Standard Operating Protocol for Growing Transgenic Sunflower Plants in Contained Environments

Search PMN

Calvin H. Pearson and Donna J. Rath, Agricultural Experiment Station, Western Colorado Research Center, 1910 L Road, Colorado State University, Fruita, CO 81521; Colleen M. McMahan, USDA-ARS, Western Regional Research Center, 800 Buchanan St., Albany, CA 94710; Katrina Cornish, Yulex Corporation, 37860 W. Smith-Enke Road, Maricopa, AZ 85238; and Maureen Whalen, USDA-ARS, Western Regional Research Center, 800 Buchanan St., Albany, CA 94710

Introduction

Biotechnology provides the tools to develop industrial products for modern society that would not be possible in any other way (3). A biotechnological approach to improve sunflower was initiated in 2001 in a collaborative research project that involved genetic transformation of sunflower. Development of transgenic sunflowers creates concern about the possible flow of transgenes into wild sunflower populations, where these populations might be genetically altered in a detrimental way (2,8,10,12). Thus, pollen from transgenic sunflowers must be contained to prevent the hybridization of transgenic sunflower with wild sunflower. Concerns about biotechnology and gene flow from transgenic crops into their wild relatives have been studied and discussed by numerous researchers and organizations including Council for Agricultural Science and Technology (4), Daniell (5), Ellstrand et al. (7), Ellstand (6), and Wolfenbarger (13).

The objective of this article is to present the standard operating protocol (SOP) we developed and have used for several years for growing transgenic sunflower plants in controlled, contained environments. This SOP may be of assistance to other researchers who work with transgenic plants when preparing their own SOP. A resource we found useful in developing our SOP was Adair et al. (1). Our SOP described in this article has been modified to include additional background information and descriptions of our operation to tailor it to the readership of this publication.

The investigations were conducted under the direction of the Principal Investigator (PI). The PI was accountable for the handling, transportation, and material control from receipt/creation of transgenic material until it was transferred or destroyed (devitalized). The PI was thus accountable to the operational institution and/or its Biosafety Committee and other regulatory authorities in this regard.

In this work, transgenic sunflower plantlets were grown at two locations. One location had a secure laboratory with a lockable plant tissue culture growth chamber. A containment greenhouse was located at the other location. Transgenic sunflower plantlets were initially grown in a tissue culture chamber and when plants were large enough or developed roots they were moved to the greenhouse. The distance between the two locations was approximately 20 miles.

Laboratory Specifications

The research laboratory that contained the tissue culture growth chamber was equipped with a broad range of standard and specialized equipment for plant tissue culture and various aspects of the research. The laboratory had several levels of physical isolation against insect, animal, or human intrusion into the plant growing space within the tissue culture chamber. These levels of physical isolation were: (i) An 8-ft-high net-wire fence surrounding the entire property. Access gates were closed and locked after working hours and when no employees were present. (ii) Lockable doors into the main building. The doors were locked after working hours and when employees were not present. (iii) Access to the laboratory was through a controlled access door within the main building. The door into the laboratory had a key pad door lock. (iv) All windows in the laboratory were lockable and had securely-fit screens. All vents and heat ducts were screened. The drain that exited from the growth chamber was screened. Sink drains in the laboratory were screened. (v) The tissue culture chamber was completely enclosed and had two access doors with key locks. The chamber doors sealed tightly when closed and were locked after working hours. (vi) Plants were grown in enclosed containers (covered Magenta boxes and covered Petri dishes sealed with parafilm) at all times. The associated research staff and PI had offices that were physically adjacent to the secure area.

Greenhouse Specifications

The greenhouse was an 1100 ft² polycarbonate structure and was divided into two, 20 × 28-ft rooms with a 3-ft-high concrete nib wall base on all sides and a 3-ft-wide door entry at both the northern and southern ends. For the duration of the research project, an internal doorway between the two rooms was closed and sealed by securing ¾-inch plywood sheeting over the entire doorway. The greenhouse had a gravel floor and was heated by radiant hot water heaters located on the inside of all outside walls. The facility had 2 × 4-ft, 2-speed fans located on the eastern wall with two fans per room. Three-foot high cooling pads were situated inside the wall and vented along the entire length of one wall. There were no roof vents in the greenhouse. Metal, space-saver rolling benches were located throughout the south room and in half of the north room. A Wadsworth Step 50 (Enviro Step 50, Wadsworth Control Systems Inc., Arvada, CO) was located in the northern room, which controlled the pad system for both rooms. Separate controls for each room existed for all other environmental controls.

