Determination of minimal inhibitory concentration of ... - MSMBB

Asia AsPacPacific J. Mol. Journal Biol. Biotechnol., of MolecularVol. Biology 15 (3), and2007 Biotechnology, 2007 Vol. 15 (3) : 133-146

Selection of transformed immature embryos of oil palm

133

Determination of minimal inhibitory concentration of selection agents for selecting transformed immature embryos of oil palm Ghulam Kadir Ahmad Parveez*, Na’imatulapidah Abdul Majid, Alizah Zainal and Omar Abdul Rasid Advanced Biotechnology and Breeding Centre, Biological Research Division, Malaysian Palm Oil Board (MPOB), P.O. Box 10620, 50720 Kuala Lumpur, Malaysia. Received 20 July 2007 / Accepted 10 September 2007 Abstract. The effectiveness of four antibiotics (kanamycin, geneticin G-418, paromomycin and hygromycin) and herbicide Basta as a selection agent for transformation of oil palm immature embryos was evaluated. The effectiveness of the selection agents was determined by identifying the minimal concentration of the selection agent required to fully inhibit the growth of oil palm immature embryos. Non-bombarded immature embryos were cultured on hormone-free germination medium containing varying concentrations (10 – 2000 mg/l) of the antibiotics and herbicide. In order to mimic the real transformation condition, bombarded immature embryos were also subjected to the same treatment. The immature embryos were subcultured into fresh medium after four (4) weeks and the growth of immature embryos were recorded every week up to eight (8) weeks. Among the five selection agents evaluated, herbicide Basta and hygromycin proved to be the most effective as they could inhibit the growth of immature embryos at 20 mg/l. Paromomycin and geneticin G-418 requires 100 mg/l and 500 mg/l, respectively, for inhibition. Kanamycin is the least effective as it only inhibits 15% of the immature embryos grown at 2000 mg/l, demonstrating a high endogenous resistance of oil palm immature embryos. It was also demonstrated that the concentration of selection agents required to inhibit non-bombarded immature embryos was the same for bombarded immature embryos. In future experiments immature embryos will be transformed with Basta and hygromycin resistance genes. Keywords. Immature embryos, transformation, selection agents, minimal inhibitory concentration.

Introduction Oil palm (Elaeis guineensis Jacq.), is an important economic perennial crop for Malaysia and its oil, palm oil, is one of the world’s main source of vegetable oils and fats. The total area planted with oil palm in Malaysia has increased from 4.05 million hectares in 2005 to 4.17 million hectares in 2006. The highest expansion was recorded in Sabah and Sarawak due to availability of land. Its production per planted area is 3 times and 10 times higher than coconut and soybean, respectively. In Malaysia, crude palm oil production is increasing annually; from 15 million tonnes in 2005 it has increased to 15.9 million tonnes in 2006, an increase of 6.1% (Basri, 2007). Similarly, the total export volume of oil palm products, such as palm oil, palm kernel oil and oleochemicals has increased by 8.1%, to 20.13 million tonnes in 2006 as compared to 18.62 million tonnes in 2005 (Basri, 2007). �� It is expected that the rate of production and export of oil palm products will continue to progress in the years to come and it will remain as one of the major sources of vegetable oils and fats to feed the

world. However, �� the oil palm industry faces challenges such as the increase in demand over supply, due to the increase in world population, limiting arable land for future expansion and competition from other oil producing crops that are far more advanced in the application of genetic manipulations (Parveez, 1998). Due to the long regeneration time, narrow gene pool and open pollination behavior of oil palm (Rajanaidu and Jalani, 1995), improvement of the crop through conventional breeding alone is limited. Therefore, genetic engineering is earmarked to face these challenges. It was estimated that f�� our to five years are required to produce useful transgenic plantlets from initial date of explant culture (Parveez, 2000). Genetic engineering involves genetically transforming foreign gene into plant genome through cell, protoplast or *Author for Correspondence. Mailing address: Advanced Biotechnolog y and Breeding Centre, Biological Research Division, Malaysian Palm Oil Board (MPOB), P.O. Box 10620, 50720 Kuala Lumpur, Malaysia. E-mail address: [email protected]

134

AsPac J. Mol. Biol. Biotechnol., Vol. 15 (3), 2007

tissue for producing transgenic plant that are physiological and biologically normal (Jenes et al., 1993). The success of the genetic transformation process is monitored through the following 3 steps: the proof for DNA integration, protein expression and transmission of the transgene into its progenies. Practically, during genetic transformation, foreign gene is transferred into target tissue, which contains thousands of cells. Following the genetic transformation process, only a few cells will become transgenic or will have the transgene stably integrated into its genome. It is very important to isolate these transformed cells from the majority of untransformed cells by using a selection agent. The transform cells should carry a selectable marker gene, which will make the cell survive on a particular selection agent. The concentration of selection agents need to be carefully chosen to avoid either being too low and thereby allowing undesirable numbers of ‘escapes’ or chimeric plants to develop, or too high so that transformants expressing moderated levels of resistance are lost. Strong selection at early stages may reduce the number of viable shoots while delayed selection may increase the number of escapes. It was reported that early selection was successfully applied in apple (James et al., 1989) and plum (Padilla et al., 2003) but late selection was efficient for almond (Miguel and Oliveira, 1999). In addition, some selection agents, such as hygromycin, have a deleterious effect on turf grass’s mature embryo development (Cao et al., 2006). On the other hand, the use of kanamycin as selection agent for apricot transformants is beneficial as it helps to improve proliferation of transformed tissues (Petri et al., 2005). Recently it was reported that there are approximately 50 selectable marker genes being used or being developed in transgenic plant research (Miki and McHugh, 2004). Selectable marker can be further divided into two categories, i.e., positive selection or negative selection. Commonly used negative selectable marker genes in plant transformation are genes that confer resistance to antibiotics, such as neomycin phosphotransferase (nptII) and hygromycin phosphotransferase (hpt) and genes that confer resistance to herbicide such as phosphinothricin acetyltransferase (bar). The strategy applied for the negative selectable marker genes is to kill non-transformed cells by supplementing an antibiotic or herbicide in the plant regeneration media and thereby ensuring that only transformed cells grow and proliferate. NptII gene was isolated from E. coli strain Tn5 (Bevan et al., 1983) and confers resistance to the following antibiotics: kanamycin, geneticin G-418, paromomycin and neomycin. NptII catalyses the phosphorylation of the antibiotics, resulting in the inactivation of the antibiotics. Kanamycin is used as a selection agent mainly for dicots plants. Many monocots especially cereals and grasses (Hauptmann et al., 1988) are highly resistant to kanamycin. The antibiotic geneticin G-418 is commonly used for monocots such as sugarcane (Bower and Birch 1992) and barley (Ritala et al., 1994)�� . Paromomycin has been reported as selection agent for transforming oat (Torbert et al., 1995). Hpt gene was isolated from E. coli (Gritz and Davies 1983)


