Growth analysis indicated that carbon gain in the alpine succulentSedum album tended to take place early during the growing season. Leaf water potential remained unchanged for a considerable length of time after the imposition of water stress in the field. Induction of crassulacean acid metabolism (CAM) by protracted water stress occurred before any signs of stress could be observed in the leaves, and appeared to be influenced by a complex interaction of environmental conditions including temperature and duration of sunshine. Increased levels of proline and betaine towards the end of the growing season appeared to reflect seasonal changes.

The ability of peanut (Arachis hypogaea L.) to grow at high concentrations of NaCl may be due to the alteration in gene expression. SDS-PAGE analysis has revealed that plants grown under NaCl showed induction (127 and 52 kDa) or repression (260 and 38 kDa) in the synthesis of few polypeptides. In addition, nine different esterase isoenzymes were detected in embryos of seeds germinated in 105 mM NaCl, whereas only five of them were detected in the embryos of untreated seeds. On the other hand, in the cotyledons, the esterase pattern was not affected by NaCl concentration. The esterase patterns of both stems and leaves were less influenced by NaCl in comparison to those of roots. The lipid contents, and fresh and dry masses were increased up to 45 mM NaCl and decreased at higher concentrations.

The effects of copper on the activity of ascorbic acid oxidasc (AAO) in detached rice leaves under both light and dark conditions and in etiolated rice seedlings were investigated. CuSO₄ increased AAO activity in detached rice leaves in both light and darkness, however, the induction in darkness was higher than in the light. In the absence of CuSO₄, irradiance (40 μmol m^-2 s^-1) resulted in a higher activity of AAO in detached rice leaves than dark treatment. Both CuSO₄ and CuCl₂ increased AAO activity in detached rice leaves, indicating that AAO is activated by Cu. Sulfate salts of Mg, Mn, Zn and Fe were ineffective in activating AAO in detached leaves. CuSO₄ was also observed to increase AAO activity in the roots but not in shoots of etiolated rice seedlings.

Organogenic callus cultures ofPapaver somniferum L. were studied with the aim of describing the morphology of the callus and observing the changes occurring during differentiation and induction of organogenesis. The morphology of the cells and the formation of meristemoid areas were studied on a light microscopic level. Histological evidence showed that from the inner meristematic centres, protracheal elements are differentiated, from the surface meristematic centres meristemoids are formed and therefrom green leaf-like organoids.

Shoots of the hybrid walnut Juglans nigra x Juglans regia contained serotonin in the micromole range and indole-3-acetic acid (IAA) in the nanomole range. The serotonin level fell by 40 % in 12 h in auxin (IBA) treated whole shoots and then reincreased to a maximum (50 %over the control) after 36 h. The same pattern was followed in the top portions of the shoot but in the shoot bases, serotonin always remained under the control level. The early decrease of serotonin was correlated with an increase in IAA-aspartate. The early decrease and peaking of the serotonin level preceded and corresponded to the increase and peaking of free IAA in the shoot bases. The initial serotonin pool in treated-to-root shoots might thus suffice for the biosynthesis of IAA and IAA-conjugated compounds. Because of its auxin-like properties, the early serotonin peak might be taken into consideration as an endogenous auxin signal for rooting in the present material. If this turns out to be so, the rooting signal for the shoot bases necessarily should come from the apices.

The effect of chloramphenicol (CP) on the differentiation of callus cells ofHaplopappus gracilis into tracheary elements (TE) was studied. CP (1 mg l^-1) added to the medium stimulating the differentiation was shown to have an inhibitory effect. This observation points to the importance of the impaired functions of mitochondria in the processes leading to the differentiation of callus cells into TE.

The effect of light-induced greening of microtubers on their storage behaviour was studied in 16 genotypes of potato. Greening improved the storage of microtubers in terms of shrinkage, biomass loss and sprout emergence. A significant genotype × treatment interaction for shrinkage was observed.

