The pattern and expression level of β-glucuronidase (gus) reporter gene regulated by six heterologous promoters were studied in transgenic Populus tremula × P. alba plants obtained by Agrobacterium-mediated transformation. Binary vector constructs used contained the following promoter sequences: the CaMV35S from cauliflower mosaic virus; its duplicated version fused to the enhancer sequence from alfalfa mosaic virus; CsVMV from cassava vein mosaic virus; ubiquitin 3 from Arabidopsis thaliana (UBQ3); S-adenosyl-L-methionine synthetase (Sam-s) from soybean; and the rolA from Agrobacterium rhizogenes. Histochemical staining of root, stem and leaf tissues showed phloem and xylem-specific gus expression under rolA promoter, and constitutive expression with the other putative constitutive promoters. Quantitative GUS expression of 10 - 15 independently transformed in vitro grown plants, containing each promoter, was determined by fluorimetric GUS assays. The UBQ3-gus fusion induced the highest average expression level, although an extensive variation in expression levels was observed between independent transgenic lines for all the constructs tested.

Shoot apical meristem-derived calli were transformed with a hLF cDNA in an attempt to produce human lactoferrin (hLF) in transgenic cell suspension cultures of sweet potato [Ipomoea batatas (L.) Lam.]. Calli were bombarded with tungsten particles coated with the binary vector pLSM1 containing a hLF cDNA under the control of the 35S promoter and the neomycin phosphotransferase gene as a selection marker. Calli were then transferred to Murashige and Skoog (MS) medium supplemented with 4.52 µM 2,4-dichlorophenoxyacetic acid (2,4-D) and 100 mg dm^-3 kanamycin. Kanamycin-resistant calli were selected at four-week intervals and subcultured. Cell suspension cultures were established in liquid MS medium with 4.52 µM 2,4-D. Southern and Northern blot analyses confirmed that hLF cDNA was incorporated into the plant genome and was properly expressed in the cells. ELISA analysis showed that transgenic cells produced hLF up to 3.2 µg mg^-1 (total protein).

In the present study, two polymerase chain reaction (PCR)-based methods namely, randomly amplified polymophic DNA (RAPD) and amplification fragment length polymorphism (AFLP) were employed to assess genetic variations, which may appeared, in tissue culture-derived date palm (Phoenix dactylifera) offshoots. Analysis of RAPD banding patterns generated by PCR amplification using 37 random primers gave no evidences for somaclonal variations and the percentage of polymorphic bands in a total of 259 scored bands was zero. Meanwhile, analysis of AFLP banding patterns generated using 13 primer combinations pointed to minor genetic variations in the AFLP banding patterns. The percentage of genetic variations (polymorphism) in tissue culture-derived date palm offshoots belonging to cultivars Sakkoty, Gandila and Bertamoda was 2.6, 0.79 and 1 %, respectively, as revealed by AFLP analysis. The low percentage of genetic variations confirms the genetic stability of tissue culture-derived dry date palm cultivars.

This report describes in vitro shoot induction and plant regeneration from mature nodal explants of Vitex trifolia L. on Murashige and Skoog (MS) medium fortified with benzylaminopurine (BAP), kinetin (KN), thidiazuron (TDZ), adenine (ADE), and 2-isopentenyladenine (2-iP) (0.25 - 10.0 μM). Multiple shoots differentiated directly without callus mediation within 3 weeks when explants were cultured on medium supplemented with cytokinins. The maximum number of shoots (9 shoots per explant) was developed on a medium supplemented with 5.0 μM BAP. Shoot cultures was established repeatedly subculturing the original nodal explant on the same medium. Rooting of shoots was achieved on half strength MS medium supplemented with 0.5 μM naphthaleneacetic acid (NAA). Rooted plantlets transferred to pots containing autoclaved soil and vermiculite mixture (1:1) showed 90 % survival when transferred to outdoor.

We have developed three protocols for the rapid micropropagation of Ruscus aculeatus. The primary explants utilised were immature embryos, aerial buds excised from rhizomes and shoot buds regenerated from organogenic calli. In order to increase the plant regeneration from the primary explants, we used organogenic calli from cladode, stem and rhizome segments. We tested more than 20 culture media for callus induction and shoot regeneration and the best results were obtained when rhizome segments were cultured on Murashige and Skoog medium supplemented with 0.5 mg dm^-3 2,4-dichlorophenoxyacetic acid and 1 mg dm^-3 kinetin.

