Experimental evaluation of the antibacterial activity of tomato callus extract and response of cultivar establishment in vitro.
Tomato has a strong traditional background in continental culinary practices.
However, it is used only to develop taste without realizing its dietary benefits. it could be exploited as a natural source of antioxidants, vitamins and other micronutrients as well. As a matter of fact, tomatoes are consumed at a higher rate in the developed countries for its nutritional value than in the developing countries. It is highly desirable to incorporate such vegetables into our diet programs as a source of nutraceuticals. Tomato being acceptable to people as a food could be a source of nutrients as well as a nutraceutic in the sub-continent.
Tomato (Lycopersicon esculentum Mill.) member of family Solanaceae, a major vegetable crop has achieved tremendous popularity over the last century. It is grown in almost every country of the world in the fields, green houses and net houses. Tomato by its nature is a perennial but commercially cultivated as an annual crop. Nutritionally tomato is important for maintenance of health and dietetics. The calorific value is less and an average sized tomato (148g) boasts only 35 calories. It also contains approximately 20-50mg of lycopene/100g of the fruit weight1. Lycopene is the most powerful antioxidant in the carotenoid family and is known to prevent cancer, blindness and certain other illnesses [2,3,4].
Tomato being acceptable to people as a food could be a source of nutrients as well as a nutraceutic in the sub-continent. Micropropagation of elite tomato cultivars can maintain their characters generation after generation. Tissue culture is used for callus production as well as micropropagation of high value commercial cultivars. By nature, tomato is a self-pollinated crop and is conventionally propagated via seeds. Various desirable genetic traits such as high carotene content are difficult to be retained due to the phenomenon of heterozygosity. Therefore, an efficient in vitro plant regeneration system may assist in the propagation of the commercially important cultivars without loosing their genetically enhanced nutraceutical constituents. In tomato, adventitious shoot regeneration can be achieved either directly  or indirectly through an intermediate callus phase [6, 7]. Indeed, both callus and shoots may be produced together . workers have studied various aspects of tomato in vitro cultures yet the antibacterial capacity of in vitro callus of tomato cultivars has been ignored.
Natural growth inhibitors and their important role in plant ecology have been well documented in plant kingdom. Antimicrobial activity of plant extracts from ginger and garlic have been studied by Ekwenye and Elegalam . Antibacterial activity of tomato pulp oil extract is reported by Vorob'ev et al. 
Flavones, flavonoids and flavonols are chemical compounds active against microorganisms . Flavonoids are also hydroxylated phenolic substances but occur as a C6-C3 unit linked to an aromatic ring, they are synthesized by plants in response to microbial infection , more lipophilic flvonoids may also disrupt microbial membrane .
Presently, the varieties under investigation have been developed by AVRDC with 10 times higher carotene content leading to higher vitamin A and lycopene content for the diet supplementation of the poor in the region. The present work aspires to develop asexual method of propagation for the above-mentioned cultivars to avoid loss of desired traits and to asses the antibacterial activity of callus.
Materials and Methods
Tomato cultivar CLN 2366C obtained from AVRDC was used in the present study. Seeds were surface sterilized by immersion in 0.1% mercuric chloride solution containing 2-3 drops of tween-20 for 4-5 min then rinsed 3-4 times with sterile distilled water. The sterilized seeds were inoculated on 1/2 strength MS  basal medium without any phytohormones or with 2.22[micro]M BA (Benzyl Adenine).
MS basal medium supplemented with various combinations and concentrations of phytohormones (details given below) was used throughout the study. All media was supplied with 3% sucrose and 0.2% gelrite. The media pH was adjusted to 5.8 prior to the addition of gelling agent. Finally, media were autoclaved at 121[degrees]C for 20 min. at 15 lbs psi. The cultures were incubated in growth room maintained at 25 [+ or -] 2[degrees]C under the photoperiod of 16/8 hours (light/dark) provided by cool white fluorescent light(3000 Lux). Each treatment was replicated thrice.
