IMPLICATIONS OF POLLEN RESEARCH TO APHIS PHARMACEUTICAL CORN REGULATIONS
Gene Stevens
Development and subsequent testing of plant-made pharmaceutical (PMP) crops is proceeding at a steady pace. Corn (maize) and tobacco have received the most attention as potential green factories for producing medicines and vaccines. Several government agencies are regulating the testing, production, and disposal of these crops to assure that safe manufacturing practices are followed. The United States Department of Agriculture's Animal and
Plant Health Inspection Service (APHIS) has developed rules for preventing dissemination into the environment of organisms engineered for pharmaceutical products. APHIS permits for field tests with PMP crops are issued on a case-by-case basis. On May 21, 2002, APHIS posted a summary of their confinement requirements for organisms containing pharmaceutical genes being field tested (barley, corn, rice, sugarcane, tobacco, and tobacco mosaic virus). The report can be downloaded at
http://www.aphis.usda. gov/ppq/biotech/pdf/pharm-2002.pdf.
An important element of the APHIS regulations is achieving reproductive isolation of PMP plants from plants of the same species or sexually compatible species. In
2002, applicants seeking APHIS permits for growing pharmaceutical corn had three options available for mitigating pollen-mediated cross-pollination or gene flow. Each option
has strict guidelines concerning isolation distance and planting dates of PMP corn relative to other cornfields in the area.
In 2001, a project was initiated by the University of Missouri-Delta Research Center in Southeast Missouri to study methods of optimizing corn pollen containment for one of the candidate plant-made pharmaceutical production systems. I am the principal investigator on the project. In the tests, we are not actually growing corn plants that contain PMP but are using other types of corn to imitate how PMP corn might be grown in the future.
Experiments were designed to determine how far corn must be planted from other corn fields and what level of detasseling (male flower removal) is sufficient to prevent gene flow. Currently, flower removal is listed among the APHIS methods for containing pollen from pharmaceutical tobacco but not corn.
Corn normally produces one tassel and one ear on each plant. A tassel is actually a male flower and an ear is the female flower. Three to five days before a tassel is
visible on a corn plant, it can be found tightly rolled in a whorl of new leaves near the top of the stalk. The immature tassel along with two or three leaves can be removed from the plant in a process called detasseling to create the equivalent of a female plant.
The objective of our research is to determine whether detasseling would be a valuable addition to a system for producing PMP corn without releasing viable pollen with the gene into the environment. Female corn lines (i.e., plants that have been detasseled) which contain the genes for a PMP will be grown in rows that alternate with rows of male corn (corn with intact tassels) without the PMP genes. Because the female plants are detasseled, they cannot produce pollen containing the gene. The silks on the ears of the female plants that contain the PMP gene will be pollinated by the rows of male corn without the genes. The resulting ears will contain the desired pharmaceutical proteins.
Detasseling has been a common practice in corn hybrid seed production for many years. Although it is a highly effective procedure, tassels are occasionally missed.
Most seed certification inspectors allow less than 1% detasseling error. Regulatory agencies need to know what kinds of separation distances must be maintained at different levels of detasseling efficiency to avoid gene flow to other corn fields. To answer this question, we planted yellow kernel inbred corn in a 10-acre block in three 160-acre cotton and bean fields at Hayti, Wardell, and Clarkton, Missouri. The block served as a source of fertile pollen to detect in white corn strips located in other parts of the field.
Within the 10-acre corn pollen blocks, four rows of yellow kernel females were planted in an alternating pattern with four rows of white kernel males. When the yellow
female rows were detasseled by our team, some of the plants were intentionally missed. The levels of detasseling were 0%, 80%, 90%, and 100% of the total plants in each row. For each detasseling treatment in the pollen block, a different yellow inbred cultivar was planted which
contained a specific transgenic trait to use as a tracer.
To aid in pollen containment, the pollen source block was surrounded by 10 feet of fallow ground and then 12 rows of male sterile corn border. The rest of the field
was planted to either lima beans or cotton, but at distances of 660 and 900 feet from the pollen block, four-row strips of white corn hybrid were planted on three different planting dates.
Yellow corn seed color is dominant over white seed color. Therefore, any yellow kernels found in the white hybrid strips were fertilized by pollen from yellow corn in
the pollen block. PCR (polymerase chain reaction) analysis was used to detect each of the specific transgenes used as tracers in the different yellow inbred corn cultivars. Using this tracer system, any yellow corn in the white corn strips could be traced back to pollen from a
specific detasseling treatment.
When the corn seeds matured, we examined over 7,000 ears of corn in the white hybrid strips at each test site looking for yellow seeds. Each ear contained approximately 500 kernels. Results were expressed as the percentage of yellow kernels among the white kernels in the corn planted at the two distances from the center block.