Calli of soybean (Glycine max Merr.) cv. Maple Arrow grew better and accumulated more proline when cultured for 5 d on 70 mM NaCl under darkness than at light. This rapid proline accumulation in salinized soybean calli appeared to play a protective role rather than to be a cause of growth failure. Throughout a 28 d-culture cycle (in control and NaCl-treated calli exposed to light or darkness), we followed the possible relationships between the proline contents and the activities of enzymes of proline biosynthesis [ornithine transaminase; NAD(P)H-pyrroline-5-carboxylate reductase], of proline catabolism [NAD(P) proline dehydrogenase], and of NAD kinase responsible of variations in NADP(H) contents. Enzyme activities of proline metabolism and NAD kinase were clearly light- and NaCl-regulated; nevertheless, relationships between enzyme activities and proline content existed only in calli grown for short-term under darkness and in presence of NaCl. The ornithine transaminase route, which was particularly enhanced in these calli during the first days of salt application, seemed to be involved in the initial proline accumulation in soybean.

It is a matter of controversy whether secondary wall deposition is dependent on lignification during the development of tracheary elements. To understand this, tracheary element differentiation was studied in the homogeneous calli obtained from the cotyledonary explants of Cucumis sativus subsequent to treatment with plant growth regulators, such as naphthalene acetic acid (NAA) and benzylamino purine (BAP), which are necessary for the induction of tracheary elements, along with metabolic blockers such as 2-aminoindan-2-phosphonic acid (AIP), 2,3,5-triiodobenzoic acid (TIBA) and nifedipine. Calli treated with AIP, a potential inhibitor of L-phenylalanine ammonia-lyase (PAL), have no PAL activity at any time during the culture period. There was a complete inhibition of lignification although secondary wall deposition was unaltered. Similar results were obtained using TIBA, an inhibitor of auxin transport, and nifedipine, a known calcium channel blocker. Thus the present study suggests that secondary wall deposition in the course of tracheary element differentiation need not to be dependent on lignification.

Supply of 1, 2, 5, 10 or 20 mM nitrate to detached roots, scutella or shoots from 5- to 6-d-old Zea mays L. seedlings increased in vitro nitrate reductase (NR) activity in all the organs and NADPH specific NR (NADPH:NR) activity in roots and scutella but not in the shoots. Usually 2 to 5 mM nitrate supported maximum enzyme activity, the higher concentration did not increase it further. The protein content in the roots, scutella and shoots increased up to 5, 2 and 20 mM medium nitrate, respectively. Nitrate uptake also increased with increasing nitrate concentration in roots and shoots, but it increased only slightly in the scutella. In both roots and scutella, methionine sulfoximine had no effect, while cycloheximide and tungstate abolished nitrate induced NADH:NR activity completely and NADPH:NR partially. Methionine sulfoximine increased nitrate uptake by roots and scutella slightly, but other inhibitors had no effect. The depletion of dissolved oxygen from the medium was lower in the presence of nitrate than in its absence or in the presence of ammonium, especially in the scutella.

Culture conditions for a fine dispersion of plated cells of Oryza sativa L. cv. IR 20, have been worked out. These plated cells developed microcalli containing large number of somatic embryos and subsequently plantlets. By using single cells and clusters of 2 - 4 cells, an efficient DNA-delivery by microprojectile bombardment into cells and its transient expression were assessed by employing a plasmid construct containing β-glucuronidase gene.

Direct somatic embryogenesis from shoot apical meristems of pea is described. Somatic embryos were induced directly (without callus intervention) from meristematic tissues grown on a medium supplemented with 2.5 µM picloram. Within 4 to 5 weeks, fully morphologically developed somatic embryos were obtained. Somatic embryos originated from apical as well as from basal parts of meristem explants. The initiation and development of somatic embryos was asynchronous, basal somatic embryos developed more quickly than apical ones. Abundant secondary embryogenesis was observed after isolation of primary somatic embryos and culturing them on media for germination. Morphologically normal somatic embryos germinated on medium without growth regulators; the conversion rate was increased by application of 10 µM thidiazuron (TDZ). TDZ was also able to induce shoot bud regeneration on embryos without differentiated a shoot apex, allowing to germinate up to 78 % of all harvested somatic embryos with various morphology. The protocol was successfully tested in 47 out of 48 P. sativum and P. arvense cultivars as well as in two wild peas (P. elatius, P. jomardi).

