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Sonication assisted Agrobacterium-mediated transformation enhances the transformation efficiency in flax (Linum usitatissimum L.)

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Abstract

A sonication-assisted, Agrobacterium-mediated, co-cultivation technique was used in an attempt to increase the transformation efficiency of flax. Hypocotyls and cotyledons excised from about 10-day-old flax seedlings grown in vitro were placed into a 10 mM MgSO4 solution, and inoculated with an A. tumefaciens vector bearing the mgfp5-ER gene driven by the CaMV 35S promoter. The explants were subjected to pulses of ultrasound delivered by a sonicator apparatus (35 kHz) for 0–150 s and co-cultivated for 2 h at 27°C. The dried hypocotyls and cotyledons were grown on a selective MS medium to promote shoot regeneration. An electron microscopic study showed that the sonication treatment resulted in thousands of microwounds on and below the surface of the explants. A stereo microscope Leica MZ 12 equipped with a GFP adaptor was used to assess the infection and transformation of plant tissues in real time. After only 48 h and for at least 30 days after bacteria elimination, signs of transgene expression could be seen as a bright fluorescence. Our results show that treatment with ultrasound facilitates an enhanced uptake of plasmid DNA into the cells of flax hypocotyls and cotyledons and that its efficiency depends on the duration of the treatment and the frequency used. SAAT could be a promising tool for enhancing transformation efficiency in flax.

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Abbreviations

BAP:

6-Benzylaminopurine

CaMV 35S:

Cauliflower mosaic virus promoter

GFP:

Green fluorescent protein

GUS:

β-Glucuronidase

Kn:

Kanamycin

SEM:

Scanning electron microscopy

NAA:

Naphthalene acetic acid

gfp :

Green fluorescent protein gene

mgfp5-ER :

Modified gene for Green fluorescent protein

nptII :

Neomycin Phosphotransferase II gene

nos (promoter):

Nopaline Synthase promoter

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Acknowledgements

The authors are grateful for the financial support received from Ministry of Education, Youth and Sport of the Czech Republic (grants 1M06030, 1PO5ME800). Linguistic revision was kindly performed by John McAvoy.

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Authors and Affiliations

  1. Faculty of Health and Social Studies, University of South Bohemia, Jírovcova 24, 370 04, Ceske Budejovice, Czech Republic

    Martina Beranová, Slavomír Rakouský, Zuzana Vávrová & Tomáš Skalický

  2. Faculty of Science, Department of Genetics, University of South Bohemia, Branišovská 31, 370 05, Ceske Budejovice, Czech Republic

    Slavomír Rakouský, Zuzana Vávrová & Tomáš Skalický

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  1. Martina Beranová
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  2. Slavomír Rakouský
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  3. Zuzana Vávrová
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  4. Tomáš Skalický
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Correspondence to Slavomír Rakouský.

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Beranová, M., Rakouský, S., Vávrová, Z. et al. Sonication assisted Agrobacterium-mediated transformation enhances the transformation efficiency in flax (Linum usitatissimum L.). Plant Cell Tiss Organ Cult 94, 253–259 (2008). https://doi.org/10.1007/s11240-007-9335-z

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