Guidelines for Handling Transgenic Plants and Associated Organisms

• Level I
• Level II
• Level III

Guidance for handling potentially biohazardous plants and associated organisms lags behind that available for vertebrates and their infectious agents. The United States Department of Agriculture, Animal and Plant Health Inspection Service (USDA-APHIS) regulates importation, interstate movement and environmental release of plant pests and transgenic plants but provides minimal guidance for management of facilities. The best available information at this time comes from the NIH Guidelines for Research Involving Recombinant DNA Molecules. While the NIH Guidelines specifically addresses recombinant DNA, the recommendations regarding effective containment are relevant equally to research using non-recombinant methods.

This document presents portions of the NIH Guidelines that pertain to containment of transgenic plants and associated organisms. The content is consistent with that of the Guidelines but the format has been rearranged to make it more readable. The current version of the NIH Guidelines can be accessed at http://oba.od.nih.gov/rdna/nih_guidelines_oba.html.

Introduction to Containment

The principle purposes of containment are:

• To avoid unintentional transmission.
• To minimize the possibility of an unanticipated deleterious effect on organisms and ecosystems outside of the experimental facility.
• To avoid the inadvertent spread of a serious pathogen from a greenhouse to a local agricultural crop.
• To avoid the unintentional introduction and establishment of an organism in a new ecosystem

Containment may be achieved by a combination of physical and biological means. Containment for transgenic plants and their associated plant pathogens relies more heavily on biological factors than is the norm for human and animal infectious agents. The goal is to protect the environment, not the researcher. The risk assessment considers the specific organism(s), geographic/ecological setting and the available mechanical barriers; the selected practices are tailored to the specific situation. It becomes especially difficult to prescribe containment when genetic modifications lead to uncertainty in characteristics like host range and competitiveness.

For research involving plants, four biosafety levels (BL1-P through BL4-P) are described. BL1-P is designed to provide a moderate level of containment for experiments for which there is convincing biological evidence that precludes the possibility of survival, transfer, or dissemination of recombinant DNA into the environment, or in which there is no recognizable and predictable risk to the environment in the event of accidental release. BL2-P is designed to provide a greater level of containment for experiments involving plants and certain associated organisms in which there is a recognized possibility of survival, transmission, or dissemination of recombinant DNA containing organisms, but the consequence of such an inadvertent release has a predictably minimal biological impact. BL3-P and BL4-P describe additional containment conditions for research with plants and certain pathogens and other organisms that require special containment because of their recognized potential for significant detrimental impact on managed or natural ecosystems. (Section II-B.)

BL1-P relies upon accepted scientific practices for conducting research in most ordinary greenhouse or growth chamber facilities and incorporates accepted procedures for good pest control and cultural practices. BL1-P facilities and procedures provide a modified and protected environment for the propagation of plants and microorganisms associated with the plants and a degree of containment that adequately controls the potential for release of biologically viable plants, plant parts, and microorganisms associated with them. BL2-P and BL3-P rely upon accepted scientific practices for conducting research in greenhouses with organisms infecting or infesting plants in a manner that minimizes or prevents inadvertent contamination of plants within or surrounding the greenhouse. BL4-P describes facilities and practices known to provide containment of certain exotic plant pathogens. (Section II-B.)

Determination of Containment Level (Section III)

Knowledge of the organisms and judgment based on accepted scientific practices should be used in all cases in selecting the appropriate level of containment. For example, if the genetic modification has the objective of increasing pathogenicity or converting a non-pathogenic organism into a pathogen, then a higher level of containment may be appropriate depending on the organism, its mode of dissemination, and its target organisms.

Experiments that fall under Section III-E require Institutional Biosafety Committee notice simultaneous with initiation. Those that fall under Section III-D require Institutional Biosafety Committee approval before initiation.

BL1-P is recommended for all experiments with recombinant DNA-containing plants and plant-associated microorganisms (excluding those covered below for Section III-D). Example: plant transformation using recombinant Agrobacterium where the genetic modification is not expected to increase adverse characteristics. [Section III-E]

Experiments requiring BL2-P or a higher level of containment involve plants that are noxious weeds, that can interbreed with noxious weeds in the immediate geographic area; or have recognized potential for rapid and widespread dissemination or for serious detrimental impact on managed or natural ecosystems.

