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Transient Expression of Green Fluorescent Protein Gene in Rice Calluses: Optimization of Parameters for Helios Gene Gun Device
Nono Carsono and Tomohiko Yoshida

Abstract : An optimized condition for particle bombardment is necessary for efficient genetic transformation. In the present study, parameters for Helios gene gun, another device for cell transformation, were optimized based on transient synthetic green fluorescent protein [sGFP (S65T)] gene expression in rice calluses of indica Fatmawati and japonica Nipponbare. Parameters that found to be the most favorable conditions for transient expression of sGFP gene in rice callus cells were as follows: 250 psi helium pressure, 0.6 ?m gold particle size, 0.25 mg gold particles per shot, and 1.5 ?g plasmid-DNA per shot. Desiccation of callus cells for eight min. was also found appropriate. The level of transient sGFP gene expression was not significantly influenced by the pre-culture for 4 to 12 days before bombardment or by callus age between 10 and 33-weeks old in Fatmawati. Optimized parameters for this particular device should improve the transient expression, thus enabling stable expression of inserted genes via Helios gene gun using callus as a target tissue.
Key words: Helios gene gun, Particle bombardment, Rice, Synthetic green fluorescent protein, Transient expression.

Among the well-known reporter genes, the green fluorescent protein (GFP) from the jellyfish (Aequorea victoria) is the most widely used reporter in biotechnology or molecular biology due to many advantages such as no necessity of any additional co-factors (Heim et al. 1994), non-invasive detection (Chalfie et al. 1994) and broad spectrum of host cells (Cubitt et al. 1995). Moreover, in plant science, GFP has been extensively applied for testing the efficacy of the promoter (Newell et al. 2003), detection of virus activity (Oparka et al. 1997), protein localization (Hibberd et al. 1998; Jang et al. 1999) and optimization of transformation methods (Tee and Maziah, 2005). Recently, many types of GFPs are available; one of them is a synthetic GFP (sGFP; S65T, replacement of the serine in position 65 with a threonine) which is 100-fold brighter than wild-type GFP in plants (Chiu et al. 1996; Niwa et al. 1999). The sGFP has been used as an efficient reporter system for direct gene transfer in maize (Chiu et al. 1996), oat (Cho et al. 2003), soybean (El-Shemy et al. 2006) and others.

Transient expression of reporter genes is useful in both plant and animal sciences particularly for improving the efficiency of transformation technology. Physical and biological parameters of transformation technology including particle bombardment could be optimized for efficient genetic transformation. Schopke et al. (1997) pointed out that the establishment of optimal parameters in particle bombardment for any plant tissues is necessary.

Particle bombardment is now emerging as the method of choice for introduction of useful agricultural genes into rice and other cereals crops. However, most bombardment methods for rice transformation utilize the PDS-1000/He Biolistic Particle Delivery System (for example, Bec et al., 1998; Ghosh Biswas et al., 1998; Jiang et al., 2000; Martinez-Trujillo et al., 2003) and currently there is no current protocol for rice callus cells bombarded using the Helios gene gun (Bio-Rad Laboratories, USA) device and transgenic plants obtained using this particular device have not been reported. In this study, we used the Helios gene gun that differs from PDS-1000/He device. It does not use a vacuum chamber and thus can be used to deliver nucleic acids into tissues, organs, and even whole organisms (Taylor and Fauquet, 2002), thereby removing limitations to the target and its size. Other advantages of the Helios gene gun are its portable size, and wide application to plant and animal cells, and also the cartridge can be stored for several months. In this study, transient expression of sGFP (Chiu et al. 1996) was used as a rapid assay to optimize the parameters of Helios gene gun for calluses of two distinct cultivars indica Fatmawati and japonica Nipponbare. These parameters are helium pressure, particle gold size, amount of gold particles per shot (MLQ), and amount of plasmid-DNA delivered per shot (DLR). In addition, the effects of desiccation treatment and pre-culture of calluses before bombardment, and callus age as the biological parameters, were also evaluated. The objective of the current work was to optimize parameters for Helios gene gun bombardment by evaluating the transient expression of sGFP using calluses of two cultivars as target materials.

