Evaluation of CaMV 35S and Sugarcane EF-1? Promoters for Use on Genetic Transformation of Oil Palm (Elaeis guineensis Jacq.) Dini Ermavitalini

• Main Author: Ermavitalini, Dini
• Format: Theses
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/34907
• Institution: Institut Teknologi Bandung

Oil palm is an oil producing plant that produce 5-7 tons of oil /ha/year. In the first step of this research, study on plant regeneration of oil palm somatic embryo was conducted. The second step was study on genetic transformation mediated by Agrobacterium tumefaciens strain GV 3101 with plasmid Cambia 1303 that harbored CaMV 35S and EF-1? promoters respectively. Cauliflower Mozaic Virus 35S (CaMV 35S) promoter is a constitutive gene promoter whereas promoter of Elongation factor- 1? (EF-1?) is a specific promoter that has activity related to active tissue that synthesize protein. The aim of this research was to evaluate the use of CaMV 35S promoter and EF-1? promoter in oil palm somatic embryo transformed by A. tumefaciens. Induction of somatic embryo was performed by culturing embryogenic callus in SIM (Suspension Initiation Medium) supplemented with 50 ppm 2,4-D and 1 ppm BAP for 4 weeks. Subculturing was then conducted on EDM I (Embryo Development Medium I) supplemented with casein hydrolysate without plant growth regulator for 4 weeks. The somatic embryos were then subcultured on EDM II (Embryo Development Medium II) supplemented with casein hydrolysate and 5 ?M BAP for 1 week. Maturation of somatic embryo was carried out on MM (Maturation Medium) supplemented with 20 mM glutamine and 5 mM arginine for 3 weeks. Germination of mature somatic embryo was performed on GM (Germination Medium) without plant growth regulator for 4 weeks. The somatic embryos on EDM I were used for transformation. Transformation with CaMV 35S promoter as positive control was carried out with 3 ways, namely soaking in A. tumefaciens culture for 25 min plus 5 min vacuum infiltration treatment, 15 min vacuum infiltration treatment and shaking on 60 rpm speed for 30 min. Cocultivation was conducted for 3 days on EDM II containing 100 ?M acetosyringone. GUS transient expression was performed by Jefferson method (1987) at 7 days after transformation. Transformation efficiency was obtained by calculating the somatic embryo that expressed GUS gene divided by total embryo x 100% while promoter strength was obtained by calculating the number of blue spots on the somatic embryo expressing GUS gene. The result showed that oil palm somatic embryo could regenerate into plantlet on 250 ppm cefotaxime without visually different with somatic embryo cultured on control medium. Hygromycine 20 ppm was a minimum lethal concentration of oil palm somatic embryo. Statistical analysis showed that there was significantly difference between soaking 25 min plus 5 min vacuum infiltration treatment and others two treatment whereas shaking for 30 min treatment was not significantly different with 15 min vacuum infiltration treatment. The best transient expression of GUS was obtained on soaking 25 min plus 5 min vacuum infiltration treatment. Transformation by EF-1? promoter was then performed by transformation method of soaking 25 min plus 5 min vacuum infiltration treatment. The mean of transformation efficiency with CaMV 35S promoter was 100% while with EF-1? promoter was 12.5%. Mean of promoter strength of CaMV 35S promoter in oil palm somatic embryo was 68.375 whereas EF-1? promoter was 0.375.