Effect of variety and plant growth regulators on callus proliferation and regeneration response of three tomato cultivars (Lycopersicon esculentum)

Abstract

The experiment was conducted to optimize a reproducible protocol for callus induction and regeneration of three tomato cultivars and also to select the cultivar which better perform under In vitro conditions for further experimentation. For callus induction hypocotyls and leaf discs were used as explant source. Explants of the tomato seedlings were cultured on MS medium supplemented with different concentrations and combinations of different plant growth regulators (PGRS) for callus proliferation. Callus induction values were significantly influenced by the variety and explant source. In all three tested cultivars maximum callus induction frequency was observed on CIM6 (MS + 0.5 mg/l IAA+2mg/l 2ip). There was also a positive correlation between the treatment and explant source. Here at T6 (2ip 2mg/l, IAA0.5 mg/l) hypocotyls gave its maximum value of (81.8%), followed by theT4 (IAA 1mg/l+kinetin 0.5 mg/l) for hypocotyls (79.48%). To observe the regeneration capacity of three tomato cultivars, 10 Regeneration Media (RM) combinations were tested. The regeneration capacity is significantly influenced by cultivar and explant type. The best regeneration media was found to be RM3 (MS +1.5 mg/l 2ip and 0.5 mg/L IAA) with (72.5-79.22%) regeneration. Among varieties Riogarande showed maximum regeneration capacity (79.22%).

Full text

Pak. J. Bot., 39(3): 857-869, 2007.
EFFECT OF VARIETY AND PLANT GROWTH REGULATORS
ON CALLUS PROLIFERATION AND REGENERATION
RESPONSE OF THREE TOMATO CULTIVARS
(LYCOPERSICON ESCULENTUM)
ZUBEDA CHAUDHRY, AMBER AFROZ AND HAMID RASHID
*
Agricultural Biotechnology Programme (ABP), Institute of Agricultural Biotechnology &
Genetic Resources (IABGR), National Agricultural Research Center (NARC),
Park Road, Islamabad, Pakistan
*
Corresponding Address: Ph #: 9251-9255217, Fax #: 9251-9255217, E-mail: abi_narc@yahoo.com
Abstract
The experiment was conducted to optimize a reproducible protocol for callus induction and
regeneration of three tomato cultivars and also to select the cultivar which better perform under In
vitro conditions for further experimentation. For callus induction hypocotyls and leaf discs were
used as explant source. Explants of the tomato seedlings were cultured on MS medium
supplemented with different concentrations and combinations of different plant growth regulators
(PGRS) for callus proliferation. Callus induction values were significantly influenced by the variety
and explant source. In all three tested cultivars maximum callus induction frequency was observed
on CIM6 (MS + 0.5 mg/l IAA+2mg/l 2ip). There was also a positive correlation between the
treatment and explant source. Here at T6
(2ip 2mg/l, IAA0.5 mg/l) hypocotyls gave its maximum
value of (81.8%), followed by theT4 (IAA 1mg/l+kinetin 0.5 mg/l) for hypocotyls (79.48%). To
observe the regeneration capacity of three tomato cultivars, 10 Regeneration Media (RM)
combinations were tested. The regeneration capacity is significantly influenced by cultivar and
explant type. The best regeneration media was found to be RM3 (MS +1.5 mg/l 2ip and 0.5 mg/L
IAA) with (72.5-79.22%) regeneration. Among varieties Riogarande showed maximum
regeneration capacity (79.22%).
Introduction
Tomato is planted in almost 4 million ha worldwide (James et al., 2004). With wide
range of adaptability to soil and climate, it is cultivated almost all over the world, It is
popular because of its high nutritive value and diversified uses Hundred gram of edible
parts of tomato contains 0.9 grams protein, 0.1 gram fat, 3.5 gram carbohydrates, 15-20
calorie energy, 500-1500 IU vitamin “A”, 0.1mg thiamine, 0.02 mg riboflavin, 0.6 mg
niacin, 20-25 mg vitamin’s”, 6-9mg calcium and 0.1-0.3 mg iron (Farid Uddin et al., 2004).
In tomato genetic transformation with regeneration In vitro has been successfully
used for genetic improvement (Lindsey 1992). Tolerance to herbicide resistance to pests
and diseases, production of edible vaccines or other novel byproducts and quality
improvement are the most important goals of genetic plant modification. One of the most
important techniques being used now a days is tissue culture for rapid and pathogen free
plants. (Averre & Gooding, 2004). Regeneration of whole fertile plants from appropriate
tissues In vitro is important in plant transformation.
The most important procedure up to date is regeneration through adventitious
organogenesis (Van Roekel et al., 1993; Frary & Eaarle 1996; Peres et al., 2001). In vitro
plant regeneration has been found to depend on many factors, of which most important are:
composition of basic medium, growth regulators, gelling agent, light intensity and quality,
photoperiod, temperature, cultivation vessels and vessel covers (Reed 1999). The choice of

