ANALYSIS OF MEEF1A6 GENE PROMOTER ACTIVITY WITH IN-VITRO AND IN-VIVO USING TRANSIENT AND TRANSGENIC TECHNIQUES IN TOBACCO PLANT (NICOTIANA TABACUM)

• Main Author: Gibral Andalusia, Galih
• Format: Theses
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/49404
• Institution: Institut Teknologi Bandung

Promoter is one part of gene that functions in carrying out the gene expression and its work activity becomes a matter of concern to ensure that expression works effectively. MeEF1A6 (Manihot esculenta Elongation Factor 1 Alfa - 6) promoter is a promoter derived from cassava plants (Manihot esculenta). Previous studies showed that the MeEF1A6 promoter was successfully isolated and inserted into the pBI121 plasmid replacing the CaMV35S promoter. This study aims to analyze the activity of MeEF1A6 promoters in -vivo and in -vitro by using transient and transgenic techniques in tobacco plants. The pBI121 plasmid containing the MeEF1A6 promoter was inserted into Agrobacterium tumefaciens strain AGL1 and LBA4404 as vectors. The promoter's work was analyzed by result from introducing into the tobacco plant using the transient method and stable transformation. Whole part of explants were used for transient study and tested in minimum two biological replicates. Furthermore, the 60 sheets of explant leaves that have been cut 1x1 cm were used for stable transformation. The promoter work analysis was carried out with the GUS (?-glukuronidase) gene histochemical test intergrated with the promoter. The transient test produced a blue color in the roots, stems and leaves on whole repetition. The transverse incision in the stem shows the same blue color as the epidermis and procambium that carried CaMV35S promoter as a control in the epidermis and procambium tissues. Stable transformation using Agrobacterium tumefaciens strain AGL1 as vector produced 43 shoots from 40 calli. A total of 43 shoots were selected with antibiotics and produced 27 planlet that were successfully grown. Some planlet are then reacted with x-gluc as histochemical GUS assay substrat and produced a blue color in the explants. The study result indicate that the MeEF1A6 promoter has been successfully introduced and could work. While the stable transformation with Agrobacterium tumefaciens strain LBA4404 as a vector produced 50 calli from a total of 60 leaf explants, but the age of the calli did not reach the shoot growth stage. GUS histochemical test has also been performed on the calli and produced blue color in the callus growth region. In conclusion, MeEF1A6 promoter could work on plant tissue in roots, stems, leaves and tissues that connect meristems such as procambium and calli tissue in tobacco plants. This reinforces the suspicion that the MeEF1A6 promoter perfoms work constitutionally as a constitutive promoter.