Nutrient-encapsulation technique using in vitro grown nodal segments was developed as an alternative method for distribution of potato germplasm. The nodal cuttings of two potato genotypes, Kufri Jyoti and Kufri Lauvkar, were encapsulated in calcium-free Murashige and Skoog's (MS) medium containing either 2 or 3 % sodium alginate and 0, 1, 2 or 3 % saccharose. The encapsulated segments were stored in tubes with or without semisolid MS medium, and incubated in the dark at 25 ± °C for 3 to 6 weeks. Presence of saccharose in the beads was found detrimental for regrowth of new shoots. In absence of saccharose, about 82 % and 53 % encapsulated segments initiated regrowth after 3 and 6 weeks of dark storage, respectively, in tubes containing MS medium. Storage in empty tubes deteriorated the encapsulated segments, and depressed shoot formation. For potato germplasm distribution, the encapsulated segments can be transported in small tubes containing semisolid MS medium.

Immature embryo and root meristem expiants of wheat were cultured on modified medium of Murashige and Skoog and Gamborg's medium supplemented with 2,4-dichlorophenoxy acetic acid. Morphogeriic callus cultures were obtained from both the expiants. The frequency of shoot formation varied from 22% to 48% from callus obtained from embryos while only root formation could be induced from root meristem expiants. Cultures from young and old non-differentiating calli, and calli with shoot and/or root formation at different intervals were analysed for isozymes of esterase, peroxidase and acid phosphatase for studying the morphogenic capacity. With the development of shoot and/or root from callus, some conspicuous isozymes appeared which indicates the involvement of these isozymes in root/shoot development rather than in the induction of morphogenesis in callus. Basic isozyme pattern of each enzyme for the callus was retained in all the callus stages.

The immature zygotic embryos of reciprocal maize hybrids (CHI-31 x GF1 and CHI-31 × GE2) were used as the initial material for induction of somatic embryogenesis in vitro. Histological analysis of somatic embryogenesis revealed high developmental variability. The arising formations were classified into 5 groups: A) somatic embryos phenotypically similar to zygotic embryos, B) polyembryos, C) formations with radicle but without meristematic plumule, D) formations with radicle without differentiated plumule, and E) formations with plumule without radicle. The formatioms A and B regenerated directly into plants. Plant regeneration from formations E required preculture on the rooting medium. Formations C and D failed to develope into plants possibly because of early loss of meristematic cell character during the embryo axis differentiation. The reverse sequence of radicle and plumule differentiation in somatic embryos in comparison with zygotic ones was noted. The epigenetic character of the scutellum, coleoptile, coleorhiza and leaves primordia development was discussed.

Adventitious root formation in vitro in 1-mm stem slices cut from microshoots of apple cv. Jork 9 was studied using light and electron microscopy. When indole-3-butyric acid (IBA) had been added to the medium, starch grains accumulated during the first 24 h of culture in cells of the cambial region and in cells in the vicinity of vascular tissue and in the primary rays. This accumulation occurred only in the basal part of explants. After that, the nuclei in these cells were activated, and the density of the cytoplasm and the number of cell organelles increased, whereas starch was broken down. Cambium cells started to divide transversely and at 96 h, after several divisions, a continuous ring of isodiametric cytoplasmic cells had appeared around the xylem near the basal cutting surface. The cells in this ring were rich in cell structures, and did not contain large starch grains and a central vacuole. Root meristemoids regenerated from the portions of the ring that were localized in the primary rays. From the other cells in the ring, callus developed. The meristemoids did not grow into the direction of the epidermis as in shoots, but along the vascular bundles. After emergence from the cutting surface, the meristemoids were transformed into small, dome-like primordia. They developed a typical root apex with root cap, root ground meristem and tracheid connection with shoot vascular tissue.

