aphis-users guide for introducing genetically engineered plants and microorganisms 1997b

/^f/r^^ J^Ä United States |t|J| Department of "^0^ Agriculture

Animal and Plant Health Inspection Service

Technical Bulletin No. 1783

User's Guide for Introducing Genetically Engineered Plants and Microorganisms

ii_

Biotechnology, Biologies, and Environmental Protection Animal and Plant Health Inspection Service U.S. Department of Agriculture Federal Building 6505 Belcrest Road Hyattsville, MD 20782

Terry L. Medley, J.D. Director (Room 850)

Michael A. LIdsky, J.D, Deputy Director for Biotechnology Coordination (Room 853)

Arnold S. Foudin, Ph.D. Deputy Director for Biotechinology Permits (Room 842)

301-436-7612 (Biotechnology Permits) 301-436-8669 (Facsimile)

Mention of commercial enterprises or brand names does not constitute endorsement or imply preference by the U.S. Department of Agriculture (USDA).

All programs of the U.S. Department of Agriculture are available to anyone without regard to race, creed, color, sex, disability, religion, national origin, or political belief.

Issued: June 1991

Foreword

To be effective, regulation must be a cooperative effort involving communication between the applicant and the reviewer. This user's guide is intended to further the cooperation necessary to transfer the products of genetic engineering from the laboratory to the field and eventually to the marketplace.

This guide has been prepared to assist you in submitting an application for a permit for importation, interstate movement, or environmental release of plants or microorganisms regulated under 7 CFR Part 340.

The guide has several sections: questions and answers; sample applications for importation, interstate movement, release, or courtesy permits; a copy of our regulations; and information concerning the submission of confidential business information.

Our Biotechnology Permits staff is ready to assist you. If you have any questions, please call us at Area Code (301) 436- 7612.

Terry L. Medley, J.D. Director Biotechnology, Biologies, and Environmental Protection

Contents

Section I. Questions and Answers

Section II. Sample Applications for Interstate Movement, Importation, and Courtesy Permits

Section III. Sample Application for Release Into the Environment

Section IV. Regulations With Preamble

Section V. Confidential Business Information (CBI) - Instructions and Policy Statement

Questions and Answers on General Regulatory Policy Under 7 CFR 340

1. When is an APHIS permit needed?

A. A permit is required to introduce a regulated article.

2. What Is a regulated article?

A. A regulated article is an organism that has been genetically engineered (via recombinant DNA techniques) from a donor organism, recipient organism, vector, or vector agent that is a plant pest or contains plant pest components. APHIS regulations in 7 CFR 340.2 list such organisms (Section IV). Other genetically engineered organisms may be regulated articles if they have been genetically engineered using an unclassified organism or if the Director of Biotechnology, Biologies, and Environmental Protection determines that the genetically engineered organism is a regulated article.

3. Are there classes of microorganisms that are not regulated articles?

A. Yes. Recipient microorganisms that are not plant pests and that result from the addition of genetic material containing only noncoding regulatory regions can be introduced without a permit. (See Section IV.)

4. What is meant by the term "introduce"?

A. Introduce means to import, move interstate, or release into the environment.

5. What is a plant pest?

A. A plant pest is defined as "any living stage (including active and dormant forms) of insects, mites, nematodes, slugs, snails, protozoa, or other invertebrate animals, bacteria, fungi, other parasitic plants or reproductive parts thereof; viruses; or any organisms similar to or allied with any of the foregoing; or any infectious agents or substances, which can directly or indirectly injure or cause disease in or damage to any plants or parts thereof; or any processed, manufactured, or other products of plants." (7 CFR 340.1)

6. Whom do I contact if I am uncertain as to whether the organism is a plant pest or a regulated article?

A. Contact the U.S. Department of Agriculture's (USDA) Animal and Plant Health Inspection Service (APHIS). Include the following information: geographical source of the organism; whether or not the organism has been genetically engineered; biological, biochemical, molecular, or genetic data that would support the claim of nonplant pest status of the organism (including published or unpublished data); and any additional information that could affect APHIS' decision on the pest status of the organism. Send this information to:

Deputy Director for Biotechnology Permits Biotechnology, Biologies, and Environmental Protection USDA-APHIS 6505 Belcrest Road, Room 844A Hyattsville, MD 20782 Area Code (301) 436-7612

7. Do the APHIS regulations apply to an organism if it is genetically engineered but no plant pest component is used in the modification process?

A. No. For APHIS regulations to apply, there must be a plant pest component involved in the modification process, or the organism used in the engineering process must be an unclassified organism, or there must be reason to believe that the resulting organism is or will be a plant pest.

8. Do the APHIS regulations cover the exportation and intrasXaXe movement of a regulated article?

A. The exportation and the intrasXaXe movement of a regulated article are not covered per se by the APHIS regulations. A permit would be required for the "in transit" movement of a regulated article in the course of the exportation of a regulated article. For example, if in exporting a regulated article to Mexico City, the article were moved via St. Louis and Dallas, an intersXaXe movement permit would be required for the movement of the article between St. Louis and Dallas. If the article were exported directly from St. Louis to Mexico City, an intersXaXe movement permit would not be required.

The USDA's Agricultural Marketing Service regulates the exportation of both genetically engineered and conventionally bred tobacco plants under the Tobacco Plant and Seed Exportation Act. Before exporting these plants, check with the Market Information and Program Analysis Branch of the Tobacco Division of the Agricultural Marketing Service in Washington, DC, at Area Code (202) 447-3489. In addition, the export of certain genetically engineered organisms to specific countries requires a license from the U.S. Depart- ment of Commerce, Bureau of Export Administration, Washington, DC, at Area Code (202) 377-5695.

The intrasXaXe movement of a regulated article may be a matter of State jurisdiction.

9. Is the introduction of an organism regulated if genetic engineering is used in combination with other techniques, such as direct injection, electroporation, embryo rescue, or somaclonal variation?

A. Yes. If during any part of the process genetic engineering is used and a plant pest component is involved, the resulting organism will be regulated.

1-1

10. Are subsequent generations of regulated articles regulated?

A. Any subsequent generation of a regulated article is subject to APHIS regulations.

11. Is it possible to have an organism exempted from the regulations? How do I accomplish this?

A. Yes. Use the petition process in APHIS regulations (7 CFR 340.4, see Section IV). Submit data and information to show that the organism in question is not a plant pest. Include copies of scientific literature, unpublished studies, and data from tests performed, even if they are unfavorable. Trade secrets or confidential business information (CBI) should not be included.

12. What is the APHIS policy on protecting CBI?

A. APHIS treats CBI as data that may be protected from disclosure under the Freedom of Information Act [5 U.S.C. 552(b)(4)]. CBI includes trade secrets and commercial or financial information found to be confidential. The applicant needs to identify the CBI in the application.

Documents containing trade secrets will be treated as CBI. "Trade secrets" refers to information relating to the production process, such as formulas, quality control tests and data, and research methodology. Such information must be (1) commercially valuable, (2) used in the applicant's business, and (3) maintained in secrecy.

In addition, documents containing financial or commercial information that the applicant does not want disclosed for competitive reasons are treated confidentially. The applicant needs to submit a detailed statement explaining the potential for competitive harm if this information is disclosed. (See Section V.)

13. If I comply with APHIS regulations, do I have to comply with regulations from other Federal agencies?

A. Yes. You must comply with all applicable regulations. Genetically engineered microorganisms may be regulated by the Environmental Protection Agency (EPA), and genetically engineered plants and microorganisms to be used as food may be regulated by the Food and Drug Administration (FDA).

14. How do I apply for approval for importation, interstate movement, or introduction of a regulated article?

A. You must submit an application form (APHIS Form 2000) to the Biotechnology Permits unit of Biotechnology, Biologies, and Environmental Protection. Form 2000 is used to apply

for a permit to import a regulated article, to move it interstate, to release it into the environment, or to request a courtesy permit. (See Sections II and III.)

15. Can an application be modified once the APHIS review process has started?

A. Yes. However, proposed modifications should be brought to the attention of the Biotechnology Permits unit as early as possible so that the review process can be completed on time.

16. What recourse do I have if a permit is denied or revoked?

A. You can appeal the decision. The procedures are found in the APHIS regulations. (See Section IV.)

1-2

Questions and Answers on Interstate Movement, Importation, and Courtesy Permits

17. Are there any regulated articles for which interstate movement permits are not required?

A. Yes. Two types of regulated articles do not require a permit for interstate movement:

1) Strains of Escherichia co//(K12 and its derivatives) that contain genetic material derived from a plant pest and

2) Plants or plant parts of Arabidopsis thaliana containing genetic material derived from a plant pest stably integrated into the plant genome.

These exemptions from permitting requirements for interstate movement are dependent upon certain conditions for biological and physical containment as specified in 7 CFR 340.2(b) of the regulations.

18. If I plan to move interstate or to import a conven- tional plant pest that has nof been modified by genetic engineering but that I plan to modify by genetic engineering techniques, do I need to apply for a permit on Form 2000?

A. No. However, to so move or import a nonindigenous, nongenetically engineered plant pest, a permit is required. To obtain this permit (PPQ Form 526) and additional information, write to:

Biological Assessment and Technical Support Staff USDA - APHIS - Plant Protection and Quarantine 6505 Belcrest Road, Room 625 Hyattsville, MD 20782

19. Is the movement of disarmed strains of Agrobacterium tumefaciens or other disarmed plant pests regulated?

A. Yes. A permit is a certification that the organism has been disarmed and does not present a risk of plant pest introduction.

20. How soon can I get a permit?

A. Interstate movement, importation, or courtesy permits are issued within 60 days of APHIS' receipt of your application.

21. After my movement permit application is received by your office, what happens next? (7 CFR 340.3a.b)

A. The application is reviewed, a preliminary pest risk analysis is made, and a letter requesting review is sent to the State regulatory official. If APHIS and State officials have not inspected the receiving facility, they will arrange for an

inspection. You will receive written notification of approval or denial. (See Section IV.)

22. Why do facilities need to be inspected? (7 CFR 340.3d)

A. The inspections verify that the facility is adequate to prevent release of a regulated article into the environment. (See Section IV.)

23. What will the inspectors look for during laboratory, growth chamber, and greenhouse inspections?

A. The inspector will review the facilities, personnel, physical security, and operational procedures and determine if National Institutes of Health guidelines for good laboratory, growth chamber, and greenhouse practices are being followed.

24. What events require mandatory reporting to APHIS after a movement or importation permit is used?

A. If an accidental or unauthorized release of a regulated article occurs, you must orally notify the Biotechnology Permits unit immediately and send written notification within 24 hours.

25. Do permits need to be renewed?

A. Permits are valid for 1 year from the date of issuance. A renewal is required if additional material is to be moved after this period.

26. Can a regulated article be given to another person in my lab?

A. Yes. When a permit is issued, one person must maintain control of the regulated article to make sure all permit conditions are met. However, this "responsible person" may give the regulated article to another person if the former agrees to maintain control of the regulated article and comply with all permit conditions.

27. Who should be the responsible person for a movement permit—the shipper or recipient of the regulated article?

A. The responsible person may be either the shipper or the recipient, but that person must ensure that all permit conditions are met. If the responsible person is the shipper, the shipper must make sure that the recipient is aware of all permit conditions.

1-3

28. If my laboratory moves, do I need to submit a new permit application for all regulated articles?

A. Yes. Because the permit is issued for work in a specific facility, moving your laboratory would require an inspection of the new facility. Contact the Biotechnology Permits unit for guidance (address and phone number are on p. 1-1).

29. Do I need a separate permit for each importation or interstate movement of a regulated article?

A. A separate permit must be requested for each importation of a regulated article. However, a permit may be issued for the importation of multiple regulated articles in the course of a single importation. APHIS may grant a single permit that is valid for multiple interstate movements of a regulated article or for the interstate movement of multiple regulated articles.

30. What is the purpose of a courtesy permit, and under what circumstances should it be requested?

A. The purpose of a courtesy permit is to facilitate the movement or the release into the environment of genetically modified organisms that are noi regulated articles.

1-4

Questions and Answers Regarding Release Into the Environment

31. How long does it take to obtain a permit for release of a regulated article into the environment?

A. Release permits are issued within 120 days of APHIS' receipt of your application.

32. After my permit application Is received, what happens next, and whom do I contact during the review process?

A. After receipt of the application, APHIS personnel review the CBI copy and the nonconfidential business information copy for completeness. If complete, the permit application is given an application number and assigned to a scientific reviewer, the responsible person is notified, and the 120-day review period begins. If the permit application is not complete, the responsible person is notified. During the review period, all correspondence (which must contain the application number) should be directed to the Deputy Director of the Biotechnology Permits unit. Notification of approval or denial will be in writing.

33. Must I notify the authorities In the State where an introduction is proposed?

A. No. Federal regulations require APHIS to notify appropriate State officials before the final decision is made on an application.

34. What is an environmental assessment, and under what law is it prepared?

A. An environmental assessment (EA) is a document that analyzes the environmental impacts associated with an environmental release permit. EA's are prepared in accordance with the National Environmental Policy Act (NEPA), Council on Environmental Quality regulations, and the USDA's NEPA procedures.

35. Can I use one permit application to support field tests in more than one State?

A. Yes.

36. Do field trials have any limits in size?

A. No, but APHIS officials take into consideration the size of the field trial when they determine the significance of the impact on the environment.

37. When will USDA inspect the field test site?

A. An APHIS representative will inspect the site either at or near the beginning of the field test, possibly during the course of the trials, and shortly after the harvest.

38. Does a permit for release into the environment have an expiration date?

A. The experiment must be started within 1 year from the date the permit is issued.

39. Is there a maximum time limit on field tests?

A. No, but periodic status reports will be required if the field test exceeds 1 year.

40. What events require mandatory reporting to APHIS after a release permit has been issued?

A. If an accidental or unauthorized release of a regulated article occurs, you must orally notify the Biotechnology Permits unit immediately and send written notification within 24 hours. APHIS must be notified in writing within 5 days if the regulated article or associated host organisms are found to have characteristics substantially different from those listed in the permit application or if they have an unantici- pated effect on nontarget organisms.

41. What conditions must be met for a subsequent application to be considered a renewal?

A. (1 ) The field trial must occur in the same county as the previous trial.

(2) The field trial must be essentially the same as the one previously approved.

(3) A new APHIS Form 2000, indicating the application is for a renewal, must be submitted with a photocopy of the previous submission. Include a statement indicating what changes in the field test design, if any, are planned.

42. How can I obtain a copy of an applicant's submission?

A. A submission can be obtained by sending a written request to:

Freedom of Information Act Coordinator USDA, APHIS, LPA, PI 6505 Belcrest Road, Room 613 FB Hyattsville, MD 20782

Include the APHIS application number or the Federal Register Notice citation, the applicant's name, name of the engineered organism, and the location and date of the test.

1-5

43. How may I obtain copies of completed environmental assessments?

A. Submit a written request to the Biotechnology Permits unit. Include the APHIS application number or the Federal Register Notice citation, the applicant's name, name of the engineered organism, and the location and date of the test.

1-6

Guidelines for Completing Interstate Movement, Importation, or Courtesy Permits for Genetically Engineered Organisms and Products

This section contains sample applications for six types of movement permits:

• Transgenic plants without confidential business information (no CBI) IJ-2

• Transgenic plants, CBI copy 11-6 • Transgenic plants, CBI-deleted copy 11-10 • Genetically engineered microorganisms 11-14 • Transgenic plants engineered by a direct method 11-19 • Courtesy permit application 11-23

Interstate movement of certain genetically engineered organisms is exempt. For details, see Section IV.

Ten steps for completing your permit application:

1. Decide if the responsible person will be the shipper or the receiver of the regulated article. The responsible person must sign APHIS Form 2000 on line 14, and his/her name must be in box 1. Although either the shipper or receiver can be the responsible person, the receiver of the article is usually preferable. Because the responsible person is legally responsible for all conditions (greenhouse, growth chambers, and the disposal of the regulated article), it is preferable to have the receiver be the responsible person since he/she will be onsite during the use of the regulated article.

2. The responsible person seeking a limited permit for interstate movement or importation must complete all items on the application except ^2, and 13a, b, e, and f.

3. The donor organism (that organism from which the genetic material is obtained) should be named on line 6a. Often there are several donor organisms (e.g., cauliflower mosaic virus 35 S promoter and Bacillus thuringiensis). Generally, list only the major gene to be transferred. The recipient organism (line 6b) is the organism that receives genetic information from the donor, and the vector or vector agent (line 6c) is the organism or method used to transfer genetic material from the donor to the recipient. Describe the regulated article in layman's terms on line 6d. If the donor organism, recipient organism, or vector/vector agent is not a plant pest but some noncoding regulatory region (e.g., promoters or terminators) or if components of the vector or vector agents are derived from a plant pest (thus triggering APHIS regulations), describe them on the appropriate line (see sample on p. 11-19).

4. Information addressing the questions on line 13c, d, g, h, and i, on the reverse side of APHIS Form 2000, must be completed for movement or importation permits. Be sure to include where the experiments will be performed (laboratory, growth chambers, or greenhouse).

5. Information on movement permits can be claimed as CBI. If a permit application does nof have any CBI, the phrase "NO CBI" should appear in the upper right corner of APHIS Form 2000. Please note how CBI material is designated on pages 11-6 to 11-9. A CBI-deleted copy of the same application is shown on pages 11-10 to 11-13. If your application contains CBI, you must submit a CBI copy and a non-CBI copy concurrently. Applicants claiming information as CBI must submit a written statement showing that competitive harm would result from disclosure.

6. Genetic maps can be drawn by hand, if legible (see p. 11-5).

7. A sample movement permit application for plants genetically engineered by a direct method is shown on pages 11-19 to 11-22. These plants are regulated because the promoter and termination sequences were derived from plant pests (cauliflower mosaic virus 35 S promoter and nopaline synthase termination signal sequences from Agrobacterium).

8. For a courtesy permit, complete items 1 to 4, 6, 12, 13b, and 14 to 16 (see sample on p. 11-23 to 11-24).

9. Permit applicants are not required to secure reprint permission when using borrowed illustrations in their applications.

10. Send completed APHIS Forms 2000 to:

Deputy Director of Biotechnology Permits Biotechnology, Biologies, and Environmental Protection 6505 Belcrest Road, Room 844A Hyattsville, MD 20782.

Sample Application: Trarrsgenic Plants, No CBI

See reverse side lor FORM APPROVED additional intormatior^ 0MB NO 05790085

U.S. DEPARTMENT OF AGRICULTURE BIOTECHNOLOGY, BIOLOGICS, ANO ENVIRONMENTAL PROTECTION

APPLICATION FOR PERMIT OR COURTESY PERMIT UNDER 7 CFR 340

(Genetically Engineered Organisms or Products)

1. NAME AND ADDRESS OF APPLICANT

Dr. James Smith 100 First Street Mersey Biotechnologies/ Minot, ND 70001

4. TELEPHONE NUMBER

(666) 555- 1212

INSTRUCTIONS: Complete this form and enclose the supporting matenals listed on the reverse side. See page 3 for detailed instructions.

