Tobacco smoking is one of principal routes tion of the genus is that by Goodspeed .... Paraguay, Uruguay and Brazil. They differ for chro- with two forms of N.
Genetic Resources and Crop Evolution 51: 323–333, 2004. 2004 Kluwer Academic Publishers. Printed in the Netherlands.
323
Variation for cadmium uptake among Nicotiana species Teresa Doroszewska* and Apoloniusz Berbec´ Institute of Soil Science and Plant Cultivation, ul. Czartoryskich, 8, 24 – 100 Pulawy, Poland; * Author for correspondence Received 19 March 2002; accepted in revised form 15 November 2002
Key words: Accumulation, Cadmium, Leaves, Nicotiana, Roots, Soil
Abstract Fifty nine species of Nicotiana were tested for the potential to take up cadmium from soil either with a natural cadmium content of 0.15 ppm or enriched with 8 mg Cd per 1 kg soil added as Cd(NO 3 ) 2 . When grown on the soil with natural cadmium content the highest leaf absorbers of cadmium were N. raimondii (6.15 ppm), N. bigelovii (5.47 ppm) and the botanical varieties of N. tabacum (3.92 – 5.75 ppm); the highest root absorber was N. rosulata (3.17 ppm). On the soil with an addition of 8 mg cadmium / kg the highest leaf cadmium contents were in N. langsdorfii (116.28 ppm), N. plumbaginifolia (99.49 ppm), N. setchellii (94.16 ppm) N. debneyi (69.12 ppm), N. velutina (62.15 ppm) and the highest root cadmium contents in N. miersii (32.2 ppm), N. clevelandii (30.45 ppm) and N. nudicaulis (30.45 ppm). Significant differences between sections were most frequently shown for the cadmium enriched soil and rarely for the control treatments. No significant differences were found between the botanical varieties of the same species when grown on the cadmium enriched vs. natural cadmium soil.
Introduction The genus Nicotiana is one of the most numerous in the egg-plant (Solanaceae) family. Its natural distribution is confined to North and South America, Australia, a few islands of the South Pacific and Africa. Currently the most widely accepted description of the genus is that by Goodspeed (1954) with some later additions by Burbidge (1960), Merxmueller and Butler (1975) Ohashi (1976) and Clarkson and Symon (1991). The genus Nicotiana is divided into 3 subgenera and 14 sections and comprises a total of 62 species. Goodspeed’s systematics of Nicotiana is based on geographical distribution, anatomy of trichomes, karyological data and chromosome pairing of interspecific F 1 hybrids. Spontaneous interspecific hybridization was probably the basic mechanism behind the evolution of the genus into the contemporary array of species. The contemporary Nicotiana species represent a wide variation in plant morphology, chromosome number, degree of cross-compatibili-
ty, response to fungal, bacterial and viral pathogens and in many other traits. A study was undertaken to assess the ability of wild Nicotiana species to take up cadmium – a potentially dangerous and very mobile pollutant (Terelak et al. 1998) - from the soil and accumulate it in the plant tissue. Tobacco smoking is one of principal routes through which cadmium can make its way into the human body and the tobacco plant is known for its ability to absorb large amounts of cadmium from the soil. The level of cadmium in tobacco varies from 0.77 to 7.02 ppm whereas in other food plants it does not exceed 0.05 ppm (Schenker 1984). There were indications that Nicotiana species and varieties differ for their potential to take up and accumulate cadmium. The understanding of that variation can be exploited either to select for low-cadmium genotypes as part of a crop improvement effort (Wagner et al. 1987) or, conversely, to look for high cadmium absorbers to be used in remediation of polluted soils. (Kumar et al. 1995). Most wild Nicotiana have mod-
324 erate soil requirements and in their natural habitats they grow on poor, rocky soils often deficient in water.
