Producing plants that protect people against pathogens
Four out of five people turn to plants to treat themselves when sick, relying virtually exclusively on traditional herbal medicine for their primary health care.¹ It is primarily in the more privileged and developed countries that synthetic chemicals are supplementing the herbalists' apothecaries. Still, a full quarter of prescription drugs are sourced directly from plant seeds, roots, leaves, stalks, and exudates. Our food biochemistry lab, established in 1978, has worked on the premise that by genetically engineering plants already equipped with useful medical compounds it may be possible to produce hard-to-get drugs. Hence, we have embarked on a program to engineer plants that promote protection against pathogens.

CD14 protecting mucosal surfaces
Plants and animals are constantly exposed to a multitude of microorganisms that colonize their surfaces. In general, a symbiotic relationship develops between the host and the microbe that provides benefits and advantages to each organism. However, pathogenic microorganisms often extend their colonizing capacities beyond mucosal surfaces. These pathogenic invasions are initially countered by innate defense mechanisms that preexist in the host and act within minutes after the initial infection. The innate immune response is based on an early recognition of microbe-specific motifs, known as pathogen-associated molecular patterns (PAMP). Most PAMPs are highly conserved surface-derived molecules, such as lipopolysaccharide or LPS, a major component of the outer membrane of Gram-negative bacteria. In mammals, this early detection is mediated by pattern recognition receptors, such as CD14 (cluster of differentiation 14), capable of detecting picomolar concentrations of LPS. By binding to LPS, CD14 and the Toll-like receptor 4 (TLR4) complex promote inflammatory responses at the site of infection through the secretion of pro-inflammatory mediators and the recruitment of immune cells. The ubiquitous presence of CD14 at mucosal surfaces and secretions (i.e., tears, cornea, breast milk, saliva, lungs, intestine, urine, sperm, and amniotic fluid) reflects its importance to the host in fighting Gram-negative infections in these constantly challenged mucosal environments.^2,3

Given its widespread mucosal distribution, numerous medical applications have been suggested for CD14, such as reducing the severity of LPS-induced septic shock and preventing Gram-negative infections at mucosal surfaces.⁴ Despite several attempts in different expression systems, recombinant human CD14 is still not mass produced for clinical evaluation due to obstacles concerning expression levels, production cost, as well as protein stability and activity (reviewed in reference 5).

Plant-produced rhCD14 could prevent ocular infections
By targeting the expression of the human CD14 coding sequence in the endosperm of tobacco seeds under the control of glutelin promoters, we were able to obtain significant levels (16 µg rhCD14/g of seeds) of recombinant human CD14 (rhCD14) proteins.⁵ Plant-produced rhCD14 was efficiently stored in a stable and biologically active form in tobacco seeds. When exposed to LPS from Pseudomonas aeruginosa, an ocular Gram-negative pathogen, plant-produced rhCD14 was able to induce an innate immune response of corneal epithelial cells similarly to CD14 naturally found in human tears.^3,5 These findings offer some promising applications for plant-made rhCD14 in preventing bacterial keratitis among contact lens users, and for use during ocular interventions, such as laser eye surgery or suppressing implant infections after orbital surgery.⁶ Such applications could include the addition of rhCD14 to contact lens solutions or artificial teardrops to mimic the immune advantages of human tears in reducing the risk of developing ocular Gram-negative infections.

Plant-made rhCD14 for the fortification of infant milk formulas
Because the tobacco endosperm glycosylation of rhCD14 might affect its stability and half-life, digestibility experiments were performed with proteolytic enzymes to determine if plant-made rhCD14 had an increased susceptibility to digestion. In vitro pepsin and pancreatin digestion, to mimic the human newborn gastrointestinal tract, revealed that plant rhCD14 has a proteolytic resistance similar to that of CD14 present in human breast milk.^2,5 The slight variation between plant rhCD14 and breast milk CD14 proteins might be attributed to the environmental milieu of the protein, such as the presence of protease inhibitors in breast milk. With its similar digestive susceptibility, rhCD14 produced in a food crop (or in a non-food plant for confinement issues) could one day be added to commercial infant milk formulas to mimic the immune advantage of breast milk for the neonate and to creams for cracked nipples to prevent mammary infections during lactation.

