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Optimization of the transient gusA gene transfer of Phalaenopsis violacea orchid via Agrobacterium tumefaciens: an assessment of factors influencing the efficiency of gene transfer mechanisms.

Introduction

Phalaenopsis orchids are among the most important commercially because of their beautiful floral shape, graceful inflorescence and fragrance. Among orchid lovers, Phalaenopsis violacea has the reputation of an almost mystic species and a very pricey rarity. We are focusing on closely related to Phalaenopsis violacea species, known as Phalaenopsis belina species, a native orchid in Borneo.

In order to improve the quality, modern techniques could be applied to transfer desired genes into orchid genome instead of using conventional breeding method which is time consuming, hindered by species barriers and a lack of genetic variability. Genetic engineering presents a new approach to the strategy and techniques to transfer potential genes into orchid. Agrobacterium-mediated transformation system in orchid plants is preferred because it is a simple technique which does not require expensive equipment, it reduces the copy number of the transgene, potentially leading to fewer problems with transgene co-suppresion and instability (Hansen et al., 1994 ; Belarmino and Mii, 2000 ; Chai et al., 2002 ; Mishiba et al., 2005). One significant limitation of this method, however, is that transformation appears to be genotype-dependent.

Early detection of plant transformation events is necessary for the optimization of gene transfer into orchid genome. Reporter gene such as E.coli gusA (uidA) gene encoding for enzyme ^-glucuronidase (GUS) is used to provide a clear indication of the expression, transient or stable, of transferred genes in plant transgenic cells. Optimization of several factors that influence transient gusA gene expression such as the effects of density of bacterial cultures (O[D.sub.600nm]), influence of PLBs sizes, duration of pre-culture and co-cultivation periods, different concentrations of L-cysteine, silver nitrate and various temperatures ([degrees]C) during co-cultivation were evaluated in order to determine the optimum condition of Agrobacterium-mediated transfer during the early stages of transformation in Phalaenopsis violacea. Protocorm-like bodies (PLBs) are used as a starting material for Agrobacterium-mediated transformation system since it is an activated cell and could be rapidly proliferate under optimum conditions. Another attribute that makes PLB an excellent target tissue is that it is an easily regenerable somatic organ. In addition, the present of coniferyl alcohol in PLBs, one of the phenolic compounds present in Dendrobium orchids which can induce vir gene expression in Agrobacterium tumefaciens and thus provides a fundamental element required for successful Agrobacterium -mediated transformation system in orchid plants (Nan et al, 1998).

The optimization of some important aspects of transformation efficiency is therefore, essential to enhance the virulence so as to increase the transformation frequency. There are no reports on systematic optimization of conditions for Phalaenopsis violacea orchid species transformation and in the present study, the effects of several parameters known to influence Agrobacterium-mediated DNA transfer were optimized using gusA gene with an intron marker system.

Materials and methods

Plant Materials

Orchid plants of Phalaenopsis violacea were obtained from Michael Ooi's orchid nursery in Sungai Dua, Seberang Jaya, Penang. The Phalaenopsis violacea PLBs were obtained from young segments of approximately 1 x 1 cm2, excised from aseptically raised three-month old in vitro seedlings. Protocorm like-bodies (PLBs) of Phalaenopsis violacea were obtained from in vitro plantlets of three months culture using 1/2 strength Murashige and Skoog (1962) medium supplemented with 5% of Mas banana (AA) extract. To produce large quantities of PLBs for transformation experiment, PLB were subcultured on the same medium with 1 mg.[L.sup.-1] of BAP cytokinin. Cultures were incubated on tissue culture room at 25[degrees]C under 16 hours photoperiod with light intensity of 40 nmol.m.sJ supplied by white fluorescent tubes. Proliferated PLBs after 2 months were used for the Agrobacterium-mediated transformation experiment.

Bacterial strain and plasmid DNA

Agrobacterium tumefaciens super-virulent strains, EHA 101 and 105 (pCAMBIA 1304) was maintained at -80[degrees]C for long term storage in 70% (v/v) glycerol. Both Agrobacterium tumefacien strains contained the disarmed plasmid pCAMBIA 1304 with an intron containing [beta]-glucuronidase gene (gusA) driven by the CaMV 35S promoter and hpt gene for resistance to the antibiotic hygromycin (Fig. 1). The gusA gene contains an intron in the coding region to ensure that GUS activity detected in plant cell is not due to residues from Agrobacterium cells.

