Cell selection with barium ions for obtaining genetically modified tobacco

L. E. Sergeeva, L. I. Bronnikova

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.


Keywords


Nicotiana tabacum; Ba2+ ions; selection in vitro; cell lines with combined stress resistance; salt tolerant plants

References


Sergeeva, L.E. (2002) New selective medium with barium ions is alternative system for obtaining salt resistant cell lines. Biotehnologia [Biotechnology], 2. P.47-52. (in Russ).

Nikolaev, L.A. (1968) Biocatalic agents and their models Higher school. – 196p. (in Russ.)

Tu, S-I, Nungesser, E. & Brauer, D. (1989). Characterization of the effect of divalent cations on the coupled activities of the H+-ATPase in tonoplast vesicles. Plant Physiol., 90, P.1636-1643.

Miller, R.J., Dumford, S.W., Koeppe D.E. & Hanson J.B. (1970). Divalent cation stimulation of substrate oxidation by corn mitochondria Ibid, 45, P.649-653.

Hasenstein, K.H., Evans, M.L., Stinemetz, C.L., Moore, R., Fondrren, M., Koon, C., Higby, M. & Smucker, A.J.M. (1988). Comparative effectiveness of metal ions in inducing curvature in primary roots of Zea mays. Ibid, 86, P. 885-889.

Rubio, F., Nieves-Cordones, M., Aleman, F. & Martinez, V. (2008). Relative contribution of AtHAK5 and AtHAK1 to K+ uptake in the high affinity range of concentrations. Physiol. Plant., 134, P.598-608.

Fan, L.M., Wu, W.-H. & Yang, Y.-Y. (1999). Identification and characterization the inward K+ channel in the plasma membrane Brassica pollen protoplasts. Plant Cell Physiol., 40 (8), P.859-865.

Wang, D.-M., Zhang, J.-L. & Flowers, T.J. (2007). Low affinity Na+ uptake in the halophyte Suaeda maritima. Plant Physiol., 145, P.559-571.

Hasegawa, P.M., Bressan, R.A., Zhu, J.K. & Bohnert, H.J. (2000). Plant cellular and molecular responses to high salinity. Annu. Rev. Plant Physiol. Plant Mol. Biol., 51, P. 463-499.

Gamborg, J.L., Miller, R.A. & Ojima K. (1968). Nutrient requirement of suspension cultures of soybean roots. Exp. Cell Res., 509, P.151-158.

Conner, A.J. & Meredith, C.P, (1985) Large scale selection of aluminum-resistant mutants from plant cell culture: expression and inheritance in seedlings. Theor. Appl. Genet., 71, P. 159-165.

Murashige, T., Skoog, F., (1962) A revised medium for rapid growth and bioassays with tobacco tissue culture Physiol. Plant. 15. P. 473-497.

Sergeeva, L.E., Mykhalska, S.I. (2019) Cell selection with heavy metal ions for obtaining salt tolerant plant cell culture Fiziologia rastenii i genttika [Plant physiol. and genetics] т.51, №4, P. 315-323 . doi.10.15407/frg2019.04.315


Full Text: PDF

Refbacks

  • There are currently no refbacks.
Archive
2014 2 36
2015 2 19
2016 1 2
2017 1 2
2018 1 2
2019 1 2
2020 1

User

Information

Journal Content

Browse

Language