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Research Journal of Agricultural Sciences 2011, 2(1): 08-12

Identification of Tomato (Lycopersicon esculentum) Varieties through Total Soluble Seed Proteins Vishwanath K, Prasanna K P R, Pallvi H M, Rajendra Prasad S, Ramegowda, Devaraju P J, *Ananthararayanan T V Department of Seed Science and Technology, University of Agricultural Sciences, GKVK, Bangalore-65, Karnataka, India *Division of Plant Genetic Recourses, Indian Institute of Horticultural Research, Hesaraghatta, Bangalore, Karnataka, India e-mail: vishwakoti@gmail.com

ABSTRACT Varietal development and its identification is one of the most important aspects of seed industry and seed trade. Due to continuous breeding programme by using elite lines, it has become difficult to identify and characterize these varieties on the basis of morphological characters alone. This has led to the exploration of new stable characters including genetic makeup to be used as markers for varietal identification. The present study includes the identification of different varieties (24) of tomato on the basis of their protein profile. Protein was extracted from sprouted seeds using electrophoresied on sodium dodecyl sulphatepolyacrylamide gel electrophoresis (SDS-PAGE). After fixation and coomaasie blue staining, all the varieties were identified according to their differences in the banding pattern and staining intensities. Key words: Lycopersicon esculantum, Seed proteins, Markers, Morphological characters Variety development is an important part of the plant breeding and the identification of these varieties by different parameters plays an important role in seed industry and seed trade. However, with the increase in the number of varieties of each crop, it is difficult to distinguish the varieties on the basis of morphological characters alone. This has led to the development of the new stable parameters such as use of their genetic material (nucleic acids and proteins) as a tool for varietal identification. Electrophoresis is a process of separation of different biomolecules under the influence of electric field and has been successfully applied for the identification of varieties. Sample characterized by different proteins bands are considered to differ genetically while sample having the same protein bands may be of the same variety (Payne 1987). Numerous studies have already been conducted for examining the protein pattern in important crops for varietal identification (Cooke 1984, Gilliland 1989). In 1986, ISTA adopted a standard reference method of PAGE for identification of varieties of wheat and barley into its international rules, involving separation of gliadin from wheat and hordein from barley (Cooper 1987). A number of new varieties are available in tomato and are very difficult to characterize based on morphological characters based on morphological characters due to continuous breeding programme.

MATERIALS AND METHODS

Table 1 Cultivars of tomato used for varietal characterization S. Cultivar Developed No. institute/ company 1. Arka Alok IIHR 2. Arka Vikas IIHR 3. Arka Ahuti IIHR 4. Arka Ashish IIHR 5. Arka Abha IIHR 6. Arka Megali IIHR 7. Arka Saurab IIHR 8. Arka Shresta* IIHR 9. Arka Abijeet* IIHR 10. Pusa Ruby IARI 11. Pusa Early Dwarf IARI 12. PKM–1 TNAU 13. Nandi UASB 14. Sankranthi UASB 15. Vybhav UASB 16. NS–2535* Namdhari Seeds 17. Mruthyunjaya–2* Sasya Seeds 18. US–618 U.S. Agriseeds* 19. J.K. Desi J.K. Agrigenetics* 20. J.K. Asha* J.K. Agrigenetics 21. Ronco* Bejo Seeds 22. A-32/ 63(Female) Indosem Seeds 23. 128/ M 131(Male) Indosem Seeds 24. M-03/ 868* (F1) Indosem Seeds

Twenty four cultivars of public and private breed cultivars were used for characterization based on protein profiles (Table 1).

Electrophoretic technique of Total soluble seed proteins SDS-PAGE of total soluble seed proteins was carried out by using 15 per cent gels according to the

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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.

