ABSTRACT. Identification of heat-tolerant winter wheat(Triticum aestivumL.) genotypes for the central and southern Great Plains of the USA as well as other ...
Published November, 1990
Heat Tolerance in Winter Wheat: I. Hardening and Genetic Effects on Membrane Thermostability M. M. Saadalla, J. F. Shanahan,* and J. S. Quick ABSTRACT Identification of heat-tolerant winter wheat (Triticum aestivum L.) genotypes for the central and southern Great Plains of the USA as well as other areas of the world is desirable. In this study, the suitability of using the membrane thermostability (MT) test for ascertaining heat tolerance of winter wheat was determined. The MT test was conducted on seven cultivars using seedlings exposed to 0, 6, 12,18, 24, and 48 h of hardening (34 °C) and flag leaf material from the same cultivars grown to anthesis and exposed to 0, 48, and 120 h of hardening (34 °C), with the results expressed as relative injury (RI). Additionally, the MT test was conducted on 90 F5 genotypes derived from two crosses (45 genotypes per cross) involving one heattolerant and two heat-sensitive parents (parents were a subset of seven previous cultivars). Plant material for the MT test was sampled at seedling (hardened for 48 h at 34 °C) and anthesis (hardened for 120 h at 34 °C) stages of growth. Large differences in RI were observed among the seven cultivars at both growth stages, ranging from 37 to 80% RI for seedlings and 31 to 69% RI at anthesis. The treatment protocols involving hardening for 48 and 120 h at seedling and anthesis stages, respectively, provided the greatest sensitivity in detecting genotypic differences in RI. The correlation of RI assessment for seedlings vs. that at anthesis for the 90 F5 genotypes was 0.79 (P < 0.01), indicating that RI determined at the two developmental stages was highly associated.
IGH-TEMPERATURE STRESS during the grain-fill H period is a major constraint to increased productivity of winter wheat grown in the central and
southern Great Plains of the USA and in other areas of the world (Gusta and Chen, 1987; Wardlaw et al., 1989). Wiegand and Cuellar (1981) and Chowdhury
M.M. Saadalla, Dep. of Agronomy, Assuit Univ., Assuit, Egypt; and J.F. Shanahan and J.S. Quick, Dep. of Agronomy, Colorado State Univ., Fort Collins, CO 80523. Contribution of Colorado Agric. Exp. Stn. Received 2 Nov. 1989. *Corresponding author. Published in Crop Sci. 30:1243-1247 (1990).
and Wardlaw (1978) reported significant yield reductions when temperature averages during grain-fill were >15°C. Identification of heat-tolerant genotypes for these areas is desirable. Current efforts, using field evaluations, are frequently inefficient because heat stress conditions during grain-fill are too inconsistent to permit selection. Thus, a laboratory procedure for measuring heat tolerance, using controlled-environment conditions, is preferable. Metabolic activities such as photosynthesis and respiration are more sensitive to heat stress in cool-season species such as wheat than in warm-season plants (Bjorkman et al., 1980). Thermal stability of warmseason species is associated with properties of the photosynthetic system, including key enzymes and the thylakoid membrane, with the thylakoid membrane being more heat-sensitive than the cell membrane (Bjorkman et al., 1980). Other reports (Bhullar and Jenner, 1985, 1986; Rijven, 1986) have shown that high temperature retards conversion of sucrose to starch in developing grains of wheat. Thus, any of a number of important metabolic functions appear sensitive to heat stress in temperate species. However, a cell-membrane system that remains functional during heat stress appears central to adaptation of plants to high temperature (Raison et al., 1980). This is especially important during the grain-fill period of wheat, since grain growth is greatly reduced by environmental conditions that adversely affect assimilate supply (Evans, 1978) or utilization (Bhullar and Jenner, 1985, 1986). Sullivan and Ross (1979) have used a test that measures the amount of electrolyte leakage from leaf discs bathed in deionized water after exposure to a heat-shock treatment. They interpreted the measurement as an indicator of cell membrane thermostability
