13 Plant cultivation
Monitoring, reporting and control of growing conditions
Eva Rosenqvist, F. López Figueroa, Iván Gómez, Pedro J. Aphalo
$$
13.1 Introduction
By applying the most constant growth conditions that we can in an experiment, we try to minimise uncontrolled variation in our measured plant data, even though this is not always achieved even with identical climate chamber protocols (Massonnet et al. 2010). However, by growing plants in stable controlled environments we will also induce differences in their response to any given stress, since natural variation in the outdoor climate contributes to plants’ ability to cope with stress. Plants grown in climate chambers are “softer” than plants grown in greenhouses, that are themselves “softer” than plants grown outdoors. This softness can be expressed as weaker stems, physically softer leaves with less developed cuticles, and a lessened ability to regulate their stomata.
Any protected cultivation will invariably differ from natural outdoor conditions, and the relative importance of these differences in aspects of the climate will vary between controlled and natural environments. The most pronounced difference is the constancy of a created climate under protected cultivation—and the lack of wind and UV-B radiation! In experiments in greenhouses during the winter and in growth chambers, the amount of radiation received by plants will be far less than they get in summer outside when most plants are active. This is unrealistic, unless dealing with obligate shade species.
13.2 Greenhouses
Greenhouses come in many shapes and materials throughout the world. For plant production the cladding material can be either glass or plastic. Normal glass is opaque to UV-B and only expensive quartz glass allows UV-B to penetrate. The latter is used in only a few places where near-ambient UV-B radiation is considered important for ecophysiological research, but the vast majority of glasshouses provide no natural UV-B inside. The use of plastic opens several opportunities for choosing materials that are transparent to UV-B. Plastics are available as opaque and UV-transparent films that transmit <1% or >70% of the radiation < 400 nm, respectively. They can be clear (transmitting 87–90% of PAR) or milky to diffuse the light (transmitting 85% of PAR). transparent films are yet primarily used for increasing the vegetable quality in horticultural production in warmer climates.
This chapter will by no means give complete instructions on how to manage the climate in a greenhouse, but we have gathered some information about possible technical solutions to various climate problems and some tips and tricks from personal experience. Greenhouses are available that provide different technical solutions for controlling the climate. In this section we will focus on glass greenhouses with fully automatic climate control regulated by a climate computer. The topic of how to apply from lamps has already been covered in section 8.2 on page .
13.2.1 Temperature
It is technically easier to heat than to cool a greenhouse and it is therefore easier to maintain a stable temperature during cold times of the year than during warm weather. As long as the boiler and heating system are correctly set-up and able to cope with the maximum expected temperature difference between outside and inside, the temperature should remain close to the point set by the thermostat. The amount of variation around the set point will depend on the sensitivity of the on/off signal to the boiler from the thermostat that regulates the heating system. Where winters are particularly cold, some greenhouses require insulation screens to enable them to maintain e.g. air temperature.
Greenhouses with conventional air conditioning are not common due to their high running costs, so cooling is normally done by passive ventilation, misting systems or fans with cooling pads. Passive cooling involves opening vents and/or using shade screens to decrease heat load from the sun. The temperature outside will naturally have a huge impact on the degree of cooling achieved by ventilation and, if the shade screens are used for cooling, it is important to keep a 10% gap between them to allow air circulation out of the greenhouse.
In a closed greenhouse in sunshine strong temperature gradients can be created. When the temperature at the floor level is ca. 25, it may be 30just 35 cm above the floor and >50just under the ridge of the roof. A few horizontal fans with moderate rotation in the ceiling can mitigate the problem. Regardless if fans are used or not it is important that the climate sensors connected to the climate computer are placed close to the plants since the plants respond to the microclimate in the canopy, not the macroclimate somewhere else in the greenhouse. It is also crucial that the temperature sensor is shielded from direct heat radiation from the sun or lamps to ensure that the true air temperature is measured.
Water-based cooling systems use the principle of evaporative cooling. As water evaporates, some sensible heat from the warm air is transferred to the water as latent heat during its vaporisation. These cooling systems work best in areas with low to moderate air humidity. Misting systems can decrease the air temperature by some in moderate humidity and can be constructed with or without fans. It is important that they create droplets that are small enough to evaporate completely before they have fallen down onto the plants. “Rain” under misting systems will drastically increase the risk of fungal disease on plants.
Fans with cooling pads regulate temperature using large industrial fans mounted in series with pads where water trickles down an enlarged surface (e.g. corrugated fibreboard, wood shavings or wheat straw), which enhance the area for evaporation. The change in latent heat in the evaporated water leads to a corresponding decrease in air temperature.
13.2.2 Light
Greenhouse conditions should mimic the outdoor conditions of the open landscape as much as possible. The light environment in a greenhouse is strongly dependent on how much shade is created by its structure. Modern greenhouses are constructed to keep shading to a minimum.
The amount of radiation received by the plants can be increased by using lamps and decreased by using shade screens but regardless of technical installations it will be difficult to create summer light conditions in a greenhouse during the winter in latitudes covering most of Europe. During the summer in Denmark daily integrals of PAR vary in the range of ca. 10–30 between rainy and sunny days inside a greenhouse without shade screens, while the values in mid-winter are 0.5–5 with some use of supplemental light until 22:00 h in the evening (Figure [fig:ppfd:greenhouse]).
