Raman Spectroscopy Study of Calcium Oxalate Extracted from Cacti Stems Claudio Frausto-Reyes,a,* Sofia Loza-Cornejo,b Tania Terrazas,c Marı´a de la Luz Miranda-Beltra´n,b Xo´chitl Aparicio-Ferna´ndez,b Brenda M. Lo´pez-Macı´as,b Sandra E. Morales-Martı´nez,c Martı´n Ortiz-Moralesa Centro de Investigaciones en O´ptica, A.C., Unidad Aguascalientes, Prol. Constitucio´n 607, Fracc. Reserva Loma Bonita, Aguascalientes, 20200 Me´xico b Laboratorio de Bioquı´mica, Centro Universitario de los Lagos, Universidad de Guadalajara, 47460, Lagos de Moreno, Jalisco, Me´xico c Instituto de Biologı´a, Universidad Nacional Auto´noma de Me´xico (UNAM), Post Office Box 70-233, Me´xico, D.F. 04510, Me´xico a

To find markers that distinguish the different Cactaceae species, by using near infrared Raman spectroscopy and scanning electron microscopy, we studied the occurrence, in the stem, of solid deposits in five Cactaceae species (Coryphantha clavata, Ferocactus latispinus, Opuntia ficus-indica, O. robusta, and O. strepthacantha) collected from their natural habitats from a region of Me´xico. The deposits in the tissues usually occurred as spheroidal aggregates, druses, or prismatic crystals. From the Raman spectra, the crystals were identified either as calcium oxalate monohydrate (CaC2O4H2O) or calcium oxalate dihydrate (CaC2O42H2O). Opuntia species (subfamily Opuntioideae) showed the presence of CaC2O4H2O, and the deposition of CaC2O42H2O was present in C. clavata and F. latispinus (subfamily Cactoideae, Cacteae tribe). As a punctual technique, Raman spectroscopy seems to be a useful tool to identify crystal composition. In addition to allowing the analysis of crystal morphology, this spectroscopic technique can be used to identify Cactaceae species and their chemotaxonomy. Index Headings: Raman spectroscopy; Cacti; Calcium oxalate monohydrate; Calcium oxalate dihydrate.

INTRODUCTION Many plants store crystals of calcium oxalate or calcium carbonate; others accumulate large amounts of silica, tannins, or phenols. Because plants have no excretory mechanism, numerous waste products are stored within the cells. Calcium oxalate is considered the most commonly occurring inorganic material in flowering plants, including dicotyledons and monocotyledons. 1 , 2 Crystal growth is a highly controlled intracellular process, and the cells where these crystals are produced are referred to as crystal idioblasts.3 The crystals of calcium oxalate are formed from environmentally derived calcium and from biologically synthesized oxalate; the crystals often form within the vacuoles of the idioblast cells in the epidermal, ground, and vascular tissues, in organs such as the roots, leaves, flowers, and seeds.4 The process occurs in more than 215 families that include the Cactaceae, Malvaceae, Orchidaceae, and Rubiaceae.1,5,6 Many crop and ornaReceived 5 February 2014; accepted 6 May 2014. * Author to whom correspondence should be sent. E-mail: cfraus@cio. mx. DOI: 10.1366/14-07485

