CURRENT ANTHROPOLOGY Volume 43, Number 4, August–October 2002
Copyr. 2002 by The Wenner-Gren Foundation for Anthropological Research. All rights reserved 0011-3204/2002/4304-0004$2.50

Crop Biotechnology Backgrounder

This was published as a CA+ enhancement to: Stone, Glenn Davis 2002 Both Sides Now: Fallacies in the Genetic-Modification Wars, Implications for Developing Countries, and Anthropological Perspectives. Current Anthropology, 43(5):611-630. This backgrounder was developed with major contributions from Dr. Nigel Taylor of the Donald Danforth Plant Science Center, St. Louis.

Introduction

     In agriculture, "biotechnology" is often used synonymously with "genetic modification." It is true that, in most respects, genetic modification is the most important type of agricultural biotechnology; yet it is not the only one. This backgrounder provides a rudimentary introduction to three forms of agricultural biotechnology, seen mainly from the perspective of developing countries. The main emphasis is on genetic modification, but descriptions of tissue culture and genetic marker assisted breeding are provided as well.

Genetic Modification of Crops for Developing Countries

      Biotechnology enthusiasts are fond of characterizing crop genetic modification as a continuation of age-old processes of alteration of nature. This is true in some limited senses; all major crop plants have been modified through their interaction with humans, most to the point of being incapable of reproducing in the wild. However, in most respects genetic modification is profoundly different. For instance, genetic modification is pivotal in establishing patent rights over crop genetic material in a way that conventional breeding never was, which is of critical importance to developing countries.

     There are fundamental biological differences as well. Conventional plant breeding, whether carried out informally by farmers or in formal programs, operates at the level of the phenotype: the plants themselves are manipulated to guide the natural processes of reproduction. Genetic modification operates at the level of the genotype: laboratory-manipulated ("recombinant") DNA is inserted into plants. This allows the transfer of genetic material across the biological kingdoms: viral and bacterial genes can be inserted in plants in a manner impossible in conventional breeding.

     The two main methods of genetically modifying plants are by the use of Agrobacterium and by particle bombardment using a "gene gun." Each method is illustrated here by projects directly relevant to agriculture in developing countries, carried out at ILTAB in the Donald Danforth Plant Science Center.

AGROBACTERIUM: AN EXAMPLE FROM A CASSAVA PROMOTER STUDY

     The soil bacterium Agrobacterium tumefaciens is one of several naturally occurring genetic engineers able to transfer and stably integrate some of its own genetic material into a plant's genome. The transferred genes ("transgenes") are then reproduced in new plant cells. It is not Agrobacterium's actual transgenes that are of interest to biotechnologists; those genes merely create a tumor on the plant that allows the bacteria to reproduce (Figure 1). Rather it is the biological capability for inserting genes, since this can be adapted in the laboratory to replace the bacterial transgenes with other genes of interest.

Figure 1.    Tumors on a tree branch produced by Agrobacterium tumefaciens.

AGROBACTERIUM: HOW IT WORKS

     Agrobacterium carries its payloadi.e., the genes that are to be inserted into the planton a plasmid. A plasmid is a circular DNA molecule that is separate from the chromosome(s), where the majority of the genes are located (Figure 2). Because the payload genes create a tumor in the infected plant, Agrobacterium's plasmid is called a TI (tumor-inducing) plasmid.

Figure 2.    Plasmid and chromosome in bacterial cell.

     Figure 3 shows a schematic display of an Agrobacterium TI plasmid with three genes. Each gene is bracketed by a promoter and a stop-sequence. The promoter is a short but crucial section of DNA, usually located directly before the gene, which controls when and under what circumstances the gene is activated. The most important and widely used promoter in plant biotechnology at this time is the "CaMV 35S" promoter, which was isolated from the cauliflower mosaic virus, and patented by Monsanto. The 35S is a constitutive promoter; it causes its gene to be continually functional in all plant tissues regardless of environmental conditions. The stop-sequence is a regulatory DNA segment that tells the genetic machinery to stop reading the gene, just as a full-stop informs the reader that a sentence is complete. (Sometimes referred to as the "terminator," the stop-sequence is an integral component of all genes and has nothing to do with the so-called "Terminator" technologies for producing sterile seeds.)

Figure 3.    Genetic elements on plasmid.

MAKING A CHIMERIC GENE CONSTRUCT

     Figure 4 shows an artificially constructed plasmid. With its genetic elements from disparate organisms, such a structure is called a chimeric gene construct (an allusion to the fire-breathing composite of a lion, goat, and serpent in Greek mythology). Acting as the agent by which the target plant cells will be infected, it is also known as the vector.

