The development of methodologies for creating transgenic animals, plants, and microbes has been invaluable for studying biological processes ranging from basic gene expression to expression of important pharmaceutical proteins. Once a potential transgenic organism is generated, one of the first steps is to verify the presence of the transgene using Southern blots or the polymerase chain reaction (PCR). Both of these methods can reveal the presence of the transgene but cannot provide information such as chromosomal location. Chromosomal localization in higher eukaryotes is typically performed using fluorescent in situ hybridization (FISH) on chromosomal spreads.

One of the potential problems with Southern blot or FISH is cross hybridization of the transgene probe to the endogenous gene when there is high sequence identity between the two genes. If this is the case, then an alternative method for detection and chromosomal localization of the transgene would be needed. Recently, researchers from Louisiana State University have developed a method for chromosomal localization of a transgene using a combination of laser pressure catapulting and PCR.

Laser microdissection has been used to isolate single cells or excise a specific group of cells from a tissue section. The laser microdissection and pressure catapulting system uses a UV-laser to ablate the area surrounding the desired sample and an additional defocused laser pulse to catapult the dissected sample directly into the cap of a sample tube. Thus samples can be readily collected without mechanical contact and without contamination from surrounding tissue.

In this study transgenic quail were generated using a transposon-based vector containing a human proinsulin gene. This vector DNA was complexed with a transfection reagent and injected into the testes of male Japanese quail. Positive males from the G₀ generation were mated with wild type females, and the G₁ offspring were tested for the presence of the transgene. Positive G₁ birds were then mated to produce G₂ birds for DNA and chromosome analysis.

Chromosome spreads were prepared from potentially transgenic G₂ quail, and individual chromosomes were laser microdissected and catapulted into separate tubes. The presence of the transgene in individual chromosomes was detected by PCR. Using this approach, one G2 quail was found to contain five chromosomes containing a transgene insertion. The transgenes were present in both the microchromosomes and the macrochromosomes.

These results demonstrate that the use of laser microdissection and catapulting in conjunction with PCR is an alternative method for detecting the presence and determining the chromosomal localization of a transgene. This method is generally applicable to transgenic mammalian, avian, and plant systems and would be particularly useful for transgenes with high sequence identity to an endogenous gene. However, given the cost of a laser microdissection system and the expertise required to collect individual chromosomes, it seems unlikely that this method would be routinely used for transgene detection or chromosomal localization in the transgenesis field. Nevertheless, this method does provide an alternative approach that would be useful for specific applications.

McNally LR, Henk WG, and Cooper RK. 2006. Chromosomal localization of a proinsulin transgene in Japanese quail by laser pressure catapulting. Transgenic Res. 15, 427-433

A major challenge in the post genome era of plant biology is to determine the functions of all the genes in a plant genome. At the level of functional gene analysis and the isolation of agronomically important genes, wheat (Triticum aestivum) is clearly lagging behind compared to other major food crops such as maize, rice, and also species such as tomato. This is mainly due to the lack of efficient tools to study gene function in polyploid species. Hexaploid wheat has a large genome (16,000 Mbp), which consists of three closely related homoeologous genomes (A, B and D) and has a high content of repetitive DNA (80%). Genes are organized in gene islands or as single genes separated by large regions of nested repetitive elements.¹ Due to the hexaploid nature of its genome, bread wheat has three (or a multiple of three) copies of most genes. Many of these homoeologous genes are expressed,² and there is, therefore, a high degree of functional gene redundancy in hexaploid wheat. Insertional mutagenesis and gene silencing are efficient tools for the determination of gene function. In contrast to gain- or loss-of-function approaches, RNA interference (RNAi)-induced gene silencing can possibly silence multigene families and homoeologous genes in polyploids by targeting sequences that are unique or shared by several genes.

