The possibility that some of the variation in callus cultures involves epigenetic changes is examined in cultures established from the hypoootyls and roots ofEuphorbia heterophylla. It is shown that the responses of the cultures are affected by the light regimes under which they are grown and that in the dark and under short photoperiods, there are differences between the two types of culture with respect to pigmentation, auxin requirement, capacity to regenerate buds and roots and in certain isozyme patterns, whereas the two cultures are similar from the first passage under continuous light. However, these differences are only maintained for 2-3 passages, after which the root callus becomes similar to the hypoc otyl callus. Evidence is presented that these differences between cultures are epigenetic. Callus cultures established from the apical meristems of shoots and roots ofE. hetero phylla show similar differences to those observed between hypocotyl and root cultures and these differences are also lost after 3 passages. These results indicate that the cells of apical meristems are not totally uncommitted, but are determined as 'shoot' and 'root' meristem colls, respectively. The practical importance of a better understanding of epigenetic effects in plant cells is strassed.

It is generally accepted that plant growth and development are regulated by the known plant hormones. Some objections to the functions of auxins and cytokinins in the induction of shoot and root primordia are reported. Instead of them oligopeptides of special amino acid sequences could be the endogenous signals. There exist structure relationships between auxins and parts of the α-helical oligopeptides of defined amino acid sequences. The same is true for cytokinins.
The most difficult part of this hypothesis is its verification. Using protonemata ofFunaria hygrometrica bud induction by various oligopeptides was investigated. The most active peptide tested is leucine-tryptophan. On the other hand endogenous oligopeptides containing [¹⁴C]-leucine in the moss protonemata during endogenous bud initiation were looked for. Three to four different oligopeptide spots seem to be related to bud induction.

The effect of GA₃, IAA and kinetin in concentration range from 5.10^-4 to 5. 10^-8M, single or in combination, was tested on the flowering of short-day plant (SDP)Chenopodium rubrum (selection 374). All substances were applied as a droplet (3 (μl) of water solution before the onset of inductive photoperiod which brought about a threshold level of induction. Flowering was enhanced only in the presence of GA₃ and the other two PGR decreased its effective concentration by one or two orders of magnitude. It is likely that the morphogenesis of the apical meristem was directly affected by such treatment. There is no need to assume that the ratio of employed plant growth regulators is important for the observed morphogenetic effects, but rather the actual concentrations are involved.

Agrobacterium tumefaciens C58M (a nopaline strain) was used for induction of crown gall tumors inCentaurium umbellatum Gilib. The formation of tumors is very rare. After elimination of bacteria the tumor tissues grow in the light on R3B medium without growth regulators. They are positive in nopaline synthase.

Transversally cut leaf segments ofCentaurium erythraea were cultivated on MS medium. Effects of segment polarity, IAA and sucrose concentrations, light and medium volume on morphogenesis were studied. Shoots generally formed at lower (1.3 × 10^-6 mol 1^-1) IAA concentrations than roots and callus (1.1 × 10^-5-3.4 × 10^-5 mol 1^-1). Leaf polarity strongly modified the effect of IAA concentration, shifting organogenesis at the segment base toward decreased IAA concentrations as compared with segment apex. Light, sucrose concentration above 3 % and high medium volume changed IAA dependence of morphogenesis in various ways and generally suppressed segment polarity.

Effects of 2,3,5-triiodobenzoic acid (TIBA) and age of etiolated pea epicotyl segments on the indol-3-ylacetic acid (IAA) stimulated transport of¹⁴C-abscisic acid (ABA) was studied. In spite of a slight decrease of IAA transport after the application of TIBA, the IAA stimulation of¹⁴C-ABA transport did not change. In segments excised from epicotyls of different age,³H-IAA transport was identical and the induction of prolongation growth by IAA in segments from the upper part of the epicotyl was observed. The IAA ap{ie226-01}ation to the growing segments was connected with intensive attraction of¹⁴C-ABA to the site {ie226-02}AA application, while the application of IAA to the older segments was growth ineffective ana no stimulation of¹⁴C-ABA transport by IAA was observed.

The current status of our knowledge of auxin effects on floral induction is summarized. The most general effect is inhibition, although the concentration of synthetic auxins added to. plants tends to be too high for us to be certain that the inhibitory effects are truly physiological. Studies of endogenous levels of auxin have focused almost entirely on IAA-like bioassay activity. Chemical identifications of endogenous IAA are needed and feasible. In addition, a search for-other auxins involved in vegetative to floral transitions, their chemical identification, and measurement of their changing levels in the plant are urgently needed.