Figure 4.    Map of the plasmid containing the PAL-840 promoter.

     This particular vector is being used at ILTAB to study the function of the PAL-840 promoter isolated from cassava. For this project the PAL-840 promoter was attached to GUSa visual marker gene that produces a a strong blue color, revealing the location and potency of the promoter's action. Note that this construct also contains the KAN gene for resistance to the antibiotic kanomycin and that this is driven by the 35S promoter.

     After this vector is created it must be multiplied so that enough copies are available for manipulation. This is done by inserting the plasmid into the bacterium E. coli. Millions of these bacteria are then produced by multiplication overnight in an appropriate culture medium containing kanomycin. The antibiotic ensures that only those cells containing the plasmid are able to survive and divide under these conditions. Simple procedures then allow the E. coli cells to be disrupted and the transformed plasmids isolated, yielding a few microliters of the desired, purified plasmid DNA.

     The plasmids are then transferred to Agrobacterium and the bacterial cells cultured in a manner similar to that for E.coli. Again kanomycin is included in order to eliminate any cells not containing the desired vector. The Agrobacterium is now ready to transfer the desired gene into a plant.

MODIFYING THE PLANT

     In the research on PAL-840, the gene construct was inserted into both Nicotiana benthamiana (a tobacco commonly used as a model species in plant biotechnology) and cassava itself. To produce transgenic plants, young leaves are cut into small disks and treated with plant hormones. The Agrobacterium is applied (Figure 5), so that the chimeric gene construct will be inserted into the plant's genome. The leaf discs are then transferred to a culture medium containing antibiotics to kill the Agrobacterium (whose job is now complete) and to select for the successful genetic transformation events. Once again kanomycin is used, this time to kill all the plant cells which have not received the transgenes. Only those containing the kanomycin-resistant gene (and the gene of interest; in this case the GUS marker gene under control of the PAL-840 promoter) can survive and grow. Plant growth hormones then stimulate regeneration of whole shoots from the transgenic cells.

Figure 5.    Tobacco leaf, cut into small discs and soaked in Agrobacterium, being cultured.

     The antibiotic-resistance gene plays a crucial role in the production of genetically engineered plants, but after this stage it has no agronomic value. Indeed, presence of antibiotic genes and other "selectable marker genes" in transgenic crops is controversial from an environmental point of view, and biotechnologists are under increasing pressure to develop alternative strategies for recovering transformed cells in culture.

     Several weeks later, thin sections of the regenerated plants can be examined under a microscope to determine the action of the promoter in the genetically modified plants (Figure 6).

Figure 6.    Expression of GUS as controlled by PAL-840 promoter. From top to bottom: petiole, young stem, semi-woody stem. Upon treatment with an appropriate chemical, the protein produced by the GUS gene produces a blue color, enabling the localised action of the PAL-840 promoter to be visualized.

PARTICLE BOMBARDMENT: AN EXAMPLE OF BACTERIA-RESISTANT RICE

     Particle bombardment with a "gene gun" (Figure 7) is a very different strategy for modifying a plant's genome. Rather than using a bacterial genetic engineer to transfer new genetic material, the DNA is literally shot into the plant cells. This method has both advantages and disadvantages when compared with Agrobacterium. This is a brief description of the method and an important example of how it has already been used to develop a crop with considerable potential to benefit Chinese agriculture.

Figure 7.    Gene gun.

HOW BOMBARDMENT WORKS

     Purified plasmids containing the gene of agronomic interest and the antibiotic resistance genes are produced as described under Agrobacterium-mediated transformation, and coated onto microscopic particles of gold. The DNA-coated particles are dried onto a plastic membrane and positioned on a holder about 5 cm above the plant tissue, within the chamber of the microparticle gun. The chamber is sealed and helium gas pumped into a small compartment situated above the gold particles. Once a predetermined pressure is reached, the gas is released and the disc is projected against a screen, which stops the plastic disc but allows the gold particles to be projected into the targeted plant cells. These act as bullets, penetrating the plant cell wall and delivering the plasmid DNA into the cell's interior (Figure 8). Thousands of such events occur for each sample of plant tissue bombarded, but in only a few percent does the inserted DNA successfully become integrated into the plant's genetic material. As with the gene transfer by Agrobacterium, the process by which the bombarded vectors are incorporated into the target plant's genome is poorly understood.

Figure 8.    Functioning of the gene gun.

