Introduction of PaCS gene encoding Citrat Synthase to Kappaphycus alvarezii using Agrobacterium tumefaciens
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Date
2013Author
Daud, Ristanti Frinra
Widyastuti, Utut
Suharsono
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Ice-ice is a disease that reduces the quality of seaweed. This disease is mainly caused by environmental stress rather than pathogen. This is supported by the fact that ice-ice disease is prevalent in the environment where the water have low quality, poor circulation, low salinity and changement of temperature, and high content of heavy metals. Citric acid is known to be a strong chelating agent for aluminum. Citric acid has a very important role in the Kreb cycle. The enzyme involved in the synthesis of citric acid is citrate synthase. Citrate synthase genes from Pseudomonas aeruginosa (PaCS) has been successfully introduced into genome of tobacco (Nicotiana tabacum) and Jatropha curcas using Agrobacterium tumefaciens. Tobacco transgenic plants containing PaCS gene are more tolerant to Al stress compared to non-transgenic plants. This research aims to introduce the citrate synthase gene into genome of seaweed Kappaphycus alvarezii by using Agrobacterium tumefaciens. Cutting Thallus of 0,5 cm were used as explants. Explants were inoculated by Agrobacterium tumefaciens for 15 minutes and then co-cultivated in PES media containing 0,3% agar for 3 days in the dark. The selection of putative transgenic K. alvarezii was carried out in PES agar selective medium containing 10 mg/l hygromicin. Based on hygromicin resistant explant on hygromicin selective medium, the transformation efficiency in K. alvarezii was 7.5 %. The efficiency of shoot regeneration of putative transgenic was 100%, and same as the regeneration efficiency of non transgenic ones. Molecular analysis by PCR showed that one of five putative transgenic K. alvarezii was confirmed as transgenic thallus containing PaCS transgene under the control of 35S CaMV promoter.