IN VITRO PLANT REGENERATION FROM LEAF AND PETIOLE TISSUES OF TOMATO (SOLANUM LYCOPERSICUM CV. SOLAN VAJR)

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1 Journal of Cell and Tissue Research Vol. 13(3) (2013) (Available online at ISSN: ; E-ISSN: Original Article IN VITRO PLANT REGENERATION FROM LEAF AND PETIOLE TISSUES OF TOMATO (SOLANUM LYCOPERSICUM CV. SOLAN VAJR)? SHARMA, P. AND SRIVASTAVA, D. K. Department of Biotechnology, Dr Y. Parmar University of Horticulture and Forestry, Solan , Himachal Pradesh. E. mail: Received: August 15, 2013; Accepted: September 28, 2013 Abstract: Plants were regenerated from leaf and petiole explants of tomato (Solanum lycopersicum cv. Solan Vajr). Three weeks old aseptically grown seedlings were used as a source of explants for plant studies. Leaf and petiole explants were induced to form callus by culturing on Murashige and Skoog (MS) medium supplemented with different combinations and concentrations of BAP, IAA, TDZ, Kinetin, NAA and Adenine. Adventitious shoot buds were induced from calluses/ explants by culturing on these media. Out of 62 different combinations of growth regulators (BAP+IAA, Kn+IAA, TDZ (alone), TDZ+Ad, TDZ+IAA, TDZ+NAA, TDZ+BAP), tried for leaf and petiole explants separately, the high frequency shoot from leaf (68%) and petiole (80%) explants was obtained on MS medium supplemented with 1mg/l BAP+ 0.5mg/l IAA and 1mg/l Kn+ 1mg/l IAA respectively. TDZ in combination with BAP gave better results on shoot in comparison to TDZ alone. High percentage of root (95%) in in vitro developed shoots was obtained on MS medium supplemented with 0.2mg/l IBA in a short interval of days. The regenerated plantlets after acclimatization were transferred to glass house conditions and both vegetative and floral characteristics were observed. An efficient and reproducible plant protocol has been standardized in tomato (Solanum lycopersicum cv. Solan Vajr). This system would be valuable for genetic transformation studies in tomato. Key words: Tomato, Leaf, Petiole, Regeneration, INTRODUCTION Tomato (Solanum lycopersicum), one of the most important vegetable crops, belongs to family Solanaceae, grown all over the world for its economic importance and nutritive value and is a model species for introduction of agronomically important genes into dicotyledonous crop plants [1]. Having its origin in Central and South America [2], in India it occupies an area of 0.86 million hectares with a high production of 16.8 million tons with 11% share of the world market [3]. Being an off season vegetable crop in the hills of India, in Himachal Pradesh the returns from its production is very lucrative, that too in short time as it finds its ready market and high prices in the plains, where it is not grown in view of high temperature. It occupies an area of 955 hectares in the state with an annual production of 336,287 metric tons [4]. Ranking first among the processing vegetables, tomato is a rich source of vitamin A, B and C. It is also a rich source of citric acid which increases the alkalinity of the blood and have a powerful antioxidant lycopene that belongs to carotenoid family, which prevents cancer, control liver problems, indigestion, arthritis, urinary troubles and protects human from free radicles that degrade various parts of the body. 3913

2 J. Cell Tissue Research Tomato requires warm season for successful cultivation and is susceptible to cold winds and frost and also the high temperature adversely affects its growth and development. Its cultivation is also limited by various biotic factors including bacteria, viruses, fungi, insect pests and nematodes [5]. Being an economically important crop, to attain sustainable tomato productions, the above mentioned constrains have been addressed by conventional breeding methods and enhanced management but has not yet resulted in much commercial success. Tomato serves as a genetic model for improving other dicotyledonous crop plants [6]. Thus the integration of plant tissue culture and genetic engineering along with