· Drought Stress - Mitigation

Managing the Dryland Crop Environment

Modern dryland farming is a system of low inputs combined with soil and water conservation practices and risk reducing strategies. The system can be sustainable if practiced properly. Water shortage is the main limiting factor, but successful dryland systems also maintain reasonable practices to eliminate other limiting factors (poor nutrient status, weeds, biotic stresses, etc'), which can reduce the effectiveness by which the crop uses the limited moisture. However, as water shortage a priori dictates a limit on yield, all other inputs must be carefully adjusted downwards to fit the expected low economic return.

The most advanced systems have been developed in the Great Plains of the USA, Canada and Australia, while traditional systems employed in Asia and the Middle East also offer important insights. In the USA, the lesson learned during the "dustbowl" years in the early 1930's prompted extensive legistration and investments in developing sustainable dryland farming systems. These systems and the associated technological progress such as plant breeding, brought about an increase in mean winter wheat yield from 0.5 ton ha^-2 in 1930 to about 2 ton ha^-2 in 1980. In Southern Australia the "ley farmimg" system was developed in the 1920's and adopted widely in the late 1940's. The system involves a rotation between a self-seeding legume grown for several years and wheat. The farmer grows wheat and raises sheep while the legume serves to sustain soil fertility (mainly nitrogen). This system has become less popular in recent years with the increase in economic pressures and other considerations.

The lesson learned from the American and Australian experience is that the development of a sustainable dryland farming system involves the following principles, not necessarily in their order of importance:

1. Improved soil and water conservation practices and the associated reduced tillage systems.

2. Optimization of the fit between crop growth cycle and the available moisture.

3. Weed control

4. Soil fertility management.

5. Optimized plant population density and spatial arrangement of plants with respect to the expected soil moisture regime.

6. Control of soil biotic stress factors that reduce root development.

7. Improved forage/livestock/grains integration and rotation.

8. Avoidance of mono cropping and enhancement of crop diversification.

9. The increase of precipitation by cloud seeding, as an ongoing experiment.

Some of the above principles of the dryland farming system constitute general knowledge in agronomy, which can be explored in our Web Resources page as well as in standard agronomy textbooks and other publications (e.g. Drought Management of Farmland - by Joan Sydney Whitmore, 2000, 360 pp., Springer, SBN 0792359984). Here only several topics will be touched upon.

Soil and water conservation

Fallow and conservation tillage

The fallow system is designed to conserve soil moisture from one season to another or from one year to the other, depending on climate and crop. Increasing storage of soil moisture by the fallow system with or without conservation tillage is standard agricultural practice in dryland farming. The benefit of fallow and conservation tillage in terms of increasing available soil moisture to the crop depends on soil water-holding capacity, climate, topography and management practices. Fallow efficiency, in terms of percent increase in soil moisture availability to the crop measured at planting date normally ranges from about 5% to 30%. While these amounts are not impressive they can make a difference between crop failure and success. The fallow carries additional benefits such as improved soil nutrients availability and the eradication of certain soil-born pests, such as nematodes.

Conservation tillage is not a novel concept or practice, which has recently gained wider and sometimes an enthusiastic acceptance. It involves the principle of minimized tillage operations to conserve soil structure and to maintain ground cover by mulch, such as stubble. These practices reduce water runoff and increase soil infiltration. Conservation tillage has become the cornerstone of dryland systems in certain regions of the USA, Canada, Australia and other regions. While the benefits of conservation tillage are well-documented it has also been noted that crop residues under this system may promote certain crop diseases. To obtain some real impressions on the subject spend an hour in a farmers’ meeting in California on the subject.

Deep tillage is a system to overcome hardpan, very high bulk density and compacted soils. It can be performed by deep plowing or deep ripping. Deep plowing involves actual plowing to depth which is an expensive operation. It is uncommon in dryland farming. Deep ripping is less expensive and often used in crop production. The important consideration in deep ripping is to operate at the correct depth in order to break the hardpan, no less and no more.

Furrow dikes and Soil pitting

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These techniques constitute a field surface tillage manipulation to minimize runoff away from the field.

Furrow dikes are furrows, which are divided into short basins by small dikes (see right side photograph). This is achieved by special equipment . The system is very amenable to row crops such as cotton, corn and sorghum and it can be integrated with or without furrow irrigation. It is generally considered effective for increasing rainfall capture and raising dryland yield where annual rainfall ranges between 500 and 800 mm.

Soil pitting (left side photograph) involves the formation of small depressions at close proximity to reduce runoff from rainstorms. The crop is planted over this modified surface. Experiments performed with wheat in nine farmer demonstration plots in Southern Israel during 1988 showed that pitting increased yield by an average of 7.5% at a mean yield of 3.25 ton/ha. Unlike furrow dikes these system is not limited to row crops.

For further online information on dryland farming and its research visit the following sites and the links therein:

§ Soil and Water conservation in Semi Arid Areas – (FAO Manual, 1987)

§ USDA-ARS Bushland Texas Experiment Station – Visit the web site of a distinguished center of excellence in dryland conservation research

§ Soil, water and reclamation publications --From Alberta, Canada

Water harvesting/spreading

This is a broad term to describe various methods to collect runoff from large contributing areas and concentrate it for use in smaller crop area. This is an ancient practice already adopted by Nabatian desert settlements in the Middle East several centuries A.D. The photo on left represents a view from the ancient city of Avdant in the Negev region of Israel. In the front there are several ancient water spreading plots while at the back is a modern experimental farm (set-up by Prof. Evenari) seeking to evaluate the effectiveness of these systems in sustaining agriculture with around 100mm of annual rainfall.

Presently, the basic water harvesting systems involve an external contributing area to induce runoff. This area is physically or chemically treated for maximizing runoff. The water is diverted into a receiving area comprising of cultivated plots, individual trees or small terraces. The contributing area may lie in the agricultural field (a system sometimes referred to as "conservation bench terrace") or outside the field in the natural watershed system. In the Avdat photo the small valley is a water-shed system experiencing flash flood once or twice a year. The size ratio between the contributing and the receiving areas is determined by the expected rainfall events, crop water requirements, soil characteristics and topography. The resulting yield increase in the receiving (crop) area is proportional to the amount of water gained.

For more detailed information in this important practice see Water Harvesting and Runoff Farming.

Diversification of farming

Diversification of farming is an ancient but an effective approach to reduce the risk associated with farming in unpredictable environments. Reduced diversification to the extent of mono-cropping is possible only with a high level of control over the crop environmental conditions. Such control method (irrigation, chemical pest control, etc’) are among the main reasons for the more recent environmental quality problems found to be associated with mono-cropping. Diversification of cropping to reduce risk is especially important under dryland conditions. It is achieved on several levels, as described by Pandey et al (#4194) for the case of traditional rain-fed rice in Eastern India.

1. Spatial diversification of fields. The farmer’s land is divided into several fields or plots which may differ in their topography, soil and hydraulic properties. Some fields may be prone to flooding while others do not hold water. Certain fields may be on a warmer slope while others on a cooler one. The different field conditions allow to achieve a better fit between the crop and the environment and to reduce the general probability of stress affecting the farmer.

2. Crop diversification is an important feature of traditional farming. It takes an advantage of the generally low correlation between crops in performance when grown in a single stress environment. Crops differ in their response to a given environment and this difference is used to reduce the risk associated with growing one crop. “Mixed cropping” or “intercropping” is an example of a traditional and a successful approach to crop diversification on a single parcel of land, where two or more crops are grown together in various possible configurations. If for some reason only one crop is grown, a certain (though lower) level of risk reduction can be achieved by varietal diversification. Planting of several crop varieties offer a better probability for reducing loss due to environmental stress, as compared with growing one variety only. For environmental stress conditions varietal diversification is based mainly on differential phenology, primarily flowering date. A typical example is a transient frost or heat wave that is likely to occur around flowering time of the specific crop. Damage reduction can be achieved when the crop is sown to several varieties of different flowering dates.

3. Temporal diversification may achieve the same result as varietal diversification, when phenology is concerned. The purpose of setting a distinct planting date is to optimize crop development with respect to seasonal climate, mainly rainfall in rain-fed agriculture. Ideally the crop is planted at the beginning of the rainy season, rainfall peaks when crop evapotranspiration peaks and it terminates just before harvest time. When such conditions are reasonable predictable, planting date can be set to optimize production. Where the timing of rainfall is very unpredictable, adopting more than one planting date for the given crop can reduce the risk involved with untimely rainfall and a given planting date.

Cloud seeding

Cloud seeding is a form of weather modification attempt. The process of cloud seeding involves deposition of cloud condensation nuclei (CCN) into a specific region of the cloud. Seeding may be achieved from above or through the clouds by aircraft, and from below where CCN are carried into the cloud by updrafts. With either method, the CCN must reach the super cooled cloud region, where water molecules remain unfrozen at temperatures below 0C. Experiments in cloud seeding have been performed for the last 60 years. The results and benefits of this practice are still under debate. Information is available in the report on Weather Modification by Cloud Seeding - A Status Report 1989-1997 by William R. Cotton, Colorado State University; and at the Oklahoma Weather Modification Demonstration Program.

