1

9th International Conference on Agricultural Biotechnology: Ten Years After

organized by the:

International Consortium on Agricultural Biotechnology Research (ICABR)

and the:

Catholic University of Leuven CEIS-University of Rome “Tor Vergata”

Centre of Sustainable Resource Development, University of California at Berkeley

Economic Growth Centre, Yale University Ravello (Italy), July 6-10, 2005

“ A Decade of Oil Palm Gene Manipulation. Where are we now?”

Ruslan Abdullah

Oil Palm Biotechnology Group, School of Bioscience & Biotechnology, Faculty of Science & Technology, Universiti Kebangsaan Malaysia,

43600 UKM-Bangi, Malaysia Telephone:+603-89215698; Telefax: +603-89252698;

Email: russzn@ukm.my, russzn@yahoo.com.my)

Abstract

The amenability of oil palm (Elaeis guineensis Jacq.) tissues to transgene transfer was first

demonstrated more than 10 years ago. Since then several techniques, both direct and vector-

mediated, were developed to introduce useful genes into different target tissues of oil palm.

In both cases, plants produced were either chimearic or transgenic. The genes introduced

include those conferring resistance to insect pests, diseases, herbicide, antibiotics, and more

recently those involved in the biosynthesis of fatty acids and bioplastics. The first batches of

transgenic plants produced were engineered to harbour the cowpea trypsin inhibitor (CpTI)

gene and the plants are now more than 8 years old. The stability of transgene integration

was continuously monitored throughout the plants life span using molecular techniques. The

transgenic CpTI plants in the planthouse took almost 4 years to flower, 2 years more than

plants planted under field conditions. Transgenes were detected in both reproductive and

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non-reproductive organs of T0 plants. Continuous bioassays on the 8 year-old CpTI plants

showed increased tolerance to bagworm larvae (Metisa plana Walker) as compared to non-

transformed controls. Using the same strategy but with different gene(s), work are in

progress to address basal stem rot caused by Ganoderma boninense in oil palm. Recently,

several genes involved in biosynthesis of fatty acids and bioplastics were successfully

transferred into target tissues and regenerated into complete plants. Preliminary assessment

indicates successful gene transfer. Functional expressions of genes leading to changes in

fatty acid profiles and the production of PHB have also been demonstrated. Results from

transmission electron microscopy (TEM) analyses on leaves from plants transformed with

PHB genes showed the presence of additional granules having properties similar to those of

PHB produced in other plants. Whilst the golden crop is being transformed with other

useful genes expressing novel traits, the techniques of gene transfer for oil palm is

continuously improved with emphasis given on their efficiency, role of different promoters,

enhancers, transgene stability and inheritance, gene targeting and also novel gene transfer

techniques.

1. Introduction

The oil palm industry in Malaysia has experienced rapid growth in the last three

decades and has emerged as one of the most important source for vegetable oils and fat in

the world. The emergence of palm oil in the world vegetable oil scenario was mainly due to

Malaysia’s capability to undertake large-scale plantings of oil palm starting from as early as

the mid 1960’s. It was aimed initially to reduce the country’s dependency on rubber and tin.

Rapid increase in the production of Malaysian palm oil has helped improved the status of

palm oil in the world’s production and export of vegetable oils. Currently, palm oil is the

second most produced vegetable oil after soya bean, amounting to almost 20% of the

world’s production. In term of export, palm oil commands at least 42% of the world’s

export in vegetable oil. Over a short period of about 40 years, palm oil production in

Malaysia has increased by leaps and bounds. From a humble production of about 92,000

tonnes in 1960, production increased significantly to almost 2.6 Million tonnes twenty years

later (1980). And in the last 5 years, palm oil production in Malaysia further showed a

steady increase from 7.81 million tones (1995), to 14.5 Million tones in 2004 and is

expected to reach 35 Million tonnes by 2020. Continuous steady increase in palm oil

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production in Malaysia is mainly due to the favourable climatic and growth conditions,

advancement in processing and down-streaming technology, rapid progress in research and

development and implementation of efficient plantation management systems. In addition,

the government is continuously providing the necessary support to the industry following its

contribution to the nation’s economy. In 2004 alone, the oil palm industry contributed to

more than USD 6.1 Billion (2000) and USD 6.9 Billion (2003) to the nations income,

second only to electronic goods. In the world market, Malaysia contributed to more than

63.9% of the world’s production of vegetable oil (Table 1,2 and Figure 1).

Table 1. World production of selected oils and fats (Million tones)

Year Palm oil

Palm kernel

oil

Soya-bean oil

Sun-flower

oil

Rape-seed oil

Other vegetable

oils

Animal oils and

fats

Total

1960 1.26 0.42 3.33 1.79 1.10 8.95 11.18 28.03 1970 1.74 0.38 5.68 3.49 1.83 10.01 14.46 38.39 1980 4.55 0.64 13.32 5.04 3.53 11.17 18.26 56.51 1990 10.95 1.45 16.10 7.87 8.16 15.02 20.20 79.75 2000 21.11 2.64 22.46 13.30 15.01 17.17 21.57 110.26

Source: MPOB, Oil World Monthly 26 Jan 2001

Table 2. Malaysian Palm Oil Position 1998 1999 2000 World oils and fats production (Million tonnes) 101.8 108.8 112.6 World palm oil production (Million tonnes) 16.8 20.3 21.3 Malaysian palm oil production (Million tonnes) 8.3

