Journal of Experimental Botany, Vol. 51, No. 342, pp. 139-146,
January 2000
© 2000 Oxford University Press
Isolation and analysis of thermotolerant mutants of wheat
Department of Plant Science, University College, Cork, Ireland
Received 15 March 1999; Accepted 17 June 1999
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Abstract |
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Thermotolerant mutants of wheat cv. Guardian were isolated by selecting survivors from 5-d-old seedlings of M2 populations exposed to 47 °C for 90 min. Progeny testing, using triphenyl tetrazolium chloride reduction as a measure of tissue viability, following heat stress treatment for 120 min at each of three temperatures (32, 38 and 50 °C), confirmed the thermotolerant nature of seedlings of 13 mutants. Mutants were isolated at frequencies of 0.1% and 0.2% following the use of sodium azide and ethyl methanesulphonate, respectively. The relative thermotolerance of ten of the mutants and ‘Guardian’ was then tested by exposing plants to heat stresses of 38 °C for 6 h in every 24 h for five successive days at one of four growth stages between seedling and anthesis. Pmax (light-saturated net photosynthetic rate) and chlorophyll content were compared in stressed and unstressed plants. The Pmaxof ‘Guardian’ was depressed by at least 23% by heat stress at each stage; this inhibition was least at ear emergence and greatest at anthesis, the latter being associated with reduced sink size as a result of lowered seed set. The stress-induced inhibition of Pmaxin ‘Guardian’ plants at anthesis had not recovered 3 d after removal of the stress. Mutant lines exhibited different developmental profiles of Pmaxthermostability. Mutant tht (thermotolerant) 10, for example, exhibited partial thermostability at each growth stage tested while the Pmaxof mutant tht 2 was completely unaffected by heat stress at second node and ear emergence, but was as inhibited as that of ‘Guardian’ at anthesis; heat stress applied at anthesis in tht 2, but not tht 10, was associated with reduced seed set. Generally, the inhibitory effect of heat stress on Pmaxin the mutants was reflected in declines in chlorophyll content. The ten mutants were grouped into nine categories, on the basis of thermotolerance characteristics.
Key words: thermotolerance, heat stress, photosynthesis, wheat, mutants, chlorophyll.
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Heat stress is one of the most important factors affecting crop yield (Wrigley et al., 1994
). Wheat is particularly susceptible to yield losses as a result of heat stress. The optimum temperature for growth and yield of wheat is in the range 18–24 °C and even short periods (5–6 d) of exposure of wheat crops to temperatures of 28–32 °C result in significant decreases in yield of 20% or more (Stone and Nicolas, 1994). Such temperatures are commonly encountered in the latter part of the growing season in major wheat growing areas such as North America, South America, Australia, and France (Wardlaw et al., 1989). All plant processes are sensitive to and can be irreversibly damaged by heat. The optimum temperature for photosynthetic activity of wheat from anthesis to maturity is 20 °C or lower (Al-Khatib and Paulsen, 1989). Elevated temperatures accelerate senescence, reduce the duration of viable leaf area and diminish photosynthetic activities (Harding et al., 1990). Effects on the thylakoid membrane lead to a loss in the number of chloroplasts per cell (Hurkman and Tanaka, 1987).
In addition to its effect on wheat yield, heat stress also affects the quality of the harvested product, reducing bread making quality (by affecting gliadin synthesis) (Blumenthal et al., 1993) and starch quality by affecting the ratio of A (large) to B (small) starch granule types (Stone and Nicolas, 1995).
Many methods of assessing thermotolerance in plants have been used by previous workers. Electrical conductivity (EC) has been used as an index of membrane stability to identify heat-tolerant genotypes in many crops. When tissues are subjected to high temperature, EC increases due to damage to the cell membrane (Blum and Ebercon, 1981). Another laboratory screening method involves measurements of chlorophyll fluorescence in vivo, monitoring cellular injury caused by environmental stresses (Smillie and Hetherington, 1983).
