Vol 65, No 5 (2018)

Transgenic plants have become one of the most important subjects of inquiry in biotechnology. Disputes about possible consequences of the use of genetically modified organisms still continue. Biological status of genetically modified plants is unsettled. Common criteria for assessment of the consequences of genetic transformation are lacking. Causes for and mechanisms of unforeseen consequences of transformation remain obscure. Methods of genetic engineering of plants are based on plant-Agrobacterium interaction. Transgenic plants are regarded as super-species systems produced as a result of artificial plant-Agrobacterium symbioses. Mechanisms of interaction between the members of symbiosis are very intricate and labile. Incorporation of foreign genes into T-DNA modifies characteristics of a bacterial symbiont. As a result, interaction between the organisms changes. The systems of inherent plant immunity are activated and plant metabolism is accordingly reorganized. These changes are the main reason for unintended effects of transgenosis. The intensity of response to transformation depends on the characteristics of the inserted target gene.

In the present study the interactions of GR24, a synthetic analog of newly discovered plant hormones strigolactones (SLs), with cytokinin (CK), benzyladenine (BA), auxin naphthaleneacetic acid (NAA), gibberellic acid (GA₃) and abscisic acid (ABA) in the regulation of axillary bud growth in pea (Pisum sativum L.) were investigated. The hormones were applied directly to buds at node 1 and 2 and the dose-response experiments were performed on 8–10-day-old SL-deficient rms1 and rms5–1 mutants, branching SL-sensitive rms2–1 mutants and wild-type plants. In the mutant plants the treatment with 5 μM GR24 completely inhibited bud growth while BA up to 100 μM stimulated it. The combined application of GR24 and BA showed that GR24 efficiency to reduce bud outgrowth constantly declines as CK-stimulated bud growth increased, with the inhibiting effect of GR24 abolished at 100 μM BA applied. GA₃ accelerated bud outgrowth, but did not interfere with GR24 inhibitory action. NAA reduced bud growth in intact SL-sensitive rms2–1 mutant and also in SL-insensitive rms3–2 and rms4–1 mutants. The NAA effect was observed already at 0.5 μM, however, even at a response saturating concentration of 500 μM its inhibiting effect was inferior to that of 5 μM GR24. At combined treatment the effectiveness of GR24 in suppressing bud growth decreased with a decrease in NAA-inhibited bud growth, suggesting that auxin might act as a suppressor of SL action. ABA strongly inhibited the bud outgrowth and, like CK and auxin, reduced the inhibitory effectiveness of GR24. The revealed ability of CK, ABA and auxin to suppress bud response to SLs is supposed to result from phytohormone signaling crosstalks.

The effect of indole butyric acid (IBA) as auxin hormone and sodium nitroprusside (SNP) on total antioxidant, carotenoid, total phenolic compounds, catalase activity and shoot dry weight in wheat (Triticum aestivum L. cv. Chamran) under 50 and 100 mM NaCl as salt stress were studied. These two experiments were done separately and in a completely randomized design, in three replications. Indole butyric acid in three levels and sodium nitroprusside in one level were used as spray on the plant to compare their effects. According to the results, the amount of carotenoid, total phenolic compounds, antioxidants and catalase activity have increased but shoot dry weight have decreased by either salinity alone and/or combined with IBA and SNP in our experiments, comparison to the control but with different level. None of the carotenoid, total phenolic compounds, antioxidants, catalase activity and shoot dry weight showed significant changes when indole butyric acid or sodium nitroprusside treatment were induced alone that shows their nontoxicity for seedlings. In connection with indole butyric acid or sodium nitroprusside treatment, these compounds appears to alleviate the stress conditions by showing significant decreasing of carotenoid, total phenolic compounds, antioxidants and catalase activity and significant increasing of shoot dry weight. Our results showed that IBA and SNP had the same effects in salt stress, so in salt stress conditions it is recommended to use IBA for crop and non-crop plants and SNP for only not edible plants.