Modern greenhouses have two or three screens for different purposes (shade, insulation, blackout) but in most greenhouses one shade screen can perform a dual function in insulation as well as shading radiation. The shade screen may also differentially remove part of the radiation, allowing the user to decide what fraction of the radiation load to remove up to the maximum shading with the screen fully closed. In many cases, the principal role of shade screens is to decrease the heat load from the sun, preventing the greenhouse from getting too hot, or as insulation screen during the cold season, but we should bear in mind that the light environment created by a medium-to-dense screen is comparable to that of an overcast day i.e. the PAR is decreased by 10–90%, depending on screen material.
For plants whose flowering is controlled by day length, blackout screens can be used to give short day treatments and incandescent bulbs (low red:far-red photon ratio) for night interruption to achieve long day treatments[^32]. Either a single pulse, or multiple pulses—e.g. 5 min per hour—can be used in practice. In this way flowering can either be induced or plants kept in a vegetative state for both short- or long-day plants. For the treatment to work, the blackout screens must provide completely black conditions without gaps, since phytochrome responds to extremely small amounts of light, and even small cracks between screens would be enough to interrupt the night length signal.
During the winter season, supplemental light from lamps is needed for most species grown in greenhouses at high latitudes. Most greenhouses in these regions are supplied with high-pressure sodium (HPS) lamps, which give a spectrum from blue to red but predominantly emit orange light (Figure [fig:HPS:greenhouse]). For photosynthesis, this spectrum is not optimal but when it is combined with natural daylight most plants grow without any problems. In research greenhouses, high-pressure lamps based on elements other than sodium are also used to give more “white” light. However, they are not used for plant production because of their lower energy efficiency than HPS lamps.
Light-emitting diodes (LEDs) are currently being developed to provide greenhouse lighting. They come in many different colours, and can emit anything from a narrow to a broad range of wavelengths. For use in greenhouses, numerous small companies have developed various LED lamps but up to now (summer 2012) large companies like Philips and Osram have as yet only released LED modules for use in growth chambers and for interlighting rows of vegetable crops (long narrow LED units placed between rows of e.g. tomatoes and cucumbers grown in greenhouses). Top light, or “ordinary” greenhouse LED lamps, will require LEDs that are sufficiently efficient to emit enough radiance while incorporating cooling for their electronics. These two factors have to be included in the “budget” when the energy efficiency is calculated for new LED lamps compared to traditional HPS lamps. Both the energy efficiency and lifetime of LEDs will decrease if their electronics are overheated.
Research on the spectral distribution of LED lamps is focusing on red light (which requires the least energy to produce per photon) and how much additional blue or white light needs to be added for good plant growth. One basic problem with this approach is that the human eye is most sensitive to green and yellow and less so to blue and red light. Since the colour of leaves is based on reflected wavelengths, leaves that are only illuminated by blue and red light, which is efficiently absorbed by chlorophyll, will appear as dark objects. It may distort our judgments of plant health and nutritional status when making comparisons, since it is often manifested as yellow chlorosis or reddish coloration of the leaves. One would guess that the technical development of specific greenhouse lamps will lead to red enriched LED lamps with some blue and white (or green) LEDs to improve colour recognition.
Beyond their spectral composition, the main difference between LED and HPS lamps is that the latter produce heat radiated in the direction of the plants while the former do not. This means that the leaf temperature will be higher under HPS lamps than under LED lamps. However, the electronics of the LEDs do become warm. When using only one row of low-efficiency LEDs, the metal mounts for the LED modules often provide enough heat sink to prevent them from over heating (which would otherwise decrease their efficiency), whereas high-efficiency LEDs require cooling. Most early LED illumination for greenhouses was water-cooled and this is one reason that its use in greenhouses has not yet been very widespread. Currently there are some high-power LED lamps available that are passively cooled by convection (Figure [fig:LEDs:greenhouse]). Some other LED lamps with passive cooling have been designed for research (Figure [fig:LEDs:greenhouse:Philips]) or with active cooling for production (Figure [fig:LEDs:greenhouse:Fionia]). The colour of the radiation emitted depends on the LEDs used, and there are currently two different approaches when designing LED lamps: (1) maximising radiation output with respect to electricity consumed, and (2) maximising plant yield and crop quality irrespective of irradiance per unit of electrical power consumed. Because of the present fast technical development of the LED technology we can expect to see numerous new LED products for plant growth on the market in the next couple of years. From the perspective of research into the effects of UV-B in nature, the best PAR sources are those with a spectrum similar to sunlight.
The light distribution in greenhouses can be enhanced by diffusing glass, diffusing plastic films or thin diffusing screens. These create a more even distribution of light and decrease the differences, such as light gradients or patchiness, that are otherwise created by shade from the structure. A resultant side effect of diffusers is enhanced growth of plants due to better penetration of light into the canopy (Markvart et al. 2010).
Commercially-available climate computers are often connected to photometric (lux) sensors that measure light visible to humans (\(\approx\)400–700 nm, see Box [box:photometric:quantities] on page ), whereas global:rad is measured radiometrically (, see Table [tab:Phys:Quants] on page ) in the range 400–1100 or 400–3000 nm to provide information for the energy-related control of greenhouses (primarily temperature). The latter is the correct way to measure irradiance linked to climate control; whereas visible light, related to photosynthesis and plant production, should be measured as PAR photon irradiance or PPFD (PPFD, , see Box [box:quantum:quantities] on page ) or PAR (energy) irradiance (). Modern climate computers can also be directly connected to PPFD sensors.
When using supplemental light from various light sources extra care need to be taken on the choice of quantum sensor since most supplemental greenhouse lamps have a dis-continuous spectrum. Some cheaper quantum sensors do not cover the full spectral range 400–700 nm and the sensor may not cover the spectrum of red LED’s, which can peak close to a wavelength of 700 nm.
