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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

Regeneration of transformed oil palm. (A) Suspension embryogenic calli (EC) cultured on EC medium. (B) EC after infection with Agrobacterium suspension cultured on medium without selection. (C) EC in selection stage using 2-DOG medium. (D) Untransformed (control) EC proliferated into whitish and green embryoids when cultured on medium without selection. (E) Resistant EC (arrows) cultured on selection medium. (F) Resistant EC were separated from dead EC for proliferation into polyembryoid. (G) Whitish polyembryoid and shoot buds. (H) Shoots. (I) Transgenic shoots in flask for proliferation. (J) Transgenic shoots in rooting stage. (K) Plantlet with strong shoots and roots were transferred into jiffy peat pellet. (L) Transgenic oil palm plant in polybag.
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Figure 3: Regeneration of transformed oil palm. (A) Suspension embryogenic calli (EC) cultured on EC medium. (B) EC after infection with Agrobacterium suspension cultured on medium without selection. (C) EC in selection stage using 2-DOG medium. (D) Untransformed (control) EC proliferated into whitish and green embryoids when cultured on medium without selection. (E) Resistant EC (arrows) cultured on selection medium. (F) Resistant EC were separated from dead EC for proliferation into polyembryoid. (G) Whitish polyembryoid and shoot buds. (H) Shoots. (I) Transgenic shoots in flask for proliferation. (J) Transgenic shoots in rooting stage. (K) Plantlet with strong shoots and roots were transferred into jiffy peat pellet. (L) Transgenic oil palm plant in polybag.

Mentions: The EC were pre-treated on a plasmolysis (PM) medium containing acetosyringone prior to bombardment (Figure 3A). Pre-treatment of the EC onto PM medium which contained acetosyringone helped to induce the vir gene transfer (Hiei et al., 1994). The function of the vir genes on the Ti plasmid is to facilitate excision of the T-DNA region of the plasmid and promote its transfer and integration into the plant genome. Lignin, flavanoid precursors and acetosyringone are compounds known to induce vir gene expression (Stachel et al., 1985). Pre-treatment by a PM containing acetosyringone has also been reported to improve transformation of other plants such as rice, orchid, garlic, Panax ginseng, gherkin (Cucumis anguria), and Artemisia annua and onion (Uzè et al., 1997; Choi et al., 2001; Men et al., 2003a; Kenel et al., 2010; Thiruvengadam et al., 2013; Tian et al., 2013; Xu et al., 2014). Prior to Agrobacterium infection, oil palm EC were bombarded with gold particles to initiate physical injury. Biolistic approach to create wounding was also reported to promote the transformation process and increase transformation efficiency in other crops such as carnation, tobacco, and rapeseed (Zuker et al., 1999; Kumar et al., 2006; Abdollahi et al., 2007). Therefore, based on the above reports, both pre-treatment and wounding were carried out with the expectation to help increase the oil palm transformation efficiency.


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)

Regeneration of transformed oil palm. (A) Suspension embryogenic calli (EC) cultured on EC medium. (B) EC after infection with Agrobacterium suspension cultured on medium without selection. (C) EC in selection stage using 2-DOG medium. (D) Untransformed (control) EC proliferated into whitish and green embryoids when cultured on medium without selection. (E) Resistant EC (arrows) cultured on selection medium. (F) Resistant EC were separated from dead EC for proliferation into polyembryoid. (G) Whitish polyembryoid and shoot buds. (H) Shoots. (I) Transgenic shoots in flask for proliferation. (J) Transgenic shoots in rooting stage. (K) Plantlet with strong shoots and roots were transferred into jiffy peat pellet. (L) Transgenic oil palm plant in polybag.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 3: Regeneration of transformed oil palm. (A) Suspension embryogenic calli (EC) cultured on EC medium. (B) EC after infection with Agrobacterium suspension cultured on medium without selection. (C) EC in selection stage using 2-DOG medium. (D) Untransformed (control) EC proliferated into whitish and green embryoids when cultured on medium without selection. (E) Resistant EC (arrows) cultured on selection medium. (F) Resistant EC were separated from dead EC for proliferation into polyembryoid. (G) Whitish polyembryoid and shoot buds. (H) Shoots. (I) Transgenic shoots in flask for proliferation. (J) Transgenic shoots in rooting stage. (K) Plantlet with strong shoots and roots were transferred into jiffy peat pellet. (L) Transgenic oil palm plant in polybag.
Mentions: The EC were pre-treated on a plasmolysis (PM) medium containing acetosyringone prior to bombardment (Figure 3A). Pre-treatment of the EC onto PM medium which contained acetosyringone helped to induce the vir gene transfer (Hiei et al., 1994). The function of the vir genes on the Ti plasmid is to facilitate excision of the T-DNA region of the plasmid and promote its transfer and integration into the plant genome. Lignin, flavanoid precursors and acetosyringone are compounds known to induce vir gene expression (Stachel et al., 1985). Pre-treatment by a PM containing acetosyringone has also been reported to improve transformation of other plants such as rice, orchid, garlic, Panax ginseng, gherkin (Cucumis anguria), and Artemisia annua and onion (Uzè et al., 1997; Choi et al., 2001; Men et al., 2003a; Kenel et al., 2010; Thiruvengadam et al., 2013; Tian et al., 2013; Xu et al., 2014). Prior to Agrobacterium infection, oil palm EC were bombarded with gold particles to initiate physical injury. Biolistic approach to create wounding was also reported to promote the transformation process and increase transformation efficiency in other crops such as carnation, tobacco, and rapeseed (Zuker et al., 1999; Kumar et al., 2006; Abdollahi et al., 2007). Therefore, based on the above reports, both pre-treatment and wounding were carried out with the expectation to help increase the oil palm transformation efficiency.

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