Cell selection with barium ions for obtaining genetically modified tobacco

Abstract

Introduction. The salinity is one of the most aggressive environmental factors. It provokes the wide range of pathological changes in the plant tissues. The drastic decrease of K⁺ cations in plant cells due to salt toxicity is the main feature of injury. The lands with secondary salinization increase. Fresh water in many regions transforms to the product of deficit even to the public. So the problem of obtaining plant forms with elevated levels of salt tolerance becomes extremely significant. Genetic effects that increased the genotype tolerance abilities are the aims of various investigations. Cell selection with heavy metal ions is the appropriate biotechnology for obtaining plant forms that challenged the salinity. The scientific interest to Ba²⁺ ions is due to its interaction with K⁺ cations. There was shown that Ba²⁺ interrupted the K⁺ inward transport

Purpose. The obtaining glycophyte-derived salt tolerant forms (cell lines – regenerants – progeny )via cell selection.

Methods. Selective systems with lethal doses of barium ions (Ba²⁺) for obtaining tobacco cell forms tolerant to salt stress are proposed and elaborated. The minimum of Ba ²⁺ doses that eliminate wild type cell population was established as lethal doses. Primary calli cultures were initiated from tobacco leaves on B5 cultural agar medium. Cell suspension was cultivated in liquid B5 medium. Cell suspension (wild type) was placed between two layers of agar selective medium with the addition of lethal doses of barium ions (“plating procedure”). Only Ba-resistant cells survive under such stress pressure. The survived cells formed primary minicolonies. Such colonies are considered to be tobacco Ba-resistant cell lines (Ba-RCL). Ba-RCL grew at Ba²⁺ ions presence during 3 passages. Then callus was cut and transferred to fresh media: basal medium (normal conditions) and selective media (stress conditions).

Resistant cell variants were tested under lethal salinity. Salt stress was simulated by the addition of sodium chloride or sodium sulfate. There were established three variants of selective systems: medium with the addition of Ba²⁺ cations, (stress I); cultural media with the addition of salinity (stress II, III). Genetic basis of combined stress resistance was confirmed via media rotations. The changes were: normal conditions → stresses I, II, III; stresses I, II, III → normal conditions; stress I → stress II or stress III 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.

Regenerants and plants of R1 seed progeny were cultivated in vitro at presence of sea water salts.

Result. Tobacco cel lines with combined resistance to lethal ion and chloride and sulfate stresses were obtained via cell selection with Ba²⁺ cation. Ba-RCL maintained their viability under any stress pressure. The calli relative fresh mass growth (RFW, Δm) was always positive.

Regenerants from selected cell lines and R1 seed progeny were cultivated at presence of sea water salts (2,5%) in vitro. During such cultivations tested plants formed new roots and leaves. After transfer to control (salt free) medium plants adapted to normal conditions.

Originality of the investigation and its priority was in the promotion of cell selection with Ba²⁺cations for obtaining tobacco variants tolerant to lethal salinity.

Conclusion. Cell selection with heavy metal ions is the perspective approach for obtaining genetically modified plant forms. Barium cation is appropriate agent for selection variants with higher tolerance to salinity. Tobacco is a classic glycophyte. From such initial  tissues tobacco plant forms (cell lines → regenerants → progeny) that survive under lethal salinity were obtained. This approach is suitable for other genotypes.