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High-throughput Agrobacterium-mediated barley transformation.

Bartlett JG, Alves SC, Smedley M, Snape JW, Harwood WA - Plant Methods (2008)

Bottom Line: Results of large scale experiments utilising the luc (firefly luciferase) gene as a reporter are described.The method presented here has been used to produce hundreds of independent, transgenic plant lines and we show that a large proportion of these lines contain single copies of the luc gene.This opens up opportunities for the development of functional genomics resources in barley.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Crop Genetics, John Innes Centre, Norwich Research Park, Colney, Norwich, NR4 7UH, UK. wendy.harwood@bbsrc.ac.uk.

ABSTRACT

Background: Plant transformation is an invaluable tool for basic plant research, as well as a useful technique for the direct improvement of commercial crops. Barley (Hordeum vulgare) is the fourth most abundant cereal crop in the world. It also provides a useful model for the study of wheat, which has a larger and more complex genome. Most existing barley transformation methodologies are either complex or have low (<10%) transformation efficiencies.

Results: A robust, simple and reproducible barley transformation protocol has been developed that yields average transformation efficiencies of 25%. This protocol is based on the infection of immature barley embryos with Agrobacterium strain AGL1, carrying vectors from the pBract series that contain the hpt gene (conferring hygromycin resistance) as a selectable marker. Results of large scale experiments utilising the luc (firefly luciferase) gene as a reporter are described. The method presented here has been used to produce hundreds of independent, transgenic plant lines and we show that a large proportion of these lines contain single copies of the luc gene.

Conclusion: This protocol demonstrates significant improvements in both efficiency and ease of use over existing barley transformation methods. This opens up opportunities for the development of functional genomics resources in barley.

No MeSH data available.


Related in: MedlinePlus

The effect of copper treatments on the number of shoots regenerated per embryo without transformation. Each bar represents the average number of shoots per embryo (recorded after 10 weeks in tissue culture) from 20–30 individual embryos. 'Standard' tissue culture regime: weeks 1–4 callus induction (CI) medium with no additional copper. 'Additional copper' tissue culture regime: weeks 1–4 CI + 5 μM CuSO4. Callus from both treatments was transferred to standard transition media (which includes CuSO4) after 4 weeks and standard regeneration medium after a further 2 weeks.
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Figure 3: The effect of copper treatments on the number of shoots regenerated per embryo without transformation. Each bar represents the average number of shoots per embryo (recorded after 10 weeks in tissue culture) from 20–30 individual embryos. 'Standard' tissue culture regime: weeks 1–4 callus induction (CI) medium with no additional copper. 'Additional copper' tissue culture regime: weeks 1–4 CI + 5 μM CuSO4. Callus from both treatments was transferred to standard transition media (which includes CuSO4) after 4 weeks and standard regeneration medium after a further 2 weeks.

Mentions: Figure 3 shows the average number of shoots regenerated per embryo with the two different tissue culture regimes. The standard protocol, gave an average of 26 shoots per embryo. The improved protocol, containing copper in the CI medium, gave an average of 53 shoots per embryo. Adding the additional copper during callus induction therefore led to approximately double the number of regenerated shoots per embryo up to the point when the shoots were scored. The shoots derived from embryos cultured on CI + Cu were also larger than those cultured without copper. It appears that the regeneration process was accelerated by the presence of copper in the CI medium as embryogenic callus formed earlier leading to the more rapid regeneration of plants.


High-throughput Agrobacterium-mediated barley transformation.

Bartlett JG, Alves SC, Smedley M, Snape JW, Harwood WA - Plant Methods (2008)

The effect of copper treatments on the number of shoots regenerated per embryo without transformation. Each bar represents the average number of shoots per embryo (recorded after 10 weeks in tissue culture) from 20–30 individual embryos. 'Standard' tissue culture regime: weeks 1–4 callus induction (CI) medium with no additional copper. 'Additional copper' tissue culture regime: weeks 1–4 CI + 5 μM CuSO4. Callus from both treatments was transferred to standard transition media (which includes CuSO4) after 4 weeks and standard regeneration medium after a further 2 weeks.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2562381&req=5

Figure 3: The effect of copper treatments on the number of shoots regenerated per embryo without transformation. Each bar represents the average number of shoots per embryo (recorded after 10 weeks in tissue culture) from 20–30 individual embryos. 'Standard' tissue culture regime: weeks 1–4 callus induction (CI) medium with no additional copper. 'Additional copper' tissue culture regime: weeks 1–4 CI + 5 μM CuSO4. Callus from both treatments was transferred to standard transition media (which includes CuSO4) after 4 weeks and standard regeneration medium after a further 2 weeks.
Mentions: Figure 3 shows the average number of shoots regenerated per embryo with the two different tissue culture regimes. The standard protocol, gave an average of 26 shoots per embryo. The improved protocol, containing copper in the CI medium, gave an average of 53 shoots per embryo. Adding the additional copper during callus induction therefore led to approximately double the number of regenerated shoots per embryo up to the point when the shoots were scored. The shoots derived from embryos cultured on CI + Cu were also larger than those cultured without copper. It appears that the regeneration process was accelerated by the presence of copper in the CI medium as embryogenic callus formed earlier leading to the more rapid regeneration of plants.

Bottom Line: Results of large scale experiments utilising the luc (firefly luciferase) gene as a reporter are described.The method presented here has been used to produce hundreds of independent, transgenic plant lines and we show that a large proportion of these lines contain single copies of the luc gene.This opens up opportunities for the development of functional genomics resources in barley.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Crop Genetics, John Innes Centre, Norwich Research Park, Colney, Norwich, NR4 7UH, UK. wendy.harwood@bbsrc.ac.uk.

ABSTRACT

Background: Plant transformation is an invaluable tool for basic plant research, as well as a useful technique for the direct improvement of commercial crops. Barley (Hordeum vulgare) is the fourth most abundant cereal crop in the world. It also provides a useful model for the study of wheat, which has a larger and more complex genome. Most existing barley transformation methodologies are either complex or have low (<10%) transformation efficiencies.

Results: A robust, simple and reproducible barley transformation protocol has been developed that yields average transformation efficiencies of 25%. This protocol is based on the infection of immature barley embryos with Agrobacterium strain AGL1, carrying vectors from the pBract series that contain the hpt gene (conferring hygromycin resistance) as a selectable marker. Results of large scale experiments utilising the luc (firefly luciferase) gene as a reporter are described. The method presented here has been used to produce hundreds of independent, transgenic plant lines and we show that a large proportion of these lines contain single copies of the luc gene.

Conclusion: This protocol demonstrates significant improvements in both efficiency and ease of use over existing barley transformation methods. This opens up opportunities for the development of functional genomics resources in barley.

No MeSH data available.


Related in: MedlinePlus