Confined Production Processes for Non-Food Corn - Seed Science ...

A necessary first step in the project that BIGMAPscientists have undertaken is the development of a setof reliable, relevant, and representative good practicestandards for levels of confinement that are consistentwith current regulatory expectations. This descriptionof BIGMAP good practices was developed by a diverseteam of experts from within the university communitywho have offered their unique talents and expertiseto develop consensus good practices for the confinedfield production of corn expressing pharmaceuticalor industrial compounds. We thank the followingindividuals for their contributions to this exercise:Bruce Babcock (meteorology)Alicia Carriquiry (statistics)Joseph Cortes (process management)Karin Doorman (statistics)Dermot Hayes (public perception, economics)Adelaida Harries (regulations)Charlie Hurburgh (grain harvesting and distribution)Scott Hurd (risk assessment)Larry Johnson (processing)Kendall Lamkey (genetics)Bob Peterson (risk assessment)Kan Wang (plant transformation)Mark Westgate (pollen flow, agronomics)FIGURE 1: BIGMAP PMP AND PMI CORN OBJECTIVES1) Differentiate unintended presence issues in seed fromthose in grain;2) Develop quantitative information describing outflows tograin and seed;3) Categorize active substances based on the hazard thatthey present,4) Describe risk for categories of substances; and5) Suggest confinement procedures tailored to fitclasses of substances.We thank Melissa Swanson for her contributionto the flowcharts.In addition, we thank the following reviewers,not directly associated with BIGMAP:Gregory Jaffe, Center for Science in Public InterestNeil Hoffman, USDA–APHIS–BRSWilliam Horan, Horan Bros. Ag EnterprisesThomas Nickson, MonsantoDaryl RollandAlthough we highly valued the suggestions made by theseindividuals, the final product cannot conform entirely totheir suggestions, and we assume responsibility for allpositions taken.Prefaces and Acknowledgments 3

The conditions for correct identification of traits dependon record keeping of the type required to meet the GoodLaboratory Practice (GLP) standards of the Food andDrug Administration (FDA) or the EnvironmentalProtection Agency (EPA). The flowcharts for laboratoryand greenhouse procedures presented here are examplesof the sound protocols required under GLP. Suchprotocols are the basis for avoiding mislabeling ofcultures and plants, and they rely heavily on labelsand location records.In addition to label and location, the development anduse of tests based on polymerase chain reaction (PCR)provide a third secure means to verify identity andconfirm a plant’s source. We assume that event-specificPCR tests would be developed before conducting fieldtests with PMPs or PMIs to provide a means ofmonitoring and mitigating losses.Misidentification of events is the main PMP and PMIconcern associated with laboratory and greenhousecontainment. It can lead to commingling or unintendedcrossing. The National Institutes of Health Guidelinesfor Research Involving Recombinant DNA Molecules,institutional biosafety committees, and GLP providemodels for the appropriate framework for safe containmentand establishing trait identity chains. The flowchartsdescribe steps that ensure correct identification of geneticmaterial to be planted in the field that can be used todevelop models to quantify the possibility of risk resultingfrom mislabeling.NURSERY PROCESSESField trials can be classified by size. Larger trials areused to produce seed, material for development work(procedural, analytical, pre-clinical and clinical trials),and commercial quantities. These larger trials usedistance offsets as the primary confinement mechanism.In contrast, smaller, research-oriented trials achieveconfinement by using methods in which the floral structuresare either bagged or removed; pollination occurs by hand;and pollen is not allowed to disperse into the air. Thesetwo scenarios lead to significantly different probabilitiesof pollen escape from trials.PMP and PMI research nurseries may contain severaltransformation events, and these events can be associatedwith varying degrees of hazard. Nurseries and researchinventory systems that manage the seed used andproduced in them are subject to the possibility ofmisidentification of events and lines, as well as geneticoutflow of pollen, seed, and plant materials.It seems prudent to classify the risk that nurseries posebased on the hazard associated with that event of greatestconcern in each nursery. Nurseries containing hazardousevents probably pose greater concern than field productionof less hazardous events, even when the area of the fieldproduction is much larger.Conventional corn research nurseries are managed withprocedures that minimize the chance of error in theidentification of lines. These procedures provide redundantsystems for identification that allows the detection andcorrection of errors as they occur. Event-specific PCRchecks allow for the specific identification of events inlines, especially when they are released from researchnurseries to be used in foundation seed, seed, and fieldproduction. Such checks also can test for the nonpresenceof events that are not intended for release for largerproduction. These latter tests are especially importantbefore lines move into larger scale trials with open pollination.Nursery information systems typically manage recordsabout plants in the nursery and seed in storage. Thenursery information management system links the plantin the field with a source of seed in a seed storage facilityor a container in storage to the row where it was grown.The information system reduces error in the records aboutplants in the field and seed in the inventory by includingboth kinds of information.The pollen isolation procedures that have been approvedby Animal and Plant Health Inspection Service (APHIS)for corn nursery are the same as for all PMP production:At any time during the field test when PMP plants areallowed to shed pollen, ensure that no other receptive cornplants are grown within a radius of 1.0 mi (1.6 km) of thetransgenic plants.Where pollination of PMP plants is controlled, ensurethat transgenic corn is planted not less than 28 daysbefore or 28 days after the planting dates of any othercorn that is growing within a zone extending from 0.5to 1 mi (0.8 to 1.6 km) of the transgenic plants to ensurethat there is no overlap in anthesis.Prefaces and Acknowledgments 5

There are no other restrictions on corn that is grownat more than 1 mi (>1.6. km) from the transgenicplants. These standards have been established using welldocumented information about pollen number, pollensize, duration of pollen shed, viability, and dispersion.For nurseries, it is possible to envision additional meansbeyond pollination control and isolation by which outcrossingcan be further mitigated if warranted by the degree ofhazard present:• In hand-pollinated nurseries, internal non-PMP or -PMIborder plantings may act as barriers and diminish theconcentration of PMP and PMI pollen from the source.• Reduction in the number of transgenic PMP plantswithin the trial may diminish the concentration ofPMP and PMI pollen at the source.• Reduction in the proportion of transgenic plants in thenursery may diminish the concentration of PMP andPMI pollen at the source.• Active daily surveillance by trained personnel of a fieldduring flowering may be used to ensure pollen control.• More comprehensive standard operating procedures tominimize potential loss of pollen, plants, or seeds fromcontainment than could be implemented for larger fields.Such nursery procedures reflect the scientifically documentedinfluence of the size of the source and borders on the rateof outcrossing in corn. Various levels of size, isolation,borders, and pollen control can be combined to achievea given goal for corn pollen confinement.A large research nursery may require thousands of handpollinations and each activity introduces some possibilityof error. The work may involve dozens of technicians, sothe frequency of human error requires special emphasis.Field production procedures, especially those using sterilefemales and nontransgenic males, are inherently simplerand therefore are less prone to human error.In PMP or PMI nurseries that contain events of uncertainhazard, isolation from nurseries with unregulated corn andfrom research and foundation seed stocks of unregulatedcorn can be an especially important consideration.Nurseries have a potential for special complexity due tothe number and variation of hazard level of the PMP andPMI traits involved and the complexity of the operationsthat are performed. Our descriptions of the systemsinvolved allow for consideration of particular managementneeded to accommodate confinement for nurseries.LARGER SCALE PRODUCTIONThe typical characteristics of the larger scale PMP or PMIproduction systems are that pollination is not conductedby hand, only a single trait is present, and harvest operationsare conducted mechanically. Process flows for larger scaletrials and seed production are grouped with field productionsystems because of their similarity.Our approach to the field production of PMPs and PMIsis based on foundation seed production practice in thecorn seed industry. We have suggested modifications ofthis practice to account for the special process and materialcontrol that may be needed for PMPs and PMIs in corn.As in other parts of the study, the existing USDA standardsfor confinement were considered the appropriate baseline,and the practices are intended to achieve confinementequivalent to this standard. No assertion is made thatthese process descriptions are optimal; however, theyare a reasonable base for considerations of managing forconfinement integrity.The geographic scale of field production means thatindividuals or small groups carry out operations awayfrom supervision. The human aspect of management ismore important in field production than in the laboratory,greenhouse, or nursery. Therefore, documentation ofactivities, responsibilities, accountability, and training areimport aspects of the processes we describe.We envision field production of the product will becarried out with sterile female plants, pollinated byfertile non-PMP or -PMI corn. Sterility can be achievedgenetically through the use of cytoplasmic male sterility,mechanically by removal of the tassels of the femaleplants before flowering, or both. For this field productionscenario, pollen released may be even lower than in thenursery where some pollen enters the air when pollen isapplied to the female plants by hand.The procedures involved in large, open-pollinated trials,seed production, and commercial PMP or PMI productionare very similar. However, PMP or PMI pollen movementmay vary based on the degree of pollen management thatis implemented; therefore, location and isolation policywill be different for production using male sterile or detasseledtrait plants compared with the pollen-shedding male plants.In certain situations, the most effective management maybe to exclude open-pollinated production from areas

where large amounts of corn production, foundation seedproduction, or corn research is conducted for the foodand feed system, but this need not be an a priori aspectof all PMP and PMI production schemes.Production of grain for processing can be on either inbredor hybrid female parents. Production on inbred femaleswith the PMP or PMI event simplifies seed productionand limits the number of foundation seed isolations withopen-pollinating PMP or PMI males, but it also increasesthe area that would be required to produce a givenamount of PMP or PMI product by 30 to 50%.The use of mechanical equipment for the harvest of PMPor PMI seed or grain provides a potential source of loss.We focus here on equipment selection, preparation, andmanagement activities that limit potential for operationallosses of seed or grain.Most of the issues relevant to confined production ofplant-manufactured pharmaceutical and industrialcompounds have analogous issues in foundation seedproduction: isolation, field shape and placement, avoidanceof loss, sampling issues, rigid quality control, record keepingfor processes and materials, and post harvest verificationof genotype. Individuals who are familiar with foundationseed production have an immediate grasp of the issuesthat impact the ability to maintain confinement in fieldproduction of PMPs and PMIs. The employment of staffmembers with experience in the production of foundationseed is one way to facilitate the preparation of the staffmembers. Although the production of foundation seedprovides a model for production of PMPs and PMIs, theexistence of this model does not imply that there are nonew issues or different answers to old problems.CONCLUSIONFormalized risk assessment makes science-basedcharacterization of risk possible. This assessment cansubsequently be integrated with broader societal concernsin policy formulation considering both the risks andbenefits of the technology.Although PMP and PMI production in corn is new, existingtechnology for corn research nurseries and foundationcorn production provides a basis for the developmentof good practices for PMP and PMI production. Theflowcharts of good practices provided in this documentare developed from this base.Establishment of practice standards allows for evaluationof process integrity through quantitative exposure assessment.Quantitative exposure assessment recognizes that foreach activity there are various likelihoods of proceduralconformity and nonconformity. For conformity andnonconformity, there are distributions of possible outcomes.Working through the flowchart, we can model all theprocess and calculate probabilities of the various systemfinal outcome states. If we can value the various possibleoutcomes, then we can calculate the cost for effectiveconfinement of a PMP or PMI production system. Themodels also can be used to evaluate the impact of changesin processes on the performance of the system as a whole.The existence of duplicate or multiple checks on systemactivities is important throughout the PMP or PMIproduction system. All of the possible checks are not shownin our process analysis. For example, the USDA-APHISnotice for PMP and PMI production provides for thepossibility of inspections at preplanting stage, plantingstage, midseason, harvest, postharvest, or other times(USDA, 2003a,b). Although the importance of theseaudits is recognized in our overall flow descriptions, theyare not explicitly indicated in the flow diagrams, becausetheir nature and timing are in the purview of APHIS andnot the PMP producer.Prefaces and Acknowledgments7

IntroductionIt is our expectation that corn that containing PMPsor PMIs will remain a regulated product. It will notbe available for sale to farmers or the general public.Production will be conducted by trained specialists. Wesupport this regulation and the accompanying oversight.This report does not address the specific nature of regulationand oversight, although these topics might be the objectof future study by BIGMAP researchers.The development and use of corn in the production ofPMPs and PMIs are complicated by the possibility forloss of genetic material, leading to the presence of grainexpressing these products in foods or feeds. Pollinationof corn outside the production area or grain loss fromthe production system could lead to the unintendedpresence of pharmaceutical and industrial compoundsin food, feed, or the environment. The consequences ofsuch unintended presence have not as yet been clarifiedin terms of their potential impact on human, animal, andenvironmental safety (National Research Council, 2004).Public acceptance of this technology is currently uneven,and there could be additional large negative economicconsequences for unintended occurrence of pharmaceuticalor industrial compounds in foods. In recognition ofthese concerns, the USDA has imposed conditions on theconfinement for field testing of PMPs and PMIs (USDA,2003a,b). These conditions are intended to prevent theunintended presence of PMP and PMI products in foodsor feeds. The conditions are especially stringent andeffectively limit the potential for these crops to be grownin the field in the major corn production regions of theUnited States.In this report, we describe good practices for achievingconfinement of PMP or PMI corn in keeping with currentexpectations of the USDA. BIGMAP scientists will usethese practices in evaluating the levels of exposureachieved when stringent confinement conditions aremet as well as when actions fail to conform to theseconditions. In specifying these practices, we have madecertain assumptions about which technologies are currentlyavailable to the industry and implicit assumptions aboutappropriate procedures. Future BIGMAP studies will testwhether our assumptions are optimal within the scope ofcurrently technology.CAUTIONS CONCERNING EXPOSURE SCENARIOSFOR PMPS AND PMISThe caution implied in the procedures presented here doesnot imply that the levels of caution will be appropriate forall PMPs or PMIs.A class of highly toxic compounds is defined by theNational Institutes Health (NIH) guidelines (NIH, 2002)in Section III-B-1: Experiments Involving the Cloning ofToxin Molecules with LD50 of Less than 100 Nanogramsper Kilogram Body Weight. Procedures developed for thisclass of highly toxic substances would be very differentfrom those appropriate for other toxic substances. We donot expect that the highly toxic substances in this classwill be produced in plant systems outside containmentfacilities. The implications of risk in such systems arenot discussed in this report. One additional reason for notdiscussing such systems is that knowledge of the procedurescould conceivably be used for the production of toxins foruse in terrorist acts.As a description of current practice, it is within our scopeto cover all possible future technology.

