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Optimized Agrobacterium-mediated sorghum transformation protocol and molecular data of transgenic sorghum plants.

Wu E, Lenderts B, Glassman K, Berezowska-Kaniewska M, Christensen H, Asmus T, Zhen S, Chu U, Cho MJ, Zhao ZY - In Vitro Cell. Dev. Biol., Plant (2013)

Bottom Line: The new transformation protocol includes the addition of elevated copper sulfate and 6-benzylaminopurine in the resting and selection media.With Agrobacterium strain AGL1, the transformation frequencies were as high as 33%.This is the first report providing molecular data for T-DNA integration patterns in a large number of independent transgenic plants in sorghum.

View Article: PubMed Central - PubMed

Affiliation: DuPont Agricultural Biotechnology, DuPont Pioneer, 8305 NW 62nd Avenue, P. O. Box 7060, Johnston, IA 50131 USA.

ABSTRACT

Agrobacterium-mediated sorghum transformation frequency has been enhanced significantly via medium optimization using immature embryos from sorghum variety TX430 as the target tissue. The new transformation protocol includes the addition of elevated copper sulfate and 6-benzylaminopurine in the resting and selection media. Using Agrobacterium strain LBA4404, the transformation frequency reached over 10% using either of two different selection marker genes, moPAT or PMI, and any of three different vectors in large-scale transformation experiments. With Agrobacterium strain AGL1, the transformation frequencies were as high as 33%. Using quantitative PCR analyses of 1,182 T0 transgenic plants representing 675 independent transgenic events, data was collected for T-DNA copy number, intact or truncated T-DNA integration, and vector backbone integration into the sorghum genome. A comparison of the transformation frequencies and molecular data characterizing T-DNA integration patterns in the transgenic plants derived from LBA4404 versus AGL1 transformation revealed that twice as many transgenic high-quality events were generated when AGL1 was used compared to LBA4404. This is the first report providing molecular data for T-DNA integration patterns in a large number of independent transgenic plants in sorghum.

No MeSH data available.


Related in: MedlinePlus

Transgenic sorghum plants derived from PHP166 grown in the greenhouse. (a) Young T0 plants, (b) self-pollinated, mature T0 plants.
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Fig4: Transgenic sorghum plants derived from PHP166 grown in the greenhouse. (a) Young T0 plants, (b) self-pollinated, mature T0 plants.

Mentions: A total of 6,077 immature embryos were treated with LBA4404/PHP166 over the course of 23 experiments, producing 755 independent putative transgenic callus events that regenerated into plants. Transformation frequencies, based on the ability to grow on mannose-containing medium, were calculated and ranged from 2.3 to 26.3% among experiments, with a mean of 12.4%. Transformation frequencies were below 10% in nine experiments, between 10 and 20% in ten experiments, and higher than 20% in four experiments. Mean transformation frequencies achieved by each of three individual transformers achieved average transformation frequencies of 9.7, 13, and 15%, which were confirmed to be statistically similar (P = 0.49). Not all events were processed for molecular analysis. Instead, two T0 plants were regenerated from a subset of 507 callus events, and all of these 1,014 T0 plants were moved to the greenhouse for later sampling and further analysis (Fig. 4). Leaf samples were collected from each T0 plant, and extracted DNA was analyzed using Q-PCR to (1) detect stable integration of the T-DNA into the sorghum genome, (2) determine copy number of T-DNA integrations, (3) determine whether integrated T-DNA was intact or truncated, and (4) detect the presence of plasmid backbone.Fig. 4


Optimized Agrobacterium-mediated sorghum transformation protocol and molecular data of transgenic sorghum plants.

Wu E, Lenderts B, Glassman K, Berezowska-Kaniewska M, Christensen H, Asmus T, Zhen S, Chu U, Cho MJ, Zhao ZY - In Vitro Cell. Dev. Biol., Plant (2013)

Transgenic sorghum plants derived from PHP166 grown in the greenhouse. (a) Young T0 plants, (b) self-pollinated, mature T0 plants.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4544465&req=5

Fig4: Transgenic sorghum plants derived from PHP166 grown in the greenhouse. (a) Young T0 plants, (b) self-pollinated, mature T0 plants.
Mentions: A total of 6,077 immature embryos were treated with LBA4404/PHP166 over the course of 23 experiments, producing 755 independent putative transgenic callus events that regenerated into plants. Transformation frequencies, based on the ability to grow on mannose-containing medium, were calculated and ranged from 2.3 to 26.3% among experiments, with a mean of 12.4%. Transformation frequencies were below 10% in nine experiments, between 10 and 20% in ten experiments, and higher than 20% in four experiments. Mean transformation frequencies achieved by each of three individual transformers achieved average transformation frequencies of 9.7, 13, and 15%, which were confirmed to be statistically similar (P = 0.49). Not all events were processed for molecular analysis. Instead, two T0 plants were regenerated from a subset of 507 callus events, and all of these 1,014 T0 plants were moved to the greenhouse for later sampling and further analysis (Fig. 4). Leaf samples were collected from each T0 plant, and extracted DNA was analyzed using Q-PCR to (1) detect stable integration of the T-DNA into the sorghum genome, (2) determine copy number of T-DNA integrations, (3) determine whether integrated T-DNA was intact or truncated, and (4) detect the presence of plasmid backbone.Fig. 4

Bottom Line: The new transformation protocol includes the addition of elevated copper sulfate and 6-benzylaminopurine in the resting and selection media.With Agrobacterium strain AGL1, the transformation frequencies were as high as 33%.This is the first report providing molecular data for T-DNA integration patterns in a large number of independent transgenic plants in sorghum.

View Article: PubMed Central - PubMed

Affiliation: DuPont Agricultural Biotechnology, DuPont Pioneer, 8305 NW 62nd Avenue, P. O. Box 7060, Johnston, IA 50131 USA.

ABSTRACT

Agrobacterium-mediated sorghum transformation frequency has been enhanced significantly via medium optimization using immature embryos from sorghum variety TX430 as the target tissue. The new transformation protocol includes the addition of elevated copper sulfate and 6-benzylaminopurine in the resting and selection media. Using Agrobacterium strain LBA4404, the transformation frequency reached over 10% using either of two different selection marker genes, moPAT or PMI, and any of three different vectors in large-scale transformation experiments. With Agrobacterium strain AGL1, the transformation frequencies were as high as 33%. Using quantitative PCR analyses of 1,182 T0 transgenic plants representing 675 independent transgenic events, data was collected for T-DNA copy number, intact or truncated T-DNA integration, and vector backbone integration into the sorghum genome. A comparison of the transformation frequencies and molecular data characterizing T-DNA integration patterns in the transgenic plants derived from LBA4404 versus AGL1 transformation revealed that twice as many transgenic high-quality events were generated when AGL1 was used compared to LBA4404. This is the first report providing molecular data for T-DNA integration patterns in a large number of independent transgenic plants in sorghum.

No MeSH data available.


Related in: MedlinePlus