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Variation in nitrogen response among spring rape (Brassica napus) cultivars and its relationship to nitrogen uptake and utilization
Field Crops Research, 16 (1987) 139-155
139
Elsevier Science Publishers B.V., Amsterdam - - Printed in The Netherlands
Variation in Nitrogen Response among Spring Rape (Brassica napus) Cultivars and its Relationship to Nitrogen Uptake and Utilization S.K. YAU* and N. THURLING
School of Agriculture, University of Western Australia, Nedlands, W.A. 6009 (Australia) *Present address: ICARDA, Aleppo, Syria (Accepted 22 September 1986)
ABSTRACT Yau, S.K. and Thurling, N., 1987. Variation in nitrogen response among spring rape (Brassica napus) cultivars and its relationship to nitrogen uptake and utilization. Field Crops Res., 16: 139-155. Growth, development and yield of 40 diverse spring rape (Brassica napus) cultivars were measured at three different levels of applied nitrogen in the field. There were significant cultivar differences in yield under a limiting N supply and in yield response to higher N application rates. Cultivars with the lowest yields at the lowest N level generally responded more markedly to increase in N application rates than cultivars with higher yields in this treatment. A greater yield response was associated with relatively greater rates of growth and development at higher N levels. Measurements of dry weights and N contents of above-groundplant parts over a period between 42 and 66 days after sowing of the field experiment were used to calculate N-uptake rates and Nutilization (change in dry weight/unit change in N content) indices. Significant cultivar differences were observed only at the lowest N level for N-utilization index and only at the intermediate level for N-uptake rate. If cultivars were grouped on the basis of their geographic origin - - Japanese, Canadian, European - - those of Japanese origin generally had the highest uptake rates and utilization indices in the respective treatments in which significant differences were detected. Two solution-culture experiments were also conducted in a daylight phytotron at 20/15 ° C with the same cultivars. Significant cultivar differences in N-uptake rate and N-utilization index were detected with an initial concentration of l m M N, but not with an initial concentration of 8 mM N. Cultivar differences in root growth and N uptake per unit root dry weight were significant at both concentrations. Again, most of the cultivars with the highest uptake rates or utilization indices were of Japanese origin. Uptake rate was correlated with uptake rate per unit dry weight, but not with initial root dry weight or increase in root weight over the course of the experiment. The significance of geographic variation in N-uptake rate in both field and controlled experiments is discussed with particular reference to the use of this character as a selection criterion in breeding programs utilizing recently developed anther culture techniques.
INTRODUCTION Rapeseed (Brassica napus and B. campestris) is becoming increasingly important in the world oilseed trade as a result of improvements in oil and
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© 1987ElsevierSciencePublishersB.V.
140 meal quality. In Australia it has considerable potential as a winter crop in southern states, although commercial production levels are still low. A concerted breeding effort has led to the development of improved cultivars adapted to local environments and resistant to the destructive blackleg disease (Roy, 1982 ), but expanded production will depend on further improvements in yield with minimal additional cost to the grower to make it competitive with cereals. Rapeseed has a high nitrogen requirement and a capacity to respond to increases in applied N above 150 kg ha-1 (Holmes and Ainsley, 1977; Mason, 1979; Holmes, 1980). Improved efficiency of N usage, either through an increase in yield at current levels of N supply or maintenance of current yields with a reduction in N supply, would benefit the grower. Several reviews ( Gerloff, 1963, 1976; Vose, 1963; Epstein and Jeffries, 1964; Gabelman, 1976) have presented evidence of genetic variation in nutrient uptake within plant species sufficient to be utilized in crop improvement. Differential yield responses of genotypes to low rates of applied N have been demonstrated in many crops including oats (Frey, 1959), wheat ( Nass et al., 1976 ) and barley (Gardener and Rathjen, 1975). Cultivar differences in yield response detected in these studies were explained in terms of variation in responses of the yield components. No studies of the relationship between differential yield responses and variation in early plant growth rate have been reported to date. Genotypic differences in total N uptake have also been observed in field studies with a wide range of crops including wheat (Woodruff, 1972; Austin et al., 1977; Fischer, 1981 ), barley (Kirby, 1968), sorghum (Maranville et al., 1980), maize (Pollmer et al., 1979; Moll et al., 1982) and rape (Grami and La Croix, 1977). Significant differences in N uptake have been detected between genotypes in solution culture experiments with maize (Chevalier and Schrader, 1977; Reed and Hageman, 1980a, b ) and perennial ryegrass (Vose and Breese, 1964; Goodman, 1981). This paper reports variation in response to applied nitrogen between B. napus cultivars in field and controlled-environment experiments. Response in the field was measured primarily in terms of seed yield and N uptake and utilization estimates were based on measurements obtained prior to flowering. Nutrient solution culture experiments with the same cultivars in controlled environments provided measurements of N uptake and utilization during early plant development. Data from these different experiments were analyzed with the main objective of assessing the feasibility of using N uptake and utilization characters as selection criteria in a rapeseed improvement program. MATERIALSAND METHODS
Field experiment Forty cultivars of spring rape ( listed in Table 1 ) were grown at three levels of applied N at the University Field Station, Perth. A fertilizer mixture was
141 TABLE 1 Brassica napus cultivars used in field and phytotron experiments
Australian
Canadian
European
Japanese
Wesbell Wesreo Wesway Wesroona Marnoo
Golden Nugget Zephyr Target Turret Oro Altex Regent Midas Tower Tanka
Valekowska Croesus Gulzover Zollengold Ceska Gulliver Bronowski Masoweicki Nilla Cyzowski Gulle Komet
Chikuzen Genkai Haya Chisaya Isuzu Tokiwa Mutu Norin 14 Norin 16 Norin 17 Norin 20 China A
applied just prior to sowing. This comprised a compound N P K fertilizer (13: 13:20) and superphosphate each applied at the rate of 100 kg ha -1, and different amounts of Agran ( 34% N ) to supply additional N at the rates of 30 (N1), 60 ( N2 ) and 120 ( N3 ) kg N h a - 1 to main plots, as appropriate. As the trial was conducted on an infertile sandy soil, further applications of the compound N P K fertilizer (100 kg h a - 1) and the same amounts of Agran as incorporated in the basal dressing were made at monthly intervals after sowing until the completion of flowering. Plots were arranged in a split-plot design with four replicates, N treatments being main plots and cultivars, sub-plots. Seeds were hand-sown between 27 and 31 May into plots comprising two rows of plants placed 15 cm apart. After thinning, 16 plants spaced 5 cm apart remained in each row. The shoots of five plants were harvested from one section of each plot at 42 and 66 days after sowing (DAS), at flowering, and at maturity. With the exception of the last harvest, plants were dried at 75 °C in a forced-draught oven for 48 h and then weighed. Plants from the final harvest were air-dried in a glasshouse, weighed and finally threshed to remove seed for weighing. Total-N ( including NO3- ) concentrations in plant materials from the first and second harvests were determined using the methods of Cataldo et al. {1974). A 90-110 mg sample was used, with N in the digest being measured by an ammonia-sensing electrode ( Orion, model 95-10) connected to a digital p H / m V meter (Orion, model 707). Aliquots for measurement comprised 0.5 ml of the diluted digest, 2 ml of 0.5M tartaric acid, and 10 ml of 0.5M sodium hydroxide. Tartaric acid was added to prevent iron from precipitating and causing errors in measurement (Woodis and Cummings, 1973). The data collected were used to calculate the following:
142 Harvest index = Seed yield/total shoot dry weight; Growth rate = (D W2 - D W1 ) / ( t 2 - tl ) ; Nitrogen-uptake rate = ( N2 - N1) / ( t2 - tl ); Nitrogen-utilization index = ( D W2 - D W, ) / ( N2 - N1 ); where D W, and D W2 are total shoot dry weights, and N1 and N2 are shoot N contents, at the first and second harvests, respectively. Phytotron experiments E x p e r i m e n t 1. With the exception of Gulliver, the same cultivars were tested
in a solution experiment in a daylight phytotron at a 20 ° C-15°C day-night temperature regime on a 12-h cycle. The experiment was conducted in a complete block design with cultivars randomized within each of three replicates. Replication was achieved through repetition of the experiment over a 4-month period (December-March). The basic plot unit was a 2.5-1 black plastic container able to support a maximum of ten plants. As some plants in the first replicate did not grow well, the data from this replicate were not included in the analyses. Seeds of the 39 cultivars were germinated in moist perlite and transferred to culture vessels, following careful washing of roots, 7-8 DAS. Seedlings were placed in holes spaced evenly around the periphery of opaque and rigid lids, and held securely in position with pre-cut pieces of foam rubber. Concentrations of nutrients in the culture solution were: 1 m M N (0.5 m M KNOa and 0.25 m M NH4N03); 1 m M CaS04; 0.5 m M K2S04; 0.1 m M MgS04"7 H20; 0.5 m M K2HP04; 0.05 m M Fe EDTA; 0.1 m M NaC1; 0.1 m M MnS04" 4 H20; 0.001 m M CUS04"5 H20; 0.001 m M ZnS04"7 H20; 0.1 m M H3B03; 2 × 1 0 -4 m M COSO4"6 H20; 6 X 10 -4 m M Na~Mo204"2 H20. Six plants with 2-3 expanded leaves were harvested from each container 10-11 days after transplanting. They were then divided into shoots and roots and over-dried for 48 h at 70 ° C before weighing. A seventh plant, the smallest remaining in each container, was also removed and discarded leaving three plants for the final harvest. The culture solution in each container was then replaced with a solution containing 8 m M N (75% NO3-N and 25% NH4-N) and other elements as before. Plants remaining in each container were harvested 6 days after the initial harvest, divided into shoots and roots, oven-dried and weighed. After the final harvest, the nutrient solution was made up to its original volume with distilled water, stirred by aeration then 30 ml was removed from each container for nitrate- and ammonium-N determination by ion-specific
143 electrodes. The depletion of N from the solution was assumed to be equal to the amount of N taken up by the plants. Data collected were used to calculate the following: Nitrogen-uptake rate per unit root dry weight = [(Nuptake) × (lnRW2- lnRW1)]/[(RW2-RW1) × (t2-tl)]; Nitrogen-utilization index = ( TW2 - TW1 ) / ( N uptake) ; Root-weight ratio at final harvest = R W2/R W1; where: T W = total plant dry weight; R W = root dry weight; and t = time of harvest. The subscripts 1 and 2 refer to the first and second harvests, respectively. Experiment 2. This experiment was done with the same cultivars used in the first phytotron experiment. However, instead of replicating the experiment in time, three replicates of cultivars were grown concurrently. Ten plants of each cultivar were again grown in 2.5-1 black plastic containers in the nutrient solution as was used in the first experiment. Three plants were harvested 10 days after the transfer of seedlings to the nutrient solution and a fourth plant, the smallest of those remaining, was removed and discarded. The original solution was replaced at this time with one containing 1 m M N ( 75% NO3-N and 25% NH4-N ) and other elements at the same concentrations as in the original solution. Nitrogen concentrations of solutions in all containers were measured 48 h after replacement of the nutrient solution. The remaining plants were harvested 6 days after the initial harvest when they had 4-5 expanded leaves and were exhibiting symptoms of N deficiency. All other procedures were as for Experiment 1.
RESULTS Field experiment
Analyses of variance of yield and related characters are summarized in Table 2. Differences between cultivars and treatments were significant for all characters measured. The interaction between cultivars and N levels was significant for all characters except harvest index (HI) and growth rate during the period between 42 and 66 DAS. Variation between cultivars in seed yield in the N1 treatment and in seed yield response to the N2 and N3 treatments is shown in diagrams in Fig. 1, with the cultivars grouped on the basis of geographic origin. The range of N1 seed yields varied little between groups, although two Japanese cultivars (Norin 17; Isuzu) yielded considerably less than any other cultivar at this N level. At
144
TABLE2 Analyses of variance of yield and related characters Source
d.f. Seed yield Total dry wt. Harvest (maturity) index
Blocks 3 Nitrogen 2 levels Residual 6 Cultivars 39 Cultivars X 78 N levels Residual 351
20.2** 1457.2"*
Total dry wt. (flowering)
Flowering Growth time rate
145.5"* 13 654.9**
10.3"* 64.1"
15.8"* 1 779.8**
461.8"* 25 39.3**
9.0** 206.6**
13.3 6.2** 1.2
105.3 44.0** 20.1"*
3.3 10.6"* 1.1
6.8 8.2** 3.7**
29.8 861.4** 24.7**
3.1 0.8** 0.3
1.2
8.7
1.0
2.1
8.3
0.4
**p 0.01. the other extreme, the European cultivar Valekowska was clearly separated from other cultivars by virtue of its high yield. Differences in response to increased N supply between cultivar groups were somewhat more distinct. Thus N2 yields of seven of the twelve European cultivars tested were greater than double their respective N1 yields, whereas three of eleven Canadian and three of eleven Japanese cultivars had N2 yields double those at N1. Differences between cultivar groups were also evident in the case of seed yield response to the N3 treatment; 6 European, 4 Japanese and 2 Canadian cultivars had N3 yields more than four times greater than their N1 yields. Similar linear relationships between yield response to increased N supply and yield at the lowest N level were evident in each group, N response tending to decline with increasing N1 yield. Coefficients of correlation between responses of different plant characters to increase in N-application rates are given in Table 3. Response was expressed in terms of the ratio of the value at N2 or N3 to that at N1 with coefficients computed over the 40 cultivars. Seed yield responses were highly correlated with responses of the total shoot dry weight of the plant at maturity to both N2 and N3 treatments. Seed yield and harvest index responses to the N2 treatment were also correlated. Total shoot dry weight (maturity) responses were, in turn, highly correlated with pre-flowering growth responses. A highly significant negative correlation between responses of shoot dry weight at flowering and flowering to the N3 treatment also reflected the influence of applied N on growth rate. Analyses of variance of N-uptake rate and N-utilization index, derived from measurements over the period between 42 and 66 DAS, are given in Table 4 for each N treatment. Significant cultivar differences in uptake rate and utilization index were detected only in the N2 and N1 treatments, respectively.
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TABLE 3 Coefficientsof correlation between responses of different plant characters to increasing nitrogen levels Total dry weight (maturity)
Seed yield
N1-N2 N1-N3 N1-N2 N1-N3
0.90*** 0.84*** 0.63*** 0.36*
Total dry weight (flowering)
N1-N2 N1-N3
0.10 0.47*
Growth rate ( 42-66 days)
N1-N2 N1-N3
0.45** 0.53***
Flowering time
N1-N2 N1-N3
-0.12 - 0.53***
Growth rate
N1-N2 N1-N3
0.21 0.40*
Harvest index Total dry weight (maturity)
Total dry weight (flowering)
Variation between cultivars in N - u p t a k e rate ( N 2 ) and N-utilization index ( N1 ) are shown in Table 5. M a n y combinations of uptake and utilization characteristics were represented in the cultivars tested and only the extreme combinations of lowest-uptake/highest-utilization index and highestuptake/lowest-utilization index were not accounted for. T h e r e was some evidence of separation in cultivars in respect of geographic origin. Most of the cultivars with a high N - upt a ke rate at N2 and a high utilization index at N1 were of Japanese origin. T h e one Australian cultivar (Wesroona) also included in this group was derived from a cross involving the Japanese cultivar Norin 20. E u r o p e a n and Canadian cultivars varied widely in N uptake, but most had low to intermediate values for N-utilization index. T h e breeding of Canadian TABLE4 Analyses of variance of nitrogen uptake and utilization for each nitrogen application rate Source
d.f.
