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Investigation of Factors in Optimizing Agrobacterium-Mediated Gene Transfer in Citrullus lanatus cv. Round Dragon



G. Kavitah, F. Taghipour and F. Huyop
 
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ABSTRACT

Agrobacterium tumefaciens strain LBA 4404 harboring a binary plasmid pCambar (containing Basta resistance (bar) gene, neomycin phosphotransferase (npt II) gene and β-Glucuronidase (GUS) gene) was used to optimize the transformation efficiency in Citrullus lanatus cv. Round Dragon. In this study, the ability of Agrobacterium tumefaciens to mediate a gene transfer in Citrullus lanatus was highly dependent on various transformation factors. In current investigation, we have established factors influencing gene expression including explant ages of cotyledon- (5 days old cotyledon), pre-culture condition (2 days), wounding technique (multi wired with massive wounding technique), Agrobacterium concentration (A600 nm 0.8), co-incubation period of explants with Agrobacterium (30 min) and acetosyringone concentration (200 μM) in co-cultivation medium. These factors gave maximized transformation efficiency. The expression of the foreign functional gene in the plant genome was confirmed by histochemical GUS assay activity after 3 days of co-cultivation period. By combining the best conditions from each evaluated factors, we successfully established an efficient and reproducible Agrobacterium-mediated transformation protocol for Citrullus lanatus cv. Round Dragon which yielded 100% of transgene expression with 181.18±0.57 blue spots per responding explant. In conclusion, the transformation system for Citrullus lanatus cv. Round Dragon was optimized. The transformation procedure was proved to influence the transformation efficiency in Citrullus lanatus at 100% transient expression. Present study also suggested that different plant varieties may affect the transformation rate. The optimized conditions will then be used in future research to make a transgenic plant.

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  How to cite this article:

G. Kavitah, F. Taghipour and F. Huyop, 2010. Investigation of Factors in Optimizing Agrobacterium-Mediated Gene Transfer in Citrullus lanatus cv. Round Dragon. Journal of Biological Sciences, 10: 209-216.

DOI: 10.3923/jbs.2010.209.216

URL: https://scialert.net/abstract/?doi=jbs.2010.209.216
 

INTRODUCTION

Genetic transformation has become a powerful tool for crop improvement in introducing desirable foreign genes into the plant genome. Among techniques used for the introduction of foreign genes in plants, Agrobacterium tumefaciens-mediated transfer remains the most popular and efficient in compatible plant species. Successful transfer of foreign genes by Agrobacterium tumefaciens have been achieved with banana (Ganapathi et al., 2001), rice (Chern et al., 2001) and white pine (Levee et al., 1999). Other findings has also been devoted to the genetic transformation of Citrullus lanatus via Agrobacterium tumefaciens using cotyledon as explant (Choi et al., 1994; Akashi et al., 2005; Park et al., 2005). However, Cho et al. (2008) suggested that establishment of genetically-engineered herbicide resistant Citrullus lanatus has been met with only limited success at present, since Citrullus species is known as one of the most recalcitrant plants and highly depends on the efficiency of Agrobacterium-mediated transformation protocol.

Previous studies on genetic improvement of crops clearly demonstrated the importance of various factors such as Agrobacterium strain (Wenck et al., 1999; Saharan et al., 2004), type and age of explant (Song and Sink, 2005), pre-culture period (Barcelo et al., 1998; Agarwal et al., 2004), wounding techniques (Velde et al., 2003), bacterial concentration (Wenck et al., 1999), pre-incubation period (Yong et al., 2006), effect of acetosyringone (Wenck et al., 1999; Lievrea et al., 2005) and co-cultivation period (Steffen et al., 1986; Cardoza and Stewart, 2003). With respect to Citrullus lanatus transformation via Agrobacterium-mediated transformation, evaluation and optimization of transformation factors plays a crucial role in order to yield high transformation efficiency. However, the systematic optimization of Agrobacterium tumefaciens-mediated transformation factors has been least reported in Citrullus lanatus. At present, only Akashi et al. (2005) focused on optimization of transformation factors in Citrullus lanatus with regard to Agrobacterium strain, bacterial concentration, pre-incubation period and effect of acetosyringone. Due to the limitation of transformation factors in Citrullus lanatus, more attention is required to optimize transformation factors in order to yield high Agrobacterium-mediated transformation efficiency.

