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Enhanced accumulation of cadmium in Linum usitatissimum L. plants due to overproduction of metallothionein α-domain as a fusion to β-glucuronidase protein

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Abstract

As most cultivars of flax and linseed (Linum usitatissimum L.) are capable of accumulating cadmium (Cd), they are suitable candidates for phytoextraction of the metal from contaminated soils. In an attempt to enhance the phytoextraction capacity of L. usitatissimum through overproduction of an efficient heterologous Cd-binding peptide, we engineered linseed breeding line AGT 917 to constitutively express genetic fusion of α-domain of mammalian metallothionein 1a (αMT1a) and β-glucuronidase gus gene under the control of CaMV 35S promoter. An improved transformation protocol was developed, which involved co-cultivation of AGT 917 hypocotyl segments with partially removed epidermis with Agrobacterium suspension for 10 min in the presence of 200 mg l−1 cellulase. The enzyme treatment increased the transformation efficiency (TE) 1.6-fold as compared to agroinfection without cellulase. Less pronounced impact on TE exerted 100 mg l−1 acetosyringone, increasing TE 1.3-fold. When tested in soils amended with Cd at 20 and 360 mg kg−1, the mature αMT1a::gus plants accumulated more Cd than parental AGT 917: the stem Cd concentrations in the best performing αMT1/2 line were 3.3- and 1.9-fold higher, respectively. Moreover, hypocotyl explants of αMT1/2 line showed 1.7-fold higher biomass than those of AGT 917 on media containing 15 mg Cd l−1, indicating that αMT1a::gus did confer higher Cd tolerance to engineered plant. Overproduction of metal-binding peptides thus appears to be a viable strategy for the production of L. usitatissimum with improved phytoremediation capacity.

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Acknowledgments

This work was supported by grants from the Ministry of Education, Youth and Sports of the Czech Republic (no. 2678424601 and 1M06030). The authors thank Dr. E. Tejklová for providing seeds of linseed breeding lines. We are also grateful to J. Matulíková, E. Fialová, D. Bencová and K. Moťková for excellent technical assistance.

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  1. Plant Biotechnology Department, AGRITEC Plant Research Ltd., Zemědělská 2520/16, 787 01, Šumperk, Czech Republic

    Miroslava Vrbová, Jiří Horáček, Petr Smýkal, Lenka Švábová, Martina Větrovcová, Iva Smýkalová & Miroslav Griga

  2. Department of Biochemistry and Microbiology, Institute of Chemical Technology, Prague, Technická 3, 166 28, Prague, Czech Republic

    Pavel Kotrba

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  1. Miroslava Vrbová
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  2. Pavel Kotrba
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Correspondence to Pavel Kotrba.

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Vrbová, M., Kotrba, P., Horáček, J. et al. Enhanced accumulation of cadmium in Linum usitatissimum L. plants due to overproduction of metallothionein α-domain as a fusion to β-glucuronidase protein. Plant Cell Tiss Organ Cult 112, 321–330 (2013). https://doi.org/10.1007/s11240-012-0239-1

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