Long-distance electrical signals generated in locally stimulated plants are linked with systemic physiological responses. The propagation of electrical signal through a plant can be measured by multiple electrodes attached to different sites of a plant body. As this signal has to be measured with the sensitivity of tens of microvolts, it can be easily disturbed by power-line hums or external electromagnetic fields. These disturbances can mimic the action potentials generated by a plant. In this work, we present a brief summary of various experimental approaches to the measurement of surface electrical potential (SEP) on a plant and a description of our multi-channel device for the SEP measurement. The main advantages of our measuring system are galvanic separation of the measuring unit, resulting in the elimination of power-line disturbances, and simple and stable contact of Ag/AgCl-peletted electrodes with the plant surface, facilitated by an ordinary gel used in human electrocardiography. These improvements enabled us to detect unperturbed variation (slow) and action (fast) potentials on a plant, as demonstrated by the four-electrode measurement of the electrical signal propagation in a locally wounded tomato plant.

Direct somatic embryogenesis of Frittilaria meleagris L. was induced using leaf base explants excised from in vitro grown shoots. Somatic embryos occurred at the basal part of leaf explants 4 weeks after culture on a Murashige and Skoog (MS) medium supplemented with various concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D) or kinetin (KIN). The highest number of somatic embryos (SEs) were formed (9.74) from leaf explant on MS medium supplemented with 0.1 mg dm^-3 2,4-D after 4 weeks of culture initiation. An initial exposure to a low concentration of KIN in the medium also enhanced SEs induction. Our observations by light and scanning electron microscopy revealed that SEs originate directly from the epidermal and subepidermal layers of leaf explant. The developmental stages of somatic embryogenesis from the first unequal cell division through the meristematic clusters, multi-cellular globular somatic embryos to the fully formed cotyledonary embryos were determined. After 4 weeks on MS medium without plant growth regulators, SEs developed into bulblets.

The major capsid protein L1 of human papillomavirus type 16 (HPV16) was transiently expressed in tobacco (Nicotiana benthamiana and Nicotiana tabacum) and tomato (Lycopersicon esculentum) leaves using Agrobacterium tumefaciens. The expression vector pTV00 was derived from tobacco rattle virus (TRV). The highest L1 expression 15 μg g^-1(f.m.) was achieved when the coding sequence of L1 was optimized for expression in humans that caused an increase of the guanine and cytosine (GC) content from 38.2 % in wild type HPV16 to 64.1 % in optimized sequence. L1 monomers readily self-assembled into capsomeres and further into virus like particles (VLPs). Immunological characterization and electron microscopy showed that 89 % of L1 retained VLP structure also in extracts prepared from freeze-dried leaves. Plant expressed L1 in crude extracts was highly immunogenic without any additional adjuvant as vaccinated mice developed strong humoral and cellular immune response, comparable to that elicited by purified VLPs derived from insect cells. Further, the induced antibodies effectively neutralized infection of 293TT cells with pseudovirions. This finding demonstrates that the TRV expression system is comparable to other plant expression systems and due to the broad host range of TRV is particularly attractive when expression in plants with low content of toxic alkaloids is desired. Moreover, a monoclonal anti-L1 antibody E2 raised in the course of immunization with crude extract from freeze-dried leaves expressing L1 is specific preferentially against HPV VLPs and could be used in direct ELISA for monitoring of VLPs assembly and VLP purification protocols.

The influence of cytokinin thidiazuron (TDZ) and auxin indole-3-acetic acid (IAA) on in vitro shoot organogenesis of fifteen Rhododendron genotypes was investigated and a protocol for high frequency adventitious shoot regeneration from leaf explants was developed. High genotypic variation was observed and regeneration frequencies ranged from 0 to 100 %. Genotype Ovation had the highest number of shoots (26.4 per explant) after 12 weeks on medium with 0.57 µM IAA and 1.20 µM TDZ, but only 65 % of explants regenerated. Catawbiense Grandiflorum had 17.7 shoots per explant and 75 % regeneration on medium with 5.70 µM IAA and 0.45 µM TDZ and Van Werden Poelman had 14.3 shoots per explant and 100 % regeneration on medium with 0 57 µM IAA and 0.45 µM TDZ.