Activities of enzymes decomposing hydrogen peroxide (H₂O₂) under long exposure to hardening low temperatures and the effect of Δ12-acyl-lipid desaturase on these processes were studied on potato (Solanum tuberosum L., cv. Desnitsa), which typically represents cold-tolerant plants. We compared nontransformed plants (control) and the line transformed with the construction carrying the target desA gene of the mentioned desaturase from cyanobacterium Synechocystis sp. PCC (desA-licBM3 plants). The plants were hardened at 5°C for six days under illumination of 50 μmol/(m² s). The hardening was found to favor plant tolerance to the subsequent frost, and the desA-licBM3 plants exceed the controls in this property. Of the studied H₂O₂-scavenging enzymes, soluble type III peroxidases (guaiacol peroxidases) displayed the most activity, and type I peroxidase (ascorbate peroxidase) was the least active in the two potato lines over the hardening period. The activity of catalase increased twofold in the control and fourfold in the transformed plants in the first day of the hardening. However, the doubled catalase activity did not appear to compensate the H₂O₂ accumulation over this period. The recorded rise in catalase activity in the desA-licBM3 plants, together with the high activity of guaiacol peroxidases, favored lowering the hydrogen peroxide level in comparison with the initial values. For the first time, electrophoresis revealed two catalase isoforms, CAT1 and CAT2, in leaves of both potato lines. The significance of CAT1 was greater than that of CAT2 in the total catalase activity during the hardening period. It is concluded that, under the long-term cold hardening of potato plants, the content of hydrogen peroxide is determined by highly active guaiacol peroxidases and Class I catalase exerting energy-independent H₂O₂ decomposing. In this case, in the transformants that are rich in membrane lipids, where polyunsaturated fatty acids predominate, the activity of H₂O₂-scavenging enzymes increased significantly more than in the control, which is why the hardening of the transformants is more effective.

To investigate the physical and ecological effect of chromones in the plant upon high-temperature, the fresh root of Saposhnikovia divaricata (Turxz.) Schischk. were exposed to high-temperature stress (30, 40, and 50°C) for 0, 3, 6 days, and the activity of antioxidant enzymes and PAL, the content of H₂O₂ and chromones were determined. The results showed that under high-temperature stress, the content of H₂O₂ and chromones were significantly increased, along with increased peroxidase and PAL activity, whereas the activity of SOD and catalase was decreased. DPPH clearance test demonstrated that chromone itself had no capacity to scavenge ROS. However, its H₂O₂ eliminating effect was significantly enhanced in the presence of peroxidase. Chromones are the main substance mediating the resistance of S. divaricata to ambient stress. The content of those secondary metabolites showed consistent trend with peroxidase activity, and they together participate in the clearance of ROS.

The present study highlights differential induction of pathogenesis related protein PR-2 (β-1,3- glucanases) in expression of rust resistance in pea using different resistant and susceptible recombinant inbred lines of pea (Pisum sativum L.). The enhanced levels of glucanase expression was noted in resistant genotypes at 24 h post inoculations that was negatively correlated (–0.54) with Area Under Disease Progress Curve (AUDPC) and positively correlated (0.67) with lignin accumulations. A significant role of structural defence mechanism in rust resistance in pea was evident from reduced colony size and lesser number of haustorium per colony in resistant lines as well as their negative correlations with lignin accumulation and AUDPC. Gene specific markers indicated constitutive nature of glucanase and peroxidase genes in test genotypes, though differential expression of the glucanase activity was observed in the resistant and susceptible genotypes. However, association of peroxidases with resistance to pea rust is yet to be established due to its non-specific role in slow rusting in pea. The result showed a significant role of β-1,3-glucanase in expression of rust resistance in pea.