Mean squares N-uptake rate
Blocks Cultivars Residual
3 39 117
***p<0.001; *p <0.05.
N-utilization index
N1
N2
N3
N1
N2
N3
55.1"** 2.3 3.3
70.5*** 17.9" 11.6
357.0*** 34.9 37.2
34.4 43.3*** 14.3
19.7 13.5 11.2
65.3*** 6.9 4.7
147 TABLE 5 Variation in nitrogen-uptake rate (N2) and nitrogen-utilization index (N1) between B. napus cuttivars in the field (origin of cultivar in parenthesis) N-uptake rate at N2 (mg per plant per day)
N-utilization index at N1 (mg d. wt. per mg N ) Mean - 2 or more S.D. (<24.9)
Mean - 1 S.D. ( 24.9- 28.6 )
Mean + 1 S.D. ( 28.6-32.3 )
M e a n - 2 or more S.D. ( < 10.81)
Croesus (E)
Altex (C) Bronowski (E) Chikuzen (J) Masoweicki (E)
Marnoo (A) Tower (C)
Mean - 1 S.D. (10.81-12.84)
Golden ( C ) Valekowska (E)
Genkai ( J ) Gulzover (E) Midas (C) Norin 16 (J) Nugget ( C ) Wesreo (A) ZoUengold (E) Gulliver (E)
Haya (J) Regent ( C ) Wesway (A)
Norin 20 (J)
Mean+l S.D. (12.84-14.87)
Wesbell (A)
Ceska (E) Cyzowski (E) Guile (E) Nilla (E) Oro (C) Target (C) Turret ( C ) Zephyr ( C )
Chisaya (J) Komet (E) Norin 14 (J)
Mutu (J) Isuzu (J)
China A (J) Tanka ( C )
Norin 17 (J)
Tkjokiwa (J) Wesroona (A)
Mean+2 or more S.D. ( > 14.87)
Mean + 2 or more S.D. (>32.3)
cultivars has been largely based on parents of European origin. A number of cultivars characterized by low glucosinolate content of the seed - Bronowski, Altex, Tower and Marnoo - were also closely associated in respect of low N uptake and intermediate utilization index. Wesroona is also a low glucosinolate cultivar, but is clearly separated from the other cultivars with this character in respect of N uptake and utilization. Coefficients of correlation between various plant characters and N-utilization index in the N1 treatment and between the same characters and N-uptake rate in the N2 treatment are given in Table 6. In the N1 treatment the Nutilization index was significantly correlated only with the growth rate over the period between 42 and 66 DAS and the total shoot dry weight at the time
148 TABLE 6 Coefficients of correlation between growth characteristics and nitrogen-uptake/utilization measurements
Growth rate (42-66 days) Time of flowering Total shoot dry weight (flowering) Total shoot dry weight (maturity) Harvest index Seed yield
Nitrogen-utilization index (N1)
Nitrogen-uptake rate (N2)
0.57*** - 0.13 0.40* 0.27 - 0.24 0.03
0.90*** 0.04 0.44** 0.65*** - 0.10 0.44**
***p < 0.001; **p < 0.01; *p < 0.05.
of flowering. These characters, as well as seed yield and total dry weight at maturity, were correlated with N-uptake rate in the N2 treatment.
Phytotron experiments Experiment 1. Cultivar differences were significant only for N uptake per unit root dry weight and root weight ratio ( Table 7 ). When cultivars were grouped on the basis of geographic origin (as in Table 8), the variation in both these characters was found to be substantially greater between cultivars within groups than between group means. The magnitude of variation in N uptake rate per unit root dry weight was somewhat greater between cultivars in the Japanese and Australian groups than in the Canadian and European groups.
Experiment 2. Cultivar differences were highly significant for all characters measured (Table 9). The variation between cultivars was similar in magnitude in each of the geographic groups and was substantially greater than that between group means (Table 10). In the case of N-utilization index, two Japanese cultivars (Haya; Norin 16) had the highest means overall, but these were not significantly greater than those of cultivars ranked highest in each of the other three groups. However, significant differences between cultivars were detected in each geographic group. Differences in N-uptake rate between cultivar groups were not significant, but were relatively greater than group differences in N-utilization index. Eight of the eleven Japanese cultivars tested had uptake rates greater than 2.3 mg plant-1 day-1, whereas only four European and one Canadian cultivar had uptake rates greater than this value. Initial root dry weight means of the three major cultivar groups were the same, but cultivar differences within these groups were significant. The pattern of variation in N-uptake rate per unit root dry weight was similar to that of the basic uptake rate, with variation within cultivar groupings being clearly
149 TABLE 7 Analyses of variance of different plant characters measured in phytotron experiment 1 Source
Blocks Cultivars Residual
d.f.
1 38 38
Mean squares Avg. growth rate
Avg. root dry wt.
23 444* 215 225
5 518" 56 36
Root weight ratio
1 759* 70* 24
Nitrogen Uptake rate
Uptake rate per unit root dry wt.
Utilization index
56.82* 0.76 0.64
4 522* 380* 122
48.57* 0.61 0.55
*p < 0.001.
TABLE 8 Root growth and nitrogen uptake characteristics of B. napus cultivars grown in solution culture (Experiment 1 ) Geographic origin
Number of cultivars
Australian Canadian European Japanese L.S.D. (p <0.05)
5 11 11 12
N-uptake rate/unit root dry weight (rag N g 1day 1)
Root weight ratio
Mean
Range
Mean
Range
201 192 189 191
177-219 182-207 162-210 166-212
0.115 0.115 0.119 0.113
0.107-0.120 0.106-0.126 0.109-0.128 0.105-0.122 0.010
22
TABLE 9 Analyses of variance of different plant characters measured in phytotron experiment 2 Source
Blocks Cultivars Residual
d.f.
2 38 76
***p < 0.001; *p < 0.05.
Mean squares Initial root dry weight
Final root weight ratio
N-uptake rate
N-uptake/ initial root dry weight
N-utilization index
1.63 5.60*** 1.99
0.145"** 0.120"** 0.009
1.509"** 0.056*** 0.019
25 980*** 6 152"** 2 288
32.8* 29.2*** 12.4
150 TABLE 10 Root size, nitrogen uptake and utilization characteristics of B. napus cultivars grown in solution culture (Experiment 2) Character
Australian
Canadian
European
Japanese
Mean
Range
Mean
Range
Mean
Range Mean
Range
Initial root dry weight ( mg plant- ' ) LSD (p < 0.05) = 2
12
1013
13
1115
13
1215
113
1016
Final root weight ratio LSD (p<0.05)=0.02
0.20
0.190.21
0.21
0.190.22
0.23
0.190.26
0.19
0.160.22
N uptake rate (mg N plant- 1 day- 1) LSD (p<0.05)=0.22
2.33
2.232.43
2.20
1.982.33
2.23
2.022.38
2.40
2.27 2.57
N uptake/unit initial root dry wt. (mg N g- 1) LSD ( p < 0 . 0 5 ) = 7 9
399
350476
347
279403
346
324385
386
298476
N utilization index (mg mg N - 1) LSD (p<0.05) =5.8
45.8
42.748.0
44.5
37.648.1
44.9
40.048.5
46.9
42.152.0
greater than that between group means, and with Japanese and Australian cultivars having the highest values. However, some changes in cultivar rankings were also evident because of the absence of an association between root dry weight and uptake rate. Coefficients of correlation given in Table 11 showed that N-utilization index was highly correlated with N-uptake rate, whether expressed in absolute terms or relative to root dry weight. The utilization index and relative uptake rate were negatively correlated with both root weight measurements, but correlations between the root weights and uptake rate were not significant. Cultivar TABLE 11 Coefficients of correlation between characters measured in phytotron experiment 2
Initial root dry weight (1) Final root weight ratio ( 2 ) N-uptake rate (3) N-uptake rate/unit initial root dry wt. (4) N-utilization index (5) ***p < 0.001; **p < 0.01; *p < 0.05.
