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2.9.4 Liquid culture Literature review In liquid culture, the explant is covered by the medium; enlarging the surface area for absorbing nutrients and plant growth regulators (ASCOUGH & FENNELL, 2004). The medium can be changed automatically, this reduces labour costs of subculturing (ASCOUGH & FENNELL, 2004). Liquid culture does however have a few side-effects (ASCOUGH & FENNELL, 2004). Because of the submersion of tissue, the explant may become oxygen deficient. This leads to the formation of elongated and hyperhydric leaves (ASCOUGH & FENNELL, 2004). There are a few methods to overcome hyperhydricity, they are however not universal and some of them retard growth and multiplication (ZIV, 1989; ASCOUGH & FENNELL, 2004). Gladiolus bud explants have been propagated in an agitated liquid medium (ZIV, 1989). ZIV (1989) concluded that liquid cultures can be used to scale up the micropropagation of Gladiolus sp. and possibly other geophytes as it allows for a faster rate of cormlet production (ZIV, 1989). 2.9.5 Embryo-excision Embryo rescue is known as one of the earliest and most widely used techniques for Iridaceae micropropagation (KRIKORIAN & KANN, 1986). Embryo culture is a well established branch of in vitro culture and is known as one of the oldest and most successful culture procedures (HU & ZANETTINI, 1995; REED, 2005). In embryo rescue, the artificial medium substitutes for the endosperm (REED, 2005). MURASHIGE & SKOOG (1962) is the most frequently used basal media for embryo culture. Embryo development occurs in two phases, a heterotrophic and an autotrophic phase (REED, 2005). In the heterotrophic phase, the young embryo, or “proembryo”, requires a complex medium. In vivo grown embryos at this stage are dependent on the endosperm. Amino acids such as glutamine and asparagine are often added to the culture medium. 75
Literature review Young embryos require a medium with high osmotic potential (PIERIK, 1997). A high osmotic potential prevents precocious development and promotes normal embryogenic development (REED, 2005). Sucrose is usually added to serve both as an osmoticum and a carbon source (PIERIK, 1997). A medium with 8 to 12% sucrose is used for the culture of heterotrophic embryos (REED, 2005). The autotrophic phase is usually initiated in the late heart-shaped embryo stage (REED, 2005). Embryos that are excised during this development stage are completely autotrophic (HU & ZANETTINI, 1995). Such embryos germinate and grow on a simple inorganic medium with a supplemental energy source (HU & ZANETTINI, 1995). An inorganic medium supplemented with 2 to 3% sucrose is used as a standard medium for the germination of autotrophic embryos (REED, 2005). Growth regulators often have inconsistent effects on embryo culture (REED, 2005). They have however been extensively used in embryo rescue protocols, especially protocols involving heterotrophic embryos (REED, 2005). Low concentrations of auxins promote normal growth whereas gibberellins cause embryo enlargement and cytokinins inhibit growth (REED, 2005). Hard-coated seeds are first soaked in water for a few hours up to a few days before dissection. Seeds are surface sterilised before and after soaking (HU & ZANETTINI, 1995). The most suitable point of incision into the ovule differs amongst species (REED, 2005). The embryos of some species can be extracted by cutting off the micropylar end of the ovule and applying gentle pressure at the opposite end of the ovule, so that the embryo is pushed through the opening (REED, 2005). Small seeds are dissected by making a longitudinal section using sterile microdissection needles (HU & ZANETTINI, 1995) After excision, large embryos should immediately be transferred into culture vessels, using a pair of forceps (HU & ZANETTINI, 1995; REED, 2005). Small embryos can be handled using the moistened tip of a dissection needle (HU & ZANETTINI, 1995). 76
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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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Page 51 and 52: Literature review R. leipoldtii occ
Page 53 and 54: Literature review Figure 2.8: Calvi
Page 55 and 56: Literature review Figure 2.14: Fras
Page 57 and 58: Figure 2.20: Nieuwoudtville weather
Page 59 and 60: Literature review Figure 2.26: Grah
Page 61 and 62: Literature review Figure 2.29: Diag
Page 63 and 64: Literature review Figure 2.30: A te
Page 65 and 66: Literature review Macronutrients ca
Page 67 and 68: Literature review The soils of the
Page 69 and 70: Literature review The embryo is the
Page 71 and 72: Literature review Phase 2 is seen a
Page 73 and 74: 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: Table 2.6: Example of a matrix to e
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 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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In vitro culture initiation and mul
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5.4 DISCUSSION In vitro culture ini
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6 In vitro corm formation and flowe
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In vitro corm formation and floweri
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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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