International Journal of Agronomy and Agricultural Research (IJAAR)

ISSN: 2223-7054 (Print) 2225-3610 (Online)

http://www.innspub.net
Vol. 5, No. 5, p. 161-169, 2014
OPEN ACCESS

RESEARCH PAPER

Standardization of high efficient and rapid regeneration

protocol for Agrobacterium mediated transformation of tomato

(Solanum lycopersicum L.) cv. Pusa Ruby, Vaibhav and Arka

Meghali.
S.V. Madhu1*, D.L. Savithramma2
1

Department of Biotechnology, Karpagam University, Coimbatore, Tamil Nadu, India

Department of Genetics and Plant Breeding, University of Agricultural Sciences, GKVK,

Bangalore, Karnataka, India

Article published on November 23, 2014
Key words: BAP-Benzyl Amino Purine, AS-Adenine Sulphate, TDZ-Thidiazuron, Zn-Zeatin, RM-Regeneration
Media, IAA- Indole-3-Acetic Acid and MS- Murashige and Skoog.

Abstract
In the present investigation, three tomato cultivars Pusa Ruby, Vaibhav and Arkamegali (Solanum lycopersicum
L.) were selected for multiple shoot induction. The explants cotyledon, hypocotyl and epicotyl of all the cultivars
of tomato were cultured on MS medium fortified with different concentrations of BAP (1.0 to 4.0 mg/l), TDZ (1.0
to 4.0 mg/l), Zeatin (0.5 to 1.0 mg/l), AS (5.0 to 20 mg/l) and 0.1 mg/l IAA . Adventitious shoot buds were
induced at the cut ends of the explants after three weeks of culture. The shoots were induced from cotyledon,
hypocotyl and epicotyl explants on different concentrations of growth regulators (RM 1 to RM 10). Among ten
media combinations tested, RM 9 was the best combination for induction of shoots from cotyledon, hypocotyl
and epicotyl explants compared to RM 2. The highest number of shoots was induced on 0.5 mg/l Zn + 10 mg/l AS
+ 0.1 mg/l IAA (RM 9) followed by 2.0 mg/l BAP + 10 mg/l AS + 0.1mg/l IAA (RM 2). Among the three genotypes
tested Pusa ruby showed highest number of shoots per explant followed by Vaibhav and Arkamegali. Highest
number of adventitious shoots was induced from cotyledonory compared to hypocotyl and epicotyl explants in
Pusa ruby and Arka meghali, where as Vaibhav induced highest shoots from epicotyl explants. Rooting was
achieved on MS medium supplemented with 0.1 mg/l IAA in all the genotypes. Hence the plant regeneration was
found to be influenced by the genotype, different explants with hormonal combinations in the media.
* Corresponding

Author: S. V. Madhu madhugowda83@gmail.com

Madhu and Savithramma

Page 161

Introduction

various explants by using different growth hormones.

Tomato is one of the most important vegetable crops

with highest production and consumption worldwide.

Material and methods

Its a perennial plant belonging to Solanaceae family

Plant material and culture conditions

grown in tropical, sub-tropical and temperate regions

Seeds of high yielding tomato cultivars from three

(Atherton & Rudich, 1986). It ranks third among

different institutes, Pusa Ruby were obtained from

vegetable crops (next to potato and sweet potato) with

the Indian Agricultural Research Institute, New

an annual production of 1617 million metric tonnes

Delhi, and Vaibhav from University of Agricultural

(FAO statistical database, 2012).

Sciences Bangalore and Arka Meghali from Indian

Institute of Horticulture Research respectively. Seeds

The production and yield of tomato is adversely

were sterilized with two different sterilants; sodium

affected by various abiotic factors such as salinity,

hypochlorite (1- 4 per cent) and mercuric chloride

drought, floods, heat and cold stress and biotic factors

(0.1 - 0.4 per cent) were tried. Seeds were treated

like pests and diseases (Bhatnagar-Mathur et al.,

with 5 per cent teepol detergent for 15 minutes,

2008). To tackle these problems conventional

soaked them under running tap water for 30-45

breeding methods are the best approaches, but time

minutes and washed with sterile water twice. Then

consuming.

with

surface sterilized for 3 minutes in 70 per cent ethanol

biotechnological tools for transfer of desired genes to

and washed with sterile water followed by sterilizing

elite varieties without modifying the genetic makeup

in 4 per cent sodium hypochlorite solution along with

will lead to development of high yielding varieties.

