HYPERHYDRICITY PHENOMENON DEVELOPED AT THE LEVEL OF SUGAR BEET (BETA VULGARIS L. VAR. SACCHARIFERA) VITROCULTURES. IV. DEUTERIUM DEPLETED WATER EFFECTS IN HYPERHYDRICITY ANNIHILATION OF VITROLEAFLET CELLS, ASPECTS VIEWED TO OPTIC AND TRANSMISSION ELECTRONIC MICROSCOPE

Headnote

ABSTRACT. In order to recovery neoplazic status, produced in tissues and cells of sugar beet (Beta vulgaris L. var. saccharifera) hyperhydric vitroplantlets, we proceeded to replace the distilled water from the culture medium, with 25 ppm deuterium depleted water. Generally, this type of water is used with therapeutic effect in improving the status of cancerous cells, in case of animal and human neoplazic tissues.

After apical explants cultivation, tacked from sugar beet vitroplantlets preleved from seed germinations in sterile conditions, on Murashige-Skoog (1962) ½ culture medium, prepared with distilled water, and with 2.5 mg/l BA addition, from this inoculs were born hyperhydric vitroplantlets. If the same kind of culture medium was prepared with deuterium depleted water (instead of distilled water), with a 25 ppm Deuterium contend (instead of 150 ppm deuterium normally held in natural and distilled water), the hyperhydricity phenomen is not installed in sugar beet vitroplantlet cells, derived from caulinare, uninodale, apicale type of explants. Moreover, to these crop types- inoculated and cultivated on a culture medium prepared with deuterium depleted water - to base on phytoinoculs was not observed the callus proliferation, although the other medium components were identical to those used in experiments made with medium ingredients which were dissolved in distilled water, situation when - to the minicutting bases - was noticed hyperhydric callus regeneration. This reaction may be a proof that, also in case of tumoral plant cells (status feature for hyperhydric phytoinoculs) water with 25 ppm deuterium content, cauzed an antiproliferative and antineoplazic effect.

Keywords: hyperhydricity, microscopy, beet, deuterium depleted water

INTRODUCTION

The morpho-physiological and biochemical evaluation of vitroplantlets hyperhydricity (vitrification), concerns many plant biotechnologists.

In the last two decades, the hyperhydricity phenomenon - to phytoinoculs - has been amply studied by specialists. Depth studies carried on physiology and biochemistry hyperhydric phytoinoculs have revealed the negative consequences, such as cancer, of these processes on the vitrocultivated plant material. Such research have brought an important contribution to the knowledge of damages, caused to the phytoinoculs, by hyperhydricity, at metabolic level - especially enzymatical and phytohormonal, a cancerous disease not caused by phytopathogens agents, as is the case of cell neoplasm generated by Agrobacterium. Moreover, Gaspar (1991, 1995) compared known phenomenons as being characteristics of human and animal cancer cells, especially consisting to maintain the embryo juvenile status, and the lack of their ability to differentiate (although they have an intense proliferation feeling etc.), with the cancer status of hyperhydric phytoinoculs cells.

The water with 25 ppm deuterium (D) content, to humans and animals inhibit tumour growth (especially in xenotransplants), stimulates vascular reactivity and immunity, and increases those resistances at low doses of gamma radiation (Nutiu and Ardelean, 2002). It can be mixed with distilled water in various proportions, obtaining thereby water with desired D content.

After Nedelcu (2002), deuterium depleted water (DDW) stop the anarchic mitosis a characteristic feature of tumour tissues. Deuterium (D), besides that affect the metabolism, it exercises a significant effect in starting or not starting the cellular division. Thus, for cells to divide, it must receive first an "impuls", which seems to be caused by a optimally report between deuterium (D) and hydrogen (H). The studies have shown that when a cell starts to divide, the membrane pump is enabled and eliminates the H from cell. By the H evacuation from the cell, it remains a surplus of D in it. The increase of D concentrations in cells, and the decrease of H levels in those, triggers the division signal.

To plants, the investigations of the treatments with DDW was made by Somlyai and collaborators (1993 and 1997), Somlyai (2001), and by Belea and his coworkers, (1997). The summoned authors found that, generally, at 5 - 6 days from placing the seed material to germinate (which was previously submerged in DDW) on a substrate wetted with DDW, with a concentration of 25 ppm deuterium, the growth of buts and vitroplantlet rootles resulting from embryos were inhibited, but the effect had disappeared after 10 - 12 days. These experiments have shown - after summoned authors - a plantlets capacity in relatively large limits, to adaptation at the medium with low deuterium concentration.

In the last few years, have also made recently research by the Berdea and collaborators (2007), which have revealed, once moreover, that DDW not only that - to plants and animals - inhibits cell division, but that to plants, it acting negatively on seed germination.