2. PERMIT REQUESTED rX" one)

Xyjxl Limited • Interstate Movement

I I Limited - Impjorlation

I I Release into the Environment

I I Courtesy Permit

3. THIS REQUEST IS ("X" one)

XXEH New

I I Renewal

I I Supplemental

5. MEANS OF MOVEMENT

X3<(xl Mail n Baggage or Handcarried

I I Common Carrier By whom

6. GIVE THE FOLLOWING (if applicable) (if more space is needed, attact) additional street)

Scientific Name Common Name Trade Nam<

Cucumber mosaic virus (CMV)

Lycopersicon esculentum cv. Packard Clipper

Agrobacterium tumefaciens and Ti plasmid pJLW120

Other Designation

a Donor Organism:

b Recipient Organism

c Vector or Vector Agent

d Regulated Organism or produci tomato expresslng CMV coat protein

e If product, list names of consliluenis

7. QUANTITY OF REGULATED ARTICLE TO BE INTRODUCED AND PROPOSED SCHEDULE AND NUMBER OF INTRODUCTIONS

100 seeds 9. COUNTRY OR POINT OF ORIGIN OF THE REGULATED ARTICLE

Paige-Sullivan Biotechnologies, Ltd. Hyattsville, MD 20782

•. DATE (or inclusive dates oí period) OF IMPORTATION, INTERSTATE MOVEMENT, OR RELEASE

October 199X- January 199X

10. PORT OF ARRIVAL, DESTINATION OF MOVEMENT, OR SPECIFIC LOCATION OF RELEASE

Mersey Biotechnologies, Minot, ND 11. ANY BIOLOGICAL MATERIAL (eg., culture medium, or tiost material) ACCOMPANYING THE REGULATED ARTICLE DURING MOVEMENT

12. APPLICANTS FOR A COURTESY PERMIT - STATE WHY YOU BELIEVE THE ORGANISM OR PRODUCT DOES NOT COME WITHIN THE DEFINITION OF A REGULATED ARTICLE

13. SEE REVERSE SIDE

I hereby certify that the informatioD in this appUcation and all atUchments is complete and accurate to the best of my knowledge and beUef.

False Statement: Falsification of any item on this application may result in a fine of not more than $10,000 or imprisonment for not more than 5 years or both (18 U S C 1001)

14. SIGNATURE OF RESPONSIBLE PERSON

State Notification Sent

Dale of Determination

Signature of BBEP Official

IS. PRINTED NAME AND TITLE

James Smith, Regulatory Affairs Officer

FOR APHIS USE ONLY

State Review Received

No. of Permit Labels Issued

APHIS FORM 2000 Reptaceê PPQ Form 1001 wttich may be uted. (JUL 89)

16. DATE

10/29/9X

I'l-nriil l-..MH>(l

□ YOS QNO

Supplemental Conditions Enclosed

□ Yes n No

Expiration Dale

(continued on reverse)

Sample Application: Transgenic Plants, No CBI

ENCLOSURES ENCLOSED rx")

IF PREVIOUSLY SUBMITTED. LIST DATE & PERMIT NO.

a

Names, addresses, and telephone numbers of the persons who developed and/or supplied the regulated article

b

A description of the anticipated or actual expression of the altered genetic material in the regulated article and how that expression differs from the expression in the

non-modified parental organism (e.g.. morphological or structural characteristics, physiological activities and processes, number of copies ol inserted genetic material and the physical state of this material inside the recipient organism (integrated or extrachromosomal), products and secretions, growth characteristics)

c A detailed description of the molecular biology of the system (e g , donor- recipient-vector) which IS or will be used to produce the regulated article

X

d Country and locality where the donor organism, recipient organism, and vector or

vector agent were collected, developed and produced X

e A detailed description of the purpose for the introduction of the regulated article including a detailed description of the proposed experimental and/or production design

f A detailed description of the processes, procedures, and safeguards which have been used or will be used in the country of origin and in the United States to prevent

contamination, release, and dissemination in the production of the: donor organism recipient organism; vector or vector agent; constituent of each regulated article which

is a product, and, regulated article

g A detailed description of the intended destination (including final and all intermediate

destinations), uses, and/or distribution of the regulated article (e g , greenhouses laboratory, or growth chamber location, field trial location, pilot proiect location, production, propagation, and manufacture location, proposed sale and distribution

location).

X

^ A detailed description of the proposed procedures, processes, and safeguards which will be used to prevent escape and dissemination of the regulated article at each of the

intended destinations. X

A detailed description of the proposed method of final disposition of the regulated article X

Public reporting burder) for this collection ol intormalion Is estimated to average 5 tiours per response, including the time for reviewing instructions, searching existing data sources, gathering and maintaining the data needed, and completing and reviewing the collection of information. Send comments regarding this burden estimate or any other aspect ol this collection of information, including suggestions tor reducing this burden, to Department of Agriculture. Clearance Officer, OIRM, Room 404-W, Washington. DC 20250, and to the Office of Information and Regulatory Affairs, Office of Management and Budget. Washington. DC 20503

APHIS FORM 2000 (Reverse)

11-3

Enclosure ^^ ^®^

13c. The donor organism was cucumber mosaic virus (CMV), strain D. CMV is an aphid-transmitted virus whose genome is composed of 4 RNA's. Only the largest 3 are required for infectivity. UNA'S 1 and 2 encode proteins thought to be involved in RNA S3mthesis. UNA 3 is a dicistronic RNA that encodes the viral coat protein and another protein thought to be involved in the movement of the virus through the plant. The fourth RNA is a subgenomic RNA coding for the viral coat protein. It is this foui*th RNA that was used to obtain a clone of viral coat protein gene.

The recipient organism is T.vnonersicon esculentum cultivar Packard Clipper. This cultivar is self- pollinating and grown domestically.

The vector system used to transfer the coat protein gene of CMV to tomato plants is based on the Ti plasnnd from Agi-obacterium tumefaciens. The vector system is "disarmed" or nonpathogenic because all the genes involved in phenotjrpic expression of the disease characteristics have been deleted. We have utilized a "two component" system of transferring genes into tomato leaf sections. This system was chosen for its use in cloning genes of interest into plasmids and for greater transformation frequencies with certain plant species and/or cultivars. A detailed description of this vector system has been described (Deblaere et al. 1985). The basic plasmid used was pGV831, which contains the marker antibiotic resistance gene neomycin phosphotransferase E imder control of NOS promoter. The cloned CMV coat protein gene imder control of the 35S cauliflower mosaic virus promoter (Odell et al. 1985) and the NOS polyadenylation and termination signal sequences were cloned into the imique BamHI restriction site, resulting in plasmid pJLWlSO (see map).

The T-region was transferred to tomato plants by leaf disk transformation (McCormick et al. 1986).

13d. The donor organism is cuc\miber mosaic virus, strain D, which was obtained from American Type Culture Collection, Rockville, MD. Tomato seeds were obtained from Dr. Tom Smith, Dept. of Horticulture, State University, Any State. A. tumefaciens and plasmid pGV831 were obtained from Dr. Marc van Montagau, Belgium.

13g. The transgenic plants will be manipulated in a laboratory, in growth chambers, or in a greenhouse. The plants will be tested for the production of viral coat protein and for the effect of challenge inoculation by various CMV strains. The plants will be allowed to flower in the greenhouse and the seeds wiU be collected.

13h. The tomato plants used in this study are exclusively self-pollinating, which precludes the escape of genes to other tomato plants. No other flowering tomato plants wiU be grown within 200 feet of the transgenic tomatoes. All constructs were prepared according to the NTH Guidelines for Research Involving Recombinant DNA Molecules. AU manipulations of recombinant bacteria were carried out in a laminar flow biosafety cabinet using "good microbiological practices." These experiments have been approved by our Institutional Biosafety Conmiittee (EBC).

A copy of your IBC's approval of the research protocol for which this organism is being requested should accompany this application.

13i. All plant material including soil will be steamed or autoclaved prior to final disposal. AU bacterial cultures will be disposed of by autoclaving.

References

Deblaere, R., Bytebier, B., DeGreve, H., Deboeck, F., Schell, J., Van Montagu, M., Leemans, J. 1985. Efficient octopine Ti plasmid-derived vectors for Agrobacterium tumefaciens-mediated gene transfer to plants. Nuc. Acids Res. 13:4777-4788.

McCormick, S., Niedermeyer, J., Fry, J., Barnason, A., Horsch, R., Fraley, R. 1986. Leaf disc transformation of cultivated tomato (L. ßSQUlgnitum) using AgTQbg^QtQnum tumgfftQiigng. Plant CeU Rep. 5:81-84.

Odell, J. T., Nag7, F., Chua, N-H. 1985. Identification of DNA sequences required for activity of the cauliflower mosaic virus 35S promoter. Nature 313:810-812.

Genetic Map of pJLW 120

Cla I Hind III

35 S promoter: CMV coat protein: NOS termination

"Mpa II

^Pst I

NOS promoter: NPT II: NOS termination

S - streptomycin resistance marker gene m

C - carbenicillin resistance marker gene

LB, RB - left and right border sequences

II-5

Sample Application: Transgenic Plants, CBI Copy

US. O»AIITM0fr Of AOMCULTURff


^imnmttcëÊfy Engin—nd Orgmnisma or Products}

INSTRUCTIONS: Complete this form and enclose the supporting materials listed on the

1. NAME AND ADDRESS OF APfH-ICANT

Dr. James Smith 100 First Street Mersey Biotechnologies/ Minot, ND 70001 Area Code ( )

2. PERMrr REOUESTEO ("X" on«)

XX^ Limited - interstate Movement

Q Limited - Importation

1 1 Release into the Environment

1 1 Courtesy Permit

3. THIS REQUEST IS CX' ons)

n Renewal □ Supplemental

4. TELEPHONE NUMBER

(666) 555- 1212


vvfyl Mail □ Baggage or Handcarried

1 1 Common Carrier By whom

6. GIVE THE FOLLOWING (H applicabie) (if more »pace IM needed, attach additional street)

Scient itic Name Common Nanrte

a Donor Organism Cucumber mosalc vlrus (CMV) I

b. Recipient Organism: Lycopersicon esculentum I

Agrobacterium tumefacien

tomato expressing CMV coat protein I C-'BJT

c. Vector or Vector Agent. Agrobacterlum tumefaclens and Ti plasmid pJLW120

d Regulated Organism or Product

e If product, list names of constituents:

Other Designation


100 seeds

1. DATE (or incluane dais» oí period) OF IMPORTATION. INTERSTATE MOVEMENT, OR RELEASE

October 199X-January 199X

9. COUNTRY OR POINT OF ORIGIN OF THE REGULATED ARTICLE


10. PORT OF ARRIVAL, DESTINATION OF MOVEMENT. OR SPECIFIC LOCATION OF RELEASE

Mersey Biotechnologies/ Minot, ND

11. ANY BIOLOGICAL MATERIAL (eg., cutture medium, or hott material) ACCOMPANYING THE REGULATED ARTICLE DURING MOVEMENT

12. APPLICANTS FOR A COURTESY PERMHT STATE WHY YOU BEUEVE THE ORGANISM OR PRODUCT DOES NOT COME WfTHIN THE DEFINHTION OF A REGULATED ARTICLE


I hereby certtfr that the laforaatioa fai this apptteatioa aad aU attachaeaU U complet« and accurate to the b««t of oty kaowlsdg« and baliaf.

False Statement: Falsification of any item on this application may result in a line of not more than $10.000 or imprisonnrtent for not more than 5 years or both. (18 U.S.C 1001)

14. SIGNATURE OF RESPONSIBLE PERSON.

^J~ßfmA ^S^ru>f^ Slate Nolilication Sent

Date of Determination

Signature of BBEP Official

15. PRINTED NAME AND TITLE


FOR APHIS liSE ONLY

Stale Review Received

No of Permit Labels Issued

APHIS FORM 2000 Repfacea PPQ Form 1001 which may be uaed ( JUL 89)

16. DATE

10/29/9X

I'l^iifiii iti<tiiii(i

□ Yes n No

Supplemental Conditions Enclosed

□ Yes QNO

Expiration Date

(conhnued on reverse)

11-6

Sample Application: Transgenic Plants, CBI Copy

ENCLOSURES ENCLOSED

rx') IF PREVKXJSLY SUBMITTEO,

LIST DATE & PERMIT NO.

a.


b

A description ot the anticipated or actual expression ot the altered genetic material in the regulated article and how that expression differs from the expression in the

non-modified parental organism (e.g., morphological or structural characteristics, physiological activities and processes, number of copies ot inserted genetic material and the physical state of this material inside the recipient organism (integrated or

extrachromosomal). products and secretions, growth characteristics)

c A detailed description of the molecular biology of the system (eg , donor-

recipient-vector) which is or will be used to produce the regulated article X




A detailed description of the processes, procedures, and safeguards which have been used or will be used in the country of origin and in the United States to prevent

contamination, release, and dissemination in the production of the; donor organism, recipient organism; vector or vector agent; constituent of each regulated article which

is a product, and, regulated article

g A detailed description of the intended destination (including final and all intermediate destinations), uses, and/or distribution of the regulated article (eg, greenhouses, laboratory, or growth chamber location; field trial location, pilot proiect location; production, propagation, and manufacture location, proposed sale and distribution

location)

X

^ A detailed description of the proposed procedures, processes, and safeguards which will t>e used to prevent escape and dissemination of the regulated article at each of the

intended destinations.

X


Public reporting burden lor this collection of information is estimated lo average 5 ttours per response, including the lime lor reviewing instructions, searching existing data sources, gathering and maintaining the data needed, and completing and reviewing the collection of information Send comments regarding this burden estimate or any other aspect of this collection of information, including suggestions for reducing this burden, to Department of Agriculture, Clearance Officar, OIRM, Room 404-W, Washington, DC 20250; and to the Office of information and Regulatory Affairs. Office ol Management and Budget, Washington, DC 20503


11-7

Enclosure CBI Copy

13c. The donor organism was cucumber mosaic virus (CMV), strain D. CMV is an aphid- transmitted virus whose genome is composed of 4 RNA's. Only the largest 3 are required for infectivity. RNA's 1 and 2 encode proteins thought to be involved in RNA synthesis. RNA 3 is a dicistronic RNA that encodes the viral coat protein and another protein thought to be involved in the movement of the virus through the plant. The fourth RNA is a subgenomic RNA coding for the viral coat protein. It is this fourth RNA that was used to obtain a clone of viral coat protein gene.

The recipient organism is Lvcopersicon esculentum cultivar Packard Clipper. This cultivar is self-pollinating and grown domestically. «

The vector system used to transfer thejcoat protein gene of CMV to tomatojplants is based on the Ti plasmdd from Agrobacterium tumefaciens. The vector system is "disarmed" or nonpathogenic because all the genes involved in phenotypic expression of the disease characteristics have been deleted. We have utilized a "two component" system of transferring genes intoômatqjfleaf sections. This system was chosen for its use in cloning genes of interest into plasmdds and for greater transformation frequencies with certain plant species and/or cultivars. A detailed description of this vector system has been described (Deblaere et al. 1985). The basic plasmid used was pGV831, which contains the marker antibiotic resistance gene neomycin phosphotransferase n under control of NOS promoter. The cloned^MV coatjprotein gene imder control of the 35S cauliflower mosaic virus promoter (Odell et al., 1985) and the NOS polyadenylation and termination signal sequences were cloned into the unique BamHI restriction site, resulting in plasmid pJLWlSO (see map).

<iÄz:

¿:&X

d&Z

c.e>j:

The T-region was transferred tcTtomatoJplants by leaf disk transformation^McCormick et al. ^^JT

13d. The donor organism isicucumber mosaic virus, strain D^Jwhich was obtained from Ameri- can Type Culture Collection, RockviUe, MD. {Tomato seeds)wêre obtained from Dr. Tom Smith, Dept. of Horticulture, State University, Any State. A. tumefaciens and plasmid pGV831 were obtained from Dr. Marc van Montagau, Belgium.

13g. The transgenic plants will be manipulated in a laboratory, in growth chambers, or in a greenhouse. The plants will be tested for the production ofîral coat proteinjand for the effect of challenge inoculation by Carious CMV strains H The plants will be allowed to flower in the greenhouse and the seeds wiU be collected.

13h. The(tomato]plants used in this study are exclusively self-pollinating, which precludes the escape of genes to otherltomatqlplants. No other flowering[tomato]plants will be grown within 200 feet of the transgenicjtomatoes!} AU constructs were prepared according to the NIH Guide- lines for Research Involving Recombinant DNA Molecules. All manipulations of recombinant bacteria were carried out in a laminar flow biosafety cabinet using "good microbiological practices." These experiments have been approved by our Institutional Biosafety Conmiittee (IBC).

A copy of your IBG's approval of the research protocol for which this organism is being requested should accompany this application.

13i. All plant material including soil will be steamed or autoclaved prior to final disposal. All bacterial cultures will be disposed of by autoclaving.

11-8

References

Deblaere, R., Bytebier, B., DeGreve, H., Deboeck, F., Schell, J., Van Montagu, M., Leemans, J. 1985. Efficient octopine Ti plasmid-derived vectors for Agrobacterium tumefaciens-mediated gene transfer to plants. Nuc. Acids Res. 13:4777-4788.

McCormick, S., Niedermeyer, J., Fry, J., Barnason, A., Horsch, R., Fraley, R. 1986. Leaf disc transformation of cultivated tomato (L. esculentum^ using Agrobacterium tumefaciens. Plant Cell Rep. 5:81-84.

Odell, J. T., Nagy, F., Chua, N-H. 1985. Identification of DNA sequences required for activity of the cauliflower mosaic virus 35S promoter. Nature 313:810-812.

Genetic Map of p JLW 120

Cla I Hind III

35 S promoter: ^ [CMV coat proteinrj C\^ NOS termination

"Mpa II

^Pst I


S - streptomycin resistance marker gene



11-9

Sample Application: Transgenic Plants, CBI-deleted

U.S. DEPARTMENT OF AGRICULTURE BIOTECHNOLOGY, BIOLOGICS. AND ENVIRONMENTAL PROTECTION


(Genetically Engineered Organisms or Products^

INSTRUCTIONS: Complete this forin and enclose the supporting materials listed on the reverse side. See page 3 for detailed instructions.

1. NAME AND ADDRESS OF APPLICANT

Dr. James Smith 100 First Street Mersey Biotechnologies, Minot, ND 70001 Area Code ( )

2. PERMIT REQUESTED ("X" one)

XX^ Limiled ■ InJerslale Movement

1 1 Limited - Importation


1 1 Courtesy Permit

3. THIS REQUEST IS rx" one)

X^HNew

1 1 Renewal

1 1 Supplemental

4. TELEPHONE NUMBER

(666) 555- 1212 5. MEANS OF MOVEMENT

XX^ Mail □ Baggage or Handcarned

Common Carrier By whom

6. GIVE THE FOLLOWING (if applicable) (if more space is needed, attach additional sheet)

Scientitic Name Common Name

a Donor Organism




Other Designation

_J to'AíLtcO Agroipacterium tumefaciens and Ti plasmid pJLW120

j 6Br-^dJÍLihd) e It product, list names of constituents


100 seeds

8. DATE (or inclusive dates of period) OF IMPORTATION, INTERSTATE MOVEMENT, OR RELEASE

October 199X-January 199X

9 COUNTRY OR POINT OF ORIGIN OF THE REGULATED ARTICLE



Mersey Biotechnologies, Minot, ND

11 ANY BIOLOGICAL MATERIAL (eg. culture medium, or host material) ACCOMPANYING THE REGULATED ARTICLE DURING MOVEMENT



I hereby certify that the information in thi« application and all attachments is complete and accurate to the best of my knowledge and belief.

