Biological Effects in Heterotic Combination with Novel Adaxially-rolling Character in Rice（Oryza sativa L.）

Xiaoping GONG，Xiaoming KUANG，Ting LUO，Jiasheng Ll，Xubing ZHAO，Bingfeng ZHAO，Sheng ZENG，Zhili ZHANG

Chongqing Yudongnan Academy of Agricultural Sciences，Fuling 408000，China

Biological Effects in Heterotic Combination with Novel Adaxially-rolling Character in Rice（Oryza sativa L.）

Xiaoping GONG，Xiaoming KUANG，Ting LUO，Jiasheng Ll，Xubing ZHAO，Bingfeng ZHAO，Sheng ZENG，Zhili ZHANG

Chongqing Yudongnan Academy of Agricultural Sciences，Fuling 408000，China

Taking the heterotic combinations which respectively had moderately-rolled leaves and flat leaves at middle and later stages as tested materials，the biological effects in the heterotic combination of the moderately-rolling leaf mutant RL（t）were studied.The results showed that （i）there was insignificant difference between the leaf morphological characteristics of the RL（t）and flat leaf heterotic ombinations at early growth stage in that both of them had flat leaves and relatively big basic leaf angles；（ii）the stomatal conductance，the intercellular CO2concentration，transpiration rate and photosynthetic rate of the three top leaves of the RL（t）heterotic combination were significantly higher than those of the flat leaf heterotic combination at middle and later stages，which laid the foundation for increasing the yield per unit area；（iii）the light transmittance of the RL（t）heterotic combination in population at the middle part was higher than that of flat leaf heterotic combination，resulted in improved light interception condition at middle and lower parts of the population and increased the utilization efficiency of light energy；and （iv）this research revealed that the RL（t）heterotic combination could not only maintain a larger amount of the effective panicles but also could present large-panicle characteristic，especially in high-density condition，and also the ability of adjusting yield components the RL（t）heterotic combination was significantly increased under different densities.This study would provide a theoretical basis for the principle of high-yielding breeding with the application of the moderately-rolling leaf mutant in rice.

Rice；Moderately rolling；Straightness；Photosynthetic rate；Light transmittance

T he leaf of rice is the main place for photosynthesis and transpiration，and plays an important role in accumulation of assimilation，and therefore，leaf character has been the focus of attention of many rice breeders all the time［1-5］.The straightness and rolling index of leaf are the main contents of forming ideal rice ideotype，and keeping rice leaves，especially the three top functional leaves“short，thick，straight”can directly improve light interception efficiency and leaf area index of population to further improve the light interception condition，to thereby stably improve the output potential of rice［6］.Previous studies showed that:the leaf rolling trait could reduce diffuse reflection and reflection of light，then lower leaves could accept more light incident from an upper level，resulting in improved light interception condition of plants as well as improved intra-population microclimate，which enabled the self-sufficiency of middle and lower rice leaves who no longer had to consume the photosynthate from upper leaves，and the overall photosynthetic efficiency in population was thus improved［5，9，11-12］.In addition，the reported leaf rolling genes of rice were mainly expressed as rolled leaf in overall growth period，resulting in weak growth vigorin early stagecaused by limited light energy utilization of rice［13-14］.The rice breeders from Chongqing Yudongnan Academy of Agricultural Sciences found at Hainan Lingshui breeding base the variant，which had flat lower leaves and moderately-rolled three top leaves in early stage and was designated as RL（t）temporarily，and they bred the threeline sterile line of RL（t）and its corresponding combinations using this variant material.In the study，we studied the biological effects in RL（t）combinations，aiming at providing theoretical basis for utilization of RL（t）in highyield breeding.

Materials and Methods

Tested materials and field cultivation

The field experiment was conducted at the test field of Chongqing Yudongnan Academy of Agricultural Sciences in 2013.The materials for the research on the physiological effects of photosynthesis in RL（t）sterile line and its sister line were RL（t）combination 0712-12-2A/Fuhui 060 and flat leaf combination 0712-5-2-2A/Fuhui 060 bred by the rice research group from Chongqing Yudongnan Academy of Agricultural Sciences，each combination had an area of 0.02×667 m2，22.5×104nests/hm2，around which were provided guard rows；and under different densities，the material for the research on the physiological effects of photosynthesis of RL（t）heterotic combination was RL（t）heterotic as combination 733-17-2-2A/0724-1（A）bred by the rice research group from Chongqing Yudongnan Academy of Agricultural Sciences，the flat leaf combination Ⅱ You 838 （B）was used as control，both of them were designed with 7 treatments of 12.0，15.0，18.0，21.0，24.0，27.0and30.0×104plants/hm2，with 2 replications，around each plot with an area of 10 m2were provided guard rows，conventional nutrient and water management was adopted，and each data was the average value of 2 replications.

