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In vitro culture initiation and multiplication There was a significant difference between the number of shoots produced on media supplemented with 2.5 M mTR and the number of shoots produced on media supplemented with all the kinetin concentrations tested including 2.5 M kinetin (Figure 5.9). Although the highest number of shoots were also produced on media supplemented with 2.5 M mTR, there was no significant difference between the number of shoots produced on this medium and the number of shoots produced on a medium supplemented with 2.5, 5.0 or 7.5 M BA, 5.0 or 7.5 M mTR, or 2.5 or 5.0 MemTR. The shoots produced on a medium supplemented with 2.5 M mTR appeared healthy and 40% of them were rooted. All other media treatments also produced healthy shoots of which at least 20% were rooted, except for media supplemented with BA, on which vitrified shoots were observed as with previous experiments. Average number of shoots per explant 8 6 4 2 0 cd Control d 2.5 µM kinetin cd 5.0 µM kinetin bcd 7.5 µM kinetin ab 2.5 µM BA abcd 5.0 µM BA abc 7.5 µM BA bcd 2.5 µM mT bcd 5.0 µM mT cd 7.5 µM mT a 2.5 µM mTR Media treatments abcd 5.0 µM mTR abcd abc Figure 5.9: Effect of three different concentrations of five cytokinins on multiplication of Romulea sabulosa shoots after 2 months. Error bars indicate standard error of the mean. Letters shows significant differences between treatments according to Duncan’s multiple range test. 7.5 µM mTR 2.5 µM MemTR abcd 5.0 µM MemTR bcd 7.5 µM MemTR 133
5.4 DISCUSSION In vitro culture initiation and multiplication Preliminary experiments with R. diversiformis, R. flava, R. leipoldtii and R. minutiflora seedlings showed that hypocotyls were the only seedling organs that gave an in vitro response for the Romulea species tested. They also show that R. leipoldtii and R. flava form the highest number of shoots per seedling hypocotyl compared to the other species tested. Because of possible vitrification and culture incompetence, the use of BA and shoots generated from callus should be avoided for this genus. The in vitro response of R. diversiformis embryos and hypocotyls, although morphologically distinct, is equally unproductive, as no shoots were formed after 2 months. A higher concentration of cytokinins, or different cytokinins, is perhaps needed to generate a more pronounced shooting response for explants of this species. The rooting of shoots formed in vitro of all species, except R. diversiformis and R. monadelpha was inhibited to a certain degree by a medium supplemented with 23.2 M kinetin. Experiments performed with the aim of finding a concentration of kinetin high enough to induce shooting, but low enough to allow for normal root growth, is necessary for the efficient production of healthy shoots of these species. Such an experiment should investigate the effect of various kinetin concentrations between 7.5 and 23.2 µM. Although R. leipoldtii embryos produced significantly more shoots on a medium with 23.2 µM mTR and 0.5 µM NAA, this medium and a medium supplemented with 2.3 µM mTR and 0.5 µM NAA resulted in a higher percentage of cultures with callus. This indicates that although these are the best media for culture initiation, they are not suitable for multiplication of shoots of R. leipoldtii. Shoots produced from this callus were vitrified. The fact that there is no significant difference between the number of shoots produced by embryos and hypocotyls of R. leipoldtii suggests that excising the embryos of even the smallest seeds of all species presented here, except for R. 134
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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
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 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 157: In vitro culture initiation and mul
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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