A protocol of high frequency shoot organogenesis and plant establishment from stem derived callus has been developed for Tylophora indica (Burm. f.) Merrill. - an endangered medicinal plant. Callus was developed on Murashige and Skoog (MS) medium supplemented with 10 μM 2,4,5-trichlorophenoxy acetic acid (2,4,5-T). Multiple shoot induction was achieved from the surface of the callus after transferring onto shoot induction medium. The highest rate (80 %) of shoot multiplication was achieved on MS medium containing 5.0 μM kinetin. The developed shoots rooted best on half-strength MS medium supplemented with 0.5 μM indole-3-butyric acid (IBA). The in vitro raised plantlets with well developed shoot and roots were acclimatized successfully and grown in greenhouse.

Treating androgenic embryos of Aesculus hippocastanum L. with low temperatures (6 °C) improved their germination and regeneration into plantlets. The embryos derived from anther cultures showed better results than those derived from microspore cultures.

We have cloned and characterized the expression of Crocus sativus AGAMOUS1 (CsAG1), a putative C-type MADS-box gene homologous to AGAMOUS (AG) from a triploid monocot species crocus (Crocus sativus L.). The typical domain structure of MIKC-type plant MADS proteins was identified. Phylogenetic analysis of the deduced amino acid sequence indicated that the isolated gene forms a clade with the AGAMOUS homologs from the monocots Hyacinthus orientalis and Phalaenopsis equestris. A differential splicing event altering the amino acid sequence at the C terminus was identified, leading to the formation of two mRNAs differing ten nucleotides in size. The presence of both differentially spliced transcripts was restricted only to mature crocus flowers and particularly to stamens and carpels.

Lead nitrate at concentration of 150 mg dm^-3 inhibits root growth of Lupinus luteus seedlings by bout 20 %, which is accompanied by an increase of K⁺ leakage from the root cells. Non-denaturing isoelectric focusing in polyacrylamide slab gel has shown that lead stimulates the activity of most lipoxygenase isoenzymes and induces one additional isoenzyme with pI 6.9.

Effect of pre-treatments of 1 and 5 μM epibrassinolide or homobrassinolide prior to water stress induction on changes in root nodulation and contents of endogenous abscisic acid (ABA) and cytokinin trans-zeatin riboside (ZR), and nitrogenase activity was investigated in the nodulated roots of Phaseolus vulgaris L. cv. Arka Suvidha. Brassinosteroids in the unstressed plants increased root nodulation, ZR content and nitrogenase activity, and also ameliorated their stress-induced decline in the nodulated roots. The ABA contents in the nodules of control or stressed plants were not altered by brassinosteroids treatment. There was an increase in pod yield by brassinosteroids treatment (5 μM) in the irrigated control as well as stressed plants without influencing pod number or pod length. Among the brassinosteroids, epibrassinolide was relatively more effective.

A screening for the enzyme L-myo-inositol-1-phosphate synthase [EC 5.5.1.4] has been made first time in both vegetative and reproductive parts of the representative members of pteridophytes: Lycopodium, Selaginella, Equisetum, Polypodium, Dryopteris, and Gleichenia. The enzyme has been partially purified following low-speed centrifugation, streptomycin sulphate precipitation, ammonium sulphate fractionation, chromatography on DEAE-cellulose and gel-filtration through Sephadex G-200, and characterised from the reproductive pinnules of Gleichenia glauca Smith. The enzyme has a pH optimum at 7.5. The K_m for glucose-6-P and NAD⁺ were 0.922 × 10^-3 M and 0.9 × 10^-4 M, respectively. A basal activity of the enzyme has been recorded in absence of exogenous NAD⁺. The enzyme activity was augmented with NH₄Cl, but heavy metals like Hg²⁺, Cu²⁺ and Zn²⁺ inactivated it.