We evaluated the combined effects of elevated CO₂ and water availability on photosynthesis in barley. Soil and plant water content decreased with water stress, but less under elevated CO₂ concentration (EC) compared with ambient CO₂ concentration (AC). During water stress, stomatal conductance, carboxylation rate, RuBP regeneration, and the rate of triose phosphate utilisation (TPU) were decreased but less when plants grew under EC. Drought treatments caused only a slight effect on maximum photochemical efficiency (variable to maximum fluorescence ratio, F_v/F_m), whereas the actual quantum yield (Φ_PS2), maximum electron transport rate (J_max) and photochemical quenching (qP) were decreased and the non photochemical quenching (NPQ) was enhanced. Under water deficit, the allocation of electrons to CO₂ assimilation was diminished by 49 % at AC and by 26 % at EC while the allocation to O₂ reduction was increased by 15 % at AC and by 12 % at EC.

It is hypothesized that since protein α-amylase inhibitor (α-AI) and stimulator might be present together in red kidney bean (Phaseolus vulgaris L.) seeds, their in vitro interactions might influence their detection and quantification. Assay of α-AI using extracts from the embryonic axes revealed an unexpected finding in that the extracts stimulated rather than inhibited α-amylase activity. The cotyledon extracts exhibited inhibitory or enhancement effect on α-amylase activity depending on whether prior to the α-amylase assay they had been boiled for 10 min or not. Phytohemagglutinin (PHA-L in particular) is implicated in the present study as a stimulator of α-amylase activity co-extracted with α-AI from red kidney bean cotyledons. The importance of these findings is discussed in relation to the possible widespread occurrence of protein α-amylase stimulator in seeds and other plant parts.

The effect of plant growth regulators (PGRs), gelling agents, sucrose and heat shock on shoot multiplication, shoot growth, rooting and subsequent survival of Chlorophytum borivilianum Sant. et Fernand was evaluated. Benzyladenine (BA) was found to be better cytokinin over kinetin (KIN) for shoot multiplication. Sucrose concentrations from 116-290 mM in the basal medium (BM) promoted shoot multiplication. Heat shock (50 °C, 1 h) also promoted shoot multiplication at these sucrose concentrations on both BM medium and BM supplemented with 5.0 μM BA. Beneficial effect of sucrose was also observed on rooting of shoots on BM as well as BM supplemented with 5.0 μM indole-3-butyric acid (IBA). Phytagel as a gelling agent was found to be more effective for shoot proliferation and growth compared to agar. Amongst various soil mixtures tested, higher survival of plants was observed in soil containing Vermicompost. It was interesting to note that a maximum plant survival (> 95 %) was observed when plants were directly transferred to net-house (irradiance reduced to 50 % with green net, without humidity and temperature control) than poly-house (with humidity and temperature control). Random amplified polymorphic DNA (RAPD) and inter simple sequence repeat (ISSR) analysis of regenerated plants showed genetic similarity to mother plant.

The aim of the study was to examine the effect of exogenous 24-epibrassinolide on its uptake and content of endogenous brassinosteroids in wheat seedlings. 24-Epibrassinolide was applied at two concentrations (0.1 and 2.0 μM) and in three different methods: by soaking seeds, by drenching and by spraying plants. Brassinosteroids were determined by high-performance liquid chromatography combined with electrospray mass spectrometry. Three important brassinosteroids, 24-epibrassinolide, brassinolide and castasterone, were detected in the wheat leaves, but their contents varied with leaf insertion and plant age. Increased 24-epibrassinolide content in the leaf tissue was found when this hormone was applied by soaking or drenching. Additionally the seed treatment influenced brassinosteroid balance in seedlings. The growth response of wheat seedlings treated with 24-epibrassinolide has been also investigated.

