Abstract: Current study provides an efficient screening system for transformed plant of Citrullus lanatus cv. Round Dragon harboring bar gene. The untransformed 5-day-old cotyledon explants were cultured on the shoot-inducing media supplemented with Basta® (0.2, 0.5, 1.0, 2.0 and 3.0 mg L-1) and without Basta® for 3 weeks and subcultured on fresh shoot-inducing media with the same media composition for another 3 weeks. The shoot growth on the cotyledon decreased, as the Basta® concentration increased. A complete inhibition of shoot growth was observed on growth medium supplemented with 2.0 and 3.0 mg L-1 of Basta®, respectively. For ex vitro condition, untransformed healthy plant leaves (derived from acclimatized in vitro plantlets) were leaf painted with an aqueous solution of Basta® at the concentration of 0.001, 0.01 and 0.1% (v/v) using writing brush. The sensitivity of untransformed plant tissues were evaluated based on tissue browning and necrosis due to herbicidal damage. Healthy plant leaves subjected to leaf painting assay showed serious necrotic within 3 days at the concentration of 0.1% (v/v) of Basta®. An efficient herbicide Basta® selection mode has been established via in vitro and ex vitro conditions of untransformed Citrullus lanatus cv. Round Dragon.
INTRODUCTION
Crop improvement through genetic engineering has become a reality with the successful introduction of agronomical desirable herbicide-resistance trait in various plant species such as in cabbage (Sretenovic-Rajicic et al., 2004) orchardgrass (Denchev et al., 1997) Norway spruce (Brukhin et al., 2000) and sugarbeet (Kishchenko et al., 2005). The availability of transgenic herbicide-resistance plant species would improve weed control and increase profitability of the economically important crops (Kishchenko et al., 2005). Application of gene transfer techniques using the bar gene as a selectable marker has been broadly used in plant species such as sugarcane (Manickavasagam et al., 2004) cassava (Sarria et al., 2000) legume (Lohar et al., 2001) and tobacco (Lutz et al., 2001). However, there was only one reported case so far concerning bar gene-based Agrobacterium tumefaciens-mediated transformation system of Citrullus lanatus (Cho et al., 2008). The bar gene was originally cloned from Streptomyces hygroscopicus and has been widely used to engineer herbicide-resistant plants (Lea et al., 1984; Thompson et al., 1987). Stable integration of bar gene into crop genome via Agrobacterium tumefaciens-mediated transformation system has yielded plants resistant to the herbicide Basta®. Basta®is a non-selective herbicide being used in agriculture sectors made up of phosphinothricin (PPT), an analogue of L-glutamic acid and two L-alanine residues (Keller et al., 1997). Basta®acts as a potent herbicide as its active ingredient, PPT interferes with amino acid synthesis through inhibition of glutamine synthetase involved in the detoxification of ammonia in plants (De Block et al., 1987). Inhibition of glutamine synthetase by PPT causes a rapid build up of intracellular ammonia levels which associated with disruption of chloroplasts structure results in inhibition of photosynthesis and plant cell death (Tachibana et al., 1986; Lutz et al., 2001). Thus, a stable introduction of bar gene that encodes the enzyme phosphinothricin acetyltransferase (PAT) into the plant genome detoxifies PPT by acetylation of the free ammonia group, thereby neutralizing its toxic effect on plant cells (Thompson et al., 1987; Strauch et al., 1988). In addition, study indicates that Agrobacterium tumefaciens-mediated transformation using the bar gene is possible to produce a transgenic plants that confers resistance to herbicide Basta®(Qing et al., 2000; Yoon et al., 2002; Choi et al., 2004).
In this study, the use of herbicide Basta®as a selective agent was described. The main objective was to establish a selection method between transgenic and non-transgenic Citrullus lanatus via plant tissue culture and leaf painting assays. This would help to select the transgenic Citrullus lanatus that express bar gene in plant tissue.
