INFORMATION SYSTEMS FOR BIOTECHNOLOGY


August 2004
COVERING AGRICULTURAL AND ENVIRONMENTAL BIOTECHNOLOGY DEVELOPMENTS


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IN THIS ISSUE:



HOW FIT ARE GENETICALLY ENGINEERED MOSQUITOES?
Mark S. Hoddle

Genetic engineering, the practice of using molecular tools to insert novel genetic material of particular interest into an organism's genome, is a research enterprise spanning many laboratories globally. This precise form of genetic insertion and manipulation has great potential for custom designing molecular-based solutions to many pest and disease problems that currently appear intractable when confronted with conventional management techniques, such as pesticides, or cultural and biological control practices that aim to eradicate or reduce pest populations to non-damaging levels.

One area of interest for molecular biologists is development of transgenic mosquitoes refractory to transmission of human diseases such as malaria, dengue fever, and yellow fever. Transformation of mosquitoes can be achieved in the laboratory through transposable elements, units of DNA that move from one DNA molecule to another where they insert at random. Transposable elements (e.g., the hermes element from Musca domestica) can be designed to carry marker (e.g., GFP from jellyfish, which glows green under UV light, facilitating rapid identification of transgenic individuals) and strategic genes (i.e., genes that make the transformant behave in the desired way, such as being unable to transmit disease-causing organisms) that are expressed following successful incorporation into the target's genome.

While significant progress is being made in developing transgenic insect technologies and effector genes to transform insect species of interest, very little work has been done with laboratory-generated genotypes to determine the likelihood of transgenic insect establishment, population growth, and persistence when competing with non-transformed wild types in nature. Transgenes conferring fitness advantages could act to promote the spread of particular genotypes while transgenes resulting in fitness costs could be a significant impediment to the establishment and competitiveness of transgenic organisms. Therefore, creating insects with appropriate fitness will be critical to the field-level success of transgenic-based control strategies. While many labs are routinely creating transgenic mosquitoes expressing various novel characters, rigorous assessment of the fitness of genetically engineered mosquitoes is lacking. Fitness of transgenic mosquitoes can be affected by type of transgene inserted, placement of the novel material within DNA and associated mutations or interruption of functional gene sequences, and founder effects resulting from inbreeding between small numbers of transformants when establishing newly transformed lines.

Fitness assessment of transgenic insects is an area of research that needs greater attention immediately if shortcomings associated with transgenesis are to be identified, and mechanisms underlying fitness costs are to be understood and ultimately remedied during the early stages of this emerging technology. University researchers located largely in the United States, England, and Italy, using various laboratory-based studies to investigate fitness of transgenic mosquitoes, are paying closer attention to evaluating fitness of transgenic mosquitoes. One study examined the fate of transgenes in Anopheles stephensi, an important vector of Plasmodium that causes malaria in humans1. In studies in which caged transgenic mosquitoes could interbreed with non-transformed mosquitoes, the frequency of transgenic alleles declined abruptly and in some instances died out. The mechanisms underlying poor performance were not elucidated but were assumed to be caused by insertion and position effects of novel genes and inbreeding depression of transgenic lines following initial creation.

Another research avenue to investigate fitness was to quantify demographic parameters for three lines of transgenic Aedes aegypti, the vector of yellow fever, and compare them with non-transformed Ae. aegypti parameters. Several factors affect the reproductive output of an insect and ultimately its population size and stability. Important demographic factors are rates of sterility or egg inviability (i.e., progeny production), sex ratio of offspring, juvenile viability, development times, and adult longevity. Workers at the University of California Riverside demonstrated that transgenic Ae. aegypti had a significantly diminished capacity for population increase in comparison to non-transformed Ae. aegypti, and that significant differences existed between lines of transgenic mosquitoes2. For example, fecundity was significantly impaired for all transgenic lines in comparison to non-transformed mosquitoes, and one transgenic line produced substantially fewer viable offspring than its non-transgenic counterparts. In this study, the authors demonstrated that negative effects of transgenesis on fitness are not uniform, and a strain that performs poorly in one area may outperform other strains when different characters are measured. For example, impaired fecundity may be correlated with a significantly shorter pre-oviposition period. Data collected from this experiment were used to calculate the intrinsic rate of increase—the rate at which a population can increase when resources are unlimited and predators are not present. This value was calculated for non-transformed and transformed Ae. Aegypti2 and inserted into a simple logistic growth equation (Fig. 1). The model began with a population of five individuals and continued for 100 generations with a maximum carrying capacity of 100 mosquitoes in the water container.

Figure 1.

