When growing in the field, plants are exposed to the effect of heavy metals as soon as the seed comes into contact with the soil solution. Therefore, we found important to study the effect of Cd and Ni on maize exposed to these heavy metals since sowing. The aim of this work was to examine which anatomical changes are induced by continuous intoxication of young maize root system with 0.1 mM Cd and Ni, thus modifying its growth and capacity for water and nutrient uptake. Concomitantly, the effect on concentration and distribution of Cd, Ni and some essential ions (Ca, Mg, Zn, Fe, Cu and Mn) was studied.

Plant regeneration via direct somatic embryogenesis from cotyledons, hypocotyls and leaves in seabuckthorn (Hippophae rhamnoides L.) was achieved. The influences of basal media, carbon sources, plant growth regulators (PGRs) with different concentrations and combinations on embryogenesis capacity of explants were studied. The highest frequency of somatic embryos production and germination was obtained on Schenk and Hildebrandt medium (SH) supplemented with 1.0 mg dm^-3 kinetin and 0.2, 0.5 mg dm^-3 indole-3-acetic acid. Granulated sugar was the optimal carbon source. The embryo-derived plantlets with well-developed roots and shoots were transferred successfully to the greenhouse with a maximum survival rate of 55 %. Histological observation revealed that the somatic embryos were similar to those of zygotic embryos.

Embryogenesis and plant regeneration was achieved from callus cultures derived from unpollinated ovaries of Psoralea corylifolia L. Callus was initiated from unpollinated ovaries on Murashige and Skoog (MS) medium supplemented with 2.2 µM N ⁶-benzyladenine (BA) and various concentrations of α-naphthaleneacetic acid (NAA (2.7 to 10.7 µM) or 2,4-dichlorophenoxyacetic acid (2,4-D (2.3 to 9 µM) alone or in combination. Highly organized embryogenic callus induction, embryo development, proliferation and maturation were achieved on transfer of callus clumps to MS medium supplemented with NAA (0.27 µM) or 2,4-D (0.23 µM) alone or in combination with BA (2.2 to 8.8 µM). Addition of abscisic acid (ABA) (0.95 to 5.8 µM) to the medium enhanced average numbers of cotyledonary stage embryos, the maximum number (34.6 ± 0.7) being obtained on MS medium containing 0.27 µM NAA, 2.2 µM BA and 3.8 µM ABA. Embryos germinated on MS medium supplemented with BA (0 to 8.8 µM). MS medium containing gibberellic acid (GA₃ (0.29 to 5.8 µM) enhanced embryo germination frequency, the highest frequency (66.7 %) occurring on MS medium containing 2.2 µM BA and 4.3 µM GA₃. Effect of several concentrations (3.0 to 6.0 %) of sucrose or maltose was also observed on germination of embryos. MS medium enriched with maltose supported high frequency of embryo germination.

The influence of different sugars (sucrose, maltose, glucose and fructose, 0.05-0.5 M) on embryogenesis and plant regeneration from cultured anthers of niger [Guizotia abyssinica (L. f.) Cass.] have been studied. Among the different sugars tested, 0.2 M sucrose was the best for embryo induction and plant regeneration. Maximum of 57 embryos per 60 anthers were induced on embryo induction medium [Gamborg's B5 medium supplemented with 10 µM 2,4-dichlorophenoxyacetic acid (2,4-D), 2 µM kinetin (KIN)] containing 0.2 M sucrose. Embryo differentiation was achieved on B5 medium supplemented with 0.5 µM benzyladenine (BA) and 0.09 M sucrose. Embryo maturation was on B5 medium containing 10 µM abscisic acid (ABA) and 0.09 M sucrose. Embryo germination was achieved on B5 medium with 0.09 M sucrose. Embryos that were developed on B5 medium supplemented with 0.2 M sucrose showed highest frequency (68 %) of plant regeneration.

Aspects of plant growth such as height, branch number, leaf number, leaf area, pod area, 100-seed mass, etc., were correlated with biochemical changes such as contents of chlorophyll (Chl), proteins, DNA, and RNA, and protease activity during development and senescent phases in leaves, flowers, and pods of Cajanus cajan L. cv. UPAS-120 after treatments with kinetin (Kn). A significant increase was noticed in branch number, leaf number, leaf area, and seed mass while other growth processes registered a small increase after Kn application. Effectiveness of 5 µM Kn was also noticed in minimizing the loss of Chls, proteins, and nucleic acids as well as reducing the protease activity during maturity and senescence. Chl a/b ratio maintained a high value up to 30-d followed by a decline in leaves while flowers registered much lower ratio at 20-d-age. Pods were unique in having relatively lower ratio of Chl a/b in comparison to leaves.

