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% Cl2 (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 μM) and 0.049 μM NAA, or TDZ (3.0; 6.0, and 9.0 μM) with 30 g/L sucrose and 9 g/L agar. The medium with 0.89 μM 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 μmol m–2 s–1, at a temperature of 24 ± 1°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 μM BAP, or 6.0 μM 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 μmol m–2 s–1 stimulated a higher-number somatic embryo formation and a temperature of 26°C affected somatic embryo development. Active formation of primary and secondary somatic embryos was also demonstrated. 2.20 μM BAP with 0.09 μM 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.

Evaluation of Endogenous Phytocomponents and Amino Acids Associated with Direct Somatic Embryogenesis of Coffee (Coffea arabica L.)

Coffee is one of the most important crops cultivated in the world for use in beverages and confectionaries. Embryogenesis is a complex process that begins with a single cell and ends with the formation of mature embryos. Somatic embryo development involves accumulation of complex metabolites and storage reserves. This present experiment identified and quantified endogenous phytocomponents and amino acids present during somatic embryogenesis of ‘Ruiru 11’. Laboratory experiments for this study were set up in the Coffee Research Institute, Kenya at Ruiru. Third leaf pair explants were excised from 8-month-old greenhouse-grown mother plants sterilized and cultured in half strength Murashige and Skoog basal salts augmented with Thidiazuron. Once embryos had developed, the cultures were analysed for phytocomponents using GCMS and HPLC. The results showed that palmitoleic and stearic acids were highest (23.3 µg/g and 69.9 µg/g respectively) in brown embryogenic cultures. Cis 7,8 epoxy-2-methyl octadecane was highest (253 µg/g) in green embryogenic cultures. (Z)-3-Tetradecene was highest (25 µg/g) in brown non-embryogenic cultures. Z, Z-3,13- Octadecedien-1-ol and (Z)-7-Hexadecenal were highest (32.1 µg/g and 70.2 µg/g respectively) in green embryogenic cultures. Alanine content was highest (4.4 µg/g) in embryos of brown cultures. Amino acids, fatty acids and their derivatives are potential biomarkers for embryogenesis. Other phytocomponents should be identified and their role in coffee somatic embryogenesis determined. Further studies regarding the status of the phytocomponents identified in the present study, especially in particular stages of embryo development are needed to propose treatments to improve coffee somatic embryo development.