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In vitro culture initiation and multiplication Seedling organs of R. flava and R. leipoldtii were placed in culture tubes with MS media with nine different plant growth regulator treatments; a control with no plant growth regulators, 4.4 µM BA, 22.2 µM BA, 4.5 µM 2,4-D, 22.6 µM 2,4-D, 4.4 µM BA and 4.5 µM 2,4-D, 4.4 µM BA and 22.6 µM 2,4-D, 22.2 µM BA and 4.5 µM 2,4-D, and 22.2 µM BA and 22.6 µM 2,4-D. Seedling organs of R. diversiformis and R. minutiflora were placed in culture tubes with MS media with 12 different plant growth regulator treatments; a control with no plant growth regulators, 2.3 µM kinetin, 23.2 µM kinetin, 5.4 µM NAA, 26.9 µM NAA, 53.7 µM NAA, 2.3 µM kinetin and 5.4 µM NAA, 23.2 µM kinetin and 5.4 µM NAA, 2.3 µM kinetin and 26.9 µM NAA, 23.2 µM kinetin and 26.9 µM NAA, 2.3 µM kinetin and 53.7 µM NAA, and 23.2 µM kinetin and 53.7 µM NAA. 5.2.2 Explants from embryos The seeds were surface sterilised as with in vitro germination experiments described in Chapter 4 and imbibed for 5 days. Decontaminated seeds were placed in a Petri dish with sterile distilled water. The seeds were left to imbibe in the laminar flow cabinet and were dissected every 24 h to test for ease of dissection. Seeds could only be dissected after 4 days, as the blade could not penetrate the periderm (fruit coat) with a clean cut with less time for imbibition. The embryos that were dissected after 7 days of imbibition did not show any growth response when placed on media. To excise the embryo some of the surrounding endosperm had to be cut away (See Figure 5.1). 117
In vitro culture initiation and multiplication Figure 5.1: General embryo excision procedure for Romulea seeds. An outer view, as one would view it through a stereo microscope, as well as a view relative to the embryo is provided so that the importance of the placing of the incisions can be seen. Step 1 is viewed from the top, Step 2 is a side view, Steps 3 and 5 are bottom views 90° to the incision made in Step 2. Step 4 is a side view. In Step 1 the seed was gripped with a pair of fine forceps and two incisions were made. The excess tissue was scrapped to the front of the Petri dish. In Step 2 the seed was turned onto one of the flat sides created by the incision done in Step 1. When turning on the bottom light of the dissection microscope the light radiated through the seed, making the embryo visible. Another incision was then made below the embryo. The seed was then turned onto the Petri dish as indicated in Step 3 to check for embryo visibility and to allow a better cutting angle. Small slices of tissue could then be removed as indicated in Step 4 until the embryo was clearly visible through the somewhat transparent endosperm tissue. An autoclaved dissection needle was then used to carefully flake away the sections indicated in Step 5 (Figure 5.1). The embryo was then lifted out of the endosperm tissue and placed in a Petri dish with sterile distilled water to rinse off residual pieces of endosperm. Excised embryos were placed into 33 ml culture tubes with 10 ml nutrient media using an autoclaved Pasteur pipette with sterilised distilled water immediately after excision. Treatments with various concentrations of plant growth regulators in the growth media were prepared for each species. 118
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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).
Page 91 and 92: Literature review PIERIK (1997) sta
Page 93 and 94: Literature review The distinguishin
Page 95 and 96: Literature review The essential fun
Page 97 and 98: 2.9.3.4 Abscisic acid Literature re
Page 99 and 100: Table 2.6: Example of a matrix to e
Page 101 and 102: Literature review Young embryos req
Page 103 and 104: Literature review and the addition
Page 105 and 106: Literature review organ is then pro
Page 107 and 108: Literature review environmental str
Page 109 and 110: 2.11 IN VITRO FLOWERING Literature
Page 111 and 112: Literature review higher regenerati
Page 113 and 114: Subfamily Ixioideae (continued) Tri
Page 115 and 116: Literature review Table 2.8: Corm i
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: 5.2 MATERIALS AND METHODS In vitro
Page 145 and 146: 5.2.3 Explant comparison In vitro c
Page 147 and 148: In vitro culture initiation and mul
Page 159 and 160: 5.4 DISCUSSION In vitro culture ini
Page 163 and 164: 6 In vitro corm formation and flowe
Page 165 and 166: In vitro corm formation and floweri
Page 167 and 168: 6.3 RESULTS 6.3.1 Corm formation In
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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