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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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Student
Level
General public
Study
biology
School/University
Monash...

About the document

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Language
documents in English
Format
Word
Type
case study
Pages
9 pages
Level
General public
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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.

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