Bingliang WAN,Zhongping ZHA,Desuo YIN,Jinbo LI,Xueshu DU,Mingyuan XIA,Huaxiong QI

1.Institute of Food Crops,Hubei Academy of Agricultural Sciences,Wuhan 430064,China;2.Hubei Key Laboratory of Food Crop Germplasm and Genetic Improvement,Wuhan 430064,China;3.Hubei Collaborative Innovation Center for Grain Industry,Jingzhou 434025,China

Rice,a major food crop for the people in China,occupies a dominant position in national strategy for food security.The hot weather in southern China,and the Yangtze River Basin from July to August in particular has resulted in serious loss to rice production.More than 30 million hm2rice planting area in the Yangtze River Basin was affected by the high temperature damage in 2013,the most serious ever,and the loss in rice production was estimated to be 51.8 million tons[1].At heading and flowering stage from July to August,rice is most sensitive to the changes in temperature,for which the appropriate temperature is from 25 to 30℃.The florets will be infertile if the average daily temperature≥32℃,and the daily maximum temperature≥35℃,and thus the seed setting rate will decline[2-3].However,high temperature damage will occur more frequently in future due to global warming.According to the meteorological data from 1951 to 2005,it was predicated that the days with a maximum temperature higher than 35℃at rice heading and flowering stage in the Yangtze River Basin will continuously increase in the next 30 to 50 years[4].

To reduce the adverse effects of high temperature on rice production,many rice germplasms tolerant to high temperature such as N22,Bala,T226 and 996,have been identified and developed from the 1970s[5-9].However,the complicated genetic mechanisms of rice’s high-temperature tolerance and difficulties in the identification of high-temperature tolerance result in the slow progress in improving rice’s tolerance to high temperature.From 1992 to 1995 Shen et al.[10]identified the tolerance of 630 new rice varieties or combinations to high temperature in greenhouse and field,and the results revealed that among the 630 rice varieties or combinations 15.4% were highly tolerant to high temperature,23.5% were moderately tolerant to high temperature,and 61.1% were sensitive to high temperature.Fu et al.[11]investigated the heat tolerance of 15 maintainer lines and 26 restorer lines those commonly used in rice breeding,and found that the seed setting rate of only one maintainer line was more than 50% at high temperature,and the seed setting rate of all the restorer lines was no more than 40%.The study of Hu et al.[12]revealed that 15 of 100 hybrid combinations of rice were tolerant to high temperature.All above studies proved that most of the commonly planted hybrid combinations and their parental varieties are sensitive to high temperature.So,it is urgent to screen some parental varieties tolerant to high temperature for hybrid rice breeding.

R1056 is a cytoplasmic-genetic male sterile restorer line developed by Hubei Key Laboratory of Food Crop Germplasm and GeneticImprovement.It has showed high tolerance to high temperature in natural environment.In this study,we further identified the tolerance of R1056 and four of its hybrid combinations to high temperature in a greenhouse equipped with heating system,and thus to evaluate its potential in hybrid rice breeding.

Materials and Methods

Plant materials

R1056,four hybrid combinations Tianfeng A/R1056,Jufeng 2A/R1056,YuetaiA/R1056andNeixiang5A/R1056 were the materials to be tested.N22(which is tolerant to high temperature),9311,Fuhui 838,and two hybrid combinations Yangliangyou 6(a control variety used in Hubei middleseason rice regional test),II You 838(a control variety used in national heattolerant rice variety test)were used as the controls.

Planting pattern

The trials were performed in Nanhu Test Base of Hubei Academy of A-gricultural Sciences in Wuhan.All the rice varieties/combinations were seeded on three different dates,May 1,12 and 25.Seedlings were transplanted to pots and field 25 d after seed sowing.Pot experiment was carried out in open air.In detail,the seedlings of every rice variety at each sowing date were transplanted into six plastic buckets of 5 L,three seedlings in each bucket.Two to three similarsized tillers of every plant were remained,and others tillers were cut off at heading stage.In field trials,the plots were arranged according to random block design.Three repetitions were set for each treatment.Each plot had three rows,and 10 plants in each row,with plants spacing 19.8 cm between rows and 16.5 cm within rows.

At the beginning of heading stage,environmental temperature was measured using an automatic temperature recorder.Among the plants sown on three different dates,the batch for which the daily minimum and maximum temperatures at flowering stage were lower than 28 and 35℃was finally selected for data analysis.

High temperature in greenhouse

Seedling treatment with high temperature was carried out in glass greenhouse,equipped with auxiliary heating system,operable windows and exhaust fans.High temperature was set at 38℃,from 9:00 am to 3:00 pm.The hopper windows and exhaust fans were closed and the heating system was started when the temperature in the greenhouse decreased below 38℃;on the contrary,the hopper windows and exhaust fans were opened and the heating system was turned off when the indoor temperature exceeded 38℃.At the remaining period of time,all the windows and exhaust fans kept open,and the indoor and then outdoor temperature was identical.At flowering stage,three buckets of plants for each variety at each sowing date were transferred into greenhouse,and the other buckets were remained in open air as control.Among the three buckets in each treatment,one was used for measuring seed setting rate,and the other two for measuring the number of dehiscent anthers and pollens.The buckets in greenhouse were transferred into open air after flowering stage to measure seed setting rate.