Materials and Methods
1. Plant material and proliferation of embryogenic callus cells
Embryogenic callus cells were induced from scutellar tissues of mature seeds of two cvs Fatmawati and Nipponbare and they were proliferated as described previously (Carsono and Yoshida, 2006).
2. Plasmid used for bombardment
Plasmid pIG001 with the sGFP gene and the hygromycin phosphotransferase (Hpt) gene conferring resistant to Hygromycin B was used in all bombardment experiments (Fig. 1). The sGFP gene was driven by maize ubiquitin promoter (Christensen and Quail, 1996), while the Hpt gene under the control of the Cauliflower Mosaic Virus (CaMV) 35S promoter. The plasmid was amplified in E. coli DH5cells and purified by the alkaline-SDS lysis method (Sambrook et al. 1989), followed by PEG/NaCl treatment. The concentration of plasmid-DNA was checked by Spectrophotometer (Gene Spec III, Naka Instruments Co., Ltd., Japan).
3. Helios gene gun bombardment procedures
The preparation of bombardment for Helios gene gun was done according to the manufacturerfs instruction and method of Helenius et al. (2000) with a minor modification for rice callus cells. In brief, 40 ?l of 50 mM spermidine was added into a microcentrifuge tube containing 12.5-25.0 mg gold particles, then sonicated for 5-8 s, and subsequently, 40 ?l of DNA plasmid (50-100 ?g) in Tris-EDTA was added and gently vortexed. While mixing, 40 ?l of 1M CaCl2 was added drop by drop, to associate the DNA with the gold particles. The suspension was allowed to settle at room temperature for 10 min.

The gold-DNA pellets were washed three times by vortexing in 1 ml of absolute ethanol, microcentrifuged at 12000 rpm for 1 min and the supernatant was removed. The pellet was then resuspended in 3 ml absolute ethanol-PVP (polyvinylpyrollidone; 0.05 mg ml-1). By using a syringe attached to an adapter, this suspension was drawn into a Tefzel tubing 76.2-cm in length. The tubing was then transferred into a tubing preparation station. After gold beads were allowed to settle, ethanol was slowly drawn off and the turner was rotated for 30 s, smearing the gold-DNA around the inside of the tubing. The residual ethanol was removed by passing nitrogen through the tubing for 3-5 min. The tubing was cut into 2.54 cm-long tubes and then loaded into the Helios gene gun device. Eight embryogenic calluses, 4-5 mm in diameter, were placed in the center of Petri dish with filter papers (Fig. 2a), and covered with a plastic bowl with cavity that suites the spacer of Helios gene gun (Fig. 2b). These calluses were bombarded once, then immediately transferred to osmotic medium and incubated in the dark at 260C. Osmotic treatment of callus cells with 0.4 M mannitol on medium NB5 was applied for 4-6 h before and 16 h after bombardment.
4. GFP monitoring and transient assessment
sGFP-expressing cells were detected using Leica MZFL III stereo-fluorescence microscope assisted with the GFP2 filter to mask the chlorophyll resulting in red fluorescent for the non-transformed tissue, thus, allowing visualization of distinctive differences for the sGFP-expressing cells. The number of sGFP positive cells emitting green fluorescent spots was recorded. The sGFP-expressing cells were always counted at the same magnification, i.e., 80X at the back and upper sides of the Petri dish 2-3 days after bombardment. Because some calluses were broken into pieces after bombardment, data on the average of green fluorescent spots for eight calluses were not available, and only the total green fluorescent spots per bombardment was recorded.

In this experiment, the Helios gene gun parameters were varied while all other parameters were maintained as standard procedure as follows: 200 psi (equal to 1,379 Kpa), 0.6 ?m gold particle diameter, 0.250 ?g gold particles per shot, and 1.0 ?g plasmid-DNA per shot, 4-day pre-culture, and 0.4 M mannitol of osmotic medium. Desiccation of callus cells was done using a vacuum drying oven (Yamato Scientific Co., Ltd., Japan) at 76 cm Hg without osmotic treatment. Callus cells in the same age were used for parameter tested.

Data were analyzed using one-way analysis of variance and the differences were analyzed using Duncanfs multiple range test (DMRT). Standard errors of the means were calculated for four replications (bombardments).