The effects of excess Cu as affected by the application of exogenous hormones (gibberellic acid - GA₃ and indole-3-acetic acid - IAA) with respect to sunflower (Helianthus annuus L.) growth, physiology, and metabolism were studied. Application of 100 μM IAA lessened the toxic effects of 80 μM Cu in roots indicating greater root length and root hair formation, while addition of 100 μM GA₃ ameliorated the toxic effect mainly to the shoot. The content of photosynthetic pigments significantly declined under Cu stress, whereas application of hormones led to a substantial preservation of chlorophylls and carotenoids. Under Cu stress, the rate constant of energy trapping by photosystem 2 (PS2) reaction centres (RCs) was reduced as a result of physical dissociation of the light-harvesting complex (LHC) from PS2 core, while application of IAA and especially GA₃ resulted in stability of the LHC of PS2 RCs. The drop in net photosynthetic (PN) and transpiration (E) rates with preserved or slightly reduced variable to maximum fluorescence ratio (F_v/F_m) in the presence of 80 μM Cu could be explained by a possible inhibition of the enzymatic processes in the Calvin cycle. Application of 100 μM IAA and 100 μM GA₃ lessened Cu effects mainly on P _N. Water use efficiency was also improved under hormone exposure.

The chickpea genotype, CSG-8962 was raised in screenhouse to study salinity induced changes in ethylene evolution, antioxidative defence system and membrane integrity in relation to changes in plant water and mineral content. At vegetative stage (60 d after sowing), the plants were exposed to single saline irrigation (0, 2.5, 5.0 and 10.0 dS m^-1). Sampling was done 3 d after saline treatments. The other sets of treated plants were re-irrigated with water and sampled after further 3 d. The Ψ_w of leaf and Ψ_s of leaf and roots decreased from -0.47 to -0.61 MPa, -0.67 to -1.23 MPa and from -0.57 to -0.95 MPa, respectively, with increasing salinity. Similarly, RWC of leaf and roots reduced from 87.5 to 72.3 % and 96.7 to 84.35 %, respectively. The decline in Ψs of roots was mainly due to accumulation of proline and total soluble sugar. With salinity, increase in ethylene evolution, 1-aminocyclopropane-1-carboxylic acid (ACC) content and ACC oxidase activity was reported. Similarly, marked increase in H₂O₂ content (20 - 182 %) and lipid peroxidation (43 - 170 %) was observed. The defense mechanism activated in roots was confirmed by the increased activities of superoxide dismutase (SOD), peroxidase (POX), ascorbate peroxidase (APX), glutathione transferase (GTase), glutathione reductase (GR) and catalase (CAT) but ascorbic acid (AA) content was decreased. About 3-fold increase in Na⁺/K⁺ ratio and 2.5 fold increase in Cl^- content was observed. Upon desalinization, a partial recovery was observed in most of the parameters studied.

Methods for induction of callus and cell suspension cultures have been developed for the medicinally important herb Centella asiatica (L.) Urban. Thin layer chromatography (TLC) and high performance liquid chromatography (HPLC) analysis showed the presence of asiaticoside in the in vitro grown leaves, callus and cell suspension cultured cells.

An efficient and reproducible protocol for regeneration of plantlets at a high frequency was developed by using sugar cane buds. Disinfected buds were firstly submerged in ethanol sodium hypochlorite solution with 0.1 % polyvinylpyrrolidone, 1.5 % ascorbic acid and 1.75 % citric acid as antioxidants and subsequently treated with solution of agrimicin:captan (1:1). The upper stalk segment was better to obtain bud in vitro culture compared to lower segments. The medium for induction of multiple shoots consisted of Murashige and Skoog basal medium (MS) supplemented with 2 mg dm^-3 thidiazuron and 1 mg dm^-3 naphthalene acetic acid. An average of 24 shoots per bud was obtained for cv. Mex 68-P23 within four weeks and 29 shoots for cv. MY 55-14 within six weeks. Indole-3-butyric acid induced more roots in both cultivars compared to the untreated plantlets. Plantlets transferred to soil showed normal growth with up to four axilliary buds in each node. It was concluded that the germplasm obtained through the above mentioned technique generated stalks with more buds in each node which would give farmers more vegetative material for plantations in field with 100 % germination.