In Fig. 1, non-transformed Ae. aegypti significantly outcompeted all transgenic lines, reaching 50% and 100% of the environmental carrying capacity 47% and 41% faster than transgenics, respectively. Based on their intrinsic rate of increase, non-transformed mosquitoes would be predicted to outcompete transgenic mosquitoes rapidly. The challenge facing molecular biologists now is to create a "super-achiever"—a mosquito that is competitively superior to wild types in nature. This would require building a transgenic mosquito with superior fitness, which would result in a shift of its logistic growth curve to the left of non-transformed mosquitoes (Fig. 1). Development of a "super-achiever" may be possible with use of promoters that drive constructs (i.e., transposable element, marker, and strategic genes) through the target population, ultimately reducing or replacing non-transformed wild type populations.

In contrast to the above study, workers in one laboratory identified no fitness costs associated with mosquito transgenesis3, and in some instances, important traits such as longevity and fecundity were actually enhanced in transgenics in comparison to non-transformed conspecifics. Consequently, it is crucial to develop a comprehensive understanding of the mechanisms driving these conflicting outcomes and for the development of standardized protocols for laboratories investigating fitness-related issues in transgenic insects. Adoption of standardized evaluation techniques would increase our ability to unambiguously quantify and compare fitness of transgenic insects among different laboratories.

Another strategy for controlling pestiferous insects is to use viruses engineered with specific activity towards insects, especially caterpillars. Transgenic baculoviruses, a group of entomopathogenic double-stranded DNA viruses with genetically enhanced toxicity for insects, has been assessed for fitness. These viruses were engineered to express insect-selective toxins produced by spider and scorpion genes and kill more rapidly than non-transformed viruses. Laboratory and field experiments have demonstrated that genetically engineered baculoviruses that express insect-selective toxins have reduced reproductive capacity and rates of transmission. Taken together, these results suggest that engineered baculoviruses are less fit than non-transformed parental wild types and will not likely persist in the environment4. It is possible that fitness costs incurred by genetically engineering insects and viruses will have similar and fundamental underpinnings that could be worth elucidating.

Transgenesis is an exciting new technology that promises to revolutionize pest and disease vector control. It is expected to become an important tool in managing many pest problems, most likely complimenting the fields of biological, cultural, and pesticidal control. Like any new emerging technology, there will be initial problems during development, and fitness related issues is just one area among many needing attention. With greater research effort, many of the current problems associated with reduced fitness may be solved once fundamental mechanisms are better understood, thereby allowing the technology to advance to field application.

References

1. Catteruccia F, Godfray HCJ, & Crisanti A. (2003) Impact of genetic manipulation of the fitness of Anopheles stephensi mosquitoes. Science 299, 1225-1227.

2. Irvin N, Hoddle MS, O'Brochta DA, Carey B, & Atkinson PW. (2004) Assessing fitness costs for transgenic Aedes aegypti expressing the GFP marker and transposase genes. PNAS 101, 891-896.

3. Moreira L, Wang J, Collins FH, & Jacobs-Lorena M. (2004) Fitness of anopheline mosquitoes expressing transgenes that inhibit plasmodium development. Genetics 166, 1337-1341.

4. Cory JS. (2000) Assessing the risks of releasing genetically modified virus insecticides: Progress to date. Crop Protection 19, 779-785.

Mark S. Hoddle
Biological Control Specialist
University of California, Riverside



ENHANCED DROUGHT TOLERANCE IN TRANSGENIC RICE
Teresa Capell

Engineering the plant polyamine biosynthetic pathway
Manipulation of metabolic pathways in plants through molecular genetics is now possible because of a significant increase in our knowledge base of how such, often complex, networks are controlled and regulated. In our ongoing efforts to implement rational molecular approaches to modulate plant metabolism, we chose the polyamine pathway as a model to unravel key factors that still present bottlenecks in engineering plant biosynthetic pathways.

The polyamine pathway is ubiquitous in living organisms. Polyamines are low molecular weight polycationic molecules, which are thought to play important roles in a number of physiological and developmental processes1. In animals and fungi, the diamine putrescine (the precursor of the higher polyamines spermidine and spermine) is synthesized directly from ornithine by the enzyme ornithine decarboxylase (ODC). Plants have an alternative route to the production of putrescine that is catalyzed by arginine decarboxylase (ADC). Additional reactions convert putrescine into spermidine and spermine. These steps are catalyzed by spermidine and spermine synthases, which add propylamino groups generated from S-adenosylmeth-ionine by S-adenosylmethionine decarboxylase (SAMDC).

Engineering of the plant polyamine biosynthetic pathway has concentrated mostly on two species, tobacco and rice2,3. We have generated a diverse rice germplasm with altered polyamine content. Transgenic rice plants expressing the Samdc cDNA accumulated spermidine and spermine in seeds at two to three-fold higher levels compared to wild type. In a different set of experiments, we were able to measure a ten-fold putrescine accumulation in transgenic rice plants harboring oat adc cDNA compared to wild type. Reduction in endogenous adc transcript levels in rice resulted in depletion of putrescine and spermidine pools, with no concomitant changes in expression of downstream genes in the pathway.