We report here the development of transgenic tobacco plants with thaumatin gene of Thaumatococcus daniellii under the control of a strong constitutive promoter-CaMV 35S. Both polymerase chain reaction and genomic Southern analysis confirmed the integration of transgene. Transgenic plants exhibited enhanced resistance with delayed disease symptoms against fungal diseases caused by Pythium aphanidermatum and Rhizoctonia solani. The leaf extract from transgenic plants effectively inhibited the mycelial growth of these pathogenic fungi in vitro. The transgenic seeds exhibited higher germination percentage and seedling survival under salinity and PEG-mediated drought stress as compared to the untransformed controls. These observations suggest that thaumatin gene can confer tolerance to both fungal pathogens and abiotic stresses.

Protein bodies (PBs) of European black pine (Pinus nigra Arn.) were isolated from mature seeds. Extracted soluble matrix proteins and crystalloid proteins PBs proteins were investigated by SDS-PAGE electrophoresis in presence and absence of 2-mercaptoethanol. The proteins of molecular masses 16, 17, 18, 61 and 65 kDa were presented only in crystalloid protein samples. Only 15 kDa protein was present in soluble matrix proteins and not in crystalloid proteins. Another protein bands were present in both soluble matrix and crystalloid proteins. 20, 37, 38, 39 and 48 kDa proteins were strongly visible among crystalloid proteins. Bands of 23 and 32 kDa were more visible in soluble matrix protein samples. Different composition in crystalloid proteins was found in absence of 2-mercaptoethanol: no proteins with molecular mass 71 kDa and more proteins in soluble matrix. In case of crystalloid proteins we detected 7 protein bands in interval from 71 to 212 kDa.

Young plants of a rhizomatous grass Calamagrostis epigejos (L.) Roth were grown from seed in nutrient solutions containing nitrogen in concentrations 0.1, 1.0, and 10 mM. After six weeks of cultivation the plants were defoliated and changes in growth parameters and in content of storage compounds were measured in the course of regrowth under highly reduced nitrogen availability. Plants grown at higher nitrogen supply before defoliation had higher amount of all types of nitrogen storage compounds (nitrates, free amino acids, soluble proteins), which was beneficial for their regrowth rate, in spite of lower content of storage saccharides. Amino acids and soluble proteins from roots and stubble bases were the most important sources of storage compounds for regrowth of the shoot. Faster growth of plants with higher N content was mediated by greater leaf area expansion and greater number of leaves. In plants with lower contents of N compounds number of green leaves decreased after defoliation significantly and senescing leaves presumably served as N source for other growing organs. Results suggest that internal N reserves can support regrowth of plants after defoliation even under fluctuating external N availability. Faster regrowth of C. epigejos with more reserves was mediated mainly by changes in plant morphogenesis.

Four rice indica genotypes of local importance were transformed with RC7, rice chitinase cDNA clone through Agrobacterium-mediated gene transfer method using mature seed derived calli as explants. The putative hygromycin resistant calli showed varied level of regeneration efficiency ranging from 2.0 to 7.6 %. The stable integration and expression of RC7 was confirmed through polymerase chain reaction (PCR) and Western analysis. Transformation efficiency ranged from 0.9 to 5.2 %. The expression of RC7 (35 kDa chitinase) in different tissues of transgenic plant (root, sheath and leaf) was proved through Western analysis and in terms of increased chitinase activity. The inheritance of transgene was studied through PCR and Western analysis in transgenic plants of Pusa Basmati 1. Bioassays with transgenic plants of local cultivars exhibited enhanced resistance up to 33.3 % to rice sheath blight pathogen Rhizoctonia solani under glasshouse conditions. Enhanced expression or 3-to 4-fold increased activity of chitinase in transgenic plants was correlated with sheath blight resistance.

Some maize (Zea mays L.) genotypes produced husk leaves without leaf blades. However, the physiological implication of this leaf deformity is unclear. Difference in protein pattern was observed between maize with and without husk leaf blades. A clear band around 38[sim ]40 kDa in seeds of maize genotypes without husk leaf blades appeared, while it was not detected in ones with husk leaf blades. These protein might be involved in leaf blade intiation.

