Dear Mr. Chairman and members of the Specialized Scientific Council, dear colleagues and guests. The theme of the thesis is: Experimental long-term in vitro cultivation of summer snowflake (Leucojum aestivum L.) - propagation potential, alkaloid profile and clonal selection. Summer snowflake is a threatened plant species included in The Red Data Book of Bulgaria and protected by Biodiversity Law of Bulgaria.
It is also included in the Red List of Bulgarian vascular plants and classified as vulnerable species. Summer snowflake is a natural source of galanthamine, an alkaloid used for the treatment of neurological disorders and neurodegenerative diseases including Alzheimer's disease. Another alkaloid of interest is lycorine which antiviral and antitumor properties are well known. The aim of the dissertation was: initiation of in vitro organ cultures from Leucojum аestivum plants of known origins which keep their capacity to synthesize alkaloids of interest and selection of clones that could be used for creation of plantations and for reintroduction as well as for the industrial scale-up of the in vitro galanthamine production. The following tasks were performed: 1. Initiation of in vitro clones via direct organogenesis from in vivo cultivated Leucojum aestivum plants of different origins. 2.Study and evaluation of the stability of morphology, propagation capacity and alkaloid profile and content of the in vitro obtained long-term cultivated clones. 3.
Selection of clones with high biomass and alkaloids production potential. 4. Ex vitro adaptation of in vitro obtained plants. 5. Study on some endogenous factors known to have an influence on the biomass accumulation and alkaloid biosynthesis. The dissertation was performed within the framework of a 5-year project financed by NATO Science for Peace Programme. A living ex situ collection of Leucojum aestivum consisting of about 3,500 individuals from 27 populations and representative of the species diversity in Bulgaria was established. Preliminary screening of 7 populations concerning the Galanthamine content was performed.
Forty-two individuals coming from 11 selected populations of different chemotypes were used as mother plants for initiation of in vitro clones. The habitats and the codes of the mother plant clusters are shown in the table. The clones which were successfully propagated in vitro are marked in red. This slide summarizes the main steps of micropropagation of Leucojum aestivum. After collection of the mother plants they were rinsed with water and disinfected. Twin-scales were excised and used as primary explants, placed vertically into the medium.
Multiple shoot-clumps were observed after 3-4 weeks in culture and after 6 months of cultivation the bulblets reached 8-10 mm in diameter. Subcultivation of detached bulblets was performed by cutting them into four sectors and placing them into a fresh medium. A part of the obtained bulblets were rooted and cultivated ex vitro. The micropropagation was speed up through a cultivation of the shoot-clumps in a liquid medium.
The disinfection was performed using 3 different sterilizing agents. Using NaDCC (Na-dichloroisocyanurate) we obtained only 1 clone of Leucojum aestivum. Three clones were obtained after sterilization with calcium hypochlorite (Ca(OCl)2). Best results were achieved using commercial bleach. Ten bulbs out of 23 were sterilized immediately after lifting and 13 got a chilling treatment at 5°C for 1–3 months in a refrigerator. 68% sterilized explants were obtained after the chilling treatment and only 35% of the explants survived when they were cut immediately after lifting so the cold storage of L.aestivum bulbs improved significantly by 33% the survival of the explants (P<0.001). Fourteen clones were obtained. Propagation coefficient was assessed for 12 out of 18 clones and for 16 clones was determined the alkaloid content. The used medium was solid or liquid after Murashige and Skoog. Different medium composition and additional compounds were used concerning the aim of the experiments: medium for long-term cultivation, for histological observations, for rooting and for alkaloid biosynthesis.
The obtained clones were transferred to a fresh medium every 4 weeks and were grown for a period of 4 years under the same conditions. Histological observations were performed aiming at studying the kinetic of the formation process of the new shoots in order to prove the direct organogenesis. The addition of Abscisic acid (a dormancy controlling hormone) to the medium led to a speed up of the process of shoot formation and to an increase of the number of the shoots. During the first week of cultivation the explants which were grown on a medium with abscisic acid showed an initial cell-division.
The explants grown on the control medium were without change. During the third week of cultivation meristem development was observed between all of the scales of the explants grown on a medium with abscisic acid and only on the outer scales of the control explants. In contrast to the slow-growing shoots on the control explants, the explants grown on abscisic acid-containing medium developed shoot apex during the fourth week of cultivation. However, at the end of the experiment were observed shapely explants on both media. The only difference was the number of the new shoots.
Significant morphological differences were found among the obtained clones during the period of in vitro cultivation. Most of the clones formed shapely shoots and bulblets. Some of them formed big-sized bulblets with a conical base and short leaves. Another group of clones formed small bulblets with longer leaves. Small number of the clones formed shapeless shoots which developed bulblets for a long period of 8-10 months. Several different-sized shoots were observed inside of these conical bulblets when the outer scale was removed or longitudinal section was performed. If these shoots were not detached the mother bulblet was exhausted for short time.
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