1
2
3
4
5
1.00
0.55*** 1.00
0.09 - 0.10 1.00
- 0.84*** - 0.57*** 0.45** 1.00
- 0.34* - 0.32* 0.59*** 0.65*** 1.00
151
differences in seed size were related to the variation in root size parameters, but no significant relationship with the utilization index or uptake rate was detected. DISCUSSION Detection of cultivar differences in yield at a low N level (N1) was encouraging in the context of breeding cultivars providing a greater economic return through reduced fertilizer costs. Seed yield in the N1 treatment was highly correlated with the total dry weight of shoots at maturity which, in turn, was related to the growth rate over the period between 42 and 66 DAS. The Nutilization index, calculated from data collected over this period, also differed significantly between cultivars in the low-N treatment and was correlated with growth rate. Thus, the ability of a spring rape genotype to yield adequately with a low N input appears to be partly dependent on a heritable capacity to utilize N efficiently for dry matter production prior to flowering. Five of the six cultivars with the highest N-utilization indices were of Japanese origin whilst the sixth, the Australian cultivar Wesroona, had the Japanese cultivar Norin 20 as one of its parents. All these Japanese cultivars had been derived from hybridization between B. napus and the related species B. campestris (Shiga, 1970), suggesting the introgression of genes for more efficient N utilization from the latter. The detection of significant cultivar differences in N-utilization index only in the low-N treatment is in accordance with the findings of a solution culture study of N response in tomatoes (O'Sullivan et al., 1974). The more efficient genotypes had lower N concentrations in old leaves but higher concentrations in the young leaves than the less efficient genotypes. Accordingly, it was concluded that N remobilization was a major determinant of N-utilization efficiency. Solution culture experiments conducted as a part of this study also showed that significant cultivar differences in N-utilization index were detectable when plants were grown in N-deficient solutions (1 m M N), but not when grown at a higher concentration (8 m M N). Nitrogen stress in the former was clearly evident from the rapid yellowing of lower leaves within five days of commencement of treatment. As whole plants were sampled for N determinations, no conclusions can be drawn regarding mechanisms responsible for cultivar differences in N-utilization efficiency. Substantial differences in response to increasing N supply were also observed between the B. napus cultivars used in this study. Overall, cultivars which yielded least in the low-N treatment tended to respond more markedly to higher rates of nitrogen supply. A greater proportion of European cultivars were low yielding under limited N supply and more responsive to higher N levels than either Canadian or Japanese cultivars. This difference possibly reflects the selection for high yield under the normally more fertile soils on which rapeseed
152 is grown in northern Europe. Responsive cultivars, measured in terms of seed yield, grew and developed at a relatively greater rate with adequate N supply than the less responsive cultivars. Thus, higher seed yields in these conditions resulted not only from a greater rate of dry matter accumulation by the plant, but also from the significant reduction in the time to flowering. The influence of the latter was evident from the correlation between the responses of yield and harvest index to higher N-application rates. Seed development on plants, which flowered earlier as a result of a higher development rate, proceeded under more favourable temperature conditions than on later-flowering plants of the same cultivar in the limited-N regime. This coincidence of flowering in B. napus with lower temperatures in Western Australia is invariably associated with a higher harvest index (Thurling, 1974). The intermediate N level in the field experiment was the only treatment in which significant cultivar differences in the rate of N uptake were detected. Failure to detect significant cultivar differences in uptake rate in the high- or low-N regimes was not unexpected. Where N supply is limited, genotypes characterized by high uptake rates would be unable to take up any more than those with low uptake rates because of the rapid exhaustion of available N. On the other hand, where the available N is more than adequate for the plant's requirements, the root growth of all genotypes would be such as to mask differences in the inherent capacity of individual roots to extract N from the soil. At the intermediate level, a wide range of N-uptake rates were apparent in each of the four geographic groups of cultivars. However, there were relatively more cultivars from the Japanese group with high uptake rates than cultivars from the other two major groups. Five of the six Japanese cultivars with high uptake rates were also included in a group of cultivars with the highest Nutilization indices in the low-N treatment. As mentioned earlier, these cultivars were all derived from hybridization of B. napus and B. campestris cultivars, the latter apparently contributing genes having pronounced effects on the N metabolism of the plant. Since there was no conscious selection for N uptake and utilization in the breeding of cultivars tested here, the results highlight the benefits of wide crossing in crop improvement and the need to exploit exotic germplasm more extensively. The solution culture experiments conducted in this study provided more detailed information on cultivar differences in N uptake. Monitoring of changes in both root weight and N concentration of the nutrient solution enabled some separation of the effects of the size of the root system and its activity on N uptake. In the two experiments reported here, significant cultivar differences in N uptake were detected in the one in which the initial N concentration was low {1 m M ) , but not in the one in which the initial concentration was high (8 mM). These findings are in accordance with the results of the field experiment which revealed significant cultivar differences only at the intermediate N level. An early solution culture study of N uptake by maize (Hoener and De Turk,
153
1938) also showed cultivar differences in uptake to be greatest at an intermediate level of nitrogen supply. However a more recent study with triticale (Mugwira et al., 1980) showed differences between two lines in one experiment to be greatest at the lowest N level (0.1 m M ) , but greatest at the highest level (10 mM) in another experiment with two different lines. Although cultivar differences in total uptake rate were not detected in this study with an 8 m M N concentration, differences in uptake rate per unit root dry weight ( root activity) and final root weight ratio ( root size) were significant. A strong negative association between these characters was primarily responsible for the absence of significant differences in total uptake rate. The same strong negative correlation between N-uptake activity of the root system and different parameters of the size of the root system was also detected in the other experiment involving a lower N concentration. In this case, total uptake rate was correlated with the measure of root activity, but not with the size of the root system. The primary objective of measuring variation in N response, uptake and utilization in this study was to assess the feasibility of using such characters as selection criteria in breeding improved rapeseed cultivars. Although significant genotypic variation in yield and N-utilization index was detected in a situation where N was deficient, yields overall were too low to give any hope of achieving suitable economic returns from breeding cultivars specifically for such environments. Moreover, it seems unlikely that a character such as the N-utilization index would be of much value as a selection criterion even if' breeding cultivars for N-deficient environments were contemplated. In contrast, N-uptake rate may well have some value in breeding cultivars for areas where moderate rates of N application are recommended. Uptake rate at the intermediate N level in the field experiment was highly correlated with the rate of dry matter increase before flowering as well as with seed yield. However, evidence from the solution culture experiments indicated that variation in uptake rate was not simply a reflection of differences between cultivars in 'growth demand'. Thus, since uptake rate measurements in the field and in solution culture were correlated (r=0.62; p < 0.01), the latter could serve a useful purpose in screening germplasm for suitable parents. The main objective of such screening would be to identify suitable combinations of existing high-yielding cultivars and cultivars with high N-uptake rates for a crossing program. Screening of parents would obviously be the major role for any procedure measuring uptake rate as it would be impractical to utilize it for selection of families in segregating generations. Nevertheless, recent developments in anther culture for the purpose of producing dihaploid lines ofB. napus (Keller and Stringham, 1978) opens up new possibilities for utilizing measurements of more complex characters such as N-uptake rate at several stages in a breeding program. An efficient anther culture system produces large numbers of pure dihaploid lines which are representative of the genetic variation generated
154 b y r e c o m b i n a t i o n a t m e i o s i s in t h e F1 hybrid. T h e s e m a y t h e n be s c r e e n e d in t h e s a m e w a y as t h e p a r e n t a l m a t e r i a l s for high N - u p t a k e rate. D i h a p l o i d lines could be e x p l o i t e d e v e n m o r e e f f e c t i v e l y t h r o u g h r e c u r r e n t s e l e c t i o n s c h e m e s in w h i c h selected lines are i n t e r c r o s s e d to g e n e r a t e s u b s e q u e n t cycles of d i h a p loid line p r o d u c t i o n a n d t e s t i n g ( C h o o a n d K a n n e n b e r g , 1978).
REFERENCES Austin, R.B., Ford, M.A., Edrich, J.A. and Blackwell, R.D., 1977. The nitrogen economy of winter wheat. J. Agric. Sci., Camb., 88: 159-167. Cataldo, D.A., Schrader, L.E. and Youngs, V.L., 1974. Analysis by digestion and calorimetric assay of total nitrogen in plant tissues high in nitrate. Crop Sci., 14: 854-856. Chevalier, P. and Schrader, L.E., 1977. Genotypic differences in nitrate absorption and partitioning of N among plant parts in maize. Crop Sci., 7: 897-901. Choo, T.M. and Kannenberg, L.W., 1978. The efficiency of using doubled haploids in recurrent selection programs. Can. J. Genet. Cytol., 20: 505-511. Epstein, E. and Jefferies, R.L., 1964. The genetic basis of selective ion transport in plants. Annu. Rev. Plant Physiol., 15: 169-184. Fischer, R.A., 1981. Developments in wheat agronomy. In: L.T. Evans and W.H. Peacock (Editors), Wheat Science - Today and Tomorrow. Cambridge University Press, pp. 249-269. Frey, K.J., 1959. Yield components in oats. II. The effect of nitrogen fertilization. Agron. J., 51: 605-608. Gabelman, W.H., 1976. Genetic potentials in nitrogen, phosphorus and potassium efficiency. In: M.J. Wright (Editor), Plant Adaptation to Mineral Stress in Problem Soils. Cornell University Press, New York, pp: 205-212. Gardener, C.J. and Rathjen, A.J., 1975. A differential response of barley genotypes to nitrogen application in a Mediterranean type climate. Aust. J. Agric. Res., 26: 219-230. Gerloff, G.C., 1963. Comparative mineral nutrition of plants. Annu. Rev. Plant. Physiol., 14: 699-704. Gerloff, G.C., 1976. Plant efficiencies in the use of nitrogen, phosphorus and potassium. In: M.J. Wright (Editor), Plant Adaptation to Mineral Stress in Problem Soils. Cornell University Press, New York, pp. 161-173. Goodman, P.J., 1981. Genetic control of nitrogen response in Lolium species. In: C.E. Wright (Editor), Plant Physiology and Herbage Production, British Grassland Society, Maidenhead, Great Britain, pp. 131-136. Grami, B. and La Croix, L.J., 1977. Cultivar variation in total nitrogen uptake in rape. Can. J. Plant Sci., 57: 619-624. Hoener, I.R. and De Turk, E.E., 1938. The absorption and utilization of nitrate nitrogen during vegetative growth by Illinois high protein and Illinois low protein corn. J. Am. Soc. Agron., 30: 232-243. Holmes, M.R.J., 1980. Nutrition of the Oilseed Rape Crop. Applied Science Publishers, London, pp. 21-67. Holmes, M.R.J. and Ainsley, A.M., 1977. Fertilizer requirements of spring oilseed rape. J. Sci. Food Agric., 28: 301-311. Keller, W.A. and Stringham, G.R., 1978. Production and utilization of microspore-derived plants. In: T.A. Thorpe {Editor), Frontiers of Plant Tissue Culture 1978. Int. Assoc. Plant Tissue Culture, Calgary, Canada, pp. 113-122. Kirby, E.J.M., 1968. The response of some barley varieties to irrigation and nitrogen fertilizer. J. Agric. Sci. Camb., 71: 47-52.
