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Introduction
Project Goals
General Strategy
Research Activities
Benefits & Beneficiaries
Project Partners
Project Events
Summary Reports
Visibility of the Project

Introduction

Summer snowflake (Leucojum aestivum L.) contains in the highest concentration the important alkaloid galanthamine (Gal), compared with the other Gal sources: Narcissus and Galanthus. In Bulgaria, natural Gal-type populations of L. aestivum have been exploited since 1960 for production of the Bulgarian patented medicines Nivalin, etc.

Overutilization of natural resources and destruction of habitats has led to protection measures: L. aestivum was included in the Red Data Book of Bulgaria as “endangered species”. Populations of industrial interest were proclaimed as protected areas and are under special usage regime.

Supplies of Gal sources are currently insufficient, whereas the market for Gal is potentially increasing.


Project Goals

  • Development of different approaches for biomass production – source of Gal;
  • Protection of natural populations of summer snowflake.

    General Strategy

    Both the economical importance of Leucojum and its conservation status were borne in mind in the project strategy. Research approach combines agronomical, biotechnological, ecological, phytochemical, etc. methods so as to comply with GLP and GAP and the legislation in Bulgaria relating to germplasm gathering.
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    Research Activities

    Ecological assessment

  • Assessment of Leucojum natural populations status;
  • Screening of individuals for Gal content in selected appropriate populations and collection of high Gal producers;
  • Germplasm gathering from diverse Leucojum populations;
  • Kariological assessment of Leucojum natural populations;
  • Establishment of ex situ collection;
  • Creation of Bulgarian Leucojum Data Base.

    Agronomical approach

  • In vivo cultivation:
    - Maintaining of genetic material in ex situ collection;
    - Selection of elite clones;
    - Development of method of rapid bulb multiplication.
  • Effect of environmental conditions on biomass productivity and alkaloid content:
  • Analysis of soil samples from habitats and the experimental field plot;
  • Comparison of biomass and Gal productivity among individuals from populations and ex situ collection.

    Biotechnological approach

  • Set up of Biotechnological Laboratory of Medicinal Plants at the Institute of Botany, BAS;
  • Establishment of analytical methodologies: Gal HPLC analysis, Gal CPC, Kariology & DNA.
  • In vitro cultivation:
    - Callogenesis;
    - Micropropagation;
    - Adaptation of plantlets obtained;
    - Establishment of liquid shoot-clumps culture;
    - Establishment of suspension culture;
    - Development of lab-scale in vitro cultures producing Gal.
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    Benefits & Beneficiaries

  • Lab-scale biotechnology for Gal production will be proposed to the end-user for further industrial transfer;
  • In vivo and in vitro multiplied original plant material and elite clones could be used for reclamation of the natural populations or establishment of new snowflake plantations;
  • Original database BLDB containing data about diversity of Leucojum and population characteristics will be proposed to the Ministry of Environment and Waters;
  • Training of young scientists in France in different lab techniques;
  • Improvement of lab equipment at the Institute of Botany and Institute of Microbiology with the Bulgarian Academy of Sciences will improve state-of-the-art research of the Bulgarian teams and will make possible student training.

    Project Partners

    Scientific coordinator:

    PPD Dr. Marina Stanilova
    Partner 1: Institute of Botany, Bulgarian Academy of Sciences, Sofia, Bulgaria; maris@bio.bas.bg

    Administrative coordinator:

    NPD Dr. Monique Burrus
    Partner 4: University P. Sabatier, Toulouse, France; burrus@cict.fr

    Codirectors:

    Prof.Mladenka Ilieva-Stoilova
    Partner 2: Institute of Microbiology, BAS, Plovdiv, Bulgaria; lbpmbas@plov.omega.bg

    Prof. Christophe Clement
    Partner 4: University of Reims, France; christophe.clement@univ-reims.fr

    Industrial Partner & End-user:

    Partner 3: Sopharma Ltd. www.sopharma.bg
    MSc Savelina Shopova, Executive Director, nihfi@abv.bg

    NATO consultants:

    Prof. Gunther Hahne
    Inst. Molec. Plant Biol., CNRS, Strasbourg, France (1999-2001)
    Prof. Michael Heinrich
    University of London, UK, michael.heinrich@ulsop.ac.uk (2000-2005)
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    Project Events

    (Project Meetings, Training of young scienties, Institution visits & Expert visits)

    9/13 February, 2001: Meeting of co-directors, Sofia, Bulgaria
    Evaluations of readiness to start the tasks; discussion of lab set-up, establishment of GLP priorities; organisation of the first co-ordination meeting

    14/19 March, 2001: Conference organised by SfP Office for PPDs "Intellectual Property and Licensing for the NATO Science for Peace Programme", Moscow, Russia; Participation of M. Stanilova