4-5 drops of Tween-20 for 10 minutes, followed by

However, transfer of genes depends on rapid and

three rinses with sterile water. The surface sterilized

efficient in-vitro regeneration protocols.

seeds were blot dried and 25 to 30 sterilized seeds

Hence,

breeding

associated

were inoculated into culture bottles containing half

Mass multiplication of tomato has been attempted

strength MS medium with 1.5 per cent sucrose, gelled

using shoot tip culture (Izadpanah and Khosh Khui,

with 0.7 per cent agar having pH 5.8. Culture bottles

1992),

and

were incubated initially for two days in dark at 25 0 C

Alderson, 2002), direct organogenesis from intact

and later exposed to light intensity of 2000 lux with

explants (Ichimura and Oda, 1998). Chaudhry et al.,

photoperiod of 16/8 hours of light and dark cycles.

somatic

embryogenesis

(Kaprakis

2007 reported the effect of genotype, explants and

plant growth hormones on callus proliferation and

Culture media, hormones

regeneration of three tomato cultivars. Shoot apex,

MS-medium

nodal segments and root segments were successfully

supplemented with various hormonal combinations.

used for callus induction and regeneration (Jatoi et

Benzylaminopurine (BAP-1.0 mg/l to 4.0 mg/l),

al., 2001). Similarly, combination of IAA + BAP for

Thidiazuron (TDZ-1.0 mg/l to 4.0 mg/l), Zeatin (Zn-

shoot regeneration from different explants of tomato

0.5 mg/l to 2.0 mg/l), Adenine sulphate (5 to 20

was found to be more effective (Gunay and Rao, 1980;

mg/l), and Indole-3-acetic acid (lAA-0.1 mg/l) labeled

Kurtz and Lineberger, 1983; Selvi and Khader, 1993;

from RM 1 to RM 10 were used for shoot and root

Villiers et al., 1993; Duzyman et al., 1994; Chen et al.,

regeneration (Table 1).

(Murashige

and

Skoog,

1962)

1999., Chandel and Katiyar, 2000;). None of the

protocols are efficient and reliable, hence an efficient

Explant selection

regeneration protocol is required for a given plant

Three types of explants, Cotyledons, hypocotyls and

species before transformation (Potrykus et al., 1995).

epicotyls from ten days old in-vitro grown seedlings

In this regard, an attempt was made to reveal the

were used for preparation of explants. Cotyledons

factors

excised at the proximal petiole edge and 1 millimeter

influencing

high

efficient

and

regeneration protocol for tomato cultivars using

rapid

width transverse strips of tissue prepared from the

Madhu and Savithramma

Page 162

proximal region of the cotyledons were incubated

primordia per explants, number of shoots per explant

with the adaxial side on shoot induction medium.

and shoot length. The regenerating explants were

Transverse hypocotyl discs of 0.51.0 millimeter

observed and revived onto fresh medium at every 15

thick were sliced from a 10 millimeter hypocotyl

days.

segment cut immediately below the cotyledonary

node and placed with their basal cut. Similarly

Result and discussion

epicotyls were cut into two pieces and cultured on the

The plant tissues cultured under in-vitro condition

regeneration medium. Each bottle was inoculated

are greatly influenced by different components of

with 10 explants of cotyledons, hypocotyls and

culture media composition and concentrations of

epicotyls separately and incubated in growth chamber

growth

with 16/8 hour photoperiod with light intensity of

differentiation of the explant. Hence evaluation of

2000 lux. The observations were recorded for

different hormones in combinations is necessary

parameters

towards better regeneration of tomato.

like

regeneration

percentage,

shoot

hormones

in

the

morphogenetic

Table 1. Combination of various growth hormones used in association with MS basal medium for
standardization of regeneration in tomato cultivars.
Media code

