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Selection and genetic transformation of a fast-growing cell line in cotton (Gossypium hirsutum) for transgene expression studies

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Abstract

A cell line, named CCL-1 has been obtained in cotton which proliferates on phytohormone free MS medium containing high KNO3. This cell line can be maintained as a friable callus on solidified medium and also readily converted to cell suspensions and maintained in liquid medium. CCL-1 cell suspensions were transformed using a disarmed Agrobacterium strain with binary vectors containing selectable marker genes- nptII, hptII, bar and ALS dm to check the efficacy of these marker genes for selecting transformed cell lines. Transformation was monitored by reporter genes, either GUS or GFP. Transformed cell lines could be readily obtained with all the marker genes, hpt being the most efficient marker for unambiguous selection in 10 day time period. Transformed cell lines showed expression of the reporter genes. We developed a protocol for using these cell lines for insect feeding bioassays by using control CCL-1 and a transformed derivative line containing the cry1Ac gene. The most optimal feeding method is to mix lyophilized transformed cells with an artificial diet to achieve various concentrations of the toxin. The most useful selectable markers for insect feeding bioassay studies are bar and ALS dm as these do not interfere with insect growth. CCL-1 will be a convenient tool for studying transgene expression in cotton and will be particularly useful for testing a large number of genes for antifeedant/ insecticidal activity.

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Abbreviations

GUS:

β-Glucuronidase

hpt :

Hygromycin phosphotransferase

nptII :

Neomycin phosphotransferase

bar :

phosphinothricin acetyl transferase

Als :

Acetolactate synthase

MS medium:

Murashige and Skoog medium

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Acknowledgments

DP acknowledges support of DST through a J. C. Bose fellowship. We thank Satish Kumar Yadava for his support with statistical analysis, Kumar Paritosh for help with reporter gene assays, Vinee Khanna for hpt::cry1Ac construct and Umakant for his help in insect feeding bioassays.

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Authors and Affiliations

  1. Centre for Genetic Manipulation of Crop Plants, University of Delhi South Campus, Benito Juarez Road, New Delhi, 110021, India

    Amarjeet Kumar Singh & Deepak Pental

  2. Department of Genetics, University of Delhi South Campus, Benito Juarez Road, New Delhi, 110021, India

    Deepak Pental

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  1. Amarjeet Kumar Singh
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  2. Deepak Pental
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Correspondence to Deepak Pental.

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Supplementary Fig. 1

In vitro culture scheme used for the development of cell line CCL-1 (PPT 117 kb)

Supplementary Fig. 2

Growth of the CCL-1 cell line in liquid medium measured by increase in fresh weight of the suspension at different time intervals (PPT 102 kb)

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Singh, A.K., Pental, D. Selection and genetic transformation of a fast-growing cell line in cotton (Gossypium hirsutum) for transgene expression studies. J. Plant Biochem. Biotechnol. 24, 225–232 (2015). https://doi.org/10.1007/s13562-014-0262-x

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  • DOI: https://doi.org/10.1007/s13562-014-0262-x

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