Interaction of 2,4-D and Ethylene in the Growth of Tobacco BY-2 suspension

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

2. The aim of the study A study of hormonal regulation of growth of BY-2 cell suspension 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: Suspension growth Cell viability Morphological changes and features of apoptosis 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

3. Study of BY-2 Suspension - Experiment 4 concentration of 2,4-D in the liquid medium Experimental: 0,1 µM; 10 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, pH 5,7 • 6-day culture on the LS medium, on INFORS AG CH-4103 shaker at 96 rpm • Daily evaluation of specified parameters

4. Dependence of BY-2 growth on 2.4-D concentration The number of cells [x103/ml] Days

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

6. Morphology of cells after 48 h 0,1 µM 100x 1 µM - control 100x 10 µM 100x 100 µM 100x

7. Morphology of cells after 72 h 0,1 µM 100x, 400x 1 µM - control100x, 400x 10 µM 100x, 400x 100 µM 100x, 400x

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

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

10. 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

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

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

13. Summary, discussion and vision for the future Concentrationsof 2,4-D isanimportantfactorforitsaction in theplant 4 studiedconcentrationsof 2,4-D show 3 differentgrowtheffects Concentrationsof 2,4-D affectthemetabolicpathwayofethylenesynthesis Cell wallsthickeningduringthe experiment, hypothesis,proposaloffurtherprocedureforrefinementoftheprocesses in thestudiedsystem

14. Thankyouforyourattention!