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.
| Language | English (US) |
|---|---|
| Pages | 20-28 |
| Number of pages | 9 |
| Journal | New Biotechnology |
| Volume | 48 |
| DOIs | |
| State | Published - Jan 25 2019 |
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Keywords
- CRISPR/Cas9
- Genetic transformation
- In vitro regeneration
- Protoplast assay
- Silene latifolia
PubMed: MeSH publication types
- Journal Article
Cite this
Agrobacterium rhizogenes-mediated transformation of a dioecious plant model Silene latifolia. / Hudzieczek, Vojtech; Cegan, Radim; Cermak, Tomas; Bacovska, Nela; Machalkova, Zuzana; Dolezal, Karel; Plihalova, Lucie; Voytas, Daniel F; Hobza, Roman; Vyskot, Boris.
In: New Biotechnology, Vol. 48, 25.01.2019, p. 20-28.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Agrobacterium rhizogenes-mediated transformation of a dioecious plant model Silene latifolia
AU - Hudzieczek, Vojtech
AU - Cegan, Radim
AU - Cermak, Tomas
AU - Bacovska, Nela
AU - Machalkova, Zuzana
AU - Dolezal, Karel
AU - Plihalova, Lucie
AU - Voytas, Daniel F
AU - Hobza, Roman
AU - Vyskot, Boris
PY - 2019/1/25
Y1 - 2019/1/25
N2 - 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.
AB - 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.
KW - CRISPR/Cas9
KW - Genetic transformation
KW - In vitro regeneration
KW - Protoplast assay
KW - Silene latifolia
UR - http://www.scopus.com/inward/record.url?scp=85045421798&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=85045421798&partnerID=8YFLogxK
U2 - 10.1016/j.nbt.2018.04.001
DO - 10.1016/j.nbt.2018.04.001
M3 - Article
VL - 48
SP - 20
EP - 28
JO - New Biotechnology
T2 - New Biotechnology
JF - New Biotechnology
SN - 1871-6784
ER -