Therefore, in this study, various factors affecting Agrobacterium-mediated genetic transformation will be evaluated to improve the transformation frequencies. As a result, successful T-DNA integration into the plant genome via Agrobacterium-mediated transformation will be detected via transient expression of gusA gene and led to an easy, stable and effective protocol of Agrobacterium tumefaciens-mediated transformation of Citrullus lanatus using cotyledon as explants.

MATERIALS AND METHODS

Explant preparation for transformation: Citrullus lanatus seeds imported from Taiwan were purchased from local supplier ACE Seed Trading (Malaysia) Sdn. Bhd. Decoated seeds were surface sterilized with 5% (v/v) sodium hypochlorite followed by five successive rinses with distilled water. Then, the sterile seeds were subjected for germination on basal MS medium supplemented with 20 g L-1 sucrose, 100 mg L-1 myo-inositol and 3.2 g L-1 phytagel for 5 days in a tissue culture chamber at 25±1°C with 16 h photoperiod under 12.16 μmol/m2/sec/ from cool white fluorescent lamps. At day 5, in vitro germinated cotyledon explants were excised into two halves and the distal portions were discarded. The proximal region of 5-day-old seedlings was used for the subsequent Agrobacterium tumefaciens-mediated transformation experiments.

Bacterial strains and plasmids: Agrobacterium tumefaciens strain LBA 4404 harboring the binary plasmid pCambar was used in the transformation experiments. The binary vector pCambar carries the Basta resistance gene (bar) as a selectable marker that confers resistance to herbicide Basta, uid A gene encoding for β-glucuronidase (gus) as a reporter gene (located at the right border of T-DNA region) with a castor bean catalase intron and npt II gene coding for neomycin phosphotransferase (located outside the T-DNA region) confers resistance to kanamycin, each driven by the cauliflower mosaic virus (CaMV 35S) promoter and NOS (nopaline synthase) terminator which provides polyadenylation signal. The binary vector pCambar is originated from plasmid pCambia 1301 but has been modified with the presence of the bar gene at the multiple cloning site.

Preparation of Agrobacterium suspension: Preparation of Agrobacterium tumefaciens strain LBA 4404 suspension for transformation was carried out according to Acereto-Escoffie et al. (2005) and Qiu et al. (2007) with some modifications. Agrobacterium tumefaciens strain LBA 4404 was inoculated into 10 mL of YEP broth containing 100 mg L-1 of streptomycin and 100 mg L-1 kanamycin. The cultures were allowed to grow with agitation at 200 rpm, 28°C for 16 h. Subsequently, the 10 mL of bacterial culture was added to 190 mL of fresh YEP broth supplemented with 100 mg L-1 of streptomycin and 100 mg L-1 kanamycin and grown at 28°C on a shaking incubator until A600 nm reached 0.6. The cells were then harvested by centrifugation at 4°C (1398 g, 20 min) and further resuspended in 20 mL of liquid co-incubation medium containing MS basal medium supplemented with 2.3 mg L-1 BAP, 30 g L-1 sucrose, 100 mg L-1 myo-inositol and 100 μM acetosyringone. This Agrobacterium suspension was used for transformation.