Biosafety Improvements to the Greenhouse

The facility complied with National Institute of Health standards for transgenic plant research at Biosafety Level 2-P containment. For more details see the website. This level of containment was the appropriate standard for growing transgenic sunflowers for this research project. The entire greenhouse met Biosafety Level 2-P containment even though only the north room was allocated for transgenic work.

The greenhouse containment measures were as follows:

1. A bioBubble with two airlocks (bioBubble Inc., Fort Collins, CO), one for entry and one for exit, was installed in half of the north room of the greenhouse.

2. Insect screening structures covered the cooling system vents. Screening was manufactured by Green-Tek, Inc. (Edgerton, WI) and was a 50 × 24 mesh screen. This had a screen hole of 266 × 818 microns. It excluded aphids, leaf miner, whitefly, and up to 80% of western flower thrips.

3. A vestibule with a keyed door lock was the first entry point into the transgenic room of the greenhouse (Fig. 1A). A person entering the greenhouse entered the vestibule first by using a key through a locked entry door. This door was closed and locked behind the person before the person opened the second locked door, using the same key. The second door opened into the greenhouse proper. Doors were locked behind the person as they entered the greenhouse to prevent an unauthorized person from entering the greenhouse while the authorized person was inside the greenhouse.

4. The two fans were enclosed with insect screening (Fig. 1B). This was the same screening used on the western wall of the greenhouse over the cooling system vents. This screening prevented insects from entering the greenhouse when fans were not operating.

5. The greenhouse was monitored daily, including weekends and holidays, for intactness of security systems. The screening, fencing, and coverings on and in the greenhouse were inspected for damage 2 to 3 times per week. These inspections were documented in the Greenhouse Log Book. The Greenhouse Log Book was stored in the greenhouse and was easily accessible to research staff and APHIS and Institutional Biosafety Committee inspectors.

6. A surveillance camera was installed in front of the greenhouse. Signage was posted on the fence surrounding the greenhouse indicating a security camera was in use.

Fig. 1. (A) Entrance into the BL2-P greenhouse, (B) insect screening over greenhouse vents, and (C) security fence around the greenhouse.

Technical experts were consulted and involved in retrofitting the greenhouse, and in developing the SOP. These experts included: a professor/plant ecologist; a greenhouse facilities manager; a greenhouse crops specialist; and a company representative from Wadsworth Control Systems Inc., Arvada, CO.

Additional details of the greenhouse system included a cooling system that overcomes problems from reduced cooling effect as a consequence of the insect screening. The cooling system was a Kuul evaporation cooling pad system (Port-A-Cool LLC, Center, TX) with a curved power rack, and rack and pinion operating system. This system allowed for greater work area in the greenhouse, improved cooling efficiency, and, combined with a 50% shade cloth roof covering, eliminated the need for whitewashing the structure in summer. Drip emitters were connected to irrigation controllers and were used to irrigate container plants of sunflower automatically. Plants in the greenhouse could also be hand-watered using a hose and garden shower wand.

Training

At the outset of the research project, the PI visited several greenhouse facilities to discuss and review biosafety, propagation/regeneration, and security protocols. These facilities included a USDA-ARS center and a major research university. The PI and Research Staff attended and completed a week long training course on biosafety and biosecurity.

Because we shipped transgenic plantlets across state lines research associate staff completed online courses for the International Air Transport Association (IATA, Montreal, Quebec, Canada) Dangerous Goods, Department of Transportation/Hazmat Employee with Packaging course, and Saf-T-Pak Shipping Dangerous Goods. According to IATA, transgenic plants that do not meet the definition of infectious substances but are capable of altering animal, plant, or microbiological substances in a way that is not normally the result of natural reproduction are considered by IATA to be dangerous goods (9).

In-service training was held monthly for personnel associated with the research project. Protocols set forth in this document as well as the manual "A Practical Guide to Containment" published by Virginia Tech (1) were used as resources for basic and specific training. The "Standard Operating Protocol for Growing Transgenic Sunflower Plants in Contained Environments" was used for specific training to meet the needs of the research project. Minutes of each training session were prepared and attendees signed the minutes to verify attendance and to acknowledge the content presented at training sessions. Signed copies of the training minutes were kept on file.