and confers resistance to the antibiotic hygromycin. It is the second most widely used selectable marker gene in transformation studies (Miki and McHugh 2004). Hygromycin �� has been used as a selection agent for producing transgenic monocots such as gramineae species (Hauptmann et al., 1988), rice (Christou et al., 1991) and maize (Weymann et al., 1993). Bar gene was isolated from Streptomyces hygroscopicus (DeBlock et al., 1987) and confers resistance to phosphinothricin, the active ingredient for the herbicides bialaphos, Liberty and Basta, an analogue of glutamate, a competitive inhibitor of the enzyme glutamine synthetase. Phosphinothricin acetyltransferase, coded by bar gene inactivates phosphinothricin by acetylation. Basta has been proven to be an effective selection agent in a number of monocot such as wheat (Vasil et al., 1992), maize (�� Fromm et al., 1990; Weymann �� et al., 1993), rice (Christou et al., 1991) and sugarcane �� (Chowdhury and Vasil, 1992). In addition to the above negative selectable marker genes, recently, there are some positive selectable marker genes, which allow the proliferation of transformed tissue and cause the untransformed cells to starve, which is followed by the suppression of their growth. In addition there is another type of positive selection marker that allows the visual isolation of transformed cells. One example of positive selectable marker is Phosphomannose isomerase (pmi) gene, which has been used to select transformed cells of sugar beat, maize and rice on medium containing mannose (Joersbo et al., 1998; Lucca et al., 2001; Negrotto et al., 2000; Wang et al., 2001). The pmi gene product converts mannose-6-phosphate to the easily metabolized fructose-6-phosphate, which could be utilized by plant cells as carbon source. The non-transformed cells will starve in medium containing mannose and allowing only the transformed cells to proliferate and produce transgenic plants. �� Green �� fluorescent protein (gfp) is an example of visual selectable marker gene. The gfp gene was isolated from jellyfish, Aequorea victoria (Heim et al., 1994). Its fluorescence is nondestructive, stable and species independent (Chalfie et al., 1994). It allows visual detection and isolation of transformed tissue. Gfp has been successfully used as a selectable marker gene for producing transgenic plants such as sugarcane (Elliott et al., 1998), rice (Vain et al., 1998), soybean (Larkin and Finer 2000) and oat (Kaeppler et al., 2000). Evaluation of selection agents for selecting transformed embryogenic calli has been reported earlier. The report revealed that herbicide Basta and antibiotic hygromycin were the most effective selection agents that can select transformants at a lower concentration, 50 mg/l (Parveez et al., 1996). Production of transgenic oil palm using embryogenic calli has been established (Parveez, 2000) in our laboratory; however utilization of immature embryos as target tissue is yet to be established. Recently, transformation of oil palm immature embryos has been reported (Ruslan et al., 2005). However, the report failed to provide any molecular evidence on the integration of the selectable marker gene, as such the effectiveness of the selection system is yet to be proven. Green


fluorescence protein gene was later evaluated as a selectable marker gene for oil palm immature embryos. Expression of gfp gene could be easily detected, under fluorescence microscope. However isolation of gfp expressing tissues and regeneration of transgenic oil palm expressing the gene have not been successful (Na’imatulapidah, 2006). It was postulated that the green fluorescence might be toxic to oil palm cells and / or cause silencing of the gene in oil palm cells. Therefore, in this study, four antibiotics and herbicide Basta were evaluated for identifying the most effective selection agent for selecting transformed oil palm immature embryos.

Materials and methods Direct Germination of Immature Embryos. Immature embryos (IE) were collected from fruits 10-12 weeks after anthesis. Loose fruits were soaked in tap water containing a few drops of Tween 20 for 15 minutes and sterilized in 100% ethanol for 15 minutes. Fruits were air dried in a laminar flow cabinet. Fruits were cut and the embryos were isolated and cultured on Shoot Induction Media (SIM) containing MS macro and micro salts (Murashige and Skoog, 1962), 100 mg/l inositol, 100 mg/l glutamine, 100 mg/l asparigine, 100 mg/l arginine, 30 g/l sucrose, 4 g/l agar and the media pH was adjusted to 5.7. Subsequently, immature embryos were subcultured on SIM media every 4 weeks. Cultures were incubated at 28°C in the presence of light until plantlets were obtained. DNA‑microcarrier Preparation and Bombardment for PDS-1000/He Apparatus. Isolation of Plasmid DNA. Large-scale plasmid DNA isolation was carried out using the QIAGEN Maxiprep Kit. Ten ml cultures were incubated with shaking at 225 rpm, 37°C for 8 hours followed by inoculation of 1 ml of the culture into 100 ml pre-warmed LB. The culture was further incubated overnight with shaking at 225 rpm and 37°C. The culture was transferred into a GSA tube and centrifuged at 7,000 rpm for 20 minutes at 4°C. The supernatant was removed and the bacterial pellet was resuspended in 10 ml Buffer P1. Mixture was vortexed until no cell clumps remained. Ten ml Buffer P2 was added, mixed gently by inverting 4-6 times and incubated for 5 minutes at room temperature. Ten ml chilled Buffer P3 was added slowly, mixed and the mixture was left on ice for 20 minutes. The mixture was centrifuged at 13,000 rpm for 40 minutes at 4°C. While centrifuging, a QIAGEN-tip 500 was equilibrated by allowing 10 ml QBT buffer to flow through the resin by gravity. The supernatant from the GSA bottle was loaded into the column promptly. The QIAGEN-tip was washed twice with 30 ml Buffer QC. A 30 ml SS48 centrifuge tube was placed below the tip and DNA was eluted using 15 ml Buffer QF. A total of 10.5 ml of


135

chilled isopropanol was added and the mixture was incubated at 4°C for 30-60 minutes. The mixture was centrifuged at 12 000 rpm for 40 minutes and the pellet obtained was washed with 5 ml 70% (v/v) ethanol. The tube was centrifuged at 12 000 rpm for 10 minutes and the pellet DNA was air dried in laminar flow. Finally, the DNA was dissolved in 1 ml TE buffer (10 mM Tris, 1 mM EDTA, pH 8). The concentration and purity of the plasmid was quantified using spectrometer. The DNA yield ranged from 300 to 500 µg with good purity i.e. 1.8-2.0 were obtained. The DNA quality was further verified by restriction digests followed by electrophoresis on 1% agarose gel. Preparation of Gold Particle. Microcarrier preparation was carried out according to the Biolistic PDS-1000/He Particle Delivery System Instruction Manual. A total of 60 mg 1.0 μm gold particle was placed in 1 ml 100% ethanol in a microtube. The microcarrier was vortexed for 1-2 minutes and spun for 2 minutes. The microtube was centrifuged at 10 000 rpm for 1 minute and the supernatant removed. The process was repeated 3 times. The microcarrier was suspended in 1 ml distilled water, centrifuged as above and the supernatant removed. The process was repeated twice. Finally microcarrier were resuspended in 1 ml sterile distilled water. While the suspension was being vortexed, it was aliquoted in 100 µl volume in microtubes. Gold aliquots were stored at 4ºC. Bombardment of immature embryos. In order to mimic real transformation condition, bombardment of immature embryos with microcarrier carrying pBluescript plasmid DNA was carried out. DNA precipitation onto gold microcarriers was carried out according to the manufacturer’s instructions for the Biolistics PDS/He 1000 (Bio-Rad) device. Five microlitres of pBluescript DNA solution (1 µg/µl), 50 µl of CaCl2 (2.5M) and 20 µl spermidine (0.1M, free base form) were added sequentially to the 50 µl gold microcarrier suspension. The mixture was vortexed for 3 minutes, spun for 10 seconds in a microfuge and the supernatant was discarded. The pellet was washed with 250µl of absolute ethanol. The final pellet was resuspended in 60 µl of absolute ethanol. Six microlitres of the solution was loaded onto the center of the macrocarrier and was air dried. Bombardments were carried out on a minimum of five replicates. Bombardment were carried out at the following conditions; 1100 Psi rupture disc pressure; 6mm rupture disc to macrocarrier distance; 11mm macrocarrier to stopping plate distance, 75mm stopping plate to target tissue distance and 67.5 mmHg vacuum pressure (Parveez, 1998). Minimal inhibitory concentration of selection agents. All antibiotics were prepared as stock solutions of 50 mg/ml except for the herbicide Basta (Bayer CropScience), which a stock solution of 20 mg/ml was used. All selection agents were filter sterilized and stored at 0°C. SIM medium was autoclaved and cooled to 50°C in a water bath prior to the

