In red clover (Trifolium pratense L.) four unique cDNAs encoding phenylalanine ammonia lyase (PAL, EC 4.3.1.5) were identified (PAL1-4). PAL2-4 encode nearly identical proteins (> 97 %) that are only 89 % identical to that encoded by PAL1. Under normal growing conditions, in young leaves and flowers PAL1 mRNA accumulates to higher levels than that of PAL2-4 whereas in mature leaves and stems, mRNA levels are similar for PAL1 and PAL2-4. Treatment of red clover seedlings with yeast elicitor preparation results in an approximately six-fold induction of PAL2-4 transcripts within 1 h of treatment but only a modest induction of PAL1 transcripts. These results suggest that while both classes of enzymes play a role in biosynthesis of phenylpropanoid compounds under normal growing conditions, PAL2-4 enzymes are also involved in pathogen defense responses.

Two maxima in flowering response to one inductive dark period of 13 h were found in the short day plant Chenopodium rubrum within three weeks of cultivation under continuous illumination either in vitro or in vivo. These maxima correlated with the number of leaf primordia and their relation to the size of the apical meristem. The first maximum in flowering responsivity corresponded with the stage when primordia of the second leaf pair had not yet overtopped the apical meristem, the second one when the primordia of the fourth leaf overgrew the meristem. Maximum responsivity to flowering reached by a mother plant was reflected in explants derived from it. The above morphological markers of responsiveness to floral induction were not linked to plant age and/or to general growth habit. The explants flowered only when part of the stem was present.

Somatic embryos differentiated from hypocotyl explant in cotton (Gossypium hirsutum L.) exhibited very divergent morphologies. Six different types of somatic embryos based on cotyledon development were observed. The growth hormones (2,4-dichlorophenoxyacetic acid and kinetin) used in induction and maintenance media did not affect embryo rooting and germination. The 95 % conversion of normal embryos (with two cotyledons) was achieved, while an overall conversion was only 38 %. Horn shaped embryos failed to exhibit shoot growth. Poorly developed apical meristems were responsible for lower conversion percentages in some of embryo classes. However, regenerated plants phenotypically resembled to seed grown control plants regardless of somatic embryo morphology.

Specific activities and isoform patterns of peroxidases, acid phosphatases, DNases and RNases were studied in relation to in vitro rooting of Petunia × hybrida microshoots in the presence of 4 µM indole-3-butyric acid (IBA). Specific activities of the above enzymes increased in the course of rooting. Rhizogenesis could be related with an increased specific activity of peroxidases during the initiation phase, in parallel with increased lignin content. Twelve peroxidases, six anionic (A1-A6) and six cationic (C1-C6), seven acid phosphatases (ACP1-ACP7), seven RNases (R1-R7) and four DNases (D1-D4) isoforms were detected following native PAGE. Variation in the number of the above isoforms and their quantity was observed during different stages of rooting. Particularly, A2, A3, C3, C4, C5, ACP2, R1, R2, R3, and D4 isoforms appeared after the induction phase and could be related to emergence of root primordia. Additionally, R3 and D4 could be associated with cell division and differentiation, since these are only expressed in rooted microshoots. Moreover, the higher number of roots in IBA-treated microshoots could be related to the higher expression of RNase and DNase isoforms during initiation and expression phases.

In six cultivars of rice (Oryza sativa L.), Pusa Basmati 1, Basmati 370, Type III, Pant Dhan 4, CSR 10 and Pokkali, embryogenic callus growth, plant regeneration, and proline and total protein contents were studied under salt stress (on agar solidified media containing 0, 0.5, 1.0, 1.5 and 2.0 % NaCl). Four weeks after inoculation the callus fresh mass decreased with increasing salt concentration in all the six cultivars. The regeneration frequency in salt stressed callus was also lower as compared to control. 15 d and 30 d after inoculation proline content increased several fold whereas total protein content decreased markedly with increase in salt concentration.