breeding programs may provide powerful tools to obtain improved or desirable traits like disease and insect resistance in this vegetable crop. Development of a high frequency in vitro system is a prerequisite and essential aspect for an efficient genetic engineering system that seeks to exploit genetically transformed plants for commercial applications. Plant studies in tomato have been previously reported via organogenesis using different explants such as leaf [7-9], cotyledon [6,10,11] and hypocotyls [10,12, 13]. Gubis et al. [9] used six different types of explants (hypocotyls, cotyledon, epicotyls, leaf, petiole and internodes) of tomato cultivars for plant studies. In the present communication, we report the effect of various combinations and concentrations of different growth regulators ( BAP+IAA, Kn+IAA, TDZ (alone), TDZ+Ad, TDZ+IAA, TDZ+NAA and TDZ+BAP) on the shoot frequency using leaf and petiole explants of tomato, cultivar Solan Vajr. We are currently using this plant system for the transformation of tomato by genetically engineered Agrobacterium tumefaciens strain containing chitinase gene in binary vector system. MATERIALS AND METHODS Plant material and culture medium: The certified seeds of tomato (Solanum lycopersicum cv. Solan Vajr) were procured from the Department of Vegetable Science, of Dr Y. Parmar University of Horticulture and Forestry, Nauni, Solan, (H.P.). The seeds were thoroughly washed with tap water and teepol and soaked for an hour. After soaking, the seeds were surface sterilized with 0.1 percent mercuric chloride for 2 minutes and then thoroughly washed (3-4 times) with sterilized distilled water to remove the traces of mercuric chloride. The seeds were then inoculated on the MS half strength basal medium [14] containing 0.5 percent sucrose for seed germination. Three weeks old seedlings were used as a source of explants (leaf and petiole) for plant studies. The explants were cultured on MS salt (macro and micro), vitamins supplemented with 100 mg/l meso-inositol, 3 percent sucrose and 0.8 percent agar agar was used as basal medium in shoot experiments. Different concentrations and combinations of cytokinins and auxins (Adenine, BAP, IAA, Kinetin, NAA, TDZ) were used in the MS basal medium. The ph of the medium was adjusted to 5.8 before adding agar agar. The medium was poured in culture vessels and sterilized at 15 lbs/ inch 2 (121 0 C) for 15 minutes in an autoclave. All the aseptic manipulations were carried out under laminar air flow chamber. Plant from leaf and petiole explants: To optimize the culture medium for high frequency shoot, leaf and petiole explants were excised from aseptically grown seedlings. Both the explants were inoculated on MS medium supplemented with various combinations and concentrations of plant growth regulators such as Kn and IAA, BAP and IAA, TDZ (high and low concentration), TDZ and Adenine, TDZ and IAA, TDZ and NAA, TDZ and BAP (mg/l), respectively. The explants were evaluated for average number of shoots s and percentage of shoot. After inoculation, the culture vessels were kept in the culture room at 26 ± 2 0 C under 16 hrs photoperiod with cool white fluorescent lamps (40µmol/m 2 /s) having 70-80% humidity. The regenerated shoots (2-3 cm) obtained from both the explants were separated and individual shoot was transferred to the MS medium containing various concentrations of different auxins IAA, NAA and IBA for root induction to get a complete plantlet. These were then evaluated for percentage of root after 4 weeks of culturing. Hardening of regenerated plantlets: After proper in vitro development, the plantlets were taken out of the tubes and flasks in such a way that no damage was caused to their root system. The roots were washed gently under running tap water to remove adhering medium. After removal of the medium, the plantlets were kept in running tap water for a few minutes so that they do not wilt after transfer to soil. 3914

