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00-front matter Transgenic book 7/16/01 10:05 PM Page iii Greenhouse Research with Transgenic Plants and Microbes Patricia L. Traynor Dann Adair Ruth Irwin Information Systems for Biotechnology Vi r g i n i a Te c h
00-front matter Transgenic book 7/16/01 10:05 PM Page v Acknowledgements Production of this manual was supported by Information Systems for Biotechnology, a program at Virginia Polytechnic Institute and State University, and funded by a grant from USDA’s Cooperative State Research, Education, and Extension Service. The authors gratefully acknowledge and thank Sue Tolin (Virginia Tech), Ralph Stoaks (USDA APHIS PPQ), Dianne Hatmaker (USDA APHIS), Dean Gabriel (University of Florida), and Richard Denis (Agritechnove, Inc.) for their valuable comments and suggestions on the manuscript. The editorial and production assistance of Joseph Moriarity (Erin Communications) is greatly appreciated. Photographs courtesy of Dave Hansen and Dann Adair, University of Minnesota, Agricultural Experiment Station. Disclaimers The information contained in this manual is accurate to the best knowledge of the authors. Any errors or omissions are their sole responsibility. The views presented here do not represent those of the University of Minnesota and Virginia Tech, USDA, or other state or federal regulatory agencies. Readers are advised to consult directly with relevant national or state agencies or authorities for official guidance. Mention of a trade name or trademarked product does not imply endorsement by the authors. v
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01-07 Transgenic book 7/16/01 10:25 PM Page 1 1 Introduction Section I. Introduction THE USE OF BIOTECHNOLOGY TO MODIFY PLANTS has become a common practice in agricultural and horticultural research. Unlike ordinary research materials used in laboratory, greenhouse, and field studies, transgenic (genetically engineered, genetically modified)1 organisms are subject to special rules intended to ensure that they are used in a way that does not pose an unacceptable risk to human health or the environment. Methods for the safe handling of transgenic materials in laboratory settings are described in the National Institutes of Health’s Guidelines for Research Involving Recombinant DNA Molecules (NIH Guidelines). Regulations and guidance for the safe release of genetically modified organisms (GMOs) into the environment are implemented by the Animal and Plant Health Inspection Service of the US Department of Agriculture (USDA/APHIS) and the Environmental Protection Agency (EPA). Genetic modifications include, but are not limited to, those made by recombinant DNA (rDNA)2 methodologies. Information about handling transgenic plants in greenhouses, however, is relatively sparse. Appendix P of the NIH Guidelines3 specifies facilities and practices for meeting containment standards appropriate for each of four biosafety levels. Presently, though, there is no single source of practical guidance on managing greenhouses containing GMOs, nor on the requirements for building or renovating plant growth facilities to make them suitable for containing transgenic plants and associated organisms. This Guide is intended as a simple and convenient reference on appropriate biosafety and containment levels for GMO research conducted in greenhouses. There may be a broad range of guesses and opinions among scientists and greenhouse managers regarding what is needed. Some may harbor a misunderstanding that all GMOs must be grown in a highly contained ‘clean-room,’ while others may be completely unaware that certain cases require specific containment measures in order to protect the surrounding environment. The Guide will help clarify what level of containment is needed and what measures are sufficient to achieve the various biosafety levels. 1 In this Guide, the terms “transgenic,” “genetically engineered,” and “genetically modified” are used interchangeably. 2 Recombinant DNA molecules are defined as: “(i) molecules that are constructed outside living cells by joining natural or synthetic DNA segments to DNA molecules that can replicate in a living cell, or (ii) molecules that result from the replication of those described in (i) above.” 3 http://www4.od.nih.gov/oba/appendix_p.htm
01-07 Transgenic book 7/16/01 10:25 PM Page 2 2 A G U I D E T O P L A N T C O N TA I N M E N T Scope information and a brief discussion of the contents. Section II covers the regulation and oversight of This Guide applies to greenhouses—controlled GMOs by government regulatory and research environment structures having a transparent or agencies, and outlines the roles and responsibilities translucent covering and used for growing plants— of institutional personnel. Section III presents that contain genetically modified plants or plant- descriptions of four biosafety levels affording associated organisms. The wide range of increasing levels of containment, together with microorganisms that are plant-associated include examples of studies that may be conducted at each viruses, bacteria, fungi, protozoa, mycoplasma-like level. Physical, biological and combination organisms, nematodes, insects, mites, and others. containment strategies are given in Section IV, Screenhouses—structures that are screened for followed by suggested management practices for insect or plant containment (or exclusion) but which greenhouses containing GMOs in Section V. Section offer little environmental control—are suitable for VI discusses options for retrofitting existing facilities temperate climates or warm seasons in zones subject to meet containment standards, and Section VII to colder temperatures. Screenhouse construction addresses the design of new facilities. Two details and upgrades are briefly described in this Appendices provide facility inspection checklists and Guide. a list of supplemental information resources. Other contained plant growth facilities, such as This Guide was written so that anyone who growth chambers, biosafety cabinets, incubators, and works in a greenhouse that houses transgenic tissue culture tables or rooms, often are an integral materials will be better informed about the purpose part of the process leading to the preparation of of containment, the variety of methods used to GMO materials for greenhouse studies or field tests. achieve it, and the facilities and practices that satisfy These facilities are mentioned in passing; a detailed the requirements of established guidelines and description is not within the scope of this Guide. regulations. It is intended as guidance and should not be a considered an authoritative source. Readers are This Guide includes: encouraged to seek additional guidance from • Relevant information on four levels of biosafety institutional authorities and USDA/APHIS officials containment; whenever questions arise. • Physical and biological strategies that provide containment; • Suggested facility modifications to achieve Audience prescribed containment levels; Greenhouse managers, facility staff, and research • Suggestions for day-to-day greenhouse scientists are the primary audience of this Guide. management; Managers, being responsible for the overall operations of a greenhouse facility, will benefit from • Methods for proper handling of GMOs; a clear description of when, where, and why • Discussions of selected design issues for new or additional containment measures should be renovated facilities; instituted, as well as practical guidance for managing • Descriptions of equipment and supplies; the facility and persons working in it. Greenhouse staff who are involved in the day-to-day care of • A sample floor plan and; transgenic organisms will gain a better • Sources for additional information. understanding of what tasks, if any, should be The Guide is organized in seven sections plus two modified when the experimental materials have been Appendices. Section I contains introductory genetically engineered. Researchers who work with
01-07 Transgenic book 7/16/01 10:25 PM Page 3 SECTION I. Introduction 3 GMOs, together with members of Institutional Biosafety Committees and students, will likely find it a simple and convenient reference on the various levels of containment and the types of experiments appropriate to each level. In addition, designers working on retrofits to existing greenhouses or on new construction will find specialized information that pertains to meeting non-standard structural requirements for containment facilities. Others who work in and around such facilities, including tradespeople, maintenance personnel, and adjacent residents, will benefit from a basic understanding of the purpose of containment. Such understanding will help ensure that GMOs are handled in an environmentally responsible manner.
