A practical guide to conta

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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

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207 Engel Hall, Blacksburg VA 24061
tel: 540-231-3747 / fax: 231-4434 / email: isb@vt.edu
http://www.isb.vt.edu

© 2001 by Information Systems for Biotechnology
All rights reserved. Published 2001.
Printed in the United States of America.

The complete text of this Guide is available on the ISB Web site
(http://www.isb.vt.edu). Print copies are available at no charge—an
order form is available on the ISB Web site, or you may send your
request by email to isb@vt.edu or by fax to 540-231-4434. Please be
sure to include a complete mailing address.

Library of Congress Cataloging-in-Publication Data
Traynor, Patricia L., Dann Adair, Ruth Irwin
Greenhouse Research with Transgenic Plants and Microbes: A Practical
Guide to Containment/Patricia Traynor, . . . [et al.].
p. cm.
Includes bibliographical references.
ISBN: 0-9703604-0-1
1. Biotechnology. 2. Transgenic plants/organisms.

Cover and interior design by Theresa Gedig, digdesign
Printed by Sexton Printing, St. Paul, Minnesota

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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.

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Table of Contents
Section I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1
Scope . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2
Audience . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2

Section II. Regulation and Oversight of GMOs . . . . . . . . . . . . . . . . . . . . . . . . . . . .5
The NIH Guidelines and Appendix P . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .5
Federal Regulatory Agencies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6
Institutional Biosafety Committee . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .8
Biological Safety Officer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .8
Principal Investigator . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .8
Greenhouse Staff . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .9

Section III. Biosafety Levels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .11
Experiments that Are Exempt . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .13
Biosafety Level 1 for Plants (BL1-P) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .13

Section IV. Elements of Containment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .15
Physical Containment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .19
Biological Containment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .21
Combining Physical and Biological Containment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .26

Section V. Management Practices . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .27
Access . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .27
Apparel and Hygiene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .28
Signage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .28
Seed Storage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .29
Transfer of Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .29
Termination and Disposal . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .29
Pest Control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .30
Training and Reference Manuals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .30
Monitoring Containment Effectiveness . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .30

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Procedures for Loss of Containment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .31
Records . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .31
Inspections . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .31
A Note about Vandalism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .32

Section VI. Retrofitting for Containment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .33
Greenhouses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .33
Screenhouses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .42
Growth Chambers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .43

Section VII. Design of New Containment Facilities . . . . . . . . . . . . . . . . . . . . . .45
Building a Design Team . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .45
Construction Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .46

Appendix I. Facility Inspection Checklists . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .49
Inspection Checklist . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50
Reinspection Checklist . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55

Appendix II. Supplemental Resources . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .57
Regulatory Contacts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .58
National Associations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .58
Greenhouse Construction Resources . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .59

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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


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


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.


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



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


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



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.


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.


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.



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.


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



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


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.



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


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.


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



FIG 1. Caulking around service intrusions

FIG 2. Sill caulking



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



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).



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.



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).



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



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


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.



Microorganisms • Treat or evaporate runoff water to eliminate viable
eggs and larvae;
Effective physical containment of bacteria,
• Avoid use of small-sized insects in experimental
viruses, and other microbes can be extremely
greenhouse cages; and
difficult because they cannot be seen and, once
dispersed, cannot be recovered. However, many will • Destroy pollinating insects in experimental cages
not survive and persist if they are dispersed. after pollen transfer to eliminate potential for
Biological measures often provide better containment dissemination of transgenic pollen into the
options. The following methods may help prevent environment.
dissemination of genetically modified
microorganisms:
COMBINING PHYSICAL AND
• Avoid creating aerosols when inoculating plants BIOLOGICAL CONTAINMENT
with transgenic microbes;
Using biological and physical containment
• Provide adequate distance between an infected measures in concert offers two advantages when
plant and another susceptible host, especially if the planning how to achieve a specified level of
microorganism can be disseminated through the containment. First, combining methods reduces the
air or by leaf contact; physical requirements to those of the next lower
• Grow experimental plants and microbes at a time biosafety level. A greenhouse experiment using
of year when nearby susceptible plants are not transgenic sunflowers, for example, located where
growing; wild sunflowers are endemic and found within the
• Eliminate vectors for insect-borne isolation distance used by breeders, requires BL2-P
microorganisms; containment so that outcrossing does not occur.
Alternatively, physically removing all wild
• Choose microorganisms having an obligate
sunflowers within the isolation distance allows BL1-
association with the host plant;
P physical standards to be used. If the experiment
• Genetically disable the microorganism to does not include seed collection for subsequent trials,
minimize survival and reproduction; and adding biological containment by removing the
• Treat or evaporate runoff water. flower heads before pollen shed similarly allows use
of the less stringent BL1-P physical standards.
Second, the ability to do BL2-P transgenic
Insects research in an existing BL1-P facility may be
achieved simply by incorporating biological
Insect and mite containment is difficult in a
containment practices. Consider an experiment
greenhouse facility. Entomologists who raise insects
designed to evaluate tomato plants genetically
on greenhouse plants work constantly to prevent
engineered for resistance to tomato spotted wilt virus
their escape and to control disease and parasites. The
(TSWV). The protocol involves three organisms:
following procedures can be used to prevent
tomatoes, the virus, and thrips, the insect vector that
dissemination of arthropods and other small animals:
transmits TSWV. Physical containment would be
• Choose or create non-flying, flight-impaired, or
provided by a greenhouse fitted with AntiVirus™
sterile strains;
screening or by conducting the experiment in insect-
• Conduct experiments at a time of year when proof cages within the greenhouse. Biological
survival of escaping organisms is impossible; containment could be added by removing alternate
• Choose organisms that have an obligate host plants for the virus both in and outside of the
association with a plant not found in the vicinity; greenhouse and by applying stringent insect control
measures in the surrounding area.


27

Management Practices
Section V. Management
Practices
CONTAINMENT STRATEGIES ARE EFFECTIVE ONLY when
greenhouse personnel understand and adhere to established procedures
for handling transgenic material. Before entering the greenhouse, all staff
working around transgenic organisms should be fully informed about the
containment measures applicable to a given research project. Prescribed
procedures and practices should be appropriate for the assigned biosafety
level; those that appear excessive for the needed level of containment
may discourage compliance.

Access
Access to greenhouses housing transgenic research materials is
restricted, regardless of the biosafety level. Such restrictions are intended
to minimize the spread of transgenic pollen, seed, or other propagative
material that could be carried by people moving between rooms or
facilities. At BL1-P, access is limited or restricted at the discretion of the
greenhouse manager or PI when experiments are in progress. At BL2-P,
the manager is required to limit greenhouse access to individuals directly
involved with the experiments, and at BL3-P, the manager, in
consultation with the PI, should determine access authorization on an
individual basis. Discretionary access is generally reserved for
maintenance personnel and accompanied visitors who have a special
interest in the research.
If the greenhouse consists of one large room as opposed to individual
compartments, access to the entire facility may need to be restricted; all
authorized personnel should have access to a key to enter. Signs must be
posted at the entries to the greenhouse indicating that access is restricted
for the experiment in progress. These signs may also contain access
instructions. An entry and exit logbook is required for BL4-P
greenhouses only. However, when exotic infectious agents are present in
the research facility, APHIS recommends keeping a record of the
personnel who regularly work there, visitor, and service personnel visits.
The log should include the names, dates, and times of everyone entering
and exiting the facility.



