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Development of disease-resistant marker-free tomato by R/RS site-specific recombination

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Abstract

The selection marker genes, imparting antibiotic or herbicide resistance, in the final transgenics have been criticized by the public and considered a hindrance in their commercialization. Multi-auto-transformation (MAT) vector system has been one of the strategies to produce marker-free transgenic plants without using selective chemicals and plant growth regulators (PGRs). In the study reported here, isopentenyltransferase (ipt) gene was used as a selection marker and wasabi defensin (WD) gene, isolated from Wasabia japonica as a target gene. WD was cloned from the binary vector, pEKH-WD to an ipt-type MAT vector, pMAT21 by gateway cloning and transferred to Agrobacterium tumefaciens strain EHA105. Infected cotyledons of tomato cv. Reiyo were cultured on PGR- and antibiotic-free MS medium. Adventitious shoots were developed by the explants infected with the pMAT21/wasabi defensin. The same PGR- and antibiotic-free MS medium was used in subcultures of the adventitious shoot lines (ASLs) to produce ipt and normal shoots. Approximately, 6 months after infection morphologically normal shoots were produced. Molecular analyses of the developed shoots confirmed the integration of gene of interest (WD) and excision of the selection marker (ipt). Expression of WD was confirmed by Northern blot and Western blot analyses. The marker-free transgenic plants exhibited enhanced resistance against Botrytis cinerea (gray mold), Alternaria solani (early blight), Fusarium oxysporum (Fusarium wilt) and Erysiphe lycopersici (powdery mildew).

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

    Raham Sher Khan, Ikuo Nakamura & Masahiro Mii

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  1. Raham Sher Khan
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  2. Ikuo Nakamura
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  3. Masahiro Mii
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Correspondence to Raham Sher Khan.

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Communicated by H. Ebinuma.

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Khan, R.S., Nakamura, I. & Mii, M. Development of disease-resistant marker-free tomato by R/RS site-specific recombination. Plant Cell Rep 30, 1041–1053 (2011). https://doi.org/10.1007/s00299-011-1011-4

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