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(MT) in response to heat stress. They have used this procedure to indentify genetic variation in heat tolerance in grain sorghum [Sorghum bicolor (L.) Moench]. Genetic variation determined by this procedure was related to differences in whole plant photosynthesis under high temperatures as well as field performance of several cultivars grown under high temperature stress. Additionally, Martineau et al. (1979a) observed genetic differences in MTwith soybean [Glycine max (L.) Merr.], and Wallner et al. (1982) noted genotypic variation in MTwith various turfgrass species using this procedure. Although the procedure holds promise for selecting heat-tolerant germplasm, apparent problems are associated with the method. For example, Blum and Ebercon (1981) noted that MTin spring wheat was affected by prior exposure of sampled plants to drought and heat stress. Shanahan et al. (1990) reported a somewhat low association in ranking of spring wheat genotypes for MTusing plants grown under two different field environments. These results may be due to variation in environmental conditions that usually occur in field situations; Chenet al. (1982) reported that genetic differences in MTare highly pendent on the type of hardening treatment or temperature to which plants are exposed prior to sampling. The objectives of this paper were to (i) determine the appropriate hardening conditions for determining genetic differences in MT, using plants grown under controlled environmental conditions and sampled at seedling and anthesis stages of growth, and (ii) determine the association between MTdetermined at the two stages of growth, since it was noted that plant tolerance to heat stress may change with age (Blum and Ebercon, 1981). MATERIALS AND METHODS Germplasm The cultivars Baca, Centurk, Nugaines, TAM 105, Trapper, Vona, and Wichita were used, since they were previously identified as having differences in membrane thermostability (MT)whenevaluated under field conditions (Shanahanet al., 1983). All except Nugaineswere adapted to the central and southern Great Plains of the USA. Seedling Propagation Approximately500 fungicide-treated seeds of each of the seven cultivars were wrapped in separate moistened germination paper and allowed to germinate under dark conditions in a low-temperatureincubator maintainedat 15 °C. Whenthe first vegetative leaf emergedfrom the seedlings (seedlings were grownfor 10-14 d, attaining ~4 cmlength), they were removedto a temperature-controlled water bath for hardening treatments. Plants were subjected to 34 °C for 0, 6, 12, 18, 24, and 48 h to provide seedlings with six levels of exposure to hardening treatment. Hardeningtreatments were not replicated. To subject seedlings to hardening treatments, roots of the seedlings (the seedlings themselvesremainedin germination paper) were immersedin water during the periods specified above. The water bath was covered with transparent plastic to minimizetemperature variation, maintain high humidity levels, and provide room-light conditions for seedling
growth. Nonetheless, seedlings did not develop green color during the hardening process. MembraneThermostability After each hardening treatment, 10 seedlings were randomly selected to comprise a sample and four samples per cultivar were used to provide replication within hardening treatments, using a completely randomexperimental design. Leaves were cut into two segments (2 cmlength); the upper segmentwas used for the heat treatment and the lower segment(excluding root tissue) was used for the control treatment. Plant material was placed in two vials containing 6 mLof deionized water and washed thoroughly with three changes of water to removeelectrolytes adhering to plant tissue, as well as electrolytes released from the cutting of plant tissue. After final rinsing, tubes were drained, maintaining sufficient water to prevent desiccation of plant material during heat treatment. Heat-treated vials were covered with plastic wrapand incubated in a water bath at 50 °C for 1 h, while control vials were maintainedin a water bath at 25 °C during the same period. Treatment temperature and duration were chosen after conducting preliminary experiments involving variation in water-bath temperature to determine treatment conditions producingthe greatest sensitivity in detecting genetic differences, as suggested by Sullivan (1972). The treatment temperatures were 50 °C for the hardeningperiods of 0, 6, 12, 18, and 24 h, and 49 and 50 °C for the 48-b hardening period, providing seven experimental protocols involving hardeningperiod and treatment temperature. After the treatment period, 10 mLof deionized water was added to both control and treated vials and the vials were held at 10 °C for 24 h more to allow diffusion of electrolytes from the material. Vials were brought to 25 °C and shaken, to mix contents; an initial conductanceof vial contents was determinedwith an electrical conductivity meter (Electroanalyzer 4400, MarksonScience, Inc., Del Mar, CA), which was calibrated weekly using a standardized NaC1solution. Vials were placed in an autoclave held at a pressure of 0.10 MPa (for 10 rain) to completelykill plant tissue and release all of the electrolytes. The vials werecooled to 25 °C, contents mixed, and a final conductance measurement made. Membrane thermostability was expressed as relative injury (RI) from the following calculation: RI (%) = 1 -- {[1 -- (T,/T2)]/[1 CI/C2)]} X 10 where T and C refer to conductance values for treatment and control vials, respectively, and