Little is known of the role of individual genes in the control of thermotolerance. Although selection of relatively thermotolerant wheat cultivars has been demonstrated, much of the success in breeding varieties for warmer climates has been due to the use of the Ppd1 gene, conferring photoperiod-insensitive development so that the plants develop more quickly, avoiding the effects of the most stressful conditions (Börner et al., 1993; Worland et al., 1994). Mechanisms of thermotolerance in crop plants are not yet fully understood. Several crop plants, including wheat, exhibit heat shock protection mechanisms, responding to sub-lethal high temperature stress by synthesizing heat shock proteins (HSPs), which can protect native proteins from denaturation (Vierling, 1991). Other possible mechanisms of thermotolerance exist; for example, several reports show that heat stress is accompanied by the formation of reactive oxygen intermediates, causing damage to membranes (Dhindsa et al., 1981). Plants can counteract this effect by producing oxygen radical scavengers, such as superoxide dismutases, peroxidases, and catalases (Holmberg and Bulow, 1998).
One approach to the identification of genes involved in determining thermotolerance involves the use of induced mutants, employing a phenotype-to-gene strategy. The isolation of one thermotolerant wheat mutant which exhibited high temperature tolerance with respect to yield under irrigated and rainfed conditions in India has been reported (Behl et al., 1986).
Here, the isolation and preliminary physiological analysis of induced wheat mutants exhibiting increased thermotolerance will be described.
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Materials and methods |
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Production of induced mutant populations
Using the chemical mutagens sodium azide and ethyl methanesulphonate (EMS), induced mutants of wheat cv. Guardian were generated. Mutagenesis was conducted as described previously (Jones, 1997
). To determine the required mutagen dosage (which caused a 15% reduction in the height of 10-d-old M1 seedlings; IAEA, 1977), dosage-response studies were conducted for both mutagens. One thousand seeds were then treated with the selected mutagen concentration (0.40 mM EMS for 8 h, 5 mM sodium azide for 6 h). Following thorough rinsing, the resulting M1 seeds were then planted at high densities and low nitrogen levels (to minimise tillering) in isolated field plots. The M1 plants were self-pollinated and the M2 seed for each mutagen was bulked and used to select for thermotolerant mutants.
Determination of selection temperature
Six thousand M0 ‘Guardian’ seeds were incubated between two sheets of Whatman 3MM chromatography paper saturated with distilled water in plastic boxes maintained in darkness at 4 °C for 24 h to aid synchronization of germination. Following this imbibition period, the boxes were transferred to 22 °C, and the seeds incubated in the dark for a further 96 h. To determine the effect of temperature on survival, these 5-d-old dark-grown seedlings were transferred to muslin bags, with 200 seedlings per bag. Each bag was then placed in a water bath at one of seven temperatures between 44 °C and 50 °C (differing in 1 °C increments) for 90 min; control seedlings were maintained at room temperature for 90 min. Following this stress period, the bags were removed, the seedlings patted dry, with absorbent towels, and placed between two sheets of moist Whatman 3MM chromatography paper in plastic boxes and allowed to grow for 5 d at 22 °C in the light. After 5 d, the boxes were removed and the seedlings scored for survivors. The lowest temperature at which no seedlings survived was identified as the lethal temperature.
Selection of thermotolerant mutants
Batches of 1000 M2 5-d-old dark-grown seedlings, produced as described above, were incubated under the lethal temperature conditions. Following the subsequent 5 d incubation period at 22 °C in the light, the survivors were counted and transferred to the glasshouse where they were grown to maturity.