Results and Discussion
Transient sGFP gene expression in rice calluses of two cultivars was observed within 48 to 72 h. after bombardment with the Helios gene gun (Fig. 2c, d). In total, 162 bombardments were performed and 1,344 embryogenic calluses from two genotypes were bombarded. The highest level of transient expressions of sGFP was obtained with a helium acceleration pressure of 250 psi in both cultivars (Figure 3). However, no significant difference was detected in the number of sGFP spots between 200 and 250 psi in Fatmawati. A low acceleration pressure resulted in low number of sGFP spots. The high helium pressure seems to be very powerful to drive DNA from the inner surface of tubing, so that it can penetrate into deeper cell layers. It is presumed that acceleration pressure affects the depth of penetration and distribution of gold particles into callus cells as already reported by Rasco-Gaunt et al. (1999) and Tee and Maziah (2005) using a PDS-1000/He device. They assumed that a high acceleration pressure bombarded a smaller area than a low pressure. Moreover, Sanford et al. (1993) explained that the highest transient expression is generally achieved with rather violent treatments giving better particle penetration, but these conditions may affect the cell division or growth. Extra tissue damage or injury (Rasco-Gaunt et al., 1999; Tee and Maziah, 2005), more necrotic, no formation of new calluses or somatic embryos (Tadesse et al., 2003) are some adverse effects of bombarded tissues with high acceleration pressure due to high velocity of gold particles. Tissue damage may be due not only to helium acceleration pressure, but also by some other parameters such as gold particles size and amount of gold particles per shot.

Gold particles, 0.6 ?m in diameter, produced significant a higher level of sGFP gene expression than 1.0 ?m particles in Fatmawati, but not significant in Nipponbare (Fig. 4). It seems that a 0.6 ?m gold particle is more suitable than a 1.0 ?m particle to carry plasmid pIG001, which is around 18 kbp. This result is in agreement with a previous report that the 0.6 ?m gold particle is best for transient expression of the uidA gene (?-glucuronidase, GUS) in the leaves of arabidopsis, tobacco and birch bombarded when using a Helios gene gun (Helenius et al., 2000). A possible explanation for this condition is that a small particle size (0.6 ?m) may cause less cells damage of bombarded target tissues due to low particle mass, thus the cells may be capable of transiently expressing sGFP gene, even capable of undergoing further division or growth.

A small amount of gold particles or microcarriers delivered per shot (0.25 mg) led to a significant increase in transient expression of sGFP, particularly in Fatmawati (Fig. 5). No significant difference in the number of sGFP expression was found when Nipponbare calluses were bombarded with different amounts of gold particles. Gold particles at more than 0.25 mg per shot produced no improvement in sGFP expression in either cultivar. A large number of cells are probably damaged or killed by the large amount of gold particles delivered to the callus cells, resulting in a reduced amount of surviving cells, therefore undesirable for developing stable transformation. Hunold et al. (1994) found that the viability of particle-containing cells decreased within two days after bombardment, which supports our result. However, a minor different was reported by Helenius et al. (2000) who found that 0.125 mg gold particles delivered per shot was effective for transient luciferase activity in arabidopsis and tobacco leaves, but for uidA gene expressions in birch leaves, 0.25 mg gold particles per shot were optimum.

The use of an appropriate concentration of DNA is important for efficient DNA-microcarrier binding (Parveez et al., 1998). Plasmid-DNA delivered with 1.5 ?g per shot produced the largest number of sGFP spots in Fatmawati, while in Nipponbare, 1.5 and 2.0 ?g plasmid-DNA per shot were not significantly different in producing sGFP spots (Fig. 6). A similar observation has been observed in oil palm (Parveez et al., 1998), in which 1.5 ?g plasmid-DNA per shot gave the highest level of the GUS expression in embryogenic callus. Increasing plasmid-DNA concentration improved the sGFP gene expression in Nipponbare, but not in Fatmawati calluses. It appears that the sGFP expression was genotype-dependent in terms of differences in callus cells types they produced, which has been described by Bec et al. (1998). Moreover, differences in the sGFP expression in calluses of the two cultivars bombarded using a Helios gene gun might be attributed to the difference in biological constraint such as callus cell density or callus compactness or callus friability that might not be exactly identical in the two cultivars.

The limitation of using Helios gene gun bombardment is the tubing that sometimes contains unequally distributed plasmid-DNA resulting in unequally spread plasmid-DNA in the target tissues. In the present experiment, the sGFP expression was mostly limited to 2 to 5 calluses out of 8 bombarded calluses. In addition, Harwood et al. (2000) found that this device was not suitable for barley transformation because the tissues jumped to other places by bombardment. However, by covering target tissues (callus cells) using a plastic bowl, this demerit could be overcome.

Bombarding target tissue at the right developmental stage is important because actively dividing cells are most receptive to the particle bombardment (Moore et al., 1994). Pre-culture of callus cells prior to bombardment did not affect the sGFP expression as shown in Fig. 7. However, it was reported that a 7 day pre-culture and 4 day pre-culture gave the highest GUS gene expression in immature embryo of oil palm (Parveez et al., 1998) and in microspores of wheat (Folling and Olesen, 2002). Moreover, in two types of orchid Dendrobium calluses, a 2 day pre-culture period was suitable for sGFP gene expression (Tee and Maziah, 2005), indicating that the effect of pre-culture duration for the efficiency of bombardment varies with the target tissue, though such variation was not significantly found in rice callus cells.