Agrobacterium tumefaciens-mediated transformation: The preliminary Agrobacterium tumefaciens-mediated transformation protocol was based on the standard protocols with some modifications (Dabauza et al., 1997; Saharan et al., 2004; Wang et al., 2008). The proximal region of the cotyledon explants were wounded once with multi-wire points of electric cord and immersed in 30 mL of liquid pre-culture medium in 50 mL centrifuge tube containing MS basal medium supplemented with 2.3 mg L-1 BAP, 30 g L-1 sucrose, 100 mg L-1 myo-inositol and 100 μM acetosyringone for two days. After two days pre-culture period, the cotyledon explants were inoculated with 10 mL of Agrobacterium suspension strain LBA 4404 at A600 nm 0.6 in the 50 mL centrifuge tube for 10 min at room temperature.

Then the cotyledon explants were removed from the Agrobacterium suspension and blotted dried before transferred to solid co-cultivation medium in 90x15 mm plastic petri dishes containing MS basal medium supplemented with 2.3 mg L-1 BAP, 30 g L-1 sucrose, 100 mg L-1 myo-inositol and 100 μM acetosyringone. The petri plates were sealed with parafilm and placed in the dark for three days. After three days of co-cultivation period, transient expression of a gus reporter gene in the explants was determined through histochemical GUS assay.

Evaluation of transformation influencing factors: Several factors affecting the Agrobacterium-mediated transformation frequency in Citrullus lanatus were evaluated. The factors are age of cotyledon explants (5, 7 and 9-day-old seedlings), pre-culture conditions (2 days pre-culture period and without pre-culture period), wounding technique (mild wounding, massive wounding and without wounding creation), Agrobacterium concentration (A600 nm 0.5, 0.6, 0.8, 1.0 and 1.2), co-incubation period of explants with Agrobacterium (1, 10, 30 and 60 min) and acetosyringone concentration in co-cultivation medium (0, 100, 200, 300 and 400 μM).

Optimized Agrobacterium tumefaciens-mediated transformation protocol: The optimized Agrobacterium-mediated transformation protocol was applied to the proximal region of 5-day-old seedlings. The cotyledon explant was gently wounded once with multi-wire points of electric cord. Wounded cotyledons were pre-cultured for 2 days in 30 mL of liquid MS medium supplemented with 2.3 mg L-1 BAP, 30 g L-1 sucrose, 100 mg L-1 myo-inositol and 100 μM acetosyringone. After the pre-culture period, the explants were submerged in 10 mL of Agrobacterium tumefaciens suspension at A600 nm 0.8 for 30 min. Following this, the cotyledon explants were blotted dry on sterile filter paper and subsequently cultured on co-cultivation medium in 90x15 mm plastic Petri dishes containing MS medium supplemented with 2.3 mg L-1 BAP, 30 g L-1 sucrose, 100 mg L-1 myo-inositol and 200 μM acetosyringone. For the negative control, the proximal region of the cotyledon explants was cultured on co-cultivation medium without immersion in Agrobacterium suspension. The co-cultivation medium was maintained in dark for 3 days prior to the histochemical GUS assay to determine transient gus expression in cotyledon explants.

Statistical analysis: All experiments were studied through statistical analysis (SPSS for Windows software, Version 15). Each evaluation experiment was replicated three times and each replicate consisted of 30 explants. Data were analyzed using one-way ANOVA (analysis of variance) and Tukey’s Honestly Significant Difference Test (HSD value) at p<0.05 level (Jackson and McLean, 1998).

RESULTS

Evaluation of transformation factors: In this study, several factors affecting transformation efficiency were evaluated. The efficiency of foreign gene delivery into the plant genome was determined through histochemical GUS assay to detect the activity of the uid A gene. The expressions of this gene appeared as blue spots and were visualized under stereoscopic microscope after 3 days of co-cultivation period.

