Seed germination and seedling radicle length were measured to determine plant sensitivity to Se. Germination was unaffected by Se treatment. The effect of Se ...
Environmentaland ExperimentalBotany, Vo|. 29, No. 4, pp. 493-498, 1989
0098 8472/89 $3.00 + 0.00 Pergamon Press plc
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EFFECTS OF S E L E N I U M ON G E R M I N A T I O N A N D R A D I C L E E L O N G A T I O N OF SELECTED A G R O N O M I C SPECIES C. L. CARLSON,* D. I. K A P L A N and D. C. ADRIANO
Biogeochemistry Division, Savannah River Ecology Laboratory, Drawer E, Aiken, SC 29801, U.S.A.
(Received 6 January 1989; acceptedin revisedform 3 April 1989) CARLSON C. L., KAPLAN D. I. and ADRIANO D. C. Effects of selenium on germination and radicle elongation qf selected agronomic species. ENVIRONMENTALAND EXPERIMENTALBOTANY 29, 493498, 1989. Certified seeds of cabbage (Brassica oleracea var. capitata L.), lettuce (Lactuca sativa L.), radish (Raphanus sativus var. radicula Perzoon.), two varieties of sorgrass (Sorghum vulgare sudanense Hitchc. var. Dub-L-Graze and var. Sugar-Graze), turnip (Brassica rapa L.), and wheat (Triticum aestivum L. var. Caldwell) were treated with fresh, pH-adjusted solutions of 0, 1, 2, 4, 8, 16, and 32 mg Se/1 in either the selenate or selenite form for 2-4 days. Seed germination and seedling radicle length were measured to determine plant sensitivity to Se. Germination was unaffected by Se treatment. The effect of Se on radicle length varied with plant species, Se form, and Se concentration. Selenite treatment generally caused the greater decrease in radicle length. Plant sensitivity to selenate varied as follows: turnip > Sugar-Graze sorgrass = lettuce = cabbage > Dub-L-Graze sorgrass = wheat > radish. For selenite, the sensitivity ranking was: turnip = cabbage > Sugar-Graze sorgrass > radish = lettuce = Dub-L-Graze sorgrass > wheat. Sensitivity to Se varied not only among plant species, but also between the two sorgrass varieties.
INTRODUCTION
SELENIUM is a trace element of a g r o n o m i c i m p o r t ance because the range between deficient a n d toxic levels in a n i m a l diets is narrow./ll'zl) T h e n u t r i t i o n a l m i n i m u m for animals in foodstuffs is 0.05-0.10 m g Se/kg; lower values could result in Se deficiency./~l) Toxic effects on animals occur when the Se c o n c e n t r a t i o n o f feed reaches 2-5 m g Se/kg.(~) I n some environments, p a r t i c u l a r l y the western U.S., concentrations of Se which are potentially toxic to animals, including fish a n d waterfowl, can occur in waters a n d soils (up to 4.2 m g Se/1 in water, 40 m g Se/kg in soil)./l't8) Selenium is also a c o m m o n c o n t a m i n a n t in coal fly ash,/~'~5! a n d p l a n t u p t a k e o f this element could
be a p r o b l e m if fly ash is used as a soil a m e n d ment.(2,17) Selenium occurs p r i m a r i l y in two forms in the n a t u r a l environment, selenate (SeO42-) a n d selenite (SeO~-)./l'8) R e d o x potential and p H to a large extent control the relative a m o u n t s and solubility of these forms, with selenate the domin a n t form u n d e r alkaline, oxidizing conditions, and selenite the d o m i n a n t form u n d e r acid, reducing conditions. (1'8) Selenite is the more toxic form to plants grown in solution culture, (~4'2°) b u t selenate is generally the m o r e toxic form in soils (5'22) since selenite is i m m o b i l i z e d in soil due to adsorption on clays a n d h y d r o u s oxides./~6) Different species a n d varieties of plants v a r y in their ability to a c c u m u l a t e Se, (7'12) with m e m b e r s
* To whom correspondence should be addressed. 493
C . L . CARLSON et al.