ALTERNATIVE TECHNOLOGIESThe most notable future technologies may be thetransformation of chloroplasts or other organellestransmitted through the female line and facultativepromoters under the control of chemical triggers. Bothtechnologies would reduce the risk associated withpollen control. They would reduce the risk associatedwith seed production. For controllable promoters, itmight be possible to remove the risk associated withisolation. In both technologies, the change in technologyradically changes the risk profile of the operations andwould necessitate separate analysis.Genetic markers could be added to the lines as avisual means to trace outcrossing. Markers could,in theory, be added to the vector to facilitate theconfinement process. For example, color markersmight aid in the observation of escape from confinement.The ideal marker would be observable in endosperm,aleurone, or germ so that seed resulting from pollinationcolored. The objective of the specific tissue expressionwould make this task difficult. Because of the difficulty,we do not assume that such markers will be availablein the management process.Natural or genetically independent transgenic colorswith the appropriate tissue-specific expression couldbe used. The color would simply be incorporated intothe recurrent parent before the backcrossing started.Genetic colors would have the advantage makingoutcrossing visible. Use of naturally occurring colorshas some drawbacks. The natural choice is purplealeurone and endosperm. These colors occur in normalcorn through disease and mutation at levels highenough to confuse the system. Any adjacent blue cornwould certainly confuse the system. Although someadvantages exist and some natural genes with theappropriate tissue specificity exist, genetic colors withexpression in the appropriate tissues are not assumedto be part of the current technology.The possible existence of alternative technologies isrelevant to the discussion of policies but goes beyondthe objectives of this report.The examples developed here assume that corn grain isthe primary product of the PMP or PMI productionsystem. Some modification would be required if theprimary product is plant material that would need tobe transported in volume.GENERAL OBJECTIVEGrowth of PMP and PMI plants must be conductedunder responsible stewardship to mitigate the loss of seeds,plants, or pollen that could lead to harm to people or theenvironment. Mitigation means reducing the probabilityof loss to levels that make it so unlikely to occur that theresidual risk is acceptable within the policy developed onthe basis of risk assessment. Prevention of loss is a goal,but risk analysis assumes that there is some probabilitythat it will not be achieved.At a very general level, mitigation measures for thelaboratory, greenhouse, and confined production includethe following:• adequate identification, packaging, and segregationmeasures during transit and at the receiving facilities toprevent or minimize mixing, spillage, and disseminationof viable plant material, including the flow of fertiletransgenic pollen to sexually compatible plants;• both physical and biological methods to minimizethe flow of fertile transgenic pollen to other sexuallycompatible plants within the contained facility or tosuch plants on the outside; and• devitalization or disposal of transgenic plant material bymeans that block dissemination when such material isno longer in use.Through the use of flowcharts, we describe the meansof implementing these general mitigation measuresthrough physical containment procedures in the laboratoryand greenhouse and through physical and biologicalconfinement procedures for field breeding operationsand field production.Implementation of production procedures requiresmanagement interventions beyond those that we candepict in the flowcharts as training and inspection, andcontrol points. Manager-employee and internal teamrelationships impact performance in ways which are difficultto foresee and document. Although for the purposes ofthis research we may accept such management variationas part of the random environment in which processesIntroduction 9

take place, we suggest that in practice management shouldtake all the appropriate human factors into account.Records for critical tasks should document who has performedactions so that responsibility in the chain of material flowsis known so that management can apply the appropriaterewards and punishments for correctly or incorrectlycompleted actions; but more importantly, managementshould be involved before actions take place to ensurethat the right person is performing the task at a timewhen that person is ready for the task. Readiness includesnot only training and capabilities but also the properemotional state to focus on the action.This report focuses on processes associated with a fieldthat is the source of outflows of pollen, grain, or plantmaterial. This is a necessary first exploration forBIGMAP, in order to further develop the nature ofexposure associated with various categories of fields thatreceive these outflows. In the future, BIGMAP researchershope to develop models that quantify risks associated withoutflows to specific kinds of fields, including seed fields.There are currently no specific laws on contamination offoundation seed. Future studies will discuss different oradditional regulation concerning the unintended presenceof PMPs and PMIs in seed.DEFINITION OF CONTAMINATIONBefore discussing outflows to seed stocks, it is usefulto discuss the definition of “contamination.” Contaminationcan have narrow or broad definitions, but it carries theconnotation of negative consequences for the presenceof a certain substance. If there are no negativeconsequences for this substance, the word “presence”may be more appropriate. Where the presence is notdue to intentional acts and it is uncertain whetherthere are negative consequences, then “unintendedpresence” is a better choice.Because negative consequences are nearly always afunction of dosage or quantity, use of the descriptorcontamination for low levels may not be directly appropriate.However, subjective reaction allows negative consequenceseven in the absence of substantive damage to healthor the environment. If the substance has a bad image,presence at physically inconsequential levels my produceadverse reactions such as revulsion and avoidance,which are a real problem to the owner of the productwith the substance in it. Society’s tolerance variesdepending on the issue. For example, generally ourreaction to rat feces as food is very negative. Despitethis sensitivity, there is a tolerance for rat feces in somefoods, and the public generally accepts them withoutconcern. A broad definition of contamination as thepresence of an unwanted substance does not addressthe issues of “unwanted by whom” and “unwanted inwhat amounts.”Contamination of food is addressed under The FederalFood, Drug, and Cosmetic Act provisions on adulteration.This act provides a narrow definition of contaminationand illustrates the concept that low-level, unintendedpresence is not necessarily contamination. The FederalFood, Drug, and Cosmetic Act (Subchapter IV Food,Sec. 342, Adulterated Food) states the following:“A food shall be deemed to be adulterated … If it bearsor contains any poisonous or deleterious substancewhich may render it injurious to health; but in case thesubstance is not an added substance such food shallnot be considered adulterated under this clause if thequantity of such substance in such food does notordinarily render it injurious to health.”Use of the word contamination for the unintendedpresence of a PMP or PMI would be appropriate on theassumption that 1) a PMP or PMI would be defined as a“poisonous or deleterious substance” by the FDA, 2)the grain or seed would be considered food under thislaw, and 3) the FDA would not define a quantity tolerance.We believe that the FDA currently applies theseassumptions to PMPs and PMIs. Currently, their unintendedpresence would be contamination. FDA statutes or theinterpretation of statutes could change for some specificproducts and make the use of the word contaminationfor unintended presence inappropriate for someplant-manufactured substances. Unintended presencebelow a tolerance would not constitute adulteration orcontamination. Contamination is a value-based classificationthat has its origin in policy decisions. Policy makersmust decide whether a definition of a tolerance has thepotential to solve economic problems associated withPMPs and PMIs.

OUTFLOWS TO SEED STOCKSThe management of confinement to high levels ofstringency with respect to foundation and research seedis of special importance. Unintended presence inresearch and foundation seed stocks has the potential tospread a trait more widely and to avoid dilution thatwould normally occur as grain passes through the bulkgrain handling system. The StarLink incident showedthat the most potentially serious kind of PMP or PMIconfinement failure is one that would result in unintendedpresence in commercial, foundation, or research seedstocks (http://www.starlinkcorn.com/starlinkcorn.htm).The regulations that governed its presence in foodestablished a zero tolerance. The StarLink failure hadeconomic implications but posed no concerns for humanor animal health and safety. This example demonstratesthat the economic consequences of confinement failureas well as risk to health and the environment per se areimportant concerns for the success of PMP and PMItechnology. The StarLink incident made low-leveltolerances look “attractive” to seed and grain producers.Isolation standards used within the seed industry includeprovision for minimum isolation from corn foundationproduction and corn research nurseries and provisionsfor verification that all such fields are known to the PMPand PMI producer. These standards explicitly state thelevels of seed purity they are meant to achieve. Undermost circumstances, they provide and upper limit to theconcentration of a PMP that could be present in seed.Because it is useful to separate the exposure associatedwith outflows into corn grain production fields from outflowsinto commercial and foundation seed productionfields, it also is useful to differentiate outflows thatcould result in the unintended presence of PMPs orPMIs in commercial and foundation seed from thosethat could result in unintended presence in breeder’sseed. Breeder’s seed is very pure seed produced underresearch conditions by self-pollination (selfing). It is usedas the source of foundation seed. In commercial seedsystems, all foundation stocks are restarted periodicallyfrom breeder’s seed. Foundation corn seed is normallylimited to three generations from breeder seed. 2Unintended presence in commercial and foundationseed can be spread to fields planted with the seed orseed produced from it, but spread from these sources isself-limiting, and the level of unintended presenceremains at about the level created when the contaminationoccurred. Unintended presence is eliminated when thestocks are restarted from breeder’s seed. Unintendedpresence in research and breeder’s seed could beself-perpetuating in some circumstances, and there is,in theory, no upper limit on the level of presence. Thispotential for a low-level tolerance in grain to lead to high-levelunintended presence in seed makes tolerance substantiallyless attractive for PMPs and PMIs and provides anargument for the use of a broader definition of contaminationwhen discussing PMPs and PMIs.SYSTEM DESCRIPTIONThis report consists of 1) a description of key rationalebehind design of the good practices; 2) flowcharts thatschematically illustrate how the good practices occur asintegrated sequences of management activities that haveimplications to materials fate in the PMP or PMI cornproduction system; and 3) a hierarchical outline of majorprocesses, subprocesses, and actions that have a bearing onmaterial flows (i.e., the fate of corn seed, grain, pollen,and plant residue) occurring in the production system.We have approached the processes involved in the productionof PMPs and PMIs in the way that these processes wouldbe approached for an exercise in process management. In abusiness situation, process management wouldbe used to manage cost and productivity, identifyimprovement opportunities, reduce employee stress,and leverage opportunities. In this report, we are mainlyconcerned with regulatory compliance and minimizingthe probability and negative consequences associated withfailure of confinement. Our focus differs from that of abusiness in two ways. First, a business would have a broaderview of optimizing their production system, going beyondregulatory needs to encompass all aspects of productdevelopment and commercial production. Second, weare concerned with monitoring outflows of material andtherefore make inflows and outflows of materials andresources a more explicit part of the model than woulda business process management exercise.2 There is no regulatory generation limit for corn foundation seed in most states. Washington limits foundation increases to 2 generations and is, in recent years, producing asignificant portion of the US foundation seed. The normal limit of 3 generations in industry practice is based on the combination of lack of need for more than 3 generationsin corn and the accumulated empirical experience in industry of incremental accumulation of impurities. With 3 generations of foundation increase, using conservative multiplicationfactors, 10 kg of breeder’s seed for a line used as a female can be increased to enough seed to foundation seed to grow hybrid seed for 37,000,000 ha, more thanthe area of the entire US corn crop.Introduction 11

DefinitionsSystem: A set of interrelated or interacting elements.Process: A list of activities that use resources to transforminputs into outputs.Subprocess: A part of a process.Activities: Component tasks that make up a process.Procedure: A specified way to perform an activityProcess Management• Identification of critical systems that must be defined,controlled, and maintained.• Critical actions must be repeatable and measurable.• System design must allow for changing needs.• Regulatory and customer requirements must beidentified and incorporated into the system.Process Management Implementation• Identify critical processes.• Analyze all processes, including inputs, outputs,and lost living material.• Document critical processes.• Train employees and communicate expectations.• Define measurement criteria.• Audit the process.Our objective is to create and study examples of the firstthree steps of process management implementation so thatindustry and regulators can use the examples when theyevaluate and implement real cases. Industry includes allsteps in process management implementation.Flowcharting is useful as a process tool because it• provides a visual representation of each step inthe process;• graphically displays functional activity and providesa visual understanding of interrelated occurrences;• provides an understanding of the entire process;• allows modifications and disconnects to be identified;and• displays a clear relationship between inputsand outputs.The flowcharts follow process control. Frequently,the process control corresponds to material flows.The symbols that we have used are standard symbolsused for process management with the addition of theinput/output oval. See Figure 2.Process TitleStart/EndFIGURE 2. SYMBOLSActivityHopefully, our examples point out the critical processfactors and control points, so that the industrialimplementation can balance biosafety, process control,and work efficiency. Those who implement processesshould not take the procedures presented here directly.When these procedures are put into action, users shouldadapt processes; add more detail; and monitor, measure,and revise activities and procedures to better achieve safeand efficient production.ConnectorInput orOutputDecisionReport orData

Processes can be described with varying levels ofaggregation and detail. Figure 3 describes a system forthe creation and production of PMPs and PMIs at avery high level of aggregation.In our situation, we have made the flowcharts for thesesystems that are not as detailed as would be required bya process management exercise, but they provide moreexplicit material flow descriptions than would be presentin typical process management designs.FIGURE 3. PMP/I PRODUCTION SYSTEM PROCESSESStartTransformationand Selection inthe LaboratoryPreliminaryEvaluation in theGreenhouseBreed ProductionCultivars in theResearch NurseryStore andInventoryResearch SeedFoundation andField ProductionGrain Transport,Processing,Storage, andShipmentTo Pharm orIndustrialProcessorIntroduction 13

Laboratory andGreenhouse ProcessesResearchers transform plants in laboratory containment.The first generations of recombinant plants are grown inlaboratories and greenhouses to ensure containmentwhile researchers do preliminary selection and gatherthe information necessary for applications for fieldrelease. The purpose of containment at these early stagesof development is to prevent or restrict the transfer ofrecombinant DNA from transgenic organisms inside thelaboratory or greenhouse to reproductively compatibleorganisms in nature. For traits that are intended to enterthe food chain, laboratory and greenhouse containmentcan be thought of as the beginning of a sequence ofsteps, gradually reducing isolation from the environment.For compounds with an inherent hazard, it is moreappropriate to think of laboratory and greenhousecontainment as part of ongoing containment orconfinement that may be adjusted to fit the natureof the risk as knowledge increases.In addition to containment, laboratory and greenhouseoperations are characterized by the possibility of theexistence of different traits (characterized by thecompound to be produced), vectors (characterizedby the genetic sequence to be inserted), and events(characterized by derivation from DNA incorporationon a specific occasion). For reasons discussed below,this report focuses mainly on the possibility of error inearly stages of development that would result in thewrong trait or event being planted in the field.NIH GUIDELINES FOR CONTAINED PROCESSESThe NIH (2002) has set out rules for contained processes.The NIH Guidelines for Research Involving RecombinantDNA Molecules “specify practices for constructing andhandling: (i) recombinant deoxyribonucleic acid (DNA)molecules, and (ii) organisms and viruses containingrecombinant DNA molecules.” These rules are mandatoryfor institutions that receive NIH funding or are affiliatedwith such institutions. They are voluntary for otherparties but are widely adapted for work with PMIs andPMPs. Some detail about the NIH rules is presentedhere as an example of the way in which organizationsparticipating in PMP and PMI development wouldaddress issues associated with biosafety.The NIH guidelines require audits of the safety systemsthat are outlined. Some form of audit of the participantsis an important part of the PMP and PMI biosafetysystem.The NIH guidelines have created a climate withinwhich laboratory biosafety is taken very seriously byparticipants. One of the major requirements is that eachorganization involved creates an Institutional BiosafetyCommittee (IBC) to oversee institutional compliance.Sometimes, responsibility for biosafety in private industryclosely follows corporate lines of authority and a separatecommittee may be superfluous. In other examples,private research may have some of the same organizationalcomplexities that are characteristic of public researchinstitutions, and there would be good rational for theequivalent of an Institutional Biosafety Committee.The oversight under NIH varies depending on the levelof risk involved. There are six categories of experimentsinvolving recombinant DNA: 1) those that requireInstitutional Biosafety Committee approval, ResearchAdvisory Committee (RAC) review, and NIH Director