Apparel and
Hygiene
Personnel entering BL1-P
and BL2-P facilities may
wear their usual street or
lab clothing. For entry into
BL3-P greenhouses,
disposable lab gowns or the
equivalent may be required
at the discretion of the
greenhouse manager. If
special clothing is required,
it must be removed before
leaving the facility and
decontaminated (usually by
autoclaving) before washing
or disposal. Users are also
required to wash their
hands before leaving BL3-P
restricted areas.
BL4-P facilities maintain
strict apparel and hygiene
protocols. All users are
required to enter only
through the dressing/shower
rooms and must shower
when leaving the facility.
Users are also required to
remove all street clothing
and don protective clothing
before entering. Likewise,
personnel leaving the
facility must remove
protective clothing before
showering and exiting. The
clothing must be stored in
the inner change room and FIG 6. GMOs marked with colored stakes
autoclaved before
laundering. Showering
before entering is required Signage
only when there is concern
that organisms will be No special signs are required for BL1-P containment greenhouses.
brought into the Entryways into BL2-P and higher facilities should be posted with signs
containment area from the indicating that access is limited to authorized personnel only. If the
outside. experiment uses organisms that pose a risk to the local ecosystem or


SECTION V. Management Practices 29

agriculture, a sign so stating must be placed on the to be transferred in a closed non-breakable container.
access doors to the greenhouse. A description of the For BL3-P and BL4-P containment, the guidelines
potential risk may be posted on the restricted access require an additional sealed secondary container for
sign as long as this is not confidential information. movement of experimental materials. The exterior
The sign should state the name and telephone surface of the secondary chamber should be
number of the responsible individual, the plants in decontaminated either chemically or in a fumigation
use, and any special requirements for using the area. chamber if the same plant, host, or vector is present
It may include contact information for the within the effective dissemination distance of the
greenhouse manager and others to be called in case propagules of the experimental organism.
of emergency.
Transgenic material in a greenhouse room must
be marked to distinguish it from non-transgenic Termination and Disposal
organisms such as plants serving as experimental
controls or those not involved with the experiment. To prevent the unintended survival of GMOs
Individual pots, bench sections, or entire benches can outside the greenhouse environment, all
be marked with stakes or signs that identify the plant experimental materials must be rendered biologically
and the primary genetic modification, for example, inactive (devitalized) before disposal. Termination
“Soybeans with viral coat protein gene” (Fig. 6). All procedures for the safe disposal of soil and plant
organisms in the room must be treated in accordance material should be part of the experimental plan for
with the highest level of containment standards a research project. The IBC may institute a policy
required by any experimental material present. that outlines acceptable disposal procedures for GM
research materials, taking into consideration the
biosafety level of the experiment and the volume of
Seed Storage material to be handled. Devitalization of plant
material and soil should be completed before it
Transgenic seed should be stored in a locked leaves a greenhouse or laboratory and goes to a
cabinet located preferably in the greenhouse room so landfill.
as to minimize handling in unconfined spaces. When Plants and associated organisms can be
stored or handled outside the area of confinement, inactivated though steam or chemical sterilization
such as in a cabinet or on a potting bench in a procedures. Steam forced into special carts or boxes
headhouse corridor, the seed should be in a spill- has traditionally been used in greenhouses for
proof container. The transgenic seed should be treating growing beds, pasteurizing or sterilizing
clearly identified and labeled to distinguish it from media, and disinfecting containers, thus it is likely to
other stored seeds or materials in the cabinet. be available. Materials from smaller experiments can
Greenhouse personnel should take ordinary be inactivated by autoclaving all plants, plant parts,
precautions to prevent seed germination in unwanted containers, and potting media. For larger volumes,
locations. composting is an acceptable treatment for
experimental plant and soil materials that pose no
recognized harm to the environment. Plants can be
Transfer of Materials devitalized through desiccation simply by
withholding water or they can be chopped or minced
The NIH Guidelines specify requirements for to pieces unable to grow independently under
transporting experimental materials to and from a natural conditions. Incineration may also be used to
greenhouse for levels BL2-4P. For BL2-P and higher destroy easily combustible, dry plant material;
facilities, transgenic material in the form of seeds or however, incineration must be used with caution
propagules, potted plants, trays of seedlings, etc. are since not all seeds are easily burned, e.g., cottonseed.



At higher containment levels, it is recommended that and parasitoids to control pests.
all materials leaving the greenhouse be sterilized in Greenhouse research commonly uses insect pests
an autoclave. as part of the experimental protocol, such as in
Disposing of very small transgenic seeds requires testing plants for disease or insect resistance. In these
special care. Fine mesh bags can be secured around cases, selective control measures are needed to
flower heads prior to disposal; a sheet of dampened eliminate the unwanted pest without killing the
white paper such as BenchKote™ placed on the required pest organism. When insect vectors are used
work surface facilitates recovery of easily scattered to transmit genetically modified viruses, particular
seeds. The gravel under benches in BL2-P facilities care should be taken to eliminate the vector once the
should be decontaminated by, for example, treatment transmission has been accomplished.
with a sodium hypochlorite (household bleach)
solution. Catching liquid in a large open pan and
allowing it to evaporate is a simple alternative. Training and Reference Manuals
Abandoned or forgotten experimental materials
Personnel instruction is an important component
are not an infrequent problem for greenhouse
of good management practices. A reference manual
managers. An IBC policy stipulating that GMO
should be prepared containing directives covering all
material is the responsibility of the PI would clarify
safety considerations pertaining to the transgenic
authority in disposing of neglected or abandoned
research being conducted. Staff are required to read,
materials. This policy would preclude a source of
understand, and follow the instructions provided in
gene escape that may occur when a PI leaves
the manual before entering the greenhouse.
transgenic material in the greenhouse due to death,
Personnel training is best accomplished through
resignation, or simple oversight.
interactive sessions that include the PI, greenhouse
manager, or other safety-management staff.
For BL2-P and higher facilities, emergency and
Pest Control
contingency plans, as well as documents pertaining
The NIH Guidelines call for a pest control to routine operations, are required to be included in
program for all biosafety levels when working with the reference manual. It is not necessary to include
transgenic organisms in a greenhouse setting. experimental protocols in the manual, however
Rodents and birds can transport transgenic seed researchers and greenhouse staff may find that a
outside the facility; insects and other organisms can copy of the experimental protocol aids compliance
transfer pollen to receptive plants located within or with containment procedures. Conversely, relevant
outside the containment area. A stringent pest portions of the manual may be included in the
control program, using physical, chemical, or project documents submitted for IBC approval.
biological control measures, alone or in
combination, should be implemented and monitored
for effectiveness. Screens are recommended for BL1- Monitoring Containment
P and required for BL2-P to exclude pollinating Effectiveness
insects and birds; BL2-P facilities must have louvers
Escaped organisms may be detected by placing
fitted on exhaust fans that are open only when fans
susceptible host plants, insect traps, or spore/pollen-
are running. The perimeters of greenhouses of every
catching devices both inside and outside the
containment level should be sealed to prevent
containment area. Traps and bioindicator plants can
rodents and other large pests from entering.
be used to detect unintended virus transmission,
Fumigation can be used to control certain insect
insect migration, and pollen or spore spread. For
pests such as whiteflies. Biological control measures
example, if an experiment involves a caged insect-
may involve the introduction of predators, parasites,


SECTION V. Management Practices 31

vectored plant disease system, uninfected plants writing to the Biological Safety Officer (if assigned),
placed in the same greenhouse but not in the caged the greenhouse manager, the Institutional Biosafety
area can be monitored for evidence of disease Committee, NIH Office of Biotechnology Activities,
transmission. Light traps placed in corridors and and/or other designated authorities. Greenhouse
operated at night are useful to indicate the presence managers should be advised that any plant material
of insects that have escaped the greenhouse rooms. governed by APHIS permit that escapes or is stolen
must be reported to Dianne Hatmaker, Biotech
17
Permits, APHIS, PPQ (telephone 301-734-5787)
Procedures for Loss of Containment within 24 hours of the incident.