The induction of defence compounds and enzymes involved in the phenylpropanoid pathway were studied in the ripe and green chilli fruits inoculated with Colletotrichum capsici and Alternaria alternata. Total phenols and the activity of phenylalanine ammonia lyase (PAL), peroxidase (PO), polyphenol oxidase (PPO) and catalase (CAT) increased in the inoculated ripe and green chilli fruits compared to the corresponding healthy fruits. Total phenols and the activities of the enzymes were at the maximum 2-3 d after inoculation and thereafter declined sharply in ripe chilli fruits, whereas slowly in green chilli fruits. In comparison with ripe chilli fruits, green chilli fruits were more resistant as they showed higher accumulation of total phenols and also higher activities of enzymes.

In vitro regeneration from leaf, cotyledon and hypocotyl explants of six cultivars belonging to three species of Capsicum was achieved by direct organogenesis. The cultivar Umorok showed the best response while Meiteimorok, Haomorok, Mashingkha and Uchithi showed intermediate response and the cultivar Chiengpi was the least responsive. Leaf and cotyledon explants regenerated more shoots than hypocotyl explants and the maximum number of shoots were produced on Murashige and Skoog (1962) medium containing 8.8 µM 6-benzylaminopurine (BAP) with 11.4 µM indole-3-acetic acid (IAA). Elongation of shoot buds derived from different explants was achieved on medium containing 2.8 µM IAA and the elongated shoots were rooted on medium containing 2.8 or 5.7 µM IAA and 2.4 or 4.9 µM indole-3-butyric acid (IBA). Four-week old rooted plantlets were hardened and transplanted to the soil. The plantlets showed 90 % survival during transplantation.

To induce somatic embryogenesis in Quercus robur L. immature zygotic embryos at different developmental stages were collected in weekly intervals from June until September in three consecutive years from four open pollinated trees at two Vienna sites. Acorns were surface sterilised and cultured firstly on P24 medium with 5μM 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.5 μM 6-benzylaminopurine (BAP) or on hormone-free P24 medium and secondly on P24 medium with 0.9 μM BAP. The formation of white-yellow globular structures of somatic embryos started during the fourth week after the induction treatment. High induction frequencies of 30 - 80 % were achieved on 2,4-D/BAP medium, whereas rates on hormone-free medium were below 20 %. The initiation of somatic embryogenesis was favoured in the heartshaped and early cotyledonary stage of the zygotic embryo in all three years and lasted until the acorns reached maximum size in August.

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.

Non-vernalized scions were grafted onto vernalized stocks in winter rape (Brassica napus L. var. oleifera, cv. Górczański). The grafted plants were subjected to electric current (30 V for 30 s or 6 V for 24 h) and the percentage of flowering scions was recorded. The negative polarity with cathode (-) attached to the scion and anode (+) left close to the roots inhibited greatly the percentage of flowering. The reverse polarity enhanced flowering markedly under short days and only slightly promoted flowering under long days. Attachment of electrodes without passing a current had no effect on flowering.

A highly reproducible system was developed for efficient rooting of cultivars Boa Casta (BC) and Peneda and a BC seedling-derived clone (BC VII) of almond (Prunus dulcis Mill.). Twenty-four accessions derived from the clone BC VII and subjected to various in vitro culture treatments were screened. The long induction pre-treatment (LIP, 5 d), the brief induction pre-treatment (BIP, 16 h) and the hormonal shock by short dipping in hormone solution (1 min), were tested. BIP was the only that allowed rooting of cultivars. In BC VII, it induced high rooting frequencies (47-100 %) when using a solution of 0.4 mM indole-3-butyric acid solidified with 2 g dm^-3 gellam gum for 16-h. The response to the auxin type was variable depending on the cultivar and the root induction pre-treatment used. Root number was significantly different between the two cultivars and BC VII. Root length was significantly higher when using 0.005 mM IBA in LIP but this concentration induced apical necrosis. The improved acclimatization procedure for up to 4 weeks increased the survival to 45 %. The initiation and development of adventitious roots were proved to be asynchronous.