01-07 Transgenic book 7/16/01 10:25 PM Page 5 5 Regulation and Oversight of GMOs Section II. Regulation and Oversight of GMOs TRANSGENIC PLANTS ARE SUBJECT TO FEDERAL GUIDELINES, regulations, and rules pertaining to their containment, movement, and release into the environment. In addition, a few states, notably Florida and California, have applicable regulations as well. Institutions where biotechnology research is conducted are expected to have an institutional biosafety committee (IBC) serving as the local authority. Ultimately, responsibility for the safe handling of transgenic materials lies with the principal investigator and other individuals who manage any part of the research. THE NIH GUIDELINES AND APPENDIX P Guidelines first published by the NIH in 1976 address the safe conduct of laboratory research involving the construction and handling of rDNA molecules and organisms containing rDNA. They are advisory in nature, rather than legally binding. However, all federal agencies that support or conduct rDNA research agreed to abide by the NIH Guidelines and require institutional compliance as a condition of funding. Thus, failure to comply may result in the suspension, limitation, or termination of financial support for rDNA research at the institution. The current version of the NIH Guidelines can be accessed on the Internet4. The NIH Guidelines discuss risk assessment and recommend containment measures for various biological experiments. They set forth facility specifications and practices for conducting experiments classified according to four levels of biosafety containment; a fifth class encompasses experiments that are exempt. Although originally focused on rDNA microorganisms, the NIH Guidelines have undergone numerous revisions and now cover plant, animal, and human gene therapy research to accommodate the wide range of federally funded research projects. The Guidelines were expanded in 1994 by the addition of Appendix P, Physical and Biological Containment for Recombinant DNA Research Involving Plants. The term “plants” includes, but is not limited to, mosses, liverworts, macroscopic algae, and vascular plants including terrestrial crop, forest, weed, and ornamental species. Recommended 4 http://www4.od.nih.gov/oba/guidelines.html
01-07 Transgenic book 7/16/01 10:25 PM Page 6 6 A G U I D E T O P L A N T C O N TA I N M E N T containment conditions for experiments involving the US Regulatory Oversight in Biotechnology site plants together with plant-associated on the Web5. microorganisms or small animals in which any organism may be genetically modified are also found in Appendix P. USDA/APHIS Plant-associated microorganisms include those known to cause plant disease, such as viroids, The USDA’s Animal and Plant Health Inspection virusoids, viruses, bacteria, and fungi, as well as Service (APHIS) has authority under the Federal protozoa, and microorganisms that have a benign or Plant Pest Act to protect US agriculture from pests beneficial association with plants, such as certain and diseases. Under the Coordinated Framework, Rhizobium species. Microorganisms that are this authority was extended to cover rDNA- modified with the objective of fostering an containing plants and other potential plant pests. association with plants are similarly subject to the USDA also regulates veterinary biologics such as terms of Appendix P. Plant-associated small animals recombinant vaccines. The Plant Protection and include those arthropods that: (1) are in obligate Quarantine division is the lead regulatory office for association with plants; (2) are plant pests; (3) are GMOs. APHIS also adheres to international plant pollinators; or (4) transmit plant disease standards created by the International Plant agents, as well as other small animals such as Protection Convention. Any introduction of a GMO, nematodes for which tests of biological properties defined as importation, interstate movement, or necessitate the use of plants. Microorganisms release to the environment, requires either associated with such small animals (e.g., pathogens notification to APHIS or application for a release or symbionts) are included. permit, depending on the nature of the plant and the Appendix P describes practices for conducting genetic modification made to it. APHIS has an experiments to construct, use experimentally, and extensive biotechnology Web site describing their propagate genetically engineered plants. It specifies regulations6. physical and biological containment measures and management protocols applicable to each of four biosafety levels designated BL1-P, the lowest level of EPA containment, through BL4-P, the highest level. The EPA regulates the use of two categories of GMOs. The first encompasses novel microorganisms (formed by deliberate combinations of genetic FEDERAL REGULATORY AGENCIES material from different taxonomic genera) that Under the Coordinated Framework for contain or express new combinations of traits and Regulation of Biotechnology, three US governmental are intended for commercial use as biofertilizers, agencies regulate GMOs: the Department of biosensors, waste treatment or pollutant Agriculture, the Environmental Protection Agency, degradation, or for commodity or specialty chemical and the Food and Drug Administration (FDA). production. The second category consists of plants Greenhouse research is not generally subject to and microbes producing pesticidal substances, such federal regulation; however, the following brief as plants expressing insect control proteins derived summary provides the broad context for regulatory from Bacillus thuringiensis (Bt). More information review of transgenic plants associated with testing in on these topics is available through the EPA’s Toxic the environment and commercialization. More Substances Control Act Biotechnology Program7 and complete information about these agencies and their their Biopesticides Program8. roles with respect to products derived from biotechnology, with links to the laws, rules, and regulations that they administer, can be accessed at 5 http://www.aphis.usda.gov/biotech/OECD/usregs.htm 6 http://www.aphis.usda.gov/bbep/bp 7 http://www.epa.gov/opptintr/biotech/index.html 8 http://www.epa.gov/oppbppd1/biopesticides/index.html
