organogenesis from elite plants of Eucalyptus tereticornis. Plant Cell Tissue Organ Cult 102:45–52

    Article  Google Scholar 

  • Alvarez R, Ordas RJ (2007) Improved genetic transformation protocol for cork oak (Quercus suber L.). Plant Cell Tissue Organ Cult 91:45–52

    Article  CAS  Google Scholar 

  • Barrueto CID LP, Machado ACM, Carvalheira SRC, Brasieleiro ACM (1999) Plant regeneration from seedling explants of Eucalyptus grandis × E. urophylla. Plant Cell Tissue Organ Cult 56:17–23

    Article  Google Scholar 

  • Binns AN, Thomashow MF (1988) Cell biology of Agrobacterium infection and transformation of plants. Ann Rev Microbiol 42:575–606

    Article  CAS  Google Scholar 

  • Brooker MIH (2000) A new classification of the genus Eucalyptus L’Her. (Myrtaceae). Aust Syst Bot 13:79–148

    Article  Google Scholar 

  • Dibax R, Eisfeld CL, Cuquel FL, Koehler H, Quoirin M (2005) Plant regeneration from cotyledonary explants of Eucalyptus camaldulensis. Sci Agric (Piracicaba, Braz) 62:406–412

    Article  Google Scholar 

  • Doyle JJ, Doyle JL (1990) Isolation of plant DNA from fresh tissue. Focus 12:13–15

    Google Scholar 

  • Dutt M, Li ZT, Dhekney SA, Gray DJ (2007) Transgenic plants from shoot apical meristems of Vitis vinifera L. “Thompson Seedless” via Agrobacterium-mediated transformation. Plant Cell Rep 26(12):2101–2110

    Article  PubMed  CAS  Google Scholar 

  • Gelvin SB (2005) Agricultural biotechnology gene exchange by design. Nature 433:583–584

    Article  PubMed  CAS  Google Scholar 

  • Godwin I, Todd G, Ford-Lloyd B, Newbury HJ (1991) The effect of acetosyringone and pH on Agrobacterium-mediated transformation vary according to plant species. Plant Cell Rep 9:671–675

    Article  CAS  Google Scholar 

  • Grattapaglia D, Kirst M (2008) Eucalyptus applied genomics from gene sequences to breeding tools. New Phytol 179(4):911–929

    Article  PubMed  CAS  Google Scholar 

  • 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 17:675–680

    Article  CAS  Google Scholar 

  • Hoekema A, Hirsch PR, Schilperoort PJJ, Hooykaas RA (1983) A binary vector strategy based on separation of vir and T-region of the Agrobacterium tumefaciens Ti-plasmid. Nature 303:179–180

    Article  CAS  Google Scholar 

  • Holsters M, De Waele D, Depicker A, Messens E, Van Montagu M, Schell J (1978) Transfection and transformation of Agrobacterium tumefaciens. Mol Gen Genet 163:181–187

    Article  PubMed  CAS  Google Scholar 

  • Hood EE, Gelvin SB, Melchers LS, Hoekema A (1993) New Agrobacterium helper plasmids for gene transfer to plants. Transgenic Res 2:208–218

    Article  CAS  Google Scholar 

  • Ivanova M, Van Staden J (2009) Nitrogen source, concentration, and NH4 +:NO3 − ratio influence shoot regeneration and hyperhydricity in tissue cultured Aloe polyphylla. Plant Cell Tissue Organ Cult 99:167–174

    Article  CAS  Google Scholar 

  • James DJ, Uratsu S, Cheng J, Negri P, Viss P, Dandekar AM (1993) Acetosyringone and osmo-protectants like betaine or proline synergistically enhance Agrobacterium-mediated transformation of apple. Plant Cell Rep 12:559–563

    Article  CAS  Google Scholar 

  • Jefferson RA, Kavanagh TA, Bevan MW (1987) GUS fusions: β-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J 6:3901–3907

    PubMed  CAS  Google Scholar 

  • Jube S, Borthakur D (2009) Development of an Agrobacterium mediated transformation protocol for the tree-legume Leucaena leucocephala using immature zygotic embryos. Plant Cell Tissue Organ Cult 96:325–333

    Article  PubMed  Google Scholar 

  • Lopez SJ, Kumar RR, Pius PK, Muralidharan N (2004) Agrobacterium tumefaciens–mediated genetic transformation in Tea (Camellia sinensis [L.] O. Kuntze). Plant Mol Biol Rep 22:201a–201j

