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Evaluation on the effectiveness of 2-deoxyglucose-6-phosphate phosphatase (DOG(R)1) gene as a selectable marker for oil palm (Elaeis guineensis Jacq.) embryogenic calli transformation mediated by Agrobacterium tumefaciens.

Izawati AM, Masani MY, Ismanizan I, Parveez GK - Front Plant Sci (2015)

Bottom Line: The plantlets were later transferred into soil and grown in a biosafety screenhouse.PCR and subsequently Southern blot analyses were carried out to confirm the integration of the transgene in the plantlets.This result demonstrates the potential of using combination of DOG(R)1 gene and 2-DOG for regenerating transgenic oil palm.

View Article: PubMed Central - PubMed

Affiliation: Advanced Biotechnology and Breeding Centre, Malaysian Palm Oil Board Selangor, Malaysia.

ABSTRACT
DOG(R)1, which encodes 2-deoxyglucose-6-phosphate phosphatase, has been used as a selectable marker gene to produce transgenic plants. In this study, a transformation vector, pBIDOG, which contains the DOG(R)1 gene, was transformed into oil palm embryogenic calli (EC) mediated by Agrobacterium tumefaciens strain LBA4404. Transformed EC were exposed to 400 mg l(-1) 2-deoxyglucose (2-DOG) as the selection agent. 2-DOG resistant tissues were regenerated into whole plantlets on various regeneration media containing the same concentration of 2-DOG. The plantlets were later transferred into soil and grown in a biosafety screenhouse. PCR and subsequently Southern blot analyses were carried out to confirm the integration of the transgene in the plantlets. A transformation efficiency of about 1.0% was obtained using DOG(R)1 gene into the genome of oil palm. This result demonstrates the potential of using combination of DOG(R)1 gene and 2-DOG for regenerating transgenic oil palm.

No MeSH data available.


Related in: MedlinePlus

Integration patterns of the DOGR1 gene determined by Southern blot analysis. Thirty μg oil palm genomic DNA was digested with HindIII and EcoRI and blotted onto nylon membrane and probed with a dATP32 labeled 1.5 kb CaMV35S-DOGR1-nos fragment isolated from pBIDOG plasmid. Copy number reconstruction experiment was performed by loading 30 μg untransformed genomic DNA spiked with 0 (lane W), 0.1 (lane A), 0.5 (lane B), and 1 (lane C) copy of the 1.5 kb CaMV35S-DOGR1-nos fragment. Lane M, 1 kb plus marker; Lane U, untransformed plant; Lanes 1–10, samples of transformed oil palm shoots.
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Figure 5: Integration patterns of the DOGR1 gene determined by Southern blot analysis. Thirty μg oil palm genomic DNA was digested with HindIII and EcoRI and blotted onto nylon membrane and probed with a dATP32 labeled 1.5 kb CaMV35S-DOGR1-nos fragment isolated from pBIDOG plasmid. Copy number reconstruction experiment was performed by loading 30 μg untransformed genomic DNA spiked with 0 (lane W), 0.1 (lane A), 0.5 (lane B), and 1 (lane C) copy of the 1.5 kb CaMV35S-DOGR1-nos fragment. Lane M, 1 kb plus marker; Lane U, untransformed plant; Lanes 1–10, samples of transformed oil palm shoots.

Mentions: Polymerase chain reaction analysis of the transgene only provides initial evidence of the presence of the transgenes in the genome of putative transgenic oil palm. For definitive evidence, Southern blot analysis is required to demonstrate integration of transgene into the genome of oil palm. Genomic DNA from transformed plantlet leaves were digested with EcoRI and HindIII and subjected to Southern hybridization using CaMV35S-DOGR1-nos fragment (Figure 1) as probe. After hybridization, it was observed that only 5 out of 10 transformed DNA samples (randomly chosen) showed hybridization to the expected 1.5 kb band (Figure 5). No signal was observed on untransformed sample (Lane U). Based on the DNA digestion profile, the ∼1.5 kb band was expected to correspond to the CaMV35S-DOGR1-nos fragment. This hybridization with expected band size demonstrates the integration of the transgene. Based on the intensity of hybridizing band compared to the copy number reconstruction lanes (Figure 5, lanes A–C), it was estimated that only two transformed DNA samples (lanes 5 and 8) carried a single copy of the DOGR1 gene while the other three DNA samples (lanes 2, 4, and 7) contained multiple copies of the DOGR1 gene.


