Nuclear Instruments and Methods in Physics Research B 267 (2009) 2884–2889

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Nuclear Instruments and Methods in Physics Research B

journal homepage: www.elsevier.com/locate/nimb

Elemental analysis of edible grains by micro-PIXE: Common buckwheat case study

K. Vogel-Mikuš a, P. Pelicon b, P. Vavpetič b, I. Kreft a,*, M. Regvar a
a
Biotechnical Faculty, University of Ljubljana, Jamnikarjeva 101, SI-1000 Ljubljana, Slovenia
b
Jožef Stefan Institute, Jamova 39, SI-1000 Ljubljana, Slovenia

a r t i c l e i n f o a b s t r a c t

Article history: The aim of this study was the adaptation of the micro-PIXE method for analysis of nutritionally relevant
Received 5 April 2009 heavy elements in different tissues of the grain of common buckwheat (Fagopyrum esculentum), as a rep-
Received in revised form 15 June 2009 resentative nutritionally interesting grain food source. At 57% of the buckwheat grain biomass, the endo-
Available online 22 June 2009
sperm was a modest nutrient source when compared to the cotyledons, at 17% of the biomass. These
latter contained high concentrations of trace elements, representing 91% of the total grain Zn, 87% for
PACS: P, 70% for S, 62% for Mg, 60% for K, 54% for Cu, 53% for Mn and 35% for Fe. The husk provided storage
82.80.Yc
for 85% of the total Ca, 84% for Al, 83% for Si, 76% for Cl, 69% for Ti and 46% for Fe. Knowledge on these
34.50.Dy
68.43.Mn
preferential elemental constitutions of the different grain tissues makes the possibility of designing target
28.52.Fa products with nutritionally optimal constitution more feasible. These data represent a basis for a more
targeted approach to nutritional improvement of grains intended for human consumption.
Keywords: Ó 2009 Elsevier B.V. All rights reserved.
Buckwheat grain
Nutritional value
Elemental distribution
Proton-induced X-ray emission (PIXE)
Nuclear microprobe

1. Introduction
Humans require more than twenty elements, which are mainly
supplied in the food. The elements most frequently lacking in diets
are Fe, Zn and I. Some of the elements, like Ca, Mg, Cu and Se, can
be deficient in diets of some groups [1]. Some of the heavy ele-
ments, like Zn, Cu and Fe, are nutritionally important in small
amounts, but can have deleterious effects for humans at higher
concentrations. Some other elements, like Al, are not desired in
food products, although as they are contained in soil, their uptake
is unavoidable, and depends on plant genotypes and growing con-
ditions [2,3].
In edible grain, like pulses, cereals and pseudocereals, which are
staple foods for many populations, the heavy elements are not dis-
tributed evenly. Indeed, different grain tissues (structures) have
highly differing elemental concentrations, depending on the tissue
origins (maternal, embryonal), physiology and/or function. From
the technological point of view, knowledge of the elemental con-
centrations in different grain structures is of particular importance
for applications such as suitable dehusking, grinding and milling
techniques, to obtain products with optimal concentrations of ben-
eficial elements.
* Corresponding author. Tel.: +386 1 4231161; fax: +386 1 4231088.
E-mail address: ivan.kreft@guest.arnes.si (I. Kreft).
Recently, a lot of attention has been paid to the elemental con-
stitution of foods, with emphasis on heavy metal concentrations.
Of the edible grains, buckwheat is a pseudocereal, and it is one of
the most important traditional old food sources in some of the
populations around the Himalayas region (south-western China,
Bhutan, Nepal, northern India, north-eastern Pakistan). It is also a
popular food in several other countries, like Slovenia, the Czech
Republic, Poland, Ukraine, Korea and Japan [4]. Buckwheat would
appear to be a good model for research into elemental constitution,
as its food products are relatively rich in trace elements, in addition
to the genetically and environmentally dependent synthesis of
phenols and other secondary metabolites by buckwheat [3,5–7].
Many of such secondary metabolites can act as chelating agents
for the heavy elements, thus influencing their distribution and con-
centrations in plant tissues [2,3].
Of the previously available analytical techniques, such as con-
ventional chemical analyses of grain, or of milling fractions from
grain, none allow for elemental localization and quantification
within the different interlaced layers of plant tissues. Similarly, it
is not possible to completely avoid the contamination of such sam-
ples by elements attached to the grain as soil or dust particles,
which are not part of the structure of the seed or fruit. Further-
more, due to the interlaced structures of grain, in most cases mill-
ing and sieving, and mechanical separation are not sufficient for
the complete separation of the grain parts that have different
compositions.