Nutrient medium

BAP (mg/L)

TDZ (mg/L)

Adenine sulphate ( mg/L)

ZR (mg/L)

IAA (mg/L)

RM 1

MS

1.0

0.1

RM 2

MS

2.0

10

0.1

RM 3

MS

3.0

15

0.1

RM 4

MS

4.0

20

RM 5

MS

1.0

0.1

RM 6

MS

2.0

10

0.1

RM 7

MS

3.0

15

0.1

RM 8

MS

4.0

20

0.1

RM 9

MS

10

0.5

0.1

RM 10

MS

15

1.0

0.1

0.1

Table 2. Percentage of regeneration in three cultivars of tomato in MS medium supplemented with growth
regulators.
Media

Arka Meghali
Hypocotyl

RM 1

2.880.03

Vaibhav
Epicotyl

Cotyledon

2.620.03 41.550.05

Mean
15.68

Hypocotyl
d

Epicotyl

Pusa Ruby

Cotyledon

5.070.03 32.850.05

7.140.04

Mean
15.02

Hypocotyl
64.560.03

53.950.03

76.490.04

91.590.04

90.720.02

95.850.04

93.55

83.563.27

80.173.33

89.542.67

84.42

69.824.47

59.984.22

73.795.20

50.934.42

30.414.42

56.755.54

46.03

86.503.05

84.722.67

86.503.05

85.91

74.823.06

79.625.16

80.174.22

8.393.33

4.573.51

7.575.77

6.84

92.700.05

92.850.05

96.950.03

96.95

88.790.04

85.140.04

94.960.02

91.18 70.080.03 90.630.08 90.320.03

83.68

RM 3

80.173.33 68.875.33 80.723.06

76.59

51.524.47 67.924.47 52.243.33

57.23

RM 4

40.364.66 27.293.27 52.243.27

30.414.42 50.123.33

RM 5

39.96

4.573.33

3.373.27 16.985.33

8.31

RM 6

81.854.47

71.785.33 84.722.67

79.45

RM 7

77.984.67 68.873.27 74.304.27

RM 8

3.373.51

3.373.51

3.80

66.074.27 74.304.27 58.345.96

66.24

61.664.67 70.793.33 54.453.06

73.72

7.084.71

4.61

RM 9

92.550.05 90.240.03 95.910.03

93.23

RM 10

90.340.04 89.030.08 92.790.04

1.842.66

2.57

80.120.03 92.730.03 90.340.02

87.73

77.550.05 85.300.02 83.550.05

90.72

3.373.51

62.30

2.493.33

1.842.94

5.976.29

39.43

3.614.47

82.13

Mean

56.33

51.16

63.84

SEm

0.01

0.01

0.02

0.01

0.02

0.02

0.02

0.01

0.02

CD

0.07

0.05

0.09

0.06

0.09

0.08

0.10

0.07

0.11

0.04

0.04

0.05

0.03

0.05

0.04

0.05

0.04

0.07

57.1

44.40

56.93

47.87

Mean

90.240.04 88.420.03 94.890.04

37.765.16

Cotyledon

RM 2

Mean
Epicotyl

49.73

71.32

65.88

75.96

65.00

31.90

89.47

67.86

78.20

f
j

72.74

49.09

19.38
77.20

d
f

71.41

4.67

92.49

87.49

89.63

71.05

(1%)
CV

Data - Mean SE of 10 replications. The data were taken 3 weeks after culture. The values followed by same
letters are not significantly different @ P= 0.05.

Madhu and Savithramma

Page 163

The results indicated that the seeds treated with

cent

mercuric chloride had low seed germination rate, long

germination rate to 95-100%, devoid of bacterial and

sodium

hypochlorite

germinating time and un-uniform germination, where

fungal contaminations. Similar results were obtained

as the seeds treated with sodium hypochlorite had

by Gubis (2004) with explant immersion time of 15

higher rate of seed germination, short germination

min in 4 per cent sodium hypochlorite in tomato

time and uniform germination, Similar results were

cultivars Hana and Premium. Similarly, Javier

observed by Gubis et al., 2003. In the present study

(2001) treated the explants with one per cent sodium

the additional step in the explant surface sterilization

hypochlorite

with 70 per cent alcohol helped in removal of

regeneration in tomato cultivars Ailsa craig,

microorganisms. The addition of tween-20 into 4 per

UC82B and Rutgers.