False Statement: Falsification ot any item on this application may result in a fine of not more than $10.000 or imprisonment tor not more than 5 years or both (18 U SC. 1001)

14. SIGNATURE OF RESPONSIBLE PERSON IS. PRINTED NAME AND TITLE


16. DATE

10/29/9X

FOR APHIS USE ONLY |

State Notification Sent State Review Received ('(Miriil IUMIIKI

Dves QNO

Date of Determination Permit No. No o» Permit Labels Issued Supplemental Con dit ions Enclosed

Signature of BBEP Otticial Date Expiration Date

APHIS FORM 2000 Rep/acet PPQ Form 1001 which may be uted. (JUL 89)

(continuod on reverse)

11-10

Sample Application: Transgenic Plants, CBI-deleted

ENCLOSURES ENCLOSED IF PREVIOUSLY SUBMITTED,


a

Names, addresses, and telephone numbers of the persons who developed and/or

supplied the regulated article

b


rujn-modified parental organism (eg., morphological or structural characteristics,

physiological activities and processes, number of copies of inserted genetic material and the physical state of this material inside the recipient organism (integrated or

extrachromosomal), products and secretions, growth characteristics)

c A detailed description of the molecular biology of the system (e g , donor-





A detailed description of the processes, procedures, and safeguards which have been used or will be used in the country of origin and m the United States to prevent contamination, release, and dissemination in the production of the donor organism, recipient organism, vector or vector agent, constituent of each regulated article which

IS a product, and, regulated article


destinations), uses, and/or distribution of the regulated article (eg greenhouses laboratory, or growth chamber location, field trial location, pilot project location,

production, propagation, and manufacture location, proposed sale and distribuiiuri

location)

X

^ A detailed description of the proposed procedures, processes, and safeguards which

will be used to prevent escape and dissemination of the regulated article at each of the

intended destinations

X


Public reporting burden for this collection of information is estimated to average 5 hours per response, including the time for reviewing instructions, searching existing data sources, gathering and maintaining the data needed, and completing and reviewing the collection of information. Send comments regarding this burden estiniate or any other aspect of this collection of information, including suggestions tor reducing this burden, to Department of Agriculture. Clearance Officer. OIRM. Room 404-W. Washington. DC 20250; and to the Office of Information and Regulatory Affairs, Office of IManagement and Budget, Washington, DC 20503


11-11

Enclosure CBI-deleted

13c.

C3Z

<x

PBLerSO The vector system used to transfer theL Jplants is based on the Ti plasmid from Agt*obacterium tumefaciens. The vector system is "disarmed" or nonpathogenic because all the genes involved in phenotypic expression of the disease characteristics have been deleted. We have utilized a "two component" system of transferring genes intoC ^leaf sections. This system was chosen for its use in cloning genes of interest into plasmids and for greater transformation frequencies with certain plant species and/or cultivars. A detailed description of this vector system has been described (Deblaere et al. 1985). The basic plasmid used was pGV831, which contains the marker antibiotic resistance gene neomycin phosphotransferase n under control of NOS promoter. The cloned[^ ÜII gene under control ^^'^^ of the 35S cauliflower mosaic virus promoter (Odell et al., 1985) and the NOS polyadenylation P^^^HÊ^tP and termination signal sequences were cloned into the imique BamHI restriction site, resulting in plasmid pJLWlSG (see map).

The T-region was transferred to£^ ^P^^^^^ ^y 1®^^ ^^^^ transformation ([^ l>- 13d. The donor organism isE Culture Collection, Rockville, MD.T culture. State University, Any State. Dr. Marc van Montagau, Belgium.

J which was obtained from American Type CÄJT 3were obtained from Dr. Tom Smith, Dept. of Horti- PßLBrBC>

A. tumefaciens and plasmid pGV831 were obtained from

13g. The transgenic plants will be manipulated in a laboratory, in growth chambers, or in a greenhouse. The plants will be tested for the production of if land for the effect of C&J' challenge inoculation byf \ The plants will be allowed to flower in the greenhouse VßiBTBC^ and the seeds wiU be collected.

13h. The|f 3plants used in this study are exclusively self-pollinating, which precludes the escape of genes to other£ ^plants. No other floweringC 3Plants will be grown within 200 feet of the transgenicC 3- AH constructs were prepared according to the NIH Guidelines for Research Involving Recombinant DNA Molecules. AU manipulations of recombinant bacteria were carried out in a laminar flow biosafety cabinet using "good microbiological practices." These experiments have been approved by our Institutional Biosafety Committee (IBC).

A copy of your IBC's approval of the research protocol for which this organism is being requested should accompany this application. __^____

13i. All plant material including soil will be steamed or autoclaved prior to final disposal. All bacterial cultures will be disposed of by autoclaving.

11-12

References

Deblaere, R., Bytebier, B., DeGreve, B[., Deboeck, P., Schell, J., Van Montagu, M., Leemans, J. 1985. Efficient octopine Ti plasmid-derived vectors for Agrobacterium tumefaoiens-mediated gene transfer to plants. Nuc. Acids Res. 13:4777-4788.

C6I

Odell, J. T., Nagy, F., Chua, N-H. 1985. Identification of DNA sequences required for activity of the cauliflower mosaic virus 35S promoter. Nature 313:810-812.

Genetic Map of pJLW ISO

Cla I Hind III

35 S promoter:

NOS termination i / / .

"Mpa II

♦"Pst I


S - streptomycin resistance marker gene



11-13

Sample Application: Genetically Engineered Microorganisms

No Cßl U.S. DEPARTMENT OF AGRICULTURE

BIOTECHNOLOGY, BIOLOGICS, AND ENVIRONMENTAL PROTECTION



1 NAME AND ADDRESS OF APPLICANT

Dr. Jane Doe Paige-Sullivan Boiotechnologies, Ltd. 6505 Belcrest Raod, Hyattsville, MD 20782

Area Code ( )

4. TELEPHONE NUMBER

(301) 436 - 7612

INSTRUCTIONS: Complete this form and enclose the supporting matenals listed on the reverse side. See page 3 for detailed instructions.


^^L] Limited - Inlerslale MovemenI

I I Limited - Importation

I I Release into the Environment

I I Courtesy Permit

3. THIS REQUEST IS CX" one)

XXi^ New

I I Renewal

I I Supplemental

S MEANS OF MOVEMENT

XX0 Mail n Baggage or Handcarried

I I Common Carrier By whom

6. GIVE THE FOLLOWING (if applicable) (if more space is needed, attacti additional street)

Scientific Name Common Name Trade Name Other Designation

Erwinia chrysanthemi CUPCPB 1237 (rif^, strp^

Escherichia coli HB 101

a Donor Organism


c vecior or Vector Agent pBR322 and tcansf oriHation

d Reguiüted organ.sm or Product E. coll expresslng pectatc lyase (pCSRl)

e If product, lisl names ot constilueiils


one 2 ml culture vial 9 COUNTRY OR POINT OF ORIGIN OF THE REGULATED ARTICLE

Dr. A. Collmer, Dept. of Plant Pathology Cornell Univeristy/ Ithaca^ NY

8. DATE (or inclusive dates of period) OF IMPORTATION. INTERSTATE MOVEMENT, OR RELEASE

Jan. 199X 10. PORT OF ARRIVAL. DESTINATION OF MOVEMENT, OR SPECIFIC LOCATION OF

RELEASE

Hyattsville, MD 11 ANY BIOLOGICAL MATERIAL (e g.. culture medium, or t^ost material) ACCOMPANYING THE REGULATED ARTICLE DURING MOVEMENT

culture media


13 SEE REVERSE SIDE

I hereby certify that the information in this application and aU atUchments is complet« and accurate to the best of my knowledge and belief.

False Statement: Falsilication ol any item on this application may result in a line ot not more than $10,000 or imprisonment for not more than 5 years or both (18 U SC 1001)

14 SIGNATURE OF RESPONSIBLE PERSON 15. PRINTED NAME AND TITLE

Jane Doe, Regulatory'Affairs Officer 16. DATE

10/29/9X

FOR APHIS USE ONLY 1

State Notification Sent State Review Received

D^- D^o

Date of Determination Permit No. No ol Permit Lat)els Issued Supplemental Conditions Enclosed

Signature ot BBEP Official Date Expiration Date

APHIS FORM 2000 Reptace$ PPQ Form 1001 which may be used (JUL 89)


11-14

Sample Application: Genetically Engineered Microorganisms

ENCLOSURES ENCLOSED

rx") IF PREVIOUSLY SUBMITTED,


a


b

A description ot the anticipated or actual expression of the altered genetic material in the regulated article and how that expression differs from the expression in the

nun-modified parental organism (e.g., morphological or structural characteristics, physiological activities and processes, number of copies ot inserted genetic material and the physical state of this material inside the recipient organism (integrated or extrachromosomal), products and secretions, growth characteristics)



d. Country and locality where the donor organism, recipient organism, and vector or vector agent were collected, developed and produced X


A detailed description of the processes, procedures, and safeguards which have been used or will be used in the country of origin and in the United States to prevent contamination, release, and dissemination in the production of the; donor organism, recipient organism; vector or vector agent, constituent of each regulated article which


g A detailed description of the intended destination (including final and all intermediate destinations), uses, and/or distribution of the regulated article (e g , greenhouses, laboratory, or growth chamber location, field trial location, pilot proioct location, production, propagation, and manufacture location, proposed sale arid distribution

location). X

^ A detailed description of the proposed procedures, processes, and safeguards which will be used to prevent escape and dissemination of the regulated article at each of the

intended destinations. X


Public reporting burden for this collection of information is estimated to average 5 hours per response, including the lime for reviewing instructions, searching existing data sources, gathering and maintaining the data needed, and completing and reviewing the collection ot information. Send comments regarding this burden estimate or any other aspect of this collection ot information, including suggestions for reducing this burden, to Department ot Agriculture. Clearance Officer, OIRM, Room 404-W, Washington, DC. 20250; and to the Office of Information and Regulatory Affairs, Office of Management and Budget. Washington. DC. 20503.


11-15

Enclosure No CBI

13c. Total DNA was isolated from E. chrysanthemi. partially digested with Sa¿ü3A, sized on a sucrose gradient, and DNA fragments (4 kb) were pooled. pBR322 DNA was digested with EamHl and dephosphorylated with alkaline phosphatase. Ligation of pBR322 and Sâli3A-digested E. chrysanthemi DNA was with T4 DNA ligase. E- QQÜ HBlOl was transformed with recombinant plasmid DNA by CaC12 (Mandel and Higa 1970). Transformants were screened for their ability to sink into pectate semisolid agar. Restriction mapping of the cloned DNA was performed by standard procedures. A genetic map of pBR322 and cloned fragments from E. chrvsanthemi DNA containing pectate lyase gene is enclosed.

13d. E. chrysanthemi was obtained from the Cornell University Collection of Phytopathogenic Bacteria. E. fiûli HBlOl was obtained from ATCC and pBR322 was obtained from Bethesda Re- search Laboratories, Gaithersburg, MD.

13g. E. fiOli expressing pectate lyase will be manipulated in a laboratory setting. No experiments will be performed in growth chambers or greenhouses. The only experiments involving plant material wiU be the maceration of potato tuber discs as described in enclosed reprint (Collmer et al. 1985).

13h. AU constructs were prepared according to the NIH Guidelines for Research Involving Recombi- nant DNA Molecules. All manipulations of recombinant bacteria were carried out in a laminar flow biosafety cabinet using "good microbiological practices." These experiments have been approved by our Institutional Biosafety Committee (IBC).

A copy of your IBC's approval of the research protocol for which this organism is being requested should accompany this application.

13i. All products containing the regulated article will be autoclaved prior to final disposal.

References

Mandel, M., Higa, A. 1970. Calcium dependent bacteriophage DNA infection. J. Mol. Biol. 53:159- 162.

Collmer, A., Schoedel, C, Roeder, D. L., Reid, J. L., Rissler, J. F. 1985. Molecular cloning in EsGherichia coli of Erwinia chrsvanthemi genes encoding multiple forms of pectate lyase. J. Bacterio! 161:913-920.

11-16

iyiapofPBRS28

£coR 1 4361 1 C/a 1 23 Aat II 4286 \ , / Hind III 29

4000 ^^'" ^JV^-H-i^^ EcoRV 185 J^ Nhe 1 229 ^^^^ *

)<^ ^'^^'-^s.ßamH 1 375 * cJoi^f!^^

Seal 3846 x\ _^-- "^^ A. 5.^

X J^"^^ ^^^ ^vSp^ 1 562 Pvül 3735 / J^xmnl 3963 ^^W X

A ^Hinc II 3907 Sa" " 471 ^^ÍL N Sa/1 651

Pst\ 3609/ ^^ Ban II 485 ^ Ace 1 651 .V H/'ncll 65 v\

\

/ X ^ Ap'

-^ ^ \ Xma III 939 k -V, Nru 1 972

/ 1 1-^ — 1000

PBR322 1- - 8spM 1 1063

4363 bp \

\ \ P//M 1 1315 ] ̂ \ .--A Pf/MI 1364 T^ ̂ /

3000 -''\^ \ PpiiMI 1438 / CVL Bsm 1 1353 PpuMI 1480^ > Sfy 11369

>>- Ava 1 1425 >A/wNI 2886^ \ / /^ 8a/1 1444

\ X /°' \ N^/ ^cc 1 2246 y / /

\ ^^^ / Xmn 1 2031 ^^ X

\^, ^--^ ^ y BspM II 1664

>^// III 2475^^'"^^'^-...^ 1 L^ \ /Vdel 2297 / ^

rmmi 2219 p,^„2066 2000

11-17

Restriction Map of Cloned Region of Erwinia DNA Containing Pectate I^rase Genes

EV EV P

B El

Cloned region-

EV EV L 1

EV SN S I 1 1

Sg HEV q HEV Sa

El El P B

-^ Plasnnid

pCSR I

pCSR2

pCSR3

Ffectdytic phenofype

pCSRSO -f pCSR5l +

pCSR52

pCSR6

pCSR7

pCSRIO

pCSRII

pCSRl2

0 L-

6 9 1

10

Kb

FIG. 2. Restriction map of the cloned region of E. chrysanthemi DNA containing PL genes. The orientation of the insert DNA in the primary clones (pCSRl, pCSRSO, and pCSR6) and their deletion derivatives is indicated by an arrow showing the direction of transcription from the vector Tet promoter. The deletion derivatives are displayed below their respective primary plasmids. Plasmids pCSRlO, pCSRll, and pCSR12 were constructed by subcloning

isolated restriction fragments into pBR322. The pectolytic phenotype was determined by pectate semisolid agar (28) and absorbance at 230 nm (38) assays. The dotted lines define the region encoding the pectolytic phenotype. Restriction enzymes: EV, £coRV; B, Bamm\ El, £CöRI; P, PVWII; A, Aval; S, Sphl\ N, Nru\\ H, Hinál\l\ Sa, Sail.

[Figure 2 is reproduced from its original source, Collmer et aL, J. Bacteriol. (1985) 161: 913-920, by permission of ASM Press.]

Permit applicants are not required to secure reprint permissions when using borrowed illustrations in the applications.

11-18

Sample Application: Transgenic Plants Engineered by a Direct Method, No CBI

No CBI U.S. DEPARTMENT OF AGRICULTURE

BIOTECHNOLCX3Y, BIOLOGICS, AND ENVIRONMENTAL PROTECTION



INSTRUCTIONS: Complete this forin and enclose the supporting materials listed on the reverse side. See page 3 for detailed instructions.


Dr. James Doe Motown Biotechnologies Detroit, MI 48553 Area Code ( )

2. PERMIT REQUESTED CX" one)

1 1 Limited Interstate Movement

XXJXJ Limited - Importation


1 1 Courtesy Permit

3. THIS REQUEST IS CX" one)

XÇ^New

1 1 Renewal

1 1 Supplemental

4. TELEPHONE NUMBER

(666) 555- 1212


1 1 Mail XJS Baggage or Handcarried

n Common Carrier By whom COITipany eiRployeeS

6. GIVE THE FOLLOWING (if applicable) (it more space is needed, attact) additional st)eet)

Scientilic Name Common Name Other Designation

a Donor Organism caullflower iTiosaic vlrus ( CaMV ) 35S promoter, nopaline synthase 3' termination signal sequences, neomycin phosphotransferase

b Recipient Organism 2ea mayS (cOm)

c Vector or Vector Agent eleCtrOpOratlon

d Regulated Organism or Product com expresslng kanamycln resistance marker

e If product, list names ot constituents.


100 plants


Ma/ 199X


Dr. B. Mersey, National Res. Council Canada Plant ßiol. Lab., Windsor, Ontario, Canada


Motown Biotechnologies, Ltd.

11 ANY BIOLOGICAL MATERIAL (e g. culture medium, or /losi material) ACCOMPANYING THE REGULATED ARTICLE DURING MOVEMENT

culture medium and containers



I hereby certify that the information in tlüj application and all attachments Ls complete and accurate to the best of my knowledge and belief.

False Statement: Falsification of any item on this application may result in a fine of not more than $10,000 or imprisonment for not more than 5 years or both (18 Ü SO. 1001)

14. SIGNATURE OF RESPONSIBLE PER SON 15. PRINTED NAME AND TfTLE 16. DATE

KJßniiU) 1M^ James Doe, Regulatory Affairs Officer 10/29/9X

\ FOR APHIS USE ONLY |

State Notification Sent Stale Review Received

Dves DNO

Date of Determination Permit No. No. of Permit Labels Issued Supplemental Conditions Enclosed

Signature of BBEP Official Dale Expiration Date

APHIS FORM 2000 Reptace» PPQ Form 1001 which may be u$ed. (JUL 89)


11-19

Sample Application: Transgenic Plants Engineered by a Direct Method, No CBI

ENCLOSURES ENCLOSED IF PREVIOUSLY SUBMITTED,


a


b


nun-modified parental organism (e.g., morphological or structural characteristics,

physiological activities and processes, number of copies of inserted genetic material and the physical stale of this material inside the recipient organism (integrated or


A detailed description of the molecular biology of the system (e g , donor-





t A detailed description of the processes, procedures, and safeguards which have been used or will be used in the country of origin and in the United States to prevent

contamination, release, and dissemination in the production of the: donor organism; recipient organism, vector or vector agent, constituent of each regulated article which



destinations), uses, and/or distribution of the regulated article (e g , greenhouses lat)üratory, or growth chamber location, field trial location, pilot project location,

production, propagation, and manufacture location, proposed sale and distribution location).

X

A detailed description of the proposed procedures, processes, and safeguards which

will be used to prevent escape and dissemination of the regulated article at each of the intended destinations X


Public reporting burden for this collection of information is estimated to average 5 hours per response, including the time for reviewing instructions, searching existing data sources, gathering and maintaining the data needed, and completing and reviewing the collection of information. Send comments regarding this burden estimate or any other aspect of this collection of information, including suggestions for reducing this burden, to Department of Agriculture. Clearance Officer, OIRM, Room 404-W, Washington, DC 20250: and to the Office of Information and Regulatory Affairs, Office of Management and Budget, Washington, DC 20503


11-20

Enclosure "^^°^^

13c. The neomycin phosphotransferase (NPT H) gene was from transposon In5. Expression of this marker under appropriate regulatory control sequences confers resistance to the antibiotic kanamycin. A plasmid (pCaMVNEO) (Fronrni et al. 1986) was constructed with 35S cauliflower mosaic virus promoter (nucleotides 7,017-7,437)(Bevan et al. 1983), NPT n, and nopaline synthase 3' region (Hohn et al. 1982) derived from a nopaline-type plasmid from A. famgfaQiens (see enclosed maps). The plasmdd was introduced into maize protoplasts (ZSâ mftys cultivar Golden Ban- tam) derived from embryonic tissues by electroporation as described (Pronmi et al. 1986). Kana- mycin-resistant calli were selected and whole plants regenerated. Integration of chimeric gene into chromosomal DNA was confirmed by Southern hybridization.