the subscripts 1 and 2 refer to initial and final conductancereadings, respectively. Propagationof Plants Seeds of the seven cultivars were allowed to germinate in moistened germination paper for 72 h and then placed in a low-temperature incubator maintained at 2 °C for a period of 8 wkto vernalize the seedlings. Five vernalized seedlings per pot were transplanted into 12 plastic pots per cultivar (22 cmdiam., 18 cmheight) containing 2.8 kg of soil, sand, and peat (2:1:2 v/v). Pots were placed on a greenhouse bench. Twoweeks after transplanting, the numberof seedlings in each pot was reduced to three. The plants were kept in the greenhouse until reaching the anthesis stage of development. The daylength was extended to 16 h using incandescent lights during the greenhouse growth period. Maximumtemperatures ranged between 18 to 27 °C and minimumtemperature ranged from 13 to 16 °C during the greenhouse growth period. Relative humidity ranged from 30 to 50%during this period. The plants were kept well watered throughout the study to avoid water-stress effects on the plants. Plants received two applications (transplant-
SAADALLAET AL.:
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ing and head emergence)of nutrient solution, consisting of 100 and 50 mgof N and P kg-I of dry soil, respectively. At anthesis, plants of all seven cultivars were movedto three growth chambers providing three separate hardening treatments. Twotreatments consisted of plants grownunder a temperature regime of 34/22 °C (16 h day/8 h night) for periods of 48 and 120 h, while the third treatment consisted of plants grownat 22/15 °C (day/night) for a period of 120 h. The latter regime was considered the unhardenedtreatment. The hardening treatments were not replicated, whereas genotypeswere represented with four replicates (four pots per genotype) in each growth chamber. A completely randomexperimental design was utilized within each growth chamber, with pots given a new randomization every 24 h. Relative humidity in the growth chamber was maintained in the range of 50 to 65%.Light was provided with a combination of cool-white fluorescent and incandescentbulbs at a intensity of 350/~molm-2 s-t (photosynthetic photonflux density) at the level of the upperleaves of the plant. After hardeningtreatments, samplingof plant material for the MTtest consisted of collecting a total of 10 flag leaf blades from each pot. Two1-cmdiam. leaf disks were taken per leaf. One disk was used as a control and the other heat treated. These disks were taken midwaybetween base and tip of leaf blade using a cork borer. Leaf disks wereplaced in control and treated vials, each containing 6 mLof deionized water, and rinsed three times. The procedure for determining RI was the same as described above, except that the treatment temperature for the MTtest was 48 °C. MembraneThermostability in Crosses Ninety genotypes derived from the crosses TAM105/ Baca and TAM 105/Vona were used in this portion of the study, as well as the cultivars Baca, TAM 105, and Vona. The cultivars Baca, TAM105, and Vonawere used as parents in the crosses because of formerlyobserveddifferences in MT(Shanahanet al., 1983) amongthese cultivars, with TAM105 being heat-tolerant and Baca and Vona intermediately heat-sensitive. The genotypes were developed by growing the two crosses as bulks during the period from 1982 to 1985 at the Colorado State University Agronomy Research Center near Fort Collins, CO,advancingthe plant material from the F2 to F5 generation. In 1985, single spikes from 650 F5 plants were harvested within each cross, maintaining genetic diversity for different morphologicalcharacteristics such as plant height, spike type, glumecolor, spike size, and numberof spikes. Seed harvested from the spikes was used to plant 650 headrowsper cross in the fall of 1985. In July of 1986, grain was harvested from the headrows. Forty-five genotypesper cross (90 total) of the potential 650 genotypes were randomlyselected. The 90 genotypes plus parents were grownto the seedling and anthesis stages of growth as described above. Seedling and anthesis-stage plants were then subjected to hardening treatments as described abovefor 48 and 120 h, respectively. Plant material for the MTtest was sampled as described above, using four replicates per genotype in a completely randomdesign. The treatment temperatures for the MTtest were 49 and 48 °C for the seedling and anthesis stages, respectively. Statistical Procedures The analysis of variance was used to determine genotypic variation for RI. Duncan’smultiple range test was used for genotypic mean comparisons. Linear correlation analysis was used to determine the relationship between RI evaluated at the seedling and anthesis stages of growth.Frequency distributions (in intervals of 5%), progenymeans, and parental meansfor RI values determined at seedling and anthesis wereplotted for both crosses.