In general, studies focusing on spatial expression of these transgenes demonstrate that more dramatic changes in polyamine content occur in storage compared to vegetative tissues, such as leaves and roots. Therefore, we believe that the polyamine biosynthetic pathway in plants is regulated strongly in a spatial manner4. In tomato, enhanced fruit juice quality and prolonged vine life of fresh fruits with increased lycopene was achieved by expression of yeast Samdc driven by the ripening-inducible E8 promoter5.

Role of polyamines in stress response
In plants, polyamines accumulate under several abiotic stress stimuli, including salt and drought. It has been suggested that this increase in polyamine concentration could be considered as an indicator of plant stress. The first demonstration of involvement of polyamines in stress responses in plants was documented by accumulation of putrescine in response to sub-optimal K+ levels6. Since then, the link between increased putrescine levels and several abiotic stresses was established. For example, Krishnamurthy and Bhagwat7 reported accumulation of spermidine and spermine in salt tolerant rice cultivars and accumulation of putrescine in rice sensitive cultivars in response to salinity stress.

The physiological role of an increase in putrescine accumulation following abiotic stresses is still unclear and is a matter of considerable debate. It has been very difficult to establish directly a cause and effect relationship between increased polyamine levels in plants and abiotic stress. The increase in putrescine levels in plants under stress might be the cause of stress-induced injury or alternatively a means of protection against stress. Earlier experiments by Roy and Wu8,9 expressing oat adc cDNA in rice under control of an ABA-inducible promoter resulted in transgenic rice plants with increased biomass when grown under salt stress. The same authors expressed Tritordeum Samdc cDNA in rice, under control of the same promoter. Under salt stress these plants showed increased seedling growth compared to wild type. Results from a number of studies suggest that polyamines, particularly spermidine and spermine, are involved in regulation of gene expression by enhancing DNA-binding activities to particular transcription factors. Polyamines are believed to have an osmoprotectant function in plan cells under water deficit.

A threshold model linking polyamines and abiotic stress response in plants
Osmoregulatory processes are important to all organisms for stabilization of the intracellular milieu against environmental fluctuations of water and ions. Despite divergence of biochemical pathways, both prokaryotic and eukaryotic organism share several physiological responses to osmotic stress.

We put forward a threshold model that is consistent with high levels of adc gene expression leading to production of putrescine. The production of putrescine needs to exceed basal levels in order to generate a large enough metabolic pool to trigger polyamine flux through the pathway leading to increases in the levels of spermidine and spermine. Transgenic rice plants expressing the Datura adc gene accumulated up to two-fold putrescine in leaf tissues compared to wild type. Such plants, when subjected to drought stress induced by 20% polyethylene glycol, exhibit a very significant divergent behavior compared to wild type under the same conditions. Following 3 and 6 days of drought stress, all wild type plants wilt and show drought-induced rolling of leaves. Such symptoms are completely absent from Dadc-transgenic plants, which exhibit significant putrescine accumulation during the same period. After the 6-day drought stress period, the phenotype of transgenic plants is indistinguishable from non-stressed wild type. Transgenic plants with 2- to 4-fold higher levels of putrescine develop and set seed normally.

Based on these observations, we put forward a model that is consistent with a mechanism linking polyamine metabolism to drought tolerance. Expression of the Dadc transgene driven by the strong maize Ubi-1 promoter would augment the putrescine pool to levels that extend beyond the critical threshold required to initiate the conversion of excess putrescine to spermidine and spermine10. Spermidine and spermine de novo synthesis in transgenic plants under drought stress is corroborated by the activation of the rice samdc gene. Transcript levels for rice samdc reach their maximum levels at 6 d after stress induction. Such increases in the endogenous spermidine and spermine pools of transgenic plants not only regulate the putrescine response, but also exert an anti-senescence effect at the whole plant level, resulting in phenotypically normal plants. Wild type plants, however, are not able to raise their spermidine and spermine levels after 6 d of drought stress and consequently exhibit the classical drought-stress response11.

Transgenic germplasm we have generated exhibiting increased tolerance to drought stress is currently being evaluated in field trials. We are very excited about the prospects of this germplasm to make a positive contribution towards sustainable rice production under stress conditions.

References

1. Malmberg RL et al. (1998) Critical Rev Plant Sci 17, 199-224.

2. Kumar A, Minocha SC (1998) Transgenic manipulation of polyamine metabolism. In: Lindsey K (ed) Transgenic research in plants. Harwood Academic Publishers UK, pp 187-199.

