Vishwanath et al. methods prescribed by Laemeli (1970) with slight modifications. M
1
2
3
4
5
6
7
8
9
10 11
brilliant blue solution overnight and destained using a mixture of 227ml of methanol, 46ml of acetic acid and 227ml of distilled water until the bands were clearly visible.
12
Rm Value
M
1
2
3
4
5
6
7
8
9
10
11 12
0.000 1 23 4 56 7 8 910 11 12 13
0.079 97.4 KD
0159
66.0 KD
0.239 0.318
43.0 KD
14
Region A Region B Region C
Region D
15
0.398 29.0 KD
0.478 0.557 0.637 16
Region E
20.0 KD
17
Region F
14.3 KD
18
0.717 0.796 0.876
19
0.956
Region G
Fig. 1. Zymograms of total soluble proteins of tomato cultivars
Fig 1 Zymograms of total soluble protein of tomato cultivars M
13
14
15
16 17
18
19
20
21
22
23
Rm Value
24
M: Marker
3: A. Ahuti
6: A Megali
9: A. Abijeet
12: PKM-1
1: A. Alok
4: A. Ashish
7: A. Saurab
10: P. Ruby
13: Nandi
2: A. Vikas
5: A. Abha
8: A. Shresta
11: PED
14: Sankranthi
M
13
14
15
16
17
18
19
20
15: Vibhav
21
22
23
24
0.000 1 2 3 4 56 7 8 910
0.077 0155 0.232
97.4 KD 43.0 KD
11
Region C
66.0 KD
0.301 0.542
Region B
12 13
0.310 0.387
Region A
14
Region D
15
Region E
29.0 KD
0.620 0.697 0.775
20.0 KD
16 0.852
Region G
17 0.930
14.3 KD
18 19
Region F
Fig. 2. Zymograms of total soluble proteins of tomato cultivars
Fig 2 Zymograms of total soluble protein of tomato cultivars M: Marker
15: Vibhav
18: US -618
21: Ronco
13: Nandi
16: NS-2535
19: JK Desi
22: A-32/63
14: Sankranthi
17: Mruthnjaya-2
20: JK Asha
23: 128/M131
24: M 03/869
RESULTS AND DISCUSSION The frequent occurrences of insufficient varietal discrimination by grow out test and the consequent inability to confirm distinctness encouraged us to 5: A. Abha 9: A. Abijeet(3days 13:old) Nandiseeds were 17: Mruthyunjaya-2 Five sprouted grounded in 21: Ronco investigate complementary methods of describing 6: centrifuge A Megali 10: Pusa Sankranthi US -618 tube byRuby using14:micro pestle18:and 200µl Tris 22: A-32/63 varieties for comparison with conventional methods. extraction buffer (25mM, pH 8.8)19:was added. The 23: 128/ 7: HCl A. Saurab 11: PED 15: Vybhav JK Desi 131 OneMapproach was to use protein electrophoresis. Many agitated thoroughly kept at 8C for 24: M-03/868 8: mixture A. Shresta was 12: PKM-1 16: NS-2535 and 20: JK Asha workers have attempted to characterize crop plants by overnight for protein extraction. Then the mixture was electrophoretic analysis of seed protein. In present study centrifuged at 10,000 rpm for 15 minutes and the attempt was made to characterize 24 tomato cultivars by supernatant was collected. This protein extract was total soluble seed proteins separated by SDS-PAGE. A dissolved in an equal volume of working buffer (0.06 M wide variation was observed in the pattern of protein Tris-HCl, pH 6.8, 2% SDS, 10% glycerol, 0.025% bands of studied cultivars. The cultivars differed in the bromophenol blue) and incubated at 60-70ºC for 10 number of bands, their relative mobility and intensity. minutes, cooled immediately for 5 minutes and The proteins separated on twelve per cent acrylamide centrifuged at 10,000 rpm for 5 minutes. The gel could be distinguished and grouped based on the supernatant was used for loading on to the gel. A standard marker (97.4 KD). By using SDS-PAGE, the current of 1.5 mA per well with a voltage of 80 V was total soluble seed protein could be fractionated into 19 applied until the tracking dye crossed the stacking gel. bands, which showed heterogeneity among different Later the current was increased to 2 mA per well and cultivars. Arka Abha exhibited maximum number of voltage up to 120 V. The electrophoresis was stopped bands (19) followed by Pusa Ruby (18 bands) and Arka when the tracking dye reached the bottom of the Vikas (18 bands) and M-03/868 exhibited least number resolving gel. Then the gel was stained using coomaasie of bands (08). The cultivars cannot be characterized Plate1.1 Total Total soluble seed protein genotypes Plate soluble seed proteinprofiles profilesofoftomato Tomato genotypes
1: A. Alok 2: A. Vikas 3: A. Ahuti 4: A. Ashish
09
www.rjas.info