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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

Vectors used. (a) and (b): pBract vectors. (a). pBract203 destination vector containing the hpt gene under the control of a CaMV 35s promoter together with the chlormaphenicol resistance gene and the negative selection ccdB gene flanked by attR Gateway recombination sites. (b). pBract215 expression vector, created by cloning a luciferase cassette into pBract203 (via Gateway recombination). This construct contains the hpt gene under the control of a CaMV 35s promoter together with the luciferase gene under the control of a maize ubiquitin promoter (ubi1). (c): pSoup containing pSa-RepA (a component of the Agrobacterium replication origin), the replicase gene trfA, a tetracycline resistance gene (tetR), a broad-host range origin of replication (pRK2) and a multiple cloning site.
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Figure 1: Vectors used. (a) and (b): pBract vectors. (a). pBract203 destination vector containing the hpt gene under the control of a CaMV 35s promoter together with the chlormaphenicol resistance gene and the negative selection ccdB gene flanked by attR Gateway recombination sites. (b). pBract215 expression vector, created by cloning a luciferase cassette into pBract203 (via Gateway recombination). This construct contains the hpt gene under the control of a CaMV 35s promoter together with the luciferase gene under the control of a maize ubiquitin promoter (ubi1). (c): pSoup containing pSa-RepA (a component of the Agrobacterium replication origin), the replicase gene trfA, a tetracycline resistance gene (tetR), a broad-host range origin of replication (pRK2) and a multiple cloning site.

Mentions: Agrobacterium strain AGL1 containing one of three pBract vectors (pBract215, pBract216 or pBract217) was used in all experiments. The vectors pBract215, pBract216 and pBract217 all derive from the Gateway destination vector pBract203, which features a T-strand region containing the hpt gene (conferring hygromycin resistance) under the control of a CaMV 35s promoter and a negative selection gene (ccdB) flanked by Gateway recombination sites to assist the cloning of genes of interest (Figure 1). In pBract215, pBract216 and pBract217, the ccdB gene has been replaced by the firefly luciferase gene (luc) driven by the maize Ubi1 promoter. These vectors are all the same apart from the intron composition of their luciferase genes. pBract216 and pBract217 contain the maize RpoT intron 4 and Arabidopsis UBQ10 intron 1 respectively within the coding sequence of the luciferase gene at position +165. The pBract vectors are detailed on the bract website [21]. All pBract vectors are based on pGreen [22] and therefore need to be co-transformed into Agrobacterium with the helper plasmid pSoup. To enable the small size of pGreen, the pSa origin of replication required for replication in Agrobacterium, is separated into its' two distinct functions. The replication origin (ori) is present on pGreen, and the trans-acting replicase gene (RepA) is present on pSoup. Both vectors are required in Agrobacterium for pGreen to replicate. Maps of pBract203, pBract215 and pSoup are shown in Figure 1.


High-throughput Agrobacterium-mediated barley transformation.

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

Vectors used. (a) and (b): pBract vectors. (a). pBract203 destination vector containing the hpt gene under the control of a CaMV 35s promoter together with the chlormaphenicol resistance gene and the negative selection ccdB gene flanked by attR Gateway recombination sites. (b). pBract215 expression vector, created by cloning a luciferase cassette into pBract203 (via Gateway recombination). This construct contains the hpt gene under the control of a CaMV 35s promoter together with the luciferase gene under the control of a maize ubiquitin promoter (ubi1). (c): pSoup containing pSa-RepA (a component of the Agrobacterium replication origin), the replicase gene trfA, a tetracycline resistance gene (tetR), a broad-host range origin of replication (pRK2) and a multiple cloning site.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Vectors used. (a) and (b): pBract vectors. (a). pBract203 destination vector containing the hpt gene under the control of a CaMV 35s promoter together with the chlormaphenicol resistance gene and the negative selection ccdB gene flanked by attR Gateway recombination sites. (b). pBract215 expression vector, created by cloning a luciferase cassette into pBract203 (via Gateway recombination). This construct contains the hpt gene under the control of a CaMV 35s promoter together with the luciferase gene under the control of a maize ubiquitin promoter (ubi1). (c): pSoup containing pSa-RepA (a component of the Agrobacterium replication origin), the replicase gene trfA, a tetracycline resistance gene (tetR), a broad-host range origin of replication (pRK2) and a multiple cloning site.
Mentions: Agrobacterium strain AGL1 containing one of three pBract vectors (pBract215, pBract216 or pBract217) was used in all experiments. The vectors pBract215, pBract216 and pBract217 all derive from the Gateway destination vector pBract203, which features a T-strand region containing the hpt gene (conferring hygromycin resistance) under the control of a CaMV 35s promoter and a negative selection gene (ccdB) flanked by Gateway recombination sites to assist the cloning of genes of interest (Figure 1). In pBract215, pBract216 and pBract217, the ccdB gene has been replaced by the firefly luciferase gene (luc) driven by the maize Ubi1 promoter. These vectors are all the same apart from the intron composition of their luciferase genes. pBract216 and pBract217 contain the maize RpoT intron 4 and Arabidopsis UBQ10 intron 1 respectively within the coding sequence of the luciferase gene at position +165. The pBract vectors are detailed on the bract website [21]. All pBract vectors are based on pGreen [22] and therefore need to be co-transformed into Agrobacterium with the helper plasmid pSoup. To enable the small size of pGreen, the pSa origin of replication required for replication in Agrobacterium, is separated into its' two distinct functions. The replication origin (ori) is present on pGreen, and the trans-acting replicase gene (RepA) is present on pSoup. Both vectors are required in Agrobacterium for pGreen to replicate. Maps of pBract203, pBract215 and pSoup are shown in Figure 1.

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