Biosafety Levels for Experiments involving Plants with rDNA/sNAs and microorganisms
Note: the following definition is used for the term “exotic plant pathogen” per NIH Guidelines:
In accordance with accepted scientific and regulatory practices of the discipline of plant pathology, an exotic plant pathogen (e.g., virus, bacteria, or fungus) is one that is unknown to occur within the U.S. (see Section V-G, Footnotes and References of Sections I-IV). Determination of whether a pathogen has a potential for serious detrimental impact on managed (agricultural, forest, grassland) or natural ecosystems should be made by the Principal Investigator and the Institutional Biosafety Committee, in consultation with scientists knowledgeable of plant diseases, crops, and ecosystems in the geographic area of the research (Section V-M).
Biosafety Level 1- Plants (BL1-P)
BL1-P includes all experiments with recombinant or synthetic nucleic acid molecule-containing plants and plant-associated microorganisms not covered in Section III-E-2-b or other sections of the NIH Guidelines. Examples of such experiments include:
- those involving recombinant or synthetic nucleic acid molecule-modified plants that are not noxious weeds or
- plants that cannot interbreed with noxious weeds in the immediate geographic area, or
- experiments involving whole plants and recombinant or synthetic nucleic acid molecule-modified non-exotic or microorganisms that have no recognized potential for rapid and widespread dissemination or for serious detrimental impact on managed or natural ecosystems (e.g., Rhizobium spp. and Agrobacterium spp.) Section III-E-2-a.
Biosafety Level 2- Plants (BL2-P)
BL-2P includes:
- Experiments involving modification of plants by recombinant or synthetic nucleic acid molecules that are noxious weeds or can interbreed with noxious weeds in the immediate geographic area. Section III-E-2-b-(1).
- Experiments in which the introduced DNA represents the complete genome of a non-exotic infectious agent into plants. Section III-E-2-b-(2).
- Plants associated with recombinant or synthetic nucleic acid molecule-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 or synthetic nucleic acid molecule-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 or synthetic nucleic acid molecule-modified arthropods or small animals associated with plants, or with arthropods or small animals with recombinant or synthetic nucleic acid molecule-modified microorganisms associated with them if the recombinant or synthetic nucleic acid molecule-modified microorganisms have no recognized potential for serious detrimental impact on managed or natural ecosystems. Section III-E-2-b-(5).
Biosafety Level 3- Plants (BL3-P)
BL3-P includes:
- Experiments involving most exotic (see Section V-M, Footnotes and References of Sections I-IV) infectious agents with recognized potential for serious detrimental impact on managed or natural ecosystems when recombinant or synthetic nucleic acid molecule techniques are associated with whole plants. Section III-D- 5-a.
- Experiments involving plants containing cloned genomes of readily transmissible exotic (see Section V-M, Footnotes and References of Sections I-IV) 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 involving sequences encoding potent vertebrate toxins introduced into plants or associated organisms. Recombinant or synthetic nucleic acid molecules containing genes for the biosynthesis of toxin molecules lethal for vertebrates at an LD50 of <100 nanograms per kilogram body weight fall under Section III-B-1, Experiments Involving the Cloning of Toxin Molecules with LD50 of Less than 100 Nanograms Per Kilogram Body Weight, and require NIH/OBA and Institutional Biosafety Committee approval before initiation. Section III-D-5-d.
- Experiments with microbial pathogens of insects or small animals associated with plants if the recombinant or synthetic nucleic acid molecule-modified organism has a recognized potential for serious detrimental impact on managed or natural ecosystems. Section III-D-5-e
Biosafety Level 4- Plants (BL4-P)
BL-4P includes:
- Experiments with a small number of readily transmissible exotic (see Section V-M, Footnotes and References of Sections I-IVa) infectious agents, such as the soybean rust fungus (Phakospora pachyrhizi) and maize streak or other viruses in the presence of their specific arthropod vectors, that have the potential of being serious pathogens of major U.S. crops
Biosafety Level 1-Plants (BL 1 – P)
| Containment Practices for Biosafety Level 1- 3 Plants | |
|---|---|
| Greenhouse Access | 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. |
| Records | A record shall be kept of experiments currently in progress in the greenhouse facility. |
| Decontamination and Inactivation | Experimental organisms shall be rendered biologically inactive by appropriate methods before disposal outside of the greenhouse facility. |
| Control of Undesired Species and Motile Macroorganisms | 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. |
| Concurrent Experiments Conducted in the Greenhouse | Experiments involving other organisms that require 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. |
Biosafety Level 2-Plants (BL 2 – P)
| Greenhouse Access | 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. |
| Records | A record shall be kept of experimental plants, microorganisms, or small animals that are brought into or removed from the greenhouse facility and shall be kept of experiments currently in progress in the greenhouse facility. Principal Investigator shall report any greenhouse accident involving the inadvertent release or spill of microorganisms to the Greenhouse Director, Institutional Biosafety Committee, NIH/ OBA and other appropriate authorities immediately (if applicable). Reports to the NIH OSP shall be sent to the Office of Science Policy, National Institutes of Health, preferably by e-mail to: NIHGuidelines@od.nih.gov; and on the OSP website (https://osp.od.nih.gov/about-us/). Documentation of any such accident shall be prepared and maintained. |
| Decontamination and Inactivation | Experimental organisms shall be rendered biologically inactive by appropriate methods before disposal outside of the greenhouse facility. |
| Control of Undesired Species and Motile Macroorganisms | 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. |
| Concurrent Experiments Conducted in the Greenhouse | 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. |
| Other Practices | A sign shall be posted indicating that a restricted experiment is in progress. The sign shall indicate the following: (i) the name of the responsible individual, (ii) the plants in use, and (iii) any special requirements for using the area. 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. 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 (BL 3 – P)
| Greenhouse Access | 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. |
| Records | A record shall be kept of experimental plants, microorganisms, or small animals that are brought into or removed from the greenhouse facility and 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/OBA, and other appropriate authorities immediately (if applicable). |
| Decontamination and Inactivation | 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. |
| Control of Undesired Species and Motile Macroorganisms | 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. |
| Concurrent Experiments Conducted in the Greenhouse | 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. |
| Other Practices | A sign shall be posted indicating that a restricted experiment is in progress. The sign shall indicate the following: (i) the name of the responsible individual, (ii) the plants in use, and (iii) 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. 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. 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. |
References:
https://osp.od.nih.gov/wp-content/uploads/NIH_Guidelines.html
01. NIH Guidelines Section V-G.
A U.S. Department of Agriculture permit, required for import and interstate transport of plant and animal pathogens, may be obtained from the U.S. Department of Agriculture, ATTN: Animal and Plant Health Inspection Service (APHIS), Veterinary Services, National Center for Import-Export, Products Program, 4700 River Road, Unit 40, Riverdale, Maryland 20737. Phone: (301) 734-8499; Fax: (301) 734-8226.
02. NIH Guidelines Section V-M.
In accordance with accepted scientific and regulatory practices of the discipline of plant pathology, an exotic plant pathogen (e.g., virus, bacteria, or fungus) is one that is unknown to occur within the U.S. (see Section V-G, Footnotes and References of Sections I-IV). Determination of whether a pathogen has a potential for serious detrimental impact on managed (agricultural, forest, grassland) or natural ecosystems should be made by the Principal Investigator and the Institutional Biosafety Committee, in consultation with scientists knowledgeable of plant diseases, crops, and ecosystems in the geographic area of the research.
