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Generation of genetically stable transformants by Agrobacterium using tomato floral buds

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Abstract

Tomato (Solanum lycopersicum) is a model crop plant for the study of fruit ripening and disease resistance. Here we present a systemic study on in planta transformation of tomato with Agrobacterium tumefaciens strain LBA4404 harboring pCAMBIA1303 binary vector bearing HPTII as a plant selectable marker and mGFP/GUS fusion as the reporter gene. We attempted the transformation of tomato at different developmental stages viz. during seed germination, seedling growth, and floral bud development. The imbibition of seeds with Agrobacterium suspension led to seed mortality. The vacuum infiltration of seedlings with Agrobacterium suspension led to sterility in surviving plants. Successful transformation could be achieved either by dipping of developing floral buds in the Agrobacterium suspension or by injecting Agrobacterium into the floral buds. Most floral buds subjected to dip as well as to injection either aborted or had arrested development. The pollination of surviving floral buds with pollen from wild-type plants yielded fruits bearing seeds. A transformation efficiency of 0.25–0.50% was obtained on floral dips/floral injections. Transgenic plants were selected by screening seedlings for hygromycin resistance. The presence of the transgene in genomic DNA was confirmed by Southern blot analysis and expression of the reporter gene up to the T4 generation. The amenability of tomato for in planta transformation simplifies the generation of transgenic tomato plants obviating intervening tissue culture.

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Funding

This work was supported by the Department of Biotechnology (Grant No. BT/PR6803/PBD/16/621/2005 and BT/PR11671/PBD/16/828/2008 to R.S. and Y.S.).

Author contributions

MSS carried out transformation and characterization of T1–T3 plants. AK carried out the characterization of T4 plants, FPNI-PCR, and multiplex PCR. AK, AKP, SS and PS contributed to the Southern analysis. MSS and RS designed the experimental protocols. MSS, AK, YS, and RS wrote the manuscript.

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    Authors and Affiliations

    1. Repository of Tomato Genomics Resources, Department of Plant Sciences, University of Hyderabad, Hyderabad, 500046, India

      Miduthuri Satya Sharada, Alka Kumari, Arun Kumar Pandey, Sulabha Sharma, Preeti Sharma, Yellamaraju Sreelakshmi & Rameshwar Sharma

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    1. Miduthuri Satya Sharada
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    2. Alka Kumari
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    3. Arun Kumar Pandey
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    4. Sulabha Sharma
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    7. Rameshwar Sharma
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    Correspondence to Rameshwar Sharma.

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    Miduthuri Satya Sharada and Alka Kumari have equally contributed.

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    Sharada, M.S., Kumari, A., Pandey, A.K. et al. Generation of genetically stable transformants by Agrobacterium using tomato floral buds. Plant Cell Tiss Organ Cult 129, 299–312 (2017). https://doi.org/10.1007/s11240-017-1178-7

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