**2. Materials and methods**

64 Oilseeds

index) has higher N efficiency also. The development of N efficient genotypes and improvement of N management will require to understand the relationship between physiological processes and biomass, yield formation of crop under no N application conditions (Dreccer et al., 2000). Mahmoud et al., (2005) compared the differences of root growth and nitrate N exhaust in cultivar culture between high N efficiency and low N efficiency oilseed rape; larger amount of root biomass, higher root uptake activity and higher exhausted nitrate N amount were found in the high N efficiency oilseed rape; Nitrate-N uptake from soil depends on root growth and uptake activity, the amount of N depleted from the compartments significantly correlated with root-length density (Mahmoud et al., 2005). Seiffert et al., (2004) suggested that, the N use efficiency can be increased significantly by strengthen the activities of asparagine synthetase and glutamine synthetase through transgenic methods. Obviously, it is necessary to 'system study' on N uptake and N use efficiency in different oilseed rape. The differences of yield, N absorption and N use efficiency in different oilseed rape genotypes should be compared, and preliminarily discussion the contribution of N absorption efficiency and N use efficiency to N efficiency under different N application levels, in order to supply scientific basic and

The differences of concentration and distribution of N in crop is depending on the differences of organs and growth stages, and N redistribution in different organs will occur at different growth stages; these differences are related to the transfer of growth center (Peoples et al., 1998; Zhang et al., 2008). Studies in wheat (Andersson et al., 2005), oilseed rape (Malagoli et al., 2005) and pea (Séverine et al., 2005) showed that the leaves and stems of crop have become important N "sources" after the flowering stage, grain N does not only come from root N uptake during the later growing period, but also from that redistributed from the vegetative organs (Zhang et al., 2010). The latter was one of the key factors that guarantee crop N requirement during the later growing period. Nutrient competition between different organs of the crop during the later growing period is fierce; the uptake ability of the root declines at this time and soil mineral nutrients are exhausted (Dong et al., 2009). In oilseed rape, all leaves drop off at harvesting stage and N in the leaves is lost. To achieve high yield and high N use efficiency in crop production, N must be redistributed from the roots to the grains, residue N in uneconomic tissues must be reduced, and N harvesting index of economic tissues must be increased (Zhang et al., 2010). Research on N redistribution in cereals reached the following conclusions. The redistribution proportion of N from the leaves, stems and reproductive organs to the grains was more than 60% in wheat (Palta et al., 1995a, 1995b). Dalling et al., (1985) showed that, although the soil can supply adequate N after the crop flowering stage, at least 50% of the N in grains is still redistributed from the vegetative organs. The effect of environmental conditions on N redistribution was considerable e.g., low N in barley (Przulj et al., 2001) and drought stress in wheat (Barbottin et al., 2005; Palta et al., 1994) can accelerate N redistribution in plants, but the process can be restrained when plants are damaged by diseases and insects e.g., wheat (Dimmock et al., 2002). However, only a few studies looked at N redistribution in oilseed rape. Malagoli et al., (2005) found that in oilseed rape the N of flowers and pods was mainly from endogenous N, comprising 64 and 73% of total N content in these tissues, respectively. The N requirements of seed filling were mainly satisfied by N mobilized from the vegetative parts. Rossato et al.,

plant materials for the future study.

#### **2.1 Comparison of Nitrogen (N) efficiency between oilseed rape genotypes**

#### **2.1.1 Plant materials and experiment design**

The experiment was conducted at Agricultural Resources and Environmental College Experiment Field in HuNan Agricultural University during Sep. 2004 to May. 2005. 16 oilseed rape varieties were used as plant materials, the numbers are shown in Table 2, plant materials were supplied by China National Oilseed Crop Improvement Center, Hunan Branch. The soil used was an alluvial for vegetable cultivation derived from river flow alluvial material, containing organic matter 27.79 g/kg, total N 1.90 g/kg, and total P 0.79 g/kg, total K 19.76 g/kg. The NaOH hydrolyzed N was 108.31 mg/kg of soil, Olsen-P 15.85 mg/kg of soil, available K 19.76 g/kg of soil with pH 5.67. Urea was used as N fertilizer, calcium magnesium phosphate as P fertilizer (containing P2O5 12%) and potassium chloride (containing K2O 60%) as K fertilizer.

The experiment had two N treatments: N application and no N application, 16 oilseed rape varieties used as plant materials, 32 treatments, 3 replicates, 96 districts, 10.5 m2 per district, randomized block. N application treatment: 225kg N, 75kg P2O5 and150 kg K2O per hectare, 50% N fertilizer used as basal fertilizer, 20% N fertilizer used as added fertilizer during winter, 30% N fertilizer used at stem elongation stage, all P and K fertilizer were used as basal fertilizer; no N application treatment: the same with N application treatment except of non-N fertilizer applied. Seedling on Sep. 25 2004, transplanted on Nov. 2 2004, transplanted density was 100 thousand plants per ha, management as normal.

Nitrogen Efficiency in Oilseed Rape and Its Physiological Mechanism 67

distinguish between N redistributed and N taken up. Sampling schedule for the other stages

Flowering

Root, Stem, Leaf, Flower

Whole plants were sampled according to the design indicated in Table 1. There were 6 replications (randomly selected were 6 plants per sampling time per variety), subsamples were taken from different tissues after clearing and drying. Biomass dried to a constant weight was measured. Dry samples were ground and sifted for isotope analysis. To measure biomass and calculate total N exactly, fallen leaves were collected in time. The concentration of N in the plant was measured using the Kjeldahl method (Kamprath et al., 1982); plant total N, grain total N, harvesting index and N harvesting index were calculated using the

Total N in plant = plant biomass × N concentration in plant tissues

Grain total N = grain yield × N concentration in grain

Harvesting index = grain yield/biomass

N harvesting index = grain total N/ total N in plant The samples were pre-processed by semi micro determination (Brenna et al., 1998). The excess 15N in each tissue was measured by the use of a continuous flow isotope mass spectrograph at the Institute of Genetics, Hebei Academy of Agricultural Science. The amount and proportion of redistributed N from the vegetative organs to the reproductive

N redistribution proportion = 15N accumulated amount in reproductive organs/15N accumulated amount in the plant at the end of the labeling treatment×100 % (Because the flower and silique are reproductive organs of the plant, labeling was done at the flowering and siliquing stages, the 15N accumulated amount in the reproductive organs should be

Sampling time

Silique

Silique

silique

stage Siliquing stage Harvesting stage

Root, stem, leaf, silique husk, grain

Root, stem, leaf, silique husk, grain

Root, stem, leaf, silique husk, grain

Root, stem, leaf, silique husk, grain

Root, stem, leaf,

Root, stem, leaf,

Root, stem, leaf,

are shown in the text and this table.