01-07 Transgenic book 7/16/01 10:25 PM Page 7 SECTION II. Regulation and Oversight of GMOs 7 FDA to consult with the FDA during the development phase for guidance on what types of data will be Commercial products modified by genetic needed at the time of product safety review. An engineering for human and animal consumption, overview of the FDA’s policies on food and feed from food additives, human and veterinary drugs are GM plants can be found on the Internet9. subject to regulation by the FDA. Their oversight does not apply to the R&D phases of product Table 1 shows a concise overview of USDA’s, EPA’s improvement. Nevertheless, developers are expected and FDA’s overlapping regulatory authorities. TABLE 1. Multiple Regulatory authorities oversee certain GMOs REGULATORY REVIEW NEW TRAIT/ORGANISM CONDUCTED BY: REVIEWED FOR: USDA Safe to grow Viral Resistance in food crop EPA Safe for the environment FDA Safe to eat Herbicide Tolerance in food USDA Safe to grow crop EPA New use of companion herbicide FDA Safe to eat Herbicide Tolerance in USDA Safe to grow ornamental crop EPA New use of companion herbicide Modified Oil Content in food USDA Safe to grow crop FDA Safe to eat Modified Flower Color in USDA Safe to grow ornamental crop Modified Pollutant Degrading EPA Safe for the environment soil bacteria 9 http://vm.cfsan.fda.gov/~lrd/biotechm.html
01-07 Transgenic book 7/16/01 10:25 PM Page 8 8 A G U I D E T O P L A N T C O N TA I N M E N T INSTITUTIONAL BIOSAFETY BIOLOGICAL SAFETY OFFICER COMMITTEE If research is conducted on organisms that require Any institution where research involving special containment conditions designated as BL3-P transgenic organisms is conducted and which or BL4-P (described later), or if large-scale microbial receives federal funding for research is required to research is conducted, a Biological Safety Officer appoint an Institutional Biosafety Committee (IBC). (BSO) must be appointed. This person, who also The committee is to consist of at least five persons, serves on the IBC, acts as a technical liaison between two of whom are “citizen members” not affiliated researchers and the IBC, develops emergency plans, with the institution. Preferably they are familiar with and periodically inspects facilities and protocols. biosafety issues and have a demonstrated Because higher containment levels require more commitment to the surrounding community, scrutiny, the BSO serves as an additional contact especially as it pertains to human and environmental beyond the IBC. protection. Local government officials, state environmental agency staff, or persons in the medical, occupational health or environmental areas PRINCIPAL INVESTIGATOR are among those individuals suitable for IBC The Principal Investigator (PI) ultimately is membership. The committee should also include at responsible for the research project and for ensuring least one member having expertise in plant, plant compliance with biosafety standards. The PI pathogen, or plant pest containment principles. functions as a project manager as well as a The IBC reviews recombinant DNA research researcher, bearing responsibility for training and programs or proposals and confirms the research supervising personnel, communicating with the IBC, leader’s assignment of the appropriate containment BSO, greenhouse manager and staff, and correcting level for the proposed work. Commonly the IBC first any operations that may result in a loss of considers the proper containment level for the containment. Based on the nature of the transgenic unmodified organism, and then considers whether or organism, the PI determines the proper containment not the proposed change to the organism could level for the project and, in accordance with the NIH increase, decrease, or leave unchanged the organism’s Guidelines, develops the necessary experimental necessary containment level. The Committee ensures protocols; he submits this information to the IBC for compliance with the NIH Guidelines by evaluating review. facilities, procedures, and the expertise of personnel For all experiments to be conducted with plants, involved in the research. In addition, the IBC is the Principal Investigator must file a notification responsible for adopting emergency plans for document with the IBC. Notification is made either responding to an accidental release from at the time the work is initiated or prior to the start containment. To facilitate timely disposal of residual of the experiment, depending on the level of transgenic experimental materials, the IBC may containment required. In some cases, the investigator adopt a closeout policy that provides the project may need to obtain further approvals before leader with written notice of project termination initiation, in addition to that of the IBC. Details of dates. The Committee is responsible for maintaining approval requirements are given in Section III of the and/or verifying documentation of rDNA research at NIH Guidelines. The IBC can assist the PI in the institution, and acts as a point of contact for obtaining requisite approvals. NIH and other agencies.