An efficient protocol was developed for in vitro plant regeneration via somatic embryogenesis from cell suspension cultures of metal tolerant grass Echinochloa colona (L.) Link. Callus was obtained by culturing leaf base on MS medium supplemented with 0.5 mg dm^-3 of 6-benzylaminopurine (BAP) and 2.0 mg dm^-3 of 1-naphthaleneacetic acid (NAA). Cell suspensions were initiated and established in MS liquid medium containing 0.5 mg dm^-3 BAP, 1.0 mg dm^-3 NAA and 2.0 mg dm^-3 2,4-dichlorophenoxyacetic acid (2,4-D). A reduction in the concentration of 2,4-D to 0.5 mg dm^-3 induced formation of somatic embryos. The embryos developed and grew into normal plants in the presence of half strength MS medium without growth regulators. The regenerated plants were hardened in the greenhouse and subsequently grown in the open. This system may be also used for isolation and culture of protoplasts as a first step in somatic hybridization.

Changes in the phenylalanine ammonia-lyase (PAL) activity, accumulation of phenolic acids and ionically-bound peroxidase activity in thein vitro selected embryogenic and nonembryogenicMedicago sativa callus cultures resistant to the filtrate ofFusarium spp. were found. The PAL activity in bothin vitro selected cultures during a 4-week cultivation on a medium with phytotoxins was higher than in the control calli grown on a medium without toxin. The filtrate fromFusarium spp. evoked an increase in the contents of all determined phenolic acids in the selected calli. They occurred predominantly bound as esters. The most pronounced portions in the elevated acids level were of ester-bound p-hydroxybenzoic, vanillic, p-coumaric and ferulic acids. The ionic cell wall-bound peroxidase activity in both selected calli cultivated on a medium with a filtrate was twice as high as the activity determined in the control cultures. The activity of soluble peroxidase was not influenced by challenge with a filtrate. No significant differences were found between thein vitro selected embryogenic and nonembryogenic alfalfa callus cultures in the response to the phytotoxic filtrate.

Comparative study on SDS-protein profiles and isoenzyme composition of non-embryogenic and embryogenic calli in two callus lines of silver fir (Abies alba Mill.) revealed the presence of abundant polypeptide fractions and an increased number of isoperoxidases in non-embryogenic calli. Non-specific esterase, on the other hand, exhibited an opposite tendency, while glutamate dehydrogenase was the only enzyme system consisting uniformly of one isoenzyme band in both types of calli investigated.

Alfalfa (Medicago sativa L.) cell suspension cultures of Verticillium albo-atrum resistant and susceptible genotypes were established from leaf callus tissues. Treatment of cultures with conidia and heat-released elicitors of V. albo-atrum induced a large increase in the activity of phenylalanine ammonia-lyase, only in the cells of the resistant genotypes with a maximum after 12 h. In co-cultivation with the fungal conidia and resistant cell lines, the production of spores were inhibited.

Possibilities of adventitious buds induction on the cotyledons obtained from sterile seedlings ofAbies concolor xAbies grandis hybrid were investigated. The following variables influencing bud induction and their further development were studied: the effect of expiant age, the effect of different growth regulators and their concentrations and duration of their application. The most suitable expiants proved to be the cotyledons of 7 d old seedlings. The most efficient cytokinin was benzylaminopurine (S mg l^-1) in combination with napthaleneacetic acid (0.01 mg l^-1). The most optimal duration of treatment was 17 to 21 d culture of explants on induction medium. Shoot growth was achieved on basal medium to which 14 mg 1^-1 spermidine was added.

The effect of low irradiance on three rice cultivars (shade tolerant cvs. Swarnaprabha and CO 43 and shade susceptible cv. IR 20) was studied. The large subunit (LSU) of ribulose-1,5-bisphosphate carboxylase/oxygenase with molecular mass of 55 kDa was reduced in cv. IR 20 grown under low irradiance (LI). Native protein profile studied showed, under LI, reduction in the contents of proteins with RF values 0.03, 0.11 and 0.37. Analysis of chloroplast polypeptides revealed an induction of light-harvesting chlorphyll-protein 2 (LHCP2) under shade. The induction was more expressed in cv. CO 43 than in cv. IR 20. Under LI, in vivo labelled protein bands in the molecular range of 26 - 27 kDa were induced. These proteins were highly turned over in the LI-grown plants of cv. CO 43 than in cv. IR 20. A signal for rbcL gene sequences in EcoRI digested lanes was also found. Isozyme analysis of peroxidase showed an induction of a new band with RF 0.43 in cv. IR 20 subjected to LI.