Hypocotyls of cotton (Gossypium hirsutum L.) cultivars cv. YZ-1, Coker 312 and Coker 201 were inoculated on Murashige and Skoog callus induction medium. YZ-1 exhibited a very high regeneration potential, with 81.9 % of the explants inoculated differentiated into embryogenic callus within 8-10 weeks. During the process of callus maintenance (subculture for 1 to 3 years), the total embryos number in Coker 312 and Coker 201 calli dropped sharply, and the percentage of embryo germination decreased. On the contrary, the callus of YZ-1 consistently maintains a high frequency of plant regeneration after long-time subculture. Transgenic kanamycin-resistant calli of Coker 201 partially lost the ability of somatic embryogenesis and plant regeneration. The stress produced by the transformation procedure slightly affected somatic embryogenesis and plant regeneration of YZ-1, which showed minimum loss of plant regeneration ability.

Alfalfa (Medicago sativa L. cv. Siwa 1) seeds were germinated in polyethylene glycol (PEG 4000) of different concentrations and with or without putrescine. The decrease in water potential of the PEG solution reduced germination rate, germination percentage, and growth criteria (e.g., hypocotyl length, fresh and dry masses of shoot and root), while the root length was increased. The decrease in water potential also reduced the contents of total soluble and reducing sugars, and proteins, and activities of α-and β-amylases and invertase, while increased protease activity. Putrescine treatment improved germination and all growth criteria and increased the activity of the hydrolytic enzymes except protease. In a pot experiment, drought stress was imposed by decreasing the soil moisture. Growth criteria, contents of proteins, chlorophyll a, b and carotenoids, as well as Hill reaction activity decreased while the hydrolytic enzyme activity and total soluble and reducing sugar contents increased under drought stress. Putrescine treatment decreased the activity of the hydrolytic enzymes and increased the polysaccharide, protein and photosynthetic pigment contents, and Hill reaction activity.

Effect of alginate matrix composition on regrowth performance of encapsulated microcuttings of hybrid aspen (Populus tremula L. × P. tremuloides Mincx.) was studied. Both high regrowth frequency and viability of explants were registered in all encapsulation mixtures tested. Some ingredients of the matrix (nutrient medium salts, sugars, growth regulators) significantly affected the initial development of the microcuttings. Sucrose appeared to play an important role in the starting stage of the regrowth event.

The effects of carbon sources (sucrose, glucose, fructose and mannitol) and auxins [indolebutyric acid (IBA) and α-naphthaleneacetic acid (NAA)] on in vitro propagation of banana (Musa spp. AAA) were studied. Over all carbon sources tested, sucrose induced highest frequency of shoot proliferation. Optimal shoot proliferation rates were achieved on the Murashige and Skoog (MS) medium supplemented with sucrose and glucose combination (1:1) at the concentration of 30 g dm^-3. Similarly, higher frequency of root induction was obtained at IBA and NAA combination (1:1; concentration of 2 mg dm^-3) than at other concentrations of IBA or NAA alone or their combinations.

Random amplified polymorphic DNA (RAPD) markers were used to assess the genetic diversity of 14 individuals belonging to 7 populations of Coscinium fenestratum (Gaertn.) Colebr. (Menispermaceae). 18 decamer primers used for the analysis generated 99 scorable bands of which 79 were found to be polymorphic. Coefficient of similarity ranged from 0.6604 to 0.9809. Variation within population was slightly higher than between populations. Similarity between individuals within and between populations was found. Dendrogram was obtained by using unwieghed pair-group method analysis (UPGMA). Distinct accession also exhibited higher percentage of medicinally active compound.

Leaf explants of Phalaenopsis amabilis var. formosa formed clusters of somatic embryos directly from epidermal cells without an intervening callus within 20 - 30 d when cultured on 1/2-strength modified Murashige and Skoog medium supplemented with 0.1, 1 and 3 mg dm^-3 TDZ. Repetitive production of embryos involved secondary embryogenesis could be obtained by culturing segments of embryogenic masses on TDZ-containing media. Plantlet conversion from embryos was successfully achieved on regulator-free growth medium.