for

20

min

has

increased

and

obtained

the

100%

Table 3. Shoot primordia per regenerating explants in three cultivars of tomato in MS medium supplemented
with growth regulators.
Media

Arka Meghali

Vaibhav

Hypocotyl

Epicotyl

Cotyledon

Mean

RM1

2.440.04

1.860.03

5.680.03

3.33

RM 2

11.420.03

8.780.03

12.710.05

RM 3

7.160.24

6.640.30

11.120.44

8.31

RM 4

3.820.93

3.600.84

9.270.26

5.56

RM 5

2.400.81

1.780.53

5.190.94

3.123

RM 6

7.760.24

7.170.20

10.980.21

8.64

RM 7

5.031.44

5.800.34

7.930.33

6.26

RM 8

1.640.42

1.600.37

1.910.35

RM 9

11.850.05

10.080.03 17.220.03

RM 10

10.000.05 8.040.04

12.950.01

10.97

e
g
h

Pusa Ruby

Hypocotyl Epicotyl

Cotyledon

Mean

2.040.03 5.040.03

2.390.04

3.16

8.590.04 11.840.04

9.840.03

5.540.26 7.560.26

6.860.23

6.65

3.280.55 6.540.30

3.260.57

4.36

1.350.47

2.400.81

2.381.27

Mean

Hypocotyl

Epicotyl

Cotyledon

Mean

7.770.03

7.590.03

9.230.03

8.20

17.880.03

14.280.03 20.420.03 17.53b

12.86

15.670.33

13.320.49

19.770.32

16.25

10.40

13.670.30

10.060.31

17.480.26

13.74

8.070.23

4.780.86

10.870.27

7.91

d
b

10.09

2.04

10.350.33

17.370.33

13.36

5.690.70 5.931.39

4.691.26

11.080.23

7.670.21

15.350.42

11.37

1.200.21

1.340.26

4.801.30

2.721.07

4.551.88

1.72

8.790.04 15.920.03

11.160.02

5.820.03 11.760.04

7.040.02

13.05

10.33

5.44
1.39

10.96

0.01

0.01

0.01

CD

0.05

0.05

0.10

0.08

0.06

0.07

0.06

0.07

0.05

0.28

0.36

0.37

0.18

0.17

0.23

0.23

0.36

0.32

5.85

0.01

7.93

2.38

8.21

19.280.03 14.34c

0.01

4.82

7.69
j

15.16

11.850.03

0.02

7.01

11.900.05

0.02

9.34

4.022

10.55

20.480.03 16.090.04 24.890.04 20.49

11.96

0.01

5.46

0.01

6.23

4.36

12.360.33

1.640.42

7.88

6.20

12.31

9.82

15.76

10.360.30 9.64c

SEm

6.860.23 11.690.53

Mean

4.89

12.6

(1%)
CV

Data - Mean SE of 10 replications. The data were taken 3 weeks after culture. The values followed by same
letters are not significantly different @ P= 0.05.
Effects of different hormonal combinations in tomato

sulphate (AS) 10 mg/l+ IAA 0.1 mg/l) with 95.85 %

shoot differentiation

and 94.89 % respectively , where as the variety

Murashige and Skoog medium supplemented with

Vaibhav showed maximum differentiation rate in

various cytokines and auxins in balanced proportion

epicotyl explant on RM 9 (92.72%) followed by RM 2

play an important role in the development of shoot

(90.63%). The induced primary shoot buds were

primordia, shoot elongation, number of shoots and

strong, less blank, less deformed. Average budding

shoot length.