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mental plants accumulate oxalate in the range of 3–80% of their dry weight.7,8 The large cactus Carnegiea gigantea, for example, contains approximately 1 3 10 5 g of calcium oxalate dehydrate (CaC 2 O 42H 2O (COD)) crystals.9 The distribution and shapes (raphides, druses, or prisms) of these crystals have been used to distinguish subfamilies, genera, and subgenera.10–12 Moreover, the diversity of the crystal shapes and sizes, as well as their prevalence and spatial distribution, has led to several hypotheses regarding the crystal functions in plants. The proposed functions include roles in ion balance, plant defense, tissue support, detoxification, and light gathering and reflection.13–19 Investigations into crystal formation and crystal function have relied primarily on biochemical, spectroscopic, and cellular approaches.6,18–23 Such studies have provided valuable information about the crystal ultrastructure and the developmental stages of crystal formation. Calcium oxalate crystals have been documented using light microscopy and polarization microscopy, X-ray diffraction,24 infrared spectroscopy,25,26 electron microscopy (scanning electron microscopy (SEM) and transmission electron microscopy (TEM)),1,6,27,28 and precipitation from high ionic strength solutions.29 Calcium oxalate is present in two hydration states in plants, in the monohydrate state (CaC2O4H2O (COM)) or in the dihydrate state (COD), also known as whewellite and weddellite, respectively.24,30–32 Several crystal shapes are formed by both hydration states: raphides, prisms, styloids, druses, and crystal sand. For some members of the family Cactaceae, the biominerals of the various species appear in the form of highly pure and well-crystallized calcium oxalates that typically grow in the form of druses, i.e., spherical aggregates of thousands of individual crystallites.6,31 These deposits were identified as either COM or COD.31,32 Studies concerning the structural differences between organic and inorganic materials suggested that the use of Raman spectroscopy and other techniques would complement each other.33 For example, infrared and Raman spectroscopic methods can be applied to the analysis of valuable plant substances or to obtain quality parameters in horticultural and agricultural crop studies. In most cases, the vibrational measurements can be made directly on the plant tissues as well as on the fractions isolated from the plant material by hydro-distillation or

0003-7028/14/6811-1260/0 Q 2014 Society for Applied Spectroscopy

APPLIED SPECTROSCOPY

TABLE I. Geographic locations and municipalities in the state of Jalisco, Me´xico, and voucher number of the species studied. Geographic location Species Opuntia ficus-indica (L.) Mill. Opuntia robusta H.L.Wendl. Opuntia strepthacantha Lem. Ferocactus latispinus (Haw.) Britton et Rose Coryphantha clavate (Scheidw.) Backeb.

Latitude (N)

Longitude (W)

Elevation (m above sea level)

Municipality

21831 0 21802 0 21824 0 21833 0 21831 0

101841 0 101827 0 102825 0 103809 0 101841 0

1930 1920 1851 2100 1930

Lagos de Moreno (S. Loza 201-205) Unio´n de San Antonio (S. Loza 206-210) Encarnacio´n de Dı´ az, (S. Loza 211-215) Ojuelos (S. Loza 216-220) Lagos de Moreno (S. Loza 221-225)

solvent extraction.4,21,34 Generally, both spectroscopic techniques provide spectra that show some bands characteristic of the individual components. These bands provide information about the chemical composition, including both primary and secondary metabolites, of the samples under investigation. In principle, spectroscopic analyses allow the discrimination between different species, and even between chemotypes of the same species, via the identification of the markers of individual plant substances.35,36

these cacti grow in desert regions.9 In this work, we studied the occurrence of calcium oxalate deposits in five Cactaceae species, gathered from their natural habitats from a central region of Me´xico. We performed a Raman spectroscopic study to characterize the chemical nature of these deposits or intracellular inclusions and to describe the crystals in terms of their morphology: single crystals, prismatic crystals, or calcium oxalate druses (spherical crystal aggregations). We also studied the distribution of the crystals in the stems (epidermis, hypodermis, cortex, vascular tissue) with SEM.

FUNCTION OF OXALATE CRYSTALS AND THEIR IMPORTANCE IN CACTI CLASSIFICATION

EXPERIMENTAL

Anatomical investigations reveal that the function of calcium oxalates, in most cacti, is not completely understood.37–41 They likely participate in the removal of heavy metals or the adsorption of water, as they do in other species.42,43 The number and distribution of crystal idioblasts within the plant body vary among taxa, and some investigators have used the distribution of crystal idioblasts in classification.27 However, according to Hartl et al.,12 the crystal idioblast distributions were not observed to follow a pattern that could be used to classify the Cactaceae; in the Pereskioideae, Maihuenioideae, and Opuntioideae, COM is predominant, whereas in the Cactoideae, COD prevails. In some genera, such as Hylocereus, Epiphyllum, Selenicereus, and Weberocereus, the COM forms were almost exclusively represented by raphides, together with different crystal forms in the epidermal cells, but in the remaining Cactoideae the crystals were quite variable. Hartl et al.12 mentioned the need to study the variation in crystal morphology at the genus and species levels in the Cactaceae in subsequent work to better understand the crystal morphology contribution to the family systematics. Cacti exhibit a wide geographic distribution from southern Canada to the southern region of Patagonia, Argentina. Me´xico is the main center of diversity of this family, with a high percentage of endemism, and most of

