ABSTRACT

We aimed to identify canola (Brassica napus L.) germplasm tolerant of aluminium (Al) and manganese (Mn) toxicity. A sub-irrigated gravel screening system for selecting Mn tolerant genotypes was used to screen B. napus and Brassica rapa (L.) germplasm. Plants were grown for 4 weeks under 9 μM (control) and 250 μM Mn in the nutrient solution. Visual assessments of Mn toxicity symptoms and differential growth response of plants at 250 μM Mn were used to determine Mn tolerance. Two screenings evaluated 33 B. napus and 3 B. rapa genotypes from diverse origins. Most of the genotypes screened were sensitive to Mn toxicity. However, within some B. napus and B. rapa genotypes, some plants expressed characteristic Mn tolerance. Tolerant individuals within B. rapa genotypes appeared to be more Mn tolerant than B. napus genotypes. Several Mn tolerant and Mn sensitive plants were selected from both screenings. Progenies from these plants were screened and their tolerance of Mn confirmed. This also confirmed that Mn tolerance was of genetic origin. Doubled haploid (DH) lines are being developed from the selected plants to obtain pure genotypes. These lines will be used for the development of DH populations for genetic studies. We are also initiating screening for Al tolerance. Results from these studies will be presented at the conference.

INTRODUCTION

Nearly half of the world's non-irrigated arable soils are acidic (Clark, 1982) and present canola (Brassica napus L.) cultivars are very sensitive to acid soils. In acid soils aluminium (Al) and manganese (Mn) toxicity are the most important limiting factors for plant growth (Foy, 1984). Fortunately, interspecific and intraspecific differences in tolerance of both Al and Mn have been identified among crop plants providing potential to develop cultivars adapted to acid soils (Foy et al., 1988). This has not been reported in canola and we aimed to identify sources of intraspecific tolerance of Al and Mn.

Although soil acidity can be ameliorated by liming, the use of lime alone will not fully correct Mn toxicity and only slowly amends sub-soil Al toxicity (Bromfield et al., 1983). Breeding for tolerance of Al and Mn maybe feasible and more economical than the use of soil amendments alone. Although canola may appear to overcomes the toxic effects of Mn as its availability drops in early winter, Mn toxicity is a stress that reduces the crop’s yield potential. Reports from both NSW and Victoria highlight the occurrence of high exchangeable soil Mn caused by extremes of environmental conditions such as summer heat exposure and waterlogging at the end of winter as soils warm (Slattery and Ronnfeldt, 1992).

We report on the selection of Mn tolerant B. napus germplasm and show that the Mn-tolerance trait is of genetic origin. Also, it appeared that the tolerance of Mn toxicity was the result of plant tissue withstanding high internal Mn concentrations.

MATERIAL AND METHODS

Experiment 1: Differential response of rapeseed genotypes to Mn toxicity

Two screening consisting of a total of 36 Brassica genotypes (B. napus, 33; B. rapa, 3) were evaluated in a sub-irrigated gravel bed system for differential tolerance of Mn toxicity. A control nutrient solution and a solution with an additional 250 mM Mn were used. The nutrient solution contained (µM): Ca, 1000; Mg, 400; K, 1000; NO3, 3400; NH4, 600; PO4, 100; SO4, 401.1; Cl 78; Na, 40.2; Fe, 20; B, 23; Mn, 9; Zn, 0.8; Cu, 0.30; and Mo, 0.1. Iron was supplied as Fe-EDTA prepared from equimolar amounts of FeCl3 and Na2EDTA. The pH of the nutrient solution was maintained at 4.7 by daily adjustment. The gravel was flushed with the nutrient solution once per hour and the nutrient solutions were renewed weekly. Four independent tanks were available and each tank contained 40 pots. All the experiments were conducted in a heated and evaporative cooled glasshouse at Wagga Wagga, New South Wales. After 4 weeks of growth plant showing differential symptoms and growth in response to Mn toxicity were selected and transplanted for seed increase. Seed from these selected plants were used in Experiment 2 and 3.

