Rice overexpressed thaumatin-like protein gene and the proteins from the leaf blades of 2-week-old transgenic rice seedlings were fractionated into cytosolic and membrane fractions, and separated by two-dimensional polyacrylamide gel electrophoresis and stained with Commassie brilliant blue. Among of 440 detected proteins, 5 proteins were up-regulated and 5 proteins were down-regulated by the overexpression of thaumatin-like protein. In the sense thaumatin-like protein transgenic rice and/or in rice inoculated with Xanthomonas oryzae pv. oryzae (Xo7435), 2-cys peroxiredoxin, thaumatin-like protein and glycine cleavage H protein were up-regulated, while oxygen evolving complex protein 2 was down-regulated. These results suggest that thaumatin-like protein-mediated disease resistance of rice against bacterial blight disease is the results of changes in proteins related to oxidative stress and energy metabolism in addition to changes in proteins related to defence.

Exposure of barley plants (Hordeum vulgare L.) to soil flooding for 2 to 24 h reduced the net photosynthetic rate and transpiration rate. Stomatal conductance also decreased in flooded plants. Stomatal closure started within 2 - 6 h and stomata remained closed up to 24 h of treatment.

The activities of antioxidative enzymes and contents of proline and total phenolics were assayed in roots of two maize (Zea mays L.) genotypes grown in a medium containing nitrate (NO₃ ^-) or both nitrogen forms, nitrate and ammonium (NH₄ ⁺/NO₃ ^-). An increase in the activities of class III peroxidases (POD), superoxide dismutase (SOD), ascorbate peroxidase (APX), ascorbate oxidase (AO) and proline content, and decrease in phenolic content were observed in NH₄ ⁺/NO₃ ^- in comparison with NO₃ ^- grown plants. When polyethylene glycol (PEG) was added to both nitrogen treatments, the content of total phenolics and proline was increased, especially in NH₄ ⁺/NO₃ ^- treatment. The PEG treatment decreased enzyme activities in NH₄ ⁺/NO₃ ^- grown plants, but in NO₃ ^- grown plants activities of POD and SOD were increased, opposite to decreased APX and AO. Isoelectric focusing demonstrated increased activities of acidic POD isoforms in PEG treated NO₃ ^- grown plants, and lower activities of both, acidic and basic isoforms in NH₄ ⁺/NO₃ ^-grown plants.

Zephyr lily (Zephyranthes grandiflora), an important ornamental plant has been micropropagated in vitro after controlling microbial contamination by a pretreatment with 0.2 % Bavistin and 0.1 % Pantomycin for 4 h before final sterilization with 0.1 % mercuric chloride. In 67 % of the sterile cultures, 11 shoots on average were regenerated directly from basal half of bulb scales in Murashige and Skoog (MS) medium containing 3 % sucrose and 2 mg dm^-3 benzylaminopurine (BAP). Shoots emerged in bunches on a basal achlorophyllous bulbous part. Combination of 2 mg dm^-3 BAP with 1 mg dm^-3 gibberellic acid (GA₃) enhanced shoot growth. Stout roots (maximum of 5-6 per shoot) were developed in presence of 1 mg dm^-3 indole-3-butyric acid (IBA). Micro-bulbs showed potential of regeneration and could be used as secondary explants. The morphologically identical plants derived by in vitro propagation were genetically identical as shown by PCR based ISSR marker analysis of genomic DNA.

In the conversion of oleic acid to linoleic acid, δ¹²-fatty acid desaturase (δ¹²-FAD) is involved. Based on the conserved oligo amino acid residues of the FAD2 genes from other plants, a new full-length cDNA (DiFAD2) encoding a δ¹²-FAD was cloned from Davidia involucrata Baill. Sequence analysis indicated that the DiFAD2 gene had an open reading frame (ORF) of 1 149 bp, coding for 382 amino acids residues of 44.3 kDa, pI of the deduced protein was 8.8. The deduced amino acid sequence of the cloned DiFAD2 showed high identities to those genes of other plant δ¹²-FAD. RT-PCR showed that DiFAD2 was expressed in all tissues and expression was abundant in young stems. Expression of DiFAD2 is not enhanced by low temperature and the altered polyunsaturated fatty acid content in leaves treated with low temperature may be due to the post-transcriptional regulation of the DiFAD2 gene or the other FAD2 gene family regulation.