BL2-P or BL1-P+ Biological Containment is recommended for:

• Plants modified by rDNA that are noxious weeds or can interbreed with noxious weed in the immediate geographic area. [Section III-E-2-b-(1)]
  
• Plants in which the introduced DNA represents the complete genome of a non-exotic infectious agent. [Section III-E-2-b-(2)]
  
• Plants associated with recombinant DNA-modified non-exotic microorganisms that have a recognized potential for serious detrimental impact on managed or natural ecosystems. [Section III-E-2-b-(3)]
  
• Plants associated with recombinant DNA-modified exotic microorganisms that have no recognized potential for serious detrimental impact on managed or natural ecosystems. [Section III-E-2-b-(4)]
  
• Experiments with recombinant DNA-modified arthropods or small animals associated with plants, or with arthropods or small animals with recombinant DNA-modified microorganisms associated with them if the recombinant DNA-modified microorganisms have no recognized potential for serious detrimental impact on managed or natural ecosystems. (Section III-E-2-b-(5))

BL3-P or BL2-P+Biological Containment is recommended for:

• Experiments involving most exotic infectious agents with recognized potential for serious detrimental impact on managed or natural ecosystems when recombinant DNA techniques are associated with whole plants. [Section III-D-5-a]
  
• Experiments involving plants containing cloned genomes of readily transmissible exotic infectious agents with recognized potential for serious detrimental effects on managed or natural ecosystems in which there exists the possibility of reconstituting the complete and functional genome of the infectious agent by genomic complementation in planta. [Section III-D-5-b]
  
• Experiments with microbial pathogens of insects or small animals associated with plants if the recombinant-DNA modified organism has a recognized potential for serious detrimental impact on managed or natural ecosystems. [Section III-D-5-e]

BL3-P is recommended for experiments involving sequences encoding potent vertebrate toxins introduced into plants or plant-associated organisms. [Section III-D-5-d]

Biological Containment in the Greenhouse (Appendix P)

The term "greenhouse" refers to a structure with walls, a roof, and a floor designed and used principally for growing plants in a controlled and protected environment. The walls and roof are usually constructed of transparent or translucent material to allow passage of sunlight for plant growth. The term "greenhouse facility" includes the actual greenhouse rooms or compartments for growing plants, including all immediately contiguous hallways and head-house areas, and is considered part of the confinement area.

Appendix P of the NIH Guidelines specifies physical and biological containment conditions and practices suitable to the greenhouse conduct of experiments involving recombinant DNA-containing plants, plant-associated microorganisms, and small animals. Appendix P applies when the research plants are of a size, number, or have growth requirements that preclude the use of laboratory containment conditions described in Appendix G, Physical Containment.

The principal purpose of plant containment is to avoid the unintentional transmission of a recombinant DNA-containing plant genome, including nuclear or organelle hereditary material or release of recombinant DNA-derived organisms associated with plants.

The containment principles are based on the recognition that the organisms that are used pose no health threat to humans or higher animals (unless deliberately modified for that purpose), and that the containment conditions minimize the possibility of an unanticipated deleterious effect on organisms and ecosystems outside of the experimental facility, e.g., the inadvertent spread of a serious pathogen from a greenhouse to a local agricultural crop or the unintentional introduction and establishment of an organism in a new ecosystem.

Four biosafety levels, referred to as Biosafety Level (BL) 1 - Plants (P), BL2-P, BL3-P, and BL4-P, are designed to provide differential levels of biosafety for plants in the absence or presence of other experimental organisms that contain recombinant DNA. These biosafety levels, in conjunction with biological containment conditions provide flexible approaches to ensure the safe conduct of research.

Biological Containment of Plants: Effective dissemination of plants by pollen or seed can be prevented by one or more of the following procedures:

• Cover the reproductive structures to prevent pollen dissemination at flowering and seed dissemination at maturity;
• Remove reproductive structures by employing male sterile strains, or harvest the plant material prior to the reproductive stage;
• Ensure that experimental plants flower at a time of year when cross-fertile plants are not flowering within the normal pollen dispersal range of the experimental plant; or (iv) ensure that cross-fertile plants are not growing within the known pollen dispersal range of the experimental plant.