Determination items and methods

Determination of leaf rolling index，straightness，basic leaf angle and leaf drooping angle For each treatment，10 plants at fixed points was chosen for，in full heading stage，the measurement on the functional leaves（from flag leaf down to the third leaf）to obtain leaf lengths，spacing between naturally-rolled leaf margins at the largest width （Ln）and leaf width after spreading （Lw）for calculation of leaf rolling index （LRI）according to:LRI（%）=（Lw-Ln）×100/Lw；the measurement of the included angle between leaf and main stem，straight-line distance（LNL）from pulvinus to leaf apex after naturally curvature and straight leaf length （LEL）for the calculation of leaf straightness （LEI）according to: LEI=LNL×100/LEL；the measurement of the included angle between leaf and stem（basic leaf angle）and the included angle between the line connecting pulvinus and leaf apex after naturally curvature and stem（drooping angle）. Determination of photosynthetic rate Ten plants at fixed points were chosen for each treatment.In rice filling stage，photosynthetic intensities of functional leaves of stem were measured with LCA-4 portable photosynthesis system in field at 10:00-12:00 in the morning by irradiating leaf surface with light having an illumination intensity of 1 600-1 800 μmol/（m2·s）.The method was according to“Comparison of leaf photosynthetic characteristics among rice hybrids with different leaf rolling index”（Zhu et al）［10］.

Determination of leaf area and light transmittance In full heading stage，an L1-3105 leaf area meter was used to determine leaf areas，and light transmittances of population at various layers were determined using layered cut method by an ST-80 illuminometer（with a linear probe）.

Determination of yield In mature stage，the 10 plants at each fixed point were measured for plant height and effective panicles，5 of them were chosen for plant laboratory test on number of grains per panicle，seed setting rate and kilo-grain weight，and the practical yield of each treatment was converted into the yield per hectare.

Table 1 Leaf rolling indexes，straightness，basic leaf angles，drooping angles of 0712-12-2A/Fuhui 060 and 0712-5-2-2A/Fuhui 060

Results and Analysis

The differences of physiological effect of photosynthesis between the RL（t）sterile line and its sister line

Expression of Leaf rolling index，straightness，basic leaf angle and leaf drooping angle 0712-12-2A/Fuhui 060 behaved as flat leaf below the third leaf，and the three top leaves were moderately rolled，while 0712-5-2-2A/Fuhui 060 behaved as flat leaf in the whole growth period.

The three top functional leaves of 0712-12-2A/Fuhui 060 had leaf rolling indexes in the range of 94.6%-99.7%，while 0712-5-2-2A/Fuhui 060 had that only of 5.6%-9.6%；The three top leaves of 0712-12-2A/Fuhui 060 had straightness up to 94.6%-99.7%，which was obvious greater that of 0712-5-2-2A/Fuhui 060，especially for the third leaf，whose straightness was significantly greater than that of 0712-5-2-2A/Fuhui060；for0712-12-2A/ Fuhui 060，the basic leaf angles of the three top leaves were 14.20，14.80 and 16.80，respectively，while for 0712-5-2-2A/Fuhui 060，they were up to 17.80，18.40 and 27.80，which were obviously greater than those of 0712-12-2A/Fuhui 060；and the three top leaves of 0712-12-2A/Fuhui 060 had the drooping angles obviously less than those of 0712-5-2-2A/Fuhui 060（Table 1）.

By comparing F1of 0712-5-2-2A/Fuhui 060 with that of its flat leaf sister line，the three top leaves obviously behaved as large leaf rolling index，high straightness，small basic leaf angle and small drooping angle，and the two combinations showed a morphological difference there between gradually increasing from flag leaf down to the third leaf.

Table 2 Photosynthetic rates of the 0712-12-2A/Fuhui 060 and 0712-5-2-2A/Fuhui 060

Table 3 Light transmittances of 0712-12-2A/Fuhui 060 and 0712-5-2-2A/Fuhui 060

Photosynthetic rate In comparison with 0712-5-2-2A/Fuhui 060，each leaf of the RL（t）combination0712-12-2A/Fuhui 060 had obviously increased stomatal conductance，intercellular CO2concentration and transpiration rate，and the photosynthetic rate of leaves increased as well，though the increase did not reach the significant level（Table 2）.

Determination of light transmittance The two combinations had about the same light transmittance at the top of panicle，while 0712-12-2A/Fuhui 060 had the light transmittances at 15，30，60 and 90 cm from the top of each panicle downwards higher than those of 0712-5-2-2A/Fuhui 060 by 8.14%，18.77%，32.97%，42.67%and 24.00%，i.e.，the difference between their light transmittances gradually increased from the top of each panicle downwards to the base and then gradually decreased（Table 3）.