3. Capell T, Christou P (2004) Current Opinion in Biotechnology 15, 148-154.

4. Trung-Nghia P et al. (2003) Planta 218, 125-134.

5. Mehta RA et al. (2002) Nature Biotech 20, 613-618.

6. Richards FJ, Coleman RG (1952) Nature 170, 460.

7. Krishnamurthy R, Bhagwat KA (1989) Plant Physiol. 91, 500-504.

8. Roy M, Wu R (2001) Plant Science 160, 869-875.

9. Roy M, Wu R (2002) Plant Science 163, 987-992.

10. Bassie L et al. (2000) Trans. Res. 9, 33-42.

11. Capell T, Bassie L, Christou P (2004) Proc. Natl. Acad. Sci. of USA 101, 9909-9914.

Teresa Capell
Department of Crop Genetics and Biotechnology
Schmallenberg, Germany
teresa.capell@ime.fraunhofer.de


BIOTECHNOLOGICAL IMPROVEMENTS OF TEA
Tapan Kumar Mondal

Tea (Camellia sinensis L.; family Theacea) is the oldest non-alcoholic caffeine-containing beverage crop in the world, and India is currently the foremost producer, consumer, and exporter of commercial tea. The plant is a woody perennial, traditionally propagated through either seeds or stem cuttings, with a life span of more than 100 years. Tea is classified morphologically into two varieties: Assam and China. The young leaves are processed into different types of tea, such as black, green, and oolong. Health benefits attributed to tea consumption are well proven.

Background
Conventional tea breeding is well established, though time-consuming and labor intensive due to its perennial nature and long gestation period (4—5 years). Vegetative propagation is standard, yet limited by slow multiplication rate, poor survivability of some clones, and need for copious initial planting material. Seed-borne plants are heterogeneous due to their highly allogamous nature; consequently, it is difficult to maintain their superior character. Additionally, tea breeding has been slowed by lack of reliable selection criteria. Although few morpho-chemical markers are available for identification of superior cultivars, these markers are greatly influenced by environmental factors and show a continuous variation with a high degree of plasticity.

To overcome these problems, a limited number of isozyme markers have been used, resulting in less polymorphism. With the advancements of molecular biology, however, efforts have shifted to using various DNA markers. Understanding genetic diversity at the molecular level of tea germplasm will help to: (1) preserve the intellectual property rights of the tea breeder; (2) identify individual tea cultivars through use of a molecular passport; (3) prevent duplicate entry of different genotypes into the tea gene pool; (4) increase efficient selection of varieties for hybridization, composite plant production, etc.; (5) classify tea genotypes taxonomically using molecular markers; and (6) improve tea varieties for agronomically important characteristics through marker assisted selection. Consequently, biotechnological tools appear to be the ideal choice to circumvent problems of conventional tea breeding.

Micropropagation
The development of micropropagation, a rapid in vitro multiplication method, of tea has passed through three phases. Until the 1980s, emphasis was on standardizing parameters of the in vitro protocol, such as using a suitable explant, overcoming microbial contamination, and optimizing media composition combined with growth regulation for better proliferation. It is now accepted that nodal segments (0.5-1 cm) cultured on MS medium with BAP (1-6 mg/l) are best for multiplication of shoots, along with either a high dose (500mg/l) pulse treatment or a low dose (1-2mg/l) long duration treatment of auxin such as IBA for in vitro rooting. Until the1990s, efforts turned toward hardening micro-shoots to achieve a higher survival percentage. Accordingly, several nonconventional approaches, such as a CO2-enriched hardening chamber, biological hardening, and micrografting, were developed. Presently, attention is increasingly focused on evaluating field performance of the micropropagated plant.

In our laboratory we developed a micropropagation protocol by using a novel plant hormone, thidiazuron, which was commercialized at the Research and Development Department of Tata Tea Ltd., India. This protocol provides much faster proliferation rates.

Somatic embryogenesis
One prerequisite for genetic transformation of tea is an efficient system of regenerating the complete plant from a single cell. Until today, somatic embryogenesis in tea was considered the most efficient regeneration system. Unlike micropropagation, tea somatic embryogenesis started in the late 1980s. Thus, emphasis was focused on standardizing parameters, such as genotypes, seed maturity, media formulation, growth regulator, physical condition, etc. We developed a complete pathway of tea somatic embryogenesis in which somatic embryos were first induced within 6—8 weeks on the cotyledon segments of mature tea seed, which were then further multiplied synchronously (Fig. 1). A germination medium was formulated that yielded a 70% conversion rate. Following this protocol, we transferred 3,000 plants to the field at the Research and Development department, Tata Tea Ltd, India1.