approval before initiation; 2) those that require NIH/Officeof Biotechnology Activities and Institutional BiosafetyCommittee approval before initiation; 3) those thatrequire Institutional Biosafety Committee andInstitutional Review Board approval and RAC reviewbefore research participant enrollment; 4) those thatrequire Institutional Biosafety Committee approvalbefore initiation; 5) those that require InstitutionalBiosafety Committee notification simultaneous withinitiation; and 6) those that are exempt from the NIHGuidelines. The NIH rules illustrate that oversightof PMPs and PMIs should vary depending on theassociated risk.In the NIH regulations, organisms are classified into riskgroups based on the hazard that they present. Biosafetylevel containment levels correspond to the risk groups.Biosafety level 4 provides the most stringent containmentconditions and biosafety level 1 the least stringent. Forexample, conventional greenhouses (with the possibleaddition of screening and impervious floors) are sufficientas level 1 containment, where recipient species that donot interbreed with weeds are used in combination withdonor species that are not exotic infections agents. Arecipient plant that intercrosses with weeds or a donorthat is an entire exotic infectious agent might requirelevel 2 greenhouses with locks, 30 mesh screen,impervious floors, collection of runoff water, andan autoclave.The NIH biosafety levels depend on the level of hazardand implicitly incorporate the principle that risk is theproduct of the hazard level with the probability ofoccurrence. To maintain a given tolerance for risk, theconditions of containment must ensure a lower probabilityof exposure if the level of hazard is higher. Conversely,where hazard is low, the degree of stringency inconfinement need not be as high. Risk managementprocedures within organizations involved in PMPs andPMIs could establish hierarchies of risk and biosafetyconfinement level that would be equivalent to thoseestablished by the NIH.The relevant containment programs can be divided intofour categories: 1) a set of standard practices that aregenerally used in microbiological laboratories; 2) speciallaboratory procedures, equipment, and installations thatprovide physical barriers that are applied in varyingdegrees according to the estimated biohazard while plantsare in culture and in the laboratory; 3) special greenhouseprocedures, equipment, and installations adapted for largeplants; and 4) biological containment measures that limitthe impact of any unplanned exposure.The following is from http://grants.nih.gov/grants/guide/notice-files/NOT-OD-02-052.html (accessed June 9, 2004):Some recombinant DNA requirements requireapproval in advance of initiation of the experiment. Insome instances, approval must be secured from theinstitutional biosafety committee (IBC), and, in others,from the IBC and the Recombinant DNA AdvisoryCommittee of NIH (RAC).Notification to either body must include:a) the source(s) of DNA;b) the nature of the inserted DNA sequences;c) the hosts and vectors to be used;d) a statement of whether a deliberate attempt willbe made to obtain expression of a foreign gene;e) containment conditions specified in the Guidelinesthemselves.Four categories of experiments are exempt from the reviewand approval requirements:a) those that are not in organisms or viruses;b) those that consist entirely of DNA segments froma single nonchromosomal or viral DNA source;c) those that consist entirely of DNA from a prokaryotichost, including its indigenous plasmids or viruseswhen propagated only in that host or whentransferred to another host by well establishedphysiological means; andd) certain specified recombinant DNA moleculesthat consist entirely of DNA segments fromdifferent species that exchange DNA by knownphysiological mechanisms.Minimum Compliance Action: Establish a five-personcommittee charged with the requirements to reviewall research involving the use of recombinant DNAmolecules.The NIH guidelines have been widely copied worldwideas the primary model for regulation of physicallyconfined research.A fully indexed, hyperlinked copy of the NIH Guidelinescan be viewed on line or downloaded athttp://www4.od.nih.gov/oba/rac/guidelines/guidelines.html.Laboratory and Greenhouse Processes 15

NIH GUIDELINES AND PMPS OR PMISWe have used the NIH Guidelines as a model in theformulation of the flowcharts for laboratory andgreenhouse procedures.The majority of the provisions of the NIH Guidelinesconcern provisions for the handling of source and donororganisms that are either plant, animal, or humaninfectious agents. The provisions concerning plantsproducing compounds where the primary concern istoxicity are relatively limited.For plants producing toxic substances, the NIH Guidelinesdifferentiate according to the toxicity of the substances.Highly toxic substances are those that are lethal to vertebratesat an LD50 of less than 100 nanograms per kilogram ofbody weight. Examples of this type of toxin are botulinumtoxins, tetanus toxin, diphtheria toxin, and Shigelladysenteriae neurotoxin. Experiments involving plants withthis class of toxin would require approval at the secondhighest level by the NIH/Office of Biotechnology Activities,and they would require level 3 containment, also the nextto highest level. The implications of risk in such systems isnot explored in this report because we expect that plantsystems producing such highly toxic substances would beso different as to require a different analysis.Below this level of toxicity, the NIH Guidelines do notdifferentiate based on toxicity. One of the primaryobjectives of future BIGMAP studies is to determinewhether further differentiation on the basis of toxicityor hazard should be used in the definition of appropriatelevels of confinement and oversight in field productionof PMPs and PMIs.Under the guidelines, the PMPs and PMIs that areconsidered here could be managed in the greenhouseunder the equivalent of one of the two lowest NIHcontainment levels, depending on the nature of the donororganism. In practice, most or all will be handled underthe equivalent of biosafety level 2, which requires• access limited to individuals involved with experiments;• personnel must read and follow instructions;• a greenhouse manual to advise of consequences and givecontingency plans;• a record of experiments and movement into and outof the greenhouse;• containment of plants during movement into or out ofthe greenhouse;• biological inactivation of experimental organisms at theend of the experiment;• decontamination of gravel periodically;• pest control program; and• sign indicating that a restricted experiment is inprogress with plant names, person responsible, andspecial requirements.If the compound being produced is of sufficiently lowtoxicity and meets the other requirements for biosafetylevel 1, the requirements are• personnel must read and follow instructions;• procedures appropriate for the organisms;• record kept of experiments in the greenhouse;• biologically inactivate experimental organisms at theend of the experiment; and pest control program.These lists are based on those presented in Adair et al.(2001). The procedures for the design and operationof the containment facilities also are summarized inthat document.LOSS FROM CONTAINMENTThe buildings are designed for containment, and with theexception of natural disaster, loss of organisms would beeither an error in the design or operation of the building.If one assumes compliance with NIH Guidelines, loss oforganisms from greenhouse containment is not generally amajor exposure concern at this stage.Containment 1evels 1 and 2 are not designed to containpollen. 3 Where greenhouses are in proximity to cornfields,there would be some probability of outcrossing. Wesuggest that this probability is low because of the size ofthe source and the restriction of pollen flow as air leavesthe greenhouse. 4 The location of greenhouses nearcornfields also would be infrequent. Physical containmentprocedures and the risks associated with their use are notreviewed in this report. The risk associated with PMP orPMI pollen outflow from greenhouses will be subject offurther study.3 Corn pollen is 70-90 µm in diameter, depending on the degree of hydration. Level 2 containment requires a 30 mesh screen. The opening in a 30 mesh screen is more than500 _m. Some corn pollen will pass through.4 Exhaust vents are normally at the top of the greenhouse and gravity works against pollen exit through the screens.

GOOD LABORATORY PRACTICEGLP conditions are government requirements for studiesthat support applications for research or marketing permitsfor products regulated by the FDA or the EPA. GLPstandards have been negotiated by the Organization forEconomic Development (OECD, 2003) and are requiredby most members for submissions concerning transgenicplants. In some countries, requirements for good laboratorypractice carry the force of law.GLP requires• Sound protocol;• Qualified, trained personnel;• Standard operating procedures;• Proper and adequate facilities;• Calibrated and maintained equipment;• Fully retrievable raw data; and• Overview by an independent quality assurance officer.The USDA does not require GLP standards for informationsubmitted for the approval of PMP or PMI production inthe field. Conformance to the spirit of GLP would providea basis for ensuring that the records are reliable. Reliablerecords ensure that events that are introduced into thefield will be correctly identified and associated with theappropriate documentation. Good manufacturing practice(GMP), a standard similar to GLP, will be applied toPMP production by the FDA. PMI production does notfall under an equivalent regulatory system, and someexploratory and development activities do not fall underGLP regulations.Institutional biosafety committees established under theNIH Guidelines provide the basis by which conformityto GLP is ensured where it is required and may serve as auseful model for institution of standards for PMPs andPMIs that are consistent with the spirit of GLP.The flowcharts for laboratory and greenhouse procedurespresented here are examples of the sound protocolrequired under GLP. Such protocols are the basis foravoiding mislabeling of cultures and plants. We assumethat the spirit of GLP will guide PMP and PMI practicethroughout all phases of exploration and development andthe commercial production of PMIs.DEVELOPMENT OF PCR 5 TESTSAfter an event-specific test has been created, PCR providesa third secure means to verify identity, in addition to labeland location, which can confirm the plant’s source.The development of event-specific PCR at the earliestpossible time is useful from the perspective of both thedevelopment of the transformation event and of riskmanagement. These reasons include exclusion ofcommingling and outcrossing and confirmation of theadequacy of containment or confinement. The flowchartsindicate times when PCR typing can be done. Requirementsfor PCR typing and its timing are possible areas for futureregulation. Our flowcharts provide points at which checksfor outcrossing can be done with PCR. Availability of thesequences for the public sector is a contested issue that isnot considered in this report.Typically, commercial transformation generates dozensof events where the same vector has been used. Some ormany may be indistinguishable in their performance inthe laboratory and their response to probes designed tocheck for the presence of the transformation cassette. Inthe greenhouse, cross-pollination between events producedfrom the same, or different, origins is possible. Theintegrity of the regulatory evaluation requires that theevents that go to the field be correctly identified. Thus,PCR tests that unambiguously identify a junction of theinsert and host genome are developed early. The PCRprobe is developed by first sequencing a DNA segment inthe transformed organism starting from the inserted DNAand continuing into the host genome. A PCR probe ofapproximately 200 base pairs is synthesized using thejunction sequence from one side of the insertion andanother site in the insert. The use of the event specificprobes before field release provides a definitive check onthe quality of the laboratory labels and location records.In theory, error in the development of an event-specifictest could create an opportunity for the release of thewrong event. In practice, the DNA sequence-specificnature of the test makes misidentification very remote.Sequence links the probe and event unambiguously. Thereare limits to the power of detection of PCR detection,but the test is sensitive to less than 1 part per 10,000(assuming tests of multiple pools).5 PCR stands for the polymerase chain reaction. It is one of a group of normally very precise procedures for identifying s specific DNA sequence and is the basis of mostDNA fingerprinting.Laboratory and Greenhouse Processes 17

The event-specific probe becomes a key tool for exposuremanagement, ensuring the identity of the event. It alsomay be an important tool in the assessment of theeffectiveness of confinement for field tests and fieldproduction, but protein tests may be cheaper and morerelevant for monitoring adulteration of grain in neighboringfields. The protein tests are not theoretically as sensitive asPCR, but lower cost allows more samples, and if samplingcan take place before dilution, an increase in samplingpermits more effective sensitivity. Errors in the developmentof protein tests could lead to undetected exposure. If sucherrors occurred, they would be probably to be at lowconcentrations, near the limits of detection.Final evaluation of the detection methods can providequantitative determination of the limits of their sensitivityand the possible sources of error in their application.Because of the possibility for evaluation before use andbecause evaluation can be made with high levels ofcertainty, the development of the detection methods isnot included in the process flow descriptions. Reliabledetection methods are assumed to be available for use.There are policy issues associated with the use ofevent-specific PCR for exposure assessment. EventspecificPCR tests are not currently required by APHISfor field-confined release. Given that we have assumedthat failure of isolation remains a possibility, it is logicalfor us to assume in our procedures that event-specificPCR tests would be developed before field tests withPMPs or PMIs to provide a means of monitoring andmitigating for the effects of such events. The outcome ofBIGMAP studies will provide a measure of the utility ofthis measure. Our procedures assume that use of PCR fordetection of exposure will be a part of the activities of theusers of PMPs and PMIs and allow for us to estimate therisk that would be associated with not using this tool.There are policy discussions about the advisability ofplacing the information for the preparation of event-specificPCR primers with government surveillance agencies orwith the public. The information on the potential forexposure will be pertinent to the discussion of policy onaccess to PCR primers and the confidential businessinformation used to prepare them.TRANSFORMATION AND REGENERATIONOur flowchart of the physically confined laboratoryprocess begins with insertion of DNA into plant cells.Error is possible in the creation of the vector. The probabilityof errors in the creation of vectors increases with thecomplexity of the vector construct. As the number ofligations increases, the possibility of including extraneousDNA also increases. This extraneous DNA poses someconcerns, but this report assumes that detection of sucherrors falls into the domain of the regulatory review andis outside the scope of this study. Insertion can cause therearrangement of the construct and incorporation ofDNA fragments from either the host or the vector. Wealso assume that irregularities created in the transformationprocess will be handled by the regulatory process or duringevent sorting. Our chief concern here is for errors thatwould result in material flows in the field or in the wrongtrait being in field plots.Human error is the more important potential source oferror associated with the laboratory and greenhouse portionsof the life cycle of PMPs and PMIs. The description ofrelevant record keeping and containment activities at thisstage in product development reflects the possibilities forhuman error.In transformation, the principal concern is that a cellline or line of plants might be mislabeled and that, as aconsequence, the wrong event will go to the field and behandled in a manner that is not appropriate for the event.PMP or PMI production will remain under regulationand mishandling would not be expected to result in anadditional risk of unintended direct exposure, except fora PMP or PMI trait misidentified as a food trait. Ourconcern is with this latter low-frequency possibility as wellas with the possibility that the hazard associated with anevent might not be correctly identified. Misclassificationof a PMP hazard would be more notable issue in a potentialfuture regulatory system that used hazard as a basis fordetermining the stringency of confinement. More emphasisis placed on record keeping in the flowchart proceduresbecause mislabeling is a greater concern than escape fromcontainment at this stage.

