Screening for Spikelet Fertility and Validation of Heat Tolerance in a Large Rice Mutant Population

Sulaiman Cheabu, Nat Panichawong, Prisana Rattanametta, Boonthong Wasuri, Poonpipope Kasemsap,Siwaret Arikit,, Apichart Vanavichit,, Chanate Malumpong

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Screening for Spikelet Fertility and Validation of Heat Tolerance in a Large Rice Mutant Population

Sulaiman Cheabu1, Nat Panichawong1, Prisana Rattanametta1, Boonthong Wasuri2, Poonpipope Kasemsap3,Siwaret Arikit1,4, Apichart Vanavichit1,4, Chanate Malumpong1

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A total of 10000 M4individuals in Jao Hom Nil (JHN) mutant population was treated with high temperature (40 oC to 45 oC) during the day time (6 h) from the booting to the harvesting stages, and ambient temperature (33 oC to 35oC) was used as the control. The results of screening and yield trials found that the mutant line M9962 had a high spikelet fertility of 78% under heat stress. In addition, the other mutant lines, including M3181 and M7988, had a spikelet fertility of approximately 70%. However, the JHN wild type, Sin Lek, RD15 and RD33 had very low spikelet fertility of 34%, 14%, 9% and 4%, respectively. The lower spikelet fertility at an elevated temperature resulted in a dramatic decrease of filled grain and contributed to a loss in 100-grain weight. M9962 is a potential genetic stock for use in a heat tolerance breeding programme. In addition, spikelet fertility at high temperature was representative of heat tolerance and can be used as a screening trait for heat tolerance during the reproductive phase on a large scale.

high temperature; rice; spikelet fertility; heat tolerance; mutant

In the past three decades, the earth’s surface temperature has become higher than it was during any preceding decade since 1850, and the worst-case scenario is that the global mean surface temperature may rise by 4.8oCcompared to the 1986–2005 period by the end of this century (IPCC, 2014). Yield reduction (7%–8%)in ricehas been shownfor each 1oCincrease in the daytime temperature between 28oCto 34oC(Baker et al, 1992). Almost 90% of the world’s rice is grown and consumed in Asia, where 50% of the population depends on rice for food. However, during the sensitive flowering and early grain-filling stages, rice is currently exposed to temperatures higher than the critical threshold of 33oCin South Asia and Southeast Asia (Wassmann et al, 2009).

Heat stress at the flowering and grain-filling stages seriously affectsspikelet fertility and grain quality in rice (Matsui et al, 2001).Previous growth chamber experiments showed that temperatures above 36oCcause high spikelet sterility(Shah et al, 2011).Heat-induced (36oC) spikelet sterility at flowering is associated with a reduction in grain yield(Prasad et al, 2006).Studies of heat-induced(38 oCto 41oC) spikelet sterility showed that the flowering stage is the most susceptible to high temperatures, followed by the booting stage (Satake and Yoshida, 1978). High temperatures above 35oCat the flowering stage cause anther dehiscence to fail and consequently less pollen shed on the stigma, resulting in incomplete fertilization (Jagadish et al, 2010).Even if a sufficient number of pollen grains are shed on the stigma, in some cases, pollen germination and pollen tube growth are poor under heat stress (Satake and Yoshida, 1978).Thus, aberrant anther dehiscence is considered a primary cause of perturbed pollen development after shedding and a secondary cause of heat-induced spikelet sterility at flowering.Exposing a flowering spikelet to heat stress (36oC) for 1h is sufficient to induce sterility (Jagadish et al, 2007), whereas heat stress (38oC) for 1h after flowering does not lead to spikelet sterility (Ishimaru et al, 2010), possibly because fertilization has been completed.