Callus culture was derived from haploid barley embryos after crossing withHordeum bulbosum. The callus tissue is cytologically heterogeneous, containing haploid, diploid and polyploid cells. Aneuploidy and karyokinetic irregularities were also observed. Some problems of chromosomal instabilities in plant tissue cultures are discussed.

The fluctuation of free IAA under 16 h dark period in shoots (receptor organs of photoperiodic induction) and roots of the short-day plant (SDP)Chenopodium rubrum and in shoots of the long-day plant (LDP)Chenopodium murale is very similar. The data reflect the general adjustment of auxin level to day-length rather than changes due to floral induction. However, the shift in phasing of the circadian rhythm of flowering was accompanied by a change in the position of the' troughs' of free IAA levels indicating a possible relationship between the two processes. Periods of higher sensitivity to application of IAA (3. 10^-4M) inhibitory to flowering have been observed both during the endogenous rhythm of flowering in the SDPC. rubrum and during induction by days of continuous illumination in the LDPC. murale. There exist common traits in the response of LDP and of SDPChenopodium to auxin treatment. Aminoethoxyvinylglycine (AVG), an inhibitor of ethylene biosynthesis, counteracted some flowering inhibitory effects of IAA when applied simultaneously with it. This suggests that auxin effects in modifying flowering might in fact be due to ethylene.

Cell division contributing to longitudinal growth of the shoot apex was investigated inChenopodium rubrum in segments marked by the axils of leaf primordia. Plants treated with two short days (16h of darkness and 8h of light) were compared with two non-induced controls (cultivated in continuous light or treated by alternations of 8 h of darkness and 4 h of light for two days). During the short-day treatments the rate of cell division contributing to the longitudinal growth decreases in all segments of the shoot apex irrespective of whether the darkness was given in inductive or non-inductive photoperiods. The rate of cell division contributing to longitudinal growth increases in the upper internodes of the shoot apex after the termination of the photoperiodic treatment and transfer of the plants to continuous light. However, cell division remains inhibited in the lowest segment of the shoot apex. This inhibition in the differentiating parts of the shoot apical meristem is a direct consequence of photoperiodic induction. It is supposed that this inhibition is related to evocation similarly as the well-known phenomenon of stimulation of cell division in the apical dome.

The anthers of three genotypes ofLycopersicon esculentum, viz. cv. HS-101, cv. HS-102 and an F₁ hybrid (Montfavet 63-4xHS-101) in different stages of development were cultured in various defined nutritive media. Only anthers containing microspores in the early uninucleate stage were found to respond with the culture medium in the formation of androgenic callus. The DGII medium with 2 mg l^-1 NAA and 1 mg 1^-1 kinetin was found to be best for callus induction but MS medium supplemented with 2 mg l^-1 2,4-D and 0.1 mg 1^-1 BAP favoured proliferation and growth of the callus. The androgenic microspores followed the 'B' type pathway of androgenesis in the formation of callus.
Induction of tracheids in the callus could be achieved by supplementing the basal medium with NAA and kinetin or 2,4-D and BAP. Initiation of vessel elements and cambium were favoured by addition of NAA and kinetin and that of the phloem in the presence of 2,4-D and BAP in the basal medium, suggesting that the hormonal requirements for production of different elements of the vascular system in androgenic callus are different. Although roots could be induced from the callus, shoot differentiation could not be achieved under cultural conditions.

The growth changes of cotyledons, leaves, hypocotyls and roots due to photoperiodic induction in short day plantChenopodium rubrum were investigated in relation to flowering. Six-day old plants were induced by photoperiods with a different number of dark hours. We found that the degree of inhibition which occurred during induction in the growth of leaves, cotyledons and roots similarly as the stimulation of hypocotyl is proportional to the length of dark period. The photoperiods with 12, 16 and 20 dark hours bring about marked inhibition of growth and at the same time induce flowering in terminal and axillary meristems. The inhibitory effect of critical period for flowering,i.e. 8 dark hours, is not apparent in all criteria used and even the flower differentiation is retarded. The photoperiods of 4 and 6 dark hours did not affect growth and were ineffective in inducing flowering even if their number has been increased. The experiments with inductive photoperiod interrupted by light break have clearly shown that growth pattern characteristic for induced plants can be evoked in purely vegetative ones. Such statement did not exclude the possible importance of growth inhibition as a modifying factor of flower differentiation. We demonstrated that the early events of flower bud differentiation are accompanied by stimulation of leaf growth. The evaluation of growth and development of axillary buds at different nodes of insertion enabled us to quantify the photoperiodic effect and to detect the effects due to differences in dark period length not exceeding 2 hours.

