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Part 2: Understanding Plant Growth In Vitro Richard R Williams Topics to be covered: 1. Fundamental Concepts / History 2. Types of Cells or Tissues 3. Cell to Cell Interaction 4. Growth Processes What is Plant Tissue Culture? “… the aseptic culture of plant protoplasts, cells, tissues or organs under conditions which lead to cell multiplication or regeneration of organs or whole plants “ 1 History Cell theory and totipotency Callus formation and growth substances Early cell culture First plant tissue cultures Animal tissue cultures Plant organ culture Cell theory …. Cork tissue as observed by Robert Hooke in 1664 Totipotency …. … each livinggenetic cell has a complete genetic complete blue print blueprint and therefore has the potential to develop into an entire plant. …cells cellslines differentiate … differentiate to form specialised tissues and organs unlikeplant animal cells, plant cells can … living cells canliving re-differentiate de-differentiate and then re-differentiate to form different cell types Callus Formation ….… Early Cell Culture …. …Haberlandt .. early 1900’s … proposed concept of totipotency … cells cultured under right conditions Callus cultured from tree cambium (Gautheret, Nobecourt, Whire in the 1930s. … cells kept alive but did not develop Early tissue culture …. - dependent on discovery of “growth regulators” Cell enlargement … role of auxins Cell division ... role of cytokinins Regeneration from tobacco pith .. (Skoog and Miller) … interaction of auxin and cytokinin gives differentiation. Further development … GA for growth of shoots Aux + Cyt + sucrose > vascular development Culture of ‘thin layers’ … many interacting factors eg pH Carrot plants from root cells – Stewart in 1964 [Steeves & Sussex 1972] Tobacco plants from single cells – Vasil & Hilderbrandt 1965 [Steeves & Sussex 1972] Plant Organ Culture …. Murashige and Skoog 1962 - mineral media micropropagation 2 Types of Cell & Tissues Many different types of cells Varying degrees of specialisation - Meristematic - Embryonic - Reproductive Meristematic tissues ... Shoot ... apical, … axillary new leaf tunica apical meristem corpus leaf trace axillary meristem procambium cortex pith Meristematic tissues ... Shoot ... apical, … axillary Leaf Root Meristematic tissues ... Shoot ... apical, … axillary Leaf Root Adventitious Cambial tissues Embryonic tissues ... Pre-formed plant Generative / somatic Juvenile Reproductive tissues ... Diploid / Haploid Female / Male Monocots versus Dicots ‘Stems’ are leaf bases … not buds Shoot apex at base Intercallary leaf growth 3 Cell to Cell Interaction Important in intact plant Isolated cell not function the same Establishment of polarity Expression of somaclonal (epigenetic) variation 4 Growth Processes Multiplication Apical dominance Differentiation Growth Cell division Cell enlargement Phasic development Juvenility Dormancy 4 Other Processes Photosynthesis Transpiration and water uptake Cytological instability 5 Controlling Growth & Development Physiological mechanisms Physical manipulation Chemical Plant Growth Regulators (PGRs) Environment Genotype 6 Growth Regulators Hormone theory Auxins Cytokinins Gibberelins (GA) Ethylene Abscisic Acid (ABA) Other PGRs Hormone Balance Auxin High Cytokinin Low Root formation on cuttings Embryogenesis Adventitious root formation in callus Callus initiation Adventitious shoot formation Axillary shoot growth Low High Auxin : Cytokinin ratio Hormone Action ….. Application + uptake Endogenous + applied Accumulation & Habituation Interactions / Sequence Pulsing vs prolonged exposure END of Part 2