155 Maranville, J.W., Clark, R.B. and Ross, W.M., 1980. Nitrogen efficiency in grain sorghum. J. Plant Nutr., 2: 577-589. Mason, M.G., 1979. Nitrogen fertilizers for rape {Brassica carnpestris and B. napus) and wheat in Western Australia. W.A. Dep. Agric., Tech. Bull., 46, 14 pp. Moll, R.H., Kamprath, E.J. and Jackson, W.A., 1982. Analyses and interpretation of factors which contribute to efficiency of nitrogen utilization. Agron. J., 74: 562-564. Mugwira, L.M., Elgawhary, S.M. and Allen, A.E., 1980. Nitrate uptake effectiveness of different cultivars of triticale, wheat and rye. Agron. J., 72: 585-588. Nass, H.G., McLeod, J.A. and Suzuki, M., 1976. Effects of nitrogen application on yield, plant characters and N levels in grain of six spring wheat cultivars. Crop. Sci., 16: 877-879. O'Sullivan, J., Gabelman, W.H. and Gerloff, G.C., 1974. Variation in efficiency of nitrogen utilization in tomatoes (Lycopersicon esculentum Mill. ) grown under nitrogen stress. J. Am. Soc. Hort. Sci., 99: 543-547. Pollmer, W.G., Eberhard, D., Klein, D. and Dhillon, B.S., 1979. Genetic control of nitrogen uptake and translocation in maize. Crop Sci., 19: 82-86. Reed, A.J. and Hageman, R.H., 1980a. Relationship between nitrate uptake, flux and reduction and the accumulation of reduced nitrogen in maize (Zea mays L.). I. Genotypic variation. Plant Physiol., 66: 1179-1183. Reed, A.J. and Hageman, R.H., 1980b. Relationship between nitrate uptake, flux and reduction and the accumulation of reduced nitrogen in maize {Zea mays L. ). II. Effect of nutrient nitrate concentration. Plant Physiol., 66:1184-1189. Roy, N.N., 1982. Can rapeseed make a comeback? J.W.A. Dep. Agric., 23: 51-52. Shiga, T., 1970. Rape breeding by interspecific crossing between Brassica napus and Brassica campestris in Japan. Jpn. Agric. Res. Q., 5: 5-10. Thurling, N., 1974. Morpho-physiological determinants of yield in rapeseed (B rassica campestris and Brassica napus). I. Growth and morphological characters. Aust. J. Agric. Res., 25: 697-710. Vose, P.B., 1963. Varietal differences in plant nutrition. Herb. Abstr., 33: 1-13. Vose, P.B. and Breese, E.L., 1964. Genetic variation in the utilization by ryegrass species Lolium perenne and L. multiflorum. Ann. Bot., 28: 251-270. Woodis, T.C. and Cummings, J.M., 1973. Determination of ammoniacal nitrogen in the presence of urea with an ammonia electrode. J. Assoc. Anal. Chem., 56: 373-374. Woodruff, D.R., 1972. Cultivar variation in nitrogen uptake and distribution in wheat. Aust. J. Exp. Agric. Anim. Husb., 12: 511-516.
139
Elsevier Science Publishers B.V., Amsterdam - - Printed in The Netherlands
Variation in Nitrogen Response among Spring Rape (Brassica napus) Cultivars and its Relationship to Nitrogen Uptake and Utilization S.K. YAU* and N. THURLING
School of Agriculture, University of Western Australia, Nedlands, W.A. 6009 (Australia) *Present address: ICARDA, Aleppo, Syria (Accepted 22 September 1986)
ABSTRACT Yau, S.K. and Thurling, N., 1987. Variation in nitrogen response among spring rape (Brassica napus) cultivars and its relationship to nitrogen uptake and utilization. Field Crops Res., 16: 139-155. Growth, development and yield of 40 diverse spring rape (Brassica napus) cultivars were measured at three different levels of applied nitrogen in the field. There were significant cultivar differences in yield under a limiting N supply and in yield response to higher N application rates. Cultivars with the lowest yields at the lowest N level generally responded more markedly to increase in N application rates than cultivars with higher yields in this treatment. A greater yield response was associated with relatively greater rates of growth and development at higher N levels. Measurements of dry weights and N contents of above-groundplant parts over a period between 42 and 66 days after sowing of the field experiment were used to calculate N-uptake rates and Nutilization (change in dry weight/unit change in N content) indices. Significant cultivar differences were observed only at the lowest N level for N-utilization index and only at the intermediate level for N-uptake rate. If cultivars were grouped on the basis of their geographic origin - - Japanese, Canadian, European - - those of Japanese origin generally had the highest uptake rates and utilization indices in the respective treatments in which significant differences were detected. Two solution-culture experiments were also conducted in a daylight phytotron at 20/15 ° C with the same cultivars. Significant cultivar differences in N-uptake rate and N-utilization index were detected with an initial concentration of l m M N, but not with an initial concentration of 8 mM N. Cultivar differences in root growth and N uptake per unit root dry weight were significant at both concentrations. Again, most of the cultivars with the highest uptake rates or utilization indices were of Japanese origin. Uptake rate was correlated with uptake rate per unit dry weight, but not with initial root dry weight or increase in root weight over the course of the experiment. The significance of geographic variation in N-uptake rate in both field and controlled experiments is discussed with particular reference to the use of this character as a selection criterion in breeding programs utilizing recently developed anther culture techniques.
INTRODUCTION Rapeseed (Brassica napus and B. campestris) is becoming increasingly important in the world oilseed trade as a result of improvements in oil and
0378-4290/87/$03.50
© 1987ElsevierSciencePublishersB.V.