number of 5.5 per explant was observed. The

combination of BAP + AS and IAA also induced

The regeneration frequency was studied using

primary shoot buds, but the number of induced

different explants, combinations of hormones in all

shoots per explant was less than that of the

the three varieties. The cotyledonory differentiation

combination of Zn + AS and IAA. It is interesting that

rate was highest in Pusa Ruby (96.95%) followed by

the combination of BAP + AS and IAA showed best

Arka Meghali (95.91%) in RM 9 (MS + Zn 0.5mg/l+

callus growth compared to other combinations with

Adenine sulphate (AS) 10 mg/l+ IAA 0.1mg/l)

respect to size and structure of the calli (Table-2). The

followed by RM 2 (MS + BAP 2mg/l+ Adenine

size of the calli was twofold larger than others, the

Madhu and Savithramma

Page 164

calli were green, swollen and loose with out bud

The MS medium supplemented with hormone Zeatin

formation. The results are in confirmation with Ling

was

et al 1998, Gubis et al., 2003 as they used 8 to 10 days

regeneration (NoGueira et al., 2001).

the

most

efficient

medium

for

tomato

old cotyledons, which were superior to other explants.

Table 4. Number of shoots per regenerating explants in three cultivars of tomato in MS medium supplemented
with growth regulators.
Media

Arka Meghali

Vaibhav

Pusa Ruby

Hypocotyl

Epicotyl

Cotyledon

Mean

Hypocotyl

Epicotyl

Cotyledon

Mean

RM 1

3.120.01

3.050.03

4.290.10

3.49

2.97

RM 2

5.630.05

4.390.01

6.160.03

5.75

RM 3

4.140.24

3.370.16

5.140.24

4.22

RM 4

2.520.22

2.630.21

3.730.24

RM 5

3.050.32

1.580.21

4.000.31

Epicotyl

Cotyledon

Mean

5.010.06

3.870.06

6.840.01

5.24

9.660.07

7.390.02

11.730.47

9.59

8.680.21

7.690.15

10.880.23

9.08

7.060.23

5.460.22

8.970.25

4.720.29

3.540.22

6.150.24

2.87

RM 6

5.050.23

3.680.29

6.450.26

5.06

4.81

6.860.23

5.810.34

8.850.31

7.18

RM 7

4.230.26

3.170.30

5.950.25

4.45

RM 8

1.320.16

1.320.16

1.580.21

6.39

4.88

1.41

RM 9

4.470.04

3.330.01

7.220.01

4.47

RM 10

3.980.02

4.380.06

5.600.03

2.070.01

4.770.08

2.070.02

4.630.04

5.840.02

5.060.05

5.11

3.370.16

4.550.22

3.730.24

3.88

3.460.16

3.700.29

2.900.25

2.050.24

3.340.34

2.630.21

2.67

3.020.23

5.650.26

4.330.26

4.33

2.810.23

4.740.24

3.840.23

3.80

1.230.15

1.580.21

1.280.22

1.37

5.920.06

7.750.08

5.860.04

6.51

5.390.02

5.630.08

4.160.02

2.96

4.84

Mean

Hypocotyl

3.35

4.26

10.990.12

8.660.04

12.820.06

10.82

7.620.03

6.680.02

10.960.14

5.13

3.17

4.72

3.52

6.87

5.54

8.97

0.02
0.09

0.02
0.08

0.02
0.10

0.04
0.19

0.03
0.13

0.02
0.09

0.02
0.09

2.70

0.99

0.55

0.83

0.94

1.36

1.00

0.87

1.06

3.70

5.52

5.320.30

4.80

3.45

8.460.26

0.04
0.19

3.100.24

2.77

3.80

8.42
7.12

4.49

4.550.22

0.13
0.66

5.73

4.350.22

3.54

6.81

6.140.29

SEm
CD
(1%)
CV

3.90

Mean

7.16

d
f

2.34
a

7.26
6.13

Mean number of shoots per regenerating explants, presented as mean SE of 10 replications. Data was recorded
at 5 weeks of culture. The values followed by same letters are not significantly different @ P= 0.05.
Table 5. Shoot length per regenerating explants in three cultivars of tomato in MS medium supplemented with
growth regulators.
Media

Arka Meghali

Vaibhav

Pusa Ruby

Hypocotyl

Epicotyl

Cotyledon

Mean
(cm)

Hypocotyl

Epicotyl

Cotyledon

Mean
(cm)

