We isolated and characterized a stress-inducible gene, designated as Slti114, encoding matrix metalloproteinase (MMP) in soybean. The derived amino acid sequences of Slti114 show the 69 % homology with MMP2 from Glycine max (AAL27029). The size of the full-length cDNA of Slti114 is 1194 bp with open reading frame comprised of 394 amino acids. RNA expression of Slti114 was induced by low temperature or wounding. During early stage, Slti114 RNA level was extremely high, but Slti114 RNA was not detectable just after cotyledons became yellowish. Green fluorescent protein fusion expression system confirmed that Slti114-smGFP and H⁺-ATPase-RFP were co-localized to the plasma membrane. Purified glutathione-S-transferase (GST)-Slti114 protein was shown to digest myelin basic protein (MBP) in vitro, but not gelatin. This report provides strong evidence that plasma membrane MMP, Slti114 protein may play a critical role during abiotic stress and senescence in plant.

Using different explants of in vitro seed grown Scutellaria baicalensis Georgi plantlets, hairy roots were induced following inoculation of Agrobacterium rhizogenes strains A₄GUS, R1000 LBA 9402 and ATCC11325. The A₄GUS proved to be more competent than other strains and the highest transformation rates were observed in cotyledonary leaf explant (42.6 %). The transformed roots appeared after 15-20 d of incubation on hormone free Murashige and Skoog medium. Growth of hairy roots was assessed on the basis of total root elongation, lateral root density and biomass accumulation. Maximum growth rate was recorded in root:medium ratio 1:100 (m/v). Hairy root lines were further established in Gamborg B₅ medium and the biomass increase was maximum from 15 to 30 d. PCR, Southern hybridization and RT-PCR confirmed integration and expression of left and right termini-linked Ri T-DNA fragment of the Ri plasmid from A₄GUS into the genome of Scutellaria baicalensis hairy roots. GUS assay was also performed for further integration and expression. All the clones showed higher growth rate them non-transformed root and accumulated considerable amounts of the root-specific flavonoids. Baicalin content was 14.1-30.0 % of dry root mass which was significantly higher then that of control field grown roots (18 %). The wogonin content varies from 0.08 to 0.18 % among the hairy root clones which was also higher than in non-transformed roots (0.07 %).

The effect of elevated CO₂ concentration (CE) on leaf chlorophyll (Chl) and nitrogen (N) contents and photosynthetic rate (P_N) was evaluated during the post-flowering stages of rice grown at CE (570 ± 50 µmol mol^-1) in open top chamber (OTC), at ambient CO₂ concentration (∼ 365 µmol mol^-1) in OTC and at open field. Thirty-five day old seedlings were transplanted in OTCs or in field and allowed to grow till maturity. Chl and N contents were highest at the time of flowering and thereafter it started to decline. The rate of decline in Chl and N contents was faster in plants grown under CE mostly in later part of growth. Irrespective of treatment difference, flag leaf contained the highest amount of Chl and N than penultimate and third leaf. The higher P_N was observed in leaves under CE than in the leaves in other two growing conditions. Considering growth stage, P_N was the highest at flowering which reduced at the later part of growth due to degradation of Chl and N content of the leaf. Under CE it was 40.02 µmol m^-2 s^-1 at flowering and it reduced to only 14.77 µmol m^-2 s^-1 at maturity stage. The beneficial effect of CE in increasing leaf P_N may be maintained by applying extra dose of nitrogen at the later stages of plant growth.

The changes in activities of soluble and cell wall-bound peroxidases and lignin contents in juglone-stressed soybean (Glycine max) seedlings and their relationships with root growth were investigated. Soybean seedlings (3-d-old) were cultivated in nutrient solution supplemented with 0.5 to 25 μM juglone for 24 h. Length and dry mass of roots decreased after 5 to 25 μM juglone treatments. Low juglone concentrations (≤ 1 μM) increased soluble peroxidase activity, while high concentrations (≥ 10 μM) inhibited activities of soluble and cell wall-bound peroxidases. Juglone (≤ 1 μM) did not affect lignin content but highly increased lignification after 5 to 25 μM treatments. Results indicate that lignification may be an important step in root growth reduction of juglone-stressed soybean.