Plant Material. This study was performed on adult plants of Opuntia ficus-indica, Opunita robusta, Opuntia strepthacantha (subfamily Opuntioideae); and Coryphantha clavata and Ferocactus latispinus (subfamily Cactoideae, Cacteae tribe). Samples of these specimens were collected from their natural habitats (Altos Norte Region in Jalisco, Me´xico) from July to September (rainy season).44 The data concerning the sources of the studied species and their voucher numbers as housed at the Instituto de Bota´nica de la Universidad de Guadalajara (IBUG) herbarium are given in Table I. Collection Sites. The main climates that prevail in this region are warm, semi-warm, and semiarid. Annual mean temperatures vary from 13.9 to 19 8C, and annual precipitation varies from 563.8 to 686 mm.44 Samples of soils were collected along the position geomorphic position per site, and they were analyzed. The dominant soils included haplic Feozem (PHha) and eutric Planosol (PLeu). General and soil fertility parameters are shown in Tables II and III. Crystal Isolation and Purification. The plant stems (three individuals per species) were carefully washed with an abundant quantity of distilled water. After the removal of the spines, trichomes, and foreign material, the stem from each cleaned plant was divided into apical, middle, and basal portions. Five samples of each portion were excised and washed several times. Thin

TABLE II. Soil parameters for the collection sites.

Sitea

Textural class

Density (g/cc)

Sand (%)

Silt (%)

Clay (%)

pH

Cation exchange capacity (mEq/100 g)

Nitric nitrogen (ppm)

LM OJ ED USA

PHh PHh PLeu PHh

2.4 2.2 2.2 2.3

58.4 60.4 38.4 54.4

28.0 26.0 38.0 28.0

13.5 13.5 23.5 17.6

6.2 6.3 5.7 5.9

11.9 13.3 17.2 16.9

3 3 3 3

a

LM, Lagos de Moreno; OJ, Ojuelos; ED, Encarnacio´n de Dı´ az; USA, Unio´n de San Antonio.

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TABLE III. Soil fertility parameters for the sites. Sitea

Available water (%)

Organic matter (%)

P (ppm)

Mn (ppm)

Ca (mEq/100 g)

Mg (mEq/100 g)

Na (mEq/100 g)

K (mEq/100 g)

LM OJ ED USA

14.0 14.0 21.0 16.0

1.3 1.8 2.0 2.3

25 25 25 25

5 5 12 5

2.3 2.5 2.7 1.9

0.78 1.18 1.57 1.17

0.32 0.31 0.32 0.22

0.92 0.90 0.92 0.81

a

LM, Lagos de Moreno; OJ, Ojuelos; ED, Encarnacio´n de Dı´ az; USA, Unio´n de San Antonio.

sections (2 mm) including epidermal-hypodermal and cortical tissues were separated and fixed in formaldehyde/acetic glacial acid/70% ethyl alcohol (5 : 3 : 2)45 until dehydration began. Crystals were isolated from all portions of plant specimens. Tissue sections were macerated using acetic acid in all cases to degrade the fresh tissue.31,32 The isolated crystals were washed several times until plant debris was no longer evident. Crystals were freed with the help of dissecting needles, and the final separation of these crystals was accomplished by manual collection under an EZ4 dissecting light microscope (Leica). Raman Spectroscopy. The Raman spectra of the samples were obtained by placing them onto an aluminum substrate and then under a DM LM microscope (Leica) integrated to the Raman system (Renishaw 1000B). The Raman system was calibrated with a silicon semiconductor using the Raman peak at 520 cm1. The excitation wavelength was 830 nm, and the laser beam was focused (spot size of approximately 2 lm) on the surface of the sample with a 503 objective. The laser power irradiation over the samples was approximately 45 mW. Scanning Electron Microscopy. The tissue samples that were fixed and dehydrated using an ethanol series (30–100%) were critical point dried. Afterward, they were fixed to aluminum specimen holders with double-sided tape and coated with gold in a Hitachi-S-2460N sputter coater before morphological observation under a JSM5310LV field-emission SEM (Jeol) at 15 kV.

