Abstract: In this study the effect of three genotypes (KalG, Kal-early and Su2270 as the commercial cultivars in Iran), two explants (cotyledon and hypocotyl), growth regulators (BAP, NAA and Zeatin) and a strain of Agrobacterium tumefaciense (pGV3850) on regeneration and transformation efficiency is reported. Although all cultivars and explants were regenerated on the different media, however our results showed that there were significant differences between genotypes and explants in the number of shoot formation per explant. Optimal regeneration of KalG, Kal-early and Su2270 cultivars were observed on MS media supplemented with 2 mg L-1 Zeatin and 2 mg L-1 BAP, respectively. The maximum number of shoot formation per explant was observed in cotyledon and hypocotyle explants in KalG and Su2270 cultivars, respectively. The transformation rate was 17% for Kal-early to 35% for KalG cultivars. Transformation were confirmed by Gus assay and PCR analysis.
INTRODUCTION
Tomato (Lycopersicon esculentum) is one of the most important vegetable crops in the world as a source of vitamins, karoteins, amino acids, sugars and other substances that have an important role in human health. Since the first report of tomato transformation (McCormick et al., 1986), there have been numerous publications of genetic manipulation on several cultivars of tomato (Chi and Phillips, 1987; Fillatti et al., 1987; Fischhoff et al., 1987; Delannay et al., 1989; Van Roekel et al., 1993; Agharbaoui et al., 1995; Frary and Earle, 1996; Ling et al., 1998; Tabaeizadeh et al., 1999; Vidya et al., 2000; Hu and Phillips, 2001). However, the transformation of the tomato using Agrobacterium tumefaciens is highly dependent on genotypes. Lack of efficient regeneration procedures is a major obstacle in the application of gene transfer technology to economically important tomato cultivars (Velcheva et al., 2005). Until now different explants such as leaf disks (McCormick et al., 1986), cotyledon (Van Roekel et al., 1993), protoplasts (Roset and Gilissen, 1989) and hypocotyls (Park et al., 2003) were used for tomato transformation. Different concentrations of BAP, IAA, NAA and Zeatin were used for direct shoot regeneration in tomato cultivars (Park et al., 2003; Velcheva et al., 2005). Also, different factors were found to play major roles in tomato transformation: Agrobacterium cell density (Velcheva et al., 2005), regeneration and co-cultivation conditions (Drorri and Altman, 2001), acetosyringone and cell competence after wounding and bacterial strain (Drorri and Altman, 2001). All these studies indicated that the regeneration efficiency and transformation of the tomato is highly dependent on the genotype and needs to be established for each cultivar (Velcheva et al., 2005).
Therefore, the aim of this study is to identify the various factors involved in the regeneration and transformation of three high yielding (70-80 ton h-1) commercial varieties of the tomato (KalG, Kal-early and Su2270) that also have some other good qualities like resistance to many disease (TMV, CMV virus and some fungal or insect pathogen) and tolerance against for various biotic and abiotic stresses which make them important and widely grown cultivars in Iran.
MATERIALS AND METHODS
Plant materials: This study was done in the Plant Molecular Department of NRCGEB during the years 2004-2005. In this study, seeds of Lycopersicon esculentum Mill. KalG, Kal-early and Su2270 cultivars were kindly provided by seed bank of Horticulture Department of Ferdowsi University in Mashad. Seeds of the tomato were first surface-sterilized in 70% (v/v) ethanol for 30 sec followed by 1% sodium hypochlorite solution for 10 min and rinsed 4 times with sterilized water. Seeds were then germinated on a MS inorganic salt medium (Murashige and Skoog, 1962) with 30 g L-1 sucrose, pH 5.7 and solidified using 8 g L-1 TC agar (Sigma, St. Louis, MO). All cultures were maintained at 25°C under fluorescent light at a 16 h light/8 h dark cycle. Shoot tips of cotyledon and hypocotyls removed from 11 day-old seedlings were used as explants for regeneration. Cotyledons were then pierced with needles in order to increase their infection rate. Cotyledon segments were placed upside down in petri dishes containing preculture medium and incubated for 2 days at 25°C in dark.
Culture media for regeneration: MS medium containing MS inorganic salts (Murashige and Skoog, 1962), 3% (w/v) sucrose, 100 mg L-1 inositol and MS vitamins with the following plant growth regulator concentrations were tested for callus induction and shoot initiation (Table 1).