The major tool available to avoid possibility ofmisidentification is use of duplicate identification systems.The most common duplication is to both maintain labelsand a grid system that identifies plants and cultures bytheir location. This system is a common and provenwarehousing technique. If the label and location identifierdo not agree, then the conflict can be investigated and thediscrepancy resolved or the plant or culture discarded.GREENHOUSE SUBPROCESSESIn the greenhouse, there is an opportunity for mislabelingof plants and also of crossing between plants that couldresult in seed in the next generation having two events,one intended and the other not intended.The primary tools for avoiding mislabeling are the use ofmultiple labels on plants or their pots and the use of gridsystems for plants in beds. For crossing between plants,the primary tool is pollination control. Flowers are covered(bagged). Pollen is not allowed to disperse in thegreenhouse. Plants are self-pollinated or pollinated fromidentified source plants. Records are kept of the source ofall pollen used in crosses.Event-specific genetic tests on progeny offer the possibilityof detecting unintended crosses.SUMMARYMisidentification of events is the main PMP and PMIsafety concern associated with laboratory and greenhousecontainment that is relevant for the general public.Misidentification can be associated with commingling oroutcrossing, as well as simple mislabeling. The NIHGuidelines, institutional biosafety committees, and GLPprovide models for the appropriate framework for safecontainment and establishing trait identity chains. Theflowchart describes steps that ensure correct identificationof events.Laboratory and Greenhouse Processes 19

PCR,Expression &Copy NumberTestsA toDiscardPlant OKforGreenhouse?NoA toDiscardYesLab Checks onPMP IdentityEvent InfoDatabaseDestroy ExcessPMP MaterialA toDiscardTransformedSmall Plant, T0A toDiscardLaboratory and Greenhouse Processes21

FIGURE 5. PMP GREENHOUSE OPERATIONSGreenhouse TeamTrain NewTeamMembersPlants &SeedsLabeledCorrectly?NoInformationAvailable?NoA toDiscardYesYesDoes TeamUnderstandSOP?NoRelabel Pots& StakesYesPrepareGreenhouseArchive EventInfo forRegulatory UseRecords forRegulatoryArchiveGreenhouseMeetsIsolationStandards?NoGrow Plants,ManageGreenhouseYesEvent SpecificInformationPrepareOperationalPlanIs Plan inConformancewith GLP andNIHGuidelines?YesNoTransformedSmall Plants,T0Seed from LaterGenerations, TNPlant SmallPlants or SeedLabel Pots andStakesPots andStakes

Lab Checks onPMP IdentityEvent InfoDatabaseEvent SpecificPCR Test if N>= 1Event InfoDatabaseRegulatoryTestsEvent InfoDatabaseEvent InfoDatabaseIs EventOK?NoIdentify as LowPriority EventEvent InfoDatabaseYesPreparePollination PlanCross and SelfWas PollenControlledto SOPStandards?NoIdentify &DiscardContaminatedPlantsYesPrepare Reportto APHISAPHIS EventReportHarvest PMPSeedA toDiscardFIGURE 5 CONTINUED ON NEXT PAGELaboratory and Greenhouse Processes 23

FIGURE 5. PMP GREENHOUSE OPERATIONS (CONTINUED)Harvest PMPSeedLabel SeedSeedLabeledCorrectly?NoYesTestExpression ofPMP in SeedEvent InfoDatabaseSeedRegulatoryTestsEvent InfoDatabaseDestroy PMPMaterialA

Event InfoDatabasePlan Action forNextCycle. EventInfo Synthesis.Report & PlanArchive EventInfo forRegulatory UseRecords forRegulatoryArchiveIs EventReady forthe Field?YesNoSeed of TransformedPlant for Recycling toGreenhouse.TN+1RegulatoryArchivePrepare Reportfor APHISReport toAPHISSeed ofTransformed Plantfor Field Trial.TN+1Figures and Tables 25

NurseryProcessesPMP and PMI field trials can be classified by size. Largertrials are used to produce foundation seed, material fordevelopment work (procedural, analytical, pre-clinical andclinical trials), and commercial quantities. These largertrials use distance offsets (isolation) as the primary meansto achieve confinement. Smaller, research-oriented trials,in contrast, achieve confinement by using methods wherethe floral structures either bagged or removed, pollinationoccurs by hand, and pollen is not allowed to disperse intothe air. The chance of pollen escape from trials representingthese two scenarios is very different. The isolation standardsfor the two kinds of production could be different,depending on the nature of the expressed product.This classification into large- and small-scale trials is largelybased on existing practice in corn research and productdevelopment and is driven by the nature of corn and ofvariety increase. Regulation will have to address all of thepossible combinations of large and small trials with variousforms of pollen control, but the procedures presented herereflect procedures that are likely to be followed.For simplicity, we refer to the smaller, research-orientedtrials as nurseries: where plants are grown under supervisionand with special care. The growing and hand-pollinationof plants in coordinate grids is a system that allows manylines or varieties to be maintained and crossed in smallareas. As plot size increases, the cost of some of the specialprocedures used in nurseries becomes prohibitive, and theneed for hand-pollen control within the trial decreases.For PMPs and PMIs, the transition size at which onedecides to divide nursery and field production differs fromthe size associated with the transition for normal corn, butthe distinction remains valid. Although corn nurseries aredesigned to maintain varietal integrity, the pollen controlprocedures that have been developed are easily adapted toprovide for confinement. Process flows for larger scaletrials, seed, and foundation seed production are groupedwith field production systems.The kinds of events under study in nurseries can bematerially different from those that would be present inthe larger trials. In nurseries, many events have existed forfewer generations and much less is known about them. Inearly trials, there is significant event sorting, where eventsmay differ in the nature of the construct and its functionalityin the transformed plant. At this stage, events may containa partial construct that is dysfunctional. Events maycontain a complete construct that is dysfunctional due togene silencing. Events may contain multiple copies ofthe construct. Events may be associated with a mutationthat causes altered growth. Events may display aberrantsegregation. Some events may not be associated withevent-specific tests. Most members of these categorieswould not be grown in larger trials.

NURSERIES AND THEIR MANAGEMENTNursery procedures have been developed from plantbreeding operations. Nurseries are normally designed asgrids. Single-plant grids are appropriate for some earlystage nurseries. More commonly, plants are be grownin rows 2-5 m in length, and all of the plants in a rowcome from the same ear and have the same parentage.This latter pattern is referred to as ear-to-row. It simplifiesrecord keeping because a single record can describeall the plants in the row. Each row is identified by itscoordinates in the rectangular grid. Typically, rows havea stake or a tag that identifies them to technicians.Rows from inbred parents my contain plants that areessentially identical. Rows that are the result of a crossor a transformation event may be segregating, and eachplant can be different, but they will all be descendedfrom at least one single plant.Nursery information systems typically manage recordsabout plants in the nursery and seed in storage. Thenursery information management system links the rowin the field with a source of seed in a seed storage facility.The seed in the seed storage facility is put in a bagidentified with a reference number and a pedigreedescribing its origin. The reference number links thestored bag back to a row in a previous nursery where itwas produced. The nursery information managementsystem records various breeding actions in the nurseryrow. Plants in the row may be self-pollinated (selfed), ortheir female shoots may be cross-pollinated (crossed)with pollen from another plant in another row. Whereplants in a row are segregating, the individual plantsinvolved in a cross are marked with a number. Any earsthat are harvested are normally given a unique inventorynumber. The nursery system generates a pedigreebased on the cross that was performed or pedigreescan be written manually. Record keeping for seed ininventory is integrated into the system. By includingboth inventory and nursery information, the systemreduces error in the records about plants in the fieldand seed in the inventory.There are two common nursery operations used in earlygenerations. In the first operation, new transformationevents are self-pollinated to move toward homozygosity(internal genetic uniformity). Homozygous lines becomestable and replicate true to type. By using comparisonwith the recipient line, a homozygous transformed linecan be useful in detection of any genetic damage thatoccurred in association with the DNA insertion event.The second operation is crossing. Crossing may be partof the process of backcrossing or production of testcrosses. In backcrossing, a line carrying an event iscrossed to a recurrent parent. Progeny carrying thetransformation event are selected either based onphenotype or the gene itself and then the cross to therecurrent parent is repeated. After four to eight generations,the genome of the recurrently parent is almost completelyrecovered in a line with the transformation event. Thebackcrossing of traits into a set of commercial lines issometimes called “trait integration.”Test crosses produce hybrid seed that can be used togrow hybrid plants and produce their seed to be usedfor the evaluation of expression levels.Typically, one or two generations of these operationsare performed in the greenhouse while information isgathered about the events and an event-specific test isdeveloped. Transformation events may frequently becrossed to two or three recurrent parents immediatelyin the greenhouse. These crosses are the first step inbackcrossing. The recurrent parents represent linesadapted to the various situations in which the eventmight be used. When the events are planted in nurseries,backcrossing is continued and extended to additionalparents. If it was not initiated in the greenhouse,backcrossing is started in the nursery. In a breedingnursery, an event may be represented by dozens ofrows, each with its own pedigree and a set of plannedcrossing operations that generate ears that will berecorded and inventoried and used to plant thenext nursery.The third major breeding operation is the increase ofseed of backcrossed lines. After sufficient similarity tothe recurrent parent is achieved, and a plant that ishomozygous for the event is located, seed is increasedby selfing until there is enough seed to grow the line inan isolation plot. This increase leads to the creation ofthe first stock of breeder seed. Corn foundation seedgroups generally like to get 10,000 to 50,000 seeds.Seed is increased by selfing, not only to ensure geneticpurity but also to allow a check that each ear ishomozygous for the trait.Subsequent stocks of breeder seed can be producedfrom a random sample of lines sampled from thefirst breeder seed stock. They are always producedby selfing.Nursery Processes 27

CONFINED NURSERIESThe procedures that have been approved by APHIS forcorn nurseries are the same as for all PMP production:• At any time during the field test when PMP plants areallowed to shed pollen, ensure that no other receptivecorn plants are grown within a radius of 1.0 mi(1.6 km) of the transgenic plants.• Where pollination of PMP plants is controlled, ensurethat transgenic corn is planted not less than 28 daysbefore or 28 days after the planting dates of any othercorn that is growing within a zone extending from0.5 to 1.0 mi (0.8 to 1.6 km) of the transgenic plantsto ensure that there is no overlap in anthesis.• There are no other restrictions on corn that is grown atmore than 1 mi (>1.6 km) from the transgenic plants.These standards have been established using informationabout pollen number (Pohl, 1937; Ogden et al., 1974;Paterniani and Stort, 1974; Poehlman and Sleper, 1995;Coe et al., 1998), pollen size (Pohl, 1937; Funkhouserand Evitt, 1959), duration of pollen shed (Lauer, 1998),viability (Shoper et al. 1987; Luna et al., 2001), anddispersion. Because pollen size and mass have not changedsignificantly, distribution measurements done in the pastremain valid (Jones and Brooks, 1950; Wych, 1998;Di-Giovanni et al., 1995; Burris, 2001). Wind speedhas been verified as a factor (Raynor et al., 1972; Burris,2001). Theoretical work has been done on the maximumdistance of pollen flow (Raynor et al., 1972; Ireland et al.,2001; Luna et al., 2001). New information and theoryare contributing to the validation of dispersion times(Fonseca, 2002). Recent research has been oriented onthe impact of transgenic pollen inflow in corn seedproduction (Ingram, 2000; Burris, 2001; Eastham andSweet, 2002).For nurseries, it is possible to envision additionalmeans beyond pollination control and isolation by whichoutcrossing and its consequential risk can be mitigated:• In hand-pollinated nurseries, internal non-PMPor -PMI border plantings may act as barriers anddiminish the concentration of PMP and PMI pollenemanating from the source.• Reduction in the number of transgenic PMP plantswithin the trial to diminish the concentration of PMPand PMI pollen at the source.• Reduction in the proportion of transgenic plants in thenursery to diminish the concentration of PMP andPMI pollen at the source.• Active daily surveillance by trained personnel of fieldduring flowering to ensure pollen control.• More comprehensive standard operating proceduresto minimize potential loss of pollen, plants, or seedsfrom confinement than could be implemented forlarger fields.Such nursery procedures reflect the scientifically documentedinfluence of the size of the source (Ministère de l’Agricultureet de la Pêche, 2002) and borders (Jones and Brooks, 1950)on the rate of outcrossing in corn. Possible differencesbetween the nursery and large plot procedures andstandards reflect the influence size of the source as well asdifferences in borders, pollen control, and the concentrationof PMP or PMI pollen at the source.Multiple mechanisms contribute to the efficacy of borderrows within the field in reducing concentration of pollinationsin a receptor field. Border rows at the periphery of thetrial work partially because pollen grains on receptive silksare competitive and deteriorate with time after they arereleased. Pollen that has traveled further is older and lesslikely to compete well with younger pollen grains fromthe border and is less likely to effectively pollinate a kernelin a receptor field. The border also acts as a partial physicalbarrier. These effects reduce the level of PMP and PMIgrains in any corn that might be pollinated from insidethe perimeter border. Border rows also diminish theconcentration of PMP and PMI pollen at the source.Reductions in this concentration reduce the probability ofescape but may not prevent escape. Reduction can reducethe risk associated with escape of a hazardous PMP orPMI. If one assumes a fixed amount of PMP or PMIpollen escapes the field, increasing the amount ofnontransgenic pollen of similar viability moving with itwill reduce the concentration of PMP or PMI in the

target field in inverse proportion. For most hazardoussubstances, risk is proportionate to concentration and isbe lowered as the concentration is lowered. If a PMP orPMI is associated with hazard, covering tassels, keepingthe ratio of transgenic to nontransgenic plants low, limitingthe number of PMP and PMI plants, or the use of malefertile nontransgenic borders that reduces the concentrationof PMP and PMI pollen at the source, all could reducethe risk associated with undetected escapes.Possible differences between the nursery and large-plotprocedures could reflect the relatively smaller amountof PMP- or PMI-pollinated material from borders thatwould have to be destroyed. Current APHIS standardsexplicitly preclude borders that are managed externalto the field trial, so as to avoid potential loss oftransgenic material from confinement through impropermanagement of that potential PMP or PMI materialthat may occur in borders separated procedurally fromthe contents of the trial. In practice, within field trialsthere are frequently plants that are neither PMP nor PMIand are allowed to open pollinate. These plants contributeto the reduction in concentration of PMP or PMI pollenin outflows. The nontransgenic male plants occurring onthe field trial periphery have the benefits of borders.These designs seem appropriate under APHIS conditions,because all materials within the field trial are managed asif transgenic.Misidentification and unintended intercrossing is aconcern in nurseries containing many regulated traits.This concern is similar to that discussed in greenhousework. Because the plants are handled in systems withwell defined record keeping and identification, such risksare minimized. Separation of transgenic nurseries withregulated events from nurseries with no regulatedevents allows for checks for unintentional crosses andmisidentification prior to the release of inbreds for largerscale trials. These checks can be made with event-specificPCR probes. Before the release of breeder’s seed, theidentity of events should be rigorously determined withevent-specific PCR tests. Event-specific PCR also can beused to test the component lines used to maintain breederseed stocks.Unintended events from the nursery system for regulatedevents pose a special problem in nurseries with noregulated events. This can be avoided by providingextra isolation of nurseries with regulated events fromnurseries with no transgenes or transgenes that havebeen deregulated and approved for food and feed use.Various levels of size, isolation, borders, and pollencontrol can be combined to achieve a given goal for cornpollen confinement. Control of the concentration ofPMP and PMI pollen at the source can be used to reduceany risk associated with escape from a hazardous PMPor PMI source. We design our model to reflect theseprocedural options.When BIGMAP results are used for policy discussions, itmay be possible to consider regulatory changes. For traitsof hazards known to be below some level, combinationsof the above-mentioned procedures may allow considerationof isolation distances less than 1 mi (

PRINCIPAL DIFFERENCES BETWEEN NURSERY ANDFIELD PRODUCTION PROCEDURESThe procedures for larger scale field production and fornurseries are generally similar, but nurseries have somesignificant differences:• Nursery operations depend more heavily on teams toplant, pollinate, and harvest.• Procedures give more attention to pollen control.• Outcrossing during hand pollination is rare.• Procedures give less attention to roguing.• Planting is nearly always in rows and ranges. Fieldproduction is less likely to be in rows and ranges.• Rows of 2-5 m in length are described by range androw coordinates.• Record keeping is required for each row.• Planting is complex and prone to error. Plantingerrors can result in misidentification of rows.• For some smaller nurseries, mechanical proceduresbecome optional, and it may be easier to performprocedures by hand than to clean the equipmentbefore and after use.• Some events are eliminated before flowering.• The entire nursery is unlikely to be cancelled inmid-season, whereas a failed production field couldbe canceled in mid-season.• Pollen inflow is less of a consideration because ofhand pollination.• Pollination is complex and prone to error.• Mixing of events is possible at pollination.• Labels on individual plants used in crosses can be lost.• Increases for one PMP event could be mixed with seed orpollen from another, resulting in incorrectly labeled seed.• Sometimes ears are harvested very early to speedplanting of the next cycle.• Not all ears are pollinated in the nursery. Many ears areopen pollinated. There is more relatively more dry matterfor destruction in relation to the grain harvested.• Errors in labeling are possible at harvest.• Seed processing is all in batch mode with easilycleaned equipment.• Errors in labeling are possible during processing.• The number of items in inventory can be very large.The reliance on teams opens the door to discussion ofquality and teamwork in management that goes beyondthe scope of this document. In practice, the relationship ofthe researcher as manager with a team has a significantimpact on errors and error rates.SUMMARYPMP and PMI nurseries and research inventory systemsare subject to both the possibility of misidentification ofevents and lines and to the possibility of genetic outflowof pollen, seed, and plant material. The unique aspect ofresearch nurseries is that they may contain a number ofevents, and these events can be associated with varyingdegrees of hazard.Conventional corn research nurseries are managed withprocedures that minimize the chance of error in theidentification of lines. When used for PMPs and PMIs,these procedures provide redundant systems for identificationthat allow the detection and correction of errors whenthey occur. Event-specific checks allow for the specificidentification of events in lines, especially when they arereleased from research nurseries to be used in foundationand field production. Event-specific tests also can beconducted to confirm that undesired genes are not present.The procedures for the prevention of outflows ofgermplasm and materials for nurseries are much the sameas for sterile field production. It seems prudent to classifythe risk that nurseries pose based on the hazard associatedwith the event with the highest LD50. Nurseries containinghazardous events likely pose greater concern than fieldproduction of less hazardous events, even when the areaof the field production is much larger.Nurseries containing hazardous events have a componentof risk that is associated with the number of tasks that areperformed in them. A research nursery may require tensof thousands of hand pollinations, and each has somepossibility of error. The work may involve dozens oftechnicians, so the frequency of human error is a concern.Field production procedures, especially those using sterilefemales and nontransgenic males, are inherently simpler.Where PMP or PMI nurseries are associated with greaterpotential risk due to uncertainties as to the nature of theexpressed product, isolation from nurseries with unregulatedcorn and from foundation seed stocks of unregulated corncan be an especially important consideration.

