Interaction of 2 4 D and Ethylene in

Interaction of 2, 4 -D and Ethylene in the Growth of Tobacco BY-2 suspension Katerina Muselikova Mendel University Brno June 2012

The aim of the study 1) A study of hormonal regulation of growth of BY-2 cell suspension 2) Determination of the importance of selected growth regulators and phytohormones (2, 4 -D and ethylene) 3) An evaluation of the action of 2, 4 -D on the cell suspension in terms of: a) Suspension growth b) Cell viability c) Morphological changes and features of apoptosis d) Induction of the production of ethylene and the precursor of ethylene (ACC) (to assess mutual interactions) e) Determination of 2, 4 -D content in cells and medium during culture

Study of BY-2 Suspension - Experiment 4 concentration of 2, 4 -D in the liquid medium Experimental: 0, 1 µM; 100 µM Control: 1 µM • Initial suspension – mesophyll of leaves of tobacco "Bright Yellow-2” variety • Subculturing of the suspension was conducted into the liquid medium LS (LINSMAYER and Skoog, 1964), 30 g / l sucrose, 1 μM 2, 4 -D, p. H 5, 7 • 6 -day culture on the LS medium, on INFORS AG CH-4103 shaker at 96 rpm • Daily evaluation of specified parameters

The number of cells [x 103/ml] Dependence of BY-2 growth on 2. 4 -D concentration Days

Viability (%) Determination of viability BY-2 Culture (days)

Morphology of cells after 48 h 0, 1 µM 100 x 1 µM - control 100 x 100 µM 100 x

Morphology of cells after 72 h 0, 1 µM 100 x, 400 x 1 µM - control 100 x, 400 x 100 µM 100 x, 400 x

BY-2 cells in the stationary phase 10 µM 400 x 1 µM - control 400 x Stationary phase Growth phase Lag phase 0, 1 µM 400 x 100 µM 400 x, 100 x

Cell morphology after 6 days 0, 1 µM 100 x 10 µM 100 x 1 µM 100 µM 100 x

Influence of 2, 4 -D on ethylene production General phenomena: Low auxin concentrations stimulate the formation of ethylene High concentrations of auxin inhibit the formation of ethylene

Content ACC in BY-2 cells Days Ethylene content in BY-2 cells Days

Accumulation of 2, 4 -D in cells and culture medium Days

Summary, discussion and vision for the future 1) Concentrations of 2, 4 -D is an important factor for its action in the plant 2) 4 studied concentrations of 2, 4 -D show 3 different growth effects 3) Concentrations of 2, 4 -D affect the metabolic pathway of ethylene synthesis 4) Cell walls thickening during the experiment, hypothesis, proposal of further procedure for refinement of the processes in the studied system

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