A Simple and Effective Bioassay Method Suitable to Comparative In Vitro Study of Tomato Salt Tolerance at Early Development Stages

Marat R Khaliluev; Liliya R Bogoutdinova; Galina N Raldugina; Ekaterina N Baranova

doi:10.3390/mps5010011

A Simple and Effective Bioassay Method Suitable to Comparative In Vitro Study of Tomato Salt Tolerance at Early Development Stages

Methods Protoc. 2022 Jan 19;5(1):11. doi: 10.3390/mps5010011.

Authors

Marat R Khaliluev^{1

2}, Liliya R Bogoutdinova³, Galina N Raldugina⁴, Ekaterina N Baranova^{3

5}

Affiliations

¹ Laboratory of Plant Cell Engineering, All-Russia Research Institute of Agricultural Biotechnology, Timiryazevskaya 42, 127550 Moscow, Russia.
² Department of Biotechnology, Institute of Agrobiotechnology, Russian State Agrarian University-Moscow Timiryazev Agricultural Academy, Timiryazevskaya 49, 127550 Moscow, Russia.
³ Laboratory of Plant Cell Biology, All-Russia Research Institute of Agricultural Biotechnology, Timiryazevskaya 42, 127550 Moscow, Russia.
⁴ Laboratory of Ion Transport and Salinity Resistance, K. A. Timiryazev Institute of Plant Physiology, Russian Academy of Sciences, Botanicheskaya Street 35, 127276 Moscow, Russia.
⁵ Laboratory of Plant Protection, N.V. Tsitsin Main Botanical Garden of Russian Academy of Sciences, Botanicheskaya 4, 127276 Moscow, Russia.

Abstract

In vitro evaluation of tomato seeds and seedlings for salt tolerance has undoubted advantages (high productivity, as well as stability and reproducibility of the obtained experimental data due to the maintenance of constant controlled conditions) in comparison with open-field system and pot experiments. However, even high-quality seeds greatly differ in the uniformity of germination capacity and germination energy. Heterogeneous germination in the habit and developmental stage of plant material significantly distorts the obtaining of relevant experimental data suitable for correct interpretation. In our study, we propose a simple and effective bioassay method suitable to comparative in vitro study of tomato salt tolerance using shoot apex of seedlings at the early first-true-leaf stage. Shoot apexes cultured the on the root induction medium (RIM) supplemented with 0.2 mg/L indole-3-butyric acid (IBA) and NaCl at different concentrations (0-250 mM NaCl) revealed significant differences between two tomato genotypes (line YaLF and cv. Rekordsmen) at the organismal (measurements of CO₂ gas exchange), organ (rhizogenesis frequency; number and length of de novo regenerated roots; root fresh (RFW) and dry (RDW) weights; shoot fresh (SFW) and dry (SDW) weights), tissue (the average cross-sectional area of epidermal and mesophylls cotyledonary cells) and cellular (ultrastructure of chloroplast and nuclear compartments) development levels. In addition, a quantitative comparison of proline and photosynthetic pigments contents under 75 and 150 mm NaCl treatments showed a different response between two tomato genotypes. The proposed methodological approach can be used for other plants with a high response to auxin-induced rhizogenesis in vitro, as well as for the comparative in vitro assessment of other abiotic stresses.

Keywords: NaCl stress in vitro; Solanum lycopersicum L.; photosynthetic pigments; proline; respiratory and photosynthetic CO2 gas exchange; ultrastructure of cotyledon spongy mesophyll.