Callus tissue cultures were established from stems of tobacco plants (N. glaucaGrah.) both healthy and mycoplasma (potato witches' broom disease) infected on a modified nutrient medium (with a lower content of mineral salts) according toMurashige andSkoog (1962) in the presence of 2,4-D (1 mg l^-1) as a growth regulator. No differences were observed in the growth and development of both tissues. Organogenesis appeared on a nutrient medium (Petrůet al. 1972) supplemented with kinetin (0.64 mg or 2.56 mg l^-1) and IAA (2 or 4 mg l^-1). Callus derived from mycoplasma diseased plants started to form numerous buds after three months whereas organogenesis in callus from healthy controls appeared only after six months. We suppose that the reason of this difference is the fact that an expressively higher content of 2,4-D was found in the calli from healthy plants in comparison with the corresponding tissue from mycoplasma diseased ones. Reconstituted plants were isolated, rooted and transferred in the soil. The infectivity of these plants was assayed by grafting their stem tips on tomato plants which indicate very reliably and sensitively this mycoplasma disease. 31 reconstituted plants were obtained in the whole from calli isolated from mycoplasma infected plants and all of them were healthy.
It was established that mycoplasma failed in the presence of 2,4-Din vitro. Stem pieces from diseased plants in which mycoplasma presence was proved, lose their infectivity after 4 weeks of cultivation on nutrient medium with this growth regulator. On the contrary 2,4-D which spreads and acts especially through phloem (Smithet al. 1947) does not kill mycoplasmain vivo even in doses evoking strong symptoms of 2,4-D effect on experimental plants.

This paper deals with some morphological and histological changes observed during regular intervals inMatricaria inodora L. tissue cultures derived from leaf expiants. The expiants were cultivated on Murashige-Skoog's culture medium supplemented with 1.0 mg 1^-1 2,4-dichlorophenoxyacetic acid. The callus formation started about a week after isolation. During the second week the meristematic centers were differentiated from which root and shoot apices were later formed. During long term cultivation under the same culture conditions the inhibition of development of shoot apices took place. Only roots of unorganized growth have been regenerated. The influence of culture conditions on morphogenetic potential is discussed.

The effect of plant tissue culture medium with different concentrations and combinations of growth regulators (kinetin, indol-3-ylacetic acid, 2,4-dichlorophenoxyacetic acid) was evaluated on mitosis ofAllium sativum meristem root tip cells. Different combinations of growth regulators at low concentrations had no effect on induction of mitotic aberrations or inhibition of mitotic activity. Inhibition of mitotic activity, a tendency to chromosome stickiness and clumping and a slight increase in the frequency of mitotic aberrations were observed at higher concentrations. It may be proposed that plant tissue culture media have no direct effect on induction of mitotic aberrations in plant tissue culturesin vitro.

Light was required for induction of nitrate reductase (NR, E.C. 1.6.6.1) in intact cotyledons of 2-day old seedlings ofLactuca sativa L. Molybdate strongly enhanced efficiency of induction. Benzyladenine (BA), gibberellin, and succinic acid-2,2-dimethylhydrazide reduced the enzyme activity. BA thrice enhanced incorporation of labelled leucine to the protein fraction. (2-chloroethyl)trimethylammonium chloride did not affect NR activity and markedly inhibited greening and protein synthesis. KNO₃ stimulated protein synthesis as well as growth of the cotyledons.

The growth ofArabidopsis thaliana calli on media without growth regulators was studied Calli under study showed autonomy for cytokmms independently whether cultivated on the light or m the dark When cultivated in intense light, they were able to grow, either transiently or permanently, on the medium without any growth regulators In the dark, they were strictly dependent on 2,4 D m the medium Both the intensity of growth and the duration of the transient growth on the medium without growth regulators m the light decreased with the duration of the previous cultivation on the medium with growth regulators The intensity of growth on the medium without grow th regulators was best and the growth was permanent m the callus clone of spontaneous origin which was never treated by growth regulators The degree of chromosomal variability (assessed as the number of chromocentres) m this callus line was lower than that in calli induced on media with growth regulators and then transferred onto medium without growth regulatois.