A reliable protocol for the regeneration of onion through repetitive somatic embryogenesis was established. Embryogenic callus was derived from mature seeds on Murashige and Skoog (MS) medium supplemented with 2 mg dm-3 2,4-dichlorophenoxyacetic acid (2,4-D). Somatic embryos aroused on the surface of calli cultures and formed plantlets after the removal of 2,4-D or its substitution with 1 mg dm-3 kinetin (Kin). Reculturing the somatic embryos on 2,4-D containing medium led to secondary embryos formation. The embryogenic cultures which were preserved for five months on maintenance medium containing 2 mg dm-3 2,4-D + 0.5 mg dm-3 Kin have retained their ability for regeneration, while those kept on 2,4-D only, failed to form plantlets. Electrophoretic analysis of total soluble proteins revealed that the competence for successful conversion of somatic embryos into plantlets is associated with the expression of new set of proteins (112, 58 and 30 kD). The regenerated plants were successfully transferred to the soil.

Changes in growth characteristics and photochemical activities inVigna unguiculata L. Walp seedlings maintained at constant temperature of 10, 20, 30 and 40 ‡C under control and ultraviolet-B enhanced radiation (UV-B) were investigated. UV-B retarded the shoot elongation and also leaf expansion to a great extent at 30 ‡C but produced only marginal changes at 20 and 40 ‡C. Similar response was also observed with respect to changes in leaf fresh and dry masses and total chlorophyll (Chl) content under these temperatures. At 10 ‡C the total Chl content was 3-fold higher under the treatment than under control conditions. In seedlings growing at 20 and 30 ‡C the overall photosynthetic electron transport (H₂O -> methyl viologen) showed a significant enhancement during the 36-h UV-B treatment and thereafter a gradual reduction. Although a similar trend was found in photosystem 1 (PS1), the inhibition even after 60 h of UV-B treatment was not statistically significant. Photosystem 2 (PS2) activity was inhibited in seedlings treated for 60 h by UV-B at 20 and 30 ‡C. However, no inhibition was observed at 40 ‡C. No detectable photochemical activity was found in seedlings grown at 10 ‡C under either control or UV-B enhanced irradiation although the chloroplasts contained Chl.

Accumulation of the extracellular proteins localized in intercellular spaces of barley primary leaves was examined after inoculation with powdery mildew (Erysiphe graminis f. sp. hordei) as biotic stress factor and after abiotic stresses such as heat shock, low temperature and heavy metal (Mg,Zn, Cu, Al, Cd and Co) treatment using native polyacrylamide gel electrophoresis. Six to eight major pathogen-induced proteins (bands on native gel) have been identified. Their accumulation at host-parasite incompatibility was more expressive than at compatibility interaction. Elevated temperature did not induce pathogenesis-related (PR) proteins while low temperature induced three of them. Cu, Al, Cd and Co induced accumulation pattern of extracellular proteins was very similar to that in powdery mildew inoculated leaves. Mg and Zn had no effect on the induction of protein accumulation in the intercellular spaces of leaves. Induction of PR proteins by different stresses indicated their general function in the resistance of plants to changing environment.

We have examined the toxicity of ethanol in tissue culture of the apple rootstock 'Jork 9'. During proliferation through axillary branching, 0.2% (v/v) ethanol slightly stimulated proliferation whereas significant inhibition occurred at concentrations of 0.4 % or higher. In adventitious root formation, significant inhibition occurred at concentrations of 0.1 % or higher. The effect of ethanol was stage-dependent: during the induction period (i.e. from 24 to 72 h after the start of the rooting treatment), there was little or no inhibition. During autoclaving, ethanol evaporated to ca. 50 %.