Cotyledonary leaves of 9-d-old tomato (Lycopersicon esculentum Mill.) were co-cultivated with Agrobacterium tumefaciens GV 3101 harboring binary vector pBI101 containing kanamycin resistance gene (npt II) as selection marker. Murashige and Skoog (MS) inorganic salts with Gamborg's B5 vitamins supplemented with optimized concentrations of zeatin riboside and indole-acetic acid resulted in enhanced regeneration efficiency. Under optimized conditions of plant regeneration, transformation frequency in cvs. Pusa Ruby, Pusa Uphar and DT-39 was greater than 37 %. Transformed shoots were selected on kanamycin medium and the presence of the transgene in the primary transformants was confirmed by PCR. Integration of the npt II gene in the tomato genome was further confirmed by Southern blot analysis. RT-PCR analysis using neomycin phospho-transferase (npt II) gene-specific primers confirmed the expression of the transgene in transgenic plants. Transformed plants were successfully transferred to phytotron, where these plants grew to maturity and produced flowers and fruits.

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.

Plants face variable environmental stresses that negatively affect plant growth and productivity. The multiplicity of responses is an important aspect of the complexity of stress signalling. Abscisic acid (ABA) is a broad-spectrum phytohormone involved not only in regulating stomatal opening, growth and development but also in coordinating various stress signal transduction pathways in plants during abiotic stresses. The both ABA-dependent and ABA-independent signal transduction pathways from stress signal perception to gene expression involve different transcription factors such as DREB, MYC/MYB, AREB/ABF, NAM, ATAF1,2, CUC and their corresponding cis-acting elements DRE, MYCRS/MYBRS, ABRE, NACRS. Genetic analysis of ABA mutants has given insight that ABA-dependent and ABA-independent pathways for osmotic stress and cold stress interact and converge. This review focuses on ABA-dependent and ABA-independent transcriptional components and cascades, their specificity and crosstalk in stress gene regulation.

Leucoanthocyanidin reductase (LAR) converts leucoanthocyanidin to (+)-catechin, a precursor of proanthocyanidins abundant in Japanese persimmon (Diospyros kaki Thunb.) fruits. A putative LAR gene (DkLAR) was isolated by rapid amplification of cDNA ends from young fruits. The full-length cDNA of DkLAR gene was 1 356 bp long and encoded an open reading frame of 349 residues. The deduced DkLAR protein was closely related to the homolog in other plant species. The expression of the DkLAR gene in Chinese pollination-constant non-astringent (PCNA) genotype was coincident with the tannin cell development, but was not in Japanese PCNA and Chinese pollination-variant astringent (PCA) genotypes.

We describe the multi-step regeneration system of medicinal plant Schisandra chinensis (Turcz.) Baill. The seeds were pre-treated with 0.005 μM thidiazuron. Subsequently the zygotic embryos of the early heart stage were cultured on medium with 50 μM of 2,4-dichlorophenoxyacetic acid (2,4-D) and after three weeks the embryogenic calli were transferred to a medium with 10 μM of 2,4-D and 4 μM of 6-benzyladenine and were sub-cultured at the 4-week intervals. Abscisic acid (30 μM) and polyethyleneglycol (3 %) significantly influenced the synchronization of development of the somatic embryos (SEs) to the globular stage. The following culture on a medium without growth regulators resulted in full developed cotyledonary stage SEs. Indole-3-butyric acid (0.05 μ) contributed to their rapid conversion to plantlets.