This latest potential application of ‘humanizing' infant milk formula with plant-produced breast milk proteins, such as rhCD14, might help reduce mother-to-child transmission of HIV in resource-constrained countries.⁷ No safe newborn feeding methods are currently available for HIV-infected mothers in these countries since breast milk is a vehicle of HIV transmission and infant formula is a source of gastrointestinal illnesses due to the lack of breast milk protective immune factors and unsanitary conditions during formula preparation. There is therefore an urgent need to ensure that breast milk substitutes embody optimal health benefits. To do so, transgenic food crops, such as rice (Fig. 1), could cheaply and safely reconstitute the beneficial and protective breast milk proteome in infant formulas to replicate the immune protection of breast milk against gastrointestinal illnesses while keeping the HIV virus at bay.⁷ At least seven breast milk proteins with immune properties have already been produced in transgenic food crops.⁷ With our current knowledge of the breast milk proteome and its fate in the newborn gastrointestinal tract, along with the maturing plant biotechnology sector, production of dozens more immune proteins in transgenic food crops is deemed feasible to supplement such fortified formulas with pharma flour.

Future perspectives
Plant-made rhCD14 was efficiently stored in a stable and biologically active form in transgenic tobacco seeds. Transgenic rice, expressing the codon-optimized version of the hCD14 coding sequence, along with stronger promoters and signal sequences, such as the KDEL retention signal, are currently being developed in our lab to further increase the yields of the recombinant protein and allow direct oral delivery (Fig. 1). This expression system constitutes a promising source of rhCD14 to continue identifying the roles of this LPS receptor protein in innate immune responses at mucosal surfaces in addition to the aforementioned potential preventive and therapeutic medical applications.

SUPER-SIZING CASSAVA
Uzo Ihemere and Richard T. Sayre

To date, transgenic approaches to biofortify or enhance the yields of crops grown primarily by subsistence farmers have been rather limited. This is particularly true for the starchy root crop cassava (Manihot esculenta Crantz). Cassava is the primary source of calories for approximately 600 million people in the tropics and ranks fourth in caloric intake among all crops directly consumed by humans.¹ Cassava is valued in many parts of the world for the food security it provides. It tolerates low soil fertility and drought and is resistant to many herbivores due to the presence of cyanogens.² In addition, the roots of cassava can be left in the ground for several years prior to harvest, providing food security against famine.¹

Unlike many of the world's major crop plants, cassava is not particularly amenable to genetic improvement through sexual crosses. Many cassava cultivars rarely flower and seed production is often low. In the field, cassava is clonally propagated by stem cuttings. This propagation method is ideal, however, for molecular approaches to cassava improvement, since each plant is clonally propagated and gene segregation or transfer through out-crossing is limited.

In 1996, the first stable genetic transformation of cassava was reported using Agrobacterium and/or microparticle-mediated delivery of DNA to plants.^3,4 Additional reports of the genetic transformation of cassava have followed; however, only recently have transgenic plants been generated with enhanced agronomic traits.⁵ In the last two years there have been several reports of genetically modified cassava with potentially enhanced agronomic traits. In 2003, Siritunga and Sayre introduced an anti-sense CYP79D1 and CYP79D2 construct into cassava to suppress expression of the cytochrome P450s that catalyze the first-dedicated step in cyanogenic glycoside synthesis.⁶ Transgenic plants having less than 1% of the normal root cyanogen levels were generated; however, these plants were unable to grow without supplemental reduced nitrogen. In 2003, Zhang et al. reported the expression of an artificial storage protein (ASP1) gene in cassava leaves and roots; however, its expression had little effect on the overall amino acid composition of leaf proteins.⁵ More recently, Siritunga et al. (2004) reported the over-expression of hydroxynitrile lyase in roots, leading to accelerated cyanogen removal and food detoxification.⁶ Cassava plants having altered amylose and amylopectin starch ratios have also been generated by RNAi silencing of the gene encoding granular bound starch synthase.

