Screening Protocols for Heat Tolerance in Rice at the Seedling and Reproductive Stages

The methods include a protocol for screening heat tolerance of rice at the seedling stage; young seedlings were exposed to heat stress and the test takes 4–5 weeks and involves the visual scoring of symptoms which allows hundreds of seedlings to be evaluated in a short time. The visual screening method was extensively validated through laboratory, glasshouse, and field-based experiments. We also adapted a protocol for screening heat tolerant mutant lines at the flowering (reproductive) stage that has been specifically adjusted for a mutation breeding program. Here, plants were treated from the first day of anthesis at different temperatures and spikelet fertility at maturity was determined as a parameter to assess the heat tolerance of the selected genotypes.

. Some cultivars that are not so sensitive to higher temperatures, e.g. cv. N22, can be used in breeding programmes as controls in screening tests. Besides spikelet fertility occurring during the flowering stage, a phenotypic marker for male gametogenesis has been reported as a screening tool for high temperature stress in rice (Jagadish et al. 2014). Authors identified a distance of 8-9 cm between the collar of the last fully opened leaf and the flag leaf collar as the environmentally stable phenotypic marker to assess heat stress (38 C) sensitivity at the micro sporogenesis stage leading to high spikelet sterility in rice. Wei-hun et al. (2012) applied heat stress at 15 day-old seedling stage 43 C/30 C (day/night and 14 h/10 h), 75% relative humidity for 7 days and observed that plant height, root length, shoot and root biomass were dramatically reduced, with heat tolerant genotypes being relatively less affected than sensitive ones. In this sense, we have included seedlings as a target for heat tolerance screening, to enable selection at the seedling stage by plant breeders. This method, as described in Sect. 2.2, is based on growing rice seedlings in glasshouse conditions using hydroponics or pots and exposing them to heat stress in a growth chamber. The seedling test is simple and rapid (4-6 days) and allows the simultaneous screening of several hundred seedlings at once depending on the size of available infrastructure. A list of equipment required for hydroponics hardware and stock solutions is given. Heat stress treatment was applied after 15 days of seedling transplantation to hydroponics or pots. Germplasm classifications for sensitive, intermediate and tolerant are also provided. Visual symptoms of heat stress include: reduced leaf area, yellowing of leaves, leaf tip burning, and entire leaf burning and leaf death. Assessment of seedlings was done according to a heat tolerance index, which is calculated for seedling biomass data using shoot, root, whole seedlings weight (fresh and dry), root, shoot length and seedling height. The test can be adapted to screen M 2 individuals as well as M 3 families from mutant populations (and other large breeding populations). A stress tolerance index (STI, Fernandez 1992) was used to identify/validate genotypes producing high yield under both stress and non-stress conditions; Blum (1988) stated that selection based on STI can result in genotypes with higher stress tolerance and yield potential to be selected. Tenorio et al. (2013) and Rang et al. (2011) developed a phenotyping protocol for use in plant breeding in which a temperature treatment of 38 C is used at the flowering stage. Our second protocol presented here is to screen for heat tolerance in rice at the flowering stage modified from Tenorio et al. (2013) and Rang et al. (2011). Flowering is most heat sensitive stage that can result in high sterility and yield loss in rice. As explained in Sect. 2.3, glasshouse grown plants are moved to a controlled environmental from the first day of anthesis for heat stress treatments, at 35.0-39.0 C/28 C for 6-4/18-20 h with relative humidity 80% for 6-4 days. After heat treatment plants may be rescued and returned to glasshouse under optimum temperature conditions to produce seed and for further evaluation. Spikelet fertility (seed set) is measured as an important predictive parameter for yield (Prasad et al. 2006;Rang et al. 2011). In our protocol also we used spikelet fertility at maturity to assess the heat tolerance of the tested genotypes.

Screening for Heat Tolerance Using Hydroponics
The equipment for hydroponics, the preparation of nutrient stock solutions and setting up of the hydroponics system are given in Bado et al. (2016).

Seedling Establishment in Hydroponics
The seedling establishment was conducted in a glasshouse set up for rice: 28/20 C and 70% humidity in 16 h photoperiod. Test tanks were filled with distilled water until the water level is about 1 mm above the mesh of the platform. Dry seed were then placed into the wet compartments of the seed support platform. For M 2 screening 20-40 seeds from one panicle is placed into one compartment (6 Â 7 cm) ( Fig. 2.1a). For M 3 and advanced lines/cultivars 3-5 seeds were placed into each 2 cm diameter compartment. Individual lines may be replicated within and among tanks (Fig. 2.1b). The test platforms were then covered with a lid for 5-6 days to promote germination in the dark. On day 3, the seed should show signs of germination (emergence of root and shoot) and the water should be replaced with half-strength Yoshida solution as vigorous seedlings require nutrients. Germinated seeds were allowed to grow for 6 days. The seedlings for subsequent heat stress treatment should be healthy, therefore odd-looking or diseased seedlings should be removed at this stage. If seed samples are not clean and rotting occurs during germination, these must also be removed. If symptoms are widespread, a new batch of seedlings and fresh hydroponics materials should be prepared.