Inoculation and co-cultivation of Agrobacterium tumefaciens, EHA 101 and EHA 105 (pCAMBIA 1304)

Three mL of culture was transferred from stock into a liquid Luria-Bertani (LB) medium containing 50 mg.[L.sup.-1] kanamycin. It was then incubated at 28[degrees]C and 120rpm overnight in order to reach an optimal density. The following day, 500[micro]L of bacterial suspension was spread over the surface of solid LB medium containing 50 mg.[L.sup.-1] kanamycin and incubated at 28[degrees]C for two days. Single colony was collected with sterile loop and suspended in 30mL sterile liquid LB medium containing 50 mg.[L.sup.-1] kanamycin. The cultures were incubated at 28C in shaker with 120 rpm agitation for 16 hours to reach an optimal density of 0.6 units at 600nm (O[D.sub.600nm]).

[FIGURE 1 OMITTED]

Experimental design: Optimization of parameters

To assess factors affecting the transformation frequency, twelve parameters were compared for each factor on transformation efficiency. A range of parameters were evaluated and for each parameter, three (3) replicates were used containing ten (10) PLBs per replicate and were repeated three (3) times. Parameters included different bacterial density of bacterial cultures O[D.sub.600nm] (0, 0.2, 0.4, 06, 0.8, 1.0 and 1.2), influence of different PLBs sizes (3, 5 and 10 mm), different duration of pre-culture (0, 1, 2, 3 and 4 days) and co-cultivation (0, 1, 2, 3, 4, 5, 6 and 7 days) periods, different concentrations of L-cysteine (0, 100, 200, 300, 400, 500 and 600 mg.[L.sup.-1]), different concentrations of silver nitrate (0, 20, 40, 60, 80, 100 and 120 [micro]M) and various temperatures (20, 22, 24, 26, 28, 30 and 32[degrees]C) during co-cultivation in half-strength MS medium supplemented with 5% of banana, Mas (AA) extract. To determine the optimum conditions for transformation, one factor of the standard conditions was changed each time and the effects on percentage of transient gusA gene expression were evaluated.

GUS histochemical assay

Comparison of the transient expression levels were made by assaying for expression gusA gene in the PLBs 3 days after co-cultivation. GUS activity was localized histochemically as described previously by Jefferson (1987). PLBs were immersed in X-Gluc solution (2mM X-Gluc, 100mM Na[H.sub.2]P[O.sub.4] (pH 7.0), 0.5mM potassium ferricyanide and 50mM ferrocyanide. The PLBs were then incubated at room temperature (37[degrees]C). After staining, the materials were treated with 70% ethanol for 3 days to remove chlorophyll before observation. A sample was scored as transient GUS positive if the 25% blue-region on a PLBs mass was at least 25%.

Statistical analysis

Data were analysed using one-way ANOVA and the differences contrasted using Duncan's multiple range test. All analyses were performed at the level 5% using SPSS 10.0 (SPSS Inc. USA).

Results and discussion

In order to optimize conditions for Phalaenopsis violacea orchid transformation, the effects of several parameters known to influence Agrobacterium-mediated DNA transformation were compared. The choice of Agrobacterium strains plant an important important role in the transformation process for the efficiency of gene transfer on recalcitrant species of orchid such as Phalaenopsis violacea in this study.

Generally, particular strain of Agrobacterium tumefaciens will show different level of competency to the orchid species. The efficiency of strain EHA 101 and 105 has been compared in transforming PLBs of Phalaenopsis violacea orchid species. By removing the kanamycin resistance gene from the bacterial chromosome, the Agrobacterium tumefaciens EHA 105 strain was created. Several studies have reported that strains EHA 101 and EHA 105 were more effective than other common strains since both are derived from supervirulent wild-type strain A281 (Hood et al., 1993), whereas strain such as LB A 4404 was derived from less virulent strain Ach5 (Hoekema et al, 1983). In our experiments, the transformation efficiency of strain EHA 105 was generally higher than EHA 101 based on the percentage of transient gusA gene expression than EHA 101 for all the parameters that were evaluated. The hypersensitive made this strain preferable as a vector for the genetic transformation of several plant species and the lack of chromosomal kanamycin resistance made it simple to later insert a kanamycin-based binary plasmid (Tzfira et al., 1997).