Pot number

 4×6=24

stage, 9 days 2×6=12 Root, stem,

3×6=18 Root, stem, leaf

leaf

3×6=18 Root, stem,

**2.2.3 Sampling, determination and calculation** 

Labeling end stage

Root, stem,

leaf, flower

leaf, silique

Table 1. Time of labeling (15N) and sampling scheme at each growth stages

Days for 15N labeling

Seedling stage, 21 days

Flowering stage, 5 days

Siliquing

following formulas:

organs were calculated as follows:

Stem elongation stage, 5 days

#### **2.1.2 Sampling, determination and calculation**

Sampled whole plant at harvesting stage, separated it according to organs after clear, dried until constant weight, measured dry weight and total N. The measurement methods followed by the general agricultural chemistry analysis methods; the plant total N measured by Kjedahl method (Hardy et al., 1968). Below formula was used to calculate N use efficiency based on total N, N use efficiency=grain yield / plant total N. Data has been analysis by T-test using SPSS statistic software (Norusis, 1990), the significant differences between the two varieties (p<0.05) are figured by stars (\*) in Table 6.

#### **2.2 Vegetative N redistribution during later growing period and its contribution to N efficiency**

#### **2.2.1 Variety tested and crop culture**

High N use efficiency variety X-36 and low N use efficiency variety X-50 were used as plant materials and grown in a glasshouse in the field experiment station of Hunan Agricultural University (Southern China) with average temperature 19 °C and natural light. These materials were selected from 30 varieties that have been evaluated in the last 10 years in Hunan Province. All the varieties were provided by the Hunan Sub-center of the Improvement Center of the National Oil Crop.

The sand culture and 15N labeling method were used in this experiment. On a 30 cm × 30 cm brown plastic bowl, sand culture (water and dilute hydrochloric acid were used to clean the growth matrix) with complete nutrient solution and normal N supply was used as growth medium. The culture solution had these components: KNO3 5 mmol·L-1, KH2PO4 1 mmol·L-1, MgSO4 7 mmol·L-1, Ca(NO3)2·4H2O 5 mmol·L-1, Fe-EDTA 3 mmol·L-1, B 0.5 mg·L-1, Mn 0.5 mg·L-1, Zn 0.05 mg·L-1, Cu 0.02 mg·L-1, Mo 0.01 mg·L-1, Ca(15NO3)2 and K15NO3 (Shanghai Chemical Engineering Corporation Research Institute) with 15N excess = 20.28% were used as N sources for the labeling treatment.

#### **2.2.2 Experimental treatment and labeling**

The seedlings were planted on Sept 25, 2005; transplanted on Nov 2, 2005; one plant per bowl, 72 bowls per variety and harvested on May 5, 2006. 15N labeled days and time of sampling are shown in Table 1. Labeling was done at the seedling stage (Dec 2-22, 2005; 21 d), stem elongation stage (Jan 21-25, 2006; 5 d), flowering stage (Mar 3-7, 2006; 5 d) and siliquing stage (Apr 15-23, 2006; 9 d) . Samples were taken at the end of each labeling stage (Dec 23, 2005; Jan 26, 2006; Mar 8, 2006; Apr 24, 2006). The other plants were transplanted into sand culture with no 15N nutrient and sampled during later growth stages (flowering stage, Mar 8, 2006; siliquing stage, Apr 24, 2006; harvesting stage, May 5, 2006; Table 1), in order to distinguish between N redistributed and N taken up.

Labeling end stage in Table 1 means the end of each labeling treatment at four growth stages. Take seedling stage for instance. Labeling treatment was done at the seedling stage (Dec 2-22, 2005; 21 d); labeling treatment application on Dec 2, 2005; and finished on Dec 22, 2005. Sampling time at labeling end stage means 6 plants per variety were sampled on Dec 23, 2005. The other plants (12) were transplanted into sand culture with no 15N nutrient and sampled at siliquing stage (Apr 24, 2006) and harvesting stage (May 5, 2006), in order to

Sampled whole plant at harvesting stage, separated it according to organs after clear, dried until constant weight, measured dry weight and total N. The measurement methods followed by the general agricultural chemistry analysis methods; the plant total N measured by Kjedahl method (Hardy et al., 1968). Below formula was used to calculate N use efficiency based on total N, N use efficiency=grain yield / plant total N. Data has been analysis by T-test using SPSS statistic software (Norusis, 1990), the significant differences

**2.2 Vegetative N redistribution during later growing period and its contribution to N** 

High N use efficiency variety X-36 and low N use efficiency variety X-50 were used as plant materials and grown in a glasshouse in the field experiment station of Hunan Agricultural University (Southern China) with average temperature 19 °C and natural light. These materials were selected from 30 varieties that have been evaluated in the last 10 years in Hunan Province. All the varieties were provided by the Hunan Sub-center of the

The sand culture and 15N labeling method were used in this experiment. On a 30 cm × 30 cm brown plastic bowl, sand culture (water and dilute hydrochloric acid were used to clean the growth matrix) with complete nutrient solution and normal N supply was used as growth medium. The culture solution had these components: KNO3 5 mmol·L-1, KH2PO4 1 mmol·L-1, MgSO4 7 mmol·L-1, Ca(NO3)2·4H2O 5 mmol·L-1, Fe-EDTA 3 mmol·L-1, B 0.5 mg·L-1, Mn 0.5 mg·L-1, Zn 0.05 mg·L-1, Cu 0.02 mg·L-1, Mo 0.01 mg·L-1, Ca(15NO3)2 and K15NO3 (Shanghai Chemical Engineering Corporation Research Institute) with 15N excess = 20.28% were used

