Stable transformation and actin visualization in callus cultures of dodder (Cuscuta europaea)

Švubová, Renáta; Blehová, Alžbeta

doi:10.2478/s11756-013-0188-0

Cited by 11 publications

(13 citation statements)

References 33 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The main bottleneck is the lack of an efficient and reproducible protocol with which transgene expression or even genetic manipulations can be performed. Previous attempts to transform Cuscuta have shown that A. tumefaciens is able to transform callus cells (Borsics et al, 2002; Svubova and Blehova, 2013). However, in our hands these transformation events were very scarce, explaining maybe why this approach has not yielded greater success.…”

Section: Discussionmentioning

confidence: 99%

“…These are low transformation efficiencies on one hand and recalcitrance of callus propagation and shoot regeneration in vitro on the other hand. Few approaches to transform C. trifolii (Borsics et al, 2002) and C. europaea (Svubova and Blehova, 2013) have been reported. Explants from seedlings were transformed using Agrobacterium tumefaciens , and expression of the transgenes was demonstrated in the calli.…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

A highly efficient protocol for transforming Cuscuta reflexa based on artificially induced infection sites

2020

View full text Add to dashboard Cite

The parasitic plant genus Cuscuta is notoriously difficult to transform and to propagate or regenerate in vitro. With it being a substantial threat to many agroecosystems, techniques allowing functional analysis of gene products involved in host interaction and infection mechanisms are, however, in high demand. We set out to explore whether Agrobacterium‐mediated transformation of different plant parts can provide efficient alternatives to the currently scarce and inefficient protocols for transgene expression in Cuscuta. We used fluorescent protein genes on the T‐DNA as markers for transformation efficiency and transformation stability. As a result, we present a novel highly efficient transformation protocol for Cuscuta reflexa cells that exploits the propensity of the infection organ to take up and express transgenes with the T‐DNA. Both, Agrobacterium rhizogenes and Agrobacterium tumefaciens carrying binary transformation vectors with reporter fluorochromes yielded high numbers of transformation events. An overwhelming majority of transformed cells were observed in the cell layer below the adhesive disk’s epidermis, suggesting that these cells are particularly susceptible to infection. Cotransformation of these cells happens frequently when Agrobacterium strains carrying different constructs are applied together. Explants containing transformed tissue expressed the fluorescent markers in in vitro culture for several weeks, offering a future possibility for development of transformed cells into callus. These results are discussed with respect to the future potential of this technique and with respect to the special characteristics of the infection organ that may explain its competence to take up the foreign DNA.

show abstract

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

A highly efficient protocol for transforming Cuscuta reflexa based on artificially induced infection sites

2020

View full text Add to dashboard Cite

show abstract

“…The main bottleneck is the lack of an efficient and reproducible protocol with which targeted genetic manipulations can be performed. Previous attempts to transform Cuscuta have shown that A. tumefaciens is able to transform callus cells (Borsics et al, 2002; Svubova and Blehova, 2013). However, in our hands these transformation events were very scarce, explaining maybe why this approach has not yielded greater success.…”

Section: Discussionmentioning

confidence: 99%

“…However, two significant bottlenecks to perform targeted genome manipulations remain in the form of low transformation efficiency and poor regeneration rates of Cuscuta tissues in vitro . Some few approaches to transform C. trifolii (Borsics et al, 2002) and C. europaea (Svubova and Blehova, 2013) have been reported. Explants from seedlings were transformed using Agrobacterium tumefaciens , and expression of the transgenes was demonstrated in the calli.…”

Section: Introductionmentioning

confidence: 99%

A highly efficient protocol for transformingCuscuta reflexabased on artificially induced infection sites

Lachner

Galstyan

Krause

2020

Preprint

View full text Add to dashboard Cite

A protocol that yields high numbers of transformed cells in the adhesive disks of Cuscuta reflexa 27 upon exposure to agrobacteria brings closer the vision of generating genetically modified 28 Cuscuta. 29 30 ABSTRACT 31 A current bottleneck in the functional analysis of the emerging parasitic model plant Cuscuta and 32 the exploitation of its recently sequenced genomes is the lack of efficient transformation tools. 33 Here, we describe the development of a novel highly efficient Agrobacterium-mediated 34 transformation protocol for Cuscuta reflexa based on the parasitic structure referred to as 35 adhesive disk. Both, Agrobacterium rhizogenes and Agrobacterium tumefaciens carrying binary 36 transformation vectors with reporter fluorochromes yielded high numbers of transformation 37 events. An overwhelming majority of transformed cells were observed in the cell layer below the 38 adhesive disk's epidermis, suggesting that these cells are particularly susceptible to infection. Co-39 transformation of these cells happens frequently when Agrobacterium strains carrying different 40 constructs are applied together. Explants containing transformed tissue expressed the fluorescent 41 markers in in vitro culture for several weeks, offering a possibility for development of 42 transformed cells into callus. 43 44 45 3 46 48 worldwide and infects a broad range of host plants. The parasite starts an attack by winding 49around the host stem. A multicellular feeding structure termed the haustorium, which can reach 50 2-3 mm in size in some species and originates from a secondary meristem in the cortex close to 51 the vascular bundles, develops within a few days and breaches the host tissue integrity using 52 mechanical pressure and enzymatic digestion of cell walls (Nagar et al., 1984; Johnsen et al., 53 2015; Kaiser et al., 2015). To make the penetration possible, a sucker-shaped attachment organ 54 provides the necessary counterforce. The development of this organ, termed "adhesive disk" or 55

show abstract

“…Published methodologies exist for obtaining callus and shoot regeneration for C. trifolii (Bakos A. et al, 1995;, C. europea (Švubová & Blehová, 2013), and C. reflexa (Srivastava & Dwivedi, 2001), and floral induction for C. reflexa (Baldev, 1962).…”

Section: Introductionmentioning

confidence: 99%

An artificial host system enables the obligate parasitic plant Cuscuta campestris to grow and complete its life cycle in vitro

Bernal-Galeano¹,

Westwood²

2021

Preprint

View full text Add to dashboard Cite

Cuscuta campestris is an obligate parasitic plant that requires a host to complete its lifecycle. Parasite-host connections occur via an haustorium, a unique organ that acts as a bridge for the uptake of water, nutrients and macromolecules. Research on Cuscuta is often complicated by host influences, but comparable systems for growing the parasite in the absence of a host do not exist. We developed an axenic method to grow C. campestris on an Artificial Host System (AHS). We evaluated the effects of nutrients and phytohormones on parasite haustoria development and growth. Haustorium morphology and gene expression were characterized. The AHS consists of an inert, fibrous stick that mimics a host stem, wicking water and nutrients to the parasite. It enables C. campestris to exhibit a parasitic habit and develop through all stages of its lifecycle, including production of new shoots and viable seeds. Phytohormones NAA and BA affect haustoria morphology, and increase parasite fresh weight and biomass. Gene expression in AHS haustoria reflect process similar to those in haustoria on actual host plants. The AHS is a methodological improvement for studying Cuscuta biology by avoiding specific host effects on parasite and giving researchers full control of the parasite environment.

show abstract

Stable transformation and actin visualization in callus cultures of dodder (Cuscuta europaea)

Cited by 11 publications

References 33 publications

A highly efficient protocol for transforming Cuscuta reflexa based on artificially induced infection sites

A highly efficient protocol for transforming Cuscuta reflexa based on artificially induced infection sites

A highly efficient protocol for transformingCuscuta reflexabased on artificially induced infection sites

An artificial host system enables the obligate parasitic plant Cuscuta campestris to grow and complete its life cycle in vitro

Contact Info

Product

Resources

About