We found that the planting date of the white hybrid corn strips had a large effect on gene flow (cross-pollination). This observation means that a narrow window of
time existed during which the viable pollen produced by the yellow corn was in synchrony with the receptive silks on the white hybrid ears. It also supports current APHIS regulations concerning using separation of planting dates of PMP corn relative to neighboring corn fields as an effective isolation tactic to prevent gene flow.
Our test site at Clarkton, Missouri, had the most gene flow in 2001. The greatest amount of gene flow (yellow corn kernels on the white corn cobs) occurred in the
northern section of white corn located 660 feet from the pollen block and was associated with pollen from corn with no detasseling. The incidence of yellow kernels was 0.0301%. Gene flow was comparatively greater in the most northerly corn strips, most likely because the prevailing wind was from the southwest.
The amount of gene flow dropped as detasseling and isolation distance increased. At 900 feet, the incidence of yellow kernels in white corn was 0.0013% from the
90% detasseled corn rows. In other words, only 1.3 seeds out of every 100,000 seeds were from pollen produced by corn with the 90% detasseled treatment. In our tests, when 100% of the corn was detasseled, no yellow kernels were detected on the white corn cobs at 900 feet. The 900
feet demarcation is less than the minimum separation of 1320 feet required by APHIS regulations for separation of controlled-pollination PMP corn from other cultivars.
Field experiments studying pollen containment were continued in 2002. We plan to begin harvesting trap plots in two weeks. If similar results are found this year, we
hope that APHIS will consider adding detasseling in combination with isolation distance as a new option in their regulations for producing PMP corn.
Gene Stevens
University of Missouri
StevensW@missouri.edu
CAST REPORT REVIEWS ENVIRONMENTAL IMPACTS OF BIOTECHNOLOGY-DERIVED CROPS
Janet Carpenter
The Council for Agricultural Science and Technology (CAST), a non-profit consortium of scientists, recently released a study summarizing available information on
the environmental impacts of biotechnology-derived soybean, corn, and cotton in comparison to their conventional counterparts. The authors of the review concluded that biotechnology-derived soybean, corn and cotton pose no environmental concerns unique or different than those historically associated with conventionally developed varieties. Three teams of researchers contributed to the report, reviewing the available scientific literature in order to evaluate the range of environmental impact issues in the context of traditional cropping practices. Authors are affiliated with the National Center for Food and Agricultural Policy, Washington State University, Clemson University, and the University of Illinois. The United Soybean Board (USB) provided funding for the report from checkoff funds.
Soybean, corn, and cotton growers in developed and developing nations have rapidly adopted biotechnology-derived commodity crops during the six years of their commercial availability. In 2001, growers planted biotechnology-derived seed on 46% of global soybean
acreage, 7% of global corn acreage, and 20% of global cotton acreage. To date, nearly all of the planted biotechnology-derived crops have either introduced tolerance to selected herbicides for weed control or protection against insect pests, or both. Of the 52.6 million hectares of
biotechnology-derived crops planted in 2001, 77% were herbicide tolerant, 15% were resistant to insect damage, and 8% were both herbicide tolerant and resistant to insect damage.
In the US, adoption of biotechnology-derived soybean climbed to 75% this year. Corn and cotton growers planted 34% and 71% of total acreage to biotechnology-derived varieties, respectively. With a technology that could be released on such vast areas, both in the US and worldwide, consideration of the potential environmental impacts is desirable. Indeed, regulatory agencies worldwide assess these potential impacts before commercialization.
Even more information has been made available in the years since these crops were first introduced. The focus of the report was on those crop traits that are currently commercialized. Some discussion of pest management traits in development for soybean, corn, and cotton is included.
Identification of Environmental Impacts
Opinions expressed in peer-reviewed literature, regulatory assessments, and the popular media and by environmental advocates have repeatedly raised questions about the environmental safety of biotechnology-derived crops. To answer these questions, the scientific literature
was reviewed and analyzed specifically for soybean, corn, and cotton. The environmental impacts of commercially available biotechnology-derived crops were assessed in relation to the current agricultural practices for crop and pest management in conventionally bred crops.
Nine potential environmental impacts were identified as follows:
1. Changes in pesticide use patterns Does the adoption of biotechnology-derived soybean, corn, and cotton impact the use of pesticides, and if so, do these changes alter grower practices in ways that affect water quality?
2. Soil management and conservation tillage Does adoption of biotechnology-derived soybean, corn, and cotton lead to changes in the adoption of no-till and other conservation tillage practices or otherwise impact soil erosion, moisture retention, soil nutrient content, water quality, fossil fuel use, and greenhouse gasses?