Material and methods A total of 59 Nicotiana species and 6 varieties of N. rustica Linnaeus, and N. tabacum Linnaeus, were evaluated for their ability to take up cadmium. The study was conducted in the greenhouse. The soil was a compost mix with a natural cadmium content of 0.15 ppm and a pH of 5.6. The cadmium was added as Cd(NO 3 ) 2 at 8 mg Cd per 1 kg soil. Each entry was grown in three replications on cadmium-enriched soil and on untreated control soil. Leaves were sampled for cadmium analysis as the plants of each entry reached flowering and seed ripening. The roots were cautiously removed from slightly dried soil, carefully washed to remove soil particles and dried at 35 – 40 8C. The cadmium contents of leaves and roots were assayed using inductively coupled plasma mass spectrometry of samples that were prepared by wet ashing with HNO 3 and H 2 O 2 . The results were analysed statistically within species grouped according to their taxonomy. Tukey test-based LSD values were calculated at 5% significance level. The data were either tabulated (for sections containing up to 4 species) or presented as graphs for larger sections.
Results The majority of Nicotiana species and botanical varieties of N. rustica and N. tabacum were analysed for cadmium content of leaves and roots. The data provided a basis for a comprehensive survey of the ability in the genus to take up cadmium while at the same time taking into account the taxonomic position of the surveyed species. Subgenus Rustica Section Paniculatae The section Paniculatae of the subgenus Rustica includes 7 species all of which come from South America (Bolivia, Argentina, Chile and Peru). They are 24-chromosome species, most of them perennials with the exception of N. paniculata Linnaeus, and N.
knightiana Goodspeed. All members of the section were included in the survey of cadmium uptake. N. raimondii Macbride was found to be the highest cadmium absorber from the untreated soil which made it stand out in the section. Cadmium content of the remaining species was very low and the differences were statistically insignificant (Figure 1). N. raimondii also had the highest cadmium content of leaves when grown on the cadmium-enriched soil. With the elevated content of soil cadmium there was a substantial variation for cadmium content among the members of Paniculatae (Figure 1). N. glauca Graham accumulated significantly more cadmium in the leaves than did N. paniculata, N. benavidesii Goodspeed and N. cordifolia Philippi. It was by far the highest accumulator of cadmium in the roots when grown on the cadmium-enriched soil (Figure 2). Sections Thyrsiflorae and Rusticae The sections Thyrsiflorae and Rusticae are classified in the subgenus Rustica and contain only one species each. N. thyrsiflora Bitter ex Goodspeed is a 24chromosome short-day species from northern Peru. N. rustica is a native of Andes – southern Peru and northern Bolivia. It has 48 chromosomes and flowers on long days. There are several botanical varieties and cultivars of that species known as makhorka or rustic tobacco. Two botanical varieties N. rustica var. brasilia Schrank and N. rustica var. pumila Schrank were analysed for cadmium content. The data on cadmium content in N. thyrsiflora are shown in (Table 1). No major differences in cadmium content of leaves or roots of the two varieties of N. rustica were found at both cadmium levels of the soil. Subgenus Tabacum Section Tomentosae The section Tomentosae belongs to the subgenus Tabacum and contains six 24-chromosome species naturally occurring in Bolivia, Peru and Ecuador. All species of the section were included in the survey of cadmium uptake. In the control treatment (natural soil) the amounts of accumulated cadmium were relatively low in all entries and showed little variation from species to species (Figure 3). On the cadmium enriched soil the reverse was true – the level of cadmium accumulated in the leaves of N. setchellii Goodspeed was three times that of the remaining species. The buildup of cadmium in the roots was similar in all entries (Figure 4).
325
Figure 1. Cadmium content of the leaves of the members of the section Paniculatae grown at natural and elevated (8 ppm) cadmium content of the soil
Figure 2. Cadmium content of the roots of the members of the section Paniculatae grown at natural and elevated (8 ppm) cadmium content of the soil.
