Oil palm (Elaeis guineensis Jacq.)is the highest oil-yieldingcrop. It is a perennial monocot with a long generation period of about 20 years. Genetic improvement of oil palm is extremely slow, thus making oil palm breeding a very long process. Even though palm oil commands a major monopoly in the worlds' vegetable oil market, but new innovative methods are needed for oil palm to remain competitive. Compared to other annual oil crops such as soybean and rapeseed, genetic manipulation of oil palm is still at its infancy.

Tissue culture technique was the first biotechnological tool introduced onto oil palm. In 1974, elite oil palm clones were successfully micro-propagated. However, clones produced from tissue culture were abnormal. When planted in the field, the abnormal palms produced abnormal flowers resulting in reduced yield. However, over the past two decades, oil palm tissue culture techniques have been greatly improved, with lower incidence of abnormalities reported. In addition, early field trials result using clonal palms showed encouraging yield improvement. Thus, the possibility of clonal palms replacing seed-derived planting materials is becoming a reality.

Early work on oil palm tissue culture involves the use of roots as explant. To date, complete plants have been successfully regenerated from almost every parts of oil palm including mature (ME) and immature embryos (IE), apical meristems (AM), embryogenic cell suspension cultures (ECS), friable embryogenic tissues (FET) and callus (PC) derived from seedlings, roots, inflorescences and young leaves. The efficiencies for complete plant regeneration from some of these explants are still inefficient (Rival et al. 1999). 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, reliable and rapid regeneration system (Ayres and Park 1994). 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) (Murphy 1999). Not all explants, however, are suitable for genetic manipulation studies.

Following recent advances in plant genetic transformation studies, it is now possible to transfer any foreign genes into any targeted plant genome. Oil palm is no exception. One routine technique is by using Agrobacterium spp. Unfortunately, until recently, the hosts for Agrobacterium have been limited to mostly dicots and only a few monocots. To date, there has been no report on the susceptibility of oil palm tissues to Agrobacterium infection. Alternatively, DNA could be delivered directly into protoplasts via electroporation. However, it is still not possible to regenerate complete plant from oil palm protoplasts. On the other hand, using particle bombardment it is now possible to transfer any gene to virtually any tissues or cell types. This ability would benefit genetic transformation of recalcitrant and perennial crops, like oil palm, the most since.

Preliminary 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 transgenes transferred. These shortcomings were addressed in this report. Here, we report the potential role of IEs in genetic transformation studies of oil palm. Results presented include studies on in vitro culture of IEs for both direct and indirect plant regeneration, and preliminary studies on the susceptibility of IEs to Agrobacterium infection, and the comparison between both systems. These new developments 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.

The main prerequisite for an efficient transformation system is the ability to produce 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. The callusing rate recorded for young leaf and root explants is about 20%. On the other hand, callusing rate for IEs is as high as 100%. IEs isolated from 9-10 weeks fruits after anthesis (WAA), germinated readily into complete plants on hormone-free medium with a germination rate reaching up to almost 100%. However, IEs isolated from 8 WAA fruits or younger failed to germinate into complete plant. Furthermore, the endosperm from 8 WAA fruits or younger are still soft, thus, resulted in poor recovery of embryos. On N₆2.5 and N₆FET media, IEs produced callus within 4-6 weeks, very much faster than other explants. Though both media were equally effective, but IEs cultured on N₆FET were less browned compared to those on N₆2.5. Cultured IEs started to swell and expanded after 3 days on callusing media and yielded primary calli within 4-6 weeks. While on the same media, PC produced embryogenic callus with distinct somatic embryos of different shapes and stages. Upon transfer onto N₆0, torpedo-shaped embryos germinated into complete plants. The whole sequence from IEs to complete plant via callus was completed in just 3-4 months as opposed to 8-52 weeks for young leaves-derived callus. This would mean shorter period in culture and would reduce the possibility for the onset of chromosomal aberrations, thus resulting in less incidence of abnormalities among clonal palms. Plants with vigorous root systems which normally take between 2-4 weeks were transferred into polybags for hardening and maintained until maturity.

Oil palm IEs are abundant. On average, between 300-500 IEs could be obtained from a single developing bunch. Large number of IEs allows for large-scale genetic transformation studies be carried out on oil palm. It was also observed that, gene transfer into oil palm IEs, are not dependent on variety for both biolistic and Agrobacterium-mediated. All three Elaeis guineensis varieties namely dura, pisifera and tenera and IEs from Elaeis oleifera were susceptible to both gene transfer systems. In addition, IEs as target tissues, enables IE-derived transgenic plants to be used directly as crossing partners for the introduction of new or elite genes into specific breeding programs. In addition it also limits the onset of fidelity-associated problems following a much shorter period needed in culture. These would further shorten the breeding cycle for oil palm. However, since IEs are the product of cross-pollination between two separate parents, they are often non-uniform in terms of genetic make up. Thus a more desirable case would be to transform a self-pollinated dura or pisifera of known parentage. Nevertheless, following theirabundance, their high response in vitro, reduced clonal fidelity-associated problems, and the ability to allow rapid introduction of elite genes, IEs are therefore considered the most suitable target tissues for transformation studies for oil palm.

In vitrotolerance of IE to antibiotics commonly used as selectable markers

Transformation efficiencies for most important plant species are still not optimal. These could be improved by using suitable selectable marker genes in the selection of putative transformants. In most cases, selectable marker genes suitable for dicots may not necessarily be suitable for monocots. As such in vitro tolerance study on various potential target tissues of oil palm were carried out. This would facilitate suitable use of antibiotics for selecting putative transformants and would be incorporated in constructs carrying gene of interest for future manipulation studies.

