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Novel recombinant binary vectors harbouring Basta (bar) gene as a plant selectable marker for genetic transformation of plants

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Abstract

Genetic transformation is one of the most widely used technique in crop improvement. However, most of the binary vectors used in this technique, especially cloning based, contain antibiotic genes as selection marker that raise serious consumer and environmental concerns; moreover, they could be transferred to non-target hosts with deleterious effects. Therefore, the goal of this study was reconstruction of the widely used pBI121 binary vector by substituting the harmful antibiotic selection marker gene with a less-harmful selection marker, Basta (herbicide resistance gene). The generated vectors were designated as pBI121NB and pBI121CB, in which Basta gene was expressed under the control of Nos or CaMV 35S promoter, respectively. The successful integration of the new inserts into both the vectors was confirmed by PCR, restriction digestion and sequencing. Both these vectors were used in transforming Arabidopsis, Egyptian wheat and barley varieties using LBA4404 and GV3101 Agrobacterium strains. The surfactant Tween-20 resulted in an efficient transformation and the number of Arabidopsis transformants was about 6–9 %. Soaked seeds of wheat and barley were transformed with Agrobacterium to introduce the bacteria to the growing shoot apices. The percentage of transgenic lines was around 16–17 and 14–15 % for wheat and barley, respectively. The quantitative studies presented in this work showed that both LBA4404 and GV3101 strains were suitable for transforming Egyptian wheat and barley.

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Abbreviations

BA:

Benzyl adenine

CaMV 35S:

Cauliflower mosaic virus 35S promoter

DMSO:

Dimethyl sulfoxide

DMF:

N,N-Dimethyl formamide

LB medium:

Luria-Bertani medium

Nos:

Nopaline synthase

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Acknowledgments

I would like to thank Dr. Gaber M. Abogadallah (Associate Professor of Plant Genetics, Botany Department, Faculty of Science, Damietta University, New Damietta 34517, Egypt) for hosting this work in his laboratory. Agrobacterium GV3101 strain was kindly provided from Dr. Mohamed S. Tawfik (Agricultural Genetic Engineering Research Institute (AGERI), Agriculture Research Center (ARC), Giza, Egypt 12619).

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  1. Department of Botany, Faculty of Science, Damietta University, New Damietta, 34517, Egypt

    Reham M. Nada

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Correspondence to Reham M. Nada.

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Nada, R.M. Novel recombinant binary vectors harbouring Basta (bar) gene as a plant selectable marker for genetic transformation of plants. Physiol Mol Biol Plants 22, 241–251 (2016). https://doi.org/10.1007/s12298-016-0360-4

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