Determination of the percentage of dehiscent anthers

Thirty open florets at the same position of plants were collected from each variety or combination at 10:00 to 12:00 am on the 3rdd of high temperature treatment,to observe anther dehiscence under a microscope after their husks were peeled off with a clamp.The percentage of dehiscent anthers was calculated using the formula:

Percentage of dehiscent anthers=Number of dehiscent anthers/Number of all anthers observed×100%.

Determination of pollen number on stigma per floret

Ten newly open florets at the same position of plants were collected from each variety or combination at 2:00-3:00 pm on the 3rdd of high temperature treatment,fixed in FAA solution,stained with aniline blue to calculate the pollens on stigma per floret under a fluorescent microscope.

Investigation of agronomic traits

At maturity,five plants in the middle rows of each plot in field trials were selected to measure their plant height,number of panicles per plant,number of grains per panicle,seed setting rate,1 000-grain weight and rice yield per plant.

Results and Analysis

Indoor and outdoor temperature at flowering stage

Since there was no air conditioning to lower the temperature in the greenhouse,the minimum indoor temperature was the same as that outside the doors.Therefore,the batch of plants for which the daily minimum and maximum temperatures at flowering stage were below 28 and 35℃were selected for data analysis.The flowering stage of the plants sown on May 1 lasted from July 22 to August 6,that of the plants sown on May 12 was from August 3 to 15,when the daily maximum temperature was above 35℃in most days,and the daily minimum temperature was higher than 28℃(Fig.1 and Fig.2),and thus the temperature in greenhouse exceeded 38℃,and was up to 41℃sometimes according to the measurements,which did not meet the requirements.So the data of the two batches of plants could not be adopted.The plants sown on May 25 bloomed from August 22 to September 6,when as the daily minimum temperature ranged from 18.2 to 26.7℃,and the daily maximum temperature ranged from 25 to 36℃,except that on August 27 exceeded 35℃(Fig.3).So the daily minimum and maximum temperature were basically consistent with the experimental design,below 28 and 35℃.During this period of time,the daily temperature in greenhouse ranged from 37 to 39℃,according to the measurements,consistent with the experimental design.Therefore,the plants sown on May 25 were selected and measured for subsequent analysis.

Seed setting rate of R1056 and its hybrid combinations at high temperature

The seed setting rate of all varieties or combinations tested showed an extremely significant decrease in high temperature treatment(P＜0.01),but the degree of decline differed among the varieties or combinations(Fig.4).The seed setting rate of R1056 and N22 were 70.4% and 72.6% at high temperature,which were 20.5% and 21.6% less than at control temperature.The seed setting rate of 9311 and Fuhui 838 was 35.8% and 47.2% at high temperature,which were 58.0% and 46.3% less than at control temperature.In high-temperature environment,the seed setting rate of R1056 and N22 was significantly higher than that of 9311 and Fuhui 838,indicating that compared with 9311 and Fuhui 838,R1056 and N22 had high tolerance to high temperature.The seed setting rate of the four hybrid combinations of R1056 was significantly lower than that of R1056 at high temperature,butsignificantly higher than that of Yangliangyou 6.The seed setting rate of three of the hybrid combinations was higher than that of II You 838,but the difference was not significant.

Anther dehiscence of R1056 and its hybrid combinations at high temperature

High temperature decreased the number of dehiscent anthers very significantly (P＜0.01),but the degree of decline was different among the varieties or combinations(Fig.5).The percentage of dehiscent anthers for the four hybrid combinations of R1056 and II You 838 was above 30% in high temperature treatment,significantly higher than that of Yangliangyou 6.Regression analysis showed that the seed setting rate and the percentage of dehiscent anthers in high temperature treatment had an extremely significant linear correlation(P＜0.01)(Fig.6),indicating that under high temperature stress the decreased percentage of dehiscent anthers was one of the factors directly influencing seed setting rate of rice.

Pollen number on stigma per floret of R1056 and its hybrid combinations

Consistent with the decrease in dehiscent anthers,the pollen number on stigma per floret was also extremely and significantly decreased at high temperature.In high temperature treatment,the pollen number on stigma per floret of R1056 and N22 were 78 and 80,both higher than that of the four hybrid combinations and II You 838 (ranging from 40 to 61),and that of the remaining three varieties was below 40.(Fig.7).Regression analysis showed that at high temperature,the pollen number on stigma per floret had extremely significant linear correlations(P＜0.01)with the percentage of dehiscent anthers and seed setting rate(Fig.8 and Fig.9).