136


addition of the selection agent. The selection agents were added to the concentrations of 10, 20, 50, 100, 300, 500, 1000 and 2000 mg/l. Five replicates (plates) were used for each treatment as well as the control. In each plate, 12 immature embryos were placed onto the solidified media and incubated in the dark at 28°C. After 4 weeks, the germinating immature embryos were transferred onto fresh plates containing the same concentrations of antibiotics or Basta. In this experiment, the germination of the untreated control was considered as 100% germination assuming that there was no inhibition or stress to reduce the germination rate. The percentages of germination for each treatment were calculated based on number of germinating immature embryos over the total immature embryos used. Immature embryos that showed some level of germination were considered as germinating. Immature embryos that did not show any sign of germination or development were considered as not germinating. The means of five replicates were used in the final calculations.

RESULTS Determination of kill curve of selection agents using nonbombarded immature embryos. One of the major steps in the production of transgenic plants is the ability to isolate and regenerate cells containing a stably integrated foreign gene. This can be achieved by using a selective agent, at a minimum concentration that will kill all the non-transformed cells and allow for the transformed cells to survive and finally regenerate into a complete transgenic plant. In this work, the minimal inhibitory concentration of 5 different selection agents (4 antibiotics: hygromycin, kanamycin, geneticin G-418, paromomycin and 1 herbicide Basta) was determined. The selection agent that can completely inhibit the proliferation of the calli at an economical concentration is the preferred choice for selecting stably transformed cells. It is extremely important to determine the optimal concentration as this can make the process of selecting transformed cells more efficient and result in either none or a very low occurrence of chimaeras or escape plants produced. Non-bombarded 10-12 weeks old immature embryos were individually isolated and cultures on SIM media containing different concentration of selection agents. The immature embryos were cultured without light in incubators and were let to proliferate at 28°C. After 8 weeks on medium containing selection agents, the percentage of immature embryos that survived were plot against the concentrations of the various selection agents tested (Figure 1). Results showed that antibiotic hygromycin and herbicide Basta are good selective agents for oil palm immature embryos. These two selection agents were shown to be able to effectively kill oil palm immature embryos at a very low concentration, 20 mg/l. This indicates that oil palm is highly susceptible to both selective


agents. Selection using Basta also showed that at a concentration as low as 5 mg/l, almost 80% of the immature embryos failed to survive. With a concentration of 20 mg/l all (at least 99%) of the tissues turned brown and died. At a higher concentration, the immature embryos became dark brown and started drying. Total browning and drying are indicators of extensive cell death. Therefore this concentration is too high to be used for selection of transformed cells. Similar results were also obtained when hygromycin was used as a selection agent. However, the browning of the immature embryos was not as dark as Basta. Kanamycin, geneticin (G‑418) and paromomycin were found to be poor selection agents for stable oil palm transformation using immature embryos. Paromomycin and geneticin G-418 completely killed the immature embryos at 100 mg/l and 500 mg/l, respectively. Paromomycin started killing the immature embryos at a lower concentration as compared to geneticin G-418, which only start killing the immature embryos at a concentration of 200 mg/l. Kanamycin on the other hand only killed around 15% of the embryos at the highest concentration tested; 2000 mg/l. Kanamycin also only started killing the immature embryos at a concentration of 1500 mg/l. These antibiotics induced only slight browning of the tissues at high concentrations. This indicates that oil palm immature embryos are highly resistant to these antibiotics. It is estimated that kanamycin concentration of more than 3000 mg/l will be needed to totally inhibit the growth of oil palm immature embryos. Selection using this high concentration of selection agents is not only economically unfeasible but also biologically ineffective. Kanamycin at a concentration of 2000 mg/l resulted in approximately 15% of the tissue turning brown at the edges, whereas at a concentration of 1000 mg/l, the majority of these tissues survived and their growth was comparable to the controls. Therefore, based on this finding, Basta and hygromycin were shown to be the most suitable selection agents for oil palm immature embryos as they were able to inhibit the growth of immature embryos at a comparatively low concentration. In addition, 20 mg/l hygromycin and Basta completely stopped the embryos development as early as 5 weeks after exposure (data not shown). Determination of kill curve of selection agents using bombarded immature embryos. The results highlighted earlier were on the exposure of non-bombarded immature embryos to various concentrations of antibiotics and one herbicide. However, in practice, immature embryos will be bombarded prior to selection on medium containing a selection agent. Therefore, in this second experiment, the kill curve was only determined for the bombarded immature embryos. All the conditions, type of selection agents and concentration of selection agents were kept constant as in the earlier experiment. The only difference was that after the immature embryos were isolated, they were bombarded with gold microcarrier carrying pBluescript DNA using the



137

Figure 1. Minimal inhibitory concentration of various selection agents on non-bombarded oil palm immature embryos. Note: Bar, Basta; Hg, hygromycin; Km, kanamycin; G-418, Geneticin G418; and Paro, paromomycin.

Figure 2. Minimal inhibitory concentration of various selection agents on bombarded oil palm immature embryos. Note: Bar, Basta; Hg, hygromycin; Km, kanamycin; G-418, Geneticin G418; and Paro, paromomycin.