Secondary embryogenesis from rapeseed microspore-derived embryos (MDEs) was studied in three Brassica napus L. cultivars Global, PF₇₀₄ and Option. The best results in terms of secondary embryogenesis percentage obtained in cultures of Global and PF₇₀₄ MDEs (75.88 and 65.97 %, respectively) and PF₇₀₄ produced the highest number of secondary embryos per each primary embryo (14.91 ± 2.18). After optimization of physical parameters, rapeseed hypocotyls of MDEs were bombarded with microcarriers coated with a plasmid containing GUS reporter gene. The highest levels of transient GUS expression were obtained using bombardment with gold particles of 1.6 µm, at helium pressure of 9.3 MPa, a bombardment distance of 9 cm, chamber vacuum pressure of 7.1 × 10^-6 kPa and single bombardment in bombardment medium containing 0.4 M mannitol.

We determined the cold (freezing) tolerance of five Spanish populations of the perennial shrub Bituminaria bituminosa (L.) C.H. Stirton (Fabaceae), as the temperature at which 50 % of leaf electrolytes are released (LT₅₀) using leaves of field-grown plants, obtained in two winters and one spring. The freezing tolerance was greater in winter and reflected the minimum temperatures at the original sites from which the populations were obtained. Tolerance in vitro was related to osmotic adjustment in the leaves; more negative osmotic potential values and more positive pressure potential values (MPa) were associated with greater tolerance. Tolerance and osmotic potential were not related to leaf cation contents but to leaf amino acids, soluble sugar and proline contents.

The genetic variability of Leucojum valentinum Pau (Amaryllidaceae), a vulnerable endemic species restricted to a small area in the region of Valencia (Eastern Spain), has been studied using random amplified polymorphic DNA (RAPD) markers. A total of 197 individuals from eleven populations were studied using 13 RAPD primers. Our results show high variability for the species, low differentiation among populations and uncorrelated levels of genetic variability and population size. Four groups in which three populations (SAG, PUG and COL) are separated from all the others were found, but without connection to geographical location.

The toxic aromatic compound 3-hydroxy-4-pyridone (HP) is an intermediate in both synthesis and degradation of mimosine, which is produced by the tree legume Leucaena leucocephala. The L. leucocephala root-nodule symbiont Rhizobium TAL1145 contains a dioxygenase (pydA) and a hydrolase (pydB) gene that produce enzymes necessary for the degradation of HP. In order to coordinately express both genes in plant tissues under a single promoter, three different pydA-pydB fusion constructs (G0, G3, and G7) with varying glycine linkers between the two genes were developed. Prior to transferring the fusion constructs into L. leucocephala, which is highly recalcitrant to genetic transformation, we tested the expression and activity of the hybrid proteins in Nicotiana tabacum, a model plant system that can be easily transformed and analyzed. Seven independent transgenic tobacco lines were generated by Agrobacterium-mediated transformation, and stable integration and expression of pydA-pydB in these transgenic lines were confirmed by polymerase chain reaction (PCR), reverse transcriptase PCR (RT-PCR) and Western analysis. Only one of the fusion constructs, G3, containing a 9-nucleotide linker between pydA and pydB, provided significant levels of resistance to 3 mM HP, indicating that the hybrid protein produced by this fusion construct could degrade HP.

A novel protocol for plant regeneration from cotyledon explants of eggplant (Solanum melongena) reducing concentration of sucrose was established. The most efficient bud induction medium consisted of Murashige and Skoog (MS) medium supplemented with 2.0 mg dm^-3 zeatin, 0.1 mg dm^-3 indoleacetic acid and 10 g dm^-3 sucrose. After 15 d, the shoot buds were fragmented and transferred to the shoot elongation MS supplemented with 1.0-2.0 mg dm^-3 gibberellic acid and 4.0-8.0 mg dm^-3 AgNO₃, which promoted shoots elongation. The genetic stability of the regenerated plants was analyzed by flow cytometry, RAPD and SSR molecular markers. The results indicated that almost no somaclonal variation was detected among the regenerants.

