In the present study, a polymerase chain reaction (PCR)-based method namely inter simple sequence repeat (ISSR) was employed to assess genetic stability in tissue culture-derived Dictyospermum ovalifolium plantlets. To study genomic stability of micropropagated plants, 14 individuals were randomly tagged among a population of 2500 regenerants and were compared with single donor mother plant. A total of 51 clear and reproducible bands ranging from 200 bp to 2.1 kb were scored corresponding to an average of 3.64 bands per primer. Two of the 51 bands were polymorphic (3.92 %) among 14 individuals, thus indicating the occurrence of low level genomic variation in the micropropagated plants. Cluster analysis indicates that genetic similarity values were 0.978 which allows classification of the plants to distinct groups. Further an attempt was made to reintroduce the micropropagated plants into their natural habitat. Over one thousand six hundred fifty plants were successfully established.

The effects of salinity on growth, leaf nutrient content, water relations, gas exchange parameters and chlorophyll fluorescence were studied in six-month-old seedlings of citrus (Citrus limonia Osbeck) and rooted cuttings of olive (Olea europaea L. cv. Arbequina). Citrus and olive were grown in a greenhouse and watered with half strength Hoagland's solution plus 0 or 50 mM NaCl for citrus, or plus 0 or 100 mM NaCl for olive. Salinity increased Cl^- and Na⁺ content in leaves and roots in both species and reduced total plant dry mass, net photosynthetic rate and stomatal conductance. Decreased growth and gas exchange was apparently due to a toxic effect of Cl^- and/or Na⁺ and not due to osmotic stress since both species were able to osmotically adjust to maintain pressure potential higher than in non-salinized leaves. Internal CO₂ concentration in the mesophyll was not reduced in either species. Salinity decreased leaf chlorophyll a content only in citrus.

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.

In this work the volatiles emitted from in vitro shoot-cultures and micropropagated plants of Lavandula viridis L'Hér. were characterized and compared with those obtained from the field-grown mother-plant, using headspace solid phase micro-extraction following by capillary gas chromatography coupled to mass spectrometry (HS-SPME-GC/MS). The headspace composition consisted mainly in oxygenated monoterpenes (66.7-79.2 %), where the major constituents emitted by the mature field-grown mother-plant, in vitro shoot-cultures and micropropagated plants were 1,8-cineole (74.0, 51.9 and 57.8 %) and camphor (2.9, 15.3 and 8.7 %), respectively. The headspace of in vitro shoot-cultures and micropropagated plants showed greater amount of α-pinene, camphene, β-pinene, β-selinene and selina-3,7(11)-diene, when compared with the field-grown mother-plant.

The influence of freezing treatment on plasma membrane (PM) H⁺-ATPase was investigated using plasma membrane vesicles isolated from calluses from Chorispora bungeana Fisch. & C.A. Mey. by the discontinuous sucrose gradient centrifugation. Freezing treatment (-4 °C) for 5 d resulted in significant increases in the ATPase activity and the activity of p-nitrophenyl phosphate (PNPP) hydrolysis, decreases in the K_m for ATP hydrolysis and PNPP hydrolysis, and the shift of optimal pH from 6.5 to 7.0. Also, the activity PNPP hydrolysis was less sensitive to vanadate after freezing treatment compared to control, while the inhibition of ATP hydrolysis by hydroxylamine was more sensitive. In addition, freezing treatment also decreased the activation effects of trypsin on PNPP hydrolysis, but increased the activation effects of lysophosphatidylcholine on ATP hydrolysis. Taken together, these results suggested that PM H⁺-ATPase might play an important role during adaptation to freezing and enhancing the frost hardness in C. bungeana.

Micropropagated plants of two annual haloxerophytic Asiatic Salsola species (S. pestifer and S. lanata) were obtained from zygotic embryos cultured on Murashige and Skoog (MS) agar medium supplemented with 0.5 µM benzylamino-purine (BAP) and 0.3 µM indole-3-acetic acid (IAA) or with 0.5 µM 6 γ, γ-dimethylallylaminopurine and 0.3 μM IAA. The callus induction from shoot and leaf explants derived from plants propagated in vitro were obtained on MS agar medium with various concentration of auxins and cytokinins. The best medium for growth and proliferation of calluses of both studied species was MS medium containing 9.0 µM 2,4-dichlorophenoxyacetic acid. It was also determined that beginning of plant regeneration from callus of S. lanata was induced by 8.8 µM BAP.

