Showing papers in "Acta Physiologiae Plantarum in 2005"

Allelopathic interactions between plants. Multi site action of allelochemicals

Abstract: Allelopathy is defined as mechanism of plant-plant, plant-microorganisms, plant-virus, plant-insect, and plant-soil-plant interactions mediated by plant- or microorganism-produced chemicals released to the environment. The majority of allelochemicals are secondary metabolites and among others belong to terpenoids, phenolic compounds, organic cyanides and longchain fatty acids. The action of allelochemicals in target plant is diverse and affects a large number of biochemical reactions resulting in modifications of different physiological functions. Thus the results of allelochemical action can be detected at different levels of plant organization: molecular, structural, biochemical, physiological and ecological. Enzyme activities, cell division and ultrastructure, membrane permeability, ion uptake and as a consequence plant growth and development are modified by allelochemicals. Significant effects on photosynthesis and respiration are the best-characterized results of allelopathic interactions. Moreover allelopathic compounds seem to induce a secondary oxidative stress expressed as enhanced free radical production and induction of cellular antioxidant system. Plant survival under allelopathy stress conditions depends on plant defense leading to allelochemical detoxication, the process which may go on in parallel to cell defense reaction to oxidative stress.

The effect of salicylic acid on barley response to water deficit

Abstract: The effect of a moderate (PEG −0.75 MPa) and severe (PEG −1.5 MPa) water deficit on SA content in leaves and roots as well as the effect of pre-treatment with SA on reaction to water stress were evaluated in two barley genotypes — the modern cv. Maresi and a wild form of Hordeum spontaneum. Water deficit increased SA content in roots, whereas SA content in leaves did not change. The level of SA in the roots of control plants was about twofold higher in ‘Maresi’ than in H. spontaneum. After 6 hours of a moderate stress the level of SA increased about twofold in H. spontaneum and about two and a half-fold in ‘Maresi’. Under severe stress conditions the level of SA increased about twofold in the both genotypes, but not before 24 hrs of the stress. Plant treatment with SA before stress reduced a damaging action of water deficit on cell membrane in leaves. A protective effect was more noticeable in H. spontaneum than in ‘Maresi’. SA treatment increased ABA content in the leaves of the studied genotypes. An increase of proline level was observed only in H. spontaneum. The obtained results suggest that ABA and proline can contribute to the development of antistress reactions induced by SA.

Antioxidative responses and proline level in leaves and roots of pea plants subjected to nickel stress

Abstract: The activities of superoxide dismutase (SOD), ascorbate peroxidase (APX), catalase (CAT), glutathione S-transferase (GST) as well as proline content were studied in leaves and roots of 14 day-old pea plants treated with NiSO4 (10, 100, 200 µm) for 1, 3, 6 and 9 days. Exposure of pea plants to nickel (Ni) resulted in the decrease in CuZnSOD as well as total SOD activities in both leaves and roots. The activity of APX in leaves of plants treated with 100 and 200 µm Ni increased following the 3rd day after metal application, while in roots at the end of the experiment the activity of this enzyme was significantly reduced. In both organs CAT activity generally did not change in response to Ni treatment. The activity of GST in plants exposed to high concentrations of Ni increased, more markedly in roots. In both leaves and roots after Ni application accumulation of free proline was observed, but in the case of leaves concentration of this amino acid increased earlier and to a greater extent than in roots. The results indicate that stimulation of GST activity and accumulation of proline in the tissues rather than antioxidative enzymes are involved in response of pea plants to Ni stress.

Oxidative stress in herbicide-treated broad bean and maize plants

Abstract: Treatments of broad bean and maize seedlings with fluometuron, atrazine or rimsulfuron affected some parameters of oxidative stress. Fluometuron significantly reduced activity of Hill reaction (PSII), chlorophyll a+b contents and activity of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco, EC 4.1.1.39) in leaves of both species and significantly increased contents of H2O2, lipid peroxides and carbonyl groups during the whole experiment. There were, moreover, significant inhibitions in activities of superoxide dismutase (SOD; E.C. 1.15.1.1), catalase (CAT; E.C. 1.11.1.6), ascorbate peroxidase (APX; E.C. 1.11.1.11) and guaiacol peroxidase (GPX; E.C. 1.11.1.7). Response to atrazine was, to some extent, similar to fluometuron throughout the entire experiment in broad bean and up mostly to the 12th day of the experiment in maize. The herbicide effect was more pronounced in broad bean than maize. These results point to indicate an occurrence of oxidative stress in both species by fluometuron and only in broad bean by atrazine. The increase in H2O2 content accompanied with drop in activities of SOD, CAT and peroxidases indicates a decline in its detoxification rather than increase in its synthesis. On the contrary, rimsulfuron seemed to have no effect on most of the tested parameters although there were transient significant increases in H2O2, lipid peroxides and carbonyl groups as well as activities of SOD, CAT, APX and GPX. These findings, based on the recovery in oxidative stress, indicate that fluometuron is involved in oxidative stress generation in both species but atrazine only in broad bean while rismulfuron is not in both species.