To investigate the potential of double-stranded RNAi in wheat, we introduced into hexaploid wheat by particle bombardment dsRNA-expressing constructs of two genes that have not yet been cloned in wheat, but with previously defined functions in other plant species and with unambiguous phenotypes in corresponding mutant plants. The first gene was Phytoene Desaturase (PDS), which is often used to evaluate VIGS (Virus-Induced Gene Silencing) efficiency because of its distinct phenotype.³ PDS is an enzyme required for synthesizing carotenoids, compounds that protect chlorophyll from photobleaching. The second candidate gene was Ethylene Insensitive 2 (EIN2), which is a central component of ethylene signaling.⁴ It has been identified in Arabidopsis thaliana where all twenty-five ein2 mutant alleles showed a clear phenotype of complete insensitivity to ethylene.⁴

Specific silencing of the PDS genes induces photo-bleaching in hexaploid wheat and interferes with mRNA accumulation of all three homoeologous genes
Seventy-eight percent of the transgenic lines recovered through the selection procedure exhibited photo-bleaching at the seedling stage, indicating specific silencing of endogenous PDS. The PDS-RNAi transgenic lines were arranged into a phenotypic series based on the severity of the photo-bleached phenotype. Most of the lines developed a strong photobleached phenotype, resulting in a lethal albino phenotype (Fig. 1A). Other lines showed an intermediate phenotype with continuous parallel streaks where photo-bleaching affected either one half of the leaves (Fig. 1B) or only the middle part of the leaf (Fig. 1C). The remaining lines produced a weak phenotype where only a small part of the leaves was affected by photo-bleaching (Fig. 1D). The severity of the photo-bleached phenotype inversely correlated with PDS mRNA expression levels in the leaves of all the transgenic RNAi lines analyzed (Fig. 2A). For real-time quantitative RT-PCR, primers were designed to specifically measure effective endogenous PDS mRNA levels of each of the homoeologous copies of the wPDS genes and not the transgene transcripts. The reduction of relative mRNA levels of wPDS was very similar in each of the three homoeologous genes (Fig. 2A). Therefore, the RNAi silencing mechanism affects all three copies of the gene in the same way.

Inheritance of the genetic interference of wPDS and detection of siRNA in silenced plants
All the T₁ progeny showed Mendelian segregation for the photo-bleached phenotype on the leaves. Surprisingly, the intermediate phenotype with continuous parallel streaks of some of the T₀ transgenic lines was never observed again in the following generations (Fig. 1, B-D). Twenty-five percent of the T₁ plants containing the hairpin transgene showed strong photo-bleaching of the leaves with large albino areas, which in a few cases eventually developed to completely albino plants (Fig. 2D).

The other T₁ plants which had integrated the transgene (~50%) showed a weak phenotype, where photo-bleaching was affecting only the base of the leaves (Fig. 2D). Quantitative real-time PCR revealed again a correlation between the level of mRNA and the severity of the photo-bleached phenotype (Fig. 2C-D). By selfing different T₁ plants showing strong/weak photo-bleaching, we could confirm that the T₁ plants that showed strong photo-bleaching were homozygous, and those T₁ plants with a weak photo-bleached phenotype were heterozygous. Small RNA gel blots were used to test whether the amounts of siRNA were different in homozygous and heterozygous lines. We detected sequence-specific small RNAs (siRNAs) of around 24 bp in both homozygous and heterozygous plants showing strong/weak photo-bleaching (Fig. 2E). Wild type plants did not accumulate small PDS RNAs. Thus, small PDS RNAs accumulated only upon silencing of the PDS genes. The relative intensity of the hybridization signals in the transgenics vs. wild type plants indicated that the homozygous plants contain around double the amount of small RNAs compared to heterozygous plants. It is therefore likely that the strong photo-bleached phenotype observed in the homozygous plants is in part related to a higher accumulation of siRNAs.

This genetic analysis indicates that RNA interference of wPDS is stably inherited over at least two generations in a Mendelian fashion of a single locus. Small RNAs specific for the silenced gene were detected and their accumulation was quantitatively different in homozygous and heterozygous lines. These results suggest that the effect of RNA interference in hexaploid wheat is probably gene-dosage dependent.

EIN2 dsRNA-mediated genetic interference in hexaploid wheat and ethylene response signaling in wheat EIN2-RNAi lines
We identified by quantitative real-time PCR six T₀ primary transgenic lines with a significant reduction of mRNA expression of the endogenous wEIN2 genes (Fig. 3A). One line (line 18) had a strong reduction of the wEIN2 mRNA level, which was only 1% of the level detected in the wild type plant. The six T₀ primary transgenic lines with lower EIN2 expression showed a normal phenotype and their ethylene response signaling was studied in the next generations.