Figure 3. Percentage of bombarded immature embryos that survive at different times (week) when exposed to various concentrations of paromomycin

Figure 4. Percentage of non-bombarded immature embryos that survive at different times (week) when exposed to various concentrations of paromomycin

standard bombardment condition optimized for oil palm (Parveez, 1998). After bombardment, the immature embryos were transferred onto new Petri plate containing SIM medium with a selection agent. The kill curve for the selection agents using bombarded immature embryos after 8 weeks of exposure is demonstrated in Figure 2. Overall, from Figure 2, there is not much difference between the kill curves of bombarded immature embryos as compared to non-bombarded immature embryos. These results similarly showed that antibiotic hygromycin and herbicide Basta are good selective agents for oil palm immature embryos transformation as they were able to effectively kill the bombarded oil palm immature embryos at a very low concentration, 20 mg/l. From the figure it can be clearly observed that the rate of killing for both selection agents was similar to the non-bombarded immature embryos (Figure 1). Similarly, kanamycin, geneticin (G‑418) and paromomycin were found to be poor selection agents

for oil palm immature embryos. Paromomycin and geneticin G-418 completely killed the bombarded immature embryos at 100 mg/l and 500 mg/l, respectively. The rate of killing also follows the pattern observed for non-bombarded immature embryos. Kanamycin, again only killed about 15% of the bombarded immature embryos at the highest concentration of the antibiotic tested, 2000 mg/l. However, kanamycin starts killing the bombarded immature embryos at a lower concentration as compared to non-bombarded immature embryos, 400 mg/l. The reason for the fast killing of bombarded immature embryos is not known, however, it can be postulated that the bombarded immature embryos were exposed to more stress, resulting in temporary loss of resistance to the antibiotic. A more detail analysis of the kill curve experiment on bombarded and non-bombarded immature embryos will be elaborated in the next section. The above observation reinforces the fact that Basta and hygromycin are the most suitable selection agents for

138



transformation of oil palm using immature embryos as target tissues as they could completely kill the bombarded immature embryos at a low concentration and as early as 5 weeks after exposure to the selection agent. Comparison of kill curve of selection agents between bom‑ barded and non-bombarded immature embryos. Previous sections have demonstrated that there was not much difference in the minimal inhibitory concentration of selection agents on bombarded immature embryos as compared to non-bombarded immature embryos. In this section, a detailed comparison of the killing pattern of one antibiotic, paromomycin, on bombarded and non-bombarded immature embryos will be demonstrated. Detailed kill curve for bombarded and non-bombarded immature embryos was plotted on weekly basis and is shown in Figures 3 and 4, respectively. The results showed that paromomycin killed bombarded and non-bombarded immature embryos at 100 mg/l. Similar profile was observed in Figures 3 and 4. However, the rate of killing by paromomycin at various concentrations will be elaborated here. Generally, it was observed that at higher concentrations of paromomycin, bombarded immature embryos were killed faster than non-bombarded immature embryos. However, at lower concentrations of paromomycin, there was not much difference in the killing of both bombarded and non-bombarded immature embryos. Paromomycin concentration of 2000 mg/l kills bombarded immature embryos at 2 weeks as compared to 4 weeks for non-bombarded immature embryos. Similarly at 1000 mg/l of paromomycin, bombarded immature embryos were killed at 3 weeks as compared to 4 weeks for non-bombarded immature embryos. The same pattern of faster killing by paromomycin on bombarded immature embryos over non-bombarded immature embryos could be seen for concentrations above 100 mg/l, where at 100 mg/l bombarded immature embryos were killed at 7 weeks as compared to non-bombarded immature embryos, which were killed at 8 weeks. However, for paromomycin concentrations below 100 mg/l, there was not much difference in the rate of killing on bombarded immature embryos as compared to non-bombarded immature embryos. Most importantly, the minimal concentration required to kills the immature embryos remain the same either for bombarded or non-bombarded. The data above suggests that bombarded immature embryos are temporarily more susceptible to selection agents as compared to non-bombarded immature embryos. There is no clear explanation for this observation. However, it is possible that as bombarded immature embryos are exposed to additional stresses, such as vacuum pressure and injuries by microcarrier particles, compared to non-bombarded immature embryos, they becomes more susceptible to other stresses, i.e. selection agents. However, the fast rate of killing does not effect the minimum concentration of selection agent required to kill the immature embryos.

Figure 5. Oil palm immature embryos exposed to different concentrations of Basta (top row) and hygromycin (bottom row). From left to right: 20, 50, 300 and 1000 mg/l.

Effects of selection agents concentrations on the inhibition and physical appearance of oil palm immature embryos. Immature embryos were exposed to different concentrations of selection agents and were evaluated weekly. After 8 weeks, it was observed that for Basta and hygromycin, initial growths and development of immature embryos were only obtained when they were exposed to a concentration of up to 20 mg/l (Figure 5). When the concentration of Basta and hygromycin was increased to 50 mg/l and above, the growth and development of immature embryos were inhibited. Furthermore, for the immature embryos exposed to Basta, they turn dark brown in color and appear dehydrated. The browning was not observed on immature embryos exposed to high concentrations of hygromycin. This may be due to the nature of the herbicide Basta that results in accumulation of ammonia in the dead cells. The observation on immature embryos exposed to kanamycin, paromomycin and geneticin G418 is different from that of Basta and hygromycin. Good development and germination of the immature embryos were observed at the end of 8 weeks when they were exposed to selection agents up to a concentration of 20 mg/l (Figure 6). For geneticin G418, similar level of development was observed, but without germination, when the immature embryos exposed to 50-100 mg/l of the selection agent. However, at concentration of 300 mg/l and higher, development of the immature embryos were fully retarded or inhibited. Similar effects of geneticin G418 were observed when immature embryos



139

Figure 7. Oil palm immature embryos after 2 weeks on medium containing various selection agents at different concentrations. From left to right: 10, 100 and 1000 mg/l.

Figure 6. Oil palm immature embryos exposed to different concentrations of kanamycin (top row) geneticin G418 (Middle row) and paromomycin (bottom row). From left to right: 20, 50, 300 and 1000 mg/l.

were exposed to paromomycin. For kanamycin, the effects on immature embryos were milder. At the highest kanamycin concentration of 2000 mg/l, low level of immature embryos development and germination could still be observed. This result indicates that kanamycin has the least inhibitory effects on oil palm immature embryos. For all the four antibiotics, no browning of the immature embryos was observed as demonstrated on immature embryos exposed to herbicide Basta. Effects of selection agent’s exposure duration and concen‑ trations on the inhibition and physical appearance of oil palm immature embryos. Oil palm immature embryos were exposed to different concentrations of selection agents for a period of 8 weeks. After the first two weeks of exposure to the selection agents, immature embryos were observed to be surviving for most of the selection agents (Figure 7). However, browning and growth inhibition of immature

embryos has started to be observed for herbicide Basta at concentration of 100 mg/l and above. After the third week of selection agent’s exposure to oil palm immature embryos, it was observed that only herbicide Basta inhibited the growth of immature embryos at concentration as low as 10 mg/l. The other selection agents did not show much effect on the development of immature embryos. At a concentration of 1000 mg/l, some level of inhibition on immature embryos development was observed for hygromycin, geneticin G418 and paromomycin. Nevertheless, for kanamycin, at a concentration of 1000 mg/l, development of immature embryos was not effected. Four weeks after exposure of immature embryos to different concentrations of selection agents, inhibition of immature embryos growth was observed for more selection agents (Figure 8). Geneticin G418 showed inhibition of immature embryos at concentrations as low as 100 mg/l. For herbicide Basta and hygromycin, inhibition on the growth of immature embryos was observed at 20 mg/l. Furthermore, for herbicide Basta, browning of immature embryos was also observed. However, for antibiotics kanamycin and paromomycin, growth of immature embryos was not much affected. This shows the inherent resistance of oil palm immature embryos to these antibiotics. At five weeks after exposure of immature embryos to the selection agents, kanamycin did not cause any signs of

140



Figure 8. Oil �� palm immature embryos after 4 weeks on medium containing various selection agents at different concentrations. From left to right: 20, 100 and 500 mg/l.

