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1.2 AIMS AND HYPOTHESES Introduction The general aim of this study was to investigate the conditions that promote growth and development in a number of Romulea species both ex vitro and in vitro to aid its commercialisation and conservation. In more detail, the aims of this study were: • To investigate the environmental factors that influence the development and growth of R. sabulosa and R. leipoldtii in their natural habitat and to replicate these conditions for ex vitro growth; • To investigate the germination physiology of R. camerooniana, R. diversiformis, R. flava, R. leipoldtii, R. minutiflora, R. monadelpha, R. rosea and R. sabulosa and to improve seed germination in some of these species that show low germination; • To obtain suitable protocols to initiate cultures for the micropropagation of R. diversiformis, R. flava, R. leipoldtii, R. monadelpha, R. minutiflora and R. sabulosa; • To establish suitable protocols for shoot multiplication for R. leipoldtii, R. minutiflora and R. sabulosa; • To investigate whether embryogenesis readily occurs in the presence of 2,4-D; • To establish protocols for in vitro corm production and ex vitro corm germination for R. leipoldtii, R. minutiflora and R. sabulosa; • To establish an in vitro flowering protocol for R. minutiflora; and • To promote the commercialisation of Romulea species It was expected that there will be a correlation between geographical distribution and suitable ex vitro and in vitro stimuli. The suitable culture conditions were expected to be similar to that of Crocus species due to their close relationship with this genus. 3
Introduction The subgenera Romulea and Spatulanthus were expected to have differentially suitable conditions for ex vitro and in vitro development and growth. 1.3 GENERAL OVERVIEW OF THESIS CONTENT Chapter 2 is a review of literature available on aspects relative to this study. It firstly covers the distribution, morphology, life-cycle, habitat and conservation status of species in the genus Romulea. It then discusses other studies performed on ecophysiology and propagation of this genus with descriptions of phylogeny and taxonomy. It further reviews the ecophysiological techniques of soil sampling and analysis. A review of seed physiology and techniques applicable to this study is included in this chapter. It also gives a review on micropropagation in general, discusses some in vitro techniques applicable to the study, placing emphasis on explant selection, culture initiation and multiplication, embryogenesis, in vitro corm formation, in vitro flowering and ex vitro acclimatization. A summary of the micropropagation of species in the family Iridaceae is included. In Chapter 3 the habitat of some Romulea species is investigated further through ecophysiological techniques of soil sampling and analysis. Chapter 4 is an examination of the germination physiology of some Romulea species. This was done firstly by examining the physical properties and viability of the seeds, and then investigating the effect of an array of physical and chemical stimuli on germination. The physical properties of the seeds; imbibition rate, moisture content and viability of seeds were determined. The seed coat and micropylar regions were examined using scanning electron microscopy. To test for suitable stimuli for germination, the effect of temperature and light, cold and warm stratification, acid and sand paper scarification, plant growth promoting substances, deficiency of nitrogen, phosphorous and potassium, and different light spectra (phytochromes) on germination were examined. Chapter 5 is an examination of the suitability of various explant types and media supplementations for culture initiation. Two explant types were used; seedling organs and embryos. It also investigates the effect of various physical and chemical 4
Page 1 and 2: Propagation of Romulea species Pier
Page 3 and 4: Contents 2.7 GERMINATION PHYSIOLOGY
Page 5 and 6: Contents 5.3.1 Explants from seedli
Page 7 and 8: Abstract The suitability of various
Page 9 and 10: Declarations I Pierre André Swart,
Page 11 and 12: Declarations DETAILS OF CONTRIBUTIO
Page 13 and 14: Publications from this thesis ASCOU
Page 15 and 16: List of Figures Figure 2.13: Suther
Page 17 and 18: was significantly different from th
Page 19 and 20: List of Tables Table 2.1: Names of
Page 21 and 22: List of Abbreviations 2,4-D 2,4-Dic
Page 23 and 24: Die vlakhaas spits sy oor hy het di
Page 25 and 26: As die mens dan ook so sy dankbaarh
Page 27: Introduction where a great number o
Page 31 and 32: 2 Literature review Leef van daad e
Page 33 and 34: Literature review Figure 2.2: Life
Page 35 and 36: Literature review Figure 2.4: Life
Page 37 and 38: Literature review The plants are sh
Page 39 and 40: Literature review flowers (DE VOS,
Page 41 and 42: Literature review often found on cl
Page 43 and 44: Literature review flowers of R. lei
Page 45 and 46: Literature review flattened upper s
Page 47 and 48: 2.2.16 Romulea tabularis Literature
Page 49 and 50: Literature review extinct, R. papyr
Page 51 and 52: Literature review R. leipoldtii occ
Page 53 and 54: Literature review Figure 2.8: Calvi
Page 55 and 56: Literature review Figure 2.14: Fras
Page 57 and 58: Figure 2.20: Nieuwoudtville weather
Page 59 and 60: Literature review Figure 2.26: Grah
Page 61 and 62: Literature review Figure 2.29: Diag
Page 63 and 64: Literature review Figure 2.30: A te
Page 65 and 66: Literature review Macronutrients ca
Page 67 and 68: Literature review The soils of the
Page 69 and 70: Literature review The embryo is the
Page 71 and 72: Literature review Phase 2 is seen a
Page 73 and 74: Literature review endogenous gibber
Page 75 and 76: Literature review tolerable level (
Page 77 and 78: Literature review these channels pe
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Literature review 2.8.7 Seed dorman
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Literature review the embryo (COPEL
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Literature review germinated under
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Literature review (COPELAND, 1976).
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2.9 BRIEF REVIEW OF IN VITRO CULTUR
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Literature review (DEBERGH, 1994).
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Literature review PIERIK (1997) sta
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Literature review The distinguishin
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Literature review The essential fun
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2.9.3.4 Abscisic acid Literature re
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Table 2.6: Example of a matrix to e
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Literature review Young embryos req
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Literature review and the addition
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Literature review organ is then pro
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Literature review environmental str
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2.11 IN VITRO FLOWERING Literature
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Literature review higher regenerati
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Subfamily Ixioideae (continued) Tri
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Literature review Table 2.8: Corm i
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3 Investigation into the habitat of
Page 119 and 120:
Soil sampling and analysis The firs
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Soil sampling and analysis Table 3.
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4 Germination physiology 4.1 INTROD
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4.2.2 Water content and imbibition
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Germination physiology (-N), phosph
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Germination physiology rosea seeds
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Germination physiology Figure 4.4:
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Germination (%) 50 40 30 20 10 0 77
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Germination (%) 100 80 60 40 20 0 c
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Germination physiology The relative
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Germination physiology seeds of R.
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5.2 MATERIALS AND METHODS In vitro
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In vitro culture initiation and mul
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5.2.3 Explant comparison In vitro c
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5.4 DISCUSSION In vitro culture ini
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6 In vitro corm formation and flowe
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In vitro corm formation and floweri
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6.3 RESULTS 6.3.1 Corm formation In
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Commercialization potential of Romu
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Commercialization potential of Romu
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BASKIN, C. C., and BASKIN, J. M. (1
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Literature cited DOWLING, P. M., CL
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Literature cited HILTON-TAYLOR, C.
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Literature cited KUMAR, A., SOOD, A
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Literature cited PIERIK, R. L. M. (
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Literature cited STEINITZ, B., COHE
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