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Tomato transformation and regeneration was optimized by using different growth regulators. Cotyledon and epicotyls explants from Solanum lycopersicum L. were used as an explants and infected by Agrobacterium tumefaciens strain AGL1 harbouring the Artificial Zinc Finger (AZF) gene. The effects of Putrescine, vitamins, explants and growth regulators on plant transformation and regeneration were studied. Optimal shoot regeneration and callus induction was obtained with 1 Ã- 108 bacterial concentration for 48 hours, 1mM Putresine, 0.5 Zeatin Riboside and 0.1 mg/ml IAA in callus induction/ shoot regeneration media. Under the above conditions transformation efficiency reached 49.2% in cotyledon explants. Transformed shoots were selected on kanamycin medium and the presence of the transgene in the primary transformants was confirmed by PCR. Transgenics are being developed in Oman against TYLCV-Om virus using this regeneration protocol.
Tomato (Solanum lycopersicum L.) is one of the most important and widely cultivated year-round in tropical and subtropical regions of the world. Tomato is a favorable food crop for In vitro studies due to its relatively small DNA content and is a genetic model for crop improvement (Arumuganathan and Earle 1991). The development of efficient and genotype independent tomato transformation procedure is crucial.
Apart from insect pests (Heliothis armigera, Bemisia tabaci, Thrip palmi), there are diseases which significantly contribute to the yield gap including fungal, bacterial and virus diseases. Viruses are the most prominent limiting factor reducing tomato yield around the globe. Tomato yellow leaf curl disease (TYLCD), caused by the Tomato yellow leaf curl virus (TYLCV) and related viruses was first reported in the Jordan valley (Cohen & Harpaz, 1964), has become a serious problem that affects tomato worldwide. TYLCV causes a considerable loss to tomato crop every year by causing a range of symptoms such as leaf curling, stunted growth, erect or upright growth and leaves curl upward and develop crumpling and distinctive interveinal chlorosis resulting in poor yield of poor fruit quality (Raquel et al., 2002).
Agronomists and plant pathologists have devoted considerable effort toward controlling virus diseases by traditional breeding, intensive use of insecticides/ pesticides and physical barriers e.g., Agryl cover. Plant genetic engineering could help plant breeders by creating and manipulating genetic variability. There is dire need to develop genetic variation in tomato that is highly resistant to several biotic and abiotic stress conditions (Kuckuck et al. 1991, Larkin, 1996 and Bhatia et al., 2004).
Artificial zinc finger protein-based sequences are based on Non Pathogen Derived Resistance (NPDR) approach have an important advantage over other mechanisms of resistance, such as truncated or mutated Reps that require high levels of expression. They have high affinity for the "Rep-specific direct repeats "and capable to bind specific stretches of both ssDNA or dsDNA to block the binding of "Rep" to "virion - ori" of geminivirus. Specificity of artificial zinc finger protein-v-ori binding site interactions, its high affinity and effective resistance might be achievable with low level expression of these proteins This approach was successfully demonstrated in Arabidopsis thaliana against Beet severe curly top virus (BSCTV) (Sera, 2005).
Since 1986 Agrobacterium-mediated tomato transformation have been developed for a variety of purposes, including characterization of gene function, production of insect- and disease-resistant plants, herbicide tolerance, improved fruit quality, delay in fruit ripening and production of foreign proteins (Davuluri et al. 2005; Fillatti et al. 1987; Janssen et al. 1998; Lin et al. 2004; Park et al. 2003; Youm et al. 2008). Factors influencing transformation efficiency such as explants (cotyledons, stem, and leaf), plant variety, growth regulators and bacterial concentration have been described in literature. Transformation efficiency reported in literature is 9% (Roekel et al. 1993), 11% (Frary and Earle 1996), 20% (Qiu et al. 2007), 41.4% (Sharma et al. 2009) and 28-48% (Sun et al. 2006).
Putrescine is naturally occurring low molecular weight organic cations Polyamines. It has been implicated in various cellular and developmental processes such as cell division, protein synthesis, DNA replication, and response to abiotic stress (Kakkar and Sawhney, 2002). Recent reports have indicated that Putrescine have a potency to enhance somatic embryogenesis in several plant species (Sakhanokho et al., 2005; Kakkar and Sawhney, 2002; Kevers et al., 2002 ).