In growth chamber experiments, heat tolerance at flowering is often tested at 37.5 oC to 38.0oCand a relative humidity of 60%–70%, and large differences in spikelet fertility between susceptible and tolerant genotypes have been noted (Matsui and Omasa, 2002; Kobayashi et al, 2011).N22, an Indian-type landrace, is identified as one of the most heat-tolerant genotypes in both growth chamber and open field experiments (Prasad et al, 2006; Jagadish et al, 2010; Ye et al, 2012; Poli et al, 2013; Manigbas et al, 2014).Of thecultivars, Akita-Komachi and Nipponbare are classified as considerably heat-tolerant genotypes(39oC) (Maruyama et al, 2013).Of thecultivars,Ciherang, ADT36, BG90-2, Dular and Todorokiwase are known as heat-tolerant genotypes at the booting stage(38oC), while Milyang23 and IR2006-P12-12-2-2 are tolerant at the flowering stage(Prasad et al, 2006; Shi et al, 2015).Giza178, an Egyptian cultivar developed from across, has considerable heat tolerance (38oC) at the booting stage as well as at the flowering stage (Tenorio et al, 2013). These cultivars can be used as control cultivars in heat tolerance tests (38 oC) (Shi et al, 2015).

The production of stress-resistant mutants has been successful in some crop plants including rice(Lee et al, 2003; Ahloowalia et al, 2004).Induced mutations andtechniques have been employed to induce salt tolerance in Basmati rice (Saleem et al, 2005). Previous research has shown thatfast neutron radiation can generate extremely Fe-tolerant mutants (Ruengphayak et al, 2015). Additionally, themutantlinehas been shown to be more tolerant to heat stress (44oC) than the wild-type N22, as evidenced to be a lower yield reduction (Poli et al, 2013). Most previous research is concerned with heat screening of native cultivars and the maximum temperature used most of the time is 38oCfor a short period during the reproductive stage, but the future global mean surface temperature may increase to a value higher than that. The aim of this study was to identify new genetic donors for heat tolerance from 10000 Jao Hom Nin (JHN) mutant lines (M4) at an extreme temperature (40 oC to 45oC) covering the entire reproductive stage from booting to harvesting.

Materials and methods

Rice materials and growth conditions

The experiments were conducted from 2012 to 2013 at the Rice Science Centre at Kasetsart University, Nakhon Patom (14o01′16.08″N, 99o58′53.63″ E), Thailand. A total of 10000 lines of JHN mutants (M4) were screened for heat tolerance. The population arose from 100000 breeder seeds of JHN, and mutations were induced by using 33 Gy fast neutrons. Successive generations from M1to M4and the family history was traceable from individual M1plants. Due to abnormal mutations affecting the seed set, several families were terminated, leaving only 21024 mutant families at M4, which formed the base population for genetic screening (Ruengphayak et al, 2015)(Supplemental Fig. 1).

The 10000 M4mutant lines were seeded in a field nursery. After 30 d, the rice seedlings were transferred into plastic pots at 1 plant/pot (30 cm in height and 25 cm in diameter with 8 kg sieved sandy loam soil). Soil containing 5.57% organic matter, total N of 0.33%, available P of 111.6 mg/kg, exchangeable K of 558.0 mg/kg, exchangeable Ca of 1882.3 mg/kg and exchangeable Mg of 118.0 mg/kg was used for the preliminary screening, whereas, for the repeat screening and validation screening, soil containing 5.50% organic matter, total N of 0.32%, available P of 110.7 mg/kg, exchangeable K of 580.0 mg/kg, exchangeable Ca of 1731.5 mg/kg, exchangeable Mg of 109.0 mg/kg and a pH of7.22was dispensed into each pot before transplanting. The other 0.5 g and 0.6 g of urea were applied to each pot at the mid-tillering (45 d) and panicle initiation (65 d) stages, respectively. Other management activities were followed a conventional high-yielding cultivation approach.

Climatic data in the experiment

Temperature, relative humidityand light intensity conditions are shown in Table 1. The capacity of greenhouse was 320 m2(40 m × 8 m). The air temperature, relative humidity and light intensity were recorded every 5 min using data loggers (WatchDog 1000 Series Micro Stations) atthree position (every 10 m in the greenhouse), and the carbon dioxide concentrationin the greenhouse, which was approximately 390 μmol-1, was also monitored with a HUATO 653 Series detector.