Study on physiological effect of photosynthesis in RL（t）combination under different densities

Expression of leaf rolling index，straightness，basic leaf angle and leaf drooping angle The leaf rolling index and straightness of 0733-17-2-2A/0724-10 were significantly greater than those of the flat leaf combination Ⅱ You 838，and meanwhile，under different densities，the leaf rolling indexes and straightness of each of the two combinations were not significantly different，indicating that leaf rolling index and straightness were less affected by planting density.Under different densities，the basic leaf angles and drooping angles of0733-17-2-2A/0724-10 were obviously smaller than those of Ⅱ You 838；and with increasing density，the basic leaf angles and drooping angles of the two combinations overall were on the decrease，while the basic leaf angle of Ⅱ You 838 showed an increase trend instead after the planting density reached 27.0×104plants/hm2，and the drooping angle also showed an increase trend after the planting density reached 24.0×104plants/hm2（Table 4）.

Determination of photosynthetic rate under different densities The third leaves of 0733-17-2-2A/0724-10 and Ⅱ You 838 had about the same photosynthetic rate under low densities in heading period，and decreased with increasing density.In the range of 12.0-30.0×104plants/hm2，the photosynthetic rate of 0733-17-2-2A/0724-10 dropped by 24.52%，and that of Ⅱ You 838 dropped by 39.92%，showing that the photosynthetic rate of 0733-17-2-2A/0724-10 had the drop less than that of Ⅱ You 838.At the density of 30.0×104plants/hm2，the photosynthetic rate of 0733-17-2-2A/0724-10 was higher than that of Ⅱ You 838 by 27.09%（Table 5）.

Determination of leaf area indexes under different densities Both the leaf area indexes of 0733-17-2-2A/ 0724-10 and Ⅱ You 838 increased with increasing density in heading period， and the increases of 0733-17-2-2A/ 0724-10 were higher than those of Ⅱ You 838 under different densities. From 12.0×104plants/hm2to 30.0×104plants/hm2，the leaf area index of 0733-17-2-2A/0724-10increased by 67.68%，while that of Ⅱ You 838 only increased by 47.58%（Table 6）.

Determination of light transmittances under different densities

Under different densities，0733-17-2-2A/0724-10 and Ⅱ You 838 had about the same light transmittances at the top of panicle，and with increasing planting density，the light transmittances at various layers of the two combinations were on the decrease from the top of panicle down to the base of plant，i.e.，the higher the density，the lower the light transmittance；under the same density，the changes in the light transmittances at various layers of 0733-17-2-2A/0724-10 were obviously less than the changes in those of Ⅱ You 838，for example，with the density of 30.0×104 plants/hm2，from the top of panicle down to 60 cm，the light transmittance of 0733-17-2-2A/0724-10 dropped by 77.19% ，while Ⅱ You 838 dropped by 90.91%；at the same height，with increasing planting density，0733-17-2-2A/0724-10 had a drop in light transmittance obviously lower than that in light transmittance of Ⅱ You 838，for example，at the level of 60 cm far away from the top of panicle，in the range of 12.0-30.0×104plants/hm2，0733-17-2-2A/0724-10 had a drop in light transmittance by 71.05%-77.19%，while the drop in the light transmittance of Ⅱ You 838 by 74.52%-90.91% （Table 7）.

Table 8 Yields in various plots of 0733-17-2-2A/0724-10 and Ⅱ You 838 under the different densities

Determination of yield under different densities By the determination of yields of 0733-17-2-2A/0724-10 and Ⅱ You 838 under difference densities，it was shown that with increasing planting densities，the yield of 0733-17-2-2A/0724-10 exhibited an overall increasing trend，and did not reach its peak value at the density of 30.0×104plants/hm2，while the yield of Ⅱ You 838 reached its peak value at the density of 21.0×104plants/hm2，and decreased with the continuously-increasing density；under different densities，the variable coefficient of the effective panicles of 0733-17-2-2A/0724-1 was of 17.39%，while that of Ⅱ You 838 was only of 11.20%，indicating that the effective panicles of 0733-17-2-2A/0724-1 could increase in a larger margin with increasing density；and meanwhile，the variable coefficients of number of grains per panicle，number of full grains per panicle，seed setting rate，kilo-grain weight and yield of 0733-17-2-2A/0724-1 were less than those of Ⅱ You 838，i.e.，the single panicle weight and yield of 0733-17-2-2A/0724-1 had good stability with increasing density and number of effective panicles（Table 8）.

