PLANT CELL ULTRASTRUCTURAL RESEARCHES AS BIO- ECONOMY, ECO-ECONOMY AND ECO-SANOGENESIS INSTRUMENT Adriana Petrus-Vancea 1, Dorina Cachită-Cosma 2

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1 PLANT CELL ULTRASTRUCTURAL RESEARCHES AS BIO- ECONOMY, ECO-ECONOMY AND ECO-SANOGENESIS INSTRUMENT Adriana Petrus-Vancea 1, Dorina Cachită-Cosma UNIVERSITY OF ORADEA AND POSTDOCTORAL RESEARCHER AT VASILE GOLDIS WESTERN UNIVERSITY ARAD; 2 - VASILE GOLDIS WESTERN UNIVERSITY ARAD Summary We aimed to study the ultrastructure of hypocotyl and foliar limb cells of a species with food interest, namely red beet (Beta vulgaris var. Conditiva), in vitro cultivated, to identify changes that occur in the hyperhydricity, a common tumoral phenomenon at this species, and quality of plant material in sanogenetic terms. At the hypocotyl cellular level, the hyperhydricity was manifested by changes in chloroplast ultrastructure, by nucleus degeneration or tonoplast destruction and by mixing of vacuolar juice with cytoplasm. The foliar limb cells showed a normal appearance, hyperhydricity not being installed. We did not identify viral or microbial formations, from ultrastructural point of view. Keywords: Beta, in vitro, hyperhydricity, ultrastructure, sanogenetic, Introduction Bio-economy is a science that is based on a Romanian origin scientist theory named Georgescu-Roegen (1971), which contrary to classical economic consideration, examines the economy work as a continuation of biological evolution with extrasomatic resources - thus cultural and social. Author's solution would reduce production, decrease and correct understanding of the development concept, which should be not a function of the production of physical goods. This would be based on eco-economics (environmental economics), a concept proposed by Brown (2001), which refers to an economy developed on long term without affecting their support system, namely the environment, without soil overloud, already been in a deterioration state (Iagăru, 2010). Eco-sanogenesis (soil, plant, animal or human health), aims to ecosystem management. Its objectives take into account the consumer interests, as well as those of manufacturers and environmental issues, raised by the principles of environmental engineering and ecological economics (Gruia, 1996). Preparation of organic agricultural products, clean and 378 unpolluted, as a raw material with such qualities, is the decisive step for obtaining proper behaviour in food products, cosmetics or pharmaceuticals. For the future, the current economy must be replaced with this economy, based on ecology and environment protection, and scientists have a major contribution in this regard (Ardelean et. al., 2011; Bogdan et. al., 2011; Purcărea and Borbely, 2011), together with decision makers (Antofie, 2010; Antofie et al., 2010). To achieve these objects using the modern biotechnology, both in animals (Ipate et al., 2011) and the plant, is a future solution, and using with maximum efficiency of biotechnological methods is based on fundamental research, which provide scientific data and certainty procedures used. In the field of bio-economy, ecoeconomy and eco-sanogenesis, the basic research has an important role in establishing eco-physiological requirements of species with an economic interest, the identification of pathological phenomena (tumours, infections etc.), that affect this organisms, both to find solutions in preventing such inconvenience - which

