Phenolic acids were separated into three fractions and determined by HPLC inMedicago sativa callus culture at the age of two, three and four weeks. The contents of free and especially of predominating ester-bound phenolic acids decreased with callus age to approx. 80 % while the content of phenolic acids nonextractable by methanol increased byca. 90 %. The proportion of benzoic acid derivatives rose from 15 to 21 % within four weeks. The determined difference in the contents of phenolic acids in the upper and lower parts of callus diminished with age. The content of bound forms was higher in the lower part regardless of the callus age. The content of free acids in two weeks old callus was half as high as in the upper part.

Growth ofCichorium intybus root explants was accompanied by an important increase of fresh mass during the course of callusing and rooting. The absence of glucose in callus forming medium was compensated for by hydrolysis of storage carbohydrates of the tissues, inducing a decrease in dry mass. Protein content showed similar slight variations in explants during the course of budding and callusing, whereas an important increase of protein content was found during the first 48 h in explants cultured on root forming medium.
Specific increase of soluble peroxidase activity during bud and root neoformation was found. A peak of peroxidase activity, proceeding the organ emergence, was always observed. This peak occurred earlier in bud forming than in root forming explants. Conversely, during callusing, the bulk peroxidase activity showed only weak variations, and no peak was detectable. Moreover one basic isoperoxidase was missing in these explants after a 6-d culture, whereas in both root and bud forming explants the isoperoxidase patterns were very similar. When explants growing on differentiation media were in darkness a supplementary basic isoperoxidase with the highest electrophoretic mobility was revealed.
The data obtained show that isoperoxidase patterns cannot be correlated to a particular organ differentiation process, but that peroxidase activity and especially the moment of peak emergence can be a reliable marker of the differentiation process. Moreover, light seems to inhibit the expression of a basic peroxidase in explants growing in media promoting organ differentiation.

Thidiazuron incorporated into MS medium stimulated rosettes formation only in some treatments. This effect was more pronounced in cultures ofMorus alba thanPrunus sp. Mulberry cultures responded to the optimal concentration of thidiazuron (0.2 mg I^-1) not only with shoot formation but also, with growth of large leaves and poor development of callus tissue. In cultures of both investigated genera the shoot elongation was inhibited. Shoots of mulberry cultures growing on proliferation medium supplemented with thidiazuron formed roots, in many cases.

Floral induction in the long day plant spinach (Spinacia oleracea) has been shown to be accompanied by a thickening of plasmalemma. This change was observed at early evocation, in both shoot apices and leaves, as well as after inducing GA₃ treatment. To get further information on this thickening, plasma membranes from spinach leaves were isolated, in the present study, using aqueous two phase partitioning and the effect of variousin vitro treatments on their thickness was investigated. The average plasmalemma thickness was unaffected by Na⁺ and K⁺ ions. It was increased upon the effect of either Ca²⁺ or gibberellic acid. A thickening of plasmalemma was also observed when plasma membranes from vegetative plants were incubated with a cytosolic preparation from photoinduced plants. The results were discussed in relation with the plasmalemma modifications previously reported in spinach.

The structure of the cells was studied in two strains of grapevine calli (Vitis riparia ×V. labrusca). These callus cultures contained only highly vacuolised cells. The pattern of division in these slightly cytosolic cells was described. In both callus strains caryological instability has been found.

SC₂ and SC₃ progenies of nineteenin vitro regenerated barley plants (SC₁) from resistant calli selected against purified culture filtrate ofHelminthosporium sativum and one parent 'Dissa' genotype were studied for stability of resistance and protein, soluble protein, maltose and saccharose contents. Cytological studies were also carried out on the SC₃ generation. Stability of resistance toHelminthosporium sativum was found in 50% of the somaclonal lines. Significant variation among different somaclonal lines and among different callus lines from which the plants were regenerated were found for yield, disease score and biochemical characters assessed except saccharose content in the somaclonal lines. Significant increase and decrease over the donor parent for most of the characters were obtained. Cytological abnormalities such as multilobed nuclei, multinucleate cells, abnormal anaphase and mixoploidy were also observed.