Electrophoretic seed protein patterns of 18 samples belonging to 14 species and 4 sections of Lathyrus are treated by principle component analysis (PCA). The morphological ground and karyological structure data of these samples are also discussed in the light of sectional and groups delimitation. The species under study of section Cicercula are separated into 3 groups and L. hirsutus referred to the most primitive species within Lathyrus species. This agrees with their previous grouping delimination based on morphological characters and with chromosomal features such as karyotype structure. L. aphaca referred to the most advanced species in this genus, which agrees with the modification of this morphological characteristics and reduction in chromosome criteria. Section Nissolia hasan intermediate position between section Cicercula and section Aphaca.

Transgenic rice plants harbouring Bacillus subtilis protoporphyrinogen oxidase (Protox) gene, which is targeted into plastid, were generated by Agrobacterium-mediated transformation using a rice (Oryza sativa L. cv. Dongjin) and their gene integration at T₁ generation by Southern and mRNA expression in T₂ generation by Northern blotting were analyzed. Their herbicide-resistant trait was further confirmed by in vitro leaf segment assay and in planta bioassays such as seed germination assay and measurement of growth inhibition. The herbicide oxyfluorfen resistance in transgenic rice plants was not very high. The results showed that the Protox from B. subtilis can not be applicable as a gene source to generate a high level oxyfluorfen tolerance in plants.

The dependency of radicle elongation in Abies numidica somatic embryos on germination media has been studied. No significant differences were detected between the Murashige and Skoog (MS) and Schenk and Hildebrandt (SH) medium. The addition of 10 g dm^-3 activated charcoal or 0.05 mg dm^-3 indole-3-butyric acid (IBA) into both media had positive influence on embryo germination. Difference between activated charcoal and IBA effects were significant. The high rooting percentage (85 %) was recorded on half SH medium with 10 g dm^-3 sucrose and activated charcoal. After IBA addition rooting percentage was increased to 95 %. During 7 months 73 % of plantlets survived transfer to soil and in 54 % of plantlets shoot growth was observed.

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

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.

Transgenic tobacco (Nicotiana tobacum) plants carrying the gene coding for potato virus A (PVA) non-structural P3 protein were prepared by inoculation with Agrobacterium tumefaciens. Seeds from self-pollinated flowers (T₁ generation) were collected. To estimate the effectiveness of vertical transfer of the introduced gene and usefulness of respective plant lines for further experiments, the T₁ generation was characterized by testing its ability to grow in the presence of kanamycin (Km) and by PCR of both neomycin phosphotransferase (nptII) and PVA P3 genes. Eight and ten of 29 lines showed Mendelian segregation of Km-resistant phenotype 3:1 and ≥15:1, respectively, the T₁ of eleven lines showed low Km resistance. Selected PCR-positive lines were tested for the presence of P3 mRNA. In most cases the transgene transcription was dependent on the presence or absence of Km in the plant growth medium. Prepared transgenic plants were furthermore tested for sensitivity to PVA and potato virus Y (PVY) infection. All of them showed identical symptom development as the non-transgenic control plants.

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.

Somatic embryogenesis of European chestnut (Castanea sativa Mill.) was obtained using juvenile tissue cultured on P24 medium with 5 μM 2,4-dichlorophenoxyacetic acid plus 0.5 μM 6-benzylaminopurine (BA) for three weeks and then cultured on 0.89 μM BA. Induction frequency with ovaries ranged from 2.0 to 19.1 % and was observed in tissue collected 2 to 8 weeks postanthesis, ovules used as a starting tissue gained 0.8 to 7.8 %, 3 to 9 weeks postanthesis. Zygotic embryos collected 5 to 10 weeks postanthesis formed 10.5 to 57.1 % somatic embryos, respectively. The culture lines were maintained via secondary embryogenesis on P24 medium with 0.89 μM BA. Development and maturation were stimulated on P24 medium with increased agar concentration (1.1 %). Five plantlets were transferred to substrate and acclimatized successfully in greenhouse.

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.

Changes of morphogenic competence in mature P. sylvestris L. buds due to frozen storage were investigated. The highest callus formation was registered on explants stored at -18°C for three months, but on explants stored for five months, it was also higher than in the control. Budding and development of needles in vitro was observed only for buds frozen three to five months. Peroxidase activity was lowest in these buds. In contrast, polyphenol oxidase activity in bud tissues continually increased during frozen storage. Within 10 months of frozen storage the content of starch and sugars in resting buds changed. It may be concluded that changes in composition of non-structural sugars in pine buds after five months of frozen storage are part of metabolic changes leading to loss of morphogenic capacity.

