Techniques for studying the nuclear condition of giant cells induced by Meloidogyne species
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Nematodes of the genus Meloidogyne are known as Âroot-knotÂ nematodes due to the characteristic knots or galls found on the roots of infected plants. Root-knot nematodes attack over 2000 species of plants and cause over 80 billion dollars lost annually. Giant cells are feeding sites of root-knot nematodes and are enlarged multinuclear cells induced by the nematodes in susceptible host roots and that function to provide nutrients to the nematode. This thesis presents data on two techniques of studying the nuclear condition in giant cells. Colchicine was used to arrest mitosis in giant cells in a previous study. Here we test the effect of colchicine on nematode activity. The results showed that colchicine did not affect nematode egg hatch, juvenile activity, or hatch of eggs produced by treated juveniles. These results confirm that colchicine can be used to arrest mitosis in giant cells without affecting the nematode parasite. A major obstacle to the study of giant cells is collecting tissue samples that are specific to giant cells. Laser capture microdissection (LCM) is a technique that allows one to sample a single giant cell. A focused laser beam was used to collect samples of giant cell cytoplasm from fixed and sectioned tissues. RNA was then extracted from those isolated samples. Using three tomato genes as test samples, specific primers were designed to measure expression level of Rb7, LHA4, and HXK1 gene by Real-Time PCR. Expression of LHA4 and Rb7 increased with time after inoculation, and immature giant cells reached levels that were 3 and 6 times, respectively, that of cortical cells, but which were not different from root meristem cells. Expression of HXK1 did not change with time after inoculation and has the same level of that in root tip and cortical tissues. These data confirmed that the techniques of LCM coupled with RT-real-time PCR can be used to quantitate expression of genes at different stages of giant cell development without contamination from surrounding cells.
He, Bin (2004). Techniques for studying the nuclear condition of giant cells induced by Meloidogyne species. Master's thesis, Texas A&M University. Texas A&M University. Available electronically from