(49%) 10.6

(52%) 10.8

(51%) World oil and fats exports (Million tonnes) 33.0 33.8 34.8 World palm oil exports (Million tonnes) 11.5 13.5 14.4 Malaysian palm oil exports (Million tonnes) 7.4

(64%) 8.8

(65%) 9.2

(64%) World planted area (Million hectares) 194.6 200.5 203.5 World oil palm area (Million hectares) 7.2 6.12 6.49 Malaysian oil palm area (Million hectares) 3.1

(43%) 3.3

(54%) 3.4

(52%) Foreign exchange earnings (commodities, Billion USD) 8.2 7.3 6.1 Foreign exchange earnings (palm oil, Billion USD) 4.7

(57%) 3.8

(52%) 2.6

(43%) Source: MPOB, Oil World Monthly 26 Jan 2001, Bank Negara Malaysia

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0500

1000150020002500300035004000

1962 1975 1980 1985 1990 1995 2000 2001 2003

Year

Hec

tara

ge ('

000)

Figure 1. Areas planted with oil palm in Malaysia

Oil palm is a perennial monocot with a long generation period. Thus, oil palm

breeding is a very slow process. Currently, oil palm improvements are mainly carried out

through conventional means. Compared to other oil producing crops, which are mainly

annuals, it may require between 12 to 14 years to improve or introduce a trait(s) into oil

palm. In addition to the long generation period, breeding of perennials such as oil palm

requires large areas for breeding trials and extensive series of backcrossing which is time

consuming. Therefore, innovative methods are needed to rapidly enhance the incorporation

of new genetic resources into oil palm. This has become more important since the early

days of oil palm breeding emphasized mainly on yield improvement and to a limited extent

on oil quality, but neglecting other important traits such as tolerance to biotic and abiotic

stresses. The need to rapidly broadening the gene pool of oil palm is extremely important

since almost all commercial hybrid oil palm trees planted in Malaysia originated from four

palms taken in from the Bogor Agricultural Garden (Indonesia) in 1870.

2. Biotechnology and Oil Palm

Biotechnology was first introduced to the oil palm industry almost three decades

ago. Initially, tissue culture techniques were used to propagate elite oil palm clones on a

large scale. The promise of a 30% increase in yield, simply by clonal high yielding palms

have resulted in the race to produce elite clonal palms in the shortest possible time,

neglecting the necessity to fully understand the basic sciences underlying the promising

technology. Unfortunately, excessive use of growth hormones particularly cytokinins to

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speed up embryoids multiplication resulted in the production of clonal palms with abnormal

features (Figure 2). Among the abnormal features observed were palms with high degree of

hermaphrodite bunches, normally seen only in young palms but at very low percentage.

Normally in seed-derived palms, incidence of hermaphrodite bunches normally subsides as

the palms mature and eventually disappeared. However, it was observed that clonal palms

derived from tissue culture exhibit severe degree of hermaphrodite bunches. Even though it

eventually subsides, but it took three times much longer time than those of seed-derived

palms. A more serious abnormal development is the production of mantled fruit bunches. In

this case, all three carpels developed into mesocarp resulting in the production of usually

seedless fruits. Normally mantled fruit bunches are not seen in seed-derived palms. The

reversion of mantled fruit to normal fruit depends greatly on its severity. Severely mantled

bunches would take between 5-8 years before it can revert back to normal. On the other

hand, less severe mantled bunches will take about 2-3 years to revert back to normal. It was

shown that, the degree of mantleness is associated to the amount and types of cytokinins

used in culture media during micropropagation, indicating the epigenetic nature of both

hermaphrodite and mantle bunches. Finally, a non-reversible abnormality classified as

androgyny, where female flower developed on male inflorescence, gave rise to almost

entirely sterile bunches with no yield. Since its occurrence in 1986, there has been no report

on complete or partial reversion of androgyny palms. In most cases, androgyny palms were

either extracted and replaced with other palm or left for further evaluation. Following these

abnormalities, oil palm tissue culture techniques have undergone continuous improvement.

After almost 30 years of continuous research and improvement, production of clonal palms

are now more reliable with minimal abnormality reported. These improvements together

with encouraging results from several field trials have made clonal propagation once again

the planting material of choice especially for elite palm production. Therefore, it is

envisaged that clonal palms would eventually replace seed-derived planting materials on a

commercial scale.

Complete plants can be successfully regenerated from various explants of oil palm.

They include mature (ME) and immature embryos (IE), apical meristems (AM),

embryogenic cell suspension cultures (ECS), friable embryogenic tissues (FET) and callus

derived from seedlings, roots, inflorescences and young leaves. The frequencies for

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Figure 2. Oil palm reproductive organs showing (A) a normal female bunch, (B) a normal male infloresence, (C) an androgyny bunch and the difference between a (D) normal and an (E) abnormal mantled fruit.

complete plant regeneration from some of these explants are still inefficient. Nevertheless, it

has become almost routine in many laboratories. As in the case for most monocots, the

introduction of foreign genes into oil palm was limited by the lack of an efficient and rapid

plant regeneration system. However, the ability to regenerate complete plants from all the

above explants has made oil palm amenable to genetic manipulation for the incorporation of

foreign gene(s). Not all explants, however, are suitable for genetic manipulation studies.