RM 1

5.060.01

5.250.01

3.570.01

4.63

5.950.01

7.800.00

7.85O.01

7.87

RM 2

4.510.04

5.510.02

6.350.07

4.820.00

9.810.02

5.770.00

RM 3

5.540.10

2.880.03

2.600.12

3.640.10

7.310.08

5.270.08

RM 4

2.450.11

3.350.10

0.630.03

2.14

2.230.11

4.760.09

3.150.09

3.38

RM 5

2.920.10

3.42 0.13

2.700.12

3.01

6.390.19

8.800.10

7.860.07

8.02

RM 6

2.060.12

2.67 0.09

1.740.12

2.16

4.500.08

8.200.11

5.980.08

6.23

RM 7

1.270.10

1.880.11

0.750.06

1.30

4.510.09

6.820.17

5.690.10

5.67

RM 8

0.690.03

1.06 0.06

0.550.03

3.760.07

5.630.10

4.280.04

RM 9

5.570.02

4.550.03

7.710.03

7.530.01

9.860.01

8.790.02

RM 10

4.510.01

5.880.04

3.040.03

6.800.02

9.430.05

8.660.01

Mean
SEm
CD
(1%)
CV

3.30
0.01
0.03

4.31
0.01
0.07

2.23
0.01
0.03

0.16

0.32

0.22

4.30
5.16

0.77
5.61

4.48
3.28

4.97
0.01
0.05

7.84
0.02
0.10

6.30
0.01
0.04

0.65

0.89

0.32

Mean

Hypocotyl

Epicotyl

Cotyledon

Mean
( cm)
4.84

4.830.03

5.710.00

3.970.03

3.260.04

4.860.02

5.720.01

3.360.10

4.750.07

3.250.07

1.610.07

3.230.07

1.150.07

1.99

2.880.11

4.120.13

2.730.07

3.91

2.810.12

4.350.10

2.510.12

3.22

2.540.11

3.240.09

1.640.09

2.47

1.590.07

2.450.11

1.190.07

5.520.03

5.500.01

6.700.03

2.870.03

5.090.01

5.320.03

6.13
5.41

4.56

8.46

8.30
6.37a

3.32
0.02
0.08

4.67
0.01
0.07

2.56
0.01
0.07

0.93

0.61

0.52

5.78

4.61

3.40

3.79

d
f

4.79

2.51

4.98

3.87

3.15

1.74

2.36
6.66

5.80

5.91

4.64
3.52b

Mean number of shoot length per regenerating explants, presented as mean SE of 10 replications. Data was
recorded at 5 weeks of culture. The values followed by same letters are not significantly different @ P= 0.05.
Shoot primordia per regenerating explant

followed by RM 2 (20.42), similarly the response of

Shoot primordia plays very important role in

Arka Meghali was highest in RM 9 (17.22) followed by

development of shoots. The shoot primordia per

RM 2 (12.71) but Vaibhav responded quite differently

explant was highest in Pusa Ruby on RM 9 (24.89)

by producing highest shoot primordia on RM 9

Madhu and Savithramma

Page 165

(15.92) followed by RM 2 (11.84) on epicotyls (Table-

the shoot regeneration response to obtain a shoot

3). The shoot bud formation started at 12 days and

length of 8-10 mm was around 22 days in cotyledons.

Fig. 1. Different stages of regeneration in tomato cultivar Pusa Ruby.

Fig. 2. Different stages of regeneration in tomato cultivar Vaibhav.

Number of shoots per explant and shoot length

(9.86) followed by cotyledonory explants of Arka

Multiple shoots arising from a single explant reduces

Meghali (7.71) and cotyledonory explants of Pusa

the time and resources for mass multiplication.