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of the Brassicaceae generally accumulating large amounts (up to 1240 mg Se/kg) /13/ and members of the Poaceae and Asteraceae accumulating much smaller amounts (up to 470/~3~ and 56/~°/ mg Se/kg, respectively),(5,10,12,13) Studies by LEVINE (14) and SPENCER and SmoEL/2°/ have indicated that Se can affect germination and radicle elongation in some plant species. However, extremely high concentrations of Se were needed to produce an effect, with germination affected by 5.50 m M Se as selenite and 28.0 m M as selenate in the study by SPENCER and S~EOrL. (2°/ The range of concentrations used by LEVINE(14) and SPENCER and SmOEL(2°/ was so wide, however, and so few levels were used, that the actual point at which Se became toxic could not be ascertained. For example, LEVINE(14)found no effect of selenite on germination of white lupin (Lupinus sp.) at a treatment concentration of 4.6 mg Se/1 (58.4 # M Se), but at his next treatment level, 46 mg Se/1 (584/~M Se), only 20O/o of the seeds germinated. A study using a relatively large number of concentrations over the relatively narrow range of Se concentrations likely to be encountered in natural and polluted systems (less than 40 mg Se/kg) (1/ was needed. This study was conducted to determine the effect of selenium form and concentration on seed germination and subsequent early radicle growth of seven agronomic species. METHODS
Experimental solutions were prepared using Na2SeO4 and Na2SeO3 salts (obtained from Fluka Chemical Corporation, Hauppauge, New York; guaranteed > 98O/o pure) and double deionized water, to obtain final concentrations of 0, 1, 2, 4, 8, 16 and 32 mg Se/1 (0, 12.7, 25.3, 50.7, 101.3, 202.6 and 405.3 #M Se) in either the selenate or selenite form. Thermodynamic solubility diagrams indicated that conversion of selenite to selenate would be minimal at p H 5.0. (8/ Accordingly, all solutions were adjusted to p H 5.0, using HC1 or N a O H as needed. Fresh solutions were prepared for each trial, with one species examined per trial. Seeds of cabbage (Brassica oleracea var. capitata L.), lettuce (Lactuca sativa L.), radish (Raphanus sativus var. radicula Perzoon.), two varieties of s o r -
grass (Sorghum vulgate sudanense Hitchc. vat. DubL-Graze and var. Sugar-Graze), turnip (Brassica raps L.), and wheat (Triticum aestivum L. var. Caldwell) were obtained from a certified seed dealer in Aiken, SC. These species were selected because previous studies/5'1°'12'13/ suggested that they would vary in sensitivity to Se. Seeds of a given species were placed, 10 seeds per dish, in 15-cm polyethylene Petri dishes, each containing one piece of W h a t m a n No. 1 filter paper. Each Petri dish was then treated with the appropriate solution, wrapped with Parafilm (TM), and placed in an environmental chamber in a randomized, complete-block design. Eight replicates were used for each treatment, for a total of 112 dishes per plant species (two forms x seven concentrations x eight replicates). The chamber was maintained on a 16/8 hr day/night photoperiod, with day and night temperatures of 28 and 21 °C, respectively. Illumination was supplied from cool white fluorescent lamps providing 64 /~E m -2 s e c t . The amount of solution added to each dish and the length of the experimental period varied with plant species as follows: cabbage, 4 ml, 3 days; lettuce, 4 ml, 3 days; radish, 3 ml, 2 days; Dub-L-Graze sorgrass, 5 ml, 3 days; Sugar-Graze sorgrass, 5 ml, 4 days; turnip, 4 ml, 3 days; and wheat, 5 ml, 3 days. Sufficient solution was added to ensure that the seeds would remain moist following imbibition. The duration of each trial was kept to a minimum to reduce the likelihood of conversion of selenite to selenate. Radicle lengths of the controls of each species were allowed to reach approximately 20 mm. Upon termination of the experiment, the number of seeds germinated for each dish and radicle length for each seedling in a dish were measured. The radicle lengths in a given Petri dish were averaged to obtain a mean length per dish. These averages were used for the statistical analysis. Per cent germination was also calculated and used in the statistical analysis. Linear regression and multiple comparisons were performed using the SAS statistical package (SAS Institute, Cary, NC). Preliminary analysis of the data indicated that significant differences in radicle length existed depending on the shelf location of the plate in the environmental chamber and the person measuring the seedling. To eliminate these differences, measurer and shelf