This study was conducted to determine the reciprocal effects for anther culture response in wheat (Triticum aestivum L.) using a set of 4 × 4 full diallel crosses. Both reciprocal and nuclear genetic effects were highly significant for anther culture response and useful for selection and breeding purposes. General combining ability (GCA) effects were predominant for all investigated anther culture traits. Also, significant differences for specific combining ability (SCA) effects were detected between reciprocal crosses. Although significant reciprocal differences for responding anther, callus number and green plant regeneration were recorded in some reciprocal crosses, there were no significant reciprocal differences for albino plant regeneration. The use of one parent as male or female could lead to change at the production of green plants from the F₁ hybrids and screening of inbred lines for response to anther culture, without reciprocal effects, could decrease the utilization of breeding material.

Two vacuolar green fluorescent proteins (GFP) were stably inserted in Nicotiana tabacum and Nicotiana benthamiana genome, with unexpected difficulties, and compared with A. thaliana cv. Wassilewskaja transgenic plants expressing the same constructs. GFP fluorescence was strong in all tissues of A. thaliana but it was barely visible in Nicotiana. Confocal microscopy analysis revealed a variable distribution of the marker in those cells where GFP fluorescence was visible. The role of light dependent proteases was the variable pointing out more inter-species diversity. GFPs degradation was much higher in Nicotiana spp. than in A. thaliana. The version of GFP used appeared not to be a good vacuolar marker for Nicotiana differentiated tissues, although it can efficiently label vacuoles in protoplasts or calli. Nevertheless the sensitivity of the reporter protein can be used as an indicator of hidden characteristics of the plant vacuoles, revealing differences otherwise invisible. One of the markers in our system, GFP-Chi, evidenced a clear morphological difference in the vacuolar system of guard cells of the three species.

To further optimize a culture medium for induction of direct embryo formation of Oncidium cvs. Gower Ramsey and Sweet Sugar, five kinds of carbon sources, cellibiose, fructose, glucose, maltose and sucrose at 10, 20, 30 and 60 g dm^-3 were tested in this study. Cellibiose supply had an inhibitory effect and resulted in high percentage of explant browning in both cultivars. By contrast, fructose, glucose and sucrose were all effective for direct embryo induction. In cv. Gower Ramsey, the suitable ranges of concentration were found at 30-60 g dm^-3 of sucrose, 10-20 g dm^-3 of glucose and 20-30 g dm^-3 of fructose, respectively. The suitable ranges for cv. Sweet Sugar were at 20-60 g dm^-3 of sucrose, 10-30 g dm^-3 of glucose, 10-20 g dm^-3 of fructose and 30-60 g dm^-3 of maltose, respectively. The highest amount of embryos was obtained at 30 g dm^-3 of sucrose for cv. Gower Ramsey and at 20 g dm^-3 of glucose for cv. Sweet Sugar.

Effect of plant growth regulators, explant size, season of explant collection, temperature (20, 25 and 30 °C) and photoperiod on in vitro lotus (Nelumbo nucifera Gaertn.) shoot formation and growth were examined. Shoots formation was greatly influenced by growth regulators, explant size and season of explant collection. The maximum number of shoots were induced from bud explants on Murashige and Skoog (MS) medium containing 4.44 µM benzyladenine (BA) + 0.54 µM α-naphthalene acetic acid (NAA). Explants formed by bud of one expanded and one unexpanded leaf, which was collected in spring gave encouraging results of shoot production. Higher temperature favoured shoot induction and subsequent growth was much better at 25 °C compared to that at 20 and 30 °C.

Immature and mature zygotic embryos were used as source of explants for induction of somatic embryogenesis in Araucaria angustifolia. Embryogenic cultures (EC) were only obtained from immature zygotic embryos. Basic medium, carbon source, and genotype showed a significant influence on the formation of stage I somatic embryos (SE). When EC were submitted to maturation conditions, SE continued their individual development until stage II, but mature embryos were not obtained. Proteins secreted by embryogenic cultures were, to a certain degree, genotype specific and included an extracellular class IV chitinase and β-1-3-glucanase.