13d. The donor organisms are cauliflower mosaic virus 35 S promoter and nopaline synthase polyadenylation and termination signal sequences. Corn seeds were obtained from Dr. Tom Smith, Dept. of Horticulture, State University, Any State. Plasnüd pCaMVNEO was obtained from Dr. L. Jackson, Dept. of Horticulture, State University, Any State.

13g. The transgenic plants wiU be manipulated in a laboratory, in growth chambers, or in a greenhouse. The plants wiU be tested for the production of NPT E and other biochemical manipulations. The plants will be destroyed prior to flowering.

13h. The corn plants used in this study will not be aUowed to flower. AU constructs were prepared according the to NIH Guidelines for Research Involving Recombinant DNA Molecules. AU mampula- tions of recombinant bacteria were carried out in a laminar flow biosafety cabinet using "good microbiological practices." These experiments have been approved by our Institutional Biosafety Committee (IBC).

A copy of your IBC^s approval of the research protocol for which this organism is being requested should accompany this application.

13i. All plant material including sou will be steamed or autoclaved prior to final disposal.

References

Bevan, M., Barnes, W. M., Chüton, M.-D. 1983. Structure and transcription of nopaline synthase gene region of T-DNA. Nuc. Acids Res. 11:369-385.

Fronmi, M. E., Taylor, L. P., Walbot, V. 1986. Stable transformation of maize after gene transfer by electroporation. Nature 319:791 -793.

Hohn, T., Richards, K., Lebeurier, G. 198S. Cauliflower mosaic virus on its way to becoming a useful plant vector. Curr. Topics. Microbiol. Immun. 96:193-236.

11-21

Genetic Map of pCaMVNEO

CaMV 35S promoter 0.43 kb

Amp^

pUCPiANT 2.7 kb

pBR322 0.029 kb

nos Poly (A) 0.25 kb

Fig. 1 Diagram of pCaMVNEO. The size in kilobase pairs is shown next to each DNA fragment composing pCaMVNEO, with the source of each fragment represented by a different background: stippled, cauliflower mosaic virus 35S promoter; hatched, neomycin phosphotransferase II; cross-hatched, nopaline synthase {nos) polyadenylation region; black, pBR322; white, pUCPiAN?. Rel- evant restriction sites are also indicated: B, Bam\\\\ Bg, ßg/II; E, £coRI; H, Hindlll; X, Xbah Methods. pUCPiAN? contains the polylinker of PÍAN7 (Biolabs) inserted into PUCS^V A 777-base pair (bp) HincW fragment (nucleotides 7,017-7,794, see ref. 18 for nucleotide numbers) from CaMV strain 1841 (ref. 32) containing the CaMV 35S promoter was inserted into the Hindi site of pUC8. A 430-bp fragment (nucleotides 7,017-7,437) was excised with EcoRI and Hphl, then inserted into EcoRI, Hindi cleaved pUC8. This 430-bp CaMV

35S promoter fragment was then excised as a BamHl-Pstl fragment and inserted into pUCPiAN7, which had been double digested with Bglll and Pstl to yield pCaMV. The 250-bp 5au3A fragment spanning the polyadenylation region of the nos gene^ was sequentially inserted into the Bglll site of pUCWAN7, then into pBR322 as a Hind 111-ßam HI fragment, followed by insertion into pCaMV as an EcoRI-ßamHI fragment. This yields the plasmid pCaMVNOS that contains a unique BamHl site between the CaMV 35S promoter and the nos polyadenylation region. A 1.0-kb Bam HI fragment of the npt-U gene spanning nucleotides 1,540- 2,518** was inserted into the J5amHI site of pCaMVNOS to yield pCaMVNEO. The nucleotide position corresponding to the 5' end of the CaMV 35S RNA'* (nucleotide 7,435) is located 17 bp upstream of the Bam HI site at the 5' end of the npt-U gene

fragment shown.

[Figure 1 is reproduced from its original source, Fromm et al., Nature (1986) 319: 791-793, by permission of Nature.]

Permit applicants are not required to secure reprint permissions when using borrowed Illustrations ¡n the applications.

-22

Sample Application: Courtesy Permit, No CBI

No cer U.S. DEPARTMENT OF AGRICULTURE

BIOTECHNOLOGY. BIOLOGICS, AND ENVIRONMENTAL PROTECTION



INSTRUCTIONS: Complete this form and ef)close the supporting materials listed on the reverse side. See page 3 for detailed instructions.

1 NAME AND ADDRESS OF APPLICANT Dr. Jane Doe Paige-Sullivan Biotechnologies, Ltd. 6505 Belcrest Road, Hyattsville, MD 20782 Area Code( )

2. PERMIT REQUESTED CX' one)

1 1 Liiniled Inlerslale Movemenl

1 1 Limited - Imporlalion

1 1 Release into Ihe Environment

XXKJ Courtesy Permit

3. THIS REQUEST IS TX one)

XQNBW

1 1 Renewal

1 1 Supplemental

4. TELEPHONE NUMBER

(301) 436- 7612 5. MEANS OF MOVEMENT

1 1 Mail 1 1 Baggage or Handcarried

1 1 Common Carrier By whom

6. GIVE THE FOLLOWING (it applicable) (it more space is needed, attacti additional st\eet)

tj Donor Organism



Scientitic Name

cucumber mosaic virus

Escherichia coli HE 101

pBR322 and transformation

Common Name Other Designation


e II product, list names ol constituents.



9. COUNTRY OR POINT OF ORIGIN OF THE REGULATED ARTICLE 10. PORT OF ARRIVAL, DESTINATION OF MOVEMENT, OR SPECIFIC LOCATION OF RELEASE

11. ANY BIOLOGICAL MATERIAL (eg., culture medium, or t)ost material) ACCOMPANYING THE REGULATED ARTICLE DURING MOVEMENT


This organism is not a regulated article since it does not contain the complete infectious genome of this virus and it is not in a desginated expression vector. It is a partial clone and will be used solely for detection techniques.

13 SEE REVERSE SIDE

False Statement: Falsilication ol any item on this application may result in a line ol not more than $10,000 or imprisonment for not more than 5 years or both (18 Ü SC 1001)

14. SIGNATURE OF RESPONSIBLE PERSON 15. PRINTED NAME AND TITLE

Jane Doe, Regulatory Affairs Officer 16. DATE

10/29/9X

1 (/ FOR APHIS USE ONLY

State Nolification Sent Stale Review Received I'l-nrnt ISMKHI

□ Yes □ No

Permit No. No ol Permit Labels Issued Supplemental Conditions Enclosed

Signature ot BBEP Ofticial Dale Expiration Dale

APHIS FORM 2000 Replace» PPQ Form 1001 wt)ich mty b» used. (JUL 89)


11-23

Sample Application: Courtesy Permit, No CBI

ENCLOSURES ENCLOSED ("Xl

IF PREVIOUSLY SUBMITTEü. LIST DATE â Pe«MIT NO.

a

Names, addresses, and telephone numbers ot the persons who developed and/or supplied the regulated article

b


non-modified parental organism (eg., morphological or structural characteristics, physiological activities and processes, number of copies ot inserted genetic material and the physical state of this material inside the recipient organism (integrated or


see below


recipient-vector) which is or will be used to produce the regulated article


vector agent were collected, developed and produced

e A detailed description of the purpose for the introduction ot the regulated article including a detailed description of the proposed experimental and/or production design.

f A detailed description of the processes, procedures, and safeguards which have been used or will be used in the country of origin and in the United States to prevent

contamination, release, and dissemination in the production ot the donor organism, recipient organism, vector or vector agent, constituent of each regulated article which



destinations), uses, and/or distribution of the regulated article (e g , greenhouses, lat)oratory, or growth chamber location, field trial location, pilot projucl location,

production, propagation, and manufacture location, proposed sale and distribution location)


will be used to prevent escape and dissemination of the regulated article at each of the intended destinations.

A detailed description of the proposed method of final disposition of the regulated article

13b. The cDNA of the CMY coat protein gene (nucleotide numbers 25 to 1948) was inserted into the BamHl cloning site of pBR322. There are multiple copies of the extrachromosomal plasmid in the recipient cell. Further details on this construct are described in a publication (J. Virol. 00:14-18 199X).

Public reporting burden for this collection ol information is eslinnated to average 5 hours per response, including the lime tor reviewing instructions, searching existing data sources, gathering and maintaining the data needed, and completing and reviewing the collection of information. Send comments regarding this burden estimate or any other aspect of this collection of information, including suggestions for reducing this burden, to Department ol Agriculture, Clearance Officer, OIRM, Room 404-W, Washington, DC 20250, and to the Office of Information and Regulatory Affairs, Office ot Itonagement and Budget, Washington, DC 20503.


11-24

Questions on the Preparation of Application for a Field Test

Comments about issues that may need to be addressed in this section are enclosed in boxes.

1. Is the person whose name appears in box 1 on APHIS Form 2000 (see form on the next page) also the person who has signed on line 14?

2. Is the application typed on 8 1/2" by 11 " paper?

To save space in this guide, copy is printed on both sides of the paper. However, permit applications must be typed on only one side.

3. Is the text of the application organized into nine sections according to points stated on line 13 a-i on APHIS Form 2000?

4. Are the pages numbered by their section number followed by their page number (e.g., 13c-12)?

5. Does the application contain any confidential business information (CBI)?

If the answer is no, do the first pages of both copies have the phrase "NO CBI"?

If the answer is yes, is there a CBI copy and a CBI-deleted copy?

6. Are the CBI and CBI-deleted copies prepared according to the following points?

a. On each page containing CBI material, is the CBI material designated by a bracket and the term "CBI" in right margins next to where the material is located, and is the phrase "CBI COPY" located on the upper right corner of the page?

b. Each page with CBI-deletions should be marked "CBI- deleted" in the upper right corner of the page. In the right margin, mark the place where the CBI matehal has been deleted with a bracket and "CBI-deleted."

d. Published literature usually cannot be claimed confidential and thus must appear in both copies.

e. If any information in an application is claimed as CBI, the applicant must support each of these claims by including a written justification.

Examples of pages containing CBI and CBI-deleted material are shown on pp. 111-28 and 111-29.

7. Reprints should not be submitted with the application unless they provide specific information required for addressing statements 13 a-i (e.g., a sequence of a gene or genetic map). Any preprints of publications cited in the permit application should be included if possible and may be claimed CBI.

c. The CBI-deleted copy should be a facsimile of the CBI copy, except for spaces occurring in the text where CBI has been deleted. Additional material (transitions, paraphrasing, generic substitutions, etc.) should not be included in the CBI- deleted copy. If several pages are CBI-deleted, a single page stating each deleted page may be substituted for several blank pages.

Sample Application: Release Into the Environment

U.S. DEPARTMENT OF AGRICULTURE BIOTECHNOLOGY, BIOLOGICS, AND ENVIRONMENTAL PROTECTION



INSTRUCTIONS: Complete this form and enclose the supporting materials listed on the reverse side. See page 3 for detailed instructions.


Dr. Edward Johnson Paige-Sullivan Biotechnologies/ Ltd. 6505 Belcrest Road, Hyattsville, MD 20782

Area Code ( )


1 1 Limued - Interstate Movement

1 1 Limited - Importation

Y Jyl Release into the Environment

1 1 Courtesy Permit

3. THIS REQUEST IS ('X" one)

XXg New

I 1 Renewal

1 1 Supplemental

4. TELEPHONE NUMBER

(301) 436- 7612


1 1 Mail x£3 Baggage or Handcarried

1 1 Common Carrier By whom COItipanV eiTip] Oye<^P 6. GIVE THE FOLLOWING (if applicable) (if more space is needed, attach additional stieet)

Scientilic Name Common Name Other Designation

a Donor Organism



Cucumber mosaic virus (CMV)

Lycopersicon esulentum cv. Packard Clipper (tomato)

Agrobaccerium tumefaciens and Ti plasmid

d Regulated Organism or Product tomato expresslng CMV coat proteln

e It product, list names ot constiluenis


enclosed


May 15, 199X


USA

10 PORT OF ARRIVAL. DESTINATION OF MOVEMENT, OR SPECIFIC LOCATION OF RELEASE

Hyattsville, FD

11. ANY BIOLOGICAL MATERIAL (e g. culture medium, or hos/ material) ACCOMPANYING THE REGULATED ARTICLE DURING MOVEMENT

soil

12. APPLICANTS FOR A COURTESY PERMIT STATE WHY YOU BELIEVE THE ORGANISM OR PRODUCT DOES NOT COME WITHIN THE DEFINITION OF A REGULATED ARTICLE


I hereby certify that the information in this application and all attachments is complete and accurate to the best of my knowledge and belief.

False Statement: Falsification ol any item on this application may result in a tine of not more than $10,000 or imprisonment lor not more than 5 years or both (18 U SC. 1001)

14. SIGNATURE OF RESPONSIBLE PER SON 15. PRINTED NAME AND TITLE 16. DATE

¿c/ UokïVS Ed Johnson, Regulatory Affairs Officer 10/29/9X

1 FOR APHIS USE ONLY | State Notification Sent Stale Review Received I'fiinil I'oMMid

Date of Determination Permit No. No. ol Permit Lat>els Issued Supplemental Conditions Enclosed

Signature of BBEP Official Dale Expiration Date

APHIS FORM 2000 Rep/ac»« PPQ Fofm lOOl which may be u»ed. (JUL 89)


1-2

Sample Application: Release Into the Environment

ENCLOSURES ENCLOSED IF PREVIOUSLY SUBMITTED.


a

Names, addresses, and telephone numbers of the persons who developed and/or

supplied the regulated article X

b


rton-modified parental organism (eg., morphological or structural characteristics, physiological activities and processes, number of copies ot inserted genetic material and the physical state of this material inside the recipient organism (integrated or


X





e A detailed description of the purpose for the introduction of the regulated article including a detailed description ot the proposed experimental and/or production design

X

A detailed description of the processes, procedures, and safeguards which have been used or will be used in the country of origin and in the United States to prevent

contamination, release, and dissemination in the production ot the: donor organism, recipient organism; vector or vector agent, constituent of each regulated article which


X

9 A detailed description of the intended destination (including final artd all intermediate

destinations), uses, and/or distribution of the regulated article (e g greenhouses, latjoratory, or growth chamt>er location, field trial location, pilot project location,

production, propagation, and manufacture location, proposed sale arid distribution

location).

X


will be used to prevent escape and dissemination of the regulated article at each of the

intended destinations X


Public reporting burder) for this collection ot information is estimated to average 5 hours per response, including the time for reviewing instructions, searching existing data sources, gathering and maintaining the data needed, and completing and reviewing the collection ot information. Send comments regarding this burden estimate or any other aspect of this collection of information, including suggestions for reducing this burden, to Department of Agriculture. Clearance Officer, OIRM. Room 404-W. Washington. DC. 20250; and to the Office of Information and Regulatory Affairs, Offica of Management and Budget. Washington, DC 20503.


Enclosure 13a-1

13a. Names of persons who developed genetically engineered organism

The transformed plants were developed by:

Dr. Ed Johnson Paige-Sullivan Biotechnologies, Ltd. 6505 Belcrest Road HyattsviUe, MD 27082 (301) 436-7612

Dr. M. C. Halasa Halasa Plant Products P.O. Box 1948 Minot, North Dakota (401) 436-7777

If resumes, curriculum vitae, or other personal information are submitted in the application, they may be claimed as confidential business information (CBI).

13b-l

13b. Description of Regulated Article

The genetically engineered tomato plants have been developed to express the coat protein gene of cucumber mosaic virus (CMV). The rationale for this experiment is given below.

PROBLEM: Viral diseases are one of the major limiting factors in tomato production. CMV is one of the most frequently detected viruses in tomatoes (Benner et al. 1985) and is an especially severe problem in China, Indonesia, and Japan. To date, no effective gene for resistance has been identified in Lycopersicon spp. by plant breeders. Therefore, we have attempted a novel approach to obtain a form of viral resistance by incorporating a gene into the tomato genome that will synthesize the CMV coat protein.

BACKGROUND: Cross protection is the mechanism whereby infection of a plant by one strain of a virus protects the plant from the effects of subsequent inoculation with another strain of the same virus (Fulton 1982). Cross protection was first demonstrated by McKiimey (1929). Tobacco plants infected with a green mosaic virus (a TMV strain) did not develop further symptoms when inoculated with a yellow mosaic virus strain. Thung (1931) confirmed these experiments and was unable to isolate the second virus from doubly inoculated plants. This suggested that the second virus had not multiplied. Salaman (1933) foimd that tobacco plants inoculated with a mild strain of potato virus X were "immimie" from subsequent inoculation with severe strains of the virus, even if the challenge was performed 5 days after inoculation. The infected plants were not inmiune to infection with unrelated viruses: tobacco mosaic virus or potato virus Y. Some viruses do not appear to induce cross protection at all (e.g., curly top virus of sugar beets). Most experiments on cross protection have been carried out using mechanical transmission, but cross protection has also been demonstrated with viruses that are transmitted in a persistent manner by insect vectors (Harrison 1958). Several theories have been put forth that explain the cross protection phenomenon. (1) The first strain uses some essential metabolite required by the second strain. (2) The virus-infected plants produce "protective substances" that inhibit replication of the challenge virus. Although inhibitory substances are detected in virus-infected plant extracts, the evidence does not support that these compounds are involved in the cross protection phenomenon. (3) Kavanau (1949) suggested that "aggregates" of virus in cells previously infected with a virus have some specific "adsorptive properties." Others (De Zoeton and Fulton 1975, Sherwood and Fulton 1982, Matthews 1982) have suggested that the viral coat protein could be the "adsorptive" molecule. They have proposed that when the challenge virus is uncoated, it is rapidly reencapsidated by viral coat protein synthesized by mild strain. This theory is consistent with observation that only closely related viruses show cross protection phenomenon. The first definitive evidence for a putative role of viral coat protein in the cross protection-like phenomenon was use of the transgenic plants expressing viral coat proteins.

GENETICALLY ENGINEERED CROSS PROTECTION: In 1986, Abel et al. produced a transgenic tobacco plant which expressed the TMV coat protein gene. Upon challenge with whole virus, plants expressing the gene showed a delay in symptom development and, in some cases, plants failed to develop symptoms for the duration of the experiment. The authors caUed this phenomenon "genetically engineered cross protection." Challenge inoculation with viral UNA rather than virus largely overcame the protective effect. This leads to the conclusion that the presence of the coat protein on the virus particle in challenge inoculum was necessary for maximum protection and that encapsidation of naked challenge UNA by coat protein was not involved in the protection phenomenon. Recently, Nelson et al. (1988) reported that if partially stripped TMV virions were used as the challenge inoculum, the protective effect was largely overcome. (Partially stripped virions have coat protein subimits removed, exposing approximately 150 nucleotides of the 5' end of viral UNA.)

13b-8

This suggests that coat protein interferes with disassembly of the incoming challenge virions. It is not known if classical and genetically engineered cross protection are based on similar mechanisms.

Other workers have engineered plants that synthesize the coat protein of alfalfa mosaic virus (Loesch-Fries et al. 1987, Tumer et al. 1987, Van Dim et al. 1987), and tobacco rattle virus (Van Dim et al. 1987). The results from these experiments further confirmed that plants expressing coat protein genes interfere with viral multiplication.

