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Gateway Test Sampling Procedures
As carried out by Quality Control, Pacific Seeds Pty Ltd.

Last updated: May 2012

A Gateway test is a procedure that allows all plants within a specified group to be screened for
the unintended presence of genetically modified (GM) material, referred to as adventitious
presence (AP). This is made possible by using leaf material taken from each plant instead of
seed for analysis, where reliance is heavily based on the representative nature of the collected
sample.

The specific population to be sampled can also be modified to suit the purpose at hand. Like seed
samples, it is not limited to plants of the same line. The sample group may comprise of
individuals across a range of varieties, provided they are of the same species. To date, the two
main uses for which Gateway testing has been implemented within Pacific Seeds, has been on
Breeder seed selections (under increase for the production of a prebasic lot) and quarantine
material prior to field release. Crops have included maize, canola and sunflower.

The protocols for quarantine and Breeder seed nurseries will be dealt with separately in this
document as there are distinct differences between the methods employed.


Sampling Quarantine Plant Material

General Information -

Research staff must notify a Genetic Purity Officer that a Quarantine planting for either Sunflower
or Corn (Field and Sweet) has been undertaken. This should be done as soon as possible after
sowing has been completed to enable QC staff to organize sampling needs and to allow a
suitable time frame for result availability before flowering begins.

The same leaf sampling techniques described in this document are to be used for sampling
quarantine material but sampling must be carried out within the quarantine facility following the
guidelines of quarantine procedure.

*NB. Sample tubes (or any containers being used) must not be sealed or packaged until leaf
material has been dried in oven in the Quarantine facility beside glasshouse. When ready to be
sealed, AQIS staff are to be notified. AQIS will then provide a Movement Direction to allow
material to be removed from the glasshouse complex.

AQIS will also require details of the Courier used for the shipment and must be notified when
samples have been received at the laboratory. (A copy of the courier docket or a tracking number
must be emailed or faxed to the Toowoomba AQIS office when package has been delivered.

Phone (Toowoomba Office): 0746 360 399
Email: exportstoowoomba@aqis.gov.au


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Sampling Breeder Seed Nurseries

General Information -

The Parent Seed Agronomist must notify a Genetic Purity Officer of any material that will require
screening by Gateway testing. This includes new material being introduced as a prebasic seed
variety for the first time and selection material not previously tested that has a risk of AP content.


Sampling Materials

The following sampling materials are used to facilitate leaf sample testing through the GMO
testing lab, ScanBi Diagnostics, Sweden.

Storage containers:

Laboratory tubes for corn small type (sourced from Scan Bi)

Laboratory tubes for canola/sunflower (Sarstedt tube flat base 12ml [product code 58.487])
Quote No: Q08-7150
Stopper for tubes for canola/sunflower (Sarstedt 23.5mm [product code 65.790]) Quote No:
Q08-7087

Specialized 3.5 mm diameter plant leaf punch unit/gun (corn leaves only)
Specialised plant leaf corer unit (canola and sunflower leaves)
Large, good quality paper bags for initial collection of leaf samples
Squeeze or spray bottle filled with methylated spirits for sterilization
Fine cotton tips and paper towel for cleaning
Permanent marking pen for labelling
Field plan or plant identification list


Sampling Methods

Leaf discs Maize (collected using a plant leaf punch unit/gun)

NB. Maximum of 25 leaf samples per tube.

1. Sterilise leaf punch unit thoroughly with methylated spirits before use. Clean out the
punch chamber using a cotton bud and sterilise with methylated spirits.
2. Attach a clean unused sample tube to the leaf punch unit.
3. Sample leaves at highest part of plants and avoid any areas of visible dirt or disease.
4. Aim for healthy leaf tissue and avoid mid rib.
5. Once sampling subset (ie maximum number of discs per individual tube) has been
completed, remove sampling tube and place in holder block. Then remove and clean
down punch cutter, clean punch chamber and reinsert cleaned punch cutter and new
sample tube.
6. Ensure sample tubes are kept cool and away from direct light. Ensure container is
protected from wind gusts which may blow samples from or between tubes. If required,
tube lids may be used, being careful to avoid excessive sweating of the leaf tissue.
7. Allow leaf material to dry out in tubes by removing tube lids and storing in a safe, dry,
environment for a few days before sending for testing. Dry leaf samples avoid bacteria
build up and prevent sample spoilage.