Figure 9. Oil �� palm immature embryos after 7 weeks on medium containing various selection agents at different concentrations. From left to right: 10, 100 and 1000 mg/l.

growth inhibition even at high concentrations. For antibiotics paromomycin and geneticin G418, good development of immature embryos was observed at the concentrations up to 100 mg/l. At higher concentrations, growth inhibition was observed. Growth of immature embryos was severely inhibited when exposed to herbicide Basta and hygromycin even at low concentrations. The observations after 6 weeks exposure of immature embryos to different concentration of selection agents, was similar to that for 5 weeks. The differences observed in a single week were not significant. After exposing the immature embryos to different concentrations of selection agents for 7 weeks, the following observations were recorded. Surprisingly, at a concentration of 1000 mg/l, kanamycin still does not inhibit the growth and germination of oil palm immature embryos (Figure 9). Germination of immature embryos was also observed for geneticin G418 and paromomycin at the lowest concentration of 10 mg/l. Some level of development was observed at 100 mg/l and complete inhibition was observed at concentrations higher than 100 mg/l. For herbicide Basta and hygromycin, inhibition of growth and germination of immature embryos were observed even at the lowest concentration tested (10 mg/l). The results remained the same after 8 weeks of treatment.

DISCUSSION The results of these experiments showed that Basta and hygromycin are the most effective selection agents for oil palm immature embryos. On the other hand, kanamycin, geneticin G‑418 and paromomycin were found to be poor selection agents. This finding is consistent with most of the published data on selection agents used to produce transgenic plants. Basta and hygromycin were effective selection agents for monocotyledonous plants. Basta was successfully used as selection agents in a number of monocots such as rice (Cao et al., 1992), wheat (Vasil et al., 1992), maize (Fromm et al., 1990, Weymann et al., 1993), sugarcane (Chowdhury and Vasil, 1992), lawngrass (Li et al., 2006) and oil palm (Parveez, 2000). In addition, Basta was also reported to inhibit growth of the non-transformants in wheat within a shorter period of time as compared to hygromycin and kanamycin (Nehra et al., 1994). Basta was also shown to induce embryogenesis in wheat tissues (Vasil et al., 1993) and showed no negative effect on the growth of transgenic rice (Cao et al., 1992). In maize, the use of Basta also produced more transformants as compared to kanamycin (Omirulleh et al., 1993). For grass Brachypodium distachyon, selection of transformants using 2-14 mg/l bialaphos (contains the same active ingredient as Basta, phosphinothricin) did not show any difference in the transformation efficiency. However it was found that the


length of callus selection phase seems to be more important than the actual concentrations of bialaphos (Christiansen et al., 2005). A prolong selection period is important to prevent production of escapes. Production of transgenic perennial grass Leymus chinenisis was also obtained using Basta as selection agent (Shu et al., 2005). In agreement with our findings, the authors also reported that the cultures turn brown and necrotic after 15 days in medium containing 15 mg/l phosphinothricin. It was also reported that prolonged exposure to selection in medium containing Basta is needed to reduce escapes. However, in Kentucky bluegrass, it was shown that transgenic plants were produced 1-3 months faster in medium containing hygromycin as compared to medium containing bialaphos (Gao et al., 2006). It was reported that regeneration of bialaphos resistant plants was more complicated due to pronounced incapability to regenerate after prolonged selection. Some of the plantlets obtained were reported to be albino. Longer exposure to Basta may also cause problems related to maturity and fertility as demonstrated in R0 plants of wheat (Vasil et al., 1992). The results observed with hygromycin are also in agreement with reports on other monocots. For example, hygromycin was successfully used as a selection agent to obtain transgenic tissue/plants in a number of gramineae (grasses) species (Hauptmann et al., 1988), rice (Christou et al., 1991; Hiei and Komari, 2006), sorghum (Hagio et al., 1991), tall fescue (Wang et al., 1992), Dendrobium orchid (Kuehnle and Sugii, 1992), maize (Weymann et al., 1993), bent grass Agrostis mongolica (Vanjildorj et al, 2006), a wetland monocot Thypa latifolia L. (Nandakumar et al., 2005), oil palm (Parveez, 2000), grass Brachypodium distachyon (Vogell et al., 2006) and turf-type perennial ryegrass (Cao et al., 2006). It was reported that grass calli were fragile and easily undergo necrosis during antibiotic selection. Since long selective cultures were shown to produce somaclonal variations, the transformed grass calli were directly regenerated to produce plantlets without selection. The regenerated plantlets were transferred to hygromycin contained rooting medium to select resistant plantlets. The well-timed selection process resulted in rapid and efficient production of normal transgenic plants (Cao et al, 2006). Moreover, it was reported that the use of hygromycin as a selection agent resulted in good growth and regeneration in wheat (Nehra et al., 1994). Hygromycin was also reported to produce albino-free transgenic barley plants (Hagio et al., 1995) unlike when geneticin G‑418 or kanamycin was used to regenerate transgenic barley plants (Ritala et al., 1994). However, hygromycin was reported to cause rooty phenotype in embryogenic calli. This calli did not yield any resistant line (Vasil et al., 1993). In castor, it was reported that hygromycin was an effective selection agent as compared to kanamycin. The later could not kill untransformed tissues at a high concentration of 500 mg/l (Sujatha and Sailaja, 2005). However, using a high concentration of the selection agent at the early stage killed the transformants. In order to minimize escape while enhancing proliferation of transgenic