A simple histo-cytochemical method, combining Evans blue staining to assess cell death and in vivo 3,3'-diaminobenzidine uptake for H₂O₂ localisation, has been used to evaluate O₃ damages in leaf tissues of three Phaseolus vulgaris L. cultivars (Cannellino, BLF, Saxa) with different sensitivity to the pollutant. Bean plants were exposed to a single pulse of O₃ (150 ± 10 mm³ m^-3 × 3 h) and leaves were examined at different time-span after fumigation. Cannellino proved to be the most sensitive, showing chlorotic spots 2 h after fumigation and chlorotic lesions 24 h later. In BLF, necrotic spots appeared 4 h after fumigation and reddish necrotic lesions (bronzing) developed in further 24 h. Saxa remained symptomless up to 10 d of observation, thus appearing tolerant. The early appearance of symptoms in Cannellino correlated with H₂O₂ accumulation in leaf tissues and consequent extensive cell death, involving both palisade and spongy mesophyll. H₂O₂ accumulation was observed also in BLF, though to a lesser extent and dead cells were rare at 2 h after fumigation. However, they increased in number 24 h later, forming small groups in the palisade mesophyll. These groups further enlarged in the next 24 h, again involving only palisade mesophyll. In Saxa leaves, H₂O₂ accumulation was found only in the epidermal cells, though the number of dead cells was very similar to BLF, at least up to 24 h after fumigation. However, in Saxa, dead cells have been always found singly scattered through the palisade mesophyll, or forming very small groups around substomatal cavity, thus remaining invisible at a macroscopic level.

We selected a informative set of twelve amplified fragment length polymorphism (AFLP) primer pairs suitable for evaluation of Chinese chestnut (Castanea mollissima Blume) genotypes. Cluster analysis based on 198 polymorphic AFLP amplified by these 12 primer pairs clearly divided investigated genotypes according to their place of origin. We showed, that genetic basis of modern genotypes is narrow also in the case of this species.

Genome size has been estimated by flow cytometry in 14 populations belonging to eight taxa (seven species, one of them with two varieties) of the genus Tripleurospermum. 2C nuclear DNA amounts range from 4.87 to 9.22 pg, and nuclear DNA amounts per basic chromosome set from 1.99 to 2.75 pg. Statistically significant differences depending on ploidy level, life cycle or environmental factors such as altitude have been found. Also, genome size is positively correlated with total karyotype length. The presence of rhizome is related to nuclear DNA content in these species.

Although it is well accepted that cytokinins (CKs) regulate processes such as leaf senescence and stomatal conductance, data on CKs in the canopy of mature trees are lacking in the literature. Here we report the first in situ sampling for determination of CKs in mature sugar maple (Acer saccharum) canopy layers. The upper canopy showed a distinct seasonal pattern in total CK content, while the lower canopy remained relatively unchanged.

Abscisic acid (ABA) and salicylic acid (SA) were sprayed on leaves of wheat genotypes C 306 and Hira at 25 and 40 d after sowing under moderate water stress (-0.8 MPa) imposed by adding PEG-6000 in nutrient solution. ABA and SA increased the activities of superoxide dismutase, ascorbate peroxidase, glutathione reductase, and catalase in comparison to unsprayed control plants. Both ABA and SA treatments decreased the contents of hydrogen peroxide and thiobarbituric acid reactive substances, a measure of lipid peroxidation, compared to unsprayed plants. The beneficial effect of increase in antioxidant enzymes activity and decrease in oxidative stress was reflected in increase in chlorophyll and carotenoid contents, relative water content, membrane stability index, leaf area and total biomass over control plants. The lower concentrations of ABA (0.5 mM) and SA (1.0 mM) were generally more effective than higher concentrations.

A simple and efficient protocol for high frequency plant regeneration of a grain legume grasspea (Lathyrus sativus L.) is described. Of different explant types tested epicotyl segments were most responsive. Murashige and Skoog's (1962) medium augmented with 17.76 µM 6-benzyladenine + 10.74 µM α-naphthaleneacetic acid showed the highest percentage of direct shoot regeneration. Among cultivars IC-120487 showed the highest regeneration frequency (80 %) with maximum shoot numbers (8.2 shoots per explant) and maximum average shoot length (4.1 cm). About 78 % of the regenerated shoots were rooted in half-strength MS medium containing 2.85 µM indole-3-acetic acid. After primary hardening the plantlets were established in soil with a survival rate of 75 %.

