Appropriate and realistic treatments are the key to successful and informative experiments. Treatments should ideally represent the natural environment, but mimicking conditions that occur naturally is not always easy in the laboratory. Many studies aim to measure a large number of genotypes simultaneously, and to compare them under the same degree of stress. There is frequently the need to screen mutant populations, or a range of genotypes representing natural diversity, for tolerance of a stressful condition. It is therefore desirable to control the development of the stress.
Protocols in this section on Experimental Treatments are designed for laboratory scientists who carry out studies representing optimal field conditions or major environmental constraints. They are divided firstly into soil and atmospheric conditions, that is, below-ground and above-ground. However, note that conditions such as drought and high temperature may involve both soil and atmospheric factors.
- Heavy metal toxicities
- Nutrient deficiencies
- Other (acidity, alkalinity, sodicity, compaction)
- High temperature
- Light (quantity and quality)
- CO2 and other greenhouse gases
- Biotic stresses (pathogens and herbivores)
The protocols presented under these treatment headings are feasible in controlled cabinets or glasshouses. The field provides its own treatment.
The first decision to make when designing an experiment is whether to grow plants in soil, what type of soil, and what type of pot. The pot size and dimension is important, as a small pot may restrict plant growth, and does not drain well. The soil at the bottom of a short pot is saturated most of the time, the air spaces being filled with water, and hypoxia results. Tall pots or artificial soil can avoid this problem.
A saturated layer at the bottom of pots is a special problem with field soils used in pots, for these typically have a high clay content and do not contain many pores large enough to be drained at the small water suctions that prevail at the base of a short pot. Even sandy soils, and especially pure sand, do not drain well, and can become compacted towards the base of the pot after frequent watering. Commercial potting mixes overcome this problem by using coarse materials in the mix, which create many large pores (>1 mm diameter) that drain at small suctions (see JB Passioura: The perils of pot experiments. Functional Plant Biology 33, 1075-1079, 2006). Pots should be tube-shaped rather than bucket-shaped, creating a suction for drainage. However use of material with large particles and little root contact may be problematic, especially in treatments for below-ground stresses. “Inorganic soils” such as fritted or calcined clay can overcome the problems of clay soils, which do not infiltrate or drain well, and of sandy soils that have a low water-holding capacity.
Drought experiments bring their own special difficulties. Soil moisture content is hard to control, as it is depleted so quickly when the plant’s leaf area increases exponentially. This is a particular problem when comparing genotypes of different vigour or rates of development. Furthermore a drying soil, as well as being very difficult to maintain at a uniform and constant water potential through the whole soil profile, may exert specific effects; for example, transmission of nutrients through the soil will be reduced at low soil water potentials. Solution culture can be used to overcome these difficulties.
If solution culture is to be used, as is common for nutrient deficiencies and toxicities including salinity, a decision must be made whether to use conventional solution culture, supported hydroponics, or sand culture. Conventional solution culture, when plants are in tanks filled with solution, has the problem of ensuring sufficient aeration without causing root damage, and the likelihood of lateral root breakage when the solution is drained and replaced. Supported hydroponics, when plant are grown in individual pots filled with fine quartz gravel or plastic beads, and placed in large tanks that are irrigated from below overcomes these two problems. Sand culture with drip irrigation from above is an alternative, but additional infrastructure is needed to collect the drained solution and recirculate it to avoid waste of chemicals.
To mimic ‘drought’, at least to control a decrease in soil water potential, soluble carbohydrates that are not taken up by roots or metabolised by plants have been used. Mannitol is such a compound. Care should be taken as these small molecules eventually enter roots in significant amounts and move in the xylem to the shoots. Carbohydrates also support bacterial growth as it is impossible to create an aseptic root environment. High-molecular-weight polyethylene glycol (PEG) was used in studies in the 1970s and 1980s to impose an immediate and controlled water deficit. However it became obvious that it caused many artefacts. It has two main problems. First is its viscosity which decreases O2 movement to roots so that the roots become O2 deficient. The latter can be overcome by bubbling with O2 rather than air. The second is that experiments must be limited to a short period of time as the PEG enters the roots and moves in the xylem where it reduces the hydraulic conductivity. Salinity is relatively easy to impose, and can substitute a water stress treatment for species that are salt tolerant. The relative benefits and disadvantages of different osmotica are discussed in the section on drought.
Sampling and statistical analysis
Overlaid on these treatments are questions of what functions to measure, and in which plant tissue. Protocols for the measurements themselves are provided in other sections, and the associated section summaries provide an indication of the studies in which these measurements provide useful information.
The associated lower level sections in box to the top left of this page provide examples of experimental designs to allow for rigorous statistical analysis.