141

plants, selection was carried out by gradually increasing the selection agent from 20 mg/l, followed by 40 mg/l and finally to 60 mg/l. The procedure allowed the proliferation and growth of transformed tissues efficiently. In carnation, 200mg/l hygromycin was shown to be required to produce transgenic plants (Kinouchi et al., 2006). Selection of transformants at 100 mg/l resulted in a high number of escapes. However in pear, 5 mg/l of hygromycin was found to be too high for selection because the tissues were very sensitive to hygromycin (Matsuda et al., 2005). Due to the narrow range of hygromycin concentration for selection, kanamycin was later used to successfully produce transgenic plants. Similarly, the finding that kanamycin, geneticin G418 and paromomycin are not effective selective agents for oil palm is in agreement with a number of published reports on the development of transformation system. Kanamycin was shown to be a poor selective agent for most monocots, as high concentrations are required to be effective. This high endogenous resistance has been reported in a number of gramineae species (Hauptmann et al., 1988), rice (Dekeyser et al., 1989), food yam (Tor et al., 1993), sugarcane (Bower and Birch, 1992); Lolium multiflorum (Potrykus et al., 1985); wheat (Vasil et al., 1991), Dendrobium orchid (Kuehnle and Sugii, 1992), maize (Spencer et al., 1990) and oil palm embryogenic calli (Parveez et al., 1996). Kanamycin was found not to be an ineffective selection agent in these species. However, a number of monocots, such as transformed white spruce embryogenic tissues and transgenic wheat plants from mature embryos have been produced using kanamycin as a selective agent (Ellis et al., 1993; Bommineni et al., 1993; Yang et al., 1994). It was postulated that the high endogenous resistance to kanamycin in monocots might be due to the inability of kanamycin to be transported through the cell wall. Therefore, it was suggested that kanamycin might be more effective if applied to protoplasts before the production of a cell wall (Wilmink and Dons, 1993). It was also suggested that the stage of cell growth, concentration of the selective agent and exposure period needed to be determined prior to the use of kanamycin as a selection agent (Zhang et al., 1988). The use of kanamycin as a selective agent for embryo culture of white spruce was reported to cause the inhibition of somatic embryo development (Bommineni et al., 1993) and also inhibition of regeneration of rice calli (Zhang et al., 1988). Long term selection on kanamycin also caused the inhibition of plantlet growth in rice (Dekeyser et al., 1989) and Dendrobium orchid (Kuehnle and Sugi, 1992). Geneticin G418 was also reported to be an ineffective selection agent for most monocots as a high concentration is required to kill the non-transformed cells. Geneticin G418 was shown to be ineffective for oil palm embryogenic calli (Parveez et al., 1996) and maize (Spencer et al., 1990). In contrast, it was reported to be effective to select transformed cells from non-transformed cells of monocots such as sugarcane (Bower and Birch 1992), barley (Ritala et al., 1994), food yam (Tor et al., 1993), wheat (Vasil et al., 1992, Nehra et al., 1994),

142


Lolium multiflorum (Potrykus et al., 1985), indica rice (Xu and Li, 1994) and bromegrass, Bromus inermis (Nakamura and Ishikawa, 2006). It was reported that non-transformed bromegrass cells were resistant to high concentrations of hygromycin and kanamycin. Selection of putative transformed cells was only possible when they were cultured on medium containing geneticin G418. Transgenic pineapple was also produced after selection using geneticin G418 (Firoozabady et al., 2006). Untransformed pineapple was shown to be highly resistant to kanamycin. This variation in the sensitivity of geneticin G418 as a selection agent in different monocots is due to the different levels of endogenous resistance. This could simply suggest that the endogenous resistance may be tissue or species specific. In this study, paromomycin was found to be not effective for selecting transformants from oil palm immature embryos. The observation augurs the findings in maize (Spencer et al., 1990). Nevertheless, paromomycin has been shown to be an effective selection agent for rice and was a superior selection agent for oat (Torbert et al., 1995) and rubber (Blanc et al., 2006). These variations, again suggests the presence of different level of endogenous resistance in the different species. Production of chimeric plants is a problem encountered in research associated with transgenic plant mainly due to mild selection of transformants. Application of lower or sub-lethal concentrations of selection agents at the early stage of selection is useful to minimize the detrimental effect on the regenerability of callus. However, this approach has resulted in cross-protection of the non‑transformed cells by the neighbouring transformed cells as demonstrated in sugarcane (Bower and Birch, 1992). Separating cell clusters at an early stage of selection and applying selection during regeneration can overcome the cross-protection problem (Fromm et al., 1990). Similarly it was reported that delaying exposure of transformed cells to selection agent is often carried out to provide time for the single transgenic cell to divide a few times making it more capable to express the resistance genes when introduced to selection stress (Ozias-Akins et al., 1993). Finally, exposing the newly transformed cells directly to the lethal dose has also been reported to increase the chance of obtaining transgenic plants by completely killing all the untransformed cells (Ellis et al., 1993; Nehra et al., 1994; Vasil et al., 1993; Hagio et al., 1995).

CONCLUSION Evaluation on the effectiveness of hygromycin, kanamycin, geneticin G-418, paromomycin and Basta as selection agent for oil palm immature embryos were carried out successfully. The results indicated that herbicide Basta and hygromycin were the most effective selection agents as they could inhibit the growth of immature embryos at a very low concentration. Paromomycin and geneticin G-418 were less effective


as they required 100 mg/l and 500 mg/l, respectively, of the selection agent for inhibiting the growth of the immature embryos. Finally, kanamycin is the least effective as it only inhibits 15% of the immature embryos growth at 2000 mg/l, demonstrating the high endogenous resistance of oil palm immature embryos. There was no difference in the effect of the selection agents on both bombarded and non-bombarded immature embryos. Therefore, Basta and hygromycin selectable marker will be used in future works to produce transgenic oil palm using immature embryos as target tissue.

ACKNOWLEDGEMENT The authors thank the Director-General of MPOB for permission to publish this paper. Thanks are also due to the personnel in the Transformation Group of MPOB for their technical assistance. Special thanks to Dr. Rajinder Singh and Dr. Abrizah Othman of MPOB for critically reviewing this paper. This research is funded by MPOB’s In Vitro Transformation of Oil Palm Programme.

REFERENCES Basri, M W (2007). Overview of the Malaysian oil palm industry 2006, Blanc, G; Baptiste, C; Oliver, G., Martin, F and Montoro, P (2006). Efficient Agrobacterium tumefaciens-mediated transformation of embryogenic calli and regeneration of Hevea brasiliensis Mu¨ ll Arg. plants Plant Cell Reports 24: 724–733. Bevan, M W; Flavell, R B and Chilton, M D (1983). A chimearic antibiotic resistance to marker gene as a selectable marker for plant cell transformation. Nature 304: 184-187. Bommineni, V R; Chibbar, R N; Datla, R S S and Tsang, E W T (1993). Transformation of white spruce (Picea glauca) somatic embryos by microprojectile bombardment. Plant Cell Reports. 13: 17‑23. Bower, R and Birch R G (1992). Transgenic sugarcane plants via microprojectile bombardment. The Plant Journal 2: 409‑416. Cao, J; Duan, X; Mcelroy, D and Wu, R (1992). Regeneration of herbicide resistant transgenic rice plants following microprojectile‑mediated transformation of suspension culture cells. Plant Cell Reports. 11: 586‑591.


Cao, M X; Huang J Q; He Y L; Liu S J; Wang C L; Jiang W Z and Wei Z M (2006). Transformation of recalcitrant turfgrass cultivars through improvement of tissue culture and selection regime. Plant Cell, Tissue and Organ Culture 85:307-316. Chalfie, M; Tu, Y; Euskirchen G; Ward, W W and Prasher, D C (1994). Gene fluorescent protein as a marker for gene expression. Science 263:802-805. Chowdhury, M K U and Vasil I K (1992). Stably transformed herbicide resistance callus of sugarcane via microprojectile bombardment of cell suspension cultures and electroporation of protoplasts. Plant Cell Reports 11: 494‑498. Christiansen, P; Andersen, C H; Didion, T; Folling, M And Kristian NIELSEN, K K (2005). A rapid and efficient transformation protocol for the grass Brachypodium distachyon. Plant Cell Reports 23:751-758. Christou, P; Ford, T and Kofron, M (1991). Production of transgenic rice (Oryza sativa L.) plants from agronomically important indica and japonica varieties via electric discharge particle acceleration of exogenous DNA into immature zygotic embryos. Bio/Technology 9: 957‑962. Deblock, M; Botterman, J; Vanderwiele, M; Montagu, M and Leemans, J (1987). Engineering herbicide resistant in plants by expression of a detoxifying enzyme. EMBO J 6:2513-2518. Dekeyser, R; Claes, B; Marichal, M; Van Montagu, M and. Caplan A (1989). Evaluation of selectable markers for rice transformation. Plant Physiology 90: 217‑223. Ellis, D D; Mccabe, D E; Mclnnis, S; Ramachandran, R; Russell, D R; Wallace, K M; Martinell, B J; Roberts, D R; Raffa, K F and Mccown B H (1993). Stable transformation of Picea glauca by particle acceleration. Bio/Technology 11: 84-89. Elliot, A R; Cambell, J A; Brettell, R I S and Grof, C P L (1998). Agrobacterium –mediated transformation of sugarcane using GFP as a screenable marker. Australian Journal of Plant Physiology 25:739-743.