Leaves of in vitro grown 1-week-old aseptically germinated seedlings were utilized for explant preparation. Explants were initially cultured on the following concentrations and combinations of BA (Benzyl Adenine) in combination with different levels of IAA (Indole Acetic Acid) and IBA (Indole Butyric Acid) added to MS basal medium for callogenesis and/or subsequent somatic embryogenesis. These concentrations and combinations of hormones supplied to MS basal medium are listed below:
1. BA (0.5[micro]M) + IAA (0.1[micro]M)
2. BA (5[micro]M) + IAA (10[micro]M)
3. BA (10[micro]M) + IAA (5[micro]M)
4. BA (0.5[micro]M) + IBA (0.1[micro]M)
5. BA (5[micro]M) + IBA (10[micro]M)
6. BA (10[micro]M) + IBA (5[micro]M)
Callus induction was observed after four weeks. After next four weeks, initiation of plantlets formation from calli was observed on the same medium with same hormonal concentrations and combinations. Then, these cultures were transferred to plant growth regulator free MS medium for plantlets development. The plantlets produced were acclimatized in vivo. After that, the in vitro plants were established in mixed soil (3:1 sand and soil).
Selected healthy, green nodular and fast growing callus cultures (Fig.i) which later on produced normal plantlets. 30g of said callus was weighed and freeze dried (EYELA-Japan, model-F.D.550). 1.0g of freeze dried callus powder was weighed and dissolved in 50 ml of 80% ethanol and vigorously stirred with a sterile glass rod. Extract was constantly shaken for 24h and then filtered through Whatman No.1 filter paper as suggested by Azoro . Extraction was repeated with 50ml of 80% ethanol. Discarded the residue left thereafter. Brownish yellow filtrate was evaporated to dryness on steam bath at 100[degrees]C. The dried alcoholic extract was sterilized under UV light for 24h. This alcoholic extract was reconstituted by adding 1ml of dimethyl sulphoxide (DMSO) making the solution of 1000mg/mL. Then the second dilution of 500mg/mL was prepared from the original 1000mg/mL concentration. Paper disc diffusion method was applied to test the antibacterial activity of the extracts. Sets of discs (5mm diameter) of Whatman No.1 filter paper were oven sterilized in tinfoil wrapping. Normal strength nutrient agar medium (OXOID, England) was prepared and autoclaved at 121[degrees]C for 15 min. at 15 psi for culture growth and determination of antibacterial activity.
Prior to inoculation for antibacterial activity test, six bacterial strains Escherichia coli, Salmonella typhi, Proteus mirabilis, Enterobacter aerogenes Staphylococcus aureus and Bacillus subtilis were sub-cultured thrice onto the fresh nutrient agar media to obtain a more vigorous population. The stock cultures were incubated at 37[degrees]C for 24 h.
Screening for antibacterial activity
Bacterial cultures were serially diluted in normal saline solution. A sterile swab stick was used to seed the nutrient medium culture plates from 10-3 dilution. Placed the extract impregnated saturated discs on pre-inoculated culture media and incubated at 37[degrees]C for 24 h. The zone of inhibition in each case was measured and recorded as the diameter of the clear zones around the discs.
Control experiment using antibiotics
This was done to compare the diameter zones of inhibition of the extracts and already standardized antibiotic. This will help to prescribe plants with antimicrobial activities. The antibiotic used was erythromycin 5 mg/mL (Abbott, Pakistan).
The results obtained in present study were statistically analyzed with one-way analysis of variance in completely randomized design. The means were separated by Duncan multiple range test at 5% level of significance as described by Steel and Torrie .
Leaf explants were subjected to in vitro establishment and studied the dedifferentiation and differentiation behaviors of the cultivar under investigation. Explants were cultured on BA+IAA/IBA combinations i.e. 0.5[micro]M BA+0.1[micro]M IAA/IBA; 5[micro]M BA+10[micro]M IAA/IBA and 10[micro]M BA+ 5[micro]M IAA/IBA. The cultures were established aseptically, formed characteristic calli and exhibited distinct morphogenic changes in 8 weeks. The results of explants behavior are presented in forth coming tables.
Leaf explants of tomato variety CLN 2366C had distinctly different behavior when grown on medium containing IAA or IBA in the presence of BA (Table i). The hormonal combinations of BA+IAA induced large amounts of calli. In the later combination, callus formation was in moderate quantities. Actively and moderately growing calli were mostly compact, hard, nodular and dark green as shown in fig i. Invariably all the explants formed calli on all the concentrations and combinations of BA+IAA/IBA. However, table i. depicts that there was difference in size, texture and color of the calli.