Calli were induced in cacao cotyledon explants on a half-strength Murashige and Skoog medium containing 6 × 10-2 g m-3 saccharose and various combinations of 2,4-dichlorophenoxyacetic acid (2,4-D) with kinetin (kin), benzylaminopurine (BAP) or 2-isopentenylphosphate (2-iP). Experiments were carried out on two clones of cacao differing in their susceptibility to black pod disease. The highest percentage of explants forming callus and the most rapid callus development were obtained with 10-6 g m-3 2,4-D and 0.5× 10-6 g m-3 kin. Somatic embryogenesis and rhizogenesis were induced by transferring 3-week-old callus in a half strength Murashige and Skoog medium containing 3 × 10-2 g m-3 saccharose and NAA or IBA in the 0 to 5 × 10-6 g m-3 concentration range. No differentiation could be observed when the medium was supplemented with kin or BAP. The conversion of callus into somatic embryos and roots was accompanied by a drop in phenol content and an increase in peroxidase and IAA-oxidase activities. Moreover, cell differentiation was characterized by the persistence in the callus of one acidic soluble isoperoxidase which was not detected in nondifferentiating callus. Although some differences were noticed between the clones, alterations responsible for cell differentiation were the same in both genotypes.

The morphogenic response of somatic (leaf and petiole) and de-differentiated tissue (callus) of two blackberry (Rubus fruticosus) and one raspberry (Rubus idaeus) cultivars have been studied in vitro. With the aim to induce regeneration the effect of two sets of plant growth regulator (PGR) combinations (high cytokinin/auxin ratios and high auxin/cytokinin ratios) in Murashige and Skoog basal medium, were analysed. The three cultivars were characterised by a qualitatively different morphogenic response to the PGR combinations. Raspberry adventitious shoot regeneration from somatic tissue was improved by the 6-benzylaminopurine (BAP)/indol-3-butyric acid (IBA) combinations. On the contrary, shoot regeneration of both blackberry cultivars was reduced by high concentrations of BAP and completely inhibited by BAP/IBA combination. Media supplemented with high auxin/cytokinin ratios promoted callus production and root differentiation according to genotype and type of auxin. All the genotypes responded to media supplemented with IBA. 2,4-dichlorophenoxyacetic acid induced good callus formation in blackberry, but was toxic to raspberry. Indirect shoot formation was observed only in callus of blackberry cultivar Hull Thornless cultivated on medium with 10 µM BAP, the same concentration able to trigger efficient direct shoot regeneration from leaf explants of the same cultivar.

Xylogenesis was induced in cultures of calli ofHaplopappus gracilis (NUTT.) A. Gray by metabolic shocks brought about by changes in nutrition. Xylogenesis was observed to occur in response to three changes in callus nutrition, caused by the following modifications of Eriksson's nutrient medium and culture conditions: i) omission of sucrose in the medium for 3 to 6 days and then transfer of the calli onto complete Eriksson's medium; ii) replacement of sucrose by 0.36*mol l_-1 xylose for the whole culture period; iii) omission of nitrogen sources(i.e. glycine and NH₄NO₃ omission and replacement of KNO₃ with equimolar KCI) for the whole culture period.
The induction of xylogenesis in response to nutritional stress inH. gracilis can be used as a suitable model system for research concerning plant cell differentiation.

DNA methylation of two repetitive sequences in tobacco nuclear genome was studied in the course ofin vitro dedifferentiation and differentiation. Using 5-mC sensitivè restriction enzymes and DNA/DNA hybridization with 25S-rDNA probe it has been shown that during the early phase of callus induction prominent changes in the methylation pattern occur which are stably maintained during subsequent callus growth. The following protoplast recovery and plant regeneration have again displayed some more modifications of the methylation status. Comparing the patterns of R₀ plants with the original plant material and the calli it can be assumed that both share in the resulting methylation status. The experiments analyzing the HRS60 family of non-transcribed highly repetitive sequences have displayed a quite monotonous methylation status thus indicating no random methylation perturbations in silent DNA sequences.