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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

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14

15

16 17

18

19

20

21

22

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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

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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 8C 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

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Identification of Tomato Varieties through Total Soluble Seed Proteins Table 2 Intensity and relative mobility of total soluble seed proteins of tomato cultivars Band Rm Region 1 2 3 4 5 6 7 8 No. value + + + + + + + + 1 0.038 + ++ ++ ++ ++ ++ + + 2 0.054 A + + + + + 3 0.062 + + + + + + + + 4 0.093 + + + + + + + + 5 0.108 ++ ++ ++ ++ ++ ++ ++ ++ B 6 0.116 + + + + + + + + 7 0.139 ++ ++ ++ ++ ++ ++ ++ ++ 8 0.162 + ++ ++ + ++ + + 9 0.178 + ++ ++ + ++ ++ + + 10 0.201 C +++ +++ ++ + + +++ 11 0.217 + + 12 0.232 + + + + ++ 13 0.255 + + + 14 0.325 D +++ +++ +++ +++ +++ +++ +++ 15 0.356 +++ +++ +++ ++ +++ +++ +++ + E 16 0.627 +++ +++ +++ ++ +++ +++ F 17 0.728 +++ +++ + +++ +++ 18 0.837 G ++ ++ ++ +++ +++ +++ 19 0.899 Note: - Absent

+ Low intensity

++ Medium intensity

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10

11

12

+ + + + ++ + ++ + ++ +++ ++ ++ +++ +++ +++

+ ++ + + + ++ + ++ + ++ + + + + +++ +++ +++ +++ -

+ ++ + + + ++ + + + ++ + + +++ + + -

+ ++ + + ++ + + + + + +++ +++ +++ +++ +++

+++ High intensity

1. Arka Alok, 2. Arka Vikas, 3. Arka Ahuti, 4. Arka Ashish, 5. Arka Abha, 6. Arka Megali, 7. Arka Saurab, 8. Arka Shresta, 9. Arka Abijeet, 10. Pusa Ruby, 11. Pusa Early Dwarf, 12. PKM–1 Table 3 Intensity and relative mobility of total soluble seed proteins of tomato cultivars Band Rm Region 13 14 15 16 17 18 19 20 No. value + + + + + + + + 1 0.024 ++ ++ ++ ++ ++ ++ ++ ++ 2 0.040 A + + + + + 3 0.084 + + + + + + + 4 0.088 + + + 5 0.096 ++ ++ ++ ++ ++ ++ ++ ++ B 6 0.120 + + + + + + 7 0.136 + 8 0.152 + + ++ ++ ++ ++ ++ 9 0.168 + + ++ ++ + + 10 0.192 C ++ + + ++ ++ ++ ++ 11 0.200 + + 12 0.272 + + 13 0.296 +++ +++ +++ +++ +++ ++ +++ D 14 0.424 +++ +++ +++ + +++ + + E 15 0.736 +++ +++ +++ +++ +++ + + 16 0.808 F ++ +++ +++ ++ +++ ++ ++ 17 0.864 ++ ++ ++ ++ ++ ++ ++ ++ 18 0.944 G ++ ++ ++ ++ ++ ++ + + 19 0.968 Note: - Absent + Low intensity ++ Medium intensity +++ High intensity

21

22

23

24

+ + + + ++ + ++ ++ + +++ +++ +++ ++ ++ ++

+ ++ + + ++ + + + + +++ +++ + ++ ++

+ ++ + + ++ + ++ ++ ++ +++ +++ +++ -

+ + + + + + ++ +++ +++ -

13. Nandi, 14. Sankranthi, 15. Vybhav, 16. NS–2535, 17. Mruthyunjaya–2, 18. US–618, 19. J.K. Desi, 20. J.K. Asha, 21. Ronco, 22. A-32/ 63 (Female), 23. 128/ M 131(Male), 24. M-03/ 868 (F1) based on number of bands, but they could be differentiated clearly by their banding intensity and relative mobility. When compared, each cultivar had its

unique profile which was different from other cultivars. Even though mobility was different between two groups (1-12 and 13-24 cultivars), number of bands remained