Confirmation of thermotolerant mutants (TTC test)
Forcible self-pollination of the individual putative mutants was conducted and the resulting M3 progeny of each plant was tested for thermotolerance. Seeds of the selected mutant lines and ‘Guardian’ were imbibed at 22 °C for 24 h in the dark in Petri dishes between two 9 cm diameter discs of Whatman number 1 filter paper. The seedlings were transplanted to potting compost and grown in the glasshouse for 10 d at a minimum temperature of 18 °C. Five hours before the stress was applied, the plants were placed in the dark to ensure that the stomata were closed. For each of the mutant lines and ‘Guardian’, three plants were stressed and three acted as unstressed controls. The primary leaf of each seedling was placed in a test tube; at the bottom of each test tube was damp cotton wool to maintain high humidity, and the tube was sealed with parafilm. The plants were incubated in the dark in a water bath at 50 °C for 120 min, while the control plants were maintained at room temperature in the dark. The ability of the seedlings to reduce triphenyl tetrazolium chloride (TTC) was then determined, using the method of Steponskus and Lanphear (Steponskus and Lanphear, 1967). The leaves were weighed individually and each leaf was cut up and placed in a test tube containing 3 ml 0.6% (w/v) TTC in 0.05 M potassium phosphate buffer (pH 7.4) with 0.05% (v/v) wetting agent (Tween 20), and infiltrated under vacuum for 2 min, to ensure solution penetration into the tissues. The test tubes were then incubated for 18 h in darkness at 22 °C. The TTC solution was drained off and the tissue rinsed once with 3 ml distilled water. The samples were extracted with 3 ml 95% (v/v) ethanol in a thermoblock at 100 °C for 10 min. The tubes were then cooled and the residue dissolved in 3 ml 95% ethanol. The absorbance of each sample was recorded at 530 nm, and the percentage heat-induced inhibition of TTC reduction determined as:
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The study was also repeated with heat stress temperatures of 38 °C and 32 °C.
Heat stress treatment of lines at four growth stages
Plants of control ‘Guardian’ and ten selected thermotolerant mutant lines were grown outdoors (mean daytime temperature of 19 °C) in 1.0 l pots of peat-based potting compost, four plants per pot, ten pots per wheat line. When plants reached one of the following growth stages, seedling (3 weeks after sowing) (GS 15, Zadoks et al., 1974), second node (GS 32), ear emergence (GS 59), anthesis (GS 69), or dough development (GS 83), one pot of each mutant and ‘Guardian’ was placed into a growth cabinet maintained at 18 °C/18 h and a heat stress temperature of 38 °C/6 h d-1 for 5 d. The change in plant temperature took place over a 30 min period. A second pot was maintained at 18 °C for the entire 24 h period and these plants represented the unstressed controls. To prevent problems associated with transpiration, the pots were watered daily, and maintained in a plastic bag containing a tray of water to keep the humidity high. The four plants in each pot represented the replicates of each treatment.
Photosynthesis (Pmax) measurements
Immediately following the 5 d treatment within the growth cabinet, the plants were removed, and the light-saturated photosynthetic rate (Pmax) was measured on the youngest fully-expanded main stem leaf at maximum photon flux rate intensity (1980 mol m-2 s-1) using an Infrared gas analyser (CIRAS-1) and a Parkinson narrow leaf cuvette (PLC) (PP Systems, Hoddesdon, UK).
Chlorophyll content measurements
Chlorophyll readings (mean of six readings per leaf) were taken on the same leaf used for Pmax measurements. A chlorophyll meter was used (Minolta SPAD meter; Minolta Camera Co., Japan), recording in SPAD units. The SPAD readings were converted to chlorophyll content (as mg chlorophyll mg -1 leaf fresh weight) using a linear regression analysis of SPAD versus chlorophyll content (determined according to the method of Hendry and Price, 1993) on wheat leaves.
Chlorophyll a and b measurements
A separate heat stress experiment was set up at four different growth stages (omitting GS 69). Immediately following the 5 d treatment period, the mutants and control ‘Guardian’ plants were sampled, with the youngest fully expanded main stem leaf being detached and weighed. The tissue was homogenized in 10 vols ammoniacal acetone (81.8% (v/v) acetone, 0.2% ammonium hydroxide). The samples were then centrifuged at 3000 g for 3 min. The absorbance of each supernatant was then determined at each of the following wavelengths: 645, 663 and 710 nm; 710 nm was used as an isobetric point, deducted from all other absorbance readings (Hendry and Price, 1993).
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Grain number measurements
Following heat stress treatment and the determination of Pmax and chlorophyll content, the plants were returned to the outdoor trial site and maintained there until final maturity. The pots were treated weekly with a proprietary liquid nutrient fertiliser. At maturity, the number of grains per main stem ear was recorded.
Each experiment described in this paper was repeated three times. For all characters tested, the effect of heat stress is presented as % change, as described for TTC reduction: negative values for % change indicate thermal injury of the parameter studied, as a result of heat stress. Similar relative results from each repetition were obtained (relative to unstressed plants of the same genotype) and data are presented from one study.
