Ruslan Abdullah

Immature embryo: A useful tool for oil palm (Elaeis guineensis Jacq.) genetic transformation studies

Oil palm (Elaeis guineensis Jacq.) is the highest yielding oil-bearing crop. However, being a perennial crop, genetic improvement of oil palm is extremely slow. Indeed, compared to other annual oil crops such as soybean and rapeseed, genetic manipulations remained less important. Therefore, to remain competitive, oil palm growers and breeders need new and novel approaches. In this report, the potential of immature embryos (IE) as a useful tool for oil palm genetic transformation studies was evaluated. It was evident that IEs were amenable to both direct and Agrobacterium-mediated gene transfer. Due to the abundant supply of IE, optimization of biolistic and Agrobacterium-mediated gene transfer into IEs were easily carried out. Transient transformation frequencies were comparable to other plant systems reported, with as high as 97.4% recorded for biolistic and 64.4% for Agrobacterium-mediated gene transfer. Like most moncots, oil palm tissues were less sensitive to kanamycin, geneticin and chloramphenicol. Instead, both hygromycin and phosphinotrycin were toxic 20 mg/l, making both suitable candidates for selecting putative transformants. IEs were also more responsive to in vitro manipulations as compared to other explants such as leaf and root tissues. Rapid in vitro response to callusing and embryogenesis or rapid and highly efficient direct germination resulted in a shorter culture period. This would minimize the production of abnormal clonal palms, which has been associated to chromosomal aberration due to prolonged time in culture. In addition, IEs also allows rapid and direct introduction of elite genes into breeding programs and in biclonal seed production.

Expression of Bacillus thuringiensis insecticidal protein gene in transgenic oil palm

Oil palm, like all other crops, is susceptible to attack from several insect pests causing significant reduction in productivity. In the past, cry genes from Bacillus thuringiensis have been reported to be effective in conferring resistance towards insect pests in crops such as corn and rice. One of the advantages of these toxin proteins is their specificity towards certain harmful insects. A rapid and efficient method was developed for the transformation and evaluation of C ryIA(b) expression in oil palm. A recombinant vector was introduced into immature embryos (IEs) of oil palm via the biolistic method. More than 700 putative transformed IEs from independent transformation events were generated. Transient transformation efficiency of 81-100 % was achieved. We found that pre-treatment of target tissues with phytohormones is essential for increasing the transformation efficiency. This finding could enable higher transformation rate in oil palm that was previously difficult to transform. PCR analysis further confirmed the presence of the CryIA(b) in the transformed tissues. Expression of CryIA(b) from PCR-positive samples was further confirmed using a rapid gene expression detection system. This novel and rapid detection system could serve as a good opportunity to analyze the impact of transgenes upon transfer to the new environment, especially for crops with long generation cycle, such as oil palm.

Transgenic Oil Palm with Stably Integrated CpTI Gene Confers Resistance to Bagworm Larvae

Insect predation of plants contributes towards reduction of crop yield worldwide. In oil palm alone, loss of yield due to insect attack is obvious. The most important insect pests of oil palm are the bagworms, particularly Metisa plana Walker. Bagworms caused severe defoliation of oil palm leaves as they fed on them. Synthetic insecticides have been applied to address losses due bagworm, but with unsatisfactory results. In addition most of the insecticide applied were wasted and it could also lead to toxicity to non-target insects. Thus efforts are in progress to produce transgenic plants resistant to insect. The use of transgenic plants offers several advantages over conventional insecticides. Earlier work on cowpea protease trypsin inhibitor (CpTI) gene on other crops showed that it has the potential to confer resistance against common insect pests especially members of the Lepidoptera, Coleoptera and Orthoptera. And most importantly, it exhibits low or no toxicity to mammals (Pusztai et al., 1992). When ingested, the larval gut protease was unable to digest proteins consumed. This eventually led to larvae death due to starvation (Gatehouse and Boulter, 1983). The CpTI gene was successfully transferred into oil palm giving rise to transgenic plants with stably intergrated gene. This report further highlights the effectiveness of CpTI gene against the common insect pest of oil palm, Metisa plana Walker.

Constructions of expression vectors of polyhydroxybutyrate-co-hydroxyvalerate (PHBV) and transient expression of transgenes in immature oil palm embryos

Polyhydroxybutyrate-co-hydroxyvalerate (PHBV) is a polyhydroxyalkanoate (PHA) bioplastic group with thermoplastic properties is thus high in quality and can be degradable. PHBV can be produced by bacteria, but the process is not economically competitive with polymers produced from petrochemicals. To overcome this problem, research on transgenic plants has been carried out as one of the solutions to produce PHBV in economically sound alternative manner. Four different genes encoded with the enzymes necessary to catalyze PHBV are bktB, phaB, phaC and tdcB. All the genes came with modified CaMV 35S promoters (except for the tdcB gene, which was promoted by the native CaMV 35S promoter), nos terminator sequences and plastid sequences in order to target the genes into the plastids. Subcloning resulted in the generation of two different orientations of the tdcB, pLMIN (left) and pRMIN (right), both 17.557 and 19.967 kb in sizes. Both plasmids were transformed in immature embryos (IE) of oil palm via Agrobacterium tumefaciens. Assays of GUS were performed on one-week-old calli and 90% of the calli turned completely blue. This preliminary test showed positive results of integration. Six-months-old calli were harvested and RNA of the calli were isolated. RT-PCR was used to confirm the transient expression of PHBV transgenes in the calli. The bands were 258, 260, 315 and 200 bp in size for bktB, phaB, phaC and tdcB transgenes respectively. The data obtained showed that the bktB, phaB, phaC and tdcB genes were successfully integrated and expressed in the oil palm genome.