MICROPROPAGATION AND SECONDARY METABOLITES OF SCLEROCARYA BIRREA MACK MOYO Submitted in fulfilment of the requirements for the degree of DOCTOR OF PHILOSOPHY Research Centre for Plant Growth and Development School of Biological and Conservation Sciences University of KwaZulu-Natal, Pietermaritzburg DECEMBER 2009 STUDENT DECLARATION Thesis title: Micropropagation and secondary metabolites of Sclerocarya birrea I, MACK MOYO, student number - 205526161, declare that: (i) The research reported in this dissertation, except where otherwise indicated, is the result of my own endeavours in the Research Centre for Plant Growth and Development, School of Biological and Conservation Sciences, University of KwaZulu-Natal Pietermaritzburg; (ii) This dissertation has not been submitted for any degrees or examination at any other University; (iii) This thesis does not contain data, figures or writing, unless specifically acknowledged, copied from other researchers; and (iv) Where I have reproduced a publication of which I am an author or co-author, I have indicated which part of the publication was contributed by me. Signed at ……………………….…………on the ………..day of……………………… 2010. __________________________ SIGNATURE i DECLARATION BY SUPERVISORS We hereby declare that we acted as Supervisors for this PhD student: Student‟s Full Name: MACK MOYO Student Number: 205526161 Thesis Title: Micropropagation and secondary metabolites of Sclerocarya birrea Regular consultation took place between the student and ourselves throughout the investigation. We advised the student to the best of our ability and approved the final document for submission to the Faculty of Science and Agriculture Higher Degrees Office for examination by the University appointed Examiners. __________________________ SUPERVISOR: PROFESSOR J VAN STADEN ________________________ CO-SUPERVISOR: DR JF FINNIE ii DECLARATION 1 - PLAGIARISM I, MACK MOYO, student number - 205526161, declare that: 1. The research reported in this thesis, except where otherwise indicated is my original research. 2. This thesis has not been submitted for any degree or examination at any other university. 3. This thesis does not contain other persons‟ data, pictures, graphs or other information, unless specifically acknowledged as being sourced from other persons. 4. This thesis does not contain other persons' writing, unless specifically acknowledged as being sourced from other researchers. Where other written sources have been quoted, then: a. Their words have been re-written but the general information attributed to them has been referenced b. Where their exact words have been used, then their writing has been placed in italics and inside quotation marks, and referenced. 5. This thesis does not contain text, graphics or tables copied and pasted from the Internet, unless specifically acknowledged, and the source being detailed in the thesis and in the References sections. Signed: iii DECLARATION 2- PUBLICATIONS DETAILS OF CONTRIBUTION TO PUBLICATIONS that form part and/or include research presented in this thesis (including publications in preparation, submitted, in press and published and details of the contributions of each author to the experimental work and writing of each publication). PUBLICATION 1 MACK MOYO, MANOJ J KULKARNI, JEFFREY F FINNIE, JOHANNES VAN STADEN (2009). After-ripening, light conditions, and cold stratification influence germination of Marula [Sclerocarya birrea (A. Rich.) Hochst. subsp. caffra (Sond.) Kokwaro] seeds. HortScience 4: 119-124. Contributions: Laboratory experiments were performed by the first author under the supervision of Prof Johannes Van Staden and Dr Jeffrey F Finnie. Dr Manoj G Kulkarni assisted with data analysis. PUBLICATION 2 MACK MOYO, JEFFREY F FINNIE, JOHANNES VAN STADEN (2009). In vitro morphogenesis of organogenic nodules derived from Sclerocarya birrea subsp. caffra leaf explants. Plant Cell, Tissue and Organ Culture 98: 273-280. Contributions: All laboratory experiments and microscopy work were done by the first author under the supervision of Prof Johannes Van Staden and Dr Jeffrey F Finnie. PUBLICATION 3 MACK MOYO, JEFFREY F FINNIE, JOHANNES VAN STADEN. Caulogenesis, rhizogenesis and tissue browning in Sclerocarya birrea. Plant Growth Regulation (In preparation). iv Contributions: All laboratory experiments and scanning electron microscopy work were done by the first author under the guidance of my PhD supervisors, Prof Johannes Van Staden and Dr Jeffrey F Finnie. Signed: v CONFERENCE PUBLICATIONS DETAILS OF CONTRIBUTION TO CONFERENCES that form part and/or include research presented in this thesis: 1. Fourth World Congress on Medicinal and Aromatic Plants (WOCMAP IV): 9–14 November 2008; Cape Town International Conference Centre, Cape Town, SOUTH AFRICA. Title of Poster: M MOYO, MG KULKARNI, JF FINNIE, J VAN STADEN (2008). After-ripening, light conditions and cold stratification of marula [Sclerocarya birrea (A. Rich) Hochst. subsp. caffra (Sond.) Kokwaro] seeds. 