oxalate crystallizes, it assumes various morphologies. Star-like conglomerates of small crystallites (druses), large single needles (styloids), and bundles of long thin crystals (raphides) are all common in higher plants.46 According to Franceschi and Horner6 and Webb,5 calcium oxalate crystals are formed within the epidermal, ground (i.e., parenchyma and mesophyll), and vascular tissues (i.e., phloem, xylem, and bundle sheath cells) in many plant organs, including the roots, stems, leaves, floral structures, and seeds. In the cacti studied, druses (round calcium oxalate crystals) were found in the hypodermal and cortical cells of the Opuntia species and within the cortical parenchyma cells in the stems and roots of C. clavata. These star-like crystallite conglomerates appear as large druses, but are in a different hydration state (as is shown in the Raman analysis). Rivera and Smith45 have found crystals with the same morphology in O. imbricata and O. engelman-

RESULTS AND DISCUSSION Crystal Morphology. The calcium oxalate crystals were mainly round aggregate druses and prismatic aggregate crystals. Both were found in the idioblastic cells of the hypodermal and cortical tissues. Prismatic crystals were observed in the cortical cells of F. latispinus. Prismatic crystals occurred singly or as aggregates (Fig. 1a). The druses were composed of hundreds of microcrystals packed tightly together in a single macrostructure. Druses were especially common in the outermost cell layer of the hypodermis in Opuntia species (Fig. 1b), whereas in C. clavata, these crystal aggregates were common in the cortex. Moreover, in Opuntia species, the druses occurred as multiple crystals that are thought to have precipitated around nucleation sites to form a crystal conglomerate. The COM druses in Opuntia species were distinguished mainly by their stellate shapes, with acute sharp points emerging from the center of the individual crystallites, as has been described previously.30,32 Most cacti contain large amounts of crystalline calcium oxalate distributed throughout their tissues. As

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FIG. 1. SEM images of (a) the prismatic crystals in the cortex of Ferocactus latispinus and (b) the druses in the stem hypodermis of Opuntia ficus-indica.

nii. In O. microdasys and other Opuntioideae, Monje and Baran30,32 have identified crystals with the same shape. Moreover, this finding is consistent with the observations of Malainine et al.47 and Tovar-Puente et al.,48 who described the druses as a group of individual tetragonal microcrystals packed tightly together in a single macrostructure. The general aspect of single, isolated druses varies among species, especially with respect to the form and the angles between the individual crystal faces. It has been suggested that these types of crystals provide additional structural support to plant tissue, and also regulate the concentration of soluble Ca in plant tissue.1,6,49 The species studied are distributed in regions where the soils are predominantly sands that are typically low in nutrients. For example, these soils were found to contain 1.3–2.3% organic matter, 12–25 parts per million (ppm) P, and 5–12 ppm Mn. Moreover, the Ca concentration in these soils is presently available and varies from 1.9 to 2.3 mEq 100 g1 (see Tables II and III). Accordingly, the species may have evolved mechanisms for acquiring adequate Ca from soils low in Ca and for tolerating periods of high Ca supply in other circumstances, similar to other plant species. Plants can grow on a range of Ca nutrition rates without developing visible deficiency or toxicity symptoms20. Based on the distribution of crystals in the cacti species studied, the crystals may regulate soluble Ca concentrations in tissues. Identification of Crystal Deposits by Raman Spectroscopy. The Raman spectra of the crystal deposits appeared very well defined, demonstrating the high purity of the isolated oxalate crystals. This purity can be seen in Fig. 2a, 2b, and 2c for the stems of O. ficus-indica, O. strepthacantha, and O. robusta, respectively. In these spectra, there are peaks at 896, 1462, and 1489 cm1. The same Raman peaks were reported by Kontoyannis et al.36 for pure COM. The similar patterns of the Raman spectral bands for the crystals from the Opuntia stems suggest that in these species, the druse crystals consist of the more stable COM state. The Raman band observed at approximately 896 cm1 is assigned to the v(C–C) stretching mode.50,51 Moreover, some Raman bands for these species can be observed for the COM (596 and 504 cm1) and the COD (596 and 507 cm1) at almost the same position. The band at 596 cm1 is broad and of low intensity and is associated in previous studies with M–O stretching modes.43,50,52–54 In their studies concerning the thermal and Raman spectroscopic analysis of COM, Frost et al. 55 showed that a band is observed in the 25 8C spectrum at 595 cm1, whereas two additional bands at 521 and 503 cm1 can be attributed to O–C–O bending modes. Although Frost et al. 55 showed that there is an intensity relationship between these two bands (521 and 503 cm1) that is temperature dependent, in this work, we observed an intensity relationship that could be related to a particular subfamily Opuntioideae member (Figs. 2a–c). Our results complement the reports that describe the presence of COM in O. ficus-indica and other species of Opuntia.12,30,32,46,47 The Raman spectra of the crystals in the stem of F. latispinus and C. clavata are shown in Fig. 2d and 2e, respectively. The Raman peaks at 1475 and 912 cm1 show that these crystals correspond to the pure state of