Plant protoplast technology: Current status

Abstract: Robust and reproducible protoplast-to-plant systems are crucial for underpinning genetic manipulation technology involving somatic hybridisation and transformation. Novel and effective approaches for maximising the efficiency of such protoplast cultures include supplementation of media with surfactants and artificial gas carriers, such as perfluorochemicals and haemoglobin. Physical parameters, particularly electrostimulation, also enhance the development of protoplasts and protoplast-derived cells in culture. DNA uptake into protoplasts is now a routine and universally accepted procedure in plant biotechnology for introducing and evaluating both short-term (transient) and long-term (stable) expression of genes in cells and regenerated plants. Importantly, protoplast fusion overcomes pre- and post-zygotic sexual incompatibility barriers and generates novel germplasm through new nuclear-cytoplasmic combinations. In this respect, considerable progress has been made in generating somatic hybrid plants, particularly in citrus, brassicas and potato. Isolated protoplasts are also a unique single cell system for evaluating aspects of ultrastructure, genetics and physiology, with potential for the biosynthesis of novel secondary products, including commercially-important recombinant proteins (e.g. antibodies), and as systems in toxicity screening. Recent advances in protoplast technology have benefited from advances in animal and microbial cell culture, with interesting parallels existing between these systems. Further innovations will necessitate the strengthening of interdisciplinary links in these research fields and the requirement for continued dialogue and co-operation between workers with diverse but complementary skills.

The role of GA, IAA and BAP in the regulation of in vitro shoot growth and microtuberization in potato

Abstract: The effect of auxin, GA and BAP on potato shoot growth and tuberization was investigated under in vitro condition. The shoot length of potato explants increased with the increasing of concentrations (0.5 – 10 mg dm−3) of IAA treatment especially with the addition of GA3 (0.5 mg dm−3), but was inhibited by BAP (5 mg dm−3). The root number and root fresh weight of potato explants increased with the increasing of IAA levels either in the presence of GA3 (treatment IAA+GA) or not (IAA alone). However, no root was observed in the treatment IAA+BAP, instead there were brown swollen calli formed around the basal cut surface of the explants. The addition of GA3 remarkably increased the fresh weight and diameter of calli. Microtubers were formed in the treatments of IAA+BAP and IAA + GA + BAP but not observed in the treatments of IAA alone or IAA + GA. IAA of higher concentrations (2.5 – 10 mg dm−3) was helpful to form sessile tubers. With the increasing of IAA levels, the fresh weight and diameter of microtubers increased progressively. At 10 mg/L IAA, the fresh weight and diameter of microtubers in the treatment of IAA + GA + BAP were 409.6 % and 184.4 % of that in the treatment of IAA + BAP respectively, indicating the interaction effect of GA and IAA in potato microtuberization.

Immature pollen-derived doubled haploid formation in barley cv. Golden Promise as a tool for transgene recombination

Abstract: Barley transformation mediated by Agrobacterium tumefaciens is routinely performed in a number of laboratories. However, elimination of selectable marker genes and formation of plants homozygous for the transgene via conventional segregation is laborious and time-consuming. Here we suggest a concept that includes the production of primary transgenic plants via infection of immature embryos with A. tumefaciens followed by androgenetic generation of a segregating population of entirely homozygous plants. Selectable marker-free, truebreeding plants carrying a single-opy transgene integrant may thus be efficiently and rapidly obtained. However, amenability to Agrobacterium-mediated transformation as well as androgenetic potential is genotype-dependent. Efficient genetic transformation by infection of immature embryos is so far confined to the spring type cultivar ‘Golden Promise’ which, however, turned out to be recalcitrant in pollen embryogenesis. To facilitate androgenetic generation of homozygous segregants from primary transformants, we have established a method for embryogenic pollen culture in cv. Golden Promise that includes conventional cold-treatment and subsequent preculture of immature pollen under starvation conditions prior to transfer to complete nutrient medium. Further we show that conditioning of the pollen culture medium by co-culture of immature wheat pistils as well as addition of pistil-preconditioned medium considerably support androgenetic development. Employment of the established method using immature pollen of primary transgenic plants demonstrates that selectable marker-free, true-breeding transgenic progeny can be rapidly obtained pursuing the concept proposed. The protocol presented will be useful in functional genomics as well as in molecular breeding approaches.