The seedlings were planted on Sept 25, 2005; transplanted on Nov 2, 2005; one plant per bowl, 72 bowls per variety and harvested on May 5, 2006. 15N labeled days and time of sampling are shown in Table 1. Labeling was done at the seedling stage (Dec 2-22, 2005; 21 d), stem elongation stage (Jan 21-25, 2006; 5 d), flowering stage (Mar 3-7, 2006; 5 d) and siliquing stage (Apr 15-23, 2006; 9 d) . Samples were taken at the end of each labeling stage (Dec 23, 2005; Jan 26, 2006; Mar 8, 2006; Apr 24, 2006). The other plants were transplanted into sand culture with no 15N nutrient and sampled during later growth stages (flowering stage, Mar 8, 2006; siliquing stage, Apr 24, 2006; harvesting stage, May 5, 2006; Table 1), in

Labeling end stage in Table 1 means the end of each labeling treatment at four growth stages. Take seedling stage for instance. Labeling treatment was done at the seedling stage (Dec 2-22, 2005; 21 d); labeling treatment application on Dec 2, 2005; and finished on Dec 22, 2005. Sampling time at labeling end stage means 6 plants per variety were sampled on Dec 23, 2005. The other plants (12) were transplanted into sand culture with no 15N nutrient and sampled at siliquing stage (Apr 24, 2006) and harvesting stage (May 5, 2006), in order to

**2.1.2 Sampling, determination and calculation** 

**2.2.1 Variety tested and crop culture** 

Improvement Center of the National Oil Crop.

as N sources for the labeling treatment.

**2.2.2 Experimental treatment and labeling** 

order to distinguish between N redistributed and N taken up.

**efficiency** 

between the two varieties (p<0.05) are figured by stars (\*) in Table 6.

Days for 15N labeling Pot number Sampling time Labeling end stage Flowering stage Siliquing stage Harvesting stage Seedling stage, 21 days 3×6=18 Root, stem, leaf Root, stem, leaf, Silique Root, stem, leaf, silique husk, grain Stem elongation stage, 5 days 4×6=24 Root, stem, leaf Root, Stem, Leaf, Flower Root, stem, leaf, Silique Root, stem, leaf, silique husk, grain Flowering stage, 5 days 3×6=18 Root, stem, leaf, flower Root, stem, leaf, silique Root, stem, leaf, silique husk, grain Siliquing stage, 9 days 2×6=12 Root, stem, leaf, silique Root, stem, leaf, silique husk, grain

distinguish between N redistributed and N taken up. Sampling schedule for the other stages are shown in the text and this table.

Table 1. Time of labeling (15N) and sampling scheme at each growth stages

#### **2.2.3 Sampling, determination and calculation**

Whole plants were sampled according to the design indicated in Table 1. There were 6 replications (randomly selected were 6 plants per sampling time per variety), subsamples were taken from different tissues after clearing and drying. Biomass dried to a constant weight was measured. Dry samples were ground and sifted for isotope analysis. To measure biomass and calculate total N exactly, fallen leaves were collected in time. The concentration of N in the plant was measured using the Kjeldahl method (Kamprath et al., 1982); plant total N, grain total N, harvesting index and N harvesting index were calculated using the following formulas:

Total N in plant = plant biomass × N concentration in plant tissues

Grain total N = grain yield × N concentration in grain

Harvesting index = grain yield/biomass

N harvesting index = grain total N/ total N in plant

The samples were pre-processed by semi micro determination (Brenna et al., 1998). The excess 15N in each tissue was measured by the use of a continuous flow isotope mass spectrograph at the Institute of Genetics, Hebei Academy of Agricultural Science. The amount and proportion of redistributed N from the vegetative organs to the reproductive organs were calculated as follows:

N redistribution proportion = 15N accumulated amount in reproductive organs/15N accumulated amount in the plant at the end of the labeling treatment×100 % (Because the flower and silique are reproductive organs of the plant, labeling was done at the flowering and siliquing stages, the 15N accumulated amount in the reproductive organs should be

Nitrogen Efficiency in Oilseed Rape and Its Physiological Mechanism 69

were lower (≤1105 kg per hm2 ). N response can be expressed by ratio of (yield with N application – yield with no N application) / N application amount. Because the N application amount of all varieties under N application treatment were the same in this study; so, the N response can be indirectly expressed by the differences of yield under N application condition and no N application condition. N responses of Xy13, Xh19 and Xh20 were higher than other varieties significantly; N responses of Xy14, Xy15 were lower than other varieties significantly. In the case of Xy15, grain yield can be increased by N

application significantly, average increased of yield in sixteen cultivars were 35%.

varieties was 7.1-12.6, the highest was Xh20 of 12.6, and the lowest was Xy9 of 7.9.

Table 3. N absorption and use efficiency of different oilseed rape varieties

Xy1 1.744a 2.200a 11.0bc 11.4abc Xy6 1.467d 2.207a 7.7ef 7.4e Xy7 1.448d 2.209a 13.2a 10.4bc Xy8 1.254e 2.151a 8.8def 7.6e Xy9 1.285e 2.186a 8.2ef 7.1e Xy11 1.456d 2.154a 7.3f 7.7e Xy12 1.545bc 2.149a 11.2abc 9.6cd Xy13 1.293e 2.245a 10.7bcd 10.8abc Xy14 1.462d 2.192a 11.4abc 8.1de Xy15 1.518cd 2.177a 11.5ab 7.9de Xy16 1.489cd 2.177a 11.4abc 10.5bc Xy17 1.500cd 2.158a 12.1ab 11.4abc Xh19 1.550bc 2.206a 11.6ab 12.3ab Xh20 1.603b 2.224a 11.9ab 12.6a Xy21 1.688a 2.224a 10.9bc 11.2abc Xy24 1.688a 2.242a 9.4cde 8.6de

N absorption (g/plant) N use efficiency (g/g) No N application N application No N application N application

Variety No.