In the present study, we compared the effects of allelochemical, water stress (WS) and their combinations on seedling growth, biochemical parameters and responses of antioxidative enzymes in Triticum aestivum L. The wheat seedlings were treated with 0.5, 1.0 and 1.5 mM concentrations of benzoic acid (BA), with and without water stress. Leaf water status, photosynthetic pigments, protein content, amount of proline and activities of nitrate reductase and antioxidant enzymes were examined. Allelopathic stress resulted in reduction of seedling height. Height of water stressed seedlings greatly decreased. The combined treatments, BA + WS further decreased the seedling height. BA treatment with and without water stress caused significant reduction in dry weight of the seedlings. BA and water stress decreased relative water content, pigments and protein content. Total soluble sugar content and nitrate reductase activity were variedly affected under all treatments. Proline content and lipid peroxidation increased in treatments with BA and water stress. Activity of superoxide dismutase increased significantly (P < 0.05) while catalase activity decreased in all treatments. Ascorbate peroxidase and guaiacol peroxidase activities were higher as compared with catalase which seems to protect wheat seedlings from oxidative stress. Water stress elevated the toxic effect of allelochemical.

The impact that the parasitic plant field dodder (Cuscuta campestris Yunk.) has on chlorophyll fluorescence and chlorophyll content of infested alfalfa (Medicago sativa L.) and sugar beet (Beta vulgaris L.) was examined under controlled conditions. Several parameters of chlorophyll fluorescence were measured in infested and non-infested alfalfa and sugar beet plants over a period of twenty days, beginning with the day of infestation. Chlorophyll contents (total, relative and ratio of chlorophyll a to b) were determined 1, 7, 14 and 20 days after infestation (DAI). Field dodder was found to affect both the total and relative chlorophyll contents in infested alfalfa and sugar beet, causing significant reduction in chlorophyll content in both host plants. This parasitic plant also affects a number of parameters of chlorophyll fluorescence (F_o, F_v/F_m, Φ_PSII, F_v and IF), showing that these parameters may be considered sensitive indicators of the impact that field dodder has on its host plants.

Plant infection with Agrobacterium rhizogenes leads to the development of a hairy root disease notable for the rapid agravitropic growth of roots on hormone-free nutrient media. In order to look into the interaction of A. rhizogenes with plants and assess opportunities of practical application of hairy root culture, new approaches to their production are elaborated. A method of bacterium-free and plasmid-free production of genetically modified roots (hairy roots) by means of biolistic transformation of leaf explants with a DNA fragment (size of 5461 bp) consisting of genes rolA, rolB, rolC, and rolD are proposed. In most cases, such transformation resulted in the emergence of only adventitious roots with transient expression of rol-genes, and the growth of such roots on hormone-free media ceased in 2–3 months in contrast to genuine hairy roots capable of unrestricted growth. Molecular analysis of different systems of target genes’ expression showed an important role of transgene rolC and host gene of cyclin-dependent protein kinase CDKB1-1 in the maintenance of rapid growth of hairy roots in vitro (in isolated cultures).

Brassinosteroids (BRs) are steroidal hormones that play crucial roles in various processes of plant growth and development. DWF1 encodes a delta(24)-sterol reductase that participates in one of the early stage in the brassinosteroids’ biosynthetic pathway: the conversion of 24-methylenecholesterol to campesterol. Here we report the isolation and expression of one DWF1 homologous gene, PeDWF1, in moso bamboo (Phyllostachys edulis (Carrière) J. Houz.). Sequence analysis revealed that the open reading frame of PeDWF1 was 1686-bp encoding a protein composed of 561 amino acid residues with a calculated molecular weight of 65.1 kD and a theoretic isoelectric point of 8.32. Phylogenetic analysis indicated that PeDWF1 was very close to the cell elongation protein Dwarf1 in rice (Oryza sativa). Furthermore, transient expression of a PeDWF1::GFP fusion protein showed that PeDWF1 was an integral membrane protein most probably associated with the endoplasmic reticulum similar to Dwarf1. Tissue specific expression analysis showed that PeDWF1 was constitutively expressed in moso bamboo with the highest level in shoots and the lowest level in mature leaves. In the early growing stage of shoots, the expression level of PeDWF1 had a rising trend with the increasing height of shoots. These results indicated that PeDWF1 might be involved in the regulation of shoot development by participating in BRs biosynthesis. Moreover, PeDWF1 was heterologously expressed in Escherichia coli and the recombinant protein was about 65 kD, which facilitated further study on the gene function of PeDWF1 in bamboo.