The effect of exogenous spermidine on superoxide dismutase activity, H2O2 and superoxide radical level in barley leaves under water deficit conditions

Abstract: Barley (Hordeum vulgare) seedlings were treated with spermidine prior to water deficit to determine whether this polyamine is able to affect the activity of superoxide dismutase -SOD (EC 1.15.1.1) responsible for hydrogen peroxide and superoxide radical level. Short-term dehydration (24h) resulted in decrease of the SOD specific activity and a distinct increase in the superoxide anion and hydrogen peroxide contents. Polyamine treatment caused a substantial reduction in the contents of these two stress-raised reactive oxygen species and thereby lowered the oxidative stress in plant cells. Antioxidant system as an important component of the water-stress-protective mechanism can be changed by polyamines, able to moderate the radical scavenging system and to lessen in this way the oxidative stress.

Effect of salinity on physiological characteristics, yield and quality of microtubers in vitro in potato

Abstract: In vitro microtuberization provides an adequate experimental model for the physiological and metabolic studies of tuberization and the preliminary screenings of potential potato genotypes. The effects of saline stress at 0–80 mmol concentration on in vitro tuberization of two potato cultivars were investigated in this study. With an increase in the salt concentration, the microtuberization of potato was either delayed by 5–10 days (20 and 40 mmol NaCl) or inhibited completely (80 mmol NaCl) in addition to the reduction in microtuber yields. The two potato genotypes studied showed different trends in total soluble sugars, sucrose and starch contents of microtubers under NaCl stress, while glucose and fructose levels remained unchanged. The vitamin C content in microtubers of two potato genotypes was reduced by salt stress. Salinity applied from 20 to 60 mmol progressively increased proline and malondialdehyde (MDA) levels in microtubers of both the potato cultivars. In genotype Zihuabai, NaCl at a low concentration (20 mmol) led to a significant increase in peroxidase (POD) and polyphenoloxiadase (PPO) activities, while in Jingshi-2, the PPO activity decreased progressively with an increase in NaCl concentrations. Genotype Zihuabai exhibited higher tolerance to salt stress than Jingshi-2 under in vitro conditions. These results could be used for preliminary selections of salt tolerance in potato breeding programmes.

Metabolism of phenolic compounds in Vitis riparia seeds during stratification and during germination under optimal and low temperature stress conditions

Abstract: We studied the alterations in phenolic compounds in grape seeds during their stratification and germination under optimal conditions (+25 °C) and at low temperature (+10 °C). Biological materials in the study were seeds of Vitis riparia. Phenolic compounds were extracted from defatted seeds using 80 % methanol or 80 % acetone. The content of total phenolics was determined with the Folin-Ciocalteau reagent, while the content of tannins was determined by vanillin assay and the protein (BSA) precipitation method. The RP-HPLC method was used to determine phenolic compounds (phenolic acids, catechins) in the extracts. High amounts of tannins, catechins, gallic acid and lesser amounts of p-coumaric acid were found in the seeds. The content of total phenolics in acetone extracts was higher than that obtained using methanol. The amounts of phenolic acids and tannins found in V. riparia seeds after stratification were much lower. It may confirm a possible role of these compounds in dormancy of V. riparia seeds. After 72 h of low temperature treatment, inhibition of grape root growth and biochemical changes in seeds were detected. The chilling stimulated increased accumulation of some phenolic compounds (free gallic acid and catechins) in the seeds. These substances can protect plants against some abiotic stressors.

Cell architecture during gametophytic and embryogenic microspore development in Brassica napus L.

Abstract: In this work, the cell architecture of the microspore following both gametophytic and embryogenic developmental pathways in vitro was compared with the gametophytic development in vivo in Brassica napus, at both light and electron microscopy level. The microspore reprogramming to embryogenesis involves defined changes affecting cell activities and structural organization which can be considered as markers of the microspore embryogenic pathway, but less is known about others developmental programmes followed by the microspore in vitro after both, inductive and non-inductive conditions. Low-temperature processing of the samples, cytochemical and immunocytochemical approaches to identify various cell components were performed. Differences in specific cellular features such as cellular size and shape, nuclear architecture, starch accumulation, presence of vacuoles and ribosomal population were studied to characterize sequential stages of microspore embryogenesis and other pathways occurring in vitro. The presence of abundant starch grains in a defined cytoplasmic region appeared as a specific feature of the in vitro gametophytic development, as well as of the non-induced microspores of in vitro cultures under embryogenic-inductive conditions.

