TRANSFORMATION OF KAPPA (κ)-CARRAGEENASE GENE FROM Pseudoalteromonas IN SEAWEED Kappaphycus alvarezii [DOTY]

Abstract

Superoxide dismutase (SOD) has an important role in the defense system of the body, especially the activity of reactive oxygen compounds that can cause stress. MmCu/Zn-SOD gene successfully regenerated in N. benthamiana and N. tabacum, rice, and Jatropha transgenic plants. The assembling of transgenic K. alvarezii has been previously performed with other genes, however, the successful introduction of genes was still low. The objective of this study was to optimize a method for the Agrobacterium mediated MmCu/Zn-SOD gene transformation based on the optical density (OD), the duration of inoculation and co-cultivation period factors to increase the effectiveness of transgenesis in K. alvarezii. The experimental method was divided into the two stages: 1) the optimization of MmCu/Zn-SOD gene transformation in Kappaphycus alvarezii based on optical density (OD), inoculation duration and co-cultivation period; and 2) the analysis of the existence of MmCu/Zn-SOD genes in Kappaphycus alvarezii using PCR. Six levels of A. tumefaciens OD600 (0.3, 0.4, 0.5, 0.6, 0.7 and 0.8), four duration of inoculation (10, 20, 30 and 60minutes) and two periods of co-cultivation (3 and 4days) were tested.
The results demonstrated that the highest putative bud (100%) and transformation (100%) efficiencies were obtained by using inoculation duration of 60minutes and 3days of co-cultivation with OD600 of 0.5. However, regeneration efficiency of explants (6.67%) was lower than the non-transgenic control (33.33%). The results of PCR analyses demonstrated the presence of MmCu/Zn-SOD gene in the transgenic explants, whereas the non-transgenic explants seaweed showed no amplification product. In conclusion, transgenic K. alvarezii could be produced and optimized by the Agrobacterium tumefaciens-mediated method based on optical density (OD), duration of inoculation and co-cultivation period factors. Transgenic explants that carry encoding genes of MmCu/ Zn-SOD tolerant to salinity of 15g /L and salinity of 45g/L so all were survive, while all the non-transgenic were died.