Thesis

Development of methodologies to be applied in the establishment of transgenic Meloidogyne incognita lines

Meloidogyne incognita (Mi) is a root knot nematode that infects various commercial crops and accounts for a crop loss of $100 billion a year worldwide. Therefore, learning about the biology of this parasite may help develop new strategies for its control. This study was focused on: ( i ) learning about the early development of Mi (up to the 16 cell stage) (ii) establishing protocols for generating transgenic Caenorhabditis elegans (C. elegans) lines using the microparticle bombardment method, and (iii) attempting to generate transgenic Mi lines by using the protocol developed for C. elegans in our laboratory. As markers for transformation using the microparticle bombardment system we used two constructs developed to monitor cell development in C. elegans: 1) two constructs with a muscle promoter fused to the green fluorescent protein (GFP); and 2) a construct with a neural promoter fused to the red fluorescent protein (RFP). Once we established transformation protocols in C. elegans, we generated 7 GFP containing lines and 5 RFP containing lines. The protocols were also used to shoot galls of Mi to determine if the constructs would express the reporter gene in the J2 state of Mi. After 3 experiments we obtained 5 J2 worms expressing GFP and 5 worms expressing RFP suggesting that our protocols can be used to generate transgenic Mi lines and that the constructs are suitable for expression in Mi.

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