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

A B S T R A C T

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 sulphate-polyacrylamide 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

Twenty four cultivars of public and private breed

cultivars were used for characterization based on

protein profiles (Table 1).

Table 1 Cultivars of tomato used for varietal

characterization

S.

No.

Cultivar Developed

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

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

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

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methods prescribed by Laemeli (1970) with slight

modifications.

Five sprouted (3days old) seeds were grounded in

centrifuge tube by using micro pestle and 200µl Tris

HCl extraction buffer (25mM, pH 8.8) was added. The

mixture was agitated thoroughly and kept at 8C for

overnight for protein extraction. Then the mixture was

centrifuged at 10,000 rpm for 15 minutes and the

supernatant was collected. This protein extract was

dissolved in an equal volume of working buffer (0.06 M

Tris-HCl, pH 6.8, 2% SDS, 10% glycerol, 0.025%

bromophenol blue) and incubated at 60-70ºC for 10

minutes, cooled immediately for 5 minutes and

centrifuged at 10,000 rpm for 5 minutes. The

supernatant was used for loading on to the gel. A

current of 1.5 mA per well with a voltage of 80 V was

applied until the tracking dye crossed the stacking gel.

Later the current was increased to 2 mA per well and

voltage up to 120 V. The electrophoresis was stopped

when the tracking dye reached the bottom of the

resolving gel. Then the gel was stained using coomaasie

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.

RESULTS AND DISCUSSION

The frequent occurrences of insufficient varietal

discrimination by grow out test and the consequent

inability to confirm distinctness encouraged us to

investigate complementary methods of describing

varieties for comparison with conventional methods.

One approach was to use protein electrophoresis. Many

workers have attempted to characterize crop plants by

electrophoretic analysis of seed protein. In present study

attempt was made to characterize 24 tomato cultivars by

total soluble seed proteins separated by SDS-PAGE. A

wide variation was observed in the pattern of protein

bands of studied cultivars. The cultivars differed in the

number of bands, their relative mobility and intensity.

The proteins separated on twelve per cent acrylamide

gel could be distinguished and grouped based on the

standard marker (97.4 KD). By using SDS-PAGE, the

total soluble seed protein could be fractionated into 19

bands, which showed heterogeneity among different

cultivars. Arka Abha exhibited maximum number of

bands (19) followed by Pusa Ruby (18 bands) and Arka

Vikas (18 bands) and M-03/868 exhibited least number

of bands (08). The cultivars cannot be characterized

Vishwanath et al.