01-07 Transgenic book 7/16/01 10:25 PM Page 9 SECTION II. Regulation and Oversight of GMOs 9 GREENHOUSE STAFF Greenhouse staff may range in experience from part time student workers who water plants to skilled tradesmen who maintain the facility’s structure and mechanical systems. Regardless of individual duties, all staff should become familiar with any differences between caring for GMOs and conventional plants that may affect their own work. In most cases, a brief orientation session is sufficient to explain the nature of the plants (or other transgenic organisms) and any special practices to be employed when handling or working around them. For example, where transgenic microbes are being tested for their ability to associate with roots, the PI may require that runoff from watering is collected and treated prior to disposal. Both the greenhouse manager and the PI should work with the staff to ensure compliance with safety procedures and standards.
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01-07 Transgenic book 7/16/01 10:25 PM Page 11 11 Biosafety Levels Section III. Biosafety Levels THE PURPOSE OF CONTAINMENT IS TO PREVENT the transfer of recombinant DNA from transgenic organisms inside the greenhouse to populations outside the greenhouse. Section III of the NIH Guidelines describes four physical containment levels for experiments involving recombinant DNA molecules. It further categorizes experiments according to specific risk criteria and assigns them to one of the four biosafety levels, BL1-P through BL4-P. Appendix P of the Guidelines specifies the physical and biological containment conditions and practices required for greenhouse experiments for each biosafety level. A brief description of the four biosafety levels and the criteria used by the NIH Guidelines for assigning experiments to each category are provided here. It is the responsibility of the IBC and PI to determine the appropriate biosafety level. When making a biosafety level assignment, consider the following criteria: • Source and nature of the introduced DNA: whether from an exotic infectious agent or pathogenic organism; and whether a fragment of DNA or complete genome; • Recipient organism: mode and ease of dissemination; invasiveness; whether a noxious weed or capable of interbreeding with noxious weeds; potential for outcrossing between recipient organisms and nearby related species; and potential for detrimental impact on natural or managed ecosystems; • Nature of expressed protein: whether a vertebrate toxin or potential or known allergen; and whether toxic to other organisms in local environment; • Local environment: nature and importance of nearby crops; presence of sexually compatible wild or weedy species; and • Experimental procedures: transfer to or from greenhouse; and necessary containment measures. Sound scientific principles and a thorough knowledge of the recipient organism and its mode of dissemination are the basis for designating a suitable level of containment. A brief comparison of criteria used in the Guidelines to assign an appropriate biosafety level is shown in Table 2.
01-07 Transgenic book 7/16/01 10:26 PM Page 12 12 A G U I D E T O P L A N T C O N TA I N M E N T The table shows that as the potential risk to the environment increases, increasingly stringent requirements for containment are indicated. When applicable, physical containment requirements may be eased by the addition of measures for biological containment, indicated by the “+” sign. (Biological containment is described in Section IV, Elements of Containment.) TABLE 2. Suggested criteria for assigning biosafety levels TRANGENIC MICROBES TRANSGENIC CRITERIA TRANSGENIC INSECTS/ANIMALS/ PLANTS ASSOC. MICROBES Exotic Non-Exotic Not a noxious weed or BL1-P cannot outcross with one Not easily disseminated BL1-P No detriment to BL2-P or BL1-P + BL1-P BL2-P or BL1-P + environment Noxious weed or can BL2-P or BL1-P + interbreed with weeds Contains complete genome BL2-P or BL1-P + of non-EIA* Contains genome of EIA BL3-P or BL2-P + Treated with an EIA BL3-P or BL2-P + Detriment to environment BL3-P-4** BL2-P or BL1-P + BL3-P or BL2-P + Involves EIA with detriment BL3-P or BL2-P + to environment May reconstitute genome BL3-P or BL2-P + of infectious agent in planta Contains Vertebrate Toxin BL3-P BL3-P BL3-P *EIA – Exotic Infectious Agent **BL4-P containment is recommended only for experiments with readily transmissible exotic infectious agents whether transgenic or not, such as air-borne fungi or viruses in the presence of their arthropod vectors that have the potential of being serious pathogens of major US crops.
