The effect of growth regulators on the in vitro morphogenesis and regeneration of Syringa vulgaris cultivars

The lilac (Syringa L.) is a genus of woody plants widely used in landscaping and also in perfumery, pharmacology, and other industries. Studies to optimize the technology of in vitro propagation and preservation of lilac are needed due to the existing genetic diversity of the genus members and cultivar specificity to growth regulators in nutrient media. In this research, we evaluated the micropropagation efficiency and morphometric parameters of Syringa vulgaris L. explants on media with different cytokinins and their mixes. The aim of the study was to determine the effect of plant growth regulators (6-benzylaminopurine, meta-Topolin, and thidiazuron) on the development of lilac explants at the micropropagation stage. The studies were conducted in the Laboratory of Plant Biotechnology of Tsitsin Main Botanical Garden in 2023. The shoot cultures of four ornamental cultivars of Syringa vulgaris with high regeneration potential ('Krasavitsa Moskvy', 'P.P. Konchalovskii', 'Frank Paterson', and 'Sensation') from the in vitro collection of the Laboratory were used in the research. For the cultivation of explants (about 5 mm long, containing 1-2 metamers), Murashige-Skoog media supplemented with 7 g/L agar, 30 g/L sucrose, and growth regulators 6-benzylaminopurine, meta-Topolin, and thidiazuron at concentrations of 0.5 or 1.0 mg/L were used in the experiments. Nine variants of a nutrient medium were tested (see Table 1). The comparison of the growth regulators showed the advantage of meta-Topolin over thidiazuron and the widely used 6-benzylaminopurine (See Figs. 1-3). When cultured on media with meta-Topolin, the greatest microshoot height (48.3 ± 1.6 mm vs. 35.6 ± 1.3 mm (on 6-benzylamino-purine) and 39.4 ± 1.3 mm (on thidiazuron)) and micropropogation rate (21.9 ± 0.8 vs. 8.0 ± 0.2 and 9.7 ± 0.4) of explants were observed. Meta-Topolin promoted the induction of axillary buds (79.8% vs. 27.6% and 33.2%) and the formation of several adventitious shoots per explant (2.3 ± 0.1 vs. 1.3 ± 0.1 and 1.6 ± 0.1). The results showed that the combinations of the growth regulators (6-benzylami-nopurine + meta-Topolin, 6-benzylaminopurine + thidiazuron, meta-Topolin + thidiazuron), added to the medium had a positive effect on micropropagation of the studied cultivars (See Table 2, Fig. 4). When cultured on the media supplemented with a mix of cytokinins, most studied cultivars produced the highest microshoots and developed more adventitious shoots per explant (2.3 ± 0.2 (6-benzylaminopurine + meta-Topolin), 2.2 ± 0.2 (6-benzylaminopurine + thidiazuron), 3.0 ± 0.2 (meta-Topolin + thidiazuron)). The combined use of cytokinins increased the micropropagation rate only relative to cultivation on medium with 6-benzylaminopurine and thidiazuron added separately. For instance, 'Frank Paterson' increased its micropropagaton rate from 10.1 ± 0.8 (1.0 mg/L thidiazuron) to 22.7 ± 1.1 on the medium with meta-Topolin + thidiazuron, and 'Sensation' increased it from 8.1 ± 0.3 (1.0 mg/L 6-benzylamino-purine) to 21.0 ± 2.2 on the medium with 6-benzylaminopurine + thidiazuron. We revealed the reduction of some negative effects of thidiazuron when used together with other growth regulators. For instance, 'Krasavitsa Moskvy' produced higher microshoots on the medium with the mix of phytohormones (86.0 ± 4.4 and 74.4 ± 3.9 mm), while 'P.P. Konchalovskii' increased its micropropagation rate from 8.1 ± 1.0 (0.5 mg/L thidiazuron) and 6.7 ± 0.6 (1.0 mg/L thidiazuron) to 13.0 ± 2.0 due to the addition of meta-Topolin to the medium. However, application of other cytokinins to thidiazuron in the medium could not completely prevent large callus formation in some lilac cultivars ('P.P. Konchalovskii' and 'Frank Paterson'). Since lilac micropropagation occurs mainly through the formation and further development of adventitious shoots at the base of explants, the observed formation of several shoots per explant on the tested media significantly increased the efficiency of propagation of Syringa vulgaris cultivars. During the research, we observed almost no root formation in explants on the media with meta-Topolin. Only 'Sensation' showed spontaneous rhizogenesis on the medium with 0.5 mg/L meta-Topolin (10%) and the medium with 6-benzylaminopurine + meta-Topolin (12.5%). When cultured on the media with 6-benzylaminopurine, the studied cultivars had 27.7% of rooted explants. When cultured on the media with thidiazuron, some cultivars showed root formation ('Krasavitsa Moskvy' (17.6%); 'Frank Paterson' (27.8%)), while other cultivars had callus formation at the base of explants. Application of cytokinin combinations to the medium suppressed root formation in explants; most cultivars on these media had no rooted explants. Thus, to obtain rooted microplants before adaptation and exclude a separate rooting stage, we recommend using media with low auxin content or media with low cytokinin content with the addition of auxins. To conclude, we investigated the morphometric parameters of microshoot development and morphogenesis of lilac cultivars on the media with different growth regulators and their combinations at the micropropagation stage. Therefore, we optimized the micropropagation technique for Syringa vulgaris cultivars. The article contains 4 Figures, 2 Tables, 50 References. The Authors declare no conflict of interest.

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