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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

Curinga transgenic tissues. a p7i2xU-Lew2gene callus proliferation with 3 mg/l bialaphos. b p7i2xU-Rubisco 1 gene shoots induction with 3 mg/l bialaphos. c p7i2xU-GLU2 gene shoots elongation with 3 mg/l bialaphos. d p7i2xU-AtNCED3 gene rooting with 5 mg/l bialaphos (scale bar in mm)
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Fig3: Curinga transgenic tissues. a p7i2xU-Lew2gene callus proliferation with 3 mg/l bialaphos. b p7i2xU-Rubisco 1 gene shoots induction with 3 mg/l bialaphos. c p7i2xU-GLU2 gene shoots elongation with 3 mg/l bialaphos. d p7i2xU-AtNCED3 gene rooting with 5 mg/l bialaphos (scale bar in mm)

Mentions: When we analyzed the number of calli needed to produce one transgenic plant depending on the construct and selection agent, the results showed significant differences (Table 3). The PPT selection worked better with the following constructs p7i2xU-AVP1, AtNCED3, p7i2xU-DA1, and p7i2xU-TOR in difference bialaphos was better for AtGLU2, p7i2xU-Lsi1, p7i2xU-PLD-alpha, and both p7i2xU-Rubisco genes. The mixed selection worked similar to the PPT only for p7i2xU-AVP1 construct. For the shoots induction, we used MS medium supplemented with 3 mg/l PPT or bialaphos and for rooting the concentration of selection agents was increased to 5 mg/l (Fig. 3a–d).Table 3


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)

Curinga transgenic tissues. a p7i2xU-Lew2gene callus proliferation with 3 mg/l bialaphos. b p7i2xU-Rubisco 1 gene shoots induction with 3 mg/l bialaphos. c p7i2xU-GLU2 gene shoots elongation with 3 mg/l bialaphos. d p7i2xU-AtNCED3 gene rooting with 5 mg/l bialaphos (scale bar in mm)
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4491359&req=5

Fig3: Curinga transgenic tissues. a p7i2xU-Lew2gene callus proliferation with 3 mg/l bialaphos. b p7i2xU-Rubisco 1 gene shoots induction with 3 mg/l bialaphos. c p7i2xU-GLU2 gene shoots elongation with 3 mg/l bialaphos. d p7i2xU-AtNCED3 gene rooting with 5 mg/l bialaphos (scale bar in mm)
Mentions: When we analyzed the number of calli needed to produce one transgenic plant depending on the construct and selection agent, the results showed significant differences (Table 3). The PPT selection worked better with the following constructs p7i2xU-AVP1, AtNCED3, p7i2xU-DA1, and p7i2xU-TOR in difference bialaphos was better for AtGLU2, p7i2xU-Lsi1, p7i2xU-PLD-alpha, and both p7i2xU-Rubisco genes. The mixed selection worked similar to the PPT only for p7i2xU-AVP1 construct. For the shoots induction, we used MS medium supplemented with 3 mg/l PPT or bialaphos and for rooting the concentration of selection agents was increased to 5 mg/l (Fig. 3a–d).Table 3

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