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Harvesting guidelines for metabolite profiling

Astrid Juncker, Rhonda Mayer, Thomas Altmann, IPK

For sampling in a standard greenhouse you need:

Two harvest teams consisting of two persons each.

Sample list with labeling instructions (provided by IPK).

Scintillation vials with caps (provided by IPK).

Cryomarker (provided by IPK).

Liquid N2 (appr. 20 liters are sufficient).

Dry ice (12.5 kg for the harvest and 12.5 for delivery to the IPK).

For each harvest team you need:

Safety goggles and gloves!

Tweezers (30 cm or more in length)

Scissors.

Dewar vessel (>20 cm height) filled with liquid N2.

Polystyrene box filled with 6 kg dry ice (>10 cm airspace above the dry ice to the upper edge of the box).

 

All plant material (= 30 samples) should be harvested at the within one hour. To account for developmental differences between plants of different genotype/growth locations, the middle 15 cm of the second youngest leaf/the fully expanded leaf that developed before the emerging one) shall be harvested and the leaf number should be noted in the sample list. As a matter of principle, the harvest should happen within the growth chamber as quick as possible without disturbing the plants or transferring them to a location of uncontrolled conditions. Shading, cooling or mechanical quenching while harvesting should be reduced to the minimum required for handling. For ease of handling and randomizing a large amount of samples coming from different facilities, a sample list with predefined labels for the vials will be supplied along with the scintillation vials to each facility. The sample information needs to be written using a cryomarker (provided by IPK) on both the vial (ID-Number/Genotype/Replicate-Number) and the cap (ID-Number) according to the list.

Please view video 1 and video 2 while reading the following section

The central 15 cm of the leaf of interested should be cut off, folded to fit through the bottleneck of a scintillation vial and transferred to the scintillation vial. Avoid quenching of the leaf while folding as much as possible. The uncapped scintillation vial should be dipped completely into the Dewar vessel containing liquid N2 (>3 seconds) with the help of a pair of tweezers. This will terminate any further biochemical reaction by flushing the material with liquid N2. The procedure from cutting the leaf until shock-freezing in liquid N2 should not take longer than 15 seconds and may be exercised before. The opened scintillation vial should then be placed on dry ice for ten minutes to allow for the evaporation of liquid N2 before closing the vial with the screwcap. There should be 10 cm cooled airspace above the vials to prevent condensation of water (just take a box “high” enough) or thawing of the sample material. Note down which leaf was harvested (leaf number counted from bottom) and clean scissors/gloves regularly if visibly contaminated with plant material. Please check after evaporation of the liquid N2, whether there is no liquid N2 left in the vials before capping. ATTENTION: Any remaining N2 may cause explosion of the vial and loss of the sample at any time later. The samples can be stored until further usage at -80°C.

IMPORTANT: Please check video 1 for metabolite profiling sampling of the central 15 cm of the sixth leaf of maize 35 DAS and video 2 for sampling of two complete leafs 28 DAS on a conveyor belt plant growth system.

>>VIDEO1

>>VIDEO2


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