Protocols for in vitro plant multiplication from somatic tissues and production of artificial seeds through encapsulation of nodes were developed for Aristolochia tagala Cham., a rare and valuable medicinal plant, as a measure of conservation and as a prerequisite for genetic transformation procedure. A maximum number of adventitious shoots were regenerated from leaf-derived callus on Murashige and Skoog (MS) medium containing 6-benzylaminopurine (BAP; 2 μM), α-naphthaleneacetic acid (NAA; 0.5 μM), and phloroglucinol (PG; 10μM). Nodes collected from in vitro established shoot cultures were encapsulated in 3 % (m/v) sodium alginate and 1 % (m/v) calcium chloride. Multiple shoots were successfully regenerated from the encapsulated nodes cultured on MS medium supplemented with 3 μM BAP and 0.5 μM kinetin (KIN). Regenerated shoots from callus and artificial seeds were successfully rooted and acclimated to greenhouse conditions. Since roots of A. tagala are primarily used in traditional medicine, a protocol for regenerating roots directly from the leaf derived callus was also developed. Maximum root length was obtained when the callus was cultured in MS medium supplemented with KIN (1 μM), indole acetic acid (IAA; 0.5 μM), NAA (0.1 μM), and PG (10 μM). Biochemical parameters were studied in calli grown with and without PG in the medium to establish a correlation between these parameters and shoot morphogenesis. An increment of antioxidant enzymes (peroxidase and catalase) and metabolites (sugars and proteins), and a decrease in the amount of polyphenol oxidase was observed in the calli which were grown in the presence of PG.

The Arabidopsis thaliana (L.) Heynh. atExt1 extensin gene is expressed in a cell and tissue-specific manner, in response to developmental cues, and is inducible by a wide range of biotic and abiotic stresses. Over-expression of this gene has been shown to alter stem morphology and to limit the invasiveness of virulent bacterial pathogens, indicating that this cell wall protein gene plays an important role in plant development and defense. A detailed sequence analysis of 3.2 kb of the atExt1 gene promoter region has identified a large number of putative 5'cis-acting elements. Based on the location of clusters of putative promoter control elements, seven atExt1 5' promoter truncations were constructed, fused upstream of the β-glucuronidase (GUS) reporter gene, and transformed into A. thaliana. Transgenic plants carrying the various promoter constructs were challenged by wounding and pathogen attack and analysed for GUS expression - this analysis revealed a complex pattern of regulation, involving positive and negative control regions. Northern analysis using wounded tissues from transgenic Arabidopsis plants carrying the 3.2 kb-promoter::GUS construct confirmed the transcriptional activation of the transgene.

Cryptic intergenomic pairing of genus Zea was induced by the use of a diluted colchicine solution in order to elucidate the phylogenetic relations and differentiation of the homoeologous genomes. Results indicate that in species and hybrids with 2n = 20, there was chromosome pairing between the homoeologous A and B genomes with a maximum of 5IV, with the exception of Zea diploperennis and their interspecific hybrids where cryptic homoeologous chromosome pairing was not induced. In almost all 2n = 30 hybrids, observed cryptic pairing increased to a maximum of 10III although Z. mays × Z. mays with 2n = 30 did not show significant differences between treated and untreated materials. Pairing was also observed in species and hybrids with 2n = 40, in which a maximum of 10IV was observed, with the exception of Z. mays with 2n = 40 where treated and untreated cells did not differ significantly.

Rice (Oryza sativa L.) seedlings were grown hydroponically in Hoagland's nutrient solution under controlled conditions to investigate the effects of NaCl pretreatment on their response to subsequent application of cadmium (Cd) alone and Cd + NaCl combination. The Cd stress caused growth retardation in all plants, significantly reduced pigment content, stomatal conductance (g_s), and net photosynthetic rate (P_N). Cd stress significantly increased malondialdehyde and proline content. Compared to Cd treatment alone, combination stress had more detrimental effects on the above parameters. However, the NaCl pretreatment was beneficial in improving the plant growth and plant tolerance to Cd alone or combination stress.