To examine whether the ethylene response was altered in the lines with lower EIN2 expression, T₁ seeds of these lines and wild type plants were germinated on MS medium in presence of ACC (1-aminocyclopropane-1-carboxylic acid), the immediate precursor of ethylene. The progenies showed Mendelian segregation for normal and stunted growth when grown in presence of ACC, whereas all wild type plants showed a stunted morphology in presence of ACC (Fig. 3, B-C). As observed with the wPDS genes, 25% of the T₁ plants showed complete insensitivity to ethylene and a normal growth in presence of ACC. These T₁ plants also showed the strongest mRNA reduction (around 70%) compared to the wild type (Fig. 3D). Around 50% of the other T₁ plants showed a growth only partially affected by the presence of ACC, and they accumulated mRNA levels around 50% of wild type, whereas all the other T₁ plants that were sensitive to ethylene and showed a stunted morphology had the same mRNA levels as the wild type (Fig. 3D). There was a very high negative linear correlation (r² = -0.976) between the length of the seedlings in the presence of ACC and the amount of wEIN2 transcript measured in the T₁ progenies. As observed with the wPDS genes, EIN2 mRNA levels declined with increasingly severe phenotypes.

These results demonstrate that the EIN2-RNAi transgenic wheat lines produced in this study are ethylene insensitive. This is consistent with the hypothesis that EIN2 is a positive signal component in ethylene signaling and that inhibiting its expression reduces the ethylene response.

Conclusions
We have demonstrated that RNAi-mediated gene silencing is effective in hexaploid wheat and can efficiently induce reduction of mRNA levels of three homoeologous genes. Expression of the three homoeologous genes was reduced to the same extent, suggesting that RNAi can resolve the issue of genetic redundancy in hexaploid wheat in an efficient way. Knowledge of the complete and exact sequence of the target genes was not essential to induce specific gene silencing, as sequence information from ESTs was sufficient. This is important for the development of high-throughput methods for functional genomics. We have shown that RNAi-induced silencing in polyploid wheat was stably inherited over at least two generations, making this approach a reliable tool not only for functional genomics but also for the genetic modification of agronomically interesting traits.

We found a strong correlation between decreased levels of mRNA and increased severity of phenotypes with homozygous plants showing the strongest mRNA reduction and the most severe phenotype. Small RNAs specific for the silenced gene were detected and their accumulation was quantitatively different in homozygous and heterozygous lines. In homozygous plants, accumulation of siRNAs was significantly higher, to produce effective gene silencing and to develop the most severe phenotype. These results suggest that the effect of RNA interference in hexaploid wheat is probably gene-dosage dependent. A phenotypic series was obtained from the RNAi lines with a full spectrum of the effect of RNAi (weak, intermediate, and strong) on gene expression, which is in agreement with the results in Arabidopsis^5,6 and tomato.⁷

The variation in the degree of silencing observed in the transformants showing both reduction and loss-of-function may be a useful feature for gene discovery and functional genomics. Complete silencing of genes encoding a key element in basic cell functions or at particular developmental stages may result in lethality, whereas the reduced gene expression may give viable plants with phenotypes indicative of the role of the target gene. Wheat seedlings with low mRNA levels for EIN2 were completely ethylene-insensitive. Thus, EIN2 is a positive regulator of the ethylene-signalling pathway in wheat, very similar to its homologs in Arabidopsis⁴ and rice.⁸

RNAi silencing has enormous potential as a tool in functional genomics of hexaploid wheat, a species for which other methods such as insertional mutagenesis are not available. However, gene transfer technology is still limited in wheat by the low frequency of generation of transgenic plants. Thus, fast and efficient systems of transformation and regeneration of transgenic plants are necessary to successfully use RNAi constructs for high-throughput wheat functional genomics.