MITIGATION OF DROUGHT STRESS BY CROP PLANT BREEDING

The following section is a summarized discussion. For a more comprehensive treatment of the subject and for additional practical considerations and directives it is recommended you read the following book by A. Blum (2011), entitled “Plant Breeding for Water Limited Environments”, published by Springer.

The nature of Drought Resistance

Drought Resistance and Crop Yield

Crop plant breeding for drought resistance has long been part of the breeding process in most crops that are grown under dryland conditions. During the period of the pre-scientific agriculture the genetic improvement of plant adaptation to dry conditions was simply attained by repeatedly selecting plants that appeared to do well when drought stress occurred. As a result of many generations of selection by generations of farmers we now encounter such materials, which are defined as “landraces” of the crop. Such landraces were shown to possess distinct drought resistance traits. Later, as scientific agriculture developed and following the emergence of Mendelian genetics, elaborate biometrical and statistical methods for quantitative genetics analysis were developed to enable selection for yield and yield stability more effectively and efficiently. An important factor of yield stability is coping with drought and other abiotic plant stresses. As crop physiology emerged and developed, yield-based selection programs were augmented by observing plants under carefully managed stress environments, followed by the development of physiological selection criteria for stress resistance. More recently, molecular methods, such as marker-assisted selection are being adopted to facilitate more efficient selection for distinct components of abiotic stress resistance. Finally, biotechnology is experimenting with genetic transformation, open the way for additional genetic solutions to breeding for drought resistance.

Fig. 1. The schematic association between yield and total seasonal precipitation for 3 different wheat cultivars.

Looking at crop drought resistance from a botanical perspective it must be realized at the onset that there is a vast difference between drought resistance in natural vegetation and in crop plants. Natural vegetation has evolved to conserve the species. Henceforth, plant survival and the capacity to produce at least one seed per life cycle despite stress is the most powerful component of natural selection. On the other hand, drought resistance in modern agriculture requires sustaining economically viable plant production despite stress. Crop survival is of a lesser consequence to economical farming. On the other hand, plant survival can be a critical factor in subsistence agriculture, where the ability of a crop to survive drought and produce some yield at all may translate into a difference between famine and livelihood. Breeding for drought resistance is therefore very tightly linked to the target environment of the crop, not only with respect to its physical and chemical features but also its social grounds.

For a variety of reasons (see discussion below and in #7963) there is a general trade-off between a genetically high yield potential and drought resistance. At the same time there is a yield advantage under drought stress brought about by a high yield potential, to a limit. This is explained very briefly here through Fig.1.

Wheat cultivar C is different from A and B in that it has a lower yield potential (yield at high moisture conditions) but as moisture becoming deficient C turns out to be superior to A and B. In terms of yield, C may be defined as drought resistant while cultivars A and B are of high yield potential but are relatively drought susceptible. The “crossover” where the advantage of C over A and B under stress begins to be expressed is at about 300 mm or at a yield level of about 300 g m^-2. Hence, drought resistance of C is expressed only when stress is sever (<300 mm). Still, it is extremely important to realize that the high yielding cultivars A and B are superior to the drought resistant C when drought stress is moderate (e.g. at 400 to 500 mm). A high yield potential therefore ascribes an advantage under moderate stress conditions. On the other hand Fig.1 indicates that by definition drought resistant cultivars have lower yield potential. Cases where drought resistance has been improved together with yield potential exist but they are very rare and exceptional and cannot be used to indicate a general rule. With the available evidence the rule seems to be with Fig.1, with exceptions. Fig.1 also implies that breeding for real drought resistance is not required if yield in the target environment is not reduced (schematically) to below 300-400 g m^-2. On the other hand, real drought resistance cannot be field- tested or evaluated if yield level is above around 300-400-g m^-2, schematically. Consider the principle not the actual numbers. The actual numbers were obtained for wheat and barley by several studies.

The components of drought resistance

Drought resistance in crop plants is conditioned by two major pathways: Dehydration avoidance and dehydration tolerance. Dehydration avoidance is the capacity to avoid plant tissues and cells dehydration under drought stress. Dehydration tolerance is the capacity to sustain function when the plant is dehydrated. Plant survival can be conditioned by either avoidance or tolerance.

As discussed above under the impact of stress, moisture stress signals the expression of certain stress responsive genes, which are responsible for a chain of events and gene “networking”, expressed at various levels of plant organization. It has been assumed almost axiomatically that stress responsive genes are involved in adaptation; henceforth that they are ‘stress adaptive’. It was later realized that not every stress responsive gene is necessarily adaptive in terms of drought resistance or survival or crop productivity under moisture stress..

Irrespective of the role and function of stress adaptive genes in plant drought resistance, it should be recognized that not only certain stress adaptive genes might determine plant performance under drought stress. Genes that are expressed irrespective of the environment also condition plant function and performance under stress. These genes are expressed constitutively and determine various plant traits irrespective of any stress. An example for a constitutive plant trait that may control drought resistance is potential root depth (maximum root length). Stress and soil conditions can affect root depth in several ways but potentially a deep rooted genotype will maintain its advantage over a potentially shallow rooted genotype under conditions of deep soil moisture. For this difference to be expressed plants do not have to be subjected to drought stress conditions. Therefore, drought resistance and plant production under drought stress is determined by constitutive and adaptive plant traits.

Dehydration Avoidance

Plant development and size

Plant size as expressed mainly in terms of single plant leaf area or leaf area index (LAI) has a major control over water-use, as explain under impact of Stress. At the same time small plants and reduced leaf area are generally conducive to low potential productivity. Botanists have long recognized small plants bearing small leaves as typical ecotypes of xeric environments. While such plants withstand drought very well their growth rate and biomass are relatively low.

In the domain of plant breeding, cultivars developed for dryland conditions by selecting mainly for yield under such conditions often resulted in plants of moderate size and water-use. For example this can be seen in dryland temperate cereals as well as upland rice, which tend to have moderate tillering. On the other hand it has also been shown that early plant (and seedling) vigor (#3903) are important traits for dry conditions. The reason is in the rapid ground cover achieved and the subsequent decrease in water loss by direct soil evaporation at this stage. However, other benefits for seedling vigor were also noted, such as the nitrogen status of the plant (#7059). Early flowering which determines ‘drought escape’ (see below) generally involves a reduction in adult plant size and leaf area leading to reduced water-use. Thus, small plant size and small leaf area are very often linked to improved dehydration avoidance and lower potential yield, a tradeoff discussed elsewhere (#7963 ).

The Root

The most important control of plant water status is with the root, whereas the roots is the main engine for meeting transpirational demand. Two major dimensions describe the root: root depth (or maximum length) and root-length density (Fig.3). The more practically and commonly important dimension for most breeding scenarios is root depth, which facilitate deep soil moisture extraction where such moisture is available. It is a primary component of drought resistance. The development of lateral roots at very shallow soil depth may have a role in capturing small amount of intermittent rainfall.


Fig. 2-3. Left panel: hydroponically grown roots of two wheat cultivars differing in root length. Right panel: roots of two sorghum cultivars in soil in a root observation box (1.8m tall), differing in root-length density on the given date.

Root depth in the cereals is generally associated with a small number of main thick axes. Such fibrous root system is typically seen in upland rice, which has a deeper root system, in contrast to lowland rice with the shallower roots. The control of root growth is not only in the root. In the cereals, tillering is associated with production of new crown roots from each developing tiller. Such profuse rooting can be at the expense of the growth of existing roots into deep soil. Hence, limited tillering in cereals and grasses has been repeatedly observed to be associated with relatively deeper root extension.

In certain soils a hardpan can limit deep root growth and the capacity for hardpan penetration by roots becomes a critical factor in drought resistance. The factors, which may support axial root force and hardpan penetration, are not known and most research in this area has been performed mostly with seedlings. In mature plants the penetration of hardpan by roots seems to be better in plants that constitutively develop fibrous and thick roots.

Many drought environments present a situation where rainfall is low and soil depth that contains moisture is permanently shallow. For example, in many of the drier Mediterranean wheat-growing regions the wetted soil depth of around 60-80 cm is shallower than the normal maximum root depth of wheat (=>100 cm). Under such conditions a deep root is not an issue. Other plant factors may then become far more important in the control and use of the limited soil moisture, such as shoot developmental characteristics (e.g. leaf area development or growth duration), osmotic adjustment, leaf surface properties, etc.' Greater root length density within this limited soil horizon might allow extracting more moisture from a given soil volume which in certain cases should provide several more days before wilting.

Another scenario of seasonal soil moisture status is where the crop is grown on stored soil moisture and there is little effective rainfall during the growing season. Under such conditions the main consideration is to manage seasonal soil moisture use such that sufficient moisture will remain for carrying the crop to maturity. It is to be expected that with the available moisture the crop might grow luxuriously leading to a large leaf area and an even greater water requirement towards the latter part of the season. Hence, short growth duration, small leaf area and perhaps a higher root hydraulic resistance can achieve the control of seasonal water use. The last option has been researched at the CSIRO Australia and an increase wheat root hydraulic resistance was effectively attained by selection for smaller root xylem element diameter. It is not known whether this approach has found its way into actual application in wheat breeding or whether such a cultivar was released.