Care Required During Plant Growth in Hydroponics
Due to evaporation from tanks and transpiration from plants there will be a gradual decrease in solution volumes over time. Every 2 days the volume needs to be brought back to the level of full capacity (touching the netting in the platform compartments) and the pH adjusted to 5. If algal contamination is found, the nutrient solution should be completely replaced. Solutions can be changed by lifting off the platforms and placing them temporarily over empty tanks and pouring the hydroponics solutions back into a drum where the bulked solution can be pH adjusted for the whole experiment in one step. Once adjusted the solution is re-distributed into the test tanks and the seedling platforms returned. These operations also act to aerate the hydroponics solution. Alternatively, the pH can be adjusted on an individual tank basis and more working solution may be added to make up the volume in each tank.

Screening for Heat Tolerance in Soil Using Pots
No special equipment is needed for screening with soil in pots and/or trays. Plastic pots, trays or tanks are filled with a clay soil which has a good water retention capacity and organic matter content. Pre-germinated, 6 day-old young healthy seedlings are transferred to the plastic pots, trays or tanks as given in Fig. 2.2a-c. Also as shown in Fig. 2.4d, in case germination problems, an excess (110-120) of seeds of each line are sown directly into the tray to have 100 seedlings in a tray 1 week after sowing. Germinated 6 day-old seedlings were transferred to pots or trays to grown in a glasshouse set up for rice: 28/20 C day/night temperatures with 70% humidity and 16 h period. The glasshouse should be disease free and well-lit by natural or artificial lighting. Standard rice management practices are applied to pots with respect to soil, fertilizer and watering until the application of heat stress.

Heat Treatment and Recovery of Seedlings
Fifteen days after transplantation, seedlings were transferred to the growth chamber to grow under tightly controlled temperature, light and relative humidity conditions. Growth chambers were adjusted to 45 C/28 C (day/night) 6/18 h with 80% relative humidity. The temperature treatment was imposed for 4-6 days; depending on seedling tolerance to heat stress application (some of them already died after 4 days). For all genotypes including control cultivars, 50/100 seedlings were exposed to heat stress and same amount of seedlings are grown in glasshouse conditions without stress as controls (

Assessment of Heat Tolerance at the Seedling Stage
Heat application to rice seedlings is illustrated in Fig. 2.5; visual symptoms of heat stress are: leaf yellowing, reduced leaf area, leaf tip burning, and entire leaf burning and leaf death. The biomass of seedlings is recorded as given   After heat stress treatment, mutants were relocated to normal conditions with control plants. Two days after treatment data were recorded for shoot and root lengths, shoot and root fresh weights, shoot and root dry weights for stressed seedlings along with their respective controls grown under normal temperature.
A heat screening scale based on the response to stress was developed using parameters showing significant correlations with each other and with relative heat tolerance. Parameters were tested under control as well as heat stress and stress index was calculated for each parameter. The HTI is calculated by dividing the sum of individual scores for each parameter by the total score and multiplied by 100%. The genotypes are graded from tolerant to sensitive.
Stress tolerance indices (STIs) for the recorded traits are calculated as follows (Fernandez 1992; Blum 1988): The genotypes were graded as tolerant, moderately tolerant, moderately sensitive and sensitive (Ashraf et al. 1999). Tables 2.2, 2.3, 2.4 and 2.5 summarize the data from one representative screening carried out on rice at the seedling stage in hydroponics and in pots and trays.

Recovery Stage After Heat Treatment
Heat tolerant lines that survive and recover after the heat stress at 45 C may also be transplanted in hydroponics or soils. Selected seedlings can be grown on to maturity in hydroponic tanks filled with Yoshida solution changed every 2 weeks. Selected tolerant seedlings were gently teased out of the test tanks with care taken to keep roots intact. The base of the aerial part of each selected seedling is then wrapped in a sponge strip (10 Â 2 Â 1 cm) and the seedlings inserted into a recovery tank (Fig. 2.6a-c).
Heat tolerant lines may also be transplanted into 5 l pots containing soil. The seedlings were maintained individually and single plants and allowed to form tillers and panicles. Preliminary data comparisons may be recorded on better lines with respect to: plant height, number of tillers, days to flowering, number of panicles, panicle length, sterility and fertility of panicle as well as yield data such as seed number, weight, and thousand grain weights for each individual plant per pot. Seeds from single plants are kept separately for multiplication in the next generation ( Fig. 2.6d).

