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Agrobacterium tumefaciens-mediated genetic transformation of haptophytes (Isochrysis species)

  • Applied genetics and molecular biotechnology
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Abstract

Isochrysis galbana and Isochrysis sp. are economically important microalgae from the division of haptophytes. Here, we report Agrobacterium-mediated stable DNA transfer into their nuclear genomes. Initial studies were performed to standardize co-cultivation media and determine the sensitivity of the microalgae to selective agents. Up to 1 mg/ml of the antibiotic hygromycin did not inhibit growth, whereas both the haptophytes bleached in artificial seawater (ASW) medium containing micromolar concentrations of the herbicide norflurazon. Co-cultivation of Isochrysis sp. and I. galbana with Agrobacterium tumefaciens strain LBA 4404 harboring the binary vector pCAMBIA 1380-pds-L504R yielded norflurazon-resistant (NR) colonies visible on selective plates after 20–30 days. pCAMBIA 1380-pds-L540R was constructed by cloning a mutated genomic phytoene desaturase (pds) gene from Haematococcus pluvialis as a selectable marker gene into the binary vector system pCAMBIA 1380. Co-cultivation of Isochrysis sp. with A. tumefaciens in ASW medium containing 200 μM of acetosyringone for 72 h produced the highest number of NR cells. For I. galbana, 100 μM of acetosyringone, ASW medium, and 48 h co-cultivation period appeared to be optimum co-cultivation parameters. The NR colonies kept their resistance phenotype for at least 24 months, even in the absence of selective pressure. The transfer of the pds gene in NR cells was shown by PCR amplification of the T-DNA sequences from the genomic DNA of NR cells and Southern blot analysis using T-DNA sequences as probes. The genetic manipulation described here will allow metabolic engineering and a better understanding of several biochemical pathways in the future.

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Acknowledgment

Binod Prasad acknowledges National Institute for International Education (NIIED, South Korea) for a fellowship. The authors are thankful to Dr. J. Steinbrenner (Universität Konstanz, Germany) and Prof. Choi PS (Nambu University, South Korea) for kindly providing the plasmid pPLAT-pds-L504R and Agrobacterium tumefaciens strain LBA 4404, respectively. This work was supported by Busan Metropolitan City, Korea, and Federal Ministry of Education and Research, Germany, which is greatly appreciated by the authors.

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Authors and Affiliations

  1. Department of Biotechnology, Dongseo University, San 47 Jurye-ro, Sasang-Gu, Busan, 617-716, South Korea

    Binod Prasad, Nithya Vadakedath, Hyun-Jeong Jeong, Thiyam General & Man-Gi Cho

  2. Institute for Biotechnology, Technical University Berlin, 13353, Berlin, Germany

    Wolfgang Lein

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  1. Binod Prasad
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  2. Nithya Vadakedath
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  3. Hyun-Jeong Jeong
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Correspondence to Man-Gi Cho.

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Prasad, B., Vadakedath, N., Jeong, HJ. et al. Agrobacterium tumefaciens-mediated genetic transformation of haptophytes (Isochrysis species). Appl Microbiol Biotechnol 98, 8629–8639 (2014). https://doi.org/10.1007/s00253-014-5900-7

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  • DOI: https://doi.org/10.1007/s00253-014-5900-7

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