ZUBEDA CHAUDHRY ET AL.,
858
an explant with regard to the tissue of origin, developmental stage and physiological state
must be determined by goals of the project. Selection of an appropriate culture medium is
based upon the nutritional and hormonal requirements of tissues literally cut off from the
metabolism of complete plant and varies from species to species.
In vitro culture is used in tomato in different biotechnological applications,
production of virus free plants (Moghaieb et al., 2004), genetic transformation (Ling et
al., 1998) and in many fundamental research programme (M Arriliaga et al., 2001). Most
of the reports about adventitious regeneration in tomato deals with induction of
regeneration in hypocotyls or cotyledon explants (Moghaieb et al., 2004, Brichkova et
al., 2002, Raziuddin et al., 2004). These In vitro seedlings derived plants under aseptic
conditions were cut in to small pieces. Many workers reported the hypocotyls (Paranhos
et al. 1996, Davis et al., 1994, Rashid et al., 1996, Jatoi et al., 1995, Park et al., 2003)
and leaf disc (Soniya et al., 2001, Raj et al., 2005, Park et al., 2003, Roy et al., 2006,
Chaudhary et al., 2004, Jabeen et al., 2005, Raj et al., 2005) as explants source. Shoot
formation from explants of apical meristem, cotyledons, stems petioles, leaves, anthers
and inflorescences has been reported in tomato (Young et al, 1987; Branca et al., 1990;
Compton & Veilleux 1991). Gubis et al., 2003 used 6 different types of explants
(hypocotyls, cotyledon, epicotyls, leaf petiole and internodes of 13 cultivars of tomato.
Takashina et al., 1998 reported that the explants nature affected callus induction and
regeneration.
De-differentiation of tomato (Lycopersicum esculentum Mill) leaf explants into
callus followed or not followed by shoot formation is dependent on genotype, culture
medium and physiological stage of the donor plant (Guillermo et al., 2003).
In the view of the above facts, the present research was designed to evaluate the
effect of variety and plant growth regulators in MS medium on Callus proliferation and
regeneration from tomato explants.
Materials and Methods
Three varieties of tomato (Lycopersicon esculentum cv.) were used. The seeds of
Roma Riogarande and money maker were kindly provided by Awan seed center and from
National Agriculture Research Centre, Islamabad.
Seeds were surface-sterilized in 0.8 %(v/v) “Clorox” bleach (Sodium hypochlorite)
for 15 minutes and rinsed three times (5 min each) with autoclaved distilled water
(Franklin & Dixon, 1993). Afterwards the seeds were placed separately in four sterilized
Petri dishes containing filter paper (Whatman No.1). Seeds were inoculated in test tubes
containing full strength MS medium. Cultures were initially kept in dark for two days at
25 ± 1°C and then maintained under 16h photoperiod at 50 u mol/ m
2
/s, with day night
temperature at 20°C-25°C.Germinated seedlings served as explant source for tissue
culture experiments.
Callus induction and growth: From the 2-3 weeks old In vitro, seedlings, hypocotyls
(Paranhos et al., 1996) and leaf discs were cut. The hypocotyls and leaf discs were
excised of uniform sizes approximately, 1 cm in length. Callus induction responses were
assessed for three varieties on 9 media formulations. The hypocotyls were cut into a
lower, middle and upper segment. The explants were placed horizontally on the medium
surface, leaf discs explants with the adaxial surface in contact with medium. Results were
secured according to the presence of callus after 28 days. Basic MS medium with various

