Ten-days-old bean plants (Phaseolus vulgaris L., cv. Cheren Starozagorski) were treated with simulated acid rain (pH 2.4, 2.2, 2.0 and 1.8). Anatomical changes in the primary leaves were studied 3, 48 and 168 h after a single treatment. This treatment induced: 1) change in the shape of palisade cells, contraction of their contact surfaces and expansion of spongy cells (pH 1.8, 3 h after treatment); 2) reduction of symplast connections among palisade cells and of apoplast in the spongy mesophyll (pH 1.8, 48 h after treatment); 3) destruction of adaxial epidermis and portions of palisade mesophyll, plasmolysis of spongy cells (pH 1.8, 168 h after treatment); 4) full destruction of mesophyll (pH 2.4, 2.2, 2.0 and 1.8, 168 h after treatment). The structure of abaxial epidermis was more stable than that of the adaxial one. With respect to anatomical parameters the studied species could be considered as comparatively resistant to acid rain.

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 hexokinase particulate and soluble isozyme composition and activities in leaves of 65- and 115-d-old tobacco plants were determined by ion exchange chromatography on DEAE cellulose. During plant ageing, the activities of glucose and of fructose phosphorylating isozymes of particulate hexokinase decreased to 9.9 and 9.2 % of initial value, respectively. The activity of soluble hexokinase decreased to a lesser extent: that of glucose phosphorylating isozyme to 49.8 % and of fructose phosphorylating isozyme to 37.8 %. The activity of soluble fructokinase isozyme dropped to 34.8 %. Thus also the ratio of particulate and soluble isozymes was dependent on the age of leaf tissue.

Foliar application of gibberellin (GA₃) to patchouli (Pogostemon cablin Benth.) increased the plant height, number of nodes per plant, leaf fresh mass and photosynthetic pigment contents. The content of chlorophyll (Chl)b increased faster than that of carotenoids (Car), Chl (a+b) and Chla. This was reflected in a decline in Chla/b and Chl (a+b)/Car ratios. There was a GA₃ concentration dependent variation in the number of branches, leaves, total leaf area, and leaf area index. These growth parameters decreased over control values up to 250 g(GA₃) m^-3 and increased at 500 g(GA₃) m^-3 concentration. The patchouli oil yield varied from 2.4 to 2.6% of the leaf dry mass.

An investigation was carried out on chickpea (Cicer arietinum L.) cv. C-235 inoculated withRhizobium sp.Cicer strain cv 4 Az^r. Nodule functioning was monitored at 15 d intervals starting from 45 days after sowing (DAS) and inoculation in order to study nodule development and senescence under natural and stress conditions (dark treatments of 18 and 66 h). Maximum rate of N₂-fixation was observed between 50 - 60 DAS. After this acetylene reducing activity (ARA) fell and it was negligible 75 DAS. This decline in ARA with ageing of plants and nodules was accompanied by a decline in leghemoglobin content and greening of the nodules. When 60 % of the nodule tissue had turned green 75 DAS, a sharp increase in nodule peroxidase activity (3.7 fold) was observed whereas the catalase activity was reduced by 50 % in comparison with the control. The glutathione-reductase and ascorbate-peroxidase activity followed a trend parallel to that in N₂-fixation, but the variation was much smaller. The changes in the total soluble carbohydrates, cytosolic proteins and nitrogen content per se were not expressive. Dark treatments induced premature senescence of the nodules as was evident from the marked decrease in ARA. However, the decline in leghemoglobin content was relatively small as compared to ARA. The changes in cytosolic proteins, total soluble carbohydrates, peroxidase activity, catalase activity, glutathione reductase activity and ascorbate peroxidase activity of nodules under dark-induced nodule senescence were almost parallel to those observed under natural senescence.

Responses of wheat (Triticum aestivum L.) to various concentrations of NaCl and levels of drought were followed. With the rise of NaCl or drought, or NaCl and drought together, growth was retarded. The water content of shoots and roots was mostly unchanged. The chlorophyll and carotenoid contents were increased in plants subjected to salinity or drought or both. Only high salinity level induced a considerable decrease in net photosynthetic rate (P_N) and dark respiration rate (R_D). P_N and R_D were decreased with the decrease of soil moisture content. The content of Na⁺ in the shoots and roots of wheat plants increased with increasing salinity or decreasing soil moisture content or both treatments. Considerable variations in the content of K⁺, Ca²⁺ or Mg²⁺ were induced by the NaCl, drought or both treatments.

