Automated imaging coupled with AI-powered analysis accelerates the assessment of plant resistance to Tetranychus urticae.

The two-spotted spider mite (TSSM), Tetranychus urticae, is among the most destructive piercing-sucking herbivores, infesting more than 1100 plant species, including numerous greenhouse and open-field crops of significant economic importance. Its prolific fecundity and short life cycle contribute to the development of resistance to pesticides. However, effective resistance loci in plants are still unknown. To advance research on plant-mite interactions and identify genes contributing to plant immunity against TSSM, efficient methods are required to screen large, genetically diverse populations. In this study, we propose an analytical pipeline utilizing high-resolution imaging of infested leaves and an artificial intelligence-based computer program, MITESPOTTER, for the precise analysis of plant susceptibility. Our system accurately identifies and quantifies eggs, feces and damaged areas on leaves without expert intervention. Evaluation of 14 TSSM-infested Arabidopsis thaliana ecotypes originating from diverse global locations revealed significant variations in symptom quantity and distribution across leaf surfaces. This analytical pipeline can be adapted to various pest and host species, facilitating diverse experiments with large specimen numbers, including screening mutagenized plant populations or phenotyping polymorphic plant populations for genetic association studies. We anticipate that such methods will expedite the identification of loci crucial for breeding TSSM-resistant plants.

Plastid retrograde regulation of miRNA expression in response to light stress.

BACKGROUND: MicroRNAs (miRNAs) are a class of endogenous noncoding RNAs that play a pivotal role in the regulation of plant development and responses to the surrounding environment. Despite the efforts made to elucidate their function in the adaptation of plants to many abiotic and biotic stresses, their role in high light (HL) stress is still vague. HL stress often arises upon plant exposure to full sunlight. Subsequent changes in nuclear gene expression are triggered by chloroplast-derived retrograde signals. RESULTS: In this study, we show that HL is involved in miRNA-dependent regulation in Arabidopsis thaliana rosettes. Microtranscriptomic screening revealed a limited number of miRNAs reacting to HL. To explain the miRNA regulation mechanisms at the different biogenesis stages, chemical and genetic approaches were applied. First, we tested the possible role of plastoquinone (PQ) redox changes using photosynthetic electron transport chain inhibitors. The results suggest that increased primary transcript abundance (pri-miRNAs) of HL-regulated miRNAs is dependent on signals upstream of PQ. This indicates that such signals may originate from photosystem II, which is the main singlet oxygen (1O2) source. Nevertheless, no changes in pri-miRNA expression upon a dark-light shift in the conditional fluorescent (flu) mutant producing 1O2 were observed when compared to wild-type plants. Thus, we explored the 1O2 signaling pathway, which is initiated independently in HL and is related to ß-carotene oxidation and production of volatile derivatives, such as ß-cyclocitral (ß-CC). Pri-miRNA induction by ß-CC, which is a component of this 1O2 pathway, as well as an altered response in the methylene blue sensitivity 1 (mbs1) mutant support the role of 1O2 signaling in miRNA regulation. CONCLUSIONS: We show that light stress triggers changes in miRNA expression. This stress response may be regulated by reactive oxygen species (ROS)-related signaling. In conclusion, our results link ROS action to miRNA biogenesis, suggesting its contribution to inconsistent pri- and mature miRNA dynamics.

Profiling the Proteome of Cyst Nematode-Induced Syncytia on Tomato Roots.