The fluctuation of endogenous cytokinins was determined in the excised cucumber (Cucumis sativa L.) cotyledons incubated with benzylaminopurine (BA) under irradiance or in darkness. The data indicated that light stimulated the cotyledon expansion compared with dark and BA further enhanced the expansion of cotyledons. However, only BA treatment markedly increased the contents of endogenous cytokinins and induced α-tubulin gene expression. Actinomycin D, a well-known inhibitor of gene transcription, strongly inhibited both light-and BA-induced cotyledon expansions and increase of endogenous cytokinin contents and α-tubulin gene expression. Colchicine, an antimicrotubular reagent, partially inhibited the cotyledon expansion without affecting the cytokinin contents.

This study describes two phenotypes of Arabidopsis thaliana (ecotype Columbia) developed in vitro under salt stress (75 mM NaCl). The phenotypes 01 and 02 appeared visibly distinguishable by rosette morphology and competence to produce flowers. Phenotype 01, sensible to salt stress, accumulated high quantities of Na⁺, showed a slight reduction in dry mass, and high protein and chlorophyll contents. Moreover, its anatomy exhibited some xeromorphic traits. Phenotype 02, clearly salt tolerant, showed a morphology similar to control plants, displaying typical phyllotactic rosette and flowering stalk production. Accumulation of Na⁺, protein and chlorophyll contents were close to control plants. Reversion experiments on NaCl free MS medium, showed a partially recovered phenotype 01. A threshold salt stress concentration that permits the simultaneous development of two phenotypes, was found.

Direct somatic embryogenesis and adventitious shoot formation were successfully achieved from root explants of Centaurium erythrea Gillib. cultured on Murashige and Skoog medium with half-strength macronutrients, full-strength micronutrients and vitamins, 3 % sucrose, 0.7 % agar, 100 mg dm^-3 myo-inositol and without growth regulators. Histological studies revealed that somatic embryos were formed directly from epidermal cells and adventitious buds were developed from meristematic cells in root cortex tissues. Somatic embryos as well as adventitious shoots developed into whole plantlets.

The effect of silicon on the growth, boron concentrations, malondialdehyde (MDA) content, lipoxygenase (LOX) activity, proline (PRO) and H₂O₂ accumulation, and the activities of major antioxidant enzymes [superoxide dismutase (SOD), catalase (CAT), and ascorbate peroxidase (APX)] and non-enzymatic antioxidants (AA) of wheat grown in soil originally with toxic B concentrations were investigated. Applied of 5.0 and 10.0 mM Si to the B toxic soil significantly increased Si concentration of the wheat and counteracted the deleterious effects of B on shoot growth. The contents of PRO, H₂O₂, MDA, and LOX activity of wheat grown in B toxic soil were significantly reduced by Si treatments. Compared with control plants, the activities of SOD, CAT, APX and content of AA were decreased by applied Si. Based on the present work, it can be concluded that Si alleviates B toxicity of wheat by preventing oxidative membrane damage and also translocation of B from root to shoot and/or soil to plant.

In vitro propagation of an anticancerous drug synthesizing plant, Ophiorrhiza prostrata D. Don, was established through indirect somatic embryogenesis. Friable embryogenic calluses were initiated from O. prostrata leaf and internode explants on Murashige and Skoog (MS) media supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D) either alone or in combination with N⁶-benzyladenine (BA) or kinetin (KIN). Somatic embryos were developed after subculture of the friable calluses onto half strength MS media containing 0.45 or 2.26 µM 2,4-D alone or in combination with BA or KIN. Medium supplemented with 2.26 µM 2,4-D and 2.22 µM BA was optimal, supporting the production of a mean of 5.8 globular embryos. Subculture of globular embryo-bearing calluses on half strength MS medium without growth regulators produced the highest embryo frequency, and the majority of them developing to early torpedo stage. Somatic embryos underwent maturation and converted to plantlets at high frequency (90 %) on half strength MS medium supplemented with 0.44 µM BA. Somatic embryo-derived plantlets with well-developed roots were established in field conditions with a 90 % survival rate.