[FIGURE 2 OMITTED]

Effect of Agrobacterium culture (O[D.sub.600nm]) density

The first parameter found to influence T-DNA delivery into PLBs of Phalaenopsis violacea using different level of transient GUS expression was the density of Agrobacterium tumefaciens suspension cultures. Transformation was investigated based on concentration of Agrobacterium suspension culture at different optical density (O[D.sub.600nm]). Observation on the effect of bacterial concentration was carried out at different density (0, 0.2, 0.4, 0.6., 0.8, 1.0 and 1.2) and the result was shown in Fig. 2.

Transformation frequency for PLBs increased with bacteria concentration, but higher concentrations also promoted the frequency of necrosis. Agrobacterium tumefaciens EHA 105 with O[D.sub.600nm] of 0.6 showed the highest percentage of GUS positive PLBs, followed by 0.2 and 0.4, which have 48% and 46% of GUS expression respectively (Fig. 2). Although O[D.sub.600nm] = 0.6 of EHA 105 gave the highest transformation efficiency, there was no significant difference (p<0.05) with the same density value by using EHA 101. Further analyses also showed that different bacterial density had different effects on transformation efficiency as shown in Fig. 2. Higher O[D.sub.600nm] (more than 0.6) significantly (p<0.05) decreased the percentage of transient gusA gene expression and even spread of GUS staining due to destruction of PLBs at overgrowth of bacteria concentrations. It can be postulated that PLBs would have stress consequence that would disturb physiology of leads to ineffective recovery of the transformed cells. However, if the concentration of Agrobacterium suspension is low, there is an insufficient bacterium cells infect and transfer T-DNA into plant cell therefore transformation efficiency is greatly low.

For Phalaenopsis hybrid orchids, transient GUS expression was highest with Agrobacterium density at O[D.sub.600nm] of 0.2 (Chai et al., 2002). Wilson et al. (2006) found that different bacterial concentration had different effects on transformation efficiency, O[D.sub.600nm] 0.8 (1 x [10.sup.7] cfu. mLJ) of EHA 105 gave highest transformation efficiency for Melastomataceae malabathricum and Tibouchina semidecandra and transformation efficiency decreased when O[D.sub.600nm] 0.8 was used in shoot and node explants of Melastomataceae malabathricum. The results imply that the efficiency of plant transformation mediated by Agrobacterium not only affected by different density of bacteria culture but also type of plants used.

It has been postulated that at higher concentrations lead to cause hypersensitive response of explants with decreased in regeneration potential, aggregation of Agrobacterium cells and difficulty in killing them after co- cultivation period. Nevertheless, when a higher density of Agrobacterium tumefaciens is necessary for recalcitrant species like Phalaenopsis violacea, transformation frequency can be improved by a short immersion period or addition of important antioxidants such as L-cysteine and ascorbic acid for protecting plants from oxidative stress in the co-culture medium.

[FIGURE 3 OMITTED]

Influence of PLBs sizes

Protocorm-likebodies (PLBs) are good explants for plant transformation because regeneration via direct shoot formation minimizes the risks of somaclonal variation. The size of the single buds, which was used as an indication of the developmental stage of tissue, was considered in order to select the right size of target that is most suitable for Agrobacterium-mediated transformation system.

Three months old single PLBs, measuring from 3, 5 and 10 mm were subjected for transient GUS gene expression experiment. The result of the effect of different sizes of PLBs for transient GUS gene expression is presented in Fig. 3 and variation could be observed in all sizes, using both EHA 101 and 105 strains. The percentage of PLBs shown transient GUS expression obtained from Agrobacterium tumefacien, EHA 105 is the highest and shown more significant (p<0.05) than EHA 101 for two of the PLBs sizes (5mm and 10mm) except for size 3mm (Fig. 3). The highest percentage of transient GUS expression exhibited by the 5mm size range was 60% by using EHA 105 and 55% with EHA 101. This could be due being smaller and delicate PLBs size, perhaps received more T-DNA strands. However, higher percentage of transient GUS expression was obtained in PLB size 3mm using Agrobacterium tumefaciens EHA 101 compared to EHA 105. The PLBs which were not inoculated with Agrobacterium, showed no GUS activity.

