Annexure-I Preparation of MS stock solutions and volume used from the stock to prepare the medium. Volume in ml to be taken for 1 lt of medium

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2 Annexure-I Preparation of MS stock solutions and volume used from the stock to prepare the medium Name of stock solution Chemical composition Quantity in mg/l Quantity in g/l of stock solution Strength of stock solution Volume in ml to be taken for 1 lt of medium A NH4NO X 20 B KNO X 20 KH2PO X 5 H3BO Na2MO4.2H2O KI COCl2.6H2O CaCl2.2H2O X 5 MgSO4.7H2O X 5 MnSO4.4H2O ZnSO4.7H2O CuSO4.5H2O FeSO4.7H2O X 5 Na2EDTA.H2O Thiamine HCl 2000X Pyridoxine HCl 2000X Nicotinic acid 2000X Glycine X 2.0 Myo-inositol 100 C D E F Sucrose 30.0 Agar-agar 6.0

3 Annexure -II Chemical composition (mg/l) of the various nutrient media used in the present study Components KH2PO4 Na2H2PO4.H2O KNO3 NH4NO3 CaCl2.2H2O Ca(NO3)4.H2O (NH4)2SO4 Na2SO4 KCl MgSO4.7H2O H3BO3 MnSO4.4H2O MnSO4.H2O ZnSO4.7H2O Na2MO4.2H2O CuSO4.5H2O COCl2.6H2O KI FeSO4.7H2O Na2EDTA.H2O Fe2(SO4)3 FeCl2.6H2O FeNaEDTA Thiamine HCl Pyridoxine HCl Nicotinic acid Glycine Myo-inositol MS B5 WPM Macronutrients Micronutrients SH HE Vitamins MS- Murashige and Skoog (1962), WPM-Woody Plant Medium (Lloyd and McCown, 1980), B5- Gamborg et al., 1968, SH-Schenk and Hilderbrandt (1972), HE-Heller, R. (1953)

4 Annexure III CHEMICAL PREPERERTION FOR ENZYME ESTIMATION (I) Glutamine synthetase activity: (A) Extraction buffer (ph 7.2) mm Tris- HCl 1.571gm Tris HCl was dissolved in 50 ml of distilled water (solution A) 2. 1mM EDTA 0.037gm EDTA was dissolved in 10 ml of distilled water (solution B) Mix A and B solution up to 100 ml and kept (ph 7.2) (B) Reaction mixture: M Glutamine Dissolve g of L- Glutamine in 10 ml of distilled water mm sodium arsenate Dissolve 0.624g Sodium arsenate in 10 ml of distilled water 3. 3 mm MnCl2 Dissolve g MnCl2 in 10ml of distilled water mm Hydroxylamine Dissolve g Hydroxylamine in 10ml of distilled water mm KOH Dissolve 0.280g KOH in 10ml of distilled water 6. 1mM ADP Dissolve 0.047g ADP in 10ml of distilled water mm Tris-HCl (8.0)

5 Dissolve 0.314g Tris HCl in 20 ml of distilled water and adjusted ph 8.0 using 1 N NaOH Mixed one by one slowly from 1-7 solution and make the volume to100 ml. (C) Ferric chloride Dissolve 10g trichloro-acetic acid and 8g ferric chloride in 250ml of N hydrochloric acid. (II) Nitrate reductase activity: 1. Phosphate buffer (ph 7.5, 0.1 M) 13.6 gm KH2PO4 was dissolved in litre distilled water (solution A) gm K2HPO4 was dissolved in litre distilled water (solution B) 160 ml solution A and 840 ml solution B were mixed together to prepare the required buffer. 2. KNO3 solution (0.2M) 2.02 gm of KNO3 was dissolved in 100 ml distilled water 3. Isopropanol (5%) 5 ml isopropanol was mixed with 95 ml of distilled water 4. Sulphanilamide (1%) 1 gm sulphanilamide was dissolved in 100 ml of 3N HCl 5. NED HCl (0.02%) 20 mg NED HCl was dissolved in 100 ml of distilled water (III) Peroxidase activity: 1. Phosphate buffer (ph 6.4) (20mM) gm Na2HPO4. 7H2O was dissolved in distilled water to make 200 ml (solution A) gm NaH2PO4. 4H2O was dissolved in distilled water to make 500 ml (solution B).

