Cadmium ions in cell selection for obtaining wheat cell forms tolerant to water stress
Introduction. Water deficit significantly decrease the plant development and crop production. Genetic effects that increased the genotype tolerance abilities are the aims of various investigations. Cell selection is the appropriate biotechnology for obtaining plant forms that challenged abiotic stresses. It is known that Cd2+ cations significantly destroy various plant compartments and tissues. There was detected that Cd2+ injures the water status of the organism.
Purpose. The aim of the investigation was the promotion of cell selection with Cd2+cations for obtaining wheat cell lines tolerant to water stress.
Methods. Selective systems with lethal doses of cadmium ions (Cd2+) for obtaining wheat cell forms tolerant to water stress are proposed and elaborated. The minimum Cd2+ concentration that eliminates wild type cell population was established as lethal doses.
The water stress was conducted by the addition of manitol. Manitol is usually used for simulation water deficit in vitro.
Callus and suspension cultures were initiated from immature embryos of winter wheat, (Triticum aestivum L.), cv. Favoritka. Cell suspension (wild type) was placed on agar cultural B5 medium with the addition of lethal doses of cadmium ions (“plating procedure”). Such doses were deduced during preliminary tests. Only Cd-resistant cell survive under lethal ion stress pressure.
In Cd2+ resistant cell lines relative fresh weight and free proline levels were estimated.
Result. Resistant cells formed primary minicolonies. Such colonies are considered to be wheat resistant cell lines (Cd-RCL). Cd-RCL grew at Cd2+ ions presence during 3 passages. Then callus was cut and transferred to fresh media: basal medium (normal conditions) and selective media (stress conditions). There were established two variants of selective systems: medium with the addition of Cd2+ cations, (stress I); cultural medium with the addition of manitol (stress II). Cd-RCL maintained their viability under any stress pressure. Genetic basis of Cd-RCL combined stress resistance was confirmed via media rotations. The changes were: normal conditions → stresses I, II; stresses I,
II → normal conditions; stress I → stress II or other way roads. The type of cultural medium and the number of passages were always free. As proliferation marker calli relative fresh mass growth (RFW, Δm) was used. It was always positive. This parameter measured under normal conditions exceeded (40-45%) biomass RFW estimated under manitol pressure. But normal data were lower (more than three times) than data measured during calli cultivation at Cd2+ presence. It is assumed that such events are the exhibitions of combined resistance.
The levels of free proline (pro) were estimated in Cd-RCL. Under normal conditions wheat cell cultures accumulated pro in various amounts. In wild type callus the proline level was the highest. In Cd-RCL cultivated at manitol presence pro contents increased. We suppose that elevated pro levels in Cd-RCL under water stress were due to activity of system of its synthesis. The wild type cell cultures eliminated at the end of any passage. Proline levels were lower than level of determination.
Conclusion. Cell lines of winter wheat with combined stress resistance were obtained via cell selection with Cd2+ cations. Cd2+-resistant cell lines tolerated both lethal ion and water stresses. Under water stress pressure callus RFW of Cd2+-resistant cell lines was lower and under Cd2+ affect was higher than normal parameters. The growth under water was cooperated with free proline accumulation. Cell selection with heavy metal ions is the perspective approach for obtaining cell variants with higher tolerance to osmotic stresses.
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