References
1. Feuillet C, Keller B (2002) Comparative genomics in the grass family: molecular characterization of grass genome structure and evolution. Ann. Bot. 89, 3-10

2. Mochida K, Yamazaki Y, Ogihara Y (2003) Discrimination of homoeologous gene expression in hexaploid wheat by SNP analysis of contigs grouped from a large number of expressed sequence tags. Mol. Gen. Genomics 270, 371-377

3. Holzberg S, Brosio P, Gross C, Pogue GP (2002) Barley stripe mosaic virus-induced gene silencing in a monocot plant. Plant J. 30, 315-327

4. Alonso JM, Hirayama T, Roman G, Nourizadeh S, Ecker JR (1999) EIN2, a bifunctional transducer of ethylene and stress responses in Arabidopsis. Science 284, 2148-2152

5. Chuang CF, Meyerowitz EM (2000) Specific and heritable genetic interference by double-stranded RNA in Arabidopsis thaliana. Proc. Natl. Acad. Sci. USA 97, 4985-4990

6. Wang T, Iyer LM, Pancholy R, Shi X, Hall TC (2005) Assessment of penetrance and expressivity of RNAi-mediated silencing of the Arabidopsis phytoene desaturase gene. New Phytologist 167, 751-760

7. Xiong AS, Yao QH, Peng RH, Li,X, Han PL, Fan HQ (2005) Different effects on ACC oxidase gene silencing triggered by RNA interference in transgenic tomato. Plant Cell Rep. 23, 639-646

8. Jun SH, Han MJ, Lee S, Seo YS, Kim WT, An G (2004) OsEIN2 is a positive component in ethylene signaling in rice. Plant Cell Physiol. 45, 281-289

In 1996, commercialization of first generation transgenic cotton that expresses Bacillus thuringiensis (Bt) toxins was approved by the US EPA. This Bt cotton was effective against many lepidopteran pests, including Heliothis virescens (Fabricius), Helicoverpa zea (Boddie) and Pectinophora gossypiella (Saunders). Since that time the acreage of cultivated Bt cotton has increased rapidly around the world. However, growing Bt cotton successively raises the potential for resistance adaptation of target insects to the Bt toxins.¹ Although no Bt crop-resistant lepidopteran pest populations have yet been documented under field conditions, some authors have reported the failing or decreased effectiveness of Bt cotton against some insect pests.^2,3,4 In addition, several species have developed resistance to Bt toxins in laboratory selection experiments.⁵ In 2002, transgenic cotton that simultaneously expresses two Bt toxins, Cry1Ac and Cry2Ab, was approved for commercial use. Although this line is more effective than single toxin Bt cotton in protecting cotton from damage by lepidopteran pests, adaptation by the target pests is still a concern.⁶

Seed mixture strategy has been proposed to delay the evolution of adaptation of insects to Bt toxins.⁷ However, Mallet and Porter⁸ warned that seed mixtures of toxic and toxin-free plants within fields would hasten the development of resistance if insect movement was independent of the toxin inside plants. Tabashnik⁹ later countered that seed mixtures could delay resistance of insects to Bt toxins regardless of whether insect movement is independent of the toxin inside the plant. However, based on the field experiments with diamondback moth, Plutella xylostella (L.), Shelton et al.¹⁰ did not endorse seed mixtures as a strategy to delay evolution of resistance to Bt toxins. Ramachandran et al.^11,12 reported that movement of P. xylostella between conventional and transgenic canola plants is independent of toxin. Furthermore, P. xylostella larvae have been shown to move to toxic plants from toxin-free plants if the population density on toxin-free plants was greater than that on toxic plants.¹³ So movement of insects between Bt plants and non-Bt plants seems to be an important factor in adapting Bt toxins of insect pests.

Cabbage looper, Trichoplusia ni, is one of the most serious pests of cruciferous vegetables and a secondary pest of cotton. It has great potential to develop resistance to Bt toxins.¹⁴ In southern Texas, cotton is planted from February to March, and grows in the field until late August. Cabbage can be planted side by side with cotton, and the two crops share many common insect pests. It is a common phenomenon that T. ni migrates from cabbage to cotton fields and vice versa.

We conducted a series of laboratory experiments to investigate the behavior response of cabbage looper larvae, originating from cabbage, to a mixture of Bollgard II (expressing Cry1Ac and Cry2Ab) and non-Bt cotton leaves.¹⁵ We also determined the effects of the mixture of Bollgard II and non-Bt cotton leaves on development of cabbage looper larvae. Our results indicate that young cabbage looper larvae were able to detect Bt cotton leaves and non-Bt cotton leaves. When the larvae were exposed to non-Bt leaves, all larvae moved to the inner part of the cotton leaves and fed there. In contrast, most larvae moved off or on the edge of the leaves (attempted escaping) when exposed to Bollgard II leaves only. When exposed to a mixture of Bollgard II and non-Bt leaves, most larvae moved to and located on the non-Bt leaves. These results indicate that young cabbage looper larvae distinguished and avoided or attempted to avoid Bt cotton leaves. The movement of T. ni larvae between Bt and non-Bt leaves was generally unidirectional, i.e., from Bt cotton leaves to non-Bt cotton leaves, and not vice versa.