Bacterial strain and plasmid: Agrobacterium tumefaciens strain, GV3850, harboring the binary vector pBI121 was used for this study. The bacteria were grown overnight in LB medium (5 g L-1 yeast extract, 10 g L-1 trypton and 10 g L-1 NaCl) with 50 mg L-1 kanamycin at 28°C on a rotary shaker (220 rpm) and then diluted to OD600 = 1.0. Bacterial suspensions were centrifuged at 3000Hg for 15 min in a 50 mL falcon tube, resuspended in liquid MS medium and finally used for co-cultivation.
Transformation procedure: Tomato explants were removed from the preculture medium and placed into sterile glass jars, containing the bacterial suspension and gently shaken for 5 min. Explants were blotted dry on sterile filter paper and cultivated on a co-culture medium at 25°C. After 2 days, explants were immersed in a washing medium (MS salts + 30 g L-1 sucrose + 500 mg L-1 cefotaxime), blotted dry on sterile filter paper and transferred to shoot Regeneration Media (RM) (Table 1) with 50 mg L-1 kanamycin at 25°C under fluorescent light at a 16 h light/8 h dark cycle. Explants were subcultured every 2 weeks. After four weeks of culture, the percentage of shoot formation per explants was evaluated in two different media and three genotypes. After 5-6 weeks, elongated shoots, emerged from the cut surfaces, were excised and placed on shoot Elongation Medium (EM) (Table 2). Most of the elongated shoots regenerated into plants and formed roots on MS basal medium lacking growth regulators (RTM).
| Table 1: | Different culture media with various plant growth regulators
used for tomato regeneration |
| Table 2: | Media compositions utilized for Agrobacterium-mediated
transformation and regeneration of the tomato |
a: Murashige and Skoog, b: Germination medium, c: Preculture
medium, d: Co-culture medium, e: Regeneration medium, f: Elongation medium,
g: Rooting medium |
|
Molecular and histochemical analysis: GUS enzyme activity of transgenic shoots were determined 2 to 8 weeks after transformation by histochemical assay as described by Jefferson (1987). The total genomic DNA was extracted from leaves using the CTAB method (Saghai-Maroof et al., 1984). Polymerase Chain Reaction (PCR) was used to detect the presence of the uidA gene in the transgenic tissue. Oligonucleotides GUS forward 5′- GGTGGTCAGTCCCTTATGTTACG-3' and GUS reverse 5′- CCGGCATAGTTAAAGAAATCATG-3′ were used as primers to check the specific amplification of uidA gene, which is in the size of 525 bp fragment.
RESULTS AND DISCUSSION
The success in tomato transformation could be largely affected by the genotype, type of explant and plant growth regulators used in culture mediums. In this study we optimized a repeatable method for the regeneration and transformation of three elite cultivars, which are widely used for commercial purposes in Iran. Hypocotyls and cotyledon explants of all three genotypes and mediums, mentioned in Table 1 and used in this experiment, were based on the results of other researches (Romero et al., 2001; Park et al., 2003; Velcheva et al., 2005).
Since the regeneration is quite dependent on the cultivar, as was mentioned in our literature review, therefore the regeneration of callus was done in the first step. The rate of callus production was different amongst different cultivars and media compositions. These media were classified based on the rate of callus induction. The maximum rate of callus induction in tested media was observed in M2 and M4 media with hypocotyls explant (Fig. 1).
| Fig. 1: | Effect of medium on the percentage of explants that produced
callus in four weeks time (p<0.05) |
| Fig. 2: | Effect of medium on the percentage of explants that produced
shoots (p<0.05) |
Present results showed that the application of callus for regeneration could not be suggested, mainly due to the somaclonal variation (Data not shown). These results are in accordance with that of Soniya et al. (2001), reported on tomatoes and the other solanacea family. Therefore, direct regeneration from transformed explants (hypocotyl and cotyledone) was used as a strategy. After four weeks, the mean number of shoots per each type of explant was scored and used as a regeneration efficiency for all three genotypes. Explants showed different responses in the presence of various growth regulators. The maximum shoot regeneration for cotyledons was observed on MS media, supplemented with 2 mg L-1 Zeatin with KalG genotype, but for hypocotyls, the high frequency of regeneration was observed in MS media containing 2 mg L-1 BAP m or 2 mg L-1 Zeatin combined with 0.1 mg L-1 NAA using Su2270 cultivar. The effect of 2 mg L-1 Zeatin was significantly different between the three genotypes (p<0.05) (Fig. 2). Many researches have previously reported that Zeatin stimulates the organogenesis of different tomato explants, such as leaves (Arillage et al., 1998), cotyledon (Drorri and Altman, 2001; Costa et al., 2000; Frary and Earle, 1996; Park et al., 2003) and hypocotyls (Frary and Earle, 1996; Park et al., 2003) which quite agree with our results obtained on BAP.