Selected factors affecting somatic embryogenesis efficiency have been studied, namely genotype, explant type and its orientation in the medium, different basal media, different auxins for somatic embryo induction, and two ways of donor plant cultivation. The key role is played by genotype and auxin used, the minimum effect was observed due to basal media. In the series of subsequent experiments we have found the best combination of individual factors as follows: cv. Altona, 10 uM 2,4-D, L2 basal medium, central part of immature cotyledon as initial expiant oriented by adaxial side down on the agar medium, and field grown donor plants. This combination exhibited 100 % embryogenic explants with 5.43 ± 0.65 somatic embryos per expiant,i.e. somatic embryogenesis efficiency 5.43.

Methylmethanesulfonate induced chromosomal aberrations in callus culture ofCrepis capillaris. The clastogenic effect was markedly decreased when calli were pretreated with dimethylsulfoxide.

Long-term callus cultures of crownvetch (Coronilla varia L.) grown on the Murashige and Skoog's medium with 2,4-D (1 mg 1^-1) and cultures of somatic embryos cultivated on the same basic medium but with IAA (1.0 mg I^-1) were exposed to ionizing irradiation. The irradiation caused a growth inhibition excepting the lowest dose of 2.5 Gy. The highest dose of 160 Gy induced browning of the culture but this colour change was not lethal. The amount of "giant cells" present in both cultures was dependent on the dose of irradiation.

The season of collection of shoot tips from field-grown plants ofChrysanthemum morifolium cv. Birbal Salmi was found to be crucial for their proliferation and establishment of plants in vitro. Shoot tips collected only during the period of March to April proliferated and survived. Shoot apices and segments of leaf, stem (nodal und internodal) and root, excised from aseptically established plants, were cultured on Murashige and Skoog's medium supplemented with different concentrations and combinations of Kn, BAP, IAA and NAA. A maximum number of 9 off-shoots differentiated, without intervening callus formation, from a shoot tip in a treatment containing 1.5 mg l^-1 BAP and 0.1 mg^-1 NAA in 60 days. A single-node stem segment produced less shoots in the same treatment. Other kinds of expiants produced only callus. About 2-cm-long shoots, excised from cultures of proliferating shoots, were rooted 100 %, acclimatized and grown in soil. They grew normally and flowered true-to-type.

The main scientific results achieved in individual departments of the Institute of Experimental Botany during 30 years of its existence are briefly summarized. They include methods of studying photosynthesis, ontogenetic changes of photosynthetic characteristics, stress factors affecting photosynthetic activities, photosynthesis of transgenic plants and duringin vitro cultivation, roles of auxins and cytokinins in plant growth and development, development and testing of new plant growth regulators, models of organogenesisin vitro, metabolic and mutagenic activities of phenolic substances, hormonal regulation of flowering, activities of promutagens (nitrosamines, 7,12-dimethylbenzanthracene), model systems of genetic damage, repair synthesis and post-replication repair, developmental pollen biology and biotechnology, extracellular nucleolytic activity of pollen, selection of apple scab immune cultivars of apple tree, chemotaxonomy ofFabaceae andAllium species, selection pressures in embryoids, somatic embryogenesis and nuclear genome changes in plant cell and callus cultures, discoveries of new plant viruses, virus spread and persistence in crops, development of polyclonal and monoclonal antibodies, role of oxidative pentosephosphate cycle in biosynthesis of viral RNA, and virus diseases of forest trees.

Embryogenic callus was initiated from immature zygotic embryos of black pine on medium DCR supplemented with 2 mg 1^-1 2,4-D and 0.5 mg 1^-1 BAP. The diploid number of chromosomes confirmed the origin of callus from zygotic embryos. The callus was white, glossy, mucilaginous and contained somatic embryos consisted of an embryonic region with densely cytoplasmic cells and suspensor region with long vacuolated cells. Although somatic embryos with green cotyledons were recognisable after ABA treatment and subsequent transfer to growth-regulator free media whole plants have not yet been obtained.

Callus tissues originating fromZea mays root meristem, induced for rhizogenesis callus, meristematic and differentiated maize root cells for isolation of nuclei and acid-soluble chromosomal proteins were used. Cytological investigations proved that rhizogenesis begins with the formation of meristematic centres, followed by root differentiation about 5-12 days after the treatment with α-naphtalene acetic acid (NAA). When applying electrophoresis in 15% polyacrylamide gel, differences between the electrophoretic profiles of acid-soluble chromosomal proteins, isolated from root cells and from callus tissues, were established. The main differences concern histone H1 and probably H4. There are no differences between electrophoretic patterns of acid-soluble chromosomal proteins of nonorganized callus and callus induced for rhizogenesis. The possible explanation of these results is discussed.

