002-ptbot-2012.p65

Morphology and anatomy of the rhizome of Polypodium ×mantoniae Rothm.
EL¯BIETA ZENKTELER1, IWONA JÊDRZEJCZYK2 1 Department of General Botany, Institute of Experimental Biology, Adam Mickiewicz University, Umultowska 89, PL-61-614 Poznañ, 2 Department of Plant Genetics and Biotechnology, University of Technology and Life Sciences, Prof. S. Kaliskiego 7 Ave., PL-85-789 Bydgoszcz; e-mail: [email protected]: Fern rhizome contains some diagnostic features which are seldom utilized to distinguish the species. An attempt is made here to display some differences in the morphology and anatomy of the rhizome between Polypodium ×mantoniae and P. vulgare. Morphometric measurements indicated that the rhizome diameters of P. ×mantoniae were bigger, annual increments were longer, axillary branching more numerous, small and large meristeles within the perforate dictyostele more numerous, and the diameter of the leaf petioles greater than in P. vulgare. Light and fluorescence microscopy analyses were taken into consideration in the assessment of the diversity of each species. Morphological analysis showed that the scales, which cover the apex of a young rhizome, provide the most discernible characters. The scales of P. ×mantoniae were of the spatulate type, broader and more convex than those of P. vulgare. Nuclear DNA content estimated by flow cytometry for P. vulgare and P. ×mantoniae ranged from 30.86 ± 0.06 pg/2C to 39.44 ± 0.25 pg/2C, respectively, and was an initial parameter for identification of those species.
ABSTRAKT: K³¹cze paproci zawiera cechy diagnostyczne, rzadko wykorzystywane przy rozró¿nianiu gatunków. W pracy wykazano ró¿nice w morfologii i anatomii k³¹cza Polypodium ×mantoniae w porównaniu do P. vulgare. Na podstawie pomiarów morfometrycznych stwierdzono, ¿e k³¹cza P.×mantoniae charakteryzowa³y siê wiêksz¹ œrednic¹, d³u¿szymi przyrostami rocznymi, wiêksz¹ liczb¹ rozga³êzieñ bocznych, wiêksz¹ liczb¹ ma³ych i du¿ych merysteli w obrêbie perforowanej diktiosteli k³¹cza oraz wiêksz¹ œrednic¹ ogonków liœciowych. Analizy w mikroskopie œwietlnym i fluorescencyjnym potwierdzi³y te obserwacje. Cechy dobrze ró¿nicuj¹ce obydwa gatunki reprezentuj¹ ³uski okrywaj¹ce wierzcho³ek k³¹cza, u P. ×mantoniae szersze i bardziej wypuk³e ni¿ u P. vulgare. J¹drowa zawartoœæ DNA oceniona za pomoc¹ cytometru przep³ywowego dla P. vulgare i P. ×mantoniae wynios³a odpowiednio 30.86 ± 0.06 pg/2C i 39.44 ± 0.25 pg/2C i by³a wyjœciowym parametrem w identy- fikacji tych gatunków.
Key words: Manton polypody, nuclear DNA content, Polypodium ×mantoniae, P. vulgare, rhizome structure Zenkteler E., Jêdrzejczyk I. 2012. Morphology and anatomy of the rhizome of Polypodium ×mantoniae Rothm. In: E. Szczêœniak, E. Gola (eds), Genus Polypodium L. in Poland. Polish Botanical Society, Wroc³aw, p. 27–38.
28 El¿bieta-Zenkteler,-Iwona-Jêdrzejczyk 28 Ferns of the genus Polypodium L. belong to desiccation–tolerant plants, which are able to protect their organs against the damage during drought periods.
P. virginianum L. can tolerate a 60% loss in weight during drying (Reynolds, Bewley 1993) and P. vulgare about 45% (Zenkteler, Bagniewska-Zadworna 2005). As the poikilohydric plants polypodies accumulate mono-, di- and oli- gosaccharides in the rhizome parenchyma, which stabilize the structure of tissues and cells during environmental stresses (low temperature, salinity) (Bagniewska- Zadworna, Zenkteler 2006). Apart from carbohydrates, rhizomes contain several bioactive compounds which possess high diuretic, antibiotic and cytostatic properties (Duke 2001). The pharmacological value and exploitation of P. vulgare and the harvesting of the fern (i.e., for Rhizoma Polypodii) is the principal cause of its present endangerment and may eventually lead to the extinction of the species.
