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Germination physiology subjected to dark treatments were inspected every second day under a green “safe light” with a PPFD of 0.3 mol m −2 s −1 . Mean germination time (MGT) was calculated using the equation: MGT = (n×d)/N, where ‘n’ is the number of seeds germinated on each day, ‘d’ is the number of days the experiment has been active and ‘N’ is the total number of seeds germinated after 90 days (ELLIS & ROBERTS, 1981). To test the effect of temperature and light on germination of four Romulea species, decontaminated seeds were placed in growth chambers set at 10, 15, 20, 25, 30 and 30/15 C (day/night) with 16 h light: 8 h dark photoperiod. Petri dishes were covered with aluminium foil for the dark treatment and were only opened under a ‘safe light’ as indicated earlier. For the stratification experiment, surface decontaminated seeds were placed between two layers of paper towelling moistened with distilled water. The towels were placed inside a plastic bag and were covered with aluminium foil to eliminate light. Seeds of all species were placed in a refrigerator at 5 C, except those of R. rosea which were placed at 30 C, as EDDY & SMITH (1975) reported pre-chilling did not improve the germination of this species. Seeds were kept under these conditions for 7, 14 and 21 days before placing them for germination in a growth chamber set at 20 C. Control seeds did not receive stratification treatment and were placed in the same growth chamber with the seeds of the 7 day stratified set. For acid scarification, seeds were placed in 50% sulphuric acid for 5 min. Seeds were also mechanically scarified by rubbing them between two pieces of sand paper (P120 grade) for 1 min. Control seeds were not subjected to scarification treatment. To test the effect of various plant growth promoting substances, seeds of R. rosea were placed in Petri dishes with filter papers moistened separately with 10 -5 M of GA3, Kinetin, KNO3, IBA, NAA and IAA. Smoke water concentration of 1:500 (v/v) and butenolide (3-methyl-2H-furo[2,3-c]pyran-2-one) solution of 10 -8 M were also used in this study. Butenolide is a compound isolated from plant-derived smoke water that enhances germination of many seeds, including Eucomis (KULKARNI et al., 2006). Plant-derived smoke water was prepared according to BAXTER et al. (1994) and butenolide was isolated by the method of VAN STADEN et al. (2004). The effect of macro-nutrient deficiency was investigated by placing seeds on filter paper moistened with 3.5 ml of 50% Hoagland’s nutrient medium deficient of either nitrogen 101
Germination physiology (-N), phosphorous (-P) or potassium (-K) respectively. The seeds that were germinated with 50% Hoagland’s medium consisting of all nutrients and with only distilled water served as controls. The filter paper was kept moist with these solutions throughout the duration of the experiment to maintain the levels of nutrients. 4.2.5 In vitro germination experiments Seeds were decontaminated in accordance with the methods of ASCOUGH et al. (2007). Seeds were placed in 1.75% sodium hypochlorite solution with a few drops of Tween 20 for 15 min, after which they were rinsed three times with sterile distilled water. All seeds were placed in 33 ml culture tubes with 10 ml 1/10 strength MS media supplemented with 100 mg.l -1 myo-inositol and no sucrose (ASCOUGH et al., 2007). Tubes were placed in a growth chamber set at 15°C and a 16 h light: 8 h dark photoperiod with PPFD of 30.1 ± 4.0 µmol m -2 s -1 . Four replicates of 10 seeds (10 tubes) each of R. autumnalis, R. citrina, R. cruciata, R. diversiformis, R. flava, R. leipoldtii, R. minutiflora, R. pearsonii, R. sabulosa and R. tabularis were used. Considering R. sabulosa’s commercial potential, additional experiments were conducted by placing four replicates of 50 seeds each at 15 and 20°C. 4.2.6 Statistical analysis Percentage germination data were arcsine transformed and analysis of variance (ANOVA) was calculated. Differences between means of percentage germination were tested with Least Significant Difference (LSD) at the 5% level. GENSTAT ® Release 4.21 statistical package was used for analysis. 102
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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
Page 75 and 76: Literature review tolerable level (
Page 77 and 78: Literature review these channels pe
Page 79 and 80: Literature review 2.8.7 Seed dorman
Page 81 and 82: Literature review the embryo (COPEL
Page 83 and 84: Literature review germinated under
Page 85 and 86: Literature review (COPELAND, 1976).
Page 87 and 88: 2.9 BRIEF REVIEW OF IN VITRO CULTUR
Page 89 and 90: 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: 4.2.2 Water content and imbibition
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 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
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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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