Biomass Production in Cassava
Several attributes of cassava's carbohydrate metabolism suggest that it has unrealized potential for enhanced starch production. Cassava has an unusually high rate of photosynthetic carbon assimilation (43 µmol CO₂/m²/s).⁷ In addition, cassava has one of the highest known rates of carbon assimilation into sucrose.⁷ It was our hypothesis that starch production in cassava roots could be substantially increased by increasing the sink strength for carbohydrate. To test this hypothesis, we generated transgenic plants with enhanced root ADP-glucose pyrophosphorylase (AGPase) activity. AGPase plays a critical role in the regulation of starch synthesis by catalyzing the first-dedicated and rate-limiting step in starch synthesis and thus playing a key role in regulating carbon flux towards starch synthesis. Plant AGPases are hetero-tetrameric enzyme complexes made up of two distinct polypeptides, both of which are catalytically active.⁸ Genetic manipulation of the plant AGPases is therefore challenging since modification of the expression or activity of the plant AGPases potentially involves manipulation of both AGPase encoding genes in transgenic plants. In contrast, bacterial AGPases are encoded by a single gene and the enzyme is catalytically more active than the plant enzyme.⁸

Transgenic Cassava with Enhanced Starch Production
To determine whether we could increase root starch production, we expressed a modified form of the bacterial gene (glgC) encoding AGPase under the control of the root-specific (in cassava) patatin promoter. This modified form of the enzyme (G336D mutant) lacks allosteric regulatory control and therefore would potentially be unaffected by the pool size of regulatory (negative) effectors. Previously, a bacterial AGPase had been expressed in transgenic plants but with mixed outcomes.

Expression of the G336D mutant form (glgC16) of the bacterial AGPase in potato tubers resulted in a 36% increase in AGPase activity and a corresponding 35% increase in tuber starch content.⁹ This increase in tuber starch production occurred only when the glgC16 gene was expressed in the tuber (driven by the patatin promoter). In contrast, expression of the glgC16 gene in all tissues led to weakly performing plants, presumably due to improper allocation of carbohydrate between phototrophic and heterotrophic tissues.⁹ Sweetlove et al., (1996) reported even higher AGPase activities (200%-400%) in transgenic potatoes expressing the glgC16 gene (under the control of the patatin promoter); however, they observed no increase in starch yield.¹⁰ They attributed this result to higher rates of starch turnover in the transgenic tubers. These results suggest that starch steady-state levels in transgenic tubers may be influenced by the sink-source demands of other (non-tuber) tissues as well.

To facilitate maximal AGPase activity in cassava roots, we modified the E. coli glgC gene encoding AGPase by site-directed mutagenesis (G336D) to eliminate negative regulation by fructose bisphosphate.¹¹ Plants expressing the modified glgC gene had between 0 – 70% higher AGPase activity than control plants when the enzyme was assayed under conditions optimal for the plant (25^oC) and not the bacterial (37^oC) form of the enzyme. Plants having the highest AGPase activities had a 260% increase in total root starch biomass when grown under greenhouse conditions. This increase in starch production was associated with an increase both in root number and root size but not an increase in root starch density. Increases in root biomass in transgenic plants were shown to be correlated with the relative increase in root AGPase activity. Interestingly, plants with the highest root AGPase activity also had significant increases (two-fold) in above ground (leaves and stems) biomass.

We propose that the enhanced top biomass production may represent a release of feedback inhibition on photosynthesis (dry matter accumulation) similar to that observed when expressing the glgC gene in potato tubers.⁹ These results demonstrated that targeted modification of enzymes regulating source-sink relationships in a crop plant having a high carbohydrate source strength is an effective strategy for increasing carbohydrate yields in sink tissues. Experiments are in progress to determine whether the cassava plants expressing the glgC16 gene will perform as well in the field as they did in the greenhouse.