0168-583X/$ - see front matter Ó 2009 Elsevier B.V. All rights reserved.
doi:10.1016/j.nimb.2009.06.104
 K. Vogel-Mikuš et al. / Nuclear Instruments and Methods in Physics Research B 267 (2009) 2884–2889 2885

However, proton-induced X-ray emission (PIXE) spectroscopy
with a focused proton beam, which is frequently referred as mi-
cro-PIXE, has long been known as an efficient technique to mea-
sure lateral elemental distributions within thin biological tissue
sections. Micro-PIXE features both high lateral resolution (of the
order of one micrometer) and high elemental sensitivity (in the
range of 1 mass ppm), and it would thus appear suitable to simul-
taneously localize and quantify elements within distinct morpho-
logical structures in plant samples, including grains. Although
more instrumentally demanding in comparison with other suitable
methods, such as the more commonly used dispersive X-ray micro
analysis available with scanning electron microscopy, the main
advantage of micro-PIXE is indeed its superior sensitivity. The
physically continuous background in the X-ray spectra, which re-
sults mainly from bremsstrahlung of the primary particle and sec-
ondary electrons, is very weak in the case of PIXE, which relates its
1 ppm limit of detection to the order of 1 lg g 1 dry weight, which
can be obtained for most elements from Na to U. Over the last dec-
ade, the PIXE instrumentation of accelerator, micro-beam-forming
system and detectors has significantly improved in reliability, and
this analytical method is now more readily available to research in
biomedical fields. PIXE has thus become a standard technique for
quantitative elemental mapping in biological tissues.
In this context, broad-beam PIXE methods were initially devel-
oped for determination of the S and N contents in cereal grains and
pulses, and thus for the estimation of their nutritional values [8–
10]. Indeed, one of these studies of S concentrations in seeds [9]
demonstrated a further advantage of micro-PIXE analysis: a part
of the seed can be used for the PIXE analysis, with the remaining
parts, including the embryonal axis, left undamaged, allowing the
seeds to germinate and to be sown to provide further multiplica-
tion of a genotype with known elemental concentrations.
The main aims of the present study were thus: (1) adaptation of
micro-PIXE for analysis of nutritionally relevant heavy elements in
different tissues of buckwheat grain; (2) calculation of the contri-
butions of such elements in particular parts of the grain to the daily
elemental intake on the basis of their concentrations and weight

Fig. 1. Above: proton flux is measured by chopper, which periodically intersects the collimated beam. Chopper is positioned between the collimating slits and microprobe-
forming triplet quadruple lens. Beam-facing chopper surface is graphite coated by 3 lm Au foil. Bellow: spectrum of the backscattering protons consists of well separated Au
peak and carbon signal in the low energy region. Signal in Au peak is integrated to obtain value proportional to the proton flux.
2886 K. Vogel-Mikuš et al. / Nuclear Instruments and Methods in Physics Research B 267 (2009) 2884–2889