Arabidopsis thaliana tumors were induced by octopine strain ofAgrobacterium tumefaciens B6S3 and its derivatives with modified T-DNA. Flowering shoots appeared sponta-neously onin vitro cultivated tumors and set seeds. R₁ and R₂ progeny of octopine synthesizing plants segregated in opine synthesis activities 3:1 and 15:1. Octopine synthase activity showed absolute linkage with agropine synthesis in most lines. In R₃ and R₄ progenies, the fraction of octopine synthase and agropine synthesis positive plants was lower than expected, but Mendelian segregation was restored if plants were cultivated on medium with 5-azacytidine. The most probable mechanism of disapearance of opine synthesis is cytosine methylation. The effect of 5-azacytidine lasted for at least next two generations.

Leaves of micropropagated highbush blueberry (Vaccinium corymbosum) cv. 'Bluetta' have been observed during the acclimatization phase. In vitro-developed leaf cells were circular and small, the spongy parenchyma was discontinuous and disorganized and formed by 1-2 layers of cells with large intercellular spaces and the palisade to spongy mesophyll thickness ratio was 1:1.5. After rooting ex vitro, the first leaves formed under natural conditions showed substantial changes in the anatomical characteristics. After 6 months, the plants produced leaves similar to those in field-grown plants. The palisade cells were rectangular, the spongy parenchyma was formed by 3-4 layers of cells and the intercellulars were around the stomata. Leaves from field-grown plants lost 24 % of water during 150 min after excision while leaves from in vitro shoots lost about 50 % of water in the same time. Leaves from in vitro shoots showed a higher number of smaller stomata (361 per mm²), with the guard cells forming a circular ring; the stomata frequency in field-grown leaves was 241 per mm² and the guard-cells were elliptical.

An improved method for the isolation of leaf mesophyll protoplasts ofSolanum tuberosum L. cvs. Xenia and Bintje, their culture and regeneration to plants is described. The improvements involve the culture of nodal stem explants on Schenk and Hildebrandt (SH) medium supplemented with AgNO₃ (7.5 mg 1¹) and Alar 85 (5 mg 1^-1), and the pre-treatment of donor plants for 2-3 weeks at a short photoperiod (6 h), a low irradiance (17 (μmol m^-2s^-1) and a low temperature (20 °C). Regeneration of cell wals was observed within 2-3 d and cell divisions within 7-12 d after resuspending freshly isolated protoplasts in Sidorov et al. (SW) medium. The resulting microcalli were cultured on solid media containing zeatin (1-2,5 mg 1^-1) and/or NAA (0.1 and/or 0.01 mg 1^-1) and subsequently on medium with BAR (0.25 mg1^-1) and GA^-3 (0.1 mg 1¹) for shoot regeneration.

A method for micropropagation ofDalbergia sissoo has been developed. Single node segments obtained from coppice shoots of a mature tree (20 - 25 year old) produced 3-4 shoots per explant on Murashige and Skoog (MS) medium containing 4.4 x 10^-6 M benzylaminopurine (BAP) and 4.4 × 10^-7 M of Β-naphthoxy acetic acid (NOA) (shoot multiplication medium) within 4 weeks. Thein vitro regenerated shoots were 3 - 4 cm in length and provided 2 to 3 culturable nodal segments which on shoot multiplication medium again produced 3-4 shoots. Following this procedure 18-24 shoots were produced from single nodal segment within 60 d. 80 % of the shoots directly produced five roots when they were firstly treated with MS medium supplemented with 10^-5 M indole-3-butyric acid (IBA) and subsequently transferred to half strength liquid MS medium containing 1 % activated charcoal followed by half strength liquid MS free hormones, vitamins and activated charcoal. Thein vitro raised plants were hardened for survival after transplantation to soil by exposing them to various humidity conditions, gradually from higher to low, with nearly 100 % transplant success.

