4. 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
6. 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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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
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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
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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.
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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
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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
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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
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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.
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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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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.
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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.
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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
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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
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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.
27. 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
28. 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
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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
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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
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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).
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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.
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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).
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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
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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
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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.
