Cytogenetic peculiarities of Larix sibirica Ledeb. embryogenic cell lines in in vitro culture

Somatic embryogenesis is a vegetative method of mass propagation of plants, which makes it possible to significantly accelerate genetic breeding studies and increase the scale of the resulting planting stock of important forestry species with breeding-significant features. This is especially relevant for slow growing coniferous plants. The cytogenetic variability of embryogenic cultures of coniferous species has been poorly studied; there are conflicting data in the reports on both the stability of chromosome numbers in in vitro culture and various genomic mutations. In this paper, we present the results of cytogenetic studies of embryogenic cell lines of Siberian larch (Larix sibirica Ledeb.) of different cultivation duration from the collection bank of embryogenic cultures of the Laboratory of Forest Genetics and Tree Breeding, VN Sukachev Institute of Forest, SB RAS (Krasnoyarsk, Russian Federation). We carried out this study to assess the level of cytogenetic stability of cell lines and to detect somaclonal variability and the selection of cell lines suitable for production of cloned plants. We used three embryogenic cellular lines of Siberian larch (CL 5, CL 6 and C 16.28), obtained from zygotic embryos of the experimental tree A4 as a result of controlled and self-pollination, as material for research. The age of the embryogenic cultures is from 1 to 8 years. In vitro zygotic embryos induction and proliferation of embryogenic cultures in Siberian larch was carried out according to the methods described earlier [Pak ME et al. 2016, Tretyakova IN et al. 2012]. Cytogenetic analysis of globular somatic embryos was carried out at the stage of proliferation using the existing methods of karyological and cytogenetic research of plants [Pravdin LF et al. 1972, Puhalskiy VA et al. 2007] with their own modifications. The material was treated with a 0.2% solution of colchicine for 18-20 hours at room temperature to reduce chromosomes and destroy the spindle apparatus, fixed with an ethanol-acetic acid solution (3:1), stained with 1% acetogematoxylin, and squash preparations were used to count the number of chromosomes. The number of chromosomes was determined in not less than 100 metaphase plates for each cell line. As a result of cytogenetic investigation of Siberian larch embryogenic cell lines in in vitro culture for the first time, somaclonal variability in the number of chromosomes was revealed for this species. Among the long-proliferating cell lines, there are both cytogenetically stable cell lines (CL 6 and CL 16.28) containing a normal diploid number of chromosomes (2n = 24) and unstable (CL 5) in the karyotype, with chromosome numbers ranging from 24 to 30, a large number of pathologies of mitosis and cells with micronuclei. Some cell lines of Siberian larch are likely to retain cytogenetic stability for many years, which allows them to be used successfully to produce cloned plants. Obviously, for successful reproduction of coniferous plants with the help of somatic embryogenesis, it is necessary to carry out cytogenetic control of embryogenic cultures that will significantly improve the quality of the obtained regenerants and increase the amount of planting stock of coniferous plants with breeding-significant features that is suitable for plantation forest growing in Siberia. The article contains 2 Figures, 1 Table, 30 References.

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