One should keep in mind, that moving an experiment from the field to a greenhouse does not make it independent of the outdoor season, since the UV-B dose should be balanced against the integrated daily PPFD if the experiment aims to mimic outdoor conditions. Daylight varies tremendously between winter and summer. Even with supplemental light the PPFD of a sunny winter day in Northern Europe will only correspond to an overcast day in the summer (Figure [fig:ppfd:greenhouse]). The contribution from lamps will be less than what is found in sunlight during the summer, even under long photoperiods (c.f. Figures [fig:ppfd:lamps] and [fig:ppfd:greenhouse]).
Except for late successional species that ecologically are adapted to obligate shade, most plants will acclimate to the prevailing light conditions (Bazzaz and Carlson 1982). This means changing chloroplast architecture and the stoichiometry of the whole photosynthetic apparatus (Anderson, Chow, and Goodchild 1988) and thus also altering the light response of photosynthesis.
Most experiments on light acclimation only discus the light environment in terms of “high light” or “low light”, regardless if it has been created as % of sunlight with permanent shade screens or as a certain PPFD in climate chambers. However, the light environment should be described by day length, maximum PPFD and integrated daily PPFD. Not much is known about which of the parameters of the light environment, actually trigger acclimation of photosynthesis. The three light parameters mentioned above have been investigated in tomato (Van den Boogaard et al. 2001), where the day length affected the chlorophyll concentration while the integrated daily PPFD affected the maximum rate of fixation at light saturation, independent of what the maximum PPFD was during the day.
If photosynthesis is being studied in greenhouses in relation to UV-B one should be aware of the effect that the seasonal changes in daylight has on the photosynthetic apparatus and therefore also on the growth of the plants, when comparing different experiments conducted at different times of the year.
13.2.3 Air humidity
Air humidity is difficult to regulate in greenhouses. If few plants are grown in a compartment the humidity tends to be low due to the large areas of potentially cold glass and concrete that act as dehumidifiers through condensation. If the leaf area index (LAI) is high, transpiration will make humidity correspondingly high despite condensation on the greenhouse surfaces.
Misting systems can increase the humidity but it is important that they create mist, not rain. Rain or condensation on the plants will increase the risk of fungal disease in the experimental plants.
The humidity will be higher in highly insulated than in single-glass greenhouses, where some of the humidity will condense on the glass. High air humidity is decreased by opening the vents at the same time as turning on the heating system to increase the exchange of air with the outside. The effect of dehumidifying ventilation will depend on the outdoor weather conditions and on whether dry or moist air is entering the greenhouse.
Using traditional climate control, the air humidity is regulated as relative humidity (RH, %). The driving force of transpiration is the absolute gradient of water vapour from the intercellular spaces inside the leaf to the outside air, i.e. the water vapour pressure difference (, Pa). As the intercellular spaces are microscopic and the cell walls saturated with water it is assumed that the RH in the leaf is 100% at the given leaf temperature. Since the absolute content of water vapour in air of the same RH is strongly temperature dependent (Figure [fig:water:vapour]), the evaporative demand can differ greatly when temperature fluctuates in the greenhouse, even if RH is stable.
In modern climate computers a measured quantity equivalent to VPD (i.e. based on absolute difference in humidity, expressed as water mass concentration, instead of difference in partial pressure) can be used for humidity control, instead of RH. It is called delta-\(\chi\) (\(\Delta \chi\), , or sometimes simply delta-X) and is the water deficit from the saturated value, but expressed based on mass concentration instead of vapour pressure. Both VPD and \(\Delta\chi\) use saturated water vapour content based on air temperature as a reference. To be correct we need information about the canopy temperature to use VPD or \(\Delta\chi\) to estimate the true evaporative demand () as when the leaves are transpiring their temperature will be somewhat lower than the air temperature, due to evaporative cooling. The canopy temperature is not easily measured but in the context of climate control the approximation based of VPD or \(\Delta\chi\) still provides a better approximation to the true evaporative demand created by the atmosphere in the greenhouse than RH.
Misting systems using ultrasonic humidifiers have been reported to also generate some hydrogen peroxide and may not be appropriate in research studies of plant growth (Arends et al. 1988). Although the evidence is not strong, one should be aware of this possible undesired effect.
However, increasing air humidity in greenhouses by forced aerosolization of water (creating small droplets by water sprays, jets, ultrasonic nebulizers etc.) may cause different problems. These processes cause charge separation of water droplets and thus forming high concentrations of nano-sized ions, which can damage electronic equipment due to electrostatic deposition. The effects of the charged droplets on plants are not known. This effect is not seen if water is being evaporated directly from a water surface to water vapour e.g. by trickling water in fans with cooling pads.
13.2.4 Elevated carbon dioxide concentration
Modern greenhouses have systems to supply elevated concentration to promote crop growth. There are several possible sources of that can be used: the three most common ones are liquid from a tank, combustion of natural gas for production and produced when burning fossil fuels for heat/power production, where the latter two require cleaning of the fumes to remove and .
A major difference between elevated supplied in full-scale greenhouses compared to open-top chambers or FACE is that the greenhouse is not supplied continuously during daytime hours. Because of the vast volume of a greenhouse compartment it would be very expensive to supply elevated when the vents are open for temperature control. Therefore in traditional greenhouse climate control elevated is given only when the vents are closed or open by at most 5%. Ethylene contamination, which can significantly reduce plant growth, has been reported in some supplies and it should be removed by scrubbing if necessary (Morison and Gifford 1984).