Cytokinin (CK) content and activities of several antioxidant enzymes were examined during plant ontogeny with the aim to elucidate their role in delayed senescence of transgenic Pssu-ipt tobacco. Control Nicotiana tabacum L. (cv. Petit Havana SR1) and transgenic tobacco with the ipt gene under the control of the promoter of small subunit of Rubisco (Pssu-ipt) were both grown either as grafts on control rootstocks or as rooted plants. Both control plant types showed a decline in total content of CKs with proceeding plant senescence. Contrary to this both transgenic plant types exhibited at least ten times higher content of CKs than controls and a significant increase of CK contents throughout the ontogeny with maximal values in the later stages of plant development. Significantly higher portion of O-glucosides was found in both transgenic plant types compared to control ones. In transgenic plants, zeatin and zeatin riboside were predominant type of CKs. Generally, Pssu-ipt tobacco exhibited elevated activities of antioxidant enzymes compared to control tobacco particularly in the later stages of plant development. While in control tobacco activity of glutathione reductase (GR) and superoxide dismutase (SOD) showed increasing activity up to the onset of flowering and then gradually decreased, in both transgenic types GR increased and SOD activity showed only small change throughout the plant ontogeny. Ascorbate peroxidase (APOD) was stimulated in both transgenic types. The manifold enhancement of syringaldazine and guaiacol peroxidase activities was observed in transgenic grafts throughout plant ontogeny in contrast to control and transgenic rooted plants, where the increase was found only in the late stages. Electron microscopic examination showed higher number of crystallic cores in peroxisomes and abnormal interactions among organelles in transgenic tobacco in comparison with control plant. The overproduction of cytokinins resulted in the stimulation of activities of AOE throughout the plant ontogeny of transgenic Pssu-ipt tobacco.

Apical parts of stems of Brassica napus L. var. oleifera cv. Gorczanski (winter rape) and cv. Mlochowski (spring rape), grown in vitro, were subjected to direct electric current (DC) of different polarity, duration and voltage. The positive orientation of DC, i.e. anode attached to the apical part and cathode to the medium, markedly enhanced the differentiation of the apical meristem in winter rape. The reverse polarity was without effect. DC treatment of positive polarity resulted in spring rape in transition of all explants to generative state while 70 % of non-treated plants remained at vegetative stage. Even negative orientation of DC brought about a rise in percentage of flowering plants with regard to control. The developmental effects of DC were dependent only to a low degree or not at all on duration and voltage of the treatment.

This paper describes rapid propagation of Sesbania drummondii using nodal explants isolated from seedlings and young plants. The nodal segments proliferated into multiple shoots on Murashige and Skoog's (MS) medium supplemented with 22.2 μM benzyladenine. MS medium containing 2.2 and 4.5 μM thidiazuron induced 5 - 6 shoots per stem node from 3-month-old plants. Nodal explants when cultured on MS medium containing combinations of benzyladenine (8.8 and 11.1 μM) and indole-3-butyric acid (0.24 - 2.46 μM) or indole-3-acetic acid (0.28 - 2.85 μM) gave lesser number of shoots. Callus induced on cotyledonary explants when subcultured on 2.2 μM thidiazuron containing medium resulted in its mass proliferation having numerous embryoid-like structures. Indole-3-butyric acid (0.24 - 2.46 μM) was found suitable for root induction. In vitro regenerated plants were acclimatized in greenhouse conditions.

This study was conducted to examine the response of date palm (Phoenix dactylifera L., cvs. Barhee and Hillali) calli to water stress. Callus derived from shoot tip explants was inoculated in liquid Murashige and Skoog medium containing 10 mg dm^-3α-naphthaleneacetic acid, 1.5 mg dm^-3 2-isopentenyladenine, and 0 to 30 % (m/v) polyethylene glycol (PEG 8000) to examine the effect of water stress. After 2 weeks, callus growth, water content, and proline accumulation were assessed. Increasing water stress caused a progressive reduction in growth as expressed in callus fresh mass, relative growth rate, and index of tolerance. Both genotypes tested followed this general trend, however, cv. Barhee was more tolerant to drought stress than cv. Hillali. Increasing PEG concentration was also associated with a progressive reduction in water content and increased content of endogenous free proline.