Total protein patterns were studied in the course of development of pea somatic embryos using simple protocol of direct regeneration from shoot apical meristems on auxin supplemented medium. Protein content and total protein spectra (SDS-PAGE) of somatic embryos in particular developmental stages were analysed in Pisum sativum, P. arvense, P. elatius and P. jomardi. Expression of seed storage proteins in somatic embryos was compared with their accumulation in zygotic embryos of selected developmental stages. Pea vegetative tissues, namely leaf and root, were used as a negative control not expressing typical seed storage proteins. The biosynthesis and accumulation of seed storage proteins was observed during somatic embryo development (since globular stage), despite of the fact that no special maturation treatment was applied. Major storage proteins typical for pea seed (globulins legumin, vicilin, convicilin and their subunits) were detected in somatic embryos. In general, the biosynthesis of storage proteins in somatic embryos was lower as compared to mature dry seed. However, in some cases the cotyledonary somatic embryos exhibited comparatively high expression of vicilin, convicilin and pea seed lectin, which was even higher than those in immature but morphologically fully developed zygotic embryos. Desiccation treatments did not affect the protein content of somatic embryos. The transfer of desiccated somatic embryos on hormone-free germination medium led to progressive storage protein degradation. The expression of true seed storage proteins may serve as an explicit marker of somatic embryogenesis pathway of regeneration as well as a measure of maturation degree of somatic embryos in pea.

The role of irradiance on the activity of antioxidant enzymes: superoxide dismutase (SOD) and catalase (CAT) was examined in the leaves of Pisum sativum L. plants grown under low (LL) or high (HL) irradiance (PPFD 50 or 600 µmol m^-2 s^-1) and exposed after detachment to 5 mM Pb (NO₃)₂ for 24 h. The activities of both enzymes increased in response to LL compared with HL and no effect of Pb ions was observed. Photosystem (PS) 1 and PS 2 activities were also investigated in chloroplasts isolated from these leaves. LL lowered PS 1 electron transport rate and changes in photochemical activity of PS 1 induced by Pb²⁺ were visible only in the chloroplasts isolated from leaves of LL grown plants. PS 2 activity was influenced similarly by Pb ions at both PPFD. This study demonstrates that leaves of HL grown plants were less sensitive to lead toxicity than those from LL grown plants. Changes in electron transport rates were the main factors responsible for the generation of reactive oxygen species in the chloroplasts and as a consequence, in induction of antioxidant enzymes.

The review summarizes the level of current knowledge of impacts of vernalization and photoperiod on the induction and maintenance of frost tolerance (FrT) in wheat and barley. The phenomenon of vernalization is briefly described and the major vernalization (VRN) loci are characterised. Vernalization requirement and the three major growth habits of Triticeae (facultative, winter and spring) are defined on the basis of the two-locus VRN-2/VRN-1 epistatic model. Major photoperiodically regulated genes, which influence the transition to flowering, are characterised and their interactions with VRN genes are briefly discussed. The phenomenon of induction of FrT during the process of cold acclimation (CA) is described and the major cold-induced Cor/Lea genes are listed. Important regulatory mechanisms, i.e., CBF pathway, controlling the expression of Cor/Lea genes under cold, are discussed. The major loci affecting the development of FrT in Triticeae, the Fr loci, are characterised. In conclusion, current progress in this research field is summarized and new questions arising in the area are formulated.

