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In vitro corm formation and flowering and ex vitro acclimatization corm formation was observed after 2 and 4 months and no tunic development was seen after 5 months. The shoots of R. minutiflora produced from seedling hypocotyls on a medium supplemented with 23.2 µM kinetin and 5.4 µM NAA were multiplied on the same medium for 6 months. These shoots were placed singly in 33 ml culture tubes on 10 ml of MS media supplemented with 3%, 6% or 9% sucrose or 5.0 g.l -1 activated charcoal. The tubes were then placed in growth chambers maintained at 10°C, 15°C, 20°C, 25°C or 30°C. The remainder of these shoots were multiplied for a further 4 months and subsequently used in an experiment to test the effect of growth retardants on corm formation in R. minutiflora. The shoots were placed in 33 ml culture tubes on 10 ml of MS medium supplemented with 3.4, 17.0 or 34.0 M paclobutrazol (PP3), or 4.9 M abscisic acid (ABA). These tubes were placed at 25°C under 4.3 µmol m –2 s –1 light supplied by Osram ® 75 W cool white fluorescent tubes. Twenty replicates were used per treatment. The shoots produced by seedling hypocotyls of R. leipoldtii on a medium supplemented with 22.2 µM BA were multiplied on this medium for 4 months. The shoots were placed in 33 ml culture tubes on 10 ml of MS medium supplemented with 3%, 6% or 9% sucrose or 5.0 g.l -1 activated charcoal. These tubes were placed at 25°C under 4.3 µmol m –2 s –1 light supplied by Osram ® 75 W cool white fluorescent tubes and 50 replicates were used per treatment. The shoots of R. sabulosa produced from excised embryos on a medium supplemented with 23.2 µM kinetin were multiplied on the same medium for 6 months. These shoots were placed singly in 33 ml culture tubes on 10 ml of MS media supplemented with 3%, 6% or 9% sucrose, 5.0 g.l -1 activated charcoal or 23.2 µM kinetin. The tubes were then placed in growth chambers were maintained at 10°C, 15°C, 20°C, 25°C or 30°C. Ten shoots were used per treatment and the experiment was repeated three times. Corm induction percentage and corm diameter for different temperatures and media compositions were analyzed for significant differences with Duncan’s multiple range 139
In vitro corm formation and flowering and ex vitro acclimatization test using Genstat. To examine the combined effect of treatments on corm induction rate and corm weight, product analysis was done. This was done by multiplying the proportion of corm induction with the mass of the corms produced (mg) and dividing this number with hundred according to the methods of ASCOUGH et al. (2008). A high value indicates that the treatment resulted in a high proportion of induction and corm mass, while a low value indicates that either the proportion of induction, the corm mass or both are low. 6.2.2 In vitro flowering After an in vitro flower was observed in the R. minutiflora corm formation treatments (9% sucrose at 20°C) further experiments to repeat this result were initiated. Information from UYEMURA & IMANISHI (1984), FLAISHMAN & KAMENETSKY (2006), LIGHT et al., (2007) and a study by KÖK (2007) on the difference in soil composition during vegetative growth and flowering in R. columnae was used to design an in vitro flowering experiment. As it is better to use larger storage organs for florogenesis studies, only the largest corms were selected (FLAISHMAN & KAMENETSKY, 2006). These corms were placed in test tubes in a growth chamber with high light (190.1 µmol m –2 s –1 ) set at 20°C. Medium treatments included a half strength MS medium, a half strength MS medium with 9% sucrose, a full strength MS medium with 9% sucrose, a full strength MS medium with 1:500 (v/v) smoke water and a full strength MS medium with 3% sucrose as a control. For each treatment 20 corms of R. minutiflora and R. sabulosa between 5 and 10 mm in diameter were used. 6.2.3 Ex vitro acclimatization and corm viability Before all corms and plantlets were used for ex vitro growth studies they were rinsed in autoclaved distilled water to remove agar from roots. Corms were partially dried in vitro by placing them inside a Petri dish on a laminar flow bench for two weeks before planting. The mist-house and greenhouse used in these experiments is situated in the University of KwaZulu Natal Botanical Garden in Pietermaritzburg (30° 24’ E, 29° 37’ S, 655 m above sea level). The mist-house had a misting interval of 15 min and a misting duration of 10 s, whereas the greenhouse did not have an automated 140
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Propagation of Romulea species Pier
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Contents 2.7 GERMINATION PHYSIOLOGY
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Contents 5.3.1 Explants from seedli
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Abstract The suitability of various
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Declarations I Pierre André Swart,
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Declarations DETAILS OF CONTRIBUTIO
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Publications from this thesis ASCOU
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List of Figures Figure 2.13: Suther
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was significantly different from th
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List of Tables Table 2.1: Names of
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List of Abbreviations 2,4-D 2,4-Dic
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Die vlakhaas spits sy oor hy het di
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As die mens dan ook so sy dankbaarh
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Introduction where a great number o
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Introduction The subgenera Romulea
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2 Literature review Leef van daad e
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Literature review Figure 2.2: Life
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Literature review Figure 2.4: Life
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Literature review The plants are sh
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Literature review flowers (DE VOS,
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Literature review often found on cl
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Literature review flowers of R. lei
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Literature review flattened upper s
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2.2.16 Romulea tabularis Literature
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Literature review extinct, R. papyr
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Literature review R. leipoldtii occ
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Literature review Figure 2.8: Calvi
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Literature review Figure 2.14: Fras
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Figure 2.20: Nieuwoudtville weather
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Literature review Figure 2.26: Grah
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Literature review Figure 2.29: Diag
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Literature review Figure 2.30: A te
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Literature review Macronutrients ca
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Literature review The soils of the
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Literature review The embryo is the
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Literature review Phase 2 is seen a
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Literature review endogenous gibber
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Literature review tolerable level (
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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
Page 113 and 114: Subfamily Ixioideae (continued) Tri
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Page 117 and 118: 3 Investigation into the habitat of
Page 119 and 120: Soil sampling and analysis The firs
Page 121 and 122: Soil sampling and analysis Table 3.
Page 123 and 124: 4 Germination physiology 4.1 INTROD
Page 125 and 126: 4.2.2 Water content and imbibition
Page 127 and 128: Germination physiology (-N), phosph
Page 129 and 130: Germination physiology rosea seeds
Page 131 and 132: Germination physiology Figure 4.4:
Page 133 and 134: Germination (%) 50 40 30 20 10 0 77
Page 135 and 136: Germination (%) 100 80 60 40 20 0 c
Page 137 and 138: Germination physiology The relative
Page 139 and 140: Germination physiology seeds of R.
Page 141 and 142: 5.2 MATERIALS AND METHODS In vitro
Page 143 and 144: In vitro culture initiation and mul
Page 145 and 146: 5.2.3 Explant comparison In vitro c
Page 159 and 160: 5.4 DISCUSSION In vitro culture ini
Page 163: 6 In vitro corm formation and flowe
Page 167 and 168: 6.3 RESULTS 6.3.1 Corm formation In
Page 169 and 170: In vitro corm formation and floweri
Page 177 and 178: Commercialization potential of Romu
Page 179 and 180: Commercialization potential of Romu
Page 181 and 182: BASKIN, C. C., and BASKIN, J. M. (1
Page 183 and 184: Literature cited DOWLING, P. M., CL
Page 185 and 186: Literature cited HILTON-TAYLOR, C.
Page 187 and 188: Literature cited KUMAR, A., SOOD, A
Page 189 and 190: Literature cited PIERIK, R. L. M. (
Page 191 and 192: Literature cited STEINITZ, B., COHE
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