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Transformation and regeneration of the holoparasitic plant Phelipanche aegyptiaca.

Fernández-Aparicio M, Rubiales D, Bandaranayake PC, Yoder JI, Westwood JH - Plant Methods (2011)

Bottom Line: The recent completion of a massive transcriptome sequencing project (the Parasitic Plant Genome Project) will fuel the use of genomic tools for studies on parasitic plants.Four months later, YFP-positive regenerated calli were inoculated onto tomato plants growing in a minirhizotron system.This work constitutes a breakthrough in holoparasitic plant research methods.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute for Sustainable Agriculture, IAS-CSIC, Dept, of Plant Breeding, Córdoba, 14080, Spain. monica.fernandez@ias.csic.es.

ABSTRACT

Background: Transformation and subsequent regeneration of holoparasitic plants has never been reported, in part due to challenges in developing transformation protocols, but also because regeneration of obligate parasites is difficult since their survival depends completely on successful haustorium penetration of a host and the formation of vascular connections. The recent completion of a massive transcriptome sequencing project (the Parasitic Plant Genome Project) will fuel the use of genomic tools for studies on parasitic plants. A reliable system for holoparasite transformation is needed to realize the full value of this resource for reverse genetics and functional genomics studies.

Results: Here we demonstrate that transformation of Phelipanche aegyptiaca is achieved by infection of 3 month-old in vitro grown P. aegyptiaca calli with Agrobacterium rhizogenes harboring the yellow fluorescent protein (YFP). Four months later, YFP-positive regenerated calli were inoculated onto tomato plants growing in a minirhizotron system. Eight days after inoculation, transgenic parasite tissue formed lateral haustoria that penetrated the host and could be visualized under UV illumination through intact host root tissue. YFP-positive shoot buds were observed one month after inoculation.

Conclusions: This work constitutes a breakthrough in holoparasitic plant research methods. The method described here is a robust system for transformation and regeneration of a holoparasitic plant and will facilitate research on unique parasitic plant capabilities such as host plant recognition, haustorial formation, penetration and vascular connection.

No MeSH data available.


Related in: MedlinePlus

Selection and regeneration of P. aegyptiaca. A) P. aegyptiaca clones with parasitic infective roots ready to be inoculated on tomato. B) Sucessful P. aegyptiaca explants infecting tomato host root in a minirhizotron system, demonstrating that the parasite is able to regenerate its whole plant structure, including anchorage roots and shoots. Black explants were not able to infect the tomato roots. C) Wild type P. aegyptiaca callus growing in MS media without kanamycin. D) Wild type P. aegyptiaca callus growing in MS media suplemented with 100 μg/ml of kanamycin. E) Wild type P. aegyptiaca callus observed under white light and F) UV illuminated image of E, showing only slight autofluorescence from the seed coat.
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Figure 2: Selection and regeneration of P. aegyptiaca. A) P. aegyptiaca clones with parasitic infective roots ready to be inoculated on tomato. B) Sucessful P. aegyptiaca explants infecting tomato host root in a minirhizotron system, demonstrating that the parasite is able to regenerate its whole plant structure, including anchorage roots and shoots. Black explants were not able to infect the tomato roots. C) Wild type P. aegyptiaca callus growing in MS media without kanamycin. D) Wild type P. aegyptiaca callus growing in MS media suplemented with 100 μg/ml of kanamycin. E) Wild type P. aegyptiaca callus observed under white light and F) UV illuminated image of E, showing only slight autofluorescence from the seed coat.

Mentions: As a first step in designing a selection and regeneration strategy for P. aegyptiaca it was necessary to determine the callus growth capacity and response to antibiotics and UV illumination. For regeneration, P. aegyptiaca calli growing in MS liquid media were divided into multiple small pieces with a scalpel and returned to liquid medium. These explants comprised in a population of clones (Figure 2A) that were inoculated onto tomato roots and which showed infectivity potential by attaching to hosts and developing to the point of shoot production (Figure 2B). To identify an optimal selective agent, one-month-old P. aegyptiaca calli were grown on media containing kanamycin. Ten wild-type P. aegyptiaca calli were grown in MS medium supplemented with a range of kanamycin concentrations from 0 μg/ml (control) (Figure 2C), to 50 μg/ml and 100 μg/ml (Figure 2D). Kanamycin did not cause death of the wild-type parasitic calli, although growth of P. aegyptiaca was slightly reduced at the highest kanamycin concentration. As an alternative to antibiotic selection, the feasibility of fluorescent markers was evaluated by observing parasites under UV light and 23 day-old P. aegyptiaca wild-type calli showed no autofluorescence except for the seed coat (Figures 2E and 2F). Therefore a fluorescent marker was determined to be more reliable than kanamycin resistance for screaning transgenic tissue.