Biosafety Issues

Containment of transgenic pollen is essential to bio-containment. Sunflower pollen is readily killed by nearly any liquid, including water. The liquid causes the pollen to burst (Jerry Miller, 2004, personal communication). Additionally, a USDA-ARS research botanist recommended ethanol for killing pollen because it is also a disinfecting agent (Gerald Seiler, 2004, personal communication). Green-Shield disinfectant (Whitmire Micro-Gen Research Laboratories Inc., St. Louis, MO) was used to devitalize sunflower pollen.

Transgenic plants were checked for reproductive parts daily. A photograph of a sunflower at the onset of reproductive stage [R-1 stage of development (11)] was kept in the Greenhouse Log Book. When a transgenic plantlet began reproductive development that resembled the photograph, the plant was moved into the bioBubble. Reproductive, transgenic sunflowers were moved into the bioBubble containment and remained there throughout flowering and seed maturation periods. Inspections were documented in the Greenhouse Log Book. Heads of transgenic sunflowers at the R-4 stage were bagged with a Delnet pollination bag (Delstar Technologies Inc., Middleton, DE) to decrease the potential for pollen movement. Plants remained bagged until fully mature and until the plant was ready for devitalization.

Due to the nature of sunflower tissue culture, precocious flowering occurred on some plantlets. Sunflower tissue culture plantlets can flower without a root system. Without a root system, plantlets would not survive outside the Magenta box and growth chamber. When precocious flowering occurred in the growth chamber, sunflower plantlets were kept inside the Magenta box sealed with parafilm until a root system developed. The Magenta box containing the flowering plantlet was not opened except in the biosafety cabinet (which was equipped with a HEPA filter) or in the bioBubble, transported there per our established SOP to the greenhouse and into the bioBubble. The biosafety cabinet was located in the laboratory. As described earlier, the laboratory has several levels of physical isolation against insects, animals, and human intrusion (see Laboratory Specifications section).

When plants were in the bioBubble, hooded Tyvek suits with feet, gloves, and dust masks for mustached individuals were worn by research personnel in the bioBubble. Rubber clogs were available to be worn over the feet of the suit. Tyvek suits, gloves, dust masks, and rubber clogs were stored in the entrance airlock of the bubble. Suits were put on in the entrance airlock along with rubber clogs and latex gloves. Gloves were worn on the outside of the sleeve of the suit to prevent pollen from entering suits through the sleeves. Clogs were worn over the feet of suits to prevent damage to the feet in the gravel. Dust masks were worn by any person who had a mustache to keep pollen from being transferred on facial hair.

When exiting the bioBubble, clogs were placed in a container of Green-Shield disinfectant that was kept in the exit airlock of the bioBubble. In the exit airlock of the bioBubble, Tyvek suits, gloves, and dust masks were placed in an autoclaveable sealed bag and placed in double-closed plastic containers (one inside the other, and secured with cargo straps) for transport back to the laboratory location, where they were autoclaved. Tyvek suits were reused several times if plants were not shedding pollen. Gloves were never reused. To reuse a Tyvek suit, it was put into an autoclave bag, which was sealed with a twist tie, and sprayed with Green-Shield. After the bioBubble exit procedures were completed, the Tyvek suit in the sprayed autoclave bag was moved to the entrance airlock in preparation for the next entry into the bioBubble. Tyvek suits, gloves, and dust masks were decontaminated in the autoclave at the greenhouse. Autoclaving was documented in the Autoclave Log book. After the clogs were disinfected in the exit airlock, they were transferred to the entrance airlock for reuse.

A procedure for cleaning the sponge filters on the HEPA-filter units in the bioBubble was established. The sponge filters were cleaned by washing them with water in a one gallon autoclavable container. The container and contaminated water was transported to the laboratory location in the designated container and autoclaved as per the SOP. Disposal of autoclaved liquids was via a septic system. As an alternative, rinseate was used for irrigating pots that were in the bioBubble.

No food, beverages, or tobacco products were used in the laboratory or greenhouse.