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Results |
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Isolation of thermotolerant mutants
The period of imbibition resulted in synchronization of seed germination; because heat sensitivity increases as dormancy ends, any late-germinating seeds (which could appear as thermotolerant) were removed before the heat stress was applied, to minimize the presence of ‘escapes’ among surviving seedlings (Abernethy et al., 1989
).
Five-day-old seedlings of wheat cv. Guardian were exposed for 90 min at temperatures between 44 °C and 50 °C to determine the lethal temperature. The results indicated that a selection temperature of 47 °C resulted in no surviving ‘Guardian’ seedlings (Fig. 1). When seedlings of each of the three populations (M0 plus the two M2 populations) were subjected to this selection strategy, no survivors were isolated from the non-mutagenized population, compared to 0.1% survivors from the sodium azide-mutagenized M2 and 0.2% from the ethyl methanesulphonate-mutagenized M2 populations.
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Confirmation of the putative thermotolerant mutant lines
A total of 14 putative thermotolerant mutants were isolated. To confirm the genetic nature of these phenotypes, forcible self-pollination of the individual M2 plants was conducted, and the resulting M3 progeny of each surviving plant was tested for thermotolerance using the TTC reduction method to compare dehydrogenase activity in stressed versus unstressed plants. Following the application of a heat stress of 50 °C for 120 min, ‘Guardian’ showed a 67% decrease in TTC reduction compared to the control (unstressed) ‘Guardian’ plants. An incubation temperature of 50 °C, rather than the selection temperature of 47 °C, was used to maximize the distinction between stressed and unstressed plants. Of the 14 putative mutants tested at this temperature, 13 suffered a lower heat-associated fall in TTC reduction lower than that exhibited by ‘Guardian’, with the difference being statistically significant in the case of four mutants (tht (thermotolerant) 3, tht 5, tht 9, and tht 8) (Fig. 2a). Further work was carried out on the mutant lines to investigate the effect of the less stressful treatments of 32 °C and 38 °C for 120 min. Results showed that following these reduced heat stresses, additional mutant lines showed less damage than ‘Guardian’ (Fig. 2b, c). For example, mutant lines tht 7, tht 3 (also tolerant at 50 °C) and tht 14 suffered significantly less damage following the heat stress of 38 °C (Fig. 2b) When the mutant lines were exposed to the lower temperature regime of 32 °C for 120 min, eight lines showed significantly less damage than the unstressed control plants, namely tht 1, tht 2, tht 5, tht 6, tht 10, tht 11, tht 12, and tht 13 (Fig. 2c). Overall, 13 out of the 14 putative mutants exhibited increased thermotolerance at at least one stress temperature, with only tht 4 failing to express thermostability of TTC reduction. In all the mutants, thermotolerance (as measured by TTC dehydrogenase activity) was temperature-specific; for example, tht 8 was only thermotolerant at 50 °C while tht 3 was thermotolerant at both 50 °C and 38 °C.
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For further studies involving the mutant lines, tht 1, tht 7 and tht 9 were omitted, following confirmation work using the TTC test, due to insufficient seed numbers.
Photosynthesis (Pmax) measurements
Pmax of ‘Guardian’ was inhibited significantly by the short heat stress treatment (38 °C for 6 h d-1 for 5 d) at each growth stage, the depression being least at GS 59 and most at GS 69 (Fig. 3a). ‘Guardian’ and selected mutant lines were exposed to the standard heat stress at GS 69 and Pmax rates were measured over a 3 d period following the removal of the stress. The inhibition of Pmax in mutants tht 14 and tht 6 following heat stress showed no sign of recovery over the 3 d period. In ‘Guardian’, the effect of heat on Pmax worsened from day 1 to day 2, then recovered until, at day 3, after removal of the stress, there was no significant difference in the Pmax levels of the three lines (Fig. 4).
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), tht 2 (•) and tht 10 (
). Any two samples in the same graph sharing a common letter were not significantly different at the P=0.05 level, using the Tukey test.
