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In vitro culture initiation and multiplication Another experiment was conducted with R. sabulosa shoots to investigate the effect of other cytokinins and different concentrations of kinetin on shoot multiplication. R. sabulosa shoots initiated and multiplied with 23.2 M kinetin were placed in 250 ml jars containing 30 ml MS media supplemented with no plant growth regulators (control) or 2.5, 5.0, 7.5 M kinetin, BA, mT, mTR or MemTR. For each treatment, 5 replicate bottles with 4 explants each were used. After 2 months the number of shoots formed was recorded. 5.2.5 Statistical analysis Data were analyzed for significant differences by one-way analysis of variance (ANOVA), and means separated using either Tukey’s HSD test or Duncan’s multiple range test (DMRT) at 5% level of significance (P 0.05). Percentage data were converted to proportion, arcsine transformed and then analyzed. GenStat ® (VSN International, Hemel Hempstead, U.K.) version 11.1 statistical package was used to analyze the data. 5.3 RESULTS 5.3.1 Explants from seedlings In the preliminary experiment with R. diversiformis, R. flava, R. leipoldtii and R. minutiflora, no shoots developed from root and shoot explants and shoots only developed from seedling hypocotyls (Results not shown due to low replication). Hypocotyls of R. leipoldtii placed on a medium supplemented with 22.2 µM BA developed the largest number of shoots. R. flava seedling hypocotyls produced less shoots per explant than those of R. leipoldtii on the same medium. R. minutiflora hypocotyls only formed shoots when placed on a medium supplemented with 23.2 µM kinetin and 5.4 µM NAA. R. diversiformis hypocotyls formed no shoots and only developed abnormal root-like structures. The shoots of R. leipoldtii appeared vitrified and when 50 shoots, multiplied on a medium supplemented with 23.2 µM kinetin, were placed in 33 ml culture tubes with 10 ml of MS media with no plant growth regulators, no rooting was observed after 2 months. 121
In vitro culture initiation and multiplication R. flava and R. leipoldtii produced callus with shoot initials on the media treatments with the highest concentrations of both cytokinins and auxins. These shoot initials and the shoots formed after one month from them, when transferred MS media with no plant growth regulators, also appeared vitrified and no roots or root initials were observed after 2 months. 5.3.2 Explants from embryos R. diversiformis embryos only showed swelling, except for one embryo which only developed one rooted shoot (Figure 5.2). R. flava embryos produced significantly more shoots when placed on a medium supplemented with either 2.3 µM kinetin or 4.7 µM kinetin compared to the control (Figure 5.3). The number of R. flava shoots observed on a medium with 4.7 µM kinetin was significantly higher than the number of shoots produced by all other treatments except on a medium supplemented with 2.3 µM kinetin. Single non-rooted shoots and swellings were observed on a medium with no plant growth regulators. Non-rooted shoots, rooted shoot clusters and swelling were observed on a medium supplemented with 2.3 µM kinetin. Only one of the shoot clusters on this medium did not produce a root, this shoot cluster had callus at its base. Non-rooted shoots, rooted and non-rooted shoot clusters and callus were produced on a medium supplemented with 4.7 µM kinetin. Shoot clusters and callus was observed on media supplemented with 23.2 µM kinetin. All but one of the shoot clusters formed on media supplemented with 23.2 µM kinetin did not root (Data not shown). Shoots generated from callus appeared vitrified and they also did not root on MS media with no plant growth regulators. The number of shoots of R. minutiflora and R. monadelpha produced on media supplemented with kinetin was much lower than the number of shoots produced by R. flava embryos and is not significantly different from the number of shoots produced on a medium with no plant growth regulators (Figures 5.4 and 5.5). Table 4.5 in Chapter 4 shows that most of the embryos of these species show an in vitro growth response. In all cases where shoots were not formed, only swelling of the embryo was observed for these two species. One swollen embryo of R. minutiflora rooted on a medium with no plant growth regulators. Single shoots and one small 122
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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
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
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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: 5.2.3 Explant comparison In vitro c
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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