Security. The greenhouse has a 6-ft-high wood privacy fence built around the outside of the structure to protect the insect screening from being damaged (Fig. 1C). Farm perimeters at both sites are completely enclosed with 8-ft-high security fencing. Gates were locked after regular working hours to prevent vehicle access and unauthorized people from entering the premises. Keys to the greenhouse were assigned only to personnel associated with the research project (See Table 1). Keys were identified as DNC (Do Not Copy). The key entry code for access into the laboratory was distributed only to specified research staff. Others who entered the area were escorted by one of the persons who were allowed access to the laboratory. A surveillance camera was mounted above the access into the greenhouse. A sign on the greenhouse gate read, "Monitored by Surveillance Camera."

No plant material was bought into or removed from the greenhouse without the knowledge and consent of the PI.

Table 1. Personnel associated with the transgenic sunflower research project, their access, and roles.

Name	Growth chamber access	Greenhouse access	Function
Principal Investigator	X	X	Research leader
Research Center Manager		X	Facilities and support staff manager
Research Associate II		X	Greenhouse operations*
Student Hourly	X	X	General operations, data collection
Research Associate I	X	X	Lab manager, data collection

* Greenhouse operations included watering, pest and disease control, and other similar activities.

Responsible Parties. A list of personnel associated with the research project is shown in Table 1.

Signage. Signs on the outer door of the greenhouse stated: "Authorized Access Only. Transgenic Plants Inside. Escaping pollen may pose risk to endemic species." The sign also indicated contact information (office phone number, cell phone number, and/or home phone number) for the PI, Research Associate II, Research Associate I, and Western Colorado Research Center Manager.

All transgenic plants in the greenhouse were clearly identified to distinguish them from non-transgenic plants. Red tags in each pot of transgenics, with written identifiers, denoted transgenic plants. White tags in each pot of non-transgenics, with written identifiers denoted non-transgenic plants. If an entire bench contained transgenic plants, a sign was placed on the end of the bench. Non-transgenic sunflower plants that were crossed with transgenic sunflower plants were tagged with a yellow marker tag containing written identifiers as to parentage, dates, and other pertinent information.

Each transgenic plant was individualized by assigning a unique number to it, as the plant was placed in a peat pellet. This also applied to non-transgenic plants that were crossed with transgenic plants. This procedure allowed each viable plantlet’s pedigree and history to be followed.

All transgenic plants in the growth chamber had plate number, plant number, and target gene, written on the outside of the Magenta box. A red dot on the outside of the Magenta box, indicated they were putative transgenic.

Transportation of Material. Plantlets and plant material were transferred between sites in double-closed plastic containers (one inside the other) and labeled as per Guidelines 7CFR340.7 and 7CFR340.8. Plant material and potting media used for growing transgenic plants were transported back to the laboratory location for devitalization in double-closed containers (one inside the other).

Transgenic seed produced to maturity was harvested in the bioBubble by cutting mature flower heads and double-sealing them in Ziploc bags with attached labels clearly identifying the seeded heads as transgenic material. The red tag with identifying information was removed from the plant container and placed in the bag with the flower head. Bagged heads were transported back to the laboratory location in double-closed plastic containers (one inside the other) and labeled.

All transgenic material (including soil and pots in which transgenics were grown in) that was transported was documented in the Transportation Log Book. Transgenic plants and associated material were in the possession of the person transporting the material at all times. In all cases, containers were secured with cargo straps and transported in the interior of the vehicle or in the trunk of the vehicle (not in the open bed of a vehicle). Transgenic plants and associated material were not left unattended in a vehicle at any time during transport.

Devitalization of Material. All transgenic sunflower heads were autoclaved after seed harvest. Any other non-useful plant material was removed from the bioBubble and greenhouse and transported back to the laboratory in well-labeled, double-closed plastic containers (one inside the other). Removal of non-useful plant material from the bioBubble and greenhouse was to help minimize a buildup of disease and insects in the bioBubble and greenhouse. Cargo straps were used to keep containers secured during transport. Containers were transported inside the vehicle or in the trunk of the vehicle. An autoclave was used to devitalize leaves, stems, and large roots of transgenic plants that could be readily separated from the soil. An autoclave was located in the greenhouse where small quantities of plant material and soil were devitalized onsite. Two other autoclaves were located in the laboratory at Fruita. An Autoclave Log Book for the autoclave at the greenhouse was located inside the greenhouse. A copy of the autoclave training was maintained in the Autoclave Log Book in the greenhouse. Established devitalization procedures were used for all three autoclaves. Pots and soil were devitalized by using one of the three autoclaves.

