BIP - Expression of Bacillus thuringiensis insecticidal protein gene in transgenic oil palm

Financial support: Ministry of Science, Technology and Innovation, Malaysia (IRPA 01-02-02-0168 and IRPA 09-02-02-0033.

Keywords: CryIA(b) gene, gene expression, insect resistance, oil palm transformation, rapid detection system, transgenic oil palm.

Oil palm (Elaeis guineensis Jacq.) is the most sought-after edible oil in the world. Advances in agricultural biotechnology, in particular through gene manipulation, have seen the monopoly enjoyed by palm oil gradually eroded by genetically modified oil-bearing crops such as soybean, sunflower seed, and rapeseed. Thus, efforts are being undertaken to increase oil palm yield to meet the ever-increasing global demands and also to broaden its spectrum of uses. Conventional oil palm breeding is inefficient and time-consuming. However, current breeding programme combines both conventional and modern biotechnology approaches such as the use of tissue culture technique, genetic transformation and marker-aided selection. Although many studies have been done on genetic manipulation of oil palm, but publications are limited. This is probably due to the high commercial implication tagged to the crop. It may also be due to the crops’ own physiological characteristics such as long generation cycle. Over the past 10 years, our group has been actively involved in genetic engineering of oil palm. Special emphasis was placed initially on developing techniques for genes transfer into oil palm, both using direct gene transfer and Agrobacterium-mediated approaches. Both techniques were then used to transfer useful genes such as cowpea trypsin inhibitor (CpTI) and Bacillus thuringiensis (Bt) crystal insecticidal protein genes, chitinases and more recently, genes for biosynthesis of bioplastics.

In most gene manipulation studies, the main bottleneck lies in assessing the functionality of transgenes in putative transformants. Very often, these could not be clearly demonstrated. Here, several expression vectors carrying CryIA(b) gene were constructed and transformed into oil palm. Successful transfer of the CryIA(b) gene into oil palm was clearly demonstrated followed by analysis on the transgenes functionality in its new environment. The approach used combines both RT-PCR and Southern Blot analysis, and was proven effective to elucidate the presence and functionality of transgenes. In addition, it also provides a rapid technique to screen for putative transformants that proved very difficult in gene manipulation of monocots, especially those having slow growing rate in vitro such as oil palm.

Construction of transformation vector

A new recombinant plasmid, pRMP, was created by cloning the CryIA(b)gene into pCAMBIA1301 while maintaining its open reading frame (ORF), its sense orientation and placed under the control of the rubisco promoter. The newly constructed pRMP carries along with it, the hptII as a selectable marker gene. The presence of the right and left border flanking these genes makes pRMP suitable for both biolistic and Agrobacterium-mediated mediated gene transfer.

Transformation of oil palm

Transformation was carried out using methods established in our laboratory as described earlier. Putative transformed IEs were randomly selected and analyzed through histochemical staining 3 days after transformation. GUS-positive blue spots or stains were detected on randomly selected IEs assayed, suggesting successful gene transfer. In contrast, no blue spot was observed in all control samples assayed. On average, 81% transient transformation efficiency was recorded for IEs bombarded once as compared to 100% for IEs subjected to double bombardment. Double or multiple bombardments is not normally practiced in other crops, as this would cause excessive wounding to target tissues resulting in eventual death. However, we believed that oil palm is a hardy plant that could withstand certain amount of wounding to its tissues. This was evident from our observation that no excessive accumulation of phenolic compound in the culture medium, which would be the case for severely injured or dying oil palm tissues, after multiple bombardments was detected. Complete plants were regenerated from bombarded IEs (single or multiple) and cultured on hormone-free N₆media. It was observed that, there were no morphological differences between plants derived from IEs bombarded once or twice where, plants from both groups exhibited similar growth rate.

Detection of transgene using PCR

Polymerase chain reaction analyses on transformants using primer sets, CRYF1 (nt 170-189) and CRYFI (nt 800-781) specific to CryIA(b)coding sequence gave rise to the expected 631 bp on all 15 samples tested. In contrast, no band was observed for all control, thus confirming successful transfer of transgene from pRMP into oil palm tissues.

Expression of cry1A(b) gene in oil palm

Although the presence of transgenes was easily detected in transformants, but its functionality could not be demonstrated. Several Northern blotting efforts failed to detect signals corresponding to the CryIA(b)gene. Similarly, Reverse Transcriptase PCR (RT-PCR) was also shown unequivocal evidence of transgene [CryIA(b)] expression. However, when the RT-PCR products showing no visible band on agarose gel were blotted onto nylon membrane and hybridized with DIG-labeled probe specific to CryIA(b), it gave rise to positive signals. The very low quantities of transgene present may be the reason why they could not be detected initially. However, following RT-PCR, the transcripts were amplified to a detectable level as evident from the RT-PCR-Southern Blotting analyses. This combination of RT-PCR and Southern Blotting therefore provide a highly sensitive detection assay as shown by the positive signals observed. These observations further showed that the transcripts were actually present on the agarose gel following RT-PCR but were beyond detectable level for visualization on the not so sensitive agarose gel. These observations, therefore, provides equivocal evidence that the mRNA of CryIA(b) is transcribed in oil palm tissues thus providing the first evidence on the functionality of a functional gene not native to oil palm following transformation. This novel detection system is important especially to study the fate of transgenes upon transfer in its new environment. This is essentially more important when multi-cellular tissues were used as target tissues, where only a minute fraction of the cells within the tissues would be transformed following bombardment. In most cases, it is quite impossible to determine the fate of the newly transferred transgene in its new environment. However, this would not be the case for transgenic plants that are derived from single cells, where every cell in the plant would have the stably integrated transgene. Similar findings were also observed in maize transformed with B.t. gene, where the expression of reporter genes at the protein level is indirectly implied. This led to the development of a direct detection method for protein expression of the gene of interest. However, our approach gives a more accurate picture on the of transcription level of the gene of interest in oil palm. For example, it was found that, the CryIA(b) transcripts was relatively little compared to the GUS protein expression observed in transformed IEs. This approach could also serve as an important tool in promoter study, as it would be more precise to evaluate promoter efficiency at the transcription level as compared to the protein level, since it will not be influence by translational factors.

Concluding Remarks

In conclusion, this detection technique of combining RT-PCR and Southern blotting is highly sensitive in detecting minute traces of transgenes expression in putative transformed tissues thus providing a tool for closer expression studies of individual genes at cellular level. Transcripts analyses could be immediately carried out following transformation, which is especially important for crops having long reproduction cycle like oil palm. In addition, this system also allows for mRNA expression reflecting biological phenomena to be detected in minute amount of cells. The detection system could also serve as an accurate evaluation of essential but lowly expressed mRNA in plant.

Acknowledgements

The authors would like to thank Prof. Illimaar Altoosaar, Ottawa University, Canada, for the cry1A(b) gene; Dr. Richard A. Jefferson, CAMBIA, Australia, for the pCAMBIA1301 cloning vector; Dr. J. K. Kim, Myongji University, Korea, for the rubisco promoter and Universiti Kebangsaan Malaysia (UKM) for research facilities.