Whatever may be the constitutive form and function of roots, the environment can modify the root in a pronounced way. Off course, soil conditions in terms of topsoil moisture and deep soil hardness alter root growth and depth. Drought stress generally inhibit total root mass (while it can modify its distribution). Root-length density may locally increase in wet regions in the soil while it might decrease in the drying parts. As soil moisture deficit develop throughout the profile, the proportion of dry to wet soil increase so that the proportion of dead to live roots increase. There is hardly evidence to show that total root mass increase with drought stress. The shoot/root mass ratios consistently decrease under drought stress, which is a universal expression of adaptation. The ratio changes mainly due to the reduction in shoot mass.

The root system is highly dynamic and as long as it is not senesced or diseased it is capable of regrowth from meristems in the root axes and meristems in the root crown (in cereals and grasses). The renewal of root branching into wet soil immediately after rainfall is considered as an important factor in plant recovery from drought stress. Root hairs are considered an important component of root length density and the capacity for soil moisture extraction via improved contact with the soil.

Roots are a major target and a candidate for marker assisted selection (MAS) for the apparent reason that phenotypic selection for root traits is a slow and impractical in large populations. Still, practical results from MAS for root traits (e.g. 8095) are limited.

Plant Surface

Plant surface structure, form and composition carry a major impact on the plant interaction with the environment. Plant surface absorbs solar energy part of which is used for photosynthesis and most of which must be dissipated. Energy is dissipated by reflection, emission and the dissipation of latent energy by transpiration. Plant surface structure determines the reflective properties of the leaves and their resistance to transpiration. Leaf resistance to transpiration is off course largely determined by stomatal activity. However, plant surface structure determines the hydraulics of leaf surface, which affect the rate of water removal from the leaf surface, upon transpiration. Therefore plant surface help to avoid dehydration by two channels: improved reflectance of incoming g radiation (i.e. decreasing net radiation) and by improved cuticular hydraulic resistance.


Fig. 4. Sorghum leaf epicuticular wax by the scanning electron microscope; left normal (Bm genotype); right low wax (bm genotype).

Fig. 5. Leaf pubescence in the wild plant Solanum elaeagnifolium (Silverleaf) (right) as compared with cotton (left).

After the stomata, the secondary site for water loss by transpiration is the cuticle. The hydraulic permeability of the cuticle is determined by the wax embedded in the cuticle matrix as well as by the wax deposited over the cuticle. High cuticular permeability not only affects non-stomatal transpiration pathway but it may also directly affect water loss from guard cells and therefore their water status and stomatal aperture. Fig.4. presents an example of a difference in epicuticular wax load between two sorghum genotypes. The lower wax (bm) genotype had far greater total leaf transpiration than the Bm genotype.

Epicuticular wax is deposited in different forms and structures, mostly as a function of its composition. The environment also affects the density of epicuticular wax. Conditions of water stress, high temperature, and high radiation increase its density. The full genetic potential for wax deposition is therefore best evaluated in plants subjected to stress.

In practical terms, the quantitative effect of wax on transpiration is finite, and for a given plant, the increase in epicuticular wax load beyond a given threshold would not reduce transpiration. Sorghum typically represents relatively high potential epicuticular wax deposition while rice represents species that lack in this respect, as estimated by quantifying epicuticular wax and by rate of cuticular transpiration. Hence, there is a potential for improving drought resistance in rice by genetically increasing epicuticular wax load.

The shape and angles of the cuticular wax deposits of may affect the spectral properties of the leaf. Thus, for example, the glaucous appearance of some wheat genotypes is determined by the structural properties of the wax deposits. Increased glaucousness was found to result in an increase in leaf reflectance of wheat and sorghum within the spectrum range of at least 400 to 700 nm and possibly also at the UV-B. This increase in reflectance may result in a reduction in net radiation and leaf temperatures in glaucous genotypes.

Leaf pubescence is a common feature in xerophytic plants (Fig. 5) as well as in some crop plants, such as soybean. Generally it increases reflectance from the leaf within the range of 400 to 700 nm and sometimes up to 900 nm, resulting in lower leaf temperatures under high irradiance. It is sometimes argued that the increased reflectance in the photosynthetically active spectrum would reduce photosynthesis under non-stress conditions. Under conditions of stress, there is a trade-off between the effect of pubescence towards the reduced stress load and its possible effect on photosynthesis. Increased leaf pubescence may increase the leaf boundary-layer resistance by up to 50%. However, it has been argued that this should carry a relatively small effect on water and C02 exchange, as compared with the effect of pubescence on the radiative properties of the leaf.

Leaf color can affect the thermal properties of the leaf. In both wheat and barley there are ‘yellow leaf’ cultivars, which have about a third less chlorophyll than the ‘normal’ ones. The ‘yellow’ cultivars tend to perform relatively better under drought stress as compared with the normal green. Yellow leaves are more reflective and their temperature is relatively lower than that of green ones. Beyond this difference in reflective properties, the low chlorophyll lines seem to sustain lower injury to the photosystem under conditions of high irradiance and water deficit (#3817).

Osmotic adjustment (OA)

Fig. 8. Differential response to sever drought stress of a high OA line (left) and a low OA line (right) of wheat.

When water deficit develop various solutes accumulate in cells and subsequently tissue osmotic potential is reduced (see Fig.1 in impact of Stress). OA is derived from the net increase in cellular osmolality caused by the accumulation of solutes such as various ions (mainly potassium), sugars, poly-sugars (e.g. fructan), amino acids (e.g. proline), glycinebetaine, etc.’ OA occurs when cellular water deficit exceeds a certain threshold, which is not universally determined. Nor has the exact signaling for OA been resolved. OA is a slow process requiring time, and very rapid desiccation in experiments or even in the field may not allow for OA. Ideally the rate of plant dehydration should not be faster than about 0.1 MPa day^-1. Practically, it should take about 2 weeks from fully hydrated state to wilting on order for the full capacity and impact of OA to be expressed in whole plant, depending on species and the growth history of the specific plant. The rate of OA varies greatly among species and cultivars. A minimal rate of OA, which can be considered as effective, is about 0.3 MPa and rates of up to 1.5 to 2.0 MPa were observed in certain cereal cultivars. Some crop plants generally tend to be better at OA than others with cowpea, japonica rice and maize generally having lower rates while indica rice, sorghum and wheat tend to express higher rates. See comparison of OA measurement methods.

OA is probably one of the most crucial components of dehydration avoidance and drought resistance in general. It help maintain cellular turgor at a given leaf water potential and thus delay wilting (Fig.8). OA enables to sustain growth and productivity at lower plant water status. Irrespective of the effect on turgor maintenance, the accumulated solutes can protect cellular proteins, various enzymes, cellular organelles, and cellular membranes against desiccation injury. Hence, cell and tissues may continue to function despite the progressing desiccation. This is why the accumulated osmotic solutes are sometimes defined as “protectants”. One consequence of OA at the whole plant level is the continued growth of roots and the extraction of deeper soil moisture. Finally, OA is crucial for the conservation of meristem viability under desiccation towards the recovery of function upon dehydration. OA in different cultivars of wheat, sorghum, various pulses and brassicas has been shown to be positively associated with biomass and/or yield under drought stress.

Upon rehydration the various solutes are recycled and metabolized to the extent that the accumulated sugars, for example, are considered as an important energy resource for recovery growth.

Extensive genetic engineering efforts are being made to use the phenomenon of OA in the design of stress resistant plants (e.g. #4635, #5897). Most experiments involve transgenic model plants that were modified to constitutively express the accumulation of osmolytes. Such transgenics that accumulate glycinebetaine, D-ononitol, mannitol, and trehalose gave positive or inconclusive results with respect to stress resistance, and work in this area is developing rapidly.

Non-senescence (delayed senescence or ‘staygreen’ -SG)

Fig. 6. “Stay green” (left) and “normal” (right) cultivars of sorghum under post-flowering drought stress.

Plant senescence is a genetically programmed process, accelerated by environmental stress such as drought, heat, and nitrogen deficiency. The primary expression of leaf senescence is the breakdown of chlorophyll and the subsequent collapse of photosynthesis. Leaf greenness as measured by chlorophyll content or by leaf reflectance properties (using the Minolta chlorophyll meter for example (see our Methods page) is becoming an acceptable estimate of senescence (and leaf nitrogen status). In various crops certain genotypes were identified as expressing delayed senescence or non-senscent or stay-green phenotype (#4440) (Fig.6). These genotypes generally sustain leaf greenness and photosynthesis for a longer time and consequently tend to yield more. Since drought stress accelerates senescence, SG genotypes are important in sustaining green leaf area under stress.

SG does not present a uniform expression across different crop plants. In sorghum for example SG can be associated with high stem soluble carbohydrate content and greater resistance to lodging caused by stem ‘charcoal rot’. In sorghum and millet at least, SG genotypes sustain higher RWC under stress as compared with normal ones. This is why SG is discussed under ‘dehydration avoidance’. Maintenance of RWC is not necessarily an expected result of delayed chlorophyll loss or delayed leaf protein breakdown. Furthermore, certain SG genotypes of sorghum are expressed better when exposed to drought stress. Hence, the phenotypic selection of SG in sorghum (and perhaps other crops) is more effective under post-flowering drought stress.