FIGURE 6. NURSERYNurseryManagement StaffRegulatorytrainingAuditNoTeam TrainingOKYesTrain StaffNursery PlanPrintEnvelopes w/Inventory IDand Range andRowInventoryRecordsStaffKnowledgeExperienceOK?NoNon-PMPInventoryEvent InfoDatabaseYesPrepareDetailedNursery PlanNursery PlanFill Envelopesfrom InventoryContainersPMP InventoryPlanOK forResources,NoObjectives?InventoryContainerAgrees w/EnvelopesNoYesRegulatoryNoYesApprovalYesFIGURE 6. CONTINUED ON NEXT PAGENursery Processes 31

FIGURE 6. NURSERY CONTINUEDFill Envelopesfrom InventoryContainersPMP InventoryInventoryContainerAgrees w/EnvelopesNoYesLabel NurseryBoxesArrangeEnvelopes inPlanting OrderField HistoriesField & TimeSelectionPlantingPositionAgrees withRange andRowNoFieldPreparationNeighborPlanting PlansYesFieldOK?NoYesRegulatoryNotificationB

Nursery PlanMark and LabelRanges andRows in FieldBRangeand RowLabelsagreew/PlanNoYesNursery PlanPrepare PlotLabelsYesRightNurseryCheck IDNursery Boxesfrom InventoryPlanting andLabeling PlotsYesPlanterCleanNoCleanPlanterPlanterNoIdentifyPlanting ErrorsPlantingTrainingPlanting CrewLabelMatchesEnvelopeandPlan?NoCanWork beDone?NoPlant ExtraRows toReplace ErrorYesYesRevise PlantingPlanRevisedNursery PlanCHART 7 CONTINUED ON NEXT PAGENursery Processes 33

FIGURE 6. NURSERY CONTINUEDLabelMatchesEnvelopeNoCanWork beNoPlant ExtraRows toandDone?Replace ErrorPlan?YesYesRevise PlantingPlanRevisedNursery PlanClean Tiresand RemovePlanterTiresNoClean?YesClean PlanterPlanter isNoClean?YesPlant Bufferand/or Non-PMP PollinatorYesPlanter isclean?CleanPlanterBuffer PlanterNoLabel, Sign and

Label, Sign andRecord PlotHistoryNoAreLabels,Signs &HistoryCorrect?NoYesPrepare Post PlantingReport for RegulatorsPost PlantingRegulatoryReportIsolation andBuffer CheckCorrect Buffersand IsolationYesAreIsolation& BuffersCorrect?NoCanIsolation&BuffersBeFixed?NoAYesFieldOperations andCleaningIsIsolationIntact?NoFIGURE 6 CONTINUED ON NEXT PAGENursery Processes 35

FIGURE 6. NURSERY CONTINUEDIsIsolationIntact?YesNursery PlanLab Checks onPMP IdentityEvent InfoDatabaseNursery PlanRegulatoryTestsEvent InfoDatabaseArchive EventInfo forRegulatoryRegulatoryArchiveEvent InfoDatabaseIs EachEventOK?YesIdentify Eventas Low PriorityNursery PlanNoEvent InfoDatabasePollination PlanNursery PlanIs FieldGoodEnough toPollinate?NoAYes

PollinationInspection.Remove Off-typesBefore PollinationTrainingPollinationCrewYesWithinStandard?NoWill RepeatWork?NoAYesNursery PlanConfirmPollinationSuppliesNursery PlanPollinationInstructionsPollinationInstruction RptReport Tasselat EmergenceMinus 5 DaysStart DailyInspectionReport TasselEmergenceYesTrainingOK?TrainingPollinationCrewFIGURE 6 CONTINUED ON NEXT PAGEAttach Tasseland ShootNoNursery Processes 37

FIGURE 6. NURSERY CONTINUEDReport TasselEmergenceYesTrainingOK?TrainingPollinationCrewAttach Tasseland ShootBagsNoAllTasselsCovered?YesNoPollinate andMark SelfsPollen OutflowCross andMark with ID ofMalePollen OutflowMark MalesUsed inSegregatingRowsAnyRevisionto PollinationPlan?YesRecordRevisions toPollinationsNursery PlanNo

Pollination ofSurrounding inTolerance?NoRemove &DestroySurroundingCropYesAllRemoved?NoYesMove PlantMaterial toContainmentYesPlantMaterialMoved?NoBNursery PlanPrepareHarvest Planand Bag LabelsHarvest PlanTrainingHarvest CrewNursery PlanLabel Bags andPlace in PlotsDoesLabeland Rowor PlantLabelAgree?NoFIGURE 6 CONTINUED ON NEXT PAGENursery Processes 39

FIGURE 6. NURSERY CONTINUEDNursery PlanLabel Bags andPlace in PlotsDoesLabeland Rowor PlantLabelAgree?NoYesHarvest,Huskand Sort. Earsinto BagsEarsRemaining?YesNoYesAll PMPPlantsRemoved?Discard PMP Field.Remove PlantsBefore FloweringANo

Contain PMP PlantMaterialYesAll PMPPlantsRemoved?Discard PMP Field.Remove PlantsAfter FloweringBNoAll PMPPlantMaterialContained?NoYesCheck FieldNumber & HistorySeedControl toInventoryField Operations toDestroy ContainedMaterial On-sitePrepare and ShipContained Materialfor Off-siteDestructionContainedMaterial to beDestroyedField Control toYear 2Summary 41

FIGURE 7. NURSERY INVENTORYSeed FromNurseryPlace HarvestBags inTransportContainers andLabelClean &Closed?NoClean & CloseTransportEquipmentYesTrainTransportTeamTransport PMPEarsReceive Corn,Verify, Label,RecordRecord Ear andTrack Weight &MoistureSeed FromNurseryPlace HarvestBags inTransportContainers andLabelClean &Closed?NoClean & CloseTransportEquipmentYesTrainTransportTeamTransport PMPEars

Receive Corn,Verify, Label,RecordRecord Ear andTrack Weight &MoistureYesDryerClean?Clean DryerDryerDry Ears to 12%NoClean DryerTrain PlantCrewDryer CrewDryerCleaned?NoYesRecord Weight andMoisture of Earsand TrashNursery PlanPrint and ApplyStorage BagLabelsNoFIGURE 7 CONTINUED ON NEXT PAGESummary 43

FIGURE 7. NURSERY INVENTORY CONTINUEDNursery PlanPrint and ApplyStorage BagLabelsNoShell CornYesShellerClean?Clean ShellerShellerDoLabels ofField andInventoryBagsAgree?NoCorrectInventory LabelYesClean ShellerIs ShellerNoClean?YesWeigh Grain andTrack MoistureUpdateInventoryRecordsNursery PlanTest Expression ofPMP in SeedEvent InfoDatabasePurity OK?NoMove Grain toContainmentCYes

Nursery PlanRegulatoryTestsEvent InfoDatabaseArchive EventInfo forRegulatoryRegulatoryArchiveStore Seed andRecordLocationUpdateInventoryRecordsCheck All Grainand Trash Weightsand MoisturesFix RecordsNoWeightConsistent?NoRecordsCorrect?YesYesFind Source ofErrorSpecialResolutionContain andDestroy All WasteFrom Field &ShellingAssembleContainedWaste FromField & PlantCWasteDestroyed?NoCompleteRegulatory ReportRegulatoryReportYesTo YearN+145

Images1. FRESH EXSERTED ANTHERS3..POLLEN GRAINS CAUGHT ONSILK TRICHOMES (HAIRS)© 2004 R. L. Nielson, Purdue UniversityStrongly pigmented silks of a popcorn hybrid.© 2004 R. L. Nielson, Purdue University2. CORN POLLEN WITH A DIME4. MULTI-COLORED “INDIAN” CORN© 2004 R. L. Nielson, Purdue UniversityPart of the color comes from the aleuron layer which is geneticallysimilar to the endosperm and can be seen through the outerpericarp layer. © 2004 R. L. Nielson, Purdue University

5. POLLEN DRIFT CONSEQUENCES,EFFECT OF DISTANCE8. POLLEN DRIFT CONSEQUENCES,EFFECT OF DISTANCEPurplish and redish kernels mixed with yellow on theears of a genetically yellow female plants due to pollendrift from adjacent row of “Indian” corn.© 2004 R. L. Nielson, Purdue UniversityPollen drift consequences, effect of distance. Purplish andredish kernels on the ears of genetically yellow plants due topollen drift from a source 11.44 m away.© 2004 R. L. Nielson, Purdue Universit6. POLLEN DRIFT CONSEQUENCES,EFFECT OF DISTANCE9. POLLEN DRIFT CONSEQUENCES,EFFECT OF DISTANCEPurplish and redish kernels on the ears of geneticallyyellow plants due to pollen drift from a source 3.8 m away.© 2004 R. L. Nielson, Purdue University7. POLLEN DRIFT CONSEQUENCES,EFFECT OF DISTANCEPollen drift consequences, effect of distance. A single purplishkernel on the ear of a genetically yellow plant, due to pollen driftfrom a source 15.2 m away. The effect of distance is increasedby the amount of yellow pollen produced by the yellow plantsbetween the source and the female rows.© 2004 R. L. Nielson, Purdue UniversityPurplish and redish kernels on the ears of genetically yellowplants due to pollen drift from a source 7.6 m away.© 2004 R. L. Nielson, Purdue UniversityImages 47

10. POLLEN DRIFT STUDIES CAN BE CONDUCTEDWITH YELLOW SOURCES IN WHITE FIELDS.12. SMALL PLOT PLANTERIn this case the yellow source was transgenic and the geneticstatus of the grain could also be measured. Photo courtesy ofthe Susana Goggi and Mark Westgate, Agronomy Department,Iowa State University.11. ONE YELLOW KERNEL ON A GENETICALLYWHITE FEMALE EAR MARKS POLLINATION.This planter plants two short rows at a time for corn nurseries.Planters come in many sizes. Image courtesy of Allan MachineCompany, Nevada, Iowa.Photo courtesy of the Susana Goggi and Mark Westgate,Agronomy Department, Iowa State University.

11. DETASSELING MACHINE16. EMPTYING AN EAR CORN DRYER IN A SEEDCORN PROCESSING PLANT.This one cuts the tassels. Others pull tassels withhydraulically powered rollers. Fields are detasseled byhand after the machines do the heavy work.14. LARGE CORN HARVESTER.Smaller driers could be used for production of non-food corn.Harvesters come in many sizes.15. SORTING TABLE IN A SEEDCORN PROCESSING PLANT.Sorting table in a seed corn processing plant. Containmentallows destruction of all waste.Images 49

Larger ScaleProductionThe primary characteristic of the larger scale production isthat pollination is not conducted by hand. PMP or PMIsystems process flows for larger scale trials, seed, andfoundation seed production are grouped with fieldproduction systems because of their similarity. We aredescribing as large production systems, fields extendingfrom foundation field size (infrequently as small as100 m 2 ) up to production fields (potentially hundreds ofhectares). Because the product is grain, the PMP or PMIplants must be pollinated. Our flowcharts describe bothproduction with PMP or PMI males, and sterile productionwith pollinators that are not PMPs or PMIs. Open pollinationby plants with the trait is required in production of foundationseed. In open pollination with trait plants, pollen flow isa much larger issue because of the amount of pollen beingreleased. Effectively, the source is larger (Ministère del’Agriculture et de la Pêche, 2002), and pollen dilutionis less of a factor (Jones and Brooks, 1950).The geographic scale of field production means thatindividuals or small groups carry out operations awayfrom supervision. The human aspect of managementin large-scale production is more important than in thelaboratory, greenhouse, or nursery. In the former, it isrelatively more important to document the person whois responsible for individual activities in the process.We suggest that those responsible for actions should berecorded and that they should sign for the completion ofactivities. This approach facilitates employee management,helps document those who can help sort out mistakes,and provides an inventory of experience for use in futureprocess improvement. The record should note any deviationfrom standard operating procedures and comments on theadequacy of the procedures themselves.As described in the flowcharts, the field production of theproduct would be carried out with sterile female plants,pollinated by fertile non-PMP or -PMI corn. Sterilitycan be achieved either genetically through the use ofcytoplasmic male sterility or mechanically by removalof the tassels of the female plants before flowering, orby both. In these production systems, the amount ofpollen released may be even lower than in the nurserywhere some pollen enters the air when pollen is appliedto the female plants by hand.Although it is not our intention at this point to recommendpolicy, regulations, or standards for approval of fieldprocedures, the use of male sterile PMP plants seems tobe an obvious choice for corn. A subsequent BIGMAPpublication will quantify the extent to which this practicecan reduce outflows. Other means of biological confinementcould conceivably make sterility unnecessary or reduceexpected outflows of genetic material by several orders ofmagnitude. However, these techniques are not currently inuse and do not qualify currently as good practice.Uncontrolled release of PMP pollen cannot be avoided inseed production in current systems. Our flowchart doesnot differentiate the procedures required for these twosystems. It does allow that the standards may be differentfor confinement compared with systems in which pollenmovement is uncontrolled.For foundation (parent) seed production, the minimumsize of an increase is about 100 m 2 containing 700 plants.Smaller parent scales of seed production would be done byhand in nursery conditions. In PMP or PMI foundationproduction, a larger minimum size might be considered toreduce the number of production fields by doing moreproduction in hand-pollinated nurseries. The maximumsize of PMP or PMI foundation production fields woulddepend mainly on the size of commercial production thatis planned and the yield of the PMP or PMI plants.