Research data has proven that the transgenic Phalaenopsis hybrid PLBs were regenerated from a single epidermal somatic somatic embryogenesis cell of the bisected PLB segments (Anzai and Tanaka, 2001; Chai et al., 2002). In addition, a transformation procedure for Phalaenopsis hybrid was also developed by using seed derived immature protocorms for Agrobacterium-mediated system (Mishiba et al., 2005). However, they were not specifically emphasized on the different sizes of protocorm that is optimum for transformation events. Therefore, subsequently 5mm size of PLBs was chosen as a suitable PLBs target size for transformation studies.

Duration of pre-culture period

Duration of pre-culture will displayed significant effects on Agrobacterium-mediated transformation efficiency as it will allowed proliferation of the plant tissues to be more competent and responsive receiving foreign gene. Pre- culture period is defined as the time between PLBs are first isolated and cultured and when the explants are inoculated with Agrobacterium. The effect of pre-culture period (0, 1, 2, 3 and 4 days) of PLBs for transformation efficiency was evaluated.

Results indicated that three days of pre-culture gave the highest transformation efficiency for both Agrobacterium tumefaciens strains (Fig. 4), followed by those pre-cultured for two days and one day. Zhang et al. (2000) reported that optimizing the pre-culture period to three days with Agrobacterium tumefaciens yielded the highest transformation frequency in Chinese cabbage. Similarly, M ohanty et al. (1999) reported that Agrobacterium cultures grown for 3 days were effective for rice callus. However, some studies reported by Sunilkumar et al. (1999) shown that tobacco the leaf discs pre-cultured for 2 days produced the highest GUS positive explants. In addition, Sreeramanan et al. (2006) reported that single bud of banana pre-cultured for two days showed the highest transient gusA expression. In contrast, for Agrobacterium mediated-transformation of Phalaenopsis hybrid callus, Belarmino and Mii (2000) have used overnight-grown culture.

[FIGURE 4 OMITTED]

In some woody fruit plants, such as plum the pre-culturing explants before the inoculation and co-cultivation with Agrobacterium have showed improved in genetic transformation frequencies (M ante et al., 1991). Besides that, number of putatively competent of Arabidopsis thaliana cells for transformation was significantly increased by a pre-culture day treatment on an auxins rich medium (Sangwan et al., 1992). It has been suggested that, stimulation of plant cell division and activation of the DNA replication machinery during the pre-culture period may have important role for T-DNA integration (Sangwan et al., 1992). In many systems, the addition of acetosyringone (phenolic compound) to the pre-culture medium has proven to be beneficial.

However, there was no significant difference (p<0.05) between 0 and 4 days of pre-culture period for Phalaenopsis violacea PLBs. On the other hand, Mondal et al. (2001) also found out that there was 40- 44% of transformation efficiency in tea without pre-culture period. Some of the plants showed the negative effect on the induction of competent cells transformation such as in the explants of citrage (Cervera et al., 1998). Besides that, Janssen and Gardner (1993) have also reported elimination of pre-culture condition was useful for kiwi fruits and apple to be transformed using Agrobacterium-mediated system.

The results clearly indicated that pre-culture period had influenced the frequency of Phalaenopsis violacea transformation by using Agrobacterium-mediated transformation system. This improvement in transformation efficiency as the result of pre-culture period can attribute to initiation of active cell division which has made possible the use of some explants from different species of monocotyledons such as orchid species, which were hitherto recalcitrant for Agrobacterium-mediated transformation studies.

Duration of co-cultivation period

The co-cultivation duration is a critical factor influencing Agrobacterium-mediated gene transfer in Phalaenopsis violacea orchid. In many plant species, usually two to four days co-cultivation period was used for Agrobacterium-mediated transformation due to the high efficiency of transformation. Two to three days of co- cultivation period have been used for general Graminea transformation (Hiei et al., 1994; Cheng et al., 1997).

Lengthening the duration of co-culture increased percentage of transient GUS expression up to a certain point. The length of co-cultivation period required for achieving maximum gene transfer was found to be 3 days with a significant difference (p<0.05) between two Agrobacterium strains for PLB explants of Phalaenopsis violacea (Fig. 5). Whereas, in 0 day and 1 day of co-cultivation period, the transformation frequency was very low for both strains especially by using Agrobacterium, EHA 101. Our results are in agreement with the observations of Khan et al. (2003) who also noted that Brassica napus was co-cultivated for two to three day shown highest transformation frequency by using Agrobacterium strain EHA 105.