Three types of transgenic plants of Solanum tuberosum cvs. Kamyk and Oreb, and Nicotiana tabacum cvs. Maryland Mammoth and Trapezond were selected according to intensity of introduced ipt gene expression and resulting amount of synthesised cytokinins (CKs). In comparison with controls, original transgenic regenerants grown in vitro showed a massive increase of CK contents, in tobacco by 379 % and in potato by 159 % (MAS). Potato grown in soil from tubers of transgenic plants demonstrated a moderate increase (44 %) of CK contents (MOD). Transgenic tobacco grown from seeds in vitro did not show any significant change in CK contents (NOT). Initial (RuBPC_i and RuBPO_i) and total (RuBPC_t) activities of ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBPCO), and the activity of phosphoenolpyruvate carboxylase (PEPC) were not significantly affected by the transformation in the NOT plants. In the MOD plants, the RuBPCO activities were stimulated by up to 34 % whereas the PEPC activity was decreased by 17 %. On the other hand, all the measured enzyme activities were 32 - 91 % lower in the MAS. Leaf area, fresh and dry masses, and chlorophyll and soluble protein contents also went down with increasing CK amounts in the transformants. Dependence of RuBPC_i/RuBPO_i and RuBPC_t/PEPC ratios on the relative CK amounts in transgenic plants revealed that the individual enzyme activities were not affected uniformly. Endogenous CK contents in the MAS thus apparently exceeded an optimum needed for positive effects on many physiological traits and became a stress factor for such plants.

A new full-length cDNA encoding 3-hydroxy-3-methylglutaryl-CoA synthase (designated as TmHMGS, GenBank Accession No. AY644708), which catalyses the condensation of acetyl CoA and acetoacetyl CoA to form 3-hydroxy-3-methylglutaryl-CoA as an early step in the taxol biosynthetic pathway, was isolated from young leaves of Taxus × media by rapid amplification of cDNA ends (RACE) for the first time. The full-length cDNA of TmHMGS contained a 1431 bp open reading frame (ORF) encoding a deduced protein of 476 amino acid residues. The deduced protein had an isoelectric point of 5.23 and a calculated molecular mass of about 53 kDa. Amino acid sequence comparison analysis showed that TmHMGS had high similarity with a number of HMGSs ranging from Schizosaccharomyces pombe to humans, with much higher identity with other HMGSs from plants than those from yeast and humans. Phylogenic analysis showed that TmHMGS had closest relationship with HMGS from Pinus sylvestris. Tissue expression pattern analysis showed that TmHMGS expressed in needles and stems at similar level, but no expression could be detected in roots. Expression of TmHMGS was all induced by under different elicitors such as silver nitrate, ammonium ceric sulphate and methyl jasmonate, revealed that TmHMGS was an elicitor-responsive gene.

Changes in the conformation of spinach thylakoid membranes were monitored in 5-doxyl stearic acid (SAL)-treated thylakoid membranes in the presence of various anions (Cl^-, Br^-, I^-, NO₂^-, SO₄^2-, PO₄^3-). The presence of anions made the thylakoid membrane more fluid. The extent of change in membrane fluidity differed with different anion and was reversible.

The localization was determined of the triterpenoids, asiaticoside and madecassoside, in different organs of glasshouse-grown plants and cultured material, including transformed roots, of two phenotypes of Centella asiatica (L.) Urban of Malaysian origin. Methanolic extracts of asiaticoside and madecassoside were prepared for gradient HPLC analysis. The two phenotypes of C. asiatica exhibited differences in terpenoid content that were tissue specific and varied between glasshouse-grown plants and tissue culture-derived material. Terpenoid content was highest in leaves, with asiaticoside (0.79 ± 0.03 and 1.15 ± 0.10 % of dry mass) and madecassoside [0.97 ± 0.06 and 1.65 ± 0.01 %(d.m.)] in the fringed (F) and smooth leaf (S) phenotypes, respectively. Roots of the F-phenotype contained the lowest content of asiaticoside [0.12 ± 0.01 %(d.m.)], whereas petioles of S-phenotype plants contained the lowest content of asiaticoside [0.16 ± 0.01 %(d.m.)] and madecassoside [0.18 ± 0.14 %(d.m.)]. Transformed roots were induced using Agrobacterium rhizogens and their growth was maximal on Murashige and Skoog basal medium supplemented with 60 g dm^-3 sucrose. However, asiaticoside and madecassoside were undetectable in transformed roots and undifferentiated callus.