portions in the grain tissues; and (3) comparison of the calculated Detection of X-ray energies from 1 keV to 25 keV was achieved
daily intake to the recommended daily intakes for humans. using two X-ray detectors: a high-purity germanium X-ray detec-
tor (active area, 95 mm2; beryllium window, 25 lm thick; polyim-
2. Experimental ide absorber, 100 lm thick) positioned at 135° with respect to the
beam direction; and a Si(Li) detector (active area, 10 mm2) posi-
Grains of the common buckwheat (Fagopyrum esculentum), cv. tioned at 125° with respect to the beam direction; this latter was
Darja, with a mean thousand-grain weight of 26 g were obtained for detection of low energy X-rays of 0.8 keV–4 keV.
from plants grown in non-polluted sandy soil in Ljubljana. As ma-
ture plant seeds usually contain less than 10% water, for the mea-
suring of thick seed sections, there was no need for any particular
fixation treatment during sample preparation. This is in contrast to
the example of thin cuttings of leaves or roots that can contain up
to 95% water, which therefore require cryo-fixation to avoid ele-
mental redistribution during drying [11–15]. The mature dry buck-
wheat grains were therefore cut into 2-mm-thick sections with a
stainless steel scalpel, which were then individually mounted be-
tween two thin layers of Pioloform foil on an aluminum frame.
The micro-PIXE analysis was performed at the nuclear micro-
probe in the tandetron accelerator laboratory of the Jožef Stefan
Institute [15,16]. The grain sections were raster scanned using a
proton microbeam (2  2 lm; ion current, approximately
400 pA). The selected beam energy was 2 MeV where the maxi-
mum scanning area of 2.5  2.5 mm2 was needed; otherwise
3 MeV was used, where the maximum scanning area was reduced
to 2.0  2.0 mm2, with significantly higher elemental sensitivity
obtained [14,17]. The samples were mounted onto a five-axis
motorised vacuum goniometer with a positioning precision of
1 lm. The positioning of the sample in the focal plane of the qua-
druple triplet lens with a precision better than 60 lm was obtained
by bringing the sample surface into the depth of field of the optical Fig. 2. PIXE spectra measured by iGe X-ray detector extracted from cotyledon and
microscope. endosperm area. Yields are normalized on the equal beam dose.

Table 1
Concentrations of elements within buckwheat endosperm, cotyledon and husk (concentration of particular element represents a mean of two representative samples).

Endosperm Cotyledon Husk

1 1 1 1 1 1
Elements Conc. [lg g ] Stat.err. [%] lod [lg g ] Conc. [lg g ] Stat.err. [%] lod [lg g ] Conc. [lg g ] Stat.err. [%] lod [lg g ]
Mg 41.8 34.1 25.7 3525.3 1.6 70.5 1306.8 5.5 115.9
Al 19.3 36.8 13.1 <lod / 46.3 214.9 15.1 57.9
Si 28.8 19.7 11 <lod / 72.4 302.8 7.6 41.3
P 109.2 5.6 11.1 6949.7 0.6 43.2 439.2 5.2 38
S 166.9 2.9 7.9 1992 1.1 24 218.8 7.3 27.2
Cl 14.1 25.3 6.5 105 10.7 19.2 319.6 4.7 23.1
K 199 2.0 4.7 8754.6 0.5 18.8 3573.9 1.0 21.6
Ca 48.9 5.9 4.9 120.9 23.1 53.6 1016.3 2.9 42.6
Ti 19.6 2.3 0.6 16 7.3 1.8 118.1 2.0 0.9
Mn <lod / 0.4 30.5 4.6 1.4 17.8 7.3 1.4
Fe 11.8 3.2 0.2 74 3.1 2.1 63.1 3.8 2.6
Ni <lod / 0.3 1.3 50.2 1 <lod / 1.7
Cu 1.3 19.7 0.2 8.1 13.2 0.9 1.7 60.9 1.5
Zn 1.2 25.1 0.3 38.8 6.2 1.6 <lod / 2.2
Br <lod / 1.5 <lod / 4.2 <lod / 7.8
Rb <lod / 2.7 11.2 47.4 7.7 <lod / 22
Mo 16.3 33.9 6.8 <lod / 42.2 <lod / 42.2

Lod, limit of detection.