01-07 Transgenic book 7/16/01 10:26 PM Page 13 SECTION III. Biosafety Levels 13 Experiments that Are Exempt natural or managed ecosystems, such as Rhizobium and Agrobacterium. A BL1-P designation would be Experiments that do not present a risk to health assigned, for example, to an experiment that uses a or the environment are exempt from the NIH transgenic strain of Rhizobium containing Guidelines and do not require the approval of the Agrobacterium genes known to affect root local IBC. For example, research using synthetic colonization, or plants using Agrobacterium DNA DNA molecules that are not part of any organism or segments as part of the transformation process. virus, or research using only DNA segments from a single nonchromosomal or viral source, are exempt. Also exempt are experiments in which the DNA Biosafety Level 2 for Plants (BL2-P) from a particular host organism is propagated only in that same organism, as would be the case for BL2-P is assigned to experiments with transgenic research designed to splice DNA segments taken plants and associated organisms, which, if released from wheat into the genome of the same or another outside the greenhouse, could be viable in the wheat variety. This exemption applies to DNA surrounding environment but would have a segments regardless of whether they were obtained negligible impact or could be readily managed. BL2- from the host chromosomes, chloroplasts, P is required for transgenic plants that may exhibit a mitochondria or plasmids, as long as the fragment is new weedy characteristic or that may be capable of propagated only in that same host, and that no other interbreeding with weeds or related species growing DNA is used, including promoters and enhancers. in the vicinity. For example, greenhouse tests of Finally, the Guidelines exempt research involving the transgenic sunflower containing wheat genes transfer of DNA between two different species if intended to confer resistance to the fungus they are known to exchange DNA by well- Sclerotinia would be classified BL2-P because established physiological means. Appendix A of the sunflower is capable both of hybridizing with wild NIH Guidelines contains a periodically revised list of relatives, and becoming established as a volunteer these natural exchangers10. Currently, most weed. organisms on this list are bacteria and yeast species, BL2-P containment is assigned to transgenic but some genera of plant pathogenic bacteria are experiments that use the entire genome of an included. indigenous infectious agent or pathogen. This level of containment is also appropriate for transgenic plant-associated microorganisms that are either Biosafety Level 1 for Plants (BL1-P) indigenous to the area and potentially harmful to the environment but manageable, or are exotic but have The BL1-P designation provides for a low level of no potential for causing serious harm to managed or containment for experiments involving transgenic natural ecosystems. The BL2-P classification likewise plants in which there is no evidence that the applies to experiments using plant-associated modified organism would be able to survive and transgenic insects or small animals as long as they spread in the environment and, if accidentally pose no threat to managed or natural ecosystems. released, would not pose an environmental risk. For example, an experiment designed to study transgenic potato plants containing cloned genes for insect Biosafety Level 3 for Plants (BL3-P) resistance obtained from primitive potato cultivars would be classified as BL1-P. BL3-P facilities are designed to prevent the BL1-P also applies to DNA-modified common accidental release of transgenic plants, plant microorganisms that cannot spread rapidly and are pathogens, or other organisms that have a not known to have any negative effects on either recognized potential for significant detrimental 10 http://www4.od.nih.gov/oba/appendix_a.htm
01-07 Transgenic book 7/16/01 10:26 PM Page 14 14 A G U I D E T O P L A N T C O N TA I N M E N T impact on the environment. This category also escape the containment facility; in this case, the applies to non-GMO plant research that involves transgenic maize plant does not itself pose a risk. exotic infectious agents capable of causing serious environmental harm. In these cases, it is the pest or pathogen that requires containment; the transgenic plant itself may pose no threat. BL3-P is also recommended for transgenic plants containing genes from an exotic infectious agent in which a complete functional genome of the infectious agent could possibly be reconstituted. Experiments using transgenic plants or organisms that contain genes coding for vertebrate toxins are likewise conducted at BL3-P. Lastly, BL3-P is recommended for experiments using transgenic microbial pathogens of insects or small animals that associate with plants, if the pathogen has the potential to cause harm to the local environment. Examples of research requiring BL3-P facilities: • Testing citrus plants engineered to be resistant to Asiatic Bacterial Canker by infecting them with the disease pathogen, which, if released in Florida, could devastate the commercial citrus crop; • Inoculating transgenic peanut plants containing fungal resistance genes with Aspergillus flavus, the organism responsible for producing the potent vertebrate mycotoxin, aflatoxin. Biosafety Level 4 for Plants (BL4-P) BL4-P is recommended for experiments on certain exotic, readily transmissible infectious agents that are potentially serious pathogens of major US crops, such as soybean rust fungus, maize streak, or other viruses, and that are performed in the presence of their arthropod vector. For example, an experiment to test the efficacy of the maize streak virus coat protein to protect corn plants against infection by that virus would necessarily use its leafhopper vector, Cicadulina spp., in challenge inoculations. This devastating virus is not found in the United States, however leafhopper species capable of transmitting it are present. Thus the experiment using both virus and vector poses a significant risk should either
01-07 Transgenic book 7/16/01 10:26 PM Page 15 15 Elemenst of Containment Section IV. Elements of Containment APPENDIX P OF THE NIH GUIDELINES addresses the containment conditions and practices required for recombinant DNA research involving plants. Achieving containment for genetically modified organisms is an exercise in risk management. The Guidelines state that the principle purpose of GMO containment is to: 1. Avoid unintentional transmission of rDNA-containing plant genomes or release of rDNA-derived organisms associated with plants; 2. Minimize the possibility of unanticipated deleterious effects on organisms and ecosystems outside of the experimental facility; 3. Avoid the inadvertent spread of a serious pathogen from a greenhouse to a local agricultural crop; and 4. Avoid the unintentional introduction and establishment of an organism in a new ecosystem. Environmental protection is the predominant goal; the key to achieving it lies in understanding the biological systems involved and accepted scientific research practices. Containment is accomplished through a combination of management practices, physical barriers, and biological methods intended to prevent GMO transfer or survival. In general, containment requirements are more stringent if plant-associated materials, such as insects and microorganisms, are included in the experiment. If insect quarantine measures are required, regardless of the presence of rDNA material, managers should contact APHIS for guidance. Research involving transgenic plants at the BL1-P or BL2-P containment levels requires little more than the basic facilities, equipment, and protocols common to most research greenhouses. However, greenhouses that offer high-level BL3-P and BL4-P containment are expensive to build and operate. The cost of greenhouse containment at these levels may be prohibitive for many institutions. Other means of attaining a high level of containment, such as use of a growth chamber or growth room, may provide a suitable alternative at a fraction of the cost. The book, Containment Facilities and Safeguards For Exotic Plant Pathogens and Pests11, offers descriptions of high security containment and quarantine facilities operating around the world. 11 Kahn, R. P. and S. B. Mathur. 1999. Containment Facilities and Safeguards: For Exotic Plant Pathogens and Pests. St. Paul, MN.: APS Press.