Current status in production and utilization of dihaploids from somatic hybrids between eggplant (Solanum melongena L.) and its wild relatives

Abstract: The major constrains for practical exploitation of the somatic hybrids between eggplant and its wild relatives have been their sterility and tetraploidy which prevented their incorporation into breeding programs. Here we demonstrate that anther culture was successfully utilized to bring back the ploidy level to the diploid status in tetraploid interspecific hybrids between eggplant and the allied species S. integrifolium and S. aethiopicum gr. gilo. Both the relative species are resistant to Fusarium oxysporum f. sp. melongenae and to some strains of bacterial wilt (Ralstonia solanacearum) which are very destructive diseases of eggplant. Dihaploid androgenetic plants were obtained from the somatic hybrids, from the “double somatic hybrid” obtained by sexual cross of the two somatic hybrids [(eggplant + S. aethiopicum) × (eggplant + S. integrifolium)], and from tetraploid backcrossed plants between the somatic hybrid with S. aethiopicum and eggplant. Phenotypical, molecular, biological and biochemical characterization, and also artificial inoculation with Fusarium oxysporum are consistent with a recombination between the genomes of the species involved in the hybridizations. Dihaploids resistant to Fusarium were successfully backcrossed with eggplant. Besides their utility as potential valuable breeding materials, the introgressed lines obtained may be utilized in genetic and molecular studies about the resistance to Fusarium from S. integrifolium and S. aethiopicum gr. gilo.

Meiotic metaphase I to telophase II as the most responsive stage during microspore development for callus induction in tomato (Solanum lycopersicum) anther cultures

Abstract: The generation of homozygous doubled haploid lines through induction of androgenesis is a promising alternative to the classical inbreeding and selection programs. However, this technology is poorly developed in tomato, where doubled haploid tomato plants have only been obtained through anther culture. Despite the fact that anther culture is routinely used in a number of economically interesting crops, there are still many drawbacks that prevent tomato breeders from adopting this technique, and improvements in methodology are required. One key issue is the correct identification of the optimal stage for anther excision and culture. In this paper we characterise in vivo microsporogenesis in tomato, defining the different microspore stages and relating them to the length of the donor flower bud. In parallel, we cultured anthers of these stages to obtain embryogenic callus, and followed the microscopic development of the callus contained within the anther. Our data suggest that the stage with the highest response, in terms of callus generation, is meiosis. In particular, we propose the window from metaphase I to telophase II, including tetrad cellularisation, as the timeframe where induction can be accomplished in tomato anther cultures.

Decrease in beech (Fagus sylvatica) seed viability caused by temperature and humidity conditions as related to membrane damage and lipid composition

Abstract: The aim of this study was to determine if the loss of germinability and viability of beech (Fagus sylvatica L.) seeds stored at different variants of temperature (4, 20, and 30 °C) and relative humidity (RH: 45 and 75 %) is associated with a loss of membrane integrity and changes in lipid composition. Beech seeds stored for 9 weeks gradually lost viability at a rate dependent on temperature and humidity. The harmful effect of temperature increased with growing humidity. The loss of seed viability was strongly correlated with an increase in membrane permeability and with production of lipid hydroxyperoxides (LHPO), which was regarded as an indicator of peroxidation of unsaturated fatty acids. The condition of membranes was assessed on the basis of their permeability and the state of lipid components: phospholipids and fatty acids. During seed storage we observed a decline in concentration of individual phospholipids and fatty acids, proportional to the loss of seeds viability. We also detected a decrease in concentrations of α-tocopherol and sterols, which play an important role in protection of membranes against the harmful influence of the environment. Our results show that the germinability of beech seeds declines rapidly at temperature above 0 °C and growing humidity. This is due mainly to the loss of membrane integrity, caused by peroxidation of unsaturated fatty acids.

Exploitation of flow cytometry for plant breeding

Abstract: In this review, the different applications of flow cytometry in plant breeding are highlighted. Four main breeding related purposes can be distinguished for flow cytometry: (i) Characterisation of available plant material, including screening of possible parent plants for breeding programs as well as evaluation of population biodiversity; (ii) Offspring screening after interspecific, interploidy or aberrant crosses; (iii) Ploidy level determination after haploidization and polyploidization treatments and (iv) Particle sorting, that allows separation of plant cells based on morphological or fluorescent characteristics. An overview and discussion of these various applications indicates that flow cytometry is a relatively quick, cheap and reliable tool for many breeding related objectives.