The integrity of containment measures is
susceptible to equipment malfunctions, acts of nature Records
such as fire, flood, and storm damage, and human
error. A loss of BL1-P containment due to any of The extent of record keeping required for
these factors would likely have only minor research using transgenic organisms is commensurate
environmental consequences, if any, and would not with the level of biosafety. Records of experiments in
require any response. At BL2-P or higher, such events progress must be kept for all biosafety levels. At
may present larger concerns. BL2-P and higher, additional records must be kept of
Facilities operated above BL1-P should be all plants and plant-associated organisms entering or
equipped with an alarm system designed to alert leaving the greenhouse. A record of the dates and
someone when mechanical or weather-related events times of personnel visits must be kept for BL4-P
causing a loss of containment occur. Greenhouse facilities.
systems that monitor automated environmental Although the NIH Guidelines do not specify who
controls should have built-in local and remote should keep records, the PI is the logical choice as
alarms. Instances of human error, such as a door left he/she is responsible for tracking experimental
open or ordinary disposal of unlabeled transgenic material. It is also appropriate that someone
materials, is actually a more common cause of stationed in the facility (e.g., the greenhouse manager
containment loss than facility malfunctions or or equivalent) has responsibility for entry and exit
structural damage. Designated people who are logs when required.
promptly alerted to problems can make timely
decisions in regards to contacting or dispatching
appropriate response personnel. Inspections
The NIH Guidelines require BL2-P and higher
Greenhouses should be inspected periodically to
facilities to have contingency plans for handling
ensure that containment measures appropriate for
emergency situations that also apply in cases of theft
the transgenic plants and other organisms held inside
or vandalism. These plans, drawn up by the BSO
are being rigorously followed. Inspections should be
and/or IBC in consultation with the PI, must include
conducted on a regular schedule and whenever new
measures to contain the breach, a personnel
types of experimental materials are brought into the
notification sequence, and decontamination
facility. Inspectors may include the greenhouse
procedures. In addition, the plans should include
manager, BSO, IBC representative, or state
names and contact information for repair personnel,
agriculture officials. Officials from USDA/APHIS
researchers, relevant authorities, and greenhouse
may, upon request, visit a facility to observe
staff.
containment features. However, USDA does not
Should an inadvertent release of transgenic
certify or otherwise designate a greenhouse’s
material at BL2-P or higher occur, the Principal
suitability for research materials requiring a specific
Investigator must immediately report the incident in

17 http://www.aphis.usda.gov/ppq/



biosafety level(s) unless there is present a plant pest designated boundary on the bench such as color-
requiring a permit from APHIS-PPQ. coded markers. Additionally, Plant Protection and
Inspection checklists help ensure that a Quarantine Officers of APHIS may conduct
greenhouse complies with all necessary physical, unannounced re-visits to facilities housing GMOs
biological, and managerial requirements for a given under federal permit. The unannounced inspections
Biosafety Level. Inspection checklists facilitate IBC occur during normal business hours and are a
approval, provide an outline for internal monitoring, Standard Permit Condition.
and serve as documentation of compliance. A sample Periodic reinspections of the greenhouse should
of an APHIS “Facility Inspection Checklist for be conducted. The presence of light, heat, and water
Containment of Genetically Engineered Organisms” within a facility promotes gradual deterioration of
is included as an Appendix. Public and private sector equipment and structural features over time.
research organizations usually develop their own in- Additionally, an inspection serves as an opportunity
house checklists. Checklists may be customized by to review any special practices that may be required,
combining items from the APHIS checklist, other as staff adherence to non-standard procedures may
lists, and the list below. Where several levels of tend to relax over time.
containment are provided by different rooms within
a single facility, checklists tailored to each level
simplify the inspections. A Note about Vandalism
Vandalism is an increasing concern for
For each room or research project, an inspection
greenhouse managers. Some individuals and
checklist may ask:
organizations opposed to recombinant DNA
• Who is the responsible party? Is their contact research have targeted greenhouse and field trial
information posted on the door? research projects, causing substantial damage.
• What is the nature of the GMO and how is it Determined individuals gain entry either by force, by
identified? defeating security hardware, or they may be
admitted inadvertently by authorized personnel—
• What is the prescribed level of containment? Do
self-closing doors may be propped open, rooms and
the physical facilities meet this level?
entries left unlocked, and strangers not always
• What specific physical and biological measures are confronted. Facility users should be advised that they
being used to achieve that level of containment? share responsibility for maintaining security.
• Are prescribed practices being followed? When the threat of vandalism is politically
motivated, a situation termed “domestic terrorism”
• Is there any evidence of deficiencies with regard to
by the US Federal Bureau of Investigation, an
containment?
institution may wish to create a response team. This
• How is the area secured? What security is group typically is composed of a high level
required? administrator, a public information officer, the
• Is there a written plan for responding to loss of facility manager, legal counsel, and relevant others
containment? What is the most likely containment whose job it is to review physical deterrents and
breach? develop public relations strategies. Because political
actions generally are designed to garner sympathy
If GMOs under APHIS permit are in a greenhouse for a cause via the news media, it is important that
with the same species of non-GMOs, APHIS an institution have an opportunity to respond
recommends that the two groups (or more) be well quickly and clearly to threats or acts of vandalism.
separated to avoid inadvertent cross pollination.
Also, it is recommended that the GMOs have some


33

Retrofitting for Containment
Section VI. Retrofitting for
Containment
GREENHOUSES
Retrofitting a conventional greenhouse to meet BL1-P and BL2-P
containment standards is far cheaper than building a new facility.
Requirements for meeting BL3-P standards are more extensive and may
involve basic structural changes; therefore, retrofitting may not be
feasible or cost-effective. Similarly, if a greenhouse is structurally
unsound or suspect, rebuilding may be the best option. BL4-P standards
require a dedicated, highly engineered, and isolated facility, which
excludes the possibility of retrofitting existing greenhouses. Accordingly,
this section primarily concerns modifications that would bring a
conventional greenhouse up to the containment standards appropriate
for the lower biosafety levels.

Existing greenhouse facilities should be carefully inspected to determine
if they are suitable for retrofitting. Structurally sound buildings in good
condition are often adequate, or nearly so, in terms of containment.
Necessary modifications, if any, are usually simple, straightforward, and
involve readily available materials. Before deciding to retrofit an existing
greenhouse, the cost should first be compared to that of building a new
structure. If retrofitting costs fall within 20% of the price of new
construction, renovation generally is not recommended. It is advisable to
contact a greenhouse builder, engineer, architect, or experienced
consultant before proceeding with any major renovation.

Upgrades needed to meet specified containment standards are shown in
Table 7.