In pea, maize and tomato plants a hitherto undescribed L-tryptophan dehydrogenase activity (TDH) has been detected. This enzyme catalyzes the reversible formation of indolepyruvic acid (IPyA) from L-tryptophan (L-trp). TDH and L-glutamate dehydrogenase (GDH), related enzymes in their mode of action, could be separated by gel chromatography. Enzymatic activity of TDH was sustained by both pyridine coenzymes NAD/NADP. With pea TDH the coenzyme NAD displays, at optimum pH 8.5 and at room temperature, only about 40-70 % of the activity of NADP. The amination of IPyA is catalysed more actively than the deamination of L-trp. L-trp/IPyA, L-glu/ketoglutarate, L-ala/pyruvate reacted as dehydrogenase substrates; L-phe/ phenylpyruvate, D-trp and D-phe did not react with pea enzyme extracts. A considerable similarity between the active centres of TDH and GDH has been found using inhibitors: absence of heavy metals, presence of a carbonyl group, indispensibility of bivalent ions for the enzyme activity. Pea TDH and GDH were distinctly inhibited by sodium azide. For the activity of TDH the presence of SH groups is less important than for GDH. The TDH activity in the investigated plants was lower than the GDH activity. The possible role of TDH in the regulation of the IPyA pool is discussed.Doc. RNDr. PhMr. M. Kutáček died on 28 November, 1989. The final form for print was prepared by dr. Ivana Machdckovd of the same Institute, who will also answer the reprint requests. Received June 6, 1990; accepted October 10, 1990

Drought created by withholding the irrigation at 30 and 45 d after sowing significantly decreased relative water content (RWC) and osmotic potential (ψ_s) ofVigna radiata (L.) Wilczek cv. MH-83-30 nodules. Potassium fed plants showed higher RWC, whereas ψ_s was further declined irrespective of soil moisture levels. The nitrogenase activity and leghemoglobin content of nodules markedly decreased under drought and nodules of potassium fed plants showed better recovery after rehydration. The proline content significantly increased under drought but declined upon reirrigation. Also, the C, N and K contents of nodules significantly declined under drought.

Sampling of natural cocksfoot (Dactylis glomerata L.) populations was carried out on the O Morrazo peninsula in NW Spain, characterized by a strong moisture gradient. The plants were kept in greenhouse under standard conditions. Nevertheless, they differ in height of plants, length and width of flag leaves, panicle size, stomatal density and size as well as in flowering period. The effects of two levels of soil water deficit and two levels of salinity on photosynthetic rate were tested. One population was exceptionally well adapted to its original environment with great tolerance to water deficit and salinity

The effect of crude volatile oils from the leaves ofEucalyptus globulus andE. citriodora and the pure terpenes - cineole and limonene from these oils, (in vapour form) was studied onPhaseolus aureus, Lens esculentum, Hordeum vulgare and Avena sativa. The parameters like germination of seeds, seedling growth, values of cell survival, and content of water and chlorophyll of the crops formed the system of bioefficacy study. The allelopathic impact of the oil vapours from the eucalypt tree becomes evident from the negative response of the parameters studied. The impact of the E. citriodora oil vapours compared to that ofE. globulus oil or the pure terpenes was seen to be relatively greater in almost all parameters under investigation. A strong reciprocal correlation that exist between the concentration and the seedling growth or the water content of the crops under study supports the dose linked allelopathic phenomenon. It is suggested that oil vapours ofEucalyptus exert their effect through impairing the respiratory as well as photosynthetic ability of the target plants.

The changes in ubiquitin mRNA level in intact plants of both susceptible (cv. Spunta) and partially resistant (cv. Pampeana-INTA) potato cultivars after inoculation with low doses (l0³ sporangia cm^-3) ofPhytophthora infestons were studied after 72 h of treatment. Inoculation leads to 5-fold accumulation of potato ubiquitin transcripts in both cultivars. This result supports the connection between ubiquitin expression and defense reaction in plants.