Biological Containment of Microorganisms: Effective dissemination of microorganisms beyond the confines of the greenhouse can be prevented by one or more of the following procedures:

• Confine all operations to injections of microorganisms or other biological procedures (including genetic manipulation) that limit replication or reproduction of viruses and microorganisms or sequences derived from microorganisms, and confine these injections to internal plant parts or adherent plant surfaces;
• Ensure that organisms, which can serve as hosts or promote the transmission of the virus or microorganism, are not present within the farthest distance that the airborne virus or microorganism may be expected to be effectively disseminated;
• Conduct experiments at a time of year when plants that can serve as hosts are either not growing or are not susceptible to productive infection;
• Use viruses and other microorganisms or their genomes that have known arthropod or animal vectors, in the absence of such vectors;
• Use microorganisms that have an obligate association with the plant; or
• Use microorganisms that are genetically disabled to minimize survival outside of the research facility and whose natural mode of transmission requires injury of the target organism, or assures that inadvertent release is unlikely to initiate productive infection of organisms outside of the experimental facility.

Biological Containment of Macroorganisms: Effective dissemination of arthropods and other small animals can be prevented by using one or more of the following procedures:

• Use non-flying, flight-impaired, or sterile arthropods;
• Use non-motile or sterile strains of small animals;
• Conduct experiments at a time of year that precludes the survival of escaping organisms;
• Use animals that have an obligate association with a plant that is not present within the dispersal range of the organism; or
• Prevent the escape of organisms present in run-off water by chemical treatment or evaporation of run-off water.

Physical Containment in the Greenhouse (Appendix P)

Biosafety Level 1 - Plants (BL1-P)

Access to the greenhouse shall be limited or restricted, at the discretion of the Greenhouse Director, when experiments are in progress.

Prior to entering the greenhouse, personnel shall be required to read and follow instructions on BL1-P greenhouse practices and procedures. All procedures shall be performed in accordance with accepted greenhouse practices that are appropriate to the experimental organism.

A record shall be kept of experiments currently in progress in the greenhouse facility.

Experimental organisms shall be rendered biologically inactive by appropriate methods before disposal outside of the greenhouse facility.

A program shall be implemented to control undesired species (e.g., weed, rodent, or arthropod pests and pathogens), by methods appropriate to the organisms and in accordance with applicable state and Federal laws.

Arthropods and other motile macroorganisms shall be housed in appropriate cages. If macroorganisms (e.g., flying arthropods or nematodes) are released within the greenhouse, precautions shall be taken to minimize escape from the greenhouse facility.

Experiments involving other organisms that require a containment level lower than BL1-P may be conducted in the greenhouse concurrently with experiments that require BL1-P containment, provided that all work is conducted in accordance with BL1-P greenhouse practices.

The greenhouse floor may be composed of gravel or other porous material. At a minimum, impervious (e.g., concrete) walkways are recommended.

Windows and other openings in the walls and roof of the greenhouse facility may be open for ventilation as needed for proper operation and do not require any special barrier to contain or exclude pollen, microorganisms, or small flying animals (e.g., arthropods and birds); however, screens are recommended.

Biosafety Level 2 - Plants (BL2-P)

Access to the greenhouse shall be limited or restricted, at the discretion of the Greenhouse Director, to individuals directly involved with the experiments when they are in progress.

Personnel shall be required to read and follow instructions on BL2-P practices and procedures. All procedures shall be conducted in accordance with accepted greenhouse practices that are appropriate to the experimental organisms.

A record shall be kept of experimental plants, microorganisms, or small animals that are brought into or removed from the greenhouse facility.

A record shall be kept of experiments currently in progress in the greenhouse facility.

The Principal Investigator shall report any greenhouse accident involving the inadvertent release or spill of microorganisms to the Greenhouse Director, Institutional Biosafety Committee, NIH/ORDA and other appropriate authorities immediately (if applicable). Documentation of any such accident shall be prepared and maintained.