143

Gao, C; Jiang, L; Folling, M; Han, L and Nielsen, K K (2006). Generation of large numbers of transgenic Kentucky bluegrass (Poa pratensis L.) plants following biolistic gene transfer. Plant Cell Reports 25:19-25. Gritz, L and Davies, J (1983). Plasmids-encodes hygromycin B resistance: the sequence of hygromycin B phosphotransferase gene and its expression in Escherichia coli and Sacchoromyces cerevisiae. Gene 25:179-188. Hagio, R; Blowers, A D and Earle, E D (1991). Stable transformation of sorghum cell cultures after bombardment with DNA‑coated microprojectiles. Plant Cell Reports 10: 260‑264. Hagio, T; Hirabayashi, T; Machii, H and Tomotsune, H (1995). Production of fertile transgenic barley (Hordeum vulgare L.) plants using the hygromycin‑resistance marker. Plant Cell Reports 14: 329‑334. Hauptmann, R M; Vasil, V; Ozias-Akins, P; Tabaeizadeh, S G; Rogers, S G; Fraley, R T; Horsch, R B and Vasil, I K (1988). Evaluation of selectable markers for obtaining stable transformants in the Gramineae. Plant Physiology 86:602-606. Heim, R; Prasher, D C and Tsien, R Y (1994). Wavelength mutation and post-translational modification of green fluorescent protein. Proceedings of the National Academy of Sciences of the United States of America 91:12501-12504. Hiei, Y and Komari, T (2006). Improved protocols for transformation of indica rice mediated by Agrobacterium tumefaciens. Plant Cell, Tissue and Organ Culture 85:271-283. James, D J; Passey, A J; Barbara, D J and Bevan, M W (1989). Genetic transformation of apple (Malus pumila Mill.) using a disarmed Ti-binary vector. Plant Cell Reports 7: 658–666 Jenes, B; Moore, H; Cao, J; Zhang, W and Wu, R (1993). Techniques for gene transfer. In Kung, S. and Wu, R. (eds.) Transgenic Plants, San Diego: Academic Press Inc. 1:125-146.

Firoozabady, E; Heckert, H and Gutterson, N (2006). Transformation and regeneration of pineapple. Plant Cell, Tissue and Organ Culture 84:1-16.

Joersbo, M; Donaldson, I; Kreiberg, J; Petersen, S G; Brunstedt, J and Okkels, F T (1998). Analysis of mannose selection used for transformation of sugar beet. Molecular Breeding 19:798-803.

Fromm, M E; Morrish, F; Armstrong, C; Williams, R; Thomas, J and Klein, T M (1990). Inheritance and expression of chimeric genes in the progeny of transgenic maize plants. Bio/Technology 8: 833‑838.

Kaeppler, H F; Menon, G K; Skadsen, R W; Nuutila, A M and Carlson, A R (2000). Transgenic oat plants via visual selection of cell expressing green fluorescent protein. Plant Cell Reports 19:561 – 666.

144


Kinouchi, T; Endo, R; Yamashita, A and Satoh, S (2006). Transformation of carnation with genes related to ethylene production and perception: towards generation of potted carnations with a longer display time. Plant Cell, Tissue and Organ Culture 86:27-35. Kuehnle, A R and Sugii, N (1992). Transformation of dendrobium orchid using particle bombardment of protocorms. Plant Cell Reports 11: 484‑488. Larkin, K M and Finer, J J (2000). Sonicated-assisted Agrobacterium-mediated transformation of embryogenic soybean tissue and subsequent monitoring of gene expression with GFP (abstract). In vitro Cell Dev. Biol. Anim. 36:64A. Li R F; Wei, J H; Wang H Z; He1, J and Sun Z Y (2006). Development of highly regenerable callus lines and Agrobacterium-mediated transformation of Chinese lawngrass (Zoysia sinica Hance) with a cold inducible transcription factor, CBF1. Plant Cell, Tissue and Organ Culture 85:297-305. Lucca, P; Yee, X and Potrykus, I (2001). Effective selection and regeneration of transgenic rice plants with mannose as selective agent. Molecular Breeding 7:43-49. Matsuda, N; Gao, M; Isuzugawa, K; Takashina, T and Nishimura, K (2005). Development of an Agrobacterium-mediated transformation method for pear (Pyrus communis L.) with leaf-section and axillary shoot-meristem explants. Plant Cell Reports 24:45-51. Miguel, C M and Oliveira, M M (1999). Transgenic almond (Prunus dulcis Mill.) plants obtained by Agrobacteriummediated transformation of leaf explants. Plant Cell Reports 18: 387–393


Nandakumar, R; Chen, L and Rogers, S M D (2005). Agrobacterium-mediated transformation of the wetland monocot Typha latifolia L. (Broadleaf cattail). Plant Cell Reports 23:744-750. Negrotto, D; Jolley, M; Beer, S; Wenck, A R and Hansen, G (2000). The use of phosphomannose-isomerase as a selectable marker to recover transgenic maize plants (Zea mays L.) via Agrobacterium transformation. Plant Cell Reports 19:798-803. Nehra, N S; Chibbar, R N: Leung, N; Caswell, K; Mallard, C; Steinhauer, L; Baga M and Kartha, K K (1994). Self‑fertile transgenic wheat plants regenerated from isolated scutellar tissues following microprojectile bombardment with two distinct gene constructs. The Plant Journal 5: 285‑297. Omirulleh, S; Abraham, M; Golovkin, M; Stefanov, I; Karabaev, M K; Mustardy, L; Morocz, S and Dudits,D (1993). Activity of a chimeric promoter with the doubled CaMV 35S enhancer element in protoplast‑derived cells and transgenic plants in maize. Plant Molecular Biology 21: 415‑428. Ozias‑Akins, P; Schnall, J A; Anderson, W F; Singsit, C; Clemente, T E; Adang, M J and Weissinger, A K (1993). Regeneration of transgenic peanut plants from stably transformed embryogenic callus. Plant Science 93: 185‑194. Padilla I M G; Webb, K and Scorza, R (2003). Early antibiotic selection and efficient rooting and acclimatization improve the production of transgenic plum plants (Prunus domestica L.). Plant Cell Reports 22: 38–45

Miki, B and McHugh, S (2004). Selectable marker genes in transgenic plants: applications, alternatives and biosafety. Journal of Biotechnology 107:193-232.