Spermine and putrescine enhance oxidative stress tolerance in maize leaves

Abstract: The protective effects of spermine (SPM) and putrescine (PUT) against paraquat (PQ), a herbicide in agriculture and oxidative stress inducer, were investigated in the leaves of maize. Maize leaves were pretreated to SPM and PUT at concentrations of 0.2 and 1 mM and treated with PQ afterwards. Pretreatment with 1 mM of SPM and PUT significantly prevented the losses in chlorophyll and carotenoid levels induced by PQ. Ascorbic acid content in the leaves pretreated with both polyamines was found to be higher than those of the leaves pretreated with water. Also, pretreatment with SPM and PUT was determined to have some effects on the activities of superoxide dismutase (SOD) and peroxidase (POD). 1 mM of SPM increased SOD activity, but PUT has no significant effect on SOD activity. On the other hand, POD activity was recorded to increase slightly in response to both concentrations of SPM and 1 mM of PUT. The results showed that such polyamine pretreated plants may become more tolerant to oxidative stress due to increases in the antioxidative enzymes and antioxidants.

Effect of arsenic on behaviour of enzymes of sugar metabolism in germinating rice seeds

Abstract: Arsenic (As) is a potential contaminant of groundwater as well as soil in many parts of the world. The effects of increasing concentration of As (25 µm and 50 µm As2O3) in the medium on the content of starch and sugars and activity levels of enzymes involved in starch and sugar metabolism i.e. α-amylase, β-amylase, starch phosphorylase and acid invertase were studied in germinating seeds of two rice cvs. Malviya-36 and Pant-12 during 0–120 h period. As toxicity in situ led to a marked decline in the activities of α-amylase, β-amylase in endosperms as well as embryoaxes of germinating rice seeds. The activity of acid invertase increased in endosperms as well as embryoaxes whereas starch phosphorylase activity declined in endosperms but increased in embryoaxes under As treatment. In endosperms a decline in starch mobilization was observed under As toxicity, however under similar conditions the content of total soluble sugars increased in embryoaxes. The observed inhibition in activities of amylolytic enzymes might contribute to delayed mobilization of endospermic starch which could affect germination of seeds in As polluted environment, while the induced acid invertase activity and increased sugar accumulation in embryoaxes could serve as a possible component for adaptation mechanism of rice seedlings grown under As containing medium.

An overview of preliminary studies on the development of doubled haploid protocols for nutraceutical species

Abstract: There is very little activity underway to improve the genetics of herbs, spices, and nutraceutical crops. Much of the industry relies on the harvest of “wild” plants; therefore, the potential for variability in performance and active ingredients is high. This presents significant challenges for an industry that is striving to achieve market credibility and meet current regulatory standards. Uniform varieties would also be beneficial for use in clinical trials. The development of plants performing consistently in cultivation under various environmental conditions and producing a stable quality and quantity of desired active ingredients cannot solely rely on traditional plant breeding, but must be supported by the development of tissue culture methods targeted to the species of interest.

Searching for mechanisms leading to albino plant formation in cereals

Abstract: Microspore embryogenesis is a method of great importance for plant production in many species. Its practical use in monocotyledonous plants, however, is limited due to the occurrence of albino plants. Molecular examinations of such albino plants showed an aberrant form of plastids and, frequently, deletions in their plastid DNA. Moreover, in our preceding work we found typical deficiencies in plastid transcription and translation. Nevertheless, there are also indications for an involvement of the nuclear genome in albino plant formation. To address these questions, we characterised, on the one hand, a plastid factor, which is possibly involved in the formation of plastid DNA deletions, and investigated, on the other hand, nuclear gene expression in albino plants. The ycf1 gene is present in the plastid genomes of dicots, while monocots only have a truncated pseudogene. The disruption of ycf1 in tobacco showed that it is essential for the survival of dicotyledonous plants. In addition, we replaced the tobacco ycf1 by the pseudogene of rice and found rearrangements in the plastid genomes of the resulting plants indicating that the ycf1 locus plays a role in the stability of plastid DNA. The study on nuclear gene expression showed that there is an extensive alteration of transcript levels in albino plants. We found that the respective genes encode proteins with a broad range of functions in plants. All these findings taken together suggest that albino plant formation is a complex phenomenon in which both, plastid and nuclear factors or their defective interactions are involved.

Carbohydrate and free amino acid contents in tomato plants grown in media with bicarbonate and nitrate or ammonium

Abstract: Tomato plants were cultivated (from 2 to 23 days after germination) in media with NO 3 − , NH 4 + , or a mixture of both forms in different proportions used as the N source given with or without 5 mol dm−3 HCO 3 − . The accumulation of soluble sugars (reducing sugars and sucrose) and free amino acids was higher in the roots and leaves of NH 4 + -fed plants than in NO 3 − -fed plants. Starch accumulation in NH 4 + -fed plants was higher in leaves (about 28%) and lower in roots (about 37%) in comparison with that of NO 3 − -fed plants. Plants cultivated in media containing a mixture of NO 3 − /NH 4 + were characterized by a lower content of sugars and amino acids accumulation in comparison with that in plants fed with NO 3 − or NH 4 + . An elevated HCO 3 − concentration in the rhizosphere stimulated the accumulation of soluble sugars and free amino acids in all the experimental variants. There were only small differences in the starch content.