A rapid and efficient in vitro plant regeneration method was developed for Aristolochia indica. Multiple shoot formation was induced from shoot tip and nodal explants on Murashige and Skoog (MS) medium with 1 - 6 mg dm^-3 2-isopentenyl-adenine (2-iP) or 1 - 4 mg dm^-3 6-benzyladenine (BA). Maximum number of shoots were induced with 5 mg dm^-3 2-iP alone (about 12 - 14 shoots). Shoot differentiation occurred directly from the leaf bases as well as from the internodes when cultured on 1 - 4 mg dm^-3 BA and 0.8 - 2 mg dm^-3 α-naphthaleneacetic acid (NAA) containing medium. Regeneration from the callus occurred when the calli initiated on MS medium containing 0.6 - 4 mg dm^-3 NAA in combination with 0.8 - 3 mg dm^-3 BA were transferred to 1 - 6 mg dm^-3 BA alone containing medium. Elongated shoots were separated and rooted in MS medium containing 1 mg dm^-3 indole-3-butyric acid. These were then transferred to soil after gradual acclimatization.

The aim of this work was to estimate genetic variability for in vitro culture response of recombinant inbred lines (RILs) of the genus Lycopersicon. The callus percentage (C), the regeneration percentage (R) and the productivity rate (PR) were evaluated 45 d after culture initiation in a set of 16 elite tomato RILs and their parents. The narrow sense heritability (h²) values were 0.38 ± 0.04 for C, 0.46 ± 0.04 for R, and 0.28 ± 0.03 for R, while the genetic correlation (r _g ) values were -0.96 ± 0.07 between C and R, 0.81 ± 0.14 between PR and R, and -0.79 ± 0.16 between PR and C. Three AFLP markers associated to the in vitro traits were identified.

This review summarizes the recent progress made towards the development of transgenic plants with improved tolerance to water stress and salinity. Of the various strategies employed, emphasis has been given to the genes engineered for the biosynthesis of osmoprotectants and osmolytes. This review also briefly discusses the importance of the use of specific stress inducible promoters and the future prospects of transgenic plants with improved agronomic traits.

The effects of osmotic dehydration on the photochemical activity, gene transcription, and protein content of photosystem 2 (PS 2) in leaves of two wheat (Triticum aestivum L.) cultivars Miannnong No. 4 and No. 5 were studied. Roots of both cultivars were submerged into polyethylene glycol (PEG) solutions with an osmotic potential of -0.5 MPa for 0, 24, 48, and 72 h. Relative water content (RWC) decreased markedly after 48 and 72 h. Simultaneously, marked increase in electrolyte leakage, decrease in contents of chlorophylls (Chl) a and b, and inhibition in PS 2 activity were observed. Northern hybridization indicated that progressive water stress remarkably reduced contents of the chloroplast gene psbA and psbD and nuclear gene cab transcripts. Urea-SDS-PAGE and Western blotting analysis showed that the contents of major PS 2 proteins, including the D1 and D2 proteins in the PS 2 reaction centre (RC) and the light-harvesting Chl a/b-protein complex (LHC 2) in periphery, declined with increasing water stress. Miannong No. 5 had less destroyed plasma membranes and higher RWC, Chl contents, and PS 2 activity during water stress than Miannong No. 4, which suggested its better drought resistance. The significant difference in steady state contents of LHC 2 proteins of two cultivars can be mainly attributed to the marked difference in transcript level of cab gene, which indicated that LHC 2 proteins protect PS 2 RC.

Multiple shoots were induced by culturing nodal explants excised from 1-month-old aseptic seedlings of red pepper (Capsicum annuum L. cv. Pusa Jwala) on Murashige and Skoog (MS) medium supplemented with (0.1-10 μM) thidiazuron (TDZ). The rate of multiple shoot induction per explant was maximum (14.4 ± 0.06) on MS medium supplemented with 1.0 μM TDZ. Regenerated shoots were elongated well on growth regulator free MS medium. Adventitious roots were induced two weeks after transfer of elongated shoots to MS medium supplemented with auxins (IAA, IBA or NAA) in different concentrations. Optimum root formation frequency was obtained in medium containing 1.0 μM IBA. Ex-vitro rooting was also achieved by pulse treatment with 300 μM IBA for 10 min. Rooted shoots were transplanted in plastic pots containing garden soil (with 90 % survival rate), where they grew well and attained maturity. Regenerated plants were phenotypically and cytologically normal.