Recently, the effectiveness of "genetically engineered cross protection" in a field situation was demonstrated (Nelson et al. 1988). In field-grown plants, less than 5 percent of the coat protein expressing plants inoculated with TMV exhibited visual symptoms by fruit harvest as compared to 99 percent of the challenged control plants. Fruit yield reduction was approximately 30 percent due to virus infection in the nonengineered controls as compared to coat protein expressing plants.

Traditional cross protection phenomenon between CMV strains has been demonstrated by Dodds and coworkers (1982, 1985). Cuozzo et al. (1988) have produced transgenic tobacco plants expressing CMV coat protein that are protected from challenge inoculation by a severe strain. Paige- Sullivan Biotechnologies has cloned and sequenced CMV coat protein and produced transgenic plants that produce high levels of CMV capsid protein (White 1988). The viral coat protein has been introduced into tomato by the leaf disc transformation technique (McCormick et al. 1986).

13c-1

13c. Description of Donor, Recipient, and Vector

Two approaches were utilized to transform plants: A. tumefaciens and the Ti plasmid or electroporation.

DESCRIPTION OP THE VECTOR SYSTEM: The vector system used to transfer the coat protein gene of cucimiber mosaic virus (CMV) to tomato plants is based on the Ti plasmid from Agrobacterium tumefaciens. The vector system is "disarmed" or nonpathogenic because all the genes involved in phenotypic expression of the disease characteristics have been deleted. We have utilized a "two- component" system of transferring genes into tomato leaf sections. This system was chosen for its use in cloning genes of interest into plasmids and for greater transformation frequencies with certain plant species and/or cultivars and was developed by Deblaere et al. (1985).

In using this system, the scientific literature supports the view that only the T-region is transferred and integrated into the plant genome (Praley et al. 1986, Cooper and Meredith 1989). The sequence that is integrated includes the genes contained between certain short, well-characterized segments of the Ti plasmid that are essential for incorporation into the plant genome. Also, border sequences (25 base pairs required for transfer) are lost during the process of insertion of T-DNA into plant genome. This means that the inserted DNA is no longer a functional T-DNA capable of being transferred by the same mechanism that originally inserted the T-DNA into plant genome (Zambryski et al. 1982). Thus, all evidence available since the delineation of T-DNA in 1978, plus the accumulated information concerning the epidemiology of crown gall disease, indicates that T-DNA transfer into plant cells by Agrobacterium is irreversible.

CONSTRUCTION OP ACCEPTOR PLASMID: The purpose of this procedure is to obtain a Ti plasmid lacking all of the T-region DNA but retaining the YíE region. An octopine plasmid pTiB6S3 containing two adjacent T-regions (T^ and T^^) was modified to contain a kanamycin resistance marker. The intermediate vector pGV746, a pBR322 derivative, contains two Ti plasmid sequences that are located respectively to the left and outside the T^ DNA segments and to the right and outside of the Tj^ DNA sequences. A double recombination between pGV746 and pGV2217 results in pGV2260 (fig. 1). In pGV2260 the entire T^ and T^^ regions are deleted and substituted by sequences derived from PBR322.

CONSTRUCTION OP THE VECTOR PLASMID: The initial plasmid pGV700 is a pBR322 derivative containing 1 kb HindlH/BgUI part of Hindin-18 fragment of pTiAch 5 and the 6.5 Kb Egin/mndin part of Hinrinr fragment of pTiACh5 (see fig. 2). This plasmid contains all T-region sequences except genes 5,7,2, and 1. A 7.5 kb Hindm fragment from pGV700 was recloned into pGV600, giving rise to pGV742. pGV600 is a pBR322 derivative lacking any EâmHI sites. The remaining T^^ DNA sequences (but not T^ 25 bp border sequences) in pGV742 were removed by deleting internal BamHI fragment giving rise to pGV744. The T^^ DNA sequences (but not the RB 25 bp sequences) were removed by deleting the internal EcoRI fragment, giving rise to pGV749. To obtain a plasmid containing only border sequences, the 1.87 kb Hindll/Nrul fragment from pGV749 was cloned into pGV710, previously digested with EcoRI/Hindin. pGV710 is a pBR322 derivative containing Sm^, Su^, Cm^, and Tc^ markers. The sticky ends obtained after digestion were flush-ended by treatment with Klenow DNA polymerase prior to Hindin treatment. The resulting plasmid pGV815 was isolated as a Sm^, Cb^, Cm^, Tc^ clone. The EQûRI and HindlII sites of this plasmid were eliminated by filling in the sticky ends and self ligation of the vector. The chimeric kanamycin was produced by inserting a 298 bp BcU/BamHI fragment from pGV230 (which contains a NOS promoter) into BfilE site of pKC7 to produce pKC7::N0S. Plasmid pCK7 is a pBR322 derivative containing 1.8 kb

13C-2

Hindn/BamHI fragment of Tn5 which contains NPT n (Rao and Rogers 1979). The NOS 3' polyadenylation and termination signal sequences were isolated and fused to NPT n (NEO) gene as previously described (White 1988). The chimeric NPT n gene was isolated as a Eûin/EâmHI fragment and cloned into BglU site of pGV825 to produce pGC831.

DESCRIPTION OP RECIPIENT: The recipient organism, L. esculentum cv. Packard Clipper, is a conmion conmiercial cultivar and is a fresh market tomato. It is not widely grown in Maryland because of its susceptibility to CMV. Additional information on the biology of this tomato can be foimd in section 13h.

DESCRIPTION OP DONOR: CMV has been linked to plant disease in all temperate regions of the world. The virus has an extremely wide host range that includes cereals, forages, woody and herbaceous ornamentals, vegetables, and fruit crops. The RNA of CMV consists of four components of different size (approximate M^: 1.01, 0.89, 0.68, and 0.33 x 10^). The three largest RNA's, which are distributed among three separate virion particles, carry aU the information needed for successful infection. The genetic information for viral coat protein is carried on RNA 3 and on a coencapsidated subgenomdc messenger RNA 4 (Kaper 1984). The viral coat protein assembles aroimd the viral RNA to form the stable virion, which protects the nucleic acid from physical, chemdcal, or biochemical degradation. The coat proteins from different CMV strains usually have different amino acid sequences that reflect differences in nucleotide sequences of RNA 4 (Kaper 1984).

CMV strain PV 29 (also called strain 1) (American Type Culture CoUection Catalogue, 14th Ed., 1988) was propagated as previously described (Lot et al. 1972) and dsRNA isolated as previously described (Diaz-Ruiz and Kaper 1978). Full-length ds cDNA copies of PV 29 CMV RNA 4 were prepared using synthetic oligonucleotide to prime RNA synthesis simultaneously from the 3' ends of both plus and minus strands of denatured ds RNA 4. The primer hybridizing to the plus strand contained an added Clal recognition site at its 5' end to facilitate forced cloning into the plasmid vector. Additional details of this construct have been published (White 1988), and a reprint appears in appendix 1.

The promoter used in this study was derived from strawberry vein banding virus (SVBV), a caulimovirus (Shepherd 1979). The replication strategy of SVBV is thought to be analogous to the weU-characterized cauliflower mosaic virus (CaMV). Upon infection of a cell with SVBV, two major RNA transcripts, designated 40S and S2S (based on their sedimentation coefficients), are produced during the replication of SVBV. These transcripts are analogous to the 35S and 19S CaMV RNAs (Hull and Covey 1983). The 40S SVBV promoter sequences have been characterized (further details in appendix 1). The 40S promoter was isolated as the Hiudni/ClaJ fragment extending from +9 to -343 with respect to the transcription start site mapped for the 40S RNA. The transcription termination and polyadenylation signal sequences were derived from the nopaline synthase (NOS) gene (Barker et al. 1983).

The 40S promoter and NOS termination signal sequences were blunt-end ligated to the cloned CMV coat protein gene and inserted into pGV831 at the unique EamHl site. A map of the resulting plasmid pJLWlSO is shown in figure 3. pJLWlSO was introduced into the acceptor Ti plasmid pGV2260 by a single homologous recombination, using Sm^ gene of pJLW180 as a selectable marker for cointegration (see fig. 4 for map of cointegrate). The mobilization of pJLWlSO from E. coll to Afpnha.ntftriinn C58ClRif^ (pGVSS60) was performed according to Van Haute et al. (1983). The structure of the T-region was confirmed by Southern blot hybridization.

ill>8

13C-3

AGRQBACTERIUM-MEDIATED TRANSFORMATION: Subterminal leaflets from 6-week-old, greenhouse-grown plants were used for transformation as described by McCormick et al. (1986). Regen- erated plantlets (R^) were challenge inoculated with CMV-CQ (10 |ag/ml) and placed in a greenhouse. Symptom development was monitored until fruit were harvested (plants were allowed to self-pollinate). A small portion of the seeds, collected from the fruits of three plants showing the mtildest viral symptoms, were germtinated on kanamycin-selective media. Results of segregation of antibiotic sensitivity suggest that one line pJLW180-l 10 (R^) contains a single CMV coat protein loci (table 1). Seedlings from seed lot pJLW180-120 will be used for the field test.

DIRECT TRANSFORMATION (ELECTROPORATION): Tomato protoplasts were isolated from fully expanded mature leaves as described by O'Gonnell and Hanson (1987). Protoplasts were electri- cally permeabilized in the presence of 50 |ig/ml pJLWlSO as described previously (Fromm et al. 1986) except that the electrical pulse was delivered 122- or 245-|iF capacitors charged to 200V (Fronmi et al. 1986). The electroporated protoplasts were cultured as previously described; callus and subsequent plantlet formation was performed as previously described by O'Gonnell and Hanson (1987). A total of 113 plantlets were regenerated; 43 were tested for CMV coat protein sjmthesis by protein dot blot analysis. Of these, 31 were positive (i.e., contained >1 ng of coat protein per \xg of protein) for coat protein production (table 2). Protein analysis was performed as described by Nelson et al. (1987).

Table 1 • Genetic analysis of progeny of self-pollination of three transformed lines with respect to kanamycin sensitivity

Kanamycin insensitive

Kanamycin sensitive

Ratio tested Chi-square*

pJLWlBO-llO 78 22 3:1 0.5 (0.25<P<0.5)

pjLwiao-iso 140 67 2:1 0.1 (0.75<P<0.9)

pJLWlBO-ieO 344 19 15:1 0.6 (0.25<P<0.5)

*H3npotlieses were rejected at the 5-percent risk level (P<0.05).

2:1 — Integration of the T-DNA (containing Km^ gene) induces a mutation that is lethal when homozygous: the expected segregation ratio is 2:1. 3:1 — The kanamycin marker segregates as one Mendelian locus, and homozygotes are viable. 15:1 — The kanamycin resistance marker segregates as two independent Mendehan loci.

III-9

Table Z. Detection of CMV coat protein in 43 direct transformants of tomato 13C-4

Direct Transformants Designation

12a,12c,12ci,12f 12g,12k,12n,12p

12b,12e,12s,12z 12y,12w,12r,12q

<1 ng Amount of CMV Coat Protein^

1-5 ng 5-10 ng >10 ng

+

+ +

l^,12s,12t,12u

12m

13m,13o,13q,13t

13u,13v,13w,13x 13y,13z,13a,13b 13c,13f,13h,13t

+ + +

13n,13p,13k,13q 13r,13s

^After extraction of protein from leaf tissue, 20 ^ig of protein was subjected to SDS-polyacrylamide gel electro- phoresis and inmiunoblot analysis (Nelson et al. 1987). The amount of CMV coat protein expressed was based on intensity of band compared to that of known concentrations of viral coat protein.

111-10

Figure 1, Constniction of p6C8860 (the disarmed Ti plasmid).

lSc-8

pTiB6S3

I '*,! " I "III: TL »n '^ I

TR

±J$mnH\ EcoRI

TR

H P E HH E E HHB PGV22I7 W

Km'^

H B HHB PCV2260

.pGV2217 Ü—til Cb'^

Restriction map of the T-region of pTiB6S3 and pGV2217. In pGV2217 [6], the TL-region is substituted by a Km marker. The intermediate vector pGV746 was constructed as follows : the 2.3-kb Hindlll/BamHI fragment from pTiAchS fragment HindIII-14 ((///]) was cloned Tnto pBff322, digested with HindiII and BamMlTihis fragment is directly adjacent to the Igft^ of the TL-region. The resulting plasmid, pGV713, was selected as a Cb Tc clone. The pTi-region adjacent to the right of the TR-region was cloned as a 4.2-kb EcoRI/Hindlll fragment, derived from pTiAchS fragment HindIII-4 ([•.'.] into pGV713 digested with EcoRI/HindllL The resulting intermediate vectoj^ is pGV746. Recombinants between pGV746 and pGV2217 wece isolated as Cb transconjugants after mobilizing pGV746 into C58ClRif (pGV2217) using the technique described [25]. The double cross-over events between pGV746j^and pGV2217, indicated by crossed lines, were obtained by screening the Cb transconjugants for the loss ofpthe Km marker present on pGV2217. The physical structure of one Rif , Cb and Km^ transconjugant, pGV2260, was verified by Southern hybridization and is depicted in the figure.

[Figure 1 is reproduced from its original source, Deblaere et al., Nue. Acids Res. (1985) 13: 4777- 4788, by permission of Oxford University Press.]

Permit applicants are not required to secure reprint permissions when using borrowed illustrations in the applications.

111-11

Figured. Construction of pGV831.

lSc-6

T.-ONA T-.ONA

M •• 3BB E8

H lit

Eco RI/DNA pol/m

Hindlll/ONApotym

1

pKC7 nos

E M

The T-region of pîiB6S3 is presented on top of the figure. The dark fragnents are those which are maintained in pGV831. The 7.5-kb Hindi 11 fragment from pGV700 (TableTT was recloned into pGV600, a pBR322 derivative lacking the BamHI site. The remaining TL-DNA genes in pGV742 were removed by deleting the internal BamHI fragments (pGV744). The left part of the TR-DNA was removed by deleting the internal EcoRI fragments (pGV749). pGV710 IT" a pBR325 derivaUva. that contains an additional Sm Su marker. To obtain pGV710 the 2.43-kb Hindlll/PstI fragment from pBR325. containing the Cm gene, was cloned in a Hindlll/ Pstl-digested cosmid pHC79 and the rrS2-kb B¿[II "cos" fragment of the resulting plasmid was substituted by a 3.45-kb BamHI fragment from the P-type plasmid R702 that ençr*Jes resistance to Sm/Sp and Su [31] In order to obtain a fragment containing only the TL-border sequences, the 1.87-kb Hindlll/ Nrul fragment from pGV749 was clonedH'nto pGV710 digested with EcoRI. and HindiII. The sticky ends obtained after EcoRI digest were flush-ended by treatment with Klenow DNA polymerase before Hindlll digestion. oQVBlS^. was iso^ Tited as a Sm*^. Cb^, Cm^ and Tc^ clone. In pGV825 the EcoRI and Hindlll sites were eliminated by TTTling-in the sticky ends and self ligation of the vector. A 298-bp Bell/ BamHI fragment from pLGV2381 [14] comprising the nopal i ne synthase promoter and cloned into the Bell site of pKC7 produced pKC7::nos. The nos promoter directs transcription of the neo gene inj)lant cells [14] This chimeric Km gene was isolated as a Bcl^I/BamHI fragment and cloned into the ^5gl 11 site of pGV825 to produce pGV831. Abbrevia- tions : B. BamHI; Be, Bc2I, Bg. Bglll, E, EcoRI; H, Hindlll; N. Nrul; Cb, carbenicillin; Cm. chlor- amphenicol; Sm. streptomycin; Sp. spectinomycin; Su, sulfathiazol; Tc. tetracycline.

[Figure 2 is reproduced from its original source, Deblaere et al., Wuc. Acids Res. (1985) 13: 4777- 4788, by permission of Oxford University Press.]

111-12

13C-7

Figure S. Map of pJLWlSO.

Cía I Hind III

40 S promoter: CMV coat protein:

NOS termination

l~Mpa II

Pst I

NOS promoter: NPT II:

NOS termination

Pst I

Eco Rl RB

S - streptomycin resistance marker

C]3 - carbenicillin resistance marker


The border regions are derived from an octopine type plasmid (Thomashow et al. 1980); the LB (1050 bp) and RB (550 bp). The chimeric CMV coat protein contains the NOS termination and polyadenylation signal sequences (nucleotides 19,995 to 20,543 (Barker et al. 1983)) and the chimeric NPT II gene contains the NOS promoter (EamHl fragment of pLGV2381 (Herrera-Estrella et al. 1983)) and the NOS polyadenylation signal sequences (White 1988). NPT II was isolated from Tn5 (Deblaere et al. 1985).

Legible Jreehanddramo^^^

111-13

Figure 4. Map of cointegrate; pJLW180::pGV8860.

lSc-8

pGV2260 P

Cb

E P

r

pJLWlSO

RB

Cointegrate

CMV COAT PROTEIN

\

NPT II

E P

*Cb LB Coat protein NPT II RB Sm Cb

11-14

13C-9

Figures.

12 3 4 5 6 7 8 9 10 11 12

Autoradiograph showing the DNA analysis of plants transformed with CMV coat protein. Southern blot analysis of Hindm-digested DNA from nine directly transformed plants (lanes 1 to 9) and vector transformed plant (pGV831; lane 10) and probed with ^^P-labeled ssRNA transcripts of the cDNA to CMV coat protein. Lanes 11 and 12 contain one or five copies, respectively, of cloned CMV coat protein per genome equivalent.

(1 ) Is there a detailed description of the derivation (whether or not they are derived from plant pests) of all new sequences that appear In the regulated articie, including promoters, polyadenylation and termination signal sequences, the engineered géne(s), marker or antibiotic resistance gene(s), and other noncoding sequences?

(2) Which of the newly acquired genes are expressed in the regulated article?

(3) is the origin of the vector/vector agent described?

(4) Is there a description of how the vector/vector agent transfers the gene(s) to the recipient?

(5) If the vector/vector agent has been derived from a plant pest, are there any genes remaining that are or have been implicated in pathogenicity of this organism?

{6) Can the mechanism by which the engineered gene{s) were introduced be reversed and mobilize the gene(s) out of the engineered organism to other organisms?

(7) If they can be mobilized out, explain the mechanism and include data, if available, on the frequency and species of organisms that could be potential recipients.

-15

13d-1

13d. Source of Regulated Article

The transformed tomato plants were developed at Paige-Sullivan Biotechnologies, Ltd., Hyattsville, Maryland. The tomato seeds of cultivar Packard Clipper were obtained from Packard Caribbean Seed Company, Riverside, California. The plasmid used to transfer the CMV coat protein gene to the tomato plants was constructed at Halasa Biotechnologies, Minot, North Dakota. The A. tnTnftfa.niftns strain used was obtained from Dr. Leonard C. Jackson, Dept. of Plant Pathology, University of California, Davis, imder USDA/APHIS permit nimiber 88-111-33.

111-16

13e-l

13e. Field Plot Design.

PURPOSE: The objective of this field trial is to test the level of tolerance of plants expressing CMV coat protein against challenge inoculation by severe strain of CMV.

FIELD SITE LOCATION: The company-owned site, in Hyattsville, Prince George's County, Maryland, is surrounded by agricultural land. The crops in adjacent fields at the expected pleunting date are soybeans and corn. The closest nonexperimental tomato plants are assumed to be at the nearest residential home, approximately 3/4 mile away.

FIELD TRIAL SUPERVISOR: Jack Baker, Field Operations Manager, (301) 436-7612.

EXPECTED PLANTING DATE: May 15.

FIELD DESIGN. Split-plot design with nine treatments as main plots and two replications.