140 meal quality. In Australia it has considerable potential as a winter crop in southern states, although commercial production levels are still low. A concerted breeding effort has led to the development of improved cultivars adapted to local environments and resistant to the destructive blackleg disease (Roy, 1982 ), but expanded production will depend on further improvements in yield with minimal additional cost to the grower to make it competitive with cereals. Rapeseed has a high nitrogen requirement and a capacity to respond to increases in applied N above 150 kg ha-1 (Holmes and Ainsley, 1977; Mason, 1979; Holmes, 1980). Improved efficiency of N usage, either through an increase in yield at current levels of N supply or maintenance of current yields with a reduction in N supply, would benefit the grower. Several reviews ( Gerloff, 1963, 1976; Vose, 1963; Epstein and Jeffries, 1964; Gabelman, 1976) have presented evidence of genetic variation in nutrient uptake within plant species sufficient to be utilized in crop improvement. Differential yield responses of genotypes to low rates of applied N have been demonstrated in many crops including oats (Frey, 1959), wheat ( Nass et al., 1976 ) and barley (Gardener and Rathjen, 1975). Cultivar differences in yield response detected in these studies were explained in terms of variation in responses of the yield components. No studies of the relationship between differential yield responses and variation in early plant growth rate have been reported to date. Genotypic differences in total N uptake have also been observed in field studies with a wide range of crops including wheat (Woodruff, 1972; Austin et al., 1977; Fischer, 1981 ), barley (Kirby, 1968), sorghum (Maranville et al., 1980), maize (Pollmer et al., 1979; Moll et al., 1982) and rape (Grami and La Croix, 1977). Significant differences in N uptake have been detected between genotypes in solution culture experiments with maize (Chevalier and Schrader, 1977; Reed and Hageman, 1980a, b ) and perennial ryegrass (Vose and Breese, 1964; Goodman, 1981). This paper reports variation in response to applied nitrogen between B. napus cultivars in field and controlled-environment experiments. Response in the field was measured primarily in terms of seed yield and N uptake and utilization estimates were based on measurements obtained prior to flowering. Nutrient solution culture experiments with the same cultivars in controlled environments provided measurements of N uptake and utilization during early plant development. Data from these different experiments were analyzed with the main objective of assessing the feasibility of using N uptake and utilization characters as selection criteria in a rapeseed improvement program. MATERIALSAND METHODS
Field experiment Forty cultivars of spring rape ( listed in Table 1 ) were grown at three levels of applied N at the University Field Station, Perth. A fertilizer mixture was
141 TABLE 1 Brassica napus cultivars used in field and phytotron experiments
Australian
Canadian
European
Japanese
Wesbell Wesreo Wesway Wesroona Marnoo
Golden Nugget Zephyr Target Turret Oro Altex Regent Midas Tower Tanka
Valekowska Croesus Gulzover Zollengold Ceska Gulliver Bronowski Masoweicki Nilla Cyzowski Gulle Komet
Chikuzen Genkai Haya Chisaya Isuzu Tokiwa Mutu Norin 14 Norin 16 Norin 17 Norin 20 China A
applied just prior to sowing. This comprised a compound N P K fertilizer (13: 13:20) and superphosphate each applied at the rate of 100 kg ha -1, and different amounts of Agran ( 34% N ) to supply additional N at the rates of 30 (N1), 60 ( N2 ) and 120 ( N3 ) kg N h a - 1 to main plots, as appropriate. As the trial was conducted on an infertile sandy soil, further applications of the compound N P K fertilizer (100 kg h a - 1) and the same amounts of Agran as incorporated in the basal dressing were made at monthly intervals after sowing until the completion of flowering. Plots were arranged in a split-plot design with four replicates, N treatments being main plots and cultivars, sub-plots. Seeds were hand-sown between 27 and 31 May into plots comprising two rows of plants placed 15 cm apart. After thinning, 16 plants spaced 5 cm apart remained in each row. The shoots of five plants were harvested from one section of each plot at 42 and 66 days after sowing (DAS), at flowering, and at maturity. With the exception of the last harvest, plants were dried at 75 °C in a forced-draught oven for 48 h and then weighed. Plants from the final harvest were air-dried in a glasshouse, weighed and finally threshed to remove seed for weighing. Total-N ( including NO3- ) concentrations in plant materials from the first and second harvests were determined using the methods of Cataldo et al. {1974). A 90-110 mg sample was used, with N in the digest being measured by an ammonia-sensing electrode ( Orion, model 95-10) connected to a digital p H / m V meter (Orion, model 707). Aliquots for measurement comprised 0.5 ml of the diluted digest, 2 ml of 0.5M tartaric acid, and 10 ml of 0.5M sodium hydroxide. Tartaric acid was added to prevent iron from precipitating and causing errors in measurement (Woodis and Cummings, 1973). The data collected were used to calculate the following:
142 Harvest index = Seed yield/total shoot dry weight; Growth rate = (D W2 - D W1 ) / ( t 2 - tl ) ; Nitrogen-uptake rate = ( N2 - N1) / ( t2 - tl ); Nitrogen-utilization index = ( D W2 - D W, ) / ( N2 - N1 ); where D W, and D W2 are total shoot dry weights, and N1 and N2 are shoot N contents, at the first and second harvests, respectively. Phytotron experiments E x p e r i m e n t 1. With the exception of Gulliver, the same cultivars were tested
in a solution experiment in a daylight phytotron at a 20 ° C-15°C day-night temperature regime on a 12-h cycle. The experiment was conducted in a complete block design with cultivars randomized within each of three replicates. Replication was achieved through repetition of the experiment over a 4-month period (December-March). The basic plot unit was a 2.5-1 black plastic container able to support a maximum of ten plants. As some plants in the first replicate did not grow well, the data from this replicate were not included in the analyses. Seeds of the 39 cultivars were germinated in moist perlite and transferred to culture vessels, following careful washing of roots, 7-8 DAS. Seedlings were placed in holes spaced evenly around the periphery of opaque and rigid lids, and held securely in position with pre-cut pieces of foam rubber. Concentrations of nutrients in the culture solution were: 1 m M N (0.5 m M KNOa and 0.25 m M NH4N03); 1 m M CaS04; 0.5 m M K2S04; 0.1 m M MgS04"7 H20; 0.5 m M K2HP04; 0.05 m M Fe EDTA; 0.1 m M NaC1; 0.1 m M MnS04" 4 H20; 0.001 m M CUS04"5 H20; 0.001 m M ZnS04"7 H20; 0.1 m M H3B03; 2 × 1 0 -4 m M COSO4"6 H20; 6 X 10 -4 m M Na~Mo204"2 H20. Six plants with 2-3 expanded leaves were harvested from each container 10-11 days after transplanting. They were then divided into shoots and roots and over-dried for 48 h at 70 ° C before weighing. A seventh plant, the smallest remaining in each container, was also removed and discarded leaving three plants for the final harvest. The culture solution in each container was then replaced with a solution containing 8 m M N (75% NO3-N and 25% NH4-N) and other elements as before. Plants remaining in each container were harvested 6 days after the initial harvest, divided into shoots and roots, oven-dried and weighed. After the final harvest, the nutrient solution was made up to its original volume with distilled water, stirred by aeration then 30 ml was removed from each container for nitrate- and ammonium-N determination by ion-specific
143 electrodes. The depletion of N from the solution was assumed to be equal to the amount of N taken up by the plants. Data collected were used to calculate the following: Nitrogen-uptake rate per unit root dry weight = [(Nuptake) × (lnRW2- lnRW1)]/[(RW2-RW1) × (t2-tl)]; Nitrogen-utilization index = ( TW2 - TW1 ) / ( N uptake) ; Root-weight ratio at final harvest = R W2/R W1; where: T W = total plant dry weight; R W = root dry weight; and t = time of harvest. The subscripts 1 and 2 refer to the first and second harvests, respectively. Experiment 2. This experiment was done with the same cultivars used in the first phytotron experiment. However, instead of replicating the experiment in time, three replicates of cultivars were grown concurrently. Ten plants of each cultivar were again grown in 2.5-1 black plastic containers in the nutrient solution as was used in the first experiment. Three plants were harvested 10 days after the transfer of seedlings to the nutrient solution and a fourth plant, the smallest of those remaining, was removed and discarded. The original solution was replaced at this time with one containing 1 m M N ( 75% NO3-N and 25% NH4-N ) and other elements at the same concentrations as in the original solution. Nitrogen concentrations of solutions in all containers were measured 48 h after replacement of the nutrient solution. The remaining plants were harvested 6 days after the initial harvest when they had 4-5 expanded leaves and were exhibiting symptoms of N deficiency. All other procedures were as for Experiment 1.
RESULTS Field experiment
Analyses of variance of yield and related characters are summarized in Table 2. Differences between cultivars and treatments were significant for all characters measured. The interaction between cultivars and N levels was significant for all characters except harvest index (HI) and growth rate during the period between 42 and 66 DAS. Variation between cultivars in seed yield in the N1 treatment and in seed yield response to the N2 and N3 treatments is shown in diagrams in Fig. 1, with the cultivars grouped on the basis of geographic origin. The range of N1 seed yields varied little between groups, although two Japanese cultivars (Norin 17; Isuzu) yielded considerably less than any other cultivar at this N level. At
144
TABLE2 Analyses of variance of yield and related characters Source
d.f. Seed yield Total dry wt. Harvest (maturity) index
Blocks 3 Nitrogen 2 levels Residual 6 Cultivars 39 Cultivars X 78 N levels Residual 351
20.2** 1457.2"*
Total dry wt. (flowering)
Flowering Growth time rate
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10.3"* 64.1"
15.8"* 1 779.8**
461.8"* 25 39.3**
9.0** 206.6**
13.3 6.2** 1.2
105.3 44.0** 20.1"*
3.3 10.6"* 1.1
6.8 8.2** 3.7**
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TABLE 3 Coefficientsof correlation between responses of different plant characters to increasing nitrogen levels Total dry weight (maturity)
Seed yield
N1-N2 N1-N3 N1-N2 N1-N3
0.90*** 0.84*** 0.63*** 0.36*
Total dry weight (flowering)
N1-N2 N1-N3
0.10 0.47*
Growth rate ( 42-66 days)
N1-N2 N1-N3
0.45** 0.53***
Flowering time
N1-N2 N1-N3
-0.12 - 0.53***
Growth rate
N1-N2 N1-N3
0.21 0.40*
Harvest index Total dry weight (maturity)
Total dry weight (flowering)
Variation between cultivars in N - u p t a k e rate ( N 2 ) and N-utilization index ( N1 ) are shown in Table 5. M a n y combinations of uptake and utilization characteristics were represented in the cultivars tested and only the extreme combinations of lowest-uptake/highest-utilization index and highestuptake/lowest-utilization index were not accounted for. T h e r e was some evidence of separation in cultivars in respect of geographic origin. Most of the cultivars with a high N - upt a ke rate at N2 and a high utilization index at N1 were of Japanese origin. T h e one Australian cultivar (Wesroona) also included in this group was derived from a cross involving the Japanese cultivar Norin 20. E u r o p e a n and Canadian cultivars varied widely in N uptake, but most had low to intermediate values for N-utilization index. T h e breeding of Canadian TABLE4 Analyses of variance of nitrogen uptake and utilization for each nitrogen application rate Source
d.f.
