Skip to main content

Advertisement

SpringerLink
Log in

Plant regeneration and genetic transformation of C. canadensis: a non-model plant appropriate for investigation of flower development in Cornus (Cornaceae)

  • Original Paper
  • Published:
Plant Cell Reports Aims and scope Submit manuscript

Abstract

Key message

Efficient Agrobacterium -mediated genetic transformation for investigation of genetic and molecular mechanisms involved in inflorescence architectures in Cornus species.

Abstract

Cornus canadensis is a subshrub species in Cornus, Cornaceae. It has recently become a favored non-model plant species to study genes involved in development and evolution of inflorescence architectures in Cornaceae. Here, we report an effective protocol of plant regeneration and genetic transformation of C. canadensis. We use young inflorescence buds as explants to efficiently induce calli and multiple adventitious shoots on an optimized induction medium consisting of basal MS medium supplemented with 1 mg/l of 6-benzylaminopurine and 0.1 mg/l of 1-naphthaleneacetic acid. On the same medium, primary adventitious shoots can produce a large number of secondary adventitious shoots. Using leaves of 8-week-old secondary shoots as explants, GFP as a reporter gene controlled by 35S promoter and hygromycin B as the selection antibiotic, a standard procedure including pre-culture of explants, infection, co-cultivation, resting and selection has been developed to transform C. canadensis via Agrobacterium strain EHA105-mediated transformation. Under a strict selection condition using 14 mg/l hygromycin B, approximately 5 % explants infected by Agrobacterium produce resistant calli, from which clusters of adventitious shoots are induced. On an optimized rooting medium consisting of basal MS medium supplemented with 0.1 mg/l of indole-3-butyric acid and 7 mg/l hygromycin B, most of the resistant shoots develop adventitious roots to form complete transgenic plantlets, which can grow normally in soil. RT-PCR analysis demonstrates the expression of GFP transgene. Green fluorescence emitted by GFP is observed in transgenic calli, roots and cells of transgenic leaves under both stereo fluorescence microscope and confocal microscope. The success of genetic transformation provides an appropriate platform to investigate the molecular mechanisms by which the various inflorescence forms are developed in Cornus plants.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

Abbreviations

AS:

Acetosyringone

BAP:

6-Benzylaminopurine

CaMV 35S:

Cauliflower mosaic virus 35S promoter

CTAB:

Cetyltrimethylammonium bromide

Hpt:

Hygromycin phosphotransferase

Hyg B:

Hygromycin B

IBA:

Indole-3-butyric acid

MS:

Murashige and Skoog

NAA:

1-Naphthaleneacetic acid

RT-PCR:

Reverse transcription polymerase chain reaction

References

  • Aggarwal D, Kumar A, Reddy MS (2011) Agrobacterium tumefaciens mediated genetic transformation of selected elite clone(s) of Eucalyptus tereticornis. Acta Physiol Plant 33:1603–1611

    Article  CAS  Google Scholar 

  • Brain JF, Denford KE (1979) The herbaceous members of the genus Cornus in Northwest North America. Bot. Not. 132:121–129

    Google Scholar 

  • Chang S, Puryear J, Cairney J (1993) A simple and efficient method for isolating RNA from pine trees. Plant Mol Biol Rep 11:113–116

    Article  CAS  Google Scholar 

  • Chiu W, Niwa Y, Zeng W, Hirano T, Kobayashi H, Sheen J (1996) Engineered GFP as a vital reporter in plants. Curr Biol 6:325–330

    Article  PubMed  CAS  Google Scholar 

  • Cutler SR, Ehrhardt DW, Griggitts JS, Somerville CR (2000) Random GFP: cDNA fusions enable visualization of subcellular structures in cells of Arabidopsis at a high frequency. PNAS 97:3718–3723

    Article  PubMed  CAS  Google Scholar 

  • Eyde RH (1988) Comprehending Cornus: puzzles and progress in the systematics of the dogwoods. Bot Rev (Lancaster) 54:233–351

    Article  Google Scholar 

  • Feng C-M, Qu R, Zhou L-L, Xie D-Y, Xiang Q-Y (2009) Shoot regeneration of dwarf dogwood (Cornus canadensis L.) and morphological characterization of the regenerated plants. Plant Cell Tiss Org Cult 97:27–37

    Article  CAS  Google Scholar 

  • Feng C-M, Xiang Q-YJ, Franks RG (2011) Phylogeny-based developmental analyses illuminate evolution of inflorescence architectures in dogwoods (Cornus s. l., Cornaceae). New Phytol 191:850–869

    Article  PubMed  Google Scholar 

  • Gervais C, Trahan R, Gagnon J (1999) IOPB chromosome data 14. Newslett Int Organ Pl Biosyst (Oslo) 30:10–15

    Google Scholar 

  • Haseloff J, Amos B (1995) GFP in plants. Trends Genet 11:328

    Article  PubMed  CAS  Google Scholar 

  • Höfgen R, Willmitzer L (1988) Storage of competent cells for Agrobacterium transformation. Nucleic Acids Res 16:9877

    Article  PubMed  Google Scholar 

  • Jenkins MA, White PS (2002) Cornus florida L. mortality and understory composition changes in western Great Smoky Mountains National park. J Torrey Bot Soc 129:194–206

    Article  Google Scholar 

  • Kean KD, Hwan KJ (1998) A furan derivative from Cornus officinalis. Arch Pharmacol Res 21:787–789