Some larvae did move to Bollgard II leaves, but fed only a little (1.8 mm² per larva compared with 22.6 mm² per larva in the control). The results indicate that the larvae moved to the Bt leaves and initiated feeding, but could not subsequently sustain feeding, and moved either to non-Bt cotton leaves or off the cotton leaves. These results were further supported by the fact that the pupae that developed from larvae that were exposed to a mixture of Bollgard II and non-Bt cotton leaves were significantly smaller than those developed from larvae that were exposed only to non-Bt cotton leaves.

The difference in total development times between male and female cabbage looper larvae and pupae fed on non-Bt cotton leaves compared to a mixture of non-Bt and Bt cotton leaves was less than 2 d, which would probably not be long enough to cause assortative mating between two populations. This result also implies that development of cabbage looper larvae is not affected as much as other lepidopteran species. Liu et al.¹⁶ found that a resistant strain of larvae of the pink bollworm, Pectinophora gossypiella, grown on Bt cotton takes 5.7 d longer to develop than susceptible larvae on non-Bt cotton. They suggested that the developmental asynchrony between the initially rare homozygous resistant adults and the more abundant homozygous susceptible adults emerging from non-Bt plants favors non-random mating and could reduce the expected benefits of the refuge strategy.

Under field conditions, lepidopteran larvae are capable of migrating from one plant to another, especially when plants are large and are touching each other; hence, non-Bt plants mixed with Bt plants may not be easily protected. In addition, if larvae that feed on Bt plants can move to non-Bt plants to complete their development, the risk of evolving resistance is increased. Another concern is that in some localities such as southern Texas, Bt insecticides are widely used on vegetables and many other crops to protect from cabbage looper damage, and Bt-resistant cabbage looper has been detected.¹⁶ Therefore, under field conditions in southern Texas it is possible that the cabbage looper could develop resistance to both Bt transgenic cotton and the Bt insecticides used on vegetable crops.

The merit of Bt and non-Bt seed mixtures at planting as a resistance management strategy needs further evaluation as part of ongoing efforts to evaluate the effects of the Bt toxins in cotton on cabbage looper and other pests.

Sources
1. Bates S L et al. (2005) Insect resistance management in GM crops: past, present and future. Nature Biotechnol. 23, 57-62

2. Adamczyk JJ Jr et al. (1998) Larval survival and development of the fall armyworm (Lepidoptera: Noctuidae) on normal and transgenic cotton expressing the Bacillus thuringiensis Cry1A(c) –endotoxin. J. Econ. Entomol. 91, 539-545

3. Burd T et al. (1999) Performance of selected Bt cotton genotypes against bollworm in North Carolina. 1999 Beltwide Cotton Conference, Orlando, USA, 3-7 January, 1999: Volume 1. National Cotton Council, Memphis, USA. 931-934

4. Allen C T et al. (2000) Effectiveness of Bollgard II cotton varieties against foliage and fruit feeding caterpillars in Arkansas. (2000) Proceedings Beltwide Cotton Conferences, San Antonio, USA, 4-8 January, 2000: Volume 2. National Cotton Council, Memphis, TN, USA. 1093-1094

5. Tabashnik B E et al. (2003) Insect resistance to transgenic Bt crops: Lessons from the laboratory and field. J. Econ. Entomol. 96: 1031-1038

6. Gould F (2003) Bt-resistance management—theory meets data. Nature Biotechnol. 21, 1450 – 1451

7. Gould F & Anderson A. (1991) Effects of Bacillus thuringiensis and HD-73 delta-endotoxin on growth, behavior, and fitness of susceptible and toxin-adapted strains of Heliothis virescens (Lepidoptera: Noctuidae). Environ. Entomol. 20, 30-38