Both morphological characteristics and amplified fragment length polymorphism (AFLP) markers were used to validate the genetic fidelity of 1 080 field-grown Echinacea purpurea plants regenerated from leaf explants of donor T5-9. Morphological diagnosis revealed that 1 067 out of 1 080 regenerants were normal, while 13 regenerants were aberrant. AFLP analysis was further performed to assess DNA variations among donor, 43 sampled normal regenerants and all 13 aberrant regenerants. Seven primer combinations generated 471 fragments among donor and normal regenerants, of which 9 fragments were polymorphic. The same primer pairs generated 484 fragments for aberrant regenerants, of which 417 fragments were polymorphic. UPGMA clustering indicated that 42 normal regenerants and donor fell into same cluster at similarity scale of > 0.99, while all 13 aberrant regenerants and one morphologically normal regenerant comprised the other clusters. AFLP analysis indicated that these 14 regenerants are off-types.

Effects of the infection with tobacco mosaic virus (TMV) and potato virus Y (PVY) on chloroplasts from susceptible tobacco plants were determined. Changes in ribonucleases (RNases), phosphomonoesterase (PME), phosphodiesterase (PDE), glucose-6-phosphate dehydrogenase (G6P DH), 6-phosphogluconate dehydrogenase (6PG DH), glucokinase (GK), and fructokinase (FK) activities in thylakoid/envelope and stroma fractions were studied. Slight increase in the activities of PME, PDE, G6P DH and 6PG DH of thylakoid/envelope fraction as well as of RNases, PME, PDE, G6P DH, 6PG DH, GK and FK of stroma fraction was found in chloroplasts isolated from leaf tissues infected with PVY. Infection with TMV produced higher increase in enzymes activities in chloroplasts; especially, PME, G6P DH and 6PG DH in fraction of thylakoid/envelope, and RNases, PME, PDE, G6P DH, 6PG DH, and GK in stroma fraction.

The photosynthetic performance and related leaf traits of Incarvillea delavayi Bur. et Franch were studied at different water regimes to assess its capacity for photosynthetic acclimation to water stress. The initial response of I. delavayi to water stress was the closure of stomata, which resulted in down-regulation of photosynthesis. The stomatal limitation (S_L) represented the main component to photosynthetic limitations but non-stomatal limitation (NS_L) increased quickly with the increasing water stress, and had similar magnitude to SL under severe water stress (soil moisture 25-30 % of field capacity). Chlorophyll (Chl) a fluorescence parameters characterizing photosystem (PS) 2 photochemical efficiency (Φ_PS2), electron transport rate (J) and photochemical quenching (qP) decreased with the increasing water stress, indicating impaired photosynthetic apparatus. However, the water-stressed plants had a increased mesophyll CO₂ diffusional conductance, Chl a/b ratio, leaf nitrogen partitioning in RuBPCO and bioenergetics in later grown parts, indicating that I. delavay had a substantial physiological plasticity and showed a good tolerance to water stress.

The effect of 5-azacytidine (5-azaC) on the alleviation of damaging effects of NaCl treatment was studied in two wheat (Triticum aestivum L.) cultivars differing in salt tolerance (salt-tolerant Dekang-961 and sensitive Lumai-15). The plants were pre-treated or not with 50 μM 5-azaC and then subjected to salt stress induced by 100 or 150 mM NaCl. Salinity caused reduction in biomass accumulation and increase in malondialdehyde content in root tissues in both cultivars, but less in pre-treated seedlings. The activities of superoxide dismutase (SOD), catalase (CAT) and peroxidase (POD) in the roots of both cultivars increased during salt stress, but the rate of increase was higher in Dekang-961. Plants treated with 5-azaC had higher root SOD, CAT and POD activities under salt stress than untreated plants. Content of 5-methylcytosine (5mC) decreased in both cultivars under salt stress, and the level of demethylation was higher in Dekang-961 than that in Lumai-15. Moreover, the degree of methylation was lower in both cultivars under salt stress after 5-azaC application compared to only salt-treated groups. These findings suggested that 5-azaC could protect plants from salt stress.