Forty-five-days old plants of Indian senna (Cassia angustifolia Vahl.) were subjected to 0-500 µM lead acetate (Pb-Ac) in pot culture. Changes in contents of thiobarbituric acid reactive substances (TBARS), ascorbate, glutathione, proline, sennosides (a+b), and activities of superoxide dismutase (SOD), ascorbate peroxidase (APX), glutathione reductase (GR), and catalase (CAT) were studied at pre-flowering (60 d after sawing, DAS), flowering (90 DAS) and post-flowering (120 DAS) stages of plant development. Compared with the controls, the Pb-Ac treated plants showed an increase in contents of TBARS, dehydroascorbate, oxidized and total glutathione at all stages of growth. However, sennoside yield and contents of ascorbate and reduced form of glutathione declined. Proline content increased at 60 DAS but declined thereafter. Activities of SOD, APX, GR and CAT were markedly increased. Sennoside content was higher at 60 and 90 DAS but lower at 120 DAS, compared to the control.

In the present study cytokinin dehydrogenase (CKX) activity was for the first time found in a conifer species, Pinus sylvestris. The activities were correlated with the endogenous cytokinin contents. Several enzyme substrates and two different electron acceptors were used to search for the enzyme activity in the extract from seeds, seedlings and plantlets. The highest specific activity was found in one-year-old plantlets with isopentenyladenine as the substrate and 2,6-dichlorophenolindophenol as the electron acceptor, at pH 8. An enhancement in the CKX specific activity corresponded to increasing contents of cytokinins, mainly isopentenyladenine and isopentenyladenosine, indicating that the enzyme activity is affected by the endogenous supply of cytokinins. CKX affinity for the ribosylated form of isopentenyladenine was dependent on the developmental stage, being higher in seeds than in seedlings, and not detectable in plantlets. The results are indicative of the presence of different isoenzymes throughout the development.

Glycinin is one of the abundant storage proteins in soybean seeds. A modified Gy1 (A1aB1b) proglycinin gene with a synthetic DNA encoding four continuous methionines (V3-1) was connected between the hpt gene and the modified green fluorescent protein sGFP(S65T) gene, and a resultant plasmid was introduced into soybean by particle bombardment in order to improve nutritional value of its seeds. After the selection with hygromycin, the efficiency of gene introduction was evaluated. More than 60 % of the regenerated plants tolerant to hygromycin yielded the hpt and V3-1 fragment by polymerase chain reaction (PCR) analysis, and the expression of sGFP was detected in about 50 % of putative transgenic soybeans. Southern hybridization confirmed the presence of transgenes in T0 plants and the transgenic soybeans hybridized with the hpt and V3-1 genes were analyzed showed different banding patterns. Most of the transgenic plants were growing, flowering normally and produced seeds. Analysis of seed obtained from transgenic soybean plants expressing hpt and V3-1 genes showed higher accumulation of glycinin compared with non-transgenic plants. In addition, protein expression in transgenic soybean plants was observed by using 2D-electrophoresis.

The regulation of proline accumulation in detached rice leaves exposed to excess NiSO₄ was investigated. NiSO₄ treatment increased proline and Ni contents but had no effect on relative water content, indicating that proline accumulation in Ni-exposed detached rice leaves was due to Ni uptake per se, rather than to water stress. Proline accumulation caused by NiSO₄ was related to protein hydrolysis, a decrease in proline dehydrogenase activity, and a decrease in proline utilization. It seems that an increase in the content of ammonia and an increase in the activities of Δ¹-pyrroline-5-carboxylate reductase and ornithine-δ-aminotransferase play minor if any role in Ni-induced proline accumulation in detached rice leaves.