 K. Vogel-Mikuš et al. / Nuclear Instruments and Methods in Physics Research B 267 (2009) 2884–2889 2887

For precise proton dose determination, an in-beam chopping gions selected on the basis of the tissue structures seen under light
device (gold-plated graphite; beam intersection frequency, microscopy and of the micro-PIXE qualitative elemental maps. The
approximately 10 Hz) was positioned in the beam line after the last spectra were analyzed using the Gupixwin package [19–21]. The
collimation of the beam, before it hit the sample, thus making the set-up used thick sample description and trace element calcula-
system insensitive to beam intensity fluctuations (Fig. 1). The spec- tions based on a starch matrix. The edges of the target oriented to-
trum of backscattered protons from the chopper was recorded in wards the corresponding X-ray detector were avoided in the
parallel with the PIXE spectra in list-mode. This high-energy part evaluation, as the description of the X-ray absorption at the X-
of the spectrum consisted of protons scattered from the Au layer ray exit path was not correctly evaluated in this particular case.
and appeared as a separate peak. During off-line data processing, The concentrations of the elements in the different grain structures
the spectrum accumulated at the in-beam chopper over an arbi- were calculated as mean concentrations of two representative
trary scanning area could be extracted from the list-mode results measured samples.
simultaneously with PIXE spectra, and used for dose information. The efficiencies of the Ge and Si(Li) X-ray detectors were deter-
The morphological structures of the buckwheat grain were mined by measuring a large series of mono-elemental targets and
determined under light microscopy, according to Kreft and Kreft certified standards. In the first step, the technical characteristics of
[18]. The X-ray spectra emitted from the distinct grain structures the detectors were loaded into the Gupixwin programme, with the
(Fig. 2, Table 1) were extracted from list-mode files over the re- results described optimally by an optimised dead-layer thickness.
These efficiency descriptions still lacked a precision better than
15% in the energy regions close to the Ge and Si absorption edges
of the respective detectors. Additional efficiency dependences
Table 2
Relative distribution of elements in buckwheat seeds. were loaded into the Gupixwin programme to account for these
discrepancies, to obtain results with the monoelemental samples
Endosperm Cotyledons Husk
significantly better than 10%. In the final calibration step, the
Mass (mg)/seed 12.7 ± 0.6 3.8 ± 10 5.8 ± 0.6
% mass 57 17 26 method was tested on Standard Reference Materials NIST Naval
Brass (C1107) and NIST Tomato Leaves (1573a). The precision was
Content (%)/seed
Mg 2.5 62.3 35.2 verified in the XRF, AES, ICPMS and micro-PIXE inter-comparative
Al 16.5 <lod 83.5 study on homogenised dry bulk plant material [22].
Si 17.3 <lod 82.7 For the determinations of the biomass of the grain tissues, as
P 4.6 87.0 8.4 husk (containing testa and aleurone), cotyledons and endosperm,
S 19.4 69.1 11.6
Cl 7.4 16.4 76.2
the grains were soaked in water overnight. The individual tissues
K 4.5 58.9 36.6 were then manually isolated from the soaked grains under a ste-
Ca 8.9 6.6 84.5 reomicroscope, and dried to a constant mass at 50 °C. Five subsam-
Ti 25.1 6.1 68.8 ples that each contained the tissues from 3 grains were weighted.
Mn 0.0 52.9 47.1
Finally, the mean weight of each tissue was multiplied by the ele-
Fe 18.8 35.3 45.8
Ni <lod 100.0 <lod ment concentrations of that particular tissue to determine the rel-
Cu 28.9 53.8 17.2 ative element distribution of the tissues of these buckwheat grains
Zn 9.4 90.6 <lod (Table 2).
Br <lod <lod <lod The elemental contents were calculated on the basis of the ele-
Rb <lod 100.0 <lod
Mo 100.0 <lod <lod
mental concentration in each tissue portion (e.g. endosperm, coty-
ledon, husk) for 100 g of buckwheat grains, as the estimated daily
Mass data as means ± se (n = 5). intake; in Table 3, these are compared to the Dietary Reference

Table 3
Calculated contents of the elements in 100 g of whole buckwheat seeds (endosperm, cotyledon and husk) taking into account the relative tissue mass within the grain compared
to the recommended adequate and tolerable.