**3.2 N absorption amount and N use efficiency in different oilseed rape varieties** 

Results of table3 showed the relationship between N absorption amount and grain yield. N absorbed amount of different oilseed rape varieties were different under different N application level conditions. The differences of N absorption amount between varieties were significant under no N application condition; however, the differences were smaller under N application condition. N absorption amount of Xy1, Xy21 and Xy24 (≥1.688g per plant) were more than other varieties Xy8, Xy9 and Xy13 (≤1.293g per plant) significantly. The relationship between N absorption amount and grain yield showed that, the correlation between N absorption amount and grain yield was significant under no N application condition, correlation coefficient was 0.685\*\* (R0.01=0.623); but under N application condition, there was no significant correlation, and the correlation coefficient was 0.415(R0.05=0.497). This indicates that, only under the no N application condition, grain yield can be increased significantly by increased N absorption amount. The differences of N use efficiency between varieties were significant according to the differences of N absorption amount, regardless of N application level. The changes scope of N use efficiency in sixteen

subtracted from the 15N accumulated amount in the reproductive organs at the end of the labeling treatment.);

N redistribution amount = N redistribution proportion × N accumulated amount in the plant at the end of labeling treatment;

N loss proportion = [15N accumulated amount in the plant at the end of the labeling treatment - 15N accumulated amount in the plant at harvesting stage (or siliquing stage)]/15N accumulated amount in the plant at the end of labeling treatment×100 %;

N loss amount = proportion of N loss × N accumulated amount in the plant at the end of the labeling treatment

The partitioning of absorbed N was calculated from the excess 15N in each tissue combined with the calculated total plant N uptake. This is based on the assumption that unlabeled N from the sand culture was taken up and allocated in the different plant tissues in a way similar to that of labeled N. All data were analyzed using the T-test by SPSS statistic software. The significant differences between the two varieties (p<0.05) were indicated by asterisks (\*) in the table.

#### **3. Results**

#### **3.1 Grain yield between different oilseed rape varieties**

The results of grain yield showed that, the differences of grain yield between different oilseed rape varieties were significant, regardless N application level (Table2). The rang of yield variation in 16 varieties were 1060-1913 kg per hm2 under no N application condition; the yields of Xy1, Xy7 and Xh20 were higher (≥2720kg per hm2), the yields of Xy8, Xy9, Xy11


Different letters in the same row indicated that differences between varieties in t-test are significant at p<0.05 level. The same as below.

Table 2. Grain yield of different oilseed rape varieties

subtracted from the 15N accumulated amount in the reproductive organs at the end of the

N redistribution amount = N redistribution proportion × N accumulated amount in the

N loss proportion = [15N accumulated amount in the plant at the end of the labeling treatment - 15N accumulated amount in the plant at harvesting stage (or siliquing stage)]/15N accumulated amount in the plant at the end of labeling treatment×100 %;

N loss amount = proportion of N loss × N accumulated amount in the plant at the end of the

The partitioning of absorbed N was calculated from the excess 15N in each tissue combined with the calculated total plant N uptake. This is based on the assumption that unlabeled N from the sand culture was taken up and allocated in the different plant tissues in a way similar to that of labeled N. All data were analyzed using the T-test by SPSS statistic software. The significant differences between the two varieties (p<0.05) were indicated by

The results of grain yield showed that, the differences of grain yield between different oilseed rape varieties were significant, regardless N application level (Table2). The rang of yield variation in 16 varieties were 1060-1913 kg per hm2 under no N application condition; the yields of Xy1, Xy7 and Xh20 were higher (≥2720kg per hm2), the yields of Xy8, Xy9, Xy11

Variety No. No N application N application N application - No N

Xy1 1913a 2490ab 577bc Xy6 1132de 1630e 498bcd Xy7 1908a 2290bc 382cd Xy8 1105e 1620e 515bcd Xy9 1060e 1550e 490bcd Xy11 1063e 1650e 587bc Xy12 1730ab 2070cd 340d Xy13 1381cd 2429abc 1048a Xy14 1659ab 1770de 111e Xy15 1742ab 1720de -22e Xy16 1696ab 2290bc 594bc Xy17 1813ab 2445ab 632b Xh19 1801ab 2720a 918a Xh20 1908a 2786a 878a Xy21 1837ab 2480ab 643b Xy24 1588bc 1920de 332d Different letters in the same row indicated that differences between varieties in t-test are significant at

application

labeling treatment.);

labeling treatment

asterisks (\*) in the table.

p<0.05 level. The same as below.

Table 2. Grain yield of different oilseed rape varieties

**3.1 Grain yield between different oilseed rape varieties** 

**3. Results** 

plant at the end of labeling treatment;

were lower (≤1105 kg per hm2 ). N response can be expressed by ratio of (yield with N application – yield with no N application) / N application amount. Because the N application amount of all varieties under N application treatment were the same in this study; so, the N response can be indirectly expressed by the differences of yield under N application condition and no N application condition. N responses of Xy13, Xh19 and Xh20 were higher than other varieties significantly; N responses of Xy14, Xy15 were lower than other varieties significantly. In the case of Xy15, grain yield can be increased by N application significantly, average increased of yield in sixteen cultivars were 35%.

#### **3.2 N absorption amount and N use efficiency in different oilseed rape varieties**

Results of table3 showed the relationship between N absorption amount and grain yield. N absorbed amount of different oilseed rape varieties were different under different N application level conditions. The differences of N absorption amount between varieties were significant under no N application condition; however, the differences were smaller under N application condition. N absorption amount of Xy1, Xy21 and Xy24 (≥1.688g per plant) were more than other varieties Xy8, Xy9 and Xy13 (≤1.293g per plant) significantly. The relationship between N absorption amount and grain yield showed that, the correlation between N absorption amount and grain yield was significant under no N application condition, correlation coefficient was 0.685\*\* (R0.01=0.623); but under N application condition, there was no significant correlation, and the correlation coefficient was 0.415(R0.05=0.497). This indicates that, only under the no N application condition, grain yield can be increased significantly by increased N absorption amount. The differences of N use efficiency between varieties were significant according to the differences of N absorption amount, regardless of N application level. The changes scope of N use efficiency in sixteen varieties was 7.1-12.6, the highest was Xh20 of 12.6, and the lowest was Xy9 of 7.9.


Table 3. N absorption and use efficiency of different oilseed rape varieties

Nitrogen Efficiency in Oilseed Rape and Its Physiological Mechanism 71

absorption efficiency in oilseed rape under the field condition, regardless of N application level. The differences of variation coefficient between N absorption amount and N use efficiency were smaller under no N application condition, compared with N application condition. It was suggested that variation coefficient of N use efficiency was declined, but variation coefficient of N absorption amount was increased under N stress condition. This indicates that, the contribution of N absorption efficiency to N efficiency was increased with N application level, but the contribution of N use efficiency to N efficiency was decreased.