EFFECTS OF TRANSGENIC COTTON ON BIODIVERSITY, PESTICIDE USE AND YIELD
P. S. Janaki Krishna

Biodiversity is threatened by agriculture in general and especially traditional methods of agriculture. There is additional concern that transgenic crops may affect biodiversity via unintended impacts on nontarget populations of arthropods. On the other hand, GE crops may positively impact agricultural species biodiversity if they enable the targeted management of weeds and insect pests, compared to conventional agriculture with widespread use of broad spectrum herbicides and pesticides.

Researchers at the University of Arizona (USA) and McGill University (Canada) conducted a two-year farm scale study to examine whether transgenic Bacillus thuringiensis (Bt) crops could increase agricultural biodiversity while minimizing the environmental impacts of agriculture.¹ They chose 81 commercial fields within a region of 6,600 km² in Arizona in which Bt cotton represented 48% and 62% of the cotton planted in the 1^st and 2^nd year of the study, respectively. Forty fields were planted to non-transgenic cotton (nonTr), 21 fields to cotton containing the Bt Cry1Ac transgene, and 20 fields to cotton containing both Bt and herbicide resistance (BtHr) transgenes.

The average number of insecticide applications applied to nonTr cotton was significantly higher than for Bt and BtHr cotton fields the first year; the difference was smaller but still significant in the second year. Use of insect growth regulators (IGRs), which are less harmful to nontarget arthropods than broad spectrum insecticides and are used to control sweet potato whitefly (Bemisia tabaci) in cotton, did not differ significantly between transgenic and nonTr cotton fields. The average number of herbicide applications, with or without glyphosate, did not differ significantly among nonTr, Bt and BtHr cotton.

The second component of the study assessed the effects of transgenic cotton on yield. Bt and BtHr cotton did not show significant yield differences, though more lint was produced in transgenic cotton than nonTR cotton. The yield gain attributed to insecticide applications was 4.2 times higher than yield gain caused by use of transgenic cotton. Yield and the number of broad spectrum insecticide applications were directly proportional, and no significant interaction was observed between cotton type and insecticide use.

The positive effect of insecticides on yield shows their importance for control of major cotton pests, B. tabaci, Lygus hesperus, and P. gossypiella. However, Bt cotton does not kill B. tabaci and Lygus hesperus, which likely contributed to a higher insecticidal use noted the second year, and indicates an increasing requirement for control of those pests. NonTr, Bt, and BtHr cotton did not show overall yield difference among them, though Bt cotton had a higher yield than nonBt for a given number of insecticide applications.

The third study goal was to examine the effects of transgenic cotton on biodiversity. The impact of nonTr and transgenic cotton cultivation on nontarget arthropods was assessed using pair-wise comparisons of ant and beetle populations in each cotton field with a directly adjacent field of noncultivated vegetation. Ant density and diversity richness were significantly less in nonTr, Bt, and BtHr cotton fields compared to noncultivated vegetation, with no significant differences among cotton types. However, the opposite was true for beetle population density and species richness, which were greater in cotton fields than in noncultivated vegetation. As with ants, the average density increase did not differ among nonTr, Bt and BtHr cotton types, indicating that cultivation of transgenic and nonTr cotton had similar effects on diversity of these species.

The authors assessed whether negative affects on ant and beetle diversity could have been masked in transgenic cotton. They used path analyses to look for differential effects on ant and beetle diversity from insecticides, IGR, and transgenic cotton. BtHr cotton had a reduced ant density compared to Bt and nonTr cottons, in which ant densities were similar. However, broad-spectrum insecticide applications significantly reduced both ant and beetle species diversity. IGRs increased beetle density but decreased diversity, resulting in overall negative impact on diversity.