Element Contents (mg in 100 g) Recommendeda Tolerableb

1 1
Endosperm Cotyledon Husk Total (mg d ) (mg d )
Mg 2 57 32 92 320–420 nd
Al 1.0 nd 5.3 6.4 nd nd
Si 1.6 nd 7.5 9.1 nd nd
P 6 113 11 130 700 4000
S 9 32 5 47 nd nd
K 11 142 88 241 4700 nd
Cl 1 2 8 10 2300 3600
Ca 3 2 25 30 1000 2500
Ti 1.1 0.3 2.9 4.2 nd nd
Mn nd 0.5 0.4 0.5 1.8–2.3 11
Fe 0.6 1.2 1.6 3.4 8–18 45
Ni nd 0.02 nd 0.02 nd 1
Cu 0.1 0.1 0.04 0.2 0.90 10
Zn 0.1 0.6 nd 0.7 8–11 40
Br nd nd nd nd nd nd
Rb nd 0.2 nd 0.2 nd nd
Mo 0.9 nd nd 0.9 0.045 2

Nd – not determined.
Dietary Reference Intakes for adults (USDA DRI, 2004) [23].
a
Recommended (bold type). Adequate (ordinary type).
b
Tolerable Dietary Reference Intakes as determined by USDA (DRI, 2004) [23].
2888 K. Vogel-Mikuš et al. / Nuclear Instruments and Methods in Physics Research B 267 (2009) 2884–2889
Intake system of nutrition recommendations from the Institute of
Medicine of the US National Academy of Sciences [23].
3. Results and discussion
The application of micro-PIXE has enabled us to generate qual-
itative and quantitative elemental maps of the most nutritionally
important elements within the tissues of buckwheat grains
(Fig. 3, Table 1). The major part (57%) of the buckwheat grain is
the starchy endosperm, which is only a moderate source of mineral
nutrients (Tables 1 and 2). This might arise from dilution effects,
whereby in the final stages of the assimilation and filling of endo-
sperm cells starch granules are formed, which are not as rich in
trace elements as other cell parts.
In buckwheat it is possible to cut and remove a section of the
grain for analysis in such a way that the embryonic axis remains
undamaged, with a part of both cotyledons still attached. Accord-
ing to our unpublished results, about 85% of such grains can still
germinate. Therefore, this indicates that despite the removal of a
part of the husk, endosperm and cotyledons, seeds can remain via-
ble. This is of importance, as by this analytical method the rest of
the grain is still viable for further propagation and application in
plant breeding, allowing for seed selection according to the most
desired elemental constitution. Among the various crop species,
there is a considerable genetic variation in their ability to concen-
trate the mineral elements [1,24,25]. By using this analytical meth-
od, the slicing can avoid the embryonic axis and include just the
outer layers (husk, testa, aleurone), some the endosperm and a part
of cotyledons in the section. These grains are thus viable, and
following the germination of those with the desired elemental
composition, the resulting plants will provide seeds for the contin-
uation of the necessary gene pool into the next generation.
K was the most abundant element within the cotyledons
according to its concentrations, followed by P, Mg and S. Concen-
trations of an order of magnitude lower were seen for these ele-
ments in the endosperm, while in the husk, K and Mg were
Fig. 3. Elemental maps of the buckwheat seed over the surface of the cut through the middle of the seed. The mapping was performed with a beam energy of 2 MeV over an
area of 2.5  2.5 mm2.
about two-fold lower than in the cotyledons, with P and S again
an order of magnitude lower (Table 1). The concentrations of the
nutritionally important trace elements of Zn, Fe, Cu and Mn were
also highest in the cotyledons. In the endosperm, Cu and Zn con-
centrations were low (1 lg g 1), with Mn not detected
(<0.4 lg g 1), while in the husk, the Fe concentration was similar
to that in the cotyledons; here, Zn was under the detection limit
(<2 lg g 1).
Again, the most abundant element within the husk according to
its concentrations was K, followed by Mg and Ca. As mentioned
previously, the mobile K and Mg were preferentially stored in the
cotyledons, while the less mobile Ca was mainly retained in the
husk (Table 1). On the other hand, Al and Si were preferentially
