Comparative study of direct and indirect evaluations of frost tolerance in barley
Introduction
By ‘winter hardiness’ of barley, is meant the ability of the plants to resist many unfavourable factors during winter. The abiotic factors include freezing, flooding, heaving, frost desiccation, enclosure of plants within an ice sheet, or their exhaustion under a long-lasting snow cover. The biotic factors are mainly represented by the possibilities of contamination, with a complex of snow moulds, of the plants. These unfavourable factors may become the main causes of damage to the wintering growth, however, they always negatively influence the frost tolerance of the plants. A significant correlation was found between the frost tolerance of barleys and their wintering in many locations e.g. in Canada (Gusta and Fowler, 1979, Bridger et al., 1995) as well as in most of the European countries from Italy, through Germany, Czech Republic, to the northern Europe countries, including extensive areas of Russia and the Ukraine (Koch and Lehmann, 1966, Remeslo, 1975, Udovenko, 1976, Prášil et al., 1994, Rizza et al., 1994, Pulli et al., 1996).
Methods, for the determination of crop frost tolerances, have become well established and standardized (e.g. Marshall et al., 1981, Prášil et al., 1994, Sãulescu and Braun, 2001). They can be divided into two main categories (Table 1). The first group involves plants exposed to the direct effects of frost, where the subsequent damages or deaths of plants, caused by the frost, are then evaluated. The second group is based upon a determination of correlations or associations between the plant frost tolerance and some characteristic(s) (the so-called diagnostic traits and markers). In this second case, the plants are not exposed to frost (indirect methods), but are evaluated based upon the presence of a given trait or a complex of traits. Lately, the study of barley's molecular markers, connected with what are the assumed to be the frost tolerance genes, ultimately for their regulation, has been greatly expanded (Tóth et al., 2004, Francia et al., 2005). The direct methods can be further sub-divided into field, field–laboratory, and laboratory methods, based upon the frost environment the plants are being acclimated in/exposed to (Table 1).
The field methods are the closest to the natural environment, but these are more dependent upon external stresses, and usually require that the tests are performed over several years. There are no guarantees of being able to distinguish between the barleys after winter in a field every year. Therefore, with the field methods, many approaches and modifications have been used to increase the chances to be able to distinguish the samples every year (e.g. wintering with snow removal) (Remeslo, 1975, Sãulescu and Braun, 2001). These approaches also include the so-called provocation pot test, in which the crop plants are wintered within pots placed at different heights above the ground, and are thus exposed to stronger frosts and the other unfavourable factors (Prášil and Rogalewicz, 1989, Prášilová and Prášil, 2001).
The field–laboratory methods are usually represented by the growth of plants in the field or in pots placed outside during the winter, and after which they are collected and exposed to frost using lab freezers. The advantage of these methods is the possibility to determine not only the actual frost tolerance of the samples, but also the frost tolerance changes during winter and, especially, early spring—when the more sensitive cultivars very quickly loose their tolerance (Udovenko, 1976, Prášil et al., 1994, Fowler et al., 1999).
When using the laboratory methods, the whole process (growing, acclimation, and frost test) takes place under controlled conditions. The aim here is to ensure for the appropriate conditions for an induction of frost tolerance of the plants, and with the possibility to distinguish the frost tolerance of individual samples. These are accomplished using combinations of the length and the intensity of light, regulation of temperature, moisture, nutrition, and the choice of an appropriate substrate (Udovenko, 1976, Marshall et al., 1981, Brule-Babel and Fowler, 1989, Mahfoozi et al., 2001, Sãulescu and Braun, 2001).
One appropriate characteristic for comparing the winter survival of cereals under field conditions is the field survival index (FSI; Fowler and Gusta, 1979). In the case of the laboratory frost test, it is the LT50 value (lethal temperature, at which 50% of samples are killed). A very high agreement has been found between the FSI and LT50 of different cultivars (Hömmö, 1994, Bridger et al., 1995, Pulli et al., 1996, Fowler, 2002).