Peroxiredoxins (Prxs) play major roles in preventing oxidative damage and their function is consistent with the presence of Prx isoforms in most, if not all, cellular compartments and their expression is dependent on environmental conditions. The aim of this study was to identify and characterize genes encoding Prxs in Vitis vinifera. Quantitative real time polymerase chain reaction (qRT PCR) was used to determine their response to irradiance, heat, and water stress. We identified seven vvprx genes, two of which were especially responsive to water stress, followed by heat stress, but no major changes were observed after high irradiance. The vvprxIIF targeted to mitochondria was the most responsive to water stress and it might be involved in drought tolerance through H₂O₂ signalling. The vvprxII-2, a putative PrxII, is targeted to the chloroplasts and was the most responsive to heat stress. It might be related with abscisic acid-dependent thermotolerance. Additional key words: abscisic acid, antioxidants, grapevine, heat stress, photosynthesis, qRT PCR, water stress.

This study investigated the effects of pH and nitrogen form and concentration on cadmium (Cd) uptake by potato (Solanum tuberosum L.) grown in hydroponic culture. Potato plants grown in a pH-buffered nutrient solution for 10 d were exposed for 24 h to 25 nM CdCl₂ labelled with ¹⁰⁹Cd. Plants showed a significantly higher Cd uptake and accumulation at pH 6.5 than at pH 4.5 and 5.5. Nitrogen supplied as nitrate (NO₃ ^-) generally resulted in a higher Cd uptake and accumulation than N supplied as ammonium (NH₄ ⁺). This effect was most pronounced at pH 6.5. The N concentration increasing from 6.5 to 26 mM resulted in a decreased Cd influx when either NO₃ ^- or NH₄ ⁺ was used. Cd translocation to the shoot was increased when NO₃ ^- was used as the sole N source. In conclusion, pH had a strong influence on Cd uptake by roots and N form is especially important for Cd translocation within the potato plant.

We isolated PaAP3, a homolog of the class B MADS-box transcription factor gene APETALA3 (AP3), from the monoecious plant London plane tree (Platanus acerifolia Willd.). PaAP3 encodes a protein that shares good levels of identity with class B genes from Arabidopsis thaliana (35 and 51 % identity with PISTILLATA (PI) and AP3, respectively), and also with class B genes of other woody species (59 % identity with PTD from Populus trichocarpa and 66 % with TraAP3 from Trochodendron aralioides). Reverse transcription polymerase chain reaction showed that PaAP3 was expressed in both the female and male flowers of P. acerifolia, but almost no signal was detected in the vegetative tissues or mature embryos. The PaAP3 expression in male flowers showed a relationship with developmental stage. There was a small transient increase during differentiation of the flower primordia in June, but maximal levels occurred during December when flower development appeared arrested. Increased PaAP3 expression was also detected in March of the following year, corresponding to meiotic divisions of the microspore mother cells, but this was lost by April when the pollen was mature.

Daucus carota is cultivated widely but grows best in cool climates. Suppression subtractive hybridization (SSH) is a PCR based method used to selectively amplify differentially expressed cDNAs and simultaneously suppress non-target cDNA. A subtraction forward library was constructed using RNA isolated from the leaves of unstressed and cold stressed carrot plants to determine the genes upregulated during cold stress. Out of the hundreds of clones obtained, sequences of 41 promising clones were submitted to the NCBI EST database. Sequence analyses revealed that these genes have significant roles in signal transduction, osmolyte synthesis and transport, regulation of transcription, translation and protein folding. Semiquantitative real-time polymerase chain reaction analysis (sqRT-PCR) of Dc cyclin, Dc WD and Dc profilin shows that the first two genes were upregulated while Dc profilin was constitutively expressed, but the analyses of the same with SSH, a much more sensitive technique showed an upregulation of all three genes.