Figure 1. Occurrence of somatic embryo on the tea cotyledon

Bioreactor technology for secondary embryogenesis
Applications of bioreactor technology further ensure the speedy, continuous, and large-scale supply of propagule. A bioreactor system for repetitive embryogenesis in tea has also been developed in Australia2 in which uniform sizes of globular somatic embryos were obtained for a bioreactor technology called the temporary immersion system (TIS). By controlling immersion cycles, synchronized multiplication (24 fold) and embryo development were achieved with greater consistency and with a high rate of plant recovery. Plantlets recovered through this method were hardy, with a well-formed taproot. Therefore, this technique was the first significant step for commercial application of bioreactor technology to produce large-scale tea somatic embryos.

Field performance of micropropagated raised plants
The ultimate success of any in vitro protocol depends upon performance of plants in the field compared to vegetative counterparts. For the last several years, researchers at the Research and Development Department of Tata Tea Ltd, India, have transferred more than 45,000 plants of eight tea cultivars to the field, from which leaves are harvested regularly to manufacture black tea.

A systematic study at 1.7, 4, and 8 year-old field-grown micropropagated and vegetatively propagated tea plants in our laboratory and elsewhere in India demonstrated that overall yields and quality were comparable. Although different physiological parameters such as photosynthetic rate, chlorophyll content, etc. remained the same, two morphological variations were noticed. First, the number of lateral shoots produced after `centering' were significantly greater in micropropagated-raised plants compared to vegetatively propagated plants. This is perhaps due to effects of various growth regulator treatments applied under in vitro conditions. Second, root volumes of tissue culture plants were also greater than in vegetatively-propagated plants. Micropropagated shoots were treated with IBA to induce rooting, which may be responsible for better root development in the field. Therefore, we concluded that the micropropagation protocol should be used only when required to produce a large number of plantings from a limited source.

Other tissue culture techniques
Other techniques have been applied in tea with specific objectives (Table 1). Efforts to improve these techniques are ongoing at laboratories worldwide.



Genetic transformation
Transgenic technology has immense potential for genetic improvement of tea; however, until 2000 there were no reports on tea transgenesis. The initial challenge was to develop a protocol for gene transfer. Recently, we reported the optimization of transformation conditions and production of transgenic tea via Agrobacterium tumefaciens3. In this study, we produced transgenic tea using GUS reporter and NPT-II marker genes under control of strong monocot gene promoters, and stepwise antibiotic selection. Using this protocol, further experiments are underway to transfer the chitinase gene for production of a fungus-resistant tea plant.

Biolistic-mediated genetic transformation is another effective method to produce transgenics in a wide variety of plant species. Although no transgenic tea plants have been grown in the field using this technique, experimental conditions have been standardized by Australian and Chinese scientists.

Genetic characterization
Morphological markers such as leaf pose, dry matter production, partitioning, flesh evenness, etc. and biochemical markers such as total catechin/polyphenol content, caffeine, etc. are used to identify the superior tea plant. However, tea breeders are often unable to use markers effectively because they are greatly influenced by environmental factors and show a continuous variation with a high degree of plasticity. Hence, to overcome these problems, research has shifted to using more sensitive DNA markers.

Work on molecular markers in tea began in our laboratory in 1994 and a significant amount of work is continuing worldwide (discussed below).

Research in India:
We have used the random amplified polymorphic DNA (RAPD) assay to characterize 25 important Indian tea cultivars and two ornamental species (Fig. 2).


Figure 2. DNA fingerprinting pattern of different tea cultivars.
Lane 1–25 denotes different tea cultivar.

In a separate study, twenty-five diverse tea cultivars were analyzed using the simple sequence repeat anchored polymerase chain reaction (SSR-anchored PCR) or Inter SSR-PCR (ISSR). In both cases, cultivars were analyzed using Shannon's diversity index, which revealed that the China type tea group is more diverse than the Assam group. Additionally, we noted that molecular classification matches conventional classification of tea. A species-specific primer was also developed for distinguishing between the Assam and China type tea cultivars.

Amplified fragment length polymorphism (AFLP) markers were also studied in depth to detect diversity and genetic differentiation of several important tea clones, including the famous `Darjeeling tea', mainly to protect cultivars for intellectual property rights purposes. Interestingly, the RFLP technique was also used to detect adulteration with cashew husk in 10 different tea samples4.

Research abroad:
Researchers in different countries have made fingerprints of tea cultivars in their countries of origin. In Kenya, fingerprints of popular tea cultivars were made through RAPD and AFLP analysis at the Tea Research Institute, Kenya. The same group also initiated a genetic linkage map of tea. Work is ongoing to develop a complete tea database with chemical as well as molecular data, which will assist with easy identification of the different cultivars.

