Construction of a Vector Containing Hygromycin (Hpt) Gene Driven by Double 35s (2xcamv35s) Promoter for Oil Palm Transformation

Abstract

Transformation vector construction is one of the important disciplines for plant genetic transformation studies. A series of vector consisting of hygromycin phosphotransferase (hpt) gene as the selective marker and green fluorescent protein (GFP) as the visual reporter gene, under the control of double cauliflower mosaic virus 35S (2XCaMV35S) promoter has been engineered for transformation into oil palm cells. These genes were cloned into different types of cloning and expression vectors. The cloning was carried out by using restriction enzyme digestion and ligation method. Five intermediate vectors have been created for insertion of 2XCaMV35S-HPT-35ST and 2XCaMV35S-GFP-35ST into modified pBINPLUS backbone vector for particle bombardment and

Genetic Transformation of Oil Palmbased on Selection With Hygromycin

“…In order to investigate the potential use of hpt gene for selection of hygromycin resistant transgenic plants, two vectors namely pBIHA-X (10 810 bp) in pCAMBIA0380 backbone, which was successfully constructed in this study and pBIHA1 (14 194 bp) in pBINPLUS backbone which was previously reported (Bahariah et al, 2017) were evaluated in this study (Figure 1).…”

“…The pBIHA-X (p2X35SHPTGFP-0380) plasmid was constructed to contain the hpt selectable marker and gfp reporter genes driven by 2XCaMV35S in . The construction was carried out by employing two intermediate plasmids, p2X35STEVHPT-GII (5.5 kb) and p2X35STEVGFP-GII (5.2 kb), created in the earlier study (Bahariah et al, 2017). The p2X35STEVHPT-GII fragment was ligated to the HindIII and EcoRI sites of pCAMBIA0380 to form 8.9 kb of p2X35STEVHPT-0380.…”

“…Thus, as part of the ongoing efforts to develop an oil palm transformation system using the antibiotic hygromycin resistance gene, two expression vectors carrying the hpt selectable marker gene and gfp reporter gene driven by 2XCaMV35S promoter in different vector backbones were evaluated in this study. The pBIHA1 vector in pBINPLUS backbone (Belknap et al, 2008) which was previously reported (Bahariah et al, 2017) and a newly constructed vector, pBIHA-X in pCAMBIA backbone (Hajdukiewicz et al, 1994) were examined for their ability to increase the transgene expression level and develop an efficient oil palm transformation system by using hygromycin selection.…”

“…The plasmids used for construction of pBIHA-X are listed in Table 2. The vector pBIHA-X is based on pCAMBIA0380, a pPZP family (Hajdukiewicz et al, 1994), was constructed by using two intermediate vectors created in a previous study (Bahariah et al, 2017). Plasmid pUC19 (Norrander et al, 1983) served as a vector for cloning DNA fragments.…”

Multiplex CRISPR/Cas9 gene-editing platform in oil palm targeting mutations in EgFAD2 and EgPAT genes

“…The CRISPR/Cas9 constructs were introduced into oil palm calli using biolistic and Agrobacterium -mediated transformation, while PEG-mediated transfection was used to introduce the constructs into oil palm protoplasts according to the established protocol [ 6 , 28 – 30 ]. The calli were then selected for regeneration on 4 mg liter −1 of hygromycin selective agent under 16/8 h light conditions at 28 °C.…”