Ernst Cebert*, Rhona Lee Miller-Cebert, Nahid Sistani and Martha Verghese

1. Accumulation and relationship of essential micro-nutrients with aluminum uptake in winter canola (Brassica napus) cultivars • ABSTRACT • Species in the Brassica genus representing canola (B. napus and B. rapa) have been classified as phyto-accumulator for their characteristics to accumulate some elements at levels which are toxic to other plant species. Since many Brassica species are consumed as green vegetables or as forage, accumulation of some elements beyond a certain threshold may be toxic to the consumers. Canola (B. napus) cultivars were grown at pH 5.3 in a Decatur silty-loam soil at the Alabama A&M University Agricultural Research Station, and were analyzed for copper (Cu), iron (Fe), manganese (Mn), zinc (Zn) and aluminum (Al) content in their leaves at the rosette, budding/bolting and blooming growth stages. Results for the essential micronutrients, Fe, Cu, and Zn indicated no significant difference among the five canola cultivars. The range content of these minerals in leaf tissues were: 23.7 – 25.3 mg/100g dry wt. for Fe; 0.27 – 0.31 mg/100g dry wt. for Cu; and 2.9 – 3.1 mg/100g dry wt. for Zn. However, result for Mn indicated significant difference among cultivars, with Jetton at 18.3 mg/100g dry wt. having significantly greater manganese content than Abilene and Wichita with 15.1 and 15.4 mg/100g dry wt. respectively. Concentration of the non-essential element Aluminum ranged between 16.7 – 24.3 mg/100g dry wt. in leaf tissues, with no significant difference among cultivars. Regression analysis also showed significant linear relationship (r2 = 0.89) between Al and Fe accumulation in leaf tissues. Fe and Al decrease, while Cu, Zn and Mn increase in the leaf tissues as plants transitioned from rosette, to budding, and blooming growth stages. • INTRODUCTION • Alabama A&M University (AAMU) has shown that winter canola could be an alternative to winter wheat and feasible for double-cropping with traditional summer crops, and become an addition to current cropping system in the southeast United States (Hopkinson et al., 2002; Kumar et al., 2007; Bishnoi et al., 2007). • Canola has been given the QHC label or qualified health claim based on evidence that consumption of about 19 grams of canola oil daily may reduce the risk of coronary heart disease, (http://www.uscanola.com). The crop has also been labeled as a phytoremediator for its ability to accumulate high content of selenium and other elements found at toxic levels in polluted soils (Salt and Kramer, 2000; Palmer et al., 2000). The use of canola as a potential leafy green vegetable was investigated by,Bhardwaj et al., (2003) andMiller-Cebert et al., 2009a, 2009b and 2009cwho reported that the crop can provide nutritional benefits similar to those of other traditional leafy greens. Canola is also being used as a forage crop for winter grazing; it is being promoted as a feedstock for biofuels; and its meal is an important source of protein for animal feed Raymer et al., (1990); Lema et al., (2004); Salt and Kramer, (2000). • Miller-Cebert et al., (2009)reported significantly higher level of Al in leaves of winter canola grown on soil pH 5.3, compared to locally purchased kale, collard and cabbage. Therefore, establishment of winter canola (Brassica napus) as a multi-purpose crop in Alabama requires further investigation of cultivars’ reaction to prevailing soil conditions such as elevated aluminum content in acid soils. • The Objectiveof this presentation was to determine the relationship of Al accumulation with essential micronutrients iron (Fe), copper (Cu), manganese (Mn) and zinc (Zn) in leaves of winter canola at three growth stages of the plant. • APPROACH: Five canola cultivars (Abilene, Kronos, Jetton, Wichita and Virginia) were grown at the Alabama A&M University (AAMU) Winfred Thomas Agricultural Research Station (WTARS), located in Hazel Green, Alabama • Soil pH = 5.3 • Samples were harvested at: Rosette, Budding/Bolting, and Blooming/100% Flowering • Microwave digested samples were analyzed using a Varian Vista MPX inductively coupled plasma optic emission spectrometer (ICP-OES) • Statistical Analysis includes the use of SAS (2006) Proc GLM to determine significance among treatments, Tukey’s mean separation (a=0.05), was used to analyze the data; Proc Reg and Graph-and-Go for regression analysis and graphical illustrations. Ernst Cebert*, Rhona