01-07 Transgenic book 7/16/01 10:26 PM Page 16 16 A G U I D E T O P L A N T C O N TA I N M E N T Growth chambers, tissue culture rooms, incubators, and biological safety cabinets are commonly used in developing GMOs. Biosafety regulations for these facilities are included in Appendix G of the NIH Guidelines, TABLE 3. Comparison of standard practices for containment of plants in greenhouses BIOSAFETY LEVEL 1-P BIOSAFETY LEVEL 2-P discretionary access access limited to individuals directly involved with experiments personnel must read and follow instructions personnel must read and follow instructions procedures followed are appropriate for greenhouse manual to advise of consequences; organisms give contingency plans record kept of experiments in facility record kept of experiments and movement in/out of greenhouse containment required for movement in/out of greenhouse biologically inactivate experimental organisms biologically inactivate experimental organisms at end of experiment at end of experiment; decontaminate gravel periodically pest control program pest control program appropriate caging and precautions for appropriate caging and precautions for escape of motile organisms escape of motile organisms sign for restricted experiment in progress with plant names, person responsible, special requirements
01-07 Transgenic book 7/16/01 10:26 PM Page 17 SECTION IV. Elements of Containment 17 which specifies physical containment standards for the laboratory. Standard practices for plants in greenhouses are summarized in Table 3. BIOSAFETY LEVEL 3-P BIOSAFETY LEVEL 4-P access restricted to required persons only access restricted; secure locked doors; record kept of all entry/exit; clothing change/shower room through air-lock is only means of entry/exit personnel must read and follow instructions all who enter advised of hazards and safeguards greenhouse manual to advise of consequences; greenhouse manual prepared and adopted; give contingency plans personnel required to follow contingency plans record kept of experiments and movement record kept of experimental material moving in/out of facility in/out of greenhouse containment required for movement in/out; special packaging containment for in/out; external decontamination airlock or decontamination for removal entry of supplies/materials through special chamber biologically inactivate experimental organisms decontaminate experimental materials prior to at end of experiment (including water runoff); removal from area by autoclave/other means decontaminate equipment & supplies all runoff water collected and decontaminated pest control program chemical control program for pests and pathogens appropriate caging and precautions for escape appropriate caging and precautions for escape of motile organisms of motile organisms sign for restricted experiment in progress; sign for restricted experiment in progress; person responsible, special requirements; special requirements, person responsible; biohazard symbol if a risk to humans biohazard symbol if a risk to humans minimize aerosol creation to reduce standard microbiological procedures to contamination decontaminate equipment and containers protective clothing worn to minimize dissemi- street clothing removed; complete change to lab nation; hands washed before leaving facility clothing which is autoclaved before laundering report/record accidents
01-07 Transgenic book 7/16/01 10:27 PM Page 18 18 A G U I D E T O P L A N T C O N TA I N M E N T FIG 1. Caulking around service intrusions FIG 2. Sill caulking
01-07 Transgenic book 7/16/01 10:27 PM Page 19 SECTION IV. Elements of Containment 19 PHYSICAL CONTAINMENT Caulking and Sealing Physical containment is achieved through facility Caulking materials are commonly used to seal design and equipment. Choices in the type of glazing, glass panes, sills, and small openings in and around sealing, screening, air flow system, and other features greenhouse structures. Caulking and sealing restricts all affect the degree to which a greenhouse is capable the passage of insects and assists with temperature of isolating transgenic plants, plant parts, and control within the greenhouse; however, it should associated organisms from the surrounding not be considered a substitute for well-fitting environment. These systems are also effective in structural components. Additional caulking and keeping unwanted pests out of the greenhouse. sealing can help to upgrade a conventional facility to meet the standards of an approved containment facility. Typical situations where the addition of Glazing caulk provides an extra measure of containment are The term glazing refers to any transparent illustrated in Figures 1 and 2. material (as glass) used for windows. Properly installed and regularly maintained greenhouse glazing of any typical material can provide a suitable Screening barrier for transgenic research materials. The type of When properly sized, installed, and maintained, glazing most commonly used consists of single panes screen can keep pests and pollinators out of a of tempered glass installed by lapping each pane over greenhouse or, conversely, keep experimental the one below. The care taken in installing and organisms in. The integrity of a screening system is maintaining the glazing determines its overall determined by several factors including the nature of effectiveness. Improperly installed or loose-fitting the material, the size and morphology of the insects glazing material can leave gaps through which being excluded, the hole shape and size, and the air transgenic materials could be released pressure applied on either side of the screen. The unintentionally. maximum hole size generally capable of restricting certain insect species is shown in Table 4. Anti- 12 Virus™ screening is a commercial product advertised to be 100% effective in excluding leafminers, melon aphids, and whiteflies. TABLE 4. Mesh sizes* for insect containment13 SCREEN HOLE SIZE ADULT INSECT mesh microns2 inches2 Leafminers 40 640 0.025 Silverleaf Whiteflies 52 460 0.018 Melon Aphids 78 340 0.013 Flower Thrips 132 190 0.0075 *The number of threads per linear inch defines the mesh size of the screen; e.g., a 30-mesh screen has 30 threads per inch. 12 Gintec Shade Technologies Inc.: http://www.gintec-shade.com/greenhouse-screens.html 13 Adapted from “Greenhouse Screening for Insect Control.” Rutgers Cooperative Extension. http://www.wvu.edu/~agexten/hortcult/greenhou/fs640.htm
01-07 Transgenic book 7/16/01 10:27 PM Page 20 20 A G U I D E T O P L A N T C O N TA I N M E N T FIG 3. Negative pressure bench-top containment unit Negative Air Pressure Cages Containment of airborne pollen, spores, and Insect cages, when properly used, can increase the insects is a significant challenge. One strategy to help containment level of a particular experiment as long achieve it is to create negative air pressure within a as the factors listed above pertaining to screen facility. Negative pressure exists when the amount of characteristics and sizing are respected. Though air exiting a space exceeds the air intake. Negative researchers may fashion their own cages out of pressure bench-top chambers can increase metal, wood, glass, or screen, commercial models are containment of pathogens and insects within also available. The Bugdorm insect cage (Fig. 4) is greenhouses, screenhouses, and laboratories. A a type of cage available from biological and chambered wood and clear plastic box fitted with a greenhouse supply companies. blower and filtration system can produce negative pressure on a small scale and at a relatively low cost (Fig. 3).