Cyst nematodes are important herbivorous pests in agriculture that obtain nutrients through specialized root structures termed syncytia. Syncytium initiation, development, and functioning are a research focus because syncytia are the primary interface for molecular interactions between the host plant and parasite. The small size and complex development (over approximately two weeks) of syncytia hinder precise analyses, therefore most studies have analyzed the transcriptome of infested whole-root systems or syncytia-containing root segments. Here, we describe an effective procedure to microdissect syncytia induced by Globodera rostochiensis from tomato roots and to analyze the syncytial proteome using mass spectrometry. As little as 15 mm2 of 10-µm-thick sections dissected from 30 syncytia enabled the identification of 100-200 proteins in each sample, indicating that mass-spectrometric methods currently in use achieved acceptable sensitivity for proteome profiling of microscopic samples of plant tissues (approximately 100 µg). Among the identified proteins, 48 were specifically detected in syncytia and 7 in uninfected roots. The occurrence of approximately 50% of these proteins in syncytia was not correlated with transcript abundance estimated by quantitative reverse-transcription PCR analysis. The functional categories of these proteins confirmed that protein turnover, stress responses, and intracellular trafficking are important components of the proteome dynamics of developing syncytia.

Unravelling the Complexity of Plant Defense Induced by a Simultaneous and Sequential Mite and Aphid Infestation.

In natural and agricultural conditions, plants are attacked by a community of herbivores, including aphids and mites. The green peach aphid and the two-spotted spider mite, both economically important pests, may share the same plant. Therefore, an important question arises as to how plants integrate signals induced by dual herbivore attack into the optimal defensive response. We showed that regardless of which attacker was first, 24 h of infestation allowed for efficient priming of the Arabidopsis defense, which decreased the reproductive performance of one of the subsequent herbivores. The expression analysis of several defense-related genes demonstrated that the individual impact of mite and aphid feeding spread systematically, engaging the salicylic acid (SA) and jasmonic acid (JA) signaling pathways. Interestingly, aphids feeding on the systemic leaf of the plant simultaneously attacked by mites, efficiently reduced the magnitude of the SA and JA activation, whereas mites feeding remotely increased the aphid-induced SA marker gene expression, while the JA-dependent response was completely abolished. We also indicated that the weaker performance of mites and aphids in double infestation essays might be attributed to aliphatic glucosinolates. Our report is the first to provide molecular data on signaling cross-talk when representatives of two distinct taxonomical classes within the phylum Arthropoda co-infest the same plant.

Arabidopsis tonoplast intrinsic protein and vacuolar H+-adenosinetriphosphatase reflect vacuole dynamics during development of syncytia induced by the beet cyst nematode Heterodera schachtii.

Plant parasitic cyst nematodes induce specific hypermetabolic syncytial nurse cell structures in host roots. A characteristic feature of syncytia is the lack of the central vacuole and the formation of numerous small and larger vesicles. We show that these structures are formed de novo via widening of ER cisternae during the entire development of syncytium, whereas in advanced stages of syncytium development, larger vacuoles are also formed via fusion of vesicles/tubules surrounding organelle-free pre-vacuole regions. Immunogold transmission electron microscopy of syncytia localised the vacuolar markers E subunit of vacuolar H+-adenosinetriphosphatase (V-ATPase) complex and tonoplast intrinsic protein (γ-TIP1;1) mostly in membranes surrounding syncytial vesicles, thus indicating that these structures are vacuoles and that some of them have a lytic character. To study the function of syncytial vacuoles, changes in expression of AtVHA-B1, AtVHA-B2 and AtVHA-B3 (coding for isoforms of subunit B of V-ATPase), and TIP1;1 and TIP1;2 (coding for γ-TIP proteins) genes were analysed. RT-qPCR revealed significant downregulation of AtVHA-B2, TIP1;1 and TIP1;2 at the examined stages of syncytium development compared to uninfected roots. Expression of VHA-B1 and VHA-B3 decreased at 3 dpi but reached the level of control at 7 dpi. These results were confirmed for TIP1;1 by monitoring At-γ-TIP-YFP reporter construct expression. Infection test conducted on tip1;1 mutant plants showed formation of larger syncytia and higher numbers of females in comparison to wild-type plants indicating that reduced levels or lack of TIP1;1 protein promote nematode development.

The Role of Programmed Cell Death Regulator LSD1 in Nematode-Induced Syncytium Formation.