Effect of explant ages and pre-culture conditions: Among different explants ages evaluated, 5-day-old seedlings showed the highest frequency of transformation in both without pre-cultured condition (52%) and 2-days pre-cultured condition (71%) compared to the 7-day-old seedlings and 9-day-old seedlings (Table 1). However, cotyledon explants of 5-day-old seedlings produced significantly higher number of spots per explant than 7-day-old seedlings and 9-day-old seedlings. On the other hand, 2 days of pre-culture period of cotyledon explants gave high frequency of transformation compared to the obtained without pre-culture condition. Thus, explants of 5-day-old seedlings with pre-culture period of 2 days in MS liquid pre-culture medium prior the transformation with Agrobacterium were recommended so far.

Effect of wounding technique: High transient GUS expression were observed in cotyledon explants with massive wounding (stabbed four to five times with multi wire points of electric cord) followed by mild wounding (stabbed once with multi wire points of electric cord) of the cotyledon explants. In contrast, unwounded cotyledon explants yielded low transformation frequency with low rate of blue spots per explant after 3 days of co-cultivation period (Table 2). The result showed that, there was a significant difference between wounding procedures studied in this experiment (p<0.05).

Table 1:Effects of explants ages and pre-culture conditions on transformation efficiency
Values within a column followed by different letters are significantly different at the p<0.05 level

Yet, massive wounding completely bleached the cotyledon explants, turned flaccid and led to necrosis in the further analysis. Based on this result, it was proposed that mild wounding of the cotyledon explants maybe the best for the Agrobacterium to transfer their DNA efficiently.

Effect of Agrobacterium concentration: Significant increase of the transient gus expression were observed at the Agrobacterium concentration up to A600 nm 0.8. However, Agrobacterium concentration at A600 nm 0.5 had the lowest transient GUS frequency (Table 3). Blue-stained area per responding explant dropped dramatically when treated with Agrobacterium concentration at A600 nm 1.0 and A600 nm 1.2 compared to explant infected with bacterial concentration at A600 nm 0.8. Therefore, Agrobacterium concentration at A600 nm 0.8 was suggested to be used in transformation study.

Effect of co-incubation period: The numbers of cotyledon explants induced transient gus expression after infection with Agrobacterium varied significantly depending on the co-incubation period. The cotyledon explants produced high frequency of transformation when co-incubated in Agrobacterium suspension for 30 min (Table 4). In contrast, the frequency of transformation on cotyledon explants infected with Agrobacterium for 10 min decreased to 43%. Furthermore, significant decrease was observed in the transformation efficiency at the co-incubation period of 60 minutes. In short, co-incubation period of cotyledon explants for 30 min in Agrobacterium suspension was the best to increase the transformation efficiency.

Table 2:Effects of wounding procedures on transformation efficiency
Values within a column followed by different letters are significantly different at the p<0.05 level

Table 3:Effects of Agrobacterium tumefaciens concentrations on transformation efficiency
Values within a column followed by different letters are significantly different at the p<0.05 level

Effect of acetosyringone: Among different concentrations of acetosyringone tested, 200 μM of acetosyringone was found to be comparatively better responsive than other concentrations (Table 5). Cotyledon explants cultured on co-cultivation medium supplemented with 200 μM of acetosyringone yielded significantly higher number of spots per explant compared to other concentrations. The transient gus expression in cotyledon explants after co-cultivation for 3 days was extremely low when acetosyringone was omitted. Further analysis of data showed addition of acetosyringone to 300 and 400 μM did not lead to an increase in transient expression and the blue-stained zone per responding explant drops gradually. Moreover, no significance differences were observed for these concentrations. In this experiment, co-cultivation medium supplemented with 200 μM acetosyringone was maintained.

Effect of optimized transformation protocol on transient expression of gus A gene: Optimized Agrobacterium transformation protocol led to high frequency of transformation (up to 100 % frequency) and yielded high blue spots per explant (181.2±0.57) after 3 days of co-cultivation in dark. Microscopic observation of the blue region on the explants revealed blue spots located on the cotyledon explants as a transient expression of gusA gene incorporated into the plant genome (Fig. 1a-e). However, no blue-stained tissue was observed in control explants (without Agrobacterium infection) after 3 days of co-cultivation. Thus, this optimized Agrobacterium tumefaciens-mediated transformation protocol that gave the highest transformation efficiency in Citrullus lanatus.