    26/27 March, 2001: First Co-ordination Meeting, Sofia, Bulgaria
    Project launching; clarification of work for the next 6 months; estimation of the state of labs' renovation; choice of equipment; Participants from all Partners and NATO consultant
    09/14 June, 2001: Training on the software for the HPLC, Vienna, Austria
    Participants: Stanislav Yanev, Bojidara Pandova

    21/22 September, 2001: Second Co-ordination Meeting, Sofia, Bulgaria
    Evaluation of work within the last 6 months, difficulties encountered; organisation of work for the next 6 months; discussion of financial issues; visit of new facilities (Biotechnological Laboratory of Medicinal Plants) set up within the frame of the Project in the Institute of Botany, BAS, Sofia. Lecture: Callogenesis - M. Ilieva; Participants from all Partners and NATO consultant

    October/December, 2001: Training of Petya Kovatcheva (Partner 2), at the University of Reims Champagne Ardenne (Partner 4); Optimisation of Gal extraction from dry plant materials and development of CPC for Gal on a small scale

    January/April, 2002: Training of Strahil Berkov (Partner 1), at the University of Reims Champagne Ardenne (Partner 4), France; To obtain knowledge and skills in the field of plant biotechnology, sterilisation of plant materials, callus induction and maintaining, micropropagation, induction of root cultures and cytological techniques for the needs of further work of Partner 1.

    16/19 March, 2002: Third Co-ordination Meeting, Plovdiv/Sofia, Bulgaria
    Acquaintance with the new Consultant; evaluation of work performed within the last 6 months and difficulties encountered; organisation of work for the next 6 months; financial issues; discussion of the end-user scientific and collaborative issues. Lectures: 1. In vitro culture of Leucojum: current status - M. Burrus; 2. In vitro liquid culture and shoot clump propagation in Amaryllidaceae - E. Couillerot; Participants from all Partners and NATO consultant

    08/11 June, 2002: Meeting organised by SfP Office for PPDs on commercialisation of the results obtained through SfP projects, Istanbul, Turkey. Participation of M. Stanilova

    24/27 October, 2002: Fourth Co-ordination Meeting, Vitosha (hotel Elitza)/Sofia, Bulgaria
    Åvaluation of work performed within the last 6 months and difficulties encountered; organisation of work for the next 6 months, financial issues. Lecture: In vitro cultivation of Amaryllidaceae - M. Stanilova; Participants from all Partners.
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    27/29 March, 2003: Fifth Co-ordination Meeting, Vitosha (hotel Kremikovtsi)/Sofia, Bulgaria
    Åvaluation of work performed within the last 6 months and organisation of work for the next 6 months, financial issues. Lecture: Application of additive models in experimental design - Emil Molle; Participants from all Partners and NATO Consultant.

    April/June 2003: Training of Atanas Pavlov (Partner 2), at the University of Reims Champagne Ardenne (Partner 4), France; To obtain knowledge and skills in the field of plant biotechnology, establishment of in vitro cultures of floral parts of L. aestivum, to obtain friable callus and plant cell suspension via enzyme hydrolysis from non-friable callus and microscopic observations of cells, for the needs of the further work of Partner 2.

    2/18 Sept. 2003: Institution visit of PPD to several Universities in France
    Visit of lab facilities and exchange of research experience and information on laboratory management with French colleagues from several Universities in France: Toulouse, Reims, Strasbourg, Besancon.

    16/19 October, 2003: Sixth (Mid-term) Co-ordination Meeting - Sofia/Bankya, Bulgaria
    Presentation of the Project to the scientific community, to the invited external expert and guests from different Bulgarian institutions, to visit the facilities of the end-user in NIHFI, to evaluate the work performed from the beginning of the project, to outline the work until the end of the project, to discuss financial issue. Lecture: Plant cell culture: a useful system to produce phenolic natural products and to look into their biosynthetic pathways - Dr. Maike Petersen; Participants form all Partners, NATO Consultant and External Expert: Maike Petersen from the University of Marburg, Germany

    16/29 February, 2004: Institution visit of Dr. T. Stoeva to several horticultural institutions in UK
    To exchange scientific experience and information on bulbous plant propagation and cultivation and ex situ conservation practices; to participate at "RHS Snowdrop & Snowflake Conference" in London; to present our SfP-project at the seminar in the HRI Wellesbourne; to visit the Royal Botanical Gardens, Royal Horticultural Society Garden, Eden Project and Millenium Seed Bank Project.