EFFECTS OF SELENIUM ON GERMINATION AND RADICLE ELONGATION were used as blocking variables in the analysis. To allow comparison among plant species, radicle lengths expressed as a per cent of the control were calculated. These radicle length percentages were then used to determine the relative sensitivity of the plants to Se. The germination numbers were expressed similarly. RESULTS
Selenium in the form ofselenate or selenite did not affect germination (P > 0.05) at concentrations up to 32 mg Se/1. The effect of selenium on early radicle elongation varied depending on plant species, Se form, and Se concentration. Treatment of radish with up to 32 mg Se/1 as selenate did not significantly (P > 0.05) affect radicle length (Fig. la). However, treatment with selenite reduced (P < 0.001) radicle length by as much as 62% at the highest concentration of 32 mg Se/1. Radicle length for wheat was significantly (P < 0.001) reduced after treatment with either form, with greater (LSD0.05 = 3.99) reductions in the presence of selenite (Fig. 1b). Both sorgrass varieties showed significant (P < 0.001) reductions in radicle length upon treatment with either selenate or selenite, with selenite being more deleterious [for Sugar-Graze, the least square difference at the 0.05 level (LSD0.05) = 8.89; for Dub-L-Graze, LSD0.05 = 9.42] for both plants (Figs lc and ld). Significant ( P < 0.001) reductions in radicle length of lettuce occurred for both forms of Se. In contrast to the other plants studied, lettuce exhibited greater (LSD0.05 = 5.31) decreases in radicle length when treated with the selenate form (Fig. le). The lettuce experiment was repeated to ascertain if this unusual response were real or a result of experimental error. The results of the second experiment confirmed the earlier results. Cabbage also exhibited significant (P < 0.001) reductions in radicle length in response to both Se forms (Fig. lf). Again, greater (LSD0.05 = 3.86) reductions in radicle length occurred when selenium was applied in the selenite form. Turnip exhibited large (P < 0.001) reductions in radicle length upon treatment with either Se form, with greater (LSD0.05 = 4.81) reductions in the presence of selenite (Fig. lg). O f the plant species tested, radish and wheat
495
were the least sensitive (based on reductions in radicle length) to selenium treatment, with cabbage and turnip the most sensitive (Fig. 2). The sensitivity ranking of the plants studied varied with Se form. For selenate, the order of sensitivity was: turnip > Sugar-Graze sorgrass = lettuce = cabbage > Dub-L-Graze sorgrass = wheat > radish. For selenite, the order or sensitivity was: turnip = cabbage > sugar-Graze sorgrass > radish = lettuce = sub-L-Graze sorgrass > wheat.
DISCUSSION
The results of this study confirm that plant species and varieties differ in their sensitivity to selenium. The relative sensitivity of cabbage (Brassicaceae), turnip (Brassicaceae), lettuce (Asteraceae), sorgrass (Poaceae), and wheat (Poaceae) generally fits well within the order of Se uptake reported for members of these families. (l°J2'13) HURD-KARRER (1~) found the highest Se uptake by members of the Brassicaceae, followed by members of the Asteraceae and the Poaceae, respectively, in soil treated with 5 mg Se/kg as selenate. FLEMING (l°) found the order of plant uptake of Se from Irish soils to be turnip > cabbage > radish > lettuce > wheat. HAMILTON and BEATH (12) also found that Se uptake by lettuce was less than that by cabbage and radish. In the present study, wheat and DubL-Graze sorgrass were less sensitive to Se than lettuce, while cabbage and turnip were more sensitive. Sugar-Graze sorgrass was similar to lettuce in sensitivity. However, not all of the members of the Brassicaceae included in the present study responded similarly to Se. Cabbage and turnip were the most sensitive to selenium; radish was the least sensitive to selenate of the plants studied but was somewhat sensitive to selenite. This varied response of the Brassicaceae species was surprising in light of reports that all three species accumulate Se in large quantities. (1°'12'13) FLEMING (10) reported that radish, cabbage, and turnips grown in soils containing 6-16 mg Se/kg (Se form not determined) contained as much as 145, 196 and 409 mg Se/kg, respectively. All three species also accumulated large amounts of S, 2700 mg S/kg for radish, 4600 mg S/kg for cabbage, and 1900 mg S/kg
496
C . L . CARLSON et al.
