Limits...
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

Five regions of the binary vector backbone, LB (T-DNA left border), SPC (spectinomycin resistant gene), TET (tetracycline resistant gene), VIR B (vir B gene), and RB (T-DNA right border) used for Q-PCR analysis to detect vector backbone integration.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC4544465&req=5

Fig2: Five regions of the binary vector backbone, LB (T-DNA left border), SPC (spectinomycin resistant gene), TET (tetracycline resistant gene), VIR B (vir B gene), and RB (T-DNA right border) used for Q-PCR analysis to detect vector backbone integration.

Mentions: Q-PCR (Yang et al. 2009) was used to estimate the copy number of the transgenes to determine if T-DNA integrations were intact or truncated, and to screen for the presence of Agrobacterium binary vector backbone integration. Genomic DNA was extracted from a single piece (200 ng) of fresh leaf tissue from each plant (Truett et al. 2000). The non-transgenic sorghum line TX430 was used as the negative control and transgenic sorghum plants known to have single-copy transgenes were used as positive controls. Quantification was based on detection of amplification of gene sequences using gene-specific forward and reverse primers along with the corresponding gene-specific FAM/Vic-based MGB fluorogenic probes (Applied Biosystems, Foster City, CA). The 2−ΔΔCT method (Livak and Schmittgen 2001; ABI’s user bulletin #2, www3.appliedbiosystems.com/cms/groups/mcb_support/documents/generaldocuments/cms_040980.pdf) was used to estimate the copy number. For plants transformed with PHP166, three regions of the T-DNA (PMI, PSY-1, and CZ19B1 terminator (Fig. 1b) were screened. Plants with single-copy scores for all of three sequences were classified as having intact single-copy T-DNA integrations. Plants that had single-copy scores for only one or two of the three sequences were classified as truncated single-copy events. Plants with multiple-copy scores for any of the three sequences were classified as having multiple-copy integrations, which could either be intact or truncated. Plants lacking all of these three sequences were classified as escapes. Plants were derived from PHP32269, primer sets that amplified segments of either the PMI or YFP; (Fig. 1c). Detection of Agrobacterium vector backbone integrations derived from PHP166 or PHP32269 was based on screening for sequences from five regions outside of the T-DNA (near RB, virG, SPC, Tet, and near LB; Fig. 2; table of primer sequences available in supplemental data). Plants with negative Q-PCR signals for all five regions were considered as backbone-negative. Otherwise, they were classified as backbone-positive. Plants with intact single-copy T-DNA integrations without vector backbone were defined as quality events (QEs).Fig. 2


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)

Five regions of the binary vector backbone, LB (T-DNA left border), SPC (spectinomycin resistant gene), TET (tetracycline resistant gene), VIR B (vir B gene), and RB (T-DNA right border) used for Q-PCR analysis to detect vector backbone integration.
© Copyright Policy
Related In: Results  -  Collection

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

Fig2: Five regions of the binary vector backbone, LB (T-DNA left border), SPC (spectinomycin resistant gene), TET (tetracycline resistant gene), VIR B (vir B gene), and RB (T-DNA right border) used for Q-PCR analysis to detect vector backbone integration.
Mentions: Q-PCR (Yang et al. 2009) was used to estimate the copy number of the transgenes to determine if T-DNA integrations were intact or truncated, and to screen for the presence of Agrobacterium binary vector backbone integration. Genomic DNA was extracted from a single piece (200 ng) of fresh leaf tissue from each plant (Truett et al. 2000). The non-transgenic sorghum line TX430 was used as the negative control and transgenic sorghum plants known to have single-copy transgenes were used as positive controls. Quantification was based on detection of amplification of gene sequences using gene-specific forward and reverse primers along with the corresponding gene-specific FAM/Vic-based MGB fluorogenic probes (Applied Biosystems, Foster City, CA). The 2−ΔΔCT method (Livak and Schmittgen 2001; ABI’s user bulletin #2, www3.appliedbiosystems.com/cms/groups/mcb_support/documents/generaldocuments/cms_040980.pdf) was used to estimate the copy number. For plants transformed with PHP166, three regions of the T-DNA (PMI, PSY-1, and CZ19B1 terminator (Fig. 1b) were screened. Plants with single-copy scores for all of three sequences were classified as having intact single-copy T-DNA integrations. Plants that had single-copy scores for only one or two of the three sequences were classified as truncated single-copy events. Plants with multiple-copy scores for any of the three sequences were classified as having multiple-copy integrations, which could either be intact or truncated. Plants lacking all of these three sequences were classified as escapes. Plants were derived from PHP32269, primer sets that amplified segments of either the PMI or YFP; (Fig. 1c). Detection of Agrobacterium vector backbone integrations derived from PHP166 or PHP32269 was based on screening for sequences from five regions outside of the T-DNA (near RB, virG, SPC, Tet, and near LB; Fig. 2; table of primer sequences available in supplemental data). Plants with negative Q-PCR signals for all five regions were considered as backbone-negative. Otherwise, they were classified as backbone-positive. Plants with intact single-copy T-DNA integrations without vector backbone were defined as quality events (QEs).Fig. 2

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