Plant Ca2+-ATPases

Abstract: Plant calcium pumps, similarly to animal Ca2+ pumps, belong to the superfamily of P-type ATPase comprising also the plasma membrane H+-ATPase of fungi and plants, Na+/K+ ATPase of animals and H+/K+ ATPase of mammalian gastric mucosa. According to their sensitivity to calmodulin the plant Ca2+-ATPases have been divided into two subgroups: type IIA (homologues of animal SERCA) and type IIB (homologues of animal PMCA). Regardless of the similarities in a protein sequence, the plant Ca2+ pumps differ from those in animals in their cellular localization, structure and sensitivity to inhibitors. Genomic investigations revealed multiplicity of plant Ca2+-ATPases; they are present not only in the plasma membranes and ER but also in membranes of most of the cell compartments, such as vacuole, plastids, nucleus or Golgi apparatus. Studies using yeast mutants made possible the functional and biochemical characterization of individual plant Ca2+-ATMPases. Plant calcium pumps play an essential role in signal transduction pathways, they are responsible for the regulation of [Ca2+] in both cytoplasm and endomembrane compartments. These Ca2+-ATPases appear to be involved in plant adaptation to stress conditions, like salinity, chilling or anoxia.

Characteristics of the interspecific somatic hybrids Solanum pinnatisectum (+) S. tuberosum H-8105

Abstract: The morphological, cytological and molecular analyses of the plants regenerated after PEG-induced fusion between mesophyll protoplasts from the dihaploid potato clone H-8105 and the wild tuberous disease-resistant species S. pinnatisectum, were performed. A single fusion experiment yielded 313 calli, although only two calli produced shoots. From the rooted shoots, two stable clones (PT-01-1 and PT-01-2) exhibiting different vigor and habitat, were developed. The plants of PT-01-1 clone grew slowly in vitro, produced tubers after transfer to soil but did not set flowers. In contrast, the plants of the vigorous clone PT-01-2 produced both tubers and flowers after transfer to soil. The flower and tuber morphology of PT-01-1 and PT-01-2 regenerants was intermediate in comparison to the parental species. Cytological analysis revealed that the PT-01-1 clone was pentaploid and the PT-01-2 clone was tetraploid. The molecular (RAPD) analysis confirmed hybridity of both clones. The preliminary tests on late blight resistance of the hybrids showed no differences with a potato parent.

Effect of temperature stress on the endogenous cytokinin content in Arabidopsis thaliana (L.) Heynh plants

Abstract: The levels of three endogenous cytokinin equivalents: zeatin (Z), iso-pentenyladenine (iP) and dihydrozeatin (dZ) in two Arabidopsis thaliana (L.) Heynh genotypes — wild type (wt) and ethylene-insensitive mutant (eti5), were compared using enzyme immunoassay (ELISA). Cytokinin content was measured after exposure to low (4 °C for 24 h in darkness) or high temperature (38 °C for 24 h in darkness). Measurements were performed immediately and 24, 48 and 120 h after treatments. It was found that at normal growth conditions eti5 plants contained more endogenous cytokinins compared to the wild type. At both temperature treatments mutant plants had decreased total cytokinin levels. Wild-type plants treated with high temperature (HT) exhibited reduced total cytokinins (with the exception of rates at 48 h), while low temperature (LT) treatment resulted in elevated total amount of the studied equivalents (except at 24 h). The obtained results suggested that HT had greater effect on cytokinin levels than LT since it caused more profound changes in the total content. We assume that this was due to the natural chilling tolerance of Arabidopsis plants.

Plant regeneration via somatic embryogenesis in Pinus kesiya (Rolye ex. Gord.) influenced by triacontanol

Abstract: Embryogenic cultures were initiated and established for the first time in 3 different genotypes of Pinus kesiya using mature zygotic embryos and triacontanol. Mature zygotic embryos produced white-mucilaginous embryogenic callus when cultured on half strength MSG (Becwar et al. 1990) basal medium supplemented with 90 mM maltose, 2.0 g l−1 Gellan gum, 9.0 M 2, 4-D and 10 g l−1triacontanol. On subculture of such embryogenic callus on the maintenance medium (II) containing 2.0 M 2,4-D and 2.0 g l−1 triacontanol induced cleavage polyembryogenesis with proembryos. The percentage of somatic embryogenesis was not similar in all the three genotypes. The highest percentage of somatic embryogenesis (88.5 %) was recorded in PK04 genotype. Somatic embryos were successfully germinated on half strength MSG basal medium without growth regulators. Somatic seedlings showed fast growth and a survival rate of 95%. This work for the first time reveals that triacontanol can be used as an effective growth regulator for inducing somatic embryogenesis in conifers.