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.

The changes in endopeptidase activity in different parts of germinating triticale cv. Malno were investigated. Haemoglobin, gliadin, azocasein and azoalbumin were used as substrates. During the first day of germination the activity of haemoglobin hydrolyzing endopeptidases predominated while after the second day, mainly in the endosperm, a rapid increase in endopeptidases activity preferring gliadin hydrolysis was observed. In all the investigated tissues azocaseinolytic activities increased with the successive days of germination. Similar changes were observed using azoalbumin with one exception: in the embryo axis this activity decreased with the progression of germination. Separation of endopeptidases on the DEAE Sepharose CL-6B reveals three activity peaks in extract from dry seeds and four peaks in extract from 3 d germinated seeds. The obtained peaks differed in substrate specificity and in sensitivities to class-specific inhibitors.

Excised embrya and subsequently divided embrya of Podophyllum peltatum were cultured on the Murashige and Skoog medium supplemented with different growth regulators, because traditional methods of breaking seed dormancy failed. The growth of excised embrya was stimulated by 1 or 0.1 mg dm-3gibberellic acid (GA3), 0.1 mg dm-3 GA3 + 0.2 mg dm-3 kinetin (kin), or 0.2mg dm-3 kin. GA3 (1 mg dm-3) showed the best effect; after 5 weeks the plantlets had 1.5 - 2 cm long cotyledons, 5 - 6 cm long roots, 88 % of embrya germinated and developed further. The addition of 0.5 mg dm-3 zeatin + 0.2 mg dm-3 naphtaleneacetic acid (NAA), 0.2 mg dm-3 NAA, and 1 mg dm-3 kinetin inhibited the growth of embrya. 1 mg dm-3 kinetin + 0.1 mg dm-3 NAA, 0.1 mg dm-3 zeatin and 0.2 mg dm-3 6-benzylaminopurine resulted in a compact appearance of plantlets and a lower germination rate. Divided embryo cultures produced plantlets via somatic embryogenesis which occurred only on the 2,4-dichlorophenoxyacetic acid containing media. The maturation of somatic embrya was observed on media without any auxin.

The effects of zirconium ascorbate (Zr-ASC), a water-soluble complex of Zr, were examined on wheat seedlings (Triticum aestivum L. cv. MV. 20). Hydroponically grown plants were exposed to 10, 33, 55, 100 and 550 µM Zr-ASC (Zr₁₀, Zr₃₃etc.). After 9 d of treatment inhibition of germination, retarded root and shoot growth, and increased activities of antioxidant enzymes (guaiacol peroxidase, ascorbate peroxidase, and glutathione reductase) showed that Zr-ASC was only harmful at and over a concentration limit of 100 µM. Chlorophyll (Chl) content of plants was only decreased by Zr₅₅₀. Zr-ASC at lower concentrations was beneficial for plant development: Zr₁₀ and Zr₃₃ enhanced root elongation, Zr₅₅ induced about 30 % increase in the total Chl content, while the activity of antioxidant enzymes was not elevated indicating that no oxidative stress was generated by the intracellularly accumulated Zr⁴⁺ ions.

Effects of NaCl on growth in vitro and contents of sugars, free proline and proteins in the seedlings and leaf explants of Nicotiana tabacum cv. Virginia were investigated. The fresh and dry mass of the seedlings decreased under salinity. These growth parameters in leaf explants decreased at 50 mM NaCl and increased up to 150 mM NaCl and then decreased at higher level of salinity. Free proline content in both seedlings and leaf explants increased and polysaccharide content decreased continuously with increasing of NaCl concentration. Reducing sugars, oligosaccharides, soluble sugars and total sugars contents in both seedlings and leaf explants decreased up to 150 mM NaCl and then increased at higher concentrations of NaCl.

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.

The purpose of this research is micropropagation of Ginkgo biloba L. Apical and nodal meristems removed from plantlets or apical buds from a tree were used as explants. Meristems produced an extensive callus and single or rare multiple shoots on Murashige and Skoog medium with different growth regulators and endosperm extract (En) obtained from mature seeds of the same species. For successful root production it was necessary to transfer the shoots to a rooting medium with En.

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.