Validation of Screening Protocols for Heat Tolerance in Rice
Selected heat tolerant mutant rice genotypes were tested for physiological and biochemical indicators associated with the pre-field screen protocols. These tests included measuring physiological and biochemical indicators associated with plant stress responses, such as electrolyte leakage, malondialdehyde level, total protein content, antioxidant enzyme activity at seedling, vegetative stages and flowering stages to understand the mechanism of the heat tolerance characteristics/traits of the selected germplasm and explore the potential of pyramiding different mutations for durable heat tolerance. The candidate heat tolerant mutant lines were also tested in hot spot areas in the different countries.
3.1 Background Analysis
An alternative to screening for rice responses to heat at seedling and reproductive discussed in Chap. 2. This chapter covers further validation of identified lines through: (1) physiological and biochemical indicators that are reported to be associated with plant stress responses to classify the mechanism of heat tolerance of screened lines/varieties; (2) Field trials under real stress conditions provide conclusive data for screening and selection for advancing promising lines to confirm the methods developed in Chap. 2. Yield data were used to assess tested rice genotypes and comparisons made to heat tolerant/susceptible standards. Tested lines were classified as sensitive, intermediate and tolerant.
Biochemical changes are another aspect of plant acclimation to heat stress and heat stress may also affect leaf chlorophyll content and membrane stability through lipid peroxidation, leading to the production of peroxide ions and MDA (Wahid et al. 2007). Increasing the temperature changes of 37 °C/30 °C (day/night) increase the electrolyte leakage percentage and MDA content in rice (Zhang et al. 2009; Liu et al. 2013).
Identified lines/varieties were subjected to physiological and biochemical tests, such as electrolyte leakage, MDA level, total protein content, antioxidant enzymes (catalase, ascorbate peroxidase and superoxide dismutase) activity at seedling, vegetative stages and flowering stages. Results of the physiological and biochemical measurements confirmed heat tolerance behaviour of selected germplasm. This work was carried out at the Stress Physiology and Molecular Biology Laboratory of School of Life Sciences, Jawaharlal Nehru University New Delhi, India.
After screening at the seedling stage and/or flowering stages, the identified heat tolerant genotypes were tested in field conditions to evaluate real performance of selected promising lines in hot spot areas at various locations (in Cuba, Pakistan and UR Tanzania), known to experience heat stress. Field trials usually involve replicated plots that take up a lot of space and often in more than one location. Therefore, breeders need to be selective in what is to be tested. The pre-field screening protocols described in Chap. 2 provide potential candidates that may be advanced for field evaluations thus saving time, space and costs on lines with no potential.
3.2 Validation of Physiological and Biochemical Indicators
3.2.1 Physiological and Biochemical Characterization of Heat Tolerant Lines at the Seedling Stage
3.2.2 Physiological and Biochemical Characterization of Heat Tolerant Lines at the Flowering Stage
Anthesis in rice is very sensitive to heat stress (Jagadish et al. 2007). Putative heat tolerant mutants performed better than their wild type parent under heat stress at the flowering stage. Phenotypic data were recorded in control optimum temperature conditions, immediately after heat stress treatment (40 °C for 4 h) for three subsequent days (to span main panicle flowering) and after recovery (72 h 28 °C). Besides healthier appearance and better growth, the putative heat tolerant mutants recorded higher spikelet fertility and yield components such as panicle length, number of tillers, and number of spikelets per panicle grain yield and plant height per plot as compared to wild type. The mutant lines had a higher vegetative and reproductive growth under both normal and heat stress conditions, suggesting that the genetic controls were constitutive.
22.214.171.124 Electrolyte Leakage Measurement
Analysis of electrolyte leakage is carried out following the protocol of Bajji et al. (2002). Leaf samples from control and heat-treated plants are harvested and washed with distilled water to remove any surface adhering ions. About 100 mg of the tissue are dipped immediately into 20 ml of de-ionized water. After incubating the leaf tissues at 37 °C for 2 h, the electrical conductivity (E1) of the immersion solution is measured using a conductivity meter (ELEINS, Inc., India). To determine the total conductivity (E2), the samples with immersion solution (effusate) are autoclaved for 15 min at 121 °C and the conductivity of the effusate is measured after cooling it to room temperature. Relative electrical conductivity is measured by the formula: percentage of electrolyte leakage = E1/E2 × 100.
126.96.36.199 Lipid Peroxidation Assay
Lipid peroxidation is estimated measuring the formation of MDA. MDA content is quantified by thiobar-bituric acid reactive substances assay (Heath and Packer 1986; Larkindale and Knight 2002). For this, about 100 mg of leaf tissue from the control and heat-treated plants is homogenized in 5 mL of 5% (w/v) trichloroaceticacid and the homogenate centrifuged at 12,000×g for 10 min at room temperature. The supernatant is mixed with an equal volume of thiobarbituric acid [0.5% in 20% (w/v) trichloroaceticacid] and the mixture is then boiled for 25 min at 100 °C, followed by centrifugation for 5 min at 7500 g to clarify the solution. Absorbance of the supernatant is measured at 532 nm. MDA equivalents are calculated by the extinction coefficient of 155 mM−1 cm−1.
