Development of Transgenic rubber plants

Dr. N Yogaratnam

Hevea brasiliensis (Para rubber tree) is the major source of commercial natural rubber (cis-1,4-polyisoprene). Rubber produced in specialized cells called laticifers is one of the most important biological molecules used for the manufacture of about 35000 products. Being a cross-pollinated perennial tree species, genetic improvement through conventional breeding is a rather slow process.

Biotechnology

Biotechnology would play an important role in the future of the rubber industry. Plant regeneration via somatic embryogenesis using a variety of explant sources like, integumental tissues, immature anther, immature inflorescences, and leaf explants are well standardized. Many genes controlling important agronomic traits and tissue-specific promoters have been characterized in rubber. Agrobacterium and biolistic-mediated genetic transformation systems are well established in this crop.

Thus, the basic technology for genetic manipulation of rubber plant at the cellular and molecular levels is available, making rubber a suitable crop for genetic engineering. In different laboratories, rubber plants were genetically transformed for recombinant protein production. Transgenic rubber plants were also produced with Mn.SOD gene to confer tolerance against a variety of environmental stresses and tapping panel dryness. Attempts are also going on to enhance the rubber yield through transgenic approaches. Since, the major harvested products are not used as food material, the biosafety concerns are less for the genetically modified rubber plants.

Rubber tree, a unique transgenesis model

The many advantages of transgenic plants for ‘bio-pharming’ notwithstanding, their one significant weakness is the difficulty in recovering the recombinant protein. Unlike transgenic animals where there is continual protein production in the milk, harvesting of the recombinant protein involves destruction of the plant or a portion of it, whether the desired protein is to be found in the seeds, leaves or shoots. After every harvest, it takes time for new growth to take place before the next harvest is possible. As a result, protein recovery is more likely to be batch-wise, rather than a continual process.

Taking into consideration the strengths of the transgenic animal (continual protein production in the milk) and the transgenic plant (low cost of maintenance, simple clonal propagation) for recombinant protein production, it would obviously be beneficial to have a production system that combines both advantages.

The ideal plant for recombinant protein production would be one that is cheap to maintain and easy to multiply clonally, while allowing for continual harvesting of the protein. This is where the transgenic rubber tree has the distinct advantage when compared with other transgenic crop plants.

In the bark of the rubber tree is a complex network of laticifers, or latex vessels, each vessel merely one-third the thickness of a human hair. These laticifers contain natural rubber latex that is exuded when the bark is cut. Rubber tapping that is routinely practised in estates and smallholdings is essentially the systematic and regulated cutting of the bark to harvest the latex. Since rubber tapping is a non-destructive method of latex extraction and harvesting, the tree can be tapped every alternate day, now even once in 3/4 days, throughout the year without pause.

Among plants, the rubber tree is unique in its capacity to produce voluminous latex upon tapping and to replenish this supply rapidly in readiness for the next tapping. If Hevea brasiliensis were transformed with a gene encoding a foreign protein, the transgenic Heveasystem would allow for continual production of the target protein, a feature lacking in any other transgenic plant system.

In the transgenic Hevea system, therefore, modern techniques in biotechnology combine with the generations-old practice of rubber tapping to add new value to the rubber tree.

Inserting foreign genes

The basic methods employed for genetic transformation of the rubber tree follow procedures well-established for other plants. As with many plants, genetic transformation of the rubber tree involves inserting the selected gene into callus tissue (unorganised clusters of cells) and then regenerating the transformed callus tissue into the complete plantlet. Hevea callus tissue cultures are established from anther walls of the rubber tree male flowers.

Foreign genes are transferred into a bacterium called Agrobacterium and this is then allowed to infect the callus tissue. The foreign gene is incorporated into the genetic make-up of the Hevea callus tissue during this process. As only a small proportion of the callus cells would be successfully transformed, a mechanism has to be available to sort out cells that are successfully transformed from those that are not.

For this reason, the DNA assembly that is used in transformation contains a second gene that confers antibiotic-resistance to transformed callus cells. When the callus tissues are transferred to culture medium containing the antibiotic, untransformed cells perish, while the transformants - armed with the means to resist the antibiotic - continue to thrive. The surviving callus cells proliferate and some develop into embryo-like structures that go on to form plantlets.

