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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 callus, shoots, roots, leaf, pollen, and seeds expressing DsRed derived from PHP149 viewed using fluorescence microscopy. (a) Transgenic callus expressing DsRed, (b) transgenic shoots and roots expressing DsRed, (c) transgenic leaf expressing DsRed, (d) pollen grains from a T0 transgenic plant with or without expression of DsRed, (e) T1 seeds from a T0 transgenic plant with or without expression of DsRed, (f) a part of a T0 transgenic panicle showing red (transgenic) and non-red (non-transgenic) seeds.
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Fig3: Transgenic callus, shoots, roots, leaf, pollen, and seeds expressing DsRed derived from PHP149 viewed using fluorescence microscopy. (a) Transgenic callus expressing DsRed, (b) transgenic shoots and roots expressing DsRed, (c) transgenic leaf expressing DsRed, (d) pollen grains from a T0 transgenic plant with or without expression of DsRed, (e) T1 seeds from a T0 transgenic plant with or without expression of DsRed, (f) a part of a T0 transgenic panicle showing red (transgenic) and non-red (non-transgenic) seeds.

Mentions: Comparison of DBC3-based media to PHI-T during the resting and PHI-UM during the selection steps demonstrated the benefit of using DBC3-based medium. Transformation frequencies were calculated based on the number of recovered T0 transformation events capable of growth on mannose-containing medium (indicating expression of PMI) and of expression of DsRed in shoots, roots, leaves, and pollen (Fig. 3) relative to the number of starting immature embryos used for transformation. Use of the baseline protocol resulted in the recovery of T0 plants from two independent transgenic events derived from 362 immature embryos, for a transformation frequency of 0.6%. By comparison, use of the DBC3 media for the resting and selection steps resulted in the recovery of T0 plants from 29 independent transgenic events derived from 361 immature embryos, for a transformation frequency of 8%. From these 29 events, 170 T0 plants (4–6 T0 plants per event) were grown and self-pollinated in the greenhouse, and 161 plants (95%) produced T1 seeds. Based on DsRed expression (Fig. 3e), a 3 red/1 non-red ratio was observed for 18 events (66.7%, Table 2). A higher ratio than expected was observed for seven events (25.9%), and a lower ratio than expected was observed for two events (7.4%). From the two transgenic events recovered using the treatment C protocol, 11 of 12 T0 plants (6 from each event) grown in the greenhouse produced T1 seed. The expected ratio of 3 red/1 non-red T1 was observed for both events.Fig. 3


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 callus, shoots, roots, leaf, pollen, and seeds expressing DsRed derived from PHP149 viewed using fluorescence microscopy. (a) Transgenic callus expressing DsRed, (b) transgenic shoots and roots expressing DsRed, (c) transgenic leaf expressing DsRed, (d) pollen grains from a T0 transgenic plant with or without expression of DsRed, (e) T1 seeds from a T0 transgenic plant with or without expression of DsRed, (f) a part of a T0 transgenic panicle showing red (transgenic) and non-red (non-transgenic) seeds.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4544465&req=5

Fig3: Transgenic callus, shoots, roots, leaf, pollen, and seeds expressing DsRed derived from PHP149 viewed using fluorescence microscopy. (a) Transgenic callus expressing DsRed, (b) transgenic shoots and roots expressing DsRed, (c) transgenic leaf expressing DsRed, (d) pollen grains from a T0 transgenic plant with or without expression of DsRed, (e) T1 seeds from a T0 transgenic plant with or without expression of DsRed, (f) a part of a T0 transgenic panicle showing red (transgenic) and non-red (non-transgenic) seeds.
Mentions: Comparison of DBC3-based media to PHI-T during the resting and PHI-UM during the selection steps demonstrated the benefit of using DBC3-based medium. Transformation frequencies were calculated based on the number of recovered T0 transformation events capable of growth on mannose-containing medium (indicating expression of PMI) and of expression of DsRed in shoots, roots, leaves, and pollen (Fig. 3) relative to the number of starting immature embryos used for transformation. Use of the baseline protocol resulted in the recovery of T0 plants from two independent transgenic events derived from 362 immature embryos, for a transformation frequency of 0.6%. By comparison, use of the DBC3 media for the resting and selection steps resulted in the recovery of T0 plants from 29 independent transgenic events derived from 361 immature embryos, for a transformation frequency of 8%. From these 29 events, 170 T0 plants (4–6 T0 plants per event) were grown and self-pollinated in the greenhouse, and 161 plants (95%) produced T1 seeds. Based on DsRed expression (Fig. 3e), a 3 red/1 non-red ratio was observed for 18 events (66.7%, Table 2). A higher ratio than expected was observed for seven events (25.9%), and a lower ratio than expected was observed for two events (7.4%). From the two transgenic events recovered using the treatment C protocol, 11 of 12 T0 plants (6 from each event) grown in the greenhouse produced T1 seed. The expected ratio of 3 red/1 non-red T1 was observed for both events.Fig. 3

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