Elsevier

New Biotechnology

Volume 48, 25 January 2019, Pages 20-28
New Biotechnology

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

https://doi.org/10.1016/j.nbt.2018.04.001Get rights and content
Under a Creative Commons license
open access

Highlights

  • In vitro regeneration of a model dioecious species Silene latifolia was significantly improved.

  • Stable genetic transformation was achieved by Agrobacterium rhizogenes.

  • Targeted mutagenesis with TALEN and CRISPR/Cas9 nucleases on plant sex chromosomes is demonstrated in protoplasts.

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.

Abbreviations

3FBAP
6-(3-fluorobenzylamino)purine
3FBAPR
6-(3-fluorobenzylamino)-9H-purine riboside
3MeOBAP
6-(3-methoxybenzylamino)purine
3MeOBAPR
6-(3-methoxybenzylamino)-9H-purine riboside
3OHBAP-9THPP
6-(3-hydroxybenzylamino)-9-(tetrahydropyran-2-yl)purine
BAP
6-benzylaminopurine
Cas9
CRISPR associated protein 9
CK
cytokinin
CRISPR
clustered regularly interspaced short palindromic repeats
GFP
green fluorescent protein
gRNA
guide RNA
GUS
β-glucuronidase
mT
meta-topolin 6-(3-hydroxybenzylamino)purine
MS
MurashigeSkoog basal salt mixture
NAA
1-naphthaleneacetic acid
nptII
neomycin phosphotransferase II
PAM
protospacer-adjacent motif
pRi
root-inducing plasmid
rolA
root locus A
SlAP3
Silene latifolia APETALA3
T-DNA
transferred DNA
TALEN
transcription activator-like effector nuclease
TDZ
thidiazuron
tZ
trans-zeatin

Keywords

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

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