Drought tolerant crops and transgenic breeding: just a utopian vision?
Alessandro Pellegrineschi
Cell Biologist
Genetic Resources Program
International Maize and Wheat Improvement Center (CIMMYT),
Mexico, D.F. Mexico
and
CRC for Molecular Plant Breeding
Australia

Agriculture has been expanding to meet growing food needs, and this has led to deteriorating growing conditions in many parts of the world. Desertification, erosion, and salinization of soils are the consequences and at the same time the causes of these developments. Currently, about 20% of farmland around the world is irrigated, and this land produces 40% of the global food supply. Drought and water shortages threaten the ability of many developing countries, especially those in Africa, to feed themselves.

Abiotic stress reactions, especially to water deficiency and high levels of salt, are complex morphological and physiological phenomena in plants (Evans et al. 1975, Wang et al. 2003). At the cellular level, water shortages cause osmotic stress. There is a flux of water from the cells as a result of alterations in extracellular solute concentrations. This water loss causes a decrease in turgor and an increase in concentrations of intracellular solutes, which puts a strain on membranes and macromolecules. Acute water deficiency impairs photosynthesis (Gallagher et al., 1975). If chloroplasts are exposed to excess excitation energy at the same time, water deficiency leads to the production of toxic substances such as superoxides and peroxides, which damage membranes and enzymes, in the cell.

The activity of osmoprotective compounds most likely mediates biochemical functions such as ion exclusion, ion export, cell wall modification, osmotic adjustments, and osmoprotection, which are involved in the response of the plant cell to osmotic stress. Furthermore, plant cells contain antioxidant enzyme systems, such as peroxidases and superoxide dismutases, which scavenge reactive oxygen intermediates (Moffat 2002; Yoshimura 2000). Plants commonly transport sodium ions to vacuoles, which are huge storage compartments and a hallmark of plant cells (Carden et al. 2003).

Several traits are responsible for plant tolerance to this stress (Reddy et al. 2002). Many characters are highly hereditary and also additive, which indicates that there is considerable room for improvement to abiotic stresses. Outstanding results obtained by breeders in different crops prove these observations.

The first molecular approach to help breeders in their efforts to increase drought tolerance has been with molecular markers, genomics and "post-genomics strategies" (Nguyen et al. 2004; Lanceras et al. 2004; Robertson 1989). The dissection of the genetic basis of quantitative traits into their single components, the socalled QTLs (Quantitative Trait Loci), provides direct access to valuable genetic diversity for important physiological processes that regulate the adaptive response to drought (Wayne and Mclntyre 2002; Masahiro and Takuji 1997). This allows scientists/breeders to deploy marker-assisted selection (MAS) for enhancing crop performance in breeding regimes. However, despite impressive progress in molecular techniques and the large number of QTLs described that influence yield in drought-stressed crops, the overall impact of MAS and other genomics applications on the release of drought-resilient cultivars has so far been marginal (Quarrie et al. 1997; Tuberosa, 2004). QTL discovery should be viewed as the first step of a longer process aimed at identifying and isolating the underlying molecular polymorphism of the functional variation revealed through QTL analysis.

The use of transgenics to provide enhanced drought tolerance is still experimental in nature, though progress is being made (Dubouzet et al., 2003; Garg et al., 2002; Kasukabe et al., 2004; Pellegrineschi et al., 2004). The obvious next step is to investigate the impact of the introduced gene by measuring the growth and yield of transgenic plants in a field environment. This is an important step because the desiccation stress applied to the transgenic plants to evaluate their response has until now been done under greenhouse conditions that do not fully represent the environment in the field. Under greenhouse conditions, transgenic plants are grown in small pots that have less soil volume than the field has, and they are subjected to rapid stress cycles that range from an hour to several days. When stress is imposed rapidly, a greater number of responses will be injury-induced than under a slower, long-term application of water-deficit stress.

The three most important elements of drought characterization for successful stress tolerance breeding are probably timing, duration, and stress intensity. For most crops, including wheat, drought tends to develop slowly as the soil dries out. Plants that are subjected to drought conditions in this gradual manner accumulate solutes that maintain cell hydration and undergo complex adjustments in their morphology and physiological characteristics. Since most experiments that have been published thus far are based on a rapid, severe water deficit treatment, it is important to conduct experiments under conditions that more closely approximate stress development in the field. Such an experiment will permit a better understanding of the potential functions of the introduced gene in stress tolerance.

Several genes have been tested that have great potential to help us understand and manipulate plant stress response (Pflieger et al. 2002). Work done in wheat at CIMMYT headquarters in El Batan, Mexico is a recent example of this strategy (Pellegrineschi et al. 2004). An earlier study focused on transgenic wheat for the DREB/CBF (DRE-binding protein/C-repeat binding factor), which showed enhanced resistance to moisture stress in greenhouse conditions. The objective of the latest study is to understand the principal effects of the DREB/CBF gene in transgenic wheat under a prolonged drought stress cycle.

Preliminary field testing of the transgenic lines showed a lower canopy temperature (1-2° Celsius less) and, in general, the transgenic lines showed a relatively higher water content, more biomass, lower chlorophyll content, and increased seeds production. The transgenic lines responded better to returning their normal phenotype after irrigation (rehydratation) and were better able to continue and complete the normal field cycle, ultimately producing viable seeds and a higher grain yield. Clearly, these results need to be verified in a larger trial with selected transgenic lines.

This trial is the first time that a food crop carrying the DREB gene has advanced to this level of testing. Following this trial, CIMMYT wishes to test other DREB genes isolated from rice by Dr. Yamaguchi-Shinozaki (JIRCAS) as well as the soluble starch synthase (SSS) gene. Conventional wheat will be transformed with these genes to determine whether the resulting plants can use water as efficiently as, or more efficiently than wheat expressing the recently tested DREB gene. Increasing the expression of the SSS gene, a key enzyme involved in amylopectin biosynthesis, is believed to increase the speed of grain filling, which is one of the major problems caused by drought stress. The synthesis of starch in the endosperm of cereals occurs via an enzymatic mechanism that uses ADPglucose as the glucosyl donor. In barley and other cereals ADPglucose is thought to be synthesized by separate ADPglucose pyrophosphorylases located in the cytoplasm and in the amyloplast, and the chain elongation (starch synthases) involving branching and debranching enzymes occur in the amyloplast.

Given that the initial DREB1A wheat transgenics do not meet CIMMYT standards for GM products (e.g., low copy, contains marker genes), scientists are now developing transgenic wheat lines that meet the Center's criteria. This will be done in collaboration with scientists in Chile and Argentina where such transgenic products could be tested and potentially deployed. CIMMYT will also develop similar transgenic lines containing the additional DREB genes obtained from JIRCAS and the SSS gene. It is anticipated that if the DREB1A wheat lines continue to perform well, a full proposal will be developed that will ultimately lead to the safe and effective deployment of these products to National Agricultural Research Systems in the Americas, Africa, and Asia.

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