In Japan, a wide range of markers has been used with various applications. The markers used for genetic characterization of different green tea cultivars are RAPD, AFLP, SSR, CAPS, and RFLP. Importantly, the RFLP technique was also applied in Japan to prevent adulteration of higher grade with lower grade tea. Several other minor tea-producing countries have used different molecular markers to characterize the tea gene pool of introduced tea cultivars available to that country. Such efforts were made using RAPD in Portugal, ISSR in Taiwan, and RAPD in South Africa. All work focused on the genetic characterization and molecular taxonomy of the introduced variety available in the respective countries. Similarly, South Korea and China tea cultivars were characterized through RAPD or AFLP, and RAPD, respectively.

Simple sequence repeats (SSR) were derived from C. japonica, a closely related species of tea in Japan. Using these primer pairs, 53 C. japonica ecotypes were genotyped and population genetic parameters calculated. Later, the same group investigated the spatial genetic structure of C. japonica using four of these microsatellite primers. Spatial distribution of individuals was also assessed to obtain an insight into spatial relationships between individuals and alleles.

Gene cloning and expression
Japanese researchers have isolated the cDNA chalcone synthase (CHS) gene as well as ß-tubulin gene from the Japanese green tea cultivar `Yabukuta'. More recently, a few important genes such as phenyl ammonia lyase (PAL), caffeine synthetase, and primeverosidase have been isolated.

References

1. Mondal TK et al. (2004) Plant Cell Tissue Org Cult, Netherlands 76, 195-254.

2. Akula A, Akula C (1999) In: Jain SM, Gupta PK, & Newton RJ (eds.), Somatic embryogenesis in Woody plants, Vol. 5, pp 239-259. Kluwer Academic Publishers: The Netherlands.

3. Mondal TK et al. (2001) Plant Cell Reports 20,712-720.

4. Dhiman & Singh (2003) Planta Med 69(9), 882-4.

Tapan Kumar Mondal
Centre for Advance Study in Tea Science and Technology
Uttar Banga Krishi Viswavidalaya, India
mondaltk@rediffmail.com



REGULATING PLANT GENES AND AGBIOTECH PRODUCTS — INTERNATIONAL STYLE
Phillip B.C. Jones

Challenges from pathogens, pests, and the climate fuel perpetual efforts to develop new crops with mixed traits. When a fungus decimated maize grown in the southern United States, for example, plant breeders devised a way to defeat the pathogen with a natural resistance trait harbored within a variety of African maize. To combat yellow dwarf disease in U.S. barley varieties, researchers harvested resistance genes from Ethiopian plants, while genes from plants originating in the Caucasus and Spain provided the keys for resisting rust disease in U.S. commercial wheat varieties.

Plant breeders can perform these feats of agricultural innovation because they can select traits from a wealth of genetic resources. And since no single country has the full range of naturally occurring genetic resources, the collection and exchange of germplasm requires international cooperation. Yet not every nation considers this exchange equitable. Accusations of biopiracy often arise from countries in tropical and subtropical regions, which possess the majority of the world's agricultural genetic diversity. At the same time, changing land use practices within these countries lead to the loss of uncollected genetic resources. The Food and Agriculture Organization (FAO) of the United Nations estimates that about three quarters of the genetic diversity found in agricultural crops has vanished over the last century.

These concerns inspired the creation of the UN's International Treaty on Plant Genetic Resources for Food and Agriculture, an agreement adopted after seven years of negotiations by delegates from 116 nations. Although drafted in November 2001, the Treaty only came into force on June 29, ninety days after forty governments ratified it. José Esquinas-Alcázar, secretary of FAO's Commission on Genetic Resources for Food and Agriculture, told BBC News Online that the "treaty will ensure the conservation and availability of raw material for agriculture."

The agreement requires each contracting party to explore and conserve its plant genetic resources for food and agriculture. Parties can work toward this objective by surveying their genetic resources and assessing any threats, and by promoting both in situ conservation and the compilation of genetic resources for preservation in public collections.

The treaty also mandates that contracting parties develop and maintain measures to advance the sustainable use of plant genetic resources. Examples of such measures include extending the genetic base of crops available to farmers and supporting plant breeding efforts that strengthen the capacity to develop varieties adapted to particular ecological conditions.

Under the Treaty, countries agree to establish a Multilateral System to facilitate access to plant genetic material and to share the benefits. The Multilateral System applies to plant genetic resources for food and agriculture listed in the first annex of the Treaty that are under the control of the contracting parties and in the public domain. The 35 itemized food crops and 29 forage crops "represent most of the important food crops on which countries rely," says Esquinas-Alcázar. According to one estimate, the Treaty's annex lists crops representing 80 percent of the world's calorie intake. The 600,000 sample gene bank collection held by the Consultative Group on International Agricultural Research will also be administered under the Treaty.

Restrictions apply to the use of these genetic resources. The material must be used to promote conservation, research, breeding, and training for food and agriculture. Any use of genetic material for chemical, pharmaceutical, and other industrial applications falls outside the scope of the Treaty. If an entity incorporates material accessed from the Multilateral System into a commercial food or agricultural product and does not permit others to use the product without restriction for research and breeding, then the Treaty requires payment of an equitable share of any resulting monetary benefits.