Following advancements in genetic transformation studies, it is possible to transfer

any foreign genes into any targeted plant genome. Oil palm is no exception. One routine

technique is by using Agrobacterium spp. Though the hosts for Agrobacterium are mainly

limited to dicots, however, more and more monocots can now be made susceptible to

A B C

D E

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Agrobacterium leading to successful gene transfer. Over the last few years, our laboratory

has been instrumental in developing conditions to make oil palm tissues become susceptible

to Agrobacterium infection. Now, following various modifications, oil palm tissues are

routinely transformed using both Agrobacterium- and biolistic mediated gene transfer

system. Though DNA could be delivered directly into protoplasts via electroporation, but

this is not possible for oil palm since the system for complete plant regeneration from

protoplasts is not fully established, with only first and second divisions observed.

Nevertheless, the introduction of DNA either through direct- or Agrobacterium-mediated

system would benefit genetic transformation of recalcitrant and perennial crops like oil

palm the most since, the technique enables the transfer of any gene to virtually any tissues

or cell types.

Studies on the influence of physical parameters and different promoters in assaying

genetic transformation events have been reported for oil palm. However, these reports lack

substantiated evidence for stable integration of the transgenes involved. Here, we report

progress made in genetic transformation of oil palm over a period of more than a decade.

Results presented include studies on the development of direct- and vector-mediated gene

transfer system leading to the production of transgenic oil palm plants resistant to insect and

also with value added products. Studies includes the evaluation of important parameters and

considerations in developing a reliable and reproducible gene transfer system using both

approaches. These new developments would provide new avenues for rapid introduction of

new and useful traits into oil palm, which until now, is dependent solely on conventional

means for improvement.

3. Important parameters for the development of gene transfer system for oil palm

a) Target tissues

The main prerequisite for an efficient genetic transformation system is the ability to

regenerate complete plants from treated target tissues. Unlike other crops, oil palm tissue

culture is a very slow process. On average at least 18 months are required to produce

complete plants from callus derived from various explants. On the other hand, IE isolated

from fruits 9-10 weeks after anthesis (WAA), readily germinate into complete plants on

hormone-free medium. Germination could reach up to almost 100%. However, IE isolated

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from 8 WAA fruits or younger normally fail to germinate. In addition to poor culture

response from IE younger than 8WAA, the endosperm from these fruits are still molten,

resulting in poor recovery of viable embryos. On an appropriate medium, such as N62.5 and

N6FET, IE could give rise to callus within 4-6 weeks, much faster than other explants.

Cultured IE started to swell and expanded after 3 days on callusing media and yielded

primary calli within 4-6 weeks. While on the same media, the primary callus produced

embryogenic callus with distinct somatic embryos of different shapes and stages. Upon

transfer to N60, torpedo-shaped embryos germinated into complete plants, completing the

whole sequence of in vitro culture of IE for complete plant regeneration via callus for just

about 3-4 months. The reduced time required for IE to produce callus (in this case 4-6

weeks) as opposed to 8-52 weeks for young leaves, would mean shorter period in culture.

This would eliminate the possibility for the onset of chromosomal aberrations that would

lead to the production of abnormal plants. Plants with rigorous root system normally

requires between 2-4 weeks to harden in plastic chamber with a relative humidity of 80-

95% and were then ready for transfer into polybags and slowly exposed to external

conditions.

IE are abundant, where on average between 300-500 IE could be extracted from a

single developing bunch. Large numbers of IE available enabled large-scale genetic

transformation studies to be carried out on oil palm. It was also observed that, both biolistic

and Agrobacterium-mediated gene transfer into oil palm IE are not dependent on clone. All

three Elaeis guineensis varieties namely dura, pisifera and tenera and IE from Elaeis

oleifera tested for both biolistic and Agrobacterium-mediated gene transfers were found

susceptible to both gene transfer systems. In addition, using IE as target tissues for genetic

transformation studies of oil palm offers an additional advantage where transgenic plants

from transformed IE could be used directly as crossing partners to introduce new or elite

genes into specific breeding programs, and with minimum fidelity-associated problems.

This would further shorten the breeding cycle for oil palm. However, since IE are often the

product of cross pollination between two separate parents, therefore, they are often non-

uniform in terms of their genetic make up, especially if it involved open pollination. A more

desirable case would be to transform a self-pollinated dura or pisifera of known parentage.

Nevertheless, following its abundance, highly responsive nature in vitro, reduced clonal

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fidelity-associated problems, and the ability to allow the introduction of elite genes rapidly,

IE are considered the most suitable target tissues for transformation studies of oil palm.

IE may not serve as an ideal target tissue for the introduction of novel genes leading

to the production of elite materials, but it could serve as an entry point for novel gene

introduction. This was evident from our continuous evaluation of chimearic plants

produced. In chimearic plants, transgenes are present in reproductive organs developing

from vegetative plant parts carrying the transgenes. Separating these reproductive organs

and culturing them in vitro would yield true transgenic plants. This can be done and has

been done by either through infloresence culture or embryo rescue, since protocols for

complete plant regeneration are fairly established, regardless of the origin of the target

tissues.

b) Selectable marker system for selecting putative transformants

Since the efficiency of plant transformation is less than optimal for many important plant

species, thus, the development of transgenic plant requires the use of suitable selectable

marker genes. Like most monocots, selectable marker genes that were suitable for dicots

may not be suitable for monocots. As such, in vitro tolerance studies were carried using

various potential target tissues of oil palm to facilitate future use of suitable antibiotics for

selecting putative transformants.