Senescence is a genetically regulated process that involves decomposition of cellular structures and distribution of the products of this degradation to other plant parts. Reactions involving reactive oxygen species are the intrinsic features of these processes and their role in senescence is suggested. The malfunction of protection against destruction induced by reactive oxygen species could be the starting point of senescence. This article reviews biochemical changes during senescence in relation to reactive oxygen species and changes in antioxidant protection.

The effect of AlCl₃ on the antioxidant system of rice roots and the role of applied antioxidants ascorbic acid (AsA) and glutathione (GSH) in AlCl₃-inhibited growth of rice roots were investigated. AlCl₃ treatment resulted in a rapid inhibition of root growth but had no effect on lipid peroxidation and antioxidative enzyme activities in rice roots. AlCl₃ treatment resulted in lower content of H₂O₂, AsA, and GSH than in controls. Exogenous AsA or GSH counteracted growth inhibition of rice roots induced by AlCl₃. AlCl₃ treatment increased syringaldazine peroxidase (SPOX) activities and lignin content in rice roots. Exogenous AsA or GSH prevented the decrease in H₂O₂ content and the increase in SPOX activities and lignin content in rice roots caused by AlCl₃. Results suggest that lignification induced by low AsA or GSH content may explain the mechanism of Al-inhibited growth of rice roots.

Embryogenic callus in Catharanthus roseus was initiated from hypocotyl on Murashige and Skoog's (MS) medium supplemented with 1.0-2.0 mg dm^-3 of 2,4-dichlorophenoxyacetic acid (2,4-D) or chlorophenoxyacetic acid (CPA). Calli from other sources were non-embryogenic. Numerous somatic embryos were induced from primary callus on MS medium suplemented with naphthalene acetic acid (NAA) within two weeks of culture. Embryo proliferation was much faster on medium supplemented with 6-benzylaminopurine (BAP). After transfer to medium with gibberellic acid (GA₃, 1.0 mg dm^{- 3}) mature green embryos were developed and germinated well into plantlets on MS liquid medium supplemented with 0.5 mg dm^-3 BAP. Later, embryos with cotyledonary leaves were subjected to different auxins treatments for the development of roots. Before transfer ex vitro, plantlets were cultivated on half strength MS medium containing 3 % sucrose and 0.5 mg dm^-3 BAP for additional 2 weeks. Additionally, the effect of liquid medium has been evaluated at different morphogenetic stages.

Water relations were studied in Norway spruce [Picea abies (L.) Karst.] trees grown at ambient (AC, 350 μmol mol^-1) and elevated (EC, 700 μmol mol^-1) CO₂ concentrations under temperate water stress. The results suggested that both crown position and variability in atmospheric CO₂ concentration are responsible for different patterns of crown water relations. Mean hourly sap flux density (FSA) showed higher values in upper crown position in comparison with the whole crown in both AC and EC treatments. Mean soil-to-leaf hydraulic conductance (G_Tsa) was 1.4 times higher for the upper crown than that calculated across the whole crown for the trees in AC. However, G_Tsa did not vary significantly with crown position in EC trees, suggesting that elevated CO₂ may mitigate differences in hydraulic supply for different canopy layers. The trees in EC treatment exhibited significantly higher values of F_SA measured on the whole crown level and slightly higher soil water content compared to AC treatment, suggesting more economical use of soil water and therefore an advantage under water-limited conditions.

