Features of regenerants morphogenesis and metabolism in vitro, obtained from different fragments of potato shoots

Features of regenerants morphogenesis and metabolism in vitro, obtained from different fragments of potato shoots Micropropagation is one of the fastest ways of plant propagation. This method allows obtaining a large number of clones that retain all characteristics of the species and varieties of plants. Among the important problems of micropropagation of potato plants, two can be distinguished: the rate ofmicroclones regeneration (rooting ofmicroclones and the development of regenerants) in vitro and the value of the propagation factor (number of layers). The plant hormones auxins and cytokinins, whose content changes along the longitudinal axis of the plant, play a key role in the regeneration of cuttings. The aim of this research was to study the specificity of micropropagation, morphogenesis and metabolism of Solanum tuberosum L. regenerants in vitro, depending on the initial explants position in the plant. The objects of the study were plants of S. tuberosum L. of the mid-season cultivar Lugovskoy. During the experiment, cuttings (explants) taken from layers 3 and 5 of the shoot consisted of internodes, buds and leaves. We chose apical cuttings containing a greater amount of endogenous auxins due to the spatial separation of the site of synthesis and the main action of these hormones in the plant as experienced clones. Cuttings from the middle of the shoot were those of control. We cultured cuttings on hormone-free solid agar 50% nutrient medium Murashige-Skoog (MS) supplemented with 3% sucrose and vitamins. The flux density of incident photons of the PAR was 200-230 pmol / (m² . s), with a 16-hour photoperiod and an air temperature of 20-22°С. Luminescent white lamps TL-D 58W / 54-765 G13 (Philips, Poland) were used as a light source. The relative light intensity was measured using an AvaSpec-102/256/1024/2048 spectrometer version 6.2 (Avantes BV, Netherlands). During 3 weeks of culturing microclones in test tubes, we described the phenology of their regeneration. We assessed the formation of adventive roots (AdR) by the dynamics of changes in the number and length of AdR primordia. The formation of regenerants was assessed by the dynamics of changes in the number of layers and the length of shoots. At the end of cultivation on the 39th day, we removed the regenerants from the test tubes and determined their growth and biochemical parameters. We used the gravimetric method to determine the wet and dry biomass of the regenerants. We measured the leaf surface area using the Moticam 2300 software (Spain) on photographs taken with a digital camera. The intensity of lipid peroxidation (LPO) and the content of free proline were measured in the leaves of the middle layers fixed with liquid nitrogen. The content of photosynthetic pigments was determined in the alcoholic extract. In the air dry material, the total content of flavonoids and anthocyanins was determined. We carried out the measurements using a Genesys 10S UV-Vis spectrophotometer (Thermo Electron, Germany). The significance of the differences between the samples was assessed using Student’s t-test (p<0.05). Figures show data as the mean of the repetitions and their confidence interval (M±1.96SEM). All experiments were performed in 20-40 (growth parameters) and five (physiological parameters) biological and three analytical replicates. We noted the emergence of roots in potato cuttings on the 4th-5th day. More active root formation and their elongation occurred after seven days of cultivation in apical clones, compared with clones obtained from the middle layer. By the 10th day of cultivation, the apical clones had formed seven roots, while the middle ones had four roots, their total length was 9.04±0.36 and 6.56±0.26 cm, respectively (SeeFig. 1, A-B). In the cuttings of the middle layer, bud opening and growth of the first shoot internode started earlier than in the apical microclone (See Fig. 1, C-D). The manifestation of donor-acceptor bonds between organs during the formation of regenerants should be noted. The predominant growth of the root system in the apical microclone caused inhibition of the growth and development of the shoot. In the middle microclone, the opposite pattern took place: shoot growth prevailed when the root growth was inhibited. We studied the growth parameters of 39-day-old potato regenerants obtained from apical (AR) and medium (MR) shoot cuttings of potato mother plants (See Fig. 2, A). In AR, root volume, shoot length, number of layers, and the total leaf area increased by 25, 14, 11 and 20% (p<0.05), respectively compared to MR (See Fig. 2, B-E). The regenerants had the same total dry biomass (See Fig. 2, F). However, the redistribution of dry matter between the organs in the plant was ambiguous. We showed that ARs had a 34% (p<0.05) lower dry root mass relative to MR, but a 13.4% higher shoot biomass due to the leaf surface. As a result, the shoot-to-root dry mass ratio was higher in AR compared to MR. In vitro potato regenerants showed predominantly heterotrophic growth since more active AR growth occurred against the background of a low content of all groups of photosynthetic pigments and the chlorophyll a / chlorophyll b ratio (See Fig. 3, E). We studied the physiological state of the regenerants by the biochemical parameters of the middle leaves. In AR leaves, the intensity of LPO relative to MR decreased by 22% (p<0.05), which indicated the formation of a higher oxidative status in MR. AR has a reduced content of free proline by 28% (p<0.05) compared to MR (SeeFig. 3, A-B). The total flavonoids content in MR was twice that in AR (See Fig. 3, C). This could indicate a decrease in the intensity of their growth processes. The level of anthocyanins in the MR and AR did not differ. The data obtained show the ability of the initial hormonal status of microclones to change the sequence and intensity of their regeneration processes, including organogenesis, growth rate and plant metabolism. The use of apical micro-clones is especially important when it is necessary to increase the reproductive rate of plants. The mechanisms underlying the different rates of regeneration of microscopic cuttings taken from different layers of the S. tuberosum shoot are discussed. The paper contains 3 Figures and 31 References. The Authors declare no conflict of interest.

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