Skip to main content
SpringerLink
Log in

Agrobacterium-mediated transformation of cauliflower: Optimization of protocol and development of Bt-transgenic cauliflower

  • Articles
  • Published:
Journal of Biosciences Aims and scope Submit manuscript

Abstract

A number of factors that are known to influence genetic transformation were evaluated to optimizeAgrobacterium-mediated transformation of hypocotyl explants of cauliflower variety Pusa Snowball K-1. The binary vector p35SGUSINT mobilized intoAgrobacterium strain GV2260 was used for transformation and transient GUS expression was used as the basis for identifying the most appropriate conditions for transformation. Explant age, preculture period, bacterial strain and density were found to be critical determinants of transformation efficiency. Using the optimized protocol, the syntheticcryIA(b) gene was mobilized into cauliflower. Molecular analyses of transgenics established the integration and expression of the transgene. Insect bioassays indicated the effectiveness of the transgene against infestation by diamondback moth (Plutella xylostella) larvae.

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

Similar content being viewed by others

References

  • Ausubel F M, Brent R, Kingston R E, Moore D D, Seidman J G, Smith J A and Struhl K 1994Current protocols in molecular biology (New York: John Wiley)

    Google Scholar 

  • Berthomieu P, Beclin C, Charlot F, Dore C and Jouanin L 1994 Routine transformation of rapid cycling cabbage (Brassicaoleracea) — molecular evidence for regeneration of chimeras;Plant Sci. 96 223–235

    Article  CAS  Google Scholar 

  • Bhalla P L and Smith N 1998Agrobacterium tumefaciens-mediated transformation of cauliflower,Brassica oleracea var.botrytis;Mol. Breed 4 531–541

    Article  CAS  Google Scholar 

  • Cheng X Y, Sardana R, Kaplan H and Altosaar I 1998Agrobacterium-transformed rice plants expressing syntheticcryIA(b) andcryIA(c) genes are highly toxic to striped stem borer and yellow stem borer;Proc. Natl. Acad. Sci. USA 95 2767–2772

    Article  CAS  Google Scholar 

  • Chilton M D, Currier T, Farrand S, Bendich A, Gordon M P and Nester E W 1974Agrobacterium tumefaciens DNA and PS8 bacteriophage DNA not deleted in crown gall tumors;Proc. Natl. Acad. Sci. USA 71 3672–3676

    Article  CAS  Google Scholar 

  • Chomcznski P and Sacchi N 1987 Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction;Annal. Biochem. 162 156–159

    Google Scholar 

  • Church G M and Gilbert W 1984 Genomic Sequencing;Proc. Natl. Acad. Sci. USA 81 1991–1995

    Article  CAS  Google Scholar 

  • Ding L C, Hu C Y, Yeh K W and Wang P J 1998 Development of insect resistant transgenic cauliflower plants expressing the trypsin inhibitor gene isolated from local sweet potato;Plant Cell Rep. 17 854–860

    Article  CAS  Google Scholar 

  • Doyle J J and Doyle J L 1990 Isolation of plant DNA from fresh tissues;Focus 12 13–15

    Google Scholar 

  • Estruch J J, Carozzi N B, Desai N, Duck N B, Warren G W and Koziel M G 1997 Transgenic plant: An emerging approach to pest control;Nat. Biotech. 15 137–141

    Article  CAS  Google Scholar 

  • Fujimoto H, Itoh K, Yamamoto M, Kyozuka J and Shimamoto K 1993 Insect resistant rice generated by introduction of a modified δ-endotoxin geneBacillus thuriengiensis;Bio/ Technology 11 1151–1155

    CAS  PubMed  Google Scholar 

  • Henzi M X, Christey M L and McNeil D L 2000 Factors that influenceAgrobacterium rhizogenes mediated transformation of broccoli (Brassica oleracea L. var.italica);Plant Cell Rep. 19 994–999

    Article  CAS  Google Scholar 

  • Hofgen R and Willmitzer Z 1990 Biochemical and genetic analysis of different potatin isoforms expressed in various organs of potato (Solanum tubersoum);Plant Sci. 66 221–230

    Article  Google Scholar 

  • Jefferson R A 1987 Assaying chimeric genes in plants: the GUS gene fusion system;Plant Mol. Biol. Rep. 5 387–405

    Article  CAS  Google Scholar 

  • Metz T D, Dixit R and Earle E D 1995Agrobacterium tumefaciens mediated transformation of broccoli (Brassica oleracea var.italica) and cabbage (B. oleracea var.capatita);Plant Cell Rep. 15 287–292