Transformation and regeneration of the holoparasitic plant Phelipanche aegyptiaca.

Fernández-Aparicio M, Rubiales D, Bandaranayake PC, Yoder JI, Westwood JH - Plant Methods (2011)

Selection and regeneration of P. aegyptiaca. A) P. aegyptiaca clones with parasitic infective roots ready to be inoculated on tomato. B) Sucessful P. aegyptiaca explants infecting tomato host root in a minirhizotron system, demonstrating that the parasite is able to regenerate its whole plant structure, including anchorage roots and shoots. Black explants were not able to infect the tomato roots. C) Wild type P. aegyptiaca callus growing in MS media without kanamycin. D) Wild type P. aegyptiaca callus growing in MS media suplemented with 100 μg/ml of kanamycin. E) Wild type P. aegyptiaca callus observed under white light and F) UV illuminated image of E, showing only slight autofluorescence from the seed coat.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3285091&req=5

Figure 2: Selection and regeneration of P. aegyptiaca. A) P. aegyptiaca clones with parasitic infective roots ready to be inoculated on tomato. B) Sucessful P. aegyptiaca explants infecting tomato host root in a minirhizotron system, demonstrating that the parasite is able to regenerate its whole plant structure, including anchorage roots and shoots. Black explants were not able to infect the tomato roots. C) Wild type P. aegyptiaca callus growing in MS media without kanamycin. D) Wild type P. aegyptiaca callus growing in MS media suplemented with 100 μg/ml of kanamycin. E) Wild type P. aegyptiaca callus observed under white light and F) UV illuminated image of E, showing only slight autofluorescence from the seed coat.
Mentions: As a first step in designing a selection and regeneration strategy for P. aegyptiaca it was necessary to determine the callus growth capacity and response to antibiotics and UV illumination. For regeneration, P. aegyptiaca calli growing in MS liquid media were divided into multiple small pieces with a scalpel and returned to liquid medium. These explants comprised in a population of clones (Figure 2A) that were inoculated onto tomato roots and which showed infectivity potential by attaching to hosts and developing to the point of shoot production (Figure 2B). To identify an optimal selective agent, one-month-old P. aegyptiaca calli were grown on media containing kanamycin. Ten wild-type P. aegyptiaca calli were grown in MS medium supplemented with a range of kanamycin concentrations from 0 μg/ml (control) (Figure 2C), to 50 μg/ml and 100 μg/ml (Figure 2D). Kanamycin did not cause death of the wild-type parasitic calli, although growth of P. aegyptiaca was slightly reduced at the highest kanamycin concentration. As an alternative to antibiotic selection, the feasibility of fluorescent markers was evaluated by observing parasites under UV light and 23 day-old P. aegyptiaca wild-type calli showed no autofluorescence except for the seed coat (Figures 2E and 2F). Therefore a fluorescent marker was determined to be more reliable than kanamycin resistance for screaning transgenic tissue.

Bottom Line: The recent completion of a massive transcriptome sequencing project (the Parasitic Plant Genome Project) will fuel the use of genomic tools for studies on parasitic plants.Four months later, YFP-positive regenerated calli were inoculated onto tomato plants growing in a minirhizotron system.This work constitutes a breakthrough in holoparasitic plant research methods.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute for Sustainable Agriculture, IAS-CSIC, Dept, of Plant Breeding, Córdoba, 14080, Spain. monica.fernandez@ias.csic.es.

ABSTRACT

Background: Transformation and subsequent regeneration of holoparasitic plants has never been reported, in part due to challenges in developing transformation protocols, but also because regeneration of obligate parasites is difficult since their survival depends completely on successful haustorium penetration of a host and the formation of vascular connections. The recent completion of a massive transcriptome sequencing project (the Parasitic Plant Genome Project) will fuel the use of genomic tools for studies on parasitic plants. A reliable system for holoparasite transformation is needed to realize the full value of this resource for reverse genetics and functional genomics studies.

Results: Here we demonstrate that transformation of Phelipanche aegyptiaca is achieved by infection of 3 month-old in vitro grown P. aegyptiaca calli with Agrobacterium rhizogenes harboring the yellow fluorescent protein (YFP). Four months later, YFP-positive regenerated calli were inoculated onto tomato plants growing in a minirhizotron system. Eight days after inoculation, transgenic parasite tissue formed lateral haustoria that penetrated the host and could be visualized under UV illumination through intact host root tissue. YFP-positive shoot buds were observed one month after inoculation.

Conclusions: This work constitutes a breakthrough in holoparasitic plant research methods. The method described here is a robust system for transformation and regeneration of a holoparasitic plant and will facilitate research on unique parasitic plant capabilities such as host plant recognition, haustorial formation, penetration and vascular connection.

No MeSH data available.


Related in: MedlinePlus