Table 1. Cadmium content of leaves and roots of N. thyrsiflora and botanical varieties of N. rustica grown on a compost soil with natural cadmium content and enriched with 8 ppm of cadmium. Section Thyrsiflorae Rusticae
Species natural soil
Chromosome number (2n) Cd-enriched soil
Leaves natural soil
Cd-enriched soil
Roots
N. thyrsiflora N. rustica var. brasilia N. rustica var. pumila LSD 5%
24 48 48
1.15 0.56 0.80 0.8
37.93 32.72 25.93 13.57
1.18 0.92 0.46 0.74
19.58 6.85 4.87 2.77
326
Figure 3. Cadmium content of the leaves of the members of the section Tomentosae grown at natural and elevated (8 ppm) cadmium content of the soil.
Section Genuinae The section Genuinae is made up of only one species N. tabacum – cultivated tobacco represented by a vast number of cultivars existing worldwide. It is a 48chromosome species of allopolyploid origin. Its genome is made up of two subgenomes thought to be contributed by ancestral forms of N. sylvestris
Spegazzini et Comes and N. tomentosiformis Goodspeed. Cultivars of N. tabacum are the subject of a separate study. In this study a few botanical varieties were included. The level of cadmium accumulated by the botanical varieties of N. tabacum was relatively high when the plants were grown on cadmium-untreated soil. There
Figure 4. Cadmium content of the roots of the members of the section Tomentosae grown at natural and elevated (8 ppm) cadmium content of the soil.
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Figure 5. Cadmium content of the leaves of the botanical varieties of N. tabacum grown at natural and elevated (8 ppm) cadmium content of the soil.
were no significant differences in the level of leaf or root cadmium among the varieties grown either at natural or elevated soil cadmium (Figure 5,6). Subgenus Petunioides The subgenus Petunioides is the most numerous in the genus as it includes 9 sections and 52 species. It is also the most diversified one both for its geographical
distribution and for the genetics of the constituent species. Chromosome number in the subgenus ranges from 18 to 48. Both polyploids and aneuploids are represented. Section Undulatae There are three species in the section Undulatae: N. undulata Ruiz et Pavon, N. arentsii Goodspeed and
Figure 6. Cadmium content of the roots of the botanical varieties of N. tabacum grown at natural and elevated (8 ppm) cadmium content of the soil.
328 Table 2. Cadmium content of the leaves and the roots of plants of the sections Undulatae and Trigonophyllae grown in compost soil with natural Cd content or enriched with 8 ppm of Cd. Section
Species
Undulatae
N. undulata N. arentsii LSD 5% N. trigonophylla N. palmerii LSD 5%
Trigonophyllae
Chromosome number (2n)
Leaves natural soil
Cd-enriched soil
Roots natural soil
Cd-enriched soil
24 48
1.37 0.71 0.89 0.40 0.73 0.45
17.8 8.17 6.63 35.51 43.88 12.86
0.86 0.81 0.60 0.55 1.27 1.41
17.8 5.03 6.43 13.2 11.32 4.35
24 24
N. wigandioides Koch et Fintelmann. They are native of Bolivia and Peru. Of the two species included in the cadmium uptake survey – N. undulata and N. arentsii - the former was a higher cadmium absorber, especially from the cadmium-enriched soil (Table 2). Section Trigonophyllae The section Trigonophyllae contains two species originating from Mexico and the southern United States. Some taxonomists treat them as botanical varieties of one species. Both showed similar rates of the uptake and accumulation of cadmium in leaves and roots which may reflect their close affinity (Table 2). Section Alatae The section Alatae is made up of seven species from South America, especially from Argentina, Bolivia, Paraguay, Uruguay and Brazil. They differ for chro-
mosome number. All are annuals and early-flowering under Poland’s conditions. N. alata Link et Otto as found to absorb relatively high amounts of cadmium from the soil with natural cadmium level. When grown on cadmium-enriched soil the species of the section varied widely for their ability to accumulate cadmium (Figure 7) with N. plumbaginifolia Viviani absorbing the highest amounts of cadmium in the roots (Figure 8). Section Repandae The section Repandae is made up of three species: N. repanda Willdenow, N. stocktonii Brandegee and N. nesophila Johnston. They originate from the southern United States (Texas), Mexico and the neighbouring islands. They are 48-chromosome annuals. Since N. stocktonii is missing from the Pulawy collection only the two remaining species were included in the study with two forms of N. repanda – a diploid and an
Figure 7. Cadmium content of the leaves of the members of the section Alatae grown at natural and elevated (8 ppm) cadmium content of the soil.
