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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 additional copper on the growth of callus from immature barley embryos. (a) and (b) show immature embryo derived callus after 27 days culture on callus induction medium. (a): standard callus induction medium. (b): Callus induction medium with 5 μM CuSO4.
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Figure 2: The effect of additional copper on the growth of callus from immature barley embryos. (a) and (b) show immature embryo derived callus after 27 days culture on callus induction medium. (a): standard callus induction medium. (b): Callus induction medium with 5 μM CuSO4.

Mentions: Our standard protocol was compared to a method that included additional copper (5 μM CuSO4) in the CI medium as well as in the transition medium. This study was carried out without any Agrobacterium treatment to examine regeneration ability from immature embryos only. Fifty-eight immature embryos with the embryonic axis removed were used in this study and were split between two different culture regimes. Thirty embryos were cultured on callus induction medium (CI) (standard protocol) and twenty-eight embryos were cultured on callus induction medium supplemented with 5 μM CuSO4 (CI + Cu). The appearance of the embryo derived callus after 27 days of culture is shown in Figure 2. There was more callus growth on the CI medium with copper (Figure 2b) than on the standard CI medium (Figure 2a). In addition, detailed examination of the callus revealed that as well as showing faster growth, the callus on the copper containing medium appeared more embryogenic. After four weeks on callus induction medium, all callus lines were transferred to transition medium for two weeks and then to regeneration medium. All callus derived from each embryo was kept separate and after 4 weeks on regeneration medium the shoots derived from each embryo were removed and counted.


High-throughput Agrobacterium-mediated barley transformation.

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

The effect of additional copper on the growth of callus from immature barley embryos. (a) and (b) show immature embryo derived callus after 27 days culture on callus induction medium. (a): standard callus induction medium. (b): Callus induction medium with 5 μM CuSO4.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: The effect of additional copper on the growth of callus from immature barley embryos. (a) and (b) show immature embryo derived callus after 27 days culture on callus induction medium. (a): standard callus induction medium. (b): Callus induction medium with 5 μM CuSO4.
Mentions: Our standard protocol was compared to a method that included additional copper (5 μM CuSO4) in the CI medium as well as in the transition medium. This study was carried out without any Agrobacterium treatment to examine regeneration ability from immature embryos only. Fifty-eight immature embryos with the embryonic axis removed were used in this study and were split between two different culture regimes. Thirty embryos were cultured on callus induction medium (CI) (standard protocol) and twenty-eight embryos were cultured on callus induction medium supplemented with 5 μM CuSO4 (CI + Cu). The appearance of the embryo derived callus after 27 days of culture is shown in Figure 2. There was more callus growth on the CI medium with copper (Figure 2b) than on the standard CI medium (Figure 2a). In addition, detailed examination of the callus revealed that as well as showing faster growth, the callus on the copper containing medium appeared more embryogenic. After four weeks on callus induction medium, all callus lines were transferred to transition medium for two weeks and then to regeneration medium. All callus derived from each embryo was kept separate and after 4 weeks on regeneration medium the shoots derived from each embryo were removed and counted.

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