In recent years, we focused our researches to examine the changes at mopho-physiological and ultrastructural level, to various phytoinoculs types affected by the hyperhydric syndrome, disease with neoplazic characteristics (Petrus-Vancea et al., 2003, 2008; Petrus et al., 2004; Radovet-Salinschi, 2004; Radovet-Salinschi and Cachita, 2005 a and b; Radovet- Salinschi et al., 2008; Beles and Cachita, 2007; Petrus and Cachita, 2008 a and b; Cachita et al., 2008 a and b; Radovet-Salinschi et al., 2008; Cachita and Ardelean, 2004).

Our research has pointed out, as is mentioned in the speciality literature (Gaspar, 1991, 1995; Ziv, 1991; George, 1996), that leaflets of hyperhydric vitroplantlets present - beside the generally changes of morpfophysiological aspect, which is characterized by a tissue transparency (vitrification), giving them a dystrophic status - the existence of a histological disorders occurred both in the epidermis (the stomata being open wide, or implosion), and in the foliar mesophyll; moreover, a cell impoverishments in chloroplasts and in assimilating pigments, a very high frequency in cells of picnotic nucleuses, the cytoplasm is dismantled, the tonoplast is inorganizable etc. were noticed.

In the present paper, we present our new studies on sugar beet (Beta vulgaris L. var saccharifera) hyperhydric vitrocultures, in the culture media preparation with DDW conditions - with a 25 ppm D content - instead of distilled water.

MATERIAL AND METHOD

In the initial part, the experiment was organized by the same procedure used in our previous work (see Cachita et al, in this volume), in which we presented our microscopy studies made on sugar beet hyperhydric callus. Compared to the previous experiment, in these studies, in the vitroculture initiation moment from uninodale apexis taken from vitroplantlets obtained after seed germination in aseptically regime, the novelty was the Murashige- Skoog (1962) (MS) ½ medium components, modified by us as follows: thiamin HCl, pyridoxine HCl and nicotinic acid, each 0.1 mg/l (instead of 0.5 ml/l, according to the original recipe), medium without glycine and with 20 g/l sucrose, instead of 30 g/l, and 7 g/l agar-agar, instead of 10 g/l; the medium components were not dissolved in distilled water, but in deuterium depleted water (DDW), with only 25 ppm deuterium concentration; in this medium was added, or not, BA 2.5 mg/l. At the control lot the vitrocultures of sugar beet apexes were realized on MS complete medium, but prepared with distilled water (DW) and with or without 2.5 mg/l BA.

The vitroculture period was 30 days; the conditions in the growing room were as follows: 23-25 ° C temperature, lighting has been assured by fluorescent tubes, issuing white light with 1700 lux intensity and 16/24 h light/day photoperiod.

Vitroplantlets foliar limbs fragments, with maximum 1 mm in diameter, were fixed 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 included in the EPON 812. The blocks with callus samples were sectionated with a Leica UC6 ultramicrotom.

Section contrastation was made with lead citrate and acetate uranil. Preparations were examined with transmission electron microscopy Joel JEM1010.

The most representative images, seen at optical or electronical microscope, are presented in the 1-6 figures.

RESULTS AND DISCUSSION

The cross-sections applied trough sugar beet (Beta vulgaris L. var saccharifera) vitroplantlet foliar limbs, 30 days old (obtained from seeds germinated in the septic regime, in the ground, or in vitro, on MS ½ culture medium, with our modifications, or from caulinare, apicale, uninodale explants taken from plantlets generated from seeds germinated in the aseptically conditions), have allowed us histological and cytology analysis, which are visible at optical or transmission electronic microscope. Microscopic examinations had as objective the decelation of similarities and differences that exist - on the optical and ultrastructural plan - between normal and hyperhydrified (vitrificated) plant cells, a phenomenon often manifested to sugar beet vitrocultures growing on medium with high cytokinine concentrations, in this experiment BA was added in 2.5 mg/l concentration. This cytokinine is, frequently, responsible for the hyperhydricity induction at phytoinoculs level (George, 1996).

In 1 and 2 (A and B) figures we illustrated the histological, cytological and ultrastructural aspects noticed to normal, nonhyperhydric, sugar beet plantlets leaflets, at 30 days old from those germination on soil, in greenhouse, same as from in vitro, on a culture medium specific to phytoinoculs (MS), but all ingredients was diluted to half. In both cases, our observations have pointed out the biostructures status of normality, both at epidermis, and in the foliar mesophyll level, which is homogeny, with an undifferentiated chlorenchyma in palisade parenchyma and lacunose parenchyma. The tissue structure is normal, the intercellular space being small. Both epidermises (upper and lower) presents stomatas. Generally, in foliar mesophyll cells, the chloroplasts abound, and they are fusiform.

