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Determination of the expression levels of DNA methyltransferase genes during a highly efficient regeneration system via shoot organogenesis in the diploid apomict Boechera divaricarpa

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Abstract

In this study, we established the first efficient method to regenerate diploid apomict Boechera divaricarpa via shoot organogenesis. Hypocotyl explants were cultured on MS medium supplemented with plant growth regulators. The morphogenic potential of B. divaricarpa hypocotyl tissue was investigated to establish an efficient adventitious shoot regeneration system. We tested hypocotyls from 7-day-old in vitro seedlings. The effect of various concentrations of cytokinin and auxin on in vitro regeneration of these explants was also investigated. We found that callus induction and shoot regeneration were significantly affected by the concentrations and types of plant growth regulators. MS medium supplemented with 17.75 µM benzylaminopurine and 0.53 µM naphthaleneacetic acid gave the highest number of shoots per explant after 4 weeks in culture. We also determined the expression levels of three DNA methyltransferase genes under tissue culture conditions to understand their activities during callus induction and shoot regeneration. BdMET1 was expressed at low levels during both callus induction and shoot regeneration, while expression of BdDRM2 was high during callus induction and low during shoot regeneration. BdCMT3 was highly expressed in both hypocotyl explants and seedlings and may be a good candidate to induce epigenetic variations in diploid apomict B. divaricarpa shoots derived from tissue culture. Our study provides an efficient in vitro regeneration method and determines the expression levels during tissue culture of methyltransferase genes; it can be used as a new tool to understand the molecular biology of apomixis in B. divaricarpa.

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Acknowledgments

We would like to thank to Prof. Dr. Timothy F. Sharbel (Leibniz Institute of Plant Genetics and Crop Plant Research, Germany) for providing partial transcript sequences of methyltransferase genes, Prof. Dr. Rod J. Scott (University of Bath, England) and Prof. Dr. M. Eric Schranz (Wageningen University, Holland) for providing the seeds, and Şebnem Güneş for primer design. KMT designed and supervised the study. KMT, AÖ, and FS performed gene expression studies. KMT, NÇ, and DE performed the in vitro regeneration work.

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The authors declare that they have no conflict of interest.

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  1. Department of Biology, Faculty of Arts and Sciences, Çanakkale Onsekiz Mart University, 17100, Çanakkale, Turkey

    Kemal M. Taskin, Aslıhan Özbilen, Fatih Sezer, Nurşen Çördük & Damla Erden

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  1. Kemal M. Taskin
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  2. Aslıhan Özbilen
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  3. Fatih Sezer
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Taskin, K.M., Özbilen, A., Sezer, F. et al. Determination of the expression levels of DNA methyltransferase genes during a highly efficient regeneration system via shoot organogenesis in the diploid apomict Boechera divaricarpa . Plant Cell Tiss Organ Cult 121, 335–343 (2015). https://doi.org/10.1007/s11240-014-0704-0

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