In vitro Regeneration of Clematis Plants in the Nikita Botanical Garden via Somatic Embryogenesis and Organogenesis

The effects of growth regulators, namely, 6-benzylaminopurine (BAP) and thidiazuron (TDZ), on the morphogenic capacity of 13 cultivars of clematis plants, in terms of their morphological structure formation, shoot regeneration, and somatic embryo development, are presented. The clematis cultivars 'Alpinist,' 'Ay-Nor,' 'Bal Tsvetov,' 'Crimson Star,' 'Crystal Fountain,' 'Kosmicheskaya Melodiya,' 'Lesnaya Opera,' 'Madame Julia Correvon,' 'Nevesta,' 'Nikitsky Rosovyi,' 'Nikolay Rubtsov,' 'Serenada Kryma,' and 'Vechniy Zov' were taken in collection plots of the Nikita Botanical Gardens for use in study. After explant sterilization with 70% ethanol (1 min), 0.3-0.4% Cl-2 (15 min), and 1% thimerosal (10 min), 1-cm long segments with a single node were introduced to an in vitro culture. The explants were established on the basal MS medium supplemented with BAP (2.20-8.90 mu M) and 0.049 mu M NAA, or TDZ (3.0; 6.0, and 9.0 mu M) with 30 g/L sucrose and 9 g/L agar. The medium with 0.89 mu M BAP served as the control. Culture vessels and test tubes with the explants were maintained in plant growth chamber-controlled conditions: with a 16-h photoperiod, under cool-white light fluorescent lamps with a light intensity of 37.5 mu mol m(-2) s(-1), at a temperature of 24 +/- 1 degrees C. Histological analysis demonstrated that adventitious bud and somatic embryo formation in studied clematis cultivars occurred at numerous areas of active meristematic cell zones. The main role of plant growth regulators and its concentrations were demonstrated. It was determined that maximum adventitious microshoot regeneration without any morphological abnormalities formed on the media supplemented with BAP or TDZ. 4.40 mu M BAP, or 6.0 mu M TDZ were optimal cytokinin concentrations for micropropagation. The explants of 'Alpinist,' 'Ay-Nor,' 'Crimson Star,' 'Crystal Fountain,' 'Nevesta,' and 'Serenada Kryma' cultivars displayed high morphogenetic capacity under in vitro culturing. During indirect somatic embryogenesis, light intensity 37.5 mu mol m(-2) s(-1) stimulated a higher-number somatic embryo formation and a temperature of 26 degrees C affected somatic embryo development. Active formation of primary and secondary somatic embryos was also demonstrated. 2.20 mu M BAP with 0.09 mu M IBA affected the high-number somatic embryo formation for eight cultivars. Secondary somatic embryogenesis by the same concentration of BAP was induced. The frequency of secondary somatic embryogenesis was higher in 'Crystal Fountain' (100%), 'Crimson Star' (100%), 'Nevesta' (97%), and 'Ay-Nor' (92%) cultivars. Based on these results, the methodology for direct somatic embryogenesis and organogenesis of studied clematis cultivars has been developed.

Automated summary

The study examined the effects of growth regulators on the morphogenic capacity of 13 cultivars of clematis plants, demonstrating that optimal concentrations of BAP and TDZ can promote shoot regeneration and somatic embryo development. Histological analysis revealed active formation of adventitious buds and somatic embryos, with specific concentrations of BAP and TDZ showing the best results for micropropagation. Additionally, light intensity and temperature were found to influence somatic embryo formation and development.