Table 1.Temperature (T), relative humidity (RH) and light intensity (LI) for the screening experiments.

High temperature treatment during panicle development of JHN

To study the sensitive stage of high temperature, the JHN wild type was subjected to 40oC–45oCfor 6 h during the daytime (10:00–16:00) in the greenhouse on April in 2012from the panicle initiation stageto booting stage(T1), the panicle initiation stageto harvesting (T2), the booting stageto harvesting (T3)and the flowering stage to harvesting(T4), respectively,and the natural treatments (control) were planted in the pots under field conditions. Five pots were used for each treatment, and main culm was labelled with the heading date. All spikelets from each panicle were subsequently examined for spikelet fertility. The experiment was set up in a completely randomized design with three replications (five pots/replication).

Screening and identifying of heat tolerance from large mutant libraries

The experiment was conducted with 10000 JHN mutant (M4) lines from May to December in 2012. The 10000 seeds from the JHN mutant population were planted in pots (three pots per line) and maintained under natural climate conditions from the seedling until the booting stage (with an auricle distance from -1 to +1 cm). The greenhouse had sufficient capacity to screen 2000 lines, so the screening for heat tolerance in this project had to be performed for five times (total 10000 lines) as preliminary screening (Supplemental Fig. 1). However, the natural treatments (control) were planted in the pots under field conditions. Spikelet fertility was investigated at the maturity stage and the heat tolerance were classified according to IRRI (2013) (Table 2).

Repeated screening and validation of heat tolerance

For repeat screening, 98 lines of JHN mutants (M5) with high spikelet fertility (>80%) were treated at high temperature (40oCto 45oC) under the same conditions as in the preliminary screening. The experiment was started in 2013 (April–May) and set up in a split-plot lattice design with two replications (five pots/replication).

The candidate lines (M6) validated for heat tolerance and 14 Thai rice cultivars (Supplemental Table 1) were treated at high temperature (40oCto 45oC). The experiment was started in 2013 (July–August), and set up in a split-plot design in a completely randomized design with three replications (five pots/replication).

Data collection

For the preliminary screening, repeat screening and validation screening, eight random panicles were selected per line.The panicle length, the number of filled and unfilled grains per panicle, and the seed weight were recorded.The spikelet fertility was estimated as the ratio of the number of filled grains to the total number of florets.The number of filled grains included both completely and partially filled grains.

Table 2. Heat tolerance scoring system in rice (IRRI, 2013).

For validation screening, plants from five pots per replication in each treatment were harvested to determine their grain yields. The yield components were recorded, including grain yield, 100-grain weight, panicle weight, grain filling and total spikelets per panicle.

For validation screening, pollen viability was estimated using 1% I2-KI stain. Pollen that stained uniformly was considered viable. For pollen viability, 10 anthers from different plants were collected early in the morning before anthesis, and the anthers were opened with a needle and the pollen was immediately brushed on a glass slide and covered with a drop of I2-KI. Pollen viability was estimated as the ratio of stained pollen grain number to pollen grain number(Prasad et al, 2006). Anther at the middle position in panicles were sampled at 8:00–9:00 am to determine either basal and apical pores open and the open ones were recorded as dehisced, and the remaining pollen grains were recorded as indehisced, with the aid of a stereomicroscope.Anther dehiscence was calculated as the ratio of the number of dehisced anthers to the total number of anthers (Rang et al, 2011).

Statistical analysis

All the data were analysed using the R program for statistical analysis.The means were separated using Tukey’s least significant difference testat the 0.05 level.

Results

High temperature stress at the reproductive stage reduced spikelet fertility of JHN

To investigate the effects of high temperature on spikelet fertility, the JHN wild type was exposed to high temperatures at 40oC to 45oCin the daytime. Fig.1 showed that the spikelet fertility was severely reduced under extreme heat stress from the booting stage, and it was highly decreased in T3 (36%), followed by T2. However,it showed no significant differences between the control andT1. Therefore, the heat screening was done during the booting stage for the large population of JHN mutants.