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Vishwanath et al. the same (19 bands in each group). Hence, profiles of all the cultivars were compared in the present study (Table 2, 3). In case of 1-12 cultivars, 10 bands were polymorphic (3, 7, 11, 12, 13, 14, 15, 17, 18 and 19) and eight bands were monomorphic (1, 2, 4, 5, 6, 8, 9, 10 and 16). While in case of 13-24 cultivars, except band numbers 1, 2 and 6 all the bands were polymorphic (Plate 1). Entire protein banding profile was divided in to seven regions (A to G) based on its decreasing molecular weight by comparing with standard protein marker (See ‘M’ in the Plate 4, 26). The seed protein weight of tomato ranged between 98 KD to 22 KD and relative mobility ranged between 0.024 to 0.968. (Fig 1, 2; Plate 1, 2). In Region A (> 97.4 KD; Phosphorylase b) both groups showed four bands, cultivar Arka Alok, US- 618 and Ronco showed unique profiles which were absent in any of cultivars and hence this region could be used to identify these three cultivars. Region B (66.0 to 97.4 KD; Bovine serum albumin) showed three bands in both the groups. Maximum number of cultivars viz Arka Alok, Arka Vikas, Arka Ahuti, Arka Abha, Arka Megali, Arka Saurab, Arka Shresta, Arka Abijeet, Pusa Ruby, Pusa Early Dwarf, Sankranthi, JK Desi, JK Asha, Ronco, A 32/63, 128/M 131 showed similar banding patterns. Only M-03/868 and PKM-1 showed unique profiles which were distinct from rest of the cultivars. Hence, this region could be useful to identify these two cultivars. In Region C (43.0 to 66.0 KD; Ovalbumin) maximum numbers of bands were appeared in both the groups. Highest number of cultivars showed unique banding pattern in this region. Arka Alok, Arka Vikas, Arka Ahuti, Arka Ashish, Arka Abha, Arka Megali, Arka Saurab, Arka Shresta, Arka Abijeet, Pusa Ruby, Pusa Early Dwarf, PKM-1, Nandi, Sankranthi, Vybav, Mruthunjaya-2, Ronco, A 32/63 and M-03/868 showed their unique protein profiles which were distinct from other cultivars based on presence or intensity of band. All these nineteen cultivars could be differentiated in this region. In Region D (29.0 to 43.0 KD; Carbonic anhydrase), group 1 (1-12 cultivars) showed two bands. Cultivars Arka Ashish was distinct by absence of both the bands in this region and which indicated that these

bands might be considered as specific marker (negative marker) for this cultivar compared with the others. While, Pusa Early Dwarf could also identified by absence of 15th band (Rm: 0.356). However, rest of the cultivars showed similar banding pattern. In group 2 (13-24 cultivars) only one band was observed in this region. Mruthunjaya-2 could be identified from other cultivars by the absence of band in this region. In Region E (20.0 to 29.0 KD; Soybean Trypsin Inhibitor), only one band was observed in both the groups. Only cultivar Arka Ashish could be identified due to its medium intensity band and rest of the cultivars showed either absence or presence of band with light/ dark intensity. In Region F (14.3 to 29.0 KD; Lysozyme), in group 2 (1-12 cultivars) only one band was observed in this region. Cultivar Pusa Early Dwarf was distinct from other cultivars by it light intensity band and Arka Ashish by its medium intensity band. In group 2 (13-24 cultivars) only Mruthunjaya-2 was distinct from all the cultivars by absence of 16 th band (Rm: 0.808) and could be used as negative marker to identify the same. In Region G (< 14.3 KD; Daltons) both groups showed two bands. Most of the studied cultivars showed similar banding pattern between each other. Only Arka Alok, Arka Ashish, Arka Abijeet, Pusa Ruby, Pusa Early Dwarf showed distinct banding pattern based on the presence or intensity. This region could be useful in identification of these five cultivars. On the basis of presence or absence and intensity of bands all the cultivars were distinguished. In conclusion, this technique was able to identify all the twenty four cultivars and can be employed effectively for identification of these tomato cultivars. Many scientists successfully showed protein electrophoresis as powerful tool to identify the crop plants (Drzewiecki 1990 in pea, Chakraborti et al. 1992 in tomato, Mudzana et al. 1995 in faba bean, Bonfitto et al. 1999 in melon, Mennella et al. 1999 in brinjal, Lucchese et al. 1999 in pepper, Wang et al. 2000 in tomato, Ahokas 2002 in barley, oat, wheat, peas and turnip, Yan-Min et al. 2003 in maize, capsicum and rice, Goyal and Sharma 2003 in cluster bean, Rahman et al. 2004 in Brassica rapa, Rani and Rathore 2006 in Brassica juncea L.).