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for turnip. This high Se accumulation has been attributed to the usually high S absorption by these members of this family./13'16) Selenium can be an analog of S and exerts its toxic effect by interfering with the S metabolism of the plant./3'7) The varied response of radish, cabbage, and turnip may reflect differences in the metabolism of Se among these species. Comparisons of accumulator and non-accumulator species have indicated differences in the compounds produced during the metabolism of Se. (3A1)It is possible that such differences also exist among species which accumulate Se, thereby explaining the responses of radish, cabbage and turnip in the present study. Selenite was generally the more toxic Se form for the plants studied (Fig. 2), supporting the results of previous solution-culture studies. (~4'2°/ The greater toxicity of selenite may be explained by the fact that selenate is converted to selenite within the plant before it exerts its toxic effect, i.e. before it is incorporated into amino acids. (3'7) The conversion ofselenate to selenite is analogous to the conversion of sulfate to sulfite prior to the incorporation of S into amino acids. ~3'7) Lettuce was the only plant studied for which radicle length was greater in the presence of selenite, although this unusual effect was only observed at concentrations less than or equal to 8 mg Se/1. Examination of the roots of lettuce
497
seedlings from the selenate and selenite treatments indicated striking morphological differences not seen in the other plants tested. The radicles of the selenite-treated plants, while longer than those for the selenate-treated plants, were much thinner, with fewer root hairs. The radicles of the selenate-treated plants were more robust, with profuse root hairs, suggesting that the selenate-treated radicles were healthier than those treated with selenite. It appears that radicle length alone is not a good indicator of Se toxicity for this particular species. This is surprising because of the important role of protein synthesis in radicle elongation,/4'~9) and the toxic effect of Se on protein synthesis. (3'7) Germination was not significantly affected by the levels of Se used in this study. This confirms the results of LEVINE(~4) and SPENCER and SIEGEL(2°) which indicated that very high (46-253 mg Se/1 as selenite) concentrations of Se were necessary to reduce seed germination. Seedling establishment, rather than seed germination, appears to be the sensitive stage. In summary, germination of radish, wheat, cabbage, lettuce, turnip, and two varieties ofsorgrass was not affected by treatment with selenate or selenite at solution concentrations as high as 32 mg Se/1. Significant differences did occur in radicle length depending on Se form, Se concentration, and plant species. Previous studies have suggested that cabbage, radish and turnip, members of the Brassicaceae family, should respond similarly to Se due to their high S absorption and demonstrated ability to accumulate Se. However, in the present study, radish was the least affected by Se treatment, with no reduction in radicle length upon treatment with selenate and some reduction upon treatment with selenite. Turnip and cabbage were the most sensitive to Se treatment, with substantial reductions in radicle length upon treatment with either Se form. Selenite was generally the more toxic form for the plants studied.
Acknowledgments--This research was supported by contract DE-AC09-76SROO-819 between the U.S. Department of Energy and the University of Georgia. Thanks are due to M. HEADEN, T. TRUAX and S. PR~SNELLfor assistance with data collection, and to P. DIXONfor assistance with the statistical analysis.