In the in vitro tolerance studies (Figure 3), freshly extracted IE, IE-derived primary

callus (PC), IE-derived embryogenic callus (EC) and IE-derived friable embryogenic tissues

were cultured on their maintenance media supplemented with different concentrations of

antibiotics commonly used as selectable markers. Antibiotics tested were kanamycin (Km),

geneticin (G418), chloramphenicol, hygromycin (Hm) and phosphinotricin (Ppt). Cultures

were sub-cultured every two weeks and observed over a period of 8 passages. IE were

insensitive to both Km and G418, where 250mg/l Km or G418 did not have any effect on its

growth even after 8 passages (16 weeks). IE were only slightly sensitive to chloramphenicol

at level higher than 100mg/l especially after the 5th passage or 10 weeks in culture with a

survival rate of 65% (at 100 and 120 mg/l) and 45% (at 150 mg/l after the 8th passage).

Unlike Km, G418 and chloramphenicol, both Hm and Ppt were toxic to oil palm tissues. All

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IE exposed to more than 50 mg/l Hm were dead within 4 passages (8 weeks), and within 7

passages (14 weeks) for exposure at 20 mg/l. Similar results were obtained for Ppt, where

all IE were killed when exposed to 20mg/l or more. In all cases, however, control IE

cultured on N60 continued to germinate and proliferate into complete plantlets. The only

difference between the effect of Hm and Ppt on IE was in the rate of IE death recorded. It

was more rapid in the case for Ppt but slightly more gradual for Hm. A slow kill is preferred

over an immediate kill. This is necessary to allow the small numbers of putative transgenic

0

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0 2 4 6 8 10 12 14 16

Control50 mg/l80 mg/l100 mg/l150 mg/l200 mg/l

% S

urvi

val

No. of Weeks in CultureA

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Control50 mg/l80 mg/l100 mg/l150 mg/l200 mg/l

% S

urvi

val

No. of Weeks in CultureC

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Control5 mg/l10 mg/l20 mg/l50 mg/l100 mg/l

% S

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Control5 mg/l10 mg/l20 mg/l50 mg/l100 mg/l

% S

urvi

val

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0 2 4 6 8 10 12 14 16

Control50 m g/l80 m g/l100 m g/l150 m g/l200 m g/lLine 7

% S

urvi

val

No. of Weeks in CultureB

Figure 3. Studies on the in vitro tolerance of oil palm immature embryos (IEs) to various antibiotics commonly used as selectable marker to select putative transformants. Sensitivity of IEs to various concentration of A) kanamycin (Km), B) geneticin (G418), C) chloramphenicol, D) hygromycin (Hm), and E) phosphinotricin.

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cells in a large population of non-transformed ones to undergo multiplication. This becomes

more critical if the percentage of transient transformation is very low and the plant systems

used have a longer doubling time (td), as in the case of oil palm.

Similar results were obtained for other target tissues tested, including primary callus,

embryogenic callus and friable embryogenic tissues, where all PC, EC and FET were

insensitive to both Km and G418, slightly sensitive to chloramphenicol but very sensitive to

both Hm and Ppt. Thus in subsequent experiments selection for putative transformants were

done either on Hm- or Ppt-containing medium.

c) Influence of genetic elements on successful gene transfer

i) Promoters

Promoter plays a major role for successful gene transfer in monocot. In our studies, the

most commonly used promoters for genetic transformation of oil palm was CaMV35S

followed by ubiquitin and actin, which proved to be functional in its new host regardless of

cell types. Target tissues tested includes primary callus, friable embryogenic tissues,

somatic embryos, immature embryos particularly its vascular bundles, vascular bundles of

leaf and roots. However, in cases that necessitate specific targeting of genes, other specific

promoters such as mesocarp- and leaf-specific promoters were used, often with targeting

sequence such as plastid targeting sequence.

ii) Single vs. co-transformation.

Current approach in gene manipulation for crop improvement often requires the insertion of

more than one gene. However, cloning of more than one gene in a construct often resulted

in the vector becoming too big, thus complicating subsequent manipulation and integration.

A co-transformation approach was therefore developed to overcome such limitation. We

have shown that more than one unlinked plasmids could be transferred into a single

bombarded target tissues. Similarly, as shown below, co-transformation protocols were also

developed for Agrobacterium-mediated gene transfer.

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iii) binary vs. superbinary vectors

Binary vectors have been widely used in gene transfer of foreign genes into plants, in

particular monocots. This is largely due to its ease in manipulation and construction.