Agronomic traits of R10566 and its hybrid combinations at high temperature

R1056 had much better agronomic traits than N22(Table 1).R1056 had a good plant shape,straight leaves,a height of 105.3 cm,about 11 panicles per plant,152 grains per panicle,1 000-grain weight of 27.3 g,good grain appearance and strong combining ability,and can be directly used to make different hybrid combinations.N22 had a plant height of 143.8 cm,thin stems,risk of lodging,less grains per panicle,lower 1 000-grain weight and weaker combining ability.The combinations generated from R1056 also exhibited excellent agronomic traits and high yield.The investigation on their agronomic traits showed that the four combinations of R1056 had a plant height of 111.6-119.2 cm,about 10 effective panicles per plant,and a seed setting rate above 90% (Table 2).The grain yield per plant of Jufeng 2A/R1056 was significantly lower than that of the control variety Yangliangyou 6,while that of the other three combinations had no significant difference with the control.The grain yield per plant of Jufeng 2A/R1056 had no significant difference with that of the other control variety II You 838,while that of the other three combinations was higher than that of the control.Among them,the yield per plant of Neixiang 5A/R1056 and Tianfeng A/R1056 was significantly or very significantly higher than that of II You 838.

Conclusions and Discussion

The most effective strategy to reduce the adverse effects of high temperature on rice is to screen and developrice varieties with tolerance to high temperature.Therefore,we measured theheattoleranceandagronomic traits of the restorer line R1056 and its combinations in this study,and the results proved that R1056 had high tolerance to high temperature at flowering stage,excellent agronomic traits and good combining ability,indicating that it is an excellent germplasm resource for heat-tolerant rice breeding.

Table 1 Comparison between R1056 and N22 for agronomic traits

Table 2 Agronomic traits and yield of R1056 and its hybrid combinations

Although high temperature damage has attracted much attention before,the progress in improving the tolerance of rice to high temperature is still very slow.The lack of germplasm resources with tolerance to high temperature may be one of the main reasons.N22,which is from India,has high tolerance to high temperature,but poor agronomic traits[5,13].R1056 is similar to N22 in tolerance to high temperature,as the seed setting rate of them was very close (70.4% and 72.6% )at high temperature.Moreover,R1056 has much better agronomic traits than N22.R1056 is also a cytoplasmic-genetic male sterile restorer line and thus can be directly used to make hybrid combinations,and its combinations also have high tolerance to high temperature according to our findings.In addition,the seed setting rate,the percentage of dehiscent anthers and other indices of the hybrid combinations from R1056 were better than Yangliangyou 6,and even the heat-tolerant combination II You 838.R1056 exhibited good combining ability,as the yield of three of the four combinations generated from R1056 was 5% higher than that of II You 838,and close to that of Yangliangyou 6,a control variety used in Hubei middleseason rice regional test.In summary,R1056 is notonly a good rice germplasm material tolerant to high temperature,butalso a superior parental variety for hybrid rice breeding.

Rice is most sensitive to high temperature at meiosis and flowering stages,when high temperature will greatly decrease its seed setting rate.Numerous studies have proven that decreased dehiscent anthers,pollens on stigma,and pollen germination rate are three important reasons causing floret infertility at high temperature[3-4].By investigating the effects of high temperature on pollen germination and fertility at flowering stage,Rang[3]found that the number of dehiscent anthers,number of pollens on stigma,the number of germinated pollens on stigma and floret sterility of the rice variety Moroberekan,which is sensitive to high temperature,were greatly decreased at high temperature,while those of N22,which is tolerant to high temperature,were higher.We also found that under high temperature stress,the percentage of dehiscent anthers,number of pollens on stigma of R1056 and N22 were significantly or very significantly higher than those of other high-temperature-sensitive varieties like 9311.Regression analysis also showed that under high temperature stress,the seed setting rate was linearly correlated to the percentage of dehiscent anthers and the number of pollens on stigma per floret,and the number of pollens on stigma per floret was also linearly correlated to the percentage of dehiscent anthers,suggesting that under high temperature stress more dehiscent anthers and germinated pollens rendered R1056 tolerance to high temperature.

The lack of effective methods for identifying rice’s tolerance to high temperature is another reason causing the slow progress in heat-tolerant rice breeding.At present,the tolerance of rice to high temperature is mainly identified in artificial climate chamber,field and common greenhouse[14-16].Artificial climate chamber can only be used to identify a few samples at a time,but cannot be used to identify a large population.In field,the uncontrollable temperature cannot completely meet the experimental design.The temperature in common greenhouse also cannot be controlled accurately.To develop an effective system for identifying rice’s tolerance to high temperature,in this study the rice varieties or combinations were sown at three different dates and transplanted into a greenhouse equipped with heating system.Then,high temperature treatment in greenhouse was performed from late August to early September when environmental temperature was the most appropriate(daily minimum temperature≤28℃and daily maximum temperature≤35℃).The results turned out that the temperature in greenhouse was precisely controlled at 38℃during this period.In summary,the method we developed in this study can be used to identify the tolerance of rice to high temperature on a large scale.

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