Furthermore the isotope composition may differ in liquid compared to natural air, which need to be checked if investigations include measurements of the ratio e.g. in relation to water balance, and when comparing with results in the literature.
13.2.5 Growth substrates, irrigation and fertilization
Transferring experiments indoors inevitably means growing the plants in pots. It is important to chose a growth substrate that has both good water holding and draining properties to meet the demand for water and oxygen for the root system. In the horticultural greenhouse industry peat mixtures are the most commonly used substrate in pots but also coco-peat (granulated husks of coconuts) is used. To ensure good aeration perlite or vermiculite is sometimes mixed into the substrate. Since the pot volume is limited the fluctuations in water content will be greater than in soil, as will be the fluctuations in soil temperature. In natural soil the temperature will be both more stable and lower than what it is in pots.
The pots are often of dark colour and they absorb heat radiation when exposed to sunlight. In commercial production the pot density is kept high enough to create shade between the pots and only the outer row of pots are potentially exposed to heat radiation from the sun. In experiments you often leave some space between the plants, not to have excessive shade between them. During sunny days without shade the pot temperature can easily increase to levels that are damaging to the root system and when knocking the soil/root out of the pot of border plants that has been exposed to direct sunlight it is not uncommon to see root death on the exposed side of the pot. The temperature of the nutrient solution/water also affects the root temperature. If the water is cold a growth retarding effect can be seen on the plants. More reflections on pot experiments are found in Passioura (2006) and a meta-analysis of the effect of pot size on plant growth in Poorter, Bühler, et al. (2012).
Several types of watering systems exist but the most common ones are drip irrigation and ebb/flow systems. Drip irrigation is based on thin tubes supplying each pot with nutrient solution from a tank. In an ebb/flow system the nutrient solution it pumped up on the bench to 1–2 cm water depth and kept there for some minutes, before being drained off, back into the tank. This system recirculates the nutrient solution and with commercial nutrient/watering computers the composition is controlled by pH and electric conductivity (EC) of the solution, topping up with stock solution or an acid to keep the nutrient concentration and pH at desired level.
Irrigation of the plants can be done by timer or on demand. In greenhouses plants will have different need for water dependent on the weather. If the irrigation is done by timer without adjustment for sunny or overcast weather there will be a risk that the plants are over-watered during dull days or water stressed on sunny days. Both stresses potentially affect the growth of the plants.
Watering by demand can be done by experience or by weighing the plants and watering after a pre-determined water loss. Load cells can be mounted under ca. 0.5 m\(^2\) watering trays or under a section of a greenhouse bench, triggering a pump in the tank with nutrient solution via a datalogger. Since plants that grow to maturity follow a sigmoid growth curve the watering needs to be adjusted accordingly during the experiment, to follow the demand.
13.2.6 Data logging of microclimatic variables
When working in greenhouses it is not enough to report the set points used for the most important climate parameters. The climate in a greenhouse needs to be measured and the observed values reported. The optimum arrangement is to have separate sensors recording the climate to those that are controlling the climate, but if this is not possible the ones connected to the climate computer can be used, if they are calibrated. Light irradiance, air temperature and humidity are the most important parameters to record, along with concentration if it is applied. Since the natural irradiance can fluctuate rapidly, it is important to have a high logging frequency, e.g. every minute. Furthermore, it is important that the temperature sensor is shielded from direct heat radiation from the sun and the lamps, while still being exposed to free air, e.g. in a ventilated box. For ideas on how to construct a radiation shield for measurement of air temperature refer to the design of weather stations (e.g. http://www.kippzonen.com, http://www.skyeinstruments.com and may others). There are several small dataloggers on the market, with built-in air temperature and humidity sensors in a plastic housing. They also have to be shielded from heat radiation when used in a greenhouse or climate chamber since the plastic housing gradually heats up and transfers the body temperature to the sensor—in climate chambers with metal halide lamps errors larger than 10have been observed when no shielding was used. Recommendations for sensors and frequency of data logging are compiled in Table [tab:growth:guidelines].