Table 6 results showed that there is no significant difference of N content in plant and grain between X-36 and X-50, in addition, biomass, harvesting indexes and total N amount of X-36 are almost the same with X-50. However, grain yield and N harvesting indexes of X-36 are higher than X-50 significantly. This indicates that the reasons for grain yield of X-36 being higher than X-50 was not greater N absorption and harvesting indexes but rather higher N

> N concentration of grain (mg◌ּ g-1DW)

X-36 119.8±9.5 31.0±2.6\* 27.0±0.9 44.2±1.8 3.22±0.16 1.37±0.07 0.26±0.024 0.43±0.029\* X-50 111.4±8.7 25.7±2.9 28.1±1.8 45.5±1.7 3.12±0.23 1.17±0.14 0.23±0.018 0.37±0.029

Indicated that differences between varieties in t-test are significant at p<0.05 level. The same as below.

Fig.1 results showed that N absorbed at seedling and stem elongation stages are mainly distributed into leaves, average proportions of transferred-N/total-N in two varieties are accounting for 83.5% and 66.3% respectively. N absorbed at flowering stage is mainly distributed into leaves and stem, proportions are accounting for 42.8% and 36.3% respectively. While 42% (average values of two cultivars) of N absorbed at siliquing stage is directly distributed into silique. Proportion of N distributed into leaves is declined on a large scale with senescence of plant, proportion of N distributed into stem and root are declined also, however proportion of N distributed into silique and grain are increased at the same time. Proportion of N absorbed at seedling stage is redistributed from vegetative organs to grain in X-50 is the lowest, but still 43% of N is distributed into grain at harvesting

In addition, proportion of N that was distributed into leaves at flowering stage in X-36 and X-50 are 47.3%, 40.7% respectively, and siliquing stages are 30.6%, 25.8% respectively. Proportion of N that was distributed into grain at harvesting stage in X-36 are higher than X-50, and proportion differences between the two varieties of N absorbed at seedling and flowering stages are significant (Fig.1 and Table7). Higher proportion of N distributed into leaves is a benefit for normal functions of leaves development, higher proportion of N

Total N in plant (g◌ּ plant1)

Total N in grain (g◌ּ plant1)

Harvesting index

N harvesting index

**3.4 Biomass and N absorption of the two oilseed rape varieties** 

N concentration of plant (mg◌ּ g-1DW)

Table 6. Physiological parameters of two oilseed rape varieties

**3.5 Distribution characters of N absorbed at different growth stages** 

distributed into grain is benefit for N harvesting indexes improvement.

redistribution amount and higher N harvesting index.

Grain yield (g◌ּ plant1)

Variety Biomass (g◌ּ plant1)

\*

stage.

#### **3.3 The differences of N efficiency in different varieties and its analysis**

Results of Table 2 and Table 3 showed that, the differences of N efficiency, N absorption efficiency and N use efficiency between varieties were significant under no N application condition; and the differences of N efficiency and N use efficiency between varieties were significant under N application condition; while there were no differences of N absorption efficiency between varieties. Grain yield of high N efficiency variety is higher than average yield of 16 varieties under no N application condition; otherwise it was low N efficiency variety. D-value of grain yield of high N response variety between no N application and N application conditions is higher than average D-value of 16 varieties; otherwise it is low N response variety. The 16 varieties can be divided into 4 genotypes according to the definitions (Table4): (1) N high efficiency – N high response; yield was higher under no N application condition; yield was increased significantly under N application condition (see varieties Xy1,Xy16,Xy17,Xh19,Xh20 and Xy21); (2) N low efficiency – N low response; yield was lower under no N application condition; yield increase was not distinct under N application condition; (see varieties Xy6, Xy8 and Xy9); (3) N high efficiency – N low response, yield was higher under no N application condition; but the yield increase was not distinct under N application condition (see varieties Xy7,Xy12,Xy14,Xy15 and Xy24); (4) N low efficiency – N high response; yield was lower under no N application condition, but the yield was increased significantly under N application condition (see varieties Xy11 and Xy13).



Results of Table5 showed that, variation coefficient of grain yield were almost the same regardless of N application level. Variation coefficient of N use efficiency was higher than variation coefficient of N absorption amount under the two N application level conditions. It was suggested that, the contribution of N use efficiency to N efficiency was higher than N


Table 5. Variation of Grain yield, N absorption and use efficiency of different oilseed rape varieties

Results of Table 2 and Table 3 showed that, the differences of N efficiency, N absorption efficiency and N use efficiency between varieties were significant under no N application condition; and the differences of N efficiency and N use efficiency between varieties were significant under N application condition; while there were no differences of N absorption efficiency between varieties. Grain yield of high N efficiency variety is higher than average yield of 16 varieties under no N application condition; otherwise it was low N efficiency variety. D-value of grain yield of high N response variety between no N application and N application conditions is higher than average D-value of 16 varieties; otherwise it is low N response variety. The 16 varieties can be divided into 4 genotypes according to the definitions (Table4): (1) N high efficiency – N high response; yield was higher under no N application condition; yield was increased significantly under N application condition (see varieties Xy1,Xy16,Xy17,Xh19,Xh20 and Xy21); (2) N low efficiency – N low response; yield was lower under no N application condition; yield increase was not distinct under N application condition; (see varieties Xy6, Xy8 and Xy9); (3) N high efficiency – N low response, yield was higher under no N application condition; but the yield increase was not distinct under N application condition (see varieties Xy7,Xy12,Xy14,Xy15 and Xy24); (4) N low efficiency – N high response; yield was lower under no N application condition, but the yield was increased

Type NHE-NHR NLE-NLR NHE-NLR NLE-NHR

Xy7 Xy12 Xy14 Xy15 Xy24 Xy11 Xy13

Xy6 Xy8 Xy9

Results of Table5 showed that, variation coefficient of grain yield were almost the same regardless of N application level. Variation coefficient of N use efficiency was higher than variation coefficient of N absorption amount under the two N application level conditions. It was suggested that, the contribution of N use efficiency to N efficiency was higher than N

N Levels Mean S CV(%)

Subtraction of both 533 277 52.1

Table 5. Variation of Grain yield, N absorption and use efficiency of different oilseed rape

Grain yield (kg/hm2) 1584 323 20.4 N absorption (g/Plant) 1.499 0.142 9.5 N use efficiency (g/g) 10.5 1.7 16.2

Grain yield (kg/hm2) 2116 424 20.0 N absorption (g/Plant) 2.194 0.031 1.4 N use efficiency (g/g) 9.7 1.9 19.5

**3.3 The differences of N efficiency in different varieties and its analysis** 

significantly under N application condition (see varieties Xy11 and Xy13).