Cyst-forming plant-parasitic nematodes are common pests of many crops. They inject secretions into host cells to induce the developmental and metabolic reprogramming that leads to the formation of a syncytium, which is the sole food source for growing nematodes. As in other host-parasite models, avirulence leads to rapid and local programmed cell death (PCD) known as the hypersensitive response (HR), whereas in the case of virulence, PCD is still observed but is limited to only some cells. Several regulators of PCD were analyzed to understand the role of PCD in compatible plant-nematode interactions. Thus, Arabidopsis plants carrying recessive mutations in LESION SIMULATING DISEASE1 (LSD1) family genes were subjected to nematode infection assays with juveniles of Heterodera schachtii. LSD1 is a negative and conditional regulator of PCD, and fewer and smaller syncytia were induced in the roots of lsd1 mutants than in wild-type Col-0 plants. Mutation in LSD ONE LIKE2 (LOL2) revealed a pattern of susceptibility to H. schachtii antagonistic to lsd1. Syncytia induced on lsd1 roots compared to Col0 showed significantly retarded growth, modified cell wall structure, increased vesiculation, and some myelin-like bodies present at 7 and 12 days post-infection. To place these data in a wider context, RNA-sequencing analysis of infected and uninfected roots was conducted. During nematode infection, the number of transcripts with changed expression in lsd1 was approximately three times smaller than in wild-type plants (1440 vs. 4206 differentially expressed genes, respectively). LSD1-dependent PCD in roots is thus a highly regulated process in compatible plant-nematode interactions. Two genes identified in this analysis, coding for AUTOPHAGY-RELATED PROTEIN 8F and 8H were down-regulated in syncytia in the presence of LSD1 and showed an increased susceptibility to nematode infection contrasting with lsd1 phenotype. Our data indicate that molecular regulators belonging to the LSD1 family play an important role in precise balancing of diverse PCD players during syncytium development required for successful nematode parasitism.

The miRNAome dynamics during developmental and metabolic reprogramming of tomato root infected with potato cyst nematode.

Cyst-forming plant-parasitic nematodes are pests threatening many crops. By means of their secretions cyst nematodes induce the developmental and metabolic reprogramming of host cells that lead to the formation of a syncytium, which is the sole food source for growing nematodes. The in depth micro RNA (miRNA) dynamics in the syncytia induced by Globodera rostochiensis in tomato roots was studied. The miRNAomes were obtained from syncytia covering the early and intermediate developmental stages, and were the subject of differential expression analysis. The expression of 1235 miRNAs was monitored. The fold change (log2FC) ranged from -7.36 to 8.38, indicating that this transcriptome fraction was very variable. Moreover, we showed that the DE (differentially expressed) miRNAs do not fully overlap between the selected time points, suggesting infection stage specific regulation by miRNA. The correctness of RNA-seq expression profiling was confirmed by qRT-PCR (quantitative Real Time Polymerase Chain Reaction) for seven miRNA species. Down- and up-regulated miRNA species, including their isomiRs, were further used to identify their potential targets. Among them there are a large number of transcription factors linked to different aspects of plant development belonging to gene families, such as APETALA2 (AP2), SQUAMOSA (MADS-box), MYB, GRAS, and AUXIN RESPONSE FACTOR (ARF). The substantial portion of potential target genes belong to the NB-LRR and RLK (RECEPTOR-LIKE KINASE) families, indicating the involvement of miRNA mediated regulation in defense responses. We also collected the evidence for target cleavage in the case of 29 miRNAs using one of three alternative methods: 5' RACE (5' Rapid Amplification of cDNA Ends), a search of tasiRNA within our datasets, and the meta-analysis of tomato degradomes in the GEO (Gene Expression Omnibus) database. Eight target transcripts showed a negative correlation with their respective miRNAs at two or three time points. These results indicate a large regulatory potential for miRNAs in tuning the development and defense responses.

Abscisic Acid Insensitive 4 transcription factor is an important player in the response of Arabidopsis thaliana to two-spotted spider mite (Tetranychus urticae) feeding.