MATERIALS AND METHODS
In vitro Basta® Sensitivity Test
In vitro sensitivity test for commercial herbicide Basta®
(Bayer Crop Science) was carried out using 5-day-old cotyledon explants of Citrullus
lanatus cv. Round Dragon. The cotyledon explants were excised 1-2 mm above
the point of attachment to the hypocotyls as described by Dong
and Jia (1991). The distal portions of the cotyledons were discarded and
the proximal region was used as explant segments.
The proximal region was cultured abaxial side down in 90x15 mm plastic petri dishes that contained MS shoot regeneration medium supplemented with 2.3 mg L-1 BAP, 30 g L-1 sucrose, 100 mg L-1 myo-inositol, 3.2 g L-1 Phytagel and various concentration of commercial herbicide Basta® (0.2, 0.5, 1.0, 2.0 and 3.0 mg L-1). The control was maintained by culturing the cotyledon explants on shoot induction medium without Basta®.
Cell growth was maintained under a 16 h photoperiod of 12.16 μmol/m2/sec from cool white fluorescent lamps at 25±1°C in a tissue culture chamber for 3 weeks. The explants were subcultured to fresh medium with the same media composition every 3 weeks and stop after 6 weeks. Each experiment was repeated three times with 30 cotyledon segments per treatment and 5 explants per Petri dish. After 6 weeks, the frequency of survived explants with shoots and number of shoots per explant were numerated by stereomicroscope. At this time, the sensitivity of cotyledon explants was evaluated based on tissue browning and necrosis.
Ex vitro Assay for Basta®Sensitivity
Ex vitro assay to test Basta®sensitivity were conducted
according to Akama et al. (1995). Cotyledon of
Citrullus lanatus was grown in plant tissue culture growth medium as
earlier described. The rooted plantlets from plant tissue culture medium (about
3-5 cm with >1 roots) were transferred to pots and transparent plastic covers
were placed over the plantlets. During 1 week, the humidity level was maintained
under a 16 h photoperiod of 12.16 μmol/m2/sec from cool white
fluorescent lamps at 25±1°C in a tissue culture chamber. After 1
week, the plastic covers were removed gradually over 3 days and watered with
nutrient solution to allow growth.
After acclimatization period (about 1 month), the untransformed plants were selected based on Basta® sensitivity via leaf painting assay. The plants were tested by painting a few leaves on the upper surface using writing brush with an aqueous solution of Basta® originally containing 13.5% (w/w) glufosinate ammonium (Bayer Crop Science) diluted to 0.001, 0.01 and 0.1% (v/v). The leaves were observed daily and scored based on herbicide damage of untransformed plants.
Statistical Analysis
Data were analyzed using one-way Analysis of Variance (ANOVA) to compare
the means for more than one treatment. Variation among treatment means was analyzed
using Tukey’s Honestly Significant Difference Test (HSD value) and the
significance were determined at the p<0.05 level (Jackson
and McLean, 1998). Statistical analysis was performed using SPSS for Windows
software (SPSS Windows Version 15). Data are presented as means and standard
errors.
RESULTS
Effect of Basta® on in vitro Shoot Regeneration
The experiment was carried out to test the sensitivity of untransformed
shoots towards commercial herbicide Basta® in order to find out
the appropriate concentration of selection agent on in vitro transformed
shoots regeneration. According to Table 1 at 2 mg L-1
of Basta® led to complete inhibition of regeneration of
shoot on MS medium. In control experiment, after 6 weeks, 93% adventitious shoots
were regenerated at the proximal region of cotyledon. Shoots regenerated per
responding explant drops dramatically on MS medium supplemented with 0.2 mg
and 0.5 mg L-1 of Basta® compared to explant regenerated
on MS medium without Basta®. At a concentration of 0.2 mg L-1
of Basta®, about 77% of cotyledon explants produced callus
after 1 week followed by shoot bud formation at week 3. The explants were subcultured
again to a fresh MS shoot regeneration medium supplemented with 0.2 mg L-1
of Basta® after 3 weeks and were capable to induce normal
shoots even at low mean number of shoots per explant (Fig. 1a-f).