Multiplying success

From a number of transgenic plants that have been produced, the ones that show the strongest protein expression are multiplied for further study. Neither new nor expensive technology is needed here. The horticultural practice of Hevea bud-grafting that is harnessed for this purpose has its roots from the 1950s.

By this approach, unlimited clonal copies - each genetically identical - can be generated from a single selected transformant. The amenability to clonal propagation has been proven through successful multiplication by bud-grafting over four successive vegetative generations of plants bearing the gus gene. Besides demonstrating the efficiency of up-scaling transgenic Hevea, these results also confirm the stability of the genetic transformation in this plant.

Proteins from Hevea latex

The fact that a gene has been successfully inserted into the rubber plant does not guarantee that the protein it encodes will be successfully synthesised. Genes, even when they are present in the transformed plant, can remain dormant. Another point to be watchful for is the fact that in nature, proteins take on characteristic patterns of folding.

Some proteins become modified, for example, by having sugars linked to them. Hence, a recombinant protein that faithfully reproduces the exact linear sequence of amino acids of the native protein that it seeks to mimic may still fail as a functional substitute if various structural modifications are not in place.

Research has shown that transgenic rubber plants can be successfully synthesised in the latex a bacterial enzyme (beta-glucuronidase or GUS) and a mouse antibody fragment.

Significantly, these proteins are functional proteins in that their operational characteristics are retained.

The recombinant GUS protein shows its characteristic enzymic properties when supplied with its designated substrate, while the antibody fragment is immuno-reactive to its matching antigenic protein. In the most recent experiments, transgenic Hevea has produced a human protein - human serum albumin - in its latex.

Affordable proteins

Its obvious commercial potential notwithstanding, the production of recombinant proteins from transgenic Hevea is not about profit making alone. Cost-efficient production by transgenic plants can alter the economics of recombinant protein synthesis. For example, hitherto prohibitively expensive chemotherapy could be brought within reach of the man in the street. Commercial proteins from transgenic plants need not be confined to high-cost pharmaceuticals either. Moderate-value proteins such as industrial enzymes or proteins used in personal care products may also be harvested from engineered plants such as the rubber tree. In fact, the low cost of maintaining transgenic plants make them especially suited to high volume production of less expensive proteins that otherwise cannot be produced cost-effectively in conventional bioreactor systems.

Advantages

There are several advantages in using transgenic Hevea for the production of commercially valuable proteins.

Among these are:

* The concept is a novel approach to cost-efficient production of high value proteins in the latex of transgenic rubber trees, which essentially serve as production lines.

* The approach is environment-friendly. The process is driven by the sun and is therefore energy-efficient and essentially pollution-free.

* Rubber trees require no special attention beyond routine horticultural maintenance.

Their use is thus highly cost-efficient as compared with conventional bioreactor systems.

* Production of the target protein is continual through a system of non-destructive harvesting (tapping) of the rubber tree.

* The latex that exudes from the rubber tree is free of animal viruses and other contagion vectors.

These include pathogenic viruses such as those causing AIDS or hepatitis, and that cause mad cow disease and its human variant.

* Successful transformation of the rubber tree for a specific gene needs to be achieved only once.

Rubber trees are amenable to vegetative propagation and an unlimited number of genetically identical plants (clones) can be generated by conventional horticultural methods.

* From the bio-safety viewpoint, the transgenic rubber tree raises far fewer objections as compared with other crops. Hevea propagation is normally by vegetative means. Hence, it is not expected to have adverse effect on the environment or on the crop. Unlike transgenic food products, recombinant proteins from Hevea are purified from the transgenic elements that are not presented to the consumer.

Conclusion

In simple terms, from the point of view of a practical rubber grower, the main interest in genetic transformation of rubber trees would be the introduction of genes controlling specific agronomic traits-- such as the genes for resisting diseases, drought and other environmental stress tolerance, enhanced rubber biosynthesis and timber yield and tolerance to tapping panel dryness etc -- to high yielding rubber clones.

The genetic transformation technique involved is the introduction of specific genes into single cells and development of whole plants from these cells.