An efficient in vitro plant regeneration system has been developed using dark preincubated leaf explants of Rhodiola crenulata, a traditional Tibetan medicinal plant. The leaf explants, preincubated in the dark for 5 d, developed an average of 9.1 shoots per explant on a medium containing 15 μM N ⁶-benzyladenine (BA) and 2.5 μM gibberellic acid (GA₃). The biochemical mechanism underlying dark-induced shoot organogenesis was investigated by measuring polyphenol oxidase (PPO) activity. Dark preincubation significantly reduced PPO activity in leaf explants during the initial period of shoot organogenesis and reduced browning compared to explants cultured in the light. Up to 88.4 % of the regenerated shoots formed roots and developed into complete plantlets on a medium containing 5 μM indoleacetic acid (IAA) within 25 d. Approximately 82 % of the regenerated plantlets survived transplantation and grew vigorously in the greenhouse.

The formation of cytomictic channels (CCs) during the tobacco microsporogenesis has been analyzed by microscopy and cytochemical methods. Starting from the pachytene stage, CCs were formed between microsporocytes with involvement of specific organelles, the so-called spherosome-like vesicles. The presence of the enzyme callase, able to degrade callose and form CCs in the cell wall of microsporocytes, has been demonstrated for the first time in the spherosome-like vesicles. An active form of callase was detectable in the spherosome-like vesicles and cell wall but not in the endoplasmic reticulum and Golgi apparatus. The release of callase from spherosome-like vesicles into the cell wall was described. Two ways in formation of the CCs in the tobacco microsporogenesis, the primary formation in the cell wall composed of pectins and cellulose (leptotene-zygotene) and secondary formation in the cell wall of callose (after the pachytene stage), were compared.

The aim of the present study was to describe the occurrence of three pectic epitopes, recognized by JIM7, LM19, and LM5 antibodies, during somatic (SE) and zygotic (ZE) embryogenesis in Arabidopsis thaliana. The epitopes recognized by JIM7 and LM19 antibodies showed different distributions during SE stages. Moreover, in the early stages of somatic embryo development, a cytoplasmic occurrence of LM19 epitope was detected. Distribution of a pectic epitope recognized by LM5 antibody corresponded to a vascular system differentiation pattern. Occurrence of LM5 epitope was the same in both zygotic and somatic embryos and often restricted to newly synthesized walls of two adjacent cells. These data suggest that both low and high methyl-esterified pectins (recognized by LM19 and JIM7 antibodies, respectively) are developmentally regulated during SE stages and (1→4)-β-D-galactan epitope (recognized by LM5 antibody) may play a role in cell cytokinesis.

The gene, designated TaMADS2, was obtained from wheat leaves infected with the wheat stripe rust fungus by in silico cloning and RT-PCR. TaMADS2 encodes a predicted 159-amino-acid polypeptide that contains a highly conserved MADS domain. Phylogenetic analysis revealed that TaMADS2 is a type I MADS-box gene. The TaMADS2 transcript was detected in wheat leaves, stems, and roots. The expression of TaMADS2 was substantially down-regulated in the compatible interaction between wheat and Puccinia striiformis f. sp. tritici (Pst) at 36 and 48 h post-inoculation (hpi), whereas in the incompatible interaction the down-regulation was only observed at 48 hpi. Exogenous salicylic acid (SA) and abscisic acid (ABA) greatly induced the expression of TaMADS2 at 12 h post treatment (hpt), whereas methyl jasmonate (MeJA) down-regulated TaMADS2 at 6 hpt by approximately two-fold.

Gibberellic acid (GA) promotes cotton fiber cell elongation under in vitro culture conditions and GA signal transduction is mediated by DELLA proteins. Our laboratory has cloned several DELLA genes from cotton and generated transgenic lines in Arabidopsis over-expressing the DELLA gene (GhRGL) and the DELLA-domain deletion mutant gene (Ghrgl). Comparative transcriptome profiling was performed on Arabidopsis transgenic lines over-expressing both GhRGL and Ghrgl. KEGG pathway analysis indicated that the differential genes participated in many important pathways, such as response to gibberellic acid, fatty acid metabolism, etc., and the expression patterns of 93 genes were validated by RT PCR in all transgenic lines and the wild-type plants. Gene ontology annotation identified that these genes were involved in sucrose synthesis, actin nucleation, root hair development, and various hormone signal responses.