In this communication, we report the binding of abscisic acid responsive elements (ABREs) of rice Osem, namely motif A and motif B, with a cognate trans-acting factor present in the nuclear extract of tobacco leaf. The binding is specific as both the complexes were disrupted with an excess of homologous non-radioactive DNA like motif A or motif B themselves or with cis-elements of rice Rab16A, motif I (ABRE) and motif IIa (non-ACGT ABRE-like sequences). Four tandem repeats of ABRE from wheat Em (4X ABRE) or two tandem repeats of Em ABRE, plus two copies of coupling element (CE1) from barley HVA22 (2X ABRC), also showed specific complexes, that were competed out by an excess of homologous competitors like motif I, motif IIa, motif A, motif B, 4X ABRE and 2X ABRC, but not by the unrelated 4X DRE sequence. Elution of the protein from all the complexes showed a single 26 kDa polypeptide band. Introgression of two of the above synthetic promoters 4X ABRE and 2X ABRC, each fused with minimal promoter of cauliflower mosaic virus 35S (CaMV 35S), could induce the expression of the reporter gene β-glucuronidase (gus) in transgenic tobacco in response to high NaCl concentration, dehydration or abscisic acid, but not at the constitutive level, proving that they can be used as efficient stress-inducible promoters. Our work shows both in vivo and in vitro activity of the promoters from monocot genes in the model dicot plant tobacco.

Effects of zinc (12-180 μM) alone and in mixtures with 12 μM Cd on metal accumulation, dry masses of roots and shoots, root respiration rate, variable to maximum fluorescence ratio (F_V/F_M), and content of photosynthetic pigments were studied in hydroponically cultivated chamomile (Matricaria recutita) plants. The content of Zn in roots and shoots increased with the increasing external Zn concentration and its accumulation in the roots was higher than that in the shoots. While at lower Zn concentrations (12 and 60 μM) the presence of 12 μM Cd decreased Zn accumulation in the roots, treatment with 120 and 180 μM Zn together with 12 μM Cd caused enhancement of Zn content in the root. Presence of Zn (12-120 μM) decreased Cd accumulation in roots. On the other hand, Cd content in the shoots of plants treated with Zn + Cd exceeded that in the plants treated only with 12 μM Cd. Only higher Zn concentrations (120 and 180 μM) and Zn + Cd mixtures negatively influenced dry mass, chlorophyll (Chl) and carotenoid content, F_V/F_M and root respiration rate. Chl b was reduced to a higher extent than Chl a.

Tubulins are basic components of microtubules and are encoded by a multigene family in eukaryotes. The expression of OsTUB4, one of eight β-tubulin isotypes identified in the rice genome, was characterized. OsTUB4 was expressed in root primodia and the shoot apical meristem in basal parts of the leaf sheath in rice seedlings. OsTUB4 transcript abundance in leaf sheath increased by treatment with gibberellic acid (GA₃) in a dose- and time-dependent manner. OsTUB4 transcript levels in gibberellin (GA)-deficient mutants were less than those of wild type rice. An OsTUB4 promoter::GUS assay also confirmed the responsiveness of OsTUB4 to exogenous GA₃, suggesting that OsTUB4 expression was regulated by GA and may be involved in GA-regulated leaf sheath growth. In addition, OsTUB4 could interact with different specific proteins in vitro as assayed by a yeast two-hybrid system, indicating that OsTUB4 may have diverse functions through interaction with different proteins.

Treatment of tomato seedlings (Lycopersicon esculentum Mill. cv. 63/5 F1) with increasing CdCl₂ concentrations in the culture medium resulted in Cd accumulation more important in roots than in leaves. Biomass production was severely inhibited, even at low Cd concentration. Cd reduced chlorophyll content in leaves and enhanced lipid peroxidation. An increase in antioxidative enzyme (superoxide dismutase, ascorbate peroxidase, guaiacol peroxidase, glutathione reductase) activities was more pronounced in leaves than in roots, while catalase activity increased only in roots. In addition, changes in isoenzyme composition were observed using the non-denaturing polyacrylamid gel electrophoresis.

A rapid plantlet regeneration system for Perilla frutescens was established from cotyledon and hypocotyl explants. A maximum of 91.06 % cotyledon and 76.4 % hypocotyl explants could directly produce shoots (3.09 ± 0.18 shoots per explants) on Murashige and Skoog (MS) medium. The optimum hormone combinations were 4.44 μM 6-benzylaminopurine (BA) for cotyledon and 2.22 μM BA + 2.85 μM indole-3-acetic acid (IAA) for hypocotyls. Rooting was induced on half-strength hormone-free MS medium. After transplantation to soil, approximate 80 % of the regenerated plantlets could survive, flower and fruit. Moreover, some morphological abnormalities were found among the regenerated plants.