In concentration range of 10^-15 to 10^-5 3-allyl-6-nitro-2-benzothiazolinone (ANB) did not affect the algaChlorella vulgaris L. and intact dicotyledonous plantVicia saliva L. However, it stimulated growth and chlorophyll production inZea mays L., showing different effects on individual plant organs, and in the callus obtained from the root ofDaucus carota L. At high concentration (10^-4 M), ANB inhibited all the characteristics studied.

Continuous treatment with spermidine or 1-aminocyclopropane-1-carboxylic acid stimulated ethylene production and ethylene-forming enzyme activity and accelerated chlorophyll breakdown in detached tobacco leaves. The treatments also induced the production of eleven major acidic pathogenesis-related proteins, which were also produced during the hypersensitive reaction to tobacco necrosis virus. A delay between the onset of the stimulated ethylene increase and the detection of PR-proteins was found; ethylene production was stimulated after a few hours of treatment, whereas one, three and all the eleven PR-proteins were detected by polyacrylamide gel electrophoresis of fluid extracts after 2, 4 and 6 days of treatments, respectively. The possible causal relationship between stimulation of ethylene production and PR-protein accumulation is discussed.

Gibberellins A₃ and A₁₃ cause floral induction inImpatiens balsamina, a qualitative short day plant, under non-inductive 24-h photoperiods (continuous illumination). However, the influence of the two inductive factors,i.e. gibberellins and short days (8-h photoperiods) on the peroxidase enzyme system is different. The total peroxidase activity decreases under both inductive and non-inductive photoperiods, with or without gibberellin treatment. The electrophoretic pattern of isoperoxidases changes only in response to gibberellin treatment. Under 24-h photoperiods, treatment with gibberellins A₃ and A₁₃ causes the appearance in the stem of three additional isoenzymes of peroxidase (Rm 0.50, 0.71 and 0.76). These bands do not appear in the leaves, which are non-essential for gibberellin-caused floral induction in this plant. Under 8-h photoperiods also, gibberellins induce the appearance of new isoenzyme bandsi.e. two in the stem (Rm 0.50 and 0.76) and one in the leaves (Rm 0.05). These may be correlated with the synergistic increase in the number of floral buds in these plants in response to simultaneous exposure to two inductive factors.

As the dynamics of changes in phytohormones may be involved in photoperiodic regulation of the rates of growth and flowering, fluctuation of cytokinins was followed in long-day and short-day tobacco. Zeatin (Z) and zeatin riboside (ZR) were identified in leaves and roots using a GC-MSC system. In plants of the long-day tobaccoNicotiana silvestris increasing the number of long-day inductive for flowering (10, 20, 30, 40 LD) resulted in a rise in ZR activity. Half the plants reached a reproductive stage on the 40th day of induction. In short-day Mam moth tobacco plants, short-day floral induction (10, 20, 30, 40 SD) caused similar but less marked changes in ZR.

This paper describes the differentiation of karyotypically stable callus strains ofCrepis capillaris (with modal chromosome numbers 7, 8 -11 and ≧ 12, in particular) during 12 years' subculturing on an identical M-S medium with supplements, including 2,4-D (1 mg 1^-1) and adenine (1 mg 1^-1). The possible modes and factors of karyotypic adaptationin vitro are discussed.

The hypocotyls, cotyledons, leaf blades, whole leaves and petioles of seedlings ofAilanthus altissima are capable of producing callus and budsin vitro. Buds and callus were also obtained from whole leaves and internodes of 2-years old plantlets grownin vitro. From the calli buds differentiated and later, both from buds developing directly without a callus phase and alsovia a callus phase, well developed shoots were formed. The cultures were mainained on MS medium in 2 combinations: A) IAA - 0.2 mg 1^-1, BAP - 1 mg 1^-1, GA₃ - 0.5 mg 1^-1, thiamine - 4 mg 1^-1 and sucrose 3 %; B) BAP - 0.5 mg 1^-1, IAA - 1 mg 1^-1, casein hydrolysate 400 mg 1^-1, thiamine 4 mg 1^-1 and sucrose 3 %. Excised shoots, which had developedde novo in culture, produced roots when incubated on the basic mineral medium of MS with the addition of IAA. The regenerative potential ofA. altissima is very high and this woody species seems to be an ideal object for various morphogenetic studies.