Xy1 Xy16 Xy17 Xy19 Xy20 Xy21

Table 4. N efficiency and response type of oilseed rape

Variety

No N application

N application

varieties

absorption efficiency in oilseed rape under the field condition, regardless of N application level. The differences of variation coefficient between N absorption amount and N use efficiency were smaller under no N application condition, compared with N application condition. It was suggested that variation coefficient of N use efficiency was declined, but variation coefficient of N absorption amount was increased under N stress condition. This indicates that, the contribution of N absorption efficiency to N efficiency was increased with N application level, but the contribution of N use efficiency to N efficiency was decreased.

## **3.4 Biomass and N absorption of the two oilseed rape varieties**

Table 6 results showed that there is no significant difference of N content in plant and grain between X-36 and X-50, in addition, biomass, harvesting indexes and total N amount of X-36 are almost the same with X-50. However, grain yield and N harvesting indexes of X-36 are higher than X-50 significantly. This indicates that the reasons for grain yield of X-36 being higher than X-50 was not greater N absorption and harvesting indexes but rather higher N redistribution amount and higher N harvesting index.


\* Indicated that differences between varieties in t-test are significant at p<0.05 level. The same as below.

Table 6. Physiological parameters of two oilseed rape varieties

#### **3.5 Distribution characters of N absorbed at different growth stages**

Fig.1 results showed that N absorbed at seedling and stem elongation stages are mainly distributed into leaves, average proportions of transferred-N/total-N in two varieties are accounting for 83.5% and 66.3% respectively. N absorbed at flowering stage is mainly distributed into leaves and stem, proportions are accounting for 42.8% and 36.3% respectively. While 42% (average values of two cultivars) of N absorbed at siliquing stage is directly distributed into silique. Proportion of N distributed into leaves is declined on a large scale with senescence of plant, proportion of N distributed into stem and root are declined also, however proportion of N distributed into silique and grain are increased at the same time. Proportion of N absorbed at seedling stage is redistributed from vegetative organs to grain in X-50 is the lowest, but still 43% of N is distributed into grain at harvesting stage.

In addition, proportion of N that was distributed into leaves at flowering stage in X-36 and X-50 are 47.3%, 40.7% respectively, and siliquing stages are 30.6%, 25.8% respectively. Proportion of N that was distributed into grain at harvesting stage in X-36 are higher than X-50, and proportion differences between the two varieties of N absorbed at seedling and flowering stages are significant (Fig.1 and Table7). Higher proportion of N distributed into leaves is a benefit for normal functions of leaves development, higher proportion of N distributed into grain is benefit for N harvesting indexes improvement.

Nitrogen Efficiency in Oilseed Rape and Its Physiological Mechanism 73

Table7 results showed that distribution proportion of N absorbed at stem elongation and flowering stages are higher than other stages, average proportion at stem elongation and flowering stages are 44.3% , 41.2% respectively, and 34.4% , 31.7% respectively at seedling and siliquing stages. Distribution amount of N absorbed at stem elongation stage is the highest, average value is 325.8 mg, following are N absorbed at flowering and seedling stages, average values are 218.0 mg and 203.2 mg respectively, the lowest is N absorbed at

Average proportion of transferred-N/grain-N in two varieties is 65.1%. Distribution proportion of N absorbed at stem elongation stage is the highest, accounting for 25.8%, following are N absorbed at flowering and seedling stages, accounting for 16.9 % and 15.9% respectively, the lowest is N absorbed at siliquing stage, accounting for 6.4%. Table3 results also showed that distribution amount of N absorbed at seedling, flowering and harvesting stages in X-36 are higher than X-50 significantly, distribution proportion of N absorbed at

N absorbed at seedling and stem elongation stages that was distributed into reproductive organs, it is real N that redistributed from vegetative organs to reproductive organs. However, part of N absorbed at flowering and siliquing stages was directly transferred into reproductive organs, therefore total N in reproductive organs is not only because of N redistribution, but also because of root absorption. So, differences amount of N, which was absorbed at flowering and siliquing stages, between absorption and redistribution can not be distinguished in this

Table 8 results showed that loss proportion and amount of N absorbed at seedling stage are the largest, average values of the two varieties are 24.0% and 141.6 mg, the next is N absorbed at stem elongation and flowering stages, average proportion values are 10.5% and 11.7%, average amount values are 79.2 mg and 43.2 mg respectively, the smallest loss are N absorbed at siliquing stage, average values are 7.3% and 16.2 mg. Table 4 also showed that N loss proportion and amount of X-36 are lower than X-50, but there are no significant different. Loss amount of N absorbed at stem elongation and flowering stages in X-36 (63.0 and 35.0 mg) are lower than X-50 (95.3 and 51.4 mg) significantly. Total loss amount of N absorbed at the four growth stages in X-36 (252.3 mg) is 18.1% lower than X-50 (307.9 mg).

> N absorbed at stem elongation stage

X-36 22.8 9.0 9.6 8.5 X-50 25.1 12.0 13.8 6.1

Table 8. Loss amount and proportions of N at harvesting stage (N absorbed at different stages)

N absorbed at flowering stage

X-36 136.4 63.0 35.0 17.9 252.3 X-50 146.7 95.3\* 51.4\* 14.5 307.9

N absorbed at siliquing stage

Sum

**3.6 Distribution proportion of N absorbed at different growth stages** 

seedling and flowering stages in X-36 are higher than X-50 significantly.

study, but the main results can not be affected by these influences significantly.

**3.7 Loss amount and proportion of N at harvesting stage** 

N absorbed at seedling stage

siliquing stage, average value is 82.0 mg.

