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High-throughput transformation pipeline for a Brazilian japonica rice with bar gene selection.

Dedicova B, Bermudez C, Prias M, Zuniga E, Brondani C - Protoplasma (2014)

Bottom Line: We tested backbone integration in 101 transgenic plants from all constructs and found 60 transgenic plants having no additional sequence integrated in the plant genome.The bar gene activity was evaluated by the chlorophenol red test and the leaf painting test using phosphinothricin with several transgenic plants.The majority of T0 plants carrying the single copy of transgene produced T1 seeds in the screen house.

View Article: PubMed Central - PubMed

Affiliation: International Center for Tropical Agriculture A.A. 6713, Cali, Colombia, beatadedicova@hotmail.com.

ABSTRACT
The goal of this work was to establish a transformation pipeline for upland Curinga rice (Oryza sativa L. ssp. japonica) with bar gene selection employing bialaphos and phosphinothricin as selection agents. The following genes of interest: AtNCED3, Lsi1, GLU2, LEW2, PLD-alpha, DA1, TOR, AVP1, and Rubisco were cloned into the binary vector p7i2x-Ubi and were transferred into Agrobacterium strain EHA 105. Embryogenic calli derived from the mature embryos were transformed, and transgenic cells and shoots were selected on the medium supplemented with bialaphos or phosphinothricin (PPT) using a stepwise selection scheme. Molecular analyses were established using polymerase chain reaction and Southern blot for the bar gene and the NOS terminator. Overall, 273 putative transgenic plants were analyzed by Southern blot with 134 events identified. In total, 77 events had a single copy of the transgene integrated in the plant genome while 29 events had two copies. We tested backbone integration in 101 transgenic plants from all constructs and found 60 transgenic plants having no additional sequence integrated in the plant genome. The bar gene activity was evaluated by the chlorophenol red test and the leaf painting test using phosphinothricin with several transgenic plants. The majority of T0 plants carrying the single copy of transgene produced T1 seeds in the screen house.

No MeSH data available.


Related in: MedlinePlus

Molecular analysis through conventional PCR. a PCR amplification of bar gene. Lanes 2–9, 11–18: PCR-positive plants. Lane 20: plasmid transgenic control. Lanes 21 and 22: genomic transgenic controls. Lane 23: not transgenic control. Lane 24: reaction control. Lanes 1, 10, and 19: 1-Kb Plus ladder (Invitrogen). Size of band, 318 bp. b PCR amplification of backbone integration. Lanes 1 and 24: 1-Kb Plus ladder (Invitrogen). Lanes 2–23: transgenic plants under backbone analysis. Lane 25: plasmid p7i2x-AtNCED3. Lane 26: plasmid p7i2x-PLD-alpha. Lanes 27–29: not transgenic controls. Lane 30: reaction control. Size of bands: 279 bp in constructs p7i2x-PLD-alpha and p7i2x-Rubisco1 and 242 bp in remaining plasmids. Note the size difference between plants transformed either with constructs p7i2x-PLD-alpha and p7i2x-Rubisco2 (Lanes 2, 7, 8, and 11) or remaining plasmids (Lanes 4, 5, 12–15)
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Fig4: Molecular analysis through conventional PCR. a PCR amplification of bar gene. Lanes 2–9, 11–18: PCR-positive plants. Lane 20: plasmid transgenic control. Lanes 21 and 22: genomic transgenic controls. Lane 23: not transgenic control. Lane 24: reaction control. Lanes 1, 10, and 19: 1-Kb Plus ladder (Invitrogen). Size of band, 318 bp. b PCR amplification of backbone integration. Lanes 1 and 24: 1-Kb Plus ladder (Invitrogen). Lanes 2–23: transgenic plants under backbone analysis. Lane 25: plasmid p7i2x-AtNCED3. Lane 26: plasmid p7i2x-PLD-alpha. Lanes 27–29: not transgenic controls. Lane 30: reaction control. Size of bands: 279 bp in constructs p7i2x-PLD-alpha and p7i2x-Rubisco1 and 242 bp in remaining plasmids. Note the size difference between plants transformed either with constructs p7i2x-PLD-alpha and p7i2x-Rubisco2 (Lanes 2, 7, 8, and 11) or remaining plasmids (Lanes 4, 5, 12–15)

Mentions: From 321 Curinga rice plants regenerated and analyzed by PCR, 143 showed amplification of the bar gene (Table 4 and Fig. 4a) and were considered as transgenic or PCR positive. Amplification of a band of 318 bp confirmed the transgenic status of plants. This first screening through PCR allowed plant selection for subsequent molecular characterization through Southern blot.Fig. 4


High-throughput transformation pipeline for a Brazilian japonica rice with bar gene selection.