    Article  Google Scholar 

  • Luna C, Collavino M, Mroginski L, Sansberro P (2008) Identification and control of bacterial contaminants from Ilex dumosa nodal segments culture in a temporal immersion bioreactor system using 16S rDNA analysis. Plant Cell Tissue Organ Cult 95:13–19

    Article  CAS  Google Scholar 

  • Maheswaran GM, Welander M, Hutchinson JF, Graham MW, Richards D (1992) Transformation of apple rootstock M26 with Agrobacterium tumefaciens. J Plant Physiol 139:560–568

    Google Scholar 

  • Manickavasagam M, Ganapathi A, Anbazhagan VR, Sudhakar B, Selvaraj N, Vasudevan A, Kasthurirengan S (2004) Agrobacterium mediated genetic transformation and development of herbicide-resistant sugarcane (Saccharum species hybrids) using axillary buds. Plant Cell Rep 23:134–143

    Article  PubMed  CAS  Google Scholar 

  • Mullins KV, Llewellyn DJ, Hartney VJ, Strauss SH, Dennis ES (1997) Regeneration and transformation of Eucalyptus camaldulensis. Plant Cell Rep 16:787–791

    Article  CAS  Google Scholar 

  • Murashige T, Skooge F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15:473–497

    Article  CAS  Google Scholar 

  • Niu X, Li X, Veronese P, Hasegawa PM, Bressan RA, Weller SC (2000) Factors affecting Agrobacterium tumefaciens-mediated transformation of peppermint. Plant Cell Rep 19:304–310

    Article  CAS  Google Scholar 

  • Nugent G, Chandler SF, Whiteman P, Stevenson TW (2001) Adventitious bud induction in Eucalyptus globulus Labill. In Vitro Cell Dev Biol Plant 37:388–391

    Article  CAS  Google Scholar 

  • Padmanabhan P, Sahi SV (2009) Genetic transformation and regeneration of Sesbania drummondii using cotyledonary nodes. Plant Cell Rep 28:31–40

    Article  PubMed  CAS  Google Scholar 

  • Peña L, Séguin A (2001) Recent advances in the genetic transformation of trees. Trends Biotech 19(12):500–506

    Article  Google Scholar 

  • Pinto G, Park YS, Silva S, Neves L, Araujo C, Santos C (2008) Factors affecting maintenance, proliferation and germination of secondary somatic embryos of Eucalyptus globulus Labill. Plant Cell Tissue Organ Cult 95:69–78

    Article  CAS  Google Scholar 

  • 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 45:429–434

    Article  CAS  Google Scholar 

  • Ramage CM, Williams RR (2002) Mineral nutrition and plant morphogenesis. In Vitro Cell Dev Biol Plant 38:116–124

    Article  CAS  Google Scholar 

  • Sangwan RS, Bourgoies Y, Brow S, Vasseur G, Sangwan-Norreel BS (1992) Characterization of competent cells and early events of Agrobacterium-mediated transformation of Arabidopsis thaliana. Planta 188:439–456

    Article  CAS  Google Scholar 

  • Serrano L, Rochange F, Semblat JP, Marque C, Teulières C, Boudet AM (1996) Genetic transformation of Eucalyptus globulus through biolistics parallel development of procedures for organogenesis from zygotic embryos and stable transformation of corresponding proliferating tissue. J Exp Bot 47(295):285–290

    Article  CAS  Google Scholar 

  • Sonia, Saini R, Singh RP, Jaiswal PK (2007) Agrobacterium tumefaciens mediated transfer of Phaseolus vulgaris α-amylase inhibitor-1 gene into mungbean: Vigna radiata (L.) Wilczek using bar as selectable marker. Plant Cell Rep 26:187–198

    Article  PubMed  CAS  Google Scholar 

  • Stachel SE, Messens E, Van Montagu M, Zambryski P (1985) Identification of signal molecules produced by wounded plant cells that activate T-DNA transfer in Agrobacterium tumefaciens. Nature 318:624–629

    Article  Google Scholar 

  • Stachel SE, Nester EW, Zambryski P (1986) A plant cell factor induces Agrobacterium tumefaciens vir gene expression. Proc Natl Acad Sci USA 83:379–383