01-07 Transgenic book 7/16/01 10:27 PM Page 21 SECTION IV. Elements of Containment 21 FIG 4. Bugdorm™ insect cage Location BIOLOGICAL CONTAINMENT The geographical location of a greenhouse provides Biological processes can provide a highly effective an element of physical containment. Research involving means of preventing unintended transmission of genetic a crop pest or noxious weed, for instance, presents a material. Biological containment methods include greater risk if the facility is located in an area adjacent reproductive, spatial, and temporal isolation. Appendix to large cropping areas susceptible to the pest. When P of the NIH Guidelines provides a partial list of the planning new facilities, it is important to determine biological containment practices appropriate for plants, what type of agricultural activities will be occurring in microbes, and insects. Scientists and technicians adjacent areas before siting. Most work with GMOs, conducting transgenic research generally best however, does not require remote or otherwise special understand the biological systems involved. They are at siting since other safeguards are usually adequate. liberty to devise other means of biological containment in their experimental protocols, subject to review by the IBC.
01-07 Transgenic book 7/16/01 10:31 PM Page 22 22 A G U I D E T O P L A N T C O N TA I N M E N T Fig 5.1—5.2 Bagging flowers for biological containment Plants • Ensure that cross-fertile plants are not within the pollen dispersal range of the experimental plant; or One or more of the following procedures can • Use genetic modification techniques that localize prevent dissemination of genetic material by pollen transgenes in non-propagative plant parts. or seed: • Cover or remove flower and seed heads to prevent pollen and seed dispersal; Bagging flowers is a standard practice used by breeders to prevent the contamination of selected • Harvest plant material prior to sexual maturity or plants with pollen from adjacent plants. Female use male sterile lines; flowers can be covered to prevent insect pollinators • Control the time of flowering so that pollen shed or windblown pollen from landing on the receptive does not coincide with the receptive period of surface. Male flowering structures can be bagged to sexually compatible plants nearby; prevent pollen from being disseminated by insect vectors, wind, or mechanical transfer (Fig. 5.1-5.2).
01-07 Transgenic book 7/16/01 10:31 PM Page 23 SECTION IV. Elements of Containment 23 TABLE 5. Isolation distances (in feet) from contaminating sources for selected groups CROP FOUNDATION REGISTERED CERTIFIED 1,2 1,2,3 1,4 Alfalfa 600 300 165 5,6 Corn (inbred lines) 660 — — 6,7 Corn (hybrid) 660 8 8 8 Cotton (hybrid) 0 0 0 9, 10, 11 9,10,11 9,10,11,12 Grasses (cross pollinated) 900 300 165 13 13 13 Mung Beans 0 0 0 Onion 5280 2640 1320 13 13 13 Peanuts 0 0 0 14 14 14 Pepper 200 100 30 15 15 Rape (self pollinated) 660 — 330 15 15 Rape (cross pollinated) 1320 — 330 16 16 16 Rice 10 10 10 13 13 13 Soybeans 0 0 0 17,18 17,18 17,18 Sunflower 2640 2640 2640 17,18 17,18 Sunflower (hybrid) 2640 — 2640 14 14 14 Tomato 200 100 30 19 19 19 Watermelon 2640 2640 1320 Source: Modified from “Genetic and Crop Standards” of the AOSCA: http://aosca.org/g&ccont.htm Paper and glassine bags are most commonly used to Crop breeders have identified numerous crops with cover flower heads. Flower heads can be removed sexually compatible wild or weedy relatives. prior to pollen or seed production in cases where the Examples of crops that outcross with wild relatives research protocol does not require seed collection. are given in Table 6. 14 Genetic and Crop Standards of the AOSCA , Depending on the location of the containment published annually by the Official Seed Certifying facility, the choice of season in which to conduct an Agencies, describes the isolation distances required experiment may constitute an appropriate biological to avoid genetic contamination by pollen dispersal. containment method for plants. For instance, Table 5 shows isolation distances for selected crops. growing transgenic sunflowers only during the In order to be considered an environmental risk, winter in northern climates insures that any escaped transgenic pollen must be able to fertilize plants of a pollen would be of no consequence to local plants or sexually compatible species growing in the vicinity. weeds. 14 Association of Official Seed Certifying Agencies: http://aosca.org/g&ccont.htm
01-07 Transgenic book 7/16/01 10:31 PM Page 24 24 A G U I D E T O P L A N T C O N TA I N M E N T 1. Distance between fields of Certified classes of the observed morphological characteristics for the field same variety may be reduced to 10 feet regardless of to be inspected. Isolation distance between upland class or size of field. and Egyptian types is 1320, 1320, and 660 for 2. This distance applies for fields over five acres. For Foundation, Registered, and Certified, respectively. alfalfa fields of five acres or less that produce the 9. Isolation between classes of the same variety may be Foundation and Registered seed classes, the reduced to 25% of the distance otherwise required. minimum distance from a different variety or a field 10. Isolation between diploids and tetraploids shall at of the same variety that does not meet the varietal least be 15 feet. purity requirements for certification shall be 900 and 450 feet, respectively. 