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CELLULAR SELECTION OF WHEAT RESISTANT TO UV-B IRRADIATION
The scientific chief - Raisa G. Butenko
Russian Academy of Science, Timiriazev Plant Physiology Institute, 35 Botanical str., 127276, Moscow, Russia.
Ultraviolet light is solar electromagnetic radiation. Human beings' eyes cannot see ultraviolet light. The ultraviolet radiation is a segment of a spectrum from 10 up to 380 nm. Respectively with properties of radiation the UV-site of a spectrum is accepted to divide to 3 parts: UV-A - 380-315 nm - short-range (long wavelength); UV-B - 315-280 nm - mean (middle wavelength) UV (area B); UV-C - 280-200 nm - distant (short-wave) radiation.
The ozone in atmosphere formed as effect of UV-radiation or electric discharge onto molecular oxygen. The atomic oxygen is a result of this reaction. The atom of oxygen at interplay with other oxygen molecule gives ozone. Thus the an ozone screen are formed - rarefied layer of ozone at the altitude 13-48km from a surface of the Earth. It's defend the Earth from UV-radiation of the Sun.
The screen of ozone in atmosphere defends the surface of the Earth from a harmful effect of UV-light. As a result of human activity in high layers of atmosphere fall the great many of chlorofluorocarbons (freons, CFCs). There under operating of a sunlight they are dissociating with formation of atoms of chlorine. The free chlorine is chemically very active. Free chlorine reacts with molecules of ozone and destroy them. As a result the quantity of UV-B irradiation on the surface of the Earth are increasing.
The "ozone hole" is a some reduction of ozone amount above some regions - par excellence in a polar areas. These holes appear over the poles mostly due to the movement of stratospheric air currents and cold air temperatures. There there are favourable conditions for destruction of ozone. Also nitrogen-containing compounds and mechanical microparticles (aerosols) cause destruction of ozone. The holes are forming because the ozone layer is being destroyed faster than is being created.
Plants have evolved as a systems which can receive and use the sun's energy. As a result of human activity the quantity of harmful solar radiation that is reaching the surface of the Earth and our food plants was increased.
The effects of short wave UV-B light on higher plants cells to be complex. The low doses of UV light form tymine dimers in DNA. High doses inhibit repair of these dimers. Higher doses also affect other physiological processes, including protein synthesis, active transport, respiration, and ion ballans. Different species of plants show different sensitivities to UV radiation.
Any portion of the plant may show UV-B damage symptoms, however, the upper leaves exhibit the most strong damage. The affected areas first appear pale green spots which turn brick red in colour later. Extensive areas on leaves, stems and fruit may become discoloured. The leaves may be metallic or bronze in color. These bronze regions on the leaf may can lead to broken leaves. In plants UV-B can affect different effects. A reduced photosynthetic capacity reduces overall productivity of the leaf. Leaf area, plant height and fruit size can be reduced. Quality: Plant pigments and other compounds produced in response to increased UV-B radiation may alter the taste and texture and of a plant product. Quantities of protein, oil and carbohydrates may be altered by UV-B radiation. Naturally compounds that protect the plant from predators and pests may be altered by UV-B radiation and loose effectiveness. Damage by UV-B may make the plants more susceptible to diseases. Ecology: When there is differential tolerance to UV-B radiation, some plants will be damaged more than others. This can change the species balance in a plant community. Weeds: Some weeds may be more tolerant to UV-B radiation than the cultivated plants. This may give the weeds a competitive advantage.
The plant resistance to UV-irradiation consist of integrated defence mechanisms. The resistance mechanisms are include photorepair, absorbtion of incident radiation by epidermal pigments, and morphologies that shield sensitives tissues. One of them is the increased synthesis of phenolic compounds. Accumulation of some phenolic compounds correlated with plant resistance to UV radiation.
Cell technologies in combination with traditional methods of breeding essential enrich the arsenal of breeder. Relaxation of stress influences on plant organisms acquired especial significance. The breeding of agricultural crops which toleranced to increase UV-irradiation is actual. In our work we are used "in vitro" culture for obtaining strain of summer common wheat which resistant to increase UV-irradiation. The final aim of this work is obtaining of whole plants from resistent callus culture.
Materials and methods
In our work we have got a purpose to obtain a cell line of wheat, steady in increased doses of UV-B on an example 2 genotypes summer common wheat T. aestivum: a "Taezhnaya" sort and "Fotos" line. At the first phase the influence on process of callusogenesis, intensity of growth and callus regeneration capacity was investigate. As a final aim of research is obtaining of whole plants from callus tissue which are capable to growth in conditions of a heightened level of UV-B. Therefore is interesting to estimate capacity of selected genotypes for regenerate the whole plant from callus tissue.