    Article  PubMed  CAS  Google Scholar 

  • Subbaiah MM, Minocha SC (1990) Shoot regeneration from stem and leaf callus of Eucalyptus tereticornis. Plant Cell Rep 9:370–373

    Article  CAS  Google Scholar 

  • Sun SB, Meng LS (2010) Genetic transformation of Gentiana dahurica Fisch by Agrobacterium tumefaciens using zygotic embryo derived callus. Acta Physiol Plant 32:629–634

    Article  CAS  Google Scholar 

  • Tazawa M, Reinert J (1969) Extracellular and intracellular chemical environments in relation to embryogenesis in vitro. Protoplasma 68:157–173

    Article  PubMed  CAS  Google Scholar 

  • Terakami S, Matsuta N, Yamamoto T, Sugaya S, Gemma H, Soejima J (2007) Agrobacterium-mediated transformation of the dwarf pomegranate (Punica granatum L.). Plant Cell Rep 26:1243–1251

    Article  PubMed  CAS  Google Scholar 

  • Thomas P, Kumari S, Swarna GK, Prakash DP, Dinesh MR (2007) Ubiquitous presence of fastidious endophytic bacteria in field shoots and index-negative apparently clean shoot-tip cultures of papaya. Plant Cell Rep 26:1491–1499

    Article  PubMed  CAS  Google Scholar 

  • 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). Transgenic Res 12:403–411

    Article  PubMed  CAS  Google Scholar 

  • Turnbull JW (1999) Eucalypt plantations. New For 17:37–52

    Article  Google Scholar 

  • Weisburg WA, Barns SM, Pelletier DA, Lane DJ (1991) 16S rDNA amplification for phylogenetic study. J Bacteriol 173:697–703

    PubMed  CAS  Google Scholar 

  • Yasmeen A (2009) An improved protocol for the regeneration and transformation of tomato (cv Rio Grande). Acta Physiol Plant 31:1271–1277

    Article  CAS  Google Scholar 

  • Yevtushenko DP, Misra S (2010) Efficient Agrobacterium-mediated transformation of commercial hybrid poplar Populus nigra L. × P. maximowiczii A. Henry. Plant Cell Rep 29:211–229

    Article  PubMed  CAS  Google Scholar 

  • Zambre M, Terryn N, Clercq JD, De Buck S, Dillen W, Montagu MV, Van Der Straeten D, Angenon G (2003) Light strongly promotes gene transfer from Agrobacterium tumefaciens to plant cells. Planta 216:580–586

    PubMed  CAS  Google Scholar 

  • Zuker A, Ahroni A, Tzfira T, Meir HB, Vainstein A (1999) Wounding by bombardment yields highly yields highly efficient Agrobacterium-mediated transformation of carnation (Dianthus caryophyllus L.). Mol Breed 5:367–375

    Article  Google Scholar 

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Acknowledgments

The authors would like to thank Prof. S.B. Gelvin, Purdae University, Purdae, USA for providing A. tumefaciens strain EHA105 and Dr. N. Das, Thapar University, Patiala for providing A. tumefaciens strain LBA4404. The authors are also thankful to Council of Scientific and Industrial Research (CSIR), Govt. of India, New Delhi for providing financial support (Scheme no. 38(1158)/07/EMR-II). TIFAC—CORE, Thapar University, Patiala is thanked for providing facilities.

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Authors and Affiliations

  1. Department of Biotechnology and Environmental Sciences, TIFAC-Center of Relevance and Excellence in Agro and Industrial Biotechnology, Thapar University, Patiala, 147004, India

    Diwakar Aggarwal, Anil Kumar & M. Sudhakara Reddy

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  1. Diwakar Aggarwal
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  2. Anil Kumar
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  3. M. Sudhakara Reddy
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Correspondence to Anil Kumar.

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Communicated by T. Moriguchi.

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Aggarwal, D., Kumar, A. & Sudhakara Reddy, M. Agrobacterium tumefaciens mediated genetic transformation of selected elite clone(s) of Eucalyptus tereticornis. Acta Physiol Plant 33, 1603–1611 (2011). https://doi.org/10.1007/s11738-010-0695-3

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  • DOI: https://doi.org/10.1007/s11738-010-0695-3

Keywords

  • Acetosyringone
  • Co-cultivation
  • Plantation forestry
  • 16S rRNA
  • GUS expression