8. Mallet J & Porter P. (1992) Preventing insect adaptation to insect-resistant crops: are seed mixtures or refugia the best strategy? Proc. R. Soc. Lond. B. 250, 165-169

9. Tabashnik B. (1994) Delaying insect adaptation to transgenic plants: seed mixtures and refugia reconsidered. Proc. R. Soc. L. B. 255, 7-12

10. Shelton A M et al.(2000) Field tests on managing resistance to Bt-engineered plants. Nature Biotechnol. 18, 339-342

11. Ramachandran S et al. (1998a) Movement and survival of diamondback moth (Lepidoptera: Plutellidae) larvae in mixtures of nontransgenic and transgenic canola containing a cryIA(c) gene of Bacillus thuringiensis. Environ. Entomol. 27, 649-656

12. Ramachandran S et al. (1998b) Survival, development, and oviposition of resistant diamondback moth (Lepidoptera: Plutellidae) on transgenic canola producing a Bacillus thuringiensis toxin. J. Econ. Entomol. 91, 1239-1244

13. Kumar H. (2004) Orientation, feeding, and ovipositional behavior of diamondback moth, Plutella xylostella (Lepidoptera: Plutellidae), on transgenic cabbage expressing Cry1Ab toxin of Bacillus thuringiensis (Berliner). Environ. Entomol. 33, 1025-1031

14. Janmaat A F & Myers J. (2003) Rapid evolution and the cost of resistance to Bacillus thuringiensis in greenhouse populations of cabbage looper, Trichoplusia ni. Proc. R. Soc. Lond. B. 270, 2263-2270

15. Li YX, Greenberg SM, Liu TX. (2006) Effects of Bt cotton expressing Cry1ac and Cry2ab and non-Bt cotton on behavior, survival and development of Trichoplusia ni (Lepidoptera: Noctuidae). Crop Protection Journal 25, 940-948

16. Liu Y B et al. (1999) Development time and resistance to Bt crops. Nature 400, 519

Roots of the Litigation
The case has its origins in a petition filed by the GE Food Alert coalition in December 2002. The coalition called for an immediate moratorium on the planting of food crops engineered to synthesize pharmaceuticals and industrial chemicals. This freeze, the petitioners stated, would provide the USDA with the chance to perform a programmatic environmental impact statement to assess the effect of the USDA’s Animal and Plant Health Inspection Service (APHIS) biopharming regulations. The petitioners also wanted the USDA and APHIS to loosen rules governing public access to confidential business information about biopharm tests.

In March 2003, APHIS requested public comments on its permitting process for the field testing of biopharm plants. A month later, the agency sent the GE Food Alert coalition a response to its petition, which the association characterized as a mere dismissal of its concerns.

In November 2003, the Center for Food Safety, the Hawaiian-Environmental Alliance (KAHEA), Friends of the Earth, and Pesticide Action Network North America filed a complaint with the Hawaii district court, which they amended twice over the next year and a half. The final complaint, filed in August 2005, alleged that APHIS had violated the Administrative Procedure Act when it failed to respond to the petition. The complaint also presented broad allegations that APHIS had violated the National Environmental Policy Act (NEPA) and the Endangered Species Act (ESA) in implementing its program to regulate the testing of biopharm crops.

The Plaintiffs further alleged that APHIS had violated NEPA and the ESA by issuing four permits for testing biopharm crops in Hawaii. From 2001 to 2003, APHIS granted permits to ProdiGene, Monsanto, the Hawaii Agriculture Research Center, and Garst Seed for performing limited field tests of corn and sugarcane engineered to produce hormones, vaccines, and other therapeutic proteins. The tests, completed by the trial date, took place on Kauai, Maui, Molokai and Oahu.

APHIS Wins One, Loses Eight
Judge J. Michael Seabright heard arguments for the case in July 2006. Here, the Plaintiffs contended that APHIS’ denial of the December 2002 petition had been arbitrary and capricious. The judge did not agree. He pointed out that APHIS had informed the Plaintiffs that a decision on whether to promulgate new biopharming regulations depended on the result of an ongoing programmatic environmental impact statement. The petition’s demands for new biopharming regulations had been premature, Seabright decided, and judicial intervention would inappropriately interfere with APHIS’s administrative proceedings. The judge also concluded that a network of laws prevented APHIS from acceding to demands for enabling the public to access confidential business information. The judge granted summary judgment for APHIS on the claim arising from the petition.