The authors concluded that growing Bt cotton in large commercial fields reduced broad-spectrum insecticide use and increased yields at fixed insecticide levels, resulting in larger benefits for producers, as the yield benefits of transgenic cotton exceeded the additional cost of transgenic seeds. However, because Bt cannot control all important cotton pests, the ultimate impact of Bt crops may largely depend on whether additional insecticides are required, a fact which should be considered before opting for Bt crops.

The ant and beetle diversity studies indicate that invertebrate taxa may react differently to agricultural practices; however, the authors did not find that transgenic cotton had a greater impact on arthropod diversity than nonTr cotton. IGR, broad-spectrum insecticides, and other agronomic and ecological factors may also significantly affect ant and beetle diversity. Additional studies are required to understand the negative impact of BtHr cotton on ant density.

The authors concluded that Bt crops could be useful in reducing environmental impacts of agricultural intensification, primarily in situations in which the replacement of insecticides by Bt crops does not reduce the control of pests unaffected by Bt protein.

1. Cattaneo MG et al. (2006) Farm Scale evaluation of the impacts of transgenic cotton on biodiversity, pesticide use and yield. PNAS 103 (20), 7571-7576

Biotechnology is among key forces reshaping world agriculture, according to a new report from North Dakota State University, authored by Jeremy Mattson and Won Koo of NDSU's Center for Agricultural Policy and Trade Studies.

The authors point out in their report, "Forces Reshaping World Agriculture," that growth of agriculture in the United States is dependent on productivity increases. Since there is little land available for expansion of agricultural production in the U.S., growth in production will require increased yields. Export competitiveness is also dependent on relative productivity growth against major competitors.

Future productivity growth will be influenced by current and future research, especially public research. "New developments that could lead to further productivity increases include improved technologies for nutrient, soil, water, and pest management; precision agriculture; and agricultural biotechnology," the report says. "The emergence of biotechnology could especially have a significant impact on productivity worldwide."

Farmers benefit from the use of GM crops through increased weed and insect control, which could lead to increased yields and decreased pesticide costs. Mattson and Koo report that despite some consumer concern, the biotechnology trend is likely to continue as it leads to productivity gains for farmers. "The introduction of GM wheat has been delayed, largely due to concern that consumers in export markets will not accept it, but it could eventually be adopted," they write.

While current biotech crops have been developed mainly to improve agricultural production, future biotech crops could be introduced that have qualities such as increased nutritional content or other characteristics that would benefit consumers. "Consumer response to the further adoption of biotech crops is uncertain, but it may become more favorable as these crops are developed with more obvious benefits for consumers." Developing countries could benefit the most from biotechnology through productivity gains and improved nutritional content of crops such as golden rice.

Mattson and Koo point out that while technological advances appear to initially benefit producers by leading to higher yields, lower costs, and increased productivity, consumers ultimately benefit from lower real food prices. According to ERS data, food expenditures by U.S. consumers as a share of disposable personal income has dropped steadily from 24.2 percent in 1930 to 10.1 percent in 2003.

ABIC 2006: AGRICULTURAL BIOTECHNOLOGY INTERNATIONAL CONFERENCE:
Unlocking the Potential of Agricultural Biotechnology
6 - 9 August 2006
Melbourne, Australia

Organized by the Agricultural Biotechnology International Conference (ABIC) in association with AusBiotech, ABIC 2006 aims to bring together leading international researchers in the AgBio sector with industry partners and investors. With the theme "Unlocking the potential of agricultural biotechnology," ABIC will organize speaker sessions that allow leading international experts to exchange ideas and nurture innovation; provide informative and educational speaker sessions that highlight the benefits of agricultural biotechnology to the non-scientist; hold forums that address key policy and risk management issues, such as commercial trials and regulatory approval for GM products; provide an opportunity for agricultural biotechnology companies and research organizations to meet with industry partners; and bring major investors in the agricultural biotechnology sector together with companies and research organizations seeking funding to develop their innovations.