FIG. 2. Raman spectra of the crystals in the species of Cactaceae: (a ) Opuntia ficus-indica, (b ) Opuntia strepthacantha, (c ) Opuntia robusta, (d ) Ferocactus latispinus, and (e ) Coryphantha clavata.

COD.36 It is possible to discriminate between the hydration states of calcium oxalate; monohydrate features two C–O symmetric stretching bands at 1462 and 1489 cm1, whereas the dihydrate has a contrasting C–O symmetric stretching band at 1475 cm1.36 Moreover, Monje and Baran30 and Hartl et al.12 noted that Cactoideae and Opuntioideae members can deposit the various hydration states of calcium oxalate that we have observed by Raman spectroscopic analysis. The less commonly observed COD has been observed in large quantities in most species of Cactoideae studied thus far.6,12,56,57 This fact is interesting because COD is the metastable form of calcium oxalate and thus is less widely distributed than the stable COM form. Our findings of the COD druses in C. clavata and the aggregated prisms in F. latispinus support the reports describing other species of these genera as Coryphantha calipensis, Ferocactus cylindraceus, and F. robustus.12 In summary, calcium oxalate crystals were observed in all the samples studied. These crystals were identified as either COM or COD. The subfamily Opuntioideae members (O. ficus-indica, O. robusta, and O. streptacantha) mineralized COM, whereas the members of the Cacteae (C. clavata and F. latispinus) tribe deposited COD.

CONCLUSIONS The intracellular inclusions in cacti species were identified as aggregates (i.e., druses) and prismatic calcium oxalate crystals by Raman spectroscopy and SEM analysis. These spectroscopic studies confirm that C. clavata and F. latispinus (subfamily Cactoideae, tribe Cacteae) deposited COD, whereas the subfamily Opuntioideae members O. ficus-indica, O. robusta, and O. streptacantha mineralized COM, and they substantiated observations of the various hydration states made by other methods such as infrared spectroscopy. In addi-

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tion, an intensity relationship in the COM spectra was observed that could be related with a particular subfamily Opuntioideae member. Our results also show that Raman spectroscopy provides excellent results in the identification of oxalate crystals in Cactaceae species.

22.

23.

ACKNOWLEDGMENT

24.

We thank Berenit Mendoza at the Instituto de Biologı´ a, UNAM, for technical assistance with SEM.

25.