| Fig. 3: | Results of the average number of shoots per explants during
in vitro regeneration of three cultivars |
The optimal medium for shoot regeneration is quite dependent on cultivars as reported by few researches. For example Velcheva et al. (2005) reported that optimal regeneration of 2 cultivars was obtained on MS medium supplemented with 1 mg L-1 Zeatin and 0.5 mg L-1 IAA but for two other cultivars 2 mg L-1 Zeatin and 0.1 mg L-1 IAA were found to be optimal. Park et al. (2003) reported that the transformation frequency of five tested cultivars was greater on media containing 2 mg L-1 Zeatin and 0.1 mg L-1 IAA, indicating that there were no significant differences in the transformation frequency amongst explants or plant growth regulators tested. It seems that all steps of the regeneration and transformation process were cultivar-dependent.
Also, results of the analysis of variance showed that there are high significant differences among explants and genotypes. Among the three genotypes, the KalG and Kal-early showed the highest and lowest values of 30 and 10 for the percentage of explants producing shoots, respectively. The highest values for the average number of shoots per explants were observed on cotyledons KalG and hypocotyls Su2270 (9 and 5, respectively) (Fig. 3). Romero et al. (2001) observed that shoot regeneration per explant were different amongst tomato cultivars. Their experiments revealed that the ability of organogenic ranged from 2.8 shoots per explant in Rutgers to 5.3 in UC828 cultivars. Comparing of the mean number of shoots per explants, in transformed and control explants, indicated that shoot formation and regeneration efficiency were critically reduced, which supports the results obtained by others (Park et al., 2003; Velcheva et al., 2005; Hamza and Chupeau, 1993; Costa et al., 2000).
Our results also showed that the application of hormones in media for the achievement of optimal shoot elongation and rooting is not apparently so critical (Table 2). On an average, 50% of the shoots were elongated after transfer to (EM) medium.
| Fig. 4: | PCR analysis for the presence of GUS gene in the regenerated
plants. GUS gene specific primers were used that give a 525 bp fragment,
K.KalG, S. Su2270, K-E. Kal-early, M. DNA standard 2500 bp lader, C. Non
transformed regenerated plant. H2O: Negative control |
On the other hand, there are a few reports which emphasized the use of growth regulators for elongation (Velcheva et al., 2005). It seems that in the hormone-free medium, explants can be elongated more easily, mainly due to the lack of the enforcing effect of hormones. But it could be also dependent on the cultivar, as we observed in the other steps of our experiment. Other researchers (Velcheva et al., 2005; Romero et al., 2001; Oktem et al., 1999; Lino et al., 2004) reported that the presence of IAA in the rooting medium is quite nessecary. Therefore it seems that root formation is also strongly genotype-dependent. More than 90% of putative transformed shoots were rooted on the MS basal medium which lacked growth regulators and supplemented with 100 mg L-1 cefotaxime and 100 mg L-1 kanamycin (RTM). However, after two weeks, all control shoots died. After 4-6 weeks the plants produced enough roots and the growth of their plantlets were supported upon their transfer to the soil.
Molecular analysis and Histochemical GUS assay of transgenic plants: The presence of the uidA gene was analyzed by PCR. Agarose gel electrophoresis of amplified fragment (with designed primer for a part of gene) showed the presence of a 525 bp band related to the gus gene for each genotype (Fig. 4). Histochemical assay of explants revealed the gus expression after 2, 4 and 8 weeks of transformation experiments with both explants (Fig. 5). The percentage of each explants having gus activity was significantly different among these three cultivars. KalG and Kal-early cultivars showed the highest and lowest values with respect to the percentage of explants with gus positive spots (35 and 17%, respectively) (Fig. 6).
| Fig. 5: | GUS assay of tomato (a) transformed explants and (b) regenerated plants |
| Fig. 6: | Transformation efficiencies of tomato based on GUS expression
(p<0.05) |
Generally our results confirmed that the cotyledon is the best explant in our tomato transformation procedure.
The type of phytohormones seems to be a crucial factor controlling tomato regeneration. The effect of Zeatin on regeneration efficiency and transformation was more significant among the three genotypes when compared with other Plant Growth Regulators studied in these experiments (p<0.05), but considering its cost, BAP could be another choice.
Our results indicated that transformation and regeneration can be simplified in tomatoes (at least in KalG cultivar) by using Zeatin for regeneration and hormone-free MS medium for elongation and rooting.
Taken together, it seems that all aspects of transformation and regeneration were cultivar-dependent. Results of this study revealed that KalG was the best genotype for the regeneration and transformation efficiency and could be used in further research for other purposes like resistance to disease or production of pharmaceuticals.