The effects of nitrogen [75 and 150 kg (N) ha^-1] and elevated CO₂ on growth, photosynthetic rate, contents of soluble leaf proteins and activities of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) and nitrate reductase (NR) were studied on wheat (Triticum aestivum L. cv. HD-2285) grown in open top chambers under either ambient (AC) or elevated (EC) CO₂ concentration (350 ± 50, 600 ± 50 μmol mol^-1) and analyzed at 40, 60 and 90 d after sowing. Plants grown under EC showed greater photosynthetic rate and were taller and attained greater leaf area along with higher total plant dry mass at all growth stages than those grown under AC. Total soluble and Rubisco protein contents decreased under EC but the activation of Rubisco was higher at EC with higher N supply. Nitrogen increased the NR activity whereas EC reduced it. Thus, EC causes increased growth and P_N ability per unit uptake of N in wheat plants, even if N is limiting.

The aim of this research was to carry out a critical study of the method of obtaining size equivalence between non-symbiotic alfalfa and alfalfa associated with Glomus and/or Rhizobium by applying fixed addition rates of nutrients to the non-symbiotic controls. The experimental design included three nutrient response curves in which the levels of added phosphorus and/or nitrogen were constant during the whole plant growth process: 1) a phosphorus response curve, in order to compare the growth of double symbiotic plants with that of only-Rhizobium inoculated ones; 2) a nitrogen response curve, that consisted of a comparison between the growth of double symbiotic alfalfa and four treatments associated only with Glomus; 3) a phosphorus and nitrogen response curve, to compare the growth of non-inoculated alfalfa with that of double symbiotic plants. Although similar size was achieved among some treatments at harvest, shoot growth over time and nutrient concentrations in tissues differed, indicating that growth equivalence did not mean functional equivalence. A second experimental design was performed taking into account the establishment of microsymbionts for determining the adequate moment to add supplemental phosphorus and/or nitrogen. It included four treatments: a) double symbiotic plants (MR); b) plants inoculated with Rhizobium only (R); c) plants inoculated with Glomus only (M), and d) non-inoculated plants (N). Great similarity in terms of plant growth and nutrient contents in tissues were obtained. Moreover, symbiotic plants were able to produce similar dry matter than non-symbiotic ones under P and N limitations.

This paper describes multiple shoot regeneration from leaf and nodal segments of a medicinally important herb Centella asiatica L. on Murashige and Skoog's (MS) medium supplemented with a range of growth regulators. The highest number of multiple shoots was observed on MS augmented with 3.0 mg dm^-3 N⁶-benzylaminopurine (BAP) and 0.05 mg dm^-3 α-naphthaleneacetic acid (NAA). Leaf explant showed maximum percentage of cultures regenerating shoots (81.6 %), with the highest shoot number (8.3 shoots per explant) and the shoot length (2.1 cm) whereas, nodal explant showed less number of shoots with callus formation at the base cut end. Successive shoot cultures were established by repeatedly sub-culturing the original explant on a fresh medium. Rooting of in vitro raised shoots was best induced on half strength MS supplemented with 0.5 mg dm^-3 indole-3-butyric acid (IBA) with highest percentage of shoot regenerating roots (76.8 %) with 3-4 roots per shoot. Plantlets were acclimated in Vermi-compost and eventually established in soil. Contents of chlorophyll, total sugars, reducing sugars and proteins were estimated in leaf tissue from both in vivo and in vitro raised plants. Chlorophyll content was higher in in vivo plants, whereas other three components were higher in in vitro plants.

Plant water potential (ψ), its components, and gas exchange data of two Mediterranean co-occurring woody species (Quercus ilex L. and Phillyrea latifolia L.) were measured in response to seasonal changes in water availability over two consecutive years. The relative contribution of physiological and morphological adjustments to drought resistance was assessed through Principal Component Analyses. There were large adjustments in stomatal conductance (∼36 % of accounted variance). Net photosynthetic rate and water use efficiency were closely tuned to water availability and accounted for ∼17 % of variance. The slope of the water potential vs. relative water content (dψ/dRWC₀) below zero pressure potential increased as a result of seasonal and ontogenic increases in apoplastic water fraction and accounted for ∼20 % variance. This tolerance mechanism was accompanied by an increased range of positive pressure potential, suggesting a functional role of sclerophylly in these Mediterranean evergreens. Similarly, changes in the slope of dψ/dRWC in the range of positive pressure potential (∼13 % of accounted variance) were associated to variations in cell wall elasticity and resulted in lower RWC at zero pressure potential. When considering the species studied separately, the results indicated the primary role of stomatal regulation in the drought resistance of Qilex, while increased apoplastic water fraction had a major contribution in the drought resistance of P. latifolia.

