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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

Southern blot analysis of eight independently-transformed pBract216 lines. Genomic DNA was digested with BamHI and probed with a 1176 bp fragment of luciferase DNA. The lanes 1–8 correspond to lines 90-04-01, 90-06-01, 70-05-01, 70-07-01, 70-21-01, 84-07-01, 87-07-01, and 81-14-01 respectively. Lane 9 corresponds to a wild-type Golden Promise control.
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Figure 5: Southern blot analysis of eight independently-transformed pBract216 lines. Genomic DNA was digested with BamHI and probed with a 1176 bp fragment of luciferase DNA. The lanes 1–8 correspond to lines 90-04-01, 90-06-01, 70-05-01, 70-07-01, 70-21-01, 84-07-01, 87-07-01, and 81-14-01 respectively. Lane 9 corresponds to a wild-type Golden Promise control.

Mentions: Luciferase copy number was determined for 260 independent hygromycin-resistant lines using real-time PCR. The distribution of copy numbers obtained is given in Figure 4. Of the transformed plant lines analysed, 8 (3%) did not contain an intact copy of the luciferase gene. These lines were not escapes, as they were shown by PCR to contain the hpt gene. Forty-six percent of the transformed lines contained a single copy of the luciferase gene, and only 8% of the lines contained more than three copies. The distribution of copy numbers obtained was consistent across the three constructs used. Southern analysis was performed to check the luc copy number and integration pattern in eight T0 lines transformed with pBract216 (Figure 5). For seven of the lines, the number of T-DNA insertion sites identified by Southern analysis was less than or equal to the number of luc copies predicted by qPCR, as would be expected (Table 4).


High-throughput Agrobacterium-mediated barley transformation.

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

Southern blot analysis of eight independently-transformed pBract216 lines. Genomic DNA was digested with BamHI and probed with a 1176 bp fragment of luciferase DNA. The lanes 1–8 correspond to lines 90-04-01, 90-06-01, 70-05-01, 70-07-01, 70-21-01, 84-07-01, 87-07-01, and 81-14-01 respectively. Lane 9 corresponds to a wild-type Golden Promise control.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: Southern blot analysis of eight independently-transformed pBract216 lines. Genomic DNA was digested with BamHI and probed with a 1176 bp fragment of luciferase DNA. The lanes 1–8 correspond to lines 90-04-01, 90-06-01, 70-05-01, 70-07-01, 70-21-01, 84-07-01, 87-07-01, and 81-14-01 respectively. Lane 9 corresponds to a wild-type Golden Promise control.
Mentions: Luciferase copy number was determined for 260 independent hygromycin-resistant lines using real-time PCR. The distribution of copy numbers obtained is given in Figure 4. Of the transformed plant lines analysed, 8 (3%) did not contain an intact copy of the luciferase gene. These lines were not escapes, as they were shown by PCR to contain the hpt gene. Forty-six percent of the transformed lines contained a single copy of the luciferase gene, and only 8% of the lines contained more than three copies. The distribution of copy numbers obtained was consistent across the three constructs used. Southern analysis was performed to check the luc copy number and integration pattern in eight T0 lines transformed with pBract216 (Figure 5). For seven of the lines, the number of T-DNA insertion sites identified by Southern analysis was less than or equal to the number of luc copies predicted by qPCR, as would be expected (Table 4).

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