| Units | Where to measure | What to report | ||||
| Climate chamber | Greenhouse | |||||
| Radiation | PPFD | Top of the canopy in the centre of growing area (in greenhouses \(> 1\) sensor is preferred) | Start, every 2 weeks and end of experiment | n.a. | Mean (daytime) and s.d. Radiation source (type, model, manufacturer) | |
| PPFD | As above | n.a. | Logging every minute | Mean max. daily PAR and s.d. Mean integrated PAR and s.d.. Radiation source (type, model, manufacturer) | ||
| Photoperiod | h | n.a. | n.a. | n.a. | Duration of light and dark period (in greenhouse for start and end of experiment). | |
| Temperature | Air | Top of canopy in the centre of growing area | Daily during each light and dark period, at least 1 h after light/dark change | Logging every 1–5 minutes | Mean and s.d. for day and night temperature, specification if it does not follow the photoperiod. | |
| Air humidity | Water vapour pressure deficit (VPD) | kPa | Top of canopy in the centre of growing area | As for air temperature | Logging every 1–5 minutes | Average and standard deviation |
| \(\Delta\chi\) | As above | As above | As above | As above | ||
| Relative humidity | % | As above | As above | As above | As above (only if VPD or \(\Delta\chi\) not available) | |
| Carbon dioxide | If elevated and part of the treatment | Top of canopy | At least hourly | Logging every 1–5 minutes (via climate computer) | Mean and s.d. | |
| Air velocity | If available and part of the treatment | At one or more representative canopy locations | At least once during the experiment | Mean and s.d. | ||
| Substrate | n.a. | n.a. | n.a. | At start of experiment | At start of experiment | Type and volume per container, components of soil(-less) substrate, container dimensions |
| Watering | n.a. | l (litre) | n.a. | Daily | Daily | Frequency, amount and type of water added |
| Nutrition or liquid culture | Acidity | pH | In bulk solution | Before and after pH correction | Before and after pH correction | Mean and s.d. |
| Electric conductivity (EC) | As above | Before and after EC correction | Before and after EC correction | Average and standard deviation | ||
| Ion composition | As above | Daily or when replenished | Daily or when replenished | Ionic concentration in initial and added solution. Aeration if any. Volume of initial solution. | ||
| Solid media | n.a. | When added or replenished | When added or replenished | Nutrients and their form added to soil media. |
13.3 Open top chambers and FACE
Open top chambers (OTC) and, less frequently, closed top chambers (CTC) are used to study the effects of elevated concentration and temperature. Most of these chambers are built using plastic films, plates, or glass that absorb radiation and sometimes also some radiation. In some cases, attenuation of radiation has been minimised by use of -transparent materials in OTCs and CTCs (e.g. Visser et al. 1997). In contrast to OTCs and CTCs, free air enhancement (FACE) systems do not significantly affect or visible radiation. In some cases, lamps have been used in OTCs and the attenuation measured. In controls with unenergised lamps, daily integrals of and radiation, and were reduced by 24% (Booker et al. 1992).
13.4 Controlled environments
In controlled environments the spectral distribution of the light emitted by the lamps used is especially important since there is no contribution from natural light. A skewed spectrum can cause strange growth patterns in plants and many lamp types produce distinct emission bands, interrupted by bands of very low emission (Figure [fig:spectra:PAR:lamps]). Fluorescent tubes and HPS lamps, in particular, emit very little far-red and produce plants with very stunted growth, compared to lamps that create a continuous spectrum resembling sunlight (Hogewoning et al. 2010). However, lamps emitting a continuous spectrum can also create unexpected differences in plants compared to sunlight. An unpublished example is that of white cabbage grown in pots outdoors then moved into a climate chamber with metal halide bulbs (see Figure [fig:spectra:PAR:lamps]), which developed a distinct red coloration after a couple of hours at constant light.
lamps emit some blue light providing a weak visual indicator that the lamp is turned on, but the contribution to the light environment will be minor in comparison to the overall from the main lamps used for . They also emit very small amounts of orange-red radiation.
Most lamps presently used also emit a substantial amount of heat radiation. Some of this heat should be removed using a heat filter. The most efficient design to achieve this is to have the lamps in a separate, ventilated compartment in the ceiling, divided from the growth chamber by a transparent glass/plexi-glass sheet. Even so, it is important that the temperature sensor measuring air temperature in a climate chamber is shielded from the heat radiation from the lamps. Water is a good absorber of infra-red radiation, and sometimes a transparent tray with circulating water[^33] is used as a barrier between lamps and the plant compartment. Most lamp types (e.g. metal halide) used in climate chambers can only be turned on/off, not dimmed. To create different photon irradiances the lamps has to be turned on in different constellations. Even though daylight shows strong and fast variations in irradiance (Figure [fig:light:regime:clouds] on page ) the overall light pattern is bell shaped. To mimic natural light conditions a gradual or stepwise change during the morning/evening hours is preferred. If it is possible to get e.g. 600 from the lamps, a 14 h-long day will give an integrated of 30 , which corresponds to a sunny day in May in Denmark (Figures [fig:ppfd:greenhouse] and [fig:ppfd:lamps]). With the present development of the LED technology the first climate chambers with LED lighting have been introduced to the market, and one can expect that LEDs will be used extensivelly for controlled environments in the future. LEDs can be dimmed without any drastic changes in their spectral profile and the lack of heat radiation in the direction of the plants removes one complicating factor for lighting in climate chambers. Figure [fig:spectra:PAR:lamps] shows the photon emission spectra of some lamps used as PAR sources in controlled environments and greenhouses.
In climate chambers it is often possible to create a fast change between the day and night temperatures, though this is unnatural to plants. Under natural conditions a 5–10decrease in air temperature normally takes hours, allowing e.g. the membrane fluidity to follow. If the temperature drop between day and night is big and fast some plants may experience transient stress linked to membrane fluidity, which would not be the case if the change were slower. This is something to bear in mind, when designing the chamber climate. It can also increase the risk of fungal disease if the air humidity is high and the plants become damp, in the same way as in greenhouses.
If experiments are done at low or high temperatures there may be restrictions on how well humidity can be controlled. At low temperature there may be limitations on the capacity for dehumidification and at high temperatures it may not be possible to sufficiently moisten the air.
Numerous plant species lose the ability to effectively regulate their stomata when grown at constant high humidity (ca. 70% RH at 20–25). To our knowledge, this has been observed in , , and . These plants lose their ability to regulate stomatal conductance when subsequently exposed to low air humidity e.g. if moved from the greenhouse to a laboratory for measurements of photosynthesis. In the worse case scenarios, the plants transpire to such an extent that small local spots of necrosis develop on the patch where gas exchange has been measured on the leaf. However, since results like these are considered as failed experiments such observations have not (to our knowledge) been scientifically published. Nevertheless, it is important to be aware of this potential problem when working with mesophytic leaves, i.e. “average” leaves that are adapted neither to dry nor to wet conditions, like the examples above.