Mean squares N-uptake rate
Blocks Cultivars Residual
3 39 117
***p<0.001; *p <0.05.
N-utilization index
N1
N2
N3
N1
N2
N3
55.1"** 2.3 3.3
70.5*** 17.9" 11.6
357.0*** 34.9 37.2
34.4 43.3*** 14.3
19.7 13.5 11.2
65.3*** 6.9 4.7
147 TABLE 5 Variation in nitrogen-uptake rate (N2) and nitrogen-utilization index (N1) between B. napus cuttivars in the field (origin of cultivar in parenthesis) N-uptake rate at N2 (mg per plant per day)
N-utilization index at N1 (mg d. wt. per mg N ) Mean - 2 or more S.D. (<24.9)
Mean - 1 S.D. ( 24.9- 28.6 )
Mean + 1 S.D. ( 28.6-32.3 )
M e a n - 2 or more S.D. ( < 10.81)
Croesus (E)
Altex (C) Bronowski (E) Chikuzen (J) Masoweicki (E)
Marnoo (A) Tower (C)
Mean - 1 S.D. (10.81-12.84)
Golden ( C ) Valekowska (E)
Genkai ( J ) Gulzover (E) Midas (C) Norin 16 (J) Nugget ( C ) Wesreo (A) ZoUengold (E) Gulliver (E)
Haya (J) Regent ( C ) Wesway (A)
Norin 20 (J)
Mean+l S.D. (12.84-14.87)
Wesbell (A)
Ceska (E) Cyzowski (E) Guile (E) Nilla (E) Oro (C) Target (C) Turret ( C ) Zephyr ( C )
Chisaya (J) Komet (E) Norin 14 (J)
Mutu (J) Isuzu (J)
China A (J) Tanka ( C )
Norin 17 (J)
Tkjokiwa (J) Wesroona (A)
Mean+2 or more S.D. ( > 14.87)
Mean + 2 or more S.D. (>32.3)
cultivars has been largely based on parents of European origin. A number of cultivars characterized by low glucosinolate content of the seed - Bronowski, Altex, Tower and Marnoo - were also closely associated in respect of low N uptake and intermediate utilization index. Wesroona is also a low glucosinolate cultivar, but is clearly separated from the other cultivars with this character in respect of N uptake and utilization. Coefficients of correlation between various plant characters and N-utilization index in the N1 treatment and between the same characters and N-uptake rate in the N2 treatment are given in Table 6. In the N1 treatment the Nutilization index was significantly correlated only with the growth rate over the period between 42 and 66 DAS and the total shoot dry weight at the time
148 TABLE 6 Coefficients of correlation between growth characteristics and nitrogen-uptake/utilization measurements
Growth rate (42-66 days) Time of flowering Total shoot dry weight (flowering) Total shoot dry weight (maturity) Harvest index Seed yield
Nitrogen-utilization index (N1)
Nitrogen-uptake rate (N2)
0.57*** - 0.13 0.40* 0.27 - 0.24 0.03
0.90*** 0.04 0.44** 0.65*** - 0.10 0.44**
***p < 0.001; **p < 0.01; *p < 0.05.
of flowering. These characters, as well as seed yield and total dry weight at maturity, were correlated with N-uptake rate in the N2 treatment.
Phytotron experiments Experiment 1. Cultivar differences were significant only for N uptake per unit root dry weight and root weight ratio ( Table 7 ). When cultivars were grouped on the basis of geographic origin (as in Table 8), the variation in both these characters was found to be substantially greater between cultivars within groups than between group means. The magnitude of variation in N uptake rate per unit root dry weight was somewhat greater between cultivars in the Japanese and Australian groups than in the Canadian and European groups.
Experiment 2. Cultivar differences were highly significant for all characters measured (Table 9). The variation between cultivars was similar in magnitude in each of the geographic groups and was substantially greater than that between group means (Table 10). In the case of N-utilization index, two Japanese cultivars (Haya; Norin 16) had the highest means overall, but these were not significantly greater than those of cultivars ranked highest in each of the other three groups. However, significant differences between cultivars were detected in each geographic group. Differences in N-uptake rate between cultivar groups were not significant, but were relatively greater than group differences in N-utilization index. Eight of the eleven Japanese cultivars tested had uptake rates greater than 2.3 mg plant-1 day-1, whereas only four European and one Canadian cultivar had uptake rates greater than this value. Initial root dry weight means of the three major cultivar groups were the same, but cultivar differences within these groups were significant. The pattern of variation in N-uptake rate per unit root dry weight was similar to that of the basic uptake rate, with variation within cultivar groupings being clearly
149 TABLE 7 Analyses of variance of different plant characters measured in phytotron experiment 1 Source
Blocks Cultivars Residual
d.f.
1 38 38
Mean squares Avg. growth rate
Avg. root dry wt.
23 444* 215 225
5 518" 56 36
Root weight ratio
1 759* 70* 24
Nitrogen Uptake rate
Uptake rate per unit root dry wt.
Utilization index
56.82* 0.76 0.64
4 522* 380* 122
48.57* 0.61 0.55
*p < 0.001.
TABLE 8 Root growth and nitrogen uptake characteristics of B. napus cultivars grown in solution culture (Experiment 1 ) Geographic origin
Number of cultivars
Australian Canadian European Japanese L.S.D. (p <0.05)
5 11 11 12
N-uptake rate/unit root dry weight (rag N g 1day 1)
Root weight ratio
Mean
Range
Mean
Range
201 192 189 191
177-219 182-207 162-210 166-212
0.115 0.115 0.119 0.113
0.107-0.120 0.106-0.126 0.109-0.128 0.105-0.122 0.010
22
TABLE 9 Analyses of variance of different plant characters measured in phytotron experiment 2 Source
Blocks Cultivars Residual
d.f.
2 38 76
***p < 0.001; *p < 0.05.
Mean squares Initial root dry weight
Final root weight ratio
N-uptake rate
N-uptake/ initial root dry weight
N-utilization index
1.63 5.60*** 1.99
0.145"** 0.120"** 0.009
1.509"** 0.056*** 0.019
25 980*** 6 152"** 2 288
32.8* 29.2*** 12.4
150 TABLE 10 Root size, nitrogen uptake and utilization characteristics of B. napus cultivars grown in solution culture (Experiment 2) Character
Australian
Canadian
European
Japanese
Mean
Range
Mean
Range
Mean
Range Mean
Range
Initial root dry weight ( mg plant- ' ) LSD (p < 0.05) = 2
12
1013
13
1115
13
1215
113
1016
Final root weight ratio LSD (p<0.05)=0.02
0.20
0.190.21
0.21
0.190.22
0.23
0.190.26
0.19
0.160.22
N uptake rate (mg N plant- 1 day- 1) LSD (p<0.05)=0.22
2.33
2.232.43
2.20
1.982.33
2.23
2.022.38
2.40
2.27 2.57
N uptake/unit initial root dry wt. (mg N g- 1) LSD ( p < 0 . 0 5 ) = 7 9
399
350476
347
279403
346
324385
386
298476
N utilization index (mg mg N - 1) LSD (p<0.05) =5.8
45.8
42.748.0
44.5
37.648.1
44.9
40.048.5
46.9
42.152.0
greater than that between group means, and with Japanese and Australian cultivars having the highest values. However, some changes in cultivar rankings were also evident because of the absence of an association between root dry weight and uptake rate. Coefficients of correlation given in Table 11 showed that N-utilization index was highly correlated with N-uptake rate, whether expressed in absolute terms or relative to root dry weight. The utilization index and relative uptake rate were negatively correlated with both root weight measurements, but correlations between the root weights and uptake rate were not significant. Cultivar TABLE 11 Coefficients of correlation between characters measured in phytotron experiment 2
Initial root dry weight (1) Final root weight ratio ( 2 ) N-uptake rate (3) N-uptake rate/unit initial root dry wt. (4) N-utilization index (5) ***p < 0.001; **p < 0.01; *p < 0.05.