CALLUS PROLIFERATION OF TOMATO CULTIVARS
859
concentrations of auxins and cytokinins were used for callus induction. Different
concentrations of NAA, BAP, IAA, Kinetin and 2ip were used. As supplement GA3 was
also used. GA3 concentration ranged from 1-2mg/l.
Healthy calli were cut into pieces and sub cultured for maintenance onto the same
medium, under the same culture conditions to assess the regeneration response.
Regeneration medium: The maintained calli were sub cultured on regeneration media.
Ten plant regeneration media, namely RM1 to RM10 (Regeneration Media), were used for
identification of regeneration capacity of different cultivars of Lycopersicon esculentum.
Rooting medium: As the tomato shoots began to regenerate from calli, they were
transferred to rooting media supplemented either with IBA, NAA, or IAA, 0.1 and 0.2
mg/l and the number of shoots that produced roots was recorded after three weeks of
incubation. All media contained 3% sucrose with pH adjusted at 5.76 and were solidified
with 4g/l of gelrite.
Results and Discussions
In vitro culture is used in tomato in different biotechnological applications,
production of virus free plants (Moghaieb et al., 1999), genetic transformation (Frary &
Earliee 1996; Ling et al., 1998) and in many fundamental research programmes (Hanus
Fajerska 2000; mArriliaga et al., 2001). Most of the reports about adventitious
regeneration in tomato deals with induction of regeneration in hypocotyls or cotyledon
explants (Asakura et al., 1995; Icjimura & Oda 1995; Moghaieb et al., 1999). We are also
using hypocotyls and leaf discs for establishment of high frequency regeneration in
tomato cultivars Roma, Riogarande and Money maker which can be used in future for
Agrobacterium mediated transformation in these tomato cultivars.
Sterilization is an important step, which affects the callus induction frequency and
regeneration. Clorox (Sodium hypochlorite) used as a surface sterilization agent, played
an important role in germination of seeds. Seeds of all three varieties Roma, Riogarande
and Money maker were treated with different concentrations of Clorox to optimize the
level of clorox suitable for in- vitro germination. High concentration had an inhibitory
effect and seeds become dead whereas when concentration was diluted from 1:0-1:8 the
percentage germination of seeds was enhanced. Our results were in line with Chaudhry et
al., (2001, 2004) and contrary to Reda et al., (2004). Reda et al, 2004 used 70% ethanol
followed by 3% Clorox, while Chaudhry et al, (2004) used 8% Clorox for the
sterilization of seeds.
Seeds were inoculated on MS medium to observe the behavior of varieties for in vitro
seed germination. Gubis et al., (2003) and Raj et al., (2005) used half strength MS medium
for in vitro germination. Moneymaker seeds showed 86.6%. Riogarande 97.2% and Roma;
showed 72.5% germination. The results show that Riogarande is the variety that gave the
maximum percentage of seed germination. The reason of difference in germination rate
might be linked to the genotypes of the varieties. JaeBok et al., (2001) also confirmed these
results that germination rate depends upon the genetic basis of the variety.
These in vitro seedlings were further used as explants source in callus induction, which
leads to regeneration (Fig. 1a,b,c). De-differentiation of tomato (
Lycopersicum esculentum
Mill) leaf explants into callus followed or not followed by shoot formation is dependent on
genotype, culture medium and physiological stage of the donor plant( Guillerno et al.,
2003).