Here, IEs were found not sensitive to both antibiotics Kanamycin (Km) and Geneticin (G418). In the presence of 250mg/l Km or G418 the growth of IEs was unaffected even after 16 weeks on the media. However, IEs were slightly sensitive to chloramphenicol with their growth affected when exposed to more than 100mg/l. The effect of chloramphenicol on IE growth was only observed after 10 weeks in culture, with a survival rate of 65% at 100 and 120 mg/l, and 45% at 150 mg/l after 16 weeks. Unlike Km, G418 and chloramphenicol, both Hygromycin (Hm) and Phosphinotricin (Ppt) were toxic to oil palm IEs. All IEs exposed to more than 50 mg/l Hm were dead within 8 weeks, and within 14 weeks for exposure at 20 mg/l. Similar result was obtained for Ppt, where all IEs were killed when exposed to 20mg/l or higher. The only difference between the effect of Hm and Ppt on IE was in the rate of IE death, where, it was more rapid for Ppt compared to Hm which was more gradual. In all cases, however, control IEs cultured on N₆0 continued to germinate and proliferate into complete plants.

Similar results were obtained for other target tissues tested, including primary callus (PC), embryogenic callus (EC) and friable embryogenic tissues (FET), where all PC, EC and FET were found not sensitive to both Km and G418, slightly sensitive to chloramphenicol but very sensitive to both Hm and Ppt.

Conditions for biolistic-mediated gene transfer for oil palm were optimized using IEs as target tissues. In the initial stage, pBI 121 was used as the DNA carrying the reporter and marker genes. Optimum conditions were determined based on histochemical assay carried out randomly on IEs, 3 days after bombardment. It was observed that, all 3 parameters evaluated did not show significant difference on the transient transformation frequency observed. Varying the macrocarrier gap from 6, to 11 or 16 mm, only slightly influence transient transformation frequency but the difference was still not significant. Even though, increasing the macrocarrier gap did not result in an increase in transient transformation frequency, but it was observed that IEs bombarded from a larger macrocarrier gap showed better survival rate. This was shown by higher number of Km^r plants recovered from IEs bombarded with a gap of 16mm as compared to 11 or 6mm. Higher survival rate is normally associated with the less detrimental effect caused by the DNA-coated gold particles on cell viability as compared to those bombarded from a gap of 11 or 6mm. Smaller gap would mean severe damage caused to target cells or tissues and this would mean 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 IEs was influenced by the degree of damage caused to the target tissue during bombardment. Here, highest number of Km^r plants was recovered from IEs 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 IEs recorded higher viability after bombardment as shown by the number of Km^r plants recovered after bombardment. In all cases,gus assay on freshly bombarded IEs showed discrete individual blue spots that became less distinct 7 days after bombardment. By then, the gus activity appeared to have spread throughout the entire IEs giving rise to IEs with blue coloration as opposed to light yellow in the case for the controls. This indicates successful transgene integration. Further observation on longitudinally sectioned-IE, indicated some degree of localization of gus activity in putative transformants. Gus activity was strongest in meristematic region and in areas known to have actively dividing cells such as shoot and root primordia. This was shown by thick blue coloration in the above regions, as opposed to light blue or pale yellow in regions known to be made up of mature or differentiated tissues.

This report presents for the first time, substantiated evidence on the susceptibility of oil palm tissues to Agrobacterium infection. However, since this study was not designed to elucidate factors affecting Agrobacterium infection on oil palm tissues, thus results presented are preliminary in nature. Nevertheless, it was evident that oil palm tissues pretreated with 2,4-Dwere susceptible to Agrobacterium infection. Unlike freshly bombarded IEs, gus assay on freshly co-cultivated IEs showed uniform and well spread gus activities. Evaluation on longitudinally sectioned-IE indicates some degree of localization of gus activity in putative transformants. This may be due to the influence of promoter used in both gene delivery systems. These were expected since both gus gene in pBI121 (biolistic) and pCAMBIA1301 (Agrobacterium) were driven by CaMV35S, a known constitutive promoter shown most efficient in meristematic region and in areas with actively dividing cells. However, it is noteworthy to note that IEs co-cultivated with pCAMBIA1301 showed stronger gus expression as compared to those bombarded with pBI121, suggesting possible influence of plasmids used in delivering the transgene into target tissues, and also possible role of pretreatment prior to transformation.

Successful transgene integration was further substantiated from callusing assays on putative transformants. It was observed that callus developing from bombarded and co-cultivated IEs subjected to callus initiation on Km-containing (for pBI121) and Hm-containing (for pCAMBIA1301) media, exhibited gus activity. Gus assays were also positive on roots and leaves isolated from putative transformants derived from bombarded and co-cultivated IEs. Furthermore, the presence gus activities on tissues 4 months or more after bombardment or co-cultivation further indicates that the transgene has been successfully and stably integrated in the genome of putative transformants produced.

Our results clearly showed that oil palm tissues especially IEs are amenable to gene transfer. This is expected of the versatile biolistic-mediated gene transfer approach. But most importantly we are able to provide substantiated evidence for successful gene transfermediated by Agrobacterium tumefaciens for oil palm, which is not a natural host for this bacterium. In addition, the frequency of gene transfer is comparable to other plants reported elsewhere. Thus it is now possible to transfer elite genes into oil palm for the production of transgenic plants with improved traits. This ability would help oil palm to remain competitive against other oil producing crops such as the annuals, in particular soybean and rapeseed.