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Figure 8. Cadmium content of the roots of the members of the section Alatae grown at natural and elevated (8 ppm) cadmium content of the soil.
artificially made autotetraploid. The species were found to vary in their ability to absorb cadmium from the cadmium-enriched soil (Table 3). Of the two N. repanda forms the tetraploid absorbed less cadmium than did the diploid.
Section Noctiflorae The section Noctiflorae contains four 24-chromosome species, N. ameghinoi Spegazzini being a herbarium specimen in Argentina and N. acaulis Spegazzini being maintained in vitro in some collections. The other two - N. noctiflora Hooker and N. petunioides (Grisebach) Millan – were found to differ significantly for their ability to accumulate cadmium both in
leaves and in roots when grown on the cadmiumenriched soil (Table 3). Section Bigelovianae Of the two 48-chromosome species of the section N. bigelovii (Torrey) Watson comes from California (USA) and N. clevelandii Gray was found in the south-western Unites States and in north-eastern Mexico. N. clevelandii was found to be the significantly higher cadmium absorber of the two (Table 3). Section Nudicaules The sole species in the section – N. nudicaulis Watson – native of north-eastern Mexico was found to absorb relatively low amounts of cadmium from natural soil
Table 3. Cadmium content of the leaves and the roots of plants of the sections Repandae, Noctiflorae, Bigelovianae, Nudicaules and Trigonophyllae grown in compost soil with natural Cd content or enriched with 8 ppm of Cd. Section
Species
Repandae
N. repanda N. repanda N. nesofila LSD 5% N. noctiflora N. petunioides LSD 5% N. bigelovii N. clevelandii LSD 5% N. nudicaulis
Noctiflorae
Bigelovianae
Nudicaules
Chromosome number (2n)
Leaves natural soil
Cd-enriched soil
Roots natural soil
Cd-enriched soil
48 96 48
0.81 0.55 0.85 0.65 1.39 1.15 0.63 5.47 1.03 2.15 0.37
42.86 34.14 12.19 15.14 26.94 61.13 12.8 16.74 39.18 9.42 30.33
0.55 1.47 0.83 1.16 1.02 0.90 0.91 1.27 1.48 0.53 1.09
20.27 12.96 10.56 3.91 6.96 25.23 8.27 11.61 30.52 4.45 30.45
24 24 48 48 48
330
Figure 9. Cadmium content of the leaves of the members of the section Acuminatae grown at natural and elevated (8 ppm) cadmium content of the soil.
and relatively high from cadmium enriched soil (Table 3).
Section Acuminatae The section Acuminatae comprises eight 24-chromosome species – all of them from South America with some reaching into the western USA and up to
southern Canada. Of six species included in the study N. attenuata Torrey ex Watson, N. miersii Remy and N. linearis Philippi were the highest cadmium absorbers (Figure 9,10). N. miersii and N. linearis are close phenotypically, short-grown (up to 50 cm). They form a large number of small narrow leaves. When exposed to elevated soil cadmium they responded with checked growth. The species varied significantly
Figure 10. Cadmium content of the roots of the members of the section Acuminatae grown at natural and elevated (8 ppm) cadmium content of the soil.
