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Production of marker-free disease-resistant potato using isopentenyl transferase gene as a positive selection marker

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Abstract

The use of antibiotic or herbicide resistant genes as selection markers for production of transgenic plants and their continuous presence in the final transgenics has been a serious problem for their public acceptance and commercialization. MAT (multi-auto-transformation) vector system has been one of the different strategies to excise the selection marker gene and produce marker-free transgenic plants. In the present study, ipt (isopentenyl transferase) gene was used as a selection marker gene. A chitinase gene, ChiC (isolated from Streptomyces griseus strain HUT 6037) was used as a gene of interest. ChiC gene was cloned from the binary vector, pEKH1 to an ipt-type MAT vector, pMAT21 by gateway cloning and transferred to Agrobacterium tumefaciens strain EHA105. The infected tuber discs of potato were cultured on hormone- and antibiotic-free MS medium. Seven of the 35 explants infected with the pMAT21/ChiC produced shoots. The same antibiotic- and hormones-free MS medium was used in subcultures of the shoots (ipt like and normal shoots). Molecular analyses of genomic DNA from transgenic plants confirmed the integration of gene of interest and excision of the selection marker in 3 of the 7 clones. Expression of ChiC gene was confirmed by Northern blot and western blot analyses. Disease-resistant assay of the marker-free transgenic, in vitro and greenhouse-grown plants exhibited enhanced resistance against Alternaria solani (early blight), Botrytis cinerea (gray mold) and Fusarium oxysporum (Fusarium wilt). From these results it could be concluded that ipt gene can be used as a selection marker to produce marker-free disease-resistant transgenic potato plants on PGR- and antibiotic-free MS medium.

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Acknowledgments

We are very grateful to Japan Society for Promotions of Sciences (JSPS) for their financial support for this research project. We are also thankful to Pulp and Paper Research group, Nippon Paper Industries, Tokyo, who kindly provided the MAT vector constructs.

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Authors and Affiliations

  1. Laboratory of Plant Cell Technology, Graduate School of Horticulture, Chiba University, 648 Matsudo, Matsudo, Chiba, 271-8510, Japan

    Raham Sher Khan, Valentine Otang Ntui, Dong Poh Chin, Ikuo Nakamura & Masahiro Mii

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  1. Raham Sher Khan
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  2. Valentine Otang Ntui
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  3. Dong Poh Chin
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  4. Ikuo Nakamura
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  5. Masahiro Mii
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Correspondence to Raham Sher Khan or Masahiro Mii.

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Communicated by H. Ebinuma.

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Khan, R.S., Ntui, V.O., Chin, D.P. et al. Production of marker-free disease-resistant potato using isopentenyl transferase gene as a positive selection marker. Plant Cell Rep 30, 587–597 (2011). https://doi.org/10.1007/s00299-010-0974-x

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  • DOI: https://doi.org/10.1007/s00299-010-0974-x

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