The FAO asserts that the Treaty benefits agricultural research because the Multilateral System will reduce transaction costs for the exchange of plant genetic material between countries. That is, researchers will no longer have to negotiate bilateral agreements with each donor country to obtain germplasm.

But before the Treaty can facilitate any transfer of plant genetic material from its germplasm clearinghouse, the countries that ratified the agreement must decide about conditions for access and benefit-sharing, details that will be embodied in a standard material transfer agreement. Conditions recited in this key document will also determine whether the United States will ratify the Treaty.

At least one mystery resides within the Treaty. Article 12 states that "recipients shall not claim any intellectual property or other rights that limit the facilitated access to the plant genetic resources for food and agriculture, or their genetic parts or components, in the form received from the Multilateral System." Even delegates involved in the drafting of the document could not agree about the meaning of this statement.

It might have been the eruption of biopiracy complaints that motivated the inclusion of the ambiguous passage. During the drafting of the Treaty, protests from India provoked the revocation of a U.S. patent covering a use of turmeric and a European patent on a compound derived from the Neem tree, while South American activists prompted the withdrawal of a U.S. patent on an ayahuasca vine variety. In 2001 alone, India, Pakistan, and Thailand voiced grievances about a U.S. patent on a type of basmati rice, and Mexico protested a U.S. patent on yellow beans.

A copy of the Treaty is available from the FAO website (http://www.fao.org/ag/cgrfa/itpgr.htm).


Labeling Food as Genetically Modified and Living Modified Organisms as Pests
In June the Codex Alimentarius Commission released its report of the 32nd Session of the Codex Committee on Food Labeling. The delegates generally supported the establishment of guidelines for end-use labeling—the labeling of foods derived from genetic engineering that have a significant change in composition, nutrient content, or intended use.

Production labeling was met with less enthusiasm. Certain delegates, including the U.S. delegation, asserted that consumers would perceive labeling based solely on the method of production as a safety warning. The Canadian delegation argued that method of production labeling fails to comply with Codex's own rule: only factors accepted worldwide should be taken into account as a basis for decision-making. A copy of the report can be obtained from the website of the Codex Alimentarius Commission (http://www.codexalimentarius.net/).

On June 1, the FAO published new guidelines for determining if a living modified organism (LMO) poses a hazard to plants. About 130 countries have adopted this assessment standard, which focuses on the risk that a foreign gene may transform a plant into a weed. The document also offers advice for determining whether genetically modified insects, fungi, or bacteria could harm plant ecosystems. If authorities deem an LMO a threat, then they can decide whether to prohibit or restrict its import and domestic use. The website of the International Plant Protection Convention offers a copy of the guidelines, entitled "Pest Risk Analysis for Quarantine Pests, Including Analysis of Environmental Risks and Living Modified Organisms" (http://www.ippc.int/).

Selected References

Anonymous. (2004) Plant gene treaty becomes law. BBC News. June 29, 2004. Available at: http://news.bbc.co.uk/2/hi/science/nature/3849489.stm.

Fowler C. (2003) The status of public and proprietary germplasm and information: An assessment of recent developments at FAO. IP Strategy Today, No. 7-2003. Available at: http://www.biodevelopments.org/.

Sullivan SN. (2004) Plant genetic resources and the law. Past, present, and future. Plant Physiology, 135: 10-15.

Phillip B.C. Jones, PhD., J.D.
Spokane, Washington
PhillJones@nasw.org


INDIA PRODUCES INDIGENOUS `GM COTTON'
P Janaki Krishna

Insects, disease, and drought present the greatest impediments to realizing expected yields in major crops. In addressing these problems, development of transgenic varieties has assumed significance, primarily through use of durable resistance genes. However, a few multinational companies in developed countries own and patent many of these genes. In developing countries, these novel genes are sometimes available to scientists as `gifts' through personal contacts. Though initial transgenic crop development is dependent on these borrowed genes, the varieties developed from them would not be available for commercial cultivation because of contractual obligations generally underlying the `gift' to investigators (as these genes generally are available for academic and experimental purposes only). At best, GM plants thus developed could be tested only for their efficacy in solving designated problems and not developed for commercial cultivation. Deployment of borrowed genes in transgenic crops might also attract patent problems under new IPR regimes.