The present investigation attempted to characterize the synergetic effect of putrescine in combination with other growth regulators on plant cell transformation, explants survival and shoots regeneration. In this study tomato was transformed with AZF gene to silence TYLCV using optimized Agrobacterium-mediated transformation by avoiding feeder layer. This method is also applicable to different tomato cultivars as well as for other Solanaceous species.
2 Materials and methods
2.1 Growth conditions
Cotyledons and epicotyl from 8-9-day-old plants were used as explants. To generate explants material, seeds of Solanum lycopersicum var. Pusa Ruby were washed 2-3 times with sterilized distilled water followed by dipping in liquid bleach 13% until seed coat is completely white. Surface sterilized seeds were washed again for 5-6 times with sterilized distilled water to remove bleach. Finally, seeds were germinated on seed germination medium (table 1) in growth chamber under fluorescent light with 12/24 photoperiod. Ten days after seed inoculation, seedlings were used for preparation of explants.
The effects of four bacterial cell densities, two different co-cultivation time periods, two different explants and six different plant growth regulator combinations on transformation efficiency were studied. To study the influence of these treatments on plant regeneration, three independent experiments were performed for each factor with a minimum of 100 explants in each experiment. All plantlets regenerated from a single callus were labelled as clones to indicate that they represented a single transformation event.
2.2 Culture media
The composition of various media is described in table 1. Media components were mixed and the pH adjusted to 5.8 using 1 M KOH prior to addition of 0.8% w/v plant tissue-culture grade phytoagar powder and autoclaved.
The growth regulators BAP, zeatin riboside and zeatin were dissolved in 1N NaoH and IAA was dissolved in 25% ethanol. 1mM putrescine stock was prepared by dissolving in MQ water. The antibiotics kanamycin, carbenicillin and cefotaxime were prepared in MQ water and filter sterilized, whereas rifampicin was dissolved in methanol. Antibiotics and growth regulators were added to the autoclaved medium after it cooled to ~50Â°C.
2.3 Bacterial strain and plasmids
Agrobacterium tumefaciens strain AGL1 was used for plant transformations. PGreen 0029 was used as plant transformation vector in which promoter pMAS (monoamine synthase promoter) and terminator tMAS (monoamine synthase terminator) was cloned at ECoRV site. AZF gene was synthesized by genescript the whole gene was 595bp long and supplied in puc57 vector. The whole gene was lifted from puc57 at SalI/EcoRI site and cloned in pGreen vector containing pMAS promoter and tMAS terminator. The clones were confirmed by SalI/EcoRI site (fig 1). AZF gene was then electroporated in AGL1 strain and colony PCR was performed to identify transforments, glycerol stock was prepared and preserved in -80oC. Single colony from freshly grown plate was picked and inoculated in 10 ml YEM medium containing 100 mg/l carbenicillin and 50 mg/l kanamycin was incubated at 28Â°C shaker for 48 h. When OD600 of primary culture reched 0.8-1.00, 50 ml of fresh YEM medium was inoculated with 5ml of primary culture and incubated in shaker at 28Â°C for 3-4 h until OD600 reach 0.5-0.8. At this point acetosyringone 200ÂµM was added and incubated for additional one hour. Agro cells were collected at 6000rpm for 10 minutes and resuspended in 50 ml fresh YEM medium and used for transformation.
Figure 1: Schematic presesntation of TDNA border of plant transformation vector pGreen0029 containing AZF gene under monoamine synthase promoter / terminator.
2.4 Plant transformation
Cotyledons from 8-day-old seedlings were cut at the tip and base. Middle pieces (~0.5 cm Ã- 0.7 cm) were precultured for 48 h at 28Â°C on pre-culture medium (table 1), with the adaxial surface in contact with the medium. Healthy explants that responded to pre-culture, as evident by swelling, were incubated in the bacterial suspension for 30 min and inverted every 10 min during incubation. The explants were then blotted on sterile tissue paper and co-cultured on the same pre-culture medium for 72 h at 28Â°C in dark. After exposure for different experimental durations, co-cultured explants were transferred to a selection medium (table 1) for regeneration. Each Petri plate (9 cm) had 20-25 explants for regeneration. Plates were cultured under a 16 h light/8 h dark cycle at 28Â°C. Explants that showed regeneration or callus formation were sub cultured onto fresh selection medium every 15 days. Regenerated shoots containing callus were transferred to shoot elongation media (table 1). Shoots were excised from callus and transferred to rooting medium (table 1). Those plantlets which attained good shoot development (~8 cm in height) and produced roots were transferred to pots containing sterilized potting mix (potting soil, peat moss, sand 1:1:1 ratio) for hardening. Pots were kept in a humidity chamber for 3-5 days in the culture room under a 16 h light/8 h dark cycle at 28Â°C and then in the greenhouse at 28 Â± 2Â°C.