The frequency of shoot regeneration on cotyledon explants decreased to 50% in
the concentration of 0.5 mg L-1 Basta®. The shoots
remained green at week 6 but grew slowly and smaller than control.
The cotyledon explants exposed with Basta® at 1 mg L-1 turned yellow with necrosis within 3 weeks. About 23% of explants survived from necrosis as well as formed callus and enable to produce shoot buds at week 6. Medium supplemented with 2 and 3 mg L-1 of Basta®, showed a complete necrosis occurred at cotyledon explants (with yellow turned to brown) within 2 week and no callus or shoot bud was initiated from any cotyledon explants.
| Table 1: | Effect of in vitro shoot regeneration on MS medium supplemented with various concentration of Basta® |
Values within a column followed by different superscripted letters are significantly different at the p<0.05 level | |
| Fig. 1: | Preliminary sensitivity test of Basta®. (a) control explants (MS medium without Basta®), (b) shoot regeneration on MS medium with 0.2 mg L-1 Basta®, (c) shoot induction on MS medium supplemented 0.5 mg L-1 Basta®, (d) necrosis occurred within 3 weeks but shoot bud formed at week 6 with 1.0 mg L-1 Basta® and (E-F) complete necrosis occurred on cotyledon explants supplied with 2 and 3 mg L-1 Basta®, respectively |
| Fig. 2: | Leaf painting of Citrullus lanatus exposed with Basta®. Untransformed plants were leaf painted with aqueous solution of diluted Basta® at (a) 0%, (b) 0.001% (c) 0.1% and (d) 0.01%, respectively. The observations were taken after day 3 |
Since, there were no significant difference was detected between the concentration of 2 and 3 mg L-1 of Basta® used, therefore, 2 mg L-1 of Basta® was chosen as the selective concentration to be used for selecting transgenic plants.
Leaf Painting Assay Using Herbicide Basta®
Herbicide leaf painting assay were conducted to assess the sensitivity
of untransformed plants of Citrullus lanatus towards commercial herbicide
Basta®. A 1 month old of untransformed healthy plant leaves were
selected. Different concentrations of Basta® were applied using
a writing brush to the upper surface of the selected leaves of Citrullus
lanatus. Untransformed plants treated with varying concentration of Basta®
showed different level of sensitivity (Fig. 2a-d).
The effect of commercial herbicide Basta®sensitivity on untransformed plants was observed within 3 days. Figure 2 showed untransformed plants treated with 0.001% (v/v) of Basta® remain green after 3 days and showed no symptoms of herbicidal damaged. In contrast, plants exposed to Basta® at the concentration of 0.01% (v/v) started to turn yellow, bleached and showed necrotic within 3 days. However, the plants leaf painted with 0.1% (v/v) of Basta® died within 3 days period. This result indicated that leaf painting with commercial herbicide Basta® at the concentration of 0.1% (v/v) yielded high sensitivity to Citrullus lanatus.
DISCUSSION
Preliminary sensitivity test of the commercial herbicide Basta® were performed, prior to the gene transfer studies on non-transgenic Citrullus lanatus to determine the best concentration of Basta® to screen and to select the transgenic plants harboring the Basta resistance gene (bar). Current study is useful for future research to establish Citrullus lanatus that confer a resistance to herbicide Basta®. A highly efficient selective condition towards herbicide Basta® from the 5-day-old cotyledon explants has been evaluated by culturing the explants on shoot induction medium supplemented with various concentrations of Basta®.
Cotyledon explants treated with 0.5 mg L-1 of Basta® enable to induce shoots but grew slowly and smaller compared to the shoots from the control. Under this non-selective condition, adventitious shoot appeared at the proximal region of the cotyledon explants indicating tissue culture technique used here similar with those Han et al. (2005) and Dang and Wei (2007). This finding supports previous research that claimed cotyledon explants exposed with 0.5 mg L-1 of Basta®eventually survived and formed adventitious shoots in bottle gourd (Han et al., 2005) and in leguminous tree (Vengadesan et al., 2006). However, it was not consistent with cotyledon explants of Perilla were unable to form any callus or shoots when treated with 0.5 mg L-1 of PPT (Kim et al., 2004). Thus, it can be concluded that the concentration of Basta®required for the selection highly dependent on the plant species.