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The Pmax of each of the thermotolerant mutants (with the exception of tht 11) proved to be more thermostable than that of ‘Guardian’ at at least one growth stage (Table 1
), although the various mutants responded differently from one another. The Pmax depression of mutant tht 10, for example, was lower than that of ‘Guardian’ at all stages tested, particularly GS 32 and GS 69 (Fig. 3a), whereas tht 2 exhibited no heat-induced reduction in Pmax when heat stress was applied at GS 32 or GS 59, but proved to be as thermosensitive as ‘Guardian’ at GS 69. The Pmax of no mutant was more thermotolerant than that of ‘Guardian’ at all temperatures, but all the thermotolerant mutants exhibited significantly less Pmax depression than ‘Guardian’ at GS 32.
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Chlorophyll content and a : b ratio measurements
Heat-related reduction in chlorophyll content in ‘Guardian’ plants followed a similar response to Pmax (with maximum thermotolerance at GS 59) (Table 1; Fig. 3b). When the heat stress was applied at GS 32, six mutants showed significantly greater thermostability of chlorophyll levels than ‘Guardian’ (Fig. 5a). When changes in chlorophyll a : b ratio were studied, two mutants (tht 2 (which also showed an increase in total chlorophyll following heat stress) and tht 5) exhibited a significantly more thermostable ratio than ‘Guardian’(Fig. 5b), while a further two exhibited a significantly less thermostable ratio, tht 10 and tht 12 (Fig. 5b).
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Grain number measurements
As an indicator of heat-stress-associated infertility, the grain number per main stem ear was compared in stressed relative to unstressed plants of the same genotype. Following application of a heat stress (38 °C for 6 h d-1 for 5 d) at GS 69, mutants tht 12, tht 10, tht 8, tht 3, and tht 6 all exhibited significantly greater thermostability of seed set than did ‘Guardian’ (Fig. 6). Mutants tht 11, tht 14 and tht 13 showed a significantly greater heat stress- induced fall in seed set than ‘Guardian’. There was no significant difference in grain number per ear observed in unstressed plants of mutants and ‘Guardian’.
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Preliminary characterization of thermotolerant mutants
Classification of ten tht mutants, according to the thermostability of five different characters, resulted in the identification of nine categories (Table 2). Only one category (I) was represented by more than one mutant.
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The fact that 13 (91%) of the putative mutants of wheat cv. Guardian selected using the seedling method exhibited greater thermotolerance of TTC reduction at at least one incubation temperature (Fig. 2a, b, c) indicates the effectiveness of the selection procedure. The frequency of the confirmed mutants (at 1–2x10-3) is rather high (IAEA, 1977
). The mutagenesis strategy used generates largely point mutations, employing chemical mutagens at low dosages; mutant frequencies for other characters from this M2 population were within the range of published values (IAEA, 1977), for example, 1x10-5 for mutants exhibiting awned ears (Crowley, 1993). This indicates that the high frequency of thermotolerant mutants is not a consequence of hyper-mutagenicity. A more likely explanation is that a large number of genes contributes to the character ‘thermotolerance’ (as indicated by the fact that each mutant exhibited a different range of characteristics; Table 2); a mutation frequency of 1x10-4–5x10-5 in each of 10–20 different genes would result in the isolation of thermotolerant mutants at frequencies similar to those observed here. The low frequency of false positives (less than 10%) can be ascribed in part to the synchronization of germination by pre-imbibition of the seeds; this undoubtedly reduced the risk of slow-germinating seedlings ‘escaping’ the heat stress. The mutants isolated were vigorous and, in the absence of heat stress, they were indistinguishable (in terms of the characters measured here) from ‘Guardian’, facilitating interpretation of the data obtained from the heat-stressed plants.
A common problem with mutant selection methods operating at the seedling stage involves non-expression of tolerance in the adult plant. In this case, the mutants continued to exhibit increased thermotolerance as the plants developed, although the thermotolerance characteristics of the different mutants varied. Thermotolerance is known to be developmentally dependent in wheat (Abernethy et al., 1989), while the various measures of thermotolerance (seedling survival, TTC reduction, Pmax) also showed differential thermosensitivity.