Data logging of the climate is needed in controlled environments to catch irregularities and failings in the climate control. This is especially important to test for differences among chambers when the climate set points changes. The Committee on Controlled Environment Technology and Use under the USDA has issued recommendations for data logging of growth chamber climate, which are compiled, with some changes, in Tables [tab:growth:guidelines] and [tab:growth:envir:describing].
| Parameter | Units | What to report |
|---|---|---|
| Manufacturer | n.a. | Name and address (n.a. for greenhouse) |
| Model | n.a. | Model descriptor if available. Greenhouse type and roof height for greenhouses |
| Size | Floor area | |
| Barrier beneath lamps | Yes/No | Indicate if present and composition. Optical properties if available (not greenhouses) |
| Cladding material | n.a. | Indicate material and optical properties if known (only for greenhouses) |
| Air flow | n.a. | Indicate whether up, down or horizontal (not greenhouses) |
Recommendations on parameters that should be logged have been published by the International Committee for Controlled Environment Guidelines - see http://www.controlledenvironments.org/, http://www.ceug.ac.uk/ICCEG.htm and http://www.controlledenvironments.org/guidelines.htm.
13.5 Material issues in greenhouses and controlled environments
A potentially problematic issue concerning materials in greenhouses and climate chambers relates to the use of different types of soft plastic materials. Plastics like cellulose acetate, cellulose nitrate or PVC are stiff by nature. It is only when they are mixed with a softener i.e. a “plasticiser” that they become soft and flexible. The plasticisers may be different alkyl esters of phthalic acid. Some of these phthalates are phytotoxic, particularly butyl phthalate (DBP) and di-isobutyl phthalate (DIBP) (Hannay and Millar 1986; Millar and Hannay 1986). The phytotoxicity is evident as poor growth, chlorosis, necrosis and plant death. At regular intervals since the 1950’s there have been examples in the greenhouse industry and plant research facilities where new plastics have been introduced for different purposes, which have caused serious growth problems (Hardwick and Cole 1987). Many species are damaged by phthalates but species of Brassicaceae seem to be particularly sensitive. Past examples of products responsible for phytotoxic problems include plastic tunnels, water hoses, drip irrigation tubes, plastic plant pots, glazing strips (that seal between the glass and aluminium frames in the greenhouse construction), plastic insulation of electrical wires and plastic boots—the latter creating growth problems in a greenhouse outside a staff changing room. Many of these problems have been created when the recipes or composition of products already in use in a greenhouse have been changed by the manufacturer. Growth chambers require some fresh air inlet for the climate control. There is an example of poor growth and bleaching of plants in a climate chamber when the air inlet was close to the ventilation outlet of a chemistry laboratory. When the air inlet was moved 200 m away and supplied with a compressor, the problem disappeared.
13.6 Gas-exchange cuvettes and chambers
Measurement of photosynthesis through gas-exchange with present requires modifications of most standard equipment. Most gas-exchange cuvettes have windows that attenuate radiation. This means that most leaf photosynthesis and stomatal conductance measurements are done in the absence of radiation, even when done in sunlight. Stomata have been shown to respond to radiation (e.g. Eisinger et al. 2000), and at high irradiances the rate of photosynthesis can also be affected (see Rozema et al. 1997 for a consice review).
For example, the GFS-3000 system from Walz, can be ordered with cuvette windows made of quartz instead of normal glass. Some cuvettes for the LI-COR LI-6400 use a plastic film as a window, and it is possible to use an -transparent material. The cuvette for CIRAS-2 from PP Systems is delivered with Calflex heat filter glass as standard, but can easily be fitted with quartz glass for transmission on request.
Frequently used light sources are tungsten-halogen lamps, which emit very little radiation, or light sources based on red LEDs in combination with blue or white LEDs, which do not emit any radiation.
When designing experiments, especially when leaves or shoots are enclosed for more than a couple of minutes in a gas-exchange cuvette, one should be aware that the leaves or shoots may be in an unnatural light environment, even when measurements are done in full sunshine. To avoid surprises, always measure irradiance and especially spectral irradiance through the cuvette window, rather than outside the cuvette, if you want to describe the radiation environment during measurements.
Plastic and rubber used for tubing have different properties of permeability to and water vapour, and absorption of water, which can lead to flawed gas-exchange data. These properties are listed in Long and Hällgren (1987).
13.7 Plants in the field
One important disturbance in experiments on natural vegetation or field crops is that produced by the researchers themselves. Some ecosystems like peat bogs get more easily damaged than others, but in most cases special precautions are needed whenever repeated access to plots is needed. The most common approach is to use catwalks made from wooden planks raised some centimetres above the ground. Sometimes when access is needed to the centre of the plots ladders are put temporarily, laying horizontally bridging two catwalks. Figure [fig:catwalks:Abisko] shows some examples from Abisko and Figure [fig:filters:photo:field1] on page shows an example from Ushuaia.
Not only the spectrum, but also the temporal variation of irradiance differs between controlled environments and natural conditions. The temporal light regime depends on the position of the sun and on clouds (Figure [fig:light:regime:clouds]), and also on the vegetation itself (Figure [fig:light:regime:sapplings]). For experiments done in the field summaries of data from an in situ or nearby weather station should be used to describe the growing conditions. To assess the light environment at different locations in a forest understorey, hemispherical photographs taken with a fish-eye lens, can be used. There is software available—e.g. Hemiview from Delta-T—that allows the prediction of the light environment based on the position of the sun in the sky at different times of the day and seasons of the year.