Table 4:Effects of co-incubation periods on transformation efficiency
Values within a column followed by different letters are significantly different at the p<0.05 level

Table 5:Effects of acetosyringone concentrations on transformation efficiency
Values within a column followed by different letters are significantly different at the p<0.05 level

Fig. 1:Transient gus expression in cotyledon explants of Citrullus lanatus. (a) control cotyledon explants (no blue region observed). (b, c), transient gus expression in cotyledon explants (stained in blue). (d, e), microscopic observation revealed blue spots on cotyledon explants. An arrow shows blue patches in cotyledon explants which subsequently lead to visualization of blue spots under microscopic observation

DISCUSSION

The results show that we have established conditions in Agrobacterium-mediated gene transfer in Citrullus lanatus cv. Round Dragon. This is the first reported study on transformation of the mentioned cultivar. Generally, different plant varieties may affect the rate of transformation. The presence of T-DNA in the explants transformed with Agrobacterium was detected by transient expression of gus A gene appeared as a blue region on the explants.

The choice of the explant ages is very important to ensure good transformation efficiency because cotyledon explants from younger seedlings (4 or 5-day old) produced more shoots than those from older seedlings. Current study suggested that cotyledon explants derived from 5-day-old seedlings showed higher gus expression than 7-day-old seedlings and 9-day-old seedlings. These results are consistent with other studies suggesting cotyledon from 5-day-old seedlings has been used as explants for transformation in hybrid watermelon (Choi et al., 1994), tomato (Qiu et al., 2007) and loblolly pine (Wei, 2001). According to Wei (2001), among different tissue that has been tested, cotyledon explants yield high gus expression compared to hypocotyls and radicles. A possible explanation for this might be that cotyledon explants retained high competence cells which enable to respond to the endogenous or exogenous growth regulator to induce shoot regeneration in plants. Other reported study indicated that 4-day-old seedlings has been used for Agrobacterium transformation in bottle gourd (Han et al., 2005) and in wild watermelon (Akashi et al., 2005). It seems possible that these are due to the small and delicate explants, perhaps capable to receive more T-DNA strands incorporated into the plant genome (Sreeramanan et al., 2005).

Pre-culture period defined as the condition between where the explants were excised and infected with Agrobacterium. Explants pre-cultured for 2 days produced the highest frequency of transformation in Citrullus lanatus than the explants transformed with Agrobacterium without pre-culture condition. These findings are consistent with Han et al. (2005) that 2 days of pre-culture period had marked an effect on transformation frequency. Similar transformation efficiency was achieved by 48 h of pre-culture in agroforestry tree (Agarwal et al., 2004). In addition, pre-culturing explants prior the transformation was efficient for barley (Shrawat et al., 2007), canola (Cardoza and Stewart, 2003) and in carnations. Sreeramanan et al. (2005) and Yong et al. (2006) also reported that explants without pre-culture condition gave low frequency of transformation and less gus spots were observed on the explants. This may be due to the fact that pre-culture condition could be attributing to the initiation of active cell division and greatly increase the number of competent cells as potential targets for transformation.

Wounding of the explants prior transformation is necessary to induce high transient gus expression in Citrullus lanatus. In this study, cotyledon explants were stabbed with multi-wire points of electric cord to create a wound. A positive effect of wounding prior the Agrobacterium transformation has been demonstrated in many plant species. Yong et al. (2006) had suggested that wounding of the explants provides the released of phenolic compound to enhance transformation frequency.