In vitro regeneration from leaf, cotyledon and hypocotyl explants of six cultivars belonging to three species of Capsicum was achieved by direct organogenesis. The cultivar Umorok showed the best response while Meiteimorok, Haomorok, Mashingkha and Uchithi showed intermediate response and the cultivar Chiengpi was the least responsive. Leaf and cotyledon explants regenerated more shoots than hypocotyl explants and the maximum number of shoots were produced on Murashige and Skoog (1962) medium containing 8.8 µM 6-benzylaminopurine (BAP) with 11.4 µM indole-3-acetic acid (IAA). Elongation of shoot buds derived from different explants was achieved on medium containing 2.8 µM IAA and the elongated shoots were rooted on medium containing 2.8 or 5.7 µM IAA and 2.4 or 4.9 µM indole-3-butyric acid (IBA). Four-week old rooted plantlets were hardened and transplanted to the soil. The plantlets showed 90 % survival during transplantation.

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.

Alterations in free and conjugated polyamines (PAs) and the enzymes involved in their biosynthesis, namely arginine decarboxylase, ornithine decarboxylase and S-adenosyl methionine decarboxylase have been reported to occur during cell division, growth, embryogenesis and rhizogenesis in an array of plant materials. However, the relationship, if any, between them and all these processes has not yet been established. It seems that specific PAs at specific concentration ranges are required during critical stages of growth and morphogenetic events. Furthermore, the effects of PA biosynthesis inhibitors vary considerably at different developmental stages of the same tissue. The present review deals with the available information about the possible role of PAs in aforesaid physiological processes.

The effects of photoperiod on the development of in vitro grown plantlets of yam (Dioscorea alata L.), were investigated. Plantlets were transplanted into pots, acclimatizated until they reached vegetative stages V₁ (3 leaves) or V₂ (8 leaves), and then grown under 12-h or 16-h photoperiod. The formation and development of underground tubers was only induced under 12-h photoperiod. Tuber initiation was not related to the initial vegetative stage of plants, and the tubers were visible at about 18 - 24 d. On the contrary, a 16-h photoperiod inhibited tuber formation and stimulated vine and leaf growth. The total dry matter production and the number of leaves per plant of V₁ stage plants were 50 and 30 % lower respectively, after 44 d under 12-h compared to 16-h photoperiod. These parameters were not influenced by photoperiod in V₂ stage plants. Consequently, the effect of 12-h photoperiod on dry matter of V₁ plants was attributed to a source limitation related to the early initiation of tuberization. The transfer of plants grown under 12-h to 16-h photoperiod stopped tuber growth and starch accumulation. On the other hand, it stimulated the shoots and the roots to grow.

An in vitro propagation system was developed for castor-bean (Ricinus communis L. cv. TMV 6) through cotyledon derived callus cultures. The impact of different concentrations of auxins, cytokinins, additives, amino acids and sugars were evaluated for callus induction and shoot proliferation. Green compact nodular organogenic callus was obtained on the medium fortified with Murashige and Skoog (MS) salts, B5 vitamins, 2.0 mg dm^-3 6-benzyladenine and 0.8 mg dm^-3 α-naphthalene acetic acid (NAA). Multiple shoot proliferation from the callus cultures was achieved on the medium with MS salts, B5 vitamins, 2.5 mg dm^-3 thidiazuron (TDZ), 0.4 mg dm^-3 NAA and 15 mg dm^-3 glutamine. During multiple shoot induction the phenolic secretion was controlled by the addition of 15 mg dm^-3 polyvinylpyrolidone. The proliferated shoots were elongated on the medium comprising MS salts, B5 vitamins, 1.5 mg dm^-3 TDZ and 0.3 mg dm^-3 gibberellic acid. The elongated shoots were rooted on the medium containing MS salts, B5 vitamins, 0.3 mg dm^-3 indole-3-butyric acid and 0.6 mg dm^-3 silver nitrate. After root induction, the plants were hardened in earthen pots containing sand, soil and vermiculite.