Experimental organisms shall be rendered biologically inactive by appropriate methods before disposal outside of the greenhouse facility.

Decontamination of run-off water is not necessarily required. If part of the greenhouse is composed of gravel or similar material, appropriate treatments should be made periodically to eliminate, or render inactive, any organisms potentially entrapped by the gravel.

A program shall be implemented to control undesired species (e.g., weed, rodent, or arthropod pests and pathogens) by methods appropriate to the organisms and in accordance with applicable state and Federal laws.

Arthropods and other motile macroorganisms shall be housed in appropriate cages. If macroorganisms (e.g., flying arthropods or nematodes) are released within the greenhouse, precautions shall be taken to minimize escape from the greenhouse facility.

Experiments involving other organisms that require a containment level lower than BL2-P may be conducted in the greenhouse concurrently with experiments that require BL2-P containment provided that all work is conducted in accordance with BL2-P greenhouse practices.

A sign shall be posted indicating that a restricted experiment is in progress. The sign shall indicate the following:

• The name of the responsible individual,
• the plants in use, and
• any special requirements for using the area.

If organisms are used that have a recognized potential for causing serious detrimental impacts on managed or natural ecosystems, their presence shall be indicated on a sign posted on the greenhouse access doors.

If there is a risk to human health, a sign shall be posted incorporating the universal biosafety symbol.

Materials containing experimental microorganisms, which are brought into or removed from the greenhouse facility in a viable or intact state, shall be transferred in a closed non-breakable container.

A greenhouse practices manual shall be prepared or adopted. This manual shall: (i) advise personnel of the potential consequences if such practices are not followed, and (ii) outline contingency plans to be implemented in the event of the unintentional release of organisms.

A greenhouse floor composed of an impervious material. Concrete is recommended, but gravel or other porous material under benches is acceptable unless propagules of experimental organisms are readily disseminated through soil. Soil beds are acceptable unless propagules of experimental organisms are readily disseminated through soil.

Windows and other openings in the walls and roof of the greenhouse facility may be open for ventilation as needed for proper operation and do not require any special barrier to exclude pollen or microorganisms; however, screens are required to exclude small flying animals (e.g., arthropods and birds).

An autoclave shall be available for the treatment of contaminated greenhouse materials.

If intake fans are used, measures shall be taken to minimize the ingress of arthropods. Louvers or fans shall be constructed such that they can only be opened when the fan is in operation.

Biosafety Level 3 - Plants (BL3-P)

Authorized entry into the greenhouse shall be restricted to individuals who are required for program or support purposes. The Greenhouse Director shall be responsible for assessing each circumstance and determining those individuals who are authorized to enter the greenhouse facility.

Prior to entering the greenhouse, personnel shall be required to read and follow instructions on BL3-P practices and procedures. All procedures shall be conducted in accordance with accepted greenhouse practices that are appropriate to the experimental organisms.

A record shall be kept of experimental plants, microorganisms, or small animals that are brought into or removed from the greenhouse facility.

A record shall be kept of experiments currently in progress in the greenhouse facility.

The Principal Investigator shall report any greenhouse accident involving the inadvertent release or spill of microorganisms to the Biological Safety Officer, Greenhouse Director, Institutional Biosafety Committee, NIH/ORDA, and other appropriate authorities immediately (if applicable). Documentation of any such accident shall be prepared and maintained.

All experimental materials shall be sterilized in an autoclave or rendered biologically inactive by appropriate methods before disposal, except those that are to remain in a viable or intact state for experimental purposes; including water that comes in contact with experimental microorganisms or with material exposed to such microorganisms, and contaminated equipment and supplies.

A program shall be implemented to control undesired species (e.g., weed, rodent, or arthropod pests and pathogens) by methods appropriate to the organisms and in accordance with applicable state and Federal laws.

Arthropods and other motile macroorganisms shall be housed in appropriate cages. When appropriate to the organism, experiments shall be conducted within cages designed to contain the motile organisms.

Experiments involving organisms that require a containment level lower than BL3-P may be conducted in the greenhouse concurrently with experiments that require BL3-P containment provided that all work is conducted in accordance with BL3-P greenhouse practices.