     As with Agrobacterium-mediated transformation, successfully transformed cells are isolated by culturing the plant tissue on medium containing kanomycin. Non-transformed tissues are killed, and plantlets regenerated from the recovered cells through standard plant tissue culture techniques. Since many plasmids can be coated onto the same gold simultaneously, microparticle bombardment can be used to introduce multiple transgenes into plant cells. Using this technology, rice plants containing twelve different transgenes have been developed at ILTAB.

MAKING XA21 GENETICALLY MODIFIED RICE

     An outstanding example of practical application of the gene gun with benefit to developing-country agriculture is the production of rice plants genetically engineered for resistance to rice bacterial leaf blight (Figure 9). Bacterial leaf blight is potentially devastating disease which affects rice throughout south east Asia. A collaborative project was initiated by ILTAB, the University of California at Davis, and the Chinese Academy of Agricultural Sciences, Beijing, and funded by the Rockefeller Foundation and the Ministry of Science and Technology of China, to use biotechnology to tackle this important disease. The Xa21 gene that imparts blight resistance in wild rice was isolated at UC Davis and transferred to a Chinese rice breeding line by particle bombardment at ILTAB.

Figure 9.    Xa21 rice exposed to bacterial leaf blight, showing strong resistance.

     Exposure of the modified rice plants to the pathogenic bacteria (under controlled greenhouse conditions in California) showed that the Xa21 transgene conferred strong resistance. The most promising transgenic plant lines were transferred to southern China, where they were incorporated into a conventional breeding program. Field trials of the resulting hybrids over seven generations have confirmed the excellent field resistance of these plants imparted by Xa21. In addition, one particular hybrid has shown a 7% yield increase over local rice cultivars and has been cleared by the biosafety committee of the Chinese Ministry of Agriculture for further field testing in multiple locations in China.

THE BIG PICTURE: LESSONS ON PUBLIC RESEARCH AND DEVELOPING COUNTRIES

     Although receiving little media attention compared to the much vaunted Golden Rice, this project has delivered a valuable product which could reach commercialization with little further development. A number of additional points in the Xa21 project are also worth highlighting (Figure 10). These plants were developed on a nonprofit basis and the germplasm is now in the hands of Chinese breeders to use as they see fit within the regulations of their own government. Xa21 rice is almost free of intellectual property encumbrances (awaiting clearance of only one tool used in its development); if released to farmers, farmers should be free to use and disperse the seeds as they see fit.

Figure 10.    Xa21 rice plants being assessed at ILTAB greenhouse.

     This presents an interesting contrast to the level of control the multinational companies attempt to retain over their products, as in the case of genetically engineered cotton and soybean. Finally, this project demonstrates that biotechnology and conventional plant improvement are not exclusive. Most will be modified by conventional breeding in order to adapt the transgenic product to local farming systems.

FURTHER INFORMATION

     Preliminary results of the plant transformations undertaken to study the function of the 840-PAL promoter were presented in

TAYLOR N. J., HONGYING L. I., J. R. BEECHING, AND C. M. FAUQUET. 2001. A PAL Promoter Isolated from Deteriorating Cassava Roots Drives Highly Specific Marker Gene Expression in Transgenic Plants. Paper at the Fifth International Meeting of the Cassava Biotechnology Network, St. Louis. Also available on the web.

TAYLOR, N.J., M.V. MASONA, P. CHELLEPAN, HONGYING LI, J.R. BEECHING AND C.M. FAUQUET. 2001. Genetic Transformation of Cassava at ILTAB. Paper at the Fifth International Meeting of the Cassava Biotechnology Network, St. Louis.

     For further information on the Xa21 rice, see:

SONG W.Y., G.L. WANG, L. CHEN, H.S. KIM, L.Y. PI, T. HOLSTEN, J. GARDNER, B. WANG, W.X. ZHAI, L.H. ZHU, C.M. FAUQUET, AND P. RONALD. 1995. A receptor Kinase-like protein encoded by the rice disease resistance gene, Xa21. Science 270:8041806.

Plant Tissue Culture

     Plant tissue culture is a form of biotechnology with great potential to aid agriculture in developing countries. It is based on the ability to grow plants in vitro by culturing sterilized tissues or seeds in test-tubes or Petri dishes. Nutrients, vitamins, and sugar are supplied in specially designed media incorporated into a gel which supports the plant tissues. Originally developed to multiply orchids, it is now possible for every major crop plant to be handled in this way. The great value of plant tissue culture is that it allows the developmental fate of the plant tissues to be manipulated toward desired ends by inclusion of growth regulators (plant hormones) and other chemicals in the medium. The ability to culture plant tissues forms the basis for several biotechnologies and is a central component of crop genetic engineering programs. However, there are also non-transgenic applications, including meristem cleaning, micropropagation, and embryo rescue.