The structure of the P. vulgare rhizome is well known in all its aspects of morphology (Hovenkamp 1990) and anatomy (Bagniewska-Zadworna, Zenkteler 2006; Bagniewska-Zadworna et al. 2007) therefore it should provide a very good model for a comparison with other more or less closely related species or forms, i.e. P. ×mantoniae Rothm., a hybrid of P. vulgare and P. interjectum Shivas.
Materials and methods The fern species were collected from wild sites situated next to the Wolin National Park (population of P. ×mantoniae from Kawcza Góra) and next to the Wielkopolski Natural Park (population of P. vulgare from the Mosina for- est). Rhizomes of both species, as the object of the study, were cleaned under tap water and detached from their leaves and roots.
Fragments of the rhizomes of both species were fixed in FAA (a mixture of formalin, glacial acetic acid and alcohol), then dehydrated in a graded ethanol - xylene series, and embedded in Paraplast Plus (Sigma). Sections (12 µm thick) were cut with a rotary microtome and stained with Safranin O and counter- stained with fast green FCF (Broda 1971). The sections were examined un- der light and fluorescent microscopes with excitation UV 350-400 nm (Axioscope Carl Zeiss, Jena, Germany) in order to estimate the rhizome anatomical structure.
The number of small and large meristeles per dictyostele was evaluated as well as the number of proto- and metaxylem elements within the vascular bundle.
Measurements were repeated 10-15 times for two rhizomes. The given diam- eters of rhizomes and meristeles are the average values.
Nuclear DNA content was estimated in young and fresh leaves of P. vulgare and P. ×mantoniae. Leaves of Pisum sativum L. cv. ‘Set’ (2C = 9.11 pg; Œliwiñska et al. 2005) were used as an internal standard. Plant material for flow cytometric analysis was prepared according to Galbarith et al. (1983), with some modifications. Plant tissues of the target species and of the inter- nal standard were co-chopped with sharp razor blade in a plastic Petri dish containing 1 mL of nucleus-isolation buffer (0.1 M Tris, 2.5 mM MgCl × 6H 0, 85 mM NaCl, 0.1% v/v Triton X-100, pH 7.0) supplemented with propidium iodide (PI; 50 ìg/mL) and ribonuclease A (RNase A; 50 ìg/mL). Addition- ally the buffer was modified by adding 1.0% (w/v) polyvinylpyrrolidone (PVP- 10) to obtain high resolution histograms and neutralize of interference of cy- tosolic compounds with PI-staining of nuclei in suspension. After chopping, the suspension was passed through a 50-ìm mesh nylon filter. Measurements of fluorescence intensities were performed in at least 7000 nuclei using CyFlow SL Green (Partec GmbH, Münster, Germany) flow cytometer, equipped with a laser with green light emission at 532 nm. Analyses were replicated at least three times depending on availability of research material. Histograms were analyzed using the FloMax (Partec GmbH, Münster, Germany) software. Total nuclear DNA content was calculated using the linear relationship between the ratio of the 2C peak positions of species studied to internal standard, on the histogram of fluorescence intensities, and the mean values and standard de- viations were presented.
Morphology. Adult sporophytes of P. vulgare and P. ×mantoniae are pe- rennial plants with a relatively short rhizome and winter-green fronds. The rhizome of species belonging to the P. vulgare complex often displays confusingly similar characteristics. Horizontally-creeping rhizomes are slightly dorsiventrally flat- tened, they branch laterally by the shoot-buds, which arise near the base of the phyllopodia. The association of branches with leaves seems to be signifi- cant. Petioles clearly abscise near their bases, leaving short phyllopodia. The leaves and roots are radially arranged on the surface of the rhizome, however, leaves grow upwards and roots downwards from the rhizome (Fig. 1: 1, 3). Com- pared to the length of the year increments and number of lateral branches of P. vulgare, the respective values of P. ×mantoniae are larger (Table 1).
Macromorphological analysis showed that P. ×mantoniae and P. vulgare rhi- zomes are very variable, depending on environmental factors.
30 El¿bieta-Zenkteler,-Iwona-Jêdrzejczyk 30 The young parts of the rhizome with the apex meristem are protected by short peltate scales whose shape, size and color are diagnostic features (Fig. 1: 2, 4). These appendages vary within a species by marginal protrusions and cell wall lignification.