Experiment 2: The effect of Mn on plant growth and Mn tissue concentration

Three rapeseed genotypes (selected in Experiment 1) were used to determine the effect of Mn on plant growth and uptake of Mn. The experimental design was a randomised complete block consisting of the 3 rapeseed genotypes, 8 concentrations of Mn in solution and 3 replicates. Each treatment contained 8 plants. The three genotypes were genotype 21, 24 and 98-51 (Table 1). Genotype 98-51 is a doubled haploid line from Canada (developed by Dr. G. Stringam, University of Alberta) and identified as Mn-tolerant in the gravel system. Genotypes 21 and 24 were identified as Mn-sensitive in the same system. Seeds were germinated and grown in plastic containers filled with the basal nutrient solution and one of eight different Mn concentrations (ie. 2, 50, 100, 150, 200, 300, 400, 500 µM). After a 14-day treatment, the plants were harvested, divided into roots and shoots, oven-dried at 65 °C for three days, and weighed. Mn tissue concentration was measured by digesting the plant samples by Kjeldahl solution, followed by analysis on AAS.

Experiment 3: Differential response of pre-screened rapeseed genotypes to Mn toxicity

Based on Experiment 2 a concentration of Mn of 125 µM was selected to determine the differential response of 12 rapeseed genotypes to Mn in a hydroponic system. The control treatment consisted of unaltered nutrient solution. For the Mn treatment, 125 µM Mn was superimposed over the basal nutrient solution. The pH of the solution was maintained at 4.7. Control and Mn treatments for each genotype within each replicate were blocked together to minimised variation. The experimental design was a split-plot with 12 genotypes, 2 Mn treatments and 3 replicates where genotypes were randomly assigned to main plots and Mn treatments to sub-plots. Germination, growth and harvest were as for Experiment 2.

Statistical analysis

All data were analysed by analysis of variance (ANOVA). Because root and shoot weight of the various genotypes differed under normal and toxic Mn levels, genotype tolerance of Mn was determined by the relative shoot and root weight (RSW and RRW) method (RSW or RRW= shoot or root weight in the presence of 125 µM Mn divided by control shoot or root weight respectively, multiplied by 100). Duncan's multiple range test using the arcsine transformation on percent data for RSW and RSW was performed according to the rules outlined by Gomez and Gomez (1984). Significance was defined at the 95% confidence level. All statistical analysis were carried out with the statistical software SAS/STATS (SAS Institute, 1992).

RESULTS AND DISCUSSION

Experiment 1

Most of the genotypes screened in the sub-irrigated system were uniformly sensitive to Mn toxicity. Foliar symptoms for Mn toxicity included leaf margin and intervenal chlorosis, brown spots, necrosis, leaf “cupping” or distortion of the leaf shape and reduction in plant growth. However, some genotypes were heterogenous and had some plants without Mn-toxicity symptoms and were vigorous. These plants were considered to be tolerant of Mn toxicity and were selected for further studies (Table 1).

Experiment 2

There was a significant differential response between the Mn sensitive genotypes 21 and 24 and the Mn-tolerant genotype 98-51 (Fig. 1.a). The growth response observed across the Mn concentrations confirmed the differential response of the 3 genotypes which was observed in the sub-irrigated gravel system.

There was a significant differential response for Mn uptake between genotypes 21, 24 and 98-51 (Fig. 1.b). However, the uptake of Mn by the three genotypes did not differ significantly below 200 μM Mn in the solution (Fig1.b). Thus, although the 3 genotypes had absorbed similar concentrations of Mn below the 200 μM Mn treatment, the growth response was different (Fig. 1.a). This suggested that the Mn tolerance mechanism was the result of the tissue of the Mn tolerant genotype (ie. 98-51) being able to withstand high concentrations of Mn relative to the sensitive genotypes (ie. 21 and 24).

