Selection and micropropagation of Solanum nigrum genotypes with varying calcium and iron content by Kimerra Goordiyal Submitted in fulfilment of the academic requirements for the degree of Master of Science in the School of Life Sciences, University of KwaZulu-Natal, Durban, South Africa January 2018 As the candidate’s supervisor I have/have not approved this dissertation for submission Supervisor Signed: Name: Dr S. Shaik Date: Co-supervisor Signed: Name: Prof. M.P Watt Date: _ ABSTRACT A direct organogenesis protocol was established for Solanum nigrum using leaf explants from seedling plants. The post acclimatisation yield of the seedling-derived leaf explants was 25 plants/explant. It included decontaminating the leaves with 1 % (v/v) sodium hypochlorite and Tween 20® (10 min), shoot multiplication on medium containing 3 mg l-1 benzylaminopurine (BAP) for 4 weeks, elongation on medium containing 0.1 mg l-1 BAP for a week, rooting on hormone-free Murashige and Skoog medium for 3 weeks and acclimatisation in pots (1 soil : 2 vermiculite [1S : 2V]) in a growth room for 2 weeks. A population of fifty 6-week old seedlings were screened using Inductively Coupled Plasma- Optical Emission Spectrometry. They varied in leaf calcium (Ca) (331.05-916.30 mg 100 g-1 dry mass [DM]) and iron (Fe) (0.64-14.95 mg 100 g-1 DM) contents. Based on these results, genotypes for high Ca (G5 and G20), high Fe (G6 and G15), low Ca (G43 and G45) and low Fe (G35 and G50) were selected for further investigation. These were micropropagated using the established protocol to determine whether their clones maintained similar levels of Ca and/or Fe to those of their parents when grown in soil. Micropropagation influenced the Ca and Fe levels of the clones of the selected genotypes, i.e. the 6-week old clones of six (i.e. G5, G20 and G45 for Ca; and G6, G15 and G50 for Fe) out of the eight selected genotypes had either significantly higher (G45 and G50) or lower (G5, G6, G15 and G20) levels of Ca and/or Fe than their 6-week old parents when grown in soil. There were also genotypic differences regarding the in vitro and ex vitro growth responses (i.e. percentage of explants with shoots, number of shoots/explant and post acclimatisation yield) and leaf Ca and Fe levels of the clones of the selected genotypes. The Ca and Fe contents of the clones of most of the selected genotypes were not affected by substrate type, suggesting that both soil and 1S : 2V were adequate to grow the S. nigrum genotypes for the benefit of high Ca and Fe. Four of the selected genotypes (viz. G15, G20, G35 and G43) were then chosen to investigate the effect of physiological age on their leaf Ca and Fe contents when grown in soil and 1S : 2V. Advancing age only affected the Fe levels of the G35 clones in soil and G15 clones in both the tested substrates. The significant differences in the Ca (G20>G43) and Fe (G15>G35) contents of the 6-week old parents were compared with those of their clones at 4, 6 and 8 weeks ex vitro in soil. A similarity (i.e. G20>G43) in the ‘ranking’, was only found at 4 weeks ex vitro for Ca. Initial attempts to establish a minimal growth protocol for storing germplasm of the eight selected genotypes showed that in vitro shoots could be kept on medium containing ¼ MS + 5 ii g l-1 sucrose for 8 weeks. After this period, the in vitro shoots of the eight selected genotypes that were placed onto shoot multiplication medium produced 7-13 shoots/explant. It can be concluded that micropropagation and genotype influenced both the Ca and Fe levels of the selected genotypes, while physiological age only influenced their Fe content. As the present study was a smaller component of a larger research program, further investigations need to be carried out on the selected S. nigrum genotypes prior to distribution to community gardens. Future work should include evaluating the effects of various environmental conditions (i.e. growth in a greenhouse, shadehouse, glasshouse and/or in the field), different light intensities, watering regimes, fertiliser treatments and soil pH on the growth, and leaf Ca and Fe levels of the clones. Regarding minimal growth storage, future work should include investigating whether the germplasm of the eight selected genotypes can be stored under the same conditions for longer than 8 weeks. iii DECLARATION I, Kimerra Goordiyal, declare that: 1. The research reported in this dissertation, except where otherwise indicated, is my original research. 2. This dissertation has not been submitted for any degree or examination at any other tertiary institution. 3. This dissertation does not contain other person’s data, graphs, pictures or other information, unless specifically acknowledged as being sourced from other persons. 4. This dissertation does not contain other persons’ writing, unless specifically acknowledged as being sourced from other researchers. Where other written sourced have been quoted, then: a. Their words have been re-written but the general information attributed to them had been referenced. b. Where their exact words have been used, then their writing has been placed in italics and within quotation marks, and referenced. 5. This dissertation does not contain texts, tables, graphs, figures or graphics copied and pasted from the internet, unless specifically acknowledged and the source being detailed in the dissertation and in the References sections. Signed_____ ________________ iv PREFACE The experimental work described in this dissertation was conducted at the University of KwaZulu-Natal, School of Life Sciences under the supervision of Prof. M.P. Watt and Dr. S. Shaik. The research was financially supported by the National Research Foundation (NRF). These studies represent original work by the author and have not otherwise been submitted in any form for any degree or diploma to any tertiary institution. Where use has been made of the work of others, they have been acknowledged. v TABLE OF CONTENTS ABSTRACT ............................................................................................................................... ii DECLARATION ...................................................................................................................... iv PREFACE .................................................................................................................................. v LIST OF TABLES .................................................................................................................... ix LIST OF FIGURES ............................................................................................................. xviii ACKNOWLEDGMENTS ...................................................................................................... xxi LIST OF ABBREVIATIONS ................................................................................................ xxii 1. Introduction and Rationale ..................................................................................................... 1 2. Literature Review................................................................................................................... 5 2.1 Background and importance of Solanum nigrum ................................................................ 5 2.2 Taxonomy, growth and genetics of Solanum nigrum .......................................................... 6 2.3 Calcium and iron content in Solanum nigrum ................................................................... 12 2.4 Factors that affect micronutrient content in ALVs ............................................................ 12 2.5 Propagation of Solanum nigrum ........................................................................................ 17 2.5.1 Conventional propagation ............................................................................................... 17 2.5.2 In vitro propagation......................................................................................................... 18 2.6 In vitro conservation .......................................................................................................... 23 3. Materials and methods ......................................................................................................... 27 3.1 Plant material ..................................................................................................................... 27 3.1.1 Whole plants ................................................................................................................... 27 3.1.2 Seeds ............................................................................................................................... 27 3.2 Plant regeneration from nodal and leaf explants................................................................ 29 3.2.1 Surface decontamination ................................................................................................. 29 3.2.2 Media preparation ........................................................................................................... 29 3.2.3 Multiplication .................................................................................................................. 29 3.2.4 Elongation and rooting .................................................................................................... 30 vi 3.2.5 Acclimatisation ............................................................................................................... 30 3.3 Determination of calcium and iron content in leaves