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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

The gene cassettes in the T-DNAs of PHP149, PHP166, and PHP32269. (A) T-DNA of PHP149 (11.8 kb) containing sequences for co-suppression of sorghum gamma-kafirin-1, gamma-kafirin-2, delta-kafirin-2, and sorghum LKR, and for expression of Ds-Red and PMI. (B) T-DNA of PHP166 (16.3 kb) containing sequences for co-suppression of sorghum gamma-kafirin-1, alpha-kafirin-A1, and sorghum MIK, and for expression of maize PSY-1, CRT-I, and PMI. The three regions for Q-PCR assays indicated with the bars underneath the vector map. (C) T-DNA of PHP32269 (7.9 kb) containing sequences for expression of PMI, the fusion protein gene moPAT, and YFP. The two regions for Q-PCR assays indicated with the bars underneath the vector map.
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Fig1: The gene cassettes in the T-DNAs of PHP149, PHP166, and PHP32269. (A) T-DNA of PHP149 (11.8 kb) containing sequences for co-suppression of sorghum gamma-kafirin-1, gamma-kafirin-2, delta-kafirin-2, and sorghum LKR, and for expression of Ds-Red and PMI. (B) T-DNA of PHP166 (16.3 kb) containing sequences for co-suppression of sorghum gamma-kafirin-1, alpha-kafirin-A1, and sorghum MIK, and for expression of maize PSY-1, CRT-I, and PMI. The three regions for Q-PCR assays indicated with the bars underneath the vector map. (C) T-DNA of PHP32269 (7.9 kb) containing sequences for expression of PMI, the fusion protein gene moPAT, and YFP. The two regions for Q-PCR assays indicated with the bars underneath the vector map.

Mentions: Agrobacterium tumefaciens strains LBA4404 and AGL1 (Lazo et al. 1991) were used in this study. Vectors pSB1 and pSB11 (Komari 1990; Komari et al. 1996) were used to construct the super-binary vectors PHP149, PHP166, and PHP32269 (Fig. 1). PHP149 contained three gene cassettes as follows: (1) the maize CZ19B1 promoter driving four inverted repeats for co-suppression of sorghum gamma-kafirin-1, gamma-kafirin-2, delta-kafirin-2, and lysine alpha-ketogluterate reductase; (2) the maize UBI promoter and UBI intron driving DsRed (Baird et al. 2000), which encodes a 28-kDa red fluorescent protein that serves as a visible marker; and (3) the maize UBI promoter and UBI intron driving PMI. PHP166 contained five gene cassettes as follows: (1) the maize CZ19B1 promoter driving inverted repeats of sorghum alpha-kafirin-A1 and gamma-kafirin-1 gene; (2) the maize oleosin promoter driving an inverted repeat of sorghum myo-inositol kinase (Shi et al. 2005); (3) the sorghum alpha-kafirin promoter driving maize phytoene synthase (Psy-1) (Buckner et al. 1996); (4) the sorghum beta-kafirin promoter driving Erwinia uredovora carotene desaturase (CrtI) (Ye et al. 2000); and (5) the maize UBI promoter and UBI intron driving PMI. PHP32269 contained two gene cassettes as follows: (1) the maize UBI promoter and UBI intron driving moPAT and yellow fluorescent protein (YFP, Nagai et al. 2002) and (2) the maize UBI promoter and UBI intron driving PMI.Fig. 1


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)

The gene cassettes in the T-DNAs of PHP149, PHP166, and PHP32269. (A) T-DNA of PHP149 (11.8 kb) containing sequences for co-suppression of sorghum gamma-kafirin-1, gamma-kafirin-2, delta-kafirin-2, and sorghum LKR, and for expression of Ds-Red and PMI. (B) T-DNA of PHP166 (16.3 kb) containing sequences for co-suppression of sorghum gamma-kafirin-1, alpha-kafirin-A1, and sorghum MIK, and for expression of maize PSY-1, CRT-I, and PMI. The three regions for Q-PCR assays indicated with the bars underneath the vector map. (C) T-DNA of PHP32269 (7.9 kb) containing sequences for expression of PMI, the fusion protein gene moPAT, and YFP. The two regions for Q-PCR assays indicated with the bars underneath the vector map.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4544465&req=5

Fig1: The gene cassettes in the T-DNAs of PHP149, PHP166, and PHP32269. (A) T-DNA of PHP149 (11.8 kb) containing sequences for co-suppression of sorghum gamma-kafirin-1, gamma-kafirin-2, delta-kafirin-2, and sorghum LKR, and for expression of Ds-Red and PMI. (B) T-DNA of PHP166 (16.3 kb) containing sequences for co-suppression of sorghum gamma-kafirin-1, alpha-kafirin-A1, and sorghum MIK, and for expression of maize PSY-1, CRT-I, and PMI. The three regions for Q-PCR assays indicated with the bars underneath the vector map. (C) T-DNA of PHP32269 (7.9 kb) containing sequences for expression of PMI, the fusion protein gene moPAT, and YFP. The two regions for Q-PCR assays indicated with the bars underneath the vector map.
Mentions: Agrobacterium tumefaciens strains LBA4404 and AGL1 (Lazo et al. 1991) were used in this study. Vectors pSB1 and pSB11 (Komari 1990; Komari et al. 1996) were used to construct the super-binary vectors PHP149, PHP166, and PHP32269 (Fig. 1). PHP149 contained three gene cassettes as follows: (1) the maize CZ19B1 promoter driving four inverted repeats for co-suppression of sorghum gamma-kafirin-1, gamma-kafirin-2, delta-kafirin-2, and lysine alpha-ketogluterate reductase; (2) the maize UBI promoter and UBI intron driving DsRed (Baird et al. 2000), which encodes a 28-kDa red fluorescent protein that serves as a visible marker; and (3) the maize UBI promoter and UBI intron driving PMI. PHP166 contained five gene cassettes as follows: (1) the maize CZ19B1 promoter driving inverted repeats of sorghum alpha-kafirin-A1 and gamma-kafirin-1 gene; (2) the maize oleosin promoter driving an inverted repeat of sorghum myo-inositol kinase (Shi et al. 2005); (3) the sorghum alpha-kafirin promoter driving maize phytoene synthase (Psy-1) (Buckner et al. 1996); (4) the sorghum beta-kafirin promoter driving Erwinia uredovora carotene desaturase (CrtI) (Ye et al. 2000); and (5) the maize UBI promoter and UBI intron driving PMI. PHP32269 contained two gene cassettes as follows: (1) the maize UBI promoter and UBI intron driving moPAT and yellow fluorescent protein (YFP, Nagai et al. 2002) and (2) the maize UBI promoter and UBI intron driving PMI.Fig. 1

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