Agrobacterium tumefaciens mediated vacuum infiltration transformation in planta has been established in pakchoi, a kind of Chinese cabbage, but the transformation frequency in harvested seeds has varied in the range of 0.5 to 3.0 × 10^-4 over several years and is much lower than the transformation frequency in Arabidopsis thaliana. To understand that, the distribution and vitality changes of A. tumefaciens in plant tissues were examined. Results revealed that there was a majority of A. tumefaciens in the flower compared with that in the stem and in the leaf at all times after infiltration. As fact of transformants in the upper part of the treated plant (T₀) stalk and fact of the survival of A. tumefaciens in the plant were proved, possibilities of optimizing the transformation conditions to increase the transformation frequency in pakchoi was discussed.

Inter simple sequence repeat (ISSR) marker assay was employed to validate the genetic fidelity of Swertia chirayita plantlets multiplied in vitro by axillary multiplication upto forty-two passages. Sixteen ISSR primers generated a total of 102 amplicons among the tissue-cultured plants. Forty-eight amplicons were amplified in the outlier (a Swertia species). The outlier (negative control) was employed to rule out the possibility that the invariant fingerprint was due to chance alone and that the ISSR technique employed was not discriminatory enough to detect the off-types. A homogenous amplification profile was observed for all the micropropagated plants. The results confirmed the clonal fidelity of the tissue culture-raised S. chirayita plantlets and corroborated the fact that axillary multiplication is the safest mode for multiplication of true to type plants.

The contents of three major steviol glycosides (SGs) (stevioside and rebaudiosides A and C) in vegetative and generative organs during ontogeny of Stevia rebaudiana Bertoni were analysed with HPLC. Plant organs contained different amounts of the SGs, which declined in the following order: leaves, flowers, stems, seeds, roots. The highest content of the SGs was found in upper young actively growing shoot sections, whereas lower senescent shoot sections exhibited the lowest amount of such compounds. During ontogeny a gradual increase in the SG content was observed in both mature stevia leaves and stems, and this process lasted up to the budding phase and the onset of flowering.

Chenopodium rubrum, a short-day plant, and C. murale, a long-day plant, were grown in vitro in continuous darkness. Control C. rubrum plants exposed to continuous darkness for 15 d at cotyledonary phase, did not flower, while 80 % of plants flowered on the medium with 5 % glucose and 10 mg dm^-3 GA₃. Control C. murale plants exposed to continuous darkness for 10 d at the age of 4^th pair of leaves, did not flower, while GA₃ (1 - 5 mg dm^-3) stimulated flowering up to 65 %.

A protocol was developed for direct differentiation of multiple shoot buds from leaf explants of Cajanus cajan. In a modified Murashige and Skoog's medium supplemented with 2.22 µM benzyladenine (BA), 0.57 µM indole-3-acetic acid (IAA) and 41 µM adenine sulphate (AdS), the segments of basal halves of the first two leaves of a young seedling incubated on filter paper bridges in liquid medium took 20 - 25 d to differentiate shoot buds. The explants after transfer to solidified medium, with lower concentration of BA (0.22 μM) resulted in fast growing healthy shoots. The developed shoots (measuring ca. 3 cm) were rooted in a medium supplemented with 1.42 µM IAA. They were subsequently grown in pots with soil with more than 80 % transplantation success.

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.

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.

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.

Potted tomato plants (Lycopersicon esculentum Mill. cv. Amalia) were submitted to three different treatments: control (C) plants were maintained at day/night temperature of 25/18 °C; preconditioned plants (PS) were submitted to two consecutive periods of 4 d each, of 30/23 and 35/28 °C before being exposed to a heat stress (40/33 °C lasting 4 d) and non-preconditioned (S) plants were maintained in the same conditions as the C plants and exposed to the heat stress. The inhibition of plant growth was observed only in PS plants. Heat stress decreased chlorophyll content, net photosynthetic rate and stomatal conductance in both PS and S plants. However, PS plants showed good osmotic adjustment, which enabled them to maintain leaf pressure potential higher than in S plants. Furthermore, at the end of the recovery period PS plants had higher pressure potential and stomatal conductance than in S plants.

