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Marker-free transgenic corn plant production through co-bombardment

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Abstract

The use of particle gun for the production of marker-free plants is scant in published literature. Perhaps this is a reflection of the widely held notion that the events generated through bombardment tend to have multiple copies of transgenes, usually integrated at a single locus, features which precludes segregating away the selectable marker gene. However, our previous studies have shown that single-copy integrants are obtained at a high frequency if limited quantity of DNA is used for bombardment. Also, the concatemerized insertion of transgenes has been demonstrated to be greatly reduced if “cassette DNA” is employed in place of whole plasmid DNA for bombardment. Based on the above findings, in the present study the feasibility of co-bombardment was evaluated for the production of marker-free plants in corn, employing a combination of limited quantity DNA and cassette DNA approaches for bombardment. Transgenic events were generated after co-bombardment of a selectable marker cassette containing the nptII gene (2.5 ng per shot) and a GUS gene cassette (15 ng per shot). Among these events single-copy integrants for nptII gene occurred at an average frequency of 68% within which the co-expression frequency of GUS and nptII genes ranged from 41% to 80%. Marker-free corn plants could be identified from the progeny of 28 out of the 103 R0 co-expressing events screened. The results demonstrate that by using cassette DNA and low quantities of DNA for bombardment, marker-free plants are produced at efficiencies comparable to that of Agrobacterium-based co-transformation methods.

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Abbreviations

P-CaMV.35S:

Cauliflower mosaic virus 35S promoter

GUS :

β-glucuronidase

nptII :

Neomycin phosphotransferase II

nos:

Nopaline synthase

Bt :

Bacillus thuringiensis

P-Os.rbcS:

Oryza sativa rubisco small subunit promoter

P-Os.Actin15:

Oryza sativa actin15 promoter

P-Os.GT-1:

Oryza sativa glutelin promoter

GOI:

Gene of interest

LWA:

Leaf whorl assay

DAG:

Days after germination

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Acknowledgments

Many people have contributed to these investigations carried out by the corn transformation and greenhouse teams at the Monsanto Research Centre, Bangalore, India. We are grateful to James D. Massucci, James F. Rice and Rodney A. Kowalewski, Monsanto Company, St. Louis, MO, USA, for performing radio-active Southern hybridization blots. We thank Beiyan Zeng, Monsanto Company, St. Louis, MO, USA, for her help with statistical analysis. The technical assistance rendered by Venkatachalapathy Muniraju and Janardhana Sundupalle is acknowledged. The authors are indebted to Mittur N. Jagadish for his encouragement throughout this study and for the critical reading of the manuscript.

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Authors and Affiliations

  1. Monsanto Research Centre, #44/2A, Vasanths’ Business Park, Bellary Road, NH:7, Hebbal, Bangalore, 560092, India

    N. Shiva Prakash, R. Bhojaraja, S. K. Shivbachan, G. G. Hari Priya, T. K. Nagraj, V. Prasad, V. Srikanth Babu, T. L. Jayaprakash & Raghava S. Boddupalli

  2. Monsanto India Limited, Ahura Complex, 5th Floor, #96, Mahakali Caves Road, Andheri (E), Mumbai, 400093, India

    Santanu Dasgupta

  3. Mystic Research, Monsanto Company, 62 Maritime Drive, Mystic, CT, 06355, USA

    T. Michael Spencer

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  1. N. Shiva Prakash
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  2. R. Bhojaraja
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  3. S. K. Shivbachan
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  4. G. G. Hari Priya
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  5. T. K. Nagraj
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  6. V. Prasad
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  7. V. Srikanth Babu
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  8. T. L. Jayaprakash
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  9. Santanu Dasgupta
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  10. T. Michael Spencer
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  11. Raghava S. Boddupalli
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Corresponding author

Correspondence to Raghava S. Boddupalli.

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Communicated by P. Kumar.

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Shiva Prakash, N., Bhojaraja, R., Shivbachan, S.K. et al. Marker-free transgenic corn plant production through co-bombardment. Plant Cell Rep 28, 1655–1668 (2009). https://doi.org/10.1007/s00299-009-0765-4

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