RESULTS AND DISCUSSION Table 1 shows RI values determined by the MTtest for seven winter wheat cultivars exposed to six different hardening periods at the seedling stage. While no statistical significance can be associated with hardening effects on RI, since hardening treatments were not replicated, it is worth noting the trends in RI across hardening levels. In general, RI declined with increased hardening duration. For example, the lowest average RI, using 50 °C for the MTtest, occurred with 48 h hardening (77°/6). Using the same hardening period and lowering the treatment temperature for the MTtest from 50 to 49 °C reduced the average RI value from 77 to 57%. The treatment protocol (48 h hardening and 49 °C for MTtest) that produced an average RI value of 57%would be the most appropriate for assaying seedlings, since this level of RI provides the greatest sensitivity in detecting genetic differences (Sullivan, 1972; Martineau et al., 1979a). This can be further illustrated by noting the range of RI values among the cultivars with increasing exposure to hardening. For example, there was no difference among cultivars in RI with zero exposure to hardening. However, when exposed to 48 h hardening and subjected to the MT test at 49 °C, the cultivars produced muchgreater differences in RI. TAM105, Vona, and Wichita produced lower RI values and Nugaines the highest RI. The other cultivars produced values between these two extremes. Chen et al. (1982) also noted that genetic differences in heat tolerance amongvarious crop species are expressed only when tested plants have been exposed to a hardening treatment. The effect of hardening on the expression of RI at anthesis was similar to that observed at the seedling stage (Table 2). Average RI was 88, 76, and 54%with 0, 48, and 120 h hardening, respectively. As was observed for seedlings, the greatest differences in RI among cultivars tested at anthesis occurred with increasing duration of hardening (120 h). Thus, the latter treatment protocol, involving 120 h hardening and 48 °C for the MTtest, apparently would be the most suitable for detecting genetic differences in RI at anthesis. The ranking of the cultivars for RI at anthesis was slightly different than the ranking at the seedling Table 1. Relative injury as determined by the membranethermostability (MT)test on seedlings of seven winter wheat cultivars exposed to six hardening periods. Hardeningperiod, h Cultivar
0
6
12
18
24
48
48~"
78b 79b 87c 68a 80b 77b 70a 77
57b 71cd 80d 37a 64bc 49a 43a 57
relative i~ury,% Baca Centurk Nugaines Tam105 Trapper Vona Wichita Mean
92a~ 90a 91a 87a 88a 91a 89a 90
86ab 87abc 94c 81a 91bc 81a 86ab 87
87abc 88b 86abc 87b 92bc 89b 85ab 81a 94c 88b 81a 87b 85ab 85ab 87 86
82bc 88cd 92d 72a 85bc 83bc 82bc 83
50 °C wasused for MTtest on all hardeningperiodsexceptfor the last 48 h period, where49 °C wasused. Withincolumns,cultivar meansfollowedby the sameletter are not significantly (P --< 0.05) differentaccordingto Duncan’s multiplerangetest.
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Table 2. Relative injury as determined by the membranethermostability (MT)test on plants at the anthesis stage for seven winter wheat cultivars exposed to three hardening periods. The MTtest was conducted using a temperature of 48 °C. Hardeningperiod, h Cultivar
0
48
120
relative injury, % Baca Centurk Nugaines Tam105 Trapper Vona Wichita Mean
92M" 88a 85a 86a 88a 84a 91a 88
7Tab 73ab 82b 69a 78ab 75ab 77ab
59b 65b 69c 31a 56b 51 b 50b 54
76
Withincolumns,cultivar meansfollowedby the sameletter are not significantly (P ___0.05) differentaccordingto Duncan’s multiplerangetest.
stage, with TAM105 producing the lowest and Nugaines the highest RI. Nonetheless, the correlation coefficient for RI betweenseedlings (hardened for 48 h and MTat 49 °C) and anthesis-stage plants (hardened for 120 h and MTat 48 °C) was 0.91 (P _< 0.01, n = 7), indicating that ranking amongcultivars for RI between growth stages was fairly consistent. The ranking of cultivars for RI in this study is similar to the ranking of the same cultivars for RI by Shanahan et al. (1983). Significant differences (P