Three short-day inductive cycles bring about inhibition followed by transitional enhancement of growth, not only in roots and leaves but also in different zones of shoot apical meristem, as shown by measurement of DNA synthesis using³H-thymidine autoradiography. The first inductive cycle resulted in marked inhibition of the cells of the central zone (CZ), rib meristem (RM), and peripheral zone (PZ). Subsequent enhancement of DNA synthesis occurs in RM during the second inductive cycle, but in CZ only in the third cycle. The growth activation in PZ is counteracted by decrease in apical dominance which results in further inhibition of leaf primordia and increases in bud primordia. In plants induced only by one cycle, which later reverse the vegetative pattern of growth and differentiation, increased DNA synthesis in RM and CZ was not observed. The significance of inhibitory and stimulatory processes in particular zones of the shoot apex is discussed considering flower morphogenesis.

Nitrate reductase (NR) induction is enhanced by exogenously supplied sucrose in excised pea roots exposed to both exogenous nitrate and exogenous nitrite. NR synthesis is preferentially supported by sugars transported to the cells at the moment, however NR induction can take place for some time without exogenous sugar influx if roots are saturated with sugars during precultivation. Steady high NR levels are dependent on steady sugar and nitrate influxes. NR induction is low in roots precultivated for 20 h without sucrose although sugar content is still high in them. This suggests that compartmentation of sugars in the cells is of major importance during NR induction. Total nitrate content in roots exposed to nitrate is not influenced by sucrose supplied together with nitrate. Some nitrite is oxidized to nitrate in roots exposed to exogenous nitrite ; we assume that this nitrate is responsible for NR induction. Our results indicate that sugars, besides many indirect effects on NR induction, may also directly influence NR synthesis either as coinducers or as derepressors of NR synthesis. Our results further show that NR is not a product-inducible enzyme.

Callogenesis was induced in mature embryos. The efficiency of induction and the growth of calli were dependent on 2,4-D concentration. No regeneration of buds or shoots was observed in 720 calli studied, but most calli showed intensive root proliferation on the regeneration medium. Most of the root meristem cells maintained diploid chromosome number. Only a low proportion (about 3.5%) of tetraploid cells was found. No other chromosomal changes were observed. Chromosomal variability does not contribute to the inability of calli derived from mature barley embryos to form buds and shoots.

The effect of silymarin complex on various types of expiants differing in their nutrition requirements was investigated. The growth of tumorous periwinkle (Catharanthus roseus [L.] G. Don) callus tissue was still identical with the control tissue at the silymarin concentration of 35 mg in 1000 ml of the nutrient medium. However, this silymarin concentration totally inhibited the growth of habituated periwinkle callus tissue; in the presence of 10 mg of silymarin, the growth of this tissue was similar to that of the corresponding tissue grown without silymarin. The growth of tobacco callus tissue (D-strain) requiring for its growth kinetin was reduced by 46.2% at the concentration 10 mg of silymarin in 1000 ml of nutrient medium, but its dry weight was increased by 21% in comparison with the control. Silymarin was most effective on the growth of callus derived from tobacco (Nicotiana glauca Grah:) stem pieces; callogenesis was observed in control tissue in 89.5% cases while in the presence of silymarin (10 mg) only in 48.6%. The primary callus growth was strongly inhibited, too (by 89.9%). The organogenesis onset was never observed on tobacco stem pieces cultured on a nutrient medium with kinetin and IAA in the presence of silymarin. When all types of expiants were transferred from the medium with silymarin on control medium, normal growth appeared very soon and the differences between the experimental and control expiants were smoothed out during two months. These results indicate that the observed changes might be due to the blocking of membrane system permeability leading to an insufficient supply of cells with nutrients and growth substances.

The growth of callus tissue cultures and the infectivity of twenty fiveSolanum laciniatum Ait. plants and of sixteenNicotiana tabacum L. cv. Samsun plants were investigated. The plants were obtained from callus tissue cultures derived from stem pieces of the respective plants infected with a mycoplasma-like organism (MLO) evoking potato witches' broom. The tissues were cultivated on synthetic nutrient medium with kinetin and IAA. Allde novo obtainedS. laciniatum plants were healthy. On the contrary twelve of the sixteen reconstituted tobacco plants showed MLO presence.
Summarizing these and previous results, the authors suppose that the most important factor influencing MLO persistence in callus tissues cultivated on the applied nutrient medium may be the callus growth rate and the organogenesis set. Both these conditions are determined by the metabolism of the investigated plant species.

Willows were rapidly propagated by repeated division of cultured rooted shoots into a larger number of nodal segments. Rapid clonal propagation of mountain-ash and black locust was achieved by induction of shoots from axillary buds and a multiple shoot culture was used for rapid multiplication. Excised shoots were rooted in an agar medium with a low concentration of auxin and rooted plantlets were transplanted to soil.