GENOTYPES:

(1) Nontransgenic control Packard Clipper

(S) Rg progeny of transgenic line pGV831

(3) Rg progeny of transgenic line pJLW180-120

(4) Direct transformants (R^) of transgenic line pçJLW180-160

TREATMENTS:

(1) Noninoculated - Packard Clipper

(5) Noninoculated - transgenic pGV831

(3) Noninoculated - transgenic pJLW180-120

(4) Challenge inoculated with CMV-CQ - nontransgenic control

(5) Challenge inoculated with CMV-CQ - transgenic pGV831

(6) Challenge inoculated with CMV-CQ - transgenic pcJLW180-120

(7) Challenge inoculated with CMV-CQ - transgemc pcJLW180-160

Applicants must name the county or counties where the field test(s) will be performed.

111-17

lSe-8

Seeds will be germinated in the greenhouse. Plants will be "hardened off" in a cold frame for up to 1 week prior to transplanting in the field after the permit for this application is issued. All challenge inoculations, which will be done mechanically, will take place 10 days after transplanting. CMV strain CQ used in this study is endemic to the Eastern United States.

If a pathogen to be used in the field test was received under a PPQ 526 movement permit, a copy of the permit should be submitted along with this application. If a plant pest is being moved interstate for the field test (e.g., CMV strain CQ for challenge inoculation in this sample application), a permit may be required (PPQ Form 526). This form can be obtained from Biological Assessment and Technical Support staff, USDA-APHIS-PPQ, Room 625,6505 Belcrest Road, Hyattsville, MD 20782.

PLOT DESIGN: Each plot contains 20-ft rows with 20 plants spaced 1 ft apart and 5 ft spacing between rows. The main plot will consist of two experimental rows and two border rows on the outside (total four rows). There will be a 20-ft implanted area between all main plots to prevent spread of the virus. The field plot will be 150 ft x 300 ft, including the disposal area.

AGRICULTURAL PRACTICES: Standard agricultural practices will be performed to control insects and pathogens. Aphid populations will be monitored closely and controlled by appropriate insecti- cides to avoid transmission of virus to control plots. Company representatives will visit the plot three times per week, and monitoring of plants will include observations of morphology, plant vigor, water status, nutrient status, physiological problems, flower initiation, disease problems, insect infestation, and damage from invertebrate and vertebrate pests.

Animals likely to visit the field include the usual faima (mice, birds).

DATA COLLECTION:

(1) Fruit count and total weight at each harvest,

(2) Biochemical and molecular monitoring of virus infection, and

(3) Visual monitoring of symptom development of engineered versus nonengineered plants.

EXPECTED TEST CONCLUSION DATE: September 15.

Initially all field test sites were enclosed by fences and some had more elaborate security measures. These kinds of security are not required but may offer some protection from vandalism. Adequate security may be invisibility from the nearest road or sheltering by surrounding crops (e.g., engineered tomatoes surrounded by border rows of corn). The duration of the field test may be longer than a single growing season; however, in these cases periodic status reports to APHIS are required.

111-18

ISe-S

Test Plot Site Design

150 feet

300 feet

Single lines - border rows

Double lines - experimental rows

(20 plants per row)

111-19

13f-l

13f. Description of Containment.

Seedlings or seeds of the transformed plants will be transported from the greenhouse to the field test location in a van under the supervision of the Paige-Sullivan Biotechnologies personnel who are directly responsible for supervising the field trial. All movements of regulated articles from Halasa Plant Products (North Dakota) to Maryland will be imder separate permit.

If a regulated article{s) is being moved interstate (prior to or after the field release is initiated), a separate APHIS Form 2000 for movement must be submitted. See Section II for sample movement permit applications.

11-20

13g-1

13g. Description of Containment.

The laboratory, growth chamber facilities, and greenhouses have been inspected and approved by APHIS under previous movement permit applications. Our laboratories meet the NTH Guidelines for Research Involving Recombinant DNA.

Seedlings wiU be transported directly from the greenhouse to the test location as described in 13f. The experimental field will be located in Prince George's Coimty, Maryland. Map 1 shows the general area of the field trial and map 2 gives greater detail of test location.

Mapl Map 2

BELCREST

ROAD

field site

PSB Laboratories

ADELPHI ROAD

Queen's Chapel Road

HYATTSVILLE

Seedlings should be transported to the field test site in an enclosed vehicle or covered in some acceptable manner if transferred in a truck. Microorganisms should be moved In containers as described in Section IV.

111-21

13h-1

13h. Detailed Description of Containment.

The field test site will be surrounded by a 3-ft-high, chain-link fence. The morphology of Packard Clipper cultivar greatly reduces the chance of cross pollination. Tomato, Lyconersicon esculentum. is a member of the family of plants called Solanaceae. It is a self-pollinating herbaceous pereimial that is usually grown as an annual crop in the United States. The natural distribution of wild species of Lyconersicon is restricted to the Andean region of South America. As with many self- pollinating species, the flower morphology of tomato greatly facilitates self-pollination (Rick 1976). The pistil is actually enveloped by a solid tube formed by the stamens. When mature, the anthers dehisce and pollen is released by lateral slits into a central cavity. Since the flowers hang down, the pollen moves by gravity towards the mouth of the tube where the stigma is located. Self- poUination then occurs. This cultivar has a much shortened style that places the stigma well within the anther tube, further expediting self-pollination and substantially reducing the opportunity for outcrossing. The Association of Official Seed Certifying Agencies (Anonymious 1971) publishes plant isolation requirements mandated to maintain the purity of seed. The separation distance for foimdation tomato seed is SCO ft. No tomato plants will be grown within 660 ft of the test site.

One enviroimiental issue is that the engineered gene could outcross to other populations of Lycopersicon or closely related species in nature. There are no other wild species of liyQQVßVßiWD. in the United States that could cross pollinate with the experimental plants. Conmiercial tomato cultivars have not been known to be weedy species. Seeds protected in soil may germinate the following spring (Rick 1976). Therefore, the field test site will be monitored for 6 months following the termination of the experiment for the presence of volxmteer tomatoes.

-22

Coiitainmeiit Points To Consider

Could the engineered organism have any Impact on: floral communities, fauna! communities, endangered or threatened organisms, humans, the health of plants or animals, and genetic resources (e.g., susceptibility of economically Important species to herbicides or pesticides) or agricultural production? What are the survival rates of the modified organism in the spectrum of conditions lil<ely to be found in the release area(s) and surrounding environment? What are the organism's reproduction rates in these areas? What ¡s the capability of the organism to disperse from the release area? What are the dispersal mechanisms? What are the consequences of the organism remaining in the environment beyond the planned period? What methods will be used to control or eliminate the organism from the site and the surrounding environment should such action be required? How effective are these methods?

Plants. One of the major concerns for plants is dissemination of the engineered genes by pollen. The Association of Seed Certifying Agencies publishes plant isolation requirements for maintaining seed-stock purity. This is a good starting point for designing containment features for many experiments as long as one takes into consideration the percentages of outcrossing assumed in those isolation distances. APHIS recommends that applicants include supporting statements from authoritative persons (e.g.. plant breeders or ecologists) stating that the experimental design, location of plot, and local conditions are sufficient to minimize escape of genes to sexually compatible plants. Having considered the pollination characteristics of the species, do wild populations of the species, or related species with which it can interbreed, exist in the vicinity of the field trial or agricultural site? Are any members of the genus of modified plants known to be weeds?

Microorganisms Associated With Plants. Is the organism able to establish itself on/in nontarget species in the surrounding environment? To what extent does the organism survive and reproduce on/in the target plant and/or other plant species in the test site and surrounding environment? Are there any effects on soil microorganisms that are beneficial to plants (e.g., Rhizobium and mycorrhizal fungi)? In the case of biological control organisms, can the organism establish itself with nontarget species? Can the modified genetic traits be transmitted to other microorganisms in the environment? What methods are used to monitor the environmental impacts, particularly the population of the modified, target, and nontarget organisms? Can the genetically engineered microorganism be disseminated by wind, water, soil, mobile organisms, or other means?

111-23

lSi-1

13i. Final Disposition of Regulated Article:

Fruit and plant material removed from the field for testing in the laboratory will be autoclaved and disposed of as trash or returned to the field for destruction with remaining live plants.

Fruits and plants harvested during the course of the experiment (and not returned to the laboratory) will be buried in the disposal site within the plot for natural decay imder compost conditions. All plants and fruits remaining at the termination of the experiment will be treated with the herbicide glyphosate. In greenhouse tests, glyphosate has been shown to kill both mature transformed and control plants. After the plants have died, the debris will be incorporated into the soil. The test site will be monitored for the next 6 months to make sure all test plants and any volunteers are killed. All tomato plants appearing during this period will be removed either by hand or by another herbicide application, depending on the number of plants involved.

11-24

REFERENCES

Abel, P. P., Nelson, R. S., De, B., Hoffmann, M., Rogers, S. G., Praley, R. T., Beachy, R. N. 1986. Delay of disease development in transgenic plants that express the tobacco mosaic virus coat protein gene. Science 832:738-743.

Anonynious. 1971. AOSCA Certification Handbook. Raleigh, NC: Association of Official Seed Certifying Agencies.

Barker, R. P., Idler, K. B. Thompson, D. V., Kemp, J. D. 1983. Nucleotide sequence of the T-DNA region from the Agrobacterium tumefaciens octopine Ti plasmid pTil5955. Plant Mol. Biol. 2:335- 350.

Benner, C. P., Kuhn, C. W., Demski, J. W., Dobson, J. W., Colditz, P. 1985. Identification and incidence of pepper viruses in northeastern Georgia. Phytopathology 69:999-1001.

Cooper, A. J., Meredith, C. P. 1989. Genetic manipulation of plant cells. Biochem. of Plants. 15:653-688.

Cuozzo, M., O'Connell, K. M., Kaneiewski, W., Fang, R. X., Chua, N-H. 1988. Viral protection in transgenic tobacco plants expressing the cucimiber mosaic virus coat protein or its antisense UNA. Bio/Technology 6:549-554.

Deblaere, R., Bjrtebier, B., De Grève, H., Deboeck, P., Van Montagu, M., Leemans, J. 1985. Effi- cient octopine Ti plasmid-derived vectors for Agrobacterium tumefaciens-mediated gene transfer to plants. Nuc. Acids Res. 13:4777-4788.

De Zoeton, G. A., Pulton, R. W. 1975. Understanding generates possibiUties. Phytopathology 65:221-222.

Diaz-Ruiz, J. R., Kaper, J. M. 1978. Isolation of viral double-stranded RNAs using LiCl fraction- ation procedure. Prep. Biochem. 8:1- 17.

Dodds, J. A. 1982. Cross-protection and interference between electrophoretically distinct strains of cucimiber mosaic virus in tomato. Virology 78:253-260.

Dodds, J. A., Lee, S. Q., Tiffany, M. 1985. Cross protection between strains of cucumber mosaic virus: effect of host and type of inoculimi on accumulation of virions and double-stranded RNA of the challenge strain. Virology 144:301-309.

Praley, R. T., Rogers, S. G., Horsch, R. B. 1986. Genetic transformation in higher plants. CRC Critical Reviews in Plant Science 4:1-46.

Fromm, M., Taylor, 1. P., Walbot, V. 1986. Stable transformation of maize after gene transfer by electroporation. Nature (London) 319:791-793.

Harrison, B. D. 1958. Ability of single aphids to transmit both avirulent and virulent strains of potato leaf roll virus. Virology 6:278-286.

Herrera-Estrella, L., De Block, M., Messens, E., Hernalsteens, J.-P., Van Montagu, M., Schell, J. 1983. Expression of chimeric genes transferred into plant cells using a Ti-plasmid derived vector. EMBO J. 2:987-995.

1-25

Hull, R., Covey, S. N. 1983. Does cauimower mosaic virus replicate by reverse transcription? Trends Biochem. Sei. 8:119-121.

Kaper, J. M. 1984. Plant disease regulation by virus-dependent satellitelike replicating. In: Con- trol of virus diseases. Kurstak, E. (ed.) pp. 317-343. New York: Marcel Dekker, Inc.

Kavanau, J. L. 1949. On correlation of the phenomena associated with chromosomes, foreign proteins and viruses. III. Virus associated phenomena, characteristics and reproduction. Am. Nat. 83:113-138.

Loesch-Fries, L. S., Merlo, D., Zinnen, T., Burhop, L., Hill, K.,Krahn, K., Jarvis, N., Nelson, S., Halk, E. 1987. Expression of alfalfa mosaic virus RNA 4 in transgenic plants confers virus resistance. EMBOJ. 6:1845-1851.

Lot, H., Marrou, J., Quiot, J. B., Esvan, Ch. 197S. Cucimiber mosaic virus (CMV). II. Rapid purification method. Ann. Phytopathol. 4:25-38.

McCormick, S., Niedermeyer, J., Pry, J., Barnason, A., Horsch, R., Fraley, R. 1986. Leaf disc transformation of cultivated tomato (L. esculentum^ using Agrobacterium tUHlgfftQigns. Plant Cell Rep. 5:81-84.

McKinney, H. H. 1929. Mosaic diseases in the Canary Islands, West Africa and Gibraltar. J. Agrie. Res. 39:557-578.

Nelson, R. S., Abel, P. P., Beachy, R. N. 1987. Lesions and virus accimiulation in inoculated transgenic tobacco plants expressing the coat protein gene of tobacco mosaic virus. Virology 158:126-132.

Nelson, R. S., McCormick, S. M., Delannay, X., Dube, P., Layton, J., Anderson, E. J., Kaniewska, M., Proksch, R. K., Horsch, R. B., Rogers, S. G., Praley, R. T., Beachy, R. N. 1988. Virus tolerance, plant growth, and field performance of transgenic tomato plants expressing coat protein from tobacco mosaic virus. Bio/Technology 6:403-409.

O'Connell, M. A., Hanson, M. R. 1987. Regeneration of somatic hybrid plants formed between Lvconersicon esculentum and L. pgmJ.QUU» Theor. Appl. Genet. 75:83-89.

Rao, R. N., Rogers, S. G. 1979. Plasmid pKC7: a vector containing ten restriction endonuclease sites suitable for cloning DNA segments. Gene 7:79-82.

Rick, C. M. 1976. Tomato (family Solanaceae). In: Sinmionds, N. W., ed. Evolution of crop plants, pp. 268-273. New York: Longman Publications.

Salaman, R. N. 1933. Protective inoculation against a plant virus. Nature (London) 131:468.

Shepherd, R. J. 1979. DNA Plant Viruses. Ann. Rev. Plant Pathol. 30:405-423.

Sherwood, J. L., Pulton, R. W. 1982. The specific involvement of coat protein in tobacco mosaic virus cross protection. Virology 119:150-158.

111-26

Thomashow, M. F., Mutter, R., Postle, K., Chilton, M-D., Blattner, P. R. Recombination between higher plant DNA and the Ti-plasmid of Agrobacterium tumefaciens. 1980. Proceedings of National Academy of Sciences (USA) 77:6448-6452.

Thung, T. H. 1931. Smetstof en plantencel by enkle virusziekten van de Tabaksplant. Rev. Appl. Mycol. 11:750-751.

Tumer, N. E., O'Connell, K. M., Nelson, R. S., Sanders, P. R., Beachy, R. N., Praley, R. T., Shah, D. M. 1987. Expression of alfalfa mosaic virus coat protein gene confers cross-protection in transgenic tobacco and tomato plants. EMBO J. 6:1181-1188.

Van Dun, C. M. P., Bol, J. P., Van Vloten-Doting, L. 1987. Expression of alfalfa mosaic virus and tobacco rattle virus coat protein genes in transgenic tobacco plants. Virology 159:299-305.

Van Haute, E., Joos, H., Maes, M. Warren, G., Van Montagu, M., Schell, J. 1983. Intergeneric transfer and exchange recombination of restriction fragments cloned in pBR322: A novel strategy for the reversed genetics of the Ti plasmids of Agrobacterium tumefaciens. EMBO J. 2:411-418.

White, J. L. 1988. The fabricated citation. Journal XX:000-000.

Zambryski, P., Depicker, A., Kruger, K., Goodman, H. 1982. Timior induction by Agrobacterium tumefaciens: Analysis of the boundaries of T-DNA. J. Mol. Appl. Gen. 1:361-370.

111-27

CBICopy

The following two pages are examf

Hindll/BamHI fragment of Tn5 which contains NPT E (Rao and Rogers 1979). The NOS 3' polyadenylation and termination signal sequences were isolated and fused to NPT n (NEO) gene as previously described (White 1988). The chimeric NPT II gene was isolated as a EfiUI/EamHI fragment and cloned into Egm site of pGV825 to produce pGG831.

DESCRIPTION OF RECIPIENT: The recipient organism, L. esculentum cv. Packard Clipper, is a nommon commercial cultivar and is a fresh market tomato. It is not widely grown in Mary- land because of its susceptibility to CMV. Additional information on the biology of this tomato can be found in section 13h.

DESCRIPTION OF DONOR: CMV has been linked to plant disease in all temperate regions of the world. The virus has an extremely wide host range that includes cereals, forages, woody and herbaceous ornamentals, vegetables, and fruit crops. The RNA of CMV consists of four components of different size (approximate M^: 1.01, 0.89, 0.68, and 0.33 x 10®). The three largest UNA'S, which are distributed among three separate virion particles, carry aU the information needed for successful infection. The genetic information for viral coat protein is carried on RNA 3 and on a coencapsidated subgenomic messenger RNA 4 (Kaper 1984). The viral coat protein assembles aroimd the viral RNA to form the stable virion, which protects the nucleic acid from physical, chemical, or biochemical degradation. The coat proteins from different CMV strains usually have different amino acid sequences that reflect differences in nucleotide sequences of RNA 4 (Kaper 1984).

CMV strain PV 29 (also called strain 1) (American Type Culture Collection Catalogue, 14th Ed., 1988) was propagated as previously described (Lot et al. 1972) and dsRNA isolated as previously described (Diaz-Ruiz and Kaper 1978). Pull-length ds cDNA copies of PV 29 CMV RNA 4 were prepared using synthetic oligonucleotide to prime RNA synthesis simultaneously from the 3' ends of both plus and minus strands of denatured ds RNA 4. The primer hybridizing to the plus strand contained an added Clal recognition site at its 5 ' end to facilitate forced cloning into the plasmid vector. Additional details of this construct have been published (White 1988), and a reprint appears in appendix 1.

The promoter used in this study was derived from strawberry vein banding virus (SVBV), a caulimovirus (Shepherd 1979). The replication strategy of SVBV is thought to be analogous to the well-characterized cauliflower mosaic virus (CaMV). Upon infection of a cell with SVBV, two major RNA transcripts, designated 40S and 22S (based on their sedimentation coefficients), are produced during the replication of SVBV. These transcripts are analogous to the 35S and 19S CaMV RNAs (HuU and Covey 1983). The 40S SVBV promoter sequences have been characterized (further details in appendix 1). The 40S promoter was isolated as the Hindin/Clal fragment extending from +9 to -343 with respect to the transcription start site mapped for the 40S RNA. The transcription termination and polyadenylation signal sequences were derived from the nopaline synthase (NOS) gene (Barker et al. 1983).

TheQlOS promoterjand NOS termination signal sequences were blunt-end ligated to the cloned J CßJL CMV coat protein gene and inserted into pGV831 at the unique EâmHl site. A map of the resulting plasmid pJLWlSO is shown in figure 3. pJLWlSO was introduced into the acceptor Ti plasmid pGV2260 by a single homologous recombination, using Sm^ gene of pJLWlSO as a selectable marker for cointegration (see fig. 4 for map of cointegrate). The mobilization of pcILW180 from E. fîûli to Agrobacterium C58ClRif^ (pGV2260) was performed according to Van Haute et al. (1983). The structure of the T-region was confirmed by Southern blot hybridization.