13.8 Cultivation of aquatic plants
When designing experimental set ups for the cultivation of aquatic plants under full solar radiation (+ + ) or under a simulated natural radiation field (see section 8.4 on page ), the spectral transmission of the vessels or photobioreactors used must be accounted for as well as the absorbtion of radiation by the water, growth medium and the aquatic plants themselves (self shading). Most studies evaluating photosynthetic activity in algae have been conducted in -opaque incubators and this has consequently led to overestimation of their photosynthetic capacity, since radiation causes photoinhibition and photodamage in aquatic plants (Häder and Figueroa 1997; Villafañe et al. 2003; Bischof et al. 2006).
The most frequently used photobioreactors have a cylindrical shape and they can be transparent or opaque to radiation (Figure [fig:culture:systems], see section 8.4 on page ). Some photobioreactors are built from rigid plastic such as polycarbonate (PC), polymethylmethacrylate (PMM) or polyvinyl chloride (PVC), and others from flexible plastic such as cellulose acetate or polyethylene.
The aims of photobioreactor design are:
high volumetric productivity (g l\(^{-1}\) d\(^{-1}\)),
high productivity per unit area (g m\(^{-2}\) d\(^{-1}\))
high cell concentration (g l\(^{-1}\))
high efficiency in the conversion of light (g mol\(_\mathrm{photons}^{-1}\))
high biomass quality
sustainable and reliable cultivation, and
low construction and operating costs.
The material for the photostage of the photobioreactors must have:
high transparency,
high mechanical strength,
high durability (resistance to weathering),
chemical stability
ease of cleaning, and
low cost.
Seaweeds can be also cultivated in large tanks (500–1000 l) made of materials such as polyethylene, glass-fibre-reinforced polyester or polypropylene—which all attenuate radiation (Figure [fig:large:tanks:photos]). Thus when and visible radiation are supplied by artificial lamps, they must be located above the open top of the tanks. Likewise when tanks receive natural solar radiation, direct:rad is available within the tank only at high solar elevation angles, whereas diffuse:rad predominates during the rest of the day. Large containers are considerably more expensive than other systems and require an ample water supply and frequent maintenance. Facilities originally designed for other purposes, e.g. biofiltration of fishpond effluents, are often used for the incubation of aquatic plants (Neori et al. 1996; Figueroa et al. 2006). These tanks can be placed outdoors under full solar radiation or enclosed inside a greenhouse.
In tanks, incident radiation is generally attenuated very rapidly and attenuation depends largely on algal density, the shape and volume of the tanks and the amount and type of particulate and dissolved material in the water. If we compare the irradiance at different depths in the water with different regions of the light-response curve of photosynthesis, we can see that PAR irradiance can be high enough to cause photoinhibition in the surface layer (Figure [fig:photosynthesis:in:tanks]). The smallest irradiance causing photoinhibition is denoted in the graph as Qh while the irradiance incident at the surface of the water is Q0. In the next layer below the surface, the incident irradiance is lower than Qh but higher than saturating irradiance for photosynthesis (Qs). About 80–90% of photons reach this layer of maximal production. The irradiance in the layer below, which is deeper than the layers above, is lower than saturating irradiance but higher than light-compensation-point irradiance (Qc)[^34]. Finally the irradiance in the deepest layer at the bottom of the tank is lower still Qc, this is considered to be a ‘dark’ layer (in general 70% of the volume of the tank). Thus, it is crucial that the algae are vigorously circulated through the tanks using air to maintain a light:dark regime adequate for growth, i.e. the algae move from the bottom of the tanks, where the tubes injecting the air are located, to the upper layers, where photon irradiance is higher.
To cultivate both fixed or free floating aquatic plants, the plants must receive air (with or without enrichment) in addition of light and nutrients. Air not only serves as a carbon source but also produces the turbulent environment needed to provide nutrients to the algae, which under a laminar regime would be less available to some of the algae leading to their starvation. Thus, the suitable movement of macrophytes within the tank results, not only in their adequate exposure to PAR and radiation, but also the provision of nutrition and gas exchange. Thus, the appropriate balance between the initial inoculum and the water volume should be accurately calculated. Due to circular movement of water within a tank, macrophytes are often exposed to variable light conditions or, when densities are high, to self-shading. The cultivation of subtidal seaweeds in outdoor tanks can cause bleaching or chronic photoinhibition because they are not normally exposed to high irradiances of . These effects of high irradiance can mask the effects of radiation. This can be avoided by the use of neutral shading screens that minimise the undesirable effects of excessive light, as exemplified in a tank cultivation of three seaweeds from the coast of Baja California (Cabello-Pasini, Aguirre-von-Wobeser, and Figueroa 2000). By neutral screens, we mean grey or black shade-cloth that has little or no effect on the radiation spectrum, attenuating all wavelengths by the same relative amount. When using more than one layer of shade-cloth, remember that the effect is multiplicative rather than additive. For example two layers of a cloth with a 50% transmittance each will yield a transmittance of approximately 25%.
As an example, in cultures of in semitransparent polyethylene tanks, the high algal biomass density caused a drastic reduction in the maximal average irradiance at 10 cm depth compared to that at the surface (Figueroa et al. 2008). At 10 cm depth, was reduced by about 87.5% at an algal density of 4 g l\(^{-1}\), 89.5% at 6 g l\(^{-1}\) and 95% at 8 g l\(^{-1}\). The attenuation of radiation was 94% at 4 g l\(^{-1}\), 96% at 6 g l\(^{-1}\) and 99% at 8 g l\(^{-1}\). radiation at noon was fully attenuated by an algal density of 4 g l\(^{-1}\) at 10 cm depth, while with this same algal density there was “complete darkness” (less than 0.1% of incident irradiance) at 25 cm depth.
The shape of the tank and the ratio of surface to volume (S:V) also have an effect on the hydrodynamics and the movement of the seaweeds (Figure [fig:culture:tanks:S:V]). Semicircular tanks of fibre glass (750 l) have better hydrodynamics, not only because they allow more water movement and a better distribution of the air bubbles in the tank, but also because of their higher S:V ratio (2.4 m\(^{-1}\)) than that of cylindric 1500 l tanks of semitransparent polyethylene (S:V = 1 m\(^{-1}\)) or 90 l (S:V = 2.2 m\(^{-1}\)).
Another aspect of good experimental design is choosing the location of plants within the culture system, i.e. the merits of fixed- versus free-floating aquatic plants. Both fixed and free floating aquatic plants absorb radiation to differing extents depending on their pigment composition and the thickness of their tissue or thallus. Aquatic angiosperms can be anchored to both natural materials, such as stones, and artificial materials (rows, epoxy, etc.). Seaweeds are usually cultured in free floating systems where they are moved by the injection of air, although in nature they are fixed to different substrates. Not all species can be cultivated floating freely and in some cases this can alter their growth forms, transforming branched forms into a spherical forms. Morphological changes affect both bio-optical properties (i.e. including absorption) and nutrient assimilation which is higher in branched forms than spherical forms due to their higher surface:volume ratio (of both cells and the whole plant). Thus, in photobiological experiments, it is crucial to optimise both algal density and the light field—by using lamps located to one side (lateral position) or above the aquarium—so as to reduce or avoid self shading.
Mesocosms have been developed to recreate natural conditions as closely as possible on a small scale. They are generally used to study plankton communities, however, their use in aquatic macrophyte ecology is increasing. For example, floating mesocosms and mesocosms connected to land—e.g. attached to a platform or pier—have been developed (Figure [fig:floating:mesocosms]).
Several mesocosm studies have used ambient radiation supplemented with radiation from fluorescent lamps to simulate an increase in radiation due to ozone depletion. Two methodologies can be applied: (a) supplementing a fixed irradiance value (“square wave systems”), or (b) supplementing a fixed percentage of ambient irradiance (“modulated systems”) (C. Belzile et al. 1998; Claude Belzile et al. 2006). Modulated systems, which were originally developed for terrestrial plants (Caldwell et al. 1983), are better than square-wave systems for simulating predicted increases in radiation (Díaz et al. 2003, 2006). See section 8.2.6 on page .
When using mesocosms, experimental designs must be as realistic as possible in simulating forcing factors as well as their variability. For example, when studying the effect of increased , experiments should be designed to reproduce diurnal variations in irradiance and episodic events such as passing clouds. Recently, experiments using mesocosms have been applied to evaluate the interactive effects on macrophytes of environmental shifts driven by climate changes (temperature, , radiation and nutrient availability). To mimic climate change conditions, Liboriussen et al. (2005) designed a flow-through mesocosm in a shallow lake which can also be applied to other aquatic ecosystems. Recently, a new automatically-operated system providing accurate simulations of the increases in radiation and temperature according to climate change scenarios has been developed (Nouguier et al. 2007). All these systems reproduce the sort of high degree of environmental variability inherent to aquatic systems.
13.9
In this section we list recommendations related to the cultivation of plants used in experiments studying the effects of radiation. See sections 8.6.1 and 8.6.2 for recommendations on manipulation of radiation, section 9.15 for recommendations on how to quantify radiation, and section 11.12 for recommendations about statistical design of experiments.
Include in the methods description in your publications enough information about the growing conditions so that the experiment can be duplicated.
When using growth chambers, growth rooms, or greenhouses with environmental control systems report in addition to model and manufacturer and its address, all the settings used:
Temperature
Air humidity
Lamps.
Shade/insulation screens.
supply
Pots, soil and fertilization.
Watering.
Plant material
Check the calibration of the climate sensors that control the greenhouse or controlled environment. For example, PAR sensors should be calibrated once a year, infrared gas analysers (IRGAs) used in control once every week or two (but depends on the instrument, so follow the manufacturer recommendations). Electronic thermometers and capacitive air humidity sensors (such as Vaisala HUMICAP) should be checked a few times per year, and recalibrated when needed.
Program the data logger for logging each minute, either a separate logger with sensor or the climate logging function of the climate computer using the same sensors as are controlling the climate. If you use a separate battery-powered datalogger, the data record will include the time during mains power interruptions or environmental control system failure.
13.10 Further reading
Poorter, Fiorani, et al. (2012) have written a practical guide on how to grow plants for reproducible results, giving several examples of how variation in the climate influence the results, including suggestions for further reading. If biomass allocation to different plant organs is studied in an experiment, the Tansley review by Poorter, Niklas, et al. (2012) should consulted as it presents a meta-analysis of how climate parameters other than UV-B affect plant morphology. This is especially important if results from different growing seasons are compared. For general introduction to the energy, water and carbon balance of plants in relation to the climate we refer to Jones (1992) and Nobel (2009), for climate physics in relation to plant canopies to Oke (1988) and for more thorough calculations of the greenhouse climate to Bakker et al. (1995; Stanhill and Enoch 1999; Beytes 2003; Stanghellini 1987).