Written Logs (Reporting). Greenhouse Log Book — Written logs were maintained for personnel entering into the greenhouse. The information in the Greenhouse Log Book (located onsite) included: date, name, purpose, time in, and time out. Changing sticky traps, checking transgenics for reproductive parts, watering and fertilizing plants, checking insect screening, bringing plants into and out of the greenhouse, checking for disease and pests, and any other management practices for the greenhouse were recorded in the Greenhouse Log Book.

Transgenic Seed Log Book — Information recorded in the Transgenic Seed Log Book included date, cultivar, target gene, number of seed, storage location, identification number, and name of person performing the seed counting process. The Transgenic Seed Log Book was maintained in the laboratory.

Transportation Log Book — Information recorded in the Transportation Log Book included date, departure/arrival time, material transported, origin/destination, transport route, and the name of individual transporting the material. The Transportation Log Book accompanied transported material. There were two Transportation Log Books, one at the laboratory location and one at the greenhouse location.

Autoclave Log Book — Information recorded in the Autoclave Log Book was date, initials, material autoclaved, and batch number (of autoclave). One autoclave indicator was attached to the log and another autoclave indicator went with the batch to the disposal site. One duplicate tag was attached to the log with a unique number on it and the second duplicate tag was attached to the devitalized material batch and remained with it. The time the material was autoclaved was logged. The Autoclave Log Book was maintained at the appropriate research center.

Log books were reviewed regularly to make sure recordkeeping was accurate. All archive log material was kept at the laboratory location.

Transgenic Sunflower Head Threshing and Seed Counting Protocol

A Tyvek suit was worn while threshing and counting seed to ensure that seed did not inadvertently fall into shirt pockets, pant cuffs, etc.

Before Removal of Seed Head from Bag — All work area counter space was cleared of all equipment, trash, paper, etc. Sink screens were put in place. The counter work area was sprayed with 70% ethanol and dried before threshing began. The counting mat was spread on the counter, sprayed with 70% ethanol, and dried before removing the seed head from the transport container.

Removal of Seed Head Bags from Transport Container — Before opening the transport container, the outside of the container was sprayed with 70% ethanol. When the transport container was opened, the inside of the transport container and inside of the lid was sprayed with 70% ethanol. As a seed head bag was removed from the transport container, each bag was sprayed with 70% ethanol. Only one seed head at a time was harvested.

Counting the Seed — Utmost care was used to remove all seeds from the head. The flower head debris was sorted through piece by piece to be sure a seed was not inadvertently left behind. Seeds were not allowed to fall off the seed counting mat.

The seed viability level was verified by the PI or research personnel. After seeds were completely separated from debris, they were placed in a seed envelope. All debris was double checked to be sure all seeds were removed. The debris was then placed in an autoclave bag. Seeds were removed from the seed envelope and counted until the same count was achieved three times in a row.

Seeds were placed in a seed envelope, labeled with the following:

(a) plate #

(b) plant #

(d) date of harvest

(e) generation (T₀, T₁, T₂ … etc.)

(f) # of seed

(g) target gene

(h) host cultivar

A red dot was placed on the outside of the envelope to indicate the seed was transgenic. Any other information that was on the red tag that accompanied the seed head was written on the outside of the envelope. The envelope was folded twice and a mini binder clip was attached to the double fold on the seed envelope. The seed envelope was placed in the larger envelope (designated as the plate envelope). The larger plate envelope was labeled with the following:

(a) plate #

(b) date of last count of entire envelope

A binder clip was used to close the plate envelope.

Leaving the Seed Counting Area — The seed counting mat and counter area were sprayed with 70% ethanol. All clothing was checked for stray seed (shirt pockets, pant cuffs, etc.).

Documentation of Transgenic Seed — The seed count for each head was entered in the Greenhouse Notebook. The Greenhouse Notebook was a different document from the Greenhouse Log Book. The Greenhouse Notebook included all research activities performed in the greenhouse and remained in the possession of the research staff at the laboratory. The seed information was also entered into Transgenic Seed Log. The Transgenic Seed log was kept on a computer in the laboratory. Once data entry was completed, a new copy of the Seed Log was printed. The seed was stored in a locked seed safe within the laboratory. All debris from plants was autoclaved and all pertinent information was entered into the Autoclave Log as per the SOP.