    Article  CAS  Google Scholar 

  • Mukhopadhyay A, Topfer R, Pradhan A K, Sodhi Y S, Steinbi H-H, Schell J and Pental D 1991 Efficient regeneration ofBrassica oleracea hypocotyl protoplasts and high efficiency genetic transformation by direct DNA uptake;Plant Cell Rep. 10 375–379

    CAS  PubMed  Google Scholar 

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

    Article  CAS  Google Scholar 

  • Ovesna J, Ptacek L and Opatrny Z 1993 Factors influencing the regeneration capacity of oilseed rape and cauliflower in transformation experiments;Biol. Plant. 35 107–112

    Article  Google Scholar 

  • Palmgren G, Mattson O and Okkels F T 1993 Treatment ofAgrobacterium or leaf discs with 5-azacytidine increases transgene expression in tobacco;Plant Mol. Biol. 21 429–435

    Article  CAS  Google Scholar 

  • Passelgue E and Kerlan C 1996 Transformation of cauliflower (Brassica oleracea var.botrytis) by transfer of cauliflower mosaic gene through cocultivation with virulent and avirulent strainsof Agrobacterium;Plant Sci. 113 79–89

    Article  Google Scholar 

  • Pius P K and Achar P N 2000 Genetic transformation and hybridization:Agrobacterium-mediated transformation and plant regeneration ofBrassica oleracea var.capitata;Plant Cell Rep. 19 888–892

    Article  CAS  Google Scholar 

  • Puddephat J, Riggs T J and Fenning T M 1996 Transformation ofBrassica oleracea L.: a critical review;Mol. Breed. 2 185–210

    Article  Google Scholar 

  • Sangwan R S, Bourgeois Y, Brown S, Vasseur G and Sangwan-Norreel B 1992 Characterization of competent cells and early events ofAgrobacterium-mediated genetic transformation inArabidopsis thaliana;Planta 188 439–456

    Article  CAS  Google Scholar 

  • Shimoda N, Toyoda-Yamamoto A, Nagamine J, Usami S, Katayama M, Sakagami Y and Machida Y 1990 Control of expression ofAgrobacterium vir genes by synergistic action of phenolic signal molecules and monosaccharides;Proc. Natl. Acad. Sci. USA 87 6684–6688

    Article  CAS  Google Scholar 

  • Srivastava V, Reddy A S and Mukherjee S G 1988 Transformation and regeneration ofBrassica oleracea mediated by an oncogenicAgrobacterium tumefaciens;Plant Cell Rep. 7 504–507

    Article  CAS  Google Scholar 

  • Tabashnik B E 1997 Seeking the root of insect resistance to transgenic plants;Proc. Natl. Acad. Sci. USA 94 3488–3490

    Article  CAS  Google Scholar 

  • VanWordragen M G and Dons H J M 1992Agrobacterium tumefaciens-mediated transformation of recalcitrant crops;Plant Mol. Biol. Rep. 10 12–36

    Article  Google Scholar 

  • Vancanneyt G, Schmidt R, O’Conner-Sanchez A, Willmitzer L and Rocha-Sosa M 1990 Construction of an introncontaining marker gene: splicing of the intron in transgenic plants and its use in monitoring early events inAgrobacterium-mediated plant transformation;Mol. Gen. Genet. 220 245–250

    Article  CAS  Google Scholar 

  • Xiang Y, Wong W K R, Ma M C and Wong R S C 2000Agrobacterium mediated transformation ofBrassica campestris ssp.parachinensis with syntheticBacillus thuringiensis cry-IAb andcryIAc genes;Plant Cell Rep. 19 251–256

    Article  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. National Research Centre on Plant Biotechnology, Indian Agricultural Research Institute, 110 012, New Delhi, India

    R. Chakrabarty, N. Viswakarma, S. R. Bhat, P. B. Kirti, B. D. Singh & V. L. Chopra

  2. Department of Plant Sciences, University of Hyderabad, 500 046, Hyderabad, India

    P. B. Kirti

  3. School of Biotechnology, Banaras Hindu University, 221 005, Varanasi, India

    B. D. Singh

Authors
  1. R. Chakrabarty
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. N. Viswakarma
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. S. R. Bhat
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. P. B. Kirti
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. B. D. Singh
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. V. L. Chopra
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to V. L. Chopra.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Chakrabarty, R., Viswakarma, N., Bhat, S.R. et al. Agrobacterium-mediated transformation of cauliflower: Optimization of protocol and development of Bt-transgenic cauliflower. J. Biosci. 27, 495–502 (2002). https://doi.org/10.1007/BF02705046

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF02705046

Keywords

Search

Navigation