After initial phase of four weeks of callus growth, the compact and nodular calli regained meristematic activity in the next 4-8 weeks. The meristemoids subsequently distinguished into shoot and root primordia. Phenomena wise the dedifferentiation and differentiation of explants was quite prominent. Maximum shoot counts were 22.67 [+ or -] [2.05.sup.a] in 10[micro]M BA+5[micro]M IAA and 15.33 [+ or -] [1.25.sup.b] in 5[micro]M BA+10[micro]M IAA (Table i). Rest of the combinations of phytohormones had shoot count of less value than 4 or none.
Antibacterial activity of callus
Results of the antibacterial study using disc diffusion method are summarized in Table ii. Both concentrations of callus extract showed good inhibitory activity against all six microbes. But the response was slightly high in 1000mg/mL concentration of tomato callus extract. The highest zone of inhibition formed by the high concentration (1000mg/mL) was [18.25.sup.a] [+ or -] 0.78mm in case of Proteus mirabilis. The resistance of Bacillus subtilis was also low as in case of Proteus mirabilis forming [16.55.sup.a] [+ or -] 0.92mm diameter zone of inhibition. No significant difference was noted in the activity of E.coli, Enterobacter aerogenes, and Salmonella typhi at 1000mg/mL of the extract by forming the clear zones of [14.25.sup.b] [+ or -] 1.69, [14.3.sup.b] [+ or -] 0.57, and [14.25.sup.b] [+ or -] 0.64mm respectively (Table ii). Whereas, Staphylococcus aureus was least inhibited showing smallest zone ([13.25.sup.bcd] [+ or -] 0.92mm).
No significant difference was observed at lower concentration of tomato callus extract (500mg/mL) in case of E.coli, S. typhi and B. subtilis creating zones of inhibition of [11.5.sup.def] [+ or -] 0.28, [11.8.sup.def] [+ or -] 1.56 and [11.05.sup.ef] [+ or -] 0.35mm respectively. In case of Proteus mirabilis and Enterobacter aerogenes the zones of inhibition were higher i.e. [13.9.sup.bc] [+ or -] 1.13 and [12.5.sup.bcde] [+ or -] 0.71mm respectively. Staphylococcus aureus had strong resistance against the extract showing only [10.25.sup.f] [+ or -] 0.35mm inhibition zone.
Sterilized seeds of tomato cultivar under investigation inoculated on 1/2 strength MS medium supplemented with or without 2.22[micro]M BA produced plantlets on both media but stunted growth was observed on 1/2 MS medium with BA. Gill et al.  also germinated tomato seeds on MS medium but containing a high concentration of BA (50 or 80[micro]M). The similar protocol was used by Kaparakis and Alderson  to successfully reproduce the same results, not only in tomato but also in aubergine and pepper.
Green morphogenic calli were induced within 4 weeks from leaf explants on MS medium supplemented with different concentrations of BA+IAA and BA+ IBA (Table i), which subsequently produced shoots and roots on the same medium composition during next 4 weeks. Kanwal  also observed callus induction and regeneration both from the leaf tissue of Momordica charantia, which occurred on single medium without involving sub culturing and media manipulation. She also obtained multiple plantlets when transferred to growth regulator free MS medium. In present study, BA+IAA (10[micro]M+5[micro]M) are found to be the only combination, which showed highest callogenesis and morphogenetic potential from leaf explants. The morphologically normal and highest numbers of shoots (22.67 [+ or -] [2.05.sup.a]) were produced from calli (Fig. i) of CLN 2366C variety on BA+IAA (10[micro]M+5[micro]M) supplemented medium (Table i). Gunay and Rao  also found that a combination of BA and IAA was best for shoot regeneration in tomato cultivar. Results reported by Novak and Maskova  are contrary to present findings, who noted that multiple shoots with simultaneous callus development were produced on media containing 5 or 10[micro]M BA alone. Explants of this variety in combination of BA+IBA produced green calli (Fig. i-ii). Only the low number of shoot regeneration (2.00 [+ or -] [0.82.sup.d]) from leaf calli was achieved on high concentrations of BA+IBA i.e. (5[micro]M+ 10[micro]M). Izadpanah and Khosh-Khui  also obtained maximum shoot proliferation from shoot tip explants in the media containing higher concentration of cytokinins i.e. BA (6.66-8.88[micro]M) or Kin (9.29-13.94[micro]M) and the highest percentage of rooting was achieved with the medium containing IBA (2.46?M). While the leaf explants calli regenerated roots on higher concentration of BA+IBA (5[micro]M+10[micro]M).