Table 1: Composition of different growth regulators used in various transformation and regeneration media.
Zeatin Riboside (mg/L)
a In the medium, various combinations of hormones were used. For details see table 3.
b Murashige and Skoog 1962
c Gamborg et al. 1968
2.6 Statistical analysis:
Six independent experiments were carried out with different culture media consisting of various combinations of plant growth regulators and 100 explants (cotyledon, hypocotyls) were tested each time. Analysis of variance (ANOVA) was performed for the effect of various plant growth regulators on plant regeneration and transformation. Means were compared using the least significant difference test (LSD) at P < 0.05.
2.7 Genetic and molecular analysis:
Plant genomic DNA was extracted from 0.1 g (fresh weight) of plant tissue using CTAB method of DNA isolation. Genomic DNA was PCR amplified by 5-forward and 3-reverse AZF gene primers.
3. Results & Discussion:
3.1Effect of bacterial concentration on transformation.:
Bacterial densities of 0.5 Ã- 108, 0.6 Ã- 108, 0.8 Ã- 108 and 1.0 Ã- 108 cells/ml culture were used for incubation and co-cultivation of cotyledon/epicotyls explants for 48 or 72 h (table 2). Optimum concentration of bacterial culture needed for successful transmission of T-DNA border was found to be 1.0 Ã- 108 cells/ml for 48 hours. Excessive growth of agrobectrium was observed if incubation time was increased from 48h to 72 h which in turn affect the overall survival of explants. Reduced bacterial density of 0.8Ã- 108 showed almost same transformation efficiency when incubated for 48h or 72 h however the rate of survival of explants was more when incubation time was 48h. Interestingly 0.8Ã- 108 and 1 Ã- 108 were having enough number of survived explants which in turn will produce transgenic shoots.
Table 2: Effect of bacterial concentration and incubation time on overall efficiency of tomato transformation.
Bacterial concentration (cells/ml)
Average % transformation efficiency
48 h 72 h
0.5 Ã- 108
28.6 Â± 1.5
32.2 Â± 2.8
0.6 Ã- 108
30.2 Â± 1.3
33.4 Â± 1.5
0.8 Ã- 108
45.7 Â± 2.3
44.8 Â± 2.5
1 Ã- 108
47.2 Â± 2.8
45. 4 Â± 2.1
3.2 Effect of Putrescine on tomato explants survival:
Callus induction as well as shoot initiation was observed from cotyledon and epicotyl explants cultured on various regeneration media. The effect of putrescine on shoot regeneration medium in combination with other growth regulators is demonstrated in (table 3). Cytokinin zeatin riboside has been previously reported by Cortina and Culianez-Macia, 2004 to have profound effect in regeneration of tomato.
Tomato explants (cotyledons, epicotyls) on MS medium (Murashiage and Skoog, 1962) containing M1 reported by Cortina and Culianez-Macia, 2004 shows 12% Transformation efficieny in cotyledon, 10% in epicotyls (Fig 3). According to current observation green callus was observed but the overall efficiency was low which is contrary to Cortina and Culianez-Macia, 2004.
Growth regulator combination M2 reported by sharma et al, 2009 showed 17% transformation efficiency in cotyledon and 16% in epicotyls (Fig 3). According to current observation zeatin riboside has found to increase transformation efficiency than zeatin. This finding is according to Cortina and Culianez-Macia, 2004.
Tomato explants on MS medium containing M5 growth regulators combination (table 3) shows dramatic increase in shoot regeneration after two weeks of cocultivation (Fig 2). In M5 combination of growth regulators transformation efficiency was 49.2% in cotyledons and 33% in epicotyls explants (Fig3). Whereas M3 combination contains zeatin instead of zeatin riboside with 1mM putrescine showed less regeneration efficiency 25.6 % cotyledons, 24.3% epicotyls (Fig 3) respectively.
Number of shoots produced per explant was also found to be highest when initiated shoots were transferred to proliferation medium with ZR (0.5 mg dm-3), IAA (0.1 mg dm-3) and Putrescine 1mM (Fig 2). No regeneration response was observed from explants cultured on MS basal medium without growth regulators.
Table 3: Effect of previously reported different growth regulators combinations on tomato regeneration and shoot development stages.