SG is at least partly regulated by endogenous plant hormones, whereas in certain cases an increase in kinetin in leaves promoted SG. In other cases SG was associated with decrease in plant ethylene content. Such hormonal regulation can involve both nitrogen and water status of leaves.

The expression of SG and plant senescence in general can be markedly influenced by intra-plant interactions which involve assimilate partitioning and endogenous hormonal balance. A simple exercise to obtain a SG phenotype in grain producing crops is by detaching the inflorescence at flowering. Grain set and grain growth generally enhance leaf senescence by enhancing carbohydrate and nitrogen export from leaves into the grain.

Very low yielding or partially sterile plants may present some delay in senescence when subjected to drought stress during grain filling. There are ongoing attempts to achieve genetic transformation of SG trait by either promotion of endogenous kinetin or by antisense suppression of ethylene. QTLs for SG are being identified in several crops (see out Biotech Issues page/ local files) and marker assisted selection for the trait is becoming possible in sorghum and probably other crops in the future.

Dehydration Tolerance

Effect of stress kinetics on differential gene expression of immature ears of maize. Plants were grown in buckets where drought stress reached the point of null photosynthesis (Pn) in 5 days. Plants were grown in the field where the same state of stress was reached after >5 weeks (From Barker et al., 2005).
Stress Phenotyping	Stress kinetics	% genes responding
Stress in a large pot	Rapid (5 days)	27
Stress in the field	Slow (4 weeks)	2

Cellular and molecular adaptive processes in response to water deficit do not occur until a certain level of water deficit has been reached. Cellular and molecular adaptive responses serve one or more of the following major functions: (a) reduce whole plant growth in order to reduce plant water-use; (b) reduce the rate of cellular water loss and retain cellular hydration; and (c) protect various cellular structures and functions as cells desiccate.

With modern genomic tools it becomes fairly straightforward to reveal hundreds of genes that are up regulated or down regulated in response to plant tissue water loss. However, research into the function of most of these genes is not as developed. Subsequently the exact function at the whole plant level of the found gene responses to cellular water deficit is not well understood to the extent that they can be used in plant breeding. However, there is slow progress in this area as can be seen in our Biotech Issues files. In terms of application to plant breeding dehydration tolerance is the capacity to function in a dehydrated state which often (but not always) means the involvement of stress responsive and adaptive genes. Most of the information that is relevant for application to breeding is derived from whole plant physiological studies while some rudimental information comes from genomics.

Plant physiology always cautioned that the evaluation of plant response to drought stress and the evaluation of plant adaptation require sufficient time under stress. Adaptive plant responses to drought stress do not only depend on the level of tissue desiccation but also on its rate (e.g. #3418). It was well established that fast or slow desiccation may have totally different impact on results in terms of adaptation. Very rapid desiccation often exercised in laboratory experiments is totally irrelevant even though statistically significant results can be obtained. Tissue desiccation under natural conditions is slow. Confirmation of this axiom is now received from a gene expression study in maize as presented in Table, which speaks for itself.

Stem Reserve Utilization

The current source of carbon for grain filling is assimilation by the light intercepting viable green leaf area. This source is normally diminishing due to natural senescence and the effect of various stresses. At the same time the demand by the growing kernel is increasing, in addition to the demand posed by maintenance respiration of the live plant biomass. When the demand by the grain is not fully supplied by the source of current assimilation, then plant reserves can provide the balance. Small grains and cereal stems as well as several other crops store carbohydrates in the form of glucose, fructose, sucrose, fructans or starch. Type of storage depends on the species. Total storage in cereal plant roots or leaves is relatively small to that in the stem (including leaf sheaths). This storage is commonly analyzed as total non-structural carbohydrates (TNC) or water-soluble carbohydrates (WSC) and it is available for translocation to the grain.

Usage of stem reserves depends on the available storage and the rate and duration of mobilization of storage to the grain. The size of the storage strongly depends on favourable growing conditions before anthesis and genotype. Developmentally, potential stem storage as a sink will also be determined by stem length and stem weight density (stem dry weight per unit stem length). Stem length, as affected by the height genes is important in affecting stem reserve storage, as demonstrated in sorghum (#3561). The Rht1 and Rht2 dwarfing genes of wheat were also found to limit the reserve storage by about one third as a consequence of a reduction in stem length. This may be one of the reasons for the general greaterer drought susceptibility of the dwarf high yielding wheat cultivars.

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ig. 7. Grain of two wheat cultivars subjected to sever drought stress during grain filling (right). Top: cultivar with superior capacity for stem reserve utilization; bottom: normal cultivar. Note the shriveled grain under stress in the latter.

Stem reserve mobilization or the percentage of stem reserves in total grain mass is affected by sink size, by the environment and by cultivar. It is not surprising therefore those different estimates of the percentage of grain yield that is accounted by stem reserves range from 9 to 100%.

The demand by the grain yield sink is a primary factor in determining stem reserve mobilization. When manual de-graining reduced sink size, more reserves were stored in the stem, as compared with intact ears. The availability of storage at grain filling does not necessarily assure mobilization. There are cases on record where despite stress conditions the available storage was not utilized. This may be traced to problems in enzymatic conversion of storage to transportable constituents or sometimes inhibition of sink processes. For example, under heat stress starch synthesis in the wheat grain might be inhibited by a thermolabile enzyme (such as soluble starch synthase) and available stem reserves would not be in demand. Heat tolerant starch synthase is therefore also essential for grain filling under heat stress. Hormonal signaling might also be involved in reserve mobilization.

The reduction in current assimilation during grain filling, under different stresses, will induce greater stem reserve mobilization to the grain. What is important is the reduction in assimilation and not the nature of stress causing the reduction. Thus, stem reserve mobilization is a solid source of carbon for grain filling under any stress (such as heat stress also), which would inhibit current photosynthesis, including biotic stresses such as late developed leaf diseases. Tolerance to Septoria leaf blotch in wheat was expressed in sustained grain filling under sever epiphytotic. It has been demonstrated that mobilized stem reserve is a major component of Septoria tolerance in wheat (#2659).

The full potential for stem reserve utilization of a cereal cultivar can be experimentally assessed by growing plants under favourable conditions and then detaching all leaf blades and shading the inflorescence at the onset of grain filling. Grain weights per inflorescence in such treated plants as compared with controls provide a reliable estimate. It appears that wheat genotypes differ in their capacity to store stem reserves (Fig.7). Cultivars that have this high capacity must also possess relatively long grain filling period in order to allow sufficient time for reserves to be mobilized into the grain.

A possible “penalty” for high stem reserve utilization capacity is accelerated shoot senescence, due to the export of C and N storage into the grain. Thus, it seems that the two factors may not be recombined and the breeder will probably have to opt for either stem reserve mobilization or delayed senescence trait as mechanisms supporting grain filling under stress.

Cellular membranes stability

The fluid mosaic model of the cellular membranes (CM) describes the membrane as a bi-layer of phospholipids and glycolipids studded and spanned by proteins partially or fully solvated by the lipid matrix. CM is central site for various cellular functions; especially those associated with membrane bound enzymes and transport of water and solutes. The function and role of the CM under extreme temperature stress is somewhat clearer than with drought stress.

The phospholipids in terms of their quantity and composition are generally considered as the more crucial components of cellular membrane stability under drought stress. The most notable factor in cellular membrane function under desiccation and heat stress is that plant previous exposure to moderate stress signal a hardening (“acclimation”) effect that is expressed in increased membrane stability under stress. Cellular membrane stability under stress has been shown to be positively correlated with yield advantage under stress, more often for heat than for drought stress. The simplest way to assess membrane stability is by measuring the leakage of cellular electrolytes under stress.

Water passage through both the plasma membrane and the tonoplast is crucial to cell life and specific proteins inserted in the membrane largely regulate it. These ‘water-channel’ proteins, also termed aquaporins respond to various signals and “molecular switches”. These pores are highly selective to water and they play an import role in cellular water relations in response to plant water deficit and osmotic stress. For example, maize root aquaporins were found to be stimulated by water deficit, resulting in improved water transport. The study of aquaporins and their function is now at the forefront of research on cell water relations.

Antioxidation

Oxidative Stress is a general term used to describe a state of damage caused by reactive oxygen species (ROS). ROS, such as free radicals and peroxides, represent a class of molecules that are derived from the metabolism of oxygen. There are many different sources of ROS that can cause oxidative damage to an organism. Most come from endogenous sources as by-products of normal and essential reactions, such as energy generation from mitochondria or the detoxification reactions. Free radicals are unstable because they have unpaired electrons in their molecular structure. This causes them to react almost instantly with any substance in their vicinity. Free radicals destroy cellular membranes, enzymes and DNA.

Antioxidants are active substances naturally occurring in all organisms which detoxify free radicals. These are for example superoxide dismutase (SOD), catalase, glutathione reductase or ascorbate peroxidase. SOD, for example, converts the O2º to H₂0₂ and Catalase converts H₂0₂ to molecular oxygen.