[FIGURE 5 OMITTED]

Further extension in co-cultivation time known to decreased the transformation frequency due to bacterial overgrowth and had detrimental effect on regeneration potential of any explants. It has been reported that more than 5 days of co-cultivation encourage an overgrowth of other bacteria with a contaminant and decreased in transformation efficiency in garden pea (De Kanthen and Jacobsen, 1990) and flax (Dong and Mchughen, 1993). Longer co-cultivation periods in Brassica napus result in explants turning necrotic and remains devoid of shoot regeneration due to excessive growth of Agrobacterium strain, EHA 101 (pIG 121-Hm) (Khan et al, 2003).

In Broccoli, the transformation frequency was low after 1 day co-cultivation, but increased rapidly when the co-cultivation period was prolong to 7 days, reaching maximum at day 3 (Metz et al, 1995a). Similar results have been reported in banana (Sreeramanan et al, 2006) and Brassica napus (Takasaki et al, 1997). However, a prolonged co-cultivation period of 5 to 7 days has been shown to increased Agrobacterium -mediated transformation in Lilium usitatissimum and Agapanthus (Dong and Mc Hughen, 1993; Suzuki et al., 2001). Seo et al. (2002) reported that GUS expression increased 2.3 times when co-cultivation period was prolonged from 3 to 10 days. Although prolong co-cultivation p eriod for more than 3 days have been successful used for certain plants (Suzuki et al., 2001; Yu et al., 2001), 2 to 3 days co-cultivation has been routinely used in most reported transformation protocols. The differential requirement of co-cultivation periods may be dependent largely upon Agrobacterium strain, type of orchids and explants used for transformation. Mondal et al. (2001) reported that the differential requirement of co-cultivation period largely depend on the Agrobacterium strain used or co-cultivation or medium for Agrobacterium culture.

Different concentrations of L-cysteine

Generally Agrobacterium tumefaciens infection caused tissue browning and necrotic on the plant explants. This is likely common defense response of plants to biotic stress (e.g. pathogen infections) or abiotic stress (e.g. mechanical wounding). The main defense mechanism firstly activated upon pathogen infection or wounding is oxidative burst (Olhoft et al, 2001). The oxidative burst involves production of reactive oxygen species which are thought to activate programmed cell death (PCD) and to generate a barrier of dead cells around the site of infection (Olhoft et al, 2001).

As a result to increase pathogen defense response and reduce wound in plants, L-cysteine, an amino acid, not only has the potential to increase the capacity of Agrobacterium to infect plant tissues and stably transfer its T-DNA but also to increase the frequency of infected cells that remain viable and become transformed (Olhoft et al, 2001). In addition, cysteine is also an inhibitor of few defense enzymes such as peroxidases (PODs), polyphenol oxiadases (PPOs) and enzymatic browning, either directly or indirectly through the action of its thiol group (Olhoft et ai, 2001; Opabode, 2006).

Therefore, the necessity of L-cysteine during co-cultivation was tested. Five different concentrations of L- cysteine (0, 100, 200, 300, 400 mg.[L.sup.-1]) in the co-cultivation medium were carried out. In this experiment, it was found that 200 mg.[L.sup.-1] L-cysteine resulted in the highest frequency of transient GUS expression of PLBs in both strains, EHA 101 (45%) and 105 (50%). The results demonstrated L-cysteine generally increase the percentage of transient GUS gene expression in Phalaenopsis violacea orchid plants (Fig. 6). However, there were 28 to 36% of transient GUS expression on PLBs observed in the absence of L-cysteine obtained by using both EHA 101 and 105 strains. Therefore, with the addition of L-cysteine in the co-cultivation medium, it was demonstrated to increase Agrobacterium-mediated PLBs transformation in Phalaenopsis violacea. In addition, it is assumed that L-cysteine and other thiol compounds inhibit wound- and plant pathogen-induced responses, rendering the PLBs explants more susceptible to Agrobacterium infection and thereby increasing the capacity of these plant totipotent cells to be transformed. Olhoft and Somers (2001) have successfully increased Agrobacterium infection from 37 to 91% of explants in the cotyledonary-node region by amending the solid co-cultivation medium with L-cysteine, which resulted in a fivefold increase in stable T-DNA transfer in newly developed shoot primordial in producing transgenic soybeans (Glycine max (L) Merrill). In addition, Zheng et al. (2003) have reported on the optimization of both Agrobacterium infection and glufosinate selection in the presence of L- cysteine for the study of expressed sequence tag (EST) and functional genomic analyses (Williams 82) applications in modern genetic analysis and multiplication of soybean (Glycine max). Similarly, Enriquez-Obregon et al. (1999) have evaluated the effect of L-cysteine and silver nitrate with known antioxidant activity on the viability of stem section taken from in vitro rice plantlets and on their interaction with Agrobacterium tumefaciens (AT 2260) containing a shuttle vector bearing the gusA and bar genes. Olhoft et al. (2001) reported that Agrobacterium- mediated transformation of soybean cells and the production of fertile transgenic soybean Glycine max (L.) Merrill plants using the cotyledonary-node method were improved by amending the solid co-cultivation medium with L-cysteine.