In Phalaenopsis, lowering irradiance has been used to delay flower stalk development but the accompanying biochemical changes remain poorly understood. We cultured two commercial Phalaenopsis-type orchids, Phalaenopsis cv. Sogo Yukidian V3, and Doritaneopsis cv. Walnut Valley Halo ES09 under reduced irradiance by under-bench shading (approximately 15 % of mean control irradiance) for 15 weeks in a greenhouse under the natural photoperiod. Besides delaying flower stalk development as expected, the treatment greatly decreased the activities of ribulose-1,5-bisphosphate carboxylase/oxygenase, phosphoenolpyruvate carboxylase, and NAD⁺-malic enzyme, and reduced the nocturnal malate accumulation and daytime starch deposition, the typical diurnal metabolite fluctuations of crassulacean acid metabolism (CAM) plants. As well, the content of sucrose and starch was reduced at dawn and dusk whereas the content of glucose and fructose only at dawn. The persistent decrease in the sucrose content under shading may be an inhibitory signal of flower stalk induction.

Leaf senescence constituted the final stage of leaf development and it is always accompanied by the leaf yellowing. The non-yellowing gene (NYE1), initially identified from Arabidopsis in our laboratory, is a key regulatory gene responsible for chlorophyll degradation during senescence. In this study, an orthologue of AtNYE1 was isolated from the bamboo (Bambusa emeiensis cv. Viridiflavus) and tentatively named BeNYE1. The full length sequence of 1 386 bp contains an open reading frame of 801 bp. The protein encoded by BeNYE1 consists of 266 amino acids. Sequence analysis revealed that BeNYE1 had high similarity with other NYE/SGR proteins from various monocotyledon species. BeNYE1 was strongly induced by natural senescence and dark-induced senescence in bamboo. Driven by a 1.5 kb upstream fragment of AtNYE1, BeNYE1 could rescue the stay-green phenotype of nye1-1. The constitutive over-expression of BeNYE1 could accelerate the chlorophyll degradation. These results indicated that BeNYE1 might play an important role in the regulation of chlorophyll degradation during leaf senescence in bamboo.

Gynogenesis of Chinese long cucumber (Cucumis sativus L.) was obtained from unpollinated ovules cultured on cucumber basal medium (CBM) supplemented with thidiazuron (TDZ) and in some experiments AgNO₃. High induction frequencies (7.85-12.14 %) were induced from unpollinated ovules at the time of anthesis at 0.03-0.07 mg dm^-3 TDZ. Histological analysis indicated that embryo sacs developed completely at the time of anthesis. Further, the highest plant regeneration rate was achieved at CBM supplemented with 0.05 mg dm^-3 a-naphthaleneacetic acid, 0.2 mg dm^-3 6-benzyladenine and 5-10 mg dm^-3 AgNO₃. Flow cytometry analysis showed that 80 % of the regenerated plants were haploid. Histological micrographs and ploidy level analyses clearly revealed initiation, development, and germination of embryos from the unpollinated ovules.

Plant microRNAs modulate diverse developmental processes by regulating expression of their target genes. To explore potential miRNA-guided gene regulation in developing rice (Oryza sativa L.) caryopses, a miRNA microarray was used to identify miRNAs present at the different developmental stages. We found that 27 miRNAs, of which 16 were conserved miRNAs, were present in developing caryopses. High expression levels were detected for miR159, miR167, and miR530 at the morphogenesis stage and for miR169, miR435, and miR528 at the stage of accumulation of metabolites. Next, 26 target genes were predicted for seven of the detected miRNAs and the expression profiles of these miRNAs and their corresponding target genes were examined in developing caryopses. Our results suggest that the miRNAs and their target genes examined at the two distinct stages could contribute to the developmental progress of rice caryopses in concert with phytohormone signalling.