In vitro organogenesis was achieved from callus derived from hypocotyl explants of Cucumis sativus L. cv. Poinsett 76. Calli were induced from hypocotyl explants excised from 7-d-old seedlings grown on Murashige and Skoog (MS) medium containing 87.64 µM sucrose, 0.8 % agar, 3.62 µM 2,4-dichlorophenoxy acetic acid and 2.22 µM 6-benzyladenine (BA). Regeneration of adventitious buds from callus (25 shoots explant^-1) was achieved on MS medium supplemented with 8.88 µM BA, 2.5 µM zeatin and 10 % coconut water after two subcultures in the same medium at 30-d interval. Gibberellic acid (1.75 µM) favoured shoot elongation and indole 3-butyric acid (7.36 µM) induced rooting. Rooted plants were hardened and successfully established in soil.

An efficient protocol of direct somatic embryogenesis (without involving intermediate callus) has been developed from stem segments and shoot tips of Capsicum annuum L. Explants were cultured on Murashige and Skoog (MS) medium supplemented with thidiazuron (TDZ). Among the various concentration of TDZ tested, 0.5 μM was proved to be best for induction of somatic embryos. Induction, maturation and germination were achieved on the same medium. The shoots developed from somatic embryos were transferred for rooting to MS medium supplemented with indole-3-butyric acid (IBA). All the regenerated plants with 85 % survival rate were normal with respect to morphology and growth characteristics.

The relationship between nutrient composition, crop biomass, and glutamate dehydrogenase (GDH) isoenzyme pattern was investigated in soybean (Glycine max) and maize (Zea mays) by monitoring the nutrient induced isomerization of the enzyme from the seedling stage to the mature crop. GDH was extracted from the leaves of the plants, and the isoenzymes were fractionated by isoelectric focusing followed by native polyacrylamide gel electrophoresis. The isomerization V_max values for soybean GDH, similar to maize GDH increased curvilinearly from 200 - 400 μmol mg^-1 min^-1 as the inorganic phosphate nutrient applied to the soil decreased from 50 - 0 mM. In soybean, combinations of N and K, P, or S nutrients induced the acidic and neutral isoenzymes, and gave biomass increases 25 - 50 % higher than the control plant. GDH isoenzymes were suppressed in soybean that received nutrients without N, K, or P and accordingly the biomass was about 30 % lower than the control. Treatment of maize with NPK nutrients increased the GDH V_max values from 138.9 at the vegetative to 256.4 μmol mg^-1 min^-1 at the reproductive phase, and suppressed the basic isoenzymes, but induced both the acidic and neutral isoenzymes thereby inducing seed production (27.0 ± 1.4 g per plant); whereas both the acidic and basic isoenzymes were suppressed in the control maize, and seeds did not develop. Simultaneous induction of the acidic, neutral, and basic isoenzymes of GDH indicated the occurrence of senescence. Therefore in maize and soybean, the induction of the acidic and basic isoenzymes of GDH led to the enhancement of biomass.

Late blight of potato, caused by Phytophthora infestans was studied by using a resistant clone of potato on one side and a susceptible clone on the other side. A gene coding putatively for an α-galactosidase has been isolated by mRNA reverse transcription polymerase chain reaction differential display and was shown to be differentially expressed between the resistant and the susceptible clone. α-Galactosidases catalyse the hydrolysis of α-1,6 linked α-galactose residues from oligosaccharides and it could be shown in the present work that raffinose content decreases at 30 h after infection by P. infestans in the resistant clone.

Seed storage proteins of Ebenus cretica were fractionated to albumins, globulins, prolamins and glutelins according to their solubility in water, 0.5 M NaCl solution, 55 % propanol-2 and 0.125 M sodium borate (pH 9.0) containing 0.5 % SDS (sodium dodecyl sulfate) solution, respectively. Glutelins consist of the major (about 81 %) fraction of the total extracted proteins. Analysis by SDS-PAGE revealed that the total extracted protein patterns from different racemes of the same plant were similar, while those from seeds of different plants were different. In addition, distinct differences were observed within protein patterns of alkaline extractable glutelin fractions and salt soluble globulin fractions. In E. cretica four ecotypes (A - D) were distinguished by SDS-PAGE of total extracted seed proteins. The last method was more simple and rapid than others and was suggested for screening analysis.

