**Procedures for Growing and Agronomic Testing of**

**Transgenic Barley (*Hordeum vulgare*) and Wheat (*Triticum aestivum*)
Plants**

**(Biosafety Level 1-P)**

**USDA-ARS, National Small Grains Germplasm Research Facility**

**Aberdeen, Idaho 83210**

OUTLINE

I. Greenhouse Growth

a\. Location

b\. Planting

c\. Maintenance

d\. Harvesting and Cleaning

e\. Seed Storage

f\. Documentation

II\. Field Tests\--Year(s) Transgenic Plants are Grown

a\. Location

b\. Seed Preparation

c\. Planting

d\. Plot Maintenance and Observations

e\. Harvest

f\. Transportation

g\. Post-harvest Operations

h\. Seed Storage

i\. Documentation

III\. Field Tests\--Years Following Growth of Transgenic Plants

a\. Elimination of Volunteer Plants

b\. Inspection and Verification

IV\. Destruction of Unwanted Seed

V. Unanticipated Events\--Response Procedures and Notifications

a\. Immediate response to unintended environmental releases

B. Notification

I. **[Greenhouse Growth]{.underline}**

> **a. [Location]{.underline}**
>
> **i. [Transgenic plants]{.underline}**\--All transgenic plants shall
> be grown in the southwest section of the west greenhouse wing unless
> otherwise specified. This section has been inspected by APHIS
> officials and determined to be suitable for such use.
>
> ii\. [Non-transgenic plants]{.underline} that are grown in the same
> greenhouse section can potentially pollinate non-transgenic plants of
> compatible species. All viable seed developing on non-transgenic
> plants of compatible species grown in the same greenhouse section is
> to be treated as potentially transgenic unless measures were taken to
> prevent uncontrolled pollination (i.e., protection via glassine bags).
> NOTE\--PROTECTION OF THE NATIONAL SMALL GRAINS COLLECTION (NSGC): NSGC
> seed increases are conducted in contiguous sections of the west
> greenhouse wing. Although the potential for pollen flow between
> greenhouse sections is minimal, it is vitally important that NSGC
> accessions not be contaminated with transgenic pollen. Therefore,
> special precautions will be taken. NSCG growouts of these species will
> be separated either temporally (i.e., widely spaced planting dates so
> that no transgenic pollen is present during the flowering of NSGC
> accessions) or spatially (NSGC accessions placed in non-contiguous
> greenhouse sections).
>
> **b. [Planting]{.underline}**\--A soilless potting mix (2:2:2
> sand:peat:vermiculite or equivalent) supplemented with an appropriate
> amount of slow-release fertilizer (type and amount will vary; follow
> manufacturer\'s instructions on rate) will be prepared in the
> headhouse and used for all transgenic plants. Transgenic seeds can be
> placed in potting mix (in pots; size will vary depending on the
> situation) either in the headhouse or the southwest-west greenhouse
> section. Care should be taken to prevent accidental dispersal of
> transgenic seeds; such accidents can be minimized or eliminated by
> following these procedures: 1) Clean a work area prior to planting; 2)
> Work with only one transgenic line (packet of seed) at a time; 3)
> Carefully handle seed packets to avoid accidentally spilling the seed,
> and all spilled seed should be picked up and repackaged prior to
> opening the next seed packet; do NOT repackage seed if there is ANY
> question to its identity; instead put such seed in a separate envelope
> and mark it for disposal 4) Transport planted pots of seed with a cart
> to the southwest-west greenhouse; 5) Clean all work areas when the
> planting process is completed to further insure that no accidentally
> spilled seed remains; 6) Any spilled seed which cannot be positively
> identified, or soil which may contain unidentifiable seed, should be
> devitalized by autoclaving for 1 hour at 121 ^o^C prior to disposal in
> normal garbage.
>
> **c. [Maintenance]{.underline}\--**maintain transgenic plants in the
> same manner as non-transgenic plants with daily watering until plants
> are mature. Transgenic plants should be treated with Marathon
> insecticide (see SOP for Marathon use) to prevent aphids; other
> insecticides and fungicides should be applied as necessary.
>
> **d. [Harvesting and Cleaning]{.underline}:** All of the areas and
> equipment used in these operations must be thoroughly cleaned,
> inspected, and reinspected prior to returning it to general service.
> Prior to such cleaning and inspections, equipment will be labeled as
> Acontaminated\--do not use@. When possible, cleaning operations will
> be performed in the small cleaning room adjoining the main clearing
> room. Use of the main cleaning room for transgenic seed cleaning will