We report here the successful micropropagation of adult Juniperus phoenicea L. with respective ploidy stability studies. Microcuttings with axillary buds were grown on five media supplemented with different growth regulator combinations. Best elongation rates were achieved on Driver and Kuniyuki (DKW) medium supplemented with kinetin alone or with naphthaleneacetic acid (NAA), while Rugini olive (OM) medium stimulated the development of new branches. Shoots growing on Murashige and Skoog (MS) medium browned and showed necrotic zones. Shoots of second to fourth subcultures usually had higher elongation rates than those of the first culture. For rooting assays, half strength DKW and OM media, different concentrations of growth regulators, auxin continuous exposure vs. dipping and the type of solid matrix were assessed. During rooting assays, two morphotypes were observed with one type having well developed internodes and the other showing hyperhydratation and no internode development. High rooting rates (40 %) were only obtained in the first morphotype shoots exposed for 5 min to 2.4 µM IBA and then transferred to OM medium without growth regulators. Plants were acclimatized in pots containing a mixture of peat and Perlite (3:2) in greenhouse with progressive reduction of relative humidity. A flow cytometric screening for major ploidy changes revealed no differences among the morphotypes and between them and the mother plant. Also the nuclear DNA content of this species was estimated for the first time using flow cytometry (2C = 24.71 pg).

Photosystem 1 and 2 and antioxidant enzyme activities were determined in wheat (Triticum aestivum L. cv. Sonalika) leaves. Seedlings from both control seeds and seeds soaked in solutions like dithiothreitol, thioglycollic acid and thiourea were subjected to water stress induced by polyethylene glycol. Photosystem 1 and 2 activities were less inhibited by water stress due to seed soaking with sulphydryl compounds. The changes in activities of antioxidant enzymes induced by water stress were higher in seedlings from thiol-pretreated seeds than from water-soaked seeds.

The apple (Malus domestica Borkh) rootstock M 4 shoots were grown in vitro for 4 weeks on Murashige and Skoog (MS) medium containing three NaCl concentrations (35, 100 and 200 mM) in combination with two CaCl₂ concentrations (5 and 10 mM). Inclusion of 10 mM CaCl₂ in the medium, in the presence of 35 mM NaCl, significantly increased the number of shoots and the fresh mass compared to 5 mM CaCl₂. The number of shoots, length of shoots, and the fresh mass of cultures were very low in the presence of 100 and 200 mM NaCl, independently of CaCl₂ concentration of the medium. By increasing NaCl and CaCl₂ concentrations in the culture medium, contents of N, Na, Cl, proline and soluble sugars in plantlets increased, whereas K, Mg, B, Zn and chlorophyll content decreased in comparison to the control.

Effect of osmotic stress and sodium nitroprusside (SNP, NO donor) pretreatment on growth and proline metabolism of wheat seedlings was investigated. Polyethylene glycol 6000 treatment for 2, 4 and 6 d could be termed as mild, moderate and severe stress, respectively, according to decrease in the relative water content. Severe osmotic stress significantly decreased the growth and photochemical efficiency, and increased proline content due to activation of its synthesis. 0.2 mM SNP pretreatment enhanced growth of wheat seedlings, increased variable to maximum fluorescence ratio (F_v/F_m) and fluorescence yield, while decreased proline content. However, 2 mM SNP retarded the seedlings growth and chlorophyll a fluorescence, and increased proline accumulation. Our results showed that NO might be involved in the regulation of osmotic stress in a concentration-dependent manner.

The interplay between nitric oxide (NO) and reactive oxygen species can lead to an induction of cell death in plants. The aim of our work was to find out if cyanide released from sodium nitroprusside (SNP; a donor of NO) could be involved in the cell death induction, which is triggered by SNP and H₂O₂. Cell suspension of Nicotiana tabacum L. (line BY-2) was treated with 0.5 mM SNP, 0.5 mM potassium ferricyanide (PFC; analogue of sodium nitroprusside which can not release NO) and/or by 0.5 mM glucose with 0.5 U cm^-3 glucose oxidase (GGO; a donor system of H₂O₂). The cell death was induced only by combination of SNP and GGO. Thus cyanide released was not involved in the induction of cell death. However, SNP showed toxic effect because of decrease in activities of intracellular oxidoreductases and esterases. The cell death caused by SNP and GGO occurred within 12 h. During cell death either length or width of the cell increased. Central vacuole was formed in 20 to 40 % of cells. Most of the dead cells showed a condensed cytoplasm. Two hallmarks of programmed cell death (PCD), chromatin condensation and blebbing of nuclear periphery, were observed. However, oligonucleosomal fragmentation of DNA, another hallmark of PCD, was not detected.