1. V.R. Franceschi, H.T. Horner Jr. ‘‘A Microscopic Comparison of Calcium Oxalate Crystal Idioblasts in Plant Parts and Callus Cultures of Psychotria punctata (Rubiaceae)’’. Z. Pflanzenphysiol. 1980. 97(5): 449-455. 2. J.C. Prychid, J.P. Rudall. ‘‘Calcium Oxalate Crystal in Monocotyledons: A Review of Their Structure and Systematic’’. Ann. Bot. 1999. 84(6): 725-739. 3. J.D. Mauseth. Botany: An Introduction to Plant Biology. Boston, MA: Jones and Bartlett Publishers, 2003. 4. R. Baranski, M. Baranska, H. Schulz, P.W. Simon, T. Nothnagel. ‘‘Single Seed Raman Measurements Allow Taxonomical Discrimination of Apiaceae Accessions Collected in Gene Banks’’. Biopolymers. 2006. 81(6): 497-505. 5. M.A. Webb. ‘‘Cell-Mediated Crystallization of Calcium Oxalate in Plants’’. Plant Cell. 1999. 11(4): 751-761. 6. V.R. Franceschi, H.T. Horner. ‘‘Calcium Oxalate Crystals in Plants’’. Bot. Rev. 1980. 46(4): 361-427. 7. M. Brizuela, T. Montenegro, P. Carjuzaa, S. Maldonado. ‘‘Insolubilization of Potassium Chloride Crystals in Tradescantia pallid’’. Protoplasma. 2007. 231(3-4): 145-149. 8. B. Libert, V.R. Franceschi. ‘‘Oxalate in Crop Plants’’. J. Agric. Food Chem. 1987. 35(6): 926-938. 9. L.A.J. Garvie. ‘‘Decay of Cacti and Carbon Cycling’’. Naturwissenschaften. 2006. 93(3): 114-118. 10. N.R. Lersten, H.T. Horner. ‘‘Calcium Oxalate Crystal Types and Trends in Their Distribution Patterns in Leaves of Prunus (Rosaceae: Prunoideae)’’. Plant Syst. Evol. 2000. 224(1-2): 83-96. 11. T. Terrazas, S. Loza-Cornejo, H.J. Arreola-Nava. ‘‘Anatomı´ a Caulinar de las Especies del Ge´nero Stenocereus (Cactaceae)’’. Acta Bot. Venez. 2005. 28(2): 321-336. 12. P.W. Hartl, H. Klapper, B. Barbier, H.J. Ensikat, R. Dronskowsky, P. Mu¨ller, G. Ostendorp, A. Tye, R. Bauer, W. Barthlott. ‘‘Diversity of Calcium Oxalate Crystals in Cactaceae’’. Can. J. Bot. 2007. 85(5): 501-517. 13. T.A. Kostman, V.R. Franceschi. ‘‘Cell and Calcium Oxalate Crystal Growth Is Coordinated to Achieve High Capacity Calcium Regulation in Plants’’. Protoplasma. 2000. 214(3-4): 166-179. 14. B. Molano-Flores. ‘‘Herbivory and Calcium Concentrations Affect Calcium Oxalate Crystal Formation in Leaves of Sida (Malvaceae)’’. Ann. Bot. 2001. 88(3): 387-391. 15. N. Ruiz, D. Ward, S. Saltz. ‘‘Calcium Oxalate Crystals in Leaves of Pancratium sickenbergeri: Constitutive or Induced Defense?’’. Funct. Ecol. 2002. 16(1): 99-105. 16. G.M. Volk, V.J. Lynch-Holm, T.A. Kostman, L.J. Goss, V.R. Franceschi. ‘‘The Role of Druse and Raphide Calcium Oxalate Crystals in Tissue Calcium Regulation in Pistia stratiotes Leaves’’. Plant Biol. 2002. 4(1): 34-45. 17. A.M.A. Mazen, D. Zhang, V.R. Franceschi. ‘‘Calcium Oxalate Formation in Lemna minor: Physiological and Ultrstructural Aspects of High Capacity Calcium Sequestration’’. New Phytol. 2003. 161(2): 435-448. 18. M.A. Webb, J.M. Cavaletto, N.C. Carpita, L.E. Lo´pez, H.J. Arnott. ‘‘The Intravacuolar Organic Matrix Associated with Calcium Oxalate Crystals in Leaves of Vitis’’. Plant J. 1995. 7(4): 633-648. 19. T.A. Kostman, V.R. Franceschi, P. Nakata. ‘‘Endoplasmic Reticulum Sub-compartments Are Involved in Calcium Sequestration Within Raphide Crystal Idioblasts of Pistia stratiotes L’’. Plant Sci. 2003. 165(1): 205-212. 20. P.A. Nakata. ‘‘Advances in Our Understanding of Calcium Oxalate Crystal Formation and Function in Plants’’. Plant Sci. 2003. 164(6): 901-909. 21. A.J. Macnish, D.E. Irving, D.C. Joyce, V. Vithanage, A.H. Wearing, R.I. Webb, R.L. Frost. ‘‘Identification of Intracellular Calcium

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APPLIED SPECTROSCOPY

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Raman spectroscopy study of calcium oxalate extracted from cacti stems.

To find markers that distinguish the different Cactaceae species, by using near infrared Raman spectroscopy and scanning electron microscopy, we studi...
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