stored in the husk, and were not detected in the cotyledons, with
only trace amounts in the endosperm. Similarly, Ti was mainly
stored in the husk, with substantially lower concentrations in the
cotyledons and endosperm.
However, as these different tissues within the grains are not
equally represented, tissue concentrations can sometimes be mis-
leading. Taking into account the relative tissue masses, it turned
out that elements such as Mg, P, S, K, Mn, Cu and Zn were prefer-
entially stored in the cotyledons, representing 17% of the total seed
biomass, while Al, Si, Ca, Cl, Ti and Fe were preferentially accumu-
lated in the husk, representing 21% of the total seed biomass. Mo
was the only element that was detected only in the starchy endo-
sperm (Table 2). According to the results of previous chemical
analyses [7], it was expected that among different parts of buck-
wheat grain there would be some differences in the concentrations
of nutritionally relevant elements; however, such big differences as
seen in the present study (Tables 1 and 2; Figs. 2 and 3) have not
been reported yet.
From a nutritional point of view, the elemental composition of
husk is less important, as the husk is mainly removed in the pro-
cess of preparation of food products, like making groats or milling
grain to flour. However, during the cooking of buckwheat grain in
water prior to conventional dehusking, some substances are trans-
 K. Vogel-Mikuš et al. / Nuclear Instruments and Methods in Physics Research B 267 (2009) 2884–2889
ported from the husk into the groat and remain there after the
mechanical removal of the husk by mechanical dehusking proce-
dures [26]. It is not yet known if, and to what extent, the heavy ele-
ments move from husk to the endosperm during the cooking of
buckwheat grain. Our results indicate that relatively high portions
of the daily intake of Mg, Ca, Mn and Fe can be lost due to dehusk-
ing. Nevertheless, the comparison of the nutritionally important
elements with the recommended daily intakes [23] indicate that
despite the presumed losses due to dehusking, fairly large amounts
of these elements can be gained through buckwheat nutrition (Ta-
ble 3), thus further supporting buckwheat as a functional food. In-
deed, a daily buckwheat intake of 100 g can cover the needs for
22% of daily Cu, 17% of P, 3% of K, 16% of Mg, 24% of Mn and 7%
of Zn, while satisfying the whole daily need for Mo.
4. Conclusions
 Micro-PIXE analysis is here shown to be suitable for determina-
tion of the concentrations of nutritionally relevant elements in
individual buckwheat grain tissues. Large differences in the ele-
ment concentrations were indeed observed when comparing
these tissues, with the buckwheat cotyledons as the richest
nutritional source of the essential elements, in line with their
role in plant physiology as supporting vital plant functions.
 With its adverse nutritional effects, Al is concentrated mainly in
the husk, which is usually removed by dehusking of the grain;
this will be similar for Si and Ti.
 Cr, Mo and Se are nutritionally relevant elements; however, only
Mo was detected in the endosperm, while the Cr and Se concen-
trations were too low to be detected by the micro-PIXE method.
 With the information about the elemental constitution of differ-
ent grain parts, the design of target products with nutritionally
optimal constituents appears to be feasible.
Acknowledgements
The authors are grateful to Ms. Erika Lah for skillful technical
assistance in preparing the grain samples, and to the anonymous
reviewers for critical reading of the manuscript. This research is
supported by the Research Programmes P1-0112 and P1-0212, Re-
search Projects J4-9673-0481-06 and J7-0352. Support of tande-
tron facility in the framework of Infrastructure Research Centres
by the Slovenian Research Agency is acknowledged.
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