Removing partial humidity of rice callus cells via desiccation treatment for 8 and 12 min. prior to bombardment gave a larger number of sGFP spots in Fatmawati (Fig. 8), but not in Nipponbare. Desiccation treatment for more than 8 min. was not effective due to a reduction in sGFP expression.

The effect of age of target tissues for bombardment experiment should be considered for obtaining highly efficient transformation. Ramesh and Gupta (2005) discovered that maximum GUS expression in rice calluses was observed when calluses were bombarded after 44 days old (6.3 wk) and tended to decrease in older callus age (68 days old or 9.7 wk). However, in Fatmawati in this study, callus age did not significantly influence the number of sGFP spots, although the sGFP expression tended to decrease in older callus (Fig. 9), indicating the same trend as previously observed. For stable expression of inserted genes for rice callus bombarded using Helios gene gun, use of helium at a pressure of 200 to 250 psi with 0.6 ?m gold particles size and 0.25 mg gold particles delivered per shot is therefore recommended. In this study, some callus cells resistant to Hygromycin B were obtained and currently a regeneration experiment of putative transgenic calluses is underway. Molecular analysis needs to be carried out to further investigate the expression of sGFP and Hpt genes in the rice genome.

Acknowledgement
Thanks are due to Dr. Hiroyuki Anzai, Gene Research Center, Ibaraki University for the kind gift of plasmid pIG001.

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Table and figure captions:

Fig. 1. Schematic map of plasmid pIG001. Hpt, hygromycin phosphotransferase gene, conferring resistance to hygromycin B; Npt II, neomycin phosphotransferase II gene, encoding resistance to kanamycin; sGFP, synthetic green fluorescent protein gene; PUbi, promoter of Ubiquitin maize; PNos, promoter of Nos (Nopaline synthase) gene; P35S, the Cauliflower mosaic virus promoter; TNos, terminator of Nos; RB, right border; LB, left border.
Fig. 2. (a) Eight embryogenic calluses ready for Helios gene gun bombardment. (b) Bombardment of rice calluses with a plastic bowl to avoid jumping the target tissues by bombardment. Transient sGFP gene expression in rice calluses cvs (c) Fatmawati and (d) Nipponbare (64X). The parameters applied were 250 psi, 0.6 ?m gold size, 0.25 mg gold particles per shot, 1 ?g plasmid-DNA delivered per shot, 0.4 M mannitol osmotic medium (4-6 h before and 16 h after bombardment), and 4-day pre-culture before bombardment.
Fig. 3. Effect of acceleration pressure on sGFP gene expression in calluses of two cultivars. Data are means with standard error of 4 replicates, each with 8 embryogenic calluses. Same letters indicate values are not significantly different according to Duncanfs multiple range test (p = 0.05).
Fig. 4. Effect of gold particles size on sGFP gene expression in calluses of two cultivars. Data are means with standard error of 4 replicates, each with 8 embryogenic calluses. Same letters indicate values are not significantly different according to Duncanfs multiple range test (p = 0.05).
Fig. 5. Effect of the amount of gold particles delivered per shot on sGFP gene expression in calluses of two cultivars. Data are means with standard error of 4 replicates, each with 8 embryogenic calluses. Same letters indicate values are not significantly different according to Duncanfs multiple range test (p = 0.05).
Fig. 6. Effect of the amount of plasmid-DNA delivered per shot on sGFP gene expression in calluses of two cultivars. Data are means with standard error of 4 replicates, each with 8 embryogenic calluses. Same letters indicate values are not significantly different according to Duncanfs multiple range test (p = 0.05).
Fig 7. Effect of pre-culture prior to bombardment on sGFP gene expression in calluses of two cultivars. Data are means with standard error of 4 replicates, each with 8 embryogenic calluses. Same letters indicate values are not significantly different according to Duncanfs multiple range test (p = 0.05).
Fig. 8. Effect of desiccation treatment prior to bombardment on sGFP gene expression in calluses of two cultivars. Data are means with standard error of 4 replicates, each with 8 embryogenic calluses. Same letters indicate values are not significantly different according to Duncanfs multiple range test (p = 0.05).
Fig. 9. Effect of callus age of Fatmawati on sGFP gene expression. Data are means with standard error of 4 replicates, each with 8 embryogenic calluses. Same letters indicate values are not significantly different according to Duncanfs multiple range test (p = 0.05).

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