Embryogenic suspension cultures of soybean (Glycine max L. cv. Iroquois) were subjected to mutagenesis using varying concentrations (1, 3, 10, and 30 mM) of ethyl methanesulfonate (EMS). Depending on the concentration of EMS used, the mean survival rate of embryogenic cultures decreased from 74 % (1 mM EMS) to 43 % after 30 mM EMS treatment. Random amplified polymorphic DNA (RAPD) analysis was used to determine whether induction of genetic variability in embryogenic cultures in response to the different EMS treatments may result in identification of polymorphic markers. Two of 35 'core' primers tested revealed polymorphisms. One of the primers, OPO-01/1150, revealed polymorphism in tissue treated with 10 mM EMS, while the other primer, OPO-05/1200, revealed polymorphism in tissue treated with either 1 or 30 mM EMS. These results suggest that RAPD markers are useful in detecting mutations in embryogenic cultures of soybean.

Treatment with methyl jasmonate (MeJA) stimulates a multicomponent defense response in leaves and suspension-cultured cells of grapevine (Vitis vinifera L. cv. Limberger). MeJA induces development of necrotic lesions, similar to that normally associated with resistance to avirulent pathogens. Sustained exposure of both leaves and cell-suspension cultures to 50 μM MeJA provoked hypersensitive cell death, stimulated medium alkalinization accompanied by massive callose deposition, but did not induce accumulation of hydrogen peroxide from the oxidative burst. Transcripts of genes encoding diverse families of the pathogenesis-related proteins accumulated rapidly after MeJA application, followed by salicylic acid production. After several days systemic accumulation of a large number of defense-associated proteins, including pathogenesis-related proteins, peroxidase, cell wall extensin and enzymes involved in the phenylpropanoid biosynthetic pathway was induced. These cumulative results suggest that grapevine cells that perceived MeJA generated a cascade of events acting at both local and long distances, and causing the sequential and coordinated expression of specific defense responses with a timing and magnitude similar to the typical hypersensitive response against pathogens.

Transformation of fenugreek (Trigonella foenumgraecum) was carried out with A281 oncogenic strain of Agrobacterium tumefaciens using root, cotyledon and hypocotyl explants excised from 1-week-old seedlings, which showed that the plant was highly susceptible to transformation. Tumors (calli) were selected on 50 mg dm^-3 kanamycin. They were analyzed for β-glucuronidase (GUS) expression. Presence of uidA (gus) gene, was confirmed by polymerase chain reaction (PCR) amplification.

Tobacco leaf discs were transformed with a plasmid pBIBnNHX1, containing the selectable marker neomycin phosphotransferase gene (nptII) and Na⁺/H⁺ vacuolar antiporter gene from Brassica napus (BnNHX1), via Agrobacterium tumefaciens-mediated transformation. Thirty-two independent transgenic plants were regenerated. Polymerase chain reaction (PCR) and Southern blot analyses confirmed that the BnNHX1 gene had integrated into plant genome and Northern blot analysis revealed the transgene expression at various levels in transgenic plants. Transgenic plants expressing BnNHX1 had enhanced salt tolerance and could grow and produce seeds normally in the presence of 200 mM NaCl. Analysis for the T₁ progenies derived from seven independent transgenic primary transformants expressing BnNHX1 showed that the transgenes in most tested independent T₁ lines were inherited at Mendelian 3:1 segregation ratios. Transgenic T₁ progenies could express _BnNHX1 and had salt tolerance at levels comparable to their T₀ parental lines. This study implicates that the BnNHX1 gene represents a promising candidate in the development of crops for enhanced salt tolerance by genetic engineering.

1-Methylcyclopropene (1-MCP) applied alone did not influence significantly the chlorophyll and carotenoid content of the older leaves of Arabidopsis thaliana (L.) Heynh., but retarded the senescence of the younger ones (6^th and 7^th leaf nodes). However, 1-MCP effectively blocks the ethylene induced senescence of excised rosette leaves. The preliminary application of 1-MCP (3 h in advance to the treatment by Ethrel) almost totally eliminated the ethylene action. Similar trend was also observed after simultaneous application of Ethrel and 1-MCP, and the effects of both treatments on the chlorophyll and carotenoid destruction are comparable.