Assuming production of PMPs or PMIs on inbred femaleplants, each 100 ha of commercial production femalewould require approximately 1 ha of foundation production.The standards for foundation production may be differentbecause of the need for it to remain open pollinated.FIGURE 8. PRODUCTION OF CYTOPLASMIC STERILE FEMALESCYTOPLASMIC MALESTERILE, FIELD 1MAINTAINER 2FIELD 2Production can be on either inbred or hybrid female parents.Production on inbred females with the PMP event simplifiesfoundation production and limits the number of foundationisolations with open-pollinating PMP males, but it alsoincreases the area that would be required to produce agiven amount of PMP product by 30 to 50%.Sterile1XMaintainer1Maintainer2Use of hybrid females heterozygous for the event is unlikely.If a female plant is heterozygous for the PMP event andthe male does not carry the gene, only 50% of the kernelswill carry the PMP event. Use of a heterozygous hybridwith a nontrait male would reduce the concentration ofthe product in the grain.From the standpoint of the breeding required, the procedurallysimple way to obtain a homozygous female is to use aninbred. There are at least three more complex schemesthat might be used:1. Fertile hybrid females where both lines carry the event.2. Cytoplasmic male sterile inbred females.3. Cytoplasmic male sterile hybrid females.Sterile1Sterile1XSterile Hybrid Production,Field 3XMaintainer1Maintainer2Maintainer2Cytoplasmic male sterile inbred females involveproducing seed of two almost identical lines. The sterileline produces no pollen. The maintainer line producespollen that fertilizes the sterile line and itself. Cytoplasmicmale sterile hybrid females involve production of thesame sterile-maintainer pair plus one other line that isa maintainer of sterility and fertile. When the secondmaintainer is crossed by the first sterile, the result isseed that produces male sterile hybrid plants.Figure 8 is a diagrammatic representation of the twoforms of cytoplasmic sterile production. It demonstratesthat use of cytoplasmic male sterile hybrids would requiretwo kinds of foundation isolations where pollen-sheddingmales would be used. Additional pollen control in thehybrid production field requires more line developmentand pollen exposure in foundation production.SterileHybridWe have used a single flowchart to describe large,open-pollinated trials, foundation production, andcommercial PMP or PMI production. The proceduresinvolved are very similar. In open-pollinated field trialsand production involving open-pollinating male fertilePMP or PMI plants, location and isolation policy aredifferent from production by using male sterile ordetasseled trait plants. For PMP or PMI productswhere the degree of hazard is above some threshold,policy makers may choose to exclude open-pollinatedproduction from areas where large amounts of cornproduction, foundation seed production, or cornresearch is conducted.Larger Scale Production 51

FOUNDATION SEED PRODUCTION AS A MODEL FORFIELD PRODUCTION OF PMPS AND PMISWith the exception of the scale, field trials, production offoundation (parent) seed, production of seed, and productionof PMP and PMI grain can be very similar. In field trials,the 2003 USDA–APHIS guidelines establish minimumprocedural standards. In seed production, there is additionalconcern about the impact of incoming pollen, but given thehigh standards for prevention of outflows, this concern isunlikely to materially change procedures for PMPs or PMIsin corn. In commercial production of PMPs, FDA GMPswould apply to ensure adherence to documented stringenciesfor confinement.The set of flowcharts developed for the field productionsection are based on foundation seed production practicein the corn seed industry. They have been modified totake into account the special process and material controlthat will be needed for some or all PMPs and PMIs incorn. In developing the good practices described here, theexisting USDA standards for confinement were consideredthe appropriate baseline; therefore, the practices are intendedto achieve confinement equivalent to this standard. Noassertion is made that they are optimal. They are a reasonablebase for models describing the relative importance ofvarious parts of the process.Foundation seed production takes seed of lines from cornbreeders in small amounts (3 to 20 kg) and increases thelines to amounts of a few hundred kilograms or more foruse to plant field where hybrid corn seed is produced. Theplants in foundation corn seed fields are the grandparents,or great grandparents, of the plants in ordinary cornfields.The scale of foundation seed production is similar to thescale that would be appropriate for many plant-manufacturedpharmaceuticals. The entire U.S. corn industry only requiresapproximately 5,000 tons of foundation seed per year.Foundation seed only represents roughly 1/50,000 of theU.S. commercial corn crop.Any accumulation of impurities in foundation seedproduction is cumulative. Thus, special procedures havebeen developed that ensure high standards of purity. Thepriority in conventional foundation seed production isthe opposite of the challenge in producing PMPs andPMIs in corn. The challenge is to prevent pollen inflowsrather than pollen outflows. Because isolation worksthe same in either direction, the purity procedures forfoundation seed production provide isolation in the waythat would be desirable for production of PMPs and PMIs.Foundation seed corn is valuable. Prices can be $15 perkilogram or higher. Thus, practices have been developedthat not only ensure its purity but also ensure that seed isnot lost in handling. In this respect, foundation seed proceduresare similar to the procedures that should be usedfor PMPs and PMIs.Foundation corn production techniques are not verywell documented in published literature, but hybrid seedproduction techniques are documented and serve as goodbackground for the processes being described here. Onegood source is Wych (1998). It includes a brief descriptionof foundation seed stock increase (pp. 568-569).McDonald and Copeland (1997) is an informative sourcefor seed production information, including sections onseed certification and corn which explain the role offoundation seed. The global corn certification standardsunder OECD also explain the higher standards thatdifferentiate foundation seed production (OECD, 2003).Foundation corn seed production can best be describedin contrast to the characteristics of hybrid corn seedproduction. Hybrid seed corn production in turn can becontrasted with commercial seed corn production.Characteristics of hybrid seed production in the fieldinclude the following:• Production takes place under the management ofexperienced professionals.• Teams are specially trained to perform procedures.• Most major production companies are ISO certified.There are written procedures for all processes.• Written records are kept of all important activities.Record keeping is supported by computer systems sothat any problems can be tracked.• Locations and contract growers are chosen carefully tomaximize quality and minimize cost. Growers are likelyto be among the most progressive farmers in the area.• Isolation standards are maintained in the productionfield. Neighboring fields are routinely inspected forproblems with isolation. Seed production has isolationstandards. Commercial field corn production does not.• Field production practices are optimized for the particularline being grown.

• Specialized equipment is used for planting and harvest.The equipment is designed so that it can be cleanedcompletely before and after use.• Female plants on which seed is produced are detasseledor male sterile so that their pollen does not contributeto the final product (self-pollinated female seed is animpurity in the hybrid).• Buffers are used to restrict the flow of outside polleninto the field.• The teams are trained to be able to identify plants thatdo not fit the description of the corn that is supposedto be in the field. Corn has a long list of characters thatcan be observed by the trained eye. Laboratory tests canbe used to verify plant genotype if needed.• Off-type plants are removed.• Teams are routinely in the field at pollination time.Teams both control pollen flow by detasseling andmonitor isolation. The field would normally be inspecteddaily at flowering time. The teams ensure that noincorrect plants flower.• Seed is usually harvested on the ear. Seed is usuallyharvested at 25–35% moisture to avoid loss in the fielddue to insects and kernels separating from the ear.• Most seed is harvested with pickers. Combines are notused. The most modern pickers leave the husk on theear so that the ear is protected from damage on its wayto the plant• Husk-on pickers dislodge very few kernels from theears in the field.• Husk-on pickers are easy to clean. It is possible toremove all ears and effectively all loose kernels.Characteristics of hybrid seed production at the plant:• Trained teams manage all activities in the process. Stepsare recorded.• At the plant the seed will be husked, sorted again foroff-type ears, dried, shelled, and stored. Since theseprocedures are performed at a central location, it ispossible to perform them in buildings where all wastecan be managed and tracked.• Facilities can be cleaned completely between lines.• When conveyors are used they are specially selectedfor ease of cleaning.• All equipment is custom designed for easy of cleaning.• Modern genetic technology allows each line to be testedto ensure that each is correctly labeled.• Integrated computer systems track weight and purity.• Purity is checked before any line is released for use.Foundation seed production differs from regular hybridseed corn production in the following respects:In planning• Production is planned for multiple years both tocompensate for some of the risks inherent in production.By increasing the size of production fields, the rate ofimpurities can be reduced.In the foundation seed production field• Record keeping standards are higher then for commercialseed production.• The foundation production field is sometimes contractedfrom the farmer for the season so that the team hasmore control of the crops produced.• Quality is of higher importance and cost considerationsare of relatively lower importance than in hybrid seedproduction.• Genetic purity standards are higher. Isolation fromspecial kinds of corn (white corn and popcorn) is higherthan isolation required for other seed production.• Procedures are sometimes altered for the productionof quality foundation seed of hybrids with traits createdthrough biotechnology to ensure trait purity.• Isolation considerations tend to be more important inchoice of location than the characteristics of the ownerbecause more actions are handled by the foundationseed production team.• Many production locations are located within easydriving distance of a management center so that teamscan visit a number of fields easily and frequently.• Buffers of other crops are used between the outsideof the foundation field and neighboring corn. Infoundation seed production. these buffers are sometimesplanted by the company’s production team rather thanthe farmer operator.• Sterile females are sometimes used in production offemale lines. Good foundation production practice canmaintain the purity of sterile female lines to a level ofa few fertile plants in 10,000.• Small plots can be harvested by hand.• Larger plots are harvested with pickers.• Small amounts of seed can be transported in customboxes that are designed to be cleaned completely.• Larger amounts of seed can be transported indedicated wagons.• Commercial trucking is used in the largestfoundation fields.Larger Scale Production 53

At the foundation seed plant• Teams are specially trained for higher standards ofconsistency and record keeping.• Equipment is specially designed for complete cleanoutbetween different varieties.• For smaller quantities, the use of boxes and batchprocessing eliminates the need for conveyors betweenactivities and the need to clean the conveyors.• Drying equipment is specialized depending on the sizeof the lot. Some seed is dried in special boxes designedto be easily and completely cleaned.• Small quantities of seed are stored in labeled boxes thatcan be completely cleaned.• High value seed may be stored for long periods, increasingthe importance of well organized record keeping.• Foundation discards are managed carefully because ofthe value of the seed.Most of the issues relevant to the confined productionof plant-manufactured pharmaceutical and -industrialcompounds have analogous issues in foundation seedproduction: isolation, field shape and placement, avoidanceof loss, sampling issues, rigid quality control, recordkeeping for processes and materials, and postharvestverification of genotype. Individuals who are familiarwith foundation seed production have an immediate graspof the issues that impact the ability to maintain containmentin field production of PMPs and PMIs.Although the production of foundation seed providesa model for production of PMPs and PMIs, it does notimply that there are no new issues; for example, theprotection of seed stocks from the possible presence ofPMPs and PMPs (see Protection of Seed Stocks above).TABLE 1. CHARACTERISTICS OF PRODUCTION CATEGORIESGRAIN SEED FOUNDATIONFIELD PRODUCTIONMultiyear Management No No YesManagement byexperienced professionals No Yes YesDistance to supervision NA Higher LowerTrained to performprocedures. No Yes YesField contractedfor season No No YesQuality objectives No Medium HighGenetic purity standards No Medium HighISO certified No Yes YesWritten andcomputer records No Medium HighLocations and growersare chosen carefully No Yes YesIsolation standards higherpriority in selection No Medium HighIsolations standardsand inspection No Yes YesStandards fertile plantsin sterile females NA Medium HighManagement of buffers NA Medium HighSpecial standards fortransgenic crops Low Medium HighProduct specific practices No Yes YesHand harvest use No No YesSpecialized equipmentfor planting and harvest No Yes YesFemale plants aredetasseled or male sterile No Yes YesBuffers restrict theflow of pollen No Yes YesOff-type plantsare removed No Yes YesTeams in the field atpollination time No Yes YesSeed is harvested onthe ear early No Yes YesPlots are harvested witheasy to clean pickers No Yes YesHusk-on pickers dislodgevery few kernels No Yes YesCustom transportequipment No No Some

TABLE 2. CHARACTERISTICS OF PRODUCTION CATEGORIESGRAIN SEED FOUNDATIONPROCESSINGTrained teams managed No Yes YesCare in record keeping Low Medium HighProcessing incontained building No Yes UsuallyFacilities and equipmentcleaned between products No Yes YesEase of cleanoutof equipment Low Medium HighTest can identify products No Yes YesBatch processing processesfor separation Low Medium HighSpecialized equipment fordrying small lots No Some YesIntegrated computersystems track weightsand purity No Yes YesLevel of careconcerning loss Low Medium HighQuality checked beforerelease of product No Yes YesSize of storage Large Medium SmallLong-term storage No Some MoreSystems to manage discards NA Some MoreLarger Scale Production 55

GOOD PRACTICES FOR PMP AND PMI PRODUCTIONThe good practices that are described here are designed forthe production of a high-hazard PMPs or PMIs. Undercurrent USDA guidelines for field testing (which are themodel for the base case standards described here), thedegree of hazard of the PMP or PMI is uncertain; therefore,the confinement conditions are highly stringent. Fieldproduction of a PMP or PMI where the hazard is lowcould reasonably require less stringency in confinementthan is described here.Because of the objective of reducing or eliminating materialsoutflows, some generalizations can be made about goodpractices for PMP and PMI production:The most important mistakes to avoid will be relativelylarge but infrequent events relating to misidentification ofseed stocks or fields. Such rare events could lead to largeoutflows, such as seed being planted in the wrong fieldwith inappropriate isolation.• Training of staff for various processes will need tobe verified.• Checks are added for cleaning of equipment leaving theproduction field to eliminate outflows of transgenicmaterial and seed.• Dominant markers, including genetic fingerprinting,permit checks on outflow levels.• If pollen isolation fails, destruction of outside cropsbecomes an option.• Hand gleaning of fields has the potential to reduce earloss to very low levels.• Duplicate systems of seed, plot, and inventoryidentification can reduce errors in the identificationof PMP and PMI material to very low levels.• Requiring husk-on harvest will reduce loss in the fieldand get the grain into a closed building earlier.• In the closed facility, inventory of material flows allowsan audit of the handling of seed and material limitedonly by sampling error in determining the weights andmoisture contents. This allows a duplicate check on theaccuracy of other activities.The isolation checks included in the flowchart includeseparate checks for isolation from commercial grain cornand from research, foundation and seed production.Specifically, APHIS PMP standards include the following:• Presence of a perimeter fallow zone of at least 50 ftaround the test site.• Dedication of planters and harvesters to use in test sitefor the duration of the test.• Cleaning of planters, harvesters, and other tillageequipment: tractors, plows, harrows, and subsoilers.• APHIS approval for the removal of dedicated equipment.• Dedicated facilities for the storage of equipment andregulated articles for the duration of the test.• APHIS approval of protocols for the cleaning of facilities.• APHIS approval for the equipment cleaning protocols.• APHIS review of protocols for seed and grain processing.• APHIS approval of team training.• At any time during the field test when PMP plants areallowed to open pollinate, ensure that no other cornplants are receptive within a radius of 1.0 mi (1.6 km)of the transgenic plants.• Ensure where pollination of PMP plants is controlledthat transgenic corn is planted not less than 28 daysbefore or 28 days after the planting dates of any othercorn that is growing within a zone extending from0.5 to 1.0 mi (0.8 to 1.6 km) of the transgenic plantsto ensure that there is no overlap in anthesis.• Ensure that no food or feed crops are produced in thetest site and fallow zone the following crop seasonwhere there is a potential for volunteer plants to beinadvertently harvested with the following crop.APHIS standards do not currently allow for buffersaround a PMP trial. We have retained them in ourflowchart to allow for consideration of their effects onpollen outflow on the supposition that buffers areretained within trials.The hand gleaning of ears dropped by harvest machineryis included in our procedures. This procedure is notrequired by current APHIS regulations. Hand gleaning isredundant in that vegetative matter is buried or turnedunder in the field, and the field is rogued for volunteerplants the following year. There may be utility in thisprocedure because it allows for early removal of potentialsource of risk by avoiding some possibility of animals andequipment moving seed. Including this procedure in ourmodel allows other BIGMAP studies to assess its utility.