The agency did not fare as well with eight allegations that APHIS had violated NEPA and the ESA when it granted each of the four biopharming permits. In his decision, Judge Seabright placed the asserted violations of environmental laws in the context of Hawaii’s ecology.

"The Fish and Wildlife Service reports on its website that there are 329 endangered and threatened plant and animal species in Hawaii," he wrote, "including thirty-two types of birds." Hawaii has more endangered and threatened species than any other state – harboring 25 percent of all listed species in the United States. "Although strict compliance with the ESA’s procedural requirements is always critically important," he observed, "these requirements are particularly crucial in Hawaii given Hawaii’s extensive number of threatened and endangered species."

To protect the environment, the ESA requires all federal agencies to obtain information from the US Fish and Wildlife Service or the National Marine Fisheries Service about any endangered and threatened species in the area of proposed agency action. APHIS failed to take this step. Nevertheless, the agency argued that it had complied with the ESA, because it had determined that its proposed actions would not affect listed species or critical habitats.

"APHIS’s argument misses the mark," the judge scolded. While a formal consultation about endangered species might not have been required, the problem is that APHIS skipped the mandatory step of obtaining information about listed species and critical habitats.

APHIS then turned to its fallback defense: the Plaintiffs’ claims must fail because the Plaintiffs had not provided any evidence to show that the biopharm crop tests had harmed a single listed species or habitat in any way. The judge characterized the argument as "absurd," because "APHIS argues that the Plaintiffs may not proceed with a lawsuit against the agency unless APHIS actually facilitates an organism’s extinction." The agency’s good fortune in avoiding such a disaster cannot absolve APHIS of its failure to follow a clear congressional mandate.

The court also concluded that APHIS had violated NEPA because its administrative record fails to indicate that, when APHIS had issued the four permits, the agency had considered the applicability of NEPA, the statute’s categorical exclusions, or the exceptions to those exclusions. After reviewing APHIS’ records, the judge found no evidence of an environmental assessment, an environmental impact statement, or an explanation as to why neither study had been required before granting the four permits. Seabright concluded that APHIS’ issuance of the four permits had been arbitrary and capricious, and he granted the Plaintiffs summary judgment on claims that APHIS had violated the ESA and NEPA when it granted the four permits.

Biopharming Moratorium?
Following Judge Seabright’s decision about the ESA and NEPA violations, environmental groups called for a moratorium on open-air tests of biopharm crops.

"We are asking the judge to enjoin the issuance of any biopharma permits anywhere in the country unless and until APHIS completes a programmatic analysis of their regulatory program," Paul H. Achitoff told The Washington Post. Achitoff, a lawyer with the Honolulu office of Earthjustice, served as lead attorney for the Plaintiffs.

On August 22, Judge Seabright held a hearing on this very matter: should the court impose a nationwide remedy? At the hearing, the Plaintiffs argued that APHIS had developed and implemented an organized, national biopharming program. Consequently, NEPA and the ESA required the agency to study the impact of this program on the environment and endangered species. APHIS’s failure to consider the cumulative effect of its national biopharming program, they argued, constitutes a violation of NEPA and the ESA.

APHIS argued against the existence of a "final agency action" for purposes of the NEPA claim and the existence of an "agency action" for purposes of the ESA claim. The court agreed.

In Judge Seabright’s view, the Plaintiffs had not pointed to any final agency action – just the issuance of the four permits. The judge could find no evidence that APHIS’ policies support an allegation that it has a biopharming program that is a final agency action sufficient for judicial review.

The judge noted that the ESA contains a broad citizen suit provision and that the Plaintiffs’ ESA claim is not limited by the "final agency action" requirement. Federal agencies must comply with the ESA’s procedural requirements when an agency proposes an "agency action." The judge could not see how APHIS’ method of regulating biopharm crops constitutes an agency action distinct from the act of issuing individual permits. The judge granted summary judgment for APHIS.

Earthjustice’s Achitoff told The Honolulu Star Bulletin that the days of rubber-stamping crop permits for genetically engineered crops have passed. He suggested that the USDA must now hold public hearings on each permit to comply with NEPA obligations.