3 Sharma and Srivastawa The survival and establishment of the plantlets were studied after transplanting the plants in sand: soil (1:1) mixture. Sand and soil were sterilized separately in an autoclave at 15 lbs/ inch 2 (121 0 C) for half an hour. After sterilization, the pots were filled half with soil and sand mixture in equal proportion and half with sand. The plantlets with washed roots were transferred to the pots. The root portion was placed inside the soil and sand mixture gently and was covered with sand. The plantlets were watered and covered with jam jars to maintain high relative humidity. These were then transferred to the culture room. Water was sprayed twice a day to maintain high relative humidity. After a week, when plants showed initial signs of establishment in pots with appearance of new leaves, the jars were temporarily removed daily for few hours. The plants were finally transferred to earthen pots and were acclimatized further in glass house. The percentage survival of the hardened plants was recorded five weeks after transfer. Data analysis: Each treatment consisted of atleast 30 explants and each experiment was repeated thrice. The data recorded for the different parameters were subjected to Complete Randomized Design. The statistical analysis based on mean values per treatment was made using analysis of variance of CRD. RESULTS The in vitro morphogenetic response of cultured plant tissue is affected by different components of the culture media and therefore, it is important to evaluate their effects on plant callus induction and potential [15]. The leaf and petiole explants on different medium compositions showed increase in size after about one week of inoculation whereas, there was no change in the colour of the medium. Callus formation was initiated within 8-10 days directly on the cut end surfaces of both the explants. Callus induction and shoot response under various media compositions was markedly affected by both the types of explants and plant growth regulators used. According to Pal et al. [16], in vitro callus induction depends on the endogenous concentration of plant growth regulator as well as exogenously supplied growth regulator. Also the growth regulator requirements for callus induction vary depending on the source of the explants as [17]. Although callus induction was observed in both the explants by all growth regulators, but subsequent organogenesis was observed in case of leaf explants, on medium containing different concentration on BAP and IAA, Kinetin and IAA, TDZ (high conc), TDZ and BAP whereas, in case of petiole explants TDZ and adenine combination also induced adventitious buds in some of the concentrations. Shoot bud induction was seen after nearly 3-4 weeks in culture from the explants and the in vitro shoots were transferred to another medium for shoot elongation. A. Shoot from leaf and petiole explants Effect of BAP and IAA on shoot : Different concentrations and combination of BAP and IAA mg/l (in MS medium) were used for shoot induction from leaf and petiole explants. In leaf explants, the highest frequency 68.08% of shoot with 1.87 shoots occurred on medium supplemented with 1.0mg/l BAP and 0.5mg/l IAA and the least shoot 22.93% was obtained with MS medium containing 1mg/l BAP + 1mg/l IAA (Table 1). In petiole explants, maximum 78.69% shoot with 2.05 shoots per explant was observed on MS medium supplemented with 2mg/l BAP + 1mg/l IAA and mimimum (36.07%) with that of 2.5 mg/l BAP + 1mg/l IAA (Table 5, Figs.1-6). Effect of Kinetin and IAA on shoot bud induction: Six different combinations of Kinetin and IAA were used for shoot induction from leaf and petiole explants. The highest percentage of shoot 66.63% and 80.01% with maximum 1.19 and 2.12 number of shoots s were observed on MS medium supplemented with 1mg/l Kinetin + 1mg/l IAA for both the leaf and petiole explants respectively. The least response for the leaf explants was observed on MS medium containing 2mg/l Kinetin + 1mg/l IAA (0.66) whereas for petiole explants, minimum number of shoots formed per explants (0.58) were recorded on MS medium containing 2.5mg/l Kinetin mg/l IAA (Tables 2, 6; Figs.1-6). Effect of TDZ on shoot bud development: TDZ was used in 14 different concentrations to get shoot from leaf and petiole explants. The TDZ (thidiazuron) at low concentration (0.10, 0.20, 0.30 and 0.40 mg/l ) were not effective on shoot from leaf and petiole explants of tomato. 3915

