Yuping ZHANG, Jing XIANG, Huizhe CHEN, Yikai ZHANG, Xianqing LIN, Defeng ZHU

State Key Laboratory of Rice Biology, China National Rice Research Institute, Hangzhou 310006, China

Rice (Oryza sativa L.) is one of the important food crops in China and its yield has direct impact on food safety[1]. Transplanta tion is an elaborate method of rice cultivation in which the appearance of the field and the population of rice plants is under controlled. The period immediately after rice transplantation is known as the turning green stage. Despite its short duration, it is an essential growth period for rice, and has direct effect on emergence of new leaf,tillering ability, canopy development and spike number. Hence, the turning green stage is an important determinant of rice yield[2-3].During the turning green stage after transplantation, rice leaf generally turns from green to yellow and then back to green, the leaf age increases, and tillering occurs.This is mainly because the root system of rice seedlings is sensitive to adverse environment and planting injury during transplantation[4]. As a re sult, absorption of essential minerals by plants that synthesize chlorophyll may be reduced,which may further influence chlorophyll renewal. As N, Mg and other minerals required for new leaf growth depend on chlorophyll transport after its decomposition in old leaves, senesced-yellowing of old leaf is accelerated[5]. When normal growthof the root system resumes, the new roots can absorb N,Mg and other minerals from the soil to sustain the synthesis of chlorophyll. With the emergence of new leaves, the parts of plants showing green color increase and the seedling enters the turning green stage. The length of the turning green stage is related to seedling quality, seedling age, and the environment[6]. In recent years, use of machines for rice transplantation has become increasingly common. However,since the seedling period is short in machine transplantation, rice seedlings are usually sensitive to low temperature.This impedes the growth and delays the “green turning”and tillering of rice seedlings. It can take them about 8 -10 days or longer to turn green under low temperature, or the seedlings may not turn green at all[7].Under high temperature, the length of the turning green stage can be as low as 2-3 days. Shortened turning green stage is beneficial for early appearance of new leaves and for tillering to occur[8]. Length of the turning green stage after transplantation also influences the competition between rice seedlings and weeds. Improving the competitiveness of rice (Oryza spp.)against weeds can be a low-cost and safe weed management strategy[2].Rapid seedling growth of rice was reported to be associated with improved ability to tolerate weed competition,which is a major constraint in some areas of China. Substantial differences in seedling growth rate have been found among rice cultivars.Some cultivars give relatively poor yields when in competition with weeds compared to those grown in weed-free conditions.Likewise, grain yields in competition with weeds in field experiments in two seasons significantly correlated with rice seedling biomass at 12 DAS 54 in greenhouse experiments[9-10].The turning green stage is an important stage during rice growth that has significant influence on tillering. Promotion of tillering and spiking in rice is a desirable goal in rice breeding and planting. However, tillering and leaf age are closely related to temperature during planting. Both high and low temperature extremes can be threatening to rice production. Therefore,studying the effect of temperature on turning green and seedling growth is of great significance for scientific field management and yield increase of rice.

Materials and Methods

Materials

The experiment was carried out at the Fuyang experimental base of China National Rice Research Institute in April and May of 2012 using green house and artificial climate chest. The experimental materials were singleseason japonica rice Xiushui 134 (V1)and early indica rice Zhongzao 39(V2).

Experimental methods

Experimental design The experiment takes a split-plot design,with the temperature as the main plot and different seedling soil as the sub-plot.Artificial climate chest was used to control temperature and day-night temperature difference was maintained at 6 ℃. Four temperature gradients (16,19, 22 and 25 ℃) were set. Three kinds of soil with 80%relative humidity(Table 1) were chosen, namely, substrate(SS),dryland soil(UD)and slurry(MF). Substrate was mixed by vermiculite, charcoal, and fine sand. Dryland soil was used for dry nursery seedlings, while slurry, which consisted of mud from paddy fields,was used for paddy nursery seedlings. Twenty days after sowing, a container was used to transplant the seedlings.Seedlings in each treatment group were transplanted into a 90 cm x 90 cm container.Soil in the container was the top soil dug out from the field.One variety was sowed in each container with 30 clumps for each variety, and a total of 3 containers was used. Two seedlings of uniform growth status were transplanted in each clump. Artificial climatic chest adjusted to different temperatures was placed into the containers after transplanting. A total of 24 treatments were set(Table 2).In order to promote turning green and tillering of rice seedlings, 8 g of urea was applied to each container after 1 week of transplanting.

Measuring items and methods

Leaf age: Leaf age of the main stem was recorded every 4 days from the day after transplanting. Each treatment group included 5 clumps and a total of 3 replicates were set up.Tillering:The number of tillers was recorded every 4 days from the day after transplanting.Each treatment group had 10 clumps and 3 replicates. Seedling height: Seedling height was measured every 4 days using a straight ruler from the day after transplanting. A total of 30 plants were measured, with 3 measurements in each treatment group.Dry matter: The dry weights of root,stem and leaf were measured at 3 time points beginning on the 20thday after transplanting 215 for 5 clumps in each treatment group. Leaf color: Leaves were sampled from the same position 20 days after transplanting.SPAD value of the unfolded leaf at the uppermost position was measured using SPAD-502 Monito,Japan.A total of 20 leaves were tested in each treatment group and 3 replicates were set up.Nitrogen content: Five plants were sampled in each treatment group 20 days after transplanting. The nitrogen contents of roots, stems and leaves were measured for three times after drying, mashing and digesting using the FOSS 2300 Automatic Instrument of Nitrogen.