Speaking for APHIS, Rachel Iadicicco reported that the agency had recently made policy changes to satisfy the court’s concerns. APHIS is also devising a sweeping programmatic environmental impact statement to address broader concerns about its oversight of genetically engineered crops.

Selected Sources

Ctr. for Food Safety v. Johanns, Civ. No. 03-00621 (D. Haw., Aug. 10, 2006).

Ctr. for Food Safety v. Johanns, Civ. No. 03-00621 (D. Haw., Aug. 31, 2006).

Finnegan, T (2006) Ruling hailed by opponents of genetically altered crops. The Honolulu Star Bulletin (August 15, 2006).

Weiss, R (2006) Gene-Altered Crops Denounced. The Washington Post, A03 (August 16, 2006).

SemBioSys Genetics Inc., a Canadian biotechnology company developing a broad pipeline of protein-based pharmaceuticals and non-pharmaceutical products, has announced it has achieved its commercial target levels of human insulin (insulin) accumulation in safflower with 1.2 percent of total seed protein.

Results from the company’s commercial plant system exceeded its target of one percent accumulation and confirm the potential of plant-produced insulin to fundamentally transform the economics and scale of insulin production.

"These results demonstrate that we have produced an authentic insulin molecule in safflower at commercially viable levels. Achieving our goal of one percent insulin accumulation in safflower confirms that SemBioSys has the potential to dramatically impact the economics of insulin manufacturing," said Andrew Baum, President and CEO of SemBioSys Genetics Inc. "At these levels we can produce over one kilogram of insulin per acre of safflower production, which is enough to supply 2,500 patients for one year of treatment. We believe that we could meet the world’s total projected insulin demand in 2010 with less than 16,000 acres of crop production. Our plan is to continue to scale-up production for sufficient material to initiate clinical trials and file an Investigational New Drug (IND) application in the second half of 2007."

SemBioSys intends to continue its preclinical program with safflower-derived insulin and assemble the components of its IND application, including toxicology, immunology profiles and demonstration of efficacy in animal models. The Company expects to be in a position to submit an IND to the US Food and Drug Administration in the second half of 2007 in preparation for a clinical trial in late 2007 or early 2008.

SemBioSys says that the combination of inhaled insulin, the increasing incidence of diabetes due to dietary trends, and the earlier diagnosis of the disease will cause demand for insulin to quadruple in the next five years.

SemBioSys believes its safflower-produced insulin can reduce capital costs compared to existing insulin manufacturing by 70% and product costs by 40%. SemBioSys believes safflower-produced insulin would require approximately $80 million in capital investment for 1,000 kilograms of insulin production capacity. Alternatively, insulin currently produced using fermentation is estimated to require $250 million in capital investment for 1,000 kilograms of production capacity. In addition, because of the ease in scaling-up crop acreage, plant-produced insulin offers significant improvements in the flexibility and speed of scale-up. SemBioSys has five years of experience growing transgenic safflower in Canada, the US, Mexico, and Chile under permits issued by the pertinent regulatory authorities.

Insulin Production
Existing commercial insulin production methods typically rely on yeast (Saccharomyces cerevisiae) or bacteria (E. coli) genetically engineered to produce synthetic human insulin. These organisms are grown in large, capital-intensive steel bioreactors and the insulin is then extracted and purified for final formulation.

SemBioSys uses safflower to produce human insulin. Through its proprietary technology, SemBioSys is able to accumulate recombinant proteins, like insulin, in safflower. As the plant grows and the seed develops, the insulin protein is produced in the seed. Safflower production is based on conventional farming practices that have been adapted to ensure product integrity and confinement. The harvested seed is then processed using SemBioSys’ proprietary extraction process. Conventional enzymatic or chemical cleavage techniques and downstream processing methods are employed to produce purified insulin.

The selection of safflower as its commercial plant system was based on safflower’s superior technical profile as well as the advantages it offers to address the strict regulatory criteria expected for plant-made pharmaceuticals. Safflower is a low acreage crop that can be easily segregated from other safflower production. This, in combination with the biology of the crop’s pollination patterns, facilitates containment of the crop.

SemBioSys’ platform offers two important capabilities – to cost effectively extract oilbodies from seeds at scale, and to use genetic engineering to attach proteins to oilbodies in seed.