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Agrobacterium-mediated transformation of rice callus with artificial miRNA construct against lipase to produce transgenic plant with lowered lipase activity

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  1. Introduction
  2. Materials and methods
  3. Construction of ami RNA targeting lipase gene
  4. Validation of success transformation
  5. Discussion
  6. Conclusion

Lipase is a lipolytic enzyme causes spoilage of rice bran. Its removal by conventional heating and chemical approaches damages bran quality. This experiment aimed to transform Nipponbare rice callus with ami RNA against lipase using Agrobacterium tumefaciens, regenerate whole transgenic rice plant with lowered lipase activity and analyzed the success of rice transformation. Callus was first inducted using seeds from Nipponbare rice line, and immersed in suspension of A. tumefaciens carrying ami RNA gene before plated onto NB plate with acetosyringone. First and second transfer to selection media containing hygromycin was done after washing with cefotaxime. Transformed callus was regenerated into transgenic plants before transferred to soil and grown in greenhouse. Southern, Northern and Western blots were performed and rice bran were analyzed its lipase content. Phenotypic analysis was also performed. Growth of both callus and A. tumefaciens were observed in non-selection NB plate. Only clear rice callus with growth but no A. tumefaciens were observed in selection NB+hygromycin+cefotaxime plate. Second selection was expected to obtain 50-90% white color transformed callus while remaining non-transformed callus turned brown or black. It was expected to detect presence of lipase and ami RNA gene in Southern blotting, presence of ami RNA transcript but low or no lipase RNA transcript in Northern blotting, low or no lipase protein in Western blotting and low lipase content in rice bran of transgenic plant. Transgenic plant had high tillering and dwarf features.

Conclusively, transgenic rice plant with lowered lipase activity was produced by Agrobacterium-mediated transformation of rice callus with ami RNA construct against lipase. Lower than expected number of transformed callus was obtained, indicated low transformation frequency. It was expected to obtain at least 10 transgenic plants. Absence or low detection of lipase mRNA transcript and lipase enzyme respectively in Northern blotting and Western blotting, as well as low lipase content in rice bran indicated successful transformation and regeneration of transgenic rice with lowered lipase activity. 40% of transformed callus expected to integrate single copy of ami RNA into genome.

[...] Northern blotting and Western blotting were performed for respectively for total RNA and protein extracted from transgenic plants after gel electrophoresis. Rice bran of transgenic plant was also analyzed. Phenotypic analysis of transgenic plant was performed. Results Figure Growth of rice callus and A. tumefaciens on NB plates containing acetosyringone after first week. Figure Growth of transformed and untransformed rice callus on NB plates+cefotaxime+hygromycin after third week. From figure most callus appeared lightly colored and covered with white plague of A. [...]


[...] Transformation of rice callus and regeneration of transgenic rice Rice callus was immersed in suspension of A. tumefaciens carrying the ami RNA construct for 10mins. Callus were drained off excess bacterial suspension before cultivated on NB+100µM acetosyringone plate at 25oC for 7 days in dark. Plate was then observed. Callus were washed using 150mg/L cefotaxime 3 times until no more visible bacteria before drying with filter paper. Washed callus was plated onto NB medium+cefotaxine+hygromycin for 10 days at 27oC in dark. [...]


[...] Monocot plant like rice however did not demonstrate such wounding response and could not initiate the bacterial infection as like in dicots (Raja et al. 2010). The lack of phenolic compound exudation might also be due to monocots responded to wounding by lignification. In this case, acetosyringone was artificially added culture medium to improve the transformation frequency by A. tumefaciens (Tripathi et al. 2010). In figure clear rice callus with growth was observed. No traces of A. tumefaciens was observed. Some callus started to show browning and blackening, while some white colored callus demonstrated unhindered growth. This was consistent with expectation. [...]


[...] Conclusion Transgenic rice plant with lowered lipase activity was produced by Agrobacterium-mediated transformation of rice callus with ami RNA construct against lipase. Lower than expected number of transformed callus was obtained, indicated low transformation frequency. It was expected to obtain at least 10 transgenic plants. Southern blotting expected to reveal 40% of transformed callus to integrate single copy of ami RNA gene into genome two and 30% three. Absence or low detection of lipase mRNA transcript and lipase enzyme respectively in Northern blotting and Western blotting, as well as low lipase content in rice bran expected for successful transformation and regeneration of transgenic rice with lowered lipase activity. [...]


[...] Grunewald Bury J & Inze D 2013, ?Biotechnology: thirty years of transgenic plants', Nature, vol no p Hiei Y & Komari T 2008, ?Agrobacterium-mediated transformation of rice using immature embryos or calli induced from mature seed', Nature Protocols, vol no pp. 824-834. Jacob SS & Veluthambi K 2003, cointegrate Ti plasmid vector for Agrobacterium tumefaciens-mediated transformation of Indica rice cv Pusa Basmati 1', Journal of Plant Biochemistry & Biotechnology, vol no pp. 1-9. Jauhar PP 2006, ?Modern biotechnology as an integral supplement to conventional plant breeding: the prospects and challenges', Crop Science, vol no pp. 1841-1859. [...]

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