3. Crop weediness Do biotechnology-derived soybean, corn, and cotton have increased fitness and acquire weediness traits?
4. Gene flow and outcrossing Do biotechnology-derived soybean, corn, and cotton unintentionally breed with local plants or crops and impact the genetic diversity in the areas where the biotechnology-derived soybean, corn, and cotton are planted?
5. Pest resistance Do biotechnology-derived soybean, corn, and cotton posses plant-protectant traits to which pests will become resistant, and if so, is the development of resistance in these three biotechnology-derived crops different from their
conventional counterparts and how is the development of resistance being managed?
6. Pest population shifts Do biotechnology-derived soybean, corn, and cotton cause changes in weed or secondary insect pest populations that impact the agricultural system or ecology of the surrounding environment?
7. Non-target and beneficial organisms Do biotechnology-derived soybean, corn, and cotton with pest protection characteristics have an impact on natural enemies of pests, (i.e., predators and parasitoids) or on other organisms in the soil
and crop canopy?
8. Land use efficiency/productivity Does the adoption of biotechnology-derived soybean, corn, and cotton impact crop yields or impact the need for cultivating forested or marginal land?
9. Human exposure Do the traits imparted by breeding herbicide tolerance and resistance to pest insects change the human exposure or other safety factors of
biotechnology-derived soybean, corn, or cotton?
General Conclusions
The authors found that the appropriateness of biotechnology-derived crops to different geographic areas depends on many economic, social, and regional factors. Nevertheless, a number of general conclusions about biotechnology-derived soybeans, corn, and cotton are supported by
the literature.
1. Biotechnology-derived corn, cotton, and soybeans provide insect, weed, and disease management options that are consistent with improved environmental stewardship in developed and developing nations.
2. Biotechnology-derived crops can provide solutions to environmental and economic problems associated with conventional crops including production security (consistent
yields), safety (worker, public and wildlife), and environmental benefits (soil, water, and ecosystems).
3. While not the only solution for all farming situations, the first commercially available biotechnology-derived crops, planted on over 100 million acres worldwide, provide benefits through enhanced conservation of soil, water, and beneficial
insect populations and through improved water and air quality.
4. The high adoption rates for commercially available biotechnology-derived crops can be attributed to economic benefits for growers.
5. When biotechnology-derived crops are available to small farmers in developing nations, they can realize environmental benefits and reduced worker exposure to pesticides.
Specific Conclusions
In addition, many specific conclusions related to the potential environmental impacts of particular biotechnology-derived crops were drawn. The most striking environmental impact of biotechnology-derived herbicide tolerant crops has been the increased adoption of conservation
tillage practices. No-till soybean acreage in the United States has increased by 35% since the introduction of glyphosate-tolerant soybean. Similar increases are observed in Argentina, which can be attributed in part to reliable and effective weed control provided by herbicide tolerant soybean. No-till cotton and corn adoption has also increased. Use of no-till farming practices results in reductions in soil erosion, dust, and pesticide run-off, increases in soil moisture retention, and improvements in air and water quality. Further, adoption of biotechnology-derived herbicide tolerant crops allows farmers to use an herbicide that
binds tightly to soil, which reduces the possibility for water contamination, compared to conventional herbicides.
Continued efficacy of weed management systems using biotechnology-derived herbicide tolerant crops, similar to conventional weed management practices, requires
effective management strategies to prevent weed population shifts and to prevent the development of weed resistance to herbicides. Emerging reports of glyphosate-resistant weed populations underscore the importance of proper technology stewardship. However, herbicide resistance in weeds is not unique to biotechnology-derived crops.
Among the conclusions regarding biotechnology-derived insect resistant crops were issues surrounding resistance management and human health. Insect Resistance Management plans were developed and implemented to prevent or delay the development of insect resistance to biotechnology-derived insect resistant crops. The reduction of naturally occurring mold toxins resulting from use of biotechnology-derived insect resistant corn can provide direct benefits to people and corn-fed livestock. Insect protected corn is less vulnerable to mold infestation. The adoption of insect-resistant cotton in China has been associated with increased safety of agricultural workers.
Recommendations
The report includes several recommendations for continued responsible use of the technology. Among the recommendations is the continued development of policies for implementation of effective insect and weed resistance management strategies in both conventional and biotechnology-derived crops. The authors also recommend that attention to gene flow between biotechnology-derived and other crops or native plants be focused on environmental
and social impacts/consequences of that gene movement.
USB supported the study in order to verify the environmental impacts of the crops through an independent assessment by scientists working in the area of biotechnology. The cross-disciplinary research team combines expertise in entomology, toxicology, weed science
and agricultural economics. CAST coordinated the research team and preparation of the report. The full report is available on the CAST and USB websites (http://www. cast-science.org
and http://www.talksoy.com).