Plants growing in constantly changeable environmental conditions are compelled to evolve regulatory mechanisms to cope with biotic and abiotic stresses. Effective defence to invaders is largely connected with phytohormone regulation, resulting in the production of numerous defensive proteins and specialized metabolites. In our work, we elucidated the role of the Abscisic Acid Insensitive 4 (ABI4) transcription factor in the plant response to the two-spotted spider mite (TSSM). This polyphagous mite is one of the most destructive herbivores, which sucks mesophyll cells of numerous crop and wild plants. Compared to the wild-type (Col-0) Arabidopsis thaliana plants, the abi4 mutant demonstrated increased susceptibility to TSSM, reflected as enhanced female fecundity and greater frequency of mite leaf damage after trypan blue staining. Because ABI4 is regarded as an important player in the plastid-to-nucleus retrograde signalling process, we investigated the plastid envelope membrane dynamics using stroma-associated fluorescent marker. Our results indicated a clear increase in the number of stroma-filled tubular structures deriving from the plastid membrane (stromules) in the close proximity of the site of mite leaf damage, highlighting the importance of chloroplast-derived signals in the response to TSSM feeding activity.

The suppression of tomato defence response genes upon potato cyst nematode infection indicates a key regulatory role of miRNAs.

Potato cyst nematode Globodera rostochiensis is an obligate parasite of solanaceous plants, triggering metabolic and morphological changes in roots which may result in substantial crop yield losses. Previously, we used the cDNA-AFLP to study the transcriptional dynamics in nematode infected tomato roots. Now, we present the rescreening of already published, upregulated transcript-derived fragment dataset using the most current tomato transcriptome sequences. Our reanalysis allowed to add 54 novel genes to 135, already found as upregulated in tomato roots upon G. rostochiensis infection (in total - 189). We also created completely new catalogue of downregulated sequences leading to the discovery of 76 novel genes. Functional classification of candidates showed that the 'wound, stress and defence response' category was enriched in the downregulated genes. We confirmed the transcriptional dynamics of six genes by qRT-PCR. To place our results in a broader context, we compared the tomato data with Arabidopsis thaliana, revealing similar proportions of upregulated and downregulated genes as well as similar enrichment of defence related transcripts in the downregulated group. Since transcript suppression is quite common in plant-nematode interactions, we assessed the possibility of miRNA-mediated inverse correlation on several tomato sequences belonging to NB-LRR and receptor-like kinase families. The qRT-PCR of miRNAs and putative target transcripts showed an opposite expression pattern in 9 cases. These results together with in silico analyses of potential miRNA targeting to the full repertoire of tomato R-genes show that miRNA mediated gene suppression may be a key regulatory mechanism during nematode parasitism.

Suppression of NGB and NAB/ERabp1 in tomato modifies root responses to potato cyst nematode infestation.

Plant-parasitic nematodes cause significant damage to major crops throughout the world. The small number of genes conferring natural plant resistance and the limitations of chemical control require the development of new protective strategies. RNA interference or the inducible over-expression of nematicidal genes provides an environment-friendly approach to this problem. Candidate genes include NGB, which encodes a small GTP-binding protein, and NAB/ERabp1, which encodes an auxin-binding protein, which were identified as being up-regulated in tomato roots in a transcriptome screen of potato cyst nematode (Globodera rostochiensis) feeding sites. Real-time reverse transcription-polymerase chain reaction (RT-PCR) and in situ hybridization confirmed the localized up-regulation of these genes in syncytia and surrounding cells following nematode infection. Gene-silencing constructs were introduced into tomato, resulting in a 20%-98% decrease in transcription levels. Nematode infection tests conducted on transgenic plants showed 57%-82% reduction in the number of G. rostochiensis females in vitro and 30%-46% reduction in pot trials. Transmission electron microscopy revealed a deterioration of cytoplasm, and degraded mitochondria and plastids, in syncytia induced in plants with reduced NAB/ERabp1 expression. Cytoplasm in syncytia induced in plants with low NGB expression was strongly electron translucent and contained very few ribosomes; however, mitochondria and plastids remained intact. Functional impairments in syncytial cytoplasm of silenced plants may result from NGB's role in ribosome biogenesis; this was confirmed by localization of yellow fluorescent protein (YFP)-labelled NGB protein in nucleoli and co-repression of NGB in plants with reduced NAB/ERabp1 expression. These results demonstrate that NGB and NAB/ERabp1 play important roles in the development of nematode-induced syncytia.