An efficient micropropagation protocol was developed for elite male and female genotypes of Simmondsia chinensis using nodal segments. Bud initiation was found to be best on Murashige and Skoog's (MS) medium supplemented with 4.44 µM 6-benzylaminopurine (BAP) and 88.8 µM adenine. Upon sub-culture, 10-15 shoots per explant were obtained when 4.44 µM BAP and 74.0 µM adenine were incorporated in the medium. Increase in KNO₃ concentration in the medium improved shoot multiplication rate and in vitro flowering in 20 % of male cultures. Elongated shoots were harvested, pulse treated for 48 h on liquid medium supplemented with 49.0 µM indole-3-butyric acid, 5.40 µM α-naphthaleneacetic acid and 5.71 µM indole-3-acetic acid for root induction and rooting (92 %) was achieved on hormonal free half-strength MS medium supplemented with 1.37 µM chlorogenic acid, 1 % activated charcoal and 2 % sucrose. After successful hardening, plantlets were transferred to greenhouse with 99 % establishment.

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 report describes in vitro plant regeneration from embryo axis explants of six cultivars of cotton. Induction of a maximum number of multiple shoots in all six cultivars could be achieved on Murashige and Skoog's (MS) salts and Gamborg's (B5) vitamins supplemented with 0.4 μM benzyladenine (BA) and 0.1 μM napthaleneacetic acid (NAA). Elongated shoots could be rooted on half strength medium supplemented with 0.5 μM NAA. Rooted shoots survived (92 %) after hardening in the greenhouse and grew to maturity (100 %) after transfer to field.

The effect of Pseudomonas fluorescens treatment and Fusarium oxysporum f. sp. cubense inoculation on induction of phenylalanine ammonia-lyase (PAL), peroxidase (POX), chitinase, β-1,3-glucanase and accumulation of phenolics in banana (Musa sp.) was studied. When banana roots were treated with P. fluorescens strain Pf10, a two-fold increase in phenolic content in leaf tissues was recorded 3 - 6 d after treatment. Challenge inoculation with F. oxysporum, the wilt pathogen, steeply increased the phenolic content in P. fluorescens-treated banana plants. Significant increase in POX activity was detected 6 - 9 d after P. fluorescens treatment. PAL, chitinase and β-1,3-glucanase activities increased significantly from 3 d after P. fluorescens treatment and reached the maximum 6 d after treatment. Challenge inoculation with F. oxysporum further increased the enzyme activities. These results suggest that the enhanced activities of defense enzymes and elevated content of phenolics may contribute to bioprotection of banana plants against F. oxysporum.

The effects of amino acids (arginine, aspargine, cysteine, glutamine, glycine and proline) and polyamines (putrescine and spermidine) on embryogenesis and plant regeneration from cultured anthers of Guizotia abyssinica (L. f.) Cass. cv. Ootacamund was studied. Supplementation of amino acids (0.5-5.0 mM) to the induction medium individually and in combination, improved embryo yield. B5 medium supplemented with 2 mM proline, 10 µM 2,4-dichlorophenoxyacetic acid, 2 µM kinetin and 0.2 M sucrose induced highest number of embryos (63 per 60 anthers cultured). Addition of polyamines (5-200 µM) to the same medium also enhanced the rate of embryogenesis.

In four genotypes of chickpea (Cicer arietinum L.) BG 362, BG 372, BG 329 and C235 the relationship between somatic embryogenesis of leaf explants and ethylene and methane evolution was studied. In BG 362, which was more embryogenic than other genotypes, a higher ethylene:methane ratio of 5.8:1 at day one after inoculation in the induction medium and a lower ethylene:methane ratio of 2.89:1 in the maturation medium was found. On the contrary, in BG 372 with the least embryogenic potential, a lower ethylene:methane ratio of 1.7:1 in the induction medium and a higher ethylene:methane ratio of 4:1 in the maturation medium was found. Thus, these ratios in induction and maturation stages seems to be markers for embryogenesis in leaf explants of chickpea.