Scientists, institutes, and seed companies have therefore decided to convene and begin searching and licensing indigenous genes and technologies for endogenous developments. Apparently this exercise is having an effect, as Monsanto's monopoly on genetically modified cotton in India will soon be broken by Swarna Bharat Biotechnics Private Ltd (SBBPL), Hyderabad, India, a consortium of seven Indian seed companies. SBBPL received licenses for two genes derived from Bacillus thuringiensis (Bt), which protect cotton against bollworm (Helicoverpa armigera) and tobacco caterpillar (Spodoptera litura). The genes are licensed from the National Botanical Research Institute (NBRI), Lucknow, India, for Rs. 7.5 ($ 0.16) million over a three year period and a royalty of 3%. SBBPL is soon likely to get license for a third gene (LecGNA 2) that directs production of lectin, a protein lethal to sucking pests such as aphids, from the publicly funded Centre for Plant Molecular Biology (CPMB), Osmania University, Hyderabad, India.

According to NBRI's Deputy Director, Dr Rakesh Tuli, SBBPL has licensed two genes—Cry1Ac and another killer gene called Cry1Ec. Of these two genes, Cry1Ec, used against a tobacco caterpillar, is designed and synthesized at NBRI. Because Cry1Ac, which confers resistance against bollworm, is not protected in India, NBRI's team altered Cry1Ac promoters to create a version with greater expression and stability. According to some experts, although NBRI has modified the gene, there could be legal implications: GM plants derived from this gene might come under the category of `essentially derived varieties' (EDVs) and cannot be registered under India's Plant Variety Protection Act - 2001. Even more, Monsanto might contest the commercialization of Indian indigenous Bt cotton after January 2005, the date when India has to comply with patents covered under the WTO rules. Tuli admits that only time might solve the problem. Meanwhile, SBBPL is seeking regulatory approval in India for both Cry1Ac and Cry1Ec, with a view toward introducing the new Bt cotton by 2006.

The consortium's aim is to enter an era of self-sustaining agribiotech development. Satish Kumar, Managing Director of SBBPL, reiterated that they are ready to source beneficial genes from any publicly funded laboratory where they are available. The consortium opines that the advantages of sourcing indigenous technology are economic and strategic. The profit generated by public sector institutes through licensing helps support reinvestment in developing more agribiotech products to serve local needs. The main benefit for consortium members is economic, as the technology access fee is shared by members of the consortium. In addition, Indian partners help with the regulatory process to obtain product approval. Kumar expects that the price of SBBPL seeds would be two-thirds of Monsanto's.

Through licensing these genes, Rs 10 ($ 0.2) billion spent on chemical pesticides can be saved by SBBPL, as 90% of cotton damage is from bollworm and sap sucking pests. Indian farmers spend about Rs 16 ($ 0.35) billion on chemical pesticides. With the introduction of novel Bt cotton varieties, SBBPL, which has a 30% share of the total Indian cottonseed market, expects to claim some of the Rs 30 ($0.66) billion market per year that is presently monopolized by the joint-venture company Monsanto-Mahyco Biotech, Mumbai, India.

Since scientific knowledge is indigenous in India, and smaller players can access costly technology monopolized by big multinational companies, many eminent scientists, activists, scholars, and institutes welcomed this initiative of SBBPL. The activist groups critical of Monsanto's monopoly are happy, as 42% of India's transgenic research has been based on Monsanto's gene. "Finally, we seem to be getting our act together," said Suman Sahai, Convener of Gene Campaign, New Delhi, India.

However, Monsanto is not daunted by the competition—they have already co-licensed the Cry 1Ac gene to nine more Indian companies whose products are at different stages. Ranjana Smetacek, the company's spokesperson in India, says Monsanto welcomes the widespread usage of Bt cotton. Meanwhile the Council for Scientific and Industrial Research (CSIR), New Delhi, India, the parent institute of NBRI, is finalizing the list of countries soon to file patents for novel genes. Experts suggest that the consortium may try to exploit those GM technologies on crops for which patents held by multinational companies are now expiring. Prabhakar Rao, Managing Director of Nujiveedu seeds (Hyderabad, India), the largest company in the consortium, said that membership would soon reach 19, as many other countries are willing to partner with them. Hence, it appears that a few multinational seed companies can no longer monopolize agribiotechnologies, and local stakeholders can play a crucial role in technology development and commercialization.

References

1. Nat. Biotechnol. 22, 255-256, 2004.

2. Nat. Biotechnol. 21, 590-591, 2003.

3. Nat. Biotechnol. 19, 895-896, 2001,

P. Janaki Krishna
Biotechnology Unit, Institute of Public Enterprise
Hyderabad, India
jankrisp@yahoo.com



MOLECULAR MARKER ASSISTED SELECTION DOCUMENT

FAO has published a biotechnology-related summary of an e-mail conference on "Molecular marker assisted selection as a potential tool for genetic improvement of crops, forest trees, livestock and fish in developing countries," which ran from 17 November to 14 December 2003. The document summarizes the main arguments and concerns raised during the moderated e-mail conference. The full summary is available at http://www.fao.org/biotech/logs/C10/summary.htm.




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