TABLE 7. Enhanced features of containment greenhouses

CONVENTIONAL BIOSAFETY LEVEL 1-P BIOSAFETY LEVEL 2-P

STRUCTURE Framing may be aluminum,
steel, wood, or pipe

ENTRY Hinged or sliding entry doors Locks on entry doors

GLAZING Standard greenhouse glass
or plastic material

SCREENING If used, standard 30 mesh Recommended 30-mesh or higher required
fly screen

VENTILATION Roof or side vents, fans,
cooling pads, fog system,
or a combination of these

BENCHING Any material; solid or porous
bottoms

FLOORS Gravel (most common), soil, or Impervious walkways Impervious material;
concrete throughout recommended collection of runoff water
may be required

DRAINS Discharge into groundwater
or sanitary/storm sewer

OTHER Automatic control and utility Autoclave available
systems meet basic operating
requirements


SECTION VI. Retrofitting for Containment 35


Rigid, wind resistant frame preferred; internal Reinforced, rigid frame required; walls, floors, and
walls, ceilings, and floors resistant to liquids ceilings form sealed internal shell, resistant to
and chemicals liquids and chemicals; see Appendix P for others

Double set of self-closing, locking doors Double set of self-closing, locking doors with
air-lock; shower and changing rooms

Laminated, strengthened, sealed Double-paned, laminated, strengthened, sealed

Not permitted Not permitted

Separate negative pressure system; Air-conditioned and HEPA filtered, closely
air supply fans with back-flow damper; monitored negative pressure, no roof or
exhaust air HEPA filtered side vent allowed

Seamless water and chemical resistant Seamless water and chemical resistant
bench tops bench tops

Impervious material; for microbes, runoff water Sealed floors as part of internal shell;
collection and decontamination runoff collection and decontamination

Provision for collection and decontamination Runoff collection required, sewer vents filtered
of runoff

Autoclave within facility; hand washing with Double-door autoclave; self-contained vacuum
hands free on/off; filtered vacuum lines; system; in-line filters and back-flow protection
disinfectant traps for liquid lines for all liquid/gas services



Layout highest level of containment required by any
organism in the room. Thus in a large room housing
A greenhouse can be an inhospitable environment BL1-P and BL2-P experiments, all plants must
for people and equipment because of the humidity, conform to BL2-P containment standards. A
temperature, light, chemicals, and soil. The compartmentalized arrangement of small rooms
headhouse, an enclosed area within or adjacent to allows the facility to provide a variety of
the greenhouse facility, provides cleaner, more containment levels as well as individualized
comfortable space for offices, labs, equipment, environmental conditions.
supplies, and control systems. In many standard greenhouses, interior space is
When upgrading a conventional greenhouse to divided into relatively large rooms with a common
accommodate transgenic materials, traffic patterns, central corridor running through them. This
process flow, and security measures should be arrangement forces personnel to pass through each
analyzed to determine if the layout should be room to get to the succeeding one, making it
modified. The configuration should be optimized to difficult, if not impossible, to restrict access to an
provide variable levels of containment and growing individual room. A more efficient and manageable
conditions, control of access, and ease of movement. layout has an array of small rooms and cubicles
The NIH Guidelines stipulate that all plant material opening off one or more common walkways (Fig. 7).
within a greenhouse room must be maintained at the

FIG 7. Floor plan of the USDA Foreign Disease and Weed Science Research Unit BLP-3 Containment
18
Facility in Frederick, Maryland

Locker 114 Growth
105

Lab Materials Room Chamber
102 Handling 109 112 Room
Main Filter Room 108 115B
Room 107 118
101 AC
Disposal Dew
Lab Lab and Lab Lab Chamber
103 104 Storage 113B 115A Room
AC
AC

113A 116
AC

Cubicle 1
Insectary

Blue = clean rooms Cubicle 2
Clear areas = contaminated rooms:
plant pathogens may be present as Greenhouse Greenhouse Greenhouse Greenhouse Cubicle 3
aerosols during research 4 3 2 1
Propagation
Rooms 105 and 112 = shower Cubicle 4
airlocks where contaminated and
clean air interface
Cubicle 5
AC = double-ended pass-through
autoclave
Cubicle 6

Note that major changes to the layout can necessitate further structural modifications, such as the addition
of partitions and/or hallways within a previously undivided greenhouse. These changes may in turn call for
revamping environmental control schemes, utilities, ventilators, and primary structural components as well.

18 Kahn, R.P. and S.B. Mathur. 1999. Containment Facilities and Safeguards for Exotic Plant Pathogens and Pests. The American
Phytopathological Society, St. Paul, MN. Reprinted with permission.



FIG 8. Neoprene door sweep

Entry doors and locks Doors should fit tightly against the jamb and have
a sweep at the threshold. The most commonly used
Standard lockable hinged doors can be used for standard door sweep consists of a neoprene or
exterior and corridor entrances. Sliding doors are rubber strip or a short plastic brush attached to an
acceptable at BL1-P and BL2-P but do not seal aluminum holder that can be fastened to any
tightly enough for higher containment levels. Both relatively flat surface (Fig. 8). Although sweeps
styles of doors can be fitted with locks to limit cannot restrict all small insects that are intent on
access. For security reasons, the distribution of entering or exiting a space, they can easily exclude
greenhouse keys should be carefully controlled and rodents, birds, and larger flying insects.
monitored. Greenhouse rooms dedicated to The NIH Guidelines stipulate a double set of
transgenic research could be re-keyed to assure self-closing and locking doors for BL3-P and BL4-P
access is limited to authorized personnel only. It is containment. Building codes prescribe the presence and
also advisable to restrict the total number of keys placement of emergency exits regardless of contain–
issued to a practical minimum and to strictly limit ment needs. Therefore local officials must be consulted
the number of master or sub-master keys made. before amending or creating entrances and exits.



Glazing accidentally or intentionally. Therefore they are not
the preferred choice for a containment greenhouse.
The condition of the glazing and bedding putty Reglazing with the new generation of film plastics
should be carefully evaluated before conducting may be a viable option; project managers are advised
transgenic research. Properly installed glazing to consult contractors and institutional officials.
provides low infiltration and generally affords a high Films such as Hostaflon™ can be installed in three
degree of containment. Bedding putty for standard layers and still transmit light as efficiently as glass.
lapped glass greenhouses, however, wears out long Longevity with some of the new films has increased
before the glass, a condition that may precipitate from four to 20 years, and they can resist hail
glass breakage and cracking. If a glass greenhouse damage better than most rigid materials.
needs new bedding putty, which is a very labor- Standards for BL3-P and BL4-P require windows
intensive job, it may be economically advantageous to be closed, sealed, and resistant to breakage. This
to consider reglazing at the same time with sheet requirement can be met by using double-paned
materials, new styles of glass, or inflated films. sealed glass or rigid, double-walled plastic panels.
Simultaneously replacing bedding putty and glazing Examples are Sedo™, a brand of double-paned glass
would provide tighter containment, better that contains an inert gas between the panes, and
environmental control, and energy cost savings. two readily available sheet materials, Lexan™ and
Standard greenhouse glazing material will satisfy Exolite™, as noted above. Reglazing with double-
the requirements for BL1-P and BL2-P. Glass glazing paned sealed glass is likely to require extensive
is the most enduring material and provides the structural renovations to bear the additional weight.
greatest amount of natural light. Laminated and The National Greenhouse Manufacturers
heat-strengthened glass is preferred or, depending on Association published glazing standards that allow
building codes, may be required. Standard tempered manufacturers to run standard tests on their
glass is more prone to spontaneous breakage and 19
products . Test results allow consumers to make
shattering which can both breach containment and comparisons between various glazing products on
create a hazard. Glass can be manufactured in the market.
lengths that extend from the eaves to the ridge,
though lengths over eight feet become impractical.
Sheets of rigid plastics such as Lexan ™ Screening
polycarbonate or Exolite ™ acrylic also are
commonly used for glazing. Polycarbonate costs less Screening is an especially important consideration
and is more fire resistant than acrylic; acrylic glazing, when retrofitting an existing structure to attain a
however, lasts longer and permits better light higher containment level. Screening should be
transmission. Double-walled sheets of rigid plastic carefully installed on all ventilation intake vents.
glazing shift significantly within their framing with Figure 9 demonstrates a method of screening around
temperature fluctuations; therefore, inspections moving vent arms. For containment purposes,
should be made seasonally for openings in these screening side vents is recommended for BL1-P and
materials. required for BL2-P. If evaporative cooling pads made
Various types of film plastic glazing are of aspen fiber or corrugated cellulose are used on the
commonly available, e.g., p1olyester, polyethylene, intake side vents, screening is still useful since insects
polyvinyl chloride, and so on. Double-layer plastics can find their way through these materials.
rely on a fan to inflate the space between the sheets, Screen mesh size should be gauged relative to the
and require regular inspections to detect loose hold- size and shape of the organisms to be contained or
down clamps and tears. Film plastics also have a excluded. A comparison of commercial screening
20
relatively short life (less than four years on average), materials indicates that in some instances screens
become brittle with age, and are easily penetrated, with a larger hole size may have exclusion

19 Book of Standards. National Greenhouse Manufacturers Association (NGMA).
20 Bell, M.L., and J.R. Baker. 1997. Choose a greenhouse screen based on its pest exclusion efficiency. North Carolina Flower Growers’
Bulletin 42(2):7-13.