ruby (6.70) (Table-5 ). Gubbis et al., 2003 reported

Variability in shoot production was recorded for

highest number of shoots produced from epicotyl and

different explant types and cultivars. The highest

hypocotyl explants but the cotyledonory explants

number of shoots per explant was produced by

produced less number of shoots in tomato cultivars of

cotyledonory explants of Pusa ruby and Arka Meghali

Hana, premium and Robura. These results are in

in RM 9 (12.82) and (7.22) respectively, followed by

confirmation with the present results of tomato

Vaibhav (7.75) with epicotyl explant and least number

cultivar Vaibhav. Moghaieb et al., 1999 also obtained

of shoots was recorded in hypocotyl explant (Table-

highest shoots per explant. In contradictory these

4). Shoot length per explant was recorded after 15 to

results obtained highest shoots (5.8-6.0) in wild

20 days of culture, shoot length was contradictory to

tomato using leaf and cotyledonory explants on

cotyledonory explants as the highest shoot length was

Zeatin supplemented medium (Arriliga et al., 2001).

recorded in epicotyl explant of Vaibhav in RM 9

The elongated shoots were excised individually and

Madhu and Savithramma

Page 166

transferred onto fresh half strength MS supplemented

controlled greenhouse with a photoperiod of 16/8

with 0.1 mg/l IAA.

Subsequently, rooted plantlets

hours conditions. Plants were hardened by removing

were acclimatized on sterilized soil Rite (Tissue

the plastic bags gradually after 7 - 10 days. The

culture grade) under in-vitro conditions for one week

similar results were reported by Lee et al., 1999, Abu-

followed by pots containing soil composed of coco

El-Heba et al., 2008, that use of half strength along

peat: sand: soil (2:1:1, v:v:v), then covered with plastic

with 0.1 mg/l IAA induced roots.

bags to increase the humidity and

grown under

Fig. 3. Different stages of regeneration in tomato cultivar Arka Meghali.

Conclusion

Agarwal S, Rao AV. 2000. Role of antioxidant

We are the first to report the effect of cytokinin

lycopene in cancer and heart disease. Journal of

Adenine sulphate in shooting response of tomato

American College Nutrition 19(5), 563-569.

cultivars Pusa ruby, Arka Meghali and Vaibhav. The

addition of Adenine sulphate increased the induction

Atherton JG, Rudich. 1986. Journal In: Tomato

of multiple shoots in cotyledonory, epicotyl and

crop Chapman and Hall, London, New York 661,

hypocotyl explants. MS medium supplemented with

Zeatin (0.5mg/l) + Adenine sulphate (10mg/l) + IAA

Arriliga I, Gis bert C, Sales E, Rogi L, Moreno

(0.1mg/l) was the best hormonal combination for all

V. 2001. in-vitro plant regeneration and gene

the explants. The above media also reduced the

transfer in the wild tomato Lycopersicon cheesmanii.

number

Journal of Horticultural Science. Biotechnology 76,

of

days

required

for

complete

plant

regeneration from 120 days to 75-80 days. Pusa Ruby

413418.

was the best cultivar followed by Vaibhav and Arka

Meghali. Hence this rapid, highly efficient and

Bhatia P, Ashwath N, Senaratna T, David M.

reproducible regeneration protocol can be utilized for

2004. Tissue culture studies of tomato (Lycopersicon

genetic transformation with desired genes to develop

esculentum) Plant Cell Tissue and Organ Culture 78,

high yielding, biotic and abiotic resistant varieties.

121.
Bhatnagar-Mathur P, Vadez _K V, Sharma K.

References

2008. Transgenic approaches for abiotic stress

Abu-El-Heba GN, Hussein GM, Abdalla NA.

tolerance in plants:retrospect and prospects., Plant

2008. A rapid and efficient tomato regeneration and

Cell Reports 27, 411424.

transformation system, Agriculture and Forestry

Research 58, 103-110.

Chandel G, Katiyar SK. 2000. Organogenesis and

Madhu and Savithramma

Page 167

somatic embryogenesis in tomato (Lycopersicon

Science 1, 1141-1143.

esculantum Mill.). Advance in Plant Science 13, 1117.

Javier Pozueta-Romero, Guy Houlne, Luis

Caas, Rodolphe Schantz, Jesus Chamarro.

Chen H, Zhang J, Zhuang T, Zhou G. 1999.