The hypocotyl and internodal segments from in vitro grown seedlings of Feronia limonia (L.) Swingle (wood apple) were cultivated on Murashige and Skoog's (1962, MS) medium supplemented with N⁶-benzyladenine (BA) or adenine (ADE) or kinetin (KN) at 0.5 to 5 µM. The optimum response was recorded on the medium containing 2 µM BA. An average of 12 and 8 shoots were developed from hypocotyl and internodal explants, respectively, after eight weeks of culture. The shoots were excised, and the residual explants were transferred to fresh medium where again they developed shoots. Up to three such passages resulted in the production of shoots from repeatedly subcultured explants and an average of 24 - 36 shoots per explant was obtained. The in vitro developed shoots produced roots when transferred to half strength MS medium supplemented with 1 µM 1-naphthaleneacetic acid (NAA). The developed plantlets were successfully transferred to mixture of soil, sand and coco-peat (1:1:1) and hardened in controlled environment. Hardened plants were transplanted to soil in greenhouse.

Organogenetic buds were induced from hypocotyl and cotyledon explants of oil crop Perilla frutescens in Murashige and Skoog (MS) medium supplemented with 5.7 μM indole-3-acetic acid (IAA) and 8.9 - 13.3 μM 6-benzylaminopurine (BA). Shoots were rooted on MS medium with 2.9 μM IAA and 1.4 μM gibberellic acid (GA3) and the regenerated plants flowered and set seeds normally.

Six pea (Pisum sativum L.) cultivars (Adept, Komet, Lantra, Olivin, Oskar, Tyrkys) were transformed via Agrobacterium tumefaciens strain EHA105 with pBIN19 plasmid carrying reporter uidA (β-glucuronidase, GUS, containing potato ST-LS1 intron) gene under the CaMV 35S promoter, and selectable marker gene nptII (neomycin phosphotransferase II) under the nos promoter. Two regeneration systems were used: continual shoot proliferation from axillary buds of cotyledonary node in vitro, and in vivo plant regeneration from imbibed germinating seed with removed testa and one cotyledon. The penetration of Agrobacterium into explants during co-cultivation was supported by sonication or vacuum infiltration treatment. The selection of putative transformants in both regeneration systems carried out on media with 100 mg dm^-3 kanamycin. The presence of introduced genes was verified histochemically (GUS assay) and by means of PCR and Southern blot analysis in T₀ putative transformants and their seed progenies (T₁ to T₃ generations). Both methods, but largely in vivo approach showed to be genotype independent, resulting in efficient and reliable transformation system for pea. The in vivo approach has in addition also benefit of time and money saving, since transgenic plants are obtained in much shorter time. All tested T₀ - T₃ plants were morphologically normal and fertile.

Watermelon (Citrullus lanatus [Trumb.] Mansfeld cv. Early Star), was used as scion grafted onto three cultivars of pumpkin (Cucurbita pepo L. cvs. Brava, Shintoza and Kamel) used as rootstocks and ungrafted Early Star plants were used as control. The rootstocks showed a high capacity for N uptake and transport to the scion where N reduction and assimilation improved growth of the scion in grafted plants with respect to the control.

Inter-simple sequence repeat (ISSR) markers were used to assess the genetic stability of long-term micropropagated plantlets of London plane tree (Platanus acerifolia Willd.). Twenty micropropagated plantlets were chosen from a clonal collection of shoots that originated from a single mother shoot. This clonal collection had been maintained under in vitro culture conditions for at least 8 years, as achieved by axillary branch multiplication. Out of 38 ISSR primers screened, 16 primers were found to produce clear reproducible bands resulting in a total of 103 distinct bands with an average of 6.44 scorable bands per primer. Of these 103 bands, 86 were monomorphic across all 20 of the plants tested and 17 showed polymorphisms (16.5 % polymorphism). Based on the ISSR band data, similarity indices between the plantlets ranged from 0.92 to 1.00. These similarity indices were used to construct an UPGMA dendrogram and demonstrated that all 20 micropropagated plants grouped together in one major cluster with a similarity level of 91 %. A total of 1771 scorable bands were obtained from the full combination of primers and plantlets and only 51 (2.88 %) were polymorphic across the plantlets which indicates that this micropropagated line of P. acerifolia is genetically stable.