APHIS standards do not currently differentiate betweenisolation distances for seed corn and other corn. As mentionedabove, BIGMAP will examine the implications of outflowsto grain production, seed production, foundation seedproduction and research nurseries, including those wherebreeder’s seed is produced.The existence of duplicate or multiple checks on systemactivities is important throughout the PMP or PMI productionsystem. Not all of the possible checks are shown.The USDA–APHIS notice for PMP and PMI productionprovides for the possibility of inspections at preplantingstage, planting stage, midseason, harvest, postharvest, orother times (USDA, 2003a,b). The importance of theseaudits is recognized in our overall flow descriptions, butthey are not explicitly indicated in the flow diagrams,because their nature and timing is in the purview ofAPHIS and not the PMP producer.The employment of staff members experienced in theproduction of foundation seed can ensure that the staffmembers have adequate background and training.Larger Scale Production 57

FIGURE 9. FIELD PRODUCTIONProductionManagement StaffRegulatoryTrainingAuditOK?NoTeam TrainingTrain StaffYesField, FallowArea & TimeSelectionStaffKnowledgeExperienceOK?NoGeographicReferenceFieldPreparationYesPrepareDetailedProductionPlanFieldOk?NoYesPlanOK forResources,Objectives?NoRegulatoryNotificationPlantingYesYesRightSeedNoPlanterCleanCheck IDCleanPlanterYesRegulatoryApprovalNoClean Tiresand RemovePlanterNoPlantingTrainingTiresClean?NoYesYes

Clean PlanterField HistoriesPlanter isClean?NoNeighborPlanting PlansYesPlant Bufferand/or Non-PMP PollinatorYesPlanter isClean?CleanPlanterBuffer PlanterNoLabel, Sign andRecord Plot &Fallow HistoryPMPSeedPlanterAre GeoRef.Labels,Signs &HistoryCorrect?NoPlanting CrewYesPrepare Post PlantingReport for RegulatorsPost PlantingRegulatoryReportFIGURE 9 CONTINUED ON NEXT PAGELarger Scale Production 59

FIGURE 9. FIELD PRODUCTION (CONTINUED)YesPrepare Post PlantingReport for RegulatorsPost PlantingRegulatoryReportIsolation andBuffer CheckCorrect Buffersand IsolationYesAreIsolation& BuffersCorrect?NoCanIsolation &BuffersBeFixed?NoAYesFieldOperations andCleaningIsIsolationIntact?NoYesIdentify RoguePlants

AreRoguePlantsNormal?NoIsContaminationAcceptable?NoAYesYesRemove RoguePlantsYesIsRoguingSuccessful?NoWillRoguing beinToleranceNoAYesIs FieldGoodEnough toPollinate?NoAYesPollinationInspection.Remove Off-typesBefore PollinationTrainingPollinationCrewYesWithinStandard?NoWill RepeatWork?NoAYesPollinationPollen EscapeFIGURE 9 CONTINUED ON NEXT PAGELarger Scale Production 61

FIGURE 9. FIELD PRODUCTION (CONTINUED)YesPollinationPollen EscapeRemove Off-typesAfter PollinationIdentifyTrainingPollinationCrewYesWithinStandard?NoWillRoguingWork?NoBYesPollination ofSurrounding inTolerance?NoRemove &DestroySurroundingCropYesAllRemoved?NoYesMove PlantMaterial toContainmentYesPlantMaterialMoved?NoB

Remove Off-typesAfter FloweringYesWithinStandard?NoWillRoguingWork?NoBYesCleanHarvesterHarvesterTrainingHarvest CrewHarvest EarsHand PickRemainingEarsEarsRemaining?YesYesAll PMPPlantsRemoved?Discard PMP Field.Remove PlantsBefore FloweringANoNoContain PMP PlantMaterialYesAll PMPPlantsRemoved?Discard PMP Field.Remove PlantsAfter FloweringBNoAll PMPPlantMaterialContained?NoFIGURE 9 CONTINUED ON NEXT PAGELarger Scale Production 63

FIGURE 9. FIELD PRODUCTION (CONTINUED)Contain PMP PlantMaterialYesAll PMPPlantsRemoved?Discard PMP Field.Remove PlantsAfter FloweringBNoAll PMPPlantMaterialContained?NoYesClean Tires ofHarvest EquipmentCheck FieldNumber & HistoryGrainControl toPlantTires Clean?NoYesField Operations toDestroy ContainedMaterial On-sitePrepare and ShipClean HarvestEquipmentContained Materialfor Off-siteDestructionCHarvestEquipmentClean?NoField Control toYear 2

Processing& StorageFIGURE 10. PROCESSING & STORAGEClean & CloseTransportEquipmentSeedControl FromFieldClean &Closed?NoYesTrainTransportTeamLabel andTransport PMPEarsReceive Corn,Verify, Label,RecordClean TiresTiresClean?NoGeneticPurity OK?YesNoMoveNon-ConformingPMP Grain toContainmentCYesRecord Weight &MoistureClean TransportEquipment, InspectYesClosed &CleanedClose & CleanHandlingEquipmentNoCleaned toStandard?NoYesClosed &CleanedClean & CloseHusking &SortingEquipmentYesNoFIGURE 10. CONTINUED ON NEXT PAGELarger Scale Production 65

FIGURE 10. PROCESSING & STORAGE (CONTINUED)Record Weight &MoistureYesClosed &CleanedClose & CleanHandlingEquipmentNoYesClosed &CleanedClean & CloseHusking &SortingEquipmentNoHusk & Sort Ears Train Sorting TeamMeetsConformityStandards?NoClean Husk & SortEquipmentRemove Husks,Non-ConformingEars & TrashCHusk &Sort Clean ?No

Record Ear andTrack Weight &MoistureYesDryerClean?Clean DryerDryerDry Ears tostandard H20%NoClean DryerTrain PlantCrewDryer CrewDryerCleaned?NoYesRecord Weight andMoisture of Earsand TrashShell CornNoShellerClean?Clean ShellerShellerYesClean ShellerRemove Cobsand TrashCFIGURE 10 CONTINUED ON NEXT PAGELarger Scale Production 67

FIGURE 10. PROCESSING & STORAGE (CONTINUED)Clean ShellerRemove Cobsand TrashCIs ShellerNoClean?YesWeigh Grain andTrack MoisturePurity CheckMove Non-Purity OK?NoConformingGrain toCContainmentYesYesBin Closed& Clean?Close & CleanBinBinStore in Bin, LabelNoRedo LabelLabelCorrect?No

Check All Grainand Trash Weightsand MoisturesFix RecordsNoWeightConsistent?NoRecordsCorrect?YesYesPMPContaminated?NoRecover LostPMPYesYesPurity Ok?NoMove toContainmentCheck All Grainand Trash Weightsare ConsistentCWeightConsistent?NoFind Source ofErrorSpecialResolutionYesConfirm POwith PMP IDPurchaseOrder FromProcessorFIGURE 10 CONTINUED ON NEXT PAGELarger Scale Production 69

FIGURE 10. PROCESSING & STORAGE (CONTINUED)WeightConsistent?NoFind Source ofErrorSpecialResolutionYesConfirm POwith PMP IDPurchaseOrder FromProcessorYesTruck Clean?Clean TruckFromProcessorNoLoad TruckProcess Invoice BillofLading/InventoryAdjustmentYesOrderMatches,Inventory, &DocumentsNoWrongDocs?NoConfirm SeedWrongYes

Clean BinCheck IncomingWeight AgainstGrain InventoryShipped &DestroyedBin is Clean?NoYesAll WeightsConsistent?NoNotifyProcessor ofProblemClean All HandlingEquipmentYesComplete ReportReportfor APHIS & FDAHandlingEquipmentClean?NoYesTo YearN+1Contain andDestroy All WasteFrom Field &Husking & PlantAssembleContainedWaste FromField & PlantCWasteDestroyed?NoLarger Scale Production 71

FIGURE 11. 2ND YEAR VOLUNTEER MANAGEMENTFromYear NPrepare PlantingPlan for Year 2Neighbor'sPlanting PlansConfirm Fieldand FallowCoordinatesOK forRegulations(Not Foodor Feed)?NoYesPlant Location WithCover CropRemoveIncorrect CropIs Location& Crop OK?NoYesRemove AllVolunteer PMPPlantsAre AllVolunteeredRemoved?NoYes

Destroy VolunteerPlant MaterialCheck SurroundingCrop Fields forVolunteers, ifDominant MarkersAvailableIsContaminationWithinTolerance?YesNoDestroy Plants inContaminated AreaYesRemovalWithinRegulations?NoPrepareMonitoring Reportfor RegulatorsMonitoring ReportPrepare FinalRegulatory RptFinalRegulatory RptEndLarger Scale Production 73

Summary: ProcessDescriptionsThe general philosophy for compliant confinementsystems is for planning the overall system and individualprocesses to comprise a logical flow of redundant tasksthat can be clearly managed and tracked. The confinementmanagement activities are described here as a hierarchyof procedures, subprocedures, activities, and subactivitiesnecessary to achieve corn confinement consistent withcurrent regulatory standards (that is “good practice”standards). These management activities are describedhere as generalized “activity group descriptions” andare more explicitly detailed in the corresponding flowdiagrams. The approach represented in these descriptionsis one where for each subprocess (for example, fieldproduction management represents a subprocess for fieldproduction) or activity grouping (for example, preplantingrepresents an activity grouping under the field productionmanagement operation), there are nests of subactivitiesdescribed entailing(i)development or confirmation of appropriate protocols,standard operating procedures (SOPs), and training;(ii) execution of management activities compliant withthe protocols, standards, and training; and(iii) quality control or quality assurance (QC/QA) actionsto confirm and document compliance with protocols,standards, and training.Effective management of confinement operationswill be most impacted by human failures in planning,implementation, and follow-up. These process descriptionsand the corresponding flow diagrams are intended as aresource for minimizing system error rates. These “goodpractices” are meant to exemplify only one of manyalternative management schemes that can meet regulatoryexpectations for confinement.PROCESS DESCRIPTION OUTLINEI. PLANT TRANSFORMATION AND GREENHOUSEA. Planning Stage• Assign staff with appropriate training andexperience in construction of cloning vector• Develop QA/QC plan for transformation vector• Prepare detailed vector development plan• Achieve appropriate resource planning, includingthe intellectual property issues associatedwith vector• Achieve necessary regulator approval, if required• Check identity of vectorB. Plant Transformation• Assign trained personal for transformation• Prepare transformation plan (Agrobacterium vs.biolistic transformation method)• Develop SOP for transformation• Implement transformation SOP fortransformation process• Implement aseptic techniques• Label individual transformation plates withvector identification• Document transformation procedure and resultsC. Selection of Transformant and Regeneration• Use trained personnel• Develop SOP for selection and regenerationof transformant• Implement aseptic strategy during the process• Identify individual transformants properly• Select stable transformant• Regenerate transformant• Collect quality information

• Properly document test results• Develop event specific detection method forevent identificationD. Greenhouse Propagation of Transformant• Use trained personnel for greenhouse process• Develop increase plan for each transformant• Use dedicated greenhouse space fortransplanting transformant• Implement containment steps in greenhouseappropriate for hazard, including pollen• Print labels based in plan, including loop (plant) labels.• Maintain identity of each individual plant in greenhousewith labels• Implement selfing and crossing strategy withpollen control• Maintain identity of seeds produced from crosseswith labels• Implement clean harvesting strategy with trackinginformation and labels• Clearly document whole process withtracking information• Document disposition of PMP materials• Confirm identity of transformant• Confirm transformant meets biosafety quality criteriaII. NURSERIESA. Nursery Management Planning• Assign staff with appropriate training and experience• Prepare detailed nursery plan consistent withcompliance best management standards• Achieve appropriate resource planning• Acquire necessary regulatory approvals• Audit for regulatory trainingB. Nursery Management1. Inventory Preparation (Preplanting)• Use trained personnel for preplant processesand cleaning• Use approved procedures for preplant processesand cleaning• Use dedicated or clean equipment forpreplant processes• Label, assemble, and fill planting envelopes consistentwith inventory records and approved procedures• Arrange planting envelopes in planting orderand arrange into planting boxes• Conduct field selection for appropriate temporaland spatial isolation in accordance with fieldhistories and neighbor planting plans• Prepare field for planting• Affirm necessary regulatory approvals andauthorizations are in place• Confirmation site coordinates with properranges and row labels• Prepare plot labels• Confirmation overall preplant process compliance2. Planting• Use trained personnel for planting processesand cleaning• Use approved procedures for planting processesand cleaning• Use dedicated or clean equipment forplanting processes• Check that crop is planted in accordancewith the nursery plan• Plant buffer or non-PMP pollinators• Assure appropriate labels, signage, and records,including adjustments for planting errors• Confirmation overall plantingoperations compliance• Prepare and submit regulatory documentation3. Stand management (preanthesis)• Use trained personnel for preanthesis processesand cleaning• Use approved procedures for preanthesis processesand cleaning• Use dedicated or clean equipment forpreanthesis processes• Re-confirm that temporal and spatial isolationstandards are being met• Perform preanthesis identification of PMP plantsand ensure agreement with nursery plan• Conduct regulatory/event testing in accordancewith nursery plan• Confirm overall preanthesis process compliance4. Pollen management (anthesis)• Use trained personnel for pollen managementprocesses and cleaning• Use approved procedures for pollen managementprocesses and cleaning• Use dedicated or clean equipment for pollenmanagement processesSummary: Process Descriptions 75

• Conduct prepollination identification and removalof off-types/breakers• Institute daily tassel inspection and management• Institute pollination control consistent withnursery plan• Document any revisions to pollination plan• Conduct postpollination identification andremoval of off-types/breakers• Confirm temporal and spatial isolation standardsare achieved throughout the pollen shed interval• Confirm overall pollen managementprocess compliance5. Harvest processes• Use trained personnel for harvest processesand cleaning• Use approved procedures for harvest processesand cleaning• Use dedicated or clean equipment forharvest processes• Conduct hand harvest operations, husk, sort intolabeled plot bags• Document disposition of PMP materials throughharvest operations• Confirm overall harvest process compliance6. Field residue management (postharvest processes)• Use trained personnel for postharvest processesand cleaning• Use approved procedures for postharvest processesand cleaning• Use dedicated or clean equipment forpostharvest processes• Contain or destroy postharvest residue in the field• Document disposition of postharvest plant residues• Confirm residue management plan is in place fornext growing season• Confirm overall postharvest process complianceC. Nursery Inventory Management1. Seed receipt and preparation• Use trained personnel for inventory management• Use approved procedures for inventory management• Use dedicated or clean equipment forinventory management• Contained transport consistent with process planand training• Segregate and confine trash throughout process• Confirm receipt of seed from nursery• Record ear weights and moisture• Dry ears to standard• Clean dryers following drying• Record dried ear and trash weights and moisture• Shell corn and transfer to inventory containers• Record grain and trash weights and moisture• Move trash to containment for destruction• Check for level expression in PMP seed• Move nonconforming grain as waste tocontainment for destruction• Confirm overall inventory preparation compliance2. Process residues management• Use trained personnel for process residues operationsand cleaning• Use approved procedures for process residues operationsand cleaning• Use dedicated or clean equipment for processresidues operations• Destroy contained process residues• Confirmation overall process residuesmanagement compliance3. Inventory disposition and accounting• Use trained personnel for inventory dispositionand accounting• Use approved procedures for inventory dispositionand accounting• Conduct regulatory/event testing in accordancewith nursery plan• Archive event information generated• Store seed• Record and archive seed disposition• Confirm nursery inventory process records• Confirm process residues destruction• Confirm overall nursery inventory process compliance• Complete regulatory reporting