Identification of genes up-regulated during somatic embryogenesis of cucumber.

Somatic embryogenesis is a method of plant regeneration, but it can also be used as a model to study plant development. A normalized library of cDNA fragments representing genes up-regulated after the induction of somatic embryogenesis in cucumber suspension cultures was constructed using the suppression subtractive hybridization technique. Candidate cDNA fragments (119) were classified according to their similarity to genes encoding known proteins and the presence of potential functional domains. Of the translation products with homology to known proteins, about 23% were possibly involved in metabolism, 13% represented proteins with a probable role in cellular communication and signal transduction, about 12% were likely to participate in protein synthesis, while around 10% were potential transcription factors. The genes corresponding to four of the cDNAs were subsequently analyzed in more detail: CsSEF2, CsSEM1 and CsSESTK1 encoding putative transcription factors or co-activators, and CsSECAD1 encoding cinnamyl alcohol dehydrogenase. Full-length cDNAs were isolated and analyzed. RT-PCR confirmed the up-regulation of these genes after the induction of somatic embryogenesis and showed the presence of their transcripts in other tissues. The in situ localization of transcripts of the CsSEF2 and CsSEM1 genes demonstrated that signalling in somatic embryo tissues involving these factors is concentrated in the cotyledon primordia and roots.

Dynamics in the tomato root transcriptome on infection with the potato cyst nematode Globodera rostochiensis.

Plant parasitic nematodes infect roots and trigger the formation of specialized feeding sites by substantial reprogramming of the developmental process of root cells. In this article, we describe the dynamic changes in the tomato root transcriptome during early interactions with the potato cyst nematode Globodera rostochiensis. Using amplified fragment length polymorphism-based mRNA fingerprinting (cDNA-AFLP), we monitored 17 600 transcript-derived fragments (TDFs) in infected and uninfected tomato roots, 1-14 days after inoculation with nematode larvae. Six hundred and twenty-four TDFs (3.5%) showed significant differential expression on nematode infection. We employed GenEST, a computer program which links gene expression profiles generated by cDNA-AFLP and databases of cDNA sequences, to identify 135 tomato sequences. These sequences were grouped into eight functional categories based on the presence of genes involved in hormone regulation, plant pathogen defence response, cell cycle and cytoskeleton regulation, cell wall modification, cellular signalling, transcriptional regulation, primary metabolism and allocation. The presence of unclassified genes was also taken into consideration. This article describes the responsiveness of numerous tomato genes hitherto uncharacterized during infection with endoparasitic cyst nematodes. The analysis of transcriptome profiles allowed the sequential order of expression to be dissected for many groups of genes and the genes to be connected with the biological processes involved in compatible interactions between the plant and nematode.

Characterization of CsSEF1 gene encoding putative CCCH-type zinc finger protein expressed during cucumber somatic embryogenesis.

Somatic embryos obtained in vitro are a form of vegetative reproduction that can be used in artificial seed technology, as well as a model to study the principles of plant development. In order to isolate the genes involved in somatic embryogenesis of the cucumber (Cucumis sativus L.), we utilized the suppression subtractive hybridization (SSH). One of the obtained sequences was the CsSEF1 clone (Cucumis sativus Somatic Embryogenesis Zinc Finger 1), with a level of expression that sharply increased with the induction of embryogenesis. The full length cDNA of CsSEF1 encodes the putative 307 amino acid long protein containing three zinc finger motifs, two with CCCH and one with the atypical CHCH pattern. The CsSEF1 protein shows significant similarity to other proteins from plants, in which the zinc fingers arrangement and patterns are very similar. Transcripts of CsSEF1 were localized in the apical part of somatic embryos, starting as early as the polarity was visible and in later developmental stages marking the cotyledon primordia and procambium tissues. As a result of transferring an antisense fragment of CsSEF1 into Arabidopsis thaliana abnormalities in zygotic embryos and also in cotyledons and root development were observed.