> not take place at the same time that non-transgenic grain cleaning
> operations are being conducted.
>
> **i. [Greenhouse operations]{.underline}**[\--]{.underline}harvest
> transgenic plants either by placing heads in envelopes or bags for
> transport to the cleaning room or for storage, or by threshing them in
> the greenhouse or in the work areas outside of the greenhouse.
> Precautions should be taken to avoid accidentally disseminating
> transgenic seeds; 1) When using the small Vogel-type thresher use a
> screened enclosure (or equivalent) to catch chaff and light seeds as
> they blow out of the thresher; 2) Place seed or heads into sound bags
> or envelopes, and close and box them immediately to prevent accidental
> spillage; this seed may be further cleaned as detailed below; 3) Sweep
> work areas immediately after harvesting operations (or several times
> during harvesting if work continues for extended periods of time); 4)
> Collect all chaff which may be contaminated with seed and autoclave
> for one hour at 121 ^o^C prior to disposal in normal garbage. Mature
> heads, removed from the plants, can also be transported to the
> cleaning room for these operations (see below); make sure all seed or
> heads are enclosed in sound bags to prevent accidental spillage prior
> to transportation; 5) Remaining plant materials which may be
> contaminated with viable seed (stems, roots, and associated soil)
> should be autoclaved for one hour at 121 ^o^C prior to disposal with
> other discarded plant materials (disposal site located at the
> northwest corner of the greenhouse).
>
> **ii. [Cleaning Room operations]{.underline}\--**As for greenhouse
> operations, the objective is to prevent escape of seed and plant
> materials into the environment. Place unthreshed heads it in a cut-off
> hot water bottle and smack it against the bench to remove grain from
> the heads. Grain threshed in this manner generally is ready for air
> cleaning (see step 2 below); barley seed threshed as specified in
> section I.d. i. with the Vogel thresher generally will need to be
> treated additionally to remove awns from seed: 1) Place seed in the
> deawning machine to remove attached awns\-- for very small quantities
> of seed use of the water bottle as specified above will be more
> efficient and effective at removing awns; 2) Pour seed slowly into an
> air separator to remove chaff\--be sure that the catch tray is in
> place prior to pouring the grain in the machine; and 3) Place cleaned
> seed into sound bags or envelopes, and close and box them immediately
> to prevent accidental spillage. The chaff may be contaminated with
> seed; collect the chaff and any contaminating seed and autoclave for
> one hour at 121 ^o^C prior to disposal in normal garbage.
>
> **e. [Seed Storage]{.underline}**\--Transgenic seed must be clearly
> labelled, and can be stored in any of the following locked areas, as
> appropriate to the length of anticipated storage, as specified by the
> supervisor: 1) In Seed Lab #1; 2) In the Tissue Culture Laboratory
> (room 113) in the drawer reserved for seed storage; 3) In the
> supervisors office (room 108); or 4) In the short- or long-term
> storage facilities of the NSGGRF.
>
> **f. [Documentation]{.underline}\--**Proper documentation will vary
> with the particular job; details will be communicated by the
> supervising project leader. In general, however, documentation will
> involve 1) Labeling envelopes or bags containing transgenic seed
> [before]{.underline} the seed is placed in the bags; 2) Labeling
> larger boxes used to store the envelopes or bags; 3) Properly storing
> the boxed seed as detailed in section I. e. above; and 4) Recording
> the devitalization (via autoclaving) of seed lots. Should any problems
> arise that could result in accidental environmental release of the
> seeds, such as spilling seed in areas in which may be difficult to
> fully clean (for example, the ground outside of the greenhouse),
> inform the supervisor. Such seed should be collected as completely as
> possible and destroyed by autoclaving for one hour at 121 ^o^C before
> disposal in normal garbage. Procedures will then be developed to
> monitor the area and destroy any transgenic seedlings that may
> volunteer. In addition, seedlots (a whole bag or envelope) that may
> have potentially been mislabeled (but not spilled) because of handling
> errors should be marked as such, and the supervisor informed of such
> potential identity problems. The supervisor will determine if such
> mislabeled seed should be destroyed or if efforts to ascertain its
> identity should be pursued.