4 J. Cell Tissue Research Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig

5 Sharma and Srivastawa Table 1: Effect of various combinations and concentrations of BAP and IAA in MS Basal medium used for shoot from leaf explants in tomato. *Values in parenthesis are the arc sine transformed values. of shoots formed 1. MS mg/l BA+ 0.5 mg/l IAA (55.61) 2. MS mg/l BA+ 0.5 mg/l IAA (42.20) 3. MS mg/l BA+ 0.5 mg/l IAA (47.02) 4. MS mg/l BA+ 1.0 mg/l IAA (28.56) 5. MS mg/l BA+ 1.0 mg/l IAA (40.93) 6. MS mg/l BA+ 1.0 mg/l IAA (43.06) CD SE± Table 2: Effect of various combinations and concentrations of Kinetin and IAA in MS Basal medium used for shoot from leaf explants in tomato. *Values in parenthesis are the arc sine transformed values. Of shoots forme d 1. MS mg/l Kn+ 0.2 mg/l IAA (49.05) 2. MS mg/l Kn+ 0.5 mg/l IAA (46.91) 3. MS mg/l Kn+ 0.5 mg/l IAA (50.35) 4. MS mg/l Kn+ 0.5 mg/l IAA (46.91) 5. MS mg/l Kn+ 1.0 mg/l IAA (54.79) 6. MS mg/l Kn+ 1.0 mg/l IAA (41.35) CD SE± Table 3: Effect of various combinations and concentrations of TDZ in MS Basal medium used for shoot from leaf explants in tomato. *Values in parenthesis are the arc sine transformed values. Of shoots formed Regeneration 1. MS mg/l TDZ (32.45) 2. MS mg/l TDZ (32.40) 3. MS mg/l TDZ (41.35) 4. MS mg/l TDZ (39.18) 5. MS mg/l TDZ (41.78) 6. MS mg/l TDZ (45.61) 7. MS mg/l TDZ (40.51) 8. MS mg/l TDZ (37.90) 9. MS mg/l TDZ (33.38) CD SE± Table 4: Effect of various combinations and concentrations of BAP and TDZ in MS Basal medium used for shoot from leaf explants in tomato. *Values in parenthesis are the arc sine transformed values. ofshoots formed 1. MS+ 0.1mg/l TDZ+ 1.0mg/l BAP (44.33) 2. MS + 0.1mg/l TDZ+ 2.0mg/l BAP (45.61) 3. MS+ 0.1 mg/l TDZ+ 2.5mg/l BAP (49.05) 4. MS+ 0.5mg/l TDZ+ 1.0mg/l BAP (46.04) 5. MS+ 0.5 mg/l TDZ+ 2.0mg/l BAP (46.91) 6. MS+ 0.5 mg/l TDZ+ 2.5mg/l BAP (48.62) 7. MS+ 2.0 mg/l TDZ+ 1.0mg/l BAP (40.39) 8. MS+ 2.0 mg/l TDZ+ 2.0mg/l BAP (40.85) 9. MS+ 2.0 mg/l TDZ+ 2.5mg/l BAP (42.63) CD SE± Table 5: Effect of various combinations and concentrations of BAP and IAA in MS Basal medium used for shoot from petiole explants in tomato. *Values in parenthesis are the arc sine transformed values. of shoots formed 1. MS mg/l BA+ 0.5 mg/l IAA (56.47) 2. MS mg/l BA+ 0.5 mg/l IAA (58.23) 3. MS mg/l BA+ 0.5 mg/l IAA (51.87) 4. MS mg/l BA+ 1.0 mg/l IAA (49.77) 5. MS mg/l BA+ 1.0 mg/l IAA (62.61) 6. MS mg/l BA+ 1.0 mg/l IAA (36.78) CD SE± Table 6: Effect of various combinations and concentrations of Kinetin and IAA in MS Basal medium used for shoot from petiole explants in tomato. *Values in parenthesis are the arc sine transformed values. Of shoots formed s 1. MS mg/l Kn+ 0.2 mg/l IAA (47.56) 2. MS mg/l Kn+ 0.5 mg/l IAA (57.05) 3. MS mg/l Kn+ 0.5 mg/l IAA (47.05) 4. MS mg/l Kn+ 0.5 mg/l IAA (39.38) 5. MS mg/l Kn+ 1.0 mg/l IAA (64.27) 6. MS mg/l Kn+ 1.0 mg/l IAA (57.64) CD SE± Explanations of figures: Fig. 1 to 6: Plant Regeneration studies in tomato (Solanum lycopersicum cv. Solan Vajr) Fig. 1. Shoot from leaf explants cultured on medium {MS (Basal medium) mg/l BAP mg/l IAA } after 4-5 weeks. Fig. 2. Shoot from petiole explants cultured on medium {MS (Basal medium) mg/l Kn mg/l IAA } after 4-5 weeks. Fig. 3. Regenerated shoots transferred to root medium. Fig. 4. In vitro well developed rooted plantlets of tomato. Fig. 5. In vitro regenerated plantlets of tomato transferred to the pots containing mixture of sand: soil (1:1) for acclimatization. Fig. 6. Acclimatized young healthy plantlets transferred to soil in earthen pots. 3917