498
C . L . CARLSON et al. REFERENCES
l. ADRIANOD. C. (1986) Trace elements in the terrestrial environment. Springer, New York. 2. ADRIANO D. C., PAGE A. L., ELSEEWI A. A., CHANGA. C. and STRAUGHANI. (1980) Utilization and disposal of fly ash and other coal residues in terrestrial ecosystems: a review J. envir. Qual. 9, 333-344. 3. ANDERSONJ.W. and SCARFA. R. (1983) Selenium and plant metabolism. Pages 241-275 in D. A. ROBB and W. S. PIERPONT, eds Metals and micronutrients: uptake and utilization by plants. Academic Press, New York. 4. BEWLEVJ. D. and BLACK M. (1985) Seeds: physiology of development and germination. Plenum Press, New York. 5. BISBJERG B. and GISSEL-NmLSEN G. (1969) The uptake of applied selenium by agricultural plants. I. The influence of soil type and plant species. Pl. Soil 31,287-298. 6. BRADB~ERJ. W. (1988) Seed dormancy and germination. Chapman and Hall, New York. 7. BROWN T. A. and SHRIFT A. (1982) Selenium: toxicity and tolerance in higher plants. Biol. Rev. 57, 59-84. 8. ELRASHIDI M. A., ADRIANO D. C., WORKMANS. M. and LINDSAYW. L. (1987) Chemical equilibria of selenium in soils: a theoretical development. Soil Sci. 144, 141-152. 9. EVANSM. L. (1984) Functions of hormones at the cellular level of organization. Pages 23 79 in T. K. SCOTT, ed. Hormonal regulation of development II, Encyclopedia of Plant Physiology New Series, Vol. 10. Springer, Berlin. 10. FLEMINGG. A. (1962) Selenium in Irish soils and plants. Soil Sci. 94, 28-35. 11. GISSEL-NIELSEN G., GUPTA U. C., LAMAND M. and WESTERMARCK T. (1984) Selenium in soils and plants and its importance in livestock and human nutrition. Adv. Agron. 37, 397-461. 12. HAMILTONJ.W. and BEATH 0 . A. (1964) Amount
13. 14. 15.
16.
17.
18.
19.
20.
21. 22.
and chemical form of selenium in vegetable plants. Agric. Food Chem. 12, 371-374. HURD-KARRERA. M. (1935) Factors affecting the absorption of selenium from soils by plants. J. agric. Res. 50, 413-427. LEVINE V. E. (1925) The effect of selenium compounds upon growth and germination in plants. Am. J. Bot. 12, 82-90. MBAGWUJ. S. C. (1983) Selenium concentrations in crops grown on low-selenium soils as affected by fly-ash amendment. Pl. Soil 74, 75-81. MIKKELSENR. L., PAGE A. L. and BINGHAMF. T. (1989) Factors affecting selenium accumulation by agricultural crops. Pages 65-93 in L. W. Jhcons, ed. Selenium in agriculture and the environment, ASA Special Publication. American Society of Agronomy, Madison, Wisconsin. PAGE A. L., ELSEEWI A. A. and STRAUGHANI. R. (1979) Physical and chemical properties of fly ash from coal-fired power plants with reference to environmental impacts. Residue Rev. 71, 83 120. PRESSER T. S. and BARNES I. (1985) Dissolved constituents including selenium in waters in the v#inity of Kesterson National Wildlife Refuge and the West Grassland, Fresno and Merced Counties, California. Water Resources Investigations Report 85-4220. U.S. Geological Survey, Washington, DC. SIMONE. W. (1984) Early events in germination. Pages 77-115 in D. R. MURRAY, ed. Seed physiology, Vol. 2, Germination and reserve mobilization. Academic Press, New York. SPENCER N. E. and SIEGEL S. M. (1978) Effects of sulfur and selenium oxyanions on Hg-toxicity in turnip seed germination. Wat. Air Soil Pollut. 9, 423-427. UNDERWOOD E . J . (1977) Trace elements in human and animal nutrition. Academic Press, New York. VAN DORST S. H. and PETERSON P. J. (1984) Selenium speciation in the soil solution and its relevance to plant uptake. J. Sci. Food Agric. 35, 601-605.