2. Thirty-fifth Annual Conference of the South African Association of Botanists (SAAB): 19–22 January 2009; Stellenbosch University, SOUTH AFRICA. Title of Paper: M MOYO, JF FINNIE, J VAN STADEN (2009). In vitro morphogenesis of organogenic nodules derived from Sclerocarya birrea subsp. caffra leaf explants. vi ABSTRACT Sclerocarya birrea (marula, Anacardiaceae) is a highly-valued indigenous tree in most parts of sub-Saharan Africa because of its medicinal and nutritional properties. The marula tree is adapted to the semi-arid conditions that characterise most parts of sub- Saharan Africa and renders them unsuitable for conventional crop agriculture. The unique nutritional properties of marula and its high tolerance to dry conditions provide opportunities for its development into a plantation crop. On the other hand, the demand for marula plant parts, mainly the bark and roots as medicinal remedies, poses a great threat to wild populations. In the long term, the growing demand of marula products in the food, pharmaceutical and cosmetic industries will not be sustainable from wild populations alone. Plant tissue culture technologies can be useful for in vitro manipulation and mass propagation of the plant in the process of domestication and conservation. The aims of the project were to determine the optimum conditions for seed germination, in vitro propagation and plant regeneration, and to evaluate the potential bioactivity of secondary metabolites from its renewable plant parts as an alternative option in the conservation of S. birrea. An ex vitro seed germination study indicated that after-ripening and cold stratification are critical factors. Cold stratification (5 °C) of marula nuts for 14 days improved germination (65%) as compared to non-stratified nuts (32%). Direct shoot organogenesis was achieved from leaf explants through the induction of nodular meristemoids on Murashige and Skoog (MS) (1962) medium and woody plant medium (WPM) supplemented with 6-benzyladenine (BA) in combination with naphthalene acetic acid (NAA), indole-3-butryric acid (IBA) and indole-3-acetic acid (IAA). Induction of nodular meristemoids from 86% of the leaf cultures was achieved on a MS medium with 4.0 µM BA and 1.0 µM NAA. High levels (78–100%) of induction were also achieved on WPM with different concentrations of BA (1.0–4.0 µM) and IBA (1.0–4.0 µM). The highest conversion of nodular meristemoids into shoots on MS initiation medium was only 22% for 4.0 µM BA and 1.0 µM NAA. This was improved to 62% when nodular clusters were cultured in MS liquid medium. Histological studies vii revealed high numbers of unipolar meristematic buds developing from globular nodules. These embryo-like structures have in the past been mistaken for true somatic embryos. The initiation of high numbers of nodular meristemoids per explant provides potential for automated large-scale clonal propagation in bioreactors, in vitro phytochemical production and the development of synthetic seed technology, similar to somatic embryogenesis. Plant regeneration through nodule culture has potential for application in mass micropropagation and plant breeding of S. birrea. Adventitious shoot and root induction are important phases in micropropagation. Plant growth regulators play an important role in these developmental processes, and the type and concentration used have major influences on the eventual organogenic pathway. Three auxins (IAA, IBA and NAA) and four aromatic cytokinins (6-benzyladenine, meta-topolin, meta-topolin riboside, and meta-methoxytopolin riboside) were evaluated for their potential to induce adventitious shoot and root formation in S. birrea shoots, hypocotyls and epicotyls. Among the evaluated cytokinins, the highest adventitious shoot induction (62%) was achieved on MS medium supplemented with meta-topolin (8.0 µM). The lowest adventitious shoot induction (2.5%) was obtained on MS basal medium containing 2.0 µM meta-methoxytopolin riboside. The highest adventitious shoot induction for hypocotyls was 55% on MS medium supplemented with 8.0 µM meta-topolin. For the tested auxins, IBA induced adventitious rooting in 91% of shoots at a concentration of 4.0 µM after 8 weeks in culture. However, the in vitro rooted plants only survived for two weeks when transferred ex vitro. A temperature of 25 °C and 16-h photoperiod were optimum for adventitious root induction. Stomatal density (number per mm2) on the abaxial leaf surfaces was higher for the 16-h photoperiod treatment (206.6 ± 15.28) compared to that for a 24-h photoperiod