6 J. Cell Tissue Research Table 7: Effect of various combinations and concentrations of TDZ in MS Basal medium used for shoot from petiole explants in tomato. *Values in parenthesis are the arc sine transformed values. Medium Composition Of shoots formed Regeneration 1. MS mg/l TDZ (31.75) 2. MS mg/l TDZ (33.50) 3. MS mg/l TDZ (37.43) 4. MS mg/l TDZ (35.25) 5. MS mg/l TDZ (38.53) 6. MS mg/l TDZ (38.53) 7. MS mg/l TDZ (42.31) 8. MS mg/l TDZ No shoot No shoot 9. MS mg/l TDZ No shoot No shoot CD SE± Table 8: Effect of various combinations and concentrations of BAP and TDZ in MS Basal medium used for shoot from petiole explants in tomato. *Values in parenthesis are the arc sine transformed values. of shoots formed 1. MS+ 0.1mg/l TDZ+ 1.0mg/l BAP (37.98) 2. MS + 0.1mg/l TDZ+ 2.0mg/l BAP (47.12) 3. MS+ 0.1 mg/l TDZ+ 2.5mg/l BAP (43.91) 4. MS+ 0.5mg/l TDZ+ 1.0mg/l BAP (41.78) 5. MS+ 0.5 mg/l TDZ+ 2.0mg/l BAP (46.59) 6. MS+ 0.5 mg/l TDZ+ 2.5mg/l BAP (43.38) 7. MS+ 2.0 mg/l TDZ+ 1.0mg/l BAP (31.75) 8. MS+ 2.0 mg/l TDZ+ 2.0mg/l BAP (37.98) 9. MS+ 2.0 mg/l TDZ+ 2.5mg/l BAP (38.53) CD SE± Table 9: Effect of different concentration of various auxins on per cent root from in vitro developed shoots Treatments Concentrations (mg/l) IAA 70± ± ± 0.67 NAA 70± ± ± 1.33 IBA 75± ± ± 0.67 The MS medium supplemented with 2 mg/l TDZ was observed the best (51.07%) for shoot and number of shoots (0.73) in case of leaf explants whereas in case of petiole explants, MS medium supplemented with 2.5 mg/l TDZ resulted in 45.33% of shoot with 0.47 number of shoots. At low concentration only 0.5mg/ l and 0.6 mg/l of TDZ was effective in shoot induction from the leaf and petiole explants (Tables 3 and 7). Effect of TDZ and adenine on shoot : Nine different combinations and concentrations of TDZ and Adenine were used for shoot from leaf and petiole explants. In leaf explants, only callus formation was observed with no further shoot differentiation. In case of petiole explants, MS medium supplemented with 2.5mg/l TDZ mM Adenine and 3.0mg/l TDZ mM Adenine and 3.5mg/l TDZ mM Adenine showed shoot with maximum number of shoots per explant (0.43) with the last combination. However, at 1.25mg/l TDZ, 1.50mg/l TDZ mM Adenine root was observed in the medium (data not shown). Effect of TDZ and IAA on shoot bud formation: Leaf and petiole explants were cultured on nine different compositions of TDZ and IAA for shoot but no shoot was observed from both the leaf and petiole explants up to 60 days of culturing. However, callus formation was observed for both the explants in all the combinations (data not shown). Effect of TDZ and NAA on shoot : TDZ and NAA were used in nine different combinations and concentrations for leaf and petiole explants. In leaf explants, first four combinations showed callus formation while in others marginal callusing was observed. In case of petiole explants, only marginal callus was recorded. No shoot was observed with both the explants (data not shown). Effect of BAP and TDZ on shoot induction: Nine combinations of BAP and TDZ (mg/l) were used in the MS medium for shoot induction and best results of shoot frequency were observed in MS medium containing 2.5mg/l BAP and 0.1mg/l TDZ (57.02%) and 2.0mg/l BAP and 0.1mg/l TDZ (53.69%) with 0.87 and 0.88 number of shoots per explant for leaf and petiole explants respectively whereas MS medium supplemented with 1mg/l BAP and 2mg/l TDZ gave the lowest shoot for both the leaf (42.00 %) and petiole (27.77%) explants respectively (Tables 4 and 8). Root from in vitro developed shoots: Root induction on regenerated shoots is essential for the successful establishment of the plantlet on the soil. Regenerated shoots (about 2-3cm in length) obtained from the leaf and petiole explants were excised and cultured separately on MS medium supplemented with various concentrations of different auxins i.e. IAA, NAA and IBA. Root 3918