Drought, as well as other stresses cause oxidative stress in plants and antioxidant abundance and activity is important for the protection of metabolism under stress. When various studies are reviewed it is unclear whether the genetic enhancement of antioxidant production in plants beyond the natural level is indeed required to alleviate drought stress at the whole plant level and whether the naturally occurring active antioxidants are not sufficient to protect the plant. It has been shown that drought induced oxidative stress related genes and that this was associated with increased levels of various antioxidants in plants. The most important information in this respect is coming from the developing work with transgenic plants, which over-express antioxidant production. When these studies are taken as a whole, no clear conclusions can be made yet with respect to the possible importance of breeding for overproduction of antioxidants towards the improvement of plant production under drought stress. More information is available on this site under The Stresses.

Stress proteins and Chaperons

Fig. 9. Rice transgenic plants over expressing the HVA1 barley embryo LEA protein and subjected to drought stress. The middle pot is the ‘wild type’ (control) plant. These transgenics were developed by the late Prof. R. Wu and associates at Cornell University. The photograph was taken from a study by Dr. H.T. Nguyen at Texas Tech University.

Stress proteins is a large group of different proteins induced by different environmental and biotic stress in various organisms ranging from prokaryotes to man. A group of relatively small molecular weight proteins is developmentally regulated in growing seed such as that of barley. Their accumulation during embryo development has a role in protecting the embryo as the seed matures and desiccates during maturation (typically to about 10% water content). These are defined as ‘late embryogenesis abundant’ (LEA) proteins. Further research found that LEA proteins consist of a family, including several similar proteins such as dehydrins. These are not limited to seed embryo and they can be induced by drought stress in various plant tissues. Some are ABA responsive while others are not. Additional information especially on LEA protein and desiccation tolerance in seed is available on line.

Work with transgenic plants indicated that the LEA family of stress protein might have a role in drought and osmotic stress resistance. Their exact function is not clear but it may involve osmotic adjustment or protection of cellular membranes or organelles during desiccation. They may also act as molecular chaperons and in that respect they are very similar to low molecular weight heat shock proteins (HSP). In this role they may conserve protein structure during stress. The LEA family of proteins may carry an important potential for enhancing stress resistance (Fig.9).

Abscisic acid (ABA) accumulation and its consequences

ABA accumulates in various plant parts subjected to desiccation. ABA responsive genes are often assumed to be stress adaptive. The rational is that if plants under stress consistently respond by producing ABA in leaves and root, then ABA must be important for coping with stress. The most prominent effect of ABA accumulation in the plant is stomatal closure and reduced transpiration. Thus, when transgenic model plants such as Arabidopsis or tobacco are engineered to over-produce ABA they maintain turgor when grown in pots subjected to dehydration. Thus it is often concluded that ABA promotes drought resistance. Turgor maintenance by stomatal closure is important for survival but not for plant production under drought stress (check Updates on Drought). Furthermore, a review of the literature indicates that ABA has numerous and critical negative effects on plants especially when crop productivity is considered (Table 1).

Table 1. Effects of ABA on plant processes involved with growth and reproduction
Growth
General growth	Inhibition
Cell division	Decrease
Cell expansion	Decrease
Leaf initiation	Inhibition
Germination	Decrease
Root growth	Increase
Tillering Dormancy	Decrease Improved
Reproduction
Flowering (annuals)	Advance
Flower induction	Inhibition
Flower abscission	Increase
Pollen viability	Decrease
Seed set	Decrease

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Fig.10. Wheat seedlings grown in vermiculite and severely desiccated after which they were irrigated. The seedling on the right received 0.1 µmol of ABA in the irrigation water before the onset of stress. Control seedlings are on the left.

Genotypes of wheat that were selected for a high ABA accumulation under drought stress were found to be no better or even worse than the normal ones in terms of function and yield under drought stress. Selection for low leaf ABA content in maize was correlated with reduced yield under conditions of limited water supply (#5393).

On the other hand ABA may have an important role in regulating an orderly shutdown of plant functions towards a state of dormancy, as the case is for the maturing seed. Dormancy is essential for surviving extreme plant desiccation (Fig.10). ABA mediated dormancy is crucial for attaining freezing tolerance, which involves cellular desiccation. The value of plant survival under sever desiccation depends on the agricultural ecosystem concerned. It can be important in subsistence agricultural where plant recovery from severe drought stress can provide some growth and production. Plant survival is of lesser consequence to commercial crop production as practiced in developed countries. Even the ability of seedling survival and recovery after a prolonged drought in commercial wheat production does not carry great impact when re-seeding the crop is a viable option. A commercial crop based on recovered seedlings is likely to be inferior to that grown from newly planted seed. The present knowledge on ABA and it role in plant adaptation to drought stress as well as in general plant production capacity does not allow yet to formulate a breeding strategy with respect to ABA.

Drought escape and plant phenology

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Fig.11. Early flowering (left) and late flowering (right) sorghum cultivars under late-season drought stress. The late cultivar will not flower at all due to stress.

Short growth duration (generally defined by early flowering) constitutes an important attribute of ‘drought escape’, especially for conditions of a late-season drought stress (Fig.11). On the other hand, longer growth duration is often associated with high yield potential. Consequently, using drought escape as a solution may involve a cost in terms of reduced yield potential. This is serious, especially when the moisture environment is absolutely unpredictable and may vary to a large extent between years. The more predictable the environment is, the easier it is to optimize phenology. The unpredictability of the environment may reach a state where short growth duration is a drawback, especially in indeterminate plants that offer a potential for regrowth and productivity upon recovery. Longer growth duration in both determinate and indeterminate plants would improve the probability for regrowth upon recovery simply because, on the same calendar day, late-maturing genotypes are younger than early ones and younger plants recover better. The final decision on the optimum growth duration has, of course, to consider additional factors, such as late-season disease and insect pressure or periods of frost.

Early maturity leads to reduced, total seasonal evapotranspiration simply because of the shorter time in the field. However, as growth duration is genetically linked with leaf number, early genotypes tend to have a small transpiring leaf-area index. Thus, early genotypes show reduced evapotranspiration during most growth stages, up to the point where a full ground cover is achieved. At most growth stages, root-length density and total root length per plant is generally greater in a late than in an early cultivar. This should be reflected in an advantage for the late genotype under conditions where extensive rooting is required.

A phenological feature specific to maize is the timing of anthesis with respect to silking, defined as anthesis-to-silking interval (ASI). Evidently a short interval is desirable whereas a large interval results in poor pollination. The maize program at CIMMYT dedicated many years of work to research the trait and explore its significance in tropical maize breeding for stress environments. Maize germplasm can vary for ASI irrespective of the effect of stress; a short ASI is a universally important trait for maize production. However, stress, and especially drought during the reproductive stage may extend ASI and thus reduce yield. Maize genotypes may vary in ASI under drought stress from few days up to a month or more. The effect on yield of change in ASI between null and 10 days is exponential. Selection for short ASI under drought stress proved to be an effective approach to improve drought resistance of tropical maize. QTLs (quantitative trait loci) controlling ASI were located and marker-assisted selection is possible.

Different crop plants may advance (e.g. wheat) or delay (e.g. rice) their flowering when stress occurs before flowering. The rate of delay is a function of plant water deficit and probably also ABA signaling. The rate of change in flowering time under stress can be taken as an index of genotypic rate of stress in the field.

Water-use efficiency (WUE)

WUE is not a component of drought resistance but the term implies greater production for a given amount of limited water. Namely “more crop per drop”. This is not necessarily the case. High WUE result (in most cases) from “less drop per crop”.

WUE was originally developed by agriculture engineers as a ratio between yield and irrigation water in order to assess returns for irrigation input and cost. WUE is an important yardstick to measure irrigation efficiency. The WUE term was later adopted by soil scientists and agronomists for a wider use in agronomy, including dryland-rainfed crop production. Physiologists found the term useful also at the leaf level in studies of gas exchange where WUE (i.e. “transpiration ratio”) is defined as the ratio of carbon fixation to transpiration. WUE can therefore be used at various levels of the crop, from the single leaf to the field.

Studies of water use efficiency at the whole plant and field level were cumbersome due to the work load and costs involved in assessing whole plant or crop water use, especially when large plant populations in plant breeding were considered. The breakthrough came with the development of better understanding of stomatal dynamics, gas exchange and photosystem function, leading to the carbon isotope discrimination (delta) assay as a heritable marker for WUE at the whole plant level (Farquhar et al. 1989; Hall et al. 1994). The reader is referred to these papers for details on the theory and the analytical method (which is not cheap). In the majority of cases low carbon isotope discrimination (low delta) as measured in the grain or the leaves was found to be well correlated with high WUE across variable genetic materials and vice versa, with few exceptions where delta was not associated with WUE.

An important contribution of the carbon isotope discrimination method was that it enhanced research on WUE and provided extensive data on the subject especially in the context of breeding and genetic diversity. At the same time the large volume of published information on delta, WUE and their implications towards selection for water limited environments created some confusion in the plant breeding community. Confusion was largely created by the fact that the relations between delta (WUE) and yield were sometimes positive and sometimes negative, depending on the crop growing conditions. Plant breeders discussing carbon isotope discrimination and WUE expressed confusion on two primary questions: (1) under what environmental conditions selection for carbon isotope discrimination is expected to result in yield gain, and (2) which direction should selection be made, high (low delta) or low (high delta) WUE.