331 for their ability to accumulate cadmium in the roots when grown on cadmium-enriched soil. Section Suaveolentes It is the largest section in the genus Nicotiana made up of 22 species the overwhelming majority of which are native to Australia and to several Pacific islands. Of the two most recent additions N. wuttkei Clarkson et Symon is indigenous to Australia whereas N. africana Merxmueller was found on isolated hillsides in central Namibia in Africa. The section comprises species that differ phenotypically, genetically and for chromosome number. Many of them are of aneuploid origin with a chromosome number that is not a multiplication of six – recognized as basic in the genus Nicotiana. Twenty Suaveolentes species were included in the survey of cadmium uptake and accumulation. The large variation among the species of the section is also reflected by the response to an abiotic factor – cadmium in this particular case. The plants of N. gossei Domin and N. excelsior Black grown at natural soil cadmium content showed high cadmium contents of leaves (Figure 11). However, they did not respond to elevated soil cadmium with a dramatic increase of cadmium content of above-ground parts which was
the case with other species. In a cadmium-rich environment the highest cadmium contents of leaves were found in N. debneyi Domin, N. velutina Wheeler, N. africana and N. occidentalis Wheeler. N. occidentalis and N. debneyi also stood out as the highest absorbers of cadmium in the roots (Figure 12).
Discussion The investigations of the ability to take up and accumulate cadmium comprised nearly all Nicotiana species and it provided an extensive survey of the response of those genotypes to different levels of cadmium in the soil. The survey showed that the species of Nicotiana varied greatly for that trait. The same conclusions were drawn by Wagner and Yeargan (1985a) from investigating a group of tobacco varieties and species. Based on the study of 12 genotypes the investigators reported high capability of cadmium accumulation by the investigated species both in the above-ground parts and in the roots. Genotypes that absorb small cadmium amounts from both low- and high-cadmium soils are of the greatest interest for crop improvement. Another po-
Figure 11. Cadmium content of the leaves of the members of the section Suaveolentes grown at natural and elevated (8 ppm) cadmium content of the soil.
332
Figure 12. Cadmium content of the roots of the members of the section Suaveolentes grown at natural and elevated (8 ppm) cadmium content of the soil.
tentially valuable group is made up of species that while taking up substantial amounts of cadmium do not translocate them to the stems and leaves. In tobacco, which is a leaf crop, the ability to accumulate low amounts of cadmium in the leaves is of the greatest importance. In the study by Wagner and Yeargan (1985b) N. rustica and N. rotundifolia Lindley were found to be low absorbers of leaf cadmium. Those findings are in agreement with the results obtained in this study, especially for N. rotundifolia. When grown on low-cadmium soil the species accumulated much cadmium in the roots but little in the leaves. However, on the high-cadmium soil the element was translocated to and built up in the leaves as well. Since this study was performed on a large group of species it was possible to find genotypes with highly restricted ability to take up and accumulate cadmium when exposed to both low- and high soil cadmium. It must be noted that the high-cadmium treatment involved an extremely high concentration of that metal which rarely occurs in natural conditions. N. benavidesii Goodspeed and N. umbratica Burbidge were found to absorb relatively little cadmium under heavy supply of that element in the soil. The results
are of interest because those species might be used in breeding work. N. benavidesii is a particularly interesting candidate because it has already a record of tobacco improvement application as a source of resistance to PVY (Berbec´ 1987). A low leaf-to-root ratio of cadmium content was found in the species N. arentsii, N. cordifolia, N. nesofila, N. tomentosa Ruiz et Pavon (Doroszewska, Berbec´ 1996). It indicates a low rate of translocation of the metal from roots to leaves. The study confirmed the opinion that cadmium has a substantial potential of being accumulated by N. tabacum (Mueller 1979). The varieties investigated for the trait showed very high contents of that metal when grown on the soil with natural cadmium content. This is a heritable trait and can be manipulated either by classical breeding methods or by Agrobacterium tumefaciens-mediated transformation (Wagner et al. 1987). The harmful effect of cadmium was manifested as growth disturbances, premature yellowing of leaves and premature aging. It was particularly conspicuous in the species of the section Alatae: N. langsdorfii Weinmann, N. alata, N. sylvestris. According to Reese and Roberts (1984) the mitotic index and the
333 level of total DNA in cadmium-exposed plants indicated that cadmium affects cell divisions which accounts for its inhibitory effect on plant growth. The species which show high cadmium accumulation are N. langsdorfii, N. plumbaginifolia, N. setchelli, N. debneyi and N. velutina. According to Mc Grath et al. (1993), Kumar et al. (1995) plant species with high potential of cadmium accumulation in their biomass can be used to clean up the most polluted soils. Some Thlaspi species are a case in point. The Nicotiana species mentioned above are able to produce much more herbage from unit area, a thing of advantage from the standpoint of phytoremediation purposes. A great variability among the species of Nicotiana was also reflected by the ability to take up and accumulate cadmium. It was shown by the huge variation of cadmium contents from species to species and by the lack of significant differences between varieties of the same species.