A sign shall be posted indicating that a restricted experiment is in progress. The sign shall indicate the following:

• The name of the responsible individual,
• The plants in use, and
• Any special requirements for using the area.

If organisms are used that have a recognized potential for causing serious detrimental impacts on managed or natural ecosystems, their presence should be indicated on a sign posted on the greenhouse access doors.

If there is a risk to human health, a sign shall be posted incorporating the universal biosafety symbol.

Experimental materials that are brought into or removed from the greenhouse facility in a viable or intact state shall be transferred to a non-breakable sealed secondary container. At the time of transfer, if the same plant species, host, or vector are present within the effective dissemination distance of propagules of the experimental organism, the surface of the secondary container shall be decontaminated. Decontamination may be accomplished by passage through a chemical disinfectant or fumigation chamber or by an alternative procedure that has demonstrated effective inactivation of the experimental organism.

A greenhouse practices manual shall be prepared or adopted. This manual shall: (i) advise personnel of the potential consequences if such practices are not followed, and (ii) outline contingency plans to be implemented in the event of the unintentional release of organisms with recognized potential for serious detrimental impact.

Disposable clothing (e.g., solid front or wrap-around gowns, scrub suits, or other appropriate clothing) shall be worn in the greenhouse if deemed necessary by the Greenhouse Director because of potential dissemination of the experimental microorganisms.

Protective clothing shall be removed before exiting the greenhouse and decontaminated prior to laundering or disposal.

Personnel are required to thoroughly wash their hands upon exiting the greenhouse.

All procedures shall be performed carefully to minimize the creation of aerosols and excessive splashing of potting material/soil during watering, transplanting, and all experimental manipulations.

The need to maintain negative pressure should be considered when constructing or renovating the greenhouse.

The greenhouse floor shall be composed of concrete or other impervious material with provision for collection and decontamination of liquid run-off.

Windows shall be closed and sealed. All glazing shall be resistant to breakage (e.g., double-pane tempered glass or equivalent).

The greenhouse shall be a closed self-contained structure with a continuous covering that is separated from areas that are open to unrestricted traffic flow. The minimum requirement for greenhouse entry shall be passage through two sets of self-closing locking doors.

The greenhouse facility shall be surrounded by a security fence or protected by equivalent security measures.

Internal walls, ceilings, and floors shall be resistant to penetration by liquids and chemicals to facilitate cleaning and decontamination of the area. All penetrations into these structures and surfaces (e.g., plumbing and utilities) shall be sealed.

Bench tops and other work surfaces should have seamless surfaces that are impervious to water and resistant to acids, alkalis, organic solvents, and moderate heat.

The greenhouse contains a foot, elbow, or automatically operated sink, which is located near the exit door for hand washing.

An autoclave shall be available for decontaminating materials within the greenhouse facility. A double-door autoclave is recommended (not required) for the decontamination of materials passing out of the greenhouse facility.

An individual supply and exhaust air ventilation system shall be provided. The system maintains pressure differentials and directional airflow, as required, to assure inward (or zero) airflow from areas outside of the greenhouse.

The exhaust air from the greenhouse facility shall be filtered through high efficiency particulate air-HEPA filters and discharged to the outside. The filter chambers shall be designed to allow in situ decontamination before filters are removed and to facilitate certification testing after they are replaced.

Air filters shall be 80-85% average efficiency by the American Society of Heating, Refrigerating, and Air Conditioning Engineers (ASHRAE) Standard 52-68 test method using atmosphere dust. Air supply fans shall be equipped with a back-flow damper that closes when the air supply fan is off. Alternatively, a HEPA filter may be used on the air supply system instead of the filters and damper. The supply and exhaust airflow shall be interlocked to assure inward (or zero) airflow at all times.

BL3-P greenhouse containment requirements may be satisfied using a growth chamber or growth room within a building provided that the location, access, airflow patterns, and provisions for decontamination of experimental materials and supplies meet the intent of the foregoing clauses.

Vacuum lines shall be protected with high efficiency particulate air/HEPA or equivalent filters and liquid disinfectant traps.