Dedicova B, Bermudez C, Prias M, Zuniga E, Brondani C - Protoplasma (2014)

Molecular analysis through conventional PCR. a PCR amplification of bar gene. Lanes 2–9, 11–18: PCR-positive plants. Lane 20: plasmid transgenic control. Lanes 21 and 22: genomic transgenic controls. Lane 23: not transgenic control. Lane 24: reaction control. Lanes 1, 10, and 19: 1-Kb Plus ladder (Invitrogen). Size of band, 318 bp. b PCR amplification of backbone integration. Lanes 1 and 24: 1-Kb Plus ladder (Invitrogen). Lanes 2–23: transgenic plants under backbone analysis. Lane 25: plasmid p7i2x-AtNCED3. Lane 26: plasmid p7i2x-PLD-alpha. Lanes 27–29: not transgenic controls. Lane 30: reaction control. Size of bands: 279 bp in constructs p7i2x-PLD-alpha and p7i2x-Rubisco1 and 242 bp in remaining plasmids. Note the size difference between plants transformed either with constructs p7i2x-PLD-alpha and p7i2x-Rubisco2 (Lanes 2, 7, 8, and 11) or remaining plasmids (Lanes 4, 5, 12–15)
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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Fig4: Molecular analysis through conventional PCR. a PCR amplification of bar gene. Lanes 2–9, 11–18: PCR-positive plants. Lane 20: plasmid transgenic control. Lanes 21 and 22: genomic transgenic controls. Lane 23: not transgenic control. Lane 24: reaction control. Lanes 1, 10, and 19: 1-Kb Plus ladder (Invitrogen). Size of band, 318 bp. b PCR amplification of backbone integration. Lanes 1 and 24: 1-Kb Plus ladder (Invitrogen). Lanes 2–23: transgenic plants under backbone analysis. Lane 25: plasmid p7i2x-AtNCED3. Lane 26: plasmid p7i2x-PLD-alpha. Lanes 27–29: not transgenic controls. Lane 30: reaction control. Size of bands: 279 bp in constructs p7i2x-PLD-alpha and p7i2x-Rubisco1 and 242 bp in remaining plasmids. Note the size difference between plants transformed either with constructs p7i2x-PLD-alpha and p7i2x-Rubisco2 (Lanes 2, 7, 8, and 11) or remaining plasmids (Lanes 4, 5, 12–15)
Mentions: From 321 Curinga rice plants regenerated and analyzed by PCR, 143 showed amplification of the bar gene (Table 4 and Fig. 4a) and were considered as transgenic or PCR positive. Amplification of a band of 318 bp confirmed the transgenic status of plants. This first screening through PCR allowed plant selection for subsequent molecular characterization through Southern blot.Fig. 4

Bottom Line: We tested backbone integration in 101 transgenic plants from all constructs and found 60 transgenic plants having no additional sequence integrated in the plant genome.The bar gene activity was evaluated by the chlorophenol red test and the leaf painting test using phosphinothricin with several transgenic plants.The majority of T0 plants carrying the single copy of transgene produced T1 seeds in the screen house.

View Article: PubMed Central - PubMed

Affiliation: International Center for Tropical Agriculture A.A. 6713, Cali, Colombia, beatadedicova@hotmail.com.

ABSTRACT
The goal of this work was to establish a transformation pipeline for upland Curinga rice (Oryza sativa L. ssp. japonica) with bar gene selection employing bialaphos and phosphinothricin as selection agents. The following genes of interest: AtNCED3, Lsi1, GLU2, LEW2, PLD-alpha, DA1, TOR, AVP1, and Rubisco were cloned into the binary vector p7i2x-Ubi and were transferred into Agrobacterium strain EHA 105. Embryogenic calli derived from the mature embryos were transformed, and transgenic cells and shoots were selected on the medium supplemented with bialaphos or phosphinothricin (PPT) using a stepwise selection scheme. Molecular analyses were established using polymerase chain reaction and Southern blot for the bar gene and the NOS terminator. Overall, 273 putative transgenic plants were analyzed by Southern blot with 134 events identified. In total, 77 events had a single copy of the transgene integrated in the plant genome while 29 events had two copies. We tested backbone integration in 101 transgenic plants from all constructs and found 60 transgenic plants having no additional sequence integrated in the plant genome. The bar gene activity was evaluated by the chlorophenol red test and the leaf painting test using phosphinothricin with several transgenic plants. The majority of T0 plants carrying the single copy of transgene produced T1 seeds in the screen house.

No MeSH data available.


Related in: MedlinePlus