FIG 9. Screen panels over ridge*

efficiencies similar to those with smaller holes. This mesh screen with a thread thickness of 0.008" has a
is because holes are not always perfectly square in total of only 23.8% open space. Dust accumulation
commercially-made screens, a factor that may or on screens can also affect their efficiency—as the
may not favor insect exclusion, depending on hole screen opening size decreases, the need to keep the
shape. Further, thread diameter and mesh material screens clean by washing or vacuuming increases.
also influence exclusion properties. Relatively rigid Regardless of where screening is placed, airflow
stainless steel mesh may offer better exclusion than considerations are paramount because of
softer mesh with a similar hole size. Fine mesh screen temperature changes associated with reduced air
requires high maintenance; therefore consideration movement. Airflow, cooling, and fan performance
should be given to ease of replacement and cleaning. are significantly affected by the installation of any
Screen size can greatly affect airflow, cooling screen, especially when using the finer mesh sizes.
efficiency, CO2 retention, humidity level, and light One solution to the airflow restriction problem is to
transmission. Proper sizing of screen to the build a “screen box” outside the cooling pad frame
ventilation system is critical, regardless of the type of (Fig. 10) to provide adequate surface area for airflow
cooling systems installed—passive, fan only, fan and though the cooling pads.
pad, or mechanical (air-conditioned). A piece of 64-

* Reprinted with permission of Agritechnove, Inc., St. Anselme, QUE., CA.



FIG 10. Typical insect screen installation shown on intake vent end of greenhouse*

LEAD-TO FLUSH PLEATED

Top view ‘cut away’ of a typical
frame holding pleated screen.

BOX GABLE/END

Pleated insect screen installations
with the same total surface area
as an un-pleated piece generate
the same airflow characteristics,
but take up less space.
Insect Screen

Ventilation, Cooling and Heating humidity measurement and proper fog delivery.
Screens should be made to fit all vent openings, fans
Motorized and/or manual hinged vents, located at
sized accordingly, and the system installed inside the
the roof ridge and/or sidewall, are a common feature
greenhouse as specified by the manufacturer.
of most greenhouse cooling and ventilating systems.
Recirculating fans and curtain systems are also used
The passive ventilation afforded by vents can be
to help control temperature.
activated with the addition of exhaust fans and
The use of mechanical cooling, i.e., air
evaporative cooling systems. Air intake screening
conditioning, is the only option for higher levels of
(but never air outlet) and motorized or gravity-
containment. Construction and operation costs are
driven exhaust fan louvers are recommended for
very high due to the enormous heat load of a
BL1-P and required for BL2-P. Motorized louvers
greenhouse.
should be interlocked so they open and close with
Typical greenhouse heating systems include hot
fan startup and shutdown. Gravity-operated louvers
water radiation, steam radiation, infrared electric,
are also adequate. The vent operator arms or racks
solar, and forced air. All are adequate for every
that pass through screen are generally fitted with
containment level.
brushes or flexible barriers to prevent rodents and
other large pests from entering the greenhouse. Fog
cooling systems, if suitable for the structure and
Benching
climate, may offer a better and more convenient
alternative to evaporative cooling pad systems. Fog Standard greenhouse benches are adequate for
cooling should always be used in conjunction with a most GMO research projects though wood is not
good control system to insure precise relative recommended. Benching made of expanded

*Book of Standards. National Greenhouse Manufacturers Association. Reprinted with permission.



collection using a sewer. Higher levels
of containment also require seamless
bench tops and other work surfaces
that are impervious to water and
chemicals and can withstand mild
heat. These requirements may make
retrofitting for high levels of
containment cost prohibitive.

Floors and Drains
Requirements for greenhouse
floors vary according to the biosafety
level indicated (Table 7). Floors and
drains may need to be renovated to
meet containment standards for
transgenic greenhouses. Gravel and
soil beds can be used under benches
in BL1-P greenhouses only if
experimental material cannot travel
through these beds and leave the
greenhouse; concrete walkways are
preferred. A BL2-P greenhouse must
have an impervious floor surface.
FIG 11. Ebb and flow bench Retrofitting a greenhouse with
concrete floors and walkways can
galvanized steel or aluminum is preferred since these materials are substantially improve containment and
resistant to water and most chemicals. In addition, such benches are sanitation practices. Coatings can be
readily available, meet higher containment standards, and allow for applied to concrete surfaces to make
thorough cleaning, which contributes positively to a pest control them easier to clean and disinfect.
program regardless of the research protocol. In some cases, benches If a new floor is to be installed, it
with solid tops have adequate framing to allow replacement with may be advantageous to install floor
expanded metal. drains designed to collect all runoff.
In cases where the IBC, as recommended by the NIH Guidelines, This is particularly true if research
stipulates collection of runoff water, a solid bench may be installed projects that use genetically
that drains runoff into a holding tank for treatment with chemicals engineered microbes are underway or
or heat before being released to the sewer or ground. A bench that expected. Retrofitting with a
collects water for recirculation, also called an Ebb and Flow bench biowaste collection and treatment
(Fig. 11), could also be modified to collect runoff for subsequent system can be prohibitively expensive
treatment, or simply desiccation, if that renders the propagules in if the existing concrete slab and
question inactive. underground piping must first be
removed and reinstalled. Such
At BL3-P, other provisions may be needed to collect and treat renovations could easily push the
runoff water. These may involve collection from the bench and cost of retrofitting an existing facility
consequent treatment but would more likely involve whole room to exceed that of new construction.



Piping systems
Heating, watering, and fertilizing systems are
typically piped into and throughout the greenhouse.
Automatic watering and fertilizing systems are
advantageous because they reduce the amount of
traffic into the greenhouse, thus decreasing the
opportunity to spread transgenic pollen, seed, and
other propagative materials. The relative ease and
affordable cost of installing these systems makes
them an attractive option. However, for containment
reasons, new piping should be installed with a
minimal number of intrusions. All new and existing
intrusions should be sealed with a durable material
to help ensure containment (see Fig. 1).

FIG 12. Sealed framing joints in a containment
screenhouse SCREENHOUSES
Screenhouses are acceptable for GMO research
only when they meet the requirements for BL1-P or
BL2-P level greenhouses, including floors, and
Control Systems contain organisms that would have minimal impact
Standard digital, analog, pneumatic, or on the environment, if released. Though they have
mechanical greenhouse control systems are suitable limited utility for research, screenhouses may offer a
for GMO research at BL1-P and BL2-P. Computer or low cost alternative to greenhouses when sited in an
other control systems that incorporate alarms and appropriate climate. Retrofitting screenhouses
interact with headhouse systems are recommended involves many of the same measures listed for
for BL3-P and BL4-P. Sensors, usually under greenhouses. Upgrades could include the addition of
computer control, are also required for high concrete floors, well-fitting, lockable doors,
containment facilities to monitor differential air individual compartments, sealed joints (Fig. 12) and
pressures. Sensor technology has become prevalent utility intrusions, and specialized screening. BL3-P
and could be employed in any modern research experiments would likely not be approved for
greenhouse. Control systems can be easily upgraded screenhouse containment.
in most situations.
Greenhouse control systems technology has
become highly advanced, reliable, and cost effective.
It is strongly recommended that any control system
used in the greenhouse itself be designed and
manufactured exclusively for greenhouses, in
contrast to building control systems, which cannot
meet the exacting specifications for a research
greenhouse.