2 can affect the ecological and economical industrial production - or procedure to suppress such situations already appeared. The actions to identify of structure and ultrastructure of the red beet (Beta vulgaris var. Conditiva) vegetative organs can contribute to a better understanding in this domain, implicitly underlying optimization applied methods for the extraction of beet red dye, and to identify hyperhydricity specific changes, respectively to find solutions to prevent or suppress it, which is a decisive factor in increasing plant mass production, it quality, with production losses minimized. By identifying possible infections of red beet aerial organs, can bring - indirectly - significant contributions to health insurance consumers. Aseptically conditions, specific to in vitro cultures, ensure - to some extent - the production of virus-free seedlings or other diseases caused by microorganisms, depending on the type of culture (meristem culture, seed germination), which is a healthy plant material higher qualitatively and quantitatively (Cachită et al., 2009). Ultrastructure study will prove once again the importance of basic research in bioeconomy and the advantages of in vitro plant cultivation, both in terms of ecosanogenesis and eco-economic. Any further contribution in this area is a step in biochemical and physiological phenomena elucidation and contributes to finding solutions by those who are involved in the multiplication problems, in the industry. Under natural conditions, Beta species may be affected by numerous diseases, including virosis (Sherf and Macnab, 1986), mycosis (Hodges, 1935), bacteriosis (Duke, 1987). Ultrastructural studies on sugar beet leaves (Beta vulgaris L.) infected with beet western yellow virus (BWYV) showed virus and tissue stages of infestation brought about viral replication in host cells (Esau and Hoefert, 1972). Such viral particles are ultrastructural highlighted in the riddled tubes even before develop external symptoms on leaves. Then, the particles appear in the parenchyma cells near plasmodesms, linking mature riddled elements with parenchyma cells. Virus invasion in the parenchyma cells is accompanied by the development of vesicles which containing similar networks to those usually interpreted as nucleic acids, which are closed in the endoplasmic reticulum cisterns, individually or in groups. Some vesicles can fuse with the nuclear membrane and the virus then occurs in the nucleus. Being released probably from the nucleus the viruses quantity increase in the cytoplasm. Those which remain in cytoplasm it degenerate, but degenerates infected cells too. Material and methods The plant material consisted of red beet (Beta vulgaris var. Conditiva) vitroplantlets obtained from seeds germinated in aseptic medium, at 60 days after culture initiation on mineral medium Murashige Skoog (1962) ½, whit thiamin HCl, pyridoxine HCl and nicotinic acid, each 0.1 mg/l, without glycine and with 20 g/l sucrose, whit 7 g/l agar-agar, without grown regulators; the ph of culture medium has been adjusted value of 5.7, prior autoclaving. From this organs of vitroplantlets, which have the two first leaflets upstairs cotyledons, the tissue samples with maximum 1 mm in diameter consist in hypocotyl and foliar limb was preleved and fixed - in standard conditions (Hayat, 2002) - in 2.7% glutaraldehyde for one hour, then fragments were postfixated in osmic acid 2% and later, were dehydrated in baths of acetone, increasing concentration, at the end they were embedded in the EPON 812. The blocks with samples were cut with a Leica UC6 ultramicrotome. Section contrastation was made with lead citrate and acetate uranil. Preparations were examined with transmission electron microscopy Jeol JEM1010, in Electron Microscopy Center of Babes-Bolyai University, Cluj-Napoca. 379

3 Results and discussions Hypocotyls ultrastructure of in vitro red beet. Red beet hypocotyl in vitro regenerated, at 60 days after culture initiation, sowed cell cultures with distorted ultrastructural appearance (fig. 1 A and B). Chloroplasts, was not only flat, but - in most cases - tonoplast was absent, and - in this case - chloroplasts were scattered in the cell lumen filled with myxoplasm which resulted from mixing juice vacuolar with cytoplasm, to the most cells (fig. 1 A and B), this fact was reported by Cachiă and collaborators (2008) too, but at sugar beet leaflets. Tonoplast disintegration leads to the formation of myxoplasm into cell lumen, which giving to cell a chaotic look, disordered cell and an early necrosis and malfunction status. These issues have different advance stages and they may lead to the installation of a total disorganization state and necrosis. Chloroplast structure is affected, they are either lenticular forms (especially in normal situations) (fig. 1 C), or are deformed (fig. 1 B), sometimes with stroma in disintegration, being lack the optical density, with vacuolization appearance in this stroma. In figure 1 D are illustrated details of the beginning of vascular differentiation, respectively by ringed or spiralled cells, transversal sectioned, with the lignin deposition, but which are sporadic and are not structured in the ribs, it appear as xylem nodules, features for old callus tissue. Ultrastructural was not highlighted extraneous. Fig. 1.Ultrastructural aspects of red beet (Beta vulgaris var. Conditiva) hypocotyls, in vitro regenerated: A disorganized parenchyma cells; B disorganized chloroplasts and myxoplasm formation; C normal chloroplast and myelinice formations began training; D conducting vessels with lignin deposits (chl chloroplasts; c.w. cell wall; d.chl - disorganized chloroplasts; l.d. lignin deposit; m.f. - myelinice formations) (bars mean: A and B - 20 µm; C and D - 10 µm). 380

4 Details of denatured chloroplasts are highlighted in figure 2 A, and in figure 2 B are captured chloroplasts almost spherical, because large deposits of starch. In our case, not only chloroplasts, but the nucleus too, appears to be equally affected by hyperhydricity (fig. 2 C). Figure 2 D highlights a nucleus loss body. Fig. 2.Ultrastructural detail from red beet (Beta vulgaris var. Conditiva) hypocotyls, in vitro regenerated (chl normal chloroplasts with large deposits of starch; c.w cell wall; d.cl. disintegrated chloroplast; d.n. degenerated nucleus; l.b. - loos body; mx myxoplasm; N nucleus; n- nucleolus; s - starch) (bar means: A, B and C - 5 µm; D - 1 µm). This type of structure fully corresponds to those described in the literature, which refer to characteristics from histological point of view - of hyperhydricity parenchyma (Cachiţă, 1987). On the other hand, Tomlinson and Webb (1978) by electron microscope examination of lettuce leaf infected with beet yellow Claytonia perfoliata showed that symptoms caused by this virus (leaf yellowing) were associated with degeneration of chloroplasts by the disappearance grains, the appearance of abnormal stroma, with large starch granules, with increased density and osmiofilic grain size. Degeneration of chloroplasts induced by the virus is related to natural degeneration of chloroplasts during leaf aging and senescence in other species which is probably controlled by endogenous hormones and mitochondria have the appearance as fibrillary inclusions 381 (Hoefert, 1985). Also, changes in chloroplast ultrastructure thereby, their function, by reducing CO2 assimilation have been reported in infections with Erysiphe polygoni DC fungi, of Beta vulgaris L. leafs (Magyarosy et al., 1976). Kutík and colleagues (1995) at sugar beet plants grown under natural conditions, treated with nitrogen, at which were either raised or lowered CO 2 concentrations, observed that chloroplast ultrastructure of parenchyma was affected by elevated CO 2 concentration and surprisingly, affected by nitrogen supply. Thus, elevated CO 2 concentrations have led to the chloroplasts increase, to the reduction to 5% of tylacoids, to lower grain size and increased volume of stroma, increased plastoglobules number. Also at sugar beet, which was grown in culture medium devoid of iron, young