ZUBEDA CHAUDHRY ET AL.,
860
During present studies the callus formation was achieved on MS medium
supplemented with IAA, BAP, NAA, GA3, 2ip and Kinetin. Nine Callus Induction Media,
viz., T1 to T9 were checked for callus induction, using explant e.g., hypocotyls, leaf discs
of three tomato cultivars. Observations in this regard are given in Tables 1 and 2.
In vitro seedlings of 2-3 weeks were used for callus induction (Fig. 1a,b,c). Hu & Philips
(2001) also reported to use 18 days old plant for callogenesis and regeneration. Unlike Reda et
al., (2004) who used 6 days old seedling for callus induction and regeneration. Soniya et al.,
2001 used leaf explants of Lycopersicum esculentum Mill cv.Sakthi from a field grown plant
(mother plant).For sterilization she used 70 % ethanol followed by 0.1% Mercuric chlorite.
These in vitro seedlings derived plants under aseptic conditions were cut into small
pieces. The explants sources were leaf discs and hypocotyls. Many workers reported the
hypocotyls (Davis et al., 1994, Rashid et al., 1995, Jatoi et al., 1995, Park et al., 2003)
and leaf disc (Soniya et al., 2001, Raj et al., 2005) as explants source. Gubis et al., (2003)
used 6 different types of explants (hypocotyls, cotyledon, epicotyls, leaf, petiole and
internodes of 13 cultivars of tomato. Takashina et al., 1998 reported that the explants
nature effect callus induction and regeneration.
Callus induction and regeneration percentage was a special matter of interest in the
studies. Our results were varying on the basis of treatments and genotype of the cultivars.
Among the nine combinations of callus induction media T1-T9. T6 (MS plus 0.5 mg/l
IAA, and 2mg/ l 2ip) was the most responsive medium. Varieties and explants source
varied significantly with different combinations used.
Callus induction values varied significantly among the treatments (Table 1). T6 gave
maximum value for the variety Riogarande (80.72%), followed by T5 (GA3 1.5 mg/l and
BAP0.5 mg/l) for Riogarande that is (80.67%). Roma gave its maximum value in T6 that
is 78.5, followed by T5 that is 77.50. Money maker had its highest value at T6 that is
(77.5%) followed by T4 (IAA 0.5 mg/l and Kinetin 0.5 mg/l), that is 72.50%. At T1 (MS
salts +vitamins without hormones) all varieties gave there minimum value (.00-.05),
followed by T8 that is (NAA 0.5 mg/l, BAP 4mg/l, IAA 0.5 mg/l and 2ip 0.5 mg/l). .Hille
et al., (1989), Gubis et al., (2003), Raj et al., (2005) and Park et al., (2003) observed that
callus is generally induced on medium with high cytokinin to moderate levels of auxin. In
our study, also as there was a decrease in the callus induction percentage as BAP
concentration was enhanced.
In this study, callus induction value for the two explants used varied significantly
with different treatments (Table 2). At T6 the hypocotyls gave the maximum Value that
is 81.8, followed by T4 that is 79.48% for the hypocotyls followed by T5 that is 79.44%
for hypocotyls. Leaf discs gave there maximum value at T6 (76%), followed by T4 that is
72.33%. Minimum values for callus induction in explants were at T1 followed by T8.
The probable reason for more callus induction percentage might be the requirement
of moderate concentrations of auxins combined with relatively higher levels of cytokinins
(Hille et al., 1989). Capote et al., (2000) also obtained more callus induction by
combination of Auxin and relatively high level of Cytokinin.Therefore, higher levels of
auxins as well as higher levels of cytokinins gave poor callus response and if the callus is
formed is completely brown or black. On the other hand CIM1 with lower level of auxins
also could not give good results for callus induction. These results are also in line with
Jatoi et al., (2001) who obtained 100% callus induction in medium with 10 µM BAP and
0.1 µM/l IAA. Park et al., (2003) used 2mg/l of zeatin alongwith 0.1mg/l of IAA in the
transformation protocol, in this experiment zeatin was replaced by 2ip. Reda et al.,
(2004) used higher auxin and low level of cytokinin for callus induction. She used 2mg/l
of 2,4-D and 0.25 mg/l of kinetin. Soniya et al, (2001) used 8.88 uM of BA and 4.13 uM
piclogram for callus induction.