Fig. 1. 1 – Morphology of the rhizome: this year increment and the shoot apex of P. ×mantoniae Rothm.; 2 – Broad, prominent and very convex scale from the rhizome of P. ×mantoniae; 3 – Morphology of the rhizome: this year increment and the shoot apex of P. vulgare L.; 4 – Narrow, medium long, flat and dark scale of P. vulgare.
Ryc. 1. 1 – Morfologia k³¹cza: przyrost tegoroczny i wierzcho³ek wzrostu u P. ×mantoniae Rothm.; 2 – Szeroka, okaza³a i wypuk³a ³uska okrywaj¹ca k³¹cze P. ×mantoniae; 3 – Morfologia k³¹cza: przyrost tegoroczny i wierzcho³ek wzrostu u P. vulgare L.; 4 – W¹ska, œredniej d³ugoœci, p³aska ³uska k³¹cza P. vulgare.
Table 1. Mean dimensions of Polypodium rhizome and stelar elements.
Tabela 1. Œrednie z wymiarów k³¹czy i elementów steli u Polypodium.
Values in mm, mean ± SD (Wartoœci w mm, œrednie ± SD) przyrostu odga³êzieñ) 4.1±0.2 2.8±0.1 233±9.93 95±11.86 P. vulgare 31.6±0.4 2.8±0.2 2.9±0.1 2.1±0.1 187±6.16 82±9.47 8.7±0.2 6.5±0.3 412±16.51 135±7.99 ×mantoniae 58.8±2.37 4.4±0.4 4.2±0.1 3.1±0.1 262±11.43 132±8.56 Data (mean ±SD) were collected after measurements of 10 specimens.
P. vulgare rhizomal scales are of a lanceolate-type, with a broad basal region.
Young scales are hyaline, but older ones, gradually impregnated with lignin in the basal part, slowly become brown. The margin of the scales is slightly den- tate without glandular trichomes (Fig. 1: 4).
P. ×mantoniae rhizomes possess scales of a spatulate-type with a round, shield-like base and a small, dark point of attachment. Dentations are often elongated with pointed apices of cells and devoid of marginal glandular tri- chomes. Another marked attribute are the large cells with deeply sinuous walls close to the margin dentations (Fig. 1: 2).
Anatomy. In a rhizome cross-section, all tissues are arranged in concen- tric cylinders covered with rhizodermis (one cell layer), hypodermis, a parenchymatous outer cortex and perforated dictyostele composed of some small and a few large meristeles surrounding the parenchyma pith (Fig. 2: 5, 6). In members of the P. vulgare complex, the rhizome appears to have a dictyostelic structure caused by leaf gaps only. A basal root trace departs before the opening of the leaf gap. Individual meristeles contain a uni-layered primary endoder- mis with Casparian strips on the anticlinal cell walls and a pericycle (one cell layer thick). Inside the meristeles, phloem surrounds protoxylem and metaxy- lem (Fig. 2: 7-10). The protoxylem strands (variable in numbers: from 3 to 5) are intermixed with xylem parenchyma cells. All cortical cells are parenchy- matous having the same shape and thin walls, apart from thick cell walls in the close vicinity of the endodermis. Most of the cells are filled with amylo- 32 El¿bieta-Zenkteler,-Iwona-Jêdrzejczyk 32 Fig.2. 5 – Cross-section through a P. ×mantoniae Rothm. rhizome showing single dictyostele encircled by storage parenchyma (light microscopy); 6 – Cross-section through a P. vulgare L. rhizome; 7 – Detail of a large meristele of P. ×mantoniae (fluorescent microscopy); 8 – One of the smaller meristeles of P. ×mantoniae; 9 – Magnification of a large meristele of P. vulgare; 10 – One of the smaller meristeles of P. vulgare.
Ryc.2. 5 – Przekrój poprzeczny przez k³¹cze P. ×mantoniae Rothm. ukazuj¹cy pojedyncz¹ diktiostelê otoczon¹ miêkiszem spichrzowym (mikroskop œwietlny); 6 – Przekrój poprzeczny przez k³¹cze P. vulgare L.; 7 – Elementy wiêkszej merysteli u P. ×mantoniae (mikroskop fluorescencyjny); 8 – Elementy mniejszej merysteli u P. ×mantoniae; 9 – Elementy wiêkszej merysteli u P. vulgare; 10 – Elementy mniejszej merysteli u P. vulgare.
plasts with numerous starch grains. The pith contains some scattered tanniferous and sclerenchymatous cells. Polypodioid ferns are characterized by 3 vascu- lar strands in the stipe, fusing upwards in the rachis into one V-shaped strand (Fig. 3). P. ×mantoniae and P. vulgare, apart from the size, do not differ in the structure of the stipes.