Plate 1. Total soluble seed protein profiles of Tomato genotypes

M 1 2 3 4 5 6 7 8 9 10 11 12

M 13 14 15 16 17 18 19 20 21 22 23 24

24: M-03/868 20: JK Asha16: NS-253512: PKM-18: A. Shresta4: A. Ashish

23: 128/ M 13119: JK Desi15: Vybhav11: PED7: A. Saurab3: A. Ahuti

22: A-32/6318: US -61814: Sankranthi10: Pusa Ruby6: A Megali2: A. Vikas

21: Ronco17: Mruthyunjaya-213: Nandi9: A. Abijeet5: A. Abha1: A. Alok

Plate 1 Total soluble seed protein profiles of tomato genotypes

Rm Value

0.000

0.079

0159

0.239

0.318

0.398

0.478

0.557

0.637

0.717

0.796

0.876

0.956

M 1 2 3 4 5 6 7 8 9 10 11 12

12

3

45

678

910

1112

13

14

15

16

17

18

19

Region A

Region B

Region C

Region D

Region E

Region F

Region G

14: Sankranthi11: PED8: A. Shresta5: A. Abha2: A. Vikas

13: Nandi10: P. Ruby7: A. Saurab4: A. Ashish1: A. Alok

15: Vibhav12: PKM-19: A. Abijeet6: A Megali3: A. AhutiM: Marker

Fig. 1. Zymograms of total soluble proteins of tomato cultivars

97.4 KD

66.0 KD

43.0 KD

29.0 KD

20.0 KD

14.3 KD

Rm Value

0.000

0.077

0155

0.232

0.310

0.387

0.301

0.542

0.620

0.697

0.775

0.852

0.930

M 13 14 15 16 17 18 19 20 21 22 23 24

123

45

678

910

11

12

13

14

15

16

17

1819

Region A

Region B

Region C

Region D

Region E

Region F

Region G

23: 128/M13120: JK Asha17: Mruthnjaya-214: Sankranthi

22: A-32/6319: JK Desi16: NS-253513: Nandi

24: M 03/86921: Ronco18: US -61815: VibhavM: Marker

Fig. 2. Zymograms of total soluble proteins of tomato cultivars

97.4 KD

66.0 KD

43.0 KD

29.0 KD

20.0 KD

14.3 KD

Fig 1 Zymograms of total soluble protein of tomato cultivars

Fig 2 Zymograms of total soluble protein of tomato cultivars

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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

Table 2 Intensity and relative mobility of total soluble seed proteins of tomato cultivars

Region Band

No.

Rm

value 1 2 3 4 5 6 7 8 9 10 11 12

A

1 0.038 + + + + + + + + + + + +

2 0.054 + ++ ++ ++ ++ ++ + + + ++ ++ ++

3 0.062 + + + + + - - - - + + -

4 0.093 + + + + + + + + + + + +

B

5 0.108 + + + + + + + + + + + +

6 0.116 ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++

7 0.139 + + + + + + + + + + + -

C

8 0.162 ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ + +

9 0.178 + ++ ++ + ++ + + - + + + +

10 0.201 + ++ ++ + ++ ++ + + ++ ++ ++ +

11 0.217 +++ +++ ++ - + + - +++ +++ + - -

12 0.232 - - - - + + - - - + - +

13 0.255 + + + + ++ - - - ++ + + -

D 14 0.325 + + - - + - - - ++ + + +

15 0.356 +++ +++ +++ - +++ +++ +++ +++ +++ +++ - +++

E 16 0.627 +++ +++ +++ ++ +++ +++ +++ + +++ +++ +++ +++

F 17 0.728 +++ +++ +++ ++ +++ +++ - - - +++ + +++

G 18 0.837 - +++ +++ + +++ +++ - - - +++ + +++

19 0.899 ++ ++ ++ +++ +++ +++ - - +++ - - +++

Note: - Absent + Low intensity ++ Medium intensity +++ 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

Region Band

No.

Rm

value 13 14 15 16 17 18 19 20 21 22 23 24

A

1 0.024 + + + + + + + + + + + +

2 0.040 ++ ++ ++ ++ ++ ++ ++ ++ + ++ ++ +

3 0.084 - + + - + - + + - - - -

4 0.088 + + + + + - + + + + + -

B

5 0.096 - + - - - - + + + + + -

6 0.120 ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ +

7 0.136 - + - + + + + + + + + +

C

8 0.152 - + - - - - - - - - - -

9 0.168 + + - ++ ++ ++ ++ ++ ++ + ++ +

10 0.192 + + - ++ - ++ + + ++ + ++ +

11 0.200 ++ + + ++ - ++ ++ ++ + + ++ -

12 0.272 + - + - - - - - - - - -

13 0.296 + - + - - - - - - - - -

D 14 0.424 +++ +++ +++ +++ - +++ ++ +++ +++ +++ +++ ++

E 15 0.736 +++ +++ - +++ + +++ + + +++ - +++ +++

F 16 0.808 +++ +++ +++ +++ - +++ + + +++ +++ +++ +++

17 0.864 ++ - +++ +++ ++ +++ ++ ++ ++ + - -

G 18 0.944 ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ - -

19 0.968 ++ ++ ++ ++ ++ ++ + + ++ ++ - -

Note: - Absent + Low intensity ++ Medium intensity +++ High intensity

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)

Identification of Tomato Varieties through Total Soluble Seed Proteins

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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 16th

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.).

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Identification of Tomato Varieties through Total Soluble Seed Proteins