Trehalose can reduce stomatal aperture by a hydrogen-peroxide-dependent pathway in Vicia faba L. (cv. Daqingpi) resulting in significantly lower values of net photosynthetic rate (P_N), stomatal conductance (g_s), and transpiration rate (E). At 8 and 24 h, the lower P_N in trehalose-treated plants was accompanied by significant decrease in intercellular CO₂ concentration (c_i) suggesting that the reduction of P_N was caused by stomatal limitation. At 48 and 72 h, trehalose decreased apparent carboxylation efficiency (P_N/c_i) and did not decrease c_i and g_s compared with controls; therefore the reduction in photosynthesis was caused by non-stomatal limitation. Trehalose treatment resulted in significantly higher effective photochemical efficiency of PS II (Φ_PSII) and did not affect maximum photochemical efficiency of PS II (F_v/F_m). At 24, 48, and 72 h, trehalose decreased non-photochemical quenching (NPQ) and increased photochemical quenching (qP). Our results suggest that trehalose did not damage photosynthetic reaction centers.

Temperature and mineral nutrition are major environmental factors regulating plant growth and development. Yet, cold impact on mineral contents and the ability of the plants to perform changes in specific elements as a part of the acclimation process received little attention. Using five Coffea genotypes previously characterized concerning their cold sensitivity, a mineral analysis was performed considering macro (N, P, K, Ca, Mg, and S) and micro (Na, Fe, Mn, Zn, Cu, and B) nutrients in order to predict their importance in cold tolerance. The results showed a cold-induced dynamics of mineral nutrients in recently mature leaves. The less cold sensitive Icatu, and partially Catuaí, accumulated N, Ca, Mn, Cu, and Zn with potential implications in the maintenance of photosynthetic performance, the reinforcement of the antioxidative defense system, lipid metabolism, and the expression of cold regulated genes, thus constituting interesting traits to evaluate the cold acclimation ability. After a principal component analysis (PCA), N, Fe, Mn, and Cu were further confirmed as strong candidates for an early cold tolerance evaluation due to their dynamics and to specific roles in the activities of Cu/Zn-SOD (Cu), APX (Fe), and PSII (Mn).

In vitro morphogenetic ability of plant cells has been demonstrated in diverse species of angiosperms and gymnosperms but no such report is available in the genus Selaginella till date. We have established an in vitro morphogenic root culture where indole butyric acid (IBA) induced profuse branched and unbranched roots in Selaginella microphylla. We observed inter-convertibility of root apical meristem (RAM) to shoot apical meristem (SAM) in presence of IBA and showed that intact roots are also capable of transformation. Friable callus was obtained from roots on prolonged (∼50 weeks) root cultures. By isolating total RNA from each of the developmental stages, we performed cDNA synthesis followed by random amplification, sequencing, and BLAST analysis of differentially expressed transcripts to correlate morphological events with the changes on molecular level. The results revealed sequence matches to genes involved in diverse cellular processes such as transcription, translation, photosynthesis, replication, secondary metabolism, stress response, and plant defense suggesting ancient origins of such proteins and the evolutionary conservation of biological function.

The effects of plant growth regulators (PRGs) on the induction of flowering and sex expression in micropropagated cucumbers are presented. The highest number of male flowers (6.0 ± 0.7 per plant) was produced by cv. Kmicic F1 on the Murashige and Skoog (MS) medium supplemented with 4.0 μM kinetin. The highest number of female flowers (3.1 ± 0.3) was also observed in cv. Kmicic F1 on either control (PRG-free) medium or medium supplemented with 6.4 μM indole-3-acetic acid (IAA). The MS medium supplemented with 4.4 μM benzyladenine inhibited flower formation. The highest percentage of flowering plantlets (67.5 ± 7.5) was observed on the control MS medium after 16 weeks of culture. Female-to-male flower ratio was influenced by the culture media and changed during cultivation. The highest pollen viability (60-70 %) was observed in anthers of plants cultured on the control medium and the medium with IAA.