Proline accumulation in two different bean (Phaseolus vulgaris L.) cultivars, one drought-sensitive (Canario 60) and one drought-resistant (Pinto Villa) was investigated. Both tolerated salt concentrations up to 150 mM NaCl, but the sensitive Canario 60 did not survive at 400 mM NaCl. In response to salt stress, both cvs. accumulated proline in all the analyzed tissues, the lowest contents were detected in roots. Pinto Villa accumulated higher proline concentrations than Canario 60 only at 400 mM NaCl. The addition of polyamines or ornithine increased proline content in plant tissues without stress, while they decreased it under salt stress.

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.

Plants of two cytotypes (2n=2x=20, and 2n=3x=30) of pinto peanut (Arachis pintoi Krapov. & W.C. Gregory) were regenerated through somatic embryogenesis. Embryogenic calli were induced from shoot tips or immature leaves dissected from in vitro growing plants. In the case of the diploid peanut the best somatic embryogenesis was achieved when shoot tips were cultured on Murashige and Skoog (MS) medium supplemented with 10 mg dm^-3 Picloram (PIC) and 0.1 mg dm^-3 6-benzylaminopurine (BAP) or when explants from immature leaves were cultured on MS + 10 mg dm^-3 PIC + 0.01 mg dm^-3 BAP. In the case of triploid peanut the highest number of somatic embryos was obtained when shoot tips were cultured on MS + 10 mg dm^-3 PIC + 0.01 mg dm^-3 BAP or when immature leaves were cultured on MS + 20 mg dm^-3 PIC + 0.01 mg dm^-3 BAP. Somatic embryos were converted into plants by culture on MS + 0.01 mg dm^-3 naphthaleneacetic acid + 0.01 mg dm^-3 BAP. Plants were successfully transferred to pots in greenhouse.

Growth parameters and cadmium accumulation were investigated in alfalfa seedlings treated with 10 μM salicylic acid (SA) at the beginning of seed imbibition. Shoot and root growths were accelerated by SA treatment and suppressed by Cd both in presence and absence of SA. Cd accumulation was stimulated by SA in alfalfa seedlings in dependence of the treatment duration. K, Mg, Ca and Fe contents in roots are decreased in the presence of Cd alone, while SA induces a decrease of Mg, Ca and Fe. Shoot K, Mg and Ca concentrations are increased by Cd only in the absence of SA, while SA induces also an increase of these concentrations, but only in the absence of Cd. High negative correlation of Cd concentration with K and Ca concentrations in root indicates a competition for the same carrier not regulated by SA. Positive correlation between Cd and Mg concentrations in shoots, which is decreased by SA pre-treatment, together with the increase of positive correlation between Cd and Fe concentrations in shoots under the influence of SA, indicates a possible mechanism of SA action through maintenance of ionic homeostasis.

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.

Exposure of pea plants to high irradiance (HI) for 60 min caused a reversible photoinhibition as shown by changes in variable to maximum fluorescence ratio (F_v/F_m). A significant decline in F_m was observed in leaves from both pea cultivars subjected to HI, the decrease being higher in JI281 than in JI399 plants. The values recovered during the post-stress period in both cultivars. In both cultivars, minimal fluorescence (F₀) increased under HI, but in cultivar JI399 F₀ recovered to initial value during the post-stress period. The expression of antioxidant enzyme genes was higher in JI399 than in JI281, both in control and stressed plants. In JI281, after 60 min of HI, an induction of the transcripts of CAT, chlMDHAR, cytAPX and cytCu,Zn-SOD was observed, whereas there was a slight increase in PHGPX, stAPX and chlCu,Zn-SOD mRNAs. After 24 h of the recovery period, the induction of some transcripts was not maintained (CAT, cytAPX and cytCu,Zn-SOD), whereas the induction of others was maintained (PHGPX and chlCu,ZnSOD) or even increased (cytGR, stAPX and chlMDHAR). In JI399, CAT and cytAPX were increased strongly after 60 min of HI, and slight increases were observed in cytGR, chlGR and chlMDHAR. In the post-stress period the expression of stAPX, cytGR and chlMDHAR was even slightly higher than after 60 min of HI, however, expression of CAT, cytAPX, cytCu,ZnSOD, chlCu,ZnSOD and chlGR decreased.