**II. Field Tests\--Year(s) Transgenic Plants are Grown**

> **a. [Location]{.underline}\--**Transgenic plants should be located in
> areas which minimize the potential for pollen dissemination to
> non-transgenic plants of compatible species in adjacent fields.
> Transgenic plants will be planted at least 100 feet from
> non-transgenic compatible species, except for non-transgenic plants
> that are planted as experimental controls. Such non-transgenic
> controls will be handled and disposed of as if it were transgenic.
> NOTE\--PROTECTION OF THE NATIONAL SMALL GRAINS COLLECTION: Transgenic
> plants are not to be grown within 600 feet of NSGC increases of any
> compatible species.
>
> **b. [Seed Preparation]{.underline}**\--Specific procedures will vary
> depending on the type of plots to be planted, as communicated by the
> supervisor, but in general will involve relatively large quantities of
> transgenic seed which must be prepared for error-free, semi-automated
> planting using small-plot grain drills. All preparation operations
> will be conducted in Seed Lab #2. All seed will be placed into sound
> envelopes and packed into wooden planting trays to facilitate easy
> transportation, prevent accidental spillage, and facilitate accurate
> planting. Off-station field locations will require transport over
> public highways and will require special packaging; see section II. f.
> below.
>
> **c. [Planting]{.underline}\--**Specific procedures will vary
> depending on the type of plots to be planted, as communicated by the
> supervisor. In all cases care must be taken to transport seed
> envelopes without spilling transgenic seed; any spillage must be
> immediately cleaned to the extent possible and recorded to facilitate
> future efforts to ensure that accidental environmental releases are
> destroyed; spilled seed should be placed in sound envelopes or bags
> and marked for destruction via autoclaving It is particularly
> important that such spills are recorded for areas outside of the test
> plot location, since such areas would not otherwise be included in
> future inspections for volunteer plants. All planting machinery should
> be thoroughly cleaned and inspected as necessary to prevent
> inadvertently transporting any seeds that have accidentally spilled on
> machinery surfaces, or which have become adhered to machinery
> surfaces, prior to removing equipment from the field in which the
> transgenic field trials are being conducted. Such seeds should be
> collected and bagged if possible, and autoclaved for one hour at 121
> ^o^C prior to disposal in normal garbage. Some seed will be impossible
> to collect, but volunteer plants arising from these seeds can be
> destroyed once they germinate, either in the test year or in
> subsequent years (see section III.) NOTE\--PROTECTION OF THE NATIONAL
> SMALL GRAINS COLLECTION: Equipment used for planting transgenic plants
> shall not be used for planting NSGC accessions.
>
> **d. [Plot Maintenance and Observations]{.underline}**\--Plot
> maintenance and observations will vary slightly depending on the
> nature of the tested materials, as communicated by the supervisor.
> However, in general field observations of transgenic plants will
> include heading date, plant height, and percent lodged plants.
> Pesticide and cultural practices that are standard for agronomic
> trials will be followed as necessary to prevent crop loss and unusable
> data. Broadleaf weeds will be controlled by applications of Bronate or
> an equivalent herbicide, and alleys between plots will be established
> using the non-selective herbicide RoundUp; follow label instructions
> for application rates, methods of applications, and timing of
> applications. Mechanical cultivation will be used to remove broadleaf
> weeds and grasses not controlled by these treatments. Observations of
> unusual plant growth or other unusual events should be recorded and
> communicated to the supervisor immediately, and appropriate action
> taken if necessary.
>
> **e. [Harvest]{.underline}**\--All harvesting operations will be
> conducted within the area designated for transgenic plots. Specific
> procedures will vary with the nature of the plots, as communicated by
> the supervisor. In general, 1) Carefully handle seed from harvested
> plots to maintain the identity of each transgenic plant or line. Seed
> may be harvested by a variety of procedures, such as: i. hand harvest
> of single heads or plants, threshing directly in the field where
> harvested or transported to the Aberdeen station for threshing and
> cleaning (see item I. d. ii above); ii. cutting entire rows and
> binding them with twine (by hand or using mechanized binders), and
> transporting them to a Vogel thresher located in the field where
> harvested; iii. harvesting with a combine\--note that without cleaning
> the combine thoroughly between plots, slight mixtures of seedlots will
> occur and such seed shall be used for data collection only, and NOT
> replanted; once data collection is complete such seed should be
> destroyed by autoclaving for one hour at 121 ^o^C; 2) Place harvested
> seed in sound bags or envelopes; 3) Box envelopes or bags in sturdy
> cardboard boxes (potato shipping boxes) and enclose the boxes with
> lid; and 4) inspect the harvesting machinery and remove seeds that
> have accidentally spilled on or become adhered to machinery surfaces
> (external and internal), prior to removing equipment from the field in
> which the transgenic field trials have been conducted. This cleaning
> operation is to done within the test plot area so that plants
> volunteering from such seed can be controlled as specified in section
> III below. NOTE\--PROTECTION OF THE NATIONAL SMALL GRAINS COLLECTION:
> Equipment used for harvesting transgenic seed will not be used for
> harvesting NSGC accesssions.
>
> **f. [Transportation]{.underline}\--**In state field test locations
> away from the Aberdeen location will require transportation via truck.
> For interstate transport, packaging will be as specified under 7 CFR
> 340.8 (b) (2). Briefly, these regulations specify that seed will be
> packed within 5-mil plastic bags, placed in a sealed metal container,
> which is in turn placed in another sealed metal container, which is in
> turn placed in a sturdy cardboard, wood, or other container of
> equivalent or better strength. For intrastate transportation, these
> regulations do not apply but reasonable precautions to prevent
> accidental release should be taken; all intrastate transportation will
> be done by project personnel and not by common or contracted carriers.
> Enclosure of bags of harvested seed in strong, covered wooden boxes or
> in sealed (55 gal openhead) drums (or equivalent) will be the minimum
> standard for containment.
>
> **g. [Post-harvest Operations]{.underline}**\--Specific procedures
> will vary with the type of plots, as communicated by the supervisor,
> but in general harvested seed will be cleaned and processed in the
> NSGGRF seed cleaning rooms and Seed Lab #2. Seed will be handled
> during cleaning as detailed above in section I. d. ii, \"Cleaning Room
> Operations\". Analyses of seed yield, test weight, and plumpness will
> be conducted in Seed Lab #2.
>
> h\. [Seed Storage]{.underline}\--see section I. e. above; in most
> cases the amount of harvested seed will require storage in the
> short-term storage facilities.
>
> i\. [Documentation]{.underline}\--see section I. f. above