Correlations within a shoot ofChenopodium rubrum L. ecotype 374 grown under continuous light or photoperiodic flower induction were studied using surgical treatments. Removal of a single pair of shoot organs had a variety of effects depending on position: significant changes in the number of leaf pair on the main axis or in axillary buds and in the height of shoot apices; or no effect on the parameters scored. Flowering was not affected by any of the treatments carried out. Decapitation brought about a significant increase in the number of leaf pairs in axillary buds and flowering was inhibited in 8- and 9-d old plants. Flowering was not affected in 21-d old plants. The role of shoot organ correlations, especially that of apical dominance, in regulation of flowering inC.rubrum is discussed.

Amphistomatous C₃ (Nicotiana tabacum L., Datura stramonium L.) and C₄ (Sorghum saccharatum Pers. and Zea mays L.) species were examined to find how (if at all) their inherent differences in water-use economy are reflected in apparent cuticular transpiration or vice versa. Transpiration efficiency (TE) was calculated from steady state photosynthesis (A) and transpiration (E) rates estimated for the upper side of the leaf after light induction of stomata opening. Apparent cuticular transpiration ('E_c) was measured as the part of transpiration which was not eliminated by convective counteraction of the air stream passing across the amphistomatous leaf: total pressure difference (AP) across the leaf was increased and the minimal value of E_ΔPτ0 was taken as the apparent cuticular transpiration rate ('E_c). 'E_c was treated relative to E at AP equal to zero (E_GDP=0), E'_cr. Measurements were carried out under two leaf-air vapour pressure differences (VPD).
E_r (i.e. E_GDPτ0/E_GDP=0) versus GDP patterns differed qualitatively between the investigated C₃ and C₄ plants. TE increased and 'E_cr decreased from tobacco, stramony, maize to sorghum for both VPD of air. 'E_cr and TE were approximately linearly related, the slope being dependent on VPD. The increase in VPD resulted in larger E and slightly smaller epidermal conductance (g) at GDP equal to zero. Both E'_cr and E'_cr decreased markedly at the same time especially, for species with high TE. The results were considered as an indirect confirmation that E'_c values estimated by the technique used reflect species-specific differences in external peristomatal and cuticular vapour loss, at least in a relative sense.

The copy number of genes encoding 5S ribosomal RNA has been found to be constant in Petunia hybrida plants regenerated from protoplast and leaf disc-derived callus cultures. However, in one somaclone a heritable change in the length of the major 5S rDNA repeat has arisen. Despite the constant copy number of 5S rRNA genes, that of the 18S-25S rRNA genes is found to very by at least ten-fold. The relevance of these findings to ribosomal RNA gene variability and to somaclonal variation is discussed.

In the present paper we deal with the possibility of morphogenesis induction in callus tissue cultures of some representatives ofMatricaria andAchillea species. Shoot regeneration from calli ofMatricaria chamomilla andM. inodora has been induced by 0.1 mg l^-1 kinetin or by combination of 0.5 mg l^-1 kinetin and 0.5 mg l^-1 NAA added to Murashige-Skoog culture medium. Rhizogenesis took place without any other addition of auxin. In callus tissue cultures ofAchillea ptarmica cultivated on Murashige-Skoog medium with 1 mg l^-1 2,4-D after a year long cultivation the whole plant has been regenerated without any change of nutrient requirements. In callus tissue ofA. nobilis under the same conditions only roots wore regenerated.

Electrophysiological processes were investigated in reception organs of photoperiodism in a model short-day plant,Chenopodium rubrum L. (selection 374), within the inductive cycle for flowering. Transorgan (surface) electric potential (E_tr) was measured as a potential difference between the first leaf surface and the roots of an intact plant, and between the surface of an excised leaf and the petiole base. The time-course of E_tr in intact plants showed irregular, or partially regular, oscillations within both phases of the inductive cycle and under continuous light. The highest amplitudes were during the postinductive light period. E_tr in excised leaves behaved practically in the same way as in intact plants. The E_tr oscillations were localized in leaves. In general, no electrophysiological changes were found in the reception organs within the inductive cycle which could be correlated with the formation and transport of floral stimulus, or with the attainment of an induced state. The results indirectly support the idea that the floral stimulus is chemical in nature.