Agrobacterium tumefaciens strain EHA105 carrying a binary vector pCAMBIA2301, which contains a neomycin phosphotransferase gene (nptII) and a β-glucuronidase (GUS) gene (uidA) interrupted with an intron, was used for transformation of Vigna mungo cotyledonary node explants. Various factors such as preculture and wounding of explants, manipulations in inoculation and co-cultivation conditions were found to play a significant role in influencing tissue competence, Agrobacterium virulence and compatibility of both, for achieving the maximum transformation frequencies. The stable transformation with 4.31 % efficiency was achieved using the optimized conditions. The transformed green shoots that were selected and rooted on medium containing kanamycin and tested positive for nptII gene by polymerase chain reaction were established in soil to collect seeds. GUS activity was detected in leaves, roots, pollen grains and T₁ seedlings. Southern analysis of T₀ plants showed the integration of nptII into the plant genome.

Foliar sprays of water or 1, 10 and 100 µM aqueous solutions of gibberellic acid (GA₃) or kinetin (KIN) were applied to 40-d-old plants of Nigella sativa (L.) to study their effects on net photosynthetic rate, nitrogen metabolism, and the seed yield. 10 µM solutions of both the hormones, especially GA₃, appreciably increased the activities of nitrate reductase and carbonic anhydrase, chlorophyll and total protein contents and net photosynthetic rate in the leaves, along with capsule number and seed yield plant^-1, at harvest.

Immunocytochemical analysis using antibody raised against human H2AX histones phosphorylated at serine 139 (γ-H2AX) demonstrates that root meristem cells of Vicia faba exposed to UV-radiation or incubated with hydroxyurea (HU) reveal discrete foci at the border of the nucleolus and perinucleolar chromatin or scattered over the whole area of cell nucleus. Western blots detected only one protein band at the position expected for the phosphorylated form of H2AX. The dose-effect relationship was demonstrated following treatment with 2.5 and 10 mM HU. Proteins extracted from root meristems incubated for 2 h either with HU and caffeine or with HU and sodium metavanadate showed unchanged amounts of bound γ-H2AX antibodies, as compared to root meristems treated with 2.5 mM HU. Higher quantities of phosphorylated H2AX histones were detected in proteins extracted from roots treated with HU and 2-aminopurine. All treatments were effective in producing evident aberrations of premature mitosis: broken and lagging chromatids, acentric fragments, chromosomal bridges and micronuclei. Our results show that phosphorylation of H2AX at the carboxy-terminal Ser-Gln-Glu sequence is among the earliest responses to double-strand breaks and, presumably, one of the key ATM/ATR-dependent signals indispensable for the repair of spontaneous and induced DNA damage in plant cells.

Plants of Miscanthus sinensis (cv. Giganteus) were grown in hydroponics for three months in nutrient solution with 0, 2.2, 4.4 and 6.6 μM CdNO₃. Growth parameters, catalase (CAT), guaiacol peroxidase (POD), ascorbate peroxidase (APX) and superoxide dismutase (SOD) activities were analysed in leaves and roots collected after 1-and 3-month exposure. Dry biomass of all miscanthus organs was affected by Cd concentration both after 1-and 3-month exposure. No visible symptoms of Cd toxicity were observed in shoots and rhizomes of plants grown in presence of Cd. In contrast, roots became shorter and thicker and the whole root system more dense and compact already after one month of treatment with 6.6 μM Cd. The lower Cd concentration increased the enzymes activities after 3 months in leaves and only after 1-month in roots, while a decrease in activity was observed at higher Cd concentrations.