Lee Miller-Cebert, Nahid Sistani and Martha Verghese Department of Natural Resources and Environmental Sciences Alabama A&M University, Normal, AL 35762 ernst.cebert@aamu.edu Table 1. Regression analysis for model: Al= Cu Fe Mn Zn, for foliage samples harvested at rosette stage of growth Rosette = , Bolting = , Flowering = . y = 2.0502x - 13.326 R² = 0.888 Figure3 - (a, b). Accumulation of Al in relation to Fe showed rapid uptake during the rosette growth stage, which represents the early establishment period of the crop (120 days after planting.) Previous report by Shen and Ma (2001) reported that early accumulation of Al in leaf tissues is not mobile. Therefore, lower level of the element in subsequent samplings indicates a depletion of its availability within the root zone. Fig 3b shows significant positive relationship between Al and Fe. Figure 1. Micro minerals in canola leaves at three different stages of plant growth indicate Al and Fe are highest at the rosette stage, while Zn and Cu show very little change from rosette to flowering. These variations during the growth cycle of canola, as previously observed by Mendham and Salisbury (1995), may be used to determine specific stages of growth for harvest in order to obtain optimal levels of desired nutrients. Fe and Al which were significantly higher (P<0.001) at the rosette stage decreased in subsequent samplings at bolting and reaching the lowest levels at the blooming stage. Shen and Ma, (2001) reported that The mobility of Al in acid soils can be taken up rapidly by plants and it creates a problem of chemical stress in plants. Table 2. Regression analysis for model: Al= Cu Fe Mn Zn, for foliage samples harvested at bolting stage of growth Rosette = , Bolting = , and Flowering = . y = -17.295x + 17.363 R² = 0.1186 Figure 4 - (a, b). In the process of high aluminum accumulation, these results show that Cu accumulates at a lower rate when compared to Fe in the presence of Al. An increase in Zn accumulation was observed as Al decreased during the bolting stage. Both elements were significantly lower during the last sampling period at flowering. Fig 3b does not indicate statistical significance in the relationship between AL and Zn based on the regression across the three harvesting periods; rosette, bolting and flowering. Table 3. Regression analysis for model: Al= Cu Fe Mn Zn, for foliage samples harvested at flowering stage of growth y = -0.4429x + 17.036 R² = 0.1446 Rosette = , Bolting = , an Flowering = . AL (mg/100g dry wt. Cultivars grouped by Harvest Rosette Bolting Flowering Level 1 Level 2 Level 3 Level 4 Tables 1,2 and 3 show regression analysis indicating the interaction of Al with essential micro-nutrients Cu, Fe, Mn and Zn is effective at the flowering (R2 = 0.87). However, at this stage negative attributes from reduced uptake would have already caused their harmful effects. Low accumulation of Cu, Mn and Zn at the rosette stage, while Al accumulates to its highest level compromises the nutritional value of canola if grown in soils with low pH. Figure 5 - (a, b). Similar to Cu, manganese (Mn) accumulation is lowest when Al is high during early establishment of the crop at the rosette stage and reached its highest at the bolting and flowering stages when Al is low. Variations for Mn among winter canola cultivars was apparent at all growth stages, indicating sensitivity of its accumulation based on genotypic characteristics of the cultivars. Further evaluation to identify cultivars which can accumulate high levels of all micro-elements is being evaluated. 150 Winter Canola Cultivars Figure 2. Although significant differences exist for Al accumulation between growth stages, among winter canola cultivars no statistically significant variation was observed within growth stages. The lowest level of 2.7 mg 100g-1 dry weight was obtained during flowering for cultivar Abilene, while the highest level of 21.5 mg 100g-1 was for Jetton at the rosette growth stage. The results, however, indicated that only Mn consistently showed significant statistical differences among cultivars for all stages of growth. In all stages, cultivar Jetton registered the highest level of Mn, while Wichitaand Abilene had the least. Other significant differences in accumulation of micronutrients among cultivars were observed for Zn at the flowering and for Cu during the rosette stages of growth. Winter canola cultivar Jetton was consistent in having higher accumulation of other micro-elements along with Al. 27.0 82.0 30.3 3,100 4,645 Zn 22.6 113.0 227.0 340.0 rosette = , bolting = , and Flowering = . Bhardwaj, H.L., Hamama, A.A., Rangappa, M., 2003. Characteristics of Nutritional Quality of Canola Greens.Hort. Science38 (6), 1156-1158. Bishnoi, U., S. Kumar, E. Cebert and R. Mentreddy. 