Beyond these questions the real issue is whether selection for high WUE is universally associated with drought resistance and improved plant production under drought stress. WUE is often equated in a simplistic manner with drought resistance without considering the fact that it is a ratio between two physiological (photosynthesis and transpiration) or agronomic (yield and crop water use) variables. This ratio it is often susceptible to misinterpretation, especially when the dynamics of the nominator and the denominator are ignored. When all studies of carbon isotope discrimination in breeding population are taken together it can be seen that higher WUE is derived from a reduction in water use rather than from an increase in production. Reduced water-use under dryland conditions is contradictory to productivity. Thus genotypes of high WUE under drought stress tend to be less productive under stress – with few specific exceptions. A more detailed discussion of WUE in the context of plant breeding for plant production under water limited environments is presented by Blum (2009). This discussion concludes that the target of plant breeding for water limited environments is effective use of water (EUW) rather than WUE.

Photosynthetic systems and plant production under stress

Plant science is still seeking ways to genetically increase productivity for a given unit of water-use under drought stress. The key is in photosynthesis. The C4 photosynthetic metabolism as compared with the more widely common C3 type photosynthetic metabolism is intimately associated with superior productivity at given water-use. The C4 pathway of photosynthesis as found in maize, sorghum, pearl millet, and various forage grasses is essentially a pumping mechanism that moves C02 from the mesophyll cells and causes high C02 concentrations in the specific biochemically active vascular-bundle sheath cells. This mechanism goes hand in hand with certain anatomical and morphological features of the C4 plant (“Kranz leaf anatomy”) that are inseparable from the system as a whole. The C02-concentrating mechanism results in a high utilization efficiency of low intercellular C02 concentrations. This is due to the PEP carboxylase enzyme in the C4 plant, which unlike RuBP carboxylase is insensitive to atmospheric 02 concentrations. Atmospheric 02 concentrations are strongly inhibitive to C02 uptake in C3 plants where C02 is fixed directly by RuBP carboxylase. In C4 plants C02 fixation is carried out in the bundle-sheath cells using C02 from decarboxylated C4 acids in the mesophyll cells. This sequence results in sufficiently high C02 concentration maintained at the bundle sheath cell. The efficiency of the C02 fixation pathway in the C4 plant bears significance toward its transpiration-ratio. For a given rate of transpiration, photosynthesis is greater in C4 than in C3 plants. This advantage is also translated into a greater plant or crop WUE, hich is not always necessarily related to drought resistance and a relatively better yield under stress. For reasons other than the biochemistry of photosynthesis (say, deep roots or OA) a certain C3 crop might produce better than a C4 one under drought stress. However, under well-watered conditions the greater WUE of the C4 plant is most likely translated into better economic returns on the cost of irrigation. The normal WUE (for grain yield) of supplemental irrigation in grain sorghum (C4) is about 20 kg mm-1 ha-1, as compared with 10 kg mm-1 ha-1 in wheat (C3).

Whereas WUE is often confused with drought resistance it is very important to take note of a comparative study of C4 and C3 Panicoid grasses (#10259). It concluded that declining C4 photosynthesis with water deficit was mainly a consequence of metabolic limitations to CO2 assimilation, whereas, in the C3 species, stomatal limitations had a prevailing role in the drought-induced decrease in photosynthesis. The drought-sensitive metabolism of the C4 plants could explain the observed slower recovery of photosynthesis upon re-watering, in comparison with C3 plants which recovered a greater proportion of photosynthesis through increased stomatal conductance. Therefore, within the Panicoid grasses, the high WUE C4 species are metabolically more sensitive to drought than the lower WUE C3 species and recover more slowly from drought.

Plant science is attempting to improve yield of C3 plants such as rice by converting their biochemistry to C4. Less ambitious but probably more closely at hand is the improvement of C3 leaf internal, or mesophyll, conductance to CO2, leading to greater leaf productivity per unit transpiration (#10465).

Conclusion about the nature of drought resistance in crop plants

It is not uncommon to come across opinions that drought resistance is “very complex” or “confusing” or “difficult” (see Blum 2011). However, while drought resistance is not perfectly simple in terms of its physiological nature, it is conceptually simple with regard to breeding if one accounts for the following main considerations.

Firstly, besides adaptive traits drought resistance is strongly dependent on plant constitutive traits that are not necessarily induced by stress and do not require stress for their expression and are often easily manipulated genetically.

Secondly, plant survival under extreme desiccation and its capacity to recover may depend on both dehydration avoidance and dehydration tolerance. In the extreme case we find resurrection plants as a model for extreme tolerance. However, survival is rarely an important feature in crop drought resistance.

Thirdly, the most important factor of drought resistance towards crop production and very possibly also in natural vegetation is dehydration avoidance, namely the ability of the plant to maintain high water status or high turgidity. This would allow sustaining function better as environmental stress increases. Plants rarely function at zero turgor.

Lastly, various plant traits, constitutive or adaptive, affect the capacity to maintain high plant water status and turgor. Depending on the drought stress profile and intensity, the most effective traits in terms of agronomic value are growth duration, plant size, root depth, osmotic adjustment, and plant surface properties. Stem reserve utilization for grain filling is an exception as it functions when plants are dehydrated to the extent that photosynthesis is inhibited.

BREEDING FOR DROUGHT RESISTANCE

Also: Check the links to Plant Breeding and Methods pages which appear on Plantstress home page.

Some Principles

The primary difficulty and the most important task in planning a breeding program for the improvement of drought resistance is the formulation of the drought resistant ideotype with respect to the target of the breeding program. This involves an educated logical integration of most of the information discussed on these web pages and their links, applied to a well-understood and defined target environment. The primary issue is the decision on the important phenological, developmental and adaptive traits that would be most effective in supporting production or survival under drought stress, depending on the agro-ecological, social and economic conditions of the target environment. The level of funding and intellectual support for the specific breeding program will determine whether the ideotype is likely to be attainable.

Conventional breeding for general yield improvement relies very strongly on selection for yield and its components as a main approach. Modern conventional breeding for drought resistance supplements selection for yield with selection for developmental and physiological attributes, that may require physiological measurements in breeding populations. Physiological methodology is generally slow and meticulous and it does not allow to measure and screen large plant populations. In most cases, indirect or rapid methods were developed as screening aids to replace the slow physiological methods. While this resulted in reduced accuracy of the measurement, it still allows partitioning the population into the desirable subpopulations. This is sufficient in the eye of the breeder who is not interested in outmost accuracy of measurements but rather in being able to reduce the population by excluding the least appropriate phenotypes.

The flaw in conventional breeding is that the breeder can identify the genotype only by measuring the phenotype. The efficiency of this approach depends on many factors, including inheritance of traits, environmental effects, measurement error and more. For certain traits, such as root depth, phenotypic measurements in very large breeding populations are technically impractical. Marker assisted selection (MAS) allows to select the desirable genotype without actually measuring the phenotype. Read more on the basics of MAS and potential application to drought resistance breeding.

The Managed Stress Environment

While the field in the target stress environment is the primary goal of the breeding program, paradoxically, it is often inappropriate for selection work. Besides the amplified spatial field variability when water is limited, stress is can also be variable from year to year. The water regime can be too sever in one year, causing complete loss of breeding materials on one hand or too favorable to constitute any stress pressure in another year. Drought stress in different seasons can also occur in different growth stages. Stress in the target field environment is typically inconsistent, causing reduced efficiency in the overall selection program. It may be argued that this variability is an inherent problem to be addressed in breeding. While this may be true, selection becomes very ineffective if it is practice under such a variable protocol. For example, if drought resistance is to be improved at two different growth stages, it must be logically addressed separately for each different stage, followed by recombination. It follows therefore that the field-screening environment must be managed for stress intensity and timing to a level that can result in a consistent selection pressure from one cycle to the next. Thus, controlled drought stress in the selection process is essential, quite analogous to the use of controlled disease infection or the use of consistent natural “hot spots” in the selection for disease resistance.

Controlled drought stress implies the appropriate duration and severity of stress at the appropriate plant growth stage. Controlling drought stress in the greenhouse or the growth chamber is relatively straightforward. In the field, however various means are required to achieve control by eliminating rainfall on one hand and by providing irrigation on the other. The ideal field selection site for drought resistance would be in a desert environment with a minimal amount of rainfall, where almost any crop water regime can be designed by irrigation. While this may not always be possible it is the conceptual basis of the managed stress environment. It follows that most breeding programs which have a component for drought resistance must develop a special phenotyping site where stress can be managed to a reasonable extent. Alternatively, certain natural drought stress conditions may be quite repeatable from year to year or very easy to manage by irrigation. This is the case for crops grown exclusively on stored soil moisture from previous season precipitation. This stress scenario is found for example in the Mediterranean summer crops or the ‘rabi’ season in parts of India.