Physiological

N Loss amount plant -1 (mg)

N Loss proportion (%)

indexes Variety

Fig. 1. Distribution proportion of N which absorbed at different growth stages in oilseed rape (vertical axis is stand for N distribution proportion among plant organs, lateral axis is stand for different growth stages)


Table 7. Translocation to grain of N which absorbed at different stages

0%

N absorbed at seedling stage N absorbed at flowering stage

0%

N absorbed at stem elongation stage

X-36 38.2\* 46.8 49.6\* 32.8 X-50 30.6 41.8 32.8 30.6

 N absorbed at stem elongation stage N absorbed at siliquing stage Fig. 1. Distribution proportion of N which absorbed at different growth stages in oilseed rape (vertical axis is stand for N distribution proportion among plant organs, lateral axis is

20%

40%

60%

80%

100%

X-36 X-50 X-36 X-50 X-36 X-50 flowering stage siliquing stage harvesting stage

root stem leaf silique/silique husk grain

X-36 X-50 X-36 X-50 siliquing stage harvesting stage

> N absorbed at siliquing stage

Sum

N absorbed at flowering stage

X-36 231.2\* 325.5 275.5\* 88.9\* 921.1 X-50 175.1 326.0 160.4 75.0 736.5

X-36 16.9 23.8 20.1\* 6.5 67.2 X-50 15.0 27.9 13.7 6.4 62.9

root stem leaf flower/silique/silique husk grain

20%

40%

60%

80%

100%

0%

0%

stem elongation stage

Physiological

Amount of N transferred (mg )

Proportion of N transferred (%)

Proportion of transferred-N/ grain-N (%)

stand for different growth stages)

indexes Variety

20% 40%

60% 80%

100%

X-36 X-50 X-36 X-50 X-36 X-50 seedling stage siliquing stage harvesting stage

root stem leaf flower/silique/silique husk grain

X-36 X-50 X-36 X-50 X-36 X-50 X-36 X-50

flowering stage siliquing stage harvesting stage

N absorbed at seedling stage

Table 7. Translocation to grain of N which absorbed at different stages

root stem leaf silique/silique husk grain

20%

40%

60%

80%

100%

#### **3.6 Distribution proportion of N absorbed at different growth stages**

Table7 results showed that distribution proportion of N absorbed at stem elongation and flowering stages are higher than other stages, average proportion at stem elongation and flowering stages are 44.3% , 41.2% respectively, and 34.4% , 31.7% respectively at seedling and siliquing stages. Distribution amount of N absorbed at stem elongation stage is the highest, average value is 325.8 mg, following are N absorbed at flowering and seedling stages, average values are 218.0 mg and 203.2 mg respectively, the lowest is N absorbed at siliquing stage, average value is 82.0 mg.

Average proportion of transferred-N/grain-N in two varieties is 65.1%. Distribution proportion of N absorbed at stem elongation stage is the highest, accounting for 25.8%, following are N absorbed at flowering and seedling stages, accounting for 16.9 % and 15.9% respectively, the lowest is N absorbed at siliquing stage, accounting for 6.4%. Table3 results also showed that distribution amount of N absorbed at seedling, flowering and harvesting stages in X-36 are higher than X-50 significantly, distribution proportion of N absorbed at seedling and flowering stages in X-36 are higher than X-50 significantly.

N absorbed at seedling and stem elongation stages that was distributed into reproductive organs, it is real N that redistributed from vegetative organs to reproductive organs. However, part of N absorbed at flowering and siliquing stages was directly transferred into reproductive organs, therefore total N in reproductive organs is not only because of N redistribution, but also because of root absorption. So, differences amount of N, which was absorbed at flowering and siliquing stages, between absorption and redistribution can not be distinguished in this study, but the main results can not be affected by these influences significantly.

#### **3.7 Loss amount and proportion of N at harvesting stage**

Table 8 results showed that loss proportion and amount of N absorbed at seedling stage are the largest, average values of the two varieties are 24.0% and 141.6 mg, the next is N absorbed at stem elongation and flowering stages, average proportion values are 10.5% and 11.7%, average amount values are 79.2 mg and 43.2 mg respectively, the smallest loss are N absorbed at siliquing stage, average values are 7.3% and 16.2 mg. Table 4 also showed that N loss proportion and amount of X-36 are lower than X-50, but there are no significant different. Loss amount of N absorbed at stem elongation and flowering stages in X-36 (63.0 and 35.0 mg) are lower than X-50 (95.3 and 51.4 mg) significantly. Total loss amount of N absorbed at the four growth stages in X-36 (252.3 mg) is 18.1% lower than X-50 (307.9 mg).


Table 8. Loss amount and proportions of N at harvesting stage (N absorbed at different stages)

Nitrogen Efficiency in Oilseed Rape and Its Physiological Mechanism 75

Grami et al. (1977) which showed that a direct relationship existed between N uptake and seed N content; they concluded that high seed N content was good for N uptake and translocation efficiency. The above results suggested that, both of N application condition and no N application conditions should be considered during the breeding and selecting

**4.3 The contribution of N absorption efficiency and N use efficiency to N efficiency**  Many studies were carried out on the in contribution of N absorption efficiency and N use efficiency to N efficiency, but the results were different (Zou et al., 2009). High seed protein content will cause low grain yield, this agrees with former results which observed negative relationship between protein content of seed and grain yield (Dudley et al., 1977; Simmonds, et al., 1995). Wheat breeders have reported selection standards combined with high yield and high protein content (Monaghan et al., 2001). Some results showed that N use efficiency was the main reason for changes of N efficiency under low N condition, and the main reason was N absorption efficiency under high N condition (Moll et al., 1982). The effect of N absorption efficiency on N efficiency was higher than N use efficiency significantly under low N condition, and the effect of N absorption efficiency and N use efficiency on N efficiency were almost the same under high N condition (Mi et al., 1998); the absorption efficiency was the main reason for changes of N efficiency regardless of N application level (Liu et al., 2002); N absorption efficiency and N use efficiency of oilseed rape were studied by Yan and Thurling (1987a), there were differences of N use efficiency between varieties under high N condition, and there were differences of N absorption efficiency between varieties under low N condition. The results of this study showed that, the changes of N use efficiency was the main reason of the changes of N efficiency under low and high N conditions, but Yan and Thurling (1987b) have different results. This study (Table 5) also showed that, variation coefficient of N absorption efficiency was increased under low N condition, while the variation coefficient of N use efficiency declined, it was suggested that, the contribution of N absorption efficiency to N efficiency was increased under low N condition. However, the results were only for the varieties which were used in this study,

procedure of crop (Liu et al., 1999; Lafitte et al., 1994).

and more varieties are necessary to be used in the future.