7 Sharma and Srivastawa was initiated within 2-3 weeks of inoculation in all the auxins used. IBA (0.20 mg/l) supplemented MS medium reported rooting at interval of days with maximum 95 % rooting, however MS medium supplemented with 0.20 mg/l NAA resulted in 90% rooting followed by 0.20mg/l IAA resulting in 85% rooting. (Table 9, Figs. 1-6) Hardening of regenerated plantlets of tomato: Since, in vitro rooted plantlets are raised in the most amiable environment conditions, hardening is vital to ensure survival of the regenerated plantlets upon transfer to soil under natural conditions. Therefore, the rooted plantlets were transferred to plastic pots containing autoclaved soil, sand and FYM and humidity was maintained by covering with plastic bags in the culture room conditions. After 3-4 weeks of transfer to the plastic pots, these plants showed 75% survival. Acclimatized plants were transferred to glass house conditions where they showed flowering and responded similar to normal plants (Figs. 1-6). DISCUSSION Tomato is one of the most studied higher plants because of its importance as a crop species, and of several advantages for genetic, molecular and physiological studies [18]. In order to establish an efficient genetic transformation system in the cultivar, we considered it necessary to test the efficiency of plant from leaf and petiole explants for experimental use. In the present study, days old seedling, with almost fully expanded green leaves and light green and turgid petioles were used as explants for shoot. The young and physiologically active explants show good response to external environment such as exogenous use of growth regulators. Further, the de-differentiation of tomato leaf and petiole explants into callus followed or not followed by shoot formation depends on the culture medium and the physiological state of the donor plant [19].The effect of age of donor seedlings on the shoot potential has also been studied in Solanum lycopersicum [7,20]. Auxins and cytokinins are involved in cell division and elongation, while; cytokinins help in the process of differentiation. So, appropriate concentration of these plant growth regulators are necessary for cell division and differentiation. Out of the seven different combinations of growth regulators (BAP, IAA, Kinetin, TDZ, Adenine and NAA) used for shoot from leaf explants the high frequency of shoot (68.08 %) was obtained on MS medium containing 1mg/l BA+ 0.5mg/l IAA. The presence of high cytokinins with low or equal amount of auxins was also confirmed by Gubis et al. [15], where they observed the capacity of six types of explants in 13 cultivars of tomato (L. esculentum). The presence of equal amounts of auxin and cytokinin in the media, were also confirmed [21]. In case of petiole explants, high frequency (80.01%) was recorded on MS medium supplemented with 1mg/l Kn + 1mg/l IAA followed by 2mg/l BAP and 1mg/l IAA (78.69%) resulting better performance of petiole in, indicating that the petiole explants of tomato is an excellent explant for plant studies. TDZ, one of the most active cytokinins, is a synthetic phenylurea used for shoot inductionn in plant tissue culture [22]. TDZ is involved in cytokinin metabolism [23] and also induce accumulation of endogenous cytokinin [24]. In the present investigation, leaf explants showed shoot (51.07 %) and (57.02 %) only on MS medium containing TDZ (alone) and TDZ+ BAP, respectively while in rest of the combinations (TDZ+ Adenine, TDZ+ IAA and TDZ +NAA) no callus differentiation was observed. MS medium supplemented with 2mg/l TDZ gave high frequency shoot which is in accordance to earlier work done [25], who showed better response at low TDZ concentration. TDZ in combination with BAP showed better results for percent shoot which is similar to results those obtained by Khalaffa et al. [10]. In petiole explants, TDZ at very low concentration up to 0.4mg/ l and at higher concentration than 3mg/l showed no shoot. MS medium supplemented with 3mg/l TDZ mM Adenine showed low levels of shoot while in other combinations of Adenine and low concentration of TDZ, no shoot was obtained whereas, TDZ along with BAP recorded 54 % shoot. Although same combination gave best results for shoot from hypocotyls explants [10] but no such previous works have been reported for the petiole explants. Previous studies however demonstrated various explants including hypocotyls, cotyledon, leaf and stems [6,10,25,26] for promoting shoot in tomato but no reports have been 3919

