http://www.bio-protocol.org/e1440 Vol 5, Iss 7, Apr 05, 2015 1 Leaf Disc Stress Tolerance Assay for Tobacco Ajit Ghosh 1 , Ashwani Pareek 2 and Sneh L Singla-Pareek 1,* 1 Plant Molecular Biology Group, International Centre for Genetic Engineering and Biotechnology, Aruna Asaf Ali Marg, New Delhi, India; 2 Stress Physiology and Molecular Biology Laboratory, School of Life Sciences, Jawaharlal Nehru University, New Delhi, India *For correspondence: sneh@icgeb.res.in [Abstract] Stress tolerance is a multigenic trait that depends on the coordinated action of several genes. Various physiological parameters, such as plant height and weight, total yield, chlorophyll content, photosynthesis rate, level of reactive oxygen species (ROS) and anti-oxidant activity could be correlated directly with the level of stress tolerance potential of any particular genotype. To evaluate the stress tolerance potential of a plant, leaf disc stress tolerance assay is a very rapid and widely acceptable experiment with minimum instrumentation facilities. Materials and Reagents 1. Young tobacco plants (to be tested) 2. Distilled water 3. Acetone (Sigma-Aldrich, catalog number: I10010) 4. Stress reagents (such as NaCl, Sigma-Aldrich, catalog number: S7653; for salinity stress) 5. Liquid nitrogen 6. Extraction buffer (see Recipes) Equipment 1. Leaf puncher/ cork borer (Sigma-Aldrich, catalog number: Z165220) 2. Petri plates (60 x 15 mm) 3. Microcentrifuge tube 4. Tissue paper 5. Graduated cylinder 6. Mortar and pestle (Sigma-Aldrich, catalog number: Z112496) 7. Glass cuvette (1 ml) (Sigma-Aldrich, catalog number: C8550-1EA) 8. Electronic balance 9. Spectrophotometer 10. Benchtop refrigerated centrifuge (Eppendorf, catalog number: 5427 R) 11. Vortexer (Sigma-Aldrich, catalog number: Z755613-1EA)