Preliminary screening

Rapid screening of 10000 lines indicated that 61% of the population (6100 lines) showing upright and semi-upright panicles were classified as highly heat susceptible (Fig. 2).Only 39% of the population (3900 lines) showing slightly drooping and strongly drooping panicles,with spikelet fertility ranging from 19% to 90%, were selected for further study. Under greenhouse conditions, 404 and 3164 lineswere classified as moderately tolerant and susceptible to heat stress, respectively. And 250 lines were classified as heat tolerant whereas 82 lines were classified as highly heat tolerant(Fig. 2).The control JHN (WT) and Sin Lek had on average spikelet fertilities of 16.8% and 0%, respectively. The spikelet fertilities of the mutant lines under field conditions ranged from 75% to 98%.

Fig. 1. Sensitivity of spikelet fertility to heat stress at 40 oC to 45 oC during the reproductive stages.

For the control, the plants were grown outside of the heat chamber under normal conditions. Different lowercase letters indicate significant difference at the 0.05 level. Bars are SE (=15).

Repeat screening

During the dry season from April–May in 2013, the 98 lines with spikelet fertility above 80% in the preliminary screening in M3 generation were investigated. The 98 lines showed a spikelet fertility ranging from 30% to 89% in greenhouse at high temperature (40oC to 45 oC) (Fig. 3-A). A differential response of susceptible and tolerant lines indicated a significant temperature-by-accession interaction. Repeat screening revealed six susceptible lines with low levels of spikelet fertility at high temperature (31%– 40%), while seven lines were identified as heat tolerant with spikelet fertility levels of 71%–90% (Fig. 3-A). Under field conditions, the spikelet fertility ranged from 61% to 95% (Fig. 3-B).

Fig. 2. Frequency distribution of 10000 mutant plants and lines selected for spikelet fertility at 40oC to 45oCdaytime temperature during the reproductive stage until harvesting.

Fig. 3. Selection of rice mutants for heat tolerance based on spikelet fertility.

The 98 mutant lines were divided into four clusters according to the spikelet fertility at high temperature after the first and second screening: highly tolerant (>80%, 1 line), tolerant (61%–80%, 29 lines), moderately tolerant (41%–60%, 63 lines) and susceptible (<40%, 5 lines) (Fig. 3). However, the highest spikelet fertility in the field and under high temperature conditions during the repeat screening was found in M9962, M3181, M7988, M8269, M8281 and M8372, which indicated that these lines were the most tolerant to heat stress during the reproductive stage. Wild type JHN was susceptible in response to heat stress (39.3%), and Sinlek was highly susceptible to heat stress (10.4%). Finally, four candidate heat-tolerant lines (M9962, M3181, M7988 and M8269) with high spikelet fertility were used in the third screening for validation (Table 3).

Validation of heat tolerant lines

Table 3.Effects of heat stress on panicle traits in the candidate heat tolerant lines.

Different lowercase letters followedthe values in the column are significantly different at< 0.05 by the Tukey’s least significant difference (LSD) test. Different uppercase letters in each row for the same trait indicate significant differenceat< 0.05 by the Tukey’s LSD test.

The spikelet fertility was decreased in all the genotypes under high temperature conditions (Fig. 4). There were significant effects(all< 0.05) of temperature, cultivar/line and an interaction between temperature and cultivar/line on spikelet fertility, which ranged from 34% to 75%. The negative effects (% decrease from control) of high temperature on spikelet fertility were the highest for RD33 (decreased by 94%) followed by RD15 (decreased by 87%), while the spikelet fertility of the negative control Sinlek decreased by 87% and was thus classified as highly susceptible to heat stress. On the other hand, the least decrease of fertility was in M9962 (4%), which indicated that it was the most heat tolerant. In addition, the cultivars/lines that were classified as moderately heat tolerantwere Thanyasirin (glutinous rice), M8269, Suphan Buri1, Phisanulok2 and Pathum Thani 1, while JHN (wild type) was heat susceptible. The three special rice cultivars Dawk Pa-yawm (upland rice), 5029A (long grain) and Homcholasit (submergence tolerant) were also susceptible to heat stress. The grainyield per plant was also reduced for all the genotypes under high temperature conditions except for M9962 and M3181. Thanyasirin (waxy rice) showed the maximum yield at high temperature stress, but its spikelet fertility was only 50%. However, high temperature was also reflected in highest decreases in grain yield for RD33 and Dawk Pa-yawm.