Cotyledon explants of Citrullus lanatus treated with massive wounding enable to induce high transient gus expression compared to the cotyledon explants with mild wounding. But this finding did not seem to be consistent with Yong et al. (2006), who claimed explants with massive wounding produced low transient gus expression compared to the explants with mild wounding. However, explants with massive wounding became flaccid and totally bleached after three days of co-cultivation and unable to survive. The results of current study seemed to confirm the findings of Araujo et al. (2004). Thus, mild wounding of the cotyledon explants might be an optimal condition at which the plant cell is most accessible to Agrobacterium infection.

To verify the ability of Agrobacterium tumefaciens to transform the cotyledon explants of Citrullus lanatus, various bacterial concentration were tested. The highest transient gus expression produced on cotyledon explants infected with A600 nm 0.8 bacterial concentration. This finding was similar to Lee et al. (2006) on orchard grass and Yong et al. (2006) on Melastomaceae transformation. However, higher bacterial concentrations above A600 nm 0.8 or less A600 nm 0.8 decreased the transformation efficiency of cotyledon explants. Higher bacterial concentrations may caused competitive inhibition which decreased the potential of the bacteria to attach at the explants for the T-DNA transfer into the plant genome (Yong et al., 2006). On the other hand low bacterial concentration resulted in low availability for transforming plant cells since least cells would attach on the explants resulted with low transient gus expression. In other cases, at higher bacterial concentration of A600 nm 1.0 allowed the successful transformation in Indian cowpea (Chaudhury et al., 2007) and sunflower (Mohamed et al., 2006). In contrast, lower bacterial concentrations at A600 nm 0.2 and A600 nm 0.35 permitted transformation in paddy straw mushroom (Wang et al., 2008) and in Medicago and Trifolium species (Ding et al., 2003). The differences of bacterial concentration required for the Agrobacterium transformation highly depends on the plant species in order to yield high transient gus expression.

Optimizing the infection time of cotyledon explants with Agrobacterium is essential to allow the maximum transient gus expression in plant genome. The result of this study showed that co-incubation period for 30 min gave the highest transient gus expression in Citrullus lanatus. However, transformation frequency and transient gus expression decreased sharply with incubation period for 60 min compared to the 10 min and 30 min. According to Tao and Li (2006), the reason could be due to the Agrobacterium could give negatively transformation efficiency by reducing bacterial affinity for competitive attachment on the cotyledon explants. In contrast, other finding suggested that 60 min was an optimum co-incubation period in Melastomataceae sp. to achieve high transformation level (Yong et al., 2006). However, very low transformation frequency observed in cotyledon explants co-incubated for 1 minute might be that too short for the infection time of Agrobacterium to allow the attachment of the bacteria on the cotyledon.

Acetosyringone plays a major role in the natural infection of plants by Agrobacterium tumefaciens and is known to induce the virulence gene of the Ti-plasmid that initiates the transfer of the T-DNA region into the plant genome (Wei, 2001). A co-cultivation medium supplemented with 200 μM of acetosyringone was found to produce higher transformation rate on cotyledon explants after 3 days of co-cultivation period in dark. The effect of the co-cultivation medium supplemented with the 200 μM of acetosyringone on transformation efficiency was well documented in orchardgrass (Lee et al., 2006) and paddy straw mushroom (Wang et al., 2008).

The transient gus expression was extremely low when acetosyringone was omitted from the co-cultivation medium. This was confirmed in previous research that low frequency of explants showed gus positive was obtained due to the absence of the acetosyringone in co-cultivation medium (Shrawat et al., 2007). Furthermore, addition of acetosyringone to 300 μM and 400 μM in co-cultivation medium did not lead to an increase in transient expression. However, supra-optimal acetosyringone concentration is toxic and can cause harmful effect (Sreeramanan et al., 2005). In any transformation study, acetosyringone has been routinely used because it is a potent vir gene inducer and can enhance the transformation efficiency (Saharan et al., 2004; Wenck et al., 1999).

ACKNOWLEDGMENT

Authors wish to thank Malaysian Government (MOSTI) Vote 79073 (e-science fund) and FRGS-MOHE fund (78307) for financial support.

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