Production and transformation of tobacco and Brassica containing macrochloroplasts

Chikkala, V 2009, Production and transformation of tobacco and Brassica containing macrochloroplasts, Doctor of Philosophy (PhD), Applied Sciences, RMIT University.

Document type: Thesis
Collection: Theses

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Title Production and transformation of tobacco and Brassica containing macrochloroplasts
Author(s) Chikkala, V
Year 2009
Abstract Plastid division, sustained by the equilibrium expression and coordination of plastid division genes is vital for the maintenance of plastid populations in dividing plant cells. Macrochloroplasts (MCP), the occurrence of one or a few chloroplasts per cell is due to the imbalance in the expression of plastid division genes. Because of the MCP size and number it was proposed that they may provide better targets for the plastid transformation than the normal (WT) chloroplasts and result in better plastid transformation frequencies.

The objective of this research was to produce transgenic plants containing macrochloroplasts by nuclear transformation and then to use these plants as a model for the development of plastid transformation of crop species. By using AtFtsZ1-1 and AtMinD1 as query sequences in the TIGR (U.S.A) and ASTRA (Australia) Brassica oleracea EST databases, this project resulted in the isolation of cauliflower FtsZ1-1 (EU684588) and MinD (EU684589) genes. In addition, AtFtsZ1-1 was used as a control gene for comparison to the cauliflower FtsZ1-1. Binary vectors were constructed to express these genes in tobacco and cauliflower either by Agrobacterium tumefaciens-mediated or PEG-mediated transformation methods. Transgenic tobacco and cauliflower plants with abnormal chloroplasts (MCP, minichloroplasts, honeycomb or doughnut shaped chloroplasts, uneven surface membrane chloroplasts) were developed. Furthermore, the transgenic tobacco and cauliflower plants were examined by PCR, RT-PCR and Southern blotting. In addition, th ese plants were also analysed for the different abnormal chloroplast phenotypes by fluorescence microscopy.

This project also generated the first plastid transformants from macrochloroplast bearing tobacco plants via biolistics. After one round of regeneration homoplasmic plastid transformants were obtained from both WT chloroplast and MCP tobacco plants. The homoplasmic nature of plastid transformants were confirmed by PCR and Southern blotting. Plastid expression of GFP in WT and MCP was confirmed by fluorescence/confocal microscopy and western blot analysis. This project showed for the first time the characterisation of cauliflower FtsZ1-1 and MinD plastid division genes in homologous and heterologous systems (cauliflower and tobacco). Moreover, obtaining homoplasmic plastid transformant shoots from one round of regeneration from the MCP containing tobacco plants is reported for the first time in this study. In addition this study explored the effect of transgene expression level on the chloroplast abnormality, highlighting the importance of analysing transgenic tobacco and cauliflower plants at the protein lev el specifically with regard to plastid division genes. The maintenance of MCP phenotype in the regenerated shoots and the requirement of standardisation of MCP containing plants via biolistics for increasing the plastid transformation frequency were also examined.
Degree Doctor of Philosophy (PhD)
Institution RMIT University
School, Department or Centre Applied Sciences
Keyword(s) Brassica -- Biotechnology
Tobacco -- Biotechnology
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