As shown in Table 4, high temperature stress during the booting to harvesting stages had significant impacts on 100-grain weight. Significant temperature ×cultivar interactions were also observed for the 100-grain weight (< 0.05). The decrease in grain weight was less in M8269 (5%), followed by RD15, Pin Kaset1, M3181, M9962, M7988, Pin Kaset 2 and Suphan Buri1, ranging between 6% and 9%. The negative effects of high temperature were the greatest in PSL2.

There were significant effects (all< 0.05) of temperature, cultivar/line and the temperature and cultivar/line interactions for filled grain per panicle(Table 4). The filled grains per panicle at high temperature ranged from 7.4 to 104.1 among different rice accessions. The mutant lines including M9962, M7988 and M3181 showed a slight decrease of filled grain number and were identified as heat tolerant lines, while the negative effects of high temperature on filled grain number were the greatest in RD33 (89%), followed by RD15 (88%) andPin Kaset 3(87%), which were classified as highly susceptible to high temperature.

The effects of temperature, cultivar, and the temperature and cultivar interactions (all< 0.05) on the panicle weight (Table 4) were similar to those obtained for spikelet fertility and filled grains per panicle. The negative effects of high temperature on the number of filled grains and the panicle weight were low in M9962 compared to the other cultivars (Table 4).

Table 4. Effects of heat stress at the booting stages on the 100-grain weight, filled grains per panicle and panicle weightin different rice cultivars in wet season 2013.

DF, Percentage decrease from the field.

Different lowercase letters followedthe values in the column are significantly different at< 0.05 by the Tukey’s least significant difference (LSD) test. Different uppercase letters in the each row for the same trait indicate significant differenceat< 0.05 by the Tukey’s LSD test.

Heat-tolerant mutants selected from JHN and commercial rice varieties were compared for pollen viability and anther dehiscence. There were significant effects(all< 0.05) of temperature, cultivar and temperature and cultivar interactions on pollen viability and anther dehiscence (Fig. 5). On average, high temperature decreased the pollen viability by 25% and the anther dehiscence by 33%. The decrease in pollen viability due to high temperature was the greatest in JHN, followed by Sinlek, while the effect was the lowest in M9962. These effects were similar to those on pollen production, for which the greatest negative effects of high temperature were seen for Sinlek, followed by JHN, while the least effects were found for the mutant lines M9962 and M3181. Anther dehiscence was thus identified as an important heat susceptible process that determines the variation of pollen numbers on the stigma (Fig. 6).

Fig. 5. Pollen viability (A) and anther dehiscence (B) under field conditions and at high temperature for the six rice genotypes.

Fig. 6. Images of six cultivars showing the pollen viability (A1–A6), anther dehiscence (apical and basal pore) (B1–B6) and pollen number on the stigma (C1–C6) under high temperature conditions.

Discussion

In general, temperatures higher than the optimal temperature induce floret sterility and thus decrease the rice yield (Nakagawa et al, 2003). Spikelet fertility is greatly decreased at temperatures higher than 35oC(Matsui et al, 1997). Jagadish et al (2007) found that exposure (less than 1 h) to temperatures above 33.7oCis sufficient to induce sterility. Matsui et al (2001) showed variations in Japanese cultivars in response to high temperature stress during the day (10:00–16:00). The spikelet fertility (seed-setting rate) is an important component of yield that is sensitive to high temperatures (Prasad et al, 2006). Severe reductions due to extreme heat stress were seen during the booting stage (Fig. 3), which is consistent with previous studies (Satake and Yoshida, 1978; Shah et al, 2011).