To study the effect of sucrose on the sink-source relationship in in vitro-grown plants, Cistus incanus seedlings and plantlets were grown horizontally in a two-compartment Petri dish (split dish), with the root system in one compartment and the shoot in the other. Shoots and roots were exposed to different sucrose concentrations (0-30 g dm^-3), two irradiance levels (25 and 160 µmol m^-2s^-1) and the presence or absence of a minimum medium containing minerals and vitamins (M medium). Root and shoot biomass of the seedlings was enhanced by an increase in irradiance when the growth medium was not supplemented with sucrose indicating the role of photosynthesis in biomass production. When sucrose was added to either organ growth was enhanced as well. In the presence of sucrose in the root compartment, sucrose applied to the shoot compartment enhanced growth of both organs under low irradiance, while under high irradiance, sucrose had no further additive effect. In the absence of sucrose in the root compartment, the enhancement of root biomass by sucrose added to the shoot compartment was lower under high irradiance than under low irradiance. The response of Cistus plantlets to sucrose and irradiance differed from that of seedlings, probably reflecting a greater susceptibility of the plantlets to sucrose feedback inhibition on photosynthesis and biomass accumulation. The decrease in root and shoot growth when M medium was added to the shoot compartment and the relatively better growth of these organs when the roots were supplied with minerals and the shoot with sucrose, indicate that growth of the two organs in our experimental set-up was regulated by opposing fluxes of C and nutrients.

Sucrose concentration in the culture medium affected chlorophyll content, trichome development and amount of solasodine in regenerated plantlets of Solanum trilobatum. High chlorophyll content and glandular trichomes were observed in the plants grown on Murashige and Skoog basal medium supplemented with 131.85 mM sucrose. The solasodine was quantified using reverse phase high performance liquid chromatography. The plantlets cultivated on this medium yielded 35.97 mg g^-1 (d.m.) solasodine whereas the field plants used as control yielded only 2.32 mg g^-1 (d.m.) of solasodine.

A new micropropagation system for Lycium barbarum (L.) was developed using root explants as starting material. Callus can be produced from root explants on Murashige and Skoog (MS) medium containing 0.2 mg dm^-3 2,4-dichlorophenoxyacetic acid. After three subcultures on the same medium, callus was then transferred onto the MS medium supplemented with 500 mg dm^-3 lactalbumin hydrolysate to induce somatic embryogenesis (SE). After 20 d, about 60 somatic embryos per 0.25 g(f.m.) of embryogenic callus were obtained but only about 10 % of embryos converted into plantlets. After acclimated in the greenhouse, all of the randomly selected plantlets had survived and were similar phenotypically to zygotic seedlings. In addition, the effects of irradiance, photoperiod, growth regulators, explant age and cold treatment on SE of root-derived callus were evaluated.

To better understand the mechanisms involved in aluminum toxicity and tolerance in plants, microarray technology was used to evaluate changes in gene expression in Arabidopsis thaliana under Al stress. With the use of Affymetrix Arabidopsis ATH1 Genechip, a comparison of RNA expression profiles was made between control and Al-treated Arabidopsis seedlings. A total of 256 genes were identified as Al-responsive. Ninety-four genes were shown to be up-regulated and 162 were down-regulated; comprising 1.1 % of the 24 000 Arabidopsis genes. Real-time RT-PCR was used to confirm the microarray data. The analysis showed that a large number of transcription factors and several putative signaling components were up-regulated by aluminum. Chloroplast structural and photosynthetic genes were, in general, down-regulated. A number of previously identified Al-responsive genes, e.g. GST, Auxin-regulated, Peroxidase, and Chitinase, were up-regulated by Al-stress, whereas Wali 3 and Wali 4 were down-regulated. We also identified several up-regulated genes involved in vacuolar signaling, sorting and docking. Three genes were also up-regulated by Al-stress, Ras GTP-binding protein, ABC-cassette binding, and the AtELP1 receptor genes, have previously been documented as responsive to drought and/or oxidative stress and may play important roles the detoxification of Al ions by transportation and storage into root vacuoles. Ultrastructural changes in the roots tips cells of Arabidopsis were evaluated using transmission electron microscopy and energy-dispersive X-ray analysis with scanning electron microscopy and results showed Al accumulation in the root tips of Arabidopsis.