From these results, it is obvious that present variety has high morphogenetic potential. The regenerated plantlets were sub-cultured on hormone free MS medium for further multiplication and development of shoots and roots (Fig. iv) as tomato contains high levels of endogenous phytohormones thus it does not seem to require any exogenous plant growth regulators for rooting .
The antibacterial activity of the callus extract at two levels (1000& 500mg/mL) was studied results are presented in table ii. Significant inhibition of the six microbes tested was observed at both levels (Table ii). Tomato being a member of family Solanaceae possesses natural insect repelling properties. During in vitro growth the callus mass retains the same properties. Furthermore, when plant growth hormones are supplied through culture medium such capabilities of plant cells to check the microbial growth. The calli of tomato plant showed inhibitory influence (Table ii) due to the secretion of inhibitory material during their growth in vitro which may inhibit the growth of microbes and this is in accordance with Lin and Mathes . This antibacterial action is linked with the presence of a complex of organic acids (succinic, citric, tartaric, etc.) as reported by Vorob'ev  (1998) who revealed in his study that tomato pulp oil extract produces a wide-spectrum antibacterial effect on Gram-positive and Gram-negative microorganisms and on fungi of the genus Candida.
From the present investigation it is found that the in vitro manipulation of the tomato cells/ plants could not only be a source of plants of desired characteristics but secondary metabolites as well as functional compounds of importance which have the property of microbial inhibition.
[FIGURE 1 OMITTED]
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Aneela Fatima, Shaista Jabeen Khan *, Nasreen Zaidi and Nusrat Ejaz
Food and Biotechnology Research Center, PCSIR Laboratories Complex, Lahore-54600 (Pakistan)
Tel No.042-9230688 Ext.288 Fax No. 042-9230705
* Corresponding author. Email: firstname.lastname@example.org
Table I: Morphogenetic response of Leaf explants high carotene cultivars of tomato variety (CLN 2366C) in vitro after 8 weeks Plant Growth Quantity Texture/color Morphogenetic Regulators ([micro]of callus of callus potential BA IAA IBA 0.5 0.1 - ++++ Dark green, 3.33 [+ or -] compact 1.25,d shoots and nodular from callus 5 10 - ++++ Green, hard, 15.33 [+ or -] compact and [1.25.sup.b] shoots nodular 10 5 - ++++ Green, 22.67 [+ or -] nodular and [2.05.sup.a] shoots compact 0.5 - 0.1 +++ Green and 8.67 [+ or -] hard [1.25.sup.c] Roots 5 - 10 +++ Light green 2.00 [+ or -] and hard [0.82.sup.d] shoots & few roots 10 - 5 +++ Green and - hard * = mean separation in columns by Duncan's multiple range test, p =0.05 Absent = - Poor = + Small = ++ Moderate = +++ Large = ++++ Table II : Antibacterial activity of different concentration of tomato callus extract against six microbes tested Samples Escherichia Salmonella Proteus coli typhi mirabilis 1000mg/mL [14.25.sup.b] [14.25.sup.b] [18.25.sup.a] extract [+ or -] 1.69 [+ or -] 0.64 [+ or -] 0.78 500mg/mL [11.5.sup.def] [11.8.sup.def] [13.9.sup.bc] extract [+ or -] 0.28 [+ or -] 1.56 [+ or -] 1.13 5mg/mL [17.33.sup.a] [13.86.sup.bc] [12.00.sup.bcd] Erythromycin [+ or -] 2.16 [+ or -] 0.36] [+ or -] 2.12 Samples Enterobacter Staphylococcus Bacillus aerogenes aureus subtilis 1000mg/mL [14.3.sup.b] [13.25.sup.bcd] [16.55.sup.a] extract [+ or -] 0.57 [+ or -] 0.92 [+ or -] 0.92 500mg/mL [12.5.sup.bcde] [10.25.sup.f] [11.05.sup.ef] extract [+ or -] 0.71 [+ or -] 0.35 [+ or -] 0.35 5mg/mL [21.66.sup.a] [25.00.sup.a] [24.00.sup.a] Erythromycin [+ or -] 2.48] [+ or -] 1.41] [+ or -] 1.22 Significance level=0.5% for Duncan's multiple range test, Diameter zone (Mean [+ or -] S.E mm) of inhibition