Transgenic sunflower seed was stored in a lockable seed safe that was clearly labeled as to its contents. Seed in the lockable seed container was stored in a storage room that was within the laboratory. The two keys for the seed safe were labeled with DNC (Do Not Copy) and held only by the PI and Research Associate. Plant material remaining following harvest of seed was devitalized by autoclaving using the autoclave in the laboratory. All tools and materials used to harvest seed were autoclaved after seed harvest. All material that was autoclaved was recorded in the Autoclave Log Book.

General Greenhouse Operations

Fumigation. Fumigation frequency was annual or between plant growing cycles. The greenhouse was fumigated prior to plant introduction. Fumigation activities were documented in the Greenhouse Log Book.

Watering (Frequency: daily). An inspection of plants occurred daily to ensure media in all plots was thoroughly wetted. Watering time of day and application amount was adjusted as needed. Changes to irrigation scheduling were recorded in the Greenhouse Log Book.

Pest and Disease Scouting (Frequency: daily). The greenhouse was monitored regularly for pests and disease. All findings were recorded in the Greenhouse Log Book. The PI was notified of any pests found or new situations related to pests. Pest and disease identification and control were discussed with the PI and an entomologist or pathologist. Pesticides were used if an insect population was determined to be established. Only pesticides registered for greenhouse application were used. All pesticide applications were recorded in the Greenhouse Log Book following State Department of Agriculture pesticide application regulations. Signage was posted on the entry door before pesticide application and removed after the re-entry time was met.

Sticky traps were used to monitor insect movement within the greenhouse. Two insect sticky traps were placed in the bioBubble, and two others were in the greenhouse outside of the bioBubble. Sticky traps were changed once each week when plants were growing in the greenhouse. Sticky traps were labeled with use dates, bagged in Ziploc plastic bags, stored in a sealable container, and archived in the laboratory. Sticky traps were transported in double-closed plastic containers (one inside the other) and labeled. Cargo straps were used to keep containers secured during transport. Containers were transported inside the vehicle or in the trunk of the vehicle (not in the bed of a vehicle). Sticky trap changes were logged in the Greenhouse Log Book.

Climate Control. Temperature — A data logger was used in conjunction with manual checks. The data logger recorded temperature and relative humidity every 5 minutes and data were downloaded onto a computer regularly as needed to capture the data.

Cooling pads (Frequency: Monthly) — Water in cooling pads was flushed to waste monthly. Water conditioner was added to fresh water. The cooling pad system was drained in late fall. This date was recorded in the Greenhouse Log Book.

Sensophone — A Sensophone in the greenhouse assisted in alerting the staff to temperature fluctuations that occurred after working hours. The upper and lower temperature thresholds that were set for the Sensophone were 100°F and 45°F, respectively. The Sensophone assisted in assuring that plants were not exposed to temperatures that were too high or too low. These thresholds also alerted staff to mechanical failures and possible loss of containment.

Contingency Plans for Loss of Containment

In the event of loss of containment, employees at the research center were to notify the PI at the time of first realization of containment loss. The PI was to be notified immediately, regardless of time of day or day of week. Notifying the PI was to be attempted with work, cell, or home telephone numbers and continued until the PI was successfully informed about the loss of containment. The Research Center Manager also was to be notified immediately. The PI (the Manager in the event the PI could not be reached after repeated attempts) would immediately report the incident to the University Biological Safety Officer or call the police dispatcher and ask to speak to the #1 Emergency Responder), the Institutional Biosafety Committee, NIH Office of Biotechnology Activities, and/or other designated authorities as required/recommended at the time of reporting/notification.

Consistent with standard permit conditions at 7 CFR 340.4(f) (10), APHIS-BRS would be notified orally immediately upon discovery and in writing within 24 h in the event of any accidental or unauthorized release of the regulated article.

In the event containment was lost, re-establishment of containment was to be attempted as rapidly as possible. Temporary containment measures were to be deployed until permanent repairs could be completed. An inventory of transgenic plant and plant part loss was to be performed. Recovery of plants and plant parts would have been done as rapidly as possible and conducted under the direction of the PI or Manager.