Cotyledon explants treated with 1.0 mg L-1 of Basta® turned yellow and partially survived with necrosis. These findings confirmed the results of other research that the embryonic tip system of soybean died when treated with 1.0 mg L-1 of PPT (Dang and Wei, 2007). A severe necrotic of cotyledon explants was observed on the shoot induction medium supplemented with 2 and 3 mg L-1 of Basta®. These findings agreed with Vengadesan et al., (2006) that showed PPT above 1.5 mg L-1 led to complete inhibition of shoot regeneration. Moreover, this findings support the idea raised in other research that 2 mg L-1 of PPT in Perilla frutescens and 3 mg L-1 of bialaphos in Dactylis glomerata (Denchev et al., 1997) caused the untransformed plant died with necrosis.
It has been reported that the concentration of PPT was gradually increased during the selection to prevent any escapes in soybean (Dang and Wei, 2007). It may be beneficial if the primary transgenic shoots cultured on the medium supplemented with 2 mg L-1 of Basta® and the secondary transgenic shoots from primary shoots cultured on the medium supplemented with 3 mg L-1 of Basta® in order to increase the selection pressure. However, the current study did not support the idea. Thus, the transformed shoots were subjected to the same level of selection pressure to eliminate possible chimeric plants and escapes. In addition, it has been suggested that exposure of the cotyledon explants on a high selection pressure for a longer period will eliminate many non-transformants (Dang and Wei, 2007).
Based on the preliminary experiment, 2 mg L-1 of Basta® was chosen as the minimal and stringent concentration for selection of transgenic plants similar to bottle gourd (Han et al., 2005). The concentration of selecting agent of Basta® for transgenic Citrullus lanatus recovery in this study were lower than those used in other plants such as rice at 4 mg L-1 (Cao et al., 1992), bean at 5 mg L-1 (Russel et al., 1993) sugarcane at 5 mg L-1(Manickavasagam et al., 2004) and woody plants at 10 mg L-1 (Choi et al., 2004). However, continuous culture of regenerating explants on selection medium supplemented with 2 mg L-1 of Basta® inhibit the number of shoots regenerating from the explant and were used for selecting transgenic Citrullus lanatus in subsequent experiments.
Leaf painting assay assessed the sensitivity of the grown untransformed plants towards commercial herbicide Basta®. Control experiment plants treated with 0.001% (v/v) of Basta® continued to grow and no changes were observed on the physical state of the plants. In contrast, plants treated with 0.01% (v/v) bleached caused necrotic after a single painting. It seems possible that these results are due to foliar damage of the plants which were sensitive to Basta® (Lohar et al., 2001).
Furthermore, a plant exposed with 0.1% (v/v) of Basta® bleached, shrank, died with severe necrosis within 3 days. This findings support previous research that untransformed cassava plants showed necrosis after a single spraying (Sarria et al., 2000). In addition, application of herbicide on plants caused the photosynthesis slowed down within 2-4 h. After spraying, the plants became yellow and died within 2-5 days (Yoon et al., 2002). Thus, the expression level of untransformed Citrullus lanatus leaf painted with Basta® at 0.1% (v/v) was sufficient to obtain a transformed plants resistance to herbicide Basta®. Han et al. (2005) also reported the similar result on the bottle gourd by spraying with a 0.1% (v/v) of Basta® solution.
In conclusion, the current study demonstrates the development of an efficient selection protocol in order to screen and to produce a transgenic plant that confers a resistance to herbicide Basta®. Preliminary study on Basta® sensitivity tests suggested that selection medium supplemented with 2.0 mg L-1 of Basta® and leaf painting assay applied with 0.1% (v/v) Basta® yielded optimum selection condition for transgenic plants with low number of escapes of non-transgenic plants.
ACKNOWLEDGMENT
We wish to thank Malaysian Government (MOSTI) Vote 79073 (e-Science fund)/MOHE FRGS (Vote 78307) for financial support.