An unknown brownish protein was purified by ammonium sulfate precipitation and DEAE-cellulose column and hydroxyapatite column chromatographies from pumpkin callus treated with a high concentration of 2,4-D. The apparent molecular mass and isoelectric point of the purified protein were estimated to be 38 kD and 4.6, respectively. The absorption spectra of the protein showed a shoulder at around 280 nm and a sharp peak at 405 nm. In order to determine what the purified protein is, a cDNA library of the callus treated with a high concentration of 2,4-D was immunoscreened with antiserum raised against the purified protein. The obtained positive cDNA clone encoded a thioredoxin h having a predicted molecular mass of 13 123 D and a predicted isoelectric point of 5.24, suggesting that the purified protein might be a trimer that was formed by oxidative polymerization of the thioredoxin h.

An efficient in vitro plant regeneration system from leaves of Ophiorrhiza japonica Blume was established for the first time. Callus formation rate was more than 90.4 % from leaf segments on Murashige and Skoog (MS) supplemented with either α-naphthaleneacetic acid (NAA) alone or in combination with 6-benzyladenine (BA). The highest shoot regeneration (78.9 %) was achieved on MS medium containing 2.0 mg dm^-3 BA and 0.2 mg dm^-3 NAA, with an average of 9.4 shoots developed per leaf segment. Shoot regeneration was also improved when the leaf explants were cultured in MS basal medium supplemented with 0.5 % (m/v) polyvinylpyrrolidone (PVP). The leaf explants from seedlings with age of about 18-27 d showed the highest shoot regeneration. The regenerated shoots were rooted on half-strength basal MS medium supplemented with 0.5 mg dm^-3 indole-3-butyric acid (IBA), which averagely produced 24.8 roots per shoot. The plantlets were transferred to soil, where 100 % survived after 1 month of acclimatization.

To characterize the low molecular mass glutenin subunit gene 177-21 (AY994364) in wheat (Triticum aestivum L. cv. Jinan 177), we developed a specific PCR primer set to decide its locus with nullisomic-tetrasomic lines of Chinese spring wheat. The result showed that it was assigned to Glu-D3. The DNA fragment of 177-21 was then subcloned into the pGEX-4T-1 expression vector and expressed in E. coli with isopropyl-1-thio-β-D-galactoside induction. The result indicated that this gene encodes about 30 kD polypeptide and deduced amino acid sequence consists of eight cysteine residues. Of the eight, six may be related with the formation of intra-molecular disulfide bonds, the last two with the formation of inter-molecular disulfide bonds, which could be a potential extender in "glutenin polymer" to have positive influence on quality of wheat flour.

Seed germination and micropropagation protocols of the medicinal species Maytenus canariensis (Loes.) G. Kunkel & Sunding were optimized. In vitro seed germination occurred (86 to 94.7 %) only after treatment of the seeds with H₂SO₄, followed by surface sterilization and culture on solid nutrient medium without any growth regulators. Micropropagation failed when explants were taken from mature trees, and browning of the nutrient medium frequently occurred despite testing many growth media. Nonetheless, adventitious shoot regeneration was achieved employing axillary or apical buds taken from 2-2.5 months old plantlets obtained after in vitro germination of seeds, following culture on nutrient media supplemented with benzylaminopurine, kinetin and naphthalenacetic acid (NAA), attaining up to 3.9 shoots per explant, after 4-6 months. Root induction was best on a medium containing 4.0 mg dm^-3 NAA, achieving a 100 % induction. After hardening of rooted plants, survival after transfer to soil was 71.43 %.

This study aims to understand the changes in the transcriptome of durum wheat (Tricitum durum cv. Balcali-85) upon exposure to varying Cd concentrations using mRNA differential display (mRNA DD) technique. Sequence analyses of the two heavily induced genes upon exposure to Cd showed high homology to NADH dehydrogenase subunit 1 (EC907725) and PsaC gene encoding a photosystem 1 (PS 1) 9 kDa subunit protein (EC907731). Additionally, three differentially expressed genes (EC907726, EC907729 and EC907730) were identified. Their sequence analyses revealed no significant homologies to known genes. The expressions of NADH dehydrogenase subunit 1 and PsaC genes were confirmed by Northern blot analysis and quantified by real time PCR. This is the first report for the induction of NADH dehydrogenase subunit 1 gene during Cd stress in wheat.