Experiment 3

A wide range of differential response was detected among the 12 genotypes screened (Table 2). Two genotypes were more Mn tolerant than genotype 98-51 which was used in Experiment 2. They were genotypes 98-28 and 98-5, both selected from the Japanese cultivar Mutu. The differential response of all genotypes in this screening agreed with the observations used to make the initial selections (Experiment 1, Table 1). Furthermore, since this seed source originated from the plants visually selected in Experiment 1, the results confirm that Mn tolerance expressed in Experiment 3 was of genetic origin.

CONCLUSIONS

The differential symptoms observed between Mn-tolerant and Mn-sensitive genotypes may provide a basis for the development of a rapid screening technique based on toxicity symptoms. Leaf chlorosis alone may offer a simple selection criterion. The germplasm identified with improved tolerance of Mn toxicity will allow the breeding of Mn tolerance into existing cultivars. With the addition of Al tolerance this would impact on the expansion of the crop where subsoil acidity and/or high levels of soil Mn can not be fully ameliorated by liming.

ACKNOWLEDGEMENTS

The research was supported by NSW Agriculture and the Acid Soil Action Initiative of the NSW Government. The assistance with plant harvesting provided by Rod Fisher and with micronutrient analysis by Graeme Poile is gratefully appreciated.

REFERENCES

1. Bromfield, S.M., R.W.M. Cumming, D.J. David and C.H. Williams. 1983. Changes in soil pH, manganese and aluminium under subterranean clover pasture. Australian Journal of Experimental Agriculture and Animal Husbandry. 23:181-191.

2. Clark, R.B. 1982. Plant response to mineral element toxicity and deficiency. p. 71-142. In M.N. Christiansen and C.F. Lewis.(ed.) Breeding Plants for Less Favorable Environments. John Wiley and Sons, New York.

3. Foy, C.D. 1984. Physiological effects of hydrogen, aluminum, and manganese toxicities in acid soil. p. 57-97. In F. Adams (ed.) Soil Acidity and Liming. Agronomy Monograph Nº 12 (2nd Ed.). ASA-CSSA-SSSA, Wis.

4. Foy, C.D., B.J. Scott, and J.A. Fisher. 1988. Genetic differences in plant tolerance to manganese toxicity. p. 293-307. In R.D. Graham, R.J. Hannam, and N.C. Uren (ed.) Manganese in Soils and Plants. Kluwer Academic Publishers, Dordrecht.

5. Gomez, K.A. and A.A. Gomez. 1984. Statistical procedures for agricultural research. Second Ed. An International Rice Research Institute book. John Wiley and Sons, New York.

6. SAS Institute. 1992. SAS/STAT user’s guide. Release 6.07 ed. SAS Inst., Cary, NC.

7. Slattery, W.J. and G.R. Ronnfeldt. 1992. Seasonal variation of pH, aluminium, and manganese in acid soils from north-eastern Victoria. Australian Journal of Experimental Agriculture. 32:1105-1112.

Table 1. Origin and some details of genotypes expressing differential response to manganese toxicity (250 μM Mn) under a sub-irrigated gravel screening system.

Table 2. Relative (%) shoot weight (RSW) and root weight (RRW) of rapeseed (Brassica napus L. and B. rapa L.) genotypes grown in nutrient solution containing 125 μM Mn compared to 9 μM Mn.

^@ B. rapa selections, all other genotypes are B. napus

& Average of three replications.

# Means followed by the same letter are not significantly different at the 5% level according to Duncan’s multiple range test.

Figure 1. The effect of manganese concentration in solution on the shoot weight (a) and manganese concentration of shoot (b) of Mn-tolerant genotype 98-51 and the Mn-sensitive genotypes 21 and 24 of Brassica napus. Mean shoot weight ± SEM is expressed as a percentage of control.