[FIGURE 6 OMITTED]

However, we have observed that browning occurred on PLBs, similar to hypersensitive symptom at higher concentration of L-cysteine, which is more than 200 mg.[L.sup.-1]. Therefore, this would affect Agrobacterium mechanisms of T-DNA transfer initially which will finally reduced percentage of the transient GUS expression. Moreover, it has been reported with a high level of L-cysteine concentration in co-cultivation medium that demonstrated some negative impacts on in vitro plant morphogenesis profiles, with 400 mg.L1 L-cysteine exhibited significance (p<0.05) decrease in maize embryos (Frame et al, 2002).

Different concentrations of silver nitrate (AgNO3)

Silver nitrate compund is known to inhibit ethylene production, which affects cell division mechanisms. It was reported that in Agrobacterium-mediated transformation, ethylene production was increased during Agrobacterium infection which resulted in a reduced efficiency of gene transfer mechanism in apple (Seong et al., 2005). In general, it is known with the inclusion of silver nitrate that will suppress the ethylene biosynthesis through Ag2+ reducing capacity to bind ethylene receptor produced.

In this study, we examined the effect of silver nitrate in different concentrations on Phalaenopsis violacea PLBs for determination of transformation efficiency based on percentage of transient GUS expression. While evaluating the different concentration of silver nitrate in both Agrobacterium strains, it was found that the highest percentage of transient GUS expression occurred at 60[micro]M and the lowest was at 120[micro]M in both Agrobacterium tumefaciens strains, EHA 101 and EHA 105, respectively (Fig. 7). This could be due to the effect of high concentrations of silver nitrate during co-cultivation is toxic to Phalaenopsis violacea PLBs. Inclusion of silver nitrate in co-culture medium has been proven for its anti-ethylene activity which is common with in vitro plant cultures. The usage of silver nitrate has shown to have other important effects in plant tissue culture, improving somatic embryogenesis, organogenesis and micropropagation in many species (Zhang et al, 2001).

[FIGURE 7 OMITTED]

Addition of silver nitrate in co-culture medium had shown to enhance stable gene transfer in maize (Armstrong and Rout, 2001; Zhao et al, 2001) and Fuji apple (Seong et al, 2005). Silver nitrate significantly suppresses the Agrobacterium growth during co-culture without compromising T-DNA delivery and subsequent T-DNA integration. The suppressed Agrobacterium growth on the target explants could facilitate plant cell recovery and result in increased efficiency of transformation. Orlikowska (1997) reported that, not only does silver nitrate stimulate direct shoot regeneration from rose (Rosa indie a) leaves taken from in vitro cultures and co-cultivated with Agrobacterium tumefaciens for genetic transformation studies, but the compound also inhibits bacterial growth after co-cultivation. They observed that at concentration of 100 mg.[L.sup.-1] silver nitrate after co- cultivation in the plant regeneration medium, will completely retards bacterial growth at least for another 3 weeks. Therefore, with addition of silver nitrate is thus able to replace certain commonly used antibiotics such as cefotaxime and carbenicillin, which are often phytotoxic to tissues at least during the first passage after co-cultivation with Agrobacterium strains.

Tissue damage by Agrobacterium tumefaciens infection has been reported before and seems to be one of the major obstacles for Agrobacterium-mediated transformation. Opabode (2006) reported that silver nitrate is an antinecrotic compound which can reduced oxidative burst during the interaction between plant tissue and Agrobacterium tumefaciens. In addition, Enriquez-Obregon et al (1999) have evaluated the effect of cysteine and silver nitrate with known antioxidant a ctivity on the viability of stem section taken from in vitro rice plantlets and on their interaction with Agrobacterium tumefaciens (AT 2260) containing a shuttle vector bearing the gusA and bar genes. Ozden et al. (2004) reported that involvement of ethylene production (enhanced by wounding during explants preparation) is leads to browning but can be reduced in the presence of silver nitrate in medium at optimum level.