1
2
3
4
5
1.00
0.55*** 1.00
0.09 - 0.10 1.00
- 0.84*** - 0.57*** 0.45** 1.00
- 0.34* - 0.32* 0.59*** 0.65*** 1.00
151
differences in seed size were related to the variation in root size parameters, but no significant relationship with the utilization index or uptake rate was detected. DISCUSSION Detection of cultivar differences in yield at a low N level (N1) was encouraging in the context of breeding cultivars providing a greater economic return through reduced fertilizer costs. Seed yield in the N1 treatment was highly correlated with the total dry weight of shoots at maturity which, in turn, was related to the growth rate over the period between 42 and 66 DAS. The Nutilization index, calculated from data collected over this period, also differed significantly between cultivars in the low-N treatment and was correlated with growth rate. Thus, the ability of a spring rape genotype to yield adequately with a low N input appears to be partly dependent on a heritable capacity to utilize N efficiently for dry matter production prior to flowering. Five of the six cultivars with the highest N-utilization indices were of Japanese origin whilst the sixth, the Australian cultivar Wesroona, had the Japanese cultivar Norin 20 as one of its parents. All these Japanese cultivars had been derived from hybridization between B. napus and the related species B. campestris (Shiga, 1970), suggesting the introgression of genes for more efficient N utilization from the latter. The detection of significant cultivar differences in N-utilization index only in the low-N treatment is in accordance with the findings of a solution culture study of N response in tomatoes (O'Sullivan et al., 1974). The more efficient genotypes had lower N concentrations in old leaves but higher concentrations in the young leaves than the less efficient genotypes. Accordingly, it was concluded that N remobilization was a major determinant of N-utilization efficiency. Solution culture experiments conducted as a part of this study also showed that significant cultivar differences in N-utilization index were detectable when plants were grown in N-deficient solutions (1 m M N), but not when grown at a higher concentration (8 m M N). Nitrogen stress in the former was clearly evident from the rapid yellowing of lower leaves within five days of commencement of treatment. As whole plants were sampled for N determinations, no conclusions can be drawn regarding mechanisms responsible for cultivar differences in N-utilization efficiency. Substantial differences in response to increasing N supply were also observed between the B. napus cultivars used in this study. Overall, cultivars which yielded least in the low-N treatment tended to respond more markedly to higher rates of nitrogen supply. A greater proportion of European cultivars were low yielding under limited N supply and more responsive to higher N levels than either Canadian or Japanese cultivars. This difference possibly reflects the selection for high yield under the normally more fertile soils on which rapeseed
152 is grown in northern Europe. Responsive cultivars, measured in terms of seed yield, grew and developed at a relatively greater rate with adequate N supply than the less responsive cultivars. Thus, higher seed yields in these conditions resulted not only from a greater rate of dry matter accumulation by the plant, but also from the significant reduction in the time to flowering. The influence of the latter was evident from the correlation between the responses of yield and harvest index to higher N-application rates. Seed development on plants, which flowered earlier as a result of a higher development rate, proceeded under more favourable temperature conditions than on later-flowering plants of the same cultivar in the limited-N regime. This coincidence of flowering in B. napus with lower temperatures in Western Australia is invariably associated with a higher harvest index (Thurling, 1974). The intermediate N level in the field experiment was the only treatment in which significant cultivar differences in the rate of N uptake were detected. Failure to detect significant cultivar differences in uptake rate in the high- or low-N regimes was not unexpected. Where N supply is limited, genotypes characterized by high uptake rates would be unable to take up any more than those with low uptake rates because of the rapid exhaustion of available N. On the other hand, where the available N is more than adequate for the plant's requirements, the root growth of all genotypes would be such as to mask differences in the inherent capacity of individual roots to extract N from the soil. At the intermediate level, a wide range of N-uptake rates were apparent in each of the four geographic groups of cultivars. However, there were relatively more cultivars from the Japanese group with high uptake rates than cultivars from the other two major groups. Five of the six Japanese cultivars with high uptake rates were also included in a group of cultivars with the highest Nutilization indices in the low-N treatment. As mentioned earlier, these cultivars were all derived from hybridization of B. napus and B. campestris cultivars, the latter apparently contributing genes having pronounced effects on the N metabolism of the plant. Since there was no conscious selection for N uptake and utilization in the breeding of cultivars tested here, the results highlight the benefits of wide crossing in crop improvement and the need to exploit exotic germplasm more extensively. The solution culture experiments conducted in this study provided more detailed information on cultivar differences in N uptake. Monitoring of changes in both root weight and N concentration of the nutrient solution enabled some separation of the effects of the size of the root system and its activity on N uptake. In the two experiments reported here, significant cultivar differences in N uptake were detected in the one in which the initial N concentration was low {1 m M ) , but not in the one in which the initial concentration was high (8 mM). These findings are in accordance with the results of the field experiment which revealed significant cultivar differences only at the intermediate N level. An early solution culture study of N uptake by maize (Hoener and De Turk,
153
1938) also showed cultivar differences in uptake to be greatest at an intermediate level of nitrogen supply. However a more recent study with triticale (Mugwira et al., 1980) showed differences between two lines in one experiment to be greatest at the lowest N level (0.1 m M ) , but greatest at the highest level (10 mM) in another experiment with two different lines. Although cultivar differences in total uptake rate were not detected in this study with an 8 m M N concentration, differences in uptake rate per unit root dry weight ( root activity) and final root weight ratio ( root size) were significant. A strong negative association between these characters was primarily responsible for the absence of significant differences in total uptake rate. The same strong negative correlation between N-uptake activity of the root system and different parameters of the size of the root system was also detected in the other experiment involving a lower N concentration. In this case, total uptake rate was correlated with the measure of root activity, but not with the size of the root system. The primary objective of measuring variation in N response, uptake and utilization in this study was to assess the feasibility of using such characters as selection criteria in breeding improved rapeseed cultivars. Although significant genotypic variation in yield and N-utilization index was detected in a situation where N was deficient, yields overall were too low to give any hope of achieving suitable economic returns from breeding cultivars specifically for such environments. Moreover, it seems unlikely that a character such as the N-utilization index would be of much value as a selection criterion even if' breeding cultivars for N-deficient environments were contemplated. In contrast, N-uptake rate may well have some value in breeding cultivars for areas where moderate rates of N application are recommended. Uptake rate at the intermediate N level in the field experiment was highly correlated with the rate of dry matter increase before flowering as well as with seed yield. However, evidence from the solution culture experiments indicated that variation in uptake rate was not simply a reflection of differences between cultivars in 'growth demand'. Thus, since uptake rate measurements in the field and in solution culture were correlated (r=0.62; p < 0.01), the latter could serve a useful purpose in screening germplasm for suitable parents. The main objective of such screening would be to identify suitable combinations of existing high-yielding cultivars and cultivars with high N-uptake rates for a crossing program. Screening of parents would obviously be the major role for any procedure measuring uptake rate as it would be impractical to utilize it for selection of families in segregating generations. Nevertheless, recent developments in anther culture for the purpose of producing dihaploid lines ofB. napus (Keller and Stringham, 1978) opens up new possibilities for utilizing measurements of more complex characters such as N-uptake rate at several stages in a breeding program. An efficient anther culture system produces large numbers of pure dihaploid lines which are representative of the genetic variation generated
154 b y r e c o m b i n a t i o n a t m e i o s i s in t h e F1 hybrid. T h e s e m a y t h e n be s c r e e n e d in t h e s a m e w a y as t h e p a r e n t a l m a t e r i a l s for high N - u p t a k e rate. D i h a p l o i d lines could be e x p l o i t e d e v e n m o r e e f f e c t i v e l y t h r o u g h r e c u r r e n t s e l e c t i o n s c h e m e s in w h i c h selected lines are i n t e r c r o s s e d to g e n e r a t e s u b s e q u e n t cycles of d i h a p loid line p r o d u c t i o n a n d t e s t i n g ( C h o o a n d K a n n e n b e r g , 1978).
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