To those sections applied through the plantlet foliar limbs derived from zygotic embryos, after in vitro seed germination on ½ MS medium, the ultrastructural images (fig. 2 A and B) showed corpuscular formations in vacuoles, spherical or ovoid, high electron dense, with unknown origin, possibly leucoanthocyans or phospholipid corpuscles (Cachita and Craciun, 1990). The presence of 10 g/ sucrose in the MS ½ culture medium led, at most plantlet vitroleaflet chloroplasts, generated from zygotic embryos, to exist starch deposits (fig. 2, A and B). In case of plant vitrocultures, the phytohormonal nutrition is heterotrophic because of the presence of the sucrose in the substrate, or myxotrophic when cells have chloroplasts. Thus, the glucose from chloroplasts resulted in the photosynthesis process is not consumed, but it is transformed in starch, and the starch shall be filed in their stroma, the necessary energy in vital processes being released - after catabolic reactions - from sucrose molecules, present in the substrate.

Hyperhydricity, at sugar beet vitroplantlet leaflets (as we've shown us in other scientific papers - see Cachita et al., 2008, a and b), causes major abnormallitys at the histological and ultrastructural level. As the hyperhydricity process moves forward (to compare fig. 3 A with fig. 3 B), in the foliar limb anatomy is installed a assimilating parenchyma dismantling phenomenon, the intercellular space is increases, turning into lacunes, cellular walls become thin, which leads to the distortion of the pectocellulosic cell membranes, a phenomenon which is installed at the epidermises level (especially at the inferior epidermis, particularly in advanced hyperhydricity stages). Also to the epidermises level is observed an implosion of the stomatic cells (fig. 3 A), or an excessive opening of their osteoles (fig. 3 B), which makes them broken.

In foliar mesophyll of hyperhydric cells, which not have such an advanced vitrification process (fig. 3 A), it is noticed the presence of huge vacuoles, the cytoplasm being pellicle; the chloroplasts are oblong, few and without starch. To vitroleaflets, with an advanced hyperhydricity process (fig. 3 B), the foliar mesophyll is extremely poor in cytoplasm and chloroplasts, the cells - mostly - have a cellular content ongoing disruption. To be recorded that to those cells, which suffer of hyperhydricity, in vacuolar juice were not able to identify large corpuscular formations.

Whereas, from our previous researches (Petrus- Vancea et al., 2008) resulted that those mediums which are prepared with deuterium depleted water (DDW) led either to hyperhdricity prevention at Petunia phytoinoculs (Petrus and Cachita, 2008), or - in the case of administration in bistratification cultures system- their caulinar zone being submersed in a supernatant consist in DDW (with 87,5 ppm D) plus glucose 1.5 % has been made a positive influence about hyperhydric vitroplantlets; to Coleus vitrocultures was observed a stem revitalizations, their apexes, up from supernatant, being unhyperhydrices.

Analyzing the images presented in 4 - 6 (A and B), figures resulted that the epidermal cells and sugar beet vitroplantlets foliar mesophyll, which was regenerated from caulinare and uninodale apexes inoculated and cultivated - for 30 days - on MS medium with 2.5 mg/l BA addition, prepared with DDW (with 25 ppm D concentration), instead of distilled water, have not an hyperhydric phenomenon; both epidermises and foliar mesophyll showing a normal structure (to compare optical microscopy aspects of fig. 1 A, with the fig. 4 A and B; and the electron microscopy aspects - fig. 2 A and B - with fig. 5 and 6, A and B imagines). All structures and cells are perfectly normal, the leading tissues (respectively the ribs - fig. 4 B) are differentiated, the xylem elements being represented. In these plantlets cells the chloroplasts and the nucleuses present a normal ultrastructure, and there are many vacuoles and rich deposits of corpuscular, electron dense, collated, which have a not predict origin.

CONCLUSIONS

The presence of the benzyladenine (BA) in the culture Murashige-Skoog (1962) culture medium, in which sugar beet uninodale and apicale explants were grown, tacked from vitroplantlets which were regenerated from zygotic embryos on aseptic seed germination, for 30 days long, on basic MS medium, without BA addition, induced hyperhydricity at vitroplantlets which reclaimed from such inoculs.

Dissolution of ingredients in deuterium (D) depleted water, with 25 ppm D concentration, even in the BA presence (in 2.5 mg/l concentration), has prevented the hyperhydricity process installation in sugar beet vitroplantlet tissues and cells regenerated from apical minicuttings. Therefore, this method proved efficiency in preventing the hyperhydricity occurrence to sugar beet vitrocultures, and callus regeneration.

References

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