FIG 13. Growth chamber with HEPA filtration*

GROWTH CHAMBERS
If the quantity of plant material is not large, use of a growth chamber or growth
room may be the best option for containment at the higher levels. A growth
chamber modified to meet BL3-P requirements is shown in Fig. 13. The two main
retrofits to the chamber are a HEPA filter and a system for collecting runoff water.
If large quantities of plant material are produced, then renovation of existing
facilities may be as cost effective as retrofitting the growth chamber.

*Reprinted with permission of Conviron, Inc., Winnepeg, Man., CA.


45

Design of New Containment Facilities
Section VII. Design of New
Containment Facilities
A new greenhouse intended for research with transgenic organisms
should be designed and built to maintain containment for the life of the
facility. A greenhouse built to BL1-P or BL2-P containment standards
costs little more than a standard research greenhouse; relatively small
differences in design details may add slightly to the total cost. For the
most part, structural features for newly constructed lower level
containment greenhouses are covered in Section V, Retrofitting for
Containment, and are not repeated here.
The main focus of this section is on the design of higher-level
containment greenhouses. Because of more stringent design
requirements, greenhouses built to BL3-P or BL4-P specifications will
cost significantly more than conventional facilities. For the same reason,
a qualified and experienced team of designers must render the detailed
plans for such facilities.

BUILDING A DESIGN TEAM
The creation of a specialized greenhouse facility requires a team of
experts. Experienced architects and/or engineers are pivotal members of
the team and generally are hired independently of the construction firm.
The design team creates the documentation that allows construction
firms to bid on the project. Construction firms specializing in
greenhouses may have an engineering staff; however, the construction of
laboratories and other specialized rooms may require the skills of an
architect as well.
Researchers and staff who will be using, operating, and maintaining
the facility should be included in the planning process. IBC members and
regulators from USDA/APHIS and state Agriculture Departments should
be notified and updated regularly and may also be invited to join the
design team. A commissioning agent with experience in testing
greenhouse systems would also be a useful team member, though such
services would be most valuable at the conclusion of the project.
Consultation with users and managers at other research greenhouses is
valuable when designing new facilities. The Association of Education and



21
Research Greenhouse Curators offers an electronic
mail forum, web pages, and annual meetings from
ARCH
which detailed information can be gathered.
VENLO

CONSTRUCTION OVERVIEW STANDARD PEAK
VINERY
Framing Materials
QUONSET
Typical construction styles for research include
even-span with a standard peak, Venlo, and ridge and
furrow with gutter connects. Roof styles include the MANSARD COLD FRAME
standard peaked, as well as arched, mansard, and
Quonset-style. Figure 14 shows examples of greenhouse
exterior structures. A headhouse and hallways that are
immediately contiguous to the greenhouse are
considered part of the containment area. EVEN SPAN UNEVEN SPAN
Modern greenhouse structures are framed with
aluminum (Fig. 15) or galvanized steel; however,
many older facilities are framed in wood or metal
pipe. Wood and pipe framing are still being used in
new construction of some plastic film greenhouses. A
SAWTOOTH HILL SIDE
reinforced, rigid frame is preferred for BL3-P and
required for BL4-P. The latter requires additional
strength and rigidity to accommodate the weight of
double-paned, break-resistant, sealed glass.
Use of aluminum or galvanized steel truss framing
allows a prefabricated frame to be quickly erected.
The rigid frame, coupled with purlins, glazing bars,
and other framing members, creates a quality, long-
lasting structure that can be covered with various
glazing materials. Environmental control and RIDGE-AND-FURROW GUTTER CONNECTED
MULTISPAN
containment is enhanced through proper installation
and fitting of all materials. Information on structural
materials, as well as other relevant topics, can be FIG 14. Greenhouse roof styles*
found in the Book of Standards authored by The
22
National Greenhouse Manufacturers Association or
in the American Society of Agricultural Engineers
23
Standards 2000 . Specifications for BL3-P and BL4-P facilities stipulate
a double set of self-closing and locking doors. High
containment facilities also require one-way
Entry doors and locks emergency exit doors for personnel safety.
Traditional cylinder locks offer good security as
The choice of greenhouse doors should receive long as good key control is implemented. Newer
careful consideration since containment and security electronic systems such as a card swipe or Marlock™
breaches occur most often at points of entry. keying provide highly restricted access and a log of

21 http://www.life.uiuc.edu/aergc
22 Book of Standards. 1995. National Greenhouse Manufacturers Association (NGMA). (Revised 1999) www.ngma.com
23 American Society of Agricultural Engineers Standards 2000. American Society of Agricultural Engineers (ASAE). asae.org

* Reprinted with permission of Hanan, Joe J., 1998. GREENHOUSES: Advanced Technology for Protected Horticulture, CRC Press LLC:
Boca Raton, FL.


SECTION VII. Design of New Containment Facilities 47

FIG 15. Aluminum-framing under construction

all entries and exits. Special keys or cards are contained area can help blow organisms and
programmed to allow individuals access to selected propagules back into containment.
areas with one tool. Using this system, fewer keys are
issued, key loss is minimized, and codes can be
changed quickly and easily. Benching
Many different types of benching can be found in
A double-door entry system, with a dark vestibule
research facilities, but when building a new high
sandwiched between the doors, aids in effective
level containment greenhouse, the design and
insect containment. UV lights may be installed in the
materials should be chosen so as to comply with BL-
vestibule. Air curtains that fan individuals exiting a
3P and BL-4P requirements. Benches must be



