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)
Selection of transformed immature embryos of oil palm
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
AsPac J. Mol. Biol. Biotechnol., Vol. 15 (3), 2007
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
Selection of transformed immature embryos of oil palm
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
AsPac J. Mol. Biol. Biotechnol., Vol. 15 (3), 2007
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
Selection of transformed immature embryos of oil palm
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
AsPac J. Mol. Biol. Biotechnol., Vol. 15 (3), 2007
Selection of transformed immature embryos of oil palm
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
AsPac J. Mol. Biol. Biotechnol., Vol. 15 (3), 2007
Selection of transformed immature embryos of oil palm
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
AsPac J. Mol. Biol. Biotechnol., Vol. 15 (3), 2007
Selection of transformed immature embryos of oil palm
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
AsPac J. Mol. Biol. Biotechnol., Vol. 15 (3), 2007
Selection of transformed immature embryos of oil palm
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
AsPac J. Mol. Biol. Biotechnol., Vol. 15 (3), 2007
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
Selection of transformed immature embryos of oil palm
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
AsPac J. Mol. Biol. Biotechnol., Vol. 15 (3), 2007
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
Selection of transformed immature embryos of oil palm
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.
AsPac J. Mol. Biol. Biotechnol., Vol. 15 (3), 2007
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.
Selection of transformed immature embryos of oil palm
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
AsPac J. Mol. Biol. Biotechnol., Vol. 15 (3), 2007
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
Selection of transformed immature embryos of oil palm
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.
AsPac J. Mol. Biol. Biotechnol., Vol. 15 (3), 2007
Selection of transformed immature embryos of oil palm
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
AsPac J. Mol. Biol. Biotechnol., Vol. 15 (3), 2007
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.
Selection of transformed immature embryos of oil palm