**4.4 N redistribution characteristics and relation to N efficiency** 

Liu, (1987) reported that N content in oilseed rape tissues at different growth stages are from 4.5% to 1.2%, N content is higher during earlier growth stages, and lower during later growth stages, N distribution and transfer in tissues can reflect plant metabolisms situation and growth center changes. The same results are showed in this study (Fig.1), N absorbed at seedling and stem elongation stages are mainly distributed into leaves, and distributed amount of N absorbed at flowering stage is lower than the former stages, 42% of N absorbed at siliquing stage was directly distributed into silique. N redistribution is happen in every part of plant tissues at different growth stages, especially after flowering stage, large amount of N are redistributed from vegetative organs to reproductive organs, these results are reported in many kinds of crops, such as wheat (Andersson et al., 2005; Palta et al., 1995a; Palta et al., 1995b; Dalling et al., 1985; Barbottin et al., 2005; Pierre et al., 2003), rice (He et al., 2002), maize (Pommel et al., 2006), pea (Séverine et al., 2005), and oilseed rape (Rossato et al., 2002; Malagoli et al., 2005). Malagoli et al., (2005) suggested that N requirements of seed filling are mainly satisfied by N mobilized from vegetative parts

Components of N can not be reflected completely in Table8. This experiment was carried out in glass room, so possibility of rain wash can be ignored. Old and dropped leaves were carefully collected during the experiment, so N loss through senescence and leaf dropped can be neglected. N loss through water exudation and dew was little also. Therefore, only N loss through N volatilization from the aboveground organs and root secretion can not be calculated in this study.

#### **4. Discussion**

#### **4.1 N efficiency in different oilseed rape varieties**

Environmental contamination was reduced by less N fertilizer application, so, breeding and spreading new crop varieties with high N efficiency was an interesting topic recently (Xu et al., 2006; Pei et al., 2007 and Wang et al., 2003). Many studies and reports were involved in N agriculture efficiency, N physiological efficiency and N absorption efficiency. N physiological efficiency and N absorption efficiency were parts of the N agriculture efficiency, there were relationships between the three efficiency indexes. Wheat breeders identified selection standards based on physiological indexes, such as high yield and high protein content (Monaghan et al., 2001). Results showed that, the differences of N efficiency in 16 oilseed rape varieties were significant, regardless of N application level. The results obtained in the present study are consistent with previous published results (Beauchamp et al., 1976; Pollmer et al., 1979; Balko et al., 1980a; Reed et al., 1980; Russell, 1984; Moll et al., 1987; Landbeck, 1995; Bertin et al., 2000). However, few oilseed rape varieties were used in these studies, more varieties were needed to be selected and estimated in the future. 16 oilseed rape varieties were used as plant materials in this experiment, N high efficiency types were better plant materials and can tolerate low N stress. These materials have high economic benefit under low N condition; N high response types were better plant materials and can tolerate high N stress, these materials have high economic benefit under high N condition. It can be seen that, the type of "N high efficiency – N high response" varieties was the ideal variety.

#### **4.2 N efficiency of oilseed rape under different N application level**

Breeding procedure was usually conducted under N application conditions recently. So, the varieties have high yield under N application condition, it was only for N application condition. It was necessary to select high N efficiency varieties under the low N stress condition, select the varieties have high yield under no N application level (Cao et al., 2010). The correlation of various agronomic traits (grain protein, yield and its component) were different with the changes of N application level (Balko et al., 1980b; Fonzo et al., 1982; Rizzi et al., 1993; Bertin et al., 2000). Therefore, the response differences of varieties under different N application level conditions were obscured; these problems are needed to be further studied in the future. Möllers et al., (2000) studies suggested that, the differences of N efficiency between different N application levels and between varieties were big, observed significant interactions between genotype and N level suggested that the high yielding genotypes in high N supply were not necessarily high yielding in the low N supply. This study has the same results, the varieties have the same yield under N application condition, but under no N application condition, the differences of yield between varieties were big. The results of the present study is different from the results of

Components of N can not be reflected completely in Table8. This experiment was carried out in glass room, so possibility of rain wash can be ignored. Old and dropped leaves were carefully collected during the experiment, so N loss through senescence and leaf dropped can be neglected. N loss through water exudation and dew was little also. Therefore, only N loss through N volatilization from the aboveground organs and root secretion can not be

Environmental contamination was reduced by less N fertilizer application, so, breeding and spreading new crop varieties with high N efficiency was an interesting topic recently (Xu et al., 2006; Pei et al., 2007 and Wang et al., 2003). Many studies and reports were involved in N agriculture efficiency, N physiological efficiency and N absorption efficiency. N physiological efficiency and N absorption efficiency were parts of the N agriculture efficiency, there were relationships between the three efficiency indexes. Wheat breeders identified selection standards based on physiological indexes, such as high yield and high protein content (Monaghan et al., 2001). Results showed that, the differences of N efficiency in 16 oilseed rape varieties were significant, regardless of N application level. The results obtained in the present study are consistent with previous published results (Beauchamp et al., 1976; Pollmer et al., 1979; Balko et al., 1980a; Reed et al., 1980; Russell, 1984; Moll et al., 1987; Landbeck, 1995; Bertin et al., 2000). However, few oilseed rape varieties were used in these studies, more varieties were needed to be selected and estimated in the future. 16 oilseed rape varieties were used as plant materials in this experiment, N high efficiency types were better plant materials and can tolerate low N stress. These materials have high economic benefit under low N condition; N high response types were better plant materials and can tolerate high N stress, these materials have high economic benefit under high N condition. It can be seen that, the type of "N high efficiency – N high response" varieties

calculated in this study.

**4.1 N efficiency in different oilseed rape varieties** 

**4.2 N efficiency of oilseed rape under different N application level** 

Breeding procedure was usually conducted under N application conditions recently. So, the varieties have high yield under N application condition, it was only for N application condition. It was necessary to select high N efficiency varieties under the low N stress condition, select the varieties have high yield under no N application level (Cao et al., 2010). The correlation of various agronomic traits (grain protein, yield and its component) were different with the changes of N application level (Balko et al., 1980b; Fonzo et al., 1982; Rizzi et al., 1993; Bertin et al., 2000). Therefore, the response differences of varieties under different N application level conditions were obscured; these problems are needed to be further studied in the future. Möllers et al., (2000) studies suggested that, the differences of N efficiency between different N application levels and between varieties were big, observed significant interactions between genotype and N level suggested that the high yielding genotypes in high N supply were not necessarily high yielding in the low N supply. This study has the same results, the varieties have the same yield under N application condition, but under no N application condition, the differences of yield between varieties were big. The results of the present study is different from the results of

**4. Discussion** 

was the ideal variety.

Grami et al. (1977) which showed that a direct relationship existed between N uptake and seed N content; they concluded that high seed N content was good for N uptake and translocation efficiency. The above results suggested that, both of N application condition and no N application conditions should be considered during the breeding and selecting procedure of crop (Liu et al., 1999; Lafitte et al., 1994).