thoroughly cleaned and disinfected in conjunction Guide. If this type of facility is required, it is highly
with transgenic research at higher biosafety levels. recommended to involve an experienced design firm
Those made of aluminum or galvanized steel provide for the project.
the longest wear, are easiest to clean, and amenable
to installing systems for runoff water collection and
treatment. An ebb and flow (also called ebb and Floors and drains
flood) bench is one that can collect water and recycle
it to the bench (see Fig. 11). This system can also be Solid concrete flooring and drains are preferred
adapted to collect and hold water prior to for new research greenhouses. Commercial
subsequent decontamination by chemicals or heat. greenhouses often use porous concrete floors to
allow passage of water. However, BL3-P and BL4-P
facilities must have non-porous floors that can be
Ventilation, Heating, and Cooling disinfected as well as a system to collect all runoff.
The floor of a BL4-P facility must be part of an
Few conventional research greenhouses are built "internal shell" system that includes the walls and
with sealed glazing, mechanically conditioned air, ceiling. Runoff is drained to a decontamination tank
differentially controlled air pressure, and exhaust air or treatment facility before entering a standard sewer
filtered through high efficiency particulate air (HEPA or other disposal system. Additionally, sewer vents
filters). Thus new construction is usually needed to on BL4-P greenhouses must be HEPA filtered.
meet the standards for BL3-P and BL4-P facilities.
Air conditioning is not strictly mandatory for higher-
level containment greenhouses; however the loss of Control Systems
cooling efficiency due to required air-handling
Normal building controls cannot readily be
measures makes it a necessity in most climates.
adapted to meet the rigorous needs of a high-level
The exhaust air produced from negative pressure
containment greenhouse; therefore dedicated
systems must be filtered to prevent contained
controls available from greenhouse control vendors
organisms from exiting. Intake air is also filtered to
are recommended. New facilities should have control
prevent introduction of organisms from the
systems that incorporate the latest digital technology,
environment into the enclosed space. Filter systems
and allow precise environmental control, logging,
can be designed to trap pollen, spores, and other
sensing, alarm, and related functions. Moreover, the
very small particles. High efficiency particulate air
security and redundancy functions that are required
(HEPA) filters can remove 0.3 micron and larger
at higher containment levels prescribe newer digital
particles but still allow gases to transfer across the
controls and should interface with the institutional
filter media.
building security system.
It is relatively difficult and expensive to equip an
Greenhouse managers and others involved in
entire greenhouse to restrict small particle
retrofitting existing greenhouses or building new
movement. Air-conditioned greenhouses, growth
facilities can draw on the experience of USDA
chambers, growth rooms, or biological safety
officials, the NIH Office of Biotechnology Activities,
cabinets are alternatives to standard research
architects, vendors, and professional colleagues. A
greenhouses with air filtration systems. Specialists
partial list of these is included in Appendix II.
should be consulted when designing or retrofitting
facilities that require a highly effective air filtration
system. The engineering specifications required for
air balancing, ventilating, and cooling BL3-P and
BL4-P greenhouses are beyond the scope of this


49

Appendix I.

Facility Inspection Checklists

Courtesy of Ralph Stoaks
C/O Diane Hatmaker
USDA/APHIS
Biotechnology Programs Operations
4700 River Road, Unit 147, Rm. 5B53
Riverdale, Maryland 20737
Telephone: (301) 734-5787



FACILITY INSPECTION CHECKLIST
FOR CONTAINMENT OF GENETICALLY ENGINEERED ORGANISMS

Address of Facility Applicant (Responsible Person )

Name

Address

( )_____________________________________ ( )________________________________________
Telephone Number Telephone Number

LOCATION OF ALL FACILITIES COVERED BY THIS INSPECTION

Building Name

Room/Laboratory

Growth Chamber Identification

Greenhouse Number or other Identification

RESEARCH QUALIFICATIONS AND GENERAL BACKGROUND

1. Does this facility operate under the National Institutes of Health (NIH) Recombinant Advisory Committee (RAC)
recombinant-DNA (r-DNA) guidelines? Yes____ No____

2. Is there a written policy regarding handling of rDNA at this establishment? Yes____ No____

3. Who is the chairperson of the local Institutional Biosafety Committee (IBC)?

Name and Title

4. Who is the scientist who will conduct the research?

Name and Title

5. Is the scientist who is conducting the research the applicant? Yes____ No____


APPENDIX I. 51

6. What other scientists and technicians will be working on the research?

Describe, in a general way, their experience and qualifications.

7. Do researchers and laboratory technicians practice and adhere to the NIH guidelines? Yes____ No____

PHYSICAL DESIGN AND SECURITY

8. Provide a short description of how the regulated article is physically marked and identified in the laboratory, growth
chamber, and greenhouse. Provide floor plan and/or map of facilities if possible.

9. Is the general area secure from public access? Yes____ No____
If not, please elaborate.

10. A. Is the general area secure from unauthorized personnel? Yes____ No____
If not, please elaborate.

B. Can individual laboratories be locked? Yes____ No____
C. Is there at least one sign posted on the facility door stating that a regulated genetically
engineered organism is present? Yes____ No____
If not, when will a sign be installed? Date _____________________

11. Who is allowed in the research areas?
Cleaning Personnel Yes____ No____ Trades Persons Yes____ No____ Other Yes____ No____

12. How distant from each other are the germination laboratories, growth chambers, and greenhouses? Be specific.

13. What kind of records, logs, or inventory are maintained regarding receipt, increase, and destruction of regulated
articles?



HANDLING OF MATERIAL—GERMINATION

14. A. Is there a cabinet to store seeds, plant material, tissue cultures, etc.? Yes____ No____
B. If yes, does it have a lock? Yes____ No____
C. Is the storage container identified with a sign stating it contains a
genetically engineered organism? Yes____ No____
D. If not, when will a sign be installed? Date ____________________

15. Where will seeds, tissue cultures, plant material, etc. be grown or germinated?

16. What medium will be used for seed germination? (e.g., germination paper, perlite, sand)

17. Is there any danger of seeds, tissue cultures, plant material, etc. being lost during this germination process, or of
ungerminated seed being transferred into subsequent research stages? Yes____ No____

18. Are there any cracks or irregular surfaces in the germination laboratory that could trap seeds? Yes____ No____
If Yes, describe size and location of cracks.

19. Are there water drains in the laboratory? Yes____ No____
20. Are the drains screened? Yes____ No____
If so, what is the size of the screen?

21. Does the drain system enter into a special waste trap? Yes____ No____

22. How will the germinated seed be moved to the growth chamber?

23. How will petri dishes, tissue cultures, spores, plant materials, etc. be moved from the laminar flow hood, to the
incubator, to the growth chamber?

24. How will the regulated articles be kept separate from other organisms?

HANDLING OF MATERIAL—GROWTH CHAMBER

25. Does growth chamber have access by authorized personnel only? Yes____ No____

26. Describe the growth chamber. lab top____ walk in____ built on site____ other ____.

27. Will the material be grown with any other plant materials in the same chamber? Yes____ No____
If yes, name the types of plants.


APPENDIX I 53

28. How will genetically engineered plants and/or containers be physically marked?

29. Does the growth chamber have water drains? Yes____ No____
If so, can they be screened? Yes____ No____

30. Does the drain system enter into a special waste trap? Yes____ No____

31. Where is the autoclave or incinerator in relation to the growth chamber?

32. Can the growth chamber be locked and separated from other growth chamber(s)? Yes____ No____

33. How will the material be transferred to the greenhouse?


HANDLING OF MATERIAL—GREENHOUSE

35. What is the name of the greenhouse manager?

36. Is the greenhouse accessed by authorized personnel only? Yes____ No____

37. A. Does the greenhouse have a double door entry system? Yes____ No____
B. Is the greenhouse entry through a "headhouse"? Yes____ No____

38. A. Do the greenhouse doors have locks? Yes____ No____
B. Is there a rear exit door? Yes____ No____

39. What type of greenhouse? Glass____Lexan____Plastic Poly____Screen____Other____
If screen, what size mesh? ___________________ If Poly, what thickness? ________________________

40. What are the approximate outside dimensions of the greenhouse(s)?

41. A. Do the roof vents open? Yes____ No____
B. If the roof vent opens, is it screened? Yes____ No_____ What size is the screen mesh? __________________

42. What kind of floor does the greenhouse have?
Concrete____Gravel____Packed Dirt____Other (Explain)_______________________________________________________

43. Does the greenhouse have water drains? Yes____ No____
Do they enter into a special waste trap? Yes____ No____



44. A. Does the greenhouse have black light traps for vectors? Yes____ No____
B. Does the greenhouse have "Sticky Board" traps for vectors? Yes____ No____
C. Does the greenhouse have other kinds of vector traps? Describe.

45. How will the plants be grown in the greenhouse? On Benches_____ In Flats_____ In Pots_____,
Other (describe)__________________________________________________________________________.