11. Border removal applies only to fields of five acres or more. These distances apply when there is no 3. Isolation distance for Certified seed production of border removal. Removal of a 9-foot border (after varieties adapted to the northern and central regions flowering) decreases the required distance to 600, shall be 500 feet from varieties adapted to the 225, and 100 feet for cross-pollinated species, and southern region. to 30, 15, and 15 feet for apomictic and self- 4. There must be at least 10 feet or a distance adequate pollinated species. Removal of a 15-foot border to prevent mechanical mixture between a field of allows a further decrease to 450, 150 and 75 feet another variety (or non-certified area within the for cross-pollinated species. same field) and the area being certified. The 165 feet 12. Application to establish pedigree must be made isolation requirement is waived if the area of the within one year of seeding. The crop will remain “isolation zone” is less than 10 percent of the field under supervision of the certifying agency as long as eligible for the Certified class. The “isolation zone” the field is eligible for certification. is that area calculated by multiplying the length of the common border(s) with other varieties of alfalfa 13. Distance adequate to prevent mechanical mixture is by the average width of the field (being certified) necessary. falling within the 165 feet isolation. Areas within the 14. The minimum distance may be reduced by 50 isolation zone nearest the contamination source percent if different classes of the same variety are shall not be certified. involved. 5. No isolation is required for the production of hand- 15. Required isolation between classes of the same pollinated seed. variety is 10 feet. 6. When the contaminant is of the same color and 16. Isolation between varieties or non-certified field of texture, the isolation distance may be modified by the same variety shall be 10 feet if ground drilled, 50 (1) adequate natural barriers, or (2) differential feet if ground broadcast, and 100 feet if aerial maturity dates provided there are no receptive silks seeded. in the seed parent at the time the contaminant is 17. Does not apply to Helianthus similes, H. ludens or shedding pollen. In addition, dent sterile popcorn H. agrestis. requires no isolation from dent corn. 18. An isolation distance of 5,280 feet is required 7. Where the contaminating source is corn of the same between oil and non-oil sunflower types and color and texture as that of the field inspected or between either type and other volunteer or wild white endosperm corn that is optically sorted, the types. isolation distance is 410 feet and may be modified by the planting of pollen parent border row. 19. The minimum distance may be reduced by 50 percent if the field is adequately protected by natural 8. Minimum isolation shall be 100 feet if the cotton or artificial barriers. plants in the contaminating source differ by easily
01-07 Transgenic book 7/16/01 10:31 PM Page 25 SECTION I. Introduction 25 15 TABLE 6. Commercially important species that hybridize with wild relatives in the USA 16 CULTIVATED SPECIES WILD RELATIVE Apium graveolens (celery) Same species Daucus carota (carrot) Same species (wild carrot) Chenopodium quinoa (quingua [a grain]) C. berlandieri Beta vulgaris (beet) B. vulgaris var. maritima (hybrid is a weed) Chicorium intybus (chicory) Same species Helianthus annuus (sunflower) Same species Lactuca sativa (lettuce) L. serriola (wild lettuce) Brassica napus (oilseed rape; canola) Same species, B. campestris, B. juncea Brassica rapa (turnip) Same species (= B. campestris) Raphanus sativus (radish) Same species, R. raphanistrum Cucurbita pepo (squash) Same species (= C. texana, wild squash) Vaccinium macrocarpon (cranberry) Same species Vaccinium angustifolium (blueberry) Same species Trifolium spp. (clover) Same species Medicago sativa (alfalfa) Same species Liquidambar styraciflua (sweetgum) Same species Juglans regia (walnut) J. hindsii Asparagus officinalis (asparagus) Same species Picea glauca (spruce) Same species Avena sativa (oat) A. fatua (wild oats) Cynodon dactylon (bermuda grass) Same species Oryza sativa (rice) Same species (red rice) Sorghum bicolor (sorghum) S. halapense (johnsongrass) Amelanchier laevis (serviceberry) Same species Fragaria sp.(strawberry) F. virginiana, F. chiloensis, others Rubus spp. (raspberry, blackberry) Same species Populus alba x P. grandidentata (poplar) Populus species. (ten species listed as weed of unknown status in U.S.) Nicotiana tabacum (tobacco) Same species (escapes cultivation) Vitus vinifera (grape) Vitus spp. (wild grape) 15 Adapted by permission of the authors (Allison Snow and Pedro Moran Palma, Department of Plant Biology, Ohio State University) from their publication titled "Commercialization of Transgenic Plants: Potential Ecological Risks." Bioscience (1997), Vol. 47, pp. 86-96. 16 This is not an exhaustive list, especially with regard to commercially important grasses and woody species, which often occur in unmanaged populations. Also, for many cultivars the extent of hybridization with wild relatives has not been studied.