We analyzed low molecular mass phenolics, lignin content and both soluble and cell wall bound peroxidase activity in the needles of three Picea omorika (Pancic) Purkyne lines grown in the generative seed orchard. The highest values of the total phenol content as well as of catechine, caffeic acid, coniferyl alcohol, isoferulic acid and lignin concentration were detected in B5 line ("semidichotomy" line). The soluble guaiacol peroxidase activity was the highest in A3 line (line "borealis"). The highest activity of cell wall bound peroxidases was measured in B5 line, and it was in correlation with lignin content.

Ageing of plant organs is accompanied by an increased production of free radicals what results in membrane lipid peroxidation. Non-polar aldehydes originating from this process interact with the cellular material to form the fluorescent end-products, lipofuscin-like pigments (LFP). Their formation was studied both qualitatively and quantitatively in ageing of bean cotyledons. The concentration of lipofuscin-like pigments increased 9-fold in 14-d-old (senescent) cotyledons in relation to 8-d-old (young) cotyledons. HPLC fractionation patterns indicate changes in their composition during ageing. The LFP increase in old cotyledons was accompanied by elevated levels of non-polar aldehydes that increased during ageing to 167 %. The composition of aldehydes was studied by mass spectrometry. The most abundant fraction in both young and old cotyledon was represented by C12 aldehydes, which comprised both saturated and unsaturated species. We have observed differences in abundances of individual aldehydes between the young and the old cotyledons that might explain the differences in the composition of lipofuscin-like pigments. These results support the involvement of free radicals in plant ageing; however, it is suggested that plant aldehydic products of lipid peroxidation differ from those found in animals.

Two genotypes (cv. Smaragd and line DP1059) of Pisum sativum with different susceptibility to Fusarium oxysporum and F. solani and influence of pathogenesis on enzyme activities were studied. The increase of activity of studied enzymes was mostly observed in both roots and shoots during pathogenesis. Only activity of acid phosphatase decreased in the root and increased in shoots. The correlation between enzyme activity change and susceptibility of pea cultivars to F. oxysporum or F. solani was observed.

In this study, a relationship between lipid peroxidation, the antioxidant defense system and salt stress in salt-sensitive cultivated tomato (Lycopersicon esculentum) and its salt-tolerant wild relative (L. pennellii) was established. Superoxide dismutase (SOD) activities were significantly higher in the leaves of L. pennellii than those of L. esculentum after 12 and 84 d. POX activity showed a gradual increase in both cultivars under 70 mM NaCl. POX activity in L. pennellii significantly increased after 6 and 84 d whereas showed no remarkable change in leaves of L. esculentum under 140 mM NaCl. A higher salinity tolerance of L. pennellii was also correlated with a lower lipid peroxidation, which might be due to a higher content of antioxidant enzymes studied.

Direct somatic embryogenesis from ray floret explants of five chrysanthemum cultivars has been obtained within 12 - 15 d on Murashige and Skoog medium supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D) and 6-benzyladenine (BA). Scanning electron microscopic observation also confirmed the direct origin of somatic embryos from explants. Somatic embryos developed asynchronously on the adaxial surface of explants. Among the five cultivars tested, Birbal Sahani was best responding (40 % explants responded on 4 mg dm^-3 2,4-D and 2 mg dm^-3 BA supplemented medium). Precocious germination of somatic embryos was noticed on the same medium. The best sucrose concentration in the medium was found to be 60 g dm^-3 where 70 % explants responded while 55 % embryogenic response was obtained on medium supplemented with 400 mg dm^-3 inositol. Plants developed from somatic embryos were transferred to soil and produced true-to-type flowers.

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.