2007. Agronomic and Economics Performance of Winter Canola Production in South East US. World Journal of Agricultural Sciences 3(3):262-268. Hopkinson, S., Bishnoi, U., Cebert, E., 2002. Sowing dates, seeding and nitrogen rate effects on yield and yield components of canola. Crop Research 24, 407-416. Kawashima, L.M., Soares, L.M., 2003. Mineral profile of raw and cooked vegetables consumed in southern Brazil. Journal of Food Composition and Analysis 16, 605-611. Suresh Kumar, U.R. Bishnoi, and E. Cebert. 2007. Impact of Rotation on Yield and Economic Performance of Summer Crops-Winter Canola Cropping Systems, Am.-Eurasian J. Sustain. Agric, 1(1):68-76. Lema, M., Cebert, E., Sapra, V.T., 2004. Evaluation of small grain cultivars for Forage in North America.Journal of Sustainable Agriculture23, 133-145. Miller-Cebert, Rhona Lee, Nahid Sistani and Ernst Cebert. 2009. Comparative Protein and Folate among Canola Cultivars and other cruciferous Leafy Greens.Journal of Food Agriculture & Environment. Vol. 7 (2): 46 - 49. 2009. Miller-Cebert, Rhona Lee, Nahid Sistani and Ernst Cebert. 2009. Comparative Mineral composition among Canola Cultivars and other cruciferous Leafy Greens.Journal of Food composition and Analysis. 22 (2009) 112–116. Miller-Cebert, Rhona Lee, Nahid Sistani and Ernst Cebert. 2009. Sensory Evaluation of Canola (Brassica napus) Greens and Other Cruciferous Vegetables. Food Research International. (Accepted for publication) Raymer, P.L., Bullock, D.G., Thomas, D.L., 1990. Potential of winter rapeseed cultivars for oilseed production in southeastern United States. Advance in New Crops 223-225. Salt, D.E., Kramer, U., 2000. Mechanisms of metal hyper-accumulation in plants. In: RASKIN, I. and ENSLEY, B.D., eds. Phytoremediation of toxic metals: using plants to clean-up the environment. John Wiley & Sons, Inc., New York. 2000, p. 231-246. SAS (2006). SAS Systems for Windows, version 9.1.3. SAS Institute, Cary, NC. U.S. Canola Association. (2006). FDA Authorizes Qualified Health Claim for Canola Oil. Retrieved 2006-11-26: http://www.uscanola.com. Shen, R. and J. F. Ma. 2001. Distribution and mobility of aluminum in an Al-accumulating plant, Fagopyrum esculentum. J. of Exp. Bot. 52(361):1683-87. y = -3.4488x + 24 R² = 0.4124 Figures 3a, b and c show the response of seedlings from three winter canola cultivars treated with selected heavy metals at four different levels of concentration. CONCLUSION Significant statistical differences among cultivars for biomass production and among nutrients tested, indicate that such genetic differences will allow the selection of canola cultivars for their higher capacity to accumulate such elements as: Zn and Fe. However, highly significant difference exists for time of harvest and plant source (roots, leaves, stems, flowers, pods and seeds) where nutrients accumulate. The impact of heavy metals on canola seedling development was adversely affected by manganese; leaf damage to all cultivars gradually increased from no damage at level 1 to severe (leaf death) at level 4 (Figure 3b). Among the three cultivars evaluated in the heavy metal study, seedlings of Titan was least affected by increasing levels of heavy metals, except for root biomass where cultivar Jetton was significantly better. These results represent preliminaryanalysis of field and greenhouse studies. They show that winter canola cultivars can be selected for conditions in the southern region of the United States and as a candidate for Phytoremediation. Figure 6- (a, b). Zinc (Zn) accumulation is similar to Cu and Mn. Its uptake is low when Al is high during early establishment of the crop and reached its highest at the bolting. However, its accumulation during flowering stage decreased significantly from the bolting, where significant variation was observed among the cultivars. Regression analysis for each growth stage would provide more specific relationship between individual micro-nutrients with Al. E. Cebert • ACKNOWLEDGMENT • The Kansas State University National Winter Canola Variety Trial (NWCVT). Anson Josephand Geoffrey Reid for their help in field and laboratory work.