In case of fire, the fire department was to be contacted by dialing 911. The fire station nearest to the greenhouse was a volunteer fire department located approximately one mile east of the research center. A fire hydrant was located on station next to the main road. The distance from the fire hydrant to the gate that goes into the greenhouse area was 275 ft. The fire station nearest to the laboratory was also a volunteer fire department located approximately three miles southwest of the research center. No fire hydrant was located near the lab. The estimated time to travel to an emergency at the laboratory was 8 minutes.

In the event of a power outage in the greenhouse, no one had permission to enter the bioBubble until power was restored to the greenhouse and the bioBubble, and five or more air exchanges occurred. (Note: According to the Clean Room Specifications provided by bioBubble Inc. for our facility, the number of air exchanges per hour was 65). Allowing for a 5-min time period once the power was back on ensured five or more air exchanges had occurred.

Green-Shield disinfectant was used in the greenhouse sump at the conclusion of an experiment or at other appropriate times (end of growing season/cycle) and was recorded in the Greenhouse Log Book.

Concluding Comments

Fortunately, no loss of containment was experienced during the course of this project. Before embarking on a project that includes rDNA it would be wise for a PI to make personal contact with regulatory authorities at the institutional and federal levels in an attempt to understand how they function and nuances that may be unique to a specific transgenic project. Throughout the life of our project we regularly referred to our SOP. A copy of the SOP was kept on site at the greenhouse and in the laboratory. Furthermore, each person associated with the research project had their own, up-to-date copy. We found our SOP to be a valuable tool and resource when conducting our transgenic plant research project. We expect other researchers would profit by creating a comprehensive and detailed SOP at the outset of their project. Additionally, researchers are encouraged to review and update their SOP regularly.

Disclaimer

Mention of a trade name or proprietary product does not imply endorsement by the authors or their institutions.

Literature Cited

1. Adair, D., Irwin, R., and Traynor, P. L. 2001. A practical guide to containment: Greenhouse research with transgenic plants and microbes. Information Systems for Biotechnology, Virginia Tech. Blacksburg, VA.

2. Arias, D. M., and Rieseberg, L. H. 1994. Gene flow between cultivated and wild sunflowers. Theor. Appl Genet. 89:655-660.

3. Brownback, S. 1993. Transforming the vision into reality: How to make it happen. Pages 2-3 in: New Industrial Uses, New Markets for US Crops: Status of Technology and Commercial Adoption. J. Harsch, ed. Office of Agric. Materials, USDA-CSREES, Washington, DC.

4. Council for Agricultural Science and Technology (CAST). 2001. Evaluation of the US regulatory process for crops developed through biotechnology. Issue Paper no. 19. CAST, Ames, IA.

5. Daniell, H. 2002. Molecular strategies for gene containment in transgenic crops. Nat. Biotechnol. 20:581-586.

6. Ellstrand, N. C. 2002. Gene flow from transgenic crops to wild relatives: what have we learned, what do we know, what do we need to know? Proc. from the Gene Flow Workshop, Ohio State Univ. Mar. 5-6, 2002.

7. Ellstrand, N. C., Prentice, H. C., and Hancock, J. F. 1999. Gene flow and introgression from domesticated plants into their wild relatives. Annu Rev. Ecol. Syst. 30:539-563.

8. Faure, N., Serieys, H., and Bervillé, A. 2002. Potential gene flow from cultivated sunflower to volunteer, wild Helianthus species in Europe. Agr. Ecosyst. Environ. 89:183-190.

9. International Air Transport Association. 2006. Dangerous Goods Regulations, 47th Edn. Int'l. Air Transport Assoc., Montreal, Quebec, Canada.

10. Reagon, M. R., and Snow, A. A. 2006. Cultivated Helianthus annuus (Asteraceae) volunteers as a genetic "bridge’ to weedy sunflower populations in North America. Amer. J. Bot. 93:127-133.

11. Schneiter, A. A., and Miller, J. F. 1981. Description of sunflower growth stages. Crop Sci. 21:901-903.

12. Whitton, J., Wolf, D. E., Arias, D. M., Snow, A. A., and Rieseberg, L. H. 1997. The persistence of cultivar alleles in wild populations of sunflower five generations after hybridization. Theor. Appl. Genet. 95:33-40.

13. Wolfenbarger, L. L., ed. 2002. Proceedings of a workshop on: Criteria for field testing of plants with engineered regulatory, metabolic and signaling pathways. Information Systems for Biotechnology, Virginia Tech. Blacksburg, VA.