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6.4 DISCUSSION In vitro corm formation and flowering and ex vitro acclimatization Temperature is the major factor that affects storage organ morphogenesis (ASCOUGH et al., 2009). Low temperature significantly increased corm formation in R. minutiflora and R. sabulosa. The highest number of instances of multiple corm formation for R. sabulosa was also observed at 10 and 15°C. In a study by ASCOUGH et al. (2008) on Watsonia vanderspuyiae (in the same subfamily, Ixioideae) the highest percentage corm induction was observed for shoots placed at low temperatures. This study showed no significant difference between corm induction percentages of shoots placed on MS media supplemented with sucrose at 10 and 20 °C. Corm formation at low temperatures has been observed in vitro for many genera in the Iridaceae (ASCOUGH et al., 2009). An exception to this is Crinum macowanii, where storage organ formation occurs above 25 °C and is inhibited at lower temperatures (SLABBERT et al., 1993). The fact that corm induction was inhibited for R. minutiflora and R. sabulosa at 25 °C in this study correlates with the results of Gladiolus spp., where corm induction was inhibited at this temperature (TAN NHUT et al., 2004). In this study the requirements for corm formation of R. leipoldtii is similar to that determined in a study involving Crocus sativus corm formation (PLESSNER et al., 1990). It was however shown by HOMES et al. (1987) that corm formation of Crocus sativus also occurred at 30°C, a temperature that totally inhibits the corm formation of R. minutiflora and R. sabulosa and that would probably inhibit the corm formation of R. leipoldtii as it occurs in the same regions as R. minutiflora (Figure 2.1). When PP3 and ABA were added to the medium on which R. minutiflora shoots were placed, these shoots developed corms although they were placed at 25°C, a temperature that totally inhibits corm formation when these retardants that reduce leaf elongation and promote storage organ formation are not present. This effect of PP3 has also been shown for Dierama and Gladiolus species (MADUBANYA, 2004; STEINITZ & LILIEN-KIPNIS, 1989; ZIV, 1989; ZIV et al., 1998). 147
In vitro corm formation and flowering and ex vitro acclimatization The fact that corms were not observed in any R. leipoldtii shoot cultures supplemented with BA after 6 months show that this chemical inhibits corm formation for this genus. The same effect is expected for the other species in this genus, as the shoots of all species generated and multiplied on BA appeared abnormal and their root growth was stunted. In Watsonia cultures, a correlation between corm mass and carbohydrate concentration was observed, with corm induction in some species decreasing as the carbohydrate concentration increases (ASCOUGH et al., 2008). In the present study this was also observed to some extent, as the treatment that delivered the highest corm mass had a 6% sucrose concentration. It was however surprising that elevated levels of carbohydrates did not have a statistically significant effect on corm mass. A two step corm formation protocol would work best for R. sabulosa, as the corms differentiate and accumulate carbohydrates under different temperatures. This two step system would involve placing corms at either 10 or 20°C for a few months and then transferring these cultures to 15°C. A similar two step program was proposed by ASCOUGH et al. (2011) for Tritonia gladiolaris. This two step system forms corms at lower temperatures (10 and 15°C) and promotes the accumulation of carbohydrates at higher temperatures (20°C). They suggest that the physiological mechanisms involved here are probably an adaptation for survival, so that the low temperature is perceived as a cue for corm induction and the onset of dormancy before the approach of unfavourable conditions. Corm production is however promoted at 10 and 20°C and corm mass increases at 15°C, a temperature flanked by the two former temperatures, for R. sabulosa. This phenomenon may be explained by comparing the life cycle of R. sabulosa with the temperature, rainfall and humidity observations for the last five years in Nieuwoudtville. R. sabulosa flowers from August to September. Before flowering there are a few weeks of vegetative growth, facilitated by the increase in rainfall in July. During July and August the averaged daily minimum is 5.1 ± 0.3°C and 4.7 ± 0.2°C and the averaged daily maximum temperature is 18.6 ± 0.6°C and 18.1 ± 0.7°C respectively. 148
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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
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Subfamily Ixioideae (continued) Tri
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Literature review Table 2.8: Corm i
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3 Investigation into the habitat of
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Soil sampling and analysis The firs
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Page 123 and 124: 4 Germination physiology 4.1 INTROD
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Page 133 and 134: Germination (%) 50 40 30 20 10 0 77
Page 135 and 136: Germination (%) 100 80 60 40 20 0 c
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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
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Page 167 and 168: 6.3 RESULTS 6.3.1 Corm formation In
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Page 177 and 178: Commercialization potential of Romu
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Page 183 and 184: Literature cited DOWLING, P. M., CL
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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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