    Article  Google Scholar 

  • Lu J, Sivamani E, Li X, Qu R (2008) Activity of the 50 regulatory regions of the rice polyubiquitin rubi3 gene in transgenic rice plants as analyzed by both GUS and GFP reporter genes. Plant Cell Rep 27:1587–1600

    Article  PubMed  CAS  Google Scholar 

  • Ma X-Y, Yi G-J, Huang X-L, Zeng J-W (2009) Leaf callus induction and suspension culture establishment in lychee (Litchi chinensis Sonn.). Acta Physiol Plant 31:401–405

    Article  Google Scholar 

  • Manchester SR, Xiang XP, Xiang Q-YJ (2010) Fruits of cornelian cherries (Cornaceae: cornus Subg. Cornus) in the paleocene and eocene of the northern hemisphere. Int J Plant Sci 171:882–891

    Article  Google Scholar 

  • McEwan RW, Mueller RN, Arthur MA, Housman HH (2000) Temporal and ecological patterns of flowering dogwood mortality in the mixed mesophytic ecological patterns of flowering dogwood mortality in the mixed mesophytic forest of eastern Kentucky. J Torrey Bot Soc 127:221–229

    Article  Google Scholar 

  • Mohebodini M, Javaran MJ, Mahboudi F, Alizadeh H (2011) Effects of genotype, explant age and growth regulators on callus induction and direct shoot regeneration of lettuce (Lactuca sativa L.). AJCS 5(1):92–95

    CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  • Murrell ZE (1994) Dwarf dogwoods: intermediacy and the morphological landscape. Syst Bot 19:539–556

    Article  Google Scholar 

  • Plante S (1995) IOPB chromosome data 10. Int Organ Pl Biosyst Newslett (Zurich) 25:9–10

    Google Scholar 

  • Sherald JL, Stidham TM, Hadidian JM, Hoeldtke JW (1996) Progression of the dogwood anthracnose epidemic and the status of flowering dogwood in Catoctin Mountain Park. Plant Dis 80:310–312

    Article  Google Scholar 

  • Shi A, Kantartzi SK, Mmbaga M, Chen P (2008) Isolation of Resistance Gene Analogues from Flowering Dogwood (Cornus florida L.). J Phytopathol 156:11–12

    Article  Google Scholar 

  • Vareed SK, Reddy MK, Schutzki RE, Nair MG (2006) Anthocyanins in Cornus alternifolia, Cornus controversa, Cornus kousa and Cornus florida fruits with health benefits. Life Sci 78:777–784

    Article  PubMed  CAS  Google Scholar 

  • Xiang Q-YJ, Boufford DE (2005) Cornaceae. Flora of China 14:206–221

    Google Scholar 

  • Xiang Q-YJ, Thomas DT (2008) Tracking character evolution and biogeographic history through time in Cornaceae—Does choice of methods matter? J Syst Evol 46:349–374

    Google Scholar 

  • Xiang Q-YJ, Thomas DT, Zhang WH, Manchester SR, Murrell Z (2006) Species level phylogeny of the genus Cornus (Cornaceae) based on molecular and morphological evidence—implication in taxonomy and tertiary intercontinental migration. Taxon 55:9–30

    Article  Google Scholar 

  • Xie D-Y, Hong Y (2002) Agrobacterium-mediated genetic transformation of Acacia mangium. Plant Cell Rep 20:917

    Article  CAS  Google Scholar 

  • Yongwen Z, Yuwu C, Shipping ZA (1999) Sedoheptulose gallate from the fruits of Cornus officinalis. Yaoxue Xuebao 34:153–155

    Google Scholar 

Download references

Acknowledgments

This project is funded by the NSF grant (IOS-1024629). We are grateful to Dr. Eva Johannes for providing help in GFP analysis, and to Yi Yu for extracting DNAs and performing some PCR reactions. We thank Nicholas Tippery for collecting C. canadensis plants, David Boufford for assistance in collecting materials, as well as NCSU Phytotron for providing space to grow plants, plant disease clinics at NCSU for identifying disease, visiting scientists Jian Zhang and Juan Liu and undergraduate student Esther Iguchi for assistance in planting and maintaining the culture.

Author information

Authors and Affiliations

  1. Department of Plant Biology, North Carolina State University, Raleigh, NC, 27695, USA

    Xiang Liu, Chun-Miao Feng, De-Yu Xie & Qiu-Yun Jenny Xiang

  2. Department of Genetics, North Carolina State University, Raleigh, NC, 27695, USA

    Robert Franks

  3. Department of Crop Sciences, North Carolina State University, Raleigh, NC, 27695, USA

    Rongda Qu

Authors
  1. Xiang Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Chun-Miao Feng
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Robert Franks
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Rongda Qu
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. De-Yu Xie
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Qiu-Yun Jenny Xiang
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to De-Yu Xie or Qiu-Yun Jenny Xiang.

Additional information

Communicated by B. Li.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Liu, X., Feng, CM., Franks, R. et al. Plant regeneration and genetic transformation of C. canadensis: a non-model plant appropriate for investigation of flower development in Cornus (Cornaceae). Plant Cell Rep 32, 77–87 (2013). https://doi.org/10.1007/s00299-012-1341-x

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00299-012-1341-x

Keywords

Search

Navigation