In the gross anatomy, there are no evident differences between the rhi- zomes of the two compared taxa, but the morphometric analysis of their mi- crostructural features revealed quantitative differences in the number and size of meristeles and the number of protoxylem/metaxylem elements (Table 2).
Table 2. Number of stelar elements in evaluated species of Polypodium.
Tabela 2. Liczba elementów steli u badanych gatunków Polypodium.
Number of stelar elements (mean ±SD) (Liczba elementów steli) (Merystela wiêksza) (Merystela mniejsza) P. vulgare P. ×mantoniae Data (mean ±SD) were collected after measurements of 10 specimens Fig. 3. Cross-sections of the petiole of P. ×mantoniae Rothm. (left) and P. vulgare L. (right).
Ryc.3. Przekrój poprzeczny przez ogonek liœciowy P. ×mantoniae Rothm. (po lewej) i P. vulgare L. (po prawej).
34 El¿bieta-Zenkteler,-Iwona-Jêdrzejczyk 34 Genome size analysis. Flow cytometry analysis of the nuclear DNA content of the tetraploid species P. vulgare (2n = 4x = 148) and the pentaploid hybrid P. ×mantoniae (2n = 5x = 185) ranged from 30.86 ± 0.06 pg/2C to 39.44 ± 0.25 pg/2C, respectively. The obtained histograms resulted in two peaks re- presenting the G /G nuclei of the internal standard and the target species (Fig. 4). The coefficients of variation (CV) ranging from 4.39% for P. vulgare to 4.61% for the hybrid, were close to the acceptable range of 5% (Doležel, Bartoš 2005).
Fig. 4. Histograms of fluorescence intensities in nuclei isolated simultaneously from young leaves of Pisum sativum (internal standard) and leaves of P. vulgare L. (a) and P. ×mantoniae Rothm. (b).
Ryc. 4. Histogramy intensywnoœci fluorescencji w j¹drach równoczeœnie izolowa- nych z m³odych liœci Pisum sativum (standard wewnêtrzny) oraz liœci P. vulgare L. (a) i P. ×mantoniae Rothm. (b).
Discussion and conclusion In gross morphological characters, the rhizomes of P. vulgare and P. ×mantoniae resemble one another apart from one exception – the scales.
The different type of scales (shield-like spatulate in P. ×mantoniae and lan- ceolate in P. vulgare) clearly reveal the usefulness of this feature in taxonomy of polypody ferns. Although some variations in the details of rhizome organi- zation exist, the basic pattern of their growth and development is similar in both species (Wardlaw 1944). It is also known that the anatomical features of tissues belonging to the rhizome are mostly uniform within the genus Polypodium (Ogura 1972; Zenkteler 2000). The stelar system presents a radial arrange- ment (White, Weidlich 1995) and shows a similar perforated dictyostelic type in both species.
Using various staining techniques for light and fluorescent microscopy, in the transverse sections we can identify the rhizodermis, hypodermis, cortex and perforated dictyostele, consisting of two-three large and a few small meristeles. In this study we demonstrated that some differences, generally in the size and number of elements of the stelar system, can be distinguished but the question as to whether they may provide any additional evidence for poly- pody systematics remains open. It is likely that not only the morphology but also the anatomy of the rhizome and the leaf petiole of P. ×mantoniae has not been examined and described in detail so far. The P. vulgare rhizome con- tains a dictyostelar system consisting of less numerous large and small meristeles, in contrary to the large rhizome of P. ×mantoniae with numerous meristeles, which in addition exceed 2.4 times the number of protoxylem and metaxylem elements of P. vulgare meristeles. It has generally been stated that the rhi- zome anatomy at the level of the species is rarely explored.
The P. vulgare complex in Europe consists of approximately 4–5 species of which three are sexual (diplo-, tetra- and hexaploid); among the remaining taxa it is difficult to morphologically distinguish sterile hybrids (pentaploid).
The latter forms (often cytotypes) survive through their rhizomes because the spores formed as a result of irregular meiosis are abortive (Shivas 1961 a, b).
