meth_1.17 Plant Sampling including leaf root and soil

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AM Project ID: IndigoAg_crop

Sample Collection

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Sample Processing

Samples were received directly over a period of ~4 weeks as cold shipments.

Samples were immediately transferred to and stored at -80 °C till processing.

Samples were removed individually and processed quickly at room temperature. Each foil parcel contained the whole plant including shoots, roots and rhizosphere. Three types of samples were collected: leaf, root and rhizosphere soil.

Note: there was considerable variation in plant size (almost ranging from ~GS31 to near anthesis), and the amount of root and associated soil provided in each sample also varied considerably (from near to none on some samples) across sites (but was relatively consistent with each batch from a site).

A portion of the shoot (~50 mg) from the most recently fully (or near fully emerged) leaf was first taken from the plant for DNA extraction. Samples were immediately returned to -80 °C.

Sterilised instruments (rinsed and flamed in ethanol) were used to cut off the roots and the attached soil was removed onto a clean foil sheet with gentle agitation.  A uniform sub-sample of this soil was collected and stored at -80 °C – as bulk soil (but this was not subsequently processed).  The level of agitation to remove the soil was done uniformly to provide a ‘consistent’ among of soil retained on the root surface (as rhizosphere soil).

A uniform sample of fresh and young roots (avoiding old and deteriorated sample) with adhering soil (~2 to 5 g) was then selected /dissected from the sample (including both seminal and nodal roots).  Depending on the sample, this subsample constituted from ~10 % to up to 100 % of the root material provided.

The root and adhering soil was transferred to 40 mL of sterile saline (50 mL sterile Falcon tube) and vigorously shaken to dislodge soil from the root.  This was done at 4 °C and samples were stored on ice. 

Fresh roots (~ 1 g) were then removed and rinsed multiple times (at least 3) in sterile 0. 9 % NaCl solution and blotted on clean filter paper.  A subsample of fresh root (~50 mg) was taken and both the subsample and the remainder of the sample were stored at -80 °C.

The Falcon tube (with dislodged soil) was then centrifuged (5k rpm / 5 min at 4 °C) to pellet the soil (= rhizosphere soil) and the supernatent was decanted and discarded.  The soil pellet (~2 g) was stored at -80 °C.

Leaf and root DNA was extracted from the 50 mg samples using the Mag-Bind Plant DNA DS kit (M1130-00) following the manufacturer’s protocol (metadata field code 3.1.12).

Soil collected from the rhizosphere (~250 mg sample) was extracted using Qiagen DNeasy® PowerSoil® Kit (12888-100), following the manufacturer’s protocol (metadata field code 3.1.2).

Samples were then quantified on the NanoDrop Spectrophotometer (metadata field code 3.3.1).

40 μL of each sample was sent (on dry ice) for sequencing (as 3 separate 96 well plates, one each for shoot, root and rhizosphere).

50 μL of the samples was retained as storage (backup) at -80 °C.