2001. Enhanced regeneration of tomato and pepper

Studies on optimum hormone levels for tomato plant

seedling

regeneration from hypocotyls explants cultured in-

transformation. Plant Cell Tissue and Organ Culture.

vitro. Acta Agriculture Scandinavica-a 15, 26-29.

67, 173180.

Chaudhary Z, Afroz A, Rashid H. 2007. Effect of

Koblitz H. 1982. Versuche zur Gewebekultur in der

variety and plant growth regulators on callus

Gattung lycopersicon Mill. Ubersicht. Kulturpflanze,

proliferation and regeneration response of three

Berlin 27-43,

explants

for

Agrobacterium-mediated

tomato cultivars (Lycopersicon esculentum). Pakistan

Journal of Botany 39(3), 857-869.

Kurtz SM, Lineberger RD. 1983. Genotypic

differences in morphogenic capacity of cultured leaf

Chaudhry Z, Feroz I, Ahmed W, Rashid H,

explants of tomato. Journal of American Society for

Mizra B, Qureshi A. 2001. Varietal response of

Horticultural Science 108, 710-714.

Lycopersicon

esculentum

to

callogenesis

and

regeneration. Online, Journal of Biological Science 1,

Lee KS, Schottler F, Collins JL, Lanzino G,

1138-1140.

Couture D, Rao A, Hiramatsu KI, Goto Y,

Hong SC, Caner H, Yamamoto H, Chen Z-F,

Compton ME, Veilleux RE. 1991. Shoot, root and

Bertram E, Berr S, Omary R, Scrable H,

flower morphogenesis on tomato influores- cence

Jackson T, Goble J, Eisenman L. 1997. A genetic

explants. Plant Cell Tissue and Organ Culture 24,

animal model of human neocortical heterotopia

223-231.

associated with seizures. Journal of Neuroscience

Research 17, 6236-6242.

Duzyman E, Tanrisever A, Gunver G. 1994.

Comparative studies on regeneration of different

Ling HQ, Kriseleit D, Ganal MW, 1998. Effect of

tissues of tomato in-vitro, Acta Horticulture 34, 235-

ticarcillin/potassium clavulanate on callus growth

242.

and shoot regeneration in Agrobacterium mediated

transformation of tomato (Lycopersicon esculentum

Gubis J, lajchova Z, farago J, jurekova Z. 2004.

Mill.). Plant Cell Reports 17, 843-847.

Effect of growth regulators on shoot induction and

plant

regeneration

in

tomato

(Lycopersicon

Moghaieb REA, Saneoka H, Fujita K. 1999.

esculentum Mill.). Biological, Bratislava 59, 405-408.

Plant regeneration from hypocotyl and cotyledon

explant of tomato (Lycopersicon esculentum Mill.).

Gunay AL, Rao PS. 1980. in-vitro propagation of

Soil Science and Plant Nutrition 45, 639646.

hybrid tomato plants (Lycopersicon esculentum L.)

using hypocotyl and cotyledon explants. Annals of

Nogueira FTS, Costa MG, Figueira ML, Otoni

Botany 45, 205-207.

WC, Finger FL. 2001. in-vitro regeneration of

Santa Clara" tomato plantlets and its natural

Jatoi SK, Sajid GM, Sappal H, Baloch MS,

mutant/Firme". Science in Agrotechnology. Lavras

Qureshi A, Anwar R. 2001. Differential in- vitro

25, 36-71.

response of tomato hybrids against a multitude of

hormonal regimes. Online Journal of Biological

Potrykus I, Spangenberg G. 1995. Gene transfer

Madhu and Savithramma

Page 168

to plants. Berlin: Springer 361,

Villiers RPD, Vuuren RJV, Ferreira DI, Staden

JV. 1993. Regeneration of adventitious buds from

Selvi

DT,

Khader

MA.

1993.

in-vitro

leaf discs of Lycopersicon esculentum cv. Rodade:

morphogenetic capacity of tomato (Lycopersicon

optimization

esculentum

conditions. Journal of South African Society of

Mill.)

var.

PKM.1.

Horticulture 41, 251-258.

South

Indian

of

culture

medium

and

growth

Horticultural Sciences 3, 24-27.

Madhu and Savithramma

Page 169