Plant age is important in thermotolerance studies due to the varying sensitivity of the plant at different growth stages. Exposing wheat plants to high temperatures at early stages (e.g. GS 32), can affect factors such as node extension and ear development, while temperature stress at anthesis and later can cause premature leaf senescence and can affect the fertility of the plant leading to reduced grain development (Wardlaw et al., 1980). The heat stress applied to the adult plants (38 °C for 6 h d-1 for 5 d) was chosen for the reason that such conditions are commonly encountered during wheat crop development in the warmer regions of developed countries such as Australia (Stone and Nicolas, 1994). The high temperature exposure of 38 °C for 6 h involved a sudden rise in temperature (over a 30 min period). Such dramatic increases in temperature would not occur under field conditions. Studies have indicated that gradual exposure to elevated temperatures confers greater protection to normal plant function than occurs when a rapid rise in incubation temperature is employed (Chen et al., 1990). All the studies here were carried out without an acclimation period, as this was the basis of the initial selection procedure. It would be valuable from a practical point of view to evaluate how the mutants respond to an acclimation period.
As indicated in the literature (Sharkova, 1994), photosynthesis was particularly sensitive to this short period of heat stress, with Pmax of ‘Guardian’ being inhibited by at least 23% when stress was applied (Fig. 3a), the effect being largely irreversible (Fig. 4). A significant positive correlation between heat-induced reduction in Pmax and chlorophyll content (r=0.604, n=13; P<0.05) at GS 59 indicates that, in those mutants exhibiting Pmax thermostability at GS 59, increased Pmax activity was associated with reduced chlorophyll damage. This would explain the irreversible nature of the heat stress effects on Pmax (Fig. 4). The main effects of heat stress on the chloroplasts are on the proteins of the thylakoid membrane. The thylakoid membranes become disoriented relative to the long axis of the chloroplasts, and the membrane unstacks, causing loss of chloroplasts (Xu et al., 1995).
An exception to this general association between chlorophyll content and Pmax occurred at GS 69 (Fig. 3b) when Pmax fell dramatically following heat stress treatment of ‘Guardian’ and most of the mutants (e.g. tht 2; Fig. 3b), whereas chlorophyll content was relatively tolerant of heat stress at this stage of development. Stage-specificity of the heat sensitivity of wheat photosynthesis is a difficult phenomenon to rationalize. A possible explanation may lie in the significant fall in seed set (possibly reflecting a decrease in fertility, due to the non-expression of certain tht mutations (e.g. tht 2) in pollen) observed in ‘Guardian’ and several mutants (Fig. 6). Reduced sink size (due to a fall in grain number per ear) could cause significant feedback inhibition of photosynthesis of the major source (i.e. the flag leaf) supplying the developing ear. Thermotolerance of Pmax in the mutants tended to be expressed in the older plants (with the exception of anthesis discussed above); the Pmax of all of the mutants (except tht 11) was significantly more tolerant than that of ‘Guardian’ at GS 32, compared with only two at the seedling stage (Table 1).
This study represents only the second report of thermotolerant mutants isolated from wheat; here, 13 mutants have been isolated (with thermotolerance stably expressed over at least three generations) compared with one by Behl et al. (1986). This opens up the possibility of significant progress being made in the elucidation of the thermotolerance pathway in wheat, especially when the number of apparently different mutants is considered (Table 2). The near-isogenic nature of the heat-tolerant (mutants) and heat-sensitive (‘Guardian’) material will facilitate interpretation of data from studies into the mechanisms of thermotolerance, permitting the establishment of cause–effect relationships. The use of low dosages of chemical mutagens (which generate primarily point mutations; IAEA, 1977) reduces the risk of gross chromosomal aberrations in the mutants; back-crossing each mutant to ‘Guardian’ would permit elimination of any multiple mutations in addition to that conferring thermotolerance, making the mutants more nearly isogenic to ‘Guardian’. Comparison of near-isogenic thermotolerant and sensitive lines will also provide direct testing of the hypothesis of cross tolerance (the ability to survive under varying stress regimes, for example, the ability to tolerate both saline and high temperature conditions) between different abiotic (and biotic) stresses (Xu et al., 1996) and will enable the identification of the individual genes involved in determining the thermotolerant phenotype (e.g. using AFLP; Vos et al., 1995; subtractive hybridization, Buchanan Wollaston and Ainsworth, 1997) in the different mutants.
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1 To whom correspondence should be addressed. Fax: +353 21 274420. E-mail: p.jones@ucc.ie
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References |
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