Protocol

Authors

Afrasyab Rahnama, Rana Munns, Michelle Watt

Overview

This root screening method can be used for measuring the growth responses of the primary axile roots and the branch roots of wheat. It gives a measure of changes in the overall root system architecture in a realistic environment, and can be extended to other species to identify variation in root elongation in response to gradients in salt, nutrients, or toxic elements.

Background

Root growth response to a salinity gradient

Salinity is rarely uniform down the soil profile, and more commonly strong gradients occur (Dang et al., 2006). When a crop is sown, salinity is mostly low at the surface and greater at depth, as sowing usually follows an irrigation or rain event. Seeds therefore germinate in low salinity, and root tips reach high salinity 1-2 weeks later. The bulk of the upper part of the root system is exposed to low salinity soil, and water is preferentially taken up from the low salinity solution. Therefore, In order to look for genetic diversity in root growth response to saline stress, we developed a rapid screening method to identify genetic variation in rates of root elongation of durum wheat in saline solution. We also reproduced a salinity gradient for roots of a germinating seed to grow into, without exposing the seed to salt, and to document genotypic differences in the salinity response in seminal root elongation and branch root elongation. Genetic variation in rate of seminal root growth and the degree of branching were found (Rahnama et al. 2011).

Materials/Equipment

For measuring EC _(paper):

• Germination paper (25 cm wide x 38 cm long) (regular weight, catalogue number #OP1015, Hoffman Manufacturing Inc., http://www.hoffmanmfg.com/entry.php id=472) (Fig 2A).
• PVC^TMtubes (37 cm long x 11 cm wide)
• Digital conductivity meter (CDM 210, Radiometer Analytical SAS, Lyon, France)
• Drying oven (60^oC)

For measuring seminal axile root length, total root length and average diameter:

• Ruler
• WINRhizo software and an Epson 1680 modified flatbed scanner (Regent instrument Inc., Quebec, CA)

Units, terms, definitions

Seminal axile roots = primary axile roots that emerge from the embryo at the base of the seed. These are the first to emerge upon seed germination and there are generally three to five in wheat. First order branch roots emerge from these axile roots. (See Fig. 1, Watt et al., 2008 for root types of small grain cereals such as wheat, barley and triticale).

Procedure

For development of salt gradient:

• Roll germination papers without seeds by making a 3 mm fold along the long side, and then rolling the paper tightly.
• Dip roll into a tray of tap water to saturate the whole roll and transfer for 15 minutes to PVC^TMtubes (37 cm long x 11 cm wide) sealed with a plumbing lid bottom with a clear base.
• Put tubes in 1500 ml of one of five concentrations of NaCl (0, 50, 100, 150, and 200 mM NaCl) in half-strength modified Hoagland’s solution (Rahnama et al., 2010) immerse the rolled papers to a depth of approximately 21 cm.
• After 15 min, transfer papers to identical PVC^TMtubes with 500 ml of the same solution, immerse the rolls to 7 cm.
• Seal the tops of the tubes with Parafilm^TMand keep in a growth cabinet at 18^∘C and a 12 h photoperiod with photosynthetic photon flux of 500 μmol m^-2s^-1at plant height.
• After 3, 4 and 5 days, take rolls out of solutions, and immediately cut into five parts (7 cm each) (Fig 1).
• Weigh fresh each part, immerse in 50 ml of distilled water, agitate several times, and after 1 hour remove and measure the electrical conductivity (EC) of the solution.
• Dry the section of the roll at 65^∘C for 24 h.
• Calculate the EC of the solution in the paper by the following equation:
  EC_(paper)= EC_(extract)x volume_(extract)/ volume_{(paper solution)}

• Convert EC in units of dS m^-1to mM NaCl by multiplying by 10.

Fig 1. Salt gradient. Diagram of a paper roll indicating the height (21 cm) dipped in salt for 15 min (sections 3 to 5) and height (7 cm) of the paper sitting in solution for the remainder of the experiment (5 d).

For plant growth conditions

• Soak seeds in tap water two hours to imbibe and then surface-sterilize.
• Make a crease 2 cm from the top of the shorter side (25 cm) for seed placement.
• Fold 3 mm of paper twice along the long side (38cm) to provide a starting fold for rolling.
• Mist along the crease by water to help secure the seeds.
• Place seeds along the crease spaced a few cm apart with the embryo facing the bottom of the page.
• Roll the paper tightly, dip whole into a tray of water and put into a PVC^TMtube with 500 ml tap water.
• Seal tubes with Parafilm^TM, refrigerate at 4^∘C in the dark for two days and then remove the Parafilm™ and transfer tubes to the growth cabinet.
• Enclose rolls in a plastic bag throughout the experiment to limit evaporation from the top of the paper roll and migration of the salt towards the seed (Fig 2C).

Fig 2. Plant growth conditions for whet plants. (A) Plants at 1.5 leaf stage, rolled in germination papers. (B) Unrolled germination leaf stage paper showing approximately five seminal axile roots (one indicated by a black arrow) emerged per seed. (C) PVC^TMtube with paper rolls with plants and sealed with a plastic bag to prevent evaporation from the top of the paper and movement of the salt gradient.

For measuring seminal axile root length, total root length and average diameter

• Harvest shoots and roots and preserve in 50% ethanol.
• Measure the length of each seminal root and shoot, the distance between the axile root tip of the longest seminal root and emergence of the most distal branch root with a ruler.
• Determine total root length and average diameter with WINRhizo software and an Epson 1680 modified flatbed scanner (Regent instrument Inc., Quebec, CA) according to Watt et al., 2005.
• Determine the length of branch root through the total length with minus the total axile root length, previously measured with a ruler.

Notes and troubleshooting tips

• Do not delay in weighing papers sections taken from the rolls out of PVC^TMtubes.
• The pots should be enclosed in a plastic bag throughout the experiment to limit evaporation from the top of the paper roll and migration of the salt towards the seed. We found that when evaporation was not minimised, salt rose to the top and reached a concentration of 600-700 mM.

Links to resources and suppliers

Anchor Germination Paper, Hoffman Manufacturing Inc., https://www.hoffmanmfg.com/products/germination-paper/

Literature references

Dang YP, Routley R, McDonald M, Dalal RC, Singh DK, Orange D, Mann M. 2006. Subsoil constraints in Vertosols: crop water use, nutrient concentration, and grain yields of bread wheat, durum wheat, barley, chickpea, and canola. Australian Journal of Agricultural Research 57, 983-998.

Rahnama A, Munns R, Poustini K, Watt M. 2011. A screening method to identify genetic variation in root growth response to a salinity gradient. Journal of Experimental Botany 62, 69-77.

Watt M, Kirkegaard JA, Rebetzke GJ. 2005. A wheat genotype developed for rapid leaf growth copes well with the physical and biological constraints of unploughed soil. Functional Plant Biology 32, 695-706. DOI: 10.1071/fp05026.

Watt M, Magee L, McCully ME (2008) Types, structure and potential for axial water flow in the deepest roots of field-grown cereals. New Phytologist 178: 135-146

Related protcols

Using WINRhizo and Photoshop to determine root length, diameter and branching

Quantifying fine root and leaf morphology (and seeds) from desktop scans