The plants of pigeonpea (Cajanus cajan L.) cv. H77-216 were subjected to moderate [soil moisture content (SMC) = 7.3 ± 0.5 %] and severe (SMC = 4.3 ± 0.5 %) drought by withholding the irrigation at vegetative stage (45 d after sowing). The control plants were maintained at SMC of 11.0 ± 0.5 %. Half of the stressed plants were re-irrigated and their recovery was studied after 2 d. Leaf water potential, osmotic potential, and relative water content of leaf and root decreased significantly while a sharp rise in proline and total soluble sugars contents were noticed. Drought induced a significant increase in 1-aminocyclopropane 1-carboxylic acid (ACC) content and ACC oxidase activity which caused a considerable increase in ethylene evolution. Malondialdehyde content and relative stress injury were increased under drought whereas reverse was true for ascorbic acid content. The membrane integrity of roots decreased during stress and recovered on rehydration. The specific activity of total superoxide dismutase, ascorbate peroxidase, glutathione reductase, and glutathione transferase decreased to 37 - 78 %, 17 - 62 %, 29 - 36 % and 57 - 79 % at moderate and severe drought, respectively. The increase in activity of catalase and peroxidase could not overcome the accumulation of H₂O₂ content in the roots.

The human papillomavirus type 16 (HPV 16) oncogene E7 fused with the gene for β-glucuronidase (gus) was used in plant transformation experiments. The E7 gene modified for lower cancerogenicity and fused with the 5' end of the gus in cassettes with cauliflower mosaic virus 35S promoter and transcription terminator produced high contents of fusion proteins in potato protoplasts. Expression vectors harbouring E7 fusion cassettes were used for Agrobacterium tumefaciens LBA4404 mediated transformation of either potato (Solanum tuberosum L. cv. Bintje) or tomato (Lycopersicon esculentum Mill. cv. Moneymaker). A fusion gene was found in all rooted regenerants using polymerase chain reaction with primers providing amplified fragments from E7 and gus genes. GUS activity was revealed in all regenerants obtained. Nevertheless, the level of GUS expression in different constructs varied much more than in transient expression experiments with potato protoplasts. Especially, expression level in plants carrying vectors with the whole E7 gene fused with gus was lowered by 2-3 orders of magnitude comparing with fusion of the first 41 codons of E7 and gus. Southern hybridisation of 18 tomato and 23 potato regenerants revealed mostly multiple tandem integration of T-DNA into the plant genome and Western blot proved the presence of the fusion protein in 9 tomato and 11 potato plants out of 41 tested individuals.

The adventitious bud development was induced in epicotyl segments of Valencia sweet orange (Citrus sinensis L. Osbeck). Seeds were cultured in vitro for three weeks in the dark, followed by one week at a 16-h photoperiod. Epicotyl segments were cultured horizontally for the induction of organogenesis in Murashige and Tucker (1969, MT) culture medium supplemented with 1.0 mg dm^-3 benzylaminopurine. Samples were observed by light and scanning electron microscopy from day zero to day 25, when buds were well grown. It was shown that the adventitious buds originated directly from the cambial region on the cut ends of the explants.

The present study evaluated the use of γ-radiation to physically remove selective marker genes previously introduced into the soybean genome. Homozygous seeds from a transgenic soybean line carrying the gus and ahas transgenes were irradiated with γ-rays. Six plants presenting a deleted gus gene were analyzed by Southern blot to confirm removal of both ahas and gus genes. Line 1A presented an absence of the gus gene cassette and presence of the ahas gene cassette.

The effect of excessive Cd on the growth and metal uptake by leafy vegetables Brassica chinensis L. (cv. Wuyueman) and Brassica pekinensis (Lour.) Rupr. (cv. Qingyan 87-114) were studied in hydroponic solution culture. The Cd concentration higher than 10 µM significantly decreased the root elongation and leaf chlorophyll contents of both plant species. The shoots of B. pekinensis had significantly higher concentrations of total and water-soluble Cd than B. chinensis. The roots of both species accumulated more Cd than the shoots in all the Cd treatments. Most of the Cd in the roots was found in the cell walls. The shoot/root ratio of Cd concentrations in B. pekinensis was always greater than that in B. chinensis. But, the concentration and proportion of Cd in the cell walls in B. chinensis were higher than that in B. pekinensis. Cadmium treatments also increased the concentrations of total non-protein thiols (NPT) in the shoots of the both species. A significant correlation was found between the concentrations of soluble Cd and NPT in plant shoots.