**III. [Field Tests\--Years Following Growth of Transgenic
Plants]{.underline}**

> **a. [Elimination of Volunteer Plants]{.underline}**\--Areas in which
> transgenic wheat or barley plants are grown will not be planted to
> small grains for at least one year (barley) or two years (wheat).
> During this time, these fields will be planted to crops other than
> small grains, or fallowed. Regular inspections will be conducted for
> the presence of volunteer, potentially transgenic plants in these
> fields, and such plants will be destroyed well before the development
> of viable seeds by cultural or chemical control methods.
> NOTE\--PROTECTION OF THE NATIONAL SMALL GRAINS COLLECTION: Fields
> planted to transgenic barley or wheat will not be used for NSGC
> increases for at least [two]{.underline} years following the growth of
> transgenic barley or [three]{.underline} years following the growth of
> transgenic wheat.
>
> **b. [Documentation]{.underline}**\--The use of the fields (fallow or
> type of crop) used to grow transgenic wheat or barley, the presence
> (or absence) of volunteer wheat or barley plants, and measures taken
> to destroy such plants will be recorded and communicated to the
> supervisor responsible for the field tests.

**IV. [Destruction of Unwanted Seed]{.underline}\--**After all data is
collected transgenic seed will be stored for a period of time to be
determined by the supervisor. However, at some point transgenic seed
will have no further utility and shall be destroyed by autoclaving for
one hour at 121 ^o^C. Seed devitalized in this manner may be disposed of
with normal garbage.

**V. [Unanticipated Events]{.underline}\--Response Procedures and
Notifications**

> **a. [Immediate response to unintended environmental
> releases]{.underline}**\--In most cases such release will involve
> accidental spillage in non-test locations, away from the test plots or
> greenhouse areas which can be adequately cleaned and monitored.
> Release via pollen will be difficult to monitor, but isolation
> procedures as described in section II.a., and the self-pollinating
> nature of barley and barley will make such releases unlikely. The
> exact response will vary depending on the situation, but in general
> the procedures to follow will be a) To collect and repackage as much
> spilled seed as practical; b) Provide notification of the release as
> specified in section V. b.; and c) Follow procedures as specified by
> the supervisor, and Federal and State agencies.
>
> **b. [Notification]{.underline}\--**Immediately notify the supervisor
> of the release. The supervisor will then notify Federal authorities as
> specified in 7CFR 340.4(b)10(i), which requires that APHIS be:
> \"Orally notified immediately upon discovery and notified in writing
> within 24 hours in the event of an accident or unauthorized release of
> the regulated article\". To comply, see the APHIS website at
> http://www.aphis.usda.gov/bbep/bp/. In your E‑mail or voice mail,
> please include the APHIS authorization number and location of the
> site. In the event that the supervisor is not available, report the
> release to other supervisory personnel and/or directly notify APHIS as
> specified above.