Culture of Plant Protoplasts for Studies of Senescence

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The most obvious signs of plant senescence are the withering and eventual death of flowers, fruits, leaves and roots. Senescence is regulated by complex processes involving environmental, physiological and genetic factors whose mechanisms are incompletely known. In this project, a unique model will be developed for the study of plant senescence, with the eventual goal of elucidating the molecular mechanisms. Plant senescence is important to the overall economy of the plant, fruit and grain ripening, crop production, and post-harvest storage of crops. Chloroplasts in mesophyll cells of Nicotiana glauca lose functionality during leaf senescence, but those in stomatal guard cells do not. In this project, mesophyll and guard cells will be cultured in ways that preserve the unique properties of the differentiated cell types. To optimize cell survival and preservation of physiological properties unique to each cell type, a variety of nutrient, osmotic, pH, light, and cell density conditions will be tested. Once cultures are established, conditions will be identified for fusing protoplasts from the two cell types and for culturing fusion hybrids. The functionality of guard cell chloroplasts in fusion hybrids will be monitored by microfluorospectrophotometry to determine whether senescing mesophyll cells contain factors capable of initiating guard cell chloroplast senescence.***//