Data processing All experimental data were processed using SAS 9.1 and Excel 2007.

Table 1 List of the temperature setting in the artificial climate chest

Results

Difference in turning green of rice seedlings under different temperatures after transplanting

Timing of appearance of new leaves New leaf appearance is an important indicator of turning green of rice. The time required for leaves to grow to thesame age was different under different temperatures, as shown in Fig.1.At temperatures ranging from 16 to 25 ℃, the time required for growing 1 leaf was 14.5, 10.4, 5.6 and 5.3 d in increasing order of temperature(Fig.1A). Thus, new leaves emerged within a shorter period under higher temperature, which was favorable for turning green of rice seedlings.Growth of 1 new leaf required 10 days under temperature lower than 19 ℃, indicating that low temperature had adverse impact on turning green. Temperature over 19℃after transplanting was required to promote early appearance of new leaves and turning green of rice seedlings.

Occurrence of tillering Occurrence of tillering is also an important manifestation of rice turning green.Obvious differences were observed in the timing of occurrence of tillering and the amount of tillers under different temperatures.Tillering did not occur under 16 ℃. Between 16 and 19 ℃, tillering occurred after 15 days of transplanting. Under 22 and 25 ℃, tillering occurred only after 10 days of transplanting. This indicated that temperature was an important factor for the timing of occurrence of tillering. Additionally, our results showed that only temperature higher than 19 ℃after transplanting could improve seedling tillering and bring about early turning green(Fig.1B).

Table 2 Treatment setting in present study

Effects of temperature on leaf age

Temperature had significant influence on leaf age.With increasing temperature,the leaves grew more quickly. However, with extension of transplanting time, leaf age increased linearly. The change could be described by the linearity equation y=kx+b, in which k is slope and b is intercept.Slope was 0.077 7, 0.110 7, 0.173 5 and 0.187 4 in increasing order of temperature between 16 to 25 ℃. K values indicated the growing speed of leaf age (Fig.2). After 20 days of transplanting, a difference of 2.3 leaf age was observed between 16 and 25℃and a 1.7 leaf age difference was noted between 19 and 25 ℃. No significant difference was found in the changing trend of leaf age between 22 and 25 ℃,indicating the close positive relationship between temperature and leaf age.

Effects of soil type and different rice varieties on tillering of rice

Tillering is one of the important agronomic traits influencing spike number and per unit area yield of rice. Its occurrence is an important indicator of the health status of rice seedlings.Early or late occurrence of tillering can affect the length of turning green stage in rice seedlings. The higher the temperature, the faster and earlier tillering will occur(Fig.1B).Occurrence of tillering is also related to the seedling substrate. In our experiments, it occurred earlier in seedlings cultivated in dry-land soil and substrate after transplanting,that is,about 7 d after transplanting with a growing speed of 0.18 tiller per day.However,it appeared 10 days after transplanting for seedlings cultivated in slurry with a growing speed of 0.16 tiller per day (Fig.3). Therefore,the seedling substrate was an important factor that led to earlier occurrence of tillering. Significant difference could also be found among different rice varieties. The time of tillering occurrence was basically the same for early indica rice and japonica rice but the latter had higher tillering speed than the former.This revealed that the tillering capacity of japonica rice was higher than that of early indica rice un-

der the same conditions and this was closely related with the characteristics of the variety itself.

Effects of temperature on seedling height

As shown in Fig.5, the seedling cultivated in slurry was the shortest before transplanting under the same temperature. The tallest seedling was one that was cultivated in dryland soil,while the height of seedling cultivated in the substrate was intermediate. After transplanting, seedling height increased slowly for the 3 kinds of substrate under 16 and 19 ℃and their changing trends were basically consistent (Fig.4). At temperatures greater than 19 ℃, plant height increased significantly. With the passage of time after transplanting, the difference in seedling height broadened on dryland soil between 22 and 25 ℃. However,seedling growth was basically identical between those cultivated in substrate and slurry. This showed that as temperature increased,seedling height did not increase indefinitely; it showed a growing trend within a certain temperature range, then plateaued. Furthermore, increase in plant height was restricted below 19 ℃. Thus, our results showed that critical temperature for plant height increase is between 19 and 22 ℃. The trend was similar for the 2 varieties, but indica rice had higher growing speed than japonica rice.

Effect of temperature on leaf growth and color

With rising temperature, the leaf SPAD value increased and the changing trends for the 3 kinds of seedling substrates and the 2 varieties of rice were consistent (Fig.5). The leaf SPAD value declined significantly under 16 ℃.Difference in SPAD value in early indica rice Zhongzao 39 between 16 and 19 ℃, and between 22 and 25℃was significant. However, such a difference was not found for the japonica rice Xiushui 134 under 19, 22 and 25 ℃. This further demonstrated the higher low-temperature resistance of japonica rice than that of indica rice.