CALLUS PROLIFERATION OF TOMATO CULTIVARS
861

ZUBEDA CHAUDHRY ET AL.,
862
The results indicated that the Riogarande produced the maximum average calli (85-
95%), which was significantly higher than all the other varieties used during the present
experiment (Fig. 1d). The callus induction in Roma was 75-80% on the average, which is
higher than that in Money Maker. These differences in callus percentage may be linked to
their genotypes. Genotypic constitution has an effect on callus induction and regeneration
(El-Farah et al., 1993). These results are also confirmed by Lu et al., (1997) who
observed that different genotypes differ significantly in callus induction and regeneration.
Within same variety, on all media combinations the results were different in all three
varieties while on same media combination among three cultivars there are some non-
significant results. This shows that media combinations or hormonal balance has strong
effect on callus induction as compared to genetic variation.
The maintained calli were subcultured on regeneration media. Different hormonal
combinations were used in Regeneration Media (RM), namely RM1 to RM10. The
results are shown in Table 3, 4, 5 and 6. According to the results, all 10 treatments of
media combinations showed different behavior in regeneration response except RM1
(without growth hormone) and RM10 (2mg/l IAA only without Cytokinin), therefore
these two media combinations showed negligible regeneration.
Varieties and explant source showed significantly different results for different
combination of media used (Table 5). At T3 (2ip 1.5 mg/l and IAA 0.5 mg/l) Riogarande
gave its maximum value that is 79.22, followed by Roma at T3 that is 76. Money maker
gave its maximum value at T3 that is 72.5.After T3 Riogarande gave its maximum value
at T2 (2mg/l BAP and 0.5 mg/l of IAA) that is 75%. At T9 (0.5 mg/l of IAA and1mg/l of
2ip) Roma gave its second highest value that is 67, followed by T7 (1mg/l of IAA, 1mg/l
of 2ip and 0.5 mg/l of Kinetin) that is 60%.
Regeneration values also varied significantly with different explants used for various
treatments (Table 5). At T3 both hypocotyls and leaf disc showed maximum regeneration
value that is 79.45 for leaf disc and 72.33 for hypocotyls followed by T9 that is 53.33%
for leaf discs and 60 for hypocotyls.
There was also significant difference for different varieties used and different explant
used for different regeneration medias used. There was significant difference in the
interaction of the treatment explant source and varieties used (Table 3). Leaf discs and
the Riogarande gave their maximum value at T3 that is 83% and 75% for hypocotyls,
followed by 80% for leaf discs and 72% for hypocotyls for Roma. For money maker 75%
in case of leaf discs and 70% for hypocotyls.
The presence of high cytokinins with low or equal amount of auxins was also
confirmed by Gubis et al., (2003), who observed the regeneration capacity of six types of
explants in 13 tomato (L. esculentum). Soniya et al., (2001) used only 17.7uM of BA for
regeneration. Raj et al., (2005) used low levels of auxin and cytokinin for regeneration of
leaf explants of the Pusa Ruby tomato e.g., 0.1 mg/l of IAA and 0.1mg/l of zeatin.
Chaudhary et al., (2004) obtained the regeneration percentage of 45.8% and 30.8% for
the hypocotyls and leaf discs respectively by using relatively higher level of auxin and
higher amount of cytokinin e.g. IAA 2mg/l, BAP 5mg/l, NAA 2mg/l and kinetin 4mg/l.
The best results were obtained when explants were cultured on a regeneration medium
containing 2 mg 2ip/litre and 0.1 mg IAA/litre. The presence of equal amounts of auxin
and cytokinin in the regeneration media, were confirmed by Botau et al., (2002). They
explored that cotyledons and hypocotyls of tomato cultivars, cultured on MS media
supplemented with different hormonal combinations and the best results were obtained in
the medium supplemented with 1 mg NAA/litre+1 mg BAP/litre. Chandel & Katiyar
(2000) tested different combinations of phytohormones , among them the MS medium

CALLUS PROLIFERATION OF TOMATO CULTIVARS
863
Fig. 1. In vitro seedlings of Lycopersicon esculentum. (a) Roma, (b) Riogarande, (c)
Money maker, (d) Hypocotyls derived calli of Riogarande and (e) Money maker (f) leaf
disc derived calli of Roma and (g) Riogarande showing green spotting and regeneration.
a
e
c
b
f
g
d