5 leaves, green and yellow became completely healthy after 6-8 days of culture (Platt-Aloia et al., 1983). Their ultrastructure appeared relatively normal, except plastids which were small and undeveloped, with a rudimentary and disorganized border and network, with vesicles groups on the periphery. After hours after reintroduction of iron in the medium, phyto-erythrine aggregates were observed in the stroma, the grain began to grow, and at 48 hours, normal ultrastructural became appearance. Also, infection of poplar leaf with rust Melampsora pinitorqua causes ultrastructural changes in chloroplasts (Młodzianowski and Siwecki, 1975). They are generally similar to changes observed in chloroplasts of other plants infected with viruses or rust or treated with different chemicals, often found in aging. However, in our case, ultrastructural have not been highlighted foreign cell formations, as microorganisms or viruses. If such infections is found (e.g. beet yellow virus), were revealed particles about 100 A diameter, with A electron-density in the cytoplasm, chloroplasts and nucleus (Cronshaw et al., 1966). Particles observed in chloroplasts may or may not be associated with lipid spheres. Particles located free in stromal regions often regularly arranged in curved rows that are parallel to one another, so as to form a three-dimensional network. Also, a decreased of ribosome numbers is concomitant with the increase in the amount of virus in a cell. During virus replication the formation of vesicles occurs in the cytoplasm, which could be derived from dictiosomes. If virus particles are close to the plasma membrane, many of them are oriented perpendicular to the cell surface. In some areas the virus particles appear to penetrate the cell wall. At the end, when the cytoplasm no longer recognizes the virus particles formed aggregations which has a crystalline structure and are surrounded by amorphous material (Cronshaw et al., 1966). Foliar limb ultrastructure of in vitro red beet. From morphologically point of view was not identified transparency, discoloration of leaf or their turns, which is specific in hyperhydricity. Although the ultrastructure of hypocotyls indicates an intense hyperhydricity, this phenomenon was not revealed in the in vitro regenerated leaflets (fig. 3 A - D), in which the tonoplast was upright and the appearance of chloroplasts, nucleus and all cellular organelles was normal. Even at the ultrastructure details (fig. 4) were not highlighted abnormalities or the presence of extraneous. Conclusions In terms of ultrastructural, at the level of hypocotyl and leaflets of in vitro red beet (Beta vulgaris var. Conditiva) were not highlighted extraneous. If the hypocotyl of in vitro red beet has intense cell degradation, caused by the hyperhydricity, at leaflet level, this phenomenon was not revealed Acknowledgements This work was cofinanced from the European Social Fund through Sectoral Operational Programme Human Resources Development , project number POSDRU/89/1.5/S/63258 Postdoctoral school for zootechnical biodiversity and food biotechnology based on the ecoeconomy and the bio-economy required by eco-sanogenesys, coordinated by the National Institute of Economic Research Costin C. Kiriţescu, Romania, in collaboration with Lucian Blaga University of Sibiu, Vasile Goldiş West University Arad, S.C. GNIR Holding S.A. and S.C. SIAT S.A. I also thank the Electorn Microscopy Center, University Babes-Bolyai Cluj- Napoca, where was made the investigations of ultrastructure, especially of Prof. Constantin Crăciun Ph.D. 382

6 Fig. 3.Ultrastructural aspects of red beet (Beta vulgaris var. Conditiva) foliar limb, in vitro regenerated (chl chloroplasts; i.sp. intercellular space; M mitochondria; N nucleus; n nucleolus; s starch; V - vacuole) (bar means: A and B - 20 µm; C and D - 10 µm). Fig. 4.Ultrastructural details of red beet (Beta vulgaris var. Conditiva) foliar limb, in vitro regenerated: A young cell; B numerous mitochondria; C normal chloroplast, without starch; D - normal nucleus (chl chloroplasts; i.sp. intercellular space; M mitochondria; N nucleus; n nucleolus; s starch; V - vacuole) (bars means: A B, C - 5 µm; D - 2 µm). 383

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