References Berbec´ A. 1987. Chromosome pairing and pollen fertility in the interspecifics F 1 hybrids Nicotiana tabacum L. x N. benavidesii Goodspeed, N. knightiana Goodspeed x N. tabacum, and N. raimondii Macbride x N. tabacum. Genetica Polonica 28: 263– 269. Burbidge N.T. 1960. The Australian species of Nicotiana. Australian J. Botany 8: 342–380. Clarkson J.R. and Symon D.E. 1991. Nicotiana wuttkei (Solanaceae), a new species from north-eastern Queensland with an unusual chromosome number. Austrobaileya 3: 389–392. ´ 1996. Uptake of cadmium by some Doroszewska, T., A. Berbec,
Nicotiana species. Inform. Bull. Yokohama CORESTA Congress: 12. Goodspeed T.H. 1954. The genus Nicotiana. Chronica Botanica, Waltham, Mass., USA, 323–489. Kumar P.B.A.N., Dushenkov V., Motto H. and Raskin I. 1995. Phytoextraction. The use of plants to remove heavy metals from soils. Environ.Sci.Technol. 29: 1232–1238. Mc Grath, S.P., C.M. Sidoli & R. D. Reeves, 1993. Plants clean up soils. (Presented by Professor Steve Mc Grath at the BAAS Science for Life meeting on August 1993). Merxmueller H. and Butler K.P. 1975. Nicotiana in der Afrikanishchen Namib. Ein pflanzengeographisches und ¨ phylogenetisches Ratsel. Mitteilungen der Botanischen Staats¨ sammlung Munchen 12: 91–104. Mueller G. 1979. Heavy metal content (Cd, Zn, Pb, Cu, Cr) of tobacco in commonly smoked cigarettes in the Federal Republic of Germany. Chem.-Zeit. 103–4. 33–39. Ohashi, Y., 1976. Nicotiana kawakamii : a new species of the genus Nicotiana. Bul. Spec. CORESTA Congress Tokyo: (P001). Reese R. and Roberts L.W. 1984. Cadmium uptake and its effects on growth of tobacco cell suspension cultures. Plant Cell Rep. 3-3. 91–94. Schenker D. 1984. Considerations on the cadmium content of tobacco products. Forum Staedte-Hyg. 35-1. 17–24. ´ Terelak H., Motowicka – Terelak T. and Stuczynski T. 1998. ´´ metali cie˛zkich ~ ~ ´ rolnych Zawartosc i siarki w glebach uzytkow Wielkopolski na tle ich wyste˛powania w glebach kraju. Zeszyty ´ Nauk Rolniczych 460: 23–39. Problemowe Poste˛pow Wagner G.J. and Yeargan R. 1985a. Species differences in shoot versus root Cd accumulation in tobacco and the influence of Cd concentrantion on the nature of accumulated Cd-forms. Plant Physiol. Suppl. 77 - 4: 119. Wagner G.J. and Yeargan R. 1985b. Cadmium in tobaccos. Genetic variation in accumulation potential and possible mechanism of accumulation. Tob. Chem. Res. Conf. 39: 38. Wagner G.J., Kandra G., Maiti B. and coll. 1987. Application of plant breeding and genetic engineering to reducing cadmium accumulation in tobacco leaves. Tob. Chem. Res. Conf. 41: 32.