ZUBEDA CHAUDHRY ET AL.,
864

CALLUS PROLIFERATION OF TOMATO CULTIVARS
865
Table 5. Regeneration response of three different varieties by
using two different explants. (Variety*Explant).
Variety Riogarande Roma Moneymaker Explant means
Hypocotyl 35.56 BC 34.47 C 30.23 D 33.42
Leaf disc 41.312 A 37.45 B 31.1 D 36.62
Variety means 38.44 35.96 30.67
Figure with same letter showed the non significant results and with different letters showed significant results.
Table 6. Regeneration response of two different explants of three tomato cultivars on
different media combinations. (Treatment*Variety* Explant.
Treatments Riogarande Roma Moneymaker
L.D Hypo L.D Hypo L.D Hypo
T1 .01 T .00 T .01 T .01 T .001 T .001 T
T2 65 EF 85 A 40 JK 30 LMN 30 LMN 20 OPO
T3 83 AB 75 BCD 80 ABC 72 CDE 75 BCD 70 DE
T4 45 IJ 58 FGH 35 KLM 40 JK 30 LMN 28 MNO
T5 30 LMN 42 IJK 30 LMN 28 MNO 24 NOP 29 LMNO
T6 28 MNO 30 LMN 10 RS 25 NO 15 PQR 12 QRS
T7 35 JLM 38 JKL 50 HI 70 DE 30 LMN 27 MNO
T8 20 OPQ 25 NO 30 LMN 35 KLM 40 JK 60 FG
T9 45 IJ 55 GH 65 EF 70 DE 50 HI 55 GH
T10 4.15 ST 5.12 ST 4.7 ST 4.5 ST 8.2 RST 10 RS
Figure with same letter showed the non-significant results and with different letters showed significant results.
*Hormones used for Regeneration medium
RM
1
MS without hormone
RM
2
0.5 mg/l BAP and 2 mg/l GA3
RM
3
0.5 mg/l 2ip and 2 mg/l GA3
RM
4
2 mg/l BAP
RM
5
NAA0.5 mg/l and BAP 5mg/l
RM
6
1 mg/l BAP and 1 mg/l Kinetin
RM
7
0.5 mg/l NAA, 0.5 mg/l and 1.5 mg/l Kinetin
RM
8
1 mg/l IAA and 1 mg/l2ip and 3mg/lKinetin
RM
9
1.5 mg/l IAA and 0.5 mg/l 2ip
RM
10
2 mg/l IAA and 0.5 mg/l 2ip
with 1.5 mg BAP and 1.5 mg IAA/litre was the most responsive for regeneration. The
presence of two cytokinins in RM3 (MS with BAP 2 mg/l and Kinetin 1 mg/l), showing
results was also indicated by Brichkova et al., (2002) who observed that presence of two
cytokinins (BAP at 5.0 mg and Zeatin at 1.0 mg/litre) in medium contributed to plant
regeneration in 74% of Vezha explants and in 89% of L-1932 explants. RM10 showed
the poorest results because of the absence of Cytokinin. Among varieties Riogarande
showed the maximum regeneration capacity (40 %) (Fig. 2C). All the three varieties
showed difference in their regeneration capacity. Moneymaker showed minimum average
regeneration capability (75%). These differences might be due different genotypes. These
results are close to that of Lu et al., (1997), who studied the effects of tomato genotype;
explant source and culture medium growth regulator composition on callus formation and
plantlet regeneration rates using 2 cultivars. They found that genotype had significant
effect on callus induction and regeneration. Mirghis et al., (1995) observed that the in
vitro reactions of the genotypes differed significantly and were dependent on the culture
media. Nandakumar et al., (1991) noted that callus formation and regeneration varied
with the genotype.

ZUBEDA CHAUDHRY ET AL.,
866
Fig. 2. (a) Leaf disc derived callus induction leading to regeneration in Money maker. (b)
Regenerated shoot of money maker placed in rooting medium, (c) Regenerated plant of Riogarande
in rooting medium, (d) Regenerated plant of Roma present in rooting medium.
Auxins and cytokinins are involved in cell division and elongation, while; cytokinins
help in the process of differentiation. So appropriate concentration of these growth
hormones is necessary for cell division and differentiation. During the present study, it
was revealed that the varieties varied significantly for their regeneration capacity on the
similar medium. Roma and moneymaker showed minimum regeneration in almost all
media combinations (Fig. 2a,b,c). This might be due to their genetic differences.
Plant establishment in the soil: Thirty rooted plants derived from each of the
hypocotyls and the leaf discs after one week of root formation were shifted into small
pots of compost in the glass house. They were covered with the polythene bag for 10-12
days to control the temperature and humidity, and were watered at 4-5 days intervals.
About 80-90% of the plants survived in the soil and all survivors flowered normally.
About 85% of the seeds collected from these plants were viable
Conclusion
For callus induction study, different Callus Induction Media (CIM) combinations
tested on tomato cultivars, all three varieties showed maximum callus induction
percentages on T4 (MS + 0.5 mg/lBAP, 2mg/l GA3, 0.5 mg/l NAA). Riogarande had
maximum callus induction (80.72%).
a b
c
d

CALLUS PROLIFERATION OF TOMATO CULTIVARS
867
From ten Regeneration combinations tested, the best regeneration media was found
to be RM3 (MS +1.5 mg/l 2ipand 0.5 mg/l IAA). Among varieties Riogarande showed
maximum regeneration capacity 79.2%. All the media combinations showed significantly
different results for regeneration. Also the varieties showed significantly different results
within the same treatment. All the treatments showed significantly different response to
two different explant, used.
Establishment of the high frequency regeneration system is a prerequisite for the
genetic transformation. The result of the present study will serve as a basis for efficient
transference of desired characters through genetic transformation procedures. The result
of the present finding Riogarande cultivar among three varieties used is recommended for
Genetic Transformation as being more responsive to the standardized conditions.
Acknowledgements
The work is present of the project entitled “Transgenic tomato with resistance to
bacterial wilt” The authors acknowledge the support from Pakistan Agricultural Research
Council Agricultural Linkages Programme (ALP) for funding the project.
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(Received for publication 10 January 2007)