In Vitro Developmental Pathways.ppt

In Vitro Developmental Pathways Explant - Piece of tissue put into culture -Tissue selection depends on purpose, species,  many factors Explants Pieces of organs Leaves Stems Roots Cotyledons Embryos Other Explants Specific cell types Leaf tissue Embryo Pollen Endosperm Nucellus Callus Unorganized, growing mass of cells Dedifferentiation of explant Loosely arranged thinned walled, outgrowths from explant No predictable site of organization or differentiation Auxin + cytokinin Often can be maintained indefinitely by subculture, but may lose ability to redifferentiate Compact vs friable Habituation Three stages of callus culture Induction: Cells in explant dedifferentiate and begin to divide Proliferative Stage: Rapid cell division Differentiation stage (sometimes): organogenesis or embryogenesis Induction Division Callus Differentiation Organogenesis Somatic embryogenesis Cell and Suspension Culture Cell Cultures? Suspension Cultures Suspension cultures Can be initiated from any part of the plant. Usually initiated from friable callus already growing in culture. Transferred into liquid medium. Agitation Breakdown of cell aggregates into smaller clumps of cells Maintains a uniform distribution of cells and cell clumps in the medium Provides gas exchange Medium Same as for callus culture? Gamborg B5 Conditioning Growth Curve Batch Cultures A certain number of cells is used to inoculate the culture, in a given volume Erlenmeyer flask: volume should be about 20% of flask capacity for aeration. Roller cultures Continuous Culture Bioreactors Closed continuous cultures: Remove some of the media and replace with fresh. Continuous removal or periodic. Terminate growth at harvest. Start over. Open continuous culture: Not only remove some of media, but cells too. Maintain cell density at optimal level. Can be grown for years. Why is it possible to regenerate in vitro? Totipotency Initial state Competence Determination Differentiation Organogenesis