Transformation of Morinda Citrifolia and reconstruction of a promoter tangging cassette

Lee, Jong Jen (2011) Transformation of Morinda Citrifolia and reconstruction of a promoter tangging cassette. Masters thesis, Universiti Malaysia Sarawak (UNIMAS).

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Abstract

Morinda cilr[/'olia is a plant that has been reported to have a wide range of medicinal properties and other characters such as flowering and fruiting throughout the year. The establishment of tin • efficient and reliable transformation protocol suitable for M. cilr(/'olia had been successfully formulated through the use of Agrobaclerium tumefaciens on direct seed transformation. A total of 2180 seeds were transformed and sown. The work includes optimi,zation of different transformation parameters and two types of binary vectors, pGSA 1131 and pB1121, were used in the transformation using A. tumefaciens strain LBA4404) Result from the study showed that the optimum condition for M. citrifolia transformation are bacterial density of 1.0, incubation period of 2 hours at room temperature and using vector pGSA 1131. Initial screening for putatively transformed seedlings was done by PCR using primers specific to the reporter gene (GUS gene) and selectable marker of the respective vector. Overall transformation efficiency using A. , lumefaciens harbouring pGSA 1131 was determined to be 75.95% while 72.27% when using ~. lumefaciens containing pB1121. There were two transgenic seedlings from the total of 182 tested gave blue coloration on histochemical assay while other seedlings were able to show expression on fluorometric assay and RT-PCR. Second part of the project involves the construction of a promoter tagging cassette which uses insertional mutagenesis to identify potential promoter or other regulatory sequences. The constructed cassette contained a reporter gene, fi-glucuroidase (GUS) as indicator. The construction of a promoter trapping cassette, containing the modified TDNA, involved a senes of molecular techniques and elements from several vectors such as pGSA 1131 and pAGS. Seedlings containing the construct were screened for possible novel genes or regulatory elements near the T-DNA insertion sites in the plant genome via DN{\.­ • walking and inverse-PCR techniques. Sequence analysis result indicated that the sequence shows some similarities to genomic sequences of 5S ribosomal RNA, mitochondrion, chloroplast and plant species such as Vilis vinifera, Zea mays, Nicoliana tabacum, Carica papaya, Lycopersicon esculentum and Triticum aestivum.

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