Abstract
Plant cell suspension cultures are used in basic research and applied biotechnology. In both cases, the transfer and stable integration of heterologous genes is a required technique. This report describes a rapid method for transformation of cell cultures of Medicago truncatula, the model species for the legume family. Accession A17 from the cultivar Jemalong is the reference genotype selected for the sequencing of the genome and therefore most studies on Medicago are carried out on this accession line. However, this line has a low embryogenic capacity and is poorly responsive to transformation protocols that rely on somatic embryogenesis. An alternative method for transformation of suspension cultures of this line, which does not depend on leaf transformation or somatic embryogenesis, was therefore needed. The method described herein uses Agrobacterium tumefaciens mediated gene transfer, allowing the transformation of Medicago callus tissue and the following establishment of liquid suspension cell cultures approximately 2 months after transformation. Kanamycin resistance was used to select for positive transformation events and the screening was facilitated by visualization of a fluorescent marker, which was fused to the gene of interest. This new protocol reduces the time between transformation and cell culture establishment, and allows the generation of transgenic suspension cultures of Medicago reference accession A17.
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Acknowledgements
This work has been funded through the ERA-IB project PRODuCE from the Portuguese Fundação para a Ciência e Tecnologia (FCT, ERA-IB/0001/2012 to RA) and the German BMBF (031A219A to AS; 031A219B to RALvdH). Additional funding from FCT was granted through UID/Multi/04551/2013 and PPBI—Portuguese Platform of BioImaging (PPBI-POCI-01-0145-FEDER-022122). RALvdH was recipient of ERC consolidator Grant 616449 ‘GreenProteases’. The authors thank James Yates for revising the manuscript.
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RBS and RA conceived research. RBS and ASP conducted experiments. AS and RALvdH contributed new reagents and tools. RBS and RA wrote the manuscript. All authors read and approved the manuscript.
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Communicated by S. J. Ochatt.
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Santos, R.B., Pires, A.S., van der Hoorn, R.A.L. et al. Generation of transgenic cell suspension cultures of the model legume Medicago truncatula: a rapid method for Agrobacterium mediated gene transfer. Plant Cell Tiss Organ Cult 136, 445–450 (2019). https://doi.org/10.1007/s11240-018-1525-3
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DOI: https://doi.org/10.1007/s11240-018-1525-3