When terminal stress (stress at the final reproductive growth stages) is considered, a delay in planting in most cases would put this stage in a dry season. Another possibility is to grow the population during a dry off-season if climate and biotic factors allow it. This approach was very successful with upland rice breeding at IRRI in the Philippines. Since growing plants in the dry offseason might expose them to somewhat different climatic conditions, an offseason nursery should be used mainly for

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Fig. 12. Forage sorghum breeding materials (tall plants at the back) grown on a line-source irrigation system. Source is indicated by arrow the and the growth of plants is seen reduced by the lack of water perpendicular to the source.

recording results on drought resistance responses but actual selection should be performed with the same (duplicate) materials during the normal season. Exceptions are noted where selection for yield under stress in an offseason stress nursery was effective in gaining progress for drought resistance. The corollary is to understand the climatic and biotic factors which might affect plant growth and yield in the offseason nursery, besides drought.

When a managed field site is impossible to achieve, the next option is the rainout shelter. A complete discussion of this option is available here on this site under Methods.

Where rainfall is limited in the natural field selection environment, such as dry season in the tropics or the summer season in the Mediterranean, managed stress environments can be designed by irrigation scheduling. Options range from having a stress and non-stress environments side by side to the ‘line-source’ irrigation system (Fig.12). This system is based on the fact that any sprinkler irrigation system spreads water in a gradient where the maximum amount is discharged at the source with a diminishing amount away from the source. Hence the amount of water available to the plants decreases perpendicular to the sprinkler irrigation line. Breeding materials can be planted in long plots or rows perpendicular to the line and be subjected to an increasing drought stress away from the line. Observations on plant response along a water supply gradient within each genotype can be very effective for revealing resistant materials. The ‘Mixed-procedure’ application (SAS) can perform the statistical analysis of data from a line-source irrigation system, if needed.

Variability of the field environment

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Fig. 13. Aerial photograph of a sorghum breeding nursery under dryland conditions planted in a farmer field (see text).

Any book on field experimentation deals extensively with the issue of spatial variability in the field, which is a major problem requiring detailed statistical design and analysis of field experiments. The field is variable in terms of topography and soil characteristics. Soil characteristics vary in all dimensions. This variability is amplified when the water regime is concerned and especially when water is lacking. Fig.13 demonstrates field variability for soil moisture seen in a sorghum breeding nursery subjected to drought stress. Plots that are generally above the drawn line on the photograph are situated on wetter soil and therefore appear more color saturated (having higher leaf water status) as compared with the more desiccated plots below the line. An experiment laid out across this line is practically useless, whatever statistics are deployed. It is therefore crucial not to find yourself in this situation to begin with.

Whichever methods and precautions are used to handle field variability as a generator of experimental error, these become especially critical in experiments involving water deficit. While suitable field topography (flat with a slight homogenous slope) can be identified, it is extremely difficult to estimate spatial variability of the site with respect to its soil moisture characteristics. It is therefore highly recommended to perform a field homogeneity test by growing a homogenous commercial crop in the candidate field before choosing it for screening work. The crop should be water stressed and then observed for variability in plant development. Photogrammetric methods can also be applied for this purpose. Machinery that surveys the field by measuring soil electro-conductivity is becoming a popular method after its use in precision agriculture application. In certain cases (typically rice in Asia) a field may be situated above a high water table. Whatever might be the rainfall regime or irrigation the crop in such a field can never be water stressed for experimental purpose. Monitoring ground water level before using such a field for drought phenotyping is essential

Yield as a Selection Criterion for Drought Resistance

The issue of selection for yield and the impact of the environment on genetic gains from selection and selection efficiency are under continuous debate as a central issue in conventional plant breeding. The comparative yield performance of two genotypes with respect to one another can vary from one environment to the other and this is basically defined as genotype by environment interaction (GxE) for yield (Fig.1 above). Generally, the ideotype preferred by most breeders and breeding textbooks is one that expresses minimal GxE and its yield is “stable” across all environments. The question is the spectrum of environmental diversity across which one variety can be stable. From Fig.1 above it can be seen that when environmental variation is extreme a GxE (crossover or other) is unavoidable. The classical solution is than to adapt different varieties to the very different environments, such as varieties A and B for one environment and C for the other in Fig.1. For more on the statistical analysis of GxE and yield stability in relations to cultivar selection see here.

However, while yield under stress is the target of the breeding program, selection for yield under stress is generally inefficient. Yield is a complex trait that is basically not directly inherited. It is the various developmental and physiological processes which make up yield that are inherited. Therefore the heritability of yield is generally not high and it becomes especially low under stress. It has been the general and repeatable observation of breeders that using yield as a selection index under stress to improve drought resistance is generally not efficient. To compensate for the low efficiency breeders screen very large populations with the expectation that the “numbers game” will allow to identify the desirable genotype. While this approach has been successful, it is expensive. The use of molecular markers to tag and select for certain yield related quantitative trait loci (QTLs) can increase the efficiency of selection for yield, but again, less effectively under stress. Again, QTL by environment interaction seems to be the rule.

However, when the breeding population contains effective genes for drought resistance (say, segregation for deep roots in upland rice population) the efficiency of selection for yield under drought stress can be increased, provided all precautions are taken to minimize spatial variability at the selection site (see above).

If selection for yield under stress is practiced, a positive GxE for the specific drought stress conditions is a strong indicator of resistance. There are many statistical models and methods that estimate GxE in different contexts and accuracies. In most cases of a planned breeding program for drought resistance the evaluation of GxE simply requires a comparison of yield performance under stress and non-stress (fully irrigated) conditions. The comparison of genotypic performance between the two environments can be simply evaluated in yield under stress as percent of yield under non-stress. Alternatively, the ‘Fischer and Maurer’ stress resistance index’ (RI) can be computed as:

RI=(Gs/Gn)/(Ms/Mn) ;

where genotype yield under stress (Gs) and non-stress (Gn) is normalized for mean yield of all genotypes under stress (Ms) and non-stress (Mn). Values above 1 indicate a relative resistance as compared with the mean of the population. It has been argued that this index is flawed because it is affected by yield potential. This is true and as we have seen above indeed genotypes of high yield potential tend to be more susceptible to drought stress. This is a reality not a mathematical flaw.

A major impediment in comparing genotypic response to a managed water stress environment is the variation among genotypes in their phenology. With such variation, different genotypes may be water-stressed at different growth stages. This has been a major pitfall in many drought resistance mapping exercises using populations such as recombinant inbred lines. The solution is to divide the population into several phenology sub-populations and compare the effect of stress only within sub-populations of similar phenology. Alternatively, staggered planting dates can be attempted where the earlier materials are planted later as compared with the late flowering materials. However, in most breeding programs, when tests for drought resistance are performed in the field at more advanced generations (e.g. =>F4), the population of lines does not express large variability for phenology.

Selection for Drought Resistance by Developmental Traits

Selection for most plant developmental traits involving drought resistance are conducive to general breeding principles, such as the case for phenology, anthesis to silking interval (ASI) in maize, tillering, plant size, etc’. Two unique developmental features are discussed here: roots and stem reserve utilization.

Roots

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Fig.14. A glass-paneled in-ground root box lysimeter installation allowing measurement of roots and transpiration. (Texas A&M Research Center at Temple, TX).

Fig.15. Rice population grown in PVC tubes for plant and root measurements (Rainout shelter site at the Huazhong Agricultural University Wuhan, China)

Root size and development is a crucial parameter in most selection programs for drought resistance. Detailed measurements of roots or even rough screening techniques for roots are generally laborious. Probably the most practical way to select for deep and effective roots is to judge their performance by observing the shoot performance under drought stress (see below). Much has been published on root measurement techniques, including books (see our Methods page)..

Very detailed root measurements can be performed with special growing containers and installations where roots can be observed in situ though a glass panel. These installations are defined as rhizotrons and they may take various forms such as individual glass-paneled soil-filled root boxes set in the ground (Fig.14). By weighing these root boxes it is also possible to estimate plant transpiration and relate it to shoot and root development. Rhizotrons and lysimeters are important tools for root research and for studying a few cultivars but they are not amenable to large scale screening work.

The simplest method for a direct selection for root length involves growing single plants in vertical soil filled disposable polyethylene tubes (used in polyethylene bags production) and then washing the root out of the soil at the time of measurement (usually at flowering). Alternatively plants can be grown in re-useable soil filled PVC tubes (~10 cm in diameter) (Fig.15). Tubes can be sawed into two halves and then taped together before planting so that they can be opened at any time and destructive measurements can be taken on roots in situ or after washing away the soil. The two methods (Fig.14 and 15) can be combined into one, where PVC tubes are set in a trench and weighed for water-use measurements while being lifted periodically.

Root penetration capacity through a hardpan is phenotyped by challenging the root to penetrate a hard layer of paraffin-wax at depth. The number of roots penetrating the wax layer is an estimate of root penetration capacity (e.g. #3034, #7965). While some criticism was expressed about the predictive power of the method, it has still gained popularity with breeders.

Stem reserve utilization for grain filling

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Fig.16. Wheat nursery rows with the center one sprayed with KI to destroy chlorophyll and eliminate current photosynthesis at the onset of grain filling.

