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Cre/lox system to develop selectable marker free transgenic tobacco plants conferring resistance against sap sucking homopteran insect

  • Genetic Transformation and Hybridization
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Abstract

A binary expression vector was constructed containing the insecticidal gene Allium sativum leaf agglutinin (ASAL), and a selectable nptII marker gene cassette, flanked by lox sites. Similarly, another binary vector was developed with the chimeric cre gene construct. Transformed tobacco plants were generated with these two independent vectors. Each of the T0 lox plants was crossed with T0 Cre plants. PCR analyses followed by the sequencing of the target T-DNA part of the hybrid T1 plants demonstrated the excision of the nptII gene in highly precised manner in certain percentage of the T1 hybrid lines. The frequency of such marker gene excision was calculated to be 19.2% in the hybrids. Marker free plants were able to express ASAL efficiently and reduce the survivability of Myzus persiceae, the deadly pest of tobacco significantly, compared to the control tobacco plants. Results of PCR and Southern blot analyses of some of the T2 plants detected the absence of cre as well as nptII genes. Thus, the crossing strategy involving Cre/lox system for the excision of marker genes appears to be very effective and easy to execute. Documentation of such marker excision phenomenon in the transgenic plants expressing the important insecticidal protein for the first time has a great significance from agricultural and biotechnological points of view.

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Acknowledgments

The authors are thankful to Swiss Agency for Development and Cooperation, Government of Switzerland and the Department of Biotechnology, Government of India under the Indo-Swiss Collaboration in Biotechnology for financial contributions and Bose Institute, Kolkata, India for providing infrastructure for carrying out the work described here. B. Hohn and D. Schuermann acknowledge the financial help of the Novartis Research Foundation. The authors are thankful to Dr. J. Mundy for providing the pCrox18 plasmid.

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Author notes

  1. David Schuermann

    Present address: Departement Klinisch–Biologische Wissenschaften DKBW, Universität Basel, Mattenstrasse 28, 4058, Basel, Switzerland

Authors and Affiliations

  1. Plant Molecular and Cellular Genetics, Bose Institute, P1/12 C.I.T. Scheme VIIM, Kankurgachi, Kolkata, 700054, India

    Dipankar Chakraborti, Anindya Sarkar, Hossain A. Mondal & Sampa Das

  2. Friedrich Miescher Institute for Biomedical Research, Novartis Research Foundation, Maulbeerstrasse 66, 4058, Basel, Switzerland

    David Schuermann & Barbara Hohn

  3. Department of Agricultural Biotechnology, Assam Agricultural University, Jorhat, 785012, India

    Bidyut K. Sarmah

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  1. Dipankar Chakraborti
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  2. Anindya Sarkar
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  3. Hossain A. Mondal
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Correspondence to Sampa Das.

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Communicated by L. Jouanin.

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299_2008_585_MOESM1_ESM.pdf

Fig. 9 (Supplementary) PCR analyses of 7 T1 hybrid progenies [1433(7), 1433(12), 1735(3), 1938(2), 1938(6), 1938(7) and 1938(14)] for the (a) absence of nptII and (b) presence of ASAL coding sequence. Lane ‘+’, plasmid pBK12.1 as positive control; lane M, DNA ladder mix (Generuler™, MBI Fermentas). (PDF 146 kb)

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Chakraborti, D., Sarkar, A., Mondal, H.A. et al. Cre/lox system to develop selectable marker free transgenic tobacco plants conferring resistance against sap sucking homopteran insect. Plant Cell Rep 27, 1623–1633 (2008). https://doi.org/10.1007/s00299-008-0585-y

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