Electrophysiological processes were investigated in the reception organ of photoperiodism, cotyledons and first leaves, in a model short-day plantChenopodium rubrum L. (selection 374) within the dark inductive cycle for flowering. Membrane potential (E_m) was measured in cotyledon and first leaf mesophyll of intact plants. The E_m time-course was fairly similar during inductive dark or postinductive light period or in non-inductive continuous light and had a character of irregular oscillations. The most distinct oscillations were found during the postinductive light period. Changes in light régime at the beginning (light off) and the end of inductive dark period (light on) triggered marked transient E_m changes having a character of damped oscillations. Cortical root cells in intact plants did not react to switching light and darkness. Changes in E_m in reception organs during the inductive cycle could not be correlated with the formation and transport of floral stimulus or with reaching the induced state. Thus, the electrophysiological nature of floral stimulus has not been confirmed.

Chromosomal variability in callus culture ofAsparagus racemosus was comparatively higher in the presence of 2,4-D than of NAA. The frequency of polyploid cells was enhanced with the increase in the concentration of 2,4-D or with the addition of coconut water. Gradual polyploidization with increasing age of the callus has been recorded.

Embryogenic callus which has maintained its embryogenic ability on media without growth regulators for three years has been induced at the base of shoots of a genotype with CMS propagated for a long timein vitro by transferring the shoots onto media richer in inorganic and organic components. The effect of two basal media (MS and PG₀) on the intensity and completeness of the proliferation of somatic embryos was examined with different combinations of growth regulators. Pollen fertility was evaluated in 87 plants regenerated from somatic embryos. Cytoplasmic male sterility was conserved in all of them.

Flower initiation takes place during a rise of peroxidase activity following a peak of minimum activity which marked the completion of the flowering inductive phase. Since basic isoperoxidases underwent an inverse variation of activity in the course of successive inductive and initiative phases, it was hypothesized that the induction of flowering led to a temporary peak of maximum auxin level in the leaves. Our analyses and available literature data support the view. They also show the different capacity of non-induced and induced material to respond to external auxin application. Since some aspects of the physiological state characterizing induced plants can be simultaneously obtained in all plant parts as a result of rapid interorgan communication, the classical florigen theory is seriously challenged.

Callus cultures were initiated from the bud apices of 10-40-year-old Scots pines (Pinus sylvestris L.) at different seasons and maintained on modified MS medium without subculturing. Separate sets of experiments were used for analyses of carbohydrate content, ethylene production, amino acid composition, protein patterns andin vitro translation. In each case the change in the colour of the calli was recorded and the fresh mass of the samples measured. The onset of tissue browning was found to be associated with changes in protein pattern, amino acid content, ethylene production and the occurrence of sucrose and accumulation of starch.In vitro translation experiments using poIy(A)⁺ RNA isolated and purified from the calli indicated that the switch in metabolism accompanying browning is paralleled by activated protein synthesis. Thus, the development of brown colour does not as such seem to be harmful to the tissue. The later, more intense tissue browning and deterioration which is reflected in a reduced capacity for protein synthesis and changes in the free amino acid pool and protein pattern is probably a secondary phenomenon.

NaCl (0 to 274 mM) was added to the culture media of normal and habituated (auxin and cytokinin independent) sugarbeet calli and its effect on growth (estimated by the increase of dry and organic matters), water content and osmotic potential was tested. Growth of normal callus was stimulated by 68 mM NaCl after a lag period of two weeks. This callus tolerated up to 137 mM NaCl without growth reduction and maintained its hydric status by readjustment of its osmotic potential in 24 h. NaCl quantities under 34 mM stimulated growth of the habituated callus from the 3rd day on; higher NaCl concentrations (68 to 274 mM) inhibited growth or were lethal. NaCl sensitivity of this habituated callus was not due to its inability to adjust its osmotic potential: this adjustment occurred from the 4th h of culture whatever the media. From the 3rd day on, however, this callus presented a water deficit which depended on NaCl concentration. It is suggested that the lowering of osmotic potential corresponds to an important water loss in relation to changes in membrane permeability. This study finally shows that mechanisms of salt tolerance may have developed at the cellular level. Lower growth and lower salt tolerance of the habituated callus need further investigation in relation to cell structure and hormone autonomy.