Temperatures ([degrees]C)

Temperature has been considered an important factor affecting the efficiency of Agrobacterium tumefaciens to transfer the T-DNA to plant cells. There were study in which temperature was evaluated in a plant-Agrobacterium tumefaciens interaction used GUS transient expression to determine transformation efficiency (Fullner and Nester, 1996 ; Dillen et al, 1997, Sunilkumar and Rathore, 2001).

In order to evaluate whether co-cultivation temperature parameter has effects on Agrobacterium -mediated transformation efficacy in Phalaenopsis violacea, seven temperature regimes were used to evaluate transformation efficiency, with the results showed in Fig. 8. The highest transformation frequency was observed at 24[degrees]C at which 60-62% PLBs showed GUS activity with no significant different (p<0.05) between the two Agrobacterium strains. In tobacco leaves, it was found that temperature at 24[degrees]C was the co-cultivation temperature regime for transformation, which confirmed the conclusion drawn by Dillen et al. (1997) for T-DNA delivery. The frequencies of percentage of transient GUS positive obtained below and above of temperature 24[degrees]C were decreased markedly (Fig. 8).

[FIGURE 8 OMITTED]

However, both Dillen et al. (1997) and Sunilkumar and Rathore (2001) reported that low temperature below 20[degrees]C during co-cultivation could enhance Agrobacterium-mediated efficiency. Similary, Kondo et al. (2000) reported that the ratio of GUS stained calli to the total calli in garlic decreased by 85% at 20[degrees]C and 69% at 24[degrees]C. Fuller et al. (1996) found that low temperatures improved pilus assembly leading to increased number of pili on the cell surface due to buffer functioning of the vir B-vir D4 part of the T-DNA transfer machinery. Fullner and Nester (1996) concluded that temperature did not only affect vir A, vir B and vir D, but the components of the T-DNA complex were sensitive at high temperature. In the present study, the highest efficiency of the PLB transformation in Phalaenopsis violacea orchid was obtained at 24[degrees]C among the seven tested temperatures by using Agrobacterium tumefaciens, EHA 105 suggesting that appropriately low temperature during co-cultivation can improve for the optimization of transformation system. Therefore, it can be concluded that temperature at 24[degrees]C appears to be more optimal for Agrobacterium transfer machinery and pilus assembly in Phalaenopsis violacea PLBs based on the highest percentage of transient GUS expression on both Agrobacterium tumefaciens strains.

Acknowlegements

The authors wish to thank Dr. Richard I.S. Bretell from CSIRO , Australia for the pCAM BIA 1304 plasm id. This research was supported financially by the Malaysian Science and Technology Toray Foundation (2005) and AIM ST University.

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Corresponding Author: S. Sreeramanan, Department of Biotechnology, AIMST University, Batu 3 1/2, Jalan Bukit Air Nasi, Bedong, 08100, Kedah, Malaysia, E-mails: sreeramanan@gmail.com / sreeramanan@usm.my Tel: 604-6533888 ext 3528 Fax: 604-6565125

(1,2) S. Sreeramanan, (1) B. Vinod, (3) S. Sashi and (1) R. Xavier

(1) Department of Biotechnology, AIMST University, Batu 3 1/2, Jalan Bukit Air Nasi, Bedong, 08100, Kedah, Malaysia,

(2) School of Biological Sciences, Universiti Sains Malaysia (USM), Minden Heights, 11800, Penang, Malaysia,

(3) Institute for Research in Molecular Medicine (INFORMM) Universiti Sains Malaysia (USM), Minden Heights, 11800, Penang, Malaysia.

(1,2) S. Sreeramanan, (1) B. Vinod, (3) S. Sashi and (1) R. Xavier, Optimization of the Transient Gusa Gene Transfer of Phalaenopsis Violacea Orchid via Agrobacterium Tumefaciens: an Assessment of Factors Influencing the Efficiency of Gene Transfer Mechanisms, Adv. in Nat. Appl. Sci., 2(2): 77-88, 2008478-482.
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Title Annotation:Original Article
Author:Sreeramanan, S.; Vinod, B.; Sashi, S.; Xavier, R.
Publication:Advances in Natural and Applied Sciences
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Date:May 1, 2008
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