The effect of plant growth regulators (PGR), 6-benzyladenine (BA), 2,4-dichlorophenoxyacetic acid (2,4-D), α-naphthaleneacetic acid (NAA) and sugars (sucrose, maltose, glucose, fructose) on the initiation of somatic embryogenesis of Pinus nigra Arn. was investigated. Megagametophytes containing immature zygotic embryos have been used as explants. The experiments were done in the years 2000 and 2001. Higher initiation frequencies were obtained in 2001 when the zygotic embryos showed uniformity, being in the precotyledonary stage of development. Embryogenic tissue initiation occurred on all the media tested, including PGR-free medium. Relatively high initiation frequencies were obtained on media containing either NAA (9.09 %) or 2,4-D (7.14 %) alone. Somatic embryos were present as bipolar structures and showed differences in morphological features among cell lines. Plantlet regeneration occurred in cell lines containing bipolar somatic embryos composed of compact meristematic embryo "head" and suspensor organized into bundles.

In this report we describe the most suitable protocol for callus formation and plant regeneration for cotton. We screened 15 cotton (Gossypium hirsutum L.) genotypes for metal resistance and two of them, Nazilli M-503 (M503) Nazilli 143 (N-143) selected as Cd, Cu and Ni resistant. The cotyledonary nodes from these genotypes were the best explants for regeneration of shoots (more than 90 %) and roots (50 to 70 %). Shoot apex also gave good shoot regeneration (more than 90 %) but their root regeneration efficiency was low (35 %). These results show that Murashige and Skoog (MS) media containing 0.44 μM naphthaleneacetic acid (NAA) and 0.98 μM indole-3-butyric acid (IBA) was the most suitable recipe for getting high shoot and root regeneration from cotyledonary nodes of N-143 and M503 cotton genotypes.

Solanum nigrum is a model system especially for newcomer to the subject of plant tissue culture. Shoot culture has been easily established from shoot cutting of germinated seeds on Gamborg (B5), or Murashige and Skoog (MS) medium without phytohormones. Direct regeneration was possible using basal media B5, B5C (B5 supplemented with 5 % coconut endosperm milk), Schenk and Hildebrandt (SH), and MS, leaf, stem, shoot tip as explants, cytokinins benzylaminopurine (BAP) or kinetin (KIN) at concentrations from 0.25 to 2 mg dm^-3, and different light treatments (dark, dim and normal light). The best culture condition for shoot formation was the culture of stem internode segments on B5 medium supplemented with 0.5 mg dm^-3 BAP at 16-h photoperiod (irradiance of 100 µmol m^-2 s^-1). Also, root formation was possible under different culture conditions. The best culture condition was the culture of microshoot segments on half strength MS medium supplemented with 1 mg dm^-3 isobutyric acid. Induction of callus formation from young and mature tissues on MS medium supplemented with 0.5 mg dm^-3 BAP, 0.1 mg dm^-3 2,4-dichlorophenoxyacetic acid and 1 mg dm^-3 naphthalene acetic acid, and subsequent plant regeneration on B5C medium supplemented with 0.5 mg dm^-3 BAP was easy. Regeneration of protoplasts isolated from shoot tips and fully expanded leaves was also simple. Finally, the transfer of rooted plantlets to the soil was successful.