The starting material in our experiments were two wheat genotipes - "Fotos" strain and "Taezhnaya" variety. The sort Taezhnaya is deduced in Krasnoyarsk agricultural Institute by a method of personal selection from a third generation of a hybrid K-441 (L - 330 е M - 341). The line Fotos (L 40959) is obtained by personal selection from a hybrid population from a crossing of sorts Moscowskaya-35 and Zhnitsa in Bashkir Bread-stuffs Institut.
Initial material for in vitro selection was mature and immature (14-16 dayes old) embryoses of this genotypes. To obtain the primary callus, isolated embryos were cultivated in modified media MS (T. Murashige & F. Scoog, 1962), containing apart from macro- and microsalts also FeNa-EDTA (37,8mg/l), nicotinic acid (0,5mg/l), pyridoxine-HCI (0,5mg/l), thiamine-HCI (1mg/l), myo-inositol (100 mg/l), L-asparagin 120 mg/l, ascorbic acid 20 mg/l, sucrose 2%, agar 0,7% and as hormonelike effector 2,4D (2,4-dichlorophenoxyacetic acid) 2mg/l. Average frequency of callus formation on 2,4-D media amounted for both of used genotypes is 98-99%. Regeneration media contained 0,5 mg/l IAA (indolylacetic acid) and 1mg/l BAP (6-benzylaminopurine).
Within the first 1-3 days watched a considerable increasing of primary explant. A beginning of callusogenesis for 2-4 days of cultivating at first for 30-40% embryoses, and by to 10-14 day reached 98-100%.
Selection for resistance was conducted both at the stage of callus formation and the the stage of callus growth. For a selective agent we are used the light of bactericidal lamp named PRK-2. This lamp apart from UV-B contains also bactericidal radiation. For exception of bactericidal radiation we have used glass filters.
The radiation intensity was regulated by changing spacing interval from a lamp. The radiation time was 2 hours each day approximately in the middle of a 16-hour light duration of exposure by white light (5Klx). The following spacing intervals from a lamp were used: 25, 40, 50, 60, 70 and 80 sm, that was equal the intensity of irradiation, accordingly, 1,66; 1,11; 0,74; 0,55; 0,46 and 0,39 W/m2 for UV-B. The control - culture under UV-B non-transparent glass on spacing interval 50 and 60 sm from this lamps.
We have used a following scheme of selection: callus induction and 3 next passages in selective conditions and then cultivation of selected callus strains in MS media without select factor. In the first passage of selection about 98% of embryoses were formed calluswith medium density, bright yellow in colour, but under the further subcultivation moustly calluses became pale yellow colour without any sign of morphogenesis.
In start experiences where used 1,66 and 0,74 W/m2 doses of UV-B the formed calluses percent from primary explant (at the end of 0 passage) and their weight (at the end of everyone passage by personal weighing) were estimated. UV negatively influences both these parameters. The different genotypes was demonstrated the difference in reacting on UV.
Callus weight was determined by personal weighing in sterile conditions on torsion weights at the moment of next passage. It has allowed to dedicate cell lines which are carrying on the genesis from one primary explant.
The percent of formed calluses, and their weight, was decreased under UV for both genotypes. % of callusogenesis has falled for the "Fotos" line more strongly: from 100 % in the control to 38 % at a dose of 0,74 W/m2 and to 36 % at a dose of 1,66 W/m2. The callus formation for Taezhnaya is less subject to operating UV-B: from 95 % in the control to 91 % at 1,66 W/m2 and to 74 % at 0,74W/m2. Callus weight for both genotypes was descended linearly at increase of a dose as contrasted to by control. So at 1,66 W/m2 callus weight of Fotos averaged 83 % from the control, and Taezhnaya - 72,8 %. And at 0,74 W/m2 - 75 % and 63,5 % accordingly.
Thus used in experience 2 genotypes variously was reacted to UV presence. For "Fotos" the considerable decrease the percent of callusogenesis has watched, whereas their weight decreased not so strongly, as for Taezhnaya. For Taezhnaya the percent of callusogenesis in UV presence was remained rather high, but weight of formed calluses was lower, than for Fotos under identical dose of UV-B.
At further cultivating was found out, that the chosen doses (1,66 and 0,74 W/m2) have appeared lethal for both genotypes - the primary callus formation from embryoses was watched only. In subsequent passages without irradiation the callus growth do not watched, while calluses was saving the external characteristic of a living tissue: light yellow colour and consistence. By results of these experiments the conditions of selection were a changed. The dose is reduced and more differentiated scale is entered.
After 4 selective subcultures under lower doses, thirteen UV-resistant clones were obtained for "Fotos" strain.
In these calluses we determined the common contents of dissoluble amino acids and phenols. Some amino acids are the predecessors of phenols. Phenyl alanine and some other amino acids are the predecessors at a biosynthesis of phenols. The heightened contents of phenolic compounds in selected lines was showed. The content of sum of phenols may be used as a biochemical index of tolerance of callus for increase of UV-irradiation.