Members of the P. vulgare complex, ancient hybrids between P. vulgare (al- lotetraploid) and P. cambricum (diploid) followed by chromosome change, mani- fested their hybrid characters by vigorous growth and larger size of all organs (Page 1997). Nevertheless, the hybrid members of the taxonomically trouble- some P. vulgare complex in Europe are difficult to distinguish owing to the great similarity of their morphological characters. Thus they require the ac- curate identification because their intermediate features between both par- ents render them unrecognizable to a high degree. According to Page (1997) members of the P. vulgare complex could be recognized using such morpho- logical features as: the number of indurated annulus cells (recommended as the most useful character), the number of basal cells and the presence of aborted spores or sporangia. The same micromorphological characters of sporangia and spores allow determination of P. ×mantoniae in Bulgaria (Ivanova 2006).
The presence of P. ×mantoniae in the Czech Republic is also confirmed by data such as the number of thick-walled cells of annulus and spore or stomata dimensions (Bureš et al. 2003). Additionally, the estimation of nuclear DNA content using flow cytometry can confirm the taxonomic identification, espe- cially when plants are present only in the vegetative stage (Morozowska et al. 2010).
36 El¿bieta-Zenkteler,-Iwona-Jêdrzejczyk 36 The genome size of P. vulgare determined in this study was close to the values published previously. Murray (1985) and Bureš et al. (2003) reported a nuclear DNA content of 30.76 pg/2C and 29.00 pg/2C, respectively. The 2C DNA amounts obtained by Redondo et al. (1999) was lower than estimated in our study and by mentioned authors, and amounted 25.00 pg/2C. It was found that differences can occur when different methodologies, buffer composition and different standars are involved in genome size estimation (Doležel et al.
1998). The nuclear DNA content obtained for pentaploid hybrid P. ×mantoniae was more-or-less midway between that of the tetraploid (P. vulgare - 30.86 pg/2C) and hexaploid (P. interjectum - 45.24 pg/2C; reported by Bureš et al. 2003). This 2C value was similar to that determined by Bureš et al. (2003) but different from values reported by Murray (1985) and Redondo et al. (1999). The differences were probably caused by using other method based on Feulgen microdensitometry and different internal standard. Our re- sults indicate that cytogenetic study as well as flow cytometry analysis of DNA content are very important tools for correct identification of plant species.
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38 El¿bieta-Zenkteler,-Iwona-Jêdrzejczyk 38 Morfologia i anatomia k³¹czy Polypodium ×mantoniae Rothm.
Chocia¿ cechy diagnostyczne k³¹czy paproci s¹ rzadko wykorzystywane przy rozró¿nianiu gatunków, w niniejszej pracy podjêto tak¹ próbê. Dobra znajomoœæ budowy anatomicznej k³¹czy P. vulgare stanowi³a zachêtê dla wyodrêbnienia cech diagnostycznych, które ró¿ni³yby ten takson od P. ×mantoniae. Na podstawie pomiarów morfometrycznych oraz analiz w mikroskopie œwietlnym i fluorescencyjnym stwierdzono, ¿e k³¹cza P. ×mantoniae charakteryzowa³y siê wiêksz¹ œrednic¹, d³u¿szymi przyrostami rocznymi, wiêksz¹ liczb¹ rozga³êzieñ bocznych, wiêksz¹ liczb¹ ma³ych i du¿ych merysteli w obrêbie perforowanej diktiosteli k³¹cza oraz wiêksz¹ œrednic¹ ogonków liœciowych ni¿ P. vulgare. By³y to ró¿nice iloœciowe, nie daj¹ce podstawy diagno- stycznej, chocia¿ bujnoœci¹ organów potwierdzaj¹ce mieszañcowy charakter pentaploidalnej formy, jak¹ jest P. ×mantoniae. Analizuj¹c fenotyp ³usek okrywaj¹cych wierzcho³ki k³¹czy uda³o siê odnaleŸæ cechy dobrze ró¿nicuj¹ce obydwa gatunki.
£uski u P. ×mantoniae by³y ponad dwukrotnie szersze, bardzo wypuk³e i jaœniej wybarwione ni¿ u P. vulgare. Ponadto ich ‘punktowe’ przytwierdzenie do epidermy ró¿ni³o siê od szerszego ‘nasadowego’ przytwierdzenia, jakim charakteryzowa³y siê ³uski P. vulgare. Zawartoœæ j¹drowego DNA dla tetraploida P. vulgare wynios³a 30.86 ± 0.06 pg a dla pentaploidalnego mieszañca P. ×mantoniae 39.44 ± 0.25 pg.

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