The rapid screening of germplasm for heat tolerance at the reproductive stage in large populations with different times did not account for factors such as interactions between cropping and night temperature, genetic variation within genotype (M4generation) and lacked replication. Additionally, number of selected lines greatly decreased after repeat screening, only seven tolerant lines remained. Previous research has mostly used spikelet fertility to screen the germplasm for heat tolerance in rice at the reproductive stages (Prasad et al, 2006; Tenorio et al, 2013; Huang et al, 2016; Moung-ngam, 2016; Prasanth et al, 2016; Sukkeoa et al, 2017), so decreased yields due to increased spikelet sterility as a result of high daytime temperatures (Prasad et al, 2006) and night time temperatures (Ziska et al, 1996) have been reported in rice.

Prasad et al (2006) identified heat tolerance in both subspecies ofand it cannot be generalized that either theorsubspecies is more tolerant than the other based on the place of origin.Studies have shown that intraspecific variation of grain yield exists among bothandtype cultivars (Ziska et al, 1996; Matsui et al, 1997, 2000; Moya et al, 1998). Thevariety N22 has consistently shown tolerance to high temperature during anthesis (Prasad et al, 2006; Jagadish et al, 2008; Mohapatra et al, 2014; González-Schain et al, 2015; Prasanth et al, 2016).This study quantified the effects of high temperature on spikelet fertility and yield components inThai rice. There were cultivar differences for heat tolerant candidates in response to high temperature. Differential cultivar responses to high temperatures were attributed to differences in the spikelet fertility and yield components.

In this study, other effects of high temperature stress during flowering were decreased pollen viability and indehiscence of anthers resulting in poor pollen shedding and a decreased number of pollen grains on the stigma. All of these phenomena can cause poor spikelet fertility. Accordingly, there were strong positive correlations between spikelet fertility and pollen viability (r= 0.62;=6;<0.05) and anther dehiscence (r= 0.64;=6;<0.05) (data not shown). Poor pollen germination after reaching the stigma might have restricted spikelet fertility. Prasad et al (2006) reported that high-temperature stress during rice flowering led to decreased pollen production and pollen shedding. The probable reasons are an inhibition of the swelling of pollen grains, indehiscence of anthers and poor release of pollen grains(Matsui et al, 2000).

Conclusions

Forward genetic screenings were used to identify heat tolerance in 10000 M4lines irradiated by fast neutrons. The fast neutrons radiation generated extremely heat tolerant mutants that showed different levels of spikelet fertility at high temperatures in the greenhouse. Additionally, a high daytime temperature decreased rice yield by decreasing spikelet fertility, 100-grain weight and panicle weight. The decrease in spikelet fertility and the differential response of cultivars at high temperature was mainly associated with impaired (decreased) pollen viability and pollen germination. The heat tolerant mutant line M9962 had the least decreases in spikelet fertility and grain yield at elevated temperature, while the susceptible cultivars (Sinlek, RD13 and RD33) had larger decreases in spikelet fertility and grain yield. Therefore, M9962 is a potential genetic stock for use in a heat tolerance breeding programme, thus effort must continue toward identifying differential genetic background with JHN by single feature polymorphism and micro array analysis method and development marker assisted selected for heat tolerance breeding program using M9962 donor.Such heat tolerance donor lines can be used to improve the heat tolerance of future rice varieties and for genetic, physiology and morphology studies to further our understanding of the mechanism of heat tolerance.

Acknowledgements

This work was supported by the Agriculture Research Development Agency and the Office of the Higher Education Commission for the Strategic Scholarships Fellowships Frontier Research Networks of Thailand.

SUPPlemental DATA

The following materials are available in the online version of this article at http://www.sciencedirect.com/science/ journal/16726308; http://www.ricescience.org.

Supplemental Table 1. Origin, grain type and special traits of various rice cultivars used during the wet season of 2013.

Supplemental Fig. 1. Schematic view of mutant screening for heat tolerance of a large fast neutron- treated population.

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31 March 2018;

22 August 2018

Chanate Malumpong(agrcnm@ku.ac.th)

Copyright ? 2019, China National Rice Research Institute. Hosting by Elsevier B V

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http://dx.doi.org/10.1016/j.rsci.2018.08.008

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