Wounding both cotyledons ofBidens pilosa (var.radiatus) induces the inhibition of hypocotyl growth. The wound signal is transmitted very rapidly from cotyledon to hypocotyl and can be visualized by the change in nucleotide pools. First we have shown that the irradiance of the plant can change the ATP level without plant wounding. Therefore, plants were harvested at the start of the light period. Under these conditions, we have determined in hypocotyl the levels of adenosine triphosphate (ATP), guanosine triphosphate (GTP) and non adenylic triphosphates (NTP), and adenylate energy charge (AEC) after wounding. We have observed a transient (2 min) increase in the ATP level followed by a decrease 5 to 30 min later. A similar result was obtained for the GTP level but with some delay. The GTP level increased in 5 min and then decreased after 60 min. For the NTP level the decrease is effective from 5 to 60 min after wounding. The calculation of AEC has shown that a very tight control in the level of ATP may be involved in response to wounding.

Agrobacterium tumefaciens binary vectors carrying kanamycin resistance gene and either C58 T-DNA gene 4 for cytokinin synthesis or genes 1 and 2 for auxin synthesis were constructed and used for transformation of a short-day tobacco Maryland Mammoth. Kanamycin resistant plants were regenerated from a small fraction of transformed tissue and the presence of T-DNA in their genome was verified by Southern blotting. The level of endogenous cytokinin in plants transgenic for gene 4 and the level of endogenous IAA in those transgenic for genes 1 and 2 increased by more than 100 %. A number of morphological characteristics distinguish them from untransformed controls.

The effects of fusaric acid, a phytotoxin produced byFusarium pathogens, on the metabolism of isolated maize root mitochondria and on maize seed germination and seedling growth were investigated. The phytotoxin inhibited basal and coupled respiration when succinate and α-ketoglutarate were the substrates. Coupled respiration dependent on NADH was inhibited, but basal respiration was not. Consistently, succinate cytochromec oxidoreductase activity was decreased whereas NADH cytochromec oxidoreductase was not affected. The ATPase activities of carbonyl cyanide p-trifluoro-methoxyphenyl hydrazone stimulated mitochondria and of freeze-thawing disrupted mitochondria were inhibited. These results indicate that the phytotoxin impairs the respiratory activity of maize mitochondria by at least three mechanisms: (1) it inhibits the flow of electrons between succinate dehydrogenase and coenzyme Q, (2) it inhibits ATPase/ATP-synthase activity and (3) it possibly inhibits α-ketoglutarate dehydrogenase. Seed germination and seedling growth were also affected by fusaric acid with the most pronounced effect on root development. These effects can possibly contribute to the diseases ofFusarium- infected plants

Buthionine sulfoximine (BSO) specifically inhibits γ-glutamylcysteine synthetase and decreases a cellular level of glutathione (GSH) in maize seedling roots. Exogenous GSH restores Zn-phytochelatins synthesis in BSO-treated maize plants.

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.

Phaseolus vulgaris L. seedlings were grown under different air humidities simulating conditions during micropropagation (very high humidity duringin vitro cultivation and low air humidity after transferex vitro). The functional stomatal apparatus developed after a short period of growth at low air humidity at the beginning of plant ontogeny or after transfer from high to low air humidity, but not in plants grown steadily under high air humidity. The ability of stomata to regulate gas exchange was not persistent and disappeared after transfer of plants from low to high humidity.

Morphology of pollen grains collected from healthy and virus infected plants ofChenopodium quinoa L.,Chenopodium album L. andNicotiana tabacum L. cv. Samsun was investigated using scanning electron microscopy (SEM). Pollen grains from tobacco plans infected with tobacco mosaic virus (TMV) were smaller, with rounded shape and conspicuous deformation of aperture unlike oval and smooth pollen grains from healthy plants. No morphological alterations were observed inC. quinoa andC. album plants infected with TMV and cucumber mosaic virus (CMV). Polyacrylamide gel electrophoresis of pollen proteins revealed substantial quantitative and qualitative differences in protein components of pollen grains collected from healthy and virus infected plants

The net photosynthetic rate (P_N), stomatal conductance (g_s) and transpiration (E) ofHardwickia binata Roxb. leaves were reduced due to decrease in the leaf water potential (ψ_w) from -2.0 to - 5.7 MPa. P_N partially recovered in the treated plants upon rewatering. Decrease in g_s due to water stress may be the main factor for reduction of P_N.