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

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.

Keywords: 2-deoxyglucose; Agrobacterium-mediated transformation; DOGR1 gene; oil palm embryogenic calli.

FIGURE 1

Schematic diagram of DOG^R1 expression cassette (line indicates the size of 1.5 kb) used as probe for Southern blot analysis. RB, right border; LB, left border; NOS, nopaline synthase gene terminator; DOG^R1, gene codes for 2-deoxyglucose-6-phosphate phosphatase; CaMV35S, cauliflower mosaic virus 35S promoter. Arrows indicates the position of primers used for PCR analysis to amplify 741 bp.

FIGURE 2

Protocol for Agrobacterium-mediated transformation and regeneration of transgenic oil palm selected on 2-DOG medium.

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.

FIGURE 4

PCR analysis for detection of DOG^R1 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.

FIGURE 5

Integration patterns of the DOG^R1 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-DOG^R1-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-DOG^R1-nos fragment. Lane M, 1 kb plus marker; Lane U, untransformed plant; Lanes 1–10, samples of transformed oil palm shoots.