Janet Carpenter
Research Associate
National Center for Food and Agricultural Policy
carpenter@ncfap.org
RETURN OF HYBRIDIZATION CLAIMS (ENZO II) AND OTHER BIOTECH PATENT NEWS
Phillip B. C. Jones
Last April, the Court of Appeals for the Federal Circuit published a decision that raised the bar for the patentability of claims to nucleic acid molecules ("Don't Bank on ATCC Deposit to Support DNA Patent Claims," ISB News Report, pages 10-11, May 2002). In this decision, the Federal Circuit had affirmed the determination of a federal district court that Enzo Biochem Inc.'s patent specification failed to provide a sufficient written description to support claims for nucleic acid probes. Three months later, the
Federal Circuit withdrew its opinion and published a new one.
The Enzo patent, US Patent No. 4,900,659, includes claims to nucleic acid probes that hybridize preferentially with particular fragments of Neisseria gonorrhoeae DNA, compared with certain fragments of N. meningitidis DNA. In the April decision, the Federal Circuit had taken the position that a claim to a nucleic acid molecule fails to meet the written description requirement if the claimed molecule is described only by its ability to hybridize with another DNA molecule. The court disregarded, some might say dissed, the US patent office written description Guidelines on this point.
In the recent opinion, however, the court found the Guidelines to be persuasive and sent the case back to the lower court. The Federal Circuit explained that the district court must determine whether a person of skill in the art would find the claimed probes, described on the basis of hybridization, consistent with the PTO Guidelines. If so, then the written description requirement would be met.
Certain Enzo patent claims specified probes deposited with American Type Culture Collection. Claim 4, for example, refers to ATCC 53409, which contains an 850 base pair
N. gonorrhoeae DNA probe. The claim also includes mutated variants of the deposited probes. In the April decision, the court had held that an ATCC deposit is not sufficient to comply with the written description requirement.
Yet now, the court agrees with Enzo that a reference in the patent specification to the ATCC deposits described the deposited probes sufficiently for the purpose of meeting the written description guidelines. The court reasoned that a person of skill in the art could obtain the claimed sequences from the ATCC depository by following standard
techniques to excise the DNA molecules from the deposited organisms and sequencing them. The Federal Circuit left it to the district court to determine whether the ATCC deposits support mutations of the deposited DNA molecules.
A copy of the latest Enzo decision can be found on the Federal Circuit's website
(http://www.fedcir.gov/dailylog.html).
Collecting Tolls on a Biological Pathway
On Christmas Eve, 1986, David Baltimore, Phillip A. Sharp, Thomas P. Maniatis, and other well-known scientists filed a patent application with the US patent office on protein factors that regulate transcription. On June 25, 2002, US Patent No. 6,410,516 issued with 203 claims for methods of treating diseases by regulating nuclear factor-kB (NF-kB). For example, claim 1 is directed to a "method for inhibiting expression, in a eukaryotic cell, of a gene whose
transcription is regulated by NF-kB, the method comprising reducing NF-kB activity in the cell such that expression of said gene is inhibited." NF-kB may regulate cytokines,
cytokine receptors, cell adhesion molecules, chemo-attractant proteins, oncoproteins, and growth factors, and NF-kB activity has been implicated in a variety of disorders,
including inflammation, tumor growth, and bone homeostasis.
ARIAD Pharmaceuticals, Inc. (Cambridge, Massachusetts), the exclusive licensee of the patent, quickly filed an infringement suit against Eli Lilly & Co.
(Indianapolis, Indiana). ARIAD claims that two of Lilly's top drugs, the osteoporosis drug Evista® and the sepsis drug Xigris®, work by the patented methods. Industry analysts
estimate that annual sales of the two drugs may exceed $1 billion. ARIAD also sent licensing offers to about 50 other companies that have products on the market or in development that work via the NF-kB pathway.
It is not uncommon for an inventor to obtain a patent for a method of using a drug even though the inventor does not know the mechanism of drug action. ARIAD's litigation with Lilly may determine whether an inventor can control all uses of a biological pathway by identifying factors that activate it.
Other Biotech Patent News
July 9: Epicyte Pharmaceutical, Inc. (San Diego, California) announced that it has exclusive rights to a patent on antibody-producing transgenic plants. US Patent No. 6,417,429, assigned to The Scripps Research Institute (La Jolla, California), lists inventors Mich Hein and Andrew Hiatt, the founders of Epicyte. The company is collaborating with The Dow Chemical Company (Midland, Michigan) to produce an antibody for treatment of herpes simplex virus.