NB: In situations where field conditions are not ideal for sampling or for sampling of large
populations, an improved & time efficient method is to cut leaf tips from plants and collect into
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labelled paper bags for leaf punching back at the lab. Examples where sampling leaf material
into bags has proven more practical include;
- Windy conditions where wind can blow leaf discs from the sampling unit
- Large sampling populations, it is more time efficient to carry out the intricate leaf
punching of samples in a lab environment. This also allows samples to be collected
quickly and kept cool.

If the plant material is low risk, leaf discs may be pooled together (maximum 100 leaf discs) after
DNA extraction to minimise testing expense. Maximum pool sizes are dictated by the testing
laboratory as per extraction methods and the sampling tubes utilized. Tubes to be pooled
together should be indicated on the testing submission forms.


Leaf discs Sunflower and Canola (collected by core unit.)

It was found after trialling the gun style leaf punch on both sunflower and canola that the leaf
punch did not yield good quality samples from these moist, flaccid, and easily desiccated plant
tissue types. The best quality samples were obtained by using a round corer with a sharp edge.

Sunflower Maximum of 45 leaf discs per tube (a maximum of 100 leaf discs may be
possible, need to confirm with testing laboratory)
Canola Maximum of 100 leaf discs per tube (max 0.5mm in diameter)

1. Sterilise hands/scissors used to collect leaves with methylated spirits. Repeat between
lines.
2. Sample leaves at highest part of plants and avoid any areas of visible dirt or disease.
3. Aim for healthy leaf tissue and avoid mid rib.
4. Collect leaves from all plants to be sampled and place in labelled paper bags.
5. Ensure leaf samples are kept cool, away from direct light. In warm conditions, it is
recommended to keep samples cool in an esky.
6. Transfer leaf samples to the cool room as soon as possible while they await leaf coring.
7. In the lab, proceed with coring the leaf samples as soon as possible. Use a hard surface
overlaid with paper towel on which to place the leaf to sample. Press the corer into the
leaf to create a leaf disc and then deliver the sample into the laboratory tube. A number
of leaves can be cored in this manner at the same time if care is taken to ensure each
leaf is properly sampled.
8. Ensure corer is cleaned and sterilized with methylated spirits and paper towel is changed
between laboratory tubes.
9. Ensure sample tubes are kept cool and away from direct light.
10. Allow leaf material to dry out in tubes by removing tube lids and storing in a safe, dry,
environment for a few days before sending for testing. The drying oven in Quarantine
room (Research Lab) may be used if available (see laboratory technician). Dry leaf
samples avoid bacteria build up and prevent sample spoilage.

If the plant material is perceived as having a low risk of AP, (eg seed imported from a non-GM
country, or seed that has already been tested at quarantine stage etc) leaf discs may be collected
up to the maximum per tube or smaller numbers (equivalent to 100 leaf samples) may be pooled
together after DNA extraction to minimise testing expense.


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Sending samples to the GMO testing lab

All samples to be clearly labelled and double bagged in Clip Seal bags.

Documentation included with package:
1. ScanBi order form
2. Letter to ScanBi contact (Lotta Holmqvist) including all details.
3. Commercial Invoice nominating samples re id, weight and number. Indicating no
commercial value but giving a low AUD amount. (eg. 5AUD) Also including wording:
Not for Breeding For Analytical Purposes Only!

Samples should be airfreighted and delivered as quickly as possible to the GM testing laboratory.
The laboratory is to be notified (email) of the package Tracking No. and should also be sent
electronic copies of all order forms etc.

Express Post International Courier is the preferred courier. Tracking service available.
http://ice.auspost.com.au/


Address and Contacts:


ATTN: Lotta Holmqvist
ScanBi Diagnostics
Elevenborgsv. 2
230 53 Alnarp
Sweden

www.scanbidiagnostics.com

Ph: 0011 +46 40 69 28 001

Email: order@scanbidiagnostics.com
name.surname@scanbidiagnostics.com


Contacts: Line Sandager
Lotta Holmqvist


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GMO Testing Supplies and Equipment

Our Australian supplier for compatible laboratory tubes and stoppers is:

Sarstedt
PO Box 90 Ingle Farm
South Australia 5098
Tel: 1800 803 308
Fax: 1800 806 073
Website: www.sarstedt.com
Qld Sales Rep: Trevor Taylor
Mobile: 0412 882 507


Storage containers:

Laboratory tubes (Sarstedt tube flat base 12ml [product code 58.487])
QuoteNo: Q08-7150

Stopper for tubes (Sarstedt 23.5mm [product code 65.790]) Quote No: Q08-7087