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Transformation of Wolffia arrhiza (L.) Horkel ex Wimm

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Abstract

To date, Lemna and Spirodela species of the family Lemnaceae are the only species that have been used to produce recombinant proteins for pharmaceutical and veterinary purposes. Wolffia arrhiza is the most evolutionarily advanced species of the Lemnaceae. A rootless duckweed, it is the most promising target for biopharmingas a candidate for submerged cultivation in a fermenter. As a first step toward future biotechnological use of Wolffia, we established a stable transformation system for it based on Agrobacterium-mediated transformation. Following inoculation with the bacteria, Wolffia cluster explants were cultured for 2 weeks on media containing 2.0 mg l−1 2,4-dichlorophenoxyacetic acid and 2.0 mg l−1 N6-benzyladenine. Explants were then transferred to growth regulator-free media in the presence of 5.0 mg l−1 hygromycin B to select antibiotic-resistant plants. Other selective agents—kanamycin and phosphinothricin—were not suitable for Wolffia, nor was application of particle bombardment for the delivery of foreign DNA to Wolffia explants. The developed agro transformation conditions yielded stably transformed lines of Wolffia, confirmed by Southern blotting, with an efficiency of 0.2–0.4 % transgenes per 100 explants.

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Abbreviations

2,4-D:

2,4-Dichlorophenoxyacetic acid

BA:

N6-benzyladenine

GFP:

Green fluorescent protein

GUS:

β-Glucuronidase

Hpt:

Hygromycin phosphotransferase

Hyg:

Hygromycin B

Km:

Kanamycin sulfate

NptII:

Neomycin phosphotransferase

PCL (Picloram):

4-Amino-3,5,6-trichloropicolinic acid

PPT (Phosphinothricin):

Glufosinate ammonium

Rif:

Rifampicin

SH:

Schenk and Hildebrandt medium

TLP:

Thaumatin-like protein

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Acknowledgments

The work was supported by a grant from the Ministry of Education and Science of the Russian Federation (Government Contract No. 14.B25.31.0027).

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Authors and Affiliations

  1. All-Russia Research Institute of Agricultural Biotechnology, Moscow, Russia

    Pavel Khvatkov, Mariya Chernobrovkina, Anna Okuneva, Alexander Pushin & Sergey Dolgov

  2. Branch of Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Puschino, Russia

    Pavel Khvatkov, Mariya Chernobrovkina, Anna Okuneva, Alexander Pushin & Sergey Dolgov

Authors
  1. Pavel Khvatkov
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  2. Mariya Chernobrovkina
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  3. Anna Okuneva
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  4. Alexander Pushin
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  5. Sergey Dolgov
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Correspondence to Pavel Khvatkov.

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Khvatkov, P., Chernobrovkina, M., Okuneva, A. et al. Transformation of Wolffia arrhiza (L.) Horkel ex Wimm. Plant Cell Tiss Organ Cult 123, 299–307 (2015). https://doi.org/10.1007/s11240-015-0834-z

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  • DOI: https://doi.org/10.1007/s11240-015-0834-z

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