Conclusions and Discussion

Conclusions

The three functional leaves of the RL （t）heterotic combination showed greater leaf rolling index，higher straightness，lesser basic leaf angle and lesser drooping angle than those of the flat leaf combination obviously，the differences gradually increased from the flag leaf to the third leaf at the same time，and therefore，the RL（t）heterotic combination had the light transmittance of population higher than that of the flat leaf combination，especially in the middle of population，where the light transmittance of the RL（t）heterotic combination was notably higher than that of the flat leaf combination；meanwhile，the stomatal conductance，intercellular CO2concentration and transpiration rate of the RL（t）heterotic combination were significantly higher than those of the flat leaf combination，and the photosynthetic rate thereof also increased compared with that of the flat leaf combination，which laid the foundation for raising the yield per unit area；and on the other hand，the light interception condition for middle and lower leaves in population was improved，thereby improving utilization efficiency of light energy，such that the self-regulation ability for the yield components of the RL（t）heterotic combination was enhanced under different densities，especially for the high densities，at which the number of panicles per unit area was higher and the big panicle characteristic could be maintained better.

As the two combinations had similar leaf morphologies with flat leaves and greater basic leaf angle in early stage，the differences between their light transmittances was notably reduced at the level near to the earth' s？surface.Therefore，RL（t）mainly influenced the leaf morphologies and light transmittances at upper and middle parts of population，and had less influence on lower part of population.

Discussion

The rice region in the upper reaches of Yangtze River is beneficial to the formation of big rice panicles as well as big grains due to low temperature and less sunshine in the early stage of rice growth and sufficient light and heat in the middle and later stages.For this reason，the rice region on the upper reaches of Yangtze River mainly conducts heavy-panicle type breeding for a long time，and the developed varieties show big panicles and big grains，loose plant type，and wide large drooping leaves，with the main defect of few panicles per unit area.The rapid development of twoline hybrid rice in the lower reaches of the Yangtze River and southern China is in close relation to the fact that most varieties have compact plant type，inwardly-rolling leaves，good light transmittance of population and more pani-cles per unit area，but it is difficult to give full play to the potential of such varieties in the upper reaches of Yangtze River due to the fact that rolled leaves result in lower utilization efficiency of light energy，which causes in turn slow growth speed of rice in the early stage.This study showed that breeding the hybrid rice combination with flat lower leaves and three moderately-rolled top functional leaves in the early stage was beneficial to form the rice leaf shape which was“loose at first and compact then”by forming dynamic plant type，by such leaf shape，then rice not only could make full use of light energy to achieve the culture purpose of early and fast growth，but also could effectively reduce stretch angle of leaf，enhance leaf straightness and improve light transmittance of population as well as improving utilization efficiency of light energy after jointing，and the number of panicles per unit area could be significantly improved while making full use of light and heat resources and ensuring heavy panicles.

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Responsible editor：Yingzhi GUANG

Responsible proofreader：Xiaoyan WU

一个新的水稻内卷叶突变体的杂优组合生物学效应

龚晓平，况晓明，罗 挺，李加胜，赵许兵，赵冰峰，曾 胜，张致力*（重庆市渝东南农科院，重庆涪陵 408000）

以生育中后期叶片适度内卷叶和平展叶组合为材料，研究了水稻适度内卷叶突变体RL（t）杂优组合的生物学效应。结果表明：①RL（t）杂优组合与平展叶组合生育前期生长叶态相似，均为平展叶且叶基角较大；②RL（t）杂优组合生育中后期叶片气孔导度、胞间CO2浓度、蒸腾速率显著高于平展叶组合，其光合速率较平展叶组合也有所提高，为其提高单位面积产量奠定了基础；③RL（t）杂优组合在群体中部透光率显著高于平展叶组合，改善了群体中下部叶片受光条件，从而提高了光能利用率；④RL（t）杂优组合在不同密度下产量构成因子自我调控能力显著增强，尤其在高密度下，具有较高的容穗量，并能较好地保持大穗特点。本研究为适度内卷叶应用于水稻高产育种提供理论依据。

水稻；适度内卷；挺直度；光合速率；透光率

重庆市“十一五”重大科技专项（6887）；重庆市科委科技攻关项目（CSTC，2009AB1061）。

龚晓平（1980-），男，重庆垫江人，博士研究生，助理研究员，主要从事水稻遗传育种、分子生物学等研究，E-mail：gong1981_120@163.com。

2015-08-30

Supported by Chongqing Major Scientific and Technological Project（6887）；Science and Technology Key Project of Chongqing Science and Technology Committee（CSTC，2009AB1061）.

*Corresponding author.E-mail:zzllbn7513@sina.com

Received:June 30，2105 Accepted:October 1，2015

*通讯作者，研究员，主要从事水稻遗传育种与抗病机理研究，E-mail：zzllbn7513@sina.com。

修回日期 2015-10-01