6 Mix 132 ml solution A and 367 ml solution B to prepare the required buffer. 2. Guaiacol solution (20mM) gm of guaiacol was dissolved in distilled water to make 100 ml solution. 3. H2O2 solution (0.2M) 2.26 ml H2O2 (30%) was dissolved in distilled water to make 100 ml solution (IV) Protein estimation: 1. NaOH solution (0.1 N) 2 gm NaOH was dissolved in distilled water to make 100 ml solution 2. Alkaline Na2CO3 solution (2%) 10 gm of Na2CO3 was dissolved in 500 ml solution of NaOH (solution A) 3. Sodium potassium tartarate solution (1%) gm sodium potassium tartarate was dissolved in distilled water to make 50 ml solution 4. CuSO4 solution (%) 0.25 gm of CuSO4 was dissolved in 50 ml sodium potassium tartarate solution 500 ml (solution B) 500ml solution (A) was mixed with 10 ml solution (B) to obtain the protein reagent (V) Gel solutions: 1. Acrylamide solution (30%): 30 gm acrylamide was dissolved in 100 ml distilled water. 1 gm bisacrylamide was dissolved in the acrylamide solution. The solution was filtered through filter paper. 2. Tris solution (1M) (ph 8.8):

7 121.1 gm tris was dissolved in litre distilled water. The solution was filtered and stored. 3. Ammonium persulphate (10%): 10 gm APS was dissolved in 100 ml distilled water. 4. Bromophenol blue (mg ml-1): 10 mg bromophenol blue was dissolved in 10 ml distilled water. 5. Glycerol (20%): 20 gm glycerol was dissolved in 100 ml distilled water. 6. H2O2 (0.06%): 1 ml (30%) H2O2 was dissolved in 100 ml distilled water. 7. Guaiacol (0.1%): 0.1 gm guaiacol was dissolved in 100 ml distilled water. 8. Glacial acetic acid (0.1%): ml glacial acetic acid was dissolved in 100 ml distilled water. 9. Phosphate buffer (ph 7.5, 0.1 M)

8 Annexure IV (A). Preparation of reagents for DNA extraction 1.Tris HCl (1M): Dissolved g Tris HCl in 800 ml of double distilled water and volume was made to 1 litre and ph of the solution was adjusted to 8.0 with NaOH pellets. 2.EDTA (M): Dissolved g of EDTA to 800 ml of double distilled water and volume was made to 1 litre and ph of the solution was adjusted to 8.0 with NaOH pellets. 3. NaCl (5M): Dissolved g of NaCl to 800 ml of double distilled water and stirred till NaCl was dissolved completely and made the volume to 1 litre. 4. Phenol, Chloroform and Iso-amyl alcohol in a ratio of 25:24:1 (v/v), respectively. 5. Chloroform and Iso-amyl alcohol in a ratio of 24:1 (v/v), respectively. 6. Ethidium bromide: 1000 ppm solution was prepared and stored in amber reagent bottle. 7. 6X loading dye: Bangalore Genei readymade 6X gel loading dye (1X working concentration). 8. TAE buffer (50 X Tris acetic acid EDTA buffer) 2M Tris: g Glacial acetic acid: 57.1 ml 0.5M Na2EDTA: 100 ml (ph 8.0) Make upto 1 litre with double distilled water.

9 (B) Preparation of DNA extraction buffer Tris HCl - 100mM EDTA - 20mM NaCl - 5M PVP - 2% β-mercaptoethanol 0.2% CTAB - 3% (C) TE buffer (ph 8.0) 10mM Tris HCl M EDTA Make up the volume to 1 litre using double distilled water.

10 Annextue V List of RAPD primers selected for the study and there sequence RAPD Primer G + C content Primer sequence (5-3 ) (%) GTCGCCGTCA 60 TCTGGTGAGG 70 TGAGCGGACA 60 GTGTGCCCCA 70 CTCTGGAGAC 60 CACCGTATCC 60 GGGGTGACGA 70 GAGAGCCAAC 60 OPD03 OPD04 OPD05 OPD08 OPD09 OPD12 OPD13 OPD18

11 Appendix -VI List of ISSR primers selected for the study and there sequence in B. nutans. ISSR Primer G+C Primer sequence (5-3 ) content (%) UBC-807 AGA GAG AGA GAG AGA GT 47 UBC-809 AGA GAG AGA GAG AGA GG 53 UBC-820 GTGTGTGTGTGTGTGTC 53 UBC-822 TCT CTC TCT CTC TCT CA 47 UBC-833 ATA TAT ATA TAT ATA TYG 5.5 UBC-834 AGA GAG AGA GAG AGA GYT UBC-835 AGA GAG AGA GAG AGA GYC 50 UBC-836 AGAGAGAGAGAGAGAGYA UBC-837 TATATATATATATATART 0.0 UBC-838 TATATATATATATATARC