188.8.131.52 Measurement of Total Proteins
Total soluble proteins are extracted from the rice leaves (100 mg) of heat treated plants at the seedling stage using Zivy’s buffer (Zivy et al. 1983). Amount of soluble proteins is estimated by Bradford’s assay (Bradford 1976) with the help of the standard curve prepared using various known concentrations of other protein, such as Bovine Serum Albumin (BSA fraction V), prepared by using different concentration of Bovine Serum Albumin (Ernst and Zor 2010).
184.108.40.206 Antioxidant Enzyme Activity Assay
About 100 mg leaf material from the heat treated plants was homogenized in ice-cold 50 mM K-PO4 buffer (pH 7.5) containing 2 mM EDTA and 0.1 mM phenylmethylsulphonylfloride (PMSF). The homogenizing buffer for APX additionally contained 2 mM of Na-ascorbate. The homogenate was centrifuged at 12,000×g for 10 min at 4 °C and the supernatant was used for enzyme assay. Total protein content in supernatant was determined following the method of Bradford (1976).The activity of SOD was measured using the method of Giannopolitis and Reis (1977). The assay mixture (1 mL) for SOD contained 79.2 mM Tris–HCI buffer (pH 8.9), containing 0.12 mM EDTA, 10.8 mM tetra ethylene diamine, bovineserum albumin (3%), 6 mM nitroblue tetrazolium (NBT), 60 μM riboflavin in 5 mM KOH and 5 μg of enzyme extract. The increase in absorbance due to formazan formation was read at 560 nm. The increase in absorbance in the absence of enzyme was taken as 100%, and 50% initial was taken as equivalent to 1 unit of SOD activity. APX activity was determined according to Nakano and Asada (1981). The reaction mixture in a total volume of 1 ml consisted of 50 mM (pH 7.5) K-PO4buffer, 0.1 mM EDTA, 0.25 mM ascorbate, 10 mM H2O2 and enzyme extract. H2O2-dependent oxidation of ascorbate was followed spectrophotometrically by recording the decrease in absorbance at 290 nm (D = 2.8 mM−1cm−1). Slope values of absorbance in 290 nm are considered for rate calculation. Catalase (CAT) activity was determined following the method of Aebi (1983) by measuring the decrease in absorbance at 240 nm due to the decomposition of H2O2 (D = 40 M−1cm−1). The slope value of the rapid decline in 240 nm absorption was considered for rate calculation. The reaction mixture in 1 ml contained 50 mM K-PO4 buffer (pH 7.0) with leaf extract equivalent to 4–5 μg protein. The reaction was initiated by adding H2O2 to a final concentration of 20 mM. All the experiments involved in physiological and biochemical analyses were repeated three times using three independent mutant plants from each line.
3.3 Validation Protocols of Rice Heat Tolerance Under Field Conditions
After screening at the seedling stage and flowering stages (as described in Chap. 2) identified heat tolerant genotypes were tested in field conditions to validate the pre-field screening protocols in hotspot test locations.
Time of day for flowering (anthesis)
Number of tillers per plant
Spikelet number of the main tiller panicle
Spikelet fertility (ratio of filled/unfilled grains)
Thousand kernel weight
Classification of heat tolerance of rice genotypes in field conditions at NIAB, Pakistan compared to medium heat tolerant standard cv. IR64
More susceptible than control cv. IR64
Equivalent to control cv. IR64
More tolerant than control cv. IR64
HS cv. ‘Super Basmati’, tested 30 mutant lines
IR 64, HT-18, HT-29, HT-31, HT-39, HT-51, HT-53, HT-98, HT-104, HT-114, HT-119, HT-132 and HT-138
HT-74, HT-81, HT-92 and HT-97
Classification of heat tolerance rice genotypes in field conditions at INCA, Cuba compared to the control parent and local cultivars
More susceptible than control parent and local cultivars
Equivalent to control parent and local cultivars
Tolerant/better than control parent and local cultivars
LP-16, A 82
8551, 8554, 8555, LP-10, LP 9, LP-7, LP 8,
Classification of heat tolerance of rice genotypes compare to parental (wild type) genotypes and HT sensitive cv. WAB 56-104 under field conditions from Sokoine University, URT Tanzania
More susceptible than control WAB 56- and parent
Equivalent to control cv WAB 56-104
More tolerant than control cv WAB 56-104
KR, WAB56-50, WAB56-104, WAB56-50-104-36-1, KR 38-1 WAB56-50-127-3, WAB56-50-98-1, KR 27-1
KR-27, WAB56-50-51, WAB56-50-97, WAB56-50-123, WAB56-50-127, WAB56-50-135, KR-37, WAB56-104-9
WAB56-50-85-3, WAB56-50-82-1, WAB56-50-74-1, WAB56-50-56-1, WAB56-104-36-1, WAB56-104-141-1, WAB56-104-141-3, WAB56-50-127-3, WAB6-104-36-1, WAB56-104-141-2, WAB56-50-97-3
Screened lines were evaluated for various agro-morphological characters in field tests and compared to standards including local cultivars and parental lines at different locations and years. Temperature and rainfall of the locations were recorded and compared with long term temperature means. Comparisons were made for yield performance as well as yield components such as spikelet number, thousand kernel weight and filled/unfilled grain ratio under natural high temperature conditions. The results of field screening confirmed the results of the laboratory (physiological and biochemical) testing and also pre-field screening results at both the seedling and flowering stage.
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