Innate immune system is employed by plants to defend against phytopathogenic microbes through specific perception of non-self molecules and subsequent initiation of resistance responses. Current researches elucidate that plants mostly rely on cell surface-located pattern recognition receptors (PRRs) and intracellular nucleotide-binding leucine-rich repeat proteins (NB-LRRs) to recognize pathogen-associated molecular patterns (PAMPs) and effector proteins from microbial pathogens, initiating PAMP- and effector-triggered immunity (PTI and ETI), respectively. Some pathogenic bacterial effector proteins are usually secreted into plant cells and play a virulence function by suppressing plant PTI, implying an evolutionary process of plant immunity from PTI to ETI. In the past several years, a great progress has been achieved to reveal fascinating molecular mechanisms underlying the pathogenic recognition, resistance signaling transduction, and plant immunity evolution. Here, we summarized the latest breakthroughs about these topics, and offered an integral understanding of plant molecular immunity.

Gentiana straminea is native to the Qinghai-Tibet plateau, where it is exposed to extremes of cold and strong UV-B radiation. Here we showed that low temperature, but not UV-B radiation, affected the accumulation of the triterpenoid oleanolic acid. Neither of these stresses altered the expression of known triterpenoid synthesis genes. However, the application of methyl jasmonate (MeJA), but not salicylic acid (SA), significantly enhanced the accumulation of oleanolic acid, and up-regulated the triterpenoid synthesis genes, especially the expression of βAS, the gene encoding β-amyrin.

The aim of this study was to evaluate the influence of methylglyoxal (MG) on organogenesis and regeneration of tobacco (Nicotiana tabacum L.) plants from callus in media containing glycine or succinate. The best improvement in shoot proliferation and shoot length was obtained in the medium supplemented with 0.1 mM MG and 0.5 mM glycine or 0.25 mM succinate. The histological studies showed vigorous development of corm like structures and shoot organogenesis from callus tissues cultured in MG supplemented media. Biochemical studies also revealed higher content of δ-aminolaevulinic acid (a precursor of chlorophyll) and of chlorophyll.

Transgenic rice (Oryza sativa L. subsp. indica cv. White Ponni) constitutively expressing the rice thaumatin-like protein gene (tlp-D34, PR-5) individually or in combination with the rice chitinase gene (chi11, PR-3) was generated using an Agrobacterium vir helper strain with multiple copies of pTiBo542 virB and virG. Transformation with the tlp-D34 gene alone and tlp-D34 + chi11 genes yielded five and seven single-copy transgenic lines, respectively. Southern blot analysis with two probes, one flanking the right T-DNA border and the second flanking the left T-DNA border, confirmed that all transgenic plants harboured single and complete T-DNA copies. Homozygous transgenic lines were first identified in the T₁ generation by Southern blot analysis and were subsequently confirmed by segregation analysis of T₂ plants. Accumulation of transcripts encoded by the transgenes was confirmed in T0 plants and homozygous T₂ plants by Northern blot analysis. The homozygous T2 plants harbouring tlp-D34 + chi11 genes showed 2.8- to 4.2-fold higher chitinase activity. Western blot analysis revealed the accumulation of thaumatin-like protein and chitinase in the respective transgenic plants. Upon infection with Rhizoctonia solani, the disease index reduced from 100 % in control plants to 65 % in a T₃ homozygous transgenic line T4 expressing the tlp-D34 gene alone. In a T2 homozygous transgenic line CT22 co-expressing tlp-D34 and chi11 genes, the disease index reduced to 39 %.

In order to produce human lactoferrin (Lf) in alfalfa (Medicago sativa L.), a construct containing human Lf cDNA under the control of cauliflower mosaic virus 35S promoter was engineered. As selectable marker bar gene, whose expression in plant cells confers tolerance to L-phosphinothricin (ppt) was used. Plants from a highly embryogenic alfalfa clone from the Bulgarian cultivar Obnova 10 were transformed using Agrobacterium tumefaciens mediated leaf disc method. Transgenic alfalfa plants were established from ppt-resistant calli via indirect somatic embryogenesis. The presence of human Lf cDNA in the genome of the selected regenerants was confirmed by polymerase chain reaction (PCR). Reverse transcriptase (RT)-PCR and Western blot showed expression of human Lf in leaf tissue. Studies on antibacterial effect of the recombinant glycoprotein were conducted and resistance of the transgenic alfalfa plants to two phytopathogens, Pseudomonas syringae pv. syringae and Clavibacter michiganensis, was demonstrated. The obtained results suggest that the expression of human Lf in alfalfa could be beneficial not only for producing recombinant protein for clinical application but also for crop quality improvement.