    5/11 April, 2004: Seventh Co-ordination Meeting: Co-Directors Meeting in Sofia and Plovdiv, Bulgaria
    NPD visited all Bulgarian participants from Partners 1 & 2. Discussion of co-directors: to evaluate the work performed within the last 6 months and the publication strategy; to organise the work for the next 6 months (scientific work, exchanges, training); to discuss financial issue.
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    May/June, 2004: Training of Yuliana Bogdanova (Partn. 1), at the University of Reims, France (Partn. 4)
    To obtain knowledge and skills in the field of plant biotechnology, histological observation of in vitro cultures of L. aestivum.

    28 October/01 November, 2004: Eighth Co-ordination Meeting - Ribaritza (hotel Argo), Sofia, Bulgaria
    To evaluate the work performed within the last 6 months and difficulties encountered; to organize the work for the next 6 months (scientific work, exchanges, training); to discuss financial issue; to visit the end-user's (NIHFI) lab, research team and R&D director. Participants from all Partners and NATO Consultant.

    22/27 April, 2005: Ninth Co-ordination Meeting: Co-Directors Meeting in Sofia and Plovdiv, Bulgaria
    NPD visited all Bulgarian participants from Partners 1 & 2 and the end-user during the second trial to transfer culture in the bioreactor of NIHFI. Discussion of co-directors on: the work performed within the last 6 months, alkaloid determination, financial issue and forthcoming publications; the work to be done in the next 6 months; the organization of the next and the final meetings.

    04/07 November 2005: Tenth Co-ordination Meeting - Plovdiv/Sofia, Bulgaria
    To evaluate the work performed within the last 6 months and difficulties encountered; to organize the work for the next 6 months (scientific work, exchanges, training); to discuss financial issue; to visit the end-user's (NIHFI) lab, research team and R&D director. Participants from all Partners and NATO Consultant.

    Summary Reports

    SfP Progress Report No 5 (31 October 2003), pp. 197-198
    SfP Progress Report No 4 (31 October 2002), pp. 195-196
    SfP Progress Report No 3 (31 October 2001), pp. 197-198
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    Visibility of the Project

    Scientific Communications

    18/20 June, 2001: VI National Conference of Botany, Sofia, Bulgaria (Poster)
    Biotechnological Laboratory of Medicinal Plants: Structure, functions and prospects. M. Stanilova, C. Gussev, Y. Bosseva

    13/19 September, 2001: X Congress of OPTIMA (Organisation of Phyto Taxonomic Investigations of the Mediterranean Area), Palermo, Italy (Poster)
    Conservation of Leucojum aestivum L. (Amaryllidaceae) in Bulgaria. C. Gussev, D. Uzunov, Y. Bosseva, T. Stoeva, M. Stanilova, M. Burrus

    05/09 June, 2002: 7th Symposium of the Flora of Southeastern Serbia and Neighboring Regions, Dimitrovgrad, Yugoslavia (Poster)
    Morphometric variability of bulbs of Leucojum aestivum L. from Bulgaria. T. Stoeva, C. Gussev, Y. Bosseva, Y. Bogdanova, M. Stanilova, M. Burrus

    29 Sept./03 Oct., 2002: 2nd Conference of AMAPSEEC (Association of Medicinal and Aromatic Plants of South-East European Countries), Chalkidiki, Greece (Oral presentation & Poster)
  • In vitro micropropagation of some Amaryllidaceae species. M. Stanilova, Y. Bogdanova, M. Burrus
  • Rapid bulb multiplication of Leucojum aestivum L. T. Stoeva, Y. Bosseva, I. Damyanova-Kirilova, Y. Bogdanova, C. Gussev, E. Molle, M. Stanilova, M. Burrus

    2003: 8th Symposium ICSN, Gif sur Yvette, France (Poster)
    Performances de la CPC pour l'isolement de la galanthamine d'origine naturelle. JH Renault, A. Maciuk, P. Kovatcheva, B. Richard, P.Thepenier, S.Hugel, M. Burrus, M. Zeches-Hanrot

    11 June 2003: International R&D Conference "Bulgaria and the system of industrial property: development and perspectives", Sofia, Bulgaria (Poster)
    Alternative approaches of bioproduction of alkaloids and active substances from Bulgarian rare and threatened medicinal plants

    14-18 July, 2003: First International Congress on Bioreactor Technology in Cell, Tissue Culture and Biomedical Applications, Tampere, Finland (Poster)
    Cultivation of plant cell suspension cultures in 3L lab bioreactor. 1. Relationship between dissolved oxygen, growth and rosmarinic acid biosynthesis by Lavandula vera MM cell suspension. A. Pavlov, M. Georgiev, M. Burrus, M. Stoilova

    8-12 Sept. 2003: Fifth International Symposium Technomat & Infotel 2003, Slunchev bryag, Bulgaria (2 oral presentations)
  • Methods of ex situ conservation of summer snowflake (Leucojum aestivum L.). Ch. Gussev, M. Stanilova, Y. Bosseva, T. Stoeva, S. Berkov, M. Burrus
  • Bulgarian Leucojum Database. V. Georgiev, Ch. Gussev, Y. Bosseva, M. Stanilova