Experiments using binary vectors such as pBI121, pCAMBIA1301, pCAMBIA1304,

pCAMBIA1305.2, pGREEN, pMRT1301, pMRC1301, pUBC, pUBB, pCAMBt1,

pBRC73, pDM402, pJLPHB3, pSAPHA1305.2 have all shown respectable percentage of

transient expression (20-70%). However, a further improvement was observed when a

super binary vector (as high as 70-90%) was used instead of a binary vector. In addition, we

also observed increased level of transgene expression in tissues co-cultivated with A.

tumefaciens carrying super binary vector as compared to those harbouring binary vectors. In

this respect, we have successfully developed our own modular superbinary vectors pMR507

and pMR607, where gene of interests can be shuttled in and out of the vector with ease.

d) Chimearic vs. transgenic plants

True transgenic plants are preferred over chimearic plants. True transgenic plants are easily

obtained from plants that are very responsive in vitro, with short period for callus initiation,

multiplication and subsequent plant regeneration. However, oil palm normally requires

more than 24 months for complete plant regeneration. Therefore, the long culture period

together with possible deterioration on the fidelity of the clones produced makes rapid

production of transgenic oil palm plant very difficult. Thus the choice of target tissue

becomes critical. The shortest time complete transgenic plant could be regenerated from

bombarded or co-cultivated IE going through callusing, subsequent multiplication and plant

regeneration was about 18-20 months. The use of other target tissues, on the other hand

would be much longer. However, it is now possible to produce true transgenic plants from

bombarded embryo without going through callusing, multiplication and plant regeneration,

but the success rate was very low. Thus, new approaches are being tested to shorten the time

taken to produce transgenic oil palm. One possible way was through Agrobacterium-

mediated transformation of apical meristems using in vitro plants.

The presence of transgenes in putatively transformed callus, plantlets and plants

were evaluated using common PCR, PCR-Southern, RT-PCR, RT-PCR-Southern, Southern

Blot and Northern Blot analyses and more recently using Real Time PCR (Figure 4 and

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Figure 5). Sequencing was also carried out on PCR products to verify the validity of the

identity of transgenes detected. In cases where possible, bioassay studies were carried out

from time to time.

Figure 4. PCR analysis on the presence of CpTI gene in transformed plantlets [lane1 and lane 8: 100 bp marker, lanes 2-5: DNA from transformed plantlets, lane 6: DNA of untransformed plantlets, lane7: plasmid DNA of pMRT 1301].

Figure 5. Southern blot analysis of DNA from regenerated plants. [lane 1: 1kb DNA ladder, lane 2: 100bp, lane 3: control positive (from the CpTI coding region), lanes 4-7: DNA of transformed plantlets digested with BamHI, lane 8: DNA of untransformed plantlet digested with BamHI]

1 2 3 4 5 6 7 8

500 400 300

I II III IV λ Hind III 100 bp Positive control Negative

control Genomic DNA

1 2 3 4 5 6 7 8

564 500

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e) Biolistic vs. Agrobacterium-mediated gene transfer.

As in most cases, plant transformation mediated by biolistic are tissue- and variety

independent. This was not the case for Agrobacterium-mediated gene transfer. However, in

oil palm, target tissues from all varieties tested (tenera, pisifera, dura and Elaeis oleifera)

could be made susceptible to Agrobacterium infection, but successful gene transfer depends

mainly on pretreatment of the target tissues prior to infection. Enhanced infection were

observed when target tissues were subjected to conditions that induced cell competency

such physical injuries and callus induction.

4. Biolistic-mediated gene transfer

Conditions for biolistic-mediated gene transfer for oil palm were optimized using IE and EC

as target tissues, and pBI 121 and pCAMBIA1301 as the DNA carrying the reporter and

marker genes (Figure 6). Determination for optimum conditions was based on histochemical

assay carried out randomly on IE and EC, 3 days after bombardment. It was observed that,

Figure 6. Sequence of activities in biolistic mediated gene transfer in oil palm followed by gus assay, using immature embryo as the target tissues.

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all 3 parameters evaluated did not significantly influence transient transformation

frequency. Varying the macrocarrier gap from 6, to 11 or 16 mm, only slightly influence

transient transformation frequency but was still not significant. Even though increasing

macrocarrier gap did not increase transient transformation frequency, but IE bombarded

further away from the macrocarrier have better survival rate. This was shown by higher

number of Kmr plants recovered from IE bombarded at a gap of 16mm as compared to 11 or

6mm. Higher survival rate could be associated to less detrimental effect caused by the

DNA-coated gold particles on cell viability during bombardment as compared to those

bombarded from an 11 or 6mm gap. Smaller gap would mean severe damage caused to

target cells or tissues resulting in reduced viability after bombardment. Increasing the

helium pressure during bombardment also did not improve transient transformation

frequency. However, as in the case for macrocarrier gap, the survival of bombarded IE was

influenced by the degree of damage caused to the target tissue during bombardment. Here,

highest number of Kmr plants was recovered from IE bombarded at 900psi, where damage

was less severe compared to those bombarded at 1100 or 1300psi. As in the case for

macrocarrier gap and helium pressure, embryo size did not influence transformation

frequency. However, larger IE recorded higher viability after bombardment as shown by the

number of Kmr plants recovered. In all cases, gus assay on freshly bombarded IE exhibited

discrete individual blue spots but the spots became less distinct 7 days after bombardment.

Instead, gus activity appeared to have spread throughout the entire IE giving rise to IE with

blue coloration as opposed to light yellow in the case for the controls, indicating successful

transgene integration. Further observation on longitudinally sectioned-IE, indicated some

degree of localization of gus activity in putative transformants. Gus activity was most

intense at meristematic regions and in areas known to comprise mainly of actively dividing

cells such as shoot and root primordia. This was shown by thick blue coloration of the

above regions, as opposed to light blue or pale yellow in regions comprising mainly of

mature or differentiated tissues.