Concentration of growth regulators (mg/L)
a Cortina and Culianez-Macia 2004
b Sharma et al 2009
c (Kakkar and Sawhney, 2002; Sakhanokho et al., 2005; Kevers et al., 2002)
d Rogozinska and Skutnik 1974
e Harry klee's lab protocol
Contrary to current observations, Rogozinska and Skutnik (1974), reported combination of BAP and NAA to be superior for shoot regeneration in tomato. Park et al. (2003) and Cortina and Culianez-Macia (2004), used NAA and BAP in pre-culture medium. However, in the present investigation rhizogenesis was observed from explants cultured on NAA and BAP combination as shown in figure 2 with M4 and M6 growth regulator combinations contrary to (Chandel and Katiyar 2000, Park et al. 2001).
Figure 2: Effect of different growth regulators combinations with/without putrescine on callus induction and shoot regeneration medium.
Figure 3: Effect of different growth regulators combination on shoot regeneration medium. Higher percentage of explants survival (cotyledon, epicotyls) was observed with 1mM putrescine and 1mg/ml zeatin riboside M5. Data represents the mean value of six independent experiment (100 explants for each independent transformation procedure) Â±SD. All the values were found to be significantly different (p< 0.05) using LSD.
Green kanamycin resistant callus along with shoot primordia was observed on the cut ends of cultured explants 2 to 3 weeks post transformation. After 4 to 8 weeks of shoot initiation, cotyledon explants were cut from the regenerating shoots and discarded. Enhanced shoot proliferation and elongation was observed after 10 weeks of culture on shoot proliferation medium supplemented with Putrescine 1mM and BAP (1.0 mg dm-3) + kanamycin (50 mg dm-3) + cefotaxime (250 mg dm-3). Half strength MSB5 medium without any growth regulator was found suitable for rooting of transformed shoots, where 80 % of shoots produced roots after 10 to 12 d. For better survival of transformed shoots, the plantlets were further transferred to liquid half-strength MSB5 medium supplemented with kanamycin (50 mg dm-3) + cefotaxime (250 mg dm-3), where hairy root formation was observed within 10 d. Transformed plants were hardened in small pots where 90 % survival rate was observed. Fully regenerated putative transgenic plants were transferred to phytotron, where these acclimatized plants grew to maturity and produced normal flowers and fruits. The transformants were observed to be morphologically normal and fertile.
3.3 Molecular analysis of T0 Transgenic plants:
DNA from non transformed control and five-selected T0 independently transformed AZF transgenic plants were extracted by using CTAB method. DNA was PCR amplified using 5-forward and 3-reverse AZF gene primers (Figure 3). A band of about 595 bp, corresponding to the predicted size of the gene fragment, confirmed the T-DNA integration in the genome of these plants. An amplicon of 595 bp corresponding to the predicted size of gene fragment confirmed the transgene integration in the plant.
L5 L4 L3 L2 L1
595bpC:\Users\Administrator\AppData\Local\Microsoft\Windows\Temporary Internet Files\Content.Word\G5, AZF,NPDR, Rnai.jpg
Figure 3: Genomic DNA from transgenic AZF plants wild type control and 5 independent lines. PCR was done to amplify AZF gene 595 bp by using AZF gene primers
Agrobacterium mediated tomato transformation has become a routine but challenging practice to obtain a number of positive plants from a reasonable amount of viable calluses. This work has developed an easy and efficient Agrobacterium mediated method for tomato transformation and regeneration. Based on previous modifications of MS medium we tested different combinations of growth regulators, bacterial density, incubation time and explants on overall survival of explants. The effect of putrescine in combination with Zeatin and zeatin riboside. Putrescine 1mM along with 1.0 mg/ml zeatin riboside in MS increased cell growth and decreased the expansion of necrotic lesions. Using cytokinin zeatin riboside instead of zeatin increased shoot regeneration rate and similer results were found by Cortina and Culianez-Macia (2004).
MS medium in combination with B5 vitamins, where the concentration of thiamine HCl was ten-fold higher as compared to MS vitamins and observed increased cell-growth and shoot proliferation with reduced necrotic lesions in transformed tissues. Similar results were found by Raj et al. (2005). The transformation efficiency was improved four times and 90% of the resistant shoots rooted on kanamycin and were confirmed as transgenic plants. In conclusion, this is simplified and improved tomato regeneration protocol to produce transformed shoots through Agrobacterium-mediated transformation.