     Meristem cleaning facilitates the production of virus- and bacteria-free plants by regeneration of plantlets from just a few cells excised from the growing tip of a new shoot. Micropropagation involves the rapid multiplication of small shoots in vitro to generate hundreds of thousands of pathogen-free plants in a matter of a few months (Figure 11). A combination of these two technologies has, among other successes, recently led to the delivery of improved sweet potato and cooking banana planting material to farmers in China and Kenya respectively. Yield improvements of up to 30% are being reported from this material in China. Both biotechnologies are relatively simple to handle and can be carried out in most developing countries possessing basic laboratory conditions.

Figure 11.     Cassava plantlet produced through micropropogation.

     Embryo rescue is a tool which greatly enhances the power of traditional plant breeding. Achieving successful sexual mating between distantly related species of the same crop, for example, African and Asian rice varieties, is problematic, often leading to abortion of the resulting embryo. Embryo rescue involves removal of the young embryo from the developing seed before abortion occurs and culturing it to maturity. One example of this capability is the new Nerica rice variety that combines the best of Asian and African traits; it has been released recently in West Africa and appears to offer up to 50% increases in yields over local varieties. Conventional breeding, without the embryo rescue biotechnology component, could not have delivered this product.

Marker-Assisted Breeding

     Biotechnology is often discussed as an alternative to conventional breeding, but it can also provide invaluable assistance to conventional breeding. Marker-assisted breeding (or Marker-assisted selection) is a form of biotechnology that does not involve genetic modification and that is already benefitting conventional breeding programs in developing countries.

     Marker-assisted breeding allows the breeder to see more clearly and more quickly what has happened each time plants are crossed. A typical breeding project may unfold as follows. An agricultural plant is being attacked by a pest, and a wild relative of the crop is found to have resistance to the pest. The breeder wishes to transfer the resistance from the wild relative but without disrupting the crop's other traits. The crop and the wild relative are crossed, and the progeny are exposed to the pest to isolate the plants that have inherited the resistance. These plants will also have inherited many other unwanted traits from the wild parent as well; therefore the breeder crosses the offspring with the crop parent again (this is called backcrossing). The pest-resistant offspring are again isolated and again backcrossed. The strategy is to continue isolating resistant offspring and backcrossing until a plant is achieved that has none of the wild plant's traits except for the desired pest resistance. The process can be extremely time-consuming, especially since it is often necessary to raise each generation to maturity in order to test for inheritance of the desired trait.

     Such breeding can be greatly expedited by the use of genetic markers. A marker is usually a distinctive stretch of DNA located close to the gene of interest. It may occur within the gene itself, in another nearby gene, or in the "non-coding" DNA between genes (markers may also be in proteins instead of the DNA, although protein markers are used less commonly). Since the marker is located very close to the gene, it will almost always be inherited along with the gene, and since it is highly recognizable it acts as a red flag indicating the presence of the gene of interest (Figure 12). Rather than raising a plant to maturity and performing an experiment on the plant (such as exposing it to an agricultural pest), the breeder can check for the marker in the plant's DNA. Marker-assisted breeding is the strategy of alternating between biotechnology (to select plants with desired traits) and conventional breeding (to produce each successive generation of plants).

Figure 12.    The marker acts as a red flag to indicate the presence of the gene of interest (represented by the green stretch of DNA).

     What makes this strategy particularly valuable is that markers can be checked quickly and inexpensively, using immature plants. Marker-assisted breeding is even applauded by staunch opponents of crop genetic modification like Britain's Soil Association, and it is already playing a significant role in public breeding. Indeed, public researchers recently wrote, "because of their relative simplicity, easy integration into conventional breeding, and minimal background intellectual property, DNA marker technology and marker-assisted selection are expected to be strong driving forces in crop improvement in the future" (Fischer, Leung and Khush 2000:20).

FISCHER, KEN, HEI LEUNG AND GURDEV KHUSH. 2000. "Molecular breeding: biotechnology at work for rice," in Promethean Science: Agricultural Biotechnology, the Environment, and the Poor. Edited by I. Serageldin and G.J. Persley, p. 20. Consultative Group on International Agricultural Research, Washington, D.C. Also available on the web.