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Effect of promoter driving selectable marker on corn transformation

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Abstract

Identification of an appropriate selection agent and its corresponding selectable marker gene is one of the first steps in establishing a transformation protocol for a given plant species. As the promoter controls expression level of the genes, the promoter driving the selectable marker gene can affect transformation. However, investigations into the direct effect of promoters driving selectable marker on transformation are lacking in the literature though many reports of relative strengths of promoters driving reporter genes like GUS or CAT or GFP are available. In the present study, we have compared rice Actin1 and CaMV.35S (commonly used promoters in monocotyledonous plant transformation) promoters driving nptII for their effectiveness in paromomycin selection of transgenic corn events. To enable statistically meaningful analysis of the results, a large sample size of nearly 5,000 immature embryos (explants) was employed producing approximately 1,250 independent events from each of the two constructs in four independent experiments. The rate of appearance of resistant calli and percentage of resistant calli recovered was higher with P-Os.Actin1/nptII/nos3′ as compared to P-CaMV.35S/nptII/nos3′ in all four experiments. There was no appreciable difference either in the frequency of plant regeneration or in the morphological characteristics of plants recovered from the two constructs. Although the escape rate trended lower with P-Os.Actin1 as compared to P-CaMV.35S, the recovery of low copy events was significantly higher with P-CaMV.35S. The higher transformation frequency with P-Os.Actin1 could be related to the strength of this promoter as compared to P-CaMV.35S in the explants and/or calli. Based on these results, we infer that the promoter driving the selectable marker is an important factor to be considered while establishing a high throughput transformation protocol as it could not only influence the transformation frequency but also the copy number of the transgene in the recovered transgenics.

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Abbreviations

bar/pat:

Phosphinothricin N-acetyltransferase

CAT:

Chloramphenicol acetyl transferase

EPSPS-CP4:

5-Enol-pyruvylshikimate-3-phosphate synthase from Agrobacterium sp. CP4

GFP:

Green fluorescent protein

GUS:

β-Glucuronidase

hptII:

Hygromycin phosphotransferase II

nos3′:

Transcriptional termination signal of Agrobacterium nopaline synthase

nptII:

Neomycin phosphotransferase II

P-CaMV.35S:

Cauliflower mosaic virus 35S promoter

P-Os.Act1:

Oryza sativa Actin1 promoter

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Acknowledgements

Many people have contributed to this research publication. The authors would like to acknowledge the Corn Transformation group in Monsanto Research Centre, Bangalore. We thank Muniraju Shamanna, Venkatachalapathy Muniraju, Nagraj K. Toranagatta, and Janardhana Sundupalle for their technical assistance and Shivbachan S. Kushwaha and Hari Priya G.G. for greenhouse care. We also thank Jay Harrison, Monsanto Company, St. Louis, for his help with statistical analysis. The authors would also like to thank Jagadish N. Mittur for his encouragement during this study and 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, V. Prasad, Thillai P. Chidambram, Shoba Cherian, T. L. Jayaprakash & Raghava S. Boddupalli

  2. Chesterfield Campus, Monsanto Company, 700 Chesterfield Parkway North, Chesterfield, MO, 63017, USA

    Santanu Dasgupta, Qi Wang & Michael T. Mann

  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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  10. Raghava S. Boddupalli
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Correspondence to Raghava S. Boddupalli.

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Shiva Prakash, N., Prasad, V., Chidambram, T.P. et al. Effect of promoter driving selectable marker on corn transformation. Transgenic Res 17, 695–704 (2008). https://doi.org/10.1007/s11248-007-9149-0

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  • DOI: https://doi.org/10.1007/s11248-007-9149-0

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