Agrobacterium rhizogenes-mediated transformation of a dioecious plant model Silene latifolia

Vojtech Hudzieczek, Radim Cegan, Tomas Cermak, Nela Bacovska, Zuzana Machalkova, Karel Dolezal, Lucie Plihalova, Daniel Voytas, Roman Hobza, Boris Vyskot

Research output: Contribution to journalArticlepeer-review

7 Scopus citations

Abstract

Silene latifolia serves as a model species to study dioecy, the evolution of sex chromosomes, dosage compensation and sex-determination systems in plants. Currently, no protocol for genetic transformation is available for this species, mainly because S. latifolia is considered recalcitrant to in vitro regeneration and infection with Agrobacterium tumefaciens. Using cytokinins and their synthetic derivatives, we markedly improved the efficiency of regeneration. Several agrobacterial strains were tested for their ability to deliver DNA into S. latifolia tissues leading to transient and stable expression of the GUS reporter. The use of Agrobacterium rhizogenes strains resulted in the highest transformation efficiency (up to 4.7% of stable transformants) in hairy root cultures. Phenotypic and genotypic analyses of the T1 generation suggested that the majority of transformation events contain a small number of independent T-DNA insertions and the transgenes are transmitted to the progeny in a Mendelian pattern of inheritance. In short, we report an efficient and reproducible protocol for leaf disc transformation and subsequent plant regeneration in S. latifolia, based on the unique combination of infection with A. rhizogenes and plant regeneration from hairy root cultures using synthetic cytokinins. A protocol for the transient transformation of S.latifolia protoplasts was also developed and applied to demonstrate the possibility of targeted mutagenesis of the sex linked gene SlAP3 by TALENs and CRISPR/Cas9.

Original languageEnglish (US)
Pages (from-to)20-28
Number of pages9
JournalNew Biotechnology
Volume48
DOIs
StatePublished - Jan 25 2019

Bibliographical note

Funding Information:
This work was financially supported by the Czech Science Foundation grant No. 17-00567S . Additional finance was provided by the Ministry of Education, Youth and Sport of the Czech National Program for Sustainability (Grant. LO1204 ).

Publisher Copyright:
© 2018 The Authors

Keywords

  • CRISPR/Cas9
  • Genetic transformation
  • In vitro regeneration
  • Protoplast assay
  • Silene latifolia

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