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.

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, which specifies physical containment standards for the laboratory. Standard practices for plants in greenhouses are summarized in Table 3.

Physical containment is achieved through facility design and equipment. Choices in the type of glazing, sealing, screening, air flow system, and other features all affect the degree to which a greenhouse is capable of isolating transgenic plants, plant parts, and associated organisms from the surrounding environment. These systems are also effective in keeping unwanted pests out of the greenhouse.

The term glazing refers to any transparent material (as glass) used for windows. Properly installed and regularly maintained greenhouse glazing of any typical material can provide a suitable barrier for transgenic research materials. The type of glazing most commonly used consists of single panes of tempered glass installed by lapping each pane over the one below. The care taken in installing and maintaining the glazing determines its overall effectiveness. Improperly installed or loose-fitting glazing material can leave gaps through which transgenic materials could be released unintentionally.

Caulking materials are commonly used to seal glass panes, sills, and small openings in and around greenhouse structures. Caulking and sealing restricts the passage of insects and assists with temperature control within the greenhouse; however, it should not be considered a substitute for well-fitting structural components. Additional caulking and sealing can help to upgrade a conventional facility to meet the standards of an approved containment facility. Typical situations where the addition of caulk provides an extra measure of containment are illustrated in Figures 1 and 2.

When properly sized, installed, and maintained, screen can keep pests and pollinators out of a greenhouse or, conversely, keep experimental organisms in. The integrity of a screening system is determined by several factors including the nature of the material, the size and morphology of the insects being excluded, the hole shape and size, and the air pressure applied on either side of the screen. The maximum hole size generally capable of restricting certain insect species is shown in Table 4. Anti-Virus® screening¹² is a commercial product advertised to be 100% effective in excluding leafminers, melon aphids, and whiteflies.

*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.

Negative Air Pressure

Containment of airborne pollen, spores, and insects is a significant challenge. One strategy to help achieve it is to create negative air pressure within a facility. Negative pressure exists when the amount of air exiting a space exceeds the air intake. Negative pressure bench-top chambers can increase containment of pathogens and insects within greenhouses, screenhouses, and laboratories. A chambered wood and clear plastic box fitted with a blower and filtration system can produce negative pressure on a small scale and at a relatively low cost (Fig. 3).

Figure 3. Negative pressure bench-top containment unit

Insect cages, when properly used, can increase the containment level of a particular experiment as long as the factors listed above pertaining to screen characteristics and sizing are respected. Though researchers may fashion their own cages out of metal, wood, glass, or screen, commercial models are also available. The Bugdorm® insect cage (Fig. 4) is a type of cage available from biological and greenhouse supply companies.

Figure 4. Bugdorm® insect cage

The geographical location of a greenhouse provides an element of physical containment. Research involving a crop pest or noxious weed, for instance, presents a greater risk if the facility is located in an area adjacent to large cropping areas susceptible to the pest. When planning new facilities, it is important to determine what type of agricultural activities will be occurring in adjacent areas before siting. Most work with GMOs, however, does not require remote or otherwise special siting since other safeguards are usually adequate.

BIOLOGICAL CONTAINMENT

Biological processes can provide a highly effective means of preventing unintended transmission of genetic material. Biological containment methods include reproductive, spatial, and temporal isolation. Appendix P of the NIH Guidelines provides a partial list of the biological containment practices appropriate for plants, microbes, and insects. Scientists and technicians conducting transgenic research generally best understand the biological systems involved. They are at liberty to devise other means of biological containment in their experimental protocols, subject to review by the IBC.

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Plants

One or more of the following procedures can prevent dissemination of genetic material by pollen or seed:

• Cover or remove flower and seed heads to prevent pollen and seed dispersal;
• Harvest plant material prior to sexual maturity or use male sterile lines;
• Control the time of flowering so that pollen shed does not coincide with the receptive period of sexually compatible plants nearby;
• Ensure that cross-fertile plants are not within the pollen dispersal range of the experimental plant; or
• Use genetic modification techniques that localize transgenes in non-propagative plant parts.

Bagging flowers is a standard practice used by breeders to prevent the contamination of selected plants with pollen from adjacent plants. Female flowers can be covered to prevent insect pollinators or windblown pollen from landing on the receptive surface. Male flowering structures can be bagged to prevent pollen from being disseminated by insect vectors, wind, or mechanical transfer (Fig. 5.1-5.2).

Figure 5.1—5.2  Bagging flowers for biological containment

Paper and glassine bags are most commonly used to cover flower heads. Flower heads can be removed prior to pollen or seed production in cases where the research protocol does not require seed collection. Genetic and Crop Standards of the AOSCA¹⁴, published annually by the Official Seed Certifying Agencies, describes the isolation distances required to avoid genetic contamination by pollen dispersal. Table 5 shows isolation distances for selected crops. In order to be considered an environmental risk, transgenic pollen must be able to fertilize plants of a sexually compatible species growing in the vicinity.

Source: Modified from Table 5, Minimum Land, Isolation, Field And Seed Standards, Adapted from Table 5, 7 CFR § 201.76 http://www.aphis.usda.gov/biotech/isolate.html

Crop breeders have identified numerous crops with sexually compatible wild or weedy relatives. Examples of crops that outcross with wild relatives are given in Table 6.

Depending on the location of the containment facility, the choice of season in which to conduct an experiment may constitute an appropriate biological containment method for plants. For instance, growing transgenic sunflowers only during the winter in northern climates insures that any escaped pollen would be of no consequence to local plants or weeds.