Parveez, G K A; Chowdhury, M K U and Saleh, N M (1996). Determination of minimal inhibitory concentration of selection agents for oil palm (Elaeis guineensis Jacq.) transformation. Asia Pacific Journal of Molecular Biology and Biotechnology 4: 219-228.

Murashige, T and Skoog T (1962). A revised method for rapid growth and bioassays with tobacco tissue cultures. Physiologia Plantarum 15: 473‑497.

Parveez, G K A (1998). Optimization of parameters involved in the transformation of oil palm using the biolistic method. Ph. D. Thesis. Universiti Putra Malaysia.

Na’imatulapidah, A M (2006). The development of gfp as selectable marker gene in oil palm transformation. Master of Science thesis submitted to Universiti Kebangsaan Malaysia.

Parveez, G K A (2000). Production of transgenic oil palm (Elaeis guineensis Jacq.) using biolistic techniques. In Molecular Biology of Woody Plants (Eds: S.M. Jain and S.C. Minocha.). Kluwer Academic Publishers, Vol2, 327-350.

Nakamura, T And Ishikawa, M (2006). Transformation of suspension cultures of bromegrass (Bromus inermis) by Agrobacterium tumefaciens. Plant Cell, Tissue and Organ Culture 84:293-299.

Potrykus, I; M.W. Saul, J. Petruska, J. Paszkowski and R. Shilito (1985). Direct gene transfer to cells of graminaceous monocot. Molecular & General Genetics 199: 183‑188.



145

Rajanaidu, N and Jalani, B S (1995). World-wide performance of DXP planting materials and future prospects. In Proceedings of 1995 PORIM National Oil Palm Conference. - Technologies in Plantation, The Way Forward. 11-12 July 1995. Kuala Lumpur: Palm Oil Research Institute of Malaysia, pp.1-29.

Vanjildorj, E; Tae-Woong, B T W; Riu, K Z; Yun, P Y; Park, S Y; Lee, C H and Kim, S Y (2006). Transgenic Agrostis mongolica Roshev. with enhanced tolerance to drought and heat stresses obtained from Agrobacterium-mediated transformation. Plant Cell, Tissue and Organ Culture 87:109–120.

Petri, C; Albuquerque, N and Burgos, L (2005). The effect of aminoglycoside antibiotics on the adventitious regeneration from apricot leaves and selection of nptIItransformed leaf tissues. Plant Cell, Tissue and Organ Culture 80: 271–276

Vasil, V; Brown, S M; Re, D; Fromm, E M and Vasil, I K (1991). Transformed callus lines from microprojectile bombardment of cell suspension cultures of wheat. Bio/Technology 9: 743-747.

Ritala, A; Aspegren, K; Kurten, U; Salmenkallio‑Marttila, M; Mannonen, L; Hannus, R; Kauppinen, V; Teeri, T H and Enari, T (1994). Fertile transgenic barley by particle bombardment of immature embryos. Plant Molecular Biology 24: 317-325. Ruslan, A; Alizah, Z; Wee, Y H; Leaw, C L; Yeap, C B; Lee, Mvp; Salwa, A S; Winnie, Y S P; Juanita, L S; Siti, A J; Muhammad, R M and Yeun, L H (2005). Immature embryo: A useful tool for oil palm (Elaeis guineensis Jacq.) genetic transformation studies. Electronic Journal of Biotechnology 8:25-34. (This paper is available on line at )

Vasil, V; Castillo, A M; Fromm, E M and Vasil, I K (1992). Herbicide resistant transgenic wheat plants obtained by microprojectile bombardment of regenerable embryogenic callus. Bio/Technology 10: 667-674. Vasil, V; Srivastava, V; Castillo, A M; Fromm, E M and Vasil, I K (1993). Rapid production of transgenic wheat plants by direct bombardment of immature embryos. Bio/Technology 11: 1553-1558. Vogel J.P; Garvin D.F; Leong O.M. and Hayden D.M. (2006). Agrobacterium-mediated transformation and inbred line development in the model grass Brachypodium distachyon. Plant Cell, Tissue and Organ Culture 84:199-211

Shu, Q Y; Liu, G S; Xu, S X; Li, X F and Li, H J (2005). Genetic transformation of Leymus chinensis with the PAT gene through microprojectile bombardment to improve resistance to the herbicide Basta. Plant Cell Reports 24:36-44. Spencer, T M; Gordon‑Kamm, W J; Daines, R J; Start, W G and Lemaux, P G (1990). Bialaphos selection of stable transformants from maize cell culture. Theoretical and Applied Genetics 79: 625‑631.

Wang, Z Y; Takamizo, T; Iglesias, V A; Osusky, M; Nagel, J; Potrykus, I and Spangenberg, (1992). Transgenic plants of tall fescue (Festuca arundinacea Schreb.) obtained by direct gene transfer to protoplasts. Bio/Technology 10: 691‑696.

Sujatha, M and Sailaja, M (2005). Stable genetic transformation of castor (Ricinus communis L.) via Agrobacterium tumefaciens-mediated gene transfer using embryo axes from mature seeds. Plant Cell Reports 23:803-810.

Wilmink, A and Dons, J H M (1993). Selective agents and marker genes for use in transformation of monocotyledonous plants. Plant Molecular Biology Reporter 11: 165-185.

Torbert, K A; Rines, H W and Somer, D A (1995). Use of paromomycin as a selective agent for oat transformation. Plant Cell Reports 14:635-640.

Weymann, K; Urban, K; Ellis, D M; Novitzky, R; Dunder, E; Jayne, S and Pace, G (1993). Isolation of transgenic progeny of maize by embryo rescue under selective conditions. In Vitro Cell Dev. Biol. 29P: 33‑37.

Tor, M; Ainsworth, C and Mantell, S H (1993). Stable transformation of the food yam Dioscorea alata L. by particle bombardment. Plant Cell Reports 12: 468‑473. Vain, P; Worland, B; Kohli, A; Snape, J W and Christou, P (1998). The Green fluorescent protein (GFP) as vital screenable marker in rice transformation. Theoretical and Applied Genetics 96:164 –169.

Wang, M B; Wesley, S V; Fennegan, E J; Smith, N A and Waterhouse, P M (2001). Replicating satellite RNA induces sequences-specific DNA methylation and truncated transcripts in plants. RNA 7:16-28.

Xu, X and Li B (1994). Fertile transgenic Indica rice plants obtained by electroporation of the seed embryo cells. Plant Cell Reports 13: 237-242. Yang, S; Zhu, Y; Qiang, Q and chen, Z (1994). Transformation of mature wheat embryos by particle bombardment. Asia Pacific Journal of Molecular Biology &

146


Biotechnology 2: 342‑345. Zhang, H M; Yang, H; Rech, E L; Golds, T J; Davis, A S; Mulligan, B J; Cocking, E C and Davey, M R (1988). Transgenic rice plants produced by electroporation‑mediated plasmid uptake into protoplasts. Plant Cell Reports 7: 379‑384.