Germination and Seedling Establishment in Hydroponics and Pots
Uniform healthy seedlings (8-10 days old) are selected and grown individually in hydroponics (Fig. 2.7). The glasshouse set up for rice: 28/20 C day/night temperatures with 70% humidity and 16 h photoperiod. The hydroponics and pot culture set ups were the same as that described in Sects. 2.2 and 2.3. The glasshouse should be diseasefree and well-lit by natural and/or artificial lighting. 15-cm pots are recommended for single plants as these give enough seed yield and panicle number for line evaluation. Every 2 weeks, the hydroponics solution needs to be changed. Hydroponics is also aerated by agitation twice in a week. Pots and boxes with soil culture were irrigated twice a week to maintain soil saturation. N, P, K fertiliser applications were done prior to transplanting and panicle emergence, which may be variable depending on the genotype. Seedling establishment of control (standard) plant materials and test plant materials, care of seedlings in hydroponic, pots and boxes are same as described in Sects. 2.2 and 2.3.

Heat Treatment at the Flowering Stage
Two treatments were applied to test genotypes (1) plants with no heat stress exposure (control) and (2) plants subjected to heat stress at flowering stage. Breeding lines and cultivars are used, with standards N22, IR64 as a heat tolerant/moderately tolerant standards; IR 52 and/or WAB 56 100 were used as heat sensitive standards for comparative purposes. Plants were transferred to controlled environmental chambers/growth room conditions at the beginning of anthesis (first day) for heat stress treatments. Various heat stress treatments were investigated, all beginning at anthesis for various durations (2-6 h to 3-6 days) and at various temperatures ranging from 35 to 39 C. Here we describe the most effective treatment, at 39 C for 4 days, 6 h per day and 35 C, 6 days, 6 h per day and 38 C, 5 days and 4 h per day). First Treatment A set of 12 mutant lines (one plant grown per pot) were taken for testing high temperature tolerance. The main tiller of each plant was tagged before exposing plants to high temperature at flowering. On the appearance of the first flower on the main tiller plants were transferred to growth chambers at 07:00 h before the anthesis process starts. Environmental control chambers are set at 39 C with 80% relative humidity during the period of 6 hours 08:00-14:00 covering the anthesis period for each day. During anthesis, opened spikelets from four replicates (five spikelets from each plant) are marked with a permanent marker pen and moved to growth chamber for heat application.
Second Treatment Each genotype replicate consisted of ten pots with one to two plants per pot. Alternatively, 10 plants per genotype were grown up in soil filled boxes. Screens consisted of three replicates per genotype. Half (5) of the 10 pots or boxes were maintained in glasshouse conditions and served as controls. The other 5 were placed in a growth chamber and exposed to high temperatures at anthesis as described below. Illustration of heat stress treatments on rice at the flowering stage is given Fig. 2.8. Heat stress treatment; At the beginning of anthesis, five pots per replicate of each genotype are transferred to growth chambers conditions in the morning 07:00 am with 80% humidity exposed to 35 C for 6 days 6 h, and 38 C 5 days 4 h respectively on consecutive days. The main tiller of each pot is tagged before exposing plants to high temperature at anthesis. At the first day of anthesis tagged plants were transferred to growth chambers set at 35-38 C with 80% relative humidity as of from 7:00 AM every day. After each daily heat stress application, plants were returned to normal glasshouse conditions (30/20 C day/night temperatures with 80% humidity). Plant phenotyping and heat stress treatment in growth chamber at flowering stage are shown in Fig. 2.9.

Screening for Heat Tolerance at the Flowering Stage
Spikelet fertility at maturity was used to screen heat tolerance of tested genotypes. Plant height and tiller number per plant were recorded. Mature panicles were harvested from five remaining pots for grain yield per plant recording, grain quality traits and calculating spikelet fertility (by counting empty and grain-filled spikelets). Main tillers were tagged and after harvest panicles were manually threshed, numbers of filled and unfilled grains per panicle were recorded. Each floret can be pressed between forefinger and thumb to determine if the grain is filled or not. Both partially and fully filled spikelets were categorized as filled spikelets.
Spikelet fertility is calculated as below; Spikelet fertility ¼ Number of filled grains Total number of reproductive sites floret ð Þ Â 100 Spikelet fertility was calculated in control and high temperature stress conditions. The negative effect (% decrease from control) was determined per genotype.
Reduced fertility of more than 65% is classed as highly susceptible, between 25-65% classed as intermediate (moderately susceptible) and less than 25% is classified as heat tolerant. This quantification has been adapted from species, ecotype and cultivar differences in spikelet fertility and harvest index of rice in response to high temperature stress (Prasad et al. 2006;Rang et al. 2011) (Tables 2.6, 2.7 and 2.8).  Open Access This chapter is licensed under the terms of the Creative Commons Attribution 3.0 IGO license (https://creativecommons.org/licenses/by/3.0/igo/), which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the International Atomic Energy Agency (IAEA), provide a link to the Creative Commons license and indicate if changes were made. Any dispute related to the use of the works of the IAEA that cannot be settled amicably shall be submitted to arbitration pursuant to the UNCITRAL rules. The use of the IAEA's name for any purpose other than for attribution, and the use of the IAEA's logo, shall be subject to a separate written license agreement between the IAEA and the user and is not authorized as part of this CC-IGO license. Note that the link provided above includes additional terms and conditions of the license.
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