The protein elicitor cerato-platanin (CP) is known to induce defence-related responses in various plants. Some of these responses occur very quickly. In the present work, transcriptional changes caused by CP in leaves from Platanus × acerifolia (Aiton) Willd. were studied. With a cDNA microarray, 131 differentially regulated transcripts were identified as responsive to CP after 24 h of treatment. Eighty-six of these were cold-or ozone-modulated transcripts, thus revealing a significant overlap between genes responsive to CP and to cold/ozone stress. The transcriptional changes caused by CP were compared with the CP-orthologous protein Pop1 in a time-course analysis performed after 3, 6, 12, and 24 h of treatment by real-time RT-PCR on five defence-related genes. Despite some differences, CP and Pop1 were both able to induce early transcriptional changes (WRKY was overexpressed after only 3 h) confirming that pathogenassociated molecular patterns (PAMPs) act very quickly on gene transcription.

Programmed cell death (PCD) is a genetically controlled and conserved process in eukaryotes during development as well as in response to pathogens and other stresses. BAX inhibitor-1 (BI-1) has been implicated as an anti-PCD factor which is highly conserved in plants. Sequence of putative cucumber BI-1 protein exhibited 77.7 % identity and 91.2 % positive value with the homologue Blast BI-1 protein of Arabidopsis thaliana (AtBI-1). This highly homologous protein to the AtBI-1 protein was named CsBI-1. This protein contains an open reading frame (ORF) of 250 amino acids with a BAX inhibitor domain and five transmembrane regions conserved among members of the BI-1 family. Primers designed by the cDNA of CsBI-1gene were used for further sequencing. Cell death in cold-stored cucumber developed concomitantly with increased expression of the CsBI-1 gene and reached maximum at day 6. However, cell death accelerated significantly after 9 d when sharp decrease of the CsBI-1 expression occurred. After warming to 20 °C, expression of the CsBI-1 gene was the highest at day 3, decreased afterwards, and the lowest expression was detected at day 9 when PCD obviously appeared. The overall results indicate that CsBI-1 is cucumber homologue of Arabidopsis thaliana AtBI-1 gene. CsBI-1 is a conserved cell death suppressor induced by cold stress and a negative regulator of PCD.

The ultrastructure of leaf mesophyll cells of in vitro cultured Hypericum perforatum L. plants regenerated after cryopreservation was studied. Electron microscopy analysis revealed that the chloroplasts in plants pretreated with abscisic acid and regenerated after cryopreservation were round, with increased amount of starch, rather small volume of the thylakoid system, and destroyed envelope. Plants pretreated with 0.3 M mannitol and cooled at rates of 0.1 or 0.3 °C min^-1 possessed chloroplasts with high starch content that resulted in a reduction of a membrane system. However, the pretreatment with 0.3 M mannitol and cooling at a rate of 0.2 °C min^-1 was the best as chloroplast ultrastructure resembled the controls regenerated without cryopreservation.

Proteins WCS120 and DHN5 are known as the major cold-inducible dehydrins in wheat and barley plants, respectively. WCS120 and DHN5 relative accumulation increased exponentially along with a growth temperature decline in the range from optimum to cold temperatures. Even at optimum growth temperatures, the most frost-tolerant wheat and barley cultivars can be distinguished from the remaining ones according to dehydrin relative accumulation. The highly tolerant wheat and barley cultivars started accumulating dehydrins at higher growth temperatures and reached higher dehydrin amounts than the less tolerant ones. Statistically significant correlations between lethal temperature for 50 % of the samples (LT50) and dehydrin relative accumulation have been found at all growth temperatures (5, 10, 15 and 20 °C) for WCS120 in wheats and at 5 and 10 °C for DHN5 in barleys. Analogous relationships between dehydrin relative accumulation at different growth temperatures and plant acquired frost tolerance have been proved for wheat WCS120 and barley DHN5.