8 J. Cell Tissue Research recorded on the use of petiole explants using TDZ (alone) or in combination with other growth regulators as a source of efficient shoot. Different auxins i.e. NAA, IAA and IBA were used in MS medium for root. Best root (95%) was observed within days on MS medium supplemented with 0.20 mg/l IBA. Devi et al. [27] reported the use of only half strength MS medium whereas, Khalaffa et al. [10] reported half strength MS medium supplemented with 1mg/l IAA as best medium for root. Swamy et al. [28] reported 100% root on MS medium supplemented with 0.5mg/l IAA. The regenerated complete plantlets were transferred to small pots containing mixture of sterilized soil: sand (1:1) for acclimatization. The acclimatized plantlets were obtained within 2-3 months. A protocol for high frequency plant has been standardized in tomato. The present protocol would be used in genetic transformation of tomato to obtain transgenic plants with chitinase gene. AKNOWLEDGEMENTS We are grateful to Professor and Head, Department of Vegetable Science, Dr Y. Parmar University of Horticulture and Forestry, Solan, India for providing the germplasm of tomato cv. Solan Vajr. Abbreviations: IAA-Indole-3-acetic acid; IBA- Indole-3-butyric acid; NAA-á-naphthalene acetic acid; BAP-N 6 benzylaminopurine, TDZ- N-phenyl- N-thiadiazol-1, 2, 3, 5- yl urea (thidiazuran), Kn- Kinetin, Ad- Adenine REFERENCES [1] Wing, A.R., Zhang, B.H. and Tanksley, D.: Molecular Genetics and Genomics, 242: (1994). [2] Vavilov, N.I.: Chronica Botanica, 1(6): 364 (1967). [3] (National Horticulture Board, 2011). [4] news/ erractic-monsoon-hits-himachaltomato-crop/ (Indo Asian News Service, 2009). [5] Jones, J.B., Jones, J.P., Stall, R.E. and Zitter, T.A.: Compendium of tomato diseases. APS press, St. Paul. MN, USA (1991). [6] Ling, H.Q., Kriseleit, D. and Ganal, M.G.: Plant Cell Reports, 17: (1998). [7] Rashid, H., Chaudhary, Z. and Afroz, A.: Pakistan J. Botany, 39(3): (2007). [8] Raj, K., Singh, R., Pandey, K. and Singh, B.P.: Current Sci., 88: (2005). [9] Gubis, J., Lajchová, Z., Faragó, J. and Jureková, Z.: J. Genetics Plant Breeding, 39: 9-14 (2003). [10] Khalafalla, M.M., Magdoleen, G.O. and Elsadig, A.E.: African J. Biotechnol., 9(28): (2010). [11] Moghaleb, R.E.A., Saneoka, H. and Fujita, K.: Soil Sci. Plant Nutrition, 45: (1999). [12] Jatoi, A., Sajid, G.M., Quraishi, A. and Munir, M.: Pakistan J. Plant Sci.,1(2): (2001). [13] Davis, M.E., Miller, A.K. and Lineberger, R.D.: J. Exp. Botany, 42: (1994). [14] Murashige, T. and Skoog, F : Physiologia Plantarum, 15: (1962). [15] Gubis, J., Lajchová, Z., Faragó, J. and Jureková, Z.: Biologia Bratislava, 59(3): (2004). [16] Pal, P., Alam, I., Anisuzzaman, M., Sarker, K.K., Sharmin A. and Alm, M.F.: Turkish J. Agricul. Forestry, 31: (2007). [17] Nikam, T.D. and Shitole, M.G.: Plant Cell Tissue Organ Culture, 55: (1998). [18] McCormick,, Niedermeyer, J., Fry, J., Barnason, A., Horsch R. and Fraley, R.: Plant Cell Reports, 5: (1986). [19] Guillermo, P., Canepa, L.N., Zorzoli, R. and Picardi, L.A.: Horticulture Sci., 38(1): (2003). [20] Reda, E., Moghaieb, A., Saneoka, H. and Fujita, K.: Cell Mol. Biol. Letters, 9: (2004). [21] Botau, D., Frantescu, M. and Darlea, A.: Biodiversitate, 10: (2002). [22] Huetteman, C.A. and Preece, J. E.: Plant Cell Tissue and Organ Culture, 33: (1993). [23] Mok, M.C., Mok, D.W., Armstrong, D.J., Shudo, K., Isogai Y. and Okamoto, T.: Phytochemistry, 21: (1982). [24] Hutchinson, M.J., Murch, J. and Saxena, P.K.: J. Plant Physio., 149: (1996). [25] Girija,, Kilankaje, A., Velu,, Malaiyandi, J. and Vaithiyanathan, G.: Asian J. Plant Sci. and Res., 1(2): (2011). [26] Van Roekel, J.C., Damm, B., Melchers, L. and Hoekema, A.: Plant Cell Reports, 12: (1993). [27] Devi, R., Gosal,, Dhaliwal, M. and Kaur, A.: Indian J. Biotechnol., 7: (2008). [28] Swamy, N.R., Godishala, V., Kairamkonda, M., Kagithoju, and Mangamoori, L.: Amer. J. Crop Sci., 6(1): (2011). 3920