The indirect methods allow one to evaluate the frost tolerance based upon measurements of selected plant characteristics (Table 1). In cereals, there are many traits utilized, starting with morphological (ground plant growth), through physiologic–biochemical (e.g. proline and osmolytes content), and ending with biophysical ones (e.g. electric conductivity of tissues) (Fowler et al., 1981, Gusta et al., 1983, Murelli et al., 1995, Sãulescu and Braun, 2001, Tantau et al., 2004). Those characteristics, correlated with barley frost tolerances, often described include the following: the level of a sugar; types of fructans; water content, and in its different forms (bound water and values of osmotic pressure); different components of cell membranes and phospholipids (e.g. fatty acids); and changes in the level of abscisic acid (Livingston et al., 1989, Olien and Clark, 1995, Murelli et al., 1995, Bravo et al., 1998). With the continuous progress in research on the molecular biology of frost tolerance have seen the description of the expression of many genes, and accumulation of proteins associated with the induction of barley frost tolerance. Of prime interest are the cold regulated (cor) genes, COR proteins, and components of their regulation pathways (Giorni et al., 1999, Stanca et al., 2003). For measuring the indirect traits and markers, plants must be pre-exposed to temperatures below 12 °C, after which the hardening, associated with the induction of frost tolerance, occurs. Only occasionally, it was possible to detect differences between frost tolerances of barley cultivars under non-induction temperatures (the so-called constitutive tolerance; Bravo et al., 1998).
Use of DNA markers does not require the tested plants being exposing to the induction conditions. Tóth et al. (2004) studied PCR-based markers, localised on the 5H chromosome in the position where the main QTLs of frost tolerance have been described. The dehydrin genes belong to those genes whose expression is closely associated with the induction of cereals’ resistance to both drought and low temperatures. In barley plants under conditions of stress, the expression of a whole group of Dhn genes (Dhn-1, -2, -3, -4, -5, -7, -8, -9, and -10) has previously been described (Van Zee et al., 1995, Zhu et al., 2000).
Frost tolerance is a complexly conditioned characteristic that is the result of the activities of many genes and the participation of many substances as well as changes in the plant cells and tissues. Therefore, the determination of a single trait alone can have limitations. Furthermore, the measurement of some characteristics is very sensitive to the previous manipulation of the plants and to the conditions used for growth and hardening of the plants (e.g. water content, endogenous level of abscisic acid, tissue conductivity). Because of this, we focused on the comparison and evaluation of several approaches in order to determine the barley's frost tolerance.
In this study, we compared the results from the wintering of 39 barleys, obtained in field provocation tests with the LT50 of samples either collected from the field in early winter, or hardened in the regulated environments of climaboxes. We then further compared these results with those determined for indirect characteristics, often described in association with barley frost tolerance (water content, osmotic potential, abscisic acid content). Our aim was also to find both the possibilities and limitations of individual methods and approaches.
Section snippets
Materials and methods
The seeds of the barley (Hordeum vulgare L.) cultivars and breeding lines were obtained from the Central Institute for Supervising and Testing in Agriculture (CISTA) in Brno, Gene Bank of Research Institute of Crop Production (RICP) in Praha, and Selgen a.s. Praha breeding company. A total of 39 barley samples were used including: 20 winter cultivars (Borwina, Camera, Cenader, Dominator, Hauters Wintergerste, Jolante, Kamil, Kromir, Kromoz, Lunet, Luran, Luxor, Marinka, Monaco, Nelly, Odesskij
The provocation pot tests
The survival of winter barleys were evaluated using the provocation pot test over a period of five winters (1999/2000–2003/2004), at two locations (RICP-Prague and Lužany breeding station), and utilizing two different options for the placement of the pots (5 and 50 cm) above the ground. This means that there were a total of 20 independent evaluations (5 winters × 2 locations × 2 variants of pot placement). For calculation of the average survival of a sample, we used only those selected variants that
Discussion
In this study, we compared the evaluation results for barley frost tolerance obtained by four direct methods (direct exposure to frost) and three indirect ones (diagnostic traits and markers). All four direct methods mutually correlated well, and resulted in a similar frost tolerance differentiation of the samples. The disadvantage, of the low frequency in successful differentiation (in frost killing) of barley in the field conditions (1× in five winters), was compensated for by growing barley
Acknowledgements
We thank Dr. M. Klemš (Mendel University of Agriculture and Forestry, Brno) for ABA determination by RIA, and Ing. D. Jurečka (Central Institute for Supervising and Testing in Agriculture, Brno) for providing the winter survival data of barley from the testing stations. The research was supported by the Ministry of Agriculture (NAZV QE1107, 1G57060 and MZe0002700602).
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