LITERATURE CITED Ahokas H. 2002. Methanol precipitation using universal protein fractionation method for cultivar identification in cereal, pea and Brassica seeds. Seed Science and Technology 30: 437-449. Bonfitto R, Galleschi L, Macchia M, Saviozzi F and Navari-Izzo. 1999. Identification of melon cultivars by gel and capillary electrophoresis. Seed Science and Technology 27: 779-783. Chakraborti A K, Das A K and Chattopadhyay. 1992. Identification of some Indian tomato cultivars by polyacrylamide gel electrophoresis of seed proteins. Seed Research 12(1): 10-13. Cooke R J. 1984. The use of SDS polyacrylamide gel electrophoresis in varietal identification. Biochemical tests for cultivar identification. Proceedings of an ISTA symposium held at the National Institute of Agricultural Botany, Cambridge, UK, on 12-15 September, 1983, pp107-108. Cooper S R. 1987. Report of the rules committee 1983-1986. Seed Science and Technology 15: 555-575.

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Identification of Tomato Varieties through Total Soluble Seed Proteins Drzewiecki J. 1990. Genuineness of pea seeds in the basis of electrophoretic investigations. Bulletin of Plant Breeding and Acclimatization Institute 36: 175-179. Gilliland T J. 1989. Electrophoresis of sexually and vegetatively propagated cultivers of allogamous species. Plant Varieties and Seed 2: 15-25. Goyal A K, and Baijal B D. 1980. Effect of GA and RNase activity and RNA contents at early seedling stage in certain rice (Oryza sativa L.) genotypes. Indian Journal of Agricultural Research 14(2): 111-114. Higgins J, Evans J L and Reed P J. 1981. Classification of western Europen cultivars of Vicia faba L. Journal of National Institute of Agricultural Botany 15: 480-487. Laemeli U K. 1970. Sodium Dodecyl Sulphate-Poly Acrylamide Gel Electrophoresis. Nature 227: 680. Lucchese C, Dinelli G, Miggiano A and Lovato. 1999. Identification of pepper (Capsicum spp) cultivars by field and electrophoresis tests. Seed Science and Technology 27: 37-47. Meennella G, Bianchi M, Sanaja V O, Tonini A and Magnifico V. 1999. Biochemical and morphological/ physiological characterization of Brassica oleracea L. Sement Elette 41(6): 9-16. Mudzana G, Pickett A A, Jarman, R J, Cooke R J and Keefe P D. 1995. Variety discrimination in faba beans (Vicia faba L.) an integrated approach. Plant Varieties and Seeds 8: 135-145. Payne R C. 1987. Seed and cultivar identification. Seed Science and Technology 15: 641-644. Rahman M M, Yutak Hirata and Shah-e-Alam. 2004. Genetic variation within Brassica rapa cultivars using SDSPAGE for seed protein and isozyme analysis. Journal of Biological Sciences 4(2): 239-242. Rani M and Rathore R K S. 2006. Identification of nine cultivars of Brassica juncea L. Czern. and Coss. Based on seed protein. Proceedings of XII National Seed Seminar, pp179. Wang X F, Konoblauch R and Leist N. 2000. Varietial discrimination of tomato (Lycopersicon esculentum Mill.) by ultra thin-layer isoerlectric focusing of seed protein. Seed Science and Technology 28: 521-526. Yan-Min, Wang Xiaofeng, Yan M and Wand X F. 2003. Varietal identification and genetic purity testing of hybrid maize, capsicum and rice in laboratory. Journal of South China Agricultural University 24(2): 6-8.

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19 identification of tomato (lycopersicon esculentum) varieties through total soluble seed proteins