Differences in accumulation of soluble alpha-galactosides during seed maturation of several Vicia species

Abstract: Composition and levels of soluble α-galactosides: raffinose family oligosaccharides (RFOs) and galactosyl cyclitols (Gal-C) in developing seeds were measured by high resolution gas chromatography (HRGC) method. The studies were performed on maturing seeds of several wild and cultivated Vicia species: Vicia angustifolia L. (common vetch), Vicia cracca L. (bird vetch), Vicia grandiflora Scop. (large yellow vetch), Vicia hirsuta (L.) S.F.Gray (tiny vetch), Vicia sativa L. (garden vetch, spring-growing cultivar Kwarta), and Vicia villosa Roth (winter vetch). In all Vicia species similar patterns in the accumulation of RFOs were observed. Galactinol — the donor of galactosyl moieties in α-galactosides biosynthesis was present in the middle stage of seed development, before appearing measurable levels of RFOs. Accumulation of RFOs started parallel with seed desiccation process. At first accumulation of the raffinose, then few days later stachyose and finally verbascose was noticed. In the final stage of seed maturation the verbascose was the main soluble α-galactoside (up to 3% of dry weight, V. sativa). Besides the RFOs seeds of three Vicia species (V. cracca, V. hirsuta, and V. villosa) accumulated d-pinitol and its α-galactosides (Gal-C). Mono-galactosylpinitols (similar to raffinose) appeared in these species 2–4 days after galactinol, di-galactosyl pinitol A (common name: ciceritol) and di-galactosyl myo-inositol were present several days later than raffinose, and accumulation of tri-galactosyl pinitol A (TGPA) began after accumulation of stachyose. Matured seeds of V. hirsuta contained much more RFOs than Gal-C, opposite to seeds of V. villosa, and V. cracca where concentration of Gal-C was 4–8-fold higher than RFOs. In V. cracca seeds RFOs were almost replaced by Gal-C. In seeds of V. cracca and V. villosa the level of d-pinitol was significantly higher, than the level of myo-inositol. Contents of both cyclitols declined rapidly at the beginning of seed desiccation, when accumulation of RFOs and Gal-C quickly increased. We suggest that α-galactosides of d-pinitol can substitute raffinose family oligosaccharides and play similar role during seed maturation and storage.

Microspore culture of small grain cereals

Abstract: Androgenesis of wheat, rice and triticale was studied in isolated microspore culture. It is the first publication which studies microspore culture reaction of Hungarian rice varieties. The effect of different basic media, lack and absence of growth regulators in culture media were tested on important parameters of microspore culture. Direct embryogenesis was observed in microspore culture of wheat and triticale genotypes. In the case of rice, calli were induced in isolated rice microspore culture and haploid rice plantlets were regenerated via organogenesis. In wheat, the effect of basic media (W14, A2, CHB3, P4-m) was compared and among them the W14, and A2 had a superior effect on embryo production and albino and green plantlet regeneration. In rice the C, CHB3 and MSm media were tested in microspore culture and the significantly highest numbers of calli were achieved by using C and CHB3 media depending on the genotypes. The lack of exogenous growth regulators was observed in isolated microspore culture of triticale and rice. Growth regulator-free medium had a positive effect on embryo production and plant regeneration of triticale genotypes, whereas in rice microspore culture multicellular structures did not continue their division without growth regulators from the third week of microspore culture. Developing of microspore-origin calli was maintained by supplement of 2,4-D and Kinetin combination in the microspore culture medium.

The effect of etiolation and shading of stock plants on rhizogenesis in stem cuttings of Cotinus coggygria

Abstract: In order to improve vegetative propagation of a difficult to root Cotinus coggygria the stock plants were subjected to: etiolation, shading and spraying with IBA, combined with the application of two commercially available rooting powders. The IBA treatment was more suitable for rooting of C. coggygria cuttings than the NAA application and it enhanced rhizogenesis regardless of the form of auxin application (foliar application to a stock plant or a rooting powder used directly on cuttings) and the amount of light provided to stock plants. Etiolation did not improve rhizogenesis in stem cuttings, however, reduction of light intensity by 50% and 96% of the ambient prior to harvest of cuttings affected rooting positively. Positive effects of shading can be ascribed to changes in shoot anatomy, i.e. a weaker sclerenchyma development. Synergistic effect of shading and foliar auxin application can result from the increase in leaf blade area and/or thinner lower epiderm. Enhanced rooting in cuttings from shoots grown out under reduced light intensity was accompanied by decrease in the contents of total soluble sugars, soluble proteins and free ABA and by increase in total chlorophyll, free amino acids, polyphenolic acids and free IAA contents.