5. Agrobacterium-mediated gene transfer

DNA delivery into target tissues by microprojectile bombardment is a commonly used

technique for gene transfer into plants. However, it often resulted in the integration of

multiple copies of the foreign genes, which are often fragmented or rearranged. Such

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complex insertion events tend to be unstable. In contrast, Agrobacterium-mediated

transformation often results in a single-copy or low-copy integration of full length T-strands

carrying intact copies of the transgenes. Methods for genetic transformation of higher plants

using Agrobacterium have been well established for most dicots and some

monocotyledonous species. Prior to this, oil palm was perceived to be recalcitrant to

Agrobacterium-mediated transformation. However, we have provided evidence on the

susceptibility of oil palm tissues to Agrobacterium infection following various

pretreatments (Figure 7). In our preliminary work using A. tumefaciens LBA4404

(pCAMBIA 1301), a transformation frequency of 11.1 to 64% was obtained. However, in

our more recent work, the transformation frequency was further enhanced to 48 to 86.6%

when a superbinary vector (A. tumefaciens LBA4404 [pTOK233]) was used instead of the

normally used binary (A. tumefaciens LBA4404 [pCAMBIA130]) vector system.

Figure 7. Sequence of activities in Agrobacterium-mediated gene transfer in oil palm followed by gus assay, using immature embryo as the target tissues.

Pre-culture of target tissues prior to Agrobacterium infection was the key to

successful Agrobacterium-mediated gene transfer in oil palm. Prior to transformation, target

tissues (IE, PEC and EC) were pre-cultured for 7 days on N6 medium. The effect of

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explants pre-culture on transformation efficiency is not clear. Even though, extended pre-

culture period was shown deleterious for transformation on some species, but in the case for

oil palm, pre-culture of target tissues prior to Agrobacterium infection resulted in high plant

regeneration after co-cultivation. On the other hand, a shorter pre-culture period of less than

7 days led to browning and eventual death of target tissues after co-cultivation. Within 2

weeks (for IE) and 2-4 months (for PEC and EC), shoots appeared from target tissues

inoculated with bacteria carrying the respective plasmids (eg pMRT 1301, pCAMBIA1301,

pTOK233, pJLPHB3, pSAPHA1305.2, pMR507 and pMR607).

Unlike freshly bombarded target tissues, gus assay on freshly co-cultivated target

tissues exhibited uniform and well spread gus activity. Further evaluation on longitudinally

sectioned-plants regenerated from the respective target tissues, also indicated some degree

of localization of gus activity in putative transformants, suggesting possible influence of the

promoter used in the delivery systems. These were expected since both gus gene in

plasmids delivered using biolistic and Agrobacterium-mediated were under the control of

constitutive promoters, which is most efficient in meristematic regions and in areas with

actively dividing cells. However, it is noteworthy to note that co-cultivated target tissues

exhibited stronger gus expression as compared to those bombarded, with the highest being

from those using superbinary vectors (pTOK233, pMR507 and pMR607), suggesting

possible influence of plasmids used in delivering transgenes into target tissues, and also the

possible role of pretreatments prior to transformation.

The Agrobacterium-mediated gene transfer system for oil palm was further

improved with the possibility of co-transforming two Agrobacterium strains with two

different plasmids demonstrated. In our study, target tissues were co-cultivated

simultaneously with two Agrobacterium strains each carrying different plasmids, A.

tumefaciens LBA4404 (pCAMBt1) and A. tumefaciens LBA4404 (pMRT1301). Following

this, we successfully produced transgenic oil palm plants carrying both cry1A(B) gene

(pCAMBt1) and CpTI gene (pMRT1301) with an overall frequency of about 20%. Further

evaluation showed that, the frequency of co-transformation of oil palm tissues mediated by

Agrobacterium is half of those observed in single transformation events.

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In addition to the above, we have also developed Agrobacterium-mediated gene

transfer protocols for apical meristems. This was done by infecting longitudinally-cut in

vitro plantlets with exposed apical meristem for infection by Agrobacterium carrying the

desirable genes in their plasmids. The advantage of this approach is that, the co-cultivated

apical meristems were able to regenerate into complete plants within 3 to 4 months. Thus

the total time required to produce complete plants from explant preparation, co-cultivation

and regeneration is only about 3.5 to 4.5 months, very much shorter compared to when

undifferentiated tissues such as immature embryos and other embryogenic cultures derived

from other explants were used as initial target tissues. It is noteworthy to highlight that the

Agrobacterium-mediated gene transfer technique developed was applicable to all oil palm

varieties tested (E. guineensis var dura, pisifera and tenera) and also including those of

E.oleifera (Table 3).

Table 3. Transient GUS expression in oil palm immature embryos co-cultivated with Agrobacterium tumefaciens LBA4404 carrying either plasmid pCAMBIA1301 or pTOK233.

GUS assay

Expt. No. Species/vaiety plasmid Transient Transformation Frequency (%)

O1 E. oleifera pCAMBIA 1301 64.4 D1 E. guineensis var dura pCAMBIA 1301 41.0 P1 E. guineensis var pisifera pCAMBIA 1301 11.1 T1 E. guineensis var tenera pCAMBIA 1301 54.0 O2 E. oleifera pTOK233 65.5 D2 E. guineensis var dura pTOK233 86.6 P2 E. guineensis var pisifera pTOK233 48.0 T2 E. guineensis var tenera pTOK233 63.3

These together with the development of our own modular superbinary vector

systems, further demonstrate the amenability of oil palms tissues to Agrobacterium-

mediated gene transfer for the introduction of novel traits for further improvement.

