A highly sensitive express immunochromatography method for molecular diagnosis of plant virus infections was elaborated on the example of a model object — tobacco mosaic virus (TMV). The analysis time does not exceed 5 min, and the lower limit of TMV detection in non-clarified leaf extract (2–4 ng/ml) is comparable with the sensitivity of the enzyme-linked immunosorbent assay of the virus. A single measurement requires 0.1–0.2 ml tested solution (extract from 10–20 mg of leaf material). The sensitivity of TMV determination in the leaf tissue extract was increased by more than one order of magnitude using signal enhancement by silver and is 0.1 ng/ml. In this case, analysis time did not exceed 25 min. The simplicity of this method makes it especially convenient in express diagnosis of numerous analyzed specimens. The prototype of a diagnostic kit for serial analyses of plant viral infections both in laboratory and field conditions was elaborated.

Express immunochromatographic test-strip assays were developed for detection of five plant viruses varying in shape and size of virions: spherical carnation mottle virus, bean mild mosaic virus, rodshaped tobacco mosaic virus, and filamentous potato viruses X and Y. Multimembrane composites (test strips) with immobilized polyclonal antibodies against viruses and colloidal gold-conjugated antibodies were used for the analysis. The immunochromatographic test strips were shown to enable the detection of viruses both in purified preparations and in leaf extracts of infected plants with a sensitivity from 0.08 to 0.5 μg/ml for 10 min. The test strips may be used for express diagnostics of plant virus diseases in field conditions.

New data are reported on the spatiotemporal distribution of free and bound forms of phytohormones (cytokinins, auxins, abscizins) in the ovaries of the amphimictic species Triticum aestivum L. and the apomictic species Taraxacum officinale Web. The data are attributed to four domains of the ovary and three intervals composing the period of ovule activity (from the moment of the ovule at relative rest to the moment of the ovule at the first division). The data were obtained by modifications of the ELISA method. The modifications were elaborated for each phytohormone. Comparative analysis of the data implies that basic events of the reproduction process in the ovary in the period of the ovule activity are under the endogenous hormonal control system. The distributions of free and bound forms of phytohormones in the ovaries of two species show both common regularities, probably characteristic of amphimicts and apomicts, and special features. The latter may be related to the specificity of the reproduction type.

An original micromethod ELISA for differential quantitative determination of free (indolyl-3-acetic acid) and bound (indolyl-3-acetyl-L-aspartic acid) forms of auxins is suggested. Our immunoassay approach is basic and may be applied to problems in biology, medicine, agriculture, and environmental protection for the identification of low-molecular compounds related in chemical structure and characterized by different biological activity.

A new ELISA method is proposed for differential quantitative determination of free (indolyl-3-acetic acid; IAA) and bound (indolyl-3-acetyl-L-aspartate) forms of natural auxins. There is similarity in results obtained by this and some traditionally used methods. The standard error of determination of the active form of IAA by our method is 1.5–2.0 times less than that using the traditional method. The method of quantitative differential determination of the main natural auxins does not require preliminary sample preparation, and this shortens assay time. The developed method has been used for practical determination of different forms of endogenous IAA in wheat and dandelion ovaries subjected to minimal treatment. This method can be used to investigate changes in the ratio of various hormonal forms of auxins that differ in their physiological activity in reproductive organs of angiosperms at various stages of reproduction.

Here we report the development of various diagnostic methods for potato virus and viroid infections including ELISA and immunochromatography on test-strips, combined technologies of DNA probing and dot ELISA as welt as RT-PCR-DNA probe-dot ELISA. Immunostrips for detection of potato viruses X and Y (PVX, PVY) were prepared using colloidal gold particle-polyclonal rabbit antibodies conjugates. Clear visual results can be achieved with 5 min incubations detecting as tow virus concentrations as 0.2 microgram per ml. Non-toxic and water-soluble agent was found out for viroid, virus and cellular RNA extraction from plant cells and viruses. Comparison of the developed procedure and SDS-phenol method for RNA extraction demonstrated identical yield and purity of RNA preparations. Several recombinant plasmids with insertions of cDNAs of potato viruses A, M, X, S, Y, PLRV and PMTV were constructed. To obtain DNA probes, the recombinant plasmids with respective cDNA insertions were chemically labelled with chlordiethylenetriamineplatinum chloride, (dien)Pt chloride. Primary antibodies against (dien)platinated DNA and secondary immunoglobulins conjugated with peroxidase were employed in DNA probe-dot ELISA detection of the target pathogen. Sensitivity of the recombinant plasmid detection by DNA probe-dot ELISA method with chemiluminescent substrate was 0.1 pg and less than 1 pg for virus RNAs. Individual molecules of PSTVd and potato virus RNAs can be found by combined RT-PCR-DNA probe-dot ELISA technique.

We performed the immunochemical study of two solid-phase competitive ELISA systems differing in their specificity toward free and bound ABA. A possible application of these systems for the quantification of natural ABA forms without their preliminary separation and purification in a single sample of plant material was demonstrated.

We quantified endogenous free and bound ABA in ovaries of the apomictic Taraxacum officinale Web. (dandelion) and amphimictic Triticum aestivum L. (wheat) species. ABA distribution was assessed in four ovary sections and during three developmental stages: from the quiescence release of the egg-cell until its first division (the period of egg-cell activity). ABA content was determined by the novel modification of the ELISA technique. The ovaries of both species contained approximately similar amounts of free ABA; whereas wheat ovaries contained 1.5 to 2-fold more bound ABA. A possible involvement of the hormonal system in the control of the chronology of the basic events of plant reproduction (egg-cell activation, the onset of endosperm development, the start of embryogenesis) is discussed.

Spatial and temporal distribution of zeatin and zeatin riboside was studied in the ovaries of amphimictic (Triticum aestivum L.) and apomictic (Taraxacum officinale Web.) species during the period from the quiescence release of the egg cell to its first division. This period was divided into three stages: (1) no signs of division of the central cell, (2) division of the central cell and no signs of division of the egg, and (3) division of the egg. Cytokinin content was determined in ovary and its four sections (basal, medial, apical, and ovule) using an original modification of ELISA. Apomicts and amphimicts were shown to be similar in the pattern of changes in the cytokinin content in basal and medial zones but differed substantially in that of apical section and ovule.

An original modification of the standard ELISA procedure for differential determination of different forms of abscisic acid (ABA) is proposed. It is shown that endogenous forms of ABA may be quantitatively determined in plant tissues subjected to minimal treatment, without purification of the hormones and their chemical modification. The modification has been approved when analyzing changes in the content of different ABA forms in plant tissues differing in physiological activity. Quantitative differential determination of changes in the content of different ABA forms has been performed in ovaries of Triticum aestivum L. and Taraxacum officinale Web. in the period of activity of the ovule (from the moment of its activation to the beginning of division). It is shown that, despite the different types of reproduction in the species studied (amphimixis and apomixis), the time course of changes in the content of different forms of ABA in ovaries is similar, which is suggestive of a correlation between the activity of endogenous hormonal system and chronology of main events (e.g., the beginning of endospermogenesis) of the reproductive cycle.