Long-term callus cultures of two barley cultivars have been investigated at the cellular level. One slowly growing, root forming callus of the barley cv. Bulgarische Nackte consists of cells the majority of which contain the diploid chromosome number. Contrarily, a fast growing callus of the cv. Elgina having no regeneration potency at all, is highly polyploid and includes more than two thirds of aneuploid cells. Tn this callus strain, a great number of multinucleate cells has been found. Some problems are discussed with respect to the relationships between growth rate, karyological stability and regeneration capacity in long-term callus cultures of cereals.

The changes in cell division rate were studied in different components of the shoot apex ofChenopodium rubrum during short-day photoperiodic induction and after the inductive treatments. Induced and vegetative apices were compared. Accumulation of metaphases by colchicine treatment was used to compare the mean cell cycle duration in different components of the apex. A direct method of evaluating the increase in cell number obtained by anticlinal or periclinal divisions was applied if the corresponding components of induced and non-induced apices had to be compared. The short-day treatment prolonged the cell cycle more in the peripheral zone than in the central zone and still more in the leaf primordia. The importance of changing growth relations for floral transition was shown particularly if the induced plants were compared with the vegetative control with interrupted dark periods. Induced plants transferred to continuous light showed further changes in the rates of cell division. The cell cycle was shortened more in the central zone than in the peripheral zone,i.e. there was a further shift in growth relations within the apical dome. The cell cycle in the leaf and bud primordia was also shortened if compared with the vegetative control, the acceleration being stronger in the bud primordia. There was a subsequent retardation in cell division in the leaf primordia formed during and after the inductive treatment if the plants were fully induced. An inhibition of the oldest bud primordia was observed in fully induced apices, as well.

Tuber callus by subculture ofDioscorea deltoidea Wall, was grown with different sources of nitrogen on N-free Murashige and Skoog's basal media supplemented with a high level of kinetin (1, 3, 6, 9 and 12 ml 1^-1) and low concentration of auxin (0.01 mg 1^-1 2,4-D) for shoot bud differentiation. Shoot and root differentiation was observed only in the case of ammonium nitrate. Other sources of nitrogen failed to produce shoot bud differentiation except in the case of ammonium citrate where tissues were slight green in colour and were recognizable as pro-embryo.

The LpDH and NpDH activities in crown gall tumors incited byAgrobacterium tumefaciens strain C58 were followed in 7 plant species and 100 tumors were assayed in each experimental variant. The NpDH activity was found in 790 out of 800 crown galls observed. The LpDH activity was tested, after induction of tumors withA. tumefaciens strain B6-806 and 37400, in three experimental variants. The LpDH activity was found in 290 out of 300 crown galls. In the small fraction of the LpDH and NpDH negative tumors, the activity was possibly actually present, but it was below the limits of the sensitivity of the detection technique used.

IAA-oxidase activity increased in the stem as well as in the leaves of plants treated with GA₃, SA and GA₃ + SA during the early stages under inductive and non-inductive photoperiods, the activity being the highest in GA₃ + SA-treated plants. An isoenzyme of IAA-oxidase with Rm 0.15 developed in the stem as well as in the leaves subsequent to 1 or 2 inductive treatments. As this band persisted till the end of the experiment, it may be associated with the initiation as well as development of floral buds. Another band (Rm 0.30) appears to be associated with the phenol (SA) as it developed in the stem as well as in the leaves of SA- and GA₃ + SA-treated plants under both photoperiods. A band with Rm 0.60 developed in the leaves but not in the stem of GA₃-, SA- and GA₃ + SA-treated plants under both photoperiods.

The technique of trees production from the undifferentiated poplar callus tissue is described. The best root formation was observed on the modifiedWolter andSkoog medium when NAA in concentration 0.2 to 0.4 mg l^-1 was used as an auxin and cytokinins were omitted. The induction of leafy shoots from the undifferentiated callus was the most effective on the modifiedLinsmaier andSkoog medium in the absence of auxin and with 0.15 to 0.70 mg l^-1 of BAP. The best development of roots at the basal end of excised shoots was achieved when shoots were transferred into the sterile mixture of perlit and sand (3: l, v/v) containing a modifiedWolter andSkoog medium.

The cells of some pea, tobacco and haplopappus callus strains reveal considerable variability in nuclear morphology (polymorphous nuclei, differences in nucleolar size and number, enlarged chromocentres) and chromosome counts. The specific features in the nuclear morphology of callus cells are related with some pecularities in the reproduction activity of these cells (amplification, amitosis, fragmentation, various deviations from normal mitosis) under cultural conditions including both the definite action of the culture system and the absence of the regulatory control by the intact organism.