Unintended consequences of plant transformation: a molecular insight.

Plant genomes are dynamic structures having both the system to maintain and accurately reproduce the information encoded therein and the ability to accept more or less random changes, which is one of the foundations of evolution. Crop improvement and various uncontrolled stress factors can induce unintended genetic and epigenetic variations. In this review it is attempted to summarize factors causing such changes and the molecular nature of these variations in transgenic plants. Unintended effects in transgenic plants can be divided into three main groups: first, pleiotropic effects of integrated DNA on the host plant genome; second, the influence of the integration site and transgene architecture on transgene expression level and stability; and third, the effect of various stresses related to tissue handling, regeneration and clonal propagation. Many of these factors are recently being redefined due to new researches, which apply modern highly sensitive analytical techniques and sequenced model organisms. The ability to inspect large portions of genomes clearly shows that tissue culture contributes to a vast majority of observed genetic and epigenetic changes. Nevertheless, monitoring of thousands transcripts, proteins and metabolites reveals that unintended variation most often falls in the range of natural differences between landraces or varieties. We expect that an increasing amount of evidence on many important crop species will support these observations in the nearest future.

In situ reverse transcription PCR on plant tissues.

In situ detection techniques allow specific nucleic acid sequences to be exposed in morphologically preserved tissue sections. In combination with immunocytochemistry, in situ detection can relate microscopic topological information to gene activity at the transcript or protein levels in specific tissues. The advantage of in situ methods over the conventional techniques (e.g., Northern blot, reverse transcription polymerase chain reaction [RT-PCR], or real-time PCR) is that they allow the investigation of the putative spatial distribution of nucleic acid products activity in a heterogeneous cell population. In this chapter, we describe a protocol for in situ RT-PCR detection of specific messenger RNA in cucumber (Cucumis sativus), although this protocol can be used for any plant species, floral buds, and somatic embryo tissue sections on glass microscope slides. A successful in situ RT-PCR procedure requires the optimization of many conditions related to the tissue types used, for example, a cell's age, size, and composition, which may influence the detection of RT-PCR products, as well as specific transcript availability. Moreover, parameters, such as the fixation time, thermal cycling set-up, and the time of detection of RT-PCR products, also should be optimized. The importance of the other factors also is estimated in the protocol. In addition several types of controls that are necessary for a trustworthy in situ RT-PCR method are being discussed.

Tissue-culture-responsive and autotetraploidy-responsive changes in metabolic profiles of cucumber (Cucumis sativus L.).

Somaclonal variation commonly occurs during in vitro plant regeneration and may introduce unintended changes in numerous plant characters. In order to assess the range of tissue-culture-responsive changes on the biochemical level, the metabolic profiles of diploid and tetraploid cucumber R1 plants regenerated from leaf-derived callus were determined. Gas chromatography and mass spectrometry were used for monitoring of 48 metabolites and many significant changes were found in metabolic profiles of these plants as compared to a seed-derived control. Most of the changes were common to diploids and tetraploids and were effects of tissue culture. However, tetraploids showed quantitative changes in 14 metabolites, as compared to regenerated diploids. These changes include increases in serine, glucose-6P, fructose-6P, oleic acid and shikimic acid levels. Basing on this study we conclude that the variation in metabolic profiles does not correlate directly with the range of genome changes in tetraploids.

The metabolic profiles of transgenic cucumber lines vary with different chromosomal locations of the transgene.