Effect of temperature on dry matter increment

Dry matter increment of plant is closely related to the temperature during its growth period. It can be seen in Figure 7 that the 100-seedling dry weight of rice rose as the temperature increased. The dry weight changed marginally when the temperature was lower than 19 ℃. This trend was observed for all three seedling substrates and rice varieties. This corresponded to growth trend as manifested in plant height and tillering. Upon comparing dry matter increment among seedlings cultivated in different substrates, higher increment of dry matter was found in seedlings cultivated in substrate and dryland soil. It was relatively lower in those cultivated in slurry. Such a difference could be attributed to different levels of nutrient absorption by plants in different substrates. Furthermore,indica rice Zhongzao 39 had higher amount of dry matter with higher increment of dry matter at any given temperature than japonica rice.

Effect of temperature on nitrogen absorption by different organs

Nitrogen absorption by different plant organs varied in the different treatment groups. Nitrogen absorption strengthened as the temperature increased. However, it increased significantly in all organs only between 19 to 22 ℃and the increase was consistent across all treatment groups. This indicated that plant organs could absorb the nutrients effectively only when the external temperature reached over 19℃after transplanting.The highest level of nitrogen absorption was found in leaves and the lowest level was in the roots in all treatment groups.Seedlings cultivated in slurry had low nitrogen absorption after transplanting but the absorption rate was significantly higher in those cultivated in dryland soil and substrate (Fig.8). This suggested that for centralized breeding,dryland soil and substrate are 141 better than plate breeding. They are not only favorable for nourishing the seedlings but also for plant growth after transplanting.

Discussions

Rice is an essential staple food for nearly 1/2 of the world population, so improving rice yield is an important goal. Transplantation is the traditional way of rice cultivation. After transplantation, rice seedlings go through a special stage known as the turning green stage before they revive from the damage caused by transplantation and tillering.The turning green stage is an important stage during the growth period of rice. Plants can grow more quickly with greater nutrient absorption if seedlings can turn green earlier.New leaf appearance is a major indicator of the timing of rice turning green. The time required for new leaf appearance and turning green are different under different temperatures.According to our study, between 22 and 25 ℃, it takes about 2-3 days for seedling to turn green regardless of variety and substrate. Under low temperature however, the turning green stage of rice seedlings is delayed.Therefore, it is advisable to keep the temperature above 19 ℃after transplanting to ensure early appearance of new leaves. With increase in temperature, leaf age showed an increasing trend, the tillering occur earlier and more dry matter was accumulated[11].Temperature is a critical factor in crop development. In rice culitvation, tillering is suspended if the temperature is too low, regardless of other optimal environmental conditions[8,12]. Studies have shown that that tillering reaches an optimum state under the average temperature of about 24℃. Temperature that is too high or too low can slow down tillering[13]. The degree of negative impact is determined by the extent to which the temperature is lower or higher than 24 ℃[14]. Songdao Shengsan et al. studied the relationship between rice tillering and temperature and reported that over the temperature range of 16-35 ℃,the higher the temperature, faster is the tillering. Others subsequently showed that significant day-night temperature difference is helpful for tillering[15]. Most studies have indicated that if the temperature is lower than 17 ℃during the turning green stage, seedling extension is restricted, resulting in low tillering,spiking and yield[16]. In our study,no tillering was found to occur in any treatment group during the turning green stage. However, after turning green, the tillering speed increased with rise of temperature over 19-25 ℃.If it waslower than 16 ℃during the tillering period, no tillering occurred,causing low spiking and low yield at later stages. Tillering was different between the 2 rice varieties and the three seedling substrates over 16-19 ℃.Only seedlings of japonica rice Xiushui 134 cultivated in dryland soil and substrate showed tillering. This revealed that japonica rice has stronger resistance to low temperature than indica rice and 19 ℃is the critical temperature for tillering after turning green.These results are consistent with those of previous studies. We found that seedling height and dry matter increased with rise of temperature.Moreover, our results revealed that 19℃is the critical temperature for growth of seedling height. Temperature lower than 19 ℃restricted seedling height and dry matter accumulation, which were both higher in indica rice than in japonica rice. No significant difference was found in nitrogen absorption by various organs at low temperatures.However, when the temperature was higher than 19 ℃, roots, stems and leaves had strong nitrogen absorption,with the strongest level observed in leaves and the lowest levels in roots.In recent years, with improvements in mechanization and centralized breeding,plate breeding area has expanded and soil needed for plate breeding is also diversified[6]. Our findings reveal that turning green and new leaf appearance of seedlings cultivated in dryland soil and substrate are accelerated, resulting in enhanced tillering and nitrogen absorption. Consistent with previous studies, we found that the advantages are especially prominent at low temperature[17]. Besides,collecting soil substrates from dryland is quite a problem, research on excellent breeding substrates is imperative.

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