6. Genetic engineering for resistance/tolerance to biotic stresses

The possibility of transferring foreign genes into oil palm was first demonstrated in 1996.

Since then efforts were put to, first, optimize the gene delivery system, and second, to

19

minimize and/or eliminate common problems associated with the development of genetic

transformation systems for the production of true transgenic oil palm plants. Technologies

developed were then applied to produce useful transgenic plants in attempts to address

common biotic stresses such as pest and disease problems in oil palm.

Insect predation of plants contributes towards reduction of crop yield worldwide. In

their natural habitat, oil palm (E. guineensis Jacq.) is less prone to pests and diseases.

However, most, if not all commercial oil palm in Malaysia came from a narrow gene pool

making it vulnerable to pest and disease outbreaks. The problem becomes more critical

since the current breeding programs intensifies aggressive introduction of new genetic

resources into elite planting materials, thus making commercial oil palm more vulnerable to

insect pests. With an economic life span of more than 30 years, the plant would be exposed

to rapidly evolving insect pests, thus yield loss in oil palm due to insect attack is of obvious

concern to oil palm growers.

To date, transgenic plants carrying useful genes such as cowpea trypsin inhibitor

(CpTI) or cry1A(B) genes or both, chitinase and ribozyme inhibiting protein (rip) have been

produced and maintained under netted planthouse conditions. CpTI gene encodes for the

production of trypsin inhibitor. The inhibitor was postulated to bind to the digestive

proteases and will in turn inhibit proteolysis in the mid-gut of the larvae. Thus, the larvae

were unable to digest protein consumed and will eventually die as in the case of larvae

feeding on leaves from the transgenic CpTI P8 plant. 8 independent bioassay studies

involving feeding trials of first instar larvae on the now 8-year old transgenic plants

carrying CpTI gene showed improved tolerance to bagworm larvae (Metisa plana Walker),

a common insect pest to the oil palm. Bagworm larvae (M. plana W) feeding on oil palm

leaf carrying CpTI genes were unable to undergo normal development and eventually

resulted in death. As a result, leaf from CpTI plants was only marginal defoliated compared

to those from control plants, indicating the effectiveness of CpTI against bagworm larvae.

This study showed that CpTI has the potential to be used as a possible defense gene for oil

palm against Metisa plana (Figure 8).

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Figure 8. Bioassay of transgenic oil palm plants carrying CpTI genes showing marginal defoliation as compared to severely defoliated leaf from control plants. Bagworms feeds on oil palm leaf for food and for raw materials to build their “bags” where they live in. Following feeding trials, bagworms feeding on CpTI plants failed to undergo normal development and eventually died. On the other hand bagworms feeding on control plants were able to undergo normal development pupate and became moth.

On the other hand, plants carrying the cry1A(B) genes are still under continuous

evaluation for its efficacy against insect pests. So are plants carrying other useful genes

such as chitinase and ribozyme inhibiting-protein. In addition, chimearic plants produced

were used as source for T1 transgenic plants using infloresence and embryo rescue

technique.

CpTi Plant

CpTi Plant

Control Plant

Control Plant

Newly hatched larva

2-day old larva

Fed with control plant

Fed with CpTi plant

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7. Genetic engineering for yield improvement

Progress had been made in attempts to produce transgenic oil palm with improved oil yield

and quality. Several important genes involved in lipids biosynthesis have been isolated,

cloned and transformed into oil palms. To date, there are some changes observed in the fatty

acid profiles of plants transformed with some of these genes. However, these fatty acid

profiling needs further evaluation as these palms mature. Similarly, attempts are under way

to use marker-assisted approach in manipulating oil palm for further yield improvements.

This approached is also utilized in selecting potential abnormal clones during tissue culture

processes.

8. Genetic engineering for value-added products

Besides oil, oil palm is a good source for value-added products such as tocopherols and

caroteniods. Being the most efficient oil-yielding crop with an annual oil production of 10

tonne/ha/year, oil palm has abundant supply of acetyl-CoA. Acetyl-CoA is also the main

building block for other compounds besides lipids such as flavonoids and isopreniods. In

the last 5 years we have embarked on attempts to produce bioplastics in oil palm taking

advantage of the abundant supply of acetyl-coA (Figure 9). In this case, genes involved in

the conversion of acetyl-CoA for the production of bioplastics were transformed into oil

palm target tissues. The conversion of acetyl-coA requires the addition of three other genes

not found in oil palm. They are acetoacetyl-CoA reductase, PHB synthase and tdcB thiolase

isolated from Ralstonia euthropa. Transgenic plants carrying these genes are now about 2

years old and are maintained in netted planthouse until maturity for further evaluation.

Again this was confirmed using standard molecular biology techniques that include PCR

and QPCR, Multiplex PCR and Multiplex QPCR, RT-PCR and Southern Blot analysis.

Evaluation using transmission electron microscopy (TEM) also revealed the presence of

additional granules similar in properties to polyhydroxybutyrate granules observed in other

plants. These granules as expected were present in the chloroplasts of transformed plants

but not found in control plants. Continuous evaluations are carried out on these plants for

further biochemical determination on the yield of the bioplastics produced.