Evaluation on the effectiveness of 2-deoxyglucose-6-phosphate phosphatase (DOG(R)1) gene as a selectable marker for oil palm (Elaeis guineensis Jacq.) embryogenic calli transformation mediated by Agrobacterium tumefaciens.

Izawati AM, Masani MY, Ismanizan I, Parveez GK - Front Plant Sci (2015)

Integration patterns of the DOGR1 gene determined by Southern blot analysis. Thirty μg oil palm genomic DNA was digested with HindIII and EcoRI and blotted onto nylon membrane and probed with a dATP32 labeled 1.5 kb CaMV35S-DOGR1-nos fragment isolated from pBIDOG plasmid. Copy number reconstruction experiment was performed by loading 30 μg untransformed genomic DNA spiked with 0 (lane W), 0.1 (lane A), 0.5 (lane B), and 1 (lane C) copy of the 1.5 kb CaMV35S-DOGR1-nos fragment. Lane M, 1 kb plus marker; Lane U, untransformed plant; Lanes 1–10, samples of transformed oil palm shoots.
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Related In: Results  -  Collection

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Figure 5: Integration patterns of the DOGR1 gene determined by Southern blot analysis. Thirty μg oil palm genomic DNA was digested with HindIII and EcoRI and blotted onto nylon membrane and probed with a dATP32 labeled 1.5 kb CaMV35S-DOGR1-nos fragment isolated from pBIDOG plasmid. Copy number reconstruction experiment was performed by loading 30 μg untransformed genomic DNA spiked with 0 (lane W), 0.1 (lane A), 0.5 (lane B), and 1 (lane C) copy of the 1.5 kb CaMV35S-DOGR1-nos fragment. Lane M, 1 kb plus marker; Lane U, untransformed plant; Lanes 1–10, samples of transformed oil palm shoots.
Mentions: Polymerase chain reaction analysis of the transgene only provides initial evidence of the presence of the transgenes in the genome of putative transgenic oil palm. For definitive evidence, Southern blot analysis is required to demonstrate integration of transgene into the genome of oil palm. Genomic DNA from transformed plantlet leaves were digested with EcoRI and HindIII and subjected to Southern hybridization using CaMV35S-DOGR1-nos fragment (Figure 1) as probe. After hybridization, it was observed that only 5 out of 10 transformed DNA samples (randomly chosen) showed hybridization to the expected 1.5 kb band (Figure 5). No signal was observed on untransformed sample (Lane U). Based on the DNA digestion profile, the ∼1.5 kb band was expected to correspond to the CaMV35S-DOGR1-nos fragment. This hybridization with expected band size demonstrates the integration of the transgene. Based on the intensity of hybridizing band compared to the copy number reconstruction lanes (Figure 5, lanes A–C), it was estimated that only two transformed DNA samples (lanes 5 and 8) carried a single copy of the DOGR1 gene while the other three DNA samples (lanes 2, 4, and 7) contained multiple copies of the DOGR1 gene.

Bottom Line: The plantlets were later transferred into soil and grown in a biosafety screenhouse.PCR and subsequently Southern blot analyses were carried out to confirm the integration of the transgene in the plantlets.This result demonstrates the potential of using combination of DOG(R)1 gene and 2-DOG for regenerating transgenic oil palm.

View Article: PubMed Central - PubMed

Affiliation: Advanced Biotechnology and Breeding Centre, Malaysian Palm Oil Board Selangor, Malaysia.

ABSTRACT
DOG(R)1, which encodes 2-deoxyglucose-6-phosphate phosphatase, has been used as a selectable marker gene to produce transgenic plants. In this study, a transformation vector, pBIDOG, which contains the DOG(R)1 gene, was transformed into oil palm embryogenic calli (EC) mediated by Agrobacterium tumefaciens strain LBA4404. Transformed EC were exposed to 400 mg l(-1) 2-deoxyglucose (2-DOG) as the selection agent. 2-DOG resistant tissues were regenerated into whole plantlets on various regeneration media containing the same concentration of 2-DOG. The plantlets were later transferred into soil and grown in a biosafety screenhouse. PCR and subsequently Southern blot analyses were carried out to confirm the integration of the transgene in the plantlets. A transformation efficiency of about 1.0% was obtained using DOG(R)1 gene into the genome of oil palm. This result demonstrates the potential of using combination of DOG(R)1 gene and 2-DOG for regenerating transgenic oil palm.

No MeSH data available.


Related in: MedlinePlus