The biotechnology of somatic embryogenesis in in vitro culture is the most promising direction in the reproduction of conifers. The use of this technology makes it possible not only to massively propagate the best genotypes of trees, but also serves a model for studying the structural, physiological and molecular and genetic mechanisms of both somatic and zygotic embryogenesis in conifers. The main aim of this research was to obtain embryogenic cultures (ECs) producing somatic embryos and embryonic suspension mass (ESM) of Picea obovata. The studies were carried out in 2014-2019 on 30 Siberian spruce trees growing in the vicinity of the city of Krasnoyarsk. To detect genotypes competent for somatic embryogenesis, new donor trees were selected every year for the experiment. 3-10 cones were collected from each tree at different stages of embryo development: globular embryo (the first decade of July), the initiation stage cotyledons (second decade of July), the stage of developed cotyledons (third decade of July) and mature embryos (August). Sterilized explants (zygotic embryos at different stages of development) were introduced into in vitro culture on basic media DCR (Gupta PK and Durzan DJ, 1985), ½LV (Litvay JD et al., 1985), MS (Murashige T and Skoog F, 1962) and AI (Tretyakova IN, 2012). All media were supplemented with myo-inositol - 100 mg/L, casein hydrolyzate - 500-1000 mg/L, L-glutamine - 500 mg/L, sucrose - 30 g/L and agar - 7 g/L. Ascorbic acid at a concentration of 400 mg/L was used as an antioxidant. The level of growth regulators was: 2,4-dichlorophenoxyacetic acid (2,4-D) - 2 mg/L and N6 -benzoaminopurine (BAP) - 1 mg/L. For the proliferation of the ESM, DCR and AI basic media containing 2,4-D (2 mg/L), BAP (0.5 mg/L) and sucrose (20 g/L) were used. The pH was adjusted to pH = 5.8. All culture medium and components were sterilized depending on their termolabile properties. Under aseptic conditions, embryos were removed from megagametophytes and inoculated into nutrient media, 10 explants per flask in 25 replicates. The cultures were incubated in the dark at 24 ± 1 °C. Subcultivation to fresh nutrient medium was carried out every 14 days. To control the quality of cell lines (CL) during subculturing, we performed cytological analyzes using temporary preparations (3-5 preparations for each CL). We evaluated the quality of the embryogenicity of the cultures by the presence of even single structures with pronounced polarity - a globular embryo with a suspensor. The results of the study showed that the induction of callus cultures of Siberian spruce is influenced by such factors as the development stage of the explant, the nutrient medium and the genotype of the donor tree. The introduction of P. obovata immature zygotic embryos into in vitro culture at the stage of the globular embryo, both with megagametophytes and extracted from them, turned out to be ineffective. The induction of callus cultures in Siberian spruce was significantly reduced when mature zygotic embryos were introduced into the culture in vitro. The highest response of explants of Siberian spruce was at the stage of developed cotyledons (See Table 1). In the DCR medium, 90% of explants formed callus (See Table 2). The mineral composition of the media did not significantly affect the induction of callus formation in Siberian spruce. The exception was the MS medium, in which callus cultures were formed only in 41% of explants (See Table 2). The growth of callus cultures was most active in the DCR medium. After 6 months of cultivation, 15-32% of calli remained viable (See Table 2). Cytological analysis of callus cultures showed that they include cells of different types (See Fig. 1 and 2). The first type of cells consisted of elongated cells reaching a length of 10 ± 3 μm, others consisted of isodiametric cells with a diameter of 60 ± 3.5 μm. The somatic embryo globule and embryonic tubes were formed from elongated cells. Isodiametric cells were actively dividing and forming callus. Only 3 cell lines (out of 300 cell lines) belonging to two donor trees had an active ability to proliferate. Globular somatic embryos were actively forming in these cell lines (See Fig. 3). An actively proliferating ESM was formed. Thus, we carried out a comprehensive assessment of the factors influencing the induction of somatic embryogenesis in Siberian spruce. The results obtained indicate that for the successful formation of somatic embryos, the determining factor is not only the choice of donor plants, but also the development stage of the explant. We found that the best stage in the development of zygotic embryos when introduced into in vitro culture of Siberian spruce is the stage of immature embryos with formed cotyledons, while the DCR, ½LV and AI nutrient medium supplemented with growth regulators (2.4-D and BAP) is optimal.
Pinus canariensis plant regeneration through somatic embryogenesis