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9. Current challenges in genetic engineering of oil palm

Even though biotechnology research on oil palm is quite recent, but it has great potential in

further improving the crop. The science is for real and is starting to show benefits. One

example is the application of in vitro technology for large-scale propagation of elite clonal

palms. The techniques currently employed may not be the best yet, but with continuous

improvement, clonal palms would be the choice for future planting materials. In addition,

from a simple tissue culture technology there exists now spin off technologies such as

biclonal seed production, large-scale propagation using bioreactor technology, marker

assisted selection being worked out in oil palm.

Similarly, even though it is now possible to produce transgenic oil palm either

through direct or vector-mediated gene transfer system, but transgenic oil palm would not

be available in the market for at least another decade. Nevertheless the technology have

brought a lot of promise that may not be possible or would take much longer time if only

conventional breeding methods were to be used. This includes the production of improved

planting materials that are resistant to pests and diseases, greater tolerance to abiotic stress,

those with higher yield and improved yield quality and also possible value added products.

Todate several useful genes have been successfully engineered into oil palm with transgenic

malonyl-CoA

FLAVONOIDS

LIPIDS

ISOPRENOIDS

acetyl-CoA

aceto-acetyl-CoA

3-hydroxybutyryl-CoA

polyhydroxybutyrate (PHB)

ACCase

ß-ketothiolase

Aceto-acetyl CoAreductase

PHB synthase

PHOTOSYNTHESIS

GLUCOSE

plastid

what happens in the plant cells

Figure 9. Channeling of byproducts in oil palm and possible manipulation for the production of bioplastics taking advantage of abundant supply of acetyl-CoA.

23

plants produced. The applicability of the techniques on all oil palm varieties and species

tested demonstrates the versatility of techniques developed (Table 4).

Table 4. Current status of genes transferred into oil palm.

Genes Species/variety Gene transfer Plant status CpTi E.guineensis (tenera) Biolistic 8-year old transgenic plants E.guineensis (tenera) Agrobacterium 2-year old transgenic plants CpTi + Cry1A(B) E.guineensis (tenera) Agrobacterium 2-year old transgenic plants Cry1A(B) E.guineensis (tenera) Biolistic 2-year old chimearic plants Chitinase E.guineensis (tenera) Agrobacterium 4-year old transgenic plants 4-year old chimearic plants E.guineensis (tenera) Biolistic 4-year old chimearic plants Rip E.guineensis (tenera) Biolistic 4-year old chimearic plants phaA, phaB, phaC, E.guineensis (tenera) Agrobacterium 2-year old transgenic plants tdcB 2-year old chimearic plants Gus E.guineensis (tenera) Biolistic 9-year old chimearic plants E.guineensis (tenera) Agrobacterium 4-year old transgenic plants 4-year old chimearic plants E.guineensis (pisifera) Agrobacterium 4-year old transgenic plants 4-year old chimearic plants E.guineensis (dura) Agrobacterium 4-year old transgenic plants 4-year old chimearic plants E. oleifera Agrobacterium 4-year old transgenic plants 4-year old chimearic plants

However, being a commodity crop with commanding cash value this may have a

disadvantage to the application of biotechnology on oil palm improvement. Biotechnology

research is very capital intensive, and being a perennial crop, the fruits of biotechnology on

oil palm improvement would take a long time. The long gestation period before any

breakthrough would normally bring disadvantage to the progress. In addition, oil palm is

mainly grown in the developing world. Therefore, gene manipulation and the products from

genetic manipulation of oil palm would not be easily accepted. Unlike other crops such as

soya bean, sunflower, maize and rapeseed, even though they are also food crops like oil

palm, but because they are grown in developed nations, gene manipulation and the products

from them are more readily accepted. Due to these reasons, oil palm biotechnology research

is not only facing scientific challenges but also from policy makers, where most research

grants are obtained. This is because of the fear of palm oil boycott, similar to that

24

experienced in 1980’s, may reduce the country’s earning from USD 6.9 billion (2003) to a

much lower income, may influenced future funding in gene manipulation of oil palm.

10. Conclusion

Genetic engineering has made possible free movement of genetic materials from one

organism to another regardless of their origin. It is therefore envisaged that the future of any

crops will depend greatly on its amenability for it to be genetically manipulated to fulfill

demands set by consumers. Thus crops such as oil palm will not be looked upon as

commodity crops in the future. Instead its role will have to change from a commodity crop

to a biofactory, engineered to produce speciality products, taking advantage of metabolites

that are abundantly produced by the plant. Therefore, developments on genetic engineering

of oil palm, though have yet to produce any commercial products to date, will have to

prepare itself to face future challenges in the field of agrobiotechnology this millennium.

Challenges from both the policy makers and the economic aspect should not surpass the real

scientific challenge involved. This is important if oil palm is to remain competitive against

other oil producing crops such as the annuals, soybean and rapeseed.

Achknowledgement The author would like to acknowledge funding from the Ministry of Science Technology

and Innovation, Malaysia and contributions from fellow co-workers from the Oil Palm

Biotechnology Research Group, School of Bioscience & Biotechnology, Faculty of Science

& Technology, Universiti Kebangsaan Malaysia, Malaysia.

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