Lignin content and peroxidase activity in soybean seed coat susceptible and resistant to mechanical damage

Abstract: Mechanical damage is one of the causes of great loss in the quality of soybean seeds during harvest and processing. Considerable interest exists in the lignin since its deposition in the seed coat tissue provides mechanical resistance and protects the cell against microorganisms. In addition, peroxidases might be involved in the oxidation of cinnamyl alcohols prior to their polymerization during lignin formation. Thus, the aim of the present work was to analyze the lignin contents and peroxidases activities of six Brazilian soybean cultivars (Savana, Paranagoiana, FT-10, Santa Rosa, Doko and Parana) and their relationships with the mechanical damage. Results showed that the lignin content and peroxidase activity in the seed coat significantly differed among the soybean cultivars. Cultivars Doko and Parana had the highest contents of lignin and peroxidases activities while the other cultivars had lowest lignin contents and enzyme activities. Lignin content and peroxidase activity may be reasonable indicators of resistance to mechanical damage in soybean seeds.

Isolated microspore culture overperforms anther culture for green plant regeneration in barley (Hordeum vulgare L.)

Abstract: Barley isolated microspore culture (IMC) was compared to anther culture (AC) for its efficiency in green plant (GP) regeneration. With six cultivars investigated, IMC resulted in significantly more GPs (3.6–287 per 100 anthers) than AC (0–29.6), which was on average 9.3-fold more efficient (113.7 vs 12.2). GPs were produced via IMC from all the genotypes tested, whereas no green shoot was generated by AC in two of the cultivars. In spite of genotype dependency for regeneration rates, the average GP percentage of IMC was just slightly higher than that of AC. Effects of microspore developmental stages and medium-sterilization methods on IMC were examined with the aim of optimizing culture conditions. We found that the optimum stage for cold pretreatment of spikes was different from that for mannitol starvation of anthers. Significant variations in microspore embryogenesis and regeneration were observed among five stages tested. Optimal stages for the two pretreatments were accordingly determined. Percentages of viable microspores were strongly influenced by protocols of medium-aseptisation. Although filter-sterilized media yielded two-time higher frequencies of living microspores and significantly more GPs than autoclaved ones, the filtration protocol was rather labor-intensive and time-consuming. Therefore a new procedure by combining filtering with autoclaving was subsequently developed as it was more effective than autoclaving and more convenient than filter-sterilization. The method described here could be useful for large-scale preparation of culture media.

Expression of several genes involved in sucrose/starch metabolism as affected by different strategies to induce phosphate deficiency in Arabidopsis

Abstract: The effects of inorganic phosphate (Pi) deficiency on expression of genes encoding ADP-glucose pyrophosphorylase small and large subunits (ApS and ApL1, ApL2, ApL3 genes), UDP-glucose pyrophosphorylase (Ugp gene), sucrose synthase (Sus1), soluble and insoluble acid invertases (Inv and Invcw) and hexokinase (Hxk1 gene), all involved in carbohydrate metabolism, were investigated in Arabidopsis thaliana (L.) Heynh. We used soil-grown pho mutants affected in Pi status, as well as wild-type (wt) plants grown under Pi deficiency conditions in liquid medium, and leaves of wt plants fed with D-mannose. Generally, ApS, ApL1, Ugp and Inv genes were upregulated, although to a varied degree, under conditions of Pi-stress. The applied conditions had differential effects on expression of other genes studied. For instance, Sus1 was downregulated in pho1 (Pi-deficient) mutant, but was unaffected in wt plants grown in liquid medium under P-deficiency. Mannose had distinct concentration-dependent effects on expression of genes under study, possibly reflecting a dual role of mannose as a sink for Pi and as glucose analog. Feeding Pi (at up to 200 mM) to the detached leaves of wt plants strongly affected the expression of ApL1, ApL2, Sus1 and Inv genes, possibly due to an osmotic effect exerted by Pi. The data suggest that ADP-glucose and UDP-glucose pyrophosphorylases (enzymes indirectly involved in Pi recycling) as well as invertases (sucrose hydrolysis) are transcriptionally regulated by Pi-deficiency, which may play a role in homeostatic mechanisms that acclimate the plant to the Pi-stress conditions.