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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

PCR analysis for detection of DOGR1 gene, PCR product is 741 bp. Lane M, 1 kb plus marker; Lane P, pBIDOG plasmid; Lane U, untransformed sample; Lanes 1–29, transformed oil palm embryoid samples.
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Figure 4: PCR analysis for detection of DOGR1 gene, PCR product is 741 bp. Lane M, 1 kb plus marker; Lane P, pBIDOG plasmid; Lane U, untransformed sample; Lanes 1–29, transformed oil palm embryoid samples.

Mentions: Regeneration of transgenic plants that are able to survive on 2-DOG provides initial evidence that the plants are transgenic; however, molecular analyses (PCR and Southern hybridization) are required to confirm stable integration of transgenes in plant genome. In this study, DNA from resistant embryoids was subjected to PCR analysis for the DOGR1 gene. DNA from untransformed embryoids was also used as negative controls. Prior to performing PCR using the DOGR1 gene, amplification of an internal control, specific to oil palm, was carried out (data not shown). A pair of primers (POR12 and POR38) which would specifically amplify a ∼1.1 kb size fragment of oil palm genomic DNA (Nurfahisza et al., 2014) was used as internal control. It is important that all samples (transgenic and negative control) used in this study amplified the 1.1 kb fragment before being used to amplify the transgene. A total of 29 putative transgenic embryoid samples and one untransformed embryoid sample were subjected to PCR analysis for the DOGR1 gene. It was observed that 21 out of the 29 samples tested were positive for DOGR1 gene as the expected band size of 741 bp was successfully amplified (Figure 4). No amplification of the band was observed for the untransformed negative control. Based on the results, it could be estimated that around 72% of the samples carried the DOGR1 gene in their genome.


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)

PCR analysis for detection of DOGR1 gene, PCR product is 741 bp. Lane M, 1 kb plus marker; Lane P, pBIDOG plasmid; Lane U, untransformed sample; Lanes 1–29, transformed oil palm embryoid samples.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4585222&req=5

Figure 4: PCR analysis for detection of DOGR1 gene, PCR product is 741 bp. Lane M, 1 kb plus marker; Lane P, pBIDOG plasmid; Lane U, untransformed sample; Lanes 1–29, transformed oil palm embryoid samples.
Mentions: Regeneration of transgenic plants that are able to survive on 2-DOG provides initial evidence that the plants are transgenic; however, molecular analyses (PCR and Southern hybridization) are required to confirm stable integration of transgenes in plant genome. In this study, DNA from resistant embryoids was subjected to PCR analysis for the DOGR1 gene. DNA from untransformed embryoids was also used as negative controls. Prior to performing PCR using the DOGR1 gene, amplification of an internal control, specific to oil palm, was carried out (data not shown). A pair of primers (POR12 and POR38) which would specifically amplify a ∼1.1 kb size fragment of oil palm genomic DNA (Nurfahisza et al., 2014) was used as internal control. It is important that all samples (transgenic and negative control) used in this study amplified the 1.1 kb fragment before being used to amplify the transgene. A total of 29 putative transgenic embryoid samples and one untransformed embryoid sample were subjected to PCR analysis for the DOGR1 gene. It was observed that 21 out of the 29 samples tested were positive for DOGR1 gene as the expected band size of 741 bp was successfully amplified (Figure 4). No amplification of the band was observed for the untransformed negative control. Based on the results, it could be estimated that around 72% of the samples carried the DOGR1 gene in their genome.

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