The metabolic profiles of five transgenic cucumber lines were compared taking into consideration their transgene integration sites. The plants analyzed were homozygous and contained transgenes integrated in a single locus on chromosomes I, II, III or IV. The transgenes were preferentially located in the euchromatic regions. Each of these locations possessed a specific metabolic profile. The number of altered compounds in the transgenic lines varied between 9 and 23 of the 47 metabolites identified. These alterations seem to be specific for each independent transgene integration. However, some changes are common: a decrease in the levels of phenylalanine, aspartate, ethanolamine and pipecolate, and an increase in the level of benzoic acid. The observed effects of transgene introduction are discussed in this paper.

Xyloglucan endotransglucosylase/hydrolase genes in cucumber (Cucumis sativus) - differential expression during somatic embryogenesis.

Defined changes in the cell wall directed by many proteins accompany every morphogenetic process in plants. Xyloglucan endotransglucosylase/hydrolase proteins (XTH; EC 2.4.1.207) have the potential to modify the hemicellulose matrix within the cell wall. Cs-XTH1 and Cs-XTH3 genes, which encode XTH proteins, were found among numerous genes that are differentially expressed after the induction of cucumber somatic embryogenesis. The expression of these genes increased during somatic embryogenesis. The Cs-XTH1 gene was localized on the second chromosome near the centromere region, whereas Cs-XTH3 was found in the middle of the fifth chromosome's longer arm. Northern blot hybridization showed that both genes were preferentially expressed in roots. We also observed higher accumulation of both transcripts in somatic embryos than in the proembryogenic mass. The localization of mRNA by in situ hybridization revealed that the Cs-XTH1 transcripts were largely accumulated in the presumptive cotyledon primordia of somatic embryos. The XTH gene family consists of a number of genes with a high degree of structural similarity. Screening a cucumber genomic library has identified other members of this gene family. The intron/exon structure, sequence similarities and the close chromosomal distance between some members suggest their common evolutionary origin. The involvement of XTH-related genes in somatic embryo formation is discussed.

The cloning of sequences differentially transcribed during the induction of somatic embryogenesis in cucumber (Cucumis sativus L.).

Somatic embryogenesis in cucumber cell suspension culture is a convenient tool to study differential gene expression, particularly during the early stages of this process. In this study, we used the cucumber somatic embryogenesis system to detect genes that were differentially transcribed during the induction of embryo development. We identified and cloned 120 candidate cDNA fragments from differential display gels. The selected cDNAs were confirmed by reverse northern, and 83 were sequenced. The obtained sequences represent 64 independent transcripts. The search for similarities in the databases gave a significant result in 16 cases. The potential involvement of these sequences in somatic embryogenesis is discussed.

Polymorphom of sexually different cucumber (Cucumis sativus L.) NIL lines.

Isolations of polymorphic sequences of two pairs of the NIL lines of cucumber (Cucumis sativus L.), which differ with respect to sex, were carried out using the subtraction hybridization methods of DSC (Differential Subtraction Chain) and GDDSC (Genetically Directed DSC). 266 DSC tags were isolated from the entire genome region, and 38 GDDSC tags were isolated from the region containing the sex genes. Based on the obtained results, the methods used may be considered highly effective. The attained sequences, like 11 AFLP clones obtained earlier [Witkowicz, J. et al. Cell. Mol. Biol. Lett. 8 (2003) 375-381], were characterized by analyzing their hybridization with differential (dhaom) and subtractive cDNA libraries (cDNAsubtractom) from 1- to 2- mm floral buds of the same lines, and by the sequencing of 28 tags. A high average degree of homology was found to exist in the genpolom to dhom and cDNAsubtractom, particularly in the case of "dominant" (when the tester used was a line in which the sex of the plants was dependent upon the dominant allele). This indicates a significant share of coding sequences in the polymorphic genomic tags as well as their share in flower formation. Many of these sequences originate from the sex gene region. Analysis of the sequenced tags showed their interesting composition, including many organelle sequences which transferred into the nucleus, and coding.