46. Will there be physical markers on each plant or container indicating that the plants are genetically engineered?
Yes____ No____

47. Where is the autoclave or incinerator in relation to where the plants will be grown?

48. Are there any openings in the greenhouse through which animals and pollinating insects could enter? Yes____ No____


GENERAL CONSIDERATIONS

What kinds of “spill response” action plan/equipment is available for items spilled in transit between labs, chambers, and
greenhouses? Items should be carried in containers so spills should not occur.

Are any similar plants growing in the area, either on the facility grounds or outside of the facility grounds?

What other factors are present which may influence the handling of seed or plants and may have an effect on
containment or risk?

Inspect for other specific conditions as stipulated on the permit.

Name of State Plant Pest Regulatory Printed Name of PPQ Officer
Official Performing Inspection Performing Inspection

Signature

Instructions to the inspector: Complete this form and return to:
Ralph Stoaks
C/O Dianne Hatmaker
USDA/APHIS
Biotechnology Program Operations
Telephone: (301) 734-5787


APPENDIX I 55

REINSPECTION CHECKLIST
FOR CONTAINMENT OF GENETICALLY ENGINEERED PLANT MATERIAL AND ORGANISMS

Address of Facility Applicant (Responsible Person )

Name

Address

( )_____________________________________ ( )________________________________________
Telephone Number Telephone Number

LOCATION OF ALL FACILITIES COVERED BY THIS INSPECTION

Building Name

Room/Laboratory

Growth Chamber Identification

Greenhouse Number or other Identification

RESEARCH QUALIFICATIONS

1. Who is the scientist responsible for conducting the research?

2. Who was the responsible scientist at the time of the initial facility inspection?

3. Do researchers and laboratory technicians regularly review, practice, and adhere to the permit protocol and the
conditions described in the permit? Yes____ No____

4. Conditions were reviewed by applicant and/or technicians on ____________________(date).



5. Have any major changes occurred or new operational procedures been instituted since the initial inspection?
Yes____ No____ If YES, initiate and complete a new facility inspection checklist.

6. Are the permit articles or any other regulated organisms derived from these articles still in use? Yes____ No____
or in storage? Yes____ No____

7. Have all of the regulated articles been properly destroyed? Yes____ No____ Date______________.
If Yes, no further action is required.

GENERAL CONSIDERATIONS

Remarks and/or observations.

Other factors which may influence the handling of seed or plants and may have an effect on continued containment or risk
of unwanted release.

Inspect or spot check for other specific conditions as stipulated in the permit.

Name of State Plant Pest Regulatory Printed Name of PPQ Officer
Official Performing Inspection Performing Inspection

Signature

Instructions to the inspector: Complete this form and return to:
Ralph Stoaks
C/O Dianne Hatmaker
USDA/APHIS
Biotechnology Program Operations
Telephone: (301) 734-5787


57

Appendix II.

Supplemental Resources

Regulatory Contacts
National Associations
Greenhouse Construction Resources



Regulatory Contacts The Association of Education and Research
Greenhouse Curators (AERGC)
c/o Department of Plant Biology
Biotechnology Evaluation University of Illinois at Urbana-Champaign
USDA-APHIS-PPQ 265 Morril Hall
4700 River Road, Unit 147 505 S. Goodwin Ave.
Riverdale, MD 20737-1236 Urbana, IL 61801-3793 USA
Phone: (301) 734-8896 Web: http://www.life.uiuc.edu/aergc/default.html
Web: http://www.aphis.usda.gov/bbep/bp
The Association consists primarily of greenhouse
and plant growth facility managers, supervisors, and
Office of Biotechnology Activities staff involved with the operation of college or
National Institutes of Health university facilities used to grow plant materials for
6705 Rockledge Drive, Suite 750, MSC 7985 research, class use or plant collections. The AERGC
Bethesda, MD 20892-7985 publishes the AERGC Newsletter and sponsors an
Phone: (301) 496-9838 Annual Meeting at a member's institution. The
Fax: (301) 496-9839 AERGC also provides the AERGC Forum, an e-mail
Web: http://www4.od.nih.gov/oba/ discussion group, as a service to its members.

National Associations
National Greenhouse Manufacturers Association
(NGMA)
USDA NCR-101 20 West Dry Creek Circle, Suite 110
Committee on Controlled Environment Technology Littleton, CO 80120
and Use Phone: (800) 792-6462
Mark Romer, Phytotron Manager Web: http://www.ngma.com/
McGill University
The National Greenhouse Manufacturers
1205 Dr. Penfield Ave.
Association is a professional trade organization for
Montreal, QC H3A 1B1 Canada
the manufacturers and suppliers of greenhouses and
Phone: (514) 398-6741
greenhouse components. The Association
Fax: (514) 398-5069
membership brings together some of the most
mark@bio1.lan.mcgill.ca
experienced and knowledgeable manufacturers in the
Web: http://www.botany.duke.edu/ncr101/
industry.
NCR-101 is a committee of the USDA's North
Central Region convened to help plant scientists
understand how to use controlled environment
technology effectively and consistently. They discuss
how to utilize growth chambers effectively to ensure
consistent and comparable growth data among
laboratories.


APPENDIX II. 59

Greenhouse Construction SUPPLIERS
Resources24 Brighton By-Products Co. Inc.
P.O. Box 23
DESIGN FIRMS New Brighton, PA 15066
Phone: (724) 846-1220 or (800) 245-3502
Agritechnove, Inc. Fax: (412) 846-7240
651 Route Begin
St-Anselme, Quebec, Canada, GOR 2NO
Phone: (418) 885-9595 E.C. Geiger, Inc.
Fax: (418) 885-4957 Route 63, Box 285
Email: agritech@total.net Harleysville, PA 19438
Phone: (215) 256-6511 or (800) 443-4437
Fax: (215) 256-6110 or (800) 432-9434
Alex Turkewitsch, P. Eng. Ltd. Web: http://www.geigerco.com/default.html
86 Glenview Avenue
Toronto, ON, Canada M4R 1P8
Phone: (416) 489-3816 McCalif Grower Supplies, Inc.
Fax: (416) 481-3883 P.O. Box 310
Ceres, California 95307
Phone: (800) 234-4559 (hard goods)
GREENHOUSE FABRICATORS Phone: (800) 473-7413 (plant sales)
Ludy Greenhouse Mfg. Corp. Fax: (209) 538-2086
P.O. Box 141 Web: http://www.mccalif.com/default.html
New Madison, OH 45346
Phone: (937) 996-1921 or (800) 255-LUDY
Fax: (937) 996-8031 Hummert International
Web: http://www.ludy.com 4500 Earth City Expressway
Earth City, MO 63045
Phone: (800) 325-3055
Nexus Greenhouse Corp. Fax: (314) 739-4510
10983 Leroy Dr. Web: http://www.hummert.com
Northglenn, CO 80233
Phone: (800) 228-9639
Fax: (303) 457-2801 Branch-Smith Publishing
Web: http://www.nexuscorp.com Online supplier search
P.O. Box 1868
Fort Worth, TX 76101
Rough Brothers, Inc. Phone: (817) 882-4120 or (800) 434-6776
5513 Vine St. Fax: (817) 882-4121
Cincinnati, OH 45217 Web: http://www.greenbeam.com/
Phone: (513) 242-0310 or (800) 543-7351 branchsmith/default.stm
Fax: (513) 242-0816
Web: http://www.roughbros.com

24 Citation of greenhouse resources is merely for the reader convenience and does not imply the authors’ endorsement of these firms and
suppliers. Readers are encouraged to independently investigate alternative resources.


Notes

Transgenic Cover final.qxd 7/16/01 10:44 PM Page 2

207 Engel Hall, Blacksburg VA 24061
tel: 540-231-3747 / fax: 540-231-4434 / email: isb@vt.edu
http://www.isb.vt.edu

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