    13-14 Sept. 2003: National poster session "Achievements in the botanical science in Bulgaria at the beginning of the 21 century", Balchik, Bulgaria (Poster)
    Alternative approaches of bioproduction of alkaloids and active substances from Bulgarian rare and threatened medicinal plants

    5-8 Sept. 2004: 3rd Conference on Medicinal and Aromatic Plants of Southeast European Countries, Nitra, Slovakia (2 Posters)
  • In vitro micropropagation and acclimatization of Leucojum aestivum L. (Amaryllidaceae) in Bulgaria. E. Genova, Y. Bogdanova, S. Berkov, M. Stanilova
  • In vitro clonal propagation of Leucojum aestivum L. M. Stanilova, Y. Bogdanova, S. Berkov, Y. Bosseva, Ch. Gussev

    5-10 Sept. 2004: XI OPTIMA Meeting, Belgrad, Serbia and Montenegro (Poster)
    Resource assessment of Leucojum aestivum L. (Amaryllidaceae) populations in Bulgaria. C. Gussev, Y. Bosseva, B. Pandova, S. Yanev, M. Stanilova

    12-15 October 2004: 10th International Symposium New Achievements in Chromatography, Opatia, Croatia (Poster)
    Rapid, selective and sensitive RP-HPLC method for separation and quantification of alkaloids from Leucojum aestivum plant, B. Pandova, V. Todorova, S. Yanev

    July 31 - August 4, 2004: International Congress on Natural Products Research, Phoenix, Arizona, USA (Poster)
    In vitro cultures from flower organs of Leucojum aestivum. A. Pavlov, D. Tuneva, E. Ceourot, C. Clement, M. Georgiev, M. Burrus, M. Ilieva

    13-14 October, 2005: Food Science, engineering and technologies' 2005, Plovdiv, Bulgaria (2 oral presentations)
  • Optimization of the nutrient medium composition for galanthamine biosynthesis by Leucojum aestivum shoot culture. A. Pavlov, M. Georgiev, V. Georgiev, T. Gocheva, D. Tuneva, M. Ilieva, M. Burrus
  • Cultivation of Leucojum aestivum shoot cultures in bioreactor. A. Pavlov, M. Georgiev, V. Georgiev, T. Gocheva, D. Tuneva, M. Ilieva, M. Burrus
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    Publications

    C Gussev, D. Uzunov, Y. Bosseva, T. Stoeva, M. Stanilova, M. Burrus. Conservation of Leucojum aestivum L. (Amaryllidaceae) in Bulgaria. Bocconea 16 (2), 815-821.

    Ch. Gussev, M. Stanilova, Y. Bosseva, T. Stoeva, S. Berkov, M. Burrus. Methods of ex situ conservation of summer snowflake (Leucojum aestivum L.). Research publications 5th Int. Symp. Technomat&Infotell 2003, Bulgaria, vol. IV, part 1: 241-247, part 2: 259-264.

    V. Georgiev, Ch. Gussev, Y. Bosseva, M. Stanilova. Bulgarian Leucojum Database. Research publications Technomat&Infotell 2003, vol. IV, part 1: 241-247, part 2: 259-264.

    S. Berkov, A. Pavlov, M. Ilieva, M. Burrus, S. Popov and M. Stanilova. CGC/MS of Alkaloids in Leucojum aestivum Plants and their in vitro Cultures, Phytochemical Analysis, 16, 98-103 (2005).

    A. Pavlov, M. Georgiev, V. Georgiev, T. Gocheva, D. Tuneva, M. Ilieva, M. Burrus. Optimization of the nutrient medium composition for galanthamine biosynthesis by Leucojum aestivum shoot culture. Proceed. Conf. Food science, engineering and technologies' 2005, Plovdiv, Bulgaria.

    A. Pavlov, M. Georgiev, V. Georgiev, T. Gocheva, D. Tuneva, M. Ilieva, M. Burrus. Cultivation of Leucojum aestivum shoot cultures in bioreactor. Proceed. Conf. Food science, engineering and technologies' 2005, Plovdiv, Bulgaria, pp. 305-310.

    C. Gussev, Y. Bosseva, B. Pandova, S. Yanev, M. Stanilova. Resource assessment of Leucojum aestivum L. (Amaryllidaceae) populations in Bulgaria. BOCCONEA (in press).

    B. Pandova, V. Todorova, S. Yanev. Rapid, selective and sensitive RP-HPLC method for separation and quantification of alkaloids from Leucojum aestivum plant. Proceed. 10th Int.l Symp. "New Achievements in Chromatography", Opatia, Croatia, 12-15 October 2004. (in press).

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