111-28

CBI

CBI-Deleted

Hindn/BamHI fragment of Tn5 which contains NPT II (Rao and Rogers 1979). The NOS 3' polyadenylation and termination signal sequences were isolated and fused to MPT n (NEO) gene as previously described (White 1988). The chimeric NPT II gene was isolated as a EfiUI/EâmHI fragment and cloned into BgUI site of pGV825 to produce pGC831.

DESCRIPTION OP RECIPIENT: The recipient organism, L- esculentum cv. Packard Clipper, is a common conmiercial cultivar and is a fresh market tomato. It is not widely grown in Mary- land because of its susceptibility to CMV. Additional information on the biology of this tomato can be found in section 13h.

DESCRIPTION OF DONOR: CMV has been linked to plant disease in all temperate regions of the world. The virus has an extremely wide host range that includes cereals, forages, woody and herbaceous ornamentals, vegetables, and fruit crops. The UNA of CMV consists of four components of different size (approximate M^: 1.01, 0.89, 0.68, and 0.33 x 10^). The three largest RNA's, which are distributed among three separate virion particles, carry all the information needed for successful infection. The genetic information for viral coat protein is carried on UNA 3 and on a coencapsidated subgenomic messenger RNA 4 (Kaper 1984). The viral coat protein assembles aroimd the viral RNA to form the stable virion, which protects the nucleic acid from physical, chemical, or biochemical degradation. The coat proteins from different CMV strains usually have different amino acid sequences that reflect differences in nucleotide sequences of RNA 4 (Kaper 1984).

CMV strain PV 29 (also called strain 1) (American Type Culture Collection Catalogue, 14th Ed., 1988) was propagated as previously described (Lot et al. 1972) and dsRNA isolated as previously described (Diaz-Ruiz and Kaper 1978). Pull-length ds cDNA copies of PV 29 CMV RNA 4 were prepared using synthetic oligonucleotide to prime RNA synthesis simultaneously from the 3' ends of both plus and minus strands of denatured ds RNA 4. The primer hybridizing to the plus strand contained an added Clal recognition site at its 5 ' end to facilitate forced cloning into the plasmid vector. Additional details of this construct have been published (White 1988), and a reprint appears in appendix 1.

C8I Df/lTfP

J The£ Jand NOS termination signal sequences were blunt-end ligated to the cloned j ^^prcry CMV coat protein gene and inserted into pGV831 at the unique EâmHl site. A map of the cLtJtU resulting plasmid pJLW180 is shown in figure 3. pJLW180 was introduced into the acceptor Ti plasmid pGV2260 by a single homologous recombination, using Sm^ gene of pJLW180 as a selectable marker for cointegration (see fig. 4 for map of cointegrate). The mobilization of pcJLW180 from E. fioli to Agrobacterium C58ClRif^ (pGV2260) was performed according to Van Haute et al. (1983). The structure of the T-region was confirmed by Southern blot hybridization.

-29

12910 Federal Register / Vol. 53, No. 76 / Wednesday, April 20, 1988 / Rules and Regulations

7 CFR Part 340

[Docket No. 88-019]

Genetically Engineered Organisms and Products; Exemption for Inter- state Movement of Certain Microor- ganisms Under Specified Conditions

AGENCY: Animal and Plant Health Inspection Service, USDA. ACTION: Final rule.

SUMMARY: This document amends the regulations pertaining to the introduction of certain genetically engineered organisms and products by exempting from regulation certain genetically engineered microorganisms which are moved interstate under specified conditions. This amendment removes unnecessary restrictions on the interstate movement of those microorganisms wrhich do not present a risk of plant pest introduction or dissemination.

EFFECTIVE DATE: April 20, 1988.

FOR FURTHER INFORMATION CONTACT: John Payne, Staff Microbiologist, Biotechnology and Environmental Coordination Staff, Animal and Plant Health Inspection Service, U.S. Department of Agriculture, Room 406, Federal Building, 6505 Belcrest Road, Hyattsville, MD 20782, 301-436-7908.

SUPPLEMENTARY INFORMATION: On September 24, 1987, the Animal and Plant Health Inspection Service (APHIS) published a proposed rule in the Federal Register (52 FR 35921- 35923) to amend the regulations in 7 CFR Part 340, pertaining to the introduction of certain genetically engineered organisms and products. The proposed rule set forth provisions which would exempt a person from having to obtain a permit for the interstate movement of certain genetically engineered microorganisms if the microorganisms were moved in accordance with certain specified conditions. APHIS indicated that the proposed exemption would remove

unnecessary restrictions on the interstate movement of those microorganisms which do not present a risk of plant pest introduction or dissemination. Specifically, it was proposed that a limited permit for interstate movement would not be required for genetic material from any plant pest contained in E. coli strain K-12, sterile strains of Saccharomyces cerevisiae, or asporogenic (nonspore forming) strains of Bacillus subtilis, provided all of the following conditions are met:

(i) The microorganisms are shipped in a container that meets the requirements of § 340.6(b)(3) of this part;

(ii) The cloned genetic material is maintained on a nonconjugation proficient plasmid and the host does not contain other conjugation proficient plasmids or generalized transduc- ing phages;

(iii) The cloned material does not include the complete infectious genome of a known plant pest;

(iv) The cloned genes are not carried on an expression vector if the cloned genes code for:

(a) A toxin to plants or plant products, or a toxin to organisms beneficial to plants; or

(b) Other factors directly involved in eliciting plant disease (i.e., cell wall degrading enzymes); or

(c) Substances acting as, or inhibitory to, plant growth regulators.

APHIS received 15 comments on the proposed rule. Commenters included academic researchers; companies engaged in genetic engineering; professional societies and trade associations; and a State department of agriculture. The majority of the commenters expressed general support for the proposed rule without suggest- ing specific wording changes. Seven commenters suggested specific wording changes either for purposes of clarification, or for purposes of broading the scope of the proposed exemption. Based on the rationale set forth in the proposed rule and in this document, APHIS has adopted the provisions of the proposed rule as a final rule with certain changes which clarify the provisions of the exemption.

Two commenters suggested specific wording changes which would have the effect of clarifying the language of the exemption. Both of these commenters noted that the wording of the exemption, as proposed, appeared to limit it solely to E. coli strain K-12 and not to its derivatives. One commenter suggested that the organism be identified as "Escherichia coli strain K-12 and its derivatives" noting that derivatives of the original strain K- 12 have been developed that do not colonize in the human intestinal tract and which will not survive in the environment. The other commenter questioned whether it was the intention of APHIS to include genotypes of E. coli strain K-12, rather than limit the exemption solely to the original K-12 strain. The commenter noted that almost all recombinant DNA research using K-12 host-vector systems is conducted with K-12 derived strains rather than the original strain. APHIS believes both commenters have provided wording changes which more specifically identify the E. coli organism. It was APHIS' intention to include the derivatives of the original E. coli strain K-12 with the original strain as organisms to be used for the interstate movement of plant pest genetic material under the specified conditions. As both the commenters noted, the derivatives are, like the original strain K-12, also inherently safe. The suggestions offered from both commenters have been accepted and incorporated into the final rule. As revised, the language in the final rule referencing E. coli now reads, ''Escherichia co7i genotype K-12 (strain K-12 and its derivatives)." It should be noted that another way of referring to the original strain K-12 and its derivatives is to use the term "genotype K- 12." This revision in wording does not constitute a substantive change, but merely clarifies APHIS' original intent which was implicit in the preamble to the proposed rule. In the preamble, APHIS cited biosafety data on strain K- 12 that in the context ofthat citation could only refer to derivatives of strain K-12 as well as the original K-12 strain.

IV-1

One commenter noted that it is often advantageous to make and use gene libraries in expression vectors. The commenter indicated that it is very difficuh to guarantee that a toxin gene is not present somew^here in the library. The commenter questioned whether the presence of a toxin gene somev^here in the library v^ould mean that the person seeking to ship the genetic material w^ould not meet the conditions for the exemption and would have to obtain a limited permit prior to interstate movement.

It appears that the commenter has misconstrued how APHIS would apply the provisions of the exemption. The presence of a toxin gene somewhere in a gene library would not be of significance in determining whether or not the provisions of the exemption had been complied with. What would be of significance is whether the toxin gene had been engineered to be expressed on the expression vector and not whether a single toxin gene inciden- tally appears somewhere in a gene library.

One commenter stated that the expression of a single gene coding for a factor directly or indirectly involved in eliciting plant disease should not be a matter or concern with respect to the risk of causing plant disease. The commenter further questioned why APHIS had included "cell wall degrading enzymes" as an example of a factor directly involved in eliciting plant disease.

APHIS agrees that most single genes from a plant pest are unlikely to confer plant pest characteristics on a previously benign recipient, but APHIS does not agree that all single genes carry no risk of conferring plant pest characteristics on an organism.

For example, the classes of genes that are referenced in § 340.2(b)(l)(iv) of the final rule, which code for toxins to plants or plant products, factors directly involved in eliciting plant disease, or substances acting as, or inhibitory to plant growth regulators, if expressed at high levels, would be capable of conferring plant pest characteristics on the three host

microorganisms specified in the exemption [E. coli, sterile strains of Saccharomyces cerevisiae, and asporogenic strains of Bacillus subtilis).

APHIS agrees with the commenter that cell wall degrading enzymes cannot cause plant disease by themselves, but cell wall degrading enzymes are important in the disease inducing mileau of many microorganisms (See: Cell Wall Composition and Metabolism, Chap. 5 in: Goodman, R.N., Kiraly, Z., and Wood, K.R., The Biochemistry and Physiology of Plant Disease, University of Missouri Press, 1986). Experimental evidence has been published in at least two reports to support the position that a highly expressed enzyme that contributes to the degradation of plant cell walls can confer on derivatives of E. coli strain K-12 the ability to damage plants or plant parts (Keen and Tamaki, Journal of Bacteriology 168:595-606, 1986; and, Payne et al.. Applied and Envi- ronmental Microbiology 53:2315-2320, 1987). Each of these studies showed that pectate lyase genes cloned from Erwinia chrysanthemi (a bacterium that incites the soft rot disease of potatoes), when expressed at high levels on an expression vector in E. coli, could confer on the E. coli the ability to cause rot symptoms in potato tubers. APHIS cannot, therefore, accept the commenter's suggestion to remove the phrase "cell wall degrading enzymes" as an example of a factor directly involved in plant disease, and of concern for its potential to confer plant pest characteristics on the host microorganisms. One commenter suggested that APHIS add another condition as an additional requirement for moving plant pest genetic material in the three host microorganisms. The commenter suggested that APHIS require that the cloned genetic material not code for a gene which extends the host range of a plant pest. This commenter further suggested that APHIS amend the exclusion so as not to require a permit for the interstate movement of Agrobacterium strains containing Ti plasmids. APHIS has not

amended the rule to include this additional condition because the Agency is unaware of and has been unable to identify any gene that extends the host range of a plant pest, which if expressed in the three host microorganisms would have the potential to confer plant pest characteristics on these three microorganisms. APHIS rejects the commenter's suggestion that a person should be allowed to move strains of Agrobacterium containing Ti plasmids without a permit. Unlike the three host microorganisms specified in the exemption, Agrobacterium tumefaciens is a plant pest as defined by the Federal Plant Pest Act [FPPA:21 U.S.C. 150aa-jj], and in implementing regulations in 7 CFR Parts 330 and 340. A. tumefaciens incites diseases that cause substantial crop losses in vineyard and orchard crops. Ti plasmids are essential to the disease causing mechanism of ^4. tumefaciens. As such, under the FPPA and implementing regulations, a permit is required for the interstate movement of this microorganism or its plasmids. It has been shown in laboratory settings that the Ti plasmids of A. tumefaciens can be effectively disarmed, that is, can be made nonpathogenic.

APHIS is reviewing permit applications for the introduction of an organism containing a disarmed Ti plasmid examines the effectiveness of the disarming techniques as well as biosafety data on the organism.

One commenter requested that Pseudomonas fluorescens, biotype A, Pseudomonas chloraphis (P. fluorescens, biotype D) and Pseudo- monas aureofaciens (P. fluorescens, biotype E) be treated like the three host microorganisms for purposes of allowing the interstate movement of plant pest genetic material without a permit. The commenter further requested that the nonplant pest status be established for: (1) Native forms of the pseudomonads referenced above selected for their inherent resistance to antiobiotics: (2) Genetically recombinant forms of the pseudomonads referenced above

IV-2

which meet the conditions specified in the exemption, and (3) Pseudomonads referenced above which have been genetically modified to contain E. coli K-12 genes which have been permanently inserted. APHIS disagrees with the commenter that the three subgroups of the pseudomonads referenced above would be suitable organisms for the movement of plant pest genetic material. While the strains oí Pseudomonas that make up the subgroups referenced by the commenter are not generally considered plant pests in and of themselves, APHIS believes that because they can persist in the environment much more readily than the three host microorganisms and because there is not sufficient biosafety data on these strains of Pseudomonas when used as cloning hosts, it would be ill advised to allow such strains to be used for the movement of plant pest genetic material, as set forth in the exemption. Accord- ingly, APHIS has not included these three subgroups of the pseudomonads in the final rule.

With respect to the commenter's request that the nonplant pest status be established for the pseudomonads referenced above, the proper way to make such a request would be to submit a petition under § 340.4 of the regulations.

Section 340.4 is entitled, "Petition to amend the list of organisms" and is intended to be used for petitioning APHIS to add or delete any genus, species, or subspecies of organisms on the list of plant pest organisms in § 340.2 of the regulations. The information submitted in the comment does not meet the requirements set forth in § 340.4 and as such does not serve as an adequate petition. APHIS will be able to consider the commenter's request for non plant pest status of these pseudomonads if the request is submitted in the form of a 340.4 petition.

One commenter suggested that § 340.2(b)(l)(iii) of the exemption be amended so as to prohibit the cloned genetic material from containing any

portion of the genome of a plant pest rather than prohibiting the genetic material from containing the complete genome from a plant pest. The commenter did not present any examples where genetic material from a portion of the genome of a plant pest had been able to incite disease nor did the commenter present citations to the scientific literature where it had been documented that a portion of the genome from a plant pest when carried by any of the three host microorganisms would present a potential risk. APHIS has been unable to identify any situation where a risk plant pest introduction or dissemination would occur by allowing less than the complete infectious genome from a known plant pest to be carried by one of three host microorganisms under the conditions specified in the exemption. It should also be noted that this exemption is limited to the movement of certain microorganisms under specified conditions. One commenter suggested that the conditions of the exemption be made more inclusive. Specifically, the commenter suggested that APHIS add working to the exemption that would prohibit a person from shipping cloned genetic material on an expression vector in one of the three host microorganisms if the cloned genes code for a substance that is beneficial to an organism that indirectly causes damage to plants. The commenter failed to provide a specific example of the type or category of substance which the commenter was referring to and which the commenter thought APHIS should protect against. In the absence of a specific example of the type of substance which the commenter is referring to, APHIS finds this comment too hypothetical and not appropriate for consideration at this time. Further, APHIS believes that the specific conditions that are included in the rule cover those cases where a specific plant pest risk could be identified. Therefore, no change has been made to the rule based on this comment.

Executive Order 12291 and Regulatory Flexibility Act

This rule is issued in conformance with Executive Order 12291 and has been determined to be not a "major rule." Based on information compiled by the Department, it has been determined that the rule will not have a significant effect on the economy; will not cause a major increase in costs or prices for consumers, individual industries. Federal, State, or local government agencies, or geographic regions; and would not have a significant adverse effect on competition, employment, investment, productivity, innovation, or on the ability of United States-based enterprises to compete with foreign-based enterprises in domestic or export markets.

The effect of this rule is to exempt a person from having to obtain a limited permit when certain genetic material from a plant pest is moved interstate in certain microorganisms in accordance with specified conditions. A limited permit requires the submission of data about the nature of the organism, how it was produced, and a description of the contained facility at destination. Such data should already be available to the researcher. The rule relieves a person from having to submit an application to APHIS for a limited permit, which results in a savings of the time which would ordinarily be associated with the preparation of such a permit application. Therefore, the deletion of the requirement to submit the data to obtain a limited permit will not have a significant economic impact on a substantial number of small entities.

The conditions that have to be complied with under the exemption are those that a researcher would normally employ when using these microorganisms as gene libraries, except that a person cannot slip genes that code for substances harmful to plants or organisms beneficial to plants, when such genes are carried on an expression vector. If such types of genetic material are expressed on an expression vector, the exemption does not apply, and a limited permit would

IV-3

have to be obtained. It is expected that this exemption will affect at least several thousand research scientists, some of whom may be operating small businesses which would be deemed "small entities" under the Regulatory Flexibility Act. The rule exempts them from the requirement of having to obtain a limited permit under the circumstances described above.

Under these circumstances, the Administrator of the Animal and Plant Health Inspection Service has determined that this action will not have a significant economic impact on a substantial number of small entities.

Effective Date Pursuant to the administrative

procedure provisions in 5 U.S.C. 553, the Acting Administrator of the Animal and Plant Health Inspection Service finds good cause for making this rule effective less than 30 days after publication in the Federal Register. The Acting Administrator has determined that this rule should become effective upon publication because it has been determined that this rule relieves unnecessary restrictions. The rule exempts persons from having to obtain a limited permit when certain genetic material from a plant pest is moved interstate in specified microorganisms under conditions which do not present a risk of plant pest introduction or dissemination.

Paperwork Reduction Act. This rule contains no information

collection or recordkeeping requirements under the Paperwork Reduction Act of 1980 (44 U.S.C. 3501 et seq.].

Executive Order 12372 This program/activity is listed in the

Catalog of Federal Domestic Assistance under No. 10.025 and is subject to the provisions of Executive Order 12372, which requires intergovernmental consultation with State and local officials. (See 7 CFR Part 3015, Part V).

List of Subjects in 7 CFR Part 340 Agricultural commodities, Biotech-

nology, Genetic engineering. Plant

diseases. Plant pests, Plants (Agricul- ture), Quarantine, Transportation.

PART 340-INTRODUCTION OF ORGANISMS AND PRODUCTS ALTERED OR PRODUCED THROUGH GENETIC ENGINEERING WHICH ARE PLANT PESTS OR WHICH THERE IS REASON TO BELIEVE ARE PLANT PESTS

Accordingly, 7 CFR Part 340 is amended as follows:

1. The authority citation for Part 340 continues to read as follows:

Authority: 7 U.S.C. 150aa-150jj, 151-167, 1622n; 31 U.S.C. 9701; 7 CFR 2.17, 2.51, and 371.2(c).

2. Section 340.1 is amended by adding the following definition of "Expression vector" in alphabetical order to the list of existing terms:

§340.1 Definitions. *****

Expression vector. A cloning vector designed so that a coding sequence inserted at a particular site will be transcribed and translated into protein.

3. The heading for § 340.2 "Groups of organism which are or contain plant pests" is revised to read "Groups of organisms which are or contain plant pests and exemptions."

