Pot Level Drought Stress Tolerance Assay in Tobacco through Plant Phenotyping and Antioxidant Assay

Materials and Reagents

1. Pipette (BrandTech Scientific, Transferpette, catalog numbers:  704770 , 704778 , 704780 )
   
2. Centrifuge tube (1.5 ml) (Corning, Axygen^®, catalog number: MCT-150-C-S )
   
3. Tobacco seed samples under study: Nicotiana tabacum L. cv. Petit Havana, wild type and transgenic lines
   Note: The complete open reading frame of histone-gene binding protein of rice (OsHBP1b) was cloned at BglII and SpeI sites in the plant expression vector (pCAMBIA1304) and used for transformation of Agrobacterium strain (LBA 4404). Fifteen days old tobacco seedlings were used for transformation through tissue culture method. The putative transgenic T₀ plants were transferred to the greenhouse for the purpose of seeds multiplication. The seeds of T₂ transgenic lines were used for experimental purpose.
   
4. Distilled H₂O (IndiaMART InterMESH Ltd, Mars Scientific Instruments Co., catalog number: BASIC/pH & XL )
   
5. Agro peat (Agro peat super) (AswiniAgrotech)
   
6. Vermiculite (Manidharma Biotech Private Limited)
   
7. Sucrose (Sigma-Aldrich, catalog number: S0389 )
   
8. Stress reagent (Polyethylene glycol 6000 or PEG 6000) (Sigma-Aldrich, catalog number: 81260 )
   
9. Ascorbate (Sigma-Aldrich, catalog number: A4034 )
   
10. Liquid Nitrogen (Advanced Instrumentation Research Facility, Jawaharlal Nehru University, New Delhi)
    
11. Hydrogen peroxide (H₂O₂) (Sigma-Aldrich, catalog number: 216763 )
    
12. Bovine serum albumin (Sigma-Aldrich, catalog number: A2153 )
    
13. Murashige & Skoog (MS) with vitamins (Caisson Laboratories, catalog number: MSP09-1lt )
    
14. K₂HPO₄ (Sigma-Aldrich, catalog number: 1551128 )
    
15. KH₂PO₄ (Sigma-Aldrich, catalog number: 1551139 )
    
16. Coomassie Brilliant Blue G-250-100 mg (Sigma-Aldrich, catalog number: 27815 )
    
17. Methanol (100%) (Sigma-Aldrich, catalog number: 34860 )
    
18. Phosphoric acid (85%) (Sigma-Aldrich, catalog number: W290017 )
    
19. Phosphate buffer (see Recipes)
    
20. Bradford reagent (see Recipes)
    
21. MS media (see Recipes)
    

Equipment

1. Plastic pots (12 cm diameter and 13 cm depth) (Garden Aids, India)
   
2. Forceps (ACE Surgical Co, catalog number: 10)
   
3. Growth chamber (Daihan LabTech India Pvt. Ltd., model: LGC-S201 )
   
4. Oven (Hicon India)
   
5. pH meter (Control Dynamic Instrumentation Pvt. Ltd., model: APX175E )
   Note: Currently, it is “CD Hightech Pvt. Ltd., model: APX175E ”.
   
6. Plastic ice tray
   
7. Liquid nitrogen container (Kailash Gases, Cryocan, model: BA-11 )
   
8. Motor and pestle (local supplier, New Delhi)
   
9. Cuvette (both plastic and quartz) (Sigma-Aldrich, catalog number: Z276677 )
   
10. Spectrophotometer (Cary 300 UV-Vis)
    
11. Image capturing device (SONY CORPORATION OF AMERICA, model: DSC-HX300 )
    
12. Weighing machine (Sartorius, model: BSA224S-CW )
    
13. Scale (30 cm plastic scale)
    

Software

1. GraphPad InStat3 software

Procedure