D. Second Year Field Management• Use trained personnel for 2nd year field management• Use approved procedures for 2nd year field management• Use dedicated or clean equipment for 2nd yearfield management• Confirm residue management plan• Confirm field coordinates• Confirm following crop field management• Use approved operations for in-fieldresidue management• Conduct preanthesis scouting and removal of volunteers• Confirm and close out field site processes• Prepare and submit regulatory documentationIIII. FIELD PRODUCTIONA. Production Planning• Assign staff with appropriate training and experience• Prepare detailed field production plan consistent withcompliance best management standards• Achieve appropriate resource planning• Acquire necessary regulatory approvalsB. Field Production Management1. Field site selection and preparation (preplanting)• Use trained personnel for preplant subprocessesand cleaning• Use approved procedures for preplant subprocessesand cleaning• Use dedicated or clean equipment forpreplant subprocesses• Conduct field selection for appropriate temporal andspatial isolation• Geographically reference the site coordinates• Establish fallow zone• Affirm necessary regulatory approvals andauthorizations are in place• Confirmation site coordinates• Confirmation overall preplant subprocess compliance2. Planting• Use trained personnel for planting subprocessesand cleaning• Use approved procedures for planting subprocessesand cleaning• Use dedicated or clean equipment forplanting subprocesses• Check that crop is planted with appropriatetemporal isolation• Confirm PMP (female) seed source and purity• Confirm non-PMP (male) seed source and purity• Establish the proportion of males to females• Assure appropriate labels, signage, and records• Confirmation overall planting subprocesses compliance• Prepare and submit regulatory documentation3. Stand management (preanthesis)• Use trained personnel for preanthesis subprocessesand cleaning• Use approved procedures for preanthesis subprocessesand cleaning• Use dedicated or clean equipment forpreanthesis operations• Reconfirm that temporal and spatial isolation standardsare being met• Perform preanthesis identification and removal ofrogue plants• Confirm overall preanthesis subprocesses compliance4. Pollen management (anthesis)• Use trained personnel for pollen managementsubprocesses and cleaning• Use approved procedures for pollen managementsubprocesses and cleaning• Use dedicated or clean equipment for pollenmanagement subprocesses• Institute appropriate pollen controls• Conduct prepollination identification and removal ofoff types/breakers• Conduct postpollination identification and removal ofoff types/breakers• Confirm temporal and spatial isolation standards areachieved throughout the pollen shed interval• Confirm overall pollen managementsubprocesses compliance5. Harvest• Use trained personnel for harvest subprocessesand cleaning• Use approved procedures for harvest subprocessesand cleaning• Use dedicated or clean equipment forharvest subprocesses• Conduct machine harvest in conformance to standards• Conduct hand harvest operations to recovermissed/dropped ears• Document disposition of PMP materials throughharvest subprocesses• Confirm overall harvest subprocesses complianceSummary: Process Descriptions 77

6. Field residue management (postharvest operations)• Use trained personnel for postharvest subprocessesand cleaning• Use approved procedures for postharvest subprocessesand cleaning• Use dedicated or clean equipment for postharvestsubprocesses Contain or destroy postharvest residuein the field• Document disposition of postharvest plant residues• Confirm residue management plan is in place for nextgrowing season• Confirm overall postharvest subprocesses complianceC. Transport• Use trained personnel for transport subprocessesand cleaning• Use approved procedures for contained transportsubprocesses and cleaning• Use dedicated or clean equipment fortransport subprocesses• Confirm overall transport subprocesses complianceD. Processing1. Husk and sort subprocess• Confirm receipt at approved facility• Use trained personnel for husk and sort subprocessesand cleaning• Use approved procedures for husk and sort operationsand cleaning• Use dedicated, closed, and clean equipment for huskand sort operations• Check for level of genetic purity• Move nonconforming ears as waste to containmentfor destruction• Record ear and husk weights and moisture• Husk and sort ears• Move trash to containment for destruction• Confirm husked ears conform to standards• Record and track husked ear weights and moisture• Confirm overall husk and sort subprocesses compliance2. Dry and shell subprocedure• Use trained personnel for dry and shell subprocessesand cleaning• Use approved procedures for dry and shell subprocessesand cleaning• Use dedicated or clean equipment for dry andshell subprocesses• Dry ears to standard• Clean dryers following• Record ear and trash weights• Move trash to containment for destruction• Shell corn• Segregate grain and trash• Record grain and trash weights and moisture• Move trash to containment for destruction• Check for level of genetic purity• Move nonconforming grain as waste to containmentfor destruction• Confirm overall dry and shell subprocesses compliance3. Residues management• Use trained personnel for residues managementsubprocesses and cleaning• Use approved procedures for residue managementsubprocesses and cleaning• Use dedicated or clean equipment for subprocessesresidues operations• Destroy contained residues• Confirmation overall residues management compliance4. Harvest accounting• Use trained personnel for harvest accounting• Use approved procedures for harvest accounting• Confirm materials balance for the harvest subprocesses• Confirm the grain purity and level of expression• Confirm residues destruction• Confirm harvest records• Confirm overall harvest compliance

E. Product storage and disposition• Use trained personnel for product storageand disposition• Use approved procedures for product storageand disposition• Use dedicated or clean equipment for product storageand disposition• Assign dedicated or clean bin• Confirm materials transfer from harvest subprocess• Confirm purchase order match to binned product• Confirm use of an approved transporter• Load truck• Clean loading area• Manage the invoice, bill of lading, and adjust inventory• Audit materials disposition• Clean and close out the bin• Confirm overall storage and disposition complianceF. Second Year Field Management• Use trained personnel for 2nd year field management• Use approved procedures for 2nd yearfield management• Use dedicated or clean equipment for 2nd yearfield management• Confirm residue management plan• Confirm field coordinates• Confirm following crop field management• Use approved procedures for in-fieldresidue management• Conduct preanthesis scouting and removal of volunteers• Confirm and close out field site• Prepare and submit regulatory documentationSummary: Process Descriptions 79

ConclusionNew technology frequently changes the value of existingresources. Technology can take something that had novalue previously and give it a value. It becomes the taskof citizens, their institutions, and their traditions to assignownership and responsibility for the new resource. Thisprocess can be assisted by knowledge about the resourceand how it is used. Our society now finds that it has aresource of PMP- and PMI-free corn. Consumers andfarmers cannot effectively preserve that resource actingindependently. We have assumed that much of theresponsibility will be divided between the PMP ownersand users and the government regulators. The task of policycreation and deciding how to divide the responsibility willbe made easier with increased knowledge of the systemsthat will be used and the risk associated with the possibilityof loss of PMPs and PMIs.Formalized risk assessment allows for a science-basedcharacterization of risk. This assessment can subsequentlybe integrated with broader societal concerns in policyformulation considering both the risks and benefits ofthe technology. Use of science-based risk characterizationmakes the convergence of opinion in the broader discussionmore likely.No system is risk free. In areas where corn is produced,some amount of outflow will occur, especially with highwinds and equipment movement. Determination of adistribution of probable loss from confinement will allowspecifications of the kind or level of hazard for which sucha system should be used. In this report, good practices arepresented in process flow diagrams and in process outlinesthat break process flows into logical systems descriptionsas a hierarchy of relevant processes, subprocesses, andactivities intended to achieve high levels of confinementintegrity. Explicitly stated practice standards are a necessaryfirst step in understanding the degree of exposure andconsequent risk that may occur with varying typesof PMP or PMI development and production underconfinement. These practices will serve as a starting pointin the analysis of the strengths and weaknesses of asystem and its possible variants.The study of this system should provide the membersof the broader public with information about theapproaches and stringencies jointly adhered to byproducers and regulators. This information will assistmembers of the public to put PMP or PMI riskin perspective.Having established these practice standards, BIGMAPwill evaluate their integrity through quantitative exposureassessment. Quantitative exposure assessment recognizesthat for each activity there are various likelihoods ofprocedural conformity and nonconformity. For conformityand nonconformity, there are probability distributions forpossible outcomes. Working through the system flowchart,we can model all the process and calculate probabilities ofthe various final outcome states. If we can value the variouspossible outcomes, then we can generate a probable“cost” for the complete PMP or PMI production system.The model can be used to evaluate the impact of changesin processes on the performance of the system as awhole. In policy making, the cost also can be comparedsubjectively or objectively with the expected benefits fromthe production of the PMP or PMI to make a judgmentas to whether the net benefit to society is positive or negative.Although the focus of this document is on the sourcefield, different kinds of recipient fields also can effect therisk distributions. The most serious form of failure ofconfinement for a toxic PMP or PMI would be one inwhich breeder’s seed or foundation seed were contaminated,because this would allow for the possibility for anundetected PMP or PMI to be conserved and replicatedin seed stocks. Understanding of the distributions ofpossible outcomes from the source field will allow thequantification of outcomes associated with various kindsof source and target fields in subsequent studies.

No system is risk free, but some systems have very lowrisks in comparison with systems that society commonlyuses without special concern, for example, automobilesand gasoline trucks. Risks for others may be higher. Thesize of the risk may vary with policies that are adopted.The objective the BIGMAP study is to make future policymaking easier and more meaningful by developing prototypemodels that can be used in determining where on thescale specific types of plant-manufactured pharmaceuticalor -industrial compound production systems shouldbe ranked.VII. REFERENCESAdair, D., Irwin R., and Traynor, P.L. 2001. A practical guide to containment.Blacksburg, VI: Information Systems for Biotechnology.Bateman, A.J. 1947. Contamination of seed crops II, wind pollination.Heredity 1:235–246.Burris, J.S. 2001. Adventitious pollen intrusion into hybrid maize seed productionfields. Association of Official Seed Certifying Agencies [AOSCA]. 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Crop Sci. 42:2201–2206.Garcia, C., Figueroa, M.J., Gomez, M.R., Townsend, L.R., and Schoper, J. 1998.Pollen control during transgenic hybrid maize development in Mexico.Crop Sci. 38:1597–1602.Hall, A.J., Villela, F., Trapani, N., and Chimenti, C. 1982. The effect of waterstress and genotype on the dynamics of pollen-shedding and silking in maize.Field Crops Res. 5:349–363.Hutchcroft, C.D. 1958. Contamination in seed fields of corn resulting fromincomplete detasseling. Agron J. 66:267–271.Ingram, J. 2000. Report on the separation distances required to ensure crosspollinationis below specified limits in non-seed crops of sugar beet, maize,and oilseed rape. MAFF Project no. RO0123. Accessed electronicallyAugust 7, 2002, http://www.defra.gov.uk/planth/pvs/gmsepdis.pdf.Ireland, D. S., Westgate, M.E., and Ashton, B.A. 2001. Combining ISCST3 andAERMOD particulate dispersion models to quantify maize pollen distribution.ASA-CSSA-SSSA Annual Meetings Abstracts, Charlotte, NC. October 21–25,2001. Madison, WI: American Society of Agronomy.Jemison, J.M. and Vayda, M.E. 2002. Cross pollination from geneticallyengineered corn: wind transport and seed source. AgBioForum 4(2) article 2.Accessed electronically January 12, 2005,http://www.agbioforum.org/v4n2/v4n2a02-jemison.htm.Jones, M.D. and Brooks, J.S. 1950. Effectiveness of distance and border rows inpreventing outcrossing in corn. Oklahoma Agricultural Experimental StationTechnical bulletin No 38. Stillwater, OK: Oklahoma State University.Kawashima et al. 2000. Estimating total corn pollen desposition forenvironmental assessment. Jpn. J. Palynol 46:103–114.Lauer, J. 1998. Successful corn pollination. Wisc Crop Manager 5:104–105.Luna, S., Figueroa, M. J., Baltazar, M. B., Gomez, L. R., Townsend, R., and Shoper,J. B. 2001. Maize pollen longevity and distance isolation requirements foreffective pollen control. Crop Sci. 41:1551–1557.McDonald, M.B. and Copeland, L. O. 1997. Seed production: principles andpractices, pp. 148–170 and 193–205. New York: Chapman & Hall.Ministère de l’Agriculture et de la Pêche. 2002. Rapport de la commissiondu genie biomoleculaire et du comite provisoire de biovigilance surl’expérimentation au champ de plantes transgeniques. Assessedelectronically June 9, 2004,http://www.conso.net/images_publications/Contribution%20CGB.rtf.Mudra, A. 1943. Ùber die reichweite des pollenstaubes bim mais in geschlossenemverband. Verband Züchter 15:29–31.Narayanaswamy, S. et al. 1997. Determination of isolation distance for hybridseed production. Curr. Res. (Univ. Bangalore) 26:193–195.[NIH] National Institutes of Health. 2002. Guidelines for Research InvolvingRecombinant DNA Molecules. April 2002, Accessed electronically June 2004,http://www4.od.nih.gov/oba.National Research Council. 2004. Biological Confinement of Genetically ModifiedOrganisms. Washington, DC: National Academies Press.[OEDC} Organization for Economic Cooperation and Development. 2003. AnnexIX: OECD scheme for the varietal certification of maize and sorghum seedmoving in international trade. OECD Seed Scheme. Accessed electronicallyJune 25, 2004, http://www.oecd.org/dataoecd/40/50/15284225.PDF.Paterniani, E. and Stort, A.C. 1974. Effective maize pollen dispersal in the field.Euphytica 23:129–134.Poehman, J.M. and Sleper, D.A. 1995. Breeding field crops, 4th ed. Ames, IA:Iowa State University Press.Pohl, F. 1937. Die pollenerzeugng der winterbluter. Bei. Bot. Centralb.56A:365–470.Raynor, G.S., Ogden, E.C., and Hayes, J.V. 1972. Dispersion and deposition ofcorn pollen from experimental sources. Agron. J. 64:420–427[USDA] United States Department of Agriculture. 2003a. Field testing of plantsto produce pharmaceutical and industrial compounds. Federal Register68:11337–11340.[USDA] United States Department of Agriculture. 2003b. 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BIGMAP102 Seed Science CenterIowa State UniversityAmes, Iowa 50011-3228Iowa State University does not discriminate on the basis of race, color, age, religion, national origin, sexual orientation, sex, marital status, disability or status as a U.S.Vietnam Era Veteran. Any persons having inquiries concerning this may contact the director of Equal Opportunity and Diversity, 3680 Beardshear Hall, (515) 294-7612.