4. In § 340.2, at the beginning of the introductory text, a new paragraph heading is added to read "(a) Groups of organisms which are or contain plant pests. "

5. In § 340.2 at the end of the text, a new paragraph (b) is added as follows:

§ 340.2 Groups of organisms whicli are or contain part pests and exemptions. *****

(b) Exemptions. (1) A limited permit for interstate movement shall not be required for genetic material from any plant pest contained in Escherichia coli genotype K-12 (strain K-12 and its derivatives), sterile strains oí Saccha- romyces cerevisiae, or asporogenic strains of Bacillus suhtilis, provided that all the following conditions are met:

(i) The microorganisms are shipped in a container that meets the requirements of § 340.6(b)(3) of this part;

(ii) The cloned genetic material is maintained on a nonconjugation proficient plasmid and the host does not contain other conjugation proficient plasmids or generalized transduc- ing phages;

(iii) The cloned material does not include the complete infectious genome of a known plant pest;

(iv) The cloned genes are not carried on an expression vector if the cloned genes code for:

(A) A toxin to plants or plant products, or a toxin to organisms beneficial to plants; or

(B) Other factors directly involved in eliciting plant disease (i.e., cell wall degrading enzymes); or

(C) Substances acting as, or inhibitory to, plant growth regulators.

Done in Washington, DC, this 15th day of April, 1988. James W. Glosser, Acting Administrator, Animal and Plant Health Inspection Service.

[FR Doc. 88-8690 Filed 4-19-88; 8:45 am] BILLING CODE 3410-34-M

IV-4

Federal Register / Vol. 55, No. 250 / Friday, December 28, 1990 / Rules and Regulations 53275

DEPARTMENT OF AGRICULTURE


7 CFR Part 340

[Docket No. 90-172]

Genetically Engineered Organisms and Products; Exemption for Inter- state Movement of the Genetically- Engineered Plant ("Arabidopsis thaliana") Under Specified Conditions

AGENCY; Animal and Plant Health Inspection Service, USDA.

ACTION: Final rule.

SUMMARY: This document amends the regulations pertaining to the introduction of certain genetically engineered organisms and products by exempting from regulation certain genetically engineered plants that are moved interstate under specified conditions. This rule removes unnecessary restrictions on the interstate movement of a genetically engineered plant, Arabidopsis thaliana, w^hich does not present a risk of plant pest introduction or dissemination.

EFFECTIVE DATE: January 28, 1991.

FOR FURTHER INFORMATION CONTACT: Dr. Catherine Joyce, Biotechnologist, Biotechnology, Biologies, and Environ- mental Protection, Animal and Plant Health Inspection Service, U.S. Department of Agriculture, Room 844, Federal Building, 6505 Belcrest Road, Hyattsville, MD 20782, (301) 436-6789.

SUPPLEMENTARY INFORMATION:

Background On July 12, 1990, a proposed rule

was published in the Federal Register (55 FR 28637-28638, Docket No. 90- 052) to exempt from permitting requirements under 7 CFR part 340 the interstate movement of genetic material from any plant pest that is incorporated in the genome of the plant Arabidopsis thaliana. A 30-day

comment period, v^hich ended August 13, 1990, was provided for in that proposal.

The Animal and Plant Health Inspection Service (APHIS) received fourteen comments regarding that proposed rule. Comments were received from members of the university research community, industrial research community, and a State Department of Agriculture. A discus- sion of these comments is presented below. APHIS has seriously considered all comments on the proposed rule.

Based on the rationale set forth in the proposed rule and this document, APHIS is adopting the provisions of the proposal as a final rule.

In the final rule, a limited permit for interstate movement is not required for genetic material from any plant pest contained in the genome of the plant Arabidopsis thaliana, provided all of the following conditions are met:

(1) The plant materials are shipped in a container that meets the requirements of § 340.6(b) (1), (2), and (3) of this part;

(2) The cloned genetic material is stably integrated into the plant genome; and

(3) The cloned material does not include the complete infectious genome of a known plant pest.

The final rule does not amend the permit requirements concerning release into the environment or importation.

The final rule includes the prepared definition of the term "stably integrated."

Public Comments Thirteen comments were in support

of the rule as proposed. One of these comments in support of the rule stated further that the cloned genetic material need not be integrated and that, except for the cloned infectious genome of exotic plant pests, the exemption should be extended to include all plants that are moved within contained facilities.

APHIS does not agree that such an exemption should be extended to all plants across the board. Rather APHIS

believes that sufficient uncertainty and unfamiliarity exists with newly engineered plant species that exemptions should only be granted on a case by case basis. Thus APHIS does not adopt the comment proposing changes to the rule.

One comment opposed the proposal and questioned the need for the rule since an exemption would allow movement without oversight to and from unnamed facilities under the assumption that all shipments are adequately packaged and contained. Moreover the comment raised the concern that Arabidopsis thaliana, while not considered a weed of any consequence, nevertheless has many characteristics (short generation time, high seed set, ease of growth, self- and cross-pollinating ability) that encour- ages its weed potential and ease of spread.

APHIS agrees that Arabidopsis thaliana has several weedy characteristics; however, the plant has been widely used in genetics research for many decades, and has been exten- sively distributed in the environment by agriculture and horticultural for much longer. None of these potential introductions have demonstrated any particular need for concern about aggressive weediness. Furthermore, the exemption from permitting requirements is only for interstate movement between contained facilities under specified shipping conditions. Any release into the environment would require a permit. Because APHIS believes that interstate movement between contained facilities of Arabidopsis thaliana does not present a risk of plant pest introduction or dissemination, the rule is adopted as proposed.

Executive Order 12291 and Regulatory Flexibility Act

This rule is issued in conformance with Executive Order 12291 and has been determined not to be a "major rule." Based on information compiled by the Department, it has been determined that this rule will have an effect on the economy of less than $100

IV-5

million; will not cause a major increase in costs or prices for consumers, individual industries, Federal, State, or local government agencies, or geographic regions; and will not cause a significant adverse effect on competition, employment, investment, productivity, innovation, or on the ability of United States-based enterprises to compete with foreign-based enterprises in domestic or export markets.

The effect of this rule will be to exempt a person from having to obtain a limited permit when certain genetic material from a plant pest, contained within the genome of the plant Arabidopsis thaliana, is moved interstate. Currently, such a limited permit requires the submission of data regarding the nature of the organism, how it is produced, and a description of the contained facility at destination. Such data should already be available to the researcher. This rule relieves a person from having to submit an application to APHIS for a limited permit, resulting in a saving of time that would ordinarily be associated with preparation of such a permit application. Therefore, the deletion of the requirement to submit the data to obtain a limited permit should not have a significant economic impact on a substantial number of small entities.

The conditions that will have to be complied with under the exemption are those that a researcher would normally employ when using Arabidopsis thaliana as an experimental organism. It is expected that this exemption will affect several hundred research scientists, some of whom may be operating small businesses that would be deemed small "entities" under the Regulatory Flexibility Act. This amendment will exempt them from the requirements of having to obtain a limited permit under the circumstances described above. Under

these circumstances, the Administrator of the Animal and Plant Health Inspection Service has determined that this action will not have significant economic impact on a substantial number of small entities.

Paperwork Reduction Act This rule contains no new informa-

tion collection or recordkeeping requirements under the Paperwork Reduction Act of 1980 (44 U.S.C. 3501 et seq.)

Executive Order 12372 This program/activity is listed in the

Catalog of Federal Domestic Assistance under No. 10.025 and is subject to Executive Order 12372, which requires intergovernmental consultation with State and local officials. (See 7 CFR part 3015, subpart V.)

List of Subjects in 7 CFR Part 340 Agricultural commodities. Biotech-

nology, Genetic engineering. Plant disease, Plant pests. Plants (Agricul- ture), Quarantine, Transportation.

Accordingly, we are amending 7 CFR part 340 as follows:

PART 340-INTRODUCTION OF ORGANISMS AND PRODUCTS ALTERED OR PRODUCED THROUGH GENETIC ENGINEERING WHICH ARE PLANT PESTS OR WHICH THERE IS REASON TO BELIEVE ARE PLANT PESTS

1. The authority citation for 7 CFR part 340 continues to read as follows:

Authority: 7 U.S.C. 150aa-150jj, 151- 167, 1622n; 31 U.S.C. 9701; 7 CFR 2.17, 2.51, and 371.2(c).

2. In § 340.1, the following definition is added in alphabetical order to read as follows:

§ 340.1 Definitions. *****

Stably integrated. The cloned genetic material is contiguous with elements of the recipient genome and is replicated exclusively by mechanisms used by recipient genomic DNA. *****

3. In § 340.2, a new paragraph (b)(2) is added to read as follows:

§ 3402 Groups of organisms wliich are or contain plant pests and exemptions. *****

(b) * * * (2) A limited permit for interstate

movement is not required for genetic material from any plant pest contained in the genome of the plant Arabidopsis thaliana, provided that all of the following conditions are met:

(i) The plants or plant materials are shipped in a container that meets the requirements of § 340.6(b) (1), (2), and (3) of this part;

(ii) The cloned genetic material is stably integrated into the plant genome;

(iii) The cloned material does not include the complete infectious genome of a known plant pest.

Done in Washington, DC, this 21 day of December 1990. James W. Glosser, Administrator, Animal and Plant Health Inspection Service. [FR Doc. 90-30382 Filed 12-27-90; 8:45 am] BILLING CODE 3410-34

IV-6

Instructions for Submitting Confidential Business Information (CBI) and CBI-Deleted Information

1. Financial or commercial information the applicant does not want disclosed for competitive reasons can be claimed as confidential business information (CBI). Applicants must submit a written justification to support each claim.

2. "Trade secrets" (information relating to the production process, such as formulas, processes, quality control tests and data, and research methodology) may be claimed as CBI. This information must be (1) commercially valuable, (2) used in the applicant's business, and (3) maintained in secrecy.

3. Each page containing CBI material must have "CBI Copy" marked in the upper right corner of the page. In the right margin, mark the CBI information with a bracket and "CBI."

4. The CBI-deleted copy should be a facsimile of the CBI copy, except for spaces occurring in the text where CBI has been deleted. Be sure that the CBI-deleted copy is pagi- nated the same as the CBI copy. (The CBI-deleted copy of the application should be made from the same copy of the application which originally contained CBI.) Additional material (transitions, paraphrasing, or generic substitutions, etc.) should not be included in the CBI-deleted copy.

5. Each page with CBI-deletions should be marked "CBI- deleted" at the upper right corner of the page. In the right margin, mark the place where the CBI material has been deleted with a bracket and "CBI-deleted."

6. If several pages are CBI-deleted, a single page designat- ing the numbers of deleted pages may be substituted for blank pages. (For example, "pages 7 through 10 have been CBI-deleted.")

7. All published references that appear in the CBI copy should be included in the reference list of the CBI-deleted copy. Published information usually cannot be claimed as confidential.

9. Direct any questions to:

Document Control Officer Biotechnology Permits Biotechnology, Biologies, and Environmental Protection 6505 Belcrest Road Hyattsville, Maryland 20782 Phone: Area Code (301) 436-7612

V-1

Federal Register / Vol. 50, No. 184 / Monday, September 23, 1985 / Notices 38561


[Docket No. 85-406]

Policy Statement on the Protection of Privileged or Confidential Business Information

This notice sets forth in full a document establishing the policy of the Animal and Plant Health Inspec- tion Service for protecting certain privileged or confidential business information. This document reads as follov^s:

APHIS POLICY STATEMENT ON THE PROTECTION OF PRIVILEGED OR CONFIDENTIAL BUSINESS INFOR- MATION

L Purpose

The purpose of this policy statement is to establish minimum requirements to control and protect documents received by the Animal and Plant Health Inspection Service (APHIS) that in its judgement contain privileged or confidential business information (CBI), as defined in section IV-E of this policy statement, concerning biotechnology and the Veterinary Biologies Program.

II. Policy

Title 7, Code of Federal Regulations, sections 1.1-1.16 contain the regulations of the United States Department of Agriculture (USDA) implementing the Freedom of Information Act (FOIA) (5 U.S.C. 552). The FOIA generally provides that federal agencies must make available to the public all records not specifically exempt from disclosure. Section (b)(4) of the FOIA exempts from disclosure "trade secrets and commercial or financial information obtained from a person and privileged or confidential." 5 U.S.C. 552(b)(4). This policy statement applies to the disclosure of CBI concerning biotechnology and the Veterinary Biologies Program. APHIS

v^ill release such CBI only if disclosure is otherwise required by Iav\^, such as a specific statute or court order, by the source of the information, or as provided herein. In addition, APHIS employees shall take whatever measures are necessary to preclude unauthorized disclosure.

APHIS employees who make unauthorized disclosures of information classified as CBI can be subject to prosecution under the Trade Secrets Act, 18 U.S.C. 1905. Under this statute, a federal employee who discloses trade secrets and certain confidential data without authorization shall be fined up to $1,000 and/or imprisoned for up to one year.

III. Applicable Statutes

A. Freedom of Information Act Section (b)(4), 5 U.S.C. 552(b)(4)

B. Trade Secrets Act, 18 U.S.C. 1905

IV. Definitions

A. Access

The ability and opportunity to gain knowledge of Confidential Business Information in any manner.

B. Administrator

The Administrator of the Animal and Plant Health Inspection Service, United States Department of Agricul- ture, or any other official of the Animal and Plant Health Inspection Service to whom the Administrator has delegated authority to act.

C. Autliorized User

An APHIS employee or other person whom the Administrator has certified as requiring access to Confidential Business Information.

D. Biotechnology

Any technique that uses living biological systems to make or modify products, to improve plants or animals.

or to develop microorganisms for specific uses.

E. Confidential Business Information (CBI)

Information that would be protected from disclosure under section (b)(4) of the Freedom of Information Act (5 U.S.C. 552(b)(4)) will be classified as Confidential Business Information (CBI). This includes trade secrets and commercial or financial information found to the confidential.

1. Trade Secrets

Documents containing trade secrets and which the person submitting asserts are trade secrets will be deemed CBI. "Trade secrets" means information relating to the production process. This includes production data, formulas, and processes, and quality control tests and data, as well as research methodology and data generated in the development of the production process. Such information must be (1) commercially valuable, (2) used in one's business and (3) maintained in secrecy.

2. Commercial or Financial Informa- tion

Documents containing commercial or financial information will be deemed confidential if review estab- lishes that substantial competitive harm would result from disclosure. Information such as safety data, efficacy or potency data, and environmental data may be such confidential information. Persons desiring protection for confidential information must submit a detailed statement containing facts to show that the person faces active competition in the area to which the information relates, and that substantial competitive harm would result from disclosure.

F. Destruction

Pulverization by a paper shredder, burning, or other approved method.

V-2

G. Information

Knowledge that can be communi- cated by any means.

H. Secured Storage Area

A room or equipment that is locked.

I. Staff Office

A staff administering a particular program within the Animal and Plant Health Inspection Service.

/. Unique Identification Number

The number permanently assigned to a document containing CBI when the document is logged in, and which enables the document to be tracked. Each document containing CBI will be assigned a separate number.

V. Identification of Confidential Business Information

A. The applicable staff office shall review documents it receives to determine whether they contain Confidential Business Information.

B. The applicable staff office shall log in Confidential Business Informa- tion it receives, and shall assign unique identification numbers to allow for tracking.

C. A red cover sheet printed with the unique identification number shall be attached to the original and each copy of a CBI document.

D. In addition to the cover sheet, each page of each copy of a CBI document shall be stamped "CONFI- DENTIAL."

E. A record of each copy of a CBI document and its disposition shall be maintained.

VI. Physical Security of Confidential Business Information

A. Storage of CBI Documents

1. CBI documents are to be stored in secured storage areas when not in use.

2. At the close of a business day, doors in secured work areas shall be locked, alarms activated if appropriate, and documents containing CBI in unsecured work areas shall be placed in secured storage.

B. Access To CBI Documents By Authorized Users

1. The APHIS Administrator's office will maintain a list of persons authorized to have access to Confidential Business Information, and will furnish this list to staff office supervisors.

2. Persons will receive training on safeguarding CBI before obtaining authorized user status.

3. Authorized users shall obtain CBI documents through a person in each applicable staff office designated to be responsible for document control.

4. Requestors must present identification when obtaining CBI material from the applicable staff office. The requestor's name must appear on the authorized user list.

5. For each person on the authorized user list, a charge-out record will be kept. Requestors shall sign the record when receiving and returning CBI documents.

6. Person who terminates USDA employment will not receive exit clearance until all CBI documents that were charged out to the employee have been returned.

7. When an authorized user no longer requires access to CBI, the locks to which the person has had access must be changed.

C Safeguards During Individual Use of CBI Documents

1. All CBI documents must be handled by authorized personnel only.

2. Authorized users shall not in any manner disclose Confidential Business Information to unauthorized persons. Authorized users shall determine whether persons are authorized to have access to CBI before discussing CBI with them.

3. When unauthorized persons are present, CBI documents must be

covered, turned face down, removed from the area, or otherwise protected.

4. All persons are individually responsible for securing any CBI documents in their possession. When persons are reviewing or processing documents containing CBI, the documents are their responsibility until they are returned to the staff person responsible for document control. Persons handling CBI documents must secure them before leaving their work area.

5. Where working areas cannot provide privacy, private meeting areas will be provided for review of CBI documents.

6. Each person must safeguard keys to files, safes, rooms, etc. Keys to CBI files must be kept in a secured place. Lost keys or suspected breaches of security must be reported immediately to the person responsible for document control, so that changes can be immediately effected.

D. Meetings

Precautions shall be taken so mat unauthorized persons are not present at meetings where CBI is discussed.

VII. Copying and Destruction

A. Photocopying

1. Reproduction of documents containing Confidential Business Information shall be kept to a minimum.

2. A record of each copy of a CBI document and its disposition shall be maintained.

3. Bad copies shall be destroyed.

B. Destruction

1. The person responsible for document control in each applicable staff office shall keep records of copies of CBI documents and their disposition.

2. The person responsible for document control shall perform any destruction of documents containing CBI.

V-3

3. When users of CBI documents have no further need for them, they shall return CBI documents to the staff office from which they obtained them. Unneeded copies will be destroyed in the staff office.

VIII. Transfer of Confidential Business Information

A. Within APHIS

1. The applicable staff office shall assign unique identification numbers to CBI documents it receives. Copies to be sent to field offices or laboratories shall also be marked with the unique identification number.

2. All transfers of CBI materials within APHIS shall be recorded by the person responsible for document control in each applicable staff office.

3. All CBI documents transferred to field offices, laboratories, or other parts of APHIS must be logged in and out by a person responsible for document control in that office.

(a) Incoming CBI documents will be entered in the log, using the previously assigned unique identification number printed on the cover sheet.

The date of receipt will be recorded. (b) Documents will then be filed in

secured file cabinets. 4. Field offices and laboratories shall

maintain security procedures equivalent to those described in this document. Field offices and laboratories shall be responsible for tracking and disposition of the CBI documents in their files.

will be notified of any requests by the public for disclosure and the scope of information to be disclosed, if any.

C. Mail

CBI documents shall be transmitted by registered mail, return receipt requested.

Effective date: September 18, 1985. Dated: September 18, 1985. Bert W. Hawkins, Administrator, Animal and Plant Health Inspection Service. [FRDoc. 85-22715 Filed 9-20-85; 8:45 am] BILLING CODE 3410-34-M

B. From APHIS to Other Parts of USD A, Other Federal Agencies, and Other Persons

1. Persons from outside of APHIS must show that they need Confidential Business Information for a proper official purpose.

2. Persons from outside of APHIS must maintain security procedures equivalent to those of APHIS before they may receive Confidential Business Information.

3. The person submitting the CBI

V-4

aphis-users guide for introducing genetically engineered plants and microorganisms 1997b

Documents

director biotechnology

director of biotechnology

recipient organism

plant pest components

donor organism

unclassified organism

regulated article

aphis regulations