The capacity for stem reserve utilization for grain filling when the photosynthetic source is completely inhibited by stress can be estimated in selection work by destroying the photosynthetic source at the onset of grain filling and measuring grain filling with no current photosynthesis in comparison with normal plants. Spraying the plants with an oxidizing chemical desiccant such as magnesium chlorate or with potassium iodide (KI) destroys chlorophyll and the photosynthetic source (Fig.16). The chemical is applied by spraying the whole plant or just the leaf canopy. The treatment is applied at the onset of the exponential stage of grain filling, which is about two weeks after anthesis in the small grains. Too early application of the chemical can kill florets and drastically reduce kernel number – which may undermine the test. Spray is therefore scheduled according to the different dates of anthesis of the different genotypes. Non-treated control plots are required. Since the capacity for stem reserve support of grain filling is measured by the difference in final kernel weight between treated and control plots of any given genotype, the control must be totally free of stress, especially drought or disease. It should be understated that the method does not simulate drought, it only simulate the effect of drought in terms of leaf killing and destruction of the photosynthetic source.

The method has been thoroughly tested and applied in wheat breeding. See a review of the principles involved and the detailed protocol in our Methods page.

Selection for Drought Resistance by Assessing Plant Water Status

Methods can vary from purely physiological to indirect assessments that are useful mainly for selection purposes. Most of the physiological methods were reviewed in previous pages. Here only methods for applied selection work in large populations are indicated. Certainly for a limited number of genotypes such as in pre-breeding work, direct measurement of leaf water potential by the pressure chamber and leaf RWC are consensus estimates of plant water status. RWC is considered here as a prefared estimate in breeding work since it accounts also for the effect of OA (see above) of leaf hydration.

Stress Symptoms

When plants reduce their water status and loose turgor under stress they display very distinct symptoms. Symptoms progress in proportion to plant water deficit and they can be visually scored and used in selection. The most notable symptom is off course leaf wilting. Leaf Rolling is an expression of wilting in the cereals and it is being widely used in field selection work. It is visually scored (typically on a 0 to 5 scale) and used in selection for drought resistance in various crops such as rice, wheat, barley, maize, millet and sorghum. Other leaf stress symptoms include leaf desiccation (“firing”), leaf tip “burning”, leaf “drooping” and leaf drop. General scores of plot appearance under stress is also used by experienced breeders who are well acquainted with the various responses of their crop to drought stress. Genotypes of delayed wilting or leaf rolling are preferred offcourse, indicating sustained turgor under stress.

Stress symptoms are also expressed in flowering time if stress occurs before normal flowering time. A delay in inflorescence appearance or exertion is typical of rice or sorghum. An advance in flowering is seen in wheat. Assessing the rate of delay or advance requires a comparison between stress and non-stress plots. The rule of thumb regarding stress symptoms and phenotypic variation for drought resistance is simple: “if you can’t see it – it is not there”.

Canopy Temperature

Selection methods were developed and applied to plant breeding following principles and techniques used in Remote Sensing in Agriculture. Most methods are based on the spectral response of leaves and its modification with plant response to the environment.

From the previous discussions it is indicated that canopy temperature is a function of transpirational cooling. As water deficit develops, canopy temperature differences among genotypes increase and plant water status becomes the main source of this variation. Canopy temperature was used to develop a crop water stress index as a tool for crop management. Canopy temperature measured under drought stress has become a most popular, fast and significant field screening method for plant water status under drought stress. Since dehydration avoidance is the major drought resistance mechanism in crop plants, canopy temperature is a most relevant screen for drought resistance. Relatively lower canopy temperatures under stress indicate a relatively better plant water status, ongoing transpiration and carbon fixation and an effective use of water. Lower canopy temperatures were generally found to be correlated with relatively higher yield under stress across diverse genetic materials. Canopy temperature can be measured remotely with the infrared thermometer, provided the correct protocol is strictly followed. Since its initial development as a screening method for dehydration avoidance by Blum et al. (1982), infrared thermometry of plant canopies under drought stress has become a popular method in breeding and phenotyping drought resistance. Twenty five years later and in tune with some 30 reports since then which verified the utility of the method in different crops, Olivares-Villegas et al. (2007) summarized their exhaustive study with wheat as follows: “Field trials under different water regimes were conducted over 3 years in Mexico and under rainfed conditions in Australia. Under drought, canopy temperature was the single-most drought-adaptive trait contributing to a higher performance, highly heritable and consistently associated with yield phenotypically and genetically. Canopy temperature epitomizes a mechanism of dehydration avoidance expressed throughout the cycle and across latitudes, which can be utilized … as an important predictor of yield performance under drought”

Leaf canopy temperature can be sensed and recorded by infrared digital cameras which present an image of the target in different colors according to temperature. With the lightweight and portable camera available today the resolution of the image is not very high but useful images can still be obtained (example). The instrument and its application is finding its way into agronomic research and some preliminary breeding work. The infrared camera has not replaced the infrared thermometer in large scale actual ground level screening work. Still the infrared camera has some potential uses in pre-breeding work and possibly when viewing breeding plots from aerial platforms, depending on its resolution and cost.

The measurement of the spectral reflectance of leaf canopies viewed from various platforms as done very early by NASA and associates, brought about the development of various spectral indices which are correlated with plant water status, leaf greenness or sometimes even yield. More details and protocols on using the method as a screen for dehydration avoidance is available on our Methods page.

Selection for Drought Resistance by Assessing Plant Function

As discussed previously the comparative assessment of plant function in different genotypes under drought stress (dehydration tolerance) must be normalized for plant water status. Else, differences in function among genotypes can be ascribed to differences in water status and not necessarily to real difference in function at a given plant stress. This is a difficult requirement in selection work especially under field conditions.

An effective approach for normalizing measurements of function against plant water status is to measure plant water status at the time of function measurement. Then develop a regression of the specific function on water status across all genotypes. Genotypes that deviate positively from the regression are more resistant while those that deviate negatively are more susceptible in terms of the specific function. Off course, these measurements cannot be performed on large breeding populations and they might be useful for pre-breeding work with potential parents or certain germplasm.

Published attempts to attain control of plant water status in laboratory studies are almost always seen to be flawed. One flawed example is to achieve a given set level of a stress low soil moisture content by frequently irrigating potted plants with small amounts of water to a given volumetric soil moisture content. This is an abnormal water regime with respect to a stressed plant, even though the accounting of soil moisture content is correct. A normally stressed plant is subjected for days to a given (or a receding) soil moisture status while these potted plants are subjected to a frequent cycle of moisture supply which is taken up mainly by the dry top soil.

One method which has gained some popularity is by growing plants in polyethylene (PEG) fortified nutrient solution. With this method (pending some limitations) plants are at least subjected to standard root medium water potential. The method is detailed in our Methods page. It is most amenable for measuring juvenile plant growth rate under a given root medium moisture stress, taking into consideration all the precautions mentioned in the described protocol.

Two examples of the more popular selection methods for function are given here, namely cell membrane stability (CMS) and chlorophyll fluorescence. Both are detailed on our Methods page.

Cell Membrane Stability (CMS)

This method is based on the fact that stress cause injury to cellular membranes (see above). This injury is expressed in leakage of various cellular solutes, including electrolytes. Electrolyte leakage can be easily measured by the electro-conductivity of the medium in which the affected leaf sample is placed. The method is being used mostly for assessing thermotolerance in heated leaf samples. Basically the method compares leakage from stress-affected leaf samples with leakage from control samples, calculating the relative injury or stability (the inverse of injury).

When CMS is used as a dehydration tolerance trait it is estimated in leaves subjected to advanced stress, typical to a set RWC of around 50-70% depending on species. Samples are taken from stressed and non-stressed (control) leaves. Samples are also taken for estimating RWC as a measure of water status. The first case of such a study was in rice where QTLs for CMS under drought stress were identified (#4793). Another way to subject leaf tissues to drought stress under this CMS protocol is to incubate leaf samples (e.g. leaf discs) in polyethylene glycol (PEG) solution as compared to non-treated well hydrated leaf samples. This method provides nice and consistent genotypic differences in drought CMS, but its relations to field performance under drought stress requires further validation.

Chlorophyll Fluorescence

The phenomenon of chlorophyll fluorescence and its value as a marker of photosystem-II function has bee discussed above. A number of instruments and imaging systems were developed for analyzing chlorophyll fluorescence. There are different levels of analysis, depending on the purpose of the study. A unique and detailed analytical probe defines as the JIP test has been developed by Prof. Strasser in Geneva. It allows a very comprehensive dissection and interpretation of the fluorescence phenomenon. This is mainly a research rather than a selection tool..

However, fast portable and simple instruments are needed for selection work and these are available from various commercial suppliers (see Web Resources page). Chlorophyll fluorescence is even entering the domain of remote sensing where vegetation function might be monitored from above ground and even aerial platforms in the near future. It must be realized however